HK1228890B - Compound useful as inhibitors of atr kinase, its preparation, and radiolabelled derivatives thereof - Google Patents

Description

Compounds useful as ATR kinase inhibitors, their preparation, and their radiolabeled derivatives

Background Art

ATR ("ATM and Rad3 related") kinase is a protein kinase involved in the cellular response to DNA damage. ATR kinase acts on ATM ("ataxia telangiectasia mutated") kinase and many other proteins to regulate the cellular response to DNA damage, commonly referred to as the DNA damage response ("DDR"). The DDR stimulates DNA repair, promotes survival, and stalls cell cycle progression by activating cell cycle checkpoints, which allows time for repair. Without the DDR, cells are much more sensitive to DNA damage and are susceptible to DNA damage-induced death, either by endogenous cellular processes such as DNA replication or by exogenous DNA damaging events commonly used in cancer therapy.

Healthy cells can rely on a diverse host of proteins for DNA repair, including the DDR kinase ATR. In some cases, these proteins can compensate for each other by functionally activating redundant DNA repair processes. In contrast, many cancer cells harbor defects in some of their DNA repair processes, such as ATM signaling, and therefore display a heavy reliance on their remaining intact DNA repair proteins, including ATR.

In addition, many cancer cells express activated oncogenes or lack key tumor suppressors, which can predispose these cancer cells to dysregulation of DNA replication, leading to DNA damage. ATR has been implicated as a key component of the DDR that responds to disrupted DNA replication. As a result, the survival of these cancer cells is more dependent on ATR activity than healthy cells. Accordingly, ATR inhibitors can be used for cancer treatment, either alone or in combination with DNA-damaging agents, because they shut down DNA repair mechanisms that are more important for cell survival in many cancer cells than in healthy normal cells.

Indeed, disruption of ATR function (e.g., due to gene deletion) has been shown to promote cancer cell death both in the presence and absence of DNA-damaging agents. This suggests that ATR inhibitors may be effective both as single agents and as potent sensitizers for radiation therapy or genotoxic chemotherapy.

For these reasons, there is a need for the development of potent and selective ATR inhibitors for cancer treatment, either as single agents or in combination with radiation therapy or genotoxic chemotherapy. Furthermore, there may be a need for a synthetic route to ATR inhibitors that is amenable to large-scale synthesis and improves upon currently known methods.

ATR peptides can be expressed and isolated using a variety of methods known in the literature (see, for example, et al., PNAS 99:10, pp6673-6678, May 14, 2002; see also Kumagai et al. Cell 124, pp943-955, March 10, 2006; Unsal-Kacmaz et al. Molecular and Cellular Biology , Feb 2004, p1292-1300; and Hall-Jackson et al. Oncogene 1999, 18, 6707-6713).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1a: XRPD of Compound I-I ethanol solvate

Figure 2a: TGA of compound I-1·ethanol solvate

Figure 3a: DSC of Compound I-1·Ethanol Solvate

Figure 4a: Solid form ¹³ C NMR spectrum of Compound I-1 ethanol solvate (12.5 kHz spinning)

Figure 5a: Solid form ¹⁹ F NMR spectrum of Compound I-1 ethanol solvate (12.5 kHz spinning)

Figure 1b: XRPD of Compound I-1·Hydrate I

Figure 2b: TGA of Compound I-1·Hydrate I

Figure 3b: DSC of Compound I-1·Hydrate I

Figure 4b: XRPD of Compound I-1·Hydrate II

Figure 5b: Solid form ¹³ C NMR spectrum of Compound I-1·Hydrate II (11 kHz spinning)

Figure 6b: Solid form ¹⁹ F NMR spectrum of Compound I-1·Hydrate II (11 kHz spinning)

Figure 1c: XRPD of Compound I-1 Anhydrous Form A

Figure 2c: TGA of Compound I-I Anhydrous Form A

Figure 3c: DSC of Compound I-I Anhydrous Form A

Figure 4c: Conformational diagram of compound I-1 anhydrous form A based on single crystal X-ray analysis

Figure 5c: Conformational diagram showing the stacking order of Compound I-1 Anhydrous Form A

Figure 6c: Solid form ¹³ C NMR spectrum of Compound I-1 Anhydrous Form A (12.5 kHz spinning)

Figure 7c: Solid form ¹⁹ F NMR spectrum of Compound I-1 Anhydrous Form A (12.5 kHz spinning)

Figure 1d: XRPD of Compound I-1 Anhydrous Form B

Figure 2d: TGA of Compound I-1 Anhydrous Form B

Figure 3d: DSC of Compound I-1 Anhydrous Form B

Figure 4d: Solid form ¹³ C NMR spectrum of Compound I-1 Anhydrous Form B (12.5 kHz spinning)

Figure 5d: Solid form ¹⁹ F NMR spectrum of Compound I-1 Anhydrous Form B (12.5 kHz spinning)

Figure 1e: XRPD of Compound I-1 Anhydrous Form C

Figure 2e: TGA of compound I-1·anhydrous form C

Figure 3e: DSC of Compound I-1 Anhydrous Form C

Figure 4e: Solid form ¹³ C NMR spectrum of Compound I-1·Anhydrous Form C (12.5 kHz spinning)

Figure 5e: Solid form ¹⁹ F NMR spectrum of Compound I-1 Anhydrous Form C (12.5 kHz spinning)

Figure 1f: XRPD of compound I-1·amorphous form

Figure 2f: DSC of compound I-1·amorphous form

Figure 3f: Solid form ¹³ C NMR spectrum of amorphous form of compound I-1 (12.5 kHz spinning)

Figure 4f: Solid form ¹⁹ F NMR spectrum of compound I-1·amorphous form (12.5 kHz spinning)

Figure 1g: XRPD of compound I-1·DMSO solvate

Figure 2g: TGA of compound I-1·DMSO solvate

Figure 3g: DSC of compound I-1·DMSO solvate

Figure 1h: XRPD of Compound I-1·DMAC solvate

Figure 2h: TGA of compound I-1·DMAC solvate

Figure 3h: DSC of compound I-1·DMAC solvate

Figure 1i: XRPD of compound I-1·acetone solvate

Figure 2i: TGA of compound I-1·acetone solvate

Figure 3i: DSC of compound I-1·acetone solvate

Figure 1j: XRPD of compound I-1·isopropanol solvate

Figure 2j: TGA of compound I-1·isopropanol solvate

Figure 3j: DSC of compound I-1·isopropanol solvate

Summary of the Invention

The present invention relates to solid forms of ATR inhibitors, compositions comprising ATR inhibitors, and deuterated ATR inhibitors. The present invention also relates to methods and intermediates for preparing compounds useful as ATR kinase inhibitors, such as amino-pyrazolopyrimidine derivatives and related molecules. Amino-pyrazolopyrimidine derivatives are useful as ATR inhibitors and can also be used to prepare ATR inhibitors.

In one aspect, the present invention provides a method for preparing a compound of formula I-A:

Another aspect comprises a method for preparing a compound of formula 1-1:

In another aspect, the present invention comprises a compound of formula I-B:

or a pharmaceutically acceptable salt or derivative thereof, wherein:

Each Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ , Y ⁸ , Y ⁹ , Y ¹⁰ , Y ¹¹ , Y ¹² , Y ¹³ , Y ¹⁴ , Y ¹⁵ , Y ¹⁶ , Y ¹⁷ , Y ¹⁸ and Y ¹⁹ is independently hydrogen or deuterium; provided that at least one of Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ , Y ⁸ , Y ⁹ , Y ¹⁰ , Y ¹¹ , Y ¹² , Y ¹³ , Y ¹⁴ , Y ¹⁵ , Y ¹⁶ , Y ¹⁷ , Y ¹⁸ and Y ¹⁹ is deuterium;

Each of X ¹ , X ² , X ⁴ , X ⁵ , X ⁶ , X ⁷ , X ⁸ and X ⁹ is independently selected from ¹² C or ¹³ C;

X ³ is independently selected from - ¹² C(O)- or - ¹³ C(O)-.

Another aspect of the present invention provides a solid form of a compound of formula I-1:

Some embodiments disclosed herein generally relate to compositions that may include an effective amount of Compound 1-1 or an anhydrous polycrystalline Form A of Compound 1-1 (hereinafter referred to as "Form A"), or a pharmaceutically acceptable salt thereof.

Other embodiments disclosed herein generally relate to methods of preparing the compositions described herein (e.g., compositions that can include an effective amount of Compound I-1 or Form A, or a pharmaceutically acceptable salt thereof). Still other embodiments disclosed herein generally relate to methods of treating cancer using the compositions described herein.

Some embodiments disclosed herein generally relate to the use of a composition described herein (e.g., a composition that can include an effective amount of Compound 1-1 or Form A or a pharmaceutically acceptable salt thereof) in the manufacture of a medicament for treating cancer.

Other aspects of the invention are set forth below.

Detailed Description of the Invention

For the purposes of this application, it will be understood that the terms embodiment, example, and aspect are used interchangeably.

method

Another aspect of the present invention comprises a process for preparing a compound of formula I-A:

Contains a compound of formula 6:

reacting under conditions suitable for forming an amide bond, wherein:

R ¹ is independently selected from fluoro, chloro or -C(J ¹ ) ₂ CN;

J ¹ is independently selected from H or C _1-2 alkyl; or

The two occurrences of ^J1 together with the carbon atoms to which they are attached form a 3-4 membered optionally substituted carbocyclic ring;

R ² is independently selected from H; halogen; -CN; NH ₂ ; C _1-2 alkyl, optionally substituted with 0-3 occurrences of fluorine; or a C _1-3 aliphatic chain, wherein up to two methylene units of the aliphatic chain are optionally replaced by -O-, -NR-, -C(O)-, or -S(O) _n ;

R ³ is independently selected from H; halogen; C _1-4 alkyl, optionally substituted with 1-3 occurrences of halogen; C _3-4 cycloalkyl; -CN; or a C _1-3 aliphatic chain, wherein up to two methylene units of the aliphatic chain are optionally replaced by -O-, -NR-, -C(O)- or -S(O) _n ;

R ⁴ is independently selected from Q ¹ or a C _1-10 aliphatic chain, wherein up to four methylene units of the aliphatic chain are optionally replaced by -O-, -NR-, -C(O)-, or -S(O) _n -; each R ⁴ is optionally substituted with 0-5 occurrences of J ^Q ; or

^R3 and ^R4, together with the atoms to which they are bound, form a 5-6 membered aromatic or non-aromatic ring having 0-2 heteroatoms selected from oxygen, nitrogen or sulfur; the ring formed by ^R3 and ^R4 is optionally substituted with 0-3 occurrences of ^JZ ;

Q ¹ is independently selected from a 3-7 membered fully saturated, partially unsaturated or aromatic monocyclic ring having 0-3 heteroatoms selected from oxygen, nitrogen or sulfur; or a 7-12 membered fully saturated, partially unsaturated or aromatic bicyclic ring having 0-5 heteroatoms selected from oxygen, nitrogen or sulfur;

J ^z is independently selected from C _1-6 aliphatic, =O, halo or →O;

J ^Q is independently selected from -CN; halogen; =0; Q ² ; or a C _1-8 aliphatic chain wherein up to three methylene units of the aliphatic chain are optionally replaced by -O-, -NR-, -C(O)-, or -S(O) _n -; each occurrence of J ^Q is optionally substituted by 0-3 occurrences of J ^R ; or

^JQ occurring twice on the same atom together with the atom to which they are attached form a 3-6 membered ring having 0-2 heteroatoms selected from oxygen, nitrogen or sulfur; wherein the ring formed by the two occurrences of ^JQ is optionally substituted with 0-3 occurrences of ^JX ; or

The two occurrences of J ^Q together with Q ¹ form a 6-10 membered saturated or partially unsaturated bridged ring system;

^Q2 is independently selected from a 3-7 membered fully saturated, partially unsaturated or aromatic monocyclic ring having 0-3 heteroatoms selected from oxygen, nitrogen or sulfur; or a 7-12 membered fully saturated, partially unsaturated or aromatic bicyclic ring having 0-5 heteroatoms selected from oxygen, nitrogen or sulfur;

J ^R is independently selected from -CN; halogen; =O; →O; Q ³ ; or a C _1-6 aliphatic chain wherein up to three methylene units of the aliphatic chain are optionally replaced by -O-, -NR-, -C(O)-, or -S(O) _n -; each J ^R is optionally substituted by 0-3 occurrences of J ^T ; or

J ^R occurring twice on the same atom together with the atom to which they are attached form a 3-6 membered ring having 0-2 heteroatoms selected from oxygen, nitrogen or sulfur; wherein the ring formed by the two occurrences of J ^R is optionally substituted with 0-3 occurrences of J ^X ; or

The two occurrences of J ^R together with Q ² form a 6-10 membered saturated or partially unsaturated bridged ring system;

^Q3 is a 3-7 membered fully saturated, partially unsaturated or aromatic monocyclic ring having 0-3 heteroatoms selected from oxygen, nitrogen or sulfur; or a 7-12 membered fully saturated, partially unsaturated or aromatic bicyclic ring having 0-5 heteroatoms selected from oxygen, nitrogen or sulfur;

J ^X is independently selected from -CN; =O; halogen; or a C _1-4 aliphatic chain, wherein up to two methylene units of the aliphatic chain are optionally replaced by -O-, -NR-, -C(O)-, or -S(O) _n -;

J ^T is independently selected from halo, -CN; →O; =O; -OH; a C _1-6 aliphatic chain wherein up to two methylene units of the aliphatic chain are optionally replaced by -O-, -NR-, -C(O)-, or -S(O) _n -; or a 3-6 membered non-aromatic ring having 0-2 heteroatoms selected from oxygen, nitrogen, or sulfur; each occurrence of J ^T is optionally substituted by 0-3 occurrences of J ^M ; or

J ^T occurring twice on the same atom together with the atom to which they are attached form a 3-6 membered ring having 0-2 heteroatoms selected from oxygen, nitrogen or sulfur; or

The two occurrences of J ^T together with Q ³ form a 6-10 membered saturated or partially unsaturated bridged ring system;

J ^M is independently selected from halo or C _1-6 aliphatic;

J is H or Cl;

n is 0, 1, or 2;

R is independently selected from H or a C _1-4 aliphatic group.

For the purposes of this application, it will be understood that if two occurrences of ^JQ are taken together with ^Q1 to form a bridged ring system, then the two occurrences of ^JQ are attached to separate atoms of ^Q1 . Additionally, if two occurrences of ^{JR are} taken together with ^Q2 to form a bridged ring system, then the two occurrences of ^JR are attached to separate atoms of ^Q2 . Furthermore, if two occurrences of ^JT are taken together with ^Q3 to form a bridged ring system, then the two occurrences of ^JT are attached to separate atoms of ^Q3 .

It will be understood by those skilled in the art that the arrow in →O represents a coordinate bond.

Reaction conditions

In some embodiments, conditions suitable for forming an amide bond include reacting a compound of Formula 6 with a substituted 3-aminopyridine in an aprotic solvent under heating. In other embodiments, the aprotic solvent is selected from NMP, an optionally substituted pyridine, or DMF. In another embodiment, the aprotic solvent is an optionally substituted pyridine. In other embodiments, the reaction temperature is at least 80° C. In another embodiment, the reaction temperature is at least 100° C.

In another embodiment, the above method further comprises preparing a compound of formula 6:

By making the compound of formula 5:

The reaction is carried out under conditions suitable for the formation of an activated ester, wherein R ¹ and J are as defined herein.

In some embodiments, conditions suitable for generating an activated ester include reacting a compound of Formula 5 with an amide coupling agent in the presence of an organic base. In other embodiments, the organic base is an aliphatic amine. In other embodiments, the organic base is independently selected from triethylamine or DIPEA. In one or more embodiments, the amide coupling agent is independently selected from TBTU, TCTU, HATU, T3P or COMU. In another embodiment, the amide coupling agent is independently selected from TBTU or TCTU. In another embodiment, the amide coupling agent is TCTU.

Another aspect of the present invention comprises a process for preparing a compound of formula I-A:

Contains a compound of formula 5:

The reaction is carried out under conditions suitable for forming an amide bond, wherein R ¹ , R ² , R ³ and R ⁴ are as defined herein.

Another aspect of the present invention comprises a method for preparing a compound of formula 5:

By making the compound of formula 4:

reacting under suitable hydrolysis conditions, wherein R ¹ is as defined herein.

In some embodiments, suitable hydrolysis conditions comprise reacting the compound of Formula 4 with a silane in the presence of a metal catalyst. In other embodiments, the silane is phenylsilane. In another embodiment, the metal catalyst is a palladium catalyst. In another embodiment, the palladium catalyst is Pd(PPh ₃ ) ₄ . In another embodiment, suitable hydrolysis conditions comprise reacting the compound of Formula 4 with 4-methylbenzenesulfinate in the presence of a metal catalyst.

In other embodiments, suitable hydrolysis conditions comprise reacting the compound of Formula 4 with an aqueous base. In some embodiments, the aqueous base is selected from LiOH, NaOH, or KOH.

Another aspect of the present invention comprises a method for preparing a compound of formula 4:

By making the compound of formula 3:

The reaction is carried out under condensation conditions suitable for forming a pyrimidine ring.

In some embodiments, the condensation conditions suitable for forming the pyrimidine ring comprise reacting the compound of Formula 3 with a 1,3-dielectrophile in the presence of a solvent. In another embodiment, the 1,3-dielectrophile is selected from a 1,3-dialdehyde or 3-(diethylamino)-prop-2-enal. In other embodiments, the solvent is selected from DMF or DMSO. In other embodiments, the 1,3-dielectrophile is generated in situ from a protected 1,3-dielectrophile. In another embodiment, the 1,3-dielectrophile is generated from a ketal in the presence of a sulfonic acid. In another embodiment, the sulfonic acid is PTSA.

Another aspect of the present invention comprises a method for preparing a compound of formula 3:

By making the compound of formula 2:

The reaction is carried out under condensation conditions suitable for forming a pyrazole ring.

In some embodiments, the condensation conditions suitable for forming the pyrazole ring include reacting the compound of Formula 2 with hydrazine or hydrazine hydrate under basic conditions in an aprotic solvent. In another embodiment, the aprotic solvent is DMF. In another embodiment, the basic conditions include reacting the compound of Formula 2 in the presence of potassium acetate or sodium acetate.

Another aspect of the present invention comprises a method for preparing a compound of formula 2:

By making the compound of formula 1:

The reaction is carried out under suitable anionic condensation conditions.

In some embodiments, suitable anion condensation conditions comprise 1) reacting a compound of Formula 1 with a base in the presence of a solvent to produce an anion of the compound of Formula 1; and 2) reacting the anion of the compound of Formula 1 with trichloroacetonitrile. In other embodiments, the base is potassium acetate. In another embodiment, the solvent is an alcohol. In other embodiments, the solvent is isopropanol.

One embodiment of the present invention comprises a method for preparing a compound of formula I-A:

Contains a compound of formula 9:

The reaction is carried out under conditions suitable for the formation of a pyrimidine ring, wherein R ¹ , R ² , R ³ and R ⁴ are as defined herein.

In some embodiments, the condensation conditions suitable for forming the pyrimidine ring comprise reacting the compound of Formula 9 with a 1,3-di-electrophile in the presence of a solvent. In another embodiment, the 1,3-di-electrophile is selected from a 1,3-dialdehyde or 3-(diethylamino)-prop-2-enal. In other embodiments, the solvent is selected from an aqueous DMF or DMSO solution. In other embodiments, the 1,3-di-electrophile is generated in situ from a protected 1,3-di-electrophile. In another embodiment, the 1,3-di-electrophile is generated from a ketal in the presence of a sulfonic acid. In another embodiment, the sulfonic acid is PTSA.

Another embodiment of the present invention comprises a method for preparing a compound of formula 9:

By making the compound of formula 8:

The reaction is carried out under condensation conditions suitable for forming a pyrazole ring.

In some embodiments, condensation conditions suitable for forming a pyrazole ring include 1) reacting a compound of Formula 8 with a base in the presence of a solvent to produce an anion of the compound of Formula 8; 2) reacting the anion with trichloroacetonitrile; and 3) reacting the product from 2) with hydrazine or hydrazine hydrate in an aprotic solvent. In another embodiment, the aprotic solvent is NMP or DMF. In some embodiments, the base is selected from sodium acetate or potassium acetate.

Another embodiment comprises a method for preparing a compound of formula 8:

By making the compound of formula 7:

The reaction is carried out under conditions suitable for forming an amide bond.

In certain embodiments, the conditions suitable for generating amide bonds are included in the presence of an aprotic solvent and an organic base, and the formula 7 compound is reacted with the 3-aminopyridine substituted and an amide coupling agent. In other embodiments, the aprotic solvent is selected from NMP or DMF. In another embodiment, the organic base is an aliphatic amine. In other embodiments, the organic base is independently selected from triethylamine or DIPEA. In another embodiment, the amide coupling agent is independently selected from TBTU or TCTU.

Synthesis of compound I-1

Another aspect of the present invention provides a method for preparing a compound of formula I-1:

Contains a compound of formula 30:

With the compound of formula 25:

A step of reacting under conditions suitable for forming an amide bond.

Other embodiments of the present invention provide methods for preparing compounds of formula 30:

By making the compound of formula 28:

The reaction is carried out under deprotection conditions suitable for the formation of a carboxylic acid.

Another embodiment provides a method for preparing a compound of formula 28:

By making the compound of formula 6a*:

With the compound of formula 27:

The reaction is carried out under conditions suitable for forming an amide bond.

In some embodiments, conditions suitable for forming an amide bond comprise reacting a compound of Formula 30 with a compound of Formula 25 in the presence of an amide coupling partner, an aprotic solvent, and a base. In other embodiments, the aprotic solvent is independently selected from NMP, DMF, or tetrahydrofuran. In other embodiments, the aprotic solvent is tetrahydrofuran. In another embodiment, the base is an aliphatic amine. In another embodiment, the base is DIPEA. In some embodiments, the amide coupling partner is independently selected from CDI, TBTU, or TCTU. In one or more embodiments, the amide coupling partner is TCTU. In another embodiment, the amide coupling partner is CDI.

In other embodiments, suitable deprotection conditions comprise reacting the compound of formula 28 with an acid in the presence of a solvent. In some embodiments, the acid is HCl. In another embodiment, the solvent is 1,4-dioxane.

In another embodiment, conditions suitable for forming an amide bond include reacting a compound of Formula 6a* with a compound of Formula 27 in an aprotic solvent under heating. In other embodiments, the aprotic solvent is independently selected from NMP, pyridine, or DMF. In another embodiment, the aprotic solvent is pyridine. In some embodiments, the reaction is carried out at a temperature of at least 80°C.

Another aspect of the present invention provides a method for preparing a compound of formula 27:

Contains a compound of formula 26:

A step of reacting under conditions suitable for forming an amine.

In some embodiments, conditions suitable for forming an amine comprise reacting a compound of Formula 27 under Buchwald-Hartwig amination conditions known to those skilled in the art.

Another embodiment provides a method for preparing a compound of formula 26:

By 1) making compound 18:

Under suitable halogen exchange conditions, the compound of formula 32 is generated.

and

2) making the compound of formula 32:

With the compound of formula 22:

React under suitable replacement conditions.

In some embodiments, suitable halogen exchange conditions include reacting a compound of Formula 18 with potassium fluoride in the presence of an aprotic solvent and a phase transfer catalyst. In other embodiments, the aprotic solvent is independently selected from DMSO, DMF, or sulfolane. In other embodiments, the phase transfer catalyst is _Me4NCI . In other embodiments, suitable replacement conditions include reacting a compound of Formula 32 with a compound of Formula 22 in the presence of a base. In another embodiment, the base is an aliphatic amine. In some embodiments, the aliphatic amine is DIPEA.

Other embodiments of the present invention provide methods for preparing compounds of formula 18:

By making the compound of formula 31:

The reaction is carried out under suitable halogenation conditions.

In some embodiments, suitable halogenation conditions comprise 1) reacting the compound of formula 31 with a base to form an anion; and 2) reacting the anion with a chlorinating agent. In another embodiment, the base is LDA. In another embodiment, the chlorinating agent is 1,1,1,2,2,2-hexachloroethane.

Some embodiments of the present invention provide methods for preparing compounds of formula I-1:

Contains a compound of formula 33:

With the compound of formula 25:

A step of reacting under conditions suitable for forming an amide bond.

In some embodiments, conditions suitable for forming an amide bond comprise reacting a compound of Formula 33 with a compound of Formula 25 in the presence of an amide coupling partner, an aprotic solvent, and a base. In other embodiments, the aprotic solvent is independently selected from NMP, DMF, or tetrahydrofuran. In other embodiments, the aprotic solvent is tetrahydrofuran. In another embodiment, the base is an aliphatic amine. In another embodiment, the base is DIPEA. In some embodiments, the amide coupling partner is independently selected from TBTU or TCTU. In one or more embodiments, the amide coupling partner is TCTU.

Another embodiment provides a method for preparing compound 33 of formula:

Contains a compound of formula 28:

The step of reacting under suitable deprotection conditions.

In some embodiments, deprotection conditions suitable for cleaving the tert-butyl ester comprise reacting the compound of formula 28 with an acid in the presence of a solvent. In one embodiment, the acid is selected from, but not limited to, methanesulfonic acid (preferred), PTSA, TFA, or HCl. In other embodiments, the solvent is selected from, but not limited to, 1,4-dioxane or acetonitrile. In another embodiment, the solvent is acetonitrile.

Another embodiment provides a method for preparing a compound of formula 4a:

The following steps are included:

a) making a compound of formula 35:

wherein R° is a C _1-6 aliphatic group,

The reaction is carried out under acidic conditions to generate a compound of formula 36:

b) reacting the compound of formula 36 with an electrophilic fluorinating agent to produce a compound of formula 38:

c) making the compound of formula 38 and the compound of formula 3:

The reaction is carried out under suitable condensation conditions to generate a compound of formula 4a.

In some embodiments, R° is independently selected from methyl, ethyl, propyl, isopropyl, butyl and pentyl. In other embodiments, R° is independently selected from methyl or ethyl.

In another embodiment, the electrophilic fluorinating agent is 1-(chloromethyl)-4-fluoro-1,4-diazobicyclo[2.2.2]octane bistetrafluoroborate. In other embodiments, the electrophilic fluorinating agent is fluorine gas.

In another embodiment, suitable condensation conditions comprise reacting a compound of Formula 38 with a compound of Formula 3 in the presence of a solvent and heating. In some embodiments, the solvent is selected from DMF or DMSO.

Another embodiment provides a method for preparing a compound of formula I-1:

The following steps are included:

a) making a compound of formula 6a*:

With the compound of formula 27:

The reaction is carried out under suitable amide bond forming conditions to generate a compound of formula 28:

b) purifying the compound of formula 28 using a suitable palladium chelating agent;

c) reacting the compound of formula 28 under suitable deprotection conditions to produce a compound of formula 30

and

d) reacting a compound of formula 30 with a compound of formula 25:

The reaction is carried out under suitable amide bond forming conditions to produce a compound of formula I-1.

In some embodiments, suitable palladium chelating agents are independently selected from propane-1,2-diamine; ethane-1,2-diamine; ethane-1,2-diamine; propane-1,3-diamine; tetramethylethylenediamine; ethylene glycol; 1,3-bis(diphenylphosphino)propane; 1,4-bis(diphenylphosphino)butane; and 1,2-bis(diphenylphosphino)ethane/Pr-1,2-diamine. In other embodiments, the suitable palladium chelating agent is propane-1,2-diamine.

Another embodiment provides a method for preparing a compound of formula 28:

The following steps are included:

a) making a compound of formula 5a

The reaction is carried out under suitable halogenation conditions to generate a compound of formula 34:

wherein X is a halogen;

b) making a compound of formula 34 and a compound of formula 27:

The reaction is carried out under suitable amide bond forming conditions to generate a compound of formula 28.

In some embodiments, X is independently selected from fluorine or chlorine. In another embodiment, X is chlorine. In some embodiments, suitable halogenation conditions comprise reacting the compound of Formula 5a with a halogenating agent and a base in the presence of a solvent. In another embodiment, the halogenating agent is SOCl ₂ . In some embodiments, the base is triethylamine. In other embodiments, the solvent is DCM.

Another aspect of the present invention provides a method for preparing a compound of formula I-1:

The following steps are included:

a) making a compound of formula 5a

The reaction is carried out under suitable halogenation conditions to generate a compound of formula 34:

wherein X is a halogen;

b) making a compound of formula 34 and a compound of formula 27:

The reaction is carried out under suitable amide bond forming conditions to generate a compound of formula 28:

c) reacting the compound of formula 28 under suitable deprotection conditions to produce a compound of formula 30

d) reacting a compound of formula 30 with a compound of formula 25:

The reaction is carried out under suitable amide bond forming conditions to produce a compound of formula I-1.

Deuterated compounds

In another embodiment, isotopes can be introduced into compound 1-1 by selecting building blocks containing isotopic atoms (commercially available or prepared according to methods known to those skilled in the art) and then ligating them into a sequence similar to that reported for the unlabeled material.

Another aspect of the present invention provides a compound of formula I-B:

or a pharmaceutically acceptable salt thereof, wherein:

Each Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ , Y ⁸ , Y ⁹ , Y ¹⁰ , Y ¹¹ , Y ¹² , Y ¹³ , Y ¹⁴ , Y ¹⁵ , Y ¹⁶ , Y ¹⁷ , Y ¹⁸ and Y ¹⁹ is independently hydrogen or deuterium; provided that at least one of Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ , Y ⁸ , Y ⁹ , Y ¹⁰ , Y ¹¹ , Y ¹² , Y ¹³ , Y ¹⁴ , Y ¹⁵ , Y ¹⁶ , Y ¹⁷ , Y ¹⁸ and Y ¹⁹ is deuterium;

Each of X ¹ , X ² , X ⁴ , X ⁵ , X ⁶ , X ⁷ , X ⁸ and X ⁹ is independently selected from ¹² C or ¹³ C;

X ³ is independently selected from - ¹² C(O)- or - ¹³ C(O)-.

The following labeled building blocks are commercially available and can be used in synthetic routes to prepare compounds of formula I-B:

2,2,3,3,5,5,6,6-octadeuteropiperazine;

2,3,5,6-tetrakis- ¹³ C-piperazine;

2,2,3,3,4,5,5,6,6-nonadeuteropiperidine-4-carboxylic acid;

1, 2- ^di13C -2-cyanoacetic acid;

1- ¹³ C-2-cyano( ¹³ C)acetic acid ethyl ester;

2- ¹³ C-2-cyano( ¹³ C)acetic acid ethyl ester.

Other labeled building blocks that can be used in the synthetic pathways for preparing compounds of Formula I-B are also known to those skilled in the art. These may include, but are not limited to, the following labeled building blocks:

2- ¹³ C-oxetan-3-one;

3- ¹³ C-oxetan-3-one;

2,2,3,3-tetradeuteropiperazine;

2,2,5,5-tetradeuteropiperazine;

4-deuterated piperidine-4-carboxylic acid ethyl ester;

2-Cyano( ¹³ C)acetic acid;

1- ¹³ C-2-cyanoacetic acid;

2- ¹³ C-2-cyanoacetic acid;

1-deuterated-3-(diethylamino)-2-fluoroacrolein;

In one or more embodiments, Y ¹² , Y ¹³ , Y ¹⁴ , Y ¹⁵ , Y ¹⁶ , Y ¹⁷ , Y ¹⁸ and Y ¹⁹ are deuterium, and Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ , Y ⁸ , Y ⁹ , Y ¹⁰ and Y ¹¹ are independently selected from hydrogen or deuterium. In another embodiment, Y ¹² , Y ¹³ , Y ¹⁴ , Y ¹⁵ , Y ¹⁶ , Y ¹⁷ , Y ¹⁸ and Y ¹⁹ are deuterium, and Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ , Y ⁸ , Y ⁹ , Y ¹⁰ and Y ¹¹ are hydrogen.

In another embodiment, X ¹ , X ² , X ⁴ , X ⁵ , X ⁶ , X ⁷ , X ⁸ and X ⁹ are ¹² C; X ³ is - ¹² C(O)-. In other embodiments, X ¹ , X ⁴ , X ⁵ , X ⁶ , X ⁷ , X ⁸ and X ⁹ are ¹² C; X ³ is - ¹³ C(O)-; X ² is ¹³ C.

In some embodiments, Y ¹¹ , Y ¹² , Y ¹³ , Y ¹⁴ , Y ¹⁵ , Y ¹⁶ , Y ¹⁷ , Y ¹⁸ and Y ¹⁹ are deuterium, and Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ , Y ⁸ , Y ⁹ and Y ¹⁰ are independently selected from hydrogen or deuterium. In other embodiments, Y ¹¹ , Y ¹² , Y ¹³ , Y ¹⁴ , Y ¹⁵ , Y ¹⁶ , Y ¹⁷ , Y ¹⁸ and Y ¹⁹ are deuterium, and Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ , Y ⁸ , Y ⁹ and Y ¹⁰ are hydrogen.

In another embodiment, Y ² , Y ¹² , Y ¹³ , Y ¹⁴ , Y ¹⁵ , Y ¹⁶ , Y ¹⁷ , Y ¹⁸ and Y ¹⁹ are deuterium, and Y ¹ , Y ³ , Y ⁴ , Y ⁵ , Y 6 , Y ⁷ , Y ⁸ , Y ⁹ , Y ^{10 and Y 11 are} independently selected from hydrogen or deuterium. In another aspect of the invention, Y ² , Y ¹² , Y ¹³ , Y ¹⁴ , Y ¹⁵ , Y ¹⁶ , Y ¹⁷ , Y ¹⁸ and Y ¹⁹ are deuterium, ^and Y ¹ , Y ³ , Y ⁴ , Y 5 , Y ⁶ , Y ⁷ , Y ⁸ , Y ⁹ , Y ¹⁰ and Y ¹¹ are hydrogen.

In some embodiments, Y ¹² , Y ¹³ , Y ¹⁸ and Y ¹⁹ are deuterium, and Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ , Y ⁸ , Y ⁹ , Y ¹⁰ , Y ¹¹ , Y ¹⁴ , Y ¹⁵ , Y ¹⁶ and Y ¹⁷ are hydrogen or deuterium. In other embodiments, Y ¹² , Y ¹³ , Y ¹⁸ and Y ¹⁹ are deuterium, and Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ , Y ⁸ , Y ⁹ , Y ¹⁰ , Y ¹¹ , Y ¹⁴ , Y ¹⁵ , Y ¹⁶ and Y ¹⁷ are hydrogen.

In one or ^{more embodiments, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10 and Y11 are deuterium , and Y1 , Y2} , ^Y12 , ^Y13 , ^Y14 , ^Y15 , ^Y16 , ^Y17 , ^Y18 and ^Y19 are independently selected from deuterium ^{or hydrogen. In another embodiment, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10 and Y11 are deuterium , and Y1 , Y2} , ^Y12 , ^Y13 , ^Y14 , ^{Y15 , Y16 , Y17 , Y18} and ^Y19 are hydrogen.

In another embodiment, ^Y2 and ^Y11 are deuterium, and Y1, Y3, ^Y4 , ^Y5 , ^Y6 , ^Y7 , ^Y8 , ^Y9 , ^Y10 , ^Y12 , ^Y13 , ^Y14 , ^Y15 , ^Y16 , ^Y17 , ^Y18 and ^Y19 are deuterium or hydrogen. In other embodiments, ^Y2 and ^Y11 are deuterium, and Y1, ^Y3 , ^Y4 , ^Y5 , ^Y6 , ^Y7 , ^Y8 , ^Y9 , ^Y10 , ^Y12 , ^Y13 , ^Y14 , ^Y15 , ^Y16 , ^Y17 , ^{Y18 and Y19 are} hydrogen.

In some embodiments, Y is ^{deuterium and} Y, Y, Y, ^Y , Y, Y, Y, Y, Y, Y, Y, ^Y , ^Y , ^Y , Y, Y, Y, Y, Y, Y, Y ^, Y ^, Y, ^Y , ^Y , ^Y , Y, Y, Y, Y, ^Y , ^Y , Y ^, Y, ^Y , ^Y , Y, ^Y , Y, ^Y , Y, Y, Y, Y, Y, ^Y , Y, Y, Y, Y, Y, Y, Y, Y, Y, Y, Y, Y, ^Y , Y, ^Y , Y, Y, Y, Y, Y, Y, Y, Y, Y, ^Y , Y, Y, ^Y , ^Y , Y, Y, Y, Y, Y, Y, Y, Y ^, Y ^, Y, ^Y , ^Y , Y, ^Y , ^Y , Y, ^Y , Y, Y, Y, Y, Y, Y, Y, Y, Y, Y, ^Y , Y, ^Y , Y, Y, Y, Y, Y, Y, ^Y , Y, ^Y , Y, Y, ^Y , Y, Y, Y, Y, ^Y , Y ^{, Y} ,

In other embodiments, X ¹ , X ² , X ⁴ , X ⁵ , X ⁶ , X ⁷ and X ⁸ are ¹² C; X ³ is - ¹² C(O)-; X ⁹ is ¹³ C. In another embodiment, X ¹ , X ² , X ⁸ and X ⁹ are ¹² C; X ³ is - ¹² C(O)-; X ⁴ , X ⁵ , X ⁶ and X ⁷ are ¹³ C. In yet another embodiment, X ² , X ⁴ , X ⁵ , X ⁶ , X ⁷ , X ⁸ and X ⁹ are ¹² C; X ³ is - ¹² C(O)-; X ¹ is ¹³ C. In other embodiments, X ² , X ⁴ , X ⁵ , X ⁶ , X ⁷ and X ⁹ are ¹² C; X ³ is - ¹³ C(O)-; and X ¹ and X ⁸ are ¹³ C.

In some embodiments, Y ¹¹ is deuterium, and Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ , Y ⁸ , Y ⁹ , Y ¹⁰ , Y ¹² , Y ¹³ , Y ¹⁴ , Y ¹⁵ , Y ¹⁶ , Y ¹⁷ , Y ¹⁸ , and Y ¹⁹ are independently selected from hydrogen or deuterium. In another embodiment, Y ¹¹ is deuterium, and Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ , Y ⁸ , Y ⁹ , Y ¹⁰ , Y ¹² , Y ¹³ , Y ¹⁴ , Y ¹⁵ , Y ¹⁶ , Y ¹⁷ , Y ¹⁸ , and Y ¹⁹ are hydrogen.

In another embodiment, X ¹ , X ⁴ , X ⁵ , X ⁶ , X ⁷ , X ⁸ and X ⁹ are ¹² C; X ³ is - ¹² C(O)-; and X ² is ¹³ C.

In another embodiment, the compounds of formula I-B of the present invention are presented in Table 1. It will be appreciated by those skilled in the art that the compounds of the present invention may exist in different tautomeric forms.

Table 1

Solid form

Another aspect of the present invention provides a solid form of a compound of formula I-1:

The form is selected from the group consisting of compound I-1·ethanol solvate, compound I-1·hydrate I, compound I-1·hydrate II, compound I-1·anhydrous form A, compound I-1·anhydrous form B, compound I-1·anhydrous form C, compound I-1·DMSO solvate, compound I-1·DMAC solvate, compound I-1·acetone solvate and compound I-1·isopropanol solvate.

Compound I-1·Ethanol solvate

In some aspects of the invention, the solid form is Compound 1-1 · ethanol solvate. In another aspect of the invention, the solid form is crystalline Compound 1-1 · ethanol solvate. In other embodiments, the crystalline Compound 1-1 · ethanol solvate has a Compound 1-1 to ethanol ratio of about 1:0.72. In another aspect of the invention, the crystalline Compound 1-1 · ethanol solvate is characterized by a weight loss of about 5.76% over a temperature range of about 166°C to about 219°C. In another aspect of the invention, the crystalline Compound 1-1 · ethanol solvate is characterized by exhibiting one or more peaks at about 17.2, 19.7, 23.8, 24.4, and 29.0 degrees in an X-ray powder diffraction pattern obtained using Cu Kα radiation, expressed as 2-θ ± 0.2. In other embodiments, the crystalline Compound 1-1 · ethanol solvate is characterized by an X-ray powder diffraction pattern substantially the same as that shown in Figure 1a. In other embodiments, the crystalline Compound I-1·ethanol solvate is characterized by having one or more peaks in the C ¹³ ssNMR spectrum corresponding to 175.4±0.3 ppm, 138.0±0.3 ppm, 123.1±0.3 ppm, 57.8±0.3 ppm, 44.0±0.3 ppm, and 19.5±0.3 ppm. In another embodiment, the crystalline Compound I-1·ethanol solvate is characterized by having one or more peaks in the F ¹⁹ ssNMR spectrum corresponding to -136.0±0.3 ppm and -151.6±0.3 ppm.

Compound I-1·Hydrate I

In some aspects of the invention, the solid form is Compound 1-1 Hydrate I. In another aspect of the invention, the solid form is crystalline Compound 1-1 Hydrate I. In other embodiments, the crystalline Compound 1-1 Hydrate I has a Compound 1-1 to H ₂ O ratio of about 1:4.5. In another embodiment, the crystalline Compound 1-1 Hydrate I is characterized by a weight loss of about 14.56% over a temperature range of about 25°C to about 100°C. In other embodiments, the crystalline Compound 1-1 Hydrate I is characterized by exhibiting one or more peaks at about 6.5, 12.5, 13.7, 18.8, and 26.0 degrees in an X-ray powder diffraction pattern obtained using Cu Ka radiation, expressed as 2-theta ± 0.2. In another embodiment, the crystalline Compound 1-1 Hydrate I is characterized by an X-ray powder diffraction pattern substantially the same as that shown in Figure 1b.

Compound I-1·Hydrate II

In some aspects of the invention, the solid form is Compound I-1 Hydrate II. In another aspect of the invention, the solid form is crystalline Compound I-1 Hydrate II. In other embodiments, the crystalline Compound I-1 Hydrate II is characterized by exhibiting one or more peaks at approximately 10.1, 11.3, 11.9, 20.2, and 25.1 degrees in an X-ray powder diffraction pattern obtained using Cu Ka radiation, expressed as 2-θ ± 0.2. In other embodiments, the crystalline Compound I-1 Hydrate II is characterized by having one or more peaks in a C ¹³ ssNMR spectrum corresponding to 177.0 ± 0.3 ppm, 158.2 ± 0.3 ppm, 142.9 ± 0.3 ppm, 85.1 ± 0.3 ppm, 58.9 ± 0.3 ppm, and 31.9 ± 0.3 ppm. In another embodiment, crystalline Compound I-1·Hydrate II is characterized by having one or more peaks in an F ¹⁹ ssNMR spectrum corresponding to -138.0±0.3 ppm and -152.7±0.3 ppm.

Compound I-1·Anhydrous Form A

In one embodiment, the solid form is Compound I-1·Anhydrous Form A. In another embodiment, the solid form is crystalline Compound I-1·Anhydrous Form A. In other embodiments, crystalline Compound I-1·Anhydrous Form A is characterized by a weight loss of about 0.96% over a temperature range of about 25°C to about 265°C. In other embodiments, crystalline Compound I-1·Anhydrous Form A is characterized by exhibiting one or more peaks at about 6.1, 12.2, 14.5, 22.3, and 31.8 degrees in an X-ray powder diffraction pattern obtained using Cu Ka radiation, expressed as 2-theta ± 0.2. In another embodiment, crystalline Compound I-1·Anhydrous Form A is characterized by an X-ray powder diffraction pattern substantially the same as that shown in Figure 1c. In other embodiments, crystalline Compound I-1 Anhydrous Form A is characterized by having one or more peaks in a C ¹³ ssNMR spectrum corresponding to 175.9 ± 0.3 ppm, 138.9 ± 0.3 ppm, 74.1 ± 0.3 ppm, 42.8 ± 0.3 ppm, and 31.5 ± 0.3 ppm. In another embodiment, crystalline Compound I-1 Anhydrous Form A is characterized by having one or more peaks in an F ¹⁹ ssNMR spectrum corresponding to -136.8 ± 0.3 ppm and -155.7 ± 0.3 ppm. One embodiment describes a method for preparing Compound I-1 Anhydrous Form A, comprising stirring a suspension containing Compound I-1 ethanol solvate and a suitable organic solvent. In another embodiment, the suitable organic solvent is tetrahydrofuran. Another aspect of the present invention describes a method for preparing Compound I-1 Anhydrous Form A, comprising stirring a suspension containing Compound I-1 Anhydrous Form, isopropanol, and water. In some embodiments, the suspension is heated to between about 65° C. and about 80° C. In another embodiment, the suspension is heated to between about 70° C. and about 75° C. In other embodiments, Compound I-1 Anhydrous Form A is characterized in that the crystalline form of Compound I-1 has a monoclinic system, a P2 _1/c centrosymmetric space group, and the following unit cell parameters:

α＝90°

β＝107.22(3)°

γ＝90°.

Compound I-1·Anhydrous Form B

As used herein, "Anhydrous Form B" refers to the THF solvate of Compound 1-1. In some embodiments, the solid form is Compound 1-1·Anhydrous Form B. In another embodiment, the solid form is crystalline Compound 1-1·Anhydrous Form B. In another embodiment, crystalline Compound 1-1·Anhydrous Form B is characterized by a weight loss of about 2.5% over a temperature range of about 25°C to about 175°C. In other embodiments, Compound 1-1·Anhydrous Form B is characterized by exhibiting one or more peaks at about 7.2, 8.3, 12.9, 19.5, and 26.6 degrees in an X-ray powder diffraction pattern obtained using Cu Kα radiation, expressed as 2-θ ± 0.2. In other embodiments, crystalline Compound 1-1·Anhydrous Form B is characterized by an X-ray powder diffraction pattern substantially the same as that shown in Figure 1d. In other embodiments, crystalline Compound I-1·Anhydrous Form B is characterized by having one or more peaks in the C ¹³ ssNMR spectrum corresponding to 173.4±0.3 ppm, 164.5±0.3 ppm, 133.5±0.3 ppm, 130.8±0.3 ppm, 67.7±0.3 ppm, 45.3±0.3 ppm, and 25.9±0.3 ppm. In another embodiment, crystalline Compound I-1·Anhydrous Form B is characterized by having one or more peaks in the F ¹⁹ ssNMR spectrum corresponding to -138.0±0.3 ppm and -153.5±0.3 ppm.

Compound I-1·Anhydrous Form C

In some embodiments, the solid form is Compound I-1·Anhydrous Form C. In another embodiment, the solid form is crystalline Compound I-1·Anhydrous Form C. In other embodiments, crystalline Compound I-1·Anhydrous Form C is characterized by exhibiting one or more peaks at approximately 6.8, 13.4, 15.9, 30.9, and 32.9 degrees in an X-ray powder diffraction pattern obtained using Cu Ka radiation, expressed as 2-θ ± 0.2. In other embodiments, crystalline Compound I-1·Anhydrous Form C is characterized by an X-ray powder diffraction pattern substantially identical to that shown in Figure 1e. In other embodiments, crystalline Compound I-1·Anhydrous Form C is characterized by having one or more peaks in a C ¹³ ssNMR spectrum corresponding to 175.2 ± 0.3 ppm, 142.5 ± 0.3 ppm, 129.6 ± 0.3 ppm, 73.5 ± 0.3 ppm, 54.0 ± 0.3 ppm, and 46.7 ± 0.3 ppm. In another embodiment, crystalline Compound 1-1·Anhydrous Form C is characterized by having one or more peaks in an F ¹⁹ ssNMR spectrum corresponding to -131.2 ± 0.3 ppm and -150.7 ± 0.3 ppm.

Compound I-1·Amorphous form

In some embodiments, the solid form is Compound I-1·amorphous. In another embodiment, the solid form is crystalline Compound I-1·amorphous. In other embodiments, the crystalline Compound I-1·amorphous is characterized by having one or more peaks in the C ¹³ ssNMR spectrum corresponding to 173.8±0.3 ppm, 144.2±0.3 ppm, 87.5±0.3 ppm, 45.6±0.3 ppm, and 29.5±0.3 ppm. In another embodiment, the crystalline Compound I-1·amorphous is characterized by having one or more peaks in the F ¹⁹ ssNMR spectrum corresponding to -137.7±0.3 ppm and -153.1±0.3 ppm.

Compound I-1·DMSO solvate

In one embodiment, the solid form is Compound I-1·DMSO solvate. In another embodiment, the solid form is crystalline Compound I-1·DMSO solvate. In other embodiments, the crystalline Compound I-1·DMSO solvate has a Compound I-1 to DMSO ratio of about 1:1. In another embodiment, the crystalline Compound I-1·DMSO solvate is characterized by a weight loss of about 12.44% over a temperature range of about 146°C to about 156°C. In some embodiments, the crystalline Compound I-1·DMSO solvate is characterized by exhibiting one or more peaks at about 8.9, 14.8, 16.5, 18.6, 20.9, 22.2, and 23.4 degrees in an X-ray powder diffraction pattern obtained using Cu Kα radiation, expressed as 2-θ ± 0.2. In other embodiments, the Compound I-1·DMSO solvate is characterized by an X-ray powder diffraction pattern substantially the same as that shown in Figure 1g.

Compound I-1·DMAC solvate

In some embodiments, the solid form is a Compound I-1·DMAC solvate. In another embodiment, the solid form is a crystalline Compound I-1·DMAC solvate. In other embodiments, the crystalline Compound I-1·DMAC solvate has a Compound I-1 to DMAC ratio of about 1:1.3. In another embodiment, the crystalline Compound I-1·DMAC solvate is characterized by a weight loss of about 17.76% over a temperature range of about 85°C to about 100°C. In other embodiments, the Compound I-1·DMAC solvate is characterized by an X-ray powder diffraction pattern obtained using Cu Kα radiation, exhibiting one or more peaks at about 6.0, 15.5, 17.7, 18.1, 20.4, and 26.6 degrees, expressed as 2-θ ± 0.2. In some embodiments, the Compound I-1·DMAC solvate is characterized by an X-ray powder diffraction pattern substantially the same as that shown in Figure 1h.

Compound I-1·Acetone solvate

In one or more embodiments, the solid form is Compound I-1 · acetone solvate. In another embodiment, the solid form is crystalline Compound I-1 · acetone solvate. In another embodiment, the crystalline Compound I-1 · acetone solvate has a Compound I-1 to acetone ratio of about 1:0.44. In other embodiments, Compound I-1 · acetone solvate is characterized by a weight loss of about 4.55% over a temperature range of about 124° C. to about 151° C. In some embodiments, Compound I-1 · acetone solvate is characterized by exhibiting one or more peaks at about 8.9, 15.5, 15.8, 16.7, 22.3, 25.7, and 29.0 degrees in an X-ray powder diffraction pattern obtained using Cu K alpha radiation, expressed as 2-θ ± 0.2. In other embodiments, Compound I-1 · acetone solvate is characterized by an X-ray powder diffraction pattern substantially the same as that shown in Figure 1i.

Compound I-1·Isopropanol solvate

In one embodiment, the solid form is Compound 1-1·isopropanol solvate. In another embodiment, the solid form is crystalline Compound 1-1·isopropanol solvate. In other embodiments, the crystalline Compound 1-1·isopropanol solvate has a Compound 1-1 to isopropanol ratio of about 1:0.35. In another embodiment, the Compound 1-1·isopropanol solvate is characterized by a weight loss of about 3.76% over a temperature range of about 136°C to about 180°C. In some embodiments, the Compound 1-1·isopropanol solvate is characterized by exhibiting one or more peaks at about 6.9, 17.1, 17.2, 19.1, 19.6, 23.7, 24.4, and 28.9 degrees in an X-ray powder diffraction pattern obtained using Cu Kα radiation, expressed as 2-θ ± 0.2. In another embodiment, the Compound 1-1·isopropanol solvate is characterized by an X-ray powder diffraction pattern substantially the same as that shown in Figure 1j.

preparation

Some embodiments disclosed herein generally relate to compositions that may include an effective amount of Compound 1-1 or a pharmaceutically acceptable salt thereof and one or more excipients. Compound 1-1 is believed to be an ATR inhibitor and is described in WO 2014/089379, which is incorporated herein by reference in its entirety.

Compound 1-1 and Form A can exist in free form or in salt form. Pharmaceutically acceptable salts can be used to administer Compound 1-1 or Form A for medical purposes. Non-pharmaceutically acceptable salts can be used to produce, isolate, purify, and/or separate stereoisomers of Compound 1-1, Form A, and/or one or more intermediates thereof.

As used herein, the term "pharmaceutically acceptable salt" refers to a compound that is suitable for use in humans and lower animals, within the scope of proper medical judgment, without undue side effects such as toxicity, irritation, allergic reactions, and is commensurate with a reasonable benefit/risk ratio. Various pharmaceutically acceptable salts can be used. For example, those disclosed in S.M.Berge et al., J.Pharmaceutical Sciences, 1977, 66, 1-19, are incorporated herein by reference. Pharmaceutically acceptable salts of the compounds described herein include those derived from suitable inorganic and organic acids and bases. Salts of the compounds described herein (e.g., Compound I-1) can be prepared in situ during the final isolation and purification of the compound.

As described above, compound I-1 can exist in different polymorphs (i.e., "solid forms"). Polymorphism is the ability of a compound to exist in more than one different crystalline or "polymorphic" species, each of which has a different molecular arrangement in the crystal lattice. Each different crystalline species is a "polymorph." Each polymorph has the same chemical formula, but exhibits different physical properties due to different lattice arrangements. Polymorphs can be characterized by analytical methods such as X-ray powder diffraction (XRPD) patterns, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), melting points, and/or other techniques known in the art.

Form A described herein may be in pure form or in admixture with other substances. Examples of other substances include, for example, other forms of Compound 1-1 (e.g., amorphous forms, other polymorphs, solvates, and hydrates); other diastereomers of Compound 1-1; and/or other substances other than Compound 1-1.

Thus, in some embodiments, the composition can include an effective amount of pure Form A. As used herein, "pure" Form A is greater than 95% (w/w) (wherein w/w is the weight of Form A/the weight of Compound I-1 (wherein the weight of Compound I-1 is the weight of Form A plus the weight of all other forms of Compound I-1)), for example, greater than 98% (w/w), greater than 99% (w/w%), greater than 99.5% (w/w%), or greater than 99.9% (w/w%). In some embodiments, the composition can include an effective amount of Form A in an amount of at least 95% (w/w%), at least 97% (w/w%), or at least 99% (w/w%), free of any other diastereomers of Compound I-1. In some embodiments, the composition can include an effective amount of Form A in an amount of at least 95% (w/w%), at least 97% (w/w%), or at least 99% (w/w%), free of any other polymorphs and amorphous forms of Compound I-1.

In some embodiments, the composition may include Form A and one or more other forms of Compound 1-1. Other forms of Compound 1-1 include, for example, hydrates, solvates, amorphous forms, other polymorphs, or combinations thereof.

In some embodiments, the composition can include Compound 1-1 or Form A (or a pharmaceutically acceptable salt thereof) in an amount ranging from a trace amount (0.1%) to 100% (w/w%), relative to the total weight of the composition. In some embodiments, the composition can include less than about 50% of Compound 1-1 or Form A, relative to the total weight of the composition (wherein the total weight includes the weight of Compound 1-1 or Form A). For example, the composition can include Compound 1-1 or Form A in an amount ranging from 0.1% to 0.5%, 0.1% to 1%, 0.1% to 2%, 0.1% to 5%, 0.1% to 10%, 0.1% to 20%, 0.1% to 30%, 0.1% to 40%, and 0.1% to <50% (w/w%), relative to the total weight of the composition (wherein the total weight includes the weight of Compound 1-1 or Form A). In other embodiments, the composition can include equal to or greater than about 50% of Compound 1-1 or Form A, relative to the total weight of the composition (wherein the total weight includes the weight of Compound 1-1 or Form A). For example, the composition can include at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% (w/w) of Compound 1-1 or Form A, relative to the total weight of the composition (wherein the total weight includes the weight of Compound 1-1 or Form A). In some embodiments, the composition can include Compound 1-1 or Form A in an amount ranging from about 1 wt% to about 50 wt%, from about 5 wt% to about 40 wt%, from about 5 wt% to about 25 wt%, or from about 5 wt% to about 15 wt% of Compound 1-1 or Form A, relative to the total weight of the composition (wherein the total weight includes the weight of Compound 1-1 or Form A).

As used herein, "excipient" is used herein in its general sense as understood by those skilled in the art and includes one or more inert substances that are included in a composition to provide, but are not limited to, body, consistency, stability, binding ability, lubricity, disintegration ability, etc. to the composition. Examples of excipients include fillers, binders, disintegrants, wetting agents, lubricants, glidants, moisturizers, and absorbents.

In some embodiments, the composition may include Compound I-1 or Form A and one or more other components selected from one or more fillers, one or more binders, one or more disintegrants, one or more wetting agents, and one or more lubricants. In some embodiments, the composition may include one or more fillers in an amount ranging from about 10 wt % to about 95 wt %; about 25 wt % to about 90 wt %; about 50 wt % to about 90 wt %; or about 70 wt % to about 90 wt % of fillers, based on the total weight of the composition (wherein the total weight includes the weight of the one or more fillers). In some embodiments, the composition may include one or more lubricants in an amount ranging from about 0.1 wt % to about 10 wt %, about 0.5 wt % to about 7 wt % or about 1 wt % to about 5 wt % of lubricants, based on the total weight of the composition (wherein the total weight includes the weight of the one or more lubricants). In some embodiments, the composition can include one or more disintegrants in an amount ranging from about 1 wt % to about 15 wt %, about 1 wt % to about 10 wt %, or about 1 wt % to about 7 wt % of disintegrant, based on the total weight of the composition (wherein the total weight includes the weight of the one or more disintegrants).

Wetting agents, binders, disintegrants, lubricants, and fillers suitable for inclusion may be compatible with the ingredients of the composition, eg, they do not substantially reduce the chemical stability of the active pharmaceutical ingredient.

The term "wetting agent" is used herein in its general sense as understood by those skilled in the art, including surfactants such as nonionic surfactants and anionic surfactants. Wetting agents can enhance the solubility of a composition. Exemplary surfactants include sodium lauryl sulfate (SLS), polyoxyethylene sorbitan fatty acids (e.g., TWEEN ^™ ), sorbitan fatty acid esters (e.g., sodium dodecylbenzene sulfonate (SDBS), dioctyl sodium sulfosuccinate (Docusate), dioxycholic acid sodium salt (DOSS), sorbitan monostearate, sorbitan tristearate, sodium N-lauroyl sarcosinate, sodium oleate, sodium myristate, sodium stearate, sodium palmitate, Gelucire 44/14, ethylenediaminetetraacetic acid (EDTA), vitamin E d-alpha tocopheryl polyethylene glycol 1000 succinate (TPGS), lecithin, MW 677-692, monosodium glutamate monohydrate, Labrasol, PEG 8 caprylic/capric glycerides, Transcutol, diethylene glycol monoethyl ether, Solutol HS-15, polyethylene glycol/hydroxystearate, taurocholic acid, polyoxypropylene and polyoxyethylene copolymers (e.g., poloxamers, trade names such as L61, F68, F108, and F127), saturated polyglycolyzed glycerides docusate sodium, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene 20 stearyl ether, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene hydrogenated castor oil, sorbitan esters of fatty acids, vitamin E or tocol derivatives, vitamin E TPGS, tocopherol esters, lecithin, phospholipids and their derivatives, stearic acid, oleic acid, oleyl alcohol, cetyl alcohol, mono- and diglycerides, propylene glycol esters of fatty acids, glycerides of fatty acids, ethylene glycol palmitostearate, polyoxyglycerol esters, propylene glycol monocaprylate, propylene glycol monolaurate, polyglyceryl oleate, and any combination thereof. Sodium lauryl sulfate is an anionic surfactant; polyoxypropylene and polyoxyethylene copolymers are nonionic surfactants. Specific examples of copolymers of polyoxypropylene and polyoxyethylene include poloxamers, such as poloxamer having a polyoxypropylene molecular weight of 1,800 g/mol and a polyoxyethylene content of 80% (eg, poloxamer 188).

The term "binder" is used herein in its general meaning as understood by those skilled in the art and includes ingredients used during granulation of an active ingredient (e.g., Compound I-1 or Form A), wherein the binder holds the active ingredient together with one or more inactive ingredients. Exemplary binders include polyvinylpyrrolidones (PVPs), pregelatinized starch, starch, microcrystalline cellulose, modified celluloses (e.g., hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), and hydroxyethyl cellulose (HEC)), and any combination thereof. PVPs are generally characterized by a "K value," which is a measure of the viscosity of a polymeric composition. PVPs are commercially available (e.g., from Tokyo Chemical Industry Co., Ltd.) under the trade names K12, K17, K25, K30, K60, and K90. Specific examples of PVPs include soluble spray-dried PVPs. PVPs can have an average molecular weight of 3,000 to 4,000 Daltons, such as K12, which has an average molecular weight of 4,000 Daltons. PVPs can be used wet or dry.

The term "filler" (or "diluent") is used herein in its general sense as understood by those skilled in the art, including microcrystalline cellulose (e.g., PH 101), lactose, sorbitol, cellulose, calcium phosphate, starch, sugars (e.g., mannitol, sucrose, etc.), glucose, maltodextrin, sorbitol, xylitol, powdered cellulose, silicified microcrystalline cellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, methylhydroxyethylcellulose, pregelatinized starch, calcium hydrogen phosphate, calcium sulfate, calcium carbonate, and any combination thereof. Specific examples of fillers include microcrystalline cellulose and lactose. Specific examples of microcrystalline cellulose include commercially available series, such as microcrystalline cellulose (e.g., PH 101) with a particle size of more than 70% at 200 mesh and less than 10% at 65 mesh. A specific example of lactose is lactose monohydrate.

The term "disintegrant" is used herein in its general sense as understood by those skilled in the art to enhance the dispersion of the composition. Examples of disintegrants include cross-linked sodium carboxymethyl cellulose, starch (e.g., corn starch, potato starch), sodium starch glycolate, cross-linked polyvinylpyrrolidone, microcrystalline cellulose, sodium alginate, calcium alginate, alginic acid, pregelatinized starch, cellulose and derivatives thereof, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, soybean polysaccharides, guar gum, ion exchange resins, effervescent systems based on food acids and alkaline carbonate components, sodium bicarbonate, and any combination thereof. Specific examples of disintegrants include cross-linked sodium carboxymethyl cellulose (e.g., croscarmellose) and sodium starch glycolate.

The term "lubricant" is used herein in its general sense as understood by those skilled in the art, and can improve the compression and projection of the composition, for example, by a hard die press. Exemplary lubricants include magnesium stearate, stearic acid (stearin), hydrogenated oil, sodium stearyl fumarate, sodium lauryl sulfate, talc, fatty acids, calcium stearate, sodium stearate, glyceryl monostearate, fatty alcohols, fatty acid esters, glyceryl behenate, mineral oil, vegetable oil, leucine, sodium benzoate, and any combination thereof. A specific example of a lubricant is sodium stearyl fumarate.

Those skilled in the art understand that a particular compound described as a wetting agent, binder, filler, disintegrant, and lubricant may serve one or more purposes. For example, microcrystalline cellulose may be used as a disintegrant and a filler.

In some embodiments, the composition may include Compound I-1 or Form A in an amount ranging from about 5 wt % to about 50 wt %, based on the total weight of the composition; and one or more fillers in an amount ranging from about 10 wt % to about 90 wt %, based on the total weight of the composition. In other embodiments, the composition may include Compound I-1 or Form A in an amount ranging from about 5 wt % to about 50 wt %, based on the total weight of the composition; one or more fillers in an amount ranging from about 10 wt % to about 90 wt %, based on the total weight of the composition; and one or more disintegrants in an amount ranging from about 1 wt % to about 15 wt %, based on the total weight of the composition. In other embodiments, the composition may include Compound I-1 or Form A in an amount ranging from about 5 wt % to about 50 wt %, based on the total weight of the composition; one or more fillers in an amount ranging from about 10 wt % to about 90 wt %, based on the total weight of the composition; one or more disintegrants in an amount ranging from about 1 wt % to about 15 wt %, based on the total weight of the composition; and one or more lubricants in an amount ranging from about 0.1 wt % to about 10 wt %, based on the total weight of the composition.

In some embodiments, the composition may include Compound I-1 or Form A in an amount ranging from about 5 wt % to about 20 wt %, based on the total weight of the composition; one or more lubricants in an amount ranging from about 1 wt % to about 5 wt %, based on the total weight of the composition; one or more disintegrants in an amount ranging from about 1 wt % to about 10 wt %, based on the total weight of the composition; and one or more fillers in an amount ranging from about 70 wt % to about 90 wt %, based on the total weight of the composition. In other embodiments, the composition may include Compound I-1 or Form A in an amount ranging from about 5 wt % to about 15 wt %, based on the total weight of the composition; one or more lubricants in an amount ranging from about 1 wt % to about 5 wt %, based on the total weight of the composition; one or more disintegrants in an amount ranging from about 1 wt % to about 5 wt %, based on the total weight of the composition; and one or more fillers in an amount ranging from about 70 wt % to about 90 wt %, based on the total weight of the composition.

In some embodiments, the composition can include Compound 1-1 or Form A in an amount of about 10 wt %, based on the total weight of the composition, lactose monohydrate in an amount of about 28 wt %, based on the total weight of the composition, Avicel PH-101 (microcrystalline cellulose) in an amount of about 55 wt %, based on the total weight of the composition, Ac-Di-Sol (croscarmellose sodium) in an amount of about 5 wt %, based on the total weight of the composition, and sodium stearyl fumarate in an amount of about 3 wt %, based on the total weight of the composition.

In some embodiments, the composition may further include one or more glidants (or "flow aids"). Glidants enhance the flow properties of the composition by reducing inter-particle friction and agglomeration. Exemplary glidants include colloidal silicon dioxide, talc, and any combination thereof. A specific example of a glidant is amorphous colloidal silicon dioxide having an average particle size of 0.2-0.3 microns, such as M5P. The amount of glidant can vary. For example, the amount of glidant can range from about 0.1 wt% to about 3 wt%, or from about 0.1 wt% to about 1 wt%, based on the total weight of the composition (wherein the total weight includes the weight of the one or more glidants).

In some embodiments, the compositions described herein may further comprise a coating.

In some embodiments, the compositions described herein can be in a solid dosage form, such as a tablet.

Some embodiments described herein relate to methods for preparing the compositions described herein. In some embodiments, a method may include providing a mixture comprising Compound 1-1 or Form A and one or more fillers to form a composition. In other embodiments, a method may include providing a mixture comprising Compound 1-1 or Form A, a lubricant, a disintegrant, and a filler to form a composition. Examples, including specific examples, of lubricants, disintegrants, and fillers are each independently as described herein.

In some embodiments, a method may include combining Compound 1-1 or Form A with one or more first excipients to form a mixture; and mixing the mixture (including Compound 1-1 or Form A and one or more first excipients) with one or more second excipients. In some embodiments, the first excipients may include one or more of the following: one or more fillers, one or more disintegrants, and one or more lubricants. In some embodiments, the second excipients may include one or more of the following: one or more disintegrants and one or more lubricants.

In other embodiments, methods of preparing the compositions described herein can include: i) combining Compound I-1 or Form A with one or more first excipients, which can include one or more fillers, one or more disintegrants, and one or more lubricants, and ii) mixing the mixture from i) with one or more second excipients, which can include one or more disintegrants and one or more lubricants, to form a composition. In some embodiments, the one or more first excipients can include one or more fillers in an amount ranging from about 70 wt % to about 90 wt %, one or more disintegrants in an amount ranging from about 1 wt % to about 15 wt %, and one or more lubricants in an amount ranging from about 1 wt % to about 5 wt %, each based on the total weight of the composition, and the second excipient can include one or more lubricants in an amount ranging from about 0.5 wt % to about 5 wt %, and one or more disintegrants in an amount ranging from about 0.5 wt % to about 5 wt %, each based on the total weight of the composition.

In some embodiments, methods of preparing the compositions described herein can include: i) providing particles of Compound I-1 or Form A by combining Compound I-1 or Form A with a first excipient, which can include one or more fillers, one or more disintegrants, and one or more lubricants; and ii) mixing the particles of Compound I-1 or Form A obtained in i) with a second excipient, which can include one or more disintegrants and one or more lubricants, and optionally one or more fillers, to form a composition. In some embodiments, the first excipient can include one or more fillers in an amount ranging from about 70 wt % to about 90 wt %, one or more disintegrants in an amount ranging from about 0.5 wt % to about 5 wt %, and a first lubricant in an amount ranging from about 1 wt % to about 5 wt %, each based on the total weight of the composition; the second excipient can include one or more second lubricants in an amount ranging from about 0.5 wt % to about 5 wt %, and one or more disintegrants in an amount ranging from about 0.5 wt % to about 5 wt %, each based on the total weight of the composition. Examples of suitable lubricants, disintegrants, and fillers, including specific examples, are described herein.

In some embodiments, the method of preparing the compositions described herein can include sieving Compound 1-1 or Form A; mixing granules of Compound 1-1 or Form A with one or more fillers, one or more disintegrants, and one or more lubricants; and blending the resulting granules with one or more disintegrants and one or more lubricants.

In some embodiments, a method of preparing a composition described herein can comprise compressing granules comprising Compound 1-1 or Form A through a tablet press to form a tablet comprising Compound 1-1 or Form A.

In some embodiments, tablets comprising Compound 1-1 or Form A (eg, tablets obtained after compression) can be film-coated.

The compositions described herein may further include one or more pharmaceutically acceptable carriers in addition to those mentioned above. As used herein, "pharmaceutically acceptable" means inert and does not unduly inhibit the biological activity of the compound. A pharmaceutically acceptable carrier should be biocompatible, for example, non-toxic, non-inflammatory, non-immunogenic, or without other unwanted reactions or side effects after administration to a subject. Furthermore, standard pharmaceutical formulation techniques can be used to incorporate one or more pharmaceutically acceptable carriers.

Some examples of materials that can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers; alumina; aluminum stearate; lecithin; serum proteins (e.g., human serum albumin); buffer substances (e.g., phosphates or glycine); partial glyceride mixtures of saturated vegetable fatty acids; water; salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts); silica gel; magnesium trisilicate; polyacrylates; waxes; polyethylene-polyoxypropylene block polymers; methylcellulose; hydroxypropyl methylcellulose; lanolin; sugars such as glucose; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and Cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; other nontoxic biocompatible lubricants; coloring agents; release agents; sweeteners; flavoring agents; aromatherapy agents; preservatives; adsorbents and antioxidants may also be present in the composition at the discretion of the pharmacist.

Some embodiments described herein relate to methods of inhibiting or reducing ATR activity in a subject, which can include administering to the subject a composition described herein comprising an effective amount of Compound 1-1 or Form A, or a pharmaceutically acceptable salt thereof.

Other embodiments described herein relate to methods of treating cancer in a subject, which can include administering to the subject a composition described herein comprising an effective amount of Compound 1-1 or Form A, or a pharmaceutically acceptable salt thereof.

Other embodiments described herein relate to the use of the composition described herein in the manufacture of a drug for treating cancer, wherein the composition contains an effective amount of Compound I-1 or Form A or a pharmaceutically acceptable salt thereof.

In some embodiments, substantially all of the weight of Compound 1-1 in the compositions described herein can be in Form A.

In some embodiments, at least 90% by weight of Compound 1-1 in a composition described herein may be in Form A.

In some embodiments, at least 95% by weight of Compound 1-1 in a composition described herein may be in Form A.

In some embodiments, at least 98% by weight of Compound 1-1 in a composition described herein may be in Form A.

In some embodiments, at least 99% by weight of Compound 1-1 in a composition described herein may be in Form A.

The compositions described herein can be administered to humans and other animals orally, rectally, parenterally, intracisternal, intravaginal, intraperitoneally, topically (as a powder, ointment, or drops), buccally, as an oral or nasal spray, etc. The term "parenteral" as used herein includes, but is not limited to, subcutaneous, intravenous, intramuscular, intraarterial, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. In some embodiments, the compositions described herein can be administered orally, intraperitoneally, and/or intravenously.

Any oral acceptable dosage form includes, but is not limited to, capsules, tablets, aqueous suspensions, or solutions. In the case of tablets, suitable carriers include, but are not limited to, lactose and corn starch. Lubricants, such as magnesium stearate, and/or wetting agents may be added. When aqueous suspensions are used, the active ingredient may be combined with an emulsifying and/or suspending agent. If desired, sweeteners, flavorings, coloring agents, and/or aromatics may be included.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid dosage form may contain inert excipients such as water or other solvents, solubilizers and emulsifiers such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan fatty acid esters, and mixtures thereof.

Solid dosage forms for oral administration include capsules (eg, soft and hard-filled gelatin capsules), tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be mixed with at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and/or a) fillers, such as starches, lactose, milk sugar, sucrose, glucose, mannitol, and silicic acid, b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia, c) humectants, such as glycerol, d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) dissolution delaying agents, such as paraffin, f) absorption accelerators, such as quaternary ammonium compounds, g) wetting agents, such as cetyl alcohol and glycerol monostearate, h) absorbents, such as kaolin and bentonite, and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also include a buffer.

Tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings known in the art of pharmaceutical preparations. They may optionally contain an opacifying agent and may also be a composition that releases the active ingredient only or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active compound can be in microencapsulated form with one or more excipients.

Sterile injectable forms can be aqueous or oily suspensions. Injectable preparations can be formulated according to known techniques, using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations can be sterile injectable solutions, suspensions, or emulsions in a nontoxic, parenterally acceptable diluent or solvent, such as a solution in propylene glycol. Acceptable vehicles and solvents that can be used are water, Ringer's solution U.S.P., and isotonic sodium chloride solution. In addition, sterile fixed oils can be used as solvents or suspending media. For this purpose, any brand of fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, can be used in the preparation of injectables because they are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants, which are commonly used to formulate pharmaceutically acceptable dosage forms, including emulsions and suspensions.

The injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use.

Topical or transdermal dosage forms include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches. The active ingredient can be mixed with a pharmaceutically acceptable carrier under aseptic conditions and can include any preservatives and/or buffers. Ophthalmic preparations, ear drops, and eye drops can be formulated. Such dosage forms can be prepared by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flow of the compound into the skin. Rate can be controlled by providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

Alternatively, the active compound and pharmaceutically acceptable compositions thereof may be administered by nasal aerosol or inhalation. Such compositions may be prepared according to techniques well known in the art of pharmaceutical formulation and may be formulated as saline solutions, using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

Surfactants, such as Tweens, Spans and other emulsifiers or bioavailability enhancers, may be included in the solid, liquid, and other dosage forms described herein.

The compositions described herein can be formulated in unit dosage form. The term "unit dosage form" refers to a physically discrete unit suitable as a single dose for a subject to be treated, each unit containing a predetermined amount of active substance calculated to produce the desired therapeutic effect, optionally in combination with a suitable pharmaceutical carrier. The unit dosage form can provide one of a single daily dose or multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form of each dose can be the same or different. The amount of active compound in the unit dosage form will depend, for example, on the host being treated and the specific mode of administration, for example, ranging from 0.01 mg/kg body weight/dose to 100 mg/kg body weight/dose.

In some embodiments, the compositions described herein can be in the form of a solid dosage form. In some embodiments, the compositions described herein can be in the form of a tablet. In other embodiments, the compositions can be in the form of a 100 mg tablet or a 500 mg tablet.

It will be appreciated that the amount of active compound (e.g., Compound I-1 or Form A) required for treatment will vary not only depending on the specific compound selected, but also on the route of administration, the nature of the condition being treated, and the age and condition of the subject, and will ultimately be determined by the attending physician or veterinarian. However, in general, a suitable dosage will be in the range of about 0.1 to about 100 mg/kg body weight per dose, for example, in the range of 0.5 to 50 mg/kg/dose, for example, in the range of 1 to 10 mg/kg/dose.

In some embodiments, the compositions described herein can be administered in an amount of about 5 mg to about 100 mg of Compound 1-1 or Form A, or a pharmaceutically acceptable salt thereof, per dose.

In some embodiments, the compositions described herein may be administered in an amount of:

a) about 5 mg of Compound 1-1 or Form A or a pharmaceutically acceptable salt thereof per dose;

b) about 10 mg of Compound 1-1 or Form A or a pharmaceutically acceptable salt thereof per dose;

c) about 20 mg of Compound 1-1 or Form A or a pharmaceutically acceptable salt thereof per dose;

d) about 30 mg of Compound 1-1 or Form A or a pharmaceutically acceptable salt thereof per dose;

e) about 50 mg of Compound 1-1 or Form A or a pharmaceutically acceptable salt thereof per dose;

f) about 60 mg of Compound 1-1 or Form A or a pharmaceutically acceptable salt thereof per dose;

g) about 80 mg of Compound 1-1 or Form A or a pharmaceutically acceptable salt thereof per dose; or

h) about 100 mg of Compound 1-1 or Form A or a pharmaceutically acceptable salt thereof per dose.

In some embodiments, the compositions described herein can be administered in a fasted state (e.g., the subject has not consumed food or liquids, except water, for at least 8 hours). In other embodiments, the compositions described herein can be administered in a fed state (e.g., with food or within 1 hour of eating).

Compound Uses

In one aspect, the present invention provides ATR kinase inhibitor compounds or compositions, which are useful for treating or lessening the severity of a disease, disorder, or condition in a subject, wherein the disease, disorder, or condition involves ATR.

Another aspect of the present invention provides compounds or compositions that can be used to treat diseases, illnesses, and conditions characterized by excessive or abnormal cell proliferation. Such diseases include proliferative or hyperproliferative diseases. Examples of proliferative and hyperproliferative diseases include, but are not limited to, cancer and myeloproliferative disorders.

In some embodiments, the compound is selected from I-1 or Form A. In other embodiments, the composition comprises Compound I-1 or Form A. The term "cancer" includes, but is not limited to, the following cancers. Oral cancer : oral cavity cancer, lip cancer, tongue cancer, mouth cancer, pharyngeal cancer; Heart cancer : sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyosarcoma, fibroma, lipoma and teratoma; Lung cancer: non-small cell lung cancer, bronchial cancer (squamous cell carcinoma or epidermoid carcinoma, undifferentiated small cell carcinoma, undifferentiated large cell carcinoma, adenocarcinoma), alveolar (bronchiolar) cancer, bronchial adenoma, sarcoma, lymphoma, enchondromatous hamartoma, mesothelioma; Stomach cancer : non-small cell lung cancer, bronchial cancer (squamous cell carcinoma or epidermoid carcinoma, undifferentiated small cell carcinoma, undifferentiated large cell carcinoma, adenocarcinoma), alveolar (bronchiolar) cancer, bronchial adenoma, sarcoma, lymphoma, enchondromatous hamartoma, mesothelioma ; Intestinal cancer : Esophageal cancer (squamous cell carcinoma, laryngeal cancer, adenocarcinoma, leiomyosarcoma, lymphoma), stomach cancer (carcinoma, lymphoma, leiomyosarcoma), pancreatic cancer (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, vipoma), small intestine cancer (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), colorectal cancer (adenocarcinoma, tubular adenoma, villous adenoma, dysplasia), Genitourinary tract cancers : kidney cancer (adenocarcinoma, Wilms tumor [Nephroblastoma], lymphoma, leukemia), bladder and urethra cancer (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate cancer (adenocarcinoma, sarcoma), testicular cancer (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatoid tumor, lipoma), and urethral cancer (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma). Liver cancer : hepatocellular carcinoma, bile duct cancer, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, biliary tract cancer; Bone cancer : osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondrofibroma (osteochronfroma), benign Nervous system cancers : skull cancers (osteomas, hemangiomas, granulomas, xanthomas, osteitis deformans), meningeal cancers (meningiomas, meningiosarcomas, gliomas), brain cancers (astrocytomas, medulloblastomas, gliomas, ependymomas, germ cell tumors [pinealomas]), glioblastomas multiforme, oligodendrogliomas, schwannomas, retinoblastomas, congenital gliomas, and gliomas). Gynecological cancers : uterine cancer (endometrial cancer), cervical cancer (cervical cancer, preneoplastic cervical atypical hyperplasia), ovarian cancer (ovarian cancer [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecoma, Severe-Lewis cell tumor, dysgerminoma, malignant teratoma), vulvar cancer (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vaginal cancer (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), Cancers of the fallopian tube (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonic rhabdomyosarcoma), fallopian tube cancer (carcinoma), breast cancer; Hematological cancers (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma], hairy cell cancer; Lymphatic disorders; Skin cancers (malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, keratoacanthoma, dysplastic nevus, lipoma, hemangioma, dermatofibroma, keloid, psoriasis); Thyroid cancers (papillary thyroid cancer, follicular thyroid cancer, anaplastic thyroid cancer, medullary thyroid cancer, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid cancer, pheochromocytoma, paraganglioma); and Adrenal gland cancers (neuroblastoma).

In some embodiments, the cancer is selected from lung or pancreatic cancer. In other embodiments, the cancer is selected from lung cancer, head and neck cancer, pancreatic cancer, stomach cancer, or brain cancer. In other embodiments, the cancer is selected from non-small cell lung cancer, small cell lung cancer, pancreatic cancer, biliary tract cancer, head and neck cancer, bladder cancer, colorectal cancer, glioblastoma, esophageal cancer, breast cancer, hepatocellular carcinoma, or ovarian cancer.

In some embodiments, the cancer is lung cancer. In other embodiments, the lung cancer is non-small cell lung cancer or small cell lung cancer. In another embodiment, the cancer is non-small cell lung cancer. In yet another embodiment, the non-small cell lung cancer is squamous non-small cell lung cancer.

Thus, the term "cancer cell" as provided herein includes cells affected by any of the conditions identified above. In some embodiments, the cancer is selected from colorectal cancer, thyroid cancer, or breast cancer. In other embodiments, the cancer is triple-negative breast cancer.

The term "myeloproliferative disorder" includes disorders such as polycythemia vera, thrombocythemia, myeloid metaplasia with myelofibrosis, hypereosinophilic syndrome, juvenile myelomonocytic leukemia, systemic mastocytosis, and hematopoietic disorders, particularly acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute promyelocytic leukemia (APL), and acute lymphoblastic leukemia (ALL).

Combination therapy

Another aspect of the present invention is directed to a method for treating cancer in a subject in need thereof, comprising administering a compound or composition of the present invention, or a pharmaceutically acceptable salt thereof, and an additional therapeutic agent. In some embodiments, the method comprises administering a compound or composition (or a pharmaceutically acceptable salt thereof) and an additional therapeutic agent sequentially or concurrently.

As used herein, the terms "combination" or "co-administration" are used interchangeably to refer to the use of more than one therapy (e.g., one or more therapeutic agents). The use of the terms does not limit the order in which the therapies (e.g., therapeutic agents) are administered to a subject or the dosage regimen of each therapeutic agent.

In some embodiments, the additional therapeutic agent is an anticancer agent. In other embodiments, the additional therapeutic agent is a DNA-damaging agent. In other embodiments, the additional therapeutic agent is selected from radiation therapy, chemotherapy, or other agents commonly used in combination with radiation therapy or chemotherapy, such as radiosensitizers and chemosensitizers. In other embodiments, the additional therapeutic agent is ionizing radiation.

As will be appreciated by those skilled in the art, radiosensitizers are ingredients that can be used in combination with radiation therapy. Radiosensitizers work in various ways, including but not limited to making cancer cells more sensitive to radiation therapy, providing an improved synergistic effect in conjunction with radiation therapy, acting additively with radiation therapy, or protecting surrounding healthy cells from damage by radiation therapy. Similarly, chemosensitizers are ingredients that can be used in combination with chemotherapy. Similarly, chemosensitizers work in various ways, including but not limited to making cancer cells more sensitive to chemotherapy, providing an improved synergistic effect in conjunction with chemotherapy, acting additively with chemotherapy, or protecting surrounding healthy cells from damage by chemotherapy.

Examples of DNA-damaging agents that can be used in combination with the compounds or compositions of the present invention include, but are not limited to, platinating agents such as cisplatin, carboplatin, nedaplatin, satraplatin, and other derivatives; Topo I inhibitors such as topotecan, irinotecan/SN38, rubitecan, and other derivatives; antimetabolites such as the folic acid family (methotrexate, pemetrexed, and related substances); purine antagonists and pyrimidine antagonists (thioguanine, fludarabine, cladribine, cytarabine, gemcitabine, 6-mercaptopurine, 5-fluorouracil (5FU), and related substances); alkylating agents such as nitrogen mustards (cyclophosphamide, melphalan, chlorambucil, dichlorvos, and chloranil). methylamine, ifosfamide, and related substances); nitrosoureas (e.g., carmustine); triazenes (dacarbazine, temozolomide); alkyl sulfonates (e.g., busulfan); procarbazine and aziridines; antibiotics such as hydroxyurea, anthracyclines (doxorubicin, daunorubicin, epirubicin, and other derivatives); anthracenediones (mitoxantrone and related substances); Streptomyces family (bleomycin, mitomycin C, actinomycin); and ultraviolet light.

In some embodiments, the additional therapeutic agent is ionizing radiation. In other embodiments, the additional therapeutic agent is cisplatin or carboplatin. In other embodiments, the additional therapeutic agent is etoposide. In other embodiments, the additional therapeutic agent is temozolomide. In other embodiments, the additional therapeutic agent is irinotecan/SN38.

In certain embodiments, the additional therapeutic agent is selected from one or more of the following: cisplatin, carboplatin, irinotecan/SN38, gemcitabine, etoposide, temozolomide, or ionizing radiation.

Other therapies or anticancer agents that can be used in combination with the compounds and compositions of the present invention include surgery, radiation therapy (gamma-irradiation, neutron beam radiation therapy, electron beam radiation therapy, proton therapy, brachytherapy, and systemic radioisotopes, to name a few), endocrine therapy, biological response modifiers (interferons, interleukins, and tumor necrosis factor (TNF), etc.), hyperthermia and cryotherapy, agents to attenuate any adverse effects (e.g., antiemetics), and other approved chemotherapeutic drugs, including but not limited to the DNA-damaging agents listed herein, spindle poisons (vinblastine, vincristine, vinorelbine, paclitaxel), podophyllotoxins (etoposide, irinotecan, topotecan), nitrosoureas (carmustine, lomustine), inorganic ions (cisplatin, carboplatin), enzymes (asparaginase), and hormones (tamoxifen, leuprorelin, flutamide, and megestrol acetate), Gleevec ^™ , doxorubicin, dexamethasone, and cyclophosphamide.

The compounds or compositions of the present invention may also be used in combination with any of the following therapeutic agents for the treatment of cancer: abarelix (Plenaxis); aldesleukin; aldesleukin alemtuzumab; alitretinoin; allopurinol; altretamine; amifostine Anastrozole, arsenic trioxide, asparaginase, azacitidine, bevacizumab, bexarotene capsules, bexarotene gel, bleomycin, bortezomib, intravenous busulfan, oral busulfan, calusterone, capecitabine, carboplatin, carmustine (Gliadel), carmustine membrane implant with polyphenylprosan 20 as carrier, celecoxib, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide (Cytoxan), cytarabine, liposomal dacarbazine, dactinomycin, actinomycin D, darbepoetin alfa Daunorubicin alfa (liposomal), daunorubicin, daunomycin, daunorubicin, denileukin diftitox, dexrazoxane, docetaxel, adriamycin (doxorubicin PFS), dromostanolone propionate (doxorubicin PFS), masterone propionate, Elliott's B Solution (sodium potassium magnesium calcium glucose injection), epirubicin, epoetin alfa, erlotinib, estramustine, etoposide phosphate, etoposide, VP-16, exemestane Filgrastim, floxuridine (intra-arterial), fludarabine, fluorouracil, 5-FU, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate (Zoladex), histrelin acetate (Histrelin), hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alfa-2a (Roferon), interferon alfa-2b (Intron), irinotecan, lenalidomide, letrozole, leucovorin, leuprorelin acetate, levamisole Lomustine, CCNU, mechlorethamine, megestrol acetate, melphalan, L-PAM, mercaptopurine, 6-MP, mesna, Mesnex; methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone phenpropionate, nelarabine, nofetumomab, oprelvekin, oxaliplatin, paclitaxel, protein-bound particles, palifermin, pamidronate, pegademase (Adagen (Pegademase Bovine)); pegapastor Pegfilgrastim, pemetrexed disodium, pentostatin, pipobroman, plicamycin, mithramycin, porfimer sodium, procarbazine, quinacrine, rasburicase, rituximab, sargramostim, sorafenib, streptozocin, sunitinib maleate, talc, tamoxifen, temozolomide, teniposide, VM-26 testolactone, thioguanine, 6-TG, thiotepa, topotecan, toremifene Tositumomab (toremifene), tositumomab/I-131, tositumomab, trastuzumab, tretinoin, ATRA, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, zoledronate, and vorinostat

For a comprehensive review of the latest cancer therapies, see the FDA-approved list of oncology drugs at http://www.nci.nih.gov/, http://www.fda.gov/cder/cancer/druglistframe.htm, and The Merck Manual, Seventeenth Edition, 1999, all of which are incorporated herein by reference.

Another embodiment provides for administering a compound or composition of the invention with an additional therapeutic agent that inhibits or regulates a base excision repair protein. In some embodiments, the base excision repair protein is selected from UNG, SMUG1, MBD4, TDG, OGG1, MYH, NTH1, MPG, NEIL1, NEIL2, NEIL3 (DNA glucosyltransferases); APE1, APEX2 (AP endonucleases); LIG1, LIG3 (DNA ligases I and III); XRCC1 (LIG3 accessory); PNK, PNKP (polynucleotide kinase and phosphatase); PARP1, PARP2 (poly (ADP-ribose) polymerase); PolB, PolG (polymerase); FEN1 (endonuclease) or Aprataxin. In other embodiments, the base excision repair protein is selected from PARP1, PARP2 or PolB. In other embodiments, the base excision repair protein is selected from PARP1 or PARP2. In some embodiments, the therapeutic agent is selected from Olaparib (also known as AZD2281 or KU-0059436), Iniparib (also known as BSI-201 or SAR240550), Veliparib (also known as ABT-888), Rucaparib (also known as PF-01367338), CEP-9722, INO-1001, MK-4827, E7016, BMN673, or AZD2461.

Treatment

In one aspect, the present invention relates to a method of inhibiting ATR kinase activity in a patient, comprising administering to the patient a compound described herein or a composition comprising the compound. In some embodiments, the method is used to treat or prevent a condition selected from a proliferative and hyperproliferative disease, such as cancer.

In some embodiments, the cancer is selected from the cancers described herein. In some embodiments, the cancer is lung cancer, head and neck cancer, pancreatic cancer, stomach cancer, or brain cancer. In other embodiments, the cancer is selected from lung or pancreatic cancer.

In other embodiments, the cancer is selected from non-small cell lung cancer, small cell lung cancer, pancreatic cancer, biliary tract cancer, head and neck cancer, bladder cancer, colorectal cancer, glioblastoma, esophageal cancer, breast cancer, hepatocellular carcinoma, or ovarian cancer.

In some embodiments, the lung cancer is small cell lung cancer and the additional therapeutic agent is cisplatin and etoposide. In other embodiments, the lung cancer is non-small cell lung cancer and the additional therapeutic agent is gemcitabine and cisplatin. In other embodiments, the non-small cell lung cancer is squamous non-small cell lung cancer. In another embodiment, the cancer is breast cancer and the additional therapeutic agent is cisplatin. In other embodiments, the cancer is triple-negative breast cancer.

In certain embodiments, an "effective amount" of a compound or pharmaceutically acceptable composition is an amount effective to treat the disease. According to the methods of the present invention, compounds and compositions may be administered using any amount and any route of administration effective to treat or lessen the severity of the disease.

One aspect provides a method of inhibiting ATR in a patient comprising administering a compound or composition as described herein. Another embodiment provides a method of treating cancer comprising administering to a patient a compound or composition as described herein, wherein the variables are as defined herein.

Another embodiment provides a method of treating pancreatic cancer by administering a compound or composition described herein in combination with another known pancreatic cancer therapeutic agent. In one aspect, the invention comprises administering a compound or composition described herein in combination with gemcitabine. In some embodiments, the pancreatic cancer comprises one of the following cell lines: PSN-1, MiaPaCa-2, or Panc-1. According to another aspect, the cancer comprises one of the following primary tumor lines: Panc-M or MRC5.

Another aspect of the invention includes administering a compound or composition described herein in combination with radiation therapy.Another aspect provides a method of eliminating the radiation-induced G2/M checkpoint by administering a compound or composition described herein in combination with radiation therapy.

On the other hand, a method for treating pancreatic cancer is provided by administering a combination of a compound or composition described herein and one or more cancer therapies to pancreatic cancer cells. In some embodiments, the compound or composition is combined with chemoradiation, chemotherapy, and/or radiation therapy. As will be appreciated by those skilled in the art, "chemoradiation" refers to a treatment regime that includes both chemotherapy (e.g., gemcitabine) and radiation. In some embodiments, chemotherapy is gemcitabine.

Another aspect provides a method of increasing the sensitivity of pancreatic cancer cells to a cancer therapy selected from gemcitabine or radiation therapy by administering a compound or composition described herein in combination with the cancer therapy.

In some embodiments, the cancer therapy is gemcitabine. In other embodiments, the cancer therapy is radiation therapy. In yet another embodiment, the cancer therapy is chemoradiation.

Another aspect provides a method of inhibiting Chk1 (Ser 345) phosphorylation in pancreatic cancer cells, comprising administering a compound or composition described herein to the pancreatic cancer cells after treatment with gemcitabine (100 nM) and/or radiation (6 Gy).

Another aspect provides methods for disrupting damage-induced cell cycle checkpoints by administering to cancer cells a compound or composition described herein in combination with radiation therapy.

Another aspect provides a method of inhibiting homologous recombination repair of DNA damage in cancer cells by administering a compound or composition described herein in combination with one or more of the following treatments: chemoradiation, chemotherapy, and radiation therapy.

In some embodiments, the chemotherapy is gemcitabine.

Another aspect provides a method of inhibiting homologous recombination repair of DNA damage in cancer cells by administering a compound or composition described herein in combination with gemcitabine and radiation therapy.

Another aspect of the present invention provides a method of treating non-small cell lung cancer comprising administering to a patient a compound or composition described herein in combination with one or more of the following additional therapeutic agents: cisplatin or carboplatin, etoposide, and ionizing radiation. Some embodiments comprise administering to a patient a compound described herein in combination with cisplatin or carboplatin, etoposide, and ionizing radiation. In some embodiments, the combination is cisplatin, etoposide, and ionizing radiation. In other embodiments, the combination is carboplatin, etoposide, and ionizing radiation.

Another embodiment provides a method of promoting cell death in cancer cells comprising administering to a patient a compound described herein or a composition comprising said compound.

Another embodiment provides a method for preventing cellular repair of DNA damage in cancer cells, comprising administering to a patient a compound as described herein or a composition comprising said compound. Another embodiment provides a method for preventing cellular repair caused by DNA damage in cancer cells, comprising administering to a patient a compound as described herein or a composition comprising said compound.

Another embodiment provides a method of sensitizing a cell to a DNA damaging agent comprising administering to a patient a compound described herein or a composition comprising said compound.

In some embodiments, the method is used to target cancer cells that have a defect in the ATM signaling cascade. In some embodiments, the defect is altered expression or activity of one or more of: ATM, p53, CHK2, MRE11, RAD50, NBS1, 53BP1, MDC1, H2AX, MCPH1/BRIT1, CTIP, or SMC1. In other embodiments, the defect is altered expression or activity of one or more of: ATM, p53, CHK2, MRE11, RAD50, NBS1, 53BP1, MDC1, or H2AX. According to another embodiment, the method is used to target cancer, cancer cells, or cells that express a DNA-damaging oncogene.

In another embodiment, the cell is a cancer cell that expresses a DNA damaging oncogene. In some embodiments, the cancer cell has altered expression or activity of one or more of K-Ras, N-Ras, H-Ras, Raf, Myc, Mos, E2F, Cdc25A, CDC4, CDK2, Cyclin E, Cyclin A, and Rb.

According to another embodiment, the method is used for cancer, cancer cells, or cells with defects in proteins involved in base excision repair ("base excision repair proteins"). Many methods are known in the art to detect whether a tumor has a base excision repair defect. For example, the genomic DNA or mRNA product of each base excision repair gene (e.g., UNG, PARP1, or LIG1) can be sequenced in a tumor sample to determine whether there are mutations that are expected to regulate the function or expression of the gene product (Wang et al., Cancer Research, Vol. 52, p. 4824, 1992). In addition to mutational inactivation, tumor cells can also regulate DNA repair genes by hypermethylating their promoter regions, causing reduced gene expression. This is most commonly assessed using methylation-specific polymerase chain reaction (PCR) to quantify the level of methylation on the promoter of the relevant base excision repair gene. Base excision repair gene promoter methylation assays are commercially available (http://www.sabiosciences.com/dna_methylation_product/HTML/ME AH-421A.html).

Finally, the expression levels of base excision repair genes can be assessed by directly quantifying the levels of each gene's mRNA and protein products using standard techniques, such as quantitative reverse transcriptase-coupled polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC), respectively (Shinmura et al., Carcinogenesis, vol. 25, p. 2311, 2004; Shinmura et al., Journal of Pathology, vol. 225, p. 414, 2011).

In some embodiments, the base excision repair protein is UNG, SMUG1, MBD4, TDG, OGG1, MYH, NTH1, MPG, NEIL1, NEIL2, NEIL3 (DNA glucosyltransferases); APE1, APEX2 (AP endonucleases); LIG1, LIG3 (DNA ligases I and III); XRCC1 (LIG3 accessory enzyme); PNK, PNKP (polynucleotide kinases and phosphatases); PARP1, PARP2 (poly (ADP-ribose) polymerases); PolB, PolG (polymerases); FEN1 (endonuclease) or Aprataxin.

In some embodiments, the base excision repair protein is PARP1, PARP2, or Pol B. In other embodiments, the base excision repair protein is PARP1 or PARP2.

The above methods (gene sequence, promoter methylation, and mRNA expression) can also be used to characterize the status (e.g., expression or mutation) of other related genes or proteins, such as DNA-damaging oncogenes expressed by tumors or defects in the cellular ATM signaling cascade.

Another embodiment provides the use of a compound or composition described herein as a radiosensitizer or chemosensitizer.

Other embodiments provide the purposes of compound described herein or composition as a single component (monotherapy) for treating cancer.In some embodiments, compound described herein or composition are used to treat patients with DNA-damage response (DDR) defective cancer.In other embodiments, the defect is a mutation or disappearance of ATM, p53, CHK2, MRE11, RAD50, NBS1, 53BP1, MDC1 or H2AX.

the term

The term "subject", "host" or "patient" includes animals and humans (eg, male or female, eg, children, teenagers or adults). Preferably, the "subject", "host" or "patient" is a human.

The compounds of the present invention include those generally described herein, as further illustrated by the classes, subclasses, and varieties disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For the purposes of the present invention, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th edition. In addition, general principles of organic chemistry are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito: 1999, and March's Advanced Organic Chemistry, 5th edition, Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, all of which are incorporated herein by reference.

As used herein, a range of numbers of atoms specified includes any integer therein. For example, a group having 1-4 atoms may have 1, 2, 3, or 4 atoms.

As described herein, the compounds of the present invention may optionally be substituted with one or more substituents, such as those generally described herein, or as exemplified by specific species, subclasses, or varieties of the present invention. It will be appreciated that the phrases "optionally substituted" and "substituted or unsubstituted" are used interchangeably. Generally speaking, the term "substituted," whether or not preceded by the term "optionally," refers to the replacement of a hydrogen radical in a given structure with a radical of a specified substituent. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group, and when one or more positions in any given structure may be substituted by one or more substituents selected from a specified group, the substituents at each position may be the same or different. The substituent combinations contemplated by the present invention are preferably those that generate stable or chemically feasible compounds.

Unless otherwise indicated, a substituent to which a bond is drawn from the center of a ring means that the substituent can be bonded at any position in the ring. In the following example i, for example, ^J can be bonded at any position on the pyridyl ring. In the case of a bicyclic ring, a bond drawn through both rings indicates that the substituent can be bonded at any position on the bicyclic ring. In the following example ii, for example, ^J can be bonded to both a 5-membered ring (e.g., on a nitrogen atom) and a 6-membered ring.

As used herein, the term "stable" refers to compounds that remain substantially unchanged when subjected to conditions that allow them to be prepared, detected, recovered, purified, and used for one or more of the purposes disclosed herein. In some embodiments, a stable compound or chemically feasible compound is one that remains substantially unchanged when kept at 40° C. or less, in the absence of moisture or other chemically reactive conditions, for at least a week.

The term "dative bond" as used herein is defined as a coordination bond formed based on intermolecular interactions, one of which acts as a donor and the other as an acceptor of an electron pair shared in the resulting complex.

As used herein, the term "aliphatic" or "aliphatic group" means a straight (i.e., unbranched), branched, or cyclic substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more units of unsaturation having a single point of attachment to the rest of the molecule.

Unless otherwise indicated, aliphatic groups contain 1-20 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. Aliphatic groups can be straight-chain or branched, substituted or unsubstituted alkyl, alkenyl, or alkynyl groups. Specific examples include, but are not limited to, methyl, ethyl, isopropyl, n-propyl, sec-butyl, vinyl, n-butenyl, ethynyl, and tert-butyl. Aliphatic groups can also be cyclic or have a combination of straight-chain or branched groups and cyclic groups. Examples of these types of aliphatic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, _{-CH2 - cyclopropyl} , and CH2CH2CH( _CH3 )-cyclohexyl.

The term "alicyclic" (or "carbocycle" or "carbocyclyl") refers to a monocyclic _C3 - _C8 hydrocarbon or a bicyclic _C8 - _C12 hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic, having a single point of attachment to the rest of the molecule, wherein any individual ring in the bicyclic ring system has 3-7 members. Examples of alicyclic groups include, but are not limited to, cycloalkyl and cycloalkenyl. Specific examples include, but are not limited to, cyclohexyl, cyclopropyl, and cyclobutyl.

As used herein, the terms "heterocycle," "heterocyclyl," or "heterocyclic" refer to a non-aromatic monocyclic, bicyclic, or tricyclic ring system in which one or more ring members are independently selected heteroatoms. In some embodiments, a "heterocycle," "heterocyclyl," or "heterocyclic" group has three to fourteen ring members, one or more of which are heteroatoms independently selected from oxygen, sulfur, nitrogen, or phosphorus, and each ring in the system contains 3 to 7 ring members.

Examples of heterocycles include, but are not limited to, 3-1H-benzimidazol-2-one, 3-(1-alkyl)-benzimidazol-2-one, 2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahydrothienyl, 3-tetrahydrothienyl, 2-morpholino, 3-morpholino, 4-morpholino, 2-thiomorpholino, 3-thiomorpholino, 4-thiomorpholino, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, 1-tetrahydropiperazinyl, 2-tetrahydropiperazinyl, 3-tetrahydropiperazinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 1-pyrazolinyl, 3-pyrazolinyl, 4-pyrazolinyl, 5-pyrazolinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 2-thiazolidinyl, 3-thiazolidinyl, 4-thiazolidinyl, 1-imidazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl, 5-imidazolidinyl, indolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, benzothiolane, benzodithiane and 1,3-dihydro-imidazol-2-one.

Cyclic groups (eg, alicyclic and heterocyclic) can be linearly fused, bridged, or spirocyclic.

The term "heteroatom" means one or more oxygen, sulfur, nitrogen, phosphorus or silicon (including any oxidized form of nitrogen, sulfur, phosphorus or silicon; the quaternized form of any basic nitrogen; or a substitutable nitrogen of a heterocycle, such as N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR ⁺ (as in N-substituted pyrrolidinyl)).

The term "unsaturated" as used herein means that a group has one or more unsaturated units. As will be appreciated by those skilled in the art, an unsaturated group can be partially unsaturated or fully unsaturated. Examples of partially unsaturated groups include, but are not limited to, butene, cyclohexene, and tetrahydropyridine. Fully unsaturated groups can be aromatic, anti-aromatic, or non-aromatic. Examples of fully unsaturated groups include, but are not limited to, phenyl, cyclooctatetraene, pyridyl, thienyl, and 1-methylpyridin-2(1H)-one.

[0066] The term "alkoxy" or "thioalkyl," as used herein, refers to an alkyl group as defined above attached through an oxygen ("alkoxy") or sulfur ("thioalkyl") atom.

The terms "haloalkyl,""haloalkenyl,""haloaliphatic," and "haloalkoxy" refer to alkyl, alkenyl, or alkoxy groups, as appropriate, substituted with one or more halogen atoms _. The term includes perfluorinated alkyl groups such as _-CF3 and _-CF2CF3 .

The terms "halogen," "halo," and "halo" denote F, Cl, Br, or I.

The term "aryl," used alone or as part of a larger group such as "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains 3 to 7 ring members. The term "aryl" can be used interchangeably with the term "aromatic ring."

The term "heteroaryl," used alone or as part of a larger group such as "heteroaralkyl" or "heteroarylalkoxy," refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, at least one ring in the system contains one or more heteroatoms, and wherein each ring in the system contains 3 to 7 ring members. The term "heteroaryl" is used interchangeably with the term "heteroaryl ring" or the term "heteroaromatic." Examples of heteroaryl rings include, but are not limited to, 2-furyl, 3-furyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, benzimidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, pyridazinyl (e.g., 3-pyridazinyl), 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, tetrazolyl (e.g., 5-tetrazolyl), triazolyl (e.g., 2-triazolyl and 5-triazolyl), 2- thiophene, 3-thiophene, benzofuranyl, benzothiophene, indolyl (e.g., 2-indolyl), pyrazolyl (e.g., 2-pyrazolyl), isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,3-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, purinyl, pyrazinyl, 1,3,5-triazinyl, quinolyl (e.g., 2-quinolyl, 3-quinolyl, 4-quinolyl), and isoquinolyl (e.g., 1-isoquinolyl, 3-isoquinolyl, or 4-isoquinolyl).

It should be understood that the term "heteroaryl" includes certain types of heteroaryl rings that exist in equilibrium between two different forms. More specifically, for example, species such as hydrogenated pyridines and pyridones (and similarly, hydroxypyrimidines and pyrimidones) are intended to be encompassed within the definition of "heteroaryl."

As used herein, the terms "protecting group" and "protective group" are used interchangeably to refer to reagents used to temporarily block one or more desired functional groups in a compound having multiple reactive sites. In certain embodiments, the protecting group has one or more, preferably all, of the following characteristics: a) it is selectively added to the functional group to form a protected substrate in good yield; b) it is stable to reactions occurring at one or more other reactive sites; and c) it can be selectively removed in good yield by reagents that do not attack the regenerated deprotected functional group. As will be understood by those skilled in the art, in some cases, these reagents do not attack other reactive groups in the compound. In other cases, these reagents may also react with other reactive groups in the compound. Examples of protecting groups are described in Greene, T.W., Wuts, P.G., "Protective Groups in Organic Synthesis", Third Edition, John Wiley & Sons, New York: 1999 (and other editions thereof), the entire contents of which are incorporated herein by reference. As used herein, the term "nitrogen protecting group" refers to a reagent used to temporarily block one or more desired nitrogen reactive sites in a multifunctional compound. Preferred nitrogen protecting groups also have the characteristics exemplified above for the protecting groups. Some exemplary nitrogen protecting groups are also described in detail in Greene, T.W., Wuts, P.G., "Protective Groups in Organic Synthesis", Third Edition, John Wiley & Sons, New York: 1999, Chapter 7, the entire contents of which are incorporated herein by reference.

In some embodiments, the methylene unit of the alkyl or aliphatic chain is optionally replaced by another atom or group. Examples of such atoms or groups include, but are not limited to, nitrogen, oxygen, sulfur, -C(O)-, -C(=N-CN)-, -C(=NR)-, -C(=NOR)-, -SO-, and _-SO2- . These atoms or groups can be combined to form larger groups. Examples of such larger groups include, but are not limited to, -OC(O)-, -C(O)CO-, _-CO2- , -C(O)NR-, -C(=N-CN), -NRCO-, -NRC(O)O-, _-SO2NR- , _-NRSO2- , -NRC(O)NR-, -OC(O)NR-, and _-NRSO2NR- , where R is, for example, H or a _C1-6 aliphatic group. It should be understood that these groups can be bonded to the methylene unit of the aliphatic chain via single, double, or triple bonds. An example of an optional substitution (a nitrogen atom, in this case) bonded to an aliphatic chain via a double bond is _-CH2CH =N- _CH3 . In some cases, especially at the terminal ends, the optional substitution may be bonded to the aliphatic group via a _{triple bond} . An example would be _{CH2CH2CH2C≡N} . It should be understood that in this case, the terminal nitrogen is not bonded to another atom.

It will also be understood that the term "methylene unit" may also refer to a branched or substituted methylene unit. For example, in the isopropyl moiety [-CH(CH ₃ ) ₂ ], a nitrogen atom (e.g., NR) would replace the first cited "methylene unit" to give dimethylamine [-N(CH ₃ ) ₂ ]. In such cases, one skilled in the art will understand that the nitrogen atom will not have any additional atoms bonded to it, and the "R" from "NR" would not be present in this case.

Unless otherwise indicated, optional substitutions result in chemically stable compounds. Optional substitutions can occur within a chain and/or at either end of the chain; that is, at the point of attachment and/or at the terminus. Two optional substitutions can also occur adjacent to each other within a chain, as long as they result in a chemically stable compound. For example, a _C3 aliphatic group can be optionally replaced by two nitrogen atoms to produce –C–N≡N. Optional substitutions can also completely replace all carbon atoms in the chain. For example, a _C3 aliphatic group can be optionally replaced by -NR—, -C(O)—, and -NR— to produce -NRC(O)NR— (urea).

Unless otherwise indicated, if a substitution occurs at a terminal end, the replacing atom is bound to a hydrogen atom at the terminal _end . For _example , if a methylene unit _{of -CH2CH2CH3} is optionally replaced by -O-, the resulting compound may _{be -OCH2CH3} , _-CH2OCH3 , or _-CH2CH2OH . It should be understood that if the _terminal atom does not contain any _free valence electrons, then _a hydrogen atom is not required at the terminal end (e.g., _-CH2CH2CH =O or _-CH2CH2C≡N ).

Unless otherwise indicated, structures depicted herein are also intended to include all isomeric (e.g., enantiomeric, diastereomeric, geometric, conformational, and rotational) forms of the structure. For example, R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers are all included in the present invention. As will be understood by those skilled in the art, substituents are able to rotate freely about any rotatable bond. For example, a substituent drawn as also represents

Therefore, single stereochemical isomers of the present compounds as well as enantiomeric, diastereomeric, geometric, conformational, and rotational mixtures of such compounds are within the scope of the invention.

Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

In the compounds of the present invention, any atom not specifically designated as a particular isotope is intended to represent any stable isotope of that atom. Unless otherwise specified, when a position is specifically designated as "H" or "hydrogen," that position is understood to be hydrogen having its natural abundance isotopic composition. Also, unless otherwise specified, when a position is specifically designated as "D" or "deuterium," that position is understood to be highly abundant deuterium, at least 3340 times greater than the natural deuterium abundance of 0.015% (i.e., at least 50.1% deuterium is bound).

Both "D" and "d" stand for deuterium.

Additionally, unless otherwise indicated, structures depicted herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by a ¹³ C- or ¹⁴ C-enriched carbon are within the scope of the invention. Such compounds are useful, for example, as analytical tools or probes in biological assays.

As used herein, the term "crystalline" refers to a solid having a particular arrangement and/or conformation of molecules in a crystal lattice.

As used herein, the term "amorphous" refers to a solid form consisting of a disordered arrangement of molecules and lacking a discernible crystal lattice.

As used herein, the term "solvate" refers to a crystalline solid adduct containing a stoichiometric or non-stoichiometric amount of a solvent bound within the crystal structure. If the bound solvent is water, such an adduct is termed a "hydrate."

abbreviation

The following abbreviations are used:

DMSO dimethyl sulfoxide

DCM dichloromethane

ATP adenosine triphosphate

TFA trifluoroacetic acid

^1HNMR proton nuclear magnetic resonance

HPLC high-performance liquid chromatography

LCMS liquid chromatography-mass spectrometry

Rt retention time

MTBE methyl tert-butyl ether

XRPD X-ray powder diffraction

DSC Differential Scanning Calorimetry

TGA Thermogravimetric Analysis

RT Room temperature

NMP N-Methyl-2-pyrrolidone

Bp boiling point

DMF dimethylformamide

PTSA p-Toluenesulfonic acid

DIPEA N,N-Diisopropylethylamine

HOBT Hydroxybenzotriazole

HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

TBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate

T3P Propylphosphonic Anhydride

COMU 1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholino)]uronium hexafluorophosphate

TCTU [(6-chlorobenzotriazol-1-yl)oxy-(dimethylamino)methylene]-dimethyl-ammonium tetrafluoroborate

HBTU O-Benzotriazole-N,N,N’,N’-tetramethyl-uronium hexafluorophosphate

ECDI 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

LDA lithium diisopropylamide

CDI 1,1'-Carbonyldiimidazole

method

The methods and compounds described herein can be used to produce ATR inhibitors containing an aminopyrazolopyrimidine core. The general synthetic procedures outlined in the schemes herein can be used to generate a wide range of chemical products that can be used in the manufacture of pharmaceutical compounds.

Process A

The compounds of the present invention can be synthesized by methods similar to those depicted in Scheme A.

Step 1

The anion of commercially available allyl cyanoacetate 1 can be reacted with, for example, trichloroacetonitrile to provide intermediate 2. In the anion condensation step, the anion of commercially available allyl cyanoacetate 1 can be generated using a base, such as potassium acetate, and a suitable solvent, such as an alcohol (e.g., isopropanol). The anion is then reacted with trichloroacetonitrile at room temperature.

Step 2

Intermediate 2 is then reacted with hydrazine to generate diaminopyrazole 3. In the pyrazole formation step, intermediate 2 is reacted with hydrazine (or its hydrate) in an aprotic solvent, such as DMF, to provide diaminopyrazole 3. The reaction occurs under basic conditions (e.g., in the presence of potassium acetate or AcONa) and heated (e.g., ≥110°C) to ensure complete cyclization.

Step 3

Intermediate 3 can be further condensed with a di-electrophilic coupling partner to generate pyrimidines 4a-c. In the pyrimidine formation step, intermediate 3 reacts with a 1,3-dialectrophile (e.g., a 1,3-diadehyde or 3-(dialkylamino)-prop-2-enal) in various solvents (e.g., DMF or DMSO/water) to afford the bicyclic cores 4a-c. If one or both of the electrophilic centers are protected/masked (e.g., an aldehyde masked as a ketal), a sulfonic acid (e.g., PTSA) is required to release the reactive functional groups.

Step 4

Deprotection of the allyl ester, for example, via hydrolysis, yields the carboxylic acid 5a-c. In the deprotection step, compounds 4a-c are subjected to hydrolysis conditions known to those skilled in the art. For example, 4a-c is treated with phenylsilane or 4-methylbenzenesulfinate in the presence of a catalytic amount of palladium (e.g., Pd(PPh ₃ ) ₄ ) to yield the corresponding carboxylic acid 5a-c. Alternatively, compounds 4a-c can be treated with an aqueous base (e.g., NaOH, LiOH, or KOH) to yield the acid 5a-c.

Step 5

In the activated ester formation step, carboxylic acids 5a-c are reacted with an amide coupling agent known to those skilled in the art. Suitable amide coupling partners include, but are not limited to, TBTU, TCTU, HATU, T3P, and COMU. If an appropriate coupling agent is selected, the reaction can proceed rapidly (~1 hr) at room temperature in the presence of an organic base, such as an aliphatic amine (e.g., triethylamine, DIPEA), to provide activated esters 6a-c. For example, if the amide coupling agents TBTU [J=H] or TCTU [J=Cl] are used, compounds 6a-c can be readily obtained by filtering the reaction mixture.

It is generally preferred to generate activated esters 6a-c to prepare I-A prior to amide bond formation, although direct conversion of 5a-c to compounds of formula I-A of the present invention is also possible. Alternatively, activated esters (isolated or generated in situ) may be employed, as will be known to those skilled in the art (e.g., using CDI, TBTU, TCTU, HATU, T3P, COMU coupling agents).

Step 6

In the amide bond formation step, activated esters 6a-c can be reacted with substituted or unsubstituted 3-aminopyridine to yield compounds of Formula I-A of the present invention. The amide coupling reaction conditions are typically in an aprotic solvent (e.g., NMP, pyridine, DMF, etc.) and heated (e.g., ≥90°C). The 3-aminopyridine can be further functionalized after amide bond formation.

Alternatively, the two steps can be combined: carboxylic acids 5a-c can be used as starting points for amide bond formation to generate activated esters in situ using the same amide coupling reagents as those described above. Compounds of the invention I-A are isolated in a manner similar to that described above.

Process B

Alternatively, the compounds disclosed herein can be prepared by methods analogous to those depicted in Scheme B.

Step 1

Amide 8 can be readily prepared from commercially available cyanoacetic acid 7. In the amide bond formation step, cyanoacetic acid 7 can be reacted with a substituted 3-aminopyridine to yield the present compound 8. The amide coupling reaction conditions are generally in an aprotic solvent (e.g., DCM, NMP, DMF, etc.) in the presence of an organic base, such as an aliphatic amine (e.g., triethylamine or DIPEA). Amide coupling agents are known to those skilled in the art, such as EDCI, TBTU, COMU, T3P, etc.

Step 2

In the pyrazole formation step, the anion of cyanoamide 8 can be generated using a base (e.g., potassium or sodium acetate) in a suitable solvent, such as an alcohol (e.g., ethanol). The anion is then reacted with trichloroacetonitrile at room temperature. The resulting solid can be collected by filtration and then reacted with hydrazine (or its hydrate) in an aprotic solvent, such as DMF or NMP, to provide diaminopyrazole 9, which is further condensed with a di-electrophilic coupling partner to form the pyrimidine portion of the compound of Formula I-A of the present invention.

Step 3

In the pyrimidine formation step, intermediate 9 reacts with a 1,3-dialectrophile (e.g., a 1,3-diadehyde or 3-(dialkylamino)-prop-2-enal) in various solvents (e.g., iPrOH/water, DMF, or DMSO/water) to afford the desired product I-A. If one or both of the electrophilic centers are protected/masked (e.g., an aldehyde masked as a ketal), a sulfonic acid (e.g., PTSA) is required to release the reactive functional group.

Preparation Examples and Examples

All commercially available solvents and reagents were used as received. Microwave reactions were performed using a CEM Discovery microwave. Flash chromatography was performed on, for example, a Combiflash ^R Companion ^™ system using a 0 to 100% EtOAc/petroleum ether gradient. Other methods known in the art were also used for flash chromatography. Samples were pre-adsorbed on silica. Supercritical fluid chromatography (SFC) was performed on a Berger Minigram SFC machine as specified. All ¹ H NMR spectra were recorded using a Bruker Avance III 500 instrument at a frequency of 500 MHz. MS samples were analyzed on a Waters SQD mass spectrometer operating electrospray ionization in both positive and negative ion modes. Samples were introduced into the mass spectrometer using a chromatogram. The purity of all final products was ≥95%, unless otherwise specified in the experimental details. HPLC purity was measured on a Waters Acquity UPLC system with a Waters SQD MS instrument equipped with a Waters UPLC BEH C8 1.7 μm, 2.1 x 50 mm column and a Vanguard BEHC8 1.7 μm, 2.1 x 5 mm guard column.

As used herein, the term "Rt(min)" refers to the HPLC retention time, in minutes, associated with a compound. Unless otherwise indicated, the HPLC method used to obtain the reported retention times is as follows:

HPLC method

Instrument: Waters Acquity UPLC-MS;

Column: Waters UPLC BEH C8 1.7 μm, 2.1 x 50 mm, with Vanguard BEH C8 1.7 μm, 2.1 x 5 mm guard column;

Column temperature: 45°C;

Mobile phase A: 10 mM ammonium formate aqueous solution: acetonitrile 95:5, pH 9;

Mobile phase B: acetonitrile;

Detection: 210-400nm;

Gradient: 0-0.40 min: 2% B, 0.40-4.85 min: 2% B to 98% B, 4.85-4.90 min: 98% B to 2% B, 4.90-5.00 min: hold at 2% B;

Flow rate: 0.6 mL/min.

Preparation Example 1: Allyl 3,5-diamino-1H-pyrazole-4-carboxylate

Step 1: Allyl 3-amino-4,4,4-trichloro-2-cyanobut-2-enoate 2

To a solution of KOAc (589.4 g, 6.006 mol) in isopropanol (3 L) was added allyl cyanoacetate (429.4 g, 403.2 mL, 3.432 mol), and the reaction mixture was cooled to 5°C. Trichloroacetonitrile (495.5 g, 3.432 mol) was added in 50 mL portions, maintaining the temperature below 15°C. The reaction mixture was then warmed to 20°C and stirred for 3 hours. Water (~4 L) was added to dissolve the inorganics and precipitate the desired product. The mixture was stirred for 20 minutes, and the solid was isolated by filtration under vacuum. The solid was filtered, washed with water (2 x 0.5 L), and dried in a vacuum oven at 40°C overnight to yield allyl 3-amino-4,4,4-trichloro-2-cyanobut-2-enoate 2 as an off-white powder (787 g, 85%).

Step 2: Allyl 3,5-diamino-1H-pyrazole-4-carboxylate 3

To a suspension of allyl 3-amino-4,4,4-trichloro-2-cyano-but-2-enoate 2 (619 g, 2.297 mol) and KOAc (676.3 g, 6.891 mol) in DMF (2.476 L) at 0°C was slowly added hydrazine hydrate (172.5 g, 167.6 mL, 3.446 mol) over 15 minutes. The reaction mixture was then stirred at ambient temperature for 2 hours, at which point ¹ H NMR indicated complete consumption of the starting material. The reaction mixture was then heated at 110°C overnight, then allowed to cool to ambient temperature and stirred for an additional 48 hours. The mixture was filtered through a sintered glass funnel to remove precipitated solids, and the filtrate was evaporated under reduced pressure to yield a thick liquid. DCM (approximately 2 L) was added, and the mixture was filtered again to remove additional precipitated solids. The filtrate was purified by passing it through a 1 kg plug of silica gel (DCM/MeOH gradient as eluent), and the solvent was removed to afford an orange solid, which was suspended in acetonitrile and heated at approximately 70° C. until the solid dissolved, at which point the solution was allowed to cool naturally to ambient temperature and then to 2° C. The resulting precipitate was isolated by filtration under vacuum, washed with cold MeCN (~50 mL), and dried in a vacuum oven to constant weight to afford the title compound as an off-white powder (171.2 g, 41%).

Preparation Example 2a: 1H-Benzo[d][1,2,3]triazol-1-yl 2-amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate

Step 1: Allyl 2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylate 4a

To a suspension of allyl 3,5-diamino-1H-pyrazole-4-carboxylate 3 (42.72 g, 234.5 mmol) in DMSO (270.8 mL)/water (270.8 mL) were added p-TsOH hydrate (46.72 g, 245.6 mmol) and 3-(diisopropylamino)-2-fluoro-prop-2-enal (described in Tetrahedron Letters, 33(3), 357-60; 1992) (38.69 g, 223.3 mmol). The reaction mixture was heated to 100°C for 3 hr, during which time a solid slowly precipitated from the solution. The orange suspension was allowed to cool to RT overnight. The solid was filtered, washed with water, and dried under vacuum to give allyl 2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylate 4a as a sandy solid (45.05 g, 85% yield).

In an alternative approach, allyl 2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylate 4a can be synthesized via the following general scheme C-1.

Process C-1

Reaction 1

The bis-acetal protected malondialdehyde (intermediate 35) can be deprotected under acidic conditions to produce intermediate 36. In some embodiments, the acidic conditions can be generated using an acid independently selected from HCl, _H2SO4 , _MeSO2H , TFA, _HBF4 , or pTSA, _with a suitable solvent such as water. Preferably, the acid used in this reaction is selected from pTSA or _MeSO2H . R° is preferably a _C1-6 aliphatic group. In some embodiments, R° is selected from methyl, ethyl, propyl, isopropyl, butyl, or pentyl. In other embodiments, R° is selected from methyl or ethyl.

Reaction 2

Intermediate 36 can react with an electrophilic fluorinating agent to produce intermediate 38. In some embodiments, the electrophilic fluorinating agent is independently selected from 1-(chloromethyl)-4-fluoro-1,4-diazobicyclo[2.2.2]octane bistetrafluoroborate N-fluorobenzenesulfonamide, a substituted 1-fluoropyridinium salt, or fluorine gas.

Reaction 3

Intermediate 38 can be reacted with intermediate 3 under suitable condensation conditions to produce intermediate 4a. In some embodiments, suitable condensation conditions may include reacting intermediate 38 with intermediate 3 in the presence of a solvent and heating to obtain the bicyclic core of 4a. The reaction can occur in various types of solvents, such as water, DMSO/water, or DMF.

In one embodiment, intermediate 4a is generated using the method described in Scheme C-2.

Process C-2

1,1,3,3-Tetramethoxypropane 35a (20 g, 121.8 mmol) was dissolved in water (200 ml). p-Toluenesulfonic acid monohydrate (23.17 g, 121.8 mmol) was added, and the mixture was stirred at 19-20°C for 90 minutes. 1-(Chloromethyl)-4-fluoro-1,4-diazobicyclo[2.2.2]octane bistetrafluoroborate 37 (Selectfluor, 1.4 eqv, 60.4 g, 170.5 mmol) was added portionwise. The addition was endothermic (20.1°C to 19.4°C), but once the addition was complete, the temperature began to rise slowly (reaching 25.4°C over 45 minutes). The selectfluor dissolved over 1 hour. The mixture was stirred at ambient temperature for 18 hours. After this time, the mixture was homogeneous. DMSO (150 ml) was added slowly over 5 minutes. The addition was exothermic, with the temperature rising from 20.4°C to 34.2°C during the addition. The mixture then began to cool. The resulting mixture was stirred for 45 minutes. Compound 3 (21.4g, 115.7mmol) was then added portionwise. The addition was not exothermic. The mixture was heated to 85°C for 4hr (Lc/Ms patterns were identical at the 2hr and 4hr time points). The stirred mixture was then allowed to cool naturally to ambient temperature overnight. The resulting reaction mixture was in a slurry state. Water (150ml) was slowly added to the resulting slurry. The temperature rose from 20.4°C to 21.5°C. The slurry was stirred for 2hr, and the product was then filtered to isolate the product. The filter cake was washed with water and dried on a sinter to a beige solid (15.5g). The product was further dried in a vacuum oven at 40°C for 20hr. Mixture 4a was obtained as a beige solid (13.5g, 50% yield). HPLC purity 97.7% area; ¹ H NMR (500 MHz, DMSO-d6) δ 4.83 (2H, d), 5.29 (1H, d), 5.49 (1H, d), 6.04-6.14 (1H, m), 6.57 (2H, brs), 8.80 (1H, m), 9.40 (1H, m); 19F NMR (500 MHz, DMSO-d6) δ -153.1.

Step 2: 2-Amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 5a

To a suspension of allyl 2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylate 4a (45 g, 190.5 mmol) in DCM (1.35 L) was added phenylsilane (41.23 g, 46.96 mL, 381.0 mmol), followed by Pd(PPh ₃ ) ₄ (8.805 g, 7.620 mmol). The reaction was stirred at room temperature for 2 hr 30 min. The reaction mixture was filtered and the solid washed with DCM to afford a light yellow solid (43.2 g). This solid was further triturated in DCM (225 mL) at room temperature for 45 min, then filtered and dried under vacuum overnight to afford 2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 5a as a light yellow solid (37.77 g, 100% yield).

In an alternative procedure, sodium 4-methylbenzenesulfinate (anhydrous, 1.2 eqv, 22.6 g, 127 mmol) was suspended in anhydrous DMSO (20 vol, 500 ml). The stirred mixture was warmed to 30°C under a nitrogen atmosphere. Once completely dissolved, Pd(PPh ₃ ) ₄ (2 mol%, 2.4 g, 2.1 mmol) was added. The mixture was stirred at 25-30°C for 10 min, after which a cloudy yellow solution appeared. Allyl 2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylate 4a (25 g, 105.8 mmol) was added portionwise, maintaining the temperature at 25-30°C. Once the addition was complete, the cloudy solution was stirred until the reaction was complete as judged by HPLC (2-3 hr). A heavy precipitate formed 15 minutes after the addition of the substrate. The mixture became thicker as the reaction proceeded. The reaction mixture was diluted with water (125 ml) and 2M HCl (66 ml) was slowly added, maintaining the temperature at 25-30°C. The slurry was stirred for 30 minutes and then filtered. The filtration was slow (2 hr). The resulting solid was washed with water and then dried on a sinter. The solid was suspended in DCM (8 vol) for 1 hr. The solid was filtered (quick filtration) and washed with DCM. The solid was resuspended in chloroform (8 vol) for 1 hr. The solid was filtered and dried on a sinter. It was further dried in a vacuum oven at 50°C for 24 hr. The product 5a was obtained as an off-white solid (18.6 g, 85%); ¹ H NMR (500 MHz, DMSO-d6) δ 12.14 (1H, brs), 9.31 (1H, dd), 8.69 (1H, m), 6.47 (2H, brS); 19F NMR (500 MHz, DMSO-d6) δ -153.65; MS (ES+) 197.1.

Step 3: 1H-Benzo[d][1,2,3]triazol-1-yl 2-amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate 6a

To a suspension of 2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 5a (20 g, 102.0 mmol) in chloroform (300 mL) was added _Et₃N (11.35 g, 15.63 mL, 112.2 mmol). The suspension was stirred for ~5 min, then (benzotriazol-1-yloxy-dimethylamino-methylene)-dimethyl-ammonium boron tetrafluoride (32.75 g, 102.0 mmol) was added. The suspension was heated to 60°C for 1 hr, then the thick suspension was allowed to cool to room temperature. The resulting suspension was filtered, washed with chloroform (200 mL), and dried under vacuum overnight to afford the title compound 6a as a light yellow powder (32.5 g, 88%).

Preparation Example 2b: (6-Chlorobenzotriazol-1-yl)-2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylate

In a 2.5 L three-necked flask equipped with a stirring rod, condenser, nitrogen line, and Hanna temperature probe, 2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 5a (60 g, 305.9 mmol), chloroform (900.0 mL), and triethylamine (32.44 g, 44.68 mL, 320.6 mmol) were placed. [(6-chlorobenzotriazol-1-yl)oxy-(dimethylamino)methylene]-dimethyl-ammonium (boron tetrafluoride ion (1)) (87.00 g, 244.7 mmol) was added portionwise over 5 min (the internal temperature dropped from 22.7 to 21.5 ° C after the addition was complete). The mixture was heated at 60 ° C (internal temperature) for 2 hours and remained a cream-colored suspension. The mixture was cooled to room temperature, and the solid was collected by filtration, washed thoroughly with chloroform (until the filtrate was essentially colorless), and dried under suction to afford product 6a* as a cream-colored solid (82.2 g, 77% yield). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 9.55 (dd, 1H), 8.91 (d, 1H), 8.22 (dd, 1H), 8.09 (dd, 1H), 7.57 (dd, 1H) and 6.87 (s, 2H). MS (ES+) 348.1.

In an alternative method, 2-amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylic acid 5a (30 g, 153 mmol) was suspended in acetonitrile (540 ml). Triethylamine (22.5 ml, 153 mmol) was added, followed by [(6-chlorobenzotriazol-1yl)oxy-(dimethylamino)methylene]-dimethylammonium tetrafluoroborate (TCTU, 54.4 g, 153 mmol). The mixture was stirred at room temperature for 2 hours. The product was isolated by filtration, and the filter cake was washed with acetonitrile (2 x 60 ml). The product was obtained as a brown solid (49.3 g, 93%); ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 9.55 (dd, 1H), 8.91 (d, 1H), 8.22 (dd, 1H), 8.09 (dd, 1H), 7.57 (dd, 1H) and 6.87 (s, 2H); 19F NMR (500 MHz, DMSO-d 6 ) δ -150.1; MS (ES+) 348.1.

Preparation Example 3: 1H-Benzo[d][1,2,3]triazol-1-yl 2-amino-6-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate

Step 1: 2-Amino-6-chloro-pyrazolo[1,5-a]pyrimidine-3-carboxylate 4b

To a suspension of allyl 3,5-diamino-1H-pyrazole-4-carboxylate 3 (1 g, 5.489 mmol) in DMF (5 mL) was added (Z)-2-chloro-3-dimethylamino-prop-2-enylidene]-dimethyl-ammonium hexafluorophosphate (1.683 g, 5.489 mmol), followed by triethylamine (722.1 mg, 994.6 μL, 7.136 mmol). The reaction mixture was heated to 60°C for 4 hours, during which time a solid slowly precipitated from solution. The brown suspension was allowed to cool to room temperature. The solid was filtered, washed with water, and dried under vacuum to afford allyl 2-amino-6-chloro-pyrazolo[1,5-a]pyrimidine-3-carboxylate 4b as a brown solid (1.092 g, 72% yield).

Step 2: 2-Amino-6-chloro-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 5b

To a suspension of allyl 2-amino-6-chloro-pyrazolo[1,5-a]pyrimidine-3-carboxylate 4b (1 g, 3.96 mmol) in DCM (15 mL) was added phenylsilane (856.6 mg, 0.9756 mL, 7.916 mmol), followed by Pd(PPh ₃ ) ₄ (182.9 mg, 0.1583 mmol). The reaction mixture was stirred at room temperature for 7 hours. The reaction mixture was filtered and the solid washed with DCM to afford a light yellow solid (43.2 g). The solid was further triturated in DCM (225 mL) at room temperature for 45 minutes, then filtered and dried under vacuum overnight to afford 2-amino-6-chloro-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 5b as a yellow solid (791 mg, 94% yield).

Step 3: 1H-Benzo[d][1,2,3]triazol-1-yl 2-amino-6-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate 6b

To a solution of 2-amino-6-chloro-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 5b (1.51 g, 7.103 mmol) in chloroform (15.1 mL) was added TBTU (2.737 g, 8.524 mmol) and TEA (862.5 mg, 1.188 mL, 8.524 mmol). The reaction mixture was stirred at 50°C for one hour. The resulting suspension was filtered, and the solid was triturated in ethyl acetate to afford the title compound 6b as a yellow solid (2.05 g, 88%).

Preparation Example 4: 1H-Benzo[d][1,2,3]triazol-1-yl 2-amino-6-(cyanomethyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate

Step 1: Allyl 2-amino-6-(cyanomethyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylate 4c

To a suspension of allyl 3,5-diamino-1H-pyrazole-4-carboxylate 3 (63.49 g, 348.5 mmol) in a mixture of DMSO (340 mL) and water (340 mL) was added 3-(dimethoxymethyl)-4,4-dimethoxy-butanenitrile (prepared according to Preparation Example 5 below) (85 g, 418.2 mmol), followed by p-toluenesulfonic acid hydrate (1) (11.27 g, 59.24 mmol). The reaction mixture was heated to 85°C and stirred overnight. The reaction mixture was cooled in an ice bath. The mixture was diluted with EtOAc (680 mL) and saturated aqueous NaHCO ₃ solution (1.36 L). The precipitate was filtered and rinsed with water and then with a mixture of water and EtOAc. The brown solid was dried under vacuum to afford allyl 2-amino-6-(cyanomethyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylate 4c as a brown solid (55.94 g, 62% yield).

Step 2: 2-Amino-6-(cyanomethyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 5c

To a suspension of allyl 2-amino-6-(cyanomethyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylate 4c (10.2 g, 39.65 mmol) in DCM (350 mL) was added phenylsilane (8.581 g, 9.773 mL, 79.3 mmol), followed by Pd(PPh ₃ ) ₄ (1.5 g, 1.298 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was filtered, and the solid was washed with DCM and dried under vacuum to afford 2-amino-6-(cyanomethyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 5c as a yellow solid (8.61 g, 100% yield).

Step 3: 1H-Benzo[d][1,2,3]triazol-1-yl 2-amino-6-(cyanomethyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate 6c

To a solution of 2-amino-6-(cyanomethyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 5c (5.11 g, 23.53 mmol) in DCM (51 mL) was added TBTU (9.067 g, 28.24 mmol) and TEA (2.858 g, 3.937 mL, 28.24 mmol). The reaction mixture was stirred at room temperature for one hour. The resulting suspension was filtered, and the solid was triturated in hot chloroform to afford the title compound 6c as a beige solid (6.59 g, 84%).

Example 1: 2-amino-6-fluoro-N-[5-fluoro-4-[4-[4-(oxetane-3-yl)piperazine-1-carbonyl]-1- Synthesis of [piperidinyl]-3-pyridinyl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound I-1)

Step 1: tert-Butyl 1-[3-[(2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carbonyl)amino]-5-fluoro-4-pyridinyl]piperidine-4-carboxylate 28

A mixture of (6-chlorobenzotriazol-1-yl) 2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylate 6a* (44.02 g, 126.6 mmol) and tert-butyl 1-(3-amino-5-fluoro-4-pyridyl)piperidine-4-carboxylate 27 (prepared according to Preparation 7b) (34 g, 115.1 mmol) in pyridine (510.0 mL) was heated at 95°C overnight (18 hr). The mixture was cooled to room temperature (with precipitation of the product), and then ethanol (340.0 mL) was added and stirred at room temperature for 10 min. The yellow solid was collected by filtration, washed thoroughly with ethanol, suction dried, and then dried under high vacuum for 1 hr to afford the product 28 as a yellow solid (32.5 g, 56% yield). ¹ H NMR (500 MHz, DMSO-d6) δ 10.45 (s, 1H), 9.58 (s, 1H), 9.51 (dd, 1H), 8.72 (dd, 1H), 8.25 (d, 1H), 6.81 (s, 2H), 3.15–2.93 (m, 4H), 2.55–2.47 (shielded signal, 1H), 2.02–1.91 (m, 4H), 1.47 (s, 9H). MS (ES+) 474.2.

Step 2: 1-[3-[(2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carbonyl)amino]-5-fluoro-4-pyridinyl]piperidine-4-carboxylic acid trifluoroacetate 29

To a suspension of tert-butyl 1-[3-[(2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carbonyl)amino]-5-fluoro-4-pyridinyl]piperidine-4-carboxylate 28 (69.7 g, 147.2 mmol) in DCM (348.5 mL) and triethylsilane (18.83 g, 25.87 mL, 161.9 mmol) was added TFA (151.1 g, 102.1 mL, 1.325 mol) (solid precipitated during the initial addition of TFA, but dissolved after complete addition). The resulting orange solution was stirred at room temperature overnight. Additional TFA (16.78 g, 11.34 mL, 147.2 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The mixture was then heated at 40°C for 20 minutes to complete the reaction. The mixture was concentrated in vacuo, chloroform (300 mL) was added, and the mixture was concentrated in vacuo again to give an orange solid suspension. The mixture was ground in DCM (approximately 200 mL), stirred for 20 min, then filtered to collect the solid, washed with a small amount of DCM, and suction dried to give a yellow solid. The filtrate was concentrated in vacuo, the residue was resuspended in DCM (approximately 50 mL), stirred for 20 min, then filtered to collect the solid, washed with a small amount of DCM, and suction dried to give a yellow solid which was merged with the first batch of solids. The solid was dried under vacuum overnight to give the desired product 29 as a yellow solid (82.8 g, 96%). ¹ H NMR (500 MHz, DMSO-d6) δ 10.44 (s, 1H), 9.59 (s, 1H), 9.50 (dd, 1H), 8.84 (dd, 1H), 8.33 (d, 1H), 3.13–3.10 (m, 4H), 2.57–2.47 (shielded signal, 1H) and 2.08–1.93 (m, 4H). MS (ES+) 418.1.

Step 3: 1-[3-[(2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carbonyl)amino]-5-fluoro-4-pyridinyl]piperidine-4-carboxylic acid hydrochloride 30

To a solution of 1-[3-[(2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carbonyl)amino]-5-fluoro-4-pyridyl]piperidine-4-carboxylic acid (trifluoroacetic acid) 29 (73 g, 124.7 mmol) in NMP (662.7 mL) was added hydrogen chloride (4M in 1,4-dioxane) (37.40 mL of 4M, 149.6 mmol). A yellow precipitate formed after a few seconds. The mixture was stirred at room temperature for 20 minutes, after which the solid was collected by filtration, washed with a small amount of NMP and then with MTBE, and suction dried to afford the pure product 30 as a light yellow solid (59.7 g, quantitative yield). MS (ES+) 418.1.

Step 4: 2-amino-6-fluoro-N-[5-fluoro-4-[4-[4-(oxetan-3-yl)piperazine-1-carbonyl]-1-piperidinyl]-3-pyridinyl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound I-1)

To a yellow suspension of 1-[3-[(2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carbonyl)amino]-5-fluoro-4-pyridyl]piperidine-4-carboxylic acid (hydrochloric acid) 30 (59.7 g, 131.5 mmol) in NMP (477.6 mL) was added DIPEA (50.99 g, 68.72 mL, 394.5 mmol) and then [(6-chlorobenzotriazol-1-yl)oxy-(dimethylamino)methylene]-dimethyl-ammonium (boron tetrafluoride ion (1)) (51.44 g, 144.7 mmol). A yellow suspension formed after a few minutes. The mixture was stirred at room temperature for 30 min and then 1-(oxetan-3-yl)piperazine 25 (prepared according to Preparation 8 below) (26.18 g, 184.1 mmol) was added. The cream/tan suspension turned into an orange solution (exotherm from 23.9 to 29.4°C).The flask was placed on an ice/water bath until the internal temperature was 24°C, then the ice bath was removed, after which the internal temperature stabilized at 24°C.

The solution is stirred at room temperature for 30min, then cooled to 10 ℃ on ice/salt/water bath, then slowly adds water (1.015L) by portion 100mL.Before adding the next 100mL water, wait for heat release to between 17 ℃ and 20 ℃ (inside), then naturally cool to between 10 and 15 ℃.Repeat until adding whole water.Once heat release stops, remove ice/salt/water bath, stir the mixture at ambient temperature for 20min (forming thick yellow/cream suspension).Collect solid by filtration through sinter funnel, fully wash with water, then suction drying 10min.Remove vacuum, on sinter funnel, solid is suspended in water, then reapply vacuum, solid suction drying spends the night, then in 40 ℃＜10mBar vacuum drying oven, dry 24hr.

The solid (54.5 g) was suspended in ethanol (545 mL, 10 vol eq.), heated at reflux for 2 hours, and then cooled to room temperature for 2 hours. The solid was collected by filtration, washed with a small amount of ethanol, and dried under vacuum for 1 hour to obtain the product as a pale yellow solid. The solid was placed in a vacuum oven at 23.5°C (<10 mBar) overnight to obtain the ethanol solvate of I-1 as a pale yellow solid (51 g, 64% yield). ¹ H NMR (500MHz, DMSO-d6)δ10.64(s,1H),9.67 (s,1H),9.48(dd,1H),9.26(dd,1H),8.26(d,1H),6.79(s,2H),4.55(t, 2H),4.47(t,2H),4.34(t,0.7H),3.61(dt,4H),3.48–3.41(m,2.5H), 3.22–3.17(m,2H),3.05–3.03(m,2H),3.99–2.93(m,1H),2.28(dt, 4H),2.17–2.10(m,2H),1.74(d,2H),1.07(t,2H).MS(ES+)542.3.

Example 2: 2-amino-6-fluoro-N-[5-fluoro-4-[4-[4-(oxetane-3-yl)piperazine-1-carbonyl]-1- Alternative synthetic route for [piperidinyl]-3-pyridinyl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound I-1)

Step 1: tert-Butyl 1-(3-(2-amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxamido)-5-fluoropyridin-4-yl)piperidine-4-carboxylate 28

6-Chloro-1H-benzo[d][1,2,3]triazol-1-yl 2-amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate 6a* (45 g, 129.4 mmol) and tert-butyl 1-(3-amino-5-fluoropyridin-4-yl)piperidine-4-carboxylate 27 (prepared according to Preparation 7b, described below) (40.1 g, 135.9 mmol) were suspended in pyridine (675 ml). The mixture was heated at 95°C under nitrogen until the reaction was complete (as determined by HPLC analysis). The mixture was cooled, and ethanol (450 ml) was added dropwise. The mixture was filtered, and the filter cake was washed with ethanol (2 x 70 ml). The wet cake was dried to give the product 28 as a yellow crystalline solid (47.7 g, 78%); ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 10.45 (s, 1H), 9.58 (s, 1H), 9.51 (dd, 1H), 8.72 (dd, 1H), 8.25 (d, 1H), 6.81 (s, 2H), 3.15–2.93 (m, 4H), 2.55–2.47 (shielded signal, 1H), 2.02–1.91 (m, 4H), 1.47 (s, 9H); 19F NMR (500 MHz, DMSO-d 6 ) δ–153.5, -136.3; MS (ES+) 474.2.

In an alternative embodiment, intermediate 28 can be purified prior to proceeding to step 2 using a procedure similar to the following:

Step 1a: Purification of tert-butyl 1-[3-[(2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carbonyl)amino]-5-fluoro-4-pyridinyl]piperidine-4-carboxylate 28

Tert-butyl 1-[3-[(2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carbonyl)amino]-5-fluoro-4-pyridinyl]piperidine-4-carboxylate 28 (530 g; 1.12 moles) was added to a mixture of NMP (5.3 L) and 1,2-diaminopropane (249 g; 3.36 moles), and the resulting thin suspension was stirred at 20-25° C. for 15 hours. Ethanol (10.4 L) was added to the suspension and stirred at 20-25° C. for 4 hours. The crystalline gold solid was collected by filtration, washed with ethanol (2 x 2.6 L), suction dried, and then dried in a vacuum oven at 35-40°C for 24 h to afford 28 as a crystalline gold solid (479 g; 90%). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 10.45 (s, 1H), 9.57 (s, 1H), 9.49 (dd, 1H), 8.71 (d, 1H), 8.24 (d, 1H), 6.79 (s, 2H), 3.44–3.33 (m, 1H), 3.34–3.20 (m, 4H), 3.07 (dt, 4H), 2.01–1.89 (m, 4H), 1.46 (s, 9H). ¹⁹ F NMR (500 MHz, DMSO-d ₆ ) δ −136.3, −153.4.

Step 2: 1-(3-(2-amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxamido)-5-fluoropyridin-4-yl)piperidine-4-carboxylic acid hydrochloride 30

Tert-butyl 1-(3-(2-amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxamido)-5-fluoropyridin-4-yl)piperidine-4-carboxylate 28 (36 g, 76 mmol) was suspended in a 4M solution of HCl in 1,4-dioxane (670 ml). Water (36 ml) was added dropwise to the rapidly stirring slurry. The mixture was stirred under nitrogen until the reaction was complete (as determined by HPLC analysis). The mixture was diluted with 1,4-dioxane (180 ml) and filtered. The filter cake was washed with TBME (2 x 72 ml). The wet cake was dried to give a light brown solid (hydrochloride salt, 32.7 g, 95%); ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 10.34 (s, 1H), 9.53-9.49 (m, 2H), 8.82 (m, 1H), 8.50 (m, 1H), 3.13–3.22 (m, 4H), 2.57–2.47 (shielded signal, 1H) and 2.08–1.93 (m, 4H); 19F NMR (500 MHz, DMSO-d 6 ) δ–152.9, -133.8; MS (ES+) 418.1.

Step 3: 2-amino-6-fluoro-N-[5-fluoro-4-[4-[4-(oxetan-3-yl)piperazine-1-carbonyl]-1-piperidinyl]-3-pyridinyl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound I-1·Amorphous Form)

To a solution of 1-(oxetan-3-yl)piperazine 25 (525 mg, 3.69 mmol) in THF (12 ml) was added DIPEA (1.72 ml, 9.91 mmol), followed by 1-(3-(2-amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxamido)-5-fluoropyridin-4-yl)piperidine-4-carboxylic acid (hydrochloride, 1.5 g, 3.3 mmol). [(6-chlorobenzotriazol-1-yl)oxy-(dimethylamino)methylene]-dimethyl-ammonium tetrafluoroborate (TCTU, 1.29 g, 3.64 mmol) was added, and the mixture was stirred under nitrogen until the reaction was complete (as determined by HPLC analysis). The mixture was cooled, and water (24 ml) was added dropwise. The mixture was allowed to warm to ambient temperature, stirred for 3 hours, and then filtered. The filter cake was washed (3 x 3 ml). The wet cake was dried under vacuum (with nitrogen flow) at 40°C to obtain an amorphous form of compound I-1 (1.54 g, 86%); ^1H NMR (500 MHz, DMSO-d6) δ10.64 (s, 1H), 9.67 (s, 1H), 9.48 (dd, 1H), 9.26 (dd, 1H), 8.26 (d, 1H), 6.79 (s, 2H), 4.55 (t, 2H), 4.47 (t, 2H), 4.34 (t, 0.7H), 3.61 (dt, 4H), 3.48–3.41 (m, 2.5H), 3.22–3.17 (m, 2H), 3.05–3.03 (m, 2H), 3.99–2.93 (m, 1H), 2.28 (dt, 4H), 2.17– 2.10(m,2H),1.74(d,2H),1.07(t,2H);19F NMR(500MHz,DMSO-d6)δ–152.8,-136.1;MS(ES+)542.3.

The amorphous form of compound I-1 can be prepared using the alternative method of step 3 in Example 2 above.

In another example, Compound I-1 amorphous form was prepared by adding N,N-diisopropylethylamine (461 μL; 342 mg; 2.64 mmol) to a suspension of 1-[3-[(2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carbonyl)amino]-5-fluoropyridin-4-yl]piperidine-4-carboxylic acid hydrochloride 30 (1.00 g; 2.20 mmol; LR) in THF (20 mL). 1,1'-Carbonyldiimidazole (CDI) (430 mg; 2.65 mmol) was added, and the mixture was heated at 40-50°C. Additional 1,1'-Carbonyldiimidazole (CDI) (213 mg total; 1.31 mmol) was added, and the mixture was heated until the reaction was complete (as determined by HPLC analysis). 1-(Oxetane-3-yl)piperazine 25 (375 mg; 2.64 mmol) was added, and the mixture was heated at 55-60°C until the reaction was complete (as determined by HPLC analysis). The reaction mixture was cooled to 20-25°C. Water (40 mL) and 2M NaOH(aq) (551 uL) were added, and the suspension was stirred for 5-10 minutes. The solid was collected by filtration, washed with water (2 x 5 mL), suction dried, and then dried in a vacuum oven at 45-50°C for 16 hours to yield I-1 as a yellow solid (869 mg; 73%).

Preparation Example 5: Tert-butyl 1-(3-(2-amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxamido)-5-fluoropyrazolo[1,5-a]pyrimidine-3-carboxamido)- Alternative synthetic route to 4-pyridin-4-yl)piperidine-4-carboxylate (Compound 28)

Step 1: 2-Amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carbonyl chloride 34

To a suspension of 2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 5a (500 mg, 2.55 mmol) in dichloromethane (7.5 mL) was added triethylamine (409 uL, 297 mg, 2.93 mmol). Thionyl chloride (205 uL, 334 mg, 2.80 mmol) was added, and the mixture was heated at 35-40°C for 2 hours. The mixture was cooled to ambient temperature and stirred at ambient temperature until the reaction was complete (monitored by HPLC). The solid was collected by filtration, washed with dichloromethane (2 x 1 mL), and dried by suction to give the product 34 as a beige solid (465 mg, 85%); ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 9.30 (dd, J=4.9, 2.7 Hz, 1H), 8.68 (d, J=2.7 Hz, 1H); 19F NMR (500 MHz, DMSO-d 6 ) δ -154.1.

Step 2: tert-Butyl 1-(3-(2-amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxamido)-5-fluoropyridin-4-yl)piperidine-4-carboxylate 28

Suspend 2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carbonyl chloride 34 (100 mg, 0.466 mmol) and tert-butyl 1-(3-amino-5-fluoropyridin-4-yl)piperidine-4-carboxylate 27 (138 mg, 0.466 mmol) in pyridine (1.5 mL). Heat the mixture to 90-100°C for 16 hours. Cool the mixture, and add ethanol (3 mL). Stir the mixture for 1-2 hours, filter, and wash the filter cake with ethanol (0.5 mL). The solid was dried by suction to give the product 28 (162 mg, 73%). 1H NMR (400 MHz, DMSO-d6) δ 10.45 (s, 1H), 9.57 (s, 1H), 9.50 (dd, J = 4.8, 2.5 Hz, 1H), 8.71 (d, J = 2.5 Hz, 1H), 8.24 (d, J = 2.5 Hz, 1H), 6.80 (s, 2H), 3.07 (dd, J = 6.5, 3.3 Hz, 4H), 2.11–1.80 (m, 4H), 1.46 (s, 9H); 19F NMR (500 MHz, DMSO-d6) δ -136.8, -153.9; MS (ES+) 474.2.

Example 3: 2-amino-6-fluoro-N-[5-fluoro-4-[4-[4-(oxetane-3-yl)piperazine-1-carbonyl]-1- Alternative synthetic route for [piperidinyl]-3-pyridinyl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound I-1)

Step 1: tert-Butyl 1-(3-(2-amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxamido)-5-fluoropyridin-4-yl)piperidine-4-carboxylate 28

6-Chloro-1H-benzo[d][1,2,3]triazol-1-yl 2-amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate 6a* (45 g, 129.4 mmol) and tert-butyl 1-(3-amino-5-fluoropyridin-4-yl)piperidine-4-carboxylate 27 (prepared according to Preparation 7b, described below) (40.1 g, 135.9 mmol) were suspended in pyridine (675 ml). The mixture was heated at 95°C under nitrogen until the reaction was complete (as determined by HPLC analysis). The mixture was cooled, and ethanol (450 ml) was added dropwise. The mixture was filtered, and the filter cake was washed with ethanol (2 x 70 ml). The wet cake was dried to give the product 28 as a yellow crystalline solid (47.7 g, 78%); ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 10.45 (s, 1H), 9.58 (s, 1H), 9.51 (dd, 1H), 8.72 (dd, 1H), 8.25 (d, 1H), 6.81 (s, 2H), 3.15-2.93 (m, 4H), 2.55–2.47 (shielded signal, 1H), 2.02–1.91 (m, 4H), 1.47 (s, 9H); 19F NMR (500 MHz, DMSO-d 6 ) δ–153.5, -136.3; MS (ES+) 474.2.

Step 2: 1-[3-[(2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carbonyl)amino]-5-fluoro-4-pyridinyl]piperidine-4-carboxylic acid methanesulfonate 33

Methanesulfonic acid (274 uL; 406 mg; 4.22 mmol) was added to a suspension of tert-butyl 1-[3-[(2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carbonyl)amino]-5-fluoro-4-pyridinyl]piperidine-4-carboxylate 28 (1.00 g; 2.11 mmol) in acetonitrile (15 mL) and the mixture was heated to 75-80 °C for 16 hours. The solid was collected by filtration, washed with acetonitrile (2 x 2 mL), and dried under vacuum to give 1-[3-[(2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carbonyl)amino]-5-fluoro-4-pyridinyl]piperidine-4-carboxylic acid methanesulfonate 33 (0.94 g; 87%). ¹ H NMR (500 MHz, DMSO-d6) δ 10.43 (s, 1H), 9.58 (s, 1H), 9.49 (dd, 1H), 8.83 (d, 1H), 8.32 (d, 1H), 6.85 (bs, 2H), 3.11 (dt, 4H), 2.31 (s, 3H), 1.99 (m, 4H); ¹⁹ F NMR (500 MHz, DMSO-d6) δ 10.43 (s, 1H), 9.58 (s, 1H), 9.49 (dd, 1H), 8.83 (d, 1H), 8.32 (d, 1H), 6.85 (bs, 2H), 3.11 (dt, 4H), 2.31 (s, 3H), 1.99 (m, 4H); MHz, DMSO-d6)δ-135.5,-153.1; MS(ES+)418.1.

Step 3: 2-amino-6-fluoro-N-[5-fluoro-4-[4-[4-(oxetane-3-yl)piperazine-1-carbonyl]-1-piperidinyl]-3-pyridinyl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (amorphous form of compound I-1)

N,N-Diisopropylethylamine (51 μL; 38 mg; 0.29 mmol) was added to a suspension of 1-[3-[(2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carbonyl)amino]-5-fluoro-4-pyridinyl]piperidine-4-carboxylic acid methanesulfonate (50 mg; 0.097 mmol) and 1-(oxetan-3-yl)piperazine (15 mg; 0.11 mmol) in THF (1.00 mL). [(6-Chlorobenzotriazol-1-yl)oxy-(dimethylamino)methylene]-dimethyl-ammonium tetrafluoroborate (TCTU, 36.3 mg; 0.10 mmol) was added, and the mixture was stirred under nitrogen until the reaction was complete (as determined by HPLC analysis). Water (2 mL) was added to the suspension, and the mixture was stirred for 5 hours. The solid was collected by filtration, washed with water (2 x 200 uL), sucked dry, and then dried in a vacuum oven at 45-50°C for 24 hours to afford 1-1 as a light yellow solid (31 mg; 59%).

Preparation Example 6: Preparation of butanenitrile intermediate

Step 1: 3-(Dimethoxymethyl)-4,4-dimethoxybutanenitrile 11

2-(Dimethoxymethyl)-3,3-dimethoxy-propan-1-ol 10 (Journal of the American Chemical Society (1973), 95(26), 8741) (92 g, 473.7 mmol) was dissolved in anhydrous THF (920 mL) and the mixture was cooled in an ice bath. Triethylamine (143.8 g, 198.1 mL, 1.421 mol) was added immediately, followed by the dropwise addition of methanesulfonyl chloride (59.69 g, 40.33 mL, 521.1 mmol) over 1 hr, maintaining the internal temperature below 5°C. The reaction mixture was stirred for 1 hr and then allowed to warm to room temperature. The mixture was diluted with ethyl acetate (920 mL) and water (920 mL). The layers were separated and the organic layer was separated and washed with saturated NaHCO ₃ solution and then with brine. The organic layer was dried over MgSO ₄ , filtered, and evaporated to give [2-(dimethoxymethyl)-3,3-dimethoxypropyl] methanesulfonate as an orange oil (125.31 g, 97%), which was used without further purification.

Tetraethylammonium cyanide (142.3 g, 910.8 mmol) was added dropwise over 10 minutes to a solution of [2-(dimethoxymethyl)-3,3-dimethoxypropyl] methanesulfonate (124 g, 455.4 mmol) in MeCN (1.24 L). The reaction mixture was stirred at room temperature for 72 hours and then partitioned between ethyl acetate (1.24 L) and water (1.24 L). The layers separated, and the organic layer was separated and washed with brine. The organic layer was dried over _MgSO , filtered, and evaporated to provide 3-(dimethoxymethyl)-4,4-dimethoxybutanenitrile 11 as a dark brown oil (86.1 g).

Step 2: 3-(Dimethoxymethyl)-4,4-dimethoxy-2-methylbutanenitrile 12a and 3-(Dimethoxymethyl)-4,4-dimethoxy-2,2-dimethylbutanenitrile 13a

To a solution of 3-(dimethoxymethyl)-4,4-dimethoxy-butanenitrile 11 (250 mg, 1.205 mmol) in THF (3 mL) was added a solution of iodomethane (513.1 mg, 225.0 μL, 3.615 mmol) in THF (1 mL) at -75°C. A solution of sodium (bis(trimethylsilyl)amide) in THF (1.808 mL, 2 M, 3.615 mmol) was then added, maintaining the temperature below -60°C. After addition, the reaction mixture was stirred at -75°C for 2 hours and then slowly quenched with saturated aqueous _NH₄Cl (5 mL). The mixture was diluted with water and diethyl ether, and the layers were separated. The organic layer was washed with brine, dried ( _Na₂SO₄ ), and concentrated in vacuo _to yield a yellow oil, which was purified by silica gel chromatography using a gradient of petroleum ether:EtOAc (100:0 to 80:20). The solvent was concentrated in vacuo to give a clear oil (194 mg). NMR confirmed that the oil was a mixture of 80% monomethyl compound 12a and 20% dimethyl compound 13a. This mixture was used directly in the next step.

Step 3: 3-(Dimethoxymethyl)-2-ethyl-4,4-dimethoxybutanenitrile 12b and 3-(Dimethoxymethyl)-2-diethyl-4,4-dimethoxybutanenitrile 13b

In a procedure similar to that described above in Preparation 6, Step 2, using ethyl iodide instead of methyl iodide, a mixture of monosubstituted compound 12b and disubstituted compound 13b was isolated and used directly in the subsequent step.

Preparation Example 7a: Synthesis of tert-butyl 1-(3-amino-5-fluoro-4-pyridyl)piperidine-4-carboxylate

Step 1: tert-Butyl 1-(3-bromo-5-fluoro-4-pyridyl)piperidine-4-carboxylate 26

A 3 L flanged flask equipped with a thermometer, condenser, nitrogen line, and overhead stirrer was heated at 40°C (external) and then charged with cyclohexanol (750 mL), disodium carbonate (129.8 g, 1.225 mol), 3-bromo-4-chloro-5-fluoro-pyridine (hydrochloric acid 18) (137.5 g, 556.8 mmol), and tert-butylpiperidine-4-carboxylate (123.8 g, 668.2 mmol), rinsed with cyclohexanol (350 mL). The mixture was heated to 120°C internal temperature overnight (18 hr). The reaction mixture was removed from the hotplate and allowed to cool to room temperature. Water (687.5 mL) and EtOAc (687.5 mL) were added, stirred for 10 min, and then transferred to a separatory funnel. Additional EtOAc (1.238 L) was added, mixed, and the aqueous phase removed. The organic phase was further washed with water (687 mL), the aqueous phase was removed, and the organic phase was collected. The aqueous phases were combined and back-extracted with EtOAc (687.5 mL), the aqueous layer was removed, and the organic phase was combined with the other organics. The organics were concentrated in vacuo (bath temperature = 60 ° C, vacuum down to 2 mBar) to obtain a viscous brown oil.

The oil was dissolved in 25% EtOAc/ gasoline, then passed through a short silica gel pad and eluted with 25% EtOAc/ gasoline until no more product came out. The filtrate was concentrated in vacuo to give a brown oil 127.1 g. The product was purified again by ISCO companion (1.5 Kg Silica, loaded in DCM, eluted with 0 to 20% EtOAc/ gasoline), and the product fractions were combined and concentrated in vacuo to give the desired product 26 as a light yellow to cream solid (98 g, 49% yield). ¹ H NMR (500MHz, DMSO-d6)δ8.47(s,1H),8.41(d,1H),3.39–3.36(m,2H), 3.12(tt,2H),2.49–2.43(m,1H),1.91–1.87(m,2H),1.71–1.64(m, 2H)and 1.43(s,9H).MS(ES+)361.0.

Step 2: tert-Butyl 1-(3-amino-5-fluoro-4-pyridinyl)piperidine-4-carboxylate 27

To a solution of tert-butyl 1-(3-bromo-5-fluoro-4-pyridyl)piperidine-4-carboxylate 26 (98 g, 272.8 mmol), benzophenone imine (59.34 g, 54.94 mL, 327.4 mmol), and Cs ₂ CO ₃ (177.8 g, 545.6 mmol) in 1,4-dioxane (1.274 L) were added the phosphorus ligand Xantphos (15.78 g, 27.28 mmol) and Pd ₂ (dba) ₃ (12.49 g, 13.64 mmol). The mixture was stirred at 95° C. under nitrogen overnight. The mixture was cooled to room temperature and then partitioned between EtOAc (1000 mL, 10 vol eq.) and water (490 mL, 5 vol eq.). The organic layer was separated after mixing. The organics were further washed with water (1 x 250 mL), brine (250 mL), dried ( _MgSO4 ), filtered and concentrated in vacuo to give the crude product as a dark red viscous oil, 185.3 g.

The resulting product oil (185.3 g) was dissolved in THF (882.0 mL), and HCl (545.5 mL, 2 M, 1.091 mol) was added, and the resulting mixture was stirred at room temperature for 20 min. The THF was removed in vacuo and additional 2M HCl (588.0 mL) was added. The aqueous phase was washed twice with EtOAc (294.0 mL). A large amount of yellow precipitate formed in both the organic and aqueous phases during the extraction, and the solids from both phases were collected by filtration and suction dried. The combined organic and aqueous filtrates were added to a separatory funnel and extracted with 2M HCl (2 x 200 mL). All aqueous phases were combined and the solid collected on the frit (product) was added to give a suspension. The pH was adjusted to 6 using 2M NaOH and extracted with DCM (3 x 600 mL). The organics were combined, dried (MgSO ₄ ), filtered, and concentrated in vacuo to give 112.2 g of a light orange waxy solid. This solid was suspended in MeCN (200 mL) and stirred for 10 min, then the solid was collected by filtration, washed with a small amount of MeCN, and suction dried to give the product 27 as a white solid (66.8 g, ⁸³ % yield). NMR(500MHz, DMSO-d6)δ7.82(d,1H),7.63(d,1H),5.22(s,2H),3.11–3.00(m, 2H),2.91(tt,2H),2.36(tt,1H),1.88–1.83(m,2H),1.79–1.71(m,2H), 1.43(s,9H).MS(ES+)297.1.

Scheme 7b: Alternative route to the synthesis of tert-butyl 1-(3-amino-5-fluoro-4-pyridinyl)piperidine-4-carboxylate

Step 1: 3-Bromo-4-chloro-5-fluoropyridine hydrochloride 18

A solution of diisopropylamine (101.2 g, 140.2 mL, 1.000 mol) in tetrahydrofuran (1.148 L) was cooled to between -25°C and -20°C. Butyllithium (2.5 M solution in hexane) (400 mL, 2.5 M, 1.000 mol) was added at a rate to maintain the reaction temperature below -20°C (addition time 20 minutes). The mixture was then allowed to warm to 4°C over 1 hour and then recooled to -78°C. A solution of 3-bromo-5-fluoro-pyridine (153.0 g, 869.6 mmol) in tetrahydrofuran (382.5 mL) was added over 40 minutes. The mixture was stirred for 90 minutes, followed by the dropwise addition of a solution of 1,1,1,2,2,2-hexachloroethane (205.9 g, 869.6 mmol) in tetrahydrofuran (350.0 mL) over 40 minutes. Once the addition is complete, the mixture is allowed to warm to ambient temperature overnight. The mixture is cooled to 0°C and then transferred to cold water (2 L). Stir for 20 min, then add MTBE (2.5 L), stir vigorously for 30 min, then transfer to a separatory funnel and separate the organic layer. The aqueous layer is transferred back to the reaction vessel and further extracted with MTBE (2.5 L), stir vigorously for 10 min, then transfer to a separatory funnel and separate the organic layer. The organics are combined, dried (MgSO ₄ ), filtered, and concentrated to a brown oil. This oil is dissolved in pentane (500 mL) and diethyl ether (300 mL). HCl (2M in diethyl ether) (434.8 mL, 2M, 869.6 mmol) is slowly added while stirring. Once the addition was complete, the mixture was stirred for 20 min, then the solid was collected by filtration, washed with ether, and dried under vacuum for 1 hr to give the product 18 as a beige solid (148.9 g, 69%); ¹ H NMR (500 MHz, DMSO-d6) δ 8.77 (2H, s); 19F NMR (500 MHz, DMSO-d6) δ -124.8; MS 210.8.

Step 2: tert-Butyl 1-(3-bromo-5-fluoropyridin-4-yl)piperidine-4-carboxylate 26

3-Bromo-4-chloro-5-fluoro-pyridine hydrochloride 18 (62 g, 251.1 mmol) was suspended in DCM (600 mL) and stirred. The mixture was cooled in an ice bath and sodium hydroxide (276.2 mL, 1 M, 276.2 mmol) was slowly added. The resulting mixture was stirred for 1 hour. The mixture phase separated. More DCM/water was added to aid the phase separation. Some tarry particles remained in the aqueous phase. The organic phase was washed with brine, dried (MgSO ₄ ), filtered, and concentrated. The residue was triturated with heptane. The heptane solution was filtered through a pad of florisil, eluting with heptane. The filtrate was concentrated to an oil, which solidified. 41 g of the free base was obtained.

A well-stirred mixture of 3-bromo-4-chloro-5-fluoropyridine free base (55 g, 0.26 mol), potassium fluoride (31 g, 0.53 mol), and _Me₄NH₄Cl (5.8 g, 53 mmol) in DMSO (400 mL) was heated to 130°C for 2 hours. The reaction mixture was cooled to room temperature, and tert-butylpiperidine-4-carboxylate hydrochloride 22 (66 g, 0.30 mol) and DIPEA (65 g, 0.50 mol) were added. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated in vacuo. The residue was partitioned between DCM/water. The organic layer was washed with water (3x), dried over _Na₂SO₄ , and filtered through silica _gel using DCM as eluent. The filtrate was evaporated to give tert-butyl 1-(3-bromo-5-fluoropyridin-4-yl)piperidine-4-carboxylate 26 (61 g, 65%) as a light yellow solid; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 8.47 (s, 1H), 8.41 (d, 1H), 3.39–3.36 (m, 2H), 3.12 (tt, 2H), 2.49–2.43 (m, 1H), 1.91–1.87 (m, 2H), 1.71–1.64 (m, 2H) and 1.43 (s, 9H); 19F NMR (500 MHz, DMSO-d 6 ) δ −135.2; MS (ES+) 361.0.

Step 3: tert-Butyl 1-(3-amino-5-fluoropyridin-4-yl)piperidine-4-carboxylate 27

Tert-butyl 1-(3-bromo-5-fluoropyridin-4-yl)piperidine-4-carboxylate 26 (800 g, 2.23 mol) was dissolved in 1,4-dioxane (7.5 L). Benzophenone imine (484 g, 2.67 mol) was added in one portion, followed by cesium carbonate (1.45 kg, 4.45 mol), the phosphorus ligand Xantphos (129 g, 223 mmol), and Pd ₂ (dba) ₃ (102 g, 111 mmol). Additional 1,4-dioxane (2.9 L) was added, and the mixture was heated to 95°C under nitrogen until the reaction was complete (as determined by HPLC analysis). The mixture was cooled to 20°C, and ethyl acetate (8 L) and water (4 L) were added. The organic phase was separated, washed with water (4 L) and brine (3.5 L), dried over magnesium sulfate, and filtered. The filtrate was concentrated to a brown oil (1.3 kg). The oil was dissolved in 2-methyltetrahydrofuran (7.2 L), 2M HCl was added at 20°C, and the mixture was stirred for 30 minutes. The aqueous layer was separated, and the organic layer was extracted with 2M HCl (1.2 L). The aqueous layers were combined and neutralized with 2M NaOH (5.4 L, pH 8-9). The product was extracted into 2-methyltetrahydrofuran (14 L then 2 x 5 L). The combined extracts were washed with water (1.6 L), and the organic solution was concentrated. The residue was suspended in acetonitrile (2 L), filtered, and dried. The product 27 was obtained as a white solid (568.7 g, 86.5%); 1H NMR (500 MHz, DMSO-d ₆ ) δ 7.82 (d, 1H), 7.63 (d, 1H), 5.22 (s, 2H), 3.11–3.00 (m, 2H), 2.91 (tt, 2H), 2.36 (tt, 1H), 1.88–1.83 (m, 2H), 1.79–1.71 (m, 2H), 1.43 (s, 9H); 19F NMR (500 MHz, DMSO-d 6 ) δ–140.0; MS (ES+) 297.1.

Preparation Example 8: Synthesis of tert-butylpiperidine-4-carboxylate

Step 1: 1-Benzyl-4-tert-butylpiperidine-1,4-dicarboxylate 21

A 5 L flanged flask was charged with a solution of 1-benzyloxycarbonylpiperidine-4-carboxylic acid 20 (200 g, 759.6 mmol) in DCM (500.0 mL), followed by additional DCM (2.000 L), tert-butanol (140.8 g, 181.7 mL, 1.899 mol), and DMAP (46.40 g, 379.8 mmol). The mixture was cooled in an ice/salt/water bath (internal -3.4°C). 3-(Ethyliminomethylamino)-N,N-dimethyl-propane-1-amine (hydrochloric acid (1)) (145.6 g, 759.6 mmol) was added dropwise over 15 min, and the addition funnel was rinsed with DCM (500.0 mL). The mixture was stirred in an ice bath for 2 hr. The ice bath was then removed (internal 3°C) and the mixture was allowed to warm to room temperature overnight. The mixture was washed with 5% citric acid (2 x 500 mL), saturated _NaHCO₃ (500 mL), and water (500 mL). The organics were dried over _MgSO₄ , filtered, and concentrated in vacuo to afford product 21 as a viscous light yellow oil that became a white solid upon standing (246.1 g, 101%). ^1H NMR (500 MHz, DMSO-d₆) δ 7.40–7.31 (m, 5H), 5.08 (s, 2H), 3.90 (dt, 2H), 2.93 (br s, 2H), 2.43 (tt, 1H), 1.80–1.76 (m, 2H), and 1.45–1.37 (m, 11H).

Step 2: tert-Butylpiperidine-4-carboxylate 22

A 3 L flask was charged with wet palladium on carbon Degussa (10% Pd, 50% water) (8.120 g, 76.30 mmol) under nitrogen, followed by EtOAc (1.706 L). The mixture was degassed via _N2 /vacuum cycles (3x), followed by the addition of a solution of 1-benzyl-4-tert-butylpiperidine-1,4-dicarboxylate 21 (243.7 g, 763.0 mmol) in EtOAc (243.7 mL). The mixture was stirred under a hydrogen atmosphere overnight. Hydrogen was replenished and the mixture was stirred for an additional 3.5 hr. Methanol (60 mL) was added to help dissolve the precipitate, which was then filtered through celite and washed thoroughly with methanol. The filtrate was concentrated in vacuo to give a brown oil slightly suspended with a white solid, 138.6 g. The solid was removed by filtration and washed with a small amount of EtOAc. The filtrate was concentrated in vacuo to give the desired product as a light brown oil (129 g, 91%). ¹ H NMR (500 MHz, DMSO-d6) δ 2.88 (dt, 2H), 2.44 (td, 2H), 2.23 (tt, 1H), 1.69-1.64 (m, 2H) and 1.41-1.33 (m, 11H).

Preparation Example 9: Synthesis of 1-(oxetane-3-yl)piperazine

Step 1: Benzyl 4-(oxetan-3-yl)piperazine-1-carboxylate 24

Benzylpiperazine-1-carboxylate 23 (27.3 mL, 142.2 mmol) was dissolved in anhydrous THF (313.1 mL), and oxetane-3-one (12.29 g, 10.93 mL, 170.6 mmol) was added. The resulting solution was cooled in an ice bath. NaBH(Oac) _₃ (59.99 g, 284.4 mmol) was added portionwise over 30 minutes, with approximately one-quarter of the volume added. The mixture was removed from the ice bath and allowed to warm to room temperature naturally, followed by a further portionwise addition of NaBH(Oac) _₃ over 30 minutes. Upon complete addition, a slow exotherm from 22°C to 32°C was observed, and the mixture was subsequently cooled in an ice bath until it reached an internal temperature of 22°C. The ice bath was removed, and the internal temperature of the reaction mixture stabilized at 22°C. The mixture was stirred at room temperature overnight.

The resulting white suspension was quenched by the addition of 2M sodium carbonate solution (approximately 150 mL) (pH = 8) and concentrated under reduced pressure to remove THF. The product was then extracted with EtOAc (3 x 250 mL). The combined organics were filtered through MgSO ₄ and concentrated under reduced pressure to afford product 24 as a white solid (32.7 g, 83% yield). ¹ H NMR (500 MHz, DMSO-d 6 ) δ 7.39–7.30 (m, 5H), 5.07 (s, 2H), 4.52 (t, 2H), 4.42 (t, 2H), 3.43–3.39 (m, 5H) and 2.22 (t, 4H). MS (ES+) 276.8.

Step 2: 1-(Oxetane-3-yl)piperazine 25

Under nitrogen, a 1 L flask was charged with Pd(OH) ₂ (1.661 g, 2.366 mmol). MeOH (130.8 mL) and EtOAc (261.6 mL) were added, and the mixture was degassed via vacuum/nitrogen cycles (3x). Benzyl 4-(oxetan-3-yl)piperazine-1-carboxylate 24 (32.7 g, 118.3 mmol) was then added, and the mixture was stirred under a hydrogen atmosphere over the weekend. The mixture was filtered through a pad of Celite, washed thoroughly with EtOAc and then methanol. The filtrate was concentrated in vacuo to afford the product 25 as an orange oil 1 (8.1 g, quantitative yield). ¹ H NMR (500 MHz, DMSO-d6) δ 4.51 (t, 2H), 4.41 (t, 2H), 3.36–3.30 (shielded signal, 1H), 2.69 (t, 4H) and 2.14 (br s, 4H).

Example 4: 2-amino-6-fluoro-N-(5-fluoro-4-(4-(2,2,3,3,5,5,6,6-octadeuterated-piperazine-1-carbonyl) Piperidin-1-yl)-3-pyridinyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound I-2) and 2-amino-6-fluoro-N-(5- 4-(4-(2,2,3,3,5,5,6,6-octadeuterated-4-(oxetan-3-yl)piperazine-1-carbonyl)piperidin-1-yl)-3-fluoro- Synthesis of pyridyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound I-3)

Step 1: tert-Butyl 1-(3-(2-amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxamido)-5-fluoropyridin-4-yl)piperidine-4-carboxylate 28

A mixture of (6-chlorobenzotriazol-1-yl) 2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylate 6a* (41.69 g, 119.9 mmol) and tert-butyl 1-(3-amino-5-fluoro-4-pyridyl)piperidine-4-carboxylate 27 (32.2 g, 109.0 mmol) in pyridine (483 mL) was heated at 90°C for 12 h. The reaction was cooled to room temperature, EtOH (322 mL) was added, and the mixture was stirred at room temperature for 10 min. The solid was collected by filtration, washed thoroughly with ethanol, and suction dried to yield 28 as a yellow solid (33 g, 64%).

Step 2: 1-(3-(2-amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxamido)-5-fluoropyridin-4-yl)piperidine-4-carboxylic acid 29

To a suspension of tert-butyl 1-[3-[(2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carbonyl)amino]-5-fluoro-4-pyridinyl]piperidine-4-carboxylate 28 (69.7 g, 147.2 mmol) in DCM (348.5 mL) was added triethylsilane (18.83 g, 25.87 mL, 161.9 mmol), followed by TFA (151.1 g, 102.1 mL, 1.325 mol). The resulting solution was stirred at room temperature for 12 h. The mixture was concentrated in vacuo to afford an orange solid, which was triturated in DCM (200 mL) for 20 min. The solid was collected by filtration, washed with a small amount of DCM, and suction dried to afford the desired trifluoroacetate salt as a yellow solid (75.2 g, 96%).

To a solution of 1-[3-[(2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carbonyl)amino]-5-fluoro-4-pyridinyl]piperidine-4-carboxylic acid trifluoroacetate (73 g, 124.7 mmol) in NMP (662.7 mL) was added hydrogen chloride (4 M in dioxane) (37.4 mL, 4 M, 149.6 mmol). The reaction was stirred at room temperature for 20 min, after which the solid was collected by filtration, washed with a small amount of NMP and then MTBE, and suction dried to afford the pure product, the hydrochloride salt 29, as a light yellow solid.

Step 3: 2-amino-6-fluoro-N-(5-fluoro-4-(4-(2,2,3,3,5,5,6,6-octadeuterated-piperazine-1-carbonyl)piperidin-1-yl)-3-pyridinyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound I-2)

(Benzotriazol-1-yloxy-dimethylamino-methylene)-dimethyl-ammonium trifluoroborate (127.3 mg, 0.3966 mmol) was added to a mixture of 1-[3-[(2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carbonyl)amino]-5-fluoro-4-pyridyl]piperidine-4-carboxylic acid hydrochloride 29 (150 mg, 0.3305 mmol), 2,2,3,3,5,5,6,6-octadeuteropiperazine (155.6 mg, 1.652 mmol), and _Et3N (83.6 mg, 115.2 μL, 0.8262 mmol) in DMF (5 mL). The reaction mixture was stirred at room temperature for 18 h. The crude mixture was purified by preparative HPLC to afford I-2 as a white solid (114 mg, 48%).

Step 4: 2-amino-6-fluoro-N-(5-fluoro-4-(4-(2,2,3,3,5,5,6,6-octadeuterated-4-(oxetan-3-yl)piperazine-1-carbonyl)piperidin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound I-3)

Sodium triacetoxyborohydride (24.67 mg, 0.1164 mmol) was added to a solution of oxetan-3-one (7.271 mg, 0.1009 mmol), 2-amino-6-fluoro-N-(5-fluoro-4-(4-(2,2,3,3,5,5,6,6-octadeuterated-piperazine-1-carbonyl)piperidin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide 13 (56 mg, 0.07761 mmol), and acetic acid (13.98 mg, 13.24 μL, 0.2328 mmol) in DMF (2 mL). The reaction mixture was stirred at room temperature for 18 h. The solution was quenched with methanol and water, and the crude mixture was purified by preparative HPLC to give the desired product I-3 (20 mg, 46%). ¹ H NMR (500 MHz, DMSO-d6) δ 10.64 (s, 1H), 9.67 (s, 1H), 9.48 (dd, 1H), 9.26 (dd, 1H), 8.26 (d, 1H), 6.79 (s, 2H), 4.55 (t, 2H), 4.47 (t, 2H), 3.63 (m, 1H), 3.20 (m, 2H), 3.15 (m, 2H), 2.95 (m, 1H), 2.10 (m, 2H), 1.74 (d, 2H); ES+ 550.4.

Compound analysis data

Solid form of compound I-1

Various solid forms of compound I-1 have been prepared, including salts, solvates, hydrates, and anhydrous forms. The solid forms of the present invention can be used in the manufacture of cancer therapeutics. One embodiment provides the use of the solid forms described herein for treating cancer. In some embodiments, the cancer is triple-negative breast cancer, pancreatic cancer, small cell lung cancer, colorectal cancer, ovarian cancer, or non-small cell lung cancer. Another embodiment provides a pharmaceutical composition comprising the solid form described herein and a pharmaceutically acceptable carrier.

Applicants have described herein a number of novel solid forms of Compound 1-1. The name and stoichiometry of each solid form are provided in Table 2 below:

Table 2

Example form Chemometrics Example 5 Compound I-1·Ethanol solvate 1:0.72 Example 6a Compound I-1·Hydrate I 1:4.5 Example 6b Compound I-1·Hydrate II ------- Example 7 Compound I-1·Anhydrous Form A N/A Example 8 Compound I-1·Anhydrous Form B ------- Example 9 Compound I-1·Anhydrous Form C N/A Example 10 Compound I-1·Amorphous form N/A Example 11 Compound I-1·DMSO solvate 1:1 Example 12 Compound I-1·DMAC solvate 1:1.3 Example 13 Compound I-1·Acetone solvate 1:0.44 Example 14 Compound I-1·Isopropanol solvate 1:0.35

ssNMR experimental method

Solid-state NMR spectra were obtained on a Bruker-Biospin 400 MHz Advance III wide-aperture spectrometer equipped with a Bruker-Biospin 4mm HFX probe. The sample was loaded into a 4mm ZrO _rotor (approximately 70 mg or less, depending on sample availability). A typical magic angle spin (MAS) speed of 12.5 kHz was used. The temperature of the probe head was set to 275 K to minimize the frictional heating effect during the spin process. ¹ H MAS T ₁ saturation recovery relaxation experiments were used to measure the proton relaxation time, so as to establish the appropriate recirculation delay for ¹³ C cross-polarization (CP) MAS experiments. ^{13 The recirculation delay for 13} C CPMAS experiments was adjusted to at least 1.2 times longer than the measured ¹ HT ₁ relaxation time, so as to maximize the carbon spectrum signal-to-noise ratio. 13 The CP contact time for ¹³ C CPMAS experiments was set to 2 milliseconds. CP proton pulses with a linear slope (from 50% to 100%) were used. The Hartmann-Hahn match is optimized for external reference sample (glycine). Fluorine spectrum uses proton decoupling MAS to be arranged, and recovery delay is set to about 5 times of measured ^FT ₁ relaxation time. Use proton decoupling 19F MAS T ₁ saturation recovery relaxation test to measure the relaxation time of fluorine. Obtain carbon spectrum and fluorine spectrum with SPINAL 64 by the field intensity decoupling of about 100kHz. Chemical shift is referenced to the external standard of the adamantane that is set to 29.5ppm with high magnetic field resonance.

Example 5: Compound I-1 (ethanol solvate)

The ethanol solvate of compound I-1 can be prepared according to the method described in Example 1, step 4.

XRPD of compound I-1 (ethanol solvate)

The XRPD pattern of Compound I-1 ethanol solvate was recorded in reflection mode at room temperature using a PANalytical diffractometer equipped with an Empyrean tube source and a PIXcel 1D detector (PANalytical, The Netherlands). The X-ray generator was operated at 45 kV and 40 mA. The powder sample was placed in a silicon holder. Data were recorded over the 2θ range of 3° to 39°, with a step size of 0.013° and a dwell time of 121 s per step. Figure 1a shows the X-ray powder diffraction pattern of the sample, which is typical of a crystalline drug substance.

Table 3a depicts representative XRPD peaks for Compound I-1 · ethanol solvate:

Table 3a: Representative XRPD peaks

Thermal analysis of compound I-1 (ethanol solvate)

Thermogravimetric analysis of Compound I-1 ethanol solvate was performed using a Discovery TGA (TA Instruments Trios) to determine the percentage weight loss as a function of temperature. A sample (8.338 mg) was added to a pre-weighed aluminum pan and heated from ambient temperature to 310°C at a rate of 20°C/min. The TGA results shown in Figure 2a show a weight loss of up to 5.76% between 166°C (start) and 219°C (end). This weight loss is equivalent to approximately 0.72 molar equivalents of ethanol. The subsequent weight loss seen at 290°C is the result of melting/degradation.

Differential Scanning Calorimetry of Compound I-1 (Ethanol Solvate)

Differential scanning calorimetry of Compound I-1 ethanol solvate was measured using a TA Instrument DSC Q2000. A sample (1.84 mg) was weighed into a pinhole-sealed aluminum pan and heated from ambient temperature to 300°C at a rate of 20°C/min. The DSC results, shown in Figure 3a, show a desolvation endotherm at 169°C (onset), followed by a single melting endotherm at 258°C (onset).

ssNMR of compound I-1 (ethanol solvate)

The solid-state ¹³ C NMR spectrum of Compound I-1·ethanol solvate is shown in Figure 4a. Table 3b provides the chemical shifts of the relevant peaks.

Table 3b: Solid-state ¹³ C NMR spectrum of Compound I-1 (ethanol solvate)

The solid-state ¹⁹ F NMR spectrum of Compound I-1·ethanol solvate is shown in Figure 5a. Table 3c provides the chemical shifts of the relevant peaks.

Table 3c: Solid-state ¹⁹ F NMR spectrum of Compound I-1 (ethanol solvate)

Example 6a: Compound I-1 (Hydrate I)

Compound I-1 ethanol solvate (1000 mg), prepared according to the method described in Step 4 of Example 1, was suspended in water (20 mL) and incubated at room temperature for 4 days. The suspension was centrifuged, and the residual solid was isolated and dried in a vacuum oven at 35°C overnight to obtain Compound I-1 Hydrate I as a yellow powder.

XRPD of Compound I-1 (Hydrate I)

The XRPD pattern of Compound I-1·Hydrate I was recorded in reflection mode at room temperature using a Bruker D8 Discover diffractometer equipped with a sealed tube source and a Hi-Star area detector (Bruker AXS, Madison, WI, AssetV012842). The X-ray generator was operated at 40 kV and 35 mA. The powder sample was placed in a nickel holder. Two frames were registered, each with an exposure time of 120 seconds. The data from the 2θ range of 3.5° to 39° were then integrated with a step size of 0.02° and fused into a single continuous pattern. Figure 1b shows the X-ray powder diffraction pattern of the sample, which is typical of a crystalline drug substance.

Table 4a depicts representative XRPD peaks of Compound I-1 Hydrate I:

Table 4a: Representative XRPD peaks

Thermal analysis of compound I-1 (hydrate I)

Thermogravimetric analysis of Compound I-1 Hydrate I was performed using a TA Instrument TGA Q5000 (Asset V014258) to determine the percentage weight loss over time. A sample (7.380 mg) was added to a pre-weighed aluminum pan and heated from ambient temperature to 350°C at a rate of 10°C/min. The TGA results, shown in Figure 2b, show a substantial initial weight loss upon reaching 100°C, followed by a small additional weight loss before melting/degradation. The initial weight loss was 14.56%, equivalent to approximately 4.5 molar equivalents of water. The onset temperature of melting/degradation was 292°C.

Differential Scanning Calorimetry of Compound I-1 (Hydrate I)

Differential scanning calorimetry of Compound I-1 Hydrate I was measured using a TA Instrument DSC Q200 (Asset V005642). A sample (5.598 mg) was weighed into a pinhole-sealed aluminum pan and heated from ambient temperature to 350°C at a rate of 10°C/min. The DSC results, shown in Figure 3b, show a broad initial endothermic event corresponding to dehydration, followed by melting to an amorphous form. After melting, the Tg reached 125°C, recrystallized at 180°C, melted at 257°C, and finally melted/degraded at 278°C.

Example 6b: Compound I-1 (Hydrate II)

Compound I-1·ethanol solvate (1000 mg) prepared according to the method described in Step 4 of Example 1 was suspended in water (20 mL) and incubated at room temperature for 4 days. The suspension was centrifuged to separate the residual solid to obtain Compound I-1·hydrate II as a yellow paste.

XRPD of Compound I-1 (Hydrate II)

The XRPD pattern of Compound I-1·Hydrate II was recorded in reflection mode at room temperature using a Bruker D8 Discover diffractometer equipped with a sealed tube source and a Hi-Star area detector (Bruker AXS, Madison, WI, Asset V012842). The X-ray generator was operated at 40 kV and 35 mA. The powder sample was placed in a nickel holder. Two frames were registered, each with an exposure time of 120 seconds. The data were then integrated over the 2θ range of 3.5°–39° with a step size of 0.02° and fused into a single continuous pattern. Figure 4b shows the X-ray powder diffraction pattern of the sample, which is typical of a crystalline drug substance.

Table 4b depicts representative XRPD peaks for Compound I-1 Hydrate II:

Table 4b: Representative XRPD peaks

ssNMR of Compound I-1 (Hydrate II)

The solid-state ¹³ C NMR spectrum of Compound I-1·Hydrate II is shown in Figure 5b. Table 4c provides the chemical shifts of the relevant peaks.

Table 4c: Solid-state ¹³ C NMR spectrum of Compound I-1 (Hydrate II)

The solid-state ¹⁹ F NMR spectrum of Compound I-1·Hydrate II is shown in Figure 6b. Table 4d provides the chemical shifts of the relevant peaks.

Table 4d: Solid-state ¹⁹ F NMR spectrum of Compound I-1 (Hydrate II)

Example 7: Compound I-1 (Anhydrous Form A)

Compound I-1 ethanol solvate (1000 mg), prepared according to the method described in Step 4 of Example 1, was suspended in THF (20 mL) and incubated at room temperature for 72 hours. The suspension was centrifuged, and the residual solid was isolated and dried in a vacuum oven at 35°C overnight to obtain Compound I-1 Anhydrous Form A ("Form A") as a yellow powder.

In an alternative method, the amorphous form of Compound I-1 (15.1 g; 0.028 mol), prepared according to the method of Step 3 of Example 2, was suspended in a mixture of 2-propanol (300 mL) and water (100 mL). The mixture was stirred, heated to 70-75°C, and filtered while hot. The resulting clear filtrate was heated and distilled, and the solvent was replaced with 2-propanol until the temperature of the contents reached 82.5°C. The resulting suspension was cooled to 15°C over 10 hours and stirred for an additional 5 hours. The solid was collected by filtration, suction dried for 1 hour, and then dried in a vacuum oven at 60°C for 20 hours to provide Compound I-1 Anhydrous Form A (13.9 g; 92%).

A variety of other solvents can be used to prepare Compound I-1 Anhydrous Form A. The following Table 5a summarizes the procedures.

Table 5a: Solvents used to prepare Form A

XRPD of compound I-1 (anhydrous form A)

The XRPD pattern of Compound I-1, Anhydrous Form A, was recorded in reflection mode at room temperature using a Bruker D8 Discover diffractometer equipped with a sealed tube source and a Hi-Star area detector (Bruker AXS, Madison, WI, AssetV012842). The X-ray generator was operated at 40 kV and 35 mA. The powder sample was placed in a nickel holder. Two frames were registered, each with an exposure time of 120 seconds. The data from the 2θ range of 3.5° to 39° was then integrated with a step size of 0.02° and fused into a single continuous pattern. Figure 1c shows the X-ray powder diffraction pattern of the sample, which is typical of a crystalline drug substance.

Table 5b depicts representative XRPD peaks of Compound I-1 Anhydrous Form A:

Table 5b: Representative XRPD peaks

Thermal analysis of compound I-1 (anhydrous form A)

Thermogravimetric analysis of Compound I-1, Anhydrous Form A, was performed using a TA Instrument TGA Q5000 (Asset V014258) to determine the percentage weight loss over time. A sample (7.377 mg) was added to a pre-weighed aluminum pan and heated from ambient temperature to 350°C at a rate of 10°C/min. The TGA results, shown in Figure 2c, show very little weight loss observed before melting or thermal degradation. The weight loss from ambient temperature to 265°C was 0.96%. The onset of degradation was 292°C.

Differential Scanning Calorimetry of Compound I-1 (Anhydrous Form A)

Differential scanning calorimetry of Compound I-1, Anhydrous Form A, was measured using a TA Instrument DSC Q2000 (Asset V014259). A sample (3.412 mg) was weighed into a pinhole-sealed aluminum pan and heated from ambient temperature to 350°C at a rate of 10°C/min. The DSC results, shown in Figure 3c, show a single endothermic melting event at 262°C. The melting event contained two distinct peaks separated by approximately 1°C.

Composition and preparation of active tablets containing anhydrous Form A

Composition of Form A 10 mg tablets

The composition of the dry granulation and compression blend formulation for the 10 mg tablets of Active Form A is described in Tables 5c and 5d. The overall composition of the tablets is described in Table 5e.

Table 5c: Form A (10 mg) intragranular blend

Table 5d: Composition of Form A (10 mg) tablets

Table 5e: Overall composition of Form A (10 mg) tablets

Composition of Form A 50 mg tablets

The composition of the dry granulation and compression blend formulation for 50 mg tablets of Active Form A is described in Tables 5f and 5g. The overall composition of the tablets is described in Table 5h.

Table 5f: Form A (50 mg) Intragranular Blend

Table 5g: Composition of Form A (50 mg) tablets

Table 5h: Overall composition of Form A (50 mg) tablets

Method for preparing Form A 10 mg and 50 mg tablets

Step I. Pre-granulation mixing:

Form A was passed through a conical mill equipped with a 24R round-mesh screen and a round-edge impeller at 1500 rpm. Lactose monohydrate, microcrystalline cellulose, and intragranular croscarmellose sodium were passed through a #30 mesh screen. The conical-milled Form A and the screened components were then blended at 26 rpm for 10 minutes. Sodium stearyl fumarate was manually passed through a 60 mesh screen and added to the blend, and all materials were blended at 26 rpm for 3 minutes. Samples were withdrawn for blend uniformity analysis.

Step II. Dry granulation:

The blend was dry granulated on a Gerteis Minipactor. The blend was passed through a roller compactor equipped with a combination of smooth and knurled rollers at a roller speed of 2 rpm, a roller pressure of 5 kN/cm, and a roller gap of 2 mm. The compacted powder was then granulated using a portable roller mill and passed through a 1 mm sieve at a grinding speed of 80 rpm.

Step III. Final blending:

Manually pass extragranular croscarmellose sodium and sodium stearyl fumarate through 30-mesh and 60-mesh sieves, respectively. Blend the extragranular croscarmellose sodium with the dry granules at 32 rpm for 5 minutes. Then, add the extragranular sodium stearyl fumarate to the main mixture and mix at 32 rpm for 3 minutes. A sample is withdrawn for blend uniformity analysis. The blend is sealed in a double-layered low-density polyethylene bag within a rigid secondary container to prevent puncture.

Step IV. Tabletting:

The tablet press (Piccola D-8 Rotatory Press) was partially processed (2 of 8) to produce 0.25" standard concave tablets for the 10 mg strength and 0.568" x 0.2885" caplets for the 50 mg strength. The turret speed was 25-35 rpm. Tablet in-process control tests included mean weight, individual weight, and hardness, as shown in Table 5i.

Table 5i: Form A (10 mg and 50 mg) Tablet Compression Process Internal Control Description

Preparation of Form A crystals

Form A was recrystallized from a DCM/heptane mixture and the solvent was slowly evaporated. Colorless, needle-shaped crystals measuring 0.10 x 0.02 x 0.02 mm were selected and diffraction experiments were performed on a Bruker APEX II CCD diffractometer using Cu Kα radiation at room temperature. The structure was solved by direct methods and refined using the SHELXTL software package.

Form A crystal experiments:

The crystals showed a monoclinic system with a P2 _1/c centrosymmetric space group. The lattice parameters were α = 90°, β = 107.22(3)°, γ = 90°, and the corrected R factor was 6.9%. The conformational diagrams of compound I-1·anhydrous form A based on single crystal X-ray analysis are shown in Figures 4c and 5c. Compound I-1·anhydrous form A presents an ordered asymmetric unit (Figure 4c). As shown in Figure 5c, the molecules of compound I-1·anhydrous form A form a one-two-dimensional chain along the b-axis, which is stabilized by intermolecular hydrogen bonds between the amine and pyridine groups. Multiple chains are stacked in three-dimensional space, with interlayer spacing of approximately

Table 5j: Crystal data of Form A

Geometry: All ESDs (except for the ESDs for dihedral angles between two L.S. planes) are estimated using the full covariance matrix. Unit cell ESDs are considered separately for estimating ESDs for distances, angles, and torsion angles; correlations between ESDs for unit cell parameters are used only when they are constrained by crystal symmetry. An approximate (isotropic) treatment of the unit cell ESD is used to estimate ESDs involving L.S. planes.

Table 5k: Data collection parameters for Form A crystals

Data collection: Apex II; unit cell modification: Apex II; data reduction: Apex II; program used for structure solution: SHELXS97 (Sheldrick, 1990); program used for structure correction: SHELXL97 (Sheldrick, 1997); molecular graphics: Mercury; software used for preparation of publication material: publCIF.

Table 5m: Corrected parameters for Form A crystals

Correction: ^F2 correction for ALL reflexes. The goodness of the weighted R-factors wR and fitS is based on ^F2 . The conventional R-factor R is based on F, with F set to zero for negative ^F2 . The threshold expression of ^F2 > 2sigma( ^F2 ) is used only for calculating R-factors (gt), etc. and is not relevant to the reflex selected for correction. The R-factor based on ^F2 is statistically twice as large as that based on F, and the R-factor based on ALL data is even larger.

ssNMR of compound I-1 (anhydrous form A)

The solid-state ¹³ C NMR spectrum of Compound I-1·Anhydrous Form A is shown in Figure 6c. Table 5n provides the chemical shifts of the relevant peaks.

Table 5n: Solid-state ¹³ C NMR spectrum of Form A

The solid-state ¹⁹ F NMR spectrum of Compound I-1·Anhydrous Form A is shown in Figure 7c. Table 5p provides the chemical shifts of the relevant peaks.

Table 5p: Solid-state ¹⁹ F NMR spectrum of Form A

Example 8: Compound I-1 (Anhydrous Form B)

Compound I-1 amorphous form (3.50 g), prepared according to the method described in Example 2, Step 3, was placed in a 250 mL 3-neck flask, THF (70 mL) was added, and the mixture was stirred overnight (e.g., for at least 12 hours) at ambient temperature using an overhead stirrer. The suspension was filtered under vacuum (4.25 cm diameter Whatman filter paper), washed with THF (7 mL), and dried under vacuum for approximately 35 minutes to yield a fairly hard yellow solid (2.25 g). The solid was dried under vacuum at 35° C. overnight, with good dehydration, to yield 1.921 g of Compound I-1 anhydrous Form B as a yellow solid.

XRPD of compound I-1 (anhydrous form B)

The XRPD pattern of Compound I-1, Anhydrous Form B, was recorded in reflection mode at room temperature using a Bruker D8 Discover diffractometer equipped with a sealed tube source and a Hi-Star area detector (Bruker AXS, Madison, WI, AssetV012842). The X-ray generator was operated at 40 kV and 35 mA. The powder sample was placed in a nickel holder. Two frames were registered, each with an exposure time of 300 seconds. The data were then integrated over the 2θ range of 3.5° to 39° with a step size of 0.02° and fused into a single continuous pattern. Figure 1d shows the X-ray powder diffraction pattern of the sample, which is typical of a crystalline drug substance.

Table 6a depicts representative XRPD peaks of Compound I-1 Anhydrous Form B:

Table 6a: Representative XRPD peaks

Thermal Analysis of Compound I-1 (Anhydrous Form B)

Thermogravimetric analysis of Compound I-1, Anhydrous Form B, was performed using a TA Instrument TGA Q500 (Asset V014840) to determine the percentage weight loss over time. A sample (2.728 mg) was added to a pre-weighed platinum pan and heated from ambient temperature to 350°C at a rate of 10°C/min. The TGA results, shown in Figure 2d, show two distinct weight loss events totaling 2.5% upon reaching 175°C. The onset temperature of melting/degradation was 284°C.

Differential Scanning Calorimetry of Compound I-1 (Anhydrous Form B)

Differential scanning calorimetry of Compound I-1, Anhydrous Form B, was measured using a TA Instrument DSC Q2000 (Asset V012390). A sample (2.125 mg) was weighed into a pinhole-sealed aluminum pan and heated from 30°C to 350°C at a rate of 3°C/min, with ±1°C adjustments every 60 seconds. The DSC results, shown in Figure 3d, show an exothermic event at 177°C (also with a slight rearrangement of the crystal structure), an endothermic melt at 257°C, recrystallization at 258°C, and final melting/degradation at 280°C.

ssNMR of compound I-1 (anhydrous form B)

The solid-state ¹³ C NMR spectrum of Compound I-1·Anhydrous Form B is shown in Figure 4d. Table 6b provides the chemical shifts of the relevant peaks.

Table 6b: Solid-state ¹³ C NMR spectrum of Compound B

The solid-state ¹⁹ F NMR spectrum of Compound I-1·Anhydrous Form B is shown in Figure 5d. Table 6c provides the chemical shifts of the relevant peaks.

Table 6c: Solid-state ¹⁹ F NMR spectrum of Compound B

Example 9: Compound I-1 (Anhydrous Form C)

Compound I-1·Anhydrous Form B (~15 mg) prepared according to the method described in Example 8 was added to an aluminum sealed pan with a pinhole and heated to 265°C via DSC at a rate of 5°C/min (3 pans, ~5 mg each) to obtain Compound I-1·Anhydrous Form C as a dark yellow powder.

XRPD of compound I-1 (anhydrous form C)

The XRPD pattern of Compound I-1, Anhydrous Form C, was recorded in reflection mode at room temperature using a Bruker D8 Discover diffractometer equipped with a sealed tube source and a Hi-Star area detector (Bruker AXS, Madison, WI, AssetV012842). The X-ray generator was operated at 40 kV and 35 mA. The powder sample was placed in a nickel holder. Two frames were registered, each with an exposure time of 120 seconds. The data from the 2θ range of 3.5° to 39° was then integrated with a step size of 0.02° and fused into a single continuous pattern. Figure 1e shows the X-ray powder diffraction pattern of the sample, which is typical of a crystalline drug substance.

Table 7a depicts representative XRPD peaks for Compound I-1 Anhydrous Form C:

Table 7a: Representative XRPD peaks

Thermal analysis of compound I-1 (anhydrous form C)

Thermogravimetric analysis of Compound I-1, Anhydrous Form C, was performed using a TA Instrument TGA Q500 (Asset V014840) to determine the percentage weight loss over time. A sample (3.363 mg) was added to a pre-weighed platinum pan and heated from ambient temperature to 350°C at a rate of 10°C/min. The TGA results, shown in Figure 2e, show no significant weight loss event prior to melting/degradation. The onset temperature of melting/degradation was 292°C.

Differential Scanning Calorimetry of Compound I-1 (Anhydrous Form C)

Differential scanning calorimetry of Compound I-1, Anhydrous Form C, was measured using a TA Instrument DSC Q2000 (Asset V012390). A sample (4.100 mg) was weighed into a pinhole-sealed aluminum pan and heated from 30°C to 350°C at a rate of 3°C/min, with ±1°C adjustments every 60 seconds. The DSC results, shown in Figure 3e, show a single endothermic melting/degradation event at 281°C.

ssNMR

The solid-state ¹³ C NMR spectrum of Compound I-1·Anhydrous Form C is shown in Figure 4e. Table 7b provides the chemical shifts of the relevant peaks.

Table 7b: Solid-state ¹³ C NMR spectrum of Compound C

The solid-state ¹⁹ F NMR spectrum of Compound I-1·Anhydrous Form C is shown in Figure 5e. Table 7c provides the chemical shifts of the relevant peaks.

Table 7c: Solid-state ¹⁹ F NMR spectrum of Compound C

Example 10: Compound I-1 (amorphous form)

Compound I-1 amorphous form was prepared according to the method described in Step 3 of Example 2 or Step 3 of Example 3.

XRPD of compound I-1 (amorphous form)

The XRPD pattern of Compound I-1 amorphous form was recorded in reflection mode at room temperature using a PANalytical diffractometer equipped with an Empyrean Cu tube source and a PIXcel 1D detector (PANalytical, The Netherlands). The X-ray generator was operated at 45 kV and 40 mA. The powder sample was placed in a silicon holder. Data were recorded over the 2θ range of 3° to 39°, with a step size of 0.013° and a dwell time of 0.5 s per step. Figure 1f shows the X-ray powder diffraction pattern of the sample, which is typical of an amorphous drug substance.

Differential Scanning Calorimetry of Compound I-1 (Amorphous Form)

Differential scanning calorimetry of Compound I-1 amorphous form was measured using a TA Instrument DSC Q2000. A sample (2.61 mg) was weighed into an aluminum, non-sealed pan and heated from ambient temperature to 350°C using ramp mode at a heating rate of 2°C/min with a ramp of ±0.5°C for 60 seconds. The DSC results, shown in Figure 2f, show a glass transition (Tg) at 128°C (onset) with a heat capacity change of 0.3 J/(g.°C). The glass transition was followed by a crystallization exotherm at 174°C (onset), followed by a melting/degradation event at 250°C.

ssNMR of compound I-1 (amorphous form)

The solid-state ¹³ C NMR spectrum of Compound I-1·amorphous form is shown in Figure 3f. Table 8a provides the chemical shifts of the relevant peaks.

Table 8a: Solid-state ¹³ C NMR spectrum of amorphous form

The solid-state ¹⁹ F NMR spectrum of Compound I-1·amorphous form is shown in Figure 4f . Table 8b provides the chemical shifts of the relevant peaks.

Table 8b: Solid-state ¹⁹ F NMR spectrum of amorphous form

Example 11: Compound I-1 (DMSO solvate)

Compound I-1, Anhydrous Form A (10.0 g; 18.47 mmol), prepared according to the method described in Example 7, was suspended in DMSO (200 mL) and heated to 55°C. The mixture was filtered while hot. The hot filtrate was stirred in a clean flask, cooled to 20-25°C, and then stirred for an additional 2 hours. The solid was collected by filtration, washed with DMSO (10 mL), suction dried, and then dried in a vacuum oven at 40-45°C for 14 hours to give compound I-1·DMSO solvate (7.23 g; 63%). ^1H NMR (500 MHz, DMSO-d6) δ10.63(s,1H),9.66(s,1H),9.47(dd,1H),9.24(dd,1H),8.24(d,1H),6.78(s,2H),4.54(t,2H), 4.46(t,2H),3.60(dt,4H),3.43(m,1H),3.18(m,2H),2.97(m,3H), 2.54(s,6H),2.26(dt,4H),2.12(qd,2H),1.73(d,2H); 19F NMR (500 MHz, DMSO-d6) delta -136.1, -152.8.

XRPD of compound I-1 (DMSO solvate)

The XRPD pattern of Compound I-1 DMSO solvate was recorded in reflection mode at room temperature using a PANalytical diffractometer equipped with an Empyrean tube source and a PIXcel 1D detector (PANalytical, The Netherlands). The X-ray generator was operated at 45 kV and 40 mA. The powder sample was placed in a silicon holder. Data were recorded over the 2θ range of 3°–39°, with a step size of 0.013° and a dwell time of 121 s per step. Figure 1g shows the X-ray powder diffraction pattern of the sample, which is typical of a crystalline drug substance.

Table 9 depicts representative XRPD peaks for Compound I-1·DMSO solvate:

Table 9: Representative XRPD peaks

Thermal analysis of compound I-1 (DMSO solvate)

Thermogravimetric analysis of Compound I-1 DMSO solvate was performed using a Discovery TGA (TA Instruments Trios) to determine the percentage weight loss as a function of temperature. A sample (3.26 mg) was added to a pre-weighed aluminum pan and heated from ambient temperature to 350°C at a rate of 10°C/min. The TGA results, shown in Figure 2g, show a significant weight loss of 12.44% between 146°C (onset) and 156°C (endpoint). This weight loss corresponds to approximately 1 molar equivalent of DMSO. A secondary weight loss of 0.52% was then observed between 254°C (onset) and 262°C (endpoint). The subsequent weight loss observed at 304°C is the result of melting/degradation.

Differential Scanning Calorimetry of Compound I-1 (DMSO Solvate)

Differential scanning calorimetry of Compound I-1 DMSO solvate was measured using a TA Instrument DSC Q2000. A sample (1.77 mg) was weighed into a pinhole-sealed aluminum pan and heated from ambient temperature to 350°C at a rate of 10°C/min. The DSC results, shown in Figure 3g, show a desolvation endotherm at 143°C (onset), followed by a single melting endotherm at 258°C (onset).

Example 12: Compound I-1 (DMAC solvate)

Compound I-1·Anhydrous Form A (100 mg; 0.18 mmol) prepared according to the method described in Example 7 was suspended in DMAC (2000 uL) and stirred at 20-25° C. for 20 hours. The solid was collected by filtration, washed with DMAC (500 uL), dried by suction, and then dried in a vacuum oven at 40-50°C to give compound I-1·DMAC solvate (84 mg). ¹ H NMR (500 MHz, DMSO-d ₆ )δ10.62(s,1H),9.66(s,1H),9.46(dd,1H),9.26– 9.22(m,1H),8.24(d,1H),6.77(s,2H),4.54(t,2H),4.46(t,2H),3.66– 3.54(m,4H),3.43(p,1H),3.18(tt,2H),2.94(s,8H),2.78(s,4H),2.26 (dt,4H),2.12(qd,2H),1.96(s,4H),1.76–1.69(m,2H).

XRPD of compound I-1 (DMAC solvate)

The XRPD pattern of Compound I-1·DMAC solvate was recorded in reflection mode at room temperature using a PANalytical diffractometer equipped with an Empyrean tube source and a PIXcel 1D detector (PANalytical, The Netherlands). The X-ray generator was operated at 45 kV and 40 mA. The powder sample was placed in a silicon holder. Data were recorded over the 2θ range of 3°–39°, with a step size of 0.013° and a dwell time of 121 s per step. Figure 1h shows the X-ray powder diffraction pattern of the sample, which is typical of a crystalline drug substance.

Table 10 depicts representative XRPD peaks for Compound I-1·DMAC solvate:

Table 10: Representative XRPD peaks

Thermal Analysis of Compound I-1 (DMAC Solvate)

Thermogravimetric analysis of Compound I-1·DMAC solvate was performed using a Discovery TGA (TA Instruments Trios) to determine the percentage weight loss as a function of temperature. A sample (5.12 mg) was added to a pre-weighed aluminum pan and heated from ambient temperature to 350°C at a rate of 10°C/min. The TGA results, shown in Figure 2h, show a significant weight loss of 17.76% between 85°C (start) and 100°C (end). This weight loss corresponds to approximately 1.3 molar equivalents of DMAC. The subsequent weight loss observed at 306°C is the result of melting/degradation.

Differential Scanning Calorimetry of Compound I-1 (DMAC Solvate)

Differential scanning calorimetry of Compound I-1·DMAC solvate was measured using a TA Instrument DSC Q2000. A sample (1.93 mg) was weighed into a pinhole-sealed aluminum pan and heated from ambient temperature to 350°C at a rate of 10°C/min. The DSC results, shown in Figure 3h, show a desolvation endotherm at 81°C (onset), followed by a single melting endotherm at 261°C (onset).

Example 13: Compound I-1 (acetone solvate)

The amorphous form of compound I-1 (100 mg; 0.18 mmol) prepared according to the method described in step 3 of Example 2 above was suspended in acetone (2000 uL) and stirred for 22 hours. The acetone solvate of compound I-1 was collected by filtration. ¹ HNMR(500MHz,DMSO-d ₆ )δ10.63(s, 1H),9.66(s,1H),9.46(dd,1H),9.24(dd,1H),8.24(d,1H),6.78(s,2H), 4.54(t,2H),4.46(t,2H),3.65–3.54(m,4H),3.43(p,1H),3.19(tt,2H),3.06–2.90(m,3H),2.26(dt,4H),2.18–2.05(m,3H),1.72(d,2H).

XRPD of compound I-1 (acetone solvate)

The XRPD pattern of Compound I-1 acetone solvate was recorded in reflection mode at room temperature using a PANalytical diffractometer equipped with an Empyrean tube source and a PIXcel 1D detector (PANalytical, The Netherlands). The X-ray generator was operated at 45 kV and 40 mA. The powder sample was placed in a silicon holder. Data were recorded over the 2θ range of 3° to 39°, with a step size of 0.013° and a dwell time of 121 s per step. Figure 1i shows the X-ray powder diffraction pattern of the sample, which is typical of a crystalline drug substance.

Table 11 depicts representative XRPD peaks of Compound I-1 · Acetone Solvate:

Table 11: Representative XRPD peaks

Thermal analysis of compound I-1 (acetone solvate)

Thermogravimetric analysis of Compound I-1 acetone solvate was performed using a Discovery TGA (TA Instruments Trios) to determine the percentage weight loss as a function of temperature. A sample (2.45 mg) was added to a pre-weighed aluminum pan and heated from ambient temperature to 350°C at a rate of 10°C/min. The TGA results, shown in Figure 2i, show an initial weight loss of 1.46%. A greater weight loss of 4.55% was then observed between 124°C (start) and 151°C (end), corresponding to approximately 0.44 molar equivalents of acetone. The subsequent weight loss observed at 302°C is the result of melting/degradation.

Differential Scanning Calorimetry of Compound I-1 (Acetone Solvate)

Differential scanning calorimetry of Compound I-1 acetone solvate was measured using a TA Instrument DSC Q2000. A sample (1.42 mg) was weighed into a pinhole-sealed aluminum pan and heated from ambient temperature to 350°C at a rate of 10°C/min. The DSC results shown in Figure 3i show a desolvation endotherm at 136°C (onset), followed by a melting endotherm at 166°C (onset). This was followed by an immediate recrystallization exotherm at 175°C. Another melting endotherm was then recorded at 259°C. This was also followed by a recrystallization exotherm at 261°C. A final melting endotherm was observed at 279°C.

Example 14: Compound I-1 (isopropanol solvate)

Compound I-1 amorphous form (100 mg; 0.18 mmol) prepared according to the method described in Step 3 of Example 2 above was suspended in 2-propanol (2000 uL) and stirred at 20-25°C for 22 hours. Compound I-1 isopropanol solvate was collected by filtration.

XRPD of compound I-1 (isopropanol solvate)

The XRPD pattern of Compound I-1 (isopropanol solvate) was recorded in reflection mode at room temperature using a PANalytical diffractometer equipped with an Empyrean tube source and a PIXcel 1D detector (PANalytical, The Netherlands). The X-ray generator was operated at 45 kV and 40 mA. The powder sample was placed in a silicon holder. Data were recorded over the 2θ range of 3° to 39°, with a step size of 0.013° and a dwell time of 121 s per step. Figure 1j shows the X-ray powder diffraction pattern of the sample, which is typical of a crystalline drug substance.

Table 12 depicts representative XRPD peaks for Compound I-1·Isopropanol Solvate:

Table 12: Representative XRPD peaks

Thermal analysis of compound I-1 (isopropanol solvate)

Thermogravimetric analysis of Compound I-1 Isopropanol Solvate was performed using a Discovery TGA (TA Instruments Trios) to determine the percentage weight loss as a function of temperature. A sample (3.39 mg) was added to a pre-weighed aluminum pan and heated from ambient temperature to 300°C at a rate of 10°C/min. The TGA results, shown in Figure 2j, show a significant weight loss of 3.76% between 136°C (onset) and 180°C (endpoint). This weight loss corresponds to approximately 0.35 molar equivalents of IPA. The subsequent weight loss observed at 278°C is the result of melting/degradation.

Differential Scanning Calorimetry of Compound I-1 (Isopropanol Solvate)

Differential scanning calorimetry of Compound I-1 isopropanol solvate was measured using a TA Instrument DSC Q2000. A sample (1.03 mg) was weighed into a T-zero aluminum pan and heated from ambient temperature to 320°C at a rate of 10°C/min. The DSC results, shown in Figure 3j, show a broad desolvation endotherm at 135°C (onset), followed by a single melting endotherm at 258°C (onset).

Example 15: Cellular ATR inhibition assay :

The ability of compounds to inhibit intracellular ATR can be screened using immunofluorescence microscopy to detect phosphorylation of the ATR substrate histone H2AX in hydroxyurea-treated cells. HT29 cells were plated at 14,000 cells/well in McCoy's 5A medium (Sigma M8403) supplemented with 10% fetal bovine serum (JRH Biosciences 12003), 1:100 diluted penicillin/streptomycin solution (Sigma P7539) and 2 mM L-glutamine (Sigma G7513) in 96-well black imaging plates (BD 353219) and allowed to adhere overnight at 37°C in 5% CO _2. Compounds were then added to the cell culture medium starting at a final concentration of 25 μM in 3-fold serial dilutions, and the cells were incubated at 37°C in 5% CO ₂ . After 15 minutes, hydroxyurea (Sigma H8627) was added to a final concentration of 2 mM.

After treatment with hydroxyurea for 45 minutes, cells were washed in PBS, fixed in 4% formaldehyde (Polysciences 18814) diluted in PBS for 10 minutes, washed with 0.2% Tween-20 in PBS (wash buffer), and permeabilized in 0.5% Triton X-100 in PBS for 10 minutes, all at room temperature. Cells were then washed once in wash buffer and blocked in 10% goat serum (Sigma G9023) diluted in wash buffer (blocking buffer) for 30 minutes at room temperature. To detect H2AX phosphorylation levels, cells were incubated with a primary antibody (mouse monoclonal anti-phospho-histone H2AX Ser139 antibody; Upstate 05-636) diluted 1:250 in blocking buffer for 1 hour at room temperature. The cells were then washed five times in wash buffer and then incubated in a mixture of secondary antibody (goat anti-mouse AlexaFluor 488-conjugated antibody; Invitrogen A11029) and Hoechst dye (Invitrogen H3570) diluted 1:500 and 1:5000 in wash buffer, respectively, for 1 hour at room temperature in the dark. The cells were then washed five times with wash buffer, and finally 100 μl of PBS was added to each well before imaging.

Cells were imaged using a BD Pathway 855 Bioimager and Attovision software (BD Biosciences, version 1.6/855) for Alexa Fluor 488 and Hoechst intensity to quantitatively stain phosphorylated H2AX Ser139 and DNA. BD Image Data Explorer software (BD Biosciences, version 2.2.15) was then used to calculate the percentage of phosphorylated H2AX-positive nuclei in a montage of 9 images at 20 times magnification for each well. Phosphorylated H2AX-positive nuclei were defined as Hoechst-positive areas containing 1.75 times the average Alexa Fluor 488 intensity in cells not treated with hydroxyurea. Finally, the percentage of H2AX-positive nuclei was plotted against the concentration of each compound and the IC50 of intracellular ATR inhibition was determined using Prism software (GraphPad Prism, version 3.0cx for Macintosh, GraphPad Software, San Diego California, USA).

The compounds described herein can also be tested according to other methods known in the art (see Sarkaria et al. , "Inhibition of ATM and ATR Kinase Activities by the Radiosensitizing Agent, Caffeine": Cancer Research, Vol. 59, pp. 4375-5382, 1999; Hickson et al., "Identification and Characterization of a Novel and Specific Inhibitor of the Ataxia-Telangiectasia Mutated Kinase ATM", Cancer Research, Vol. 64, pp. 9152-9159, 2004; Kim et al., "Substrate Specificities and Identification of Putative Substrates of ATM Kinase Family Members", The Journal of Biological Chemistry, vol. 274, no. 53, pp. 37538-37543, 1999; and Chiang et al., “Determination of the catalytic activities of mTOR and other members of the phosphoinositide-3-kinase-related kinase family,” Methods Mol. Biol., vol. 281, pp. 125-141, 2004.

Example 16: ATR inhibition assay:

Compounds can be screened for their ability to inhibit ATR kinase using a radiophosphate incorporation assay. The assay is performed in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl ₂ , and 1 mM DTT. The final substrate concentrations are 10 μM [γ- 33 P] ATP (3 mCi 33 P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK).

The assay was performed at 25°C in the presence of 5 nM full-length ATR. Prepare a stock assay buffer solution containing all the reagents listed above, except for ATP and the test compound of interest. Place 13.5 μL of the stock solution into a 96-well plate, then add 2 μL of a DMSO stock solution containing a serial dilution of the test compound (typically starting with a 3-fold serial dilution at a final concentration of 15 μM) in duplicate (final DMSO concentration of 7%). Preincubate the plate at 25°C for 10 minutes, and initiate the reaction by adding 15 μL of [γ-33P]ATP (final concentration 10 μM).

After 24 hours, the reaction was terminated by adding 30 μL of 0.1M phosphoric acid containing 2 mM ATP. A multiscreen phosphocellulose filter 96-well plate (Millipore, catalog number MAPHN 0B50) was pretreated with 100 μL of 0.2M phosphoric acid, followed by the addition of 45 μL of the assay mixture. The plate was washed with 5 × 200 μL of 0.2M phosphoric acid. After drying, 100 μL of Optiphase 'SuperMix' liquid scintillation cocktail (Perkin Elmer) was added to the wells, followed by scintillation counting (1450 Microbeta liquid scintillation counter, Wallac).

Ki(app) data were calculated from nonlinear regression analysis of the initial ratio data using the Prism software package (GraphPad Prism, Version 3.0cx for Macintosh, GraphPad Software, Inc., San Diego, CA, USA) after removing the average background value of all data points.

In general, the compounds of the present invention are effective in inhibiting ATR. Compounds I-1 and I-3 inhibit ATR with Ki values below 1 μM.

Example 17: Cisplatin Sensitization Assay

A 96-hour cell viability (MTS) assay can be used to screen compounds for their ability to increase the sensitivity of HCT116 colorectal cancer cells to cisplatin. HCT116 cells deficient in ATM signaling in response to cisplatin ( see Kim et al., Oncogene, vol. 21, p. 3864, 2002; see also Takemura et al., JBC, vol. 281, p. 30814, 2006) were plated at 470 cells/well in 96-well polystyrene plates (Costar 3596) in 150 μl of McCoy's 5A medium (Sigma M8403) supplemented with 10% fetal bovine serum (JRH Biosciences 12003), a 1:100 dilution of penicillin/streptomycin solution (Sigma P7539), and 2 mM L-glutamine (Sigma G7513) and allowed to attach overnight at 37°C in 5% _CO2 . Both the compound and cisplatin were then added simultaneously to the cell culture medium as a full set of concentrations in a two-fold serial dilution starting from a maximum final concentration of 10 μM, with a final cell volume of 200 μl, and the cells were incubated at 37° C. in 5% CO _2. After 96 hours, 40 μl of MTS reagent (Promega G358a) was added to each well, and the cells were incubated for 1 hour at 37° C. in 5% CO _2. Finally, the absorbance was measured at 490 nm using a SpectraMax Plus 384 reader (Molecular Devices) and the compound concentration required to reduce the IC50 of cisplatin alone by at least 3-fold (to 1 decimal place) was reported.

In general, the compounds of the present invention are effective in sensitizing cancer cells to cisplatin. The cisplatin sensitization values of compounds I-1 and I-3 are <0.2 μM.

Example 18: Single Agent HCT116 Activity

Compounds can be screened for single-agent activity against HCT116 colorectal cancer cells using a 96-hour cell viability (MTS) assay. HCT116 cells were plated at 470 cells/well in 96-well polystyrene plates (Costar 3596) in 150 μl McCoy's 5A medium (Sigma M8403), supplemented with 10% fetal bovine serum (JRH Biosciences 12003), a 1:100 dilution of penicillin/streptomycin solution (Sigma P7539), and 2 mM L-glutamine (Sigma G7513), and allowed to adhere overnight at 37°C in 5% CO _2. Compounds were then added to the cell culture medium as a full set of concentrations in a 2-fold serial dilution starting from the highest final concentration of 10 μM, with a final cell volume of 200 μl, and the cells were incubated at 37°C in 5% CO ₂ . After 96 hours, 40 μl of MTS reagent (Promega G358a) was added to each well, and the cells were incubated for 1 hour at 37° C. in 5% CO _2. Finally, the absorbance was measured at 490 nm using a SpectraMax Plus 384 reader (Molecular Devices) and the IC50 value was calculated.

Example 19: ATR-complex inhibition assay

Compounds were screened for their ability to inhibit ATR kinase using a radioactive phosphate binding assay in the presence of the chaperone proteins ATRIP, CLK2, and TopBP1. The assay was performed in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM _MgCl₂ , and 1 mM DTT. The final substrate concentrations were 10 μM [g-⁻¹⁺P]ATP (3.5 μCi ⁻¹³P ATP/nmol ATP, PerkinElmer, Massachusetts, USA) and 800 μM target peptide (ASELPASQPQPFSAKKK, Isca Biochemicals, Cambridgeshire, UK).

The assay was performed at 25°C in the presence of 4 nM full-length ATR, 40 nM full-length ATRIP, 40 nM full-length CLK2, and 600 nM TopBP1 (A891-S1105). An enzyme stock buffer solution was prepared containing all the reagents listed above, except for the target peptide, ATP, and the relevant test compound. This enzyme stock solution was preincubated at 25°C for 30 minutes. 8.5 μL of the enzyme stock solution was placed in a 96-well plate, followed by the addition of 5 μL of the target peptide and 2 μL of a DMSO stock solution containing serial dilutions of the test compound (typically starting at a final concentration of 1.5 μM and serially diluted in 2.5-fold increments) in duplicate (final DMSO concentration 7%). The plate was preincubated at 25°C for 10 minutes, and the reaction was initiated by the addition of 15 μL of [g-33P]ATP (final concentration 10 μM).

After 20 hours, the reaction was terminated by adding 30 μL of 0.3 M phosphoric acid containing 2 mM ATP. A 96-well phosphocellulose filter plate (Multiscreen HTS MAPHNOB50, Merck-Millipore, Massachusetts, USA) was pretreated with 100 μL of 0.1 M phosphoric acid, and then 45 μL of the terminated assay mixture was added. The plate was washed 5 times with 200 μL of 0.1 M phosphoric acid. After drying, 50 μL of Optiphase 'SuperMix' liquid scintillation cocktail (Perkin Elmer, Massachusetts, USA) was added to the wells, and scintillation counting was performed (Wallac 1450 Microbeta Liquid Scintillation Counter, Perkin Elmer, Massachusetts, USA).

Ki(app) data were calculated from nonlinear regression analysis of the raw rate data using the Prism software package (GraphPad Prism version 6.0c for Macintosh, GraphPad Software Inc., San Diego, USA) after removing the average background value of all data points.

Although we have described a number of embodiments of the present invention, it will be apparent that our basic examples can be altered to provide other embodiments that utilize the compounds, methods, and processes of the present invention. It will be appreciated, therefore, that the scope of the invention is to be defined by the appended claims rather than by the specific embodiments that have been presented herein by way of example.

Claims (108)

1. Compound of formula I-B: or a pharmaceutically acceptable salt thereof, wherein: Each Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ , Y ⁸ , Y ⁹ , Y ¹⁰ , Y ¹¹ , Y ¹² , Y ¹³ , Y ¹⁴ , Y ¹⁵ , Y ¹⁶ , Y ¹⁷ , Y ¹⁸ and Y ¹⁹ is independently hydrogen or deuterium; provided that at least one of Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ , Y ⁸ , Y ⁹ , Y ¹⁰ , Y ¹¹ , Y ¹² , Y ¹³ , Y ¹⁴ , Y ¹⁵ , Y ¹⁶ , Y ¹⁷ , Y ¹⁸ and Y ¹⁹ is deuterium; Each of X ¹ , X ² , X ⁴ , X ⁵ , X ⁶ , X ⁷ , X ⁸ and X ⁹ is independently selected from ¹² C or ¹³ C; and X ³ is independently selected from - ¹² C(O)- or - ¹³ C(O)-. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein ^Y12 , Y13, ^Y14 , ^Y15 , ^Y16 , ^Y17 , ^Y18 and ^{Y19 are} deuterium, and ^Y1 , ^Y2 , Y3, ^Y4 , ^Y5 , ^Y6 , ^Y7 , ^Y8 , ^Y9 , ^Y10 and ^Y11 are independently selected ^from hydrogen or deuterium. 3. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein ^Y12 , ^{Y13, Y14} , ^Y15 , ^Y16 , ^Y17 , ^Y18 and ^{Y19 are} deuterium, ^and Y1, ^Y2 , Y3, ^Y4 , ^Y5 , ^Y6 , ^Y7 , ^Y8 , ^Y9 , ^Y10 and ^{Y11 are} hydrogen. 4. The compound of claim 3 or a pharmaceutically acceptable salt thereof, wherein: X ¹ , X ² , X ⁴ , X ⁵ , X ⁶ , X ⁷ , X ⁸ and X ⁹ are ¹² C; X ³ is - ¹² C(O)-. 5. The compound of claim 3 or a pharmaceutically acceptable salt thereof, wherein: X ¹ , X ⁴ , X ⁵ , X ⁶ , X ⁷ , X ⁸ and X ⁹ are ¹² C; X ³ is - ¹³ C(O)-; and X ² is ¹³ C. 6. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein ^Y11 , Y12, ^Y13 , ^Y14 , ^Y15 , ^Y16 , ^Y17 , ^Y18 and ^Y19 are deuterium, and ^Y1 , ^Y2 , ^Y3 , ^Y4 , ^Y5 , ^Y6 , ^Y7 , ^Y8 , ^{Y9 and Y10} are independently selected from hydrogen or deuterium. 7. The compound of claim 6, or a pharmaceutically acceptable salt thereof, wherein ^Y11 , Y12, ^Y13 , ^Y14 , ^Y15 , ^Y16 , ^Y17 , ^Y18 and ^Y19 are deuterium, and ^Y1 , ^Y2 , ^Y3 , ^Y4 , ^Y5 , ^Y6 , ^Y7 , ^Y8 , ^{Y9 and Y10} are hydrogen. 8. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein ^Y2 , Y12, ^Y13 , ^Y14 , ^Y15 , ^Y16 , ^Y17 , ^Y18 and ^Y19 are deuterium, and ^Y1 , ^Y3 , ^Y4 , ^Y5 , ^Y6 , ^Y7 , ^Y8 , ^Y9 , ^{Y10 and Y11} are independently selected from hydrogen or deuterium. 9. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein ^Y2 , Y12, ^Y13 , ^Y14 , ^Y15 , ^Y16 , ^Y17 , ^Y18 and ^Y19 are deuterium, and ^Y1 , ^Y3 , ^Y4 , ^Y5 , ^Y6 , ^Y7 , ^Y8 , ^Y9 , ^{Y10 and Y11} are hydrogen. 10. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein ^Y12 , ^Y13 , ^Y18 and ^Y19 are deuterium, ^{and Y1} , ^Y2 , ^Y3 , ^Y4 , ^Y5 , ^Y6 , ^Y7 , Y8, ^Y9 , ^Y10 , ^Y11 , ^Y14 , ^Y15 , ^Y16 and ^Y17 are hydrogen or deuterium. 11. The compound of claim 10, or a pharmaceutically acceptable salt thereof, wherein ^Y12 , ^Y13 , ^Y18 and ^Y19 are deuterium, and ^Y1 , ^Y2 , ^Y3 , ^Y4 , ^Y5 , ^Y6 , ^Y7 , ^Y8 , Y9, ^Y10 , ^Y11 , ^Y14 , ^Y15 , ^Y16 and ^Y17 are ^hydrogen . 12. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein ^{Y3 , Y4} , Y5, ^Y6 , ^Y7 , ^Y8 , ^Y9 , ^Y10 and ^Y11 are deuterium, and ^Y1 , ^Y2 , ^Y12 , ^Y13 , ^Y14 , ^Y15 , ^Y16 , ^Y17 , ^Y18 and ^Y19 are independently selected from deuterium or hydrogen. 13. The compound of claim 12, or a pharmaceutically acceptable salt thereof, wherein ^{Y3 , Y4} , Y5, ^Y6 , ^Y7 , ^Y8 , ^Y9 , ^Y10 and ^Y11 are deuterium, and ^Y1 , ^Y2 , ^Y12 , ^Y13 , ^Y14 , ^Y15 , ^Y16 , ^Y17 , ^Y18 and ^Y19 are hydrogen. 14. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein ^Y2 and ^Y11 are deuterium, and ^Y1 , ^Y3 , ^Y4 , ^Y5 , ^Y6 , ^Y7 , ^Y8 , ^Y9 , ^Y10 , ^Y12 , Y13, ^Y14 , ^Y15 , ^Y16 , ^Y17 , ^Y18 and ^{Y19 are} deuterium or hydrogen. 15. The compound of claim 14, or a pharmaceutically acceptable salt thereof, wherein ^Y2 and ^Y11 are deuterium, and ^Y1 , ^Y3 , ^Y4 , ^Y5 , ^Y6 , ^Y7 , ^Y8 , ^Y9 , ^Y10 , ^Y12 , Y13, ^Y14 , ^Y15 , ^Y16 , ^Y17 , ^Y18 and ^Y19 are ^hydrogen . 16. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein ^Y2 is deuterium, and ^Y1 , ^Y3 , ^Y4 , ^Y5 , ^Y6 , ^Y7 , ^Y8 , ^Y9 , ^Y10 , ^Y11 , ^Y12 , Y13, ^Y14 , ^Y15 , ^Y16 , ^{Y17 , Y18} and ^Y19 are deuterium or hydrogen. 17. The compound of claim 16, or a pharmaceutically acceptable salt thereof, wherein ^Y2 is deuterium, and ^Y1 , ^Y3 , ^Y4 , ^Y5 , ^Y6 , ^Y7 , ^Y8 , ^Y9 , ^Y10 , ^Y11 , ^Y12 , Y13, ^Y14 , ^Y15 , ^Y16 , ^Y17 , ^Y18 , and ^{Y19 are} hydrogen. 18. The compound of claim 17 or a pharmaceutically acceptable salt thereof, wherein: X ¹ , X ² , X ⁴ , X ⁵ , X ⁶ , X ⁷ and X ⁸ are ¹² C; X ³ is - ¹² C(O)-; and X ⁹ is ¹³ C. 19. The compound of claim 17 or a pharmaceutically acceptable salt thereof, wherein: X ¹ , X ² , X ⁸ and X ⁹ are ¹² C; X ³ is - ¹² C(O)-; and X ⁴ , X ⁵ , X ⁶ and X ⁷ are ¹³ C. 20. The compound of claim 17 or a pharmaceutically acceptable salt thereof, wherein: X ² , X ⁴ , X ⁵ , X ⁶ , X ⁷ , X ⁸ and X ⁹ are ¹² C; X ³ is - ¹² C(O)-; and X ¹ is ¹³ C. 21. The compound of claim 17 or a pharmaceutically acceptable salt thereof, wherein: X ² , X ⁴ , X ⁵ , X ⁶ , X ⁷ and X ⁹ are ¹² C; X ³ is - ¹³ C(O)-; and ^X1 and ^X8 are ^13C . 22. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein Y ¹¹ is deuterium, and Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ , Y ⁸ , Y ⁹ , Y ¹⁰ , Y ¹² , Y ¹³ , Y ¹⁴ , Y ¹⁵ , Y ¹⁶ , Y ¹⁷ , Y ¹⁸ and Y ¹⁹ are independently selected from hydrogen or deuterium. 23. The compound of claim 21, or a pharmaceutically acceptable salt thereof, wherein Y ¹¹ is deuterium, and Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ , Y ⁸ , Y ⁹ , Y ¹⁰ , Y ¹² , Y ¹³ , Y ¹⁴ , Y ¹⁵ , Y ¹⁶ , Y ¹⁷ , Y ¹⁸ , and Y ¹⁹ are hydrogen. 24. The compound of claim 22 or a pharmaceutically acceptable salt thereof, wherein: X ¹ , X ⁴ , X ⁵ , X ⁶ , X ⁷ , X ⁸ and X ⁹ are ¹² C; X ³ is - ¹² C(O)-; and X ² is ¹³ C. 25. The compound according to any one of claims 1 to 24 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of: 26. Solid form of the compound of formula I-1: wherein the form is crystalline Compound I-1 Hydrate I, wherein the ratio of Compound I-1 to H ₂ O of the crystalline Compound I-1 Hydrate I is 1:4.5, and is characterized by exhibiting peaks at 6.5, 12.5, 13.7, 18.8, and 26.0 degrees in an X-ray powder diffraction pattern obtained using Cu Kα radiation, expressed as 2-θ ± 0.2. 27. The solid form of claim 26, characterized by a weight loss of 14.56% in the temperature range of 25°C to 100°C. 28. The solid form of claim 26, characterized by an X-ray powder diffraction pattern substantially the same as that shown in Figure 1b. 29. Solid form of the compound of formula I-1: Among these forms is crystalline Compound I-1·Hydrate II, characterized by exhibiting peaks at 10.1, 11.3, 11.9, 20.2, and 25.1 degrees in an X-ray powder diffraction pattern obtained using Cu Kα radiation, expressed as 2-θ ± 0.2. 30. The solid form of claim 29, characterized by having one or more peaks in a ^C13 ssNMR spectrum corresponding to 177.0 ± 0.3 ppm, 158.2 ± 0.3 ppm, 142.9 ± 0.3 ppm, 85.1 ± 0.3 ppm, 58.9 ± 0.3 ppm, and 31.9 ± 0.3 ppm. 31. The solid form of claim 29, characterized by having one or more peaks in the ^F19 ssNMR spectrum corresponding to -138.0 ± 0.3 ppm and -152.7 ± 0.3 ppm. 32. Solid form of the compound of formula I-1: Among them, the form is crystalline Compound I-1·Anhydrous Form A, which is characterized by peaks at 6.1, 12.2, 14.5, 22.3 and 31.8 degrees in an X-ray powder diffraction pattern obtained using Cu Kα radiation, expressed as 2-θ±0.2. 33. The solid form of claim 32, characterized by a weight loss of 0.96% over a temperature range of 25°C to 265°C. 34. The solid form of claim 32, characterized by an X-ray powder diffraction pattern substantially the same as that shown in Figure 1c. 35. The solid form of claim 32, characterized by having one or more peaks in a ^C13 ssNMR spectrum corresponding to 175.9 ± 0.3 ppm, 138.9 ± 0.3 ppm, 74.1 ± 0.3 ppm, 42.8 ± 0.3 ppm, and 31.5 ± 0.3 ppm. 36. The solid form of claim 32, characterized by having one or more peaks in the ^F19 ssNMR spectrum corresponding to -136.8 ± 0.3 ppm and -155.7 ± 0.3 ppm. 37. Solid form of the compound of formula I-1: Among them, the form is crystalline Compound I-1·Anhydrous Form B, which is characterized by peaks at 7.2, 8.3, 12.9, 19.5 and 26.6 degrees in an X-ray powder diffraction pattern obtained using Cu Kα radiation, expressed as 2-θ±0.2. 38. The solid form of claim 37, characterized by a 2.5% weight loss over a temperature range of 25°C to 175°C. 39. The solid form of claim 37, characterized by an X-ray powder diffraction pattern substantially the same as that shown in Figure 1d. 40. The solid form of claim 37, characterized by having one or more peaks in a ^C13 ssNMR spectrum corresponding to 173.4±0.3 ppm, 164.5±0.3 ppm, 133.5±0.3 ppm, 130.8±0.3 ppm, 67.7±0.3 ppm, 45.3±0.3 ppm, and 25.9±0.3 ppm. 41. The solid form of claim 37, characterized by having one or more peaks in an ^F19 ssNMR spectrum corresponding to -138.0 ± 0.3 ppm and -153.5 ± 0.3 ppm. 42. Solid form of the compound of formula I-1: Among these forms is crystalline Compound I-1·Anhydrous Form C, characterized by peaks at 6.8, 13.4, 15.9, 30.9, and 32.9 degrees in an X-ray powder diffraction pattern obtained using Cu Kα radiation, expressed as 2-θ ± 0.2. 43. The solid form of claim 42, characterized by an X-ray powder diffraction pattern substantially the same as that shown in Figure 1e. 44. The solid form of claim 42, characterized by having one or more peaks in a ^C13 ssNMR spectrum corresponding to 175.2±0.3 ppm, 142.5±0.3 ppm, 129.6±0.3 ppm, 73.5±0.3 ppm, 54.0±0.3 ppm, and 46.7±0.3 ppm. 45. The solid form of claim 42, characterized by having one or more peaks in an ^F19 ssNMR spectrum corresponding to -131.2 ± 0.3 ppm and -150.7 ± 0.3 ppm. 46. Solid form of the compound of formula I-1: The form is a crystalline compound I-1·DMSO solvate, wherein the ratio of compound I-1 to DMSO in the crystalline compound I-1·DMSO solvate is 1:1, and is characterized in that in the X-ray powder diffraction pattern obtained using Cu Kα radiation, peaks appear at 8.9, 14.8, 16.5, 18.6, 20.9, 22.2 and 23.4 degrees, expressed as 2-θ±0.2. 47. The solid form of claim 46, characterized by a weight loss of 12.44% over a temperature range of 146°C to 156°C. 48. The solid form of claim 46, characterized by an X-ray powder diffraction pattern substantially the same as that shown in Figure 1g. 49. Solid form of the compound of formula I-1: The form is a crystalline Compound I-1·DMAC solvate, wherein the ratio of Compound I-1 to DMAC in the crystalline Compound I-1·DMAC solvate is 1:1.3, characterized in that in an X-ray powder diffraction pattern obtained using Cu Kα radiation, peaks are exhibited at 6.0, 15.5, 17.7, 18.1, 20.4 and 26.6 degrees, expressed as 2-θ±0.2. 50. The solid form of claim 49, characterized by a weight loss of 17.76% over a temperature range of 85°C to 100°C. 51. The solid form of claim 49, characterized by an X-ray powder diffraction pattern substantially the same as that shown in Figure 1h. 52. Solid form of the compound of formula I-1: The form is a crystalline compound I-1·acetone solvate, wherein the ratio of compound I-1 to acetone in the crystalline compound I-1·acetone solvate is 1:0.44, and is characterized in that in the X-ray powder diffraction pattern obtained using Cu Kα radiation, peaks appear at 8.9, 15.5, 15.8, 16.7, 22.3, 25.7 and 29.0 degrees, expressed as 2-θ±0.2. 53. The solid form of claim 52, characterized by a weight loss of 4.55% over a temperature range of 124°C to 151°C. 54. The solid form of claim 52, characterized by an X-ray powder diffraction pattern substantially the same as that shown in Figure 1i. 55. Solid form of the compound of formula I-1: The form is a crystalline compound I-1·isopropanol solvate, wherein the ratio of compound I-1 to isopropanol in the crystalline compound I-1·isopropanol solvate is 1:0.35, and is characterized in that in the X-ray powder diffraction pattern obtained using Cu Kα radiation, peaks appear at 6.9, 17.1, 17.2, 19.1, 19.6, 23.7, 24.4 and 28.9 degrees, expressed as 2-θ±0.2. 56. The solid form of claim 55, characterized by a weight loss of 3.76% over a temperature range of 136°C to 180°C. 57. The solid form of claim 55, characterized by an X-ray powder diffraction pattern substantially the same as that shown in Figure 1j. 58. A composition comprising: a) Compound I-1 or a pharmaceutically acceptable salt thereof, wherein Compound I-1 is represented by the following structural formula: and b) one or more excipients; c) wherein at least 90% by weight of Compound 1-1 is in the anhydrous Form A of claim 32. 59. The composition of claim 58, wherein the one or more excipients comprise one or more selected from the group consisting of one or more fillers, one or more wetting agents, one or more lubricants, and one or more disintegrants. 60. The composition of claim 59, wherein the one or more excipients comprise one or more fillers. 61. The composition of claim 60, wherein the one or more fillers are present in an amount ranging from 10 wt% to 88 wt%, based on the total weight of the composition. 62. The composition of claim 60, wherein the one or more fillers are selected from the group consisting of mannitol, lactose, sucrose, glucose, maltodextrin, sorbitol, xylitol, powdered cellulose, microcrystalline cellulose, silicified microcrystalline cellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, methylhydroxyethylcellulose, starch, pregelatinized starch, dibasic calcium phosphate, calcium sulfate, and calcium carbonate. 63. The composition of claim 62, wherein the one or more fillers are selected from microcrystalline cellulose and lactose. 64. The composition of claim 58, wherein the one or more excipients comprise one or more disintegrants. 65. The composition of claim 64, wherein the one or more disintegrants are present in an amount ranging from 1 wt% to 15 wt%, based on the total weight of the composition. 66. The composition of claim 65, wherein the one or more disintegrants are selected from the group consisting of croscarmellose sodium, sodium alginate, calcium alginate, alginic acid, starch, pregelatinized starch, sodium starch glycolate, cross-linked polyvidone, cellulose and its derivatives, carboxymethylcellulose calcium, carboxymethylcellulose sodium, soy polysaccharides, guar gum, ion exchange resins, effervescent systems based on food acids and alkaline carbonate components, and sodium bicarbonate. 67. The composition of claim 66, wherein the one or more disintegrants is croscarmellose sodium. 68. The composition of claim 58, wherein the one or more excipients comprise one or more lubricants. 69. The composition of claim 68, wherein the one or more lubricants are present in an amount ranging from 0.1 wt% to 10 wt%, based on the total weight of the composition. 70. The composition of claim 69, wherein the one or more lubricants are selected from the group consisting of talc, fatty acids, stearic acid, magnesium stearate, calcium stearate, sodium stearate, glyceryl monostearate, sodium lauryl sulfate, sodium stearyl fumarate, hydrogenated oils, fatty alcohols, fatty acid esters, glyceryl behenate, mineral oil, vegetable oil, leucine, sodium benzoate, and combinations thereof. 71. The composition of claim 70, wherein the one or more lubricants is sodium stearyl fumarate. 72. The composition of claim 58, comprising: a) Compound I-1 or a pharmaceutically acceptable salt thereof, in an amount ranging from 5 wt % to 50 wt %, based on the total weight of the composition; b) one or more lubricants, present in an amount ranging from 0.1 wt % to 10 wt %, based on the total weight of the composition; c) one or more disintegrants in an amount ranging from 1 wt% to 15 wt%, based on the total weight of the composition; and d) one or more fillers, present in an amount ranging from 10 wt% to 90 wt%, based on the total weight of the composition. 73. The composition of claim 58, comprising: a) Compound I-1 or a pharmaceutically acceptable salt thereof, in an amount of 10 wt %, based on the total weight of the composition; b) lactose monohydrate, in an amount of 28 wt %, based on the total weight of the composition; c) Avicel PH-101 (microcrystalline cellulose), in an amount of 55 wt %, based on the total weight of the composition; d) Ac-Di-Sol (croscarmellose sodium) in an amount of 5 wt %, based on the total weight of the composition; and e) sodium stearyl fumarate, in an amount of 3 wt %, based on the total weight of the composition. 74. The composition of claim 58, wherein substantially all of the weight of Compound 1-1 is in anhydrous Form A. 75. The composition of claim 58, wherein at least 95% by weight of Compound 1-1 is in anhydrous Form A. 76. The composition of claim 75, wherein at least 98% by weight of Compound 1-1 is in anhydrous Form A. 77. The solid form of claim 32, wherein the form is crystalline Compound I-1·Anhydrous Form A having a monoclinic crystal system, a P2 _1/c centrosymmetric space group, and the following unit cell parameters: α＝90° β＝107.22(3)° γ＝90°. 78. A method for preparing Compound I-1·Anhydrous Form A according to claim 32, comprising stirring a suspension containing Compound I-1·ethanol solvate and tetrahydrofuran. 79. A method for preparing Compound I-1· Anhydrous Form A of claim 32, comprising stirring a suspension containing Compound I-1· amorphous form, isopropanol and water. 80. The method of claim 79, wherein the suspension is heated to between 65°C and 80°C. 81. The method of claim 80, wherein the suspension is heated to between 70°C and 75°C. 82. A method for preparing a compound of formula 4a: The following steps are included: a) making a compound of formula 35: wherein R° is a C _1-6 aliphatic group, The reaction is carried out under acidic conditions to generate a compound of formula 36: b) reacting the compound of formula 36 with an electrophilic fluorinating agent to produce a compound of formula 38: c) making the compound of formula 38 and the compound of formula 3: The reaction is carried out under suitable condensation conditions to generate a compound of formula 4a. 83. The method of claim 82, wherein R° is independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, and pentyl. 84. The method of claim 83, wherein R° is independently selected from methyl or ethyl. 85. The method of claim 82, wherein the electrophilic fluorinating agent is 1-(chloromethyl)-4-fluoro-1,4-diazobicyclo[2.2.2]octane bistetrafluoroborate. 86. The method of claim 82, wherein the electrophilic fluorinating agent is fluorine gas. 87. The method of claim 82, wherein suitable condensation conditions comprise reacting the compound of formula 38 with the compound of formula 3 in the presence of a solvent. 88. The method of claim 87, wherein the solvent is selected from DMF or DMSO. 89. Method for preparing the compound of formula I-1: The following steps are included: a) making a compound of formula 6a*: With the compound of formula 27: The reaction is carried out under suitable amide bond forming conditions to generate a compound of formula 28: b) purifying the compound of formula 28 using a suitable palladium chelating agent; c) reacting the compound of formula 28 under suitable deprotection conditions to produce a compound of formula 30 d) reacting a compound of formula 30 with a compound of formula 25: The reaction is carried out under suitable amide bond forming conditions to produce a compound of formula I-1. 90. The method of claim 89, wherein conditions suitable for forming an amide bond comprise reacting a compound of formula 30 with a compound of formula 25 in the presence of an amide coupling partner, an aprotic solvent, and a base. 91. The method of claim 90, wherein the aprotic solvent is independently selected from NMP, DMF, or tetrahydrofuran. 92. The method of claim 91, wherein the aprotic solvent is tetrahydrofuran. 93. The method of claim 90, wherein the base is an aliphatic amine. 94. The method of claim 93, wherein the base is DIPEA. 95. The method of any one of claims 91-94, wherein the amide coupling partner is independently selected from CDI, TBTU, or TCTU. 96. The method of claim 95, wherein the amide coupling partner is TCTU. 97. The method of claim 95, wherein the amide coupling partner is CDI. 98. The method of claim 89, wherein suitable deprotection conditions comprise reacting the compound of formula 28 with an acid in the presence of a solvent. 99. The method of claim 98, wherein the acid is HCl. 100. The method of claim 98, wherein the solvent is 1,4-dioxane. 101. The method of claim 89, wherein conditions suitable for forming an amide bond comprise reacting a compound of Formula 6a* with a compound of Formula 27 in an aprotic solvent under heating. 102. The method of claim 101, wherein the aprotic solvent is independently selected from NMP, pyridine, or DMF. 103. The method of claim 102, wherein the aprotic solvent is pyridine. 104. The method of claim 103, wherein the reaction is carried out at a temperature of at least 80°C. 105. The method of claim 89, wherein the palladium chelating agent is independently selected from the group consisting of propane-1,2-diamine; ethane-1,2-diamine; ethane-1,2-diamine; propane-1,3-diamine; tetramethylethylenediamine; ethylene glycol; 1,3-bis(diphenylphosphino)propane; 1,4-bis(diphenylphosphino)butane; and 1,2-bis(diphenylphosphino)ethane/Pr-1,2-diamine. 106. The method of claim 105, wherein the palladium chelator is propane-1,2-diamine. 107. A method for preparing a compound of formula 28: The following steps are included: a) making a compound of formula 5a The reaction is carried out under suitable halogenation conditions to generate a compound of formula 34: wherein X is a halogen; b) making a compound of formula 34 and a compound of formula 27: The reaction is carried out under suitable amide bond forming conditions to generate a compound of formula 28. 108. A method for preparing a compound of formula I-1: The following steps are included: a) making a compound of formula 5a The reaction is carried out under suitable halogenation conditions to generate a compound of formula 34: wherein X is a halogen; b) making a compound of formula 34 and a compound of formula 27: The reaction is carried out under suitable amide bond forming conditions to generate a compound of formula 28: c) reacting the compound of formula 28 under suitable deprotection conditions to produce a compound of formula 30 d) reacting a compound of formula 30 with a compound of formula 25: The reaction is carried out under suitable amide bond forming conditions to produce a compound of formula I-1.