WO2019167973A1

WO2019167973A1 - Cell cycle progression inhibitor

Info

Publication number: WO2019167973A1
Application number: PCT/JP2019/007414
Authority: WO
Inventors: Motonari Uesugi; Amelie PERRON; Yuzo KODAMA
Original assignee: Kyoto University
Priority date: 2018-03-01
Filing date: 2019-02-27
Publication date: 2019-09-06

Abstract

The present invention provides a cell cycle progression inhibitor, a cytostatic agent, and an anticancer agent. The cell cycle progression inhibitor, the cytostatic agent, and the anticancer agent each contains a Hes1 protein-PHB2 protein binding enhancer.

Description

CELL CYCLE PROGRESSION INHIBITOR

The present disclosure relates to a cell cycle progression inhibitor, and the like.

Cancers are the top causes of death worldwide, and it is known that, among the cancers, pancreatic cancer is difficult to be detected early, progresses quickly, and has bad prognosis. Therefore, more effective anticancer agents have been desired to be developed.

An agent that inhibits cell cycle progression using, as a target, a signal transduction pathway that relates to, for example, cell growth has been reported as an anticancer agent. For example, a certain type of γ secretase inhibitor causes the stop of cell cycle in G0/G1 phase and accordingly exerts an anticancer effect.

It is known that Hes1 protein is a transcription factor positioned on the downstream side of Notch signal transduction pathway and is involved in, for instance, self biogenesis of pancreas precursor cell.

Figure 1A shows procedure for the selection and validation of compounds from the chemical library. HEK293 cells were transfected with the indicated reporter genes expressed under the control of Hes1 promoter, together with Hes1 under the control of a constitutive promoter (pCMV-Hes1) to repress transcription. The compounds were screened according to their ability to inhibit Hes1-mediated transcriptional repression. Figure 1B shows effect of validated compounds (2.5 μM) on Hes1-mediated repression of luciferase gene expression. Data are displayed as mean ±SD of triplicates. Two-tailed Student’s t-test was used for statistical analysis (*p<0.01 and **p<0.001, compared to DMSO control). Figure 1C shows effect of validated compounds (10 μM) on Hes1-mediated repression of EGFPd2 gene expression. Data are mean ± SD from a minimum of 9 individual fields representing at least 366 cells per condition. Figure 1D shows chemical structures of validated compounds. Figure 2A: HEK293 cells were incubated with 2.5 μM D8C prior to cell proliferation analysis with the tetrazolium salt WST-8. Data are normalized to DMSO treatment and displayed as mean ±SD. Figure 2B shows quantification of viable cells by Trypan Blue exclusion upon incubation with 2.5 μM D8C or DMSO for the indicated periods of time (mean ± SD). Figure 2C shows dose-response curve of the effect of D8C derivatives on cell proliferation following a 24-h incubation. Figure 2D shows chemical structures of D8C derivatives and corresponding EC50 values (n/a = not applicable). Figure 3A: HEK293 cells expressing the Fucci fluorescent probes mCherry-hCdt1 (30/120) (red) and AmCyan-hGeminin (green) were treated with 1 μM JI051 for 24 h prior to confocal microscopy imaging in live cells. An increase in Geminin/Cdt1 ratio indicates an increase in the cell population in G2/M phase of the cell cycle. Nocodazole (1000 ng/ml) was used as a positive control. Data are mean ± SD from triplicates (9 fields each) representing a minimum of 762 cells per condition. Scale bar, 50 μm. Figure 3B shows calculation of the mitotic index (% of cells with condensed chromatin) following a 24-h incubation with 1 μM JI051 or DMSO. Nuclei were stained with Hoechst 33342. Arrowheads indicate condensed chromatin. Data represent mean ± SD from a minimum of 22 individual fields representing at least 373 cells per condition. Scale bar, 10 μm. Figure 4A shows target identification by microsequencing analysis. Silver staining of proteins isolated by streptavidin beads upon incubation with 10 μM of a biotinylated version of JI051 (JITV14) before or after displacement with 10-fold excess of JI130, a JI051 derivative with improved solubility. DMSO and JIN05, a biotinylated derivative without the JI051 moiety, were used as negative controls. Bands indicated by arrows were analyzed by microsequencing and corresponding proteins are shown. Figure 4B shows validation of PHB2 as a target for JI051 by pulldown with a photoreactive probe. Flag-PHB2-expressing HEK293 cells were incubated with NeutrAvidin beads together with 20 μM JIN04 (negative control), JITV14 (no photoreactive moiety) or JITV10, with or without a 2.5-fold excess of JI051, prior to western blot with anti-Flag antibody. Input represents the supernatant fraction of cell lysates. Figure 4C shows KD determination of JITV10. Human recombinant PHB2 protein was incubated with JITV10 prior to UV exposure and western blot with HRP-conjugated streptavidin or PHB2 antibody. The densitometry analysis of HRP-streptavidin signal was normalized to PHB2 (data represent mean from 2 independent experiments). Figure 5A: Lysates from HEK293 cells expressing Flag-PHB2 alone or together with Hes1 were incubated with JI051 at the indicated concentrations together with anti-Flag antibody, prior to pulldown with protein A-Sepharose beads. Western blot analysis was carried out with either Hes1 or PHB2 antibodies. Blots are representative of 3 individual experiments. Figure 5B shows Pearson correlation coefficient analysis showing the colocalization of Hes1 and PHB2 following a 24-h incubation with 1 μM JI051 or DMSO prior to dual immunocytochemistry with Hes1 and PHB2 antibodies. Data are mean ± SD from a minimum of 22 individual fields representing at least 399 cells per condition. Scale bar, 25 μm. Figure 6A: Cells were treated for the indicated periods of time with 1 μM JI130 or DMSO prior to cell proliferation analysis with the tetrazolium salt MTS. Data represent mean ± SD. Figure 6B shows dose-response curve of the effect of JI130 and the chemotherapy drug Gemcitabine on cell proliferation following a 72-h incubation. Data are expressed as mean ± SD. Figure 6C shows chemical structure of JI130 and corresponding EC50 value. Figure 6D shows effect of a 24-h incubation with JI130 (100 nM and 1 μM) or DMSO on nucleus appearance. Nuclei were stained with Hoechst 33342. Arrowheads indicate cells with micronuclei. DIC, differential interference contrast. Scale bar, 25 μm. Figure 7A: MIA PaCa-2 cells were implanted in 28-day-old nude mice. Mice were treated with JI130 at a concentration of 50 mg/kg of body weight 5 days/week for a total of 3 weeks. Figure 7B shows tumor volume of the mice treated with JI130 or DMSO. Data are mean ± SEM from 7 mice in each group. Two-tailed Student’s t-test was used for statistical analysis (*p<0.01 and **p<0.002 compared to DMSO). Figure 7C shows boxed plot illustrating the tumor weight following treatment with JI130 or DMSO. Data are mean ± SD from 7 mice in each group. Figure 7D shows representative pictures of mice treated with JI130 or DMSO. Figure 7E shows transition of body weight of the mice treated with JI130 or DMSO. Data are mean ± SD from 7 mice in each group.

The present invention is intended to provide a cell cycle progression inhibitor, a cytostatic agent, and an anticancer agent.

The inventors of the present invention conducted earnest studies based on the problem and found a novel binding partner (PHB2 protein) for Hes1 protein. They also found that the Hes1 protein-PHB2 protein binding enhancer has a cell cycle progression effect, a cytostatic effect, and an anticancer effect. They also found that a compound having a specific structure that has an indole ring has a Hes1 protein-PHB2 protein binding enhancing effect and also has a cell cycle progression effect, a cytostatic effect, and an anticancer effect.
The present invention can provide a cell cycle progression inhibitor, a cytostatic agent, and an anticancer agent.
The present disclosure includes the following embodiments.

(Item 1)
A cell cycle progression inhibitor comprising an Hes1 protein-PHB2 protein binding enhancer.
(Item 1A)
An Hes1 protein-PHB2 protein binding enhancer for use in the inhibition of cell cycle progression.
(Item 1B1)
A method for inhibiting cell cycle progression, comprising adding an Hes1 protein-PHB2 protein binding enhancer to a cell.
(Item 1B2)
A method for inhibiting cell cycle progression, comprising administering an Hes1 protein-PHB2 protein binding enhancer to a patient in need thereof.
(Item 1C)
Use of an Hes1 protein-PHB2 protein binding enhancer for the production of a cell cycle progression inhibitor.

(Item 2)
The cell cycle progression inhibitor according to item 1, which induces G2/M phase cell cycle arrest.

(Item 3)
The cell cycle progression inhibitor according to item 1 or 2, wherein the binding enhancer is at least one member selected from the group consisting of a compound represented by Formula (1):

wherein R¹¹ and R¹² are identical or different, and each represents -(O)_n-R¹⁰ wherein R¹⁰ represents alkyl, alkenyl, or alkynyl, and n is 0 or 1,
m is 0 or an integer of 1 to 3,
R² represents a single bond or a linker,
R¹¹ and R², taken together with the carbon atoms to which they are attached, may form a ring,
R³ represents -NH-CO- or -CO-NH-,
R⁴ represents a single bond or a linker, and
R⁵ represents substituted or unsubstituted indolyl, and
salt, hydrate, and solvate thereof.

(Item 4)
The cell cycle progression inhibitor according to item 3, wherein the compound is a compound represented by Formula (1A):

wherein R¹¹, R¹², m, R², and R⁴ are as defined above;
R⁵¹, R⁵², R⁵³, R⁵⁴, and R⁵⁵ are identical or different, and each represents hydrogen, hydroxyl, halogen, or -(O)_p-R^50a wherein R^50a represents substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, or substituted or unsubstituted aryl, and p is 0 or 1; and
R⁵⁶ represents hydrogen or -(R^50b)_q-R^50c wherein R^50b represents -C(=O)-, -C(=O)-O-, -S(O)-, or -S(O)₂-, and R^50c represents substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, or substituted or unsubstituted aryl, and q is 0 or 1.

(Item 5)
A cytostatic agent (A cell proliferation inhibitor) comprising an Hes1 protein-PHB2 protein binding enhancer.
(Item 5A)
An Hes1 protein-PHB2 protein binding enhancer for use in the inhibition of cell proliferation.
(Item 5B1)
A method for inhibiting cell proliferation, comprising adding an Hes1 protein-PHB2 protein binding enhancer to a cell.
(Item 5B2)
A method for inhibiting cell proliferation, comprising administering an Hes1 protein-PHB2 protein binding enhancer to a patient in need thereof.
(Item 5C)
Use of an Hes1 protein-PHB2 protein binding enhancer for the production of a cytostatic agent (a cell proliferation inhibitor).

(Item 6)
An anticancer agent (An agent for preventing or treating cancer) comprising an Hes1 protein-PHB2 protein binding enhancer.
(Item 6A)
An Hes1 protein-PHB2 protein binding enhancer for use in the preventing or treating cancer.
(Item 6B)
A method for preventing or treating cancer, comprising administering an Hes1 protein-PHB2 protein binding enhancer to a patient in need thereof.
(Item 6C)
Use of an Hes1 protein-PHB2 protein binding enhancer for the production of an anticancer agent (an agent for preventing or treating cancer).

(Item 7)
A compound represented by Formula (1):

wherein R¹¹ and R¹² are identical or different, and each represents -(O)_n-R¹⁰ wherein R¹⁰ represents alkyl, alkenyl, or alkynyl, and n is 0 or 1,
m is 0 or an integer of 1 to 3,
R² represents a single bond or a linker,
R¹¹ and R², taken together with the carbon atoms to which they are attached, may form a ring,
R³ represents -NH-CO- or -CO-NH-,
R⁴ represents a single bond or a linker, and
R⁵ represents substituted or unsubstituted indolyl.

1. Definition
In the present specification, the expressions “containing” and “including” include the concepts of “containing”, “including”, “being substantially composed of”, and “consisting of”.
The “identity” of the amino acid sequence means the extent of agreement between at least two comparable amino acid sequences. Thus, the higher the agreement between two amino acid sequences is, the higher the identity or the similarity between them is. The level of the identity of the amino acid sequence can be determined using, for example, FASTA that is a sequence analysis tool and the default parameter or can be determined using the Karlin-Altschul algorithm, BLAST (KarlinS, Altschul SF. “Methods for assessing the statistical significance of molecular sequence features by using general scoringschemes” Proc Natl Acad Sci USA. 87:2264-2268 (1990), KarlinS, Altschul SF. “Applications and statistics for multiple high-scoring segments in molecular sequences.” Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX based on the algorithm of such BLAST has been developed. Specific techniques of these analysis methods are known, and the website of National Center of Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) can be referred to. The “identity” of the base sequence is defined in accordance with the description above.

In the present specification, the “conservative substitution” means substitution of an amino-acid residue by an amino-acid residue having a similar side chain. For example, substitution between amino-acid residues each having a basic side chain such as lysine, arginine, or histidine is the conservative substitution. In addition, substitution between amino-acid residues each having an acidic side chain such as aspartic acid or glutamic acid; substitution between amino-acid residues each having an uncharged polar side chain such as glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine; substitution between amino-acid residues each having a non-polar side chain such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan; substitution between amino-acid residues each having a β-branched side chain such as threonine, valine, or isoleucine; and substitution between amino-acid residues each having an aromatic side chain such as tyrosine, phenylalanine, tryptophan, or histidine are also conservative substitutions.

2. Cell cycle progression inhibitor, Cytostatic agent, Anticancer agent
An embodiment of the present disclosure relates to a cell cycle progression inhibitor, a cytostatic agent, an anticancer agent, and the like, each containing a Hes1 protein-PHB2 protein binding enhancer (hereinafter also merely referred to as “the binding enhancer”) (also referred to as “the agent of the present disclosure” in the present specification). The following describes these.

Hes1 protein is required to be expressed in biological species (for example, various mammals such as human, monkeys, mice, rats, dogs, cats, rabbits, swine, horses, cattle, sheep, goats, and deer) of applicable objects of the agent of the present disclosure and is not limited to particular proteins.

Amino acid sequences of Hes1 proteins derived from various biological species are known. Specifically, human Hes1 protein can be, for example, a protein including an amino acid sequence represented by SEQ ID NO: 1 (NCBI Reference Sequence: NP_005515), and mouse Hes1 protein can be, for example, a protein including an amino acid sequence represented by SEQ ID NO: 2 (NCBI Reference Sequence: XP_006521860). Moreover, Hes1 protein may be a protein obtained by deleting a signal peptide from Hes1 protein.

Hes1 protein is required to have original activity, transcriptional regulation activity, and binding activity to PHB2 protein and may have an amino acid mutation such as substitution, deletion, addition, or insertion. The mutation is preferably substitution, more preferably conservative substitution, from the viewpoint of being difficult to impair the activity.

As a specific example, preferred Hes1 protein to be subjected to inhibition can be at least one selected from the group consisting of the following proteins (a) and (b):
(a) a protein including an amino acid sequence represented by SEQ ID NO: 1 or 2; and
(b) a protein including an amino acid sequence having an identity of at least 85% to the amino acid sequence represented by SEQ ID NO: 1 or 2 and having transcription activity and binding activity to PHB2 protein.

In the (b) above, the identity is more preferably at least 90%, yet more preferably at least 95%, yet more preferably at least 98%.

PHB2 protein is required to be expressed in biological species (for example, various mammals such as human, monkeys, mice, rats, dogs, cats, rabbits, swine, horses, cattle, sheep, goats, and deer) of applicable objects of the agent of the present disclosure and is not limited to particular proteins.

Amino acid sequences of PHB2 proteins derived from various biological species are known. Specifically, human PHB2 protein can be, for example, a protein including an amino acid sequence represented by SEQ ID NO: 3 (NCBI Reference Sequence: NP_001138303), and mouse PHB2 protein can be, for example, a protein including an amino acid sequence represented by SEQ ID NO: 4 (NCBI Reference Sequence: NP_031557). Moreover, PHB2 protein may be a protein obtained by deleting a signal peptide from PHB2 protein.

PHB2 protein is required to have original activity, transcriptional regulation activity, and binding activity to PHB2 protein and may have an amino acid mutation such as substitution, deletion, addition, or insertion. The mutation is preferably substitution, more preferably conservative substitution, from the viewpoint of being difficult to impair the activity.

As a specific example, preferred PHB2 protein to be subjected to inhibition can be at least one selected from the group consisting of the following proteins (c) and (d):
(c) a protein including an amino acid sequence represented by SEQ ID NO: 3 or 4; and
(d) a protein including an amino acid sequence having an identity of at least 85% to the amino acid sequence represented by SEQ ID NO: 3 or 4 and having transcription activity and binding activity to Hes1 protein.

In the protein (d), the identity is more preferably at least 90%, yet more preferably at least 95%, yet more preferably at least 98%.

The binding enhancer is required to be a substance that enhances Hes1 protein-PHB2 protein binding and is not limited to particular substances and includes a substance that enhances Hes1 protein-PHB2 protein association, a substance that stabilizes Hes1 protein-PHB2 protein binding, and a substance that inhibits Hes1 protein-PHB2 protein dissociation. Examples of the binding enhancer include low-molecular-weight compounds, antibodies, antigens, receptors, ligands, aptamers, nucleic acids, sugars, lipids, and complex substances thereof. More specifically, examples of the binding enhancer include low-molecular-weight compounds described in the following section “3. Low-molecular-weight compound”.

The binding enhancer has, for example, a cell cycle progression inhibiting effect, a cytostatic effect, and an anticancer effect. The binding enhancer can be used as an active ingredient of a cell cycle progression inhibitor, a cytostatic agent, and an anticancer agent. The use field of these is not limited to particular fields, and these can be used as pharmaceuticals, reagents, food compositions (including health food and supplements) cosmetics, and oral compositions.

The agent of the present disclosure is required to contain the binding enhancer, is not limited to particular agents, and may contain another component. Another component is required to be a pharmaceutically acceptable component and is not limited to particular components, and examples thereof include bases, carriers, solvents, dispersants, emulsifiers, buffer agents, stabilizing agents, excipients, binders, disintegrants, lubricants, thickeners, humectants, coloring agents, flavors, and chelating agents.

The mode of use of the agent of the present disclosure is not limited to particular modes, and an appropriate mode of use can be employed according to the type. The agent of the present disclosure can be used, for example, in vitro (for example, by adding a medium containing cultured cells) or in vivo (for example, by administering animals).

An applicable object of the agent of the present disclosure is not limited to particular objects, and examples thereof include various mammals such as human, monkeys, mice, rats, dogs, cats, rabbits, swine, horses, cattle, sheep, goats, and deer; and animal cells. The kinds of the animal cells are not limited to particular cells, and examples thereof include blood cells, hematopoietic stem cells/progenitor cells, gamete (sperms, ova), fibroblast, epithelial cells, vascular endothelial cells, neuron, liver cells, keratinocyte, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, and cancer cells.

In the use of the agent of the present disclosure as an anticancer agent, the kind of the cancer cell is not limited to particular cancer cells, and examples thereof include a pancreatic cancer cell, a renal cancer cell, a leukemia cell, an esophageal cancer, a stomach cancer cell, a large bowel cancer cell, a liver cancer cell, a lung cancer cell, a prostate cancer cell, a skin cancer cell, a breast cancer cell, and a cervical cancer cell.

The dosage form of the agent of the present disclosure is not limited to particular dosage forms, and an appropriate dosage form can be employed according to the use form. In administration of the agent of the present disclosure into animals, examples of the dosage form include oral agents such as a tablet, a capsule, a granule, a powder, a fine granule, a syrup, an enteric agent, a sustained release capsule, a chewable tablet, a drop, a pill, a liquid for internal use, a lozenge, a sustained release agent, and a sustained release granule; and topical agents such as a nasal drop, an inhalant, a rectal suppository, an intercalating agent, an enema, and a jelly. In addition, the agent of the present disclosure may be any of a solid preparation, a semisolid preparation, or a liquid.

The content of the binding enhancer in the agent of the present disclosure depends on, for example, the mode of use, an applicable object, and the state of the applicable object and is not limited and can be, for example, 0.0001% to 100% by weight, preferably 0.001% to 50% by weight.

When the agent of the present disclosure is applied to a biological body, the applicable (for example, administration, intake, inoculum) amount thereof is not limited as long as it is an effective amount at which a desired effect can be exerted and is, in general, 0.1 to 1000 mg/kg per day as an amount of the active ingredient. As to the administration amount, the agent of the present disclosure is preferably administered one or two to three times a day, and the administration amount can be increased or decreased, as appropriate, according to age, condition, and symptom.

3. Low-molecular-weight compound
An embodiment of the present disclosure relates to a compound (also referred to as “the compound of the present disclosure” in the present specification) represented by the Formula (1):

and a salt, a hydrate, and a solvate thereof. These have an action of enhancing binding between Hes1 protein and PHB2 protein and can be favorably used as active ingredients of the cell cycle progression inhibitor, the cytostatic agent, the anticancer agent, and the like. The following describes these.

<3-1 R¹¹, R¹²>
R¹¹ and R¹² are identical or different, and each represents -(O)_n-R¹⁰ wherein R¹⁰ represents alkyl, alkenyl, or alkynyl, and n is 0 or 1. R¹⁰ is preferably alkyl or alkenyl. n is preferably 1.

The alkyl represented by R¹⁰ is straight-chain, branched, or ring-shaped (preferably straight-chain or branched, more preferably straight-chain) alkyl. The carbon number in the alkyl is not limited to particular numbers and is, for example, 1 to 6, preferably 2 to 4, more preferably 2 to 3, yet more preferably 2. Specific examples of the alkyl include methyl, ethyl, n-propyl group, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, n-hexyl, and 3-methylpentyl.

The alkenyl represented by R¹⁰ is straight-chain or branched (preferably straight-chain) alkenyl. The carbon number in the alkenyl is not limited to particular numbers and is, for example 2 to 6, preferably 2 to 4, more preferably 2 to 3, yet more preferably 3. Specific examples of the alkenyl include vinyl, allyl, 1-propenyl, isopropenyl, butenyl, pentenyl, hexenyl.

The alkynyl represented by R¹⁰ is straight-chain or branched (preferably straight-chain) alkynyl. The carbon number in the alkynyl is not limited to particular numbers and is, for example 2 to 6, preferably 2 to 4, more preferably 2 to 3, yet more preferably 3. Specific examples of the alkynyl include ethynyl, propynyl (for example, 1-propynyl, 2-propynyl (propargyl)), butynyl, and pentynyl, hexynyl.

m is 0 or an integer of 1 to 3. m is preferably 0 or 1, more preferably 0.

<3-2 R²>
R² represents a single bond or a linker. R² is preferably a linker.
The linker represented by R² is not limited to particular linkers, and examples thereof include alkylene, alkenylene, alkylene, or a group obtained by substituting one or more (preferably 1 to 2, more preferably 1) carbon atoms on a main chain of alkenylene with hetero atoms (for example, oxygen atoms, nitrogen atoms, or sulfur atoms, preferably oxygen atoms or nitrogen atoms). The linker is preferably an alkylene.

The alkylene is straight-chain, branched, or ring-shaped (preferably straight chain or branched, more preferably straight-chain) alkylene. The carbon number in the alkylene is not limited to particular numbers and is, for example, 1 to 6, preferably 1 to 4, more preferably 2 to 3, yet more preferably 2. Specific examples of the alkylene include methylene, ethylene, n-propylene, isopropylene, cycloprolylene, n-butylene, and isobutylene.

The alkenylene is straight-chain or branched (preferably straight-chain) alkenylene. The carbon number in the alkenylene is not limited to particular numbers and is, for example, 1 to 6, preferably 1 to 4, more preferably 2 to 3, yet more preferably 2. Specific examples of the alkenylene include vinylene, 1-propenylene, 2-propenylene, isopropenylene, butenylene, pentenylene, and hexenylene.

R¹¹ and R², taken together with the carbon atoms to which they are attached, may form a ring. The ring is preferably a single ring. The formed ring and the benzene ring to which R¹¹ and R² are linked form a condensed ring. The condensed ring can be, specifically, a condensed ring in a structure of the compound JI010 or the compound JI094 in the examples (table x) described below. In one embodiment of the present disclosure, R¹¹ and R² preferably do not form a ring.

<3-3 R³>
R³ represents -NH-CO- or -CO-NH-. When R³ is -NH-CO-, the nitrogen atom in R³ links to R², and when R³ is -CO-NH-, the nitrogen atom in R³ links to R⁴. R³ is preferably -NH-CO-.

<3-4 R⁴>
R⁴ represents a single bond or a linker. R⁴ is preferably a linker.
The linker represented by R⁴ is not particularly limited, and examples thereof include alkylene, alkenylene, and a group formed by substituting one or more (preferably 1 to 2, more preferably 1) carbon atoms on a main chain of the alkylene or the alkenylene with hetero atoms (for example, oxygen atoms, nitrogen atoms, or sulfur atoms, preferably oxygen atoms or nitrogen atoms). The linker is preferably alkenylene.
The alkylene and the alkenylene are the same as those for R².

<3-5 R⁵>
R⁵ represents substituted or unsubstituted indolyl.

The indolyl represented by R⁵ is not limited to particular indolyls, and examples thereof include 1-indolyl, 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, and 7-indolyl, and the indolyl is preferably 3-indolyl.

The substituent in the substituted indolyl group represented by R⁵ is not limited to particular substituents, and examples thereof include hydroxyl, a halogen atom, -(O)_p-R^50a, and -(R^50b)_q-R^50c. The number of substituents is not limited to particular numbers and is, for example, 0 to 3, preferably 0 to 1.

Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, and the halogen atom is preferably a bromine atom.
p represents 0 or 1 and is preferably 1.

R^50a represents, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, or substituted or unsubstituted aryl. R^50a is preferably substituted or unsubstituted alkyl.

The substituted or unsubstituted alkyl group represented by R^50a cab be straight-chain, branched, or ring-shaped (preferably straight-chain or branched, more preferably straight-chain) alkyl that is substituted or unsubstituted with a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, or the like), hydroxyl, or a lower alkyl and has a carbon number of 1 to 12, preferably 1 to 8, more preferably 1 to 4, yet more preferably 1 to 2, yet more preferably 1. The number of the substituents is not limited to particular numbers and is, for example, 0 to 3, preferably 0. Examples of such substituted or unsubstituted alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, perfluoromethyl, and perfluoroethyl.

The substituted or unsubstituted alkenyl represented by R^50a cab be straight-chain or branched (preferably straight-chain) alkenyl that is substituted or unsubstituted with a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, or the like), hydroxyl, or lower alkyl and has a carbon number of 2 to 12, preferably 2 to 8, more preferably 2 to 4, yet more preferably 2. The number of the substituents is not limited to particular numbers and is, for example, 0 to 3, preferably 0. Examples of such substituted or unsubstituted alkenyl include vinyl, allyl, 1-propenyl, isopropenyl, butenyl, pentenyl, and hexenyl.

The substituted or unsubstituted alkynyl represented by R^50a can be straight-chain or branched (preferably straight-chain) alkynyl that is substituted with a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, or the like), or hydroxyl, lower alkyl and has a carbon number of 2 to 12, preferably 2 to 8, more preferably 2 to 4, yet more preferably 2. The number of substituents is not limited to particular numbers and is, for example, 0 to 3, preferably 0. Examples of such substituted or unsubstituted alkynyl include ethynyl, propynyl (for example, 1-propynyl, 2-propynyl (propargyl)), butynyl, pentynyl, and hexynyl.

The substituted or unsubstituted aryl represented by R^50a can be aryl that is substituted or unsubstituted a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, or the like), hydroxyl, or lower alkyl and has a carbon number of 6 to 12, preferably 6 to 8, more preferably 6. The number of the substituents is not limited to particular numbers and is, for example, 0 to 3, preferably 0. Examples of such substituted or unsubstituted aryl include phenyl, naphthyl, biphenyl, pentalenyl, indenyl, anthranyl, tetracenyl, pentacenyl, pyrenyl, perylenyl, fluorenyl, and phenanthryl.
R^50b represents -C(=O)-, -C(=O)-O-, -S(O)-, or -S(O)₂-.
q represents 0 or 1.
R^50c represents substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, or substituted or unsubstituted aryl. These are the same as those defined for R^50a.

<3-6 Preferred compound represented by Formula (1)>
In an embodiment of the present disclosure, the compound represented by Formula (1) is preferably a compound represented by Formula (1A):

wherein R¹¹, R¹², m, R², and R⁴ are as defined above;
R⁵¹, R⁵², R⁵³, R⁵⁴, and R⁵⁵ are identical or different, and each represents hydrogen, hydroxyl, halogen, or -(O)_p-R^50a wherein R^50a represents substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, or substituted or unsubstituted aryl, and p is 0 or 1; and
R⁵⁶ represents hydrogen or -(R^50b)_q-R^50c wherein R^50b represents -C(=O)-, -C(=O)-O-, -S(O)-, or -S(O)₂-, and R^50c represents substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, or substituted or unsubstituted aryl, and q is 0 or 1, more preferably a compound represented by Formula (1B):

wherein R¹¹, R¹², m, R², R⁴, R⁵¹, R⁵², and R⁵⁶ are as defined above, yet more preferably a compound represented by Formula (1C):

wherein R¹¹, R¹², m, R², R⁴, and R⁵¹ are as defined above.
In Formula (1A), it is preferred that one of R⁵¹, R⁵², R⁵³, R⁵⁴, R⁵⁵, and R⁵⁶ is a group other than a hydrogen atom, and the others are hydrogen atoms.
In Formula (1B), it is preferred that one of R⁵¹, R⁵², and R⁵⁶ is a group other than a hydrogen atom, and the others are hydrogen atoms.

<3-7 Isomer, Salt, Hydrate, Solvate>
The compound represented by Formula (1) includes a stereoisomer and an optical isomer thereof.

A salt of the compound represented by Formula (1) is not limited to particular salts. As the salt, either one of an acidic salt or a basic salt can be employed. Examples of the acidic salt include inorganic acid salts such as hydrochlorides, hydrobromides, sulfates, nitrates, phosphates; and organic acid salts such as acetates, propionates, tartrate, fumarates, maleates, malates, citrates, methanesulfonates, and p-toluenesulfonates. Examples of the basic salt include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts; and organic amine salts such as morpholine, piperidine, pyrrolidine, monoalkylamine, dialkylamine, trialkylamine, mono(hydroxyalkyl)amine, di(hydroxyalkyl)amine, and tri(hydroxyalkyl)amine.

Examples of the solvent that forms the solvate of the compound represented by Formula (1) include water and organic solvents (for example, ethanol, glycerol, acetic acid).

<3-8 Production method>
The compound represented by Formula (1) can be synthesized by various methods. The compound represented by Formula (1) can be synthesized according to, for example, the following reaction formula:

wherein R¹¹, R¹², m, R², R⁴, and R⁵ are as defined above;
R^3A and R^3B are different from each other and each represents amino or carboxy.
In the present reaction, the compound represented by Formula (1a) and the compound represented by Formula (1b) are caused to react with each other to obtain the compound represented by Formula (1).

In general, the amount of the compound represented by Formula (1b) to be used is, from the viewpoint of yield, preferably 0.3 to 3 mole, more preferably 0.5 to 2 mole, yet more preferably 0.8 to 1.2 mole, relative to 1 mole of the compound represented by Formula (1a).

The present reaction is performed preferably in the presence of a condensation agent. The condensation agent is not limited to particular agents, and any of known condensation agents can be used widely, for example. Specific examples of the condensation agent include 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N,N’-dicyclohexylcarbodiimide (DCC), and the condensation agent is preferably EDC. These condensation agents may be used alone or in a combination of two or more of them.

The amount of the condensation agent to be used is, for example, preferably 0.5 to 3 mole, more preferably 1 to 2 mole, relative to 1 mole of the compound represented by Formula (1a), although it depends on the kind of the condensation agent.

The present reaction is performed preferably in the presence of a base. The base is not limited to particular bases, and any of known bases can be used widely, for example. Specific examples of the base include 4-dimethylamino pyridine (DMAP) and diisopropyl ethyl amine (DIPEA), and the base is preferably DMAP. These bases may be used alone or in a combination of two or more of them.

The amount of the base to be used is, for example, preferably 0.5 to 3 moles, more preferably 1 to 2 mole, relative to 1 mole of the compound represented by Formula (1a), although it depends on the kind of the base.

In addition, the present reaction is performed preferably in the presence of various additives such as 1-hydroxybenzotriazole.

In general, the present reaction is performed in the presence of a reaction solvent. The reaction solvent is not limited to particular solvents, and examples thereof include dichloromethane, N,N-dimethylformamide, acetonitrile, tetrahydrofuran, acetone, and toluene, and the reaction solvent is preferably dichloromethane. These solvents may be used alone or in a combination of multiple of them.

As to reaction temperature, the present reaction can be performed while heating, at normal temperature, or while cooling, and in general, the present reaction is performed preferably at 0°C to 50°C (specifically 10°C to 30°C). The reaction time is not limited to particular times and can be, in general, 8 to 48 hours, specifically 12 to 24 hours.

The development of the reaction can be checked by a general method such as chromatography. After the reaction, solvent is removed, and a product can be isolated and purified by a general method such as chromatography or recrystallization method. The structure of the product can be identified by element analysis, MS(FD-MS) analysis, IR analysis, ¹H-NMR, or ¹³C-NMR.

When any functional group that inhibits the reaction is present in the compound represented by Formula (1a) and/or the compound represented by Formula (1b) in the reaction, compounds obtained by protecting functional groups of these compounds can be used in addition to these additives. In this case, a deprotection treatment is performed if necessary after the reaction.

The following describes the present invention in detail on the basis of examples. The present invention, however, is by no means limited thereby.

Synthesis Example 1
Synthesis Example 1-1
N-(2-Ethoxyphenethyl)acrylamide (2; JI046)

To a stirred solution of 1 (0.48g, 2.90 mmol) in dichloromethane (10 ml) were addedtrimethylamine (0.29 g, 2.88 mmol) and acryloyl chloride (0.26 g, 2.87 mmol) at 0°C. The mixture was stirred overnight at RT. The mixture was washed with saturated NaHCO₃ solution, water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified by column chromatography (ethyl acetate/n-hexane) to yield 2 (0.35 g, 1.60 mmol, 55%). ¹H-NMR (CDCl₃) : δ = 1.44 (t, J=7.2 Hz, 3H), 2.88 (t, J=6.3 Hz, 2H), 3.59 (q, J=6.4 Hz, 2H), 4.07 (q, J=7.1 Hz, 2H), 5.59 (d, J=10.2 Hz, 1H), 5.88 (brs, 1H), 5.97-6.06 (m, 1H), 6.22 (d, J=15.4 Hz, 1H), 6.85-6.92 (m, 2H), 7.12-7.28 (m, 2H).

Synthesis Example 1-2
I-N-(2-Ethoxyphenethyl)-3-(7-methoxy-1H-indol-3-yl)acrylamide (7; JI051)

Phosphoryl chloride (1.15 g, 7.50 mmol) was added dropwise to a stirred mixture of N,N-dimethylformamide (2.8 g, 38.3 mmol) and 3 (1.0 g, 6.79 mmol) at 0°C. The mixture was stirred overnight at RT, then poured into crushed ice and neutralized with 2N NaOH solution. The mixture was extracted with ethyl acetate, and washed with water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo to give pure 4 (1.18 g, 6.74 mmol, 99%).

Methyl(triphenylphosphoranylidene)acetate (2.72 g, 8.13 mmol) was added to a stirred mixture of 4 (1.18 g, 6.74 mmol) in benzene (50 ml) at RT. The mixture was refluxed for 3 h. The mixture was then concentrated in vacuo and purified by column chromatography (ethyl acetate/n-hexane) to yield 5 (1.0 g, 4.32 mmol, 64%). Lithium hydroxide monohydrate (0.36 g, 8.58 mmol) was added to a mixture of 5 (1.0 g, 4.32 mmol) in tetrahydrofuran (10 ml), methanol (10 ml), and water (5 ml) at 0°C. The mixture was refluxed for 2 h. The mixture was acidified with 1N HCl solution, and the solid was collected by filtration and dried under reduced pressure to yield 6 (0.68 g, 3.13 mmol, 72%).

To a mixture of 6 (0.3 g, 1.38 mmol) and 1 (0.23 g, 1.39 mmol) in dichloromethane (10 ml) were added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.32 g, 1.67 mmol), 4-dimethylaminopyridine (0.20 g, 1.64 mmol), and 1-hydroxybenzotriazole monohydrate (0.22 g, 1.63 mmol) at 0°C. The mixture was stirred overnight at RT. The mixture was washed with saturated NaHCO₃ solution, water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified by column chromatography (ethyl acetate/n-hexane) to yield 7 (0.26g, 0.71mmol, 52%). ¹H-NMR (CDCl₃) : δ= 1.47 (t, J=6.9 Hz, 3H), 2.92 (t, J=6.6 Hz, 2H), 3.66 (q, J=6.3 Hz, 2H), 3.96 (s, 3H), 4.10 (q, J=7.1 Hz, 2H), 5.80 (brs, 1H), 6.33(d, J= 15.7 Hz, 1H), 6.71 (d, J=7.7 Hz, 1H), 6.86-6.93 (m, 2H), 7.10-7.28 (m, 3H), 7.39-7.45 (m, 2H), 7.82 (d, J=15.4 Hz, 1H), 8.61 (brs, 1H).

Synthesis Example 1-3
I-N-(2-(Allyloxy)phenethyl)-3-(7-methoxy-1H-indol-3-yl)acrylamide (9; JI130)

To a mixture of 6 (0.46 g, 2.24 mmol) and 8 (0.40 g, 2.26 mmol) in dichloromethane (20 ml) were added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.52 g, 2.71 mmol), 4-dimethylaminopyridine (0.33 g, 2.70 mmol), and 1-hydroxybenzotriazole monohydrate (0.37 g, 2.74 mmol) at 0°C. The mixture was stirred overnight at RT. The mixture was washed with saturated NaHCO₃ solution, water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified by column chromatography (ethyl acetate/n-hexane) to yield 9 (0.49 g, 1.30 mmol, 58%). ¹H-NMR (CDCl₃) : δ= 2.95 (t, J=6.6 Hz, 2H), 3.67 (q, J=6.3 Hz, 2H), 3.96 (s, 3H), 4.56-4.61 (m, 2H), 5.29-5.51 (m, 2H), 5.85 (brs, 1H), 6.07-6.21 (m,1H), 6.32 (d, J=15.7 Hz, 1H), 6.71 (d, 1H), 6.86-6.96 (m, 2H), 7.10-7.24 (m, 3H), 7.38-7.45 (m, 2H), 7.81 (d, J=15.4 Hz), 8.62 (brs, 1H).

Synthesis Example 2
Synthesis Example 2-1
(S)-5-Methoxy-5-oxo-2-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamido)pentanoic acid (3)

To a stirred solution of 1 (0.5 g, 2.17 mmol) and N-hydroxysuccinimide (0.25 g, 2.17 mmol) in dichloromethane (15 ml) was added N,N’-Dicyclohexylcarbodiimide (0.45 g, 2.17 mmol) at 0°C. The mixture was stirred overnight at RT, then filtered and concentrated under reduced pressure to give 2 without further purification. To a stirred solution of 5-methyl L-glutamate (0.38 g, 2.36 mmol) in acetonitrile (10 ml) and water (3 ml) were added 2 and trimethylamine (0.66 g, 6.52 mmol). The mixture was stirred overnight at RT. The mixture was evaporated, and the residue was dissolved in ethyl acetate washed with 1N HCl solution, water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo to give crude 3 (0.89 g), which was used in next step without further purification.

Synthesis Example 2-2
Methyl-(S)-2,2-dimethyl-4,20-dioxo-21-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamido)-3,9,12,15-tetraoxa-5,19-diazatetracosan-24-oate (5)

To a mixture of crude 2 (0.89 g) and 4 (0.69 g, 2.15 mmol) in dichloromethane (10 ml) were added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.5 g, 2.61 mmol), 4-dimethylaminopyridine (0.32 g, 2.85 mmol) and 1-hydroxybenzotriazole monohydrate (0.35 g, 2.59 mmol) at 0°C. The mixture was stirred overnight at RT, then washed with saturated NaHCO₃ solution, water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified by column chromatography (ethyl acetate/n-hexane) to yield 5 (0.76 g, 1.13 mmol, 52% (from 1)).

Synthesis Example 2-3
Methyl-(S)-1-amino-15-oxo-16-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamido)-4,7,10-trioxa-14-azanonadecan-19-oate (6)

To a solution of crude 5 (0.76g, 1.13 mmol) in dichloromethane (10 ml) was added trifluoroacetic acid (2 ml) dropwise at 0°C. The mixture was stirred at RT for 2 h. The mixture was evaporated, and the residue was dissolved in chloroform, washed with saturated Na₂CO₃ solution, water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo to produce 6 (0.60 g, 1.04 mmol, 92%), which was used in next step without further purification.

Synthesis Example 2-4
Methyl-(S)-5,21-dioxo-25-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]rimethyl-4-yl)-4-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamido)-10,13,16-trioxa-6,20-diazapentacosanoate (8)

To a stirred solution of 6 (0.60 g, 1.04 mmol) in N,N-dimethylformamide (5 ml) were added 7 (0.36 g, 1.04 mmol) and trimethylamine (0.32 g, 3.12 mmol). The mixture was stirred overnight at RT. The mixture was evaporated, and the residue was purified by column chromatography (methanol/chloroform) to yield 8 (0.70 g, 0.87 mmol, 84%).

Synthesis Example 2-5
(S)-5,21-Dioxo-25-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]rimethyl-4-yl)-4-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamido)-10,13,16-trioxa-6,20-diazapentacosanoic acid (10)

To a mixture of 9 (0.56 g, 0.70 mmol) in tetrahydrofuran (3 ml) and water (1 ml) was added lithium hydroxide monohydrate (59 mg, 1.40 mmol) at 0°C. The mixture was stirred at RT for 2 h. The mixture was acidified with 1N HCl solution and extracted with chloroform. The combined extracts were dried over anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified by column chromatography (methanol/chloroform) to yield 10 (0.46 g, 0.59 mmol, 84%).

Synthesis Example 2-6
(S)-N1-(15-Oxo-19-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]rimethyl-4-yl)-4,7,10-trioxa-14-azanonadecyl)-N5-(3-phenoxypropyl)-2-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamido) pentanediamide (12; JIN04)

To a stirred solution of 10 (20 mg, 0.0254 mmol) and 11 (7.7 mg, 0.0509 mmol) in methanol (1 ml) was added 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (14.1 mg, 0.0509 mmol) at 0°C. The mixture was stirred overnight at RT. The mixture was evaporated, and the residue was purified by column chromatography (methanol/chloroform) to yield 12 (11.2 mg, 0.0122 mmol, 48%). ¹H-NMR (CD3OD) :δ= 1.29-1.34 (m, 2H), 1.49-1.69 (m, 8H), 2.08 (t, J=8.0 Hz, 2H), 2.24-2.41 (m, 4H), 2.52-2.85 (m, 4H), 3.12-3.54 (m, 19H), 3.87 (t, J=6.1 Hz, 2H), 4.01-4.05 (m, 1H), 4.16-4.21 (m, 1H), 4.35-4.40 (m, 2H), 6.60-6.85 (m, 3H), 7.11-7.25 (m, 4H), 7.88 (d, J=8.8 Hz, 2H).

Synthesis Example 2-7
I-N-(5-(3-Aminopropoxy)-2-ethoxyphenethyl)-3-(7-methoxy-1H-indol-3-yl)acrylamide (23)

To a stirred solution of 13 (3.0 g, 27.3 mmol) in acetonitrile (150 ml) were added potassium carbonate (5.72 g, 41.4 mmol) and allyl bromide (3.30 g, 27.3 mmol) at 0°C. The mixture was refluxed overnight, then filtered and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate/n-hexane) to yield 14 (1.53 g, 10.2 mmol, 37%). To a stirred mixture of 14 (1.53 g, 10.2 mmol) in water (4 ml) was added sodium hydroxide (2.70 g, 67.6 mmol) at RT. The mixture was warmed to 70°C and chloroform was added (2.43 g, 20.4 mmol). The mixture was then refluxed for 1 h, poured into crushed ice, and neutralized with 1N HCl solution. The mixture was extracted with ethyl acetate and washed with water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified by column chromatography (ethyl acetate/n-hexane) to yield 15 (0.64 g, 3.6 mmol, 35%).

To a stirred solution of 15 (1.12 g, 6.29 mmol) in acetonitrile (30 ml) were added potassium carbonate (0.99 g, 7.16 mmol) and ethyl iodide (1.12 g, 7.18 mmol) at 0°C. The mixture was refluxed for 3 h, then filtered and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate/n-hexane) to yield 16 (1.21 g, 5.87 mmol, 93%). To a stirred solution of 16 (1.21 g, 5.87 mmol) in acetic acid (5 ml) were added ammonium acetate (018 g, 2.35 mmol) and nitromethane (1.07 g, 17.6 mmol) at RT. The mixture was stirred at 110°C for 3 h, then evaporated and neutralized with saturated NaHCO₃ solution. The mixture was extracted with ethyl acetate and washed with water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified by column chromatography (ethyl acetate/n-hexane) to yield 17 (1.30 g, 5.22 mmol, 89%).

To a stirred solution of 17 (1.30 g, 5.22 mmol) in tetrahydrofuran (10 ml) was added lithium aluminium hydride (0.50 g, 13.2 mmol) at 0°C. The mixture was stirred at RT for 5 h. The reaction was quenched with water, dried over anhydrous magnesium sulfate, and concentrated in vacuo to give pure 18 (1.09 g, 4.93 mmol, 94%). To a mixture of 18 (0.39 g, 1.76 mmol) and 19 (0.32 g, 1.47 mmol) in dichloromethane (20 ml) were added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.37 g, 1.93 mmol), 4-dimethylaminopyridine (0.23 g, 1.88 mmol), and 1-hydroxybenzotriazole monohydrate (0.26 g, 1.92 mmol) at 0°C. The mixture was stirred overnight at RT, then washed with saturated NaHCO₃ solution, water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified by column chromatography (ethyl acetate/n-hexane) to yield 20 (0.54 g, 1.28 mmol, 87%).

To a stirred mixture of 20 (0.54 g, 1.28 mmol) in methanol (10 ml) were added tetrakis(triphenylphosphine)palladium(0) (14.8 mg, 0.013 mmol) and potassium carbonate (0.53 g, 3.83 mmol) at room temperature. The mixture was refluxed for 2 h, then evaporated and extracted with ethyl acetate, and washed with water and brine. The mixture was dried over anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified by column chromatography (ethyl acetate/n-hexane) to yield 21 (0.48 g, 1.26 mmol, 98%). To a stirred solution of 21 (0.48 g, 1.26 mmol) in acetonitrile (15 ml) were added potassium carbonate (0.38 g, 2.75 mmol) and N-(3-bromopropyl)phthalimide (0.41 g, 1.53 mmol) at 0°C. The mixture was refluxed for 6 h. Then the mixture was filtered and concentrated under reduced pressure, and the residue was purified by column chromatography (ethyl acetate/n-hexane) to yield 22 (0.50 g, 0.88 mmol, 70%).

To a stirred mixture of 22 (0.50 g, 0.88 mmol) in methanol (5 ml) was added hydrazine monohydrate (0.13 g, 2.60 mmol) at RT. The mixture was refluxed for 2 h, then filtered and concentrated under reduced pressure to give 23 (0.35 g, 0.80 mmol, 91%) without further purification. ¹H-NMR (CDCl₃) :δ= 1.44 (t, J=7.1 Hz, 3H), 1.86-1.92 (m, 2H), 2.82-2.91(m, 4H), 3.59-3.65 (m, 2H), 3.94-4.06 (m, 7H), 6.30 (brs, 1H), 6.38 (d, J=15.7 Hz, 1H), 6.68-6.81 (m, 4H), 7.08-7.14 (m, 1H), 7.34-7.50 (m, 2H), 7.79 (d, J=15.4 Hz), 9.91 (brs, 1H).

Synthesis Example 2-8
(S)-N5-(3-(4-Ethoxy-3-(2-(I-3-(7-methoxy-1H-indol-3-yl)rimethyla)ethyl)phenoxy) propyl)-N1-(15-oxo-19-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]rimethyl-4-yl)-4,7,10-trioxa-14-azanonadecyl)-2-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamido) pentanediamide (24; JITV10)

To a stirred solution of 10 (24.1 mg, 0.0306 mmol) and 23 (20 mg, 0.0457 mmol) in methanol (1 ml) was added 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (12.7 mg, 0.0459 mmol) at 0°C. The mixture was stirred overnight at RT, then evaporated, and the residue was purified by column chromatography (methanol/chloroform) to yield 24 (14.5 mg, 0.012 mmol, 39%). ¹H-NMR (CDCl₃) :δ= 1.25-1.47 (m, 5H), 1.70-1.90 (m, 5H), 2.05-2.32 (m, 6H), 2.84-2.92 (m, 2H), 3.04-3.11 (m, 2H), 3.34-3.66 (m, 21H), 3.92-4.03 (m, 5H), 4.20-4.59 (m, 3H), 6.37 (d, J=15.7 Hz, 2H), 6.67-6.79 (m, 4H), 6.86-6.93 (m, 1H), 7.10-7.45 (m, 4H), 7.79 (d, J=15.4 Hz, 1H), 7.85 (d, J=8.8 Hz, 2H).

Synthesis Example 3
Synthesis Example 3-1
1-(5-((3aS,4S,6aR)-2-Oxohexahydro-1H-thieno[3,4-d]rimethyl-4-yl)pentanamido)-N-(3-phenoxypropyl)-3,6,9,12-tetraoxapentadecan-15-amide (3; JIN05)

To a stirred solution of 1 (20 mg, 0.034 mmol) and 2 (7.7 mg, 0.051 mmol) in tetrahydrofuran (1 ml) was added trimethylamine (10.3 mg, 0.102 mmol) at 0°C. The mixture was stirred overnight at RT. The mixture was evaporated, and the residue was purified by column chromatography (methanol/chloroform) to yield 3 (11.8 mg, 0.019 mmol, 56%). ¹H-NMR (CDCl₃) :δ= 1.42-1.48 (m, 2H), 1.64-1.73 (m, 4H), 1.98-2.05 (m, 2H), 2.22 (t, J=6.0 Hz, 2H), 2.48 (t, J=5.8 Hz, 2H), 2.69-2.75 (m, 1H), 2.88-2.94 (m, 1H), 3.11-3.18 (m, 1H), 3.41-3.76 (m, 18H), 4.02 (t, J=6.0 Hz, 2H), 4.29-4.35(m, 1H), 4.49-4.53 (m, 1H), 5.24 (brs, 1H), 5.94 (brs, 1H), 6.54 (brs, 1H), 6.88-6.97 (m, 3H), 7.25-7.36 (m, 2H).

Synthesis Example 3-2
N-(3-(4-Ethoxy-3-(2-(I-3-(7-methoxy-1H-indol-3-yl)rimethyla)ethyl)phenoxy)propyl)-1-(5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]rimethyl-4-yl)pentanamido)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide (9; JITV14)

To a mixture of 4 (0.10 g, 0.15 mmol) and 5 (0.06 g, 0.14 mmol) in dichloromethane (3 ml) were added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.053 g, 0.28 mmol), 4-dimethylaminopyridine (0.034 g, 0.28 mmol) and 1-hydroxybenzotriazole monohydrate (0.037 g, 0.27 mmol) at 0°C. The mixture was stirred overnight at RT, then washed with saturated NaHCO₃ solution, water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified by column chromatography (ethyl acetate/n-hexane) to yield 6 (0.08 g, 0.074 mmol, 53%). To a mixture of 6 (0.08 g, 0.074 mmol) in N,N-dimethylformamide (2 ml) was added piperidine (0.2 ml) at RT. The mixture was stirred for 4 h at RT, then evaporated to give crude 7. To 7 stirred in tetrahydrofuran (3 ml) were added 8 (0.047 g, 0.14 mmol) and trimethylamine (0.042 mg, 0.42 mmol) at 0°C. The mixture was stirred overnight at RT. The mixture was evaporated, and the residue was purified by column chromatography (methanol/chloroform) to yield 9 (0.05 g, 0.046 mmol, 62%). ¹H-NMR (CDCl₃) :δ= 1.26-1.46 (m, 5H), 1.56-1.72 (m, 4H), 1.90-1.95 (m, 4H), 2.15-2.23 (m, 2H), 2.39-2.44 (m, 2H), 2.64-2.91 (m, 4H), 3.09-3.14 (m, 1H), 3.37-3.70 (m, 36H), 3.93-4.08 (m, 7H), 4.21-4.28 (m,1H), 4.44-4.51 (m, 1H), 4.97 (brs, 1H), 5.65 (brs, 1H), 6.17 (brs, 1H), 6.39 (d, J=15.9 Hz, 1H), 6.68-6.80 (m, 4H), 7.08-7.14 (m, 1H), 7.43-7.46 (m, 2H), 7.80 (d, J=15.7 Hz, 1H), 9.21 (brs, 1H).

Materials and Methods
Materials and Methods 1:Cell Culture and Transfection
HEK293 cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco) supplemented with 5% fetal bovine serum (FBS; HyClone) and 100 units/ml penicillin and streptomycin (PS; Invitrogen). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂. Cells were transfected with FuGENE HD transfection reagent (Promega) according to the manufacturer’s instructions. Human pancreatic cancer cell lines (MIA PaCa-2, CFPAC-1, PK9 and KP4-1) were maintained at 37°C and 5% CO₂ in DMEM or Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco) supplemented with 10% FBS (Gibco) and 100 units/ml PS.

Materials and Methods 2:Animals and Xenograft Model
Immune-incompetent nude (Nu/Nu) mice were purchased from SLC (Japan). All animal care and experiments were conducted following the guidelines for the Japan’s Act on Welfare and Management of Animals. All animal experiments described herein were approved by Kyoto University Graduate School. All procedure was performed when mice were anesthetized by chloral hydrate, isoflurane, or diethyl ether, and all efforts were made to minimize the number of animals used and their suffering. 1 x 10⁶ MIA PaCa-2 pancreatic cancer cells, suspended with 50 μl DMEM without FBS and antibiotics, were injected subcutaneously into the right flank of 4-week old female immune-incompetent nude (Nu/Nu) mice. Tumors were measured with digital caliper in two dimensions and volume was calculated (π/6 x length x width2). When the tumor volume had reached 100 mm³, mice were assigned into the control group (DMSO) or the treatment group (JI130). JI130 dissolved in DMSO (100 mg/ml) was administered intraperitoneally at 50 mg/kg of body weight 5 days a week for 3 weeks. Tumor volume was calculated 3 times a week and body weight was measured at the same time. After 3 weeks treatment, mice were sacrificed and tumor weight was measured.

Materials and Methods 3:Plasmid cDNA
pC1-Hes1, pHes1-EGFPd2, pHes1-Ub-Luciferase, and pEF-NICD were provided by Prof. Ryoichiro Kageyama (Kyoto University, Japan). pCMV3-FLAG-PHB2 and pcDNA3.1 were purchased from Sino Biological Inc. and Thermo Fisher Scientific, respectively. pMXs-mCherry-hCdh1 (30/120) and pCSII-AmCyan-hGeminin (1/110) were provided by Prof. Atsushi Miyawaki (RIKEN BSI, Japan). pCMV-Hes1-DsRed was generated by amplifying Hes1 (from pC1-Hes1 plasmid) with sense and antisense primers comprising a NheI and an AgeI site, respectively, prior to insertion into pDsRed Monomer (a monomeric mutant derived from thetetrameric Discosoma sp. Red fluorescent protein (Matz et al., 1999, Nat. Biotechnol. 10, 969-973.)) Golgi vector (Clontech) predigested with NheI and AgeI to remove the Golgi-targeting signal peptide.pCMV2-FLAG-Gro/TLE1was provided by Prof. Stefano Stifani (McGill University, Canada). pCMV2-EGFP-TLE1 was obtained by amplifying EGFP from p177 Grg-1s EGFP (provided by Ramesh Shivdasani;Addgene plasmid #11066) with sense and antisense primers comprising a NheI and an ClaI site, respectively, prior to insertion into pCMV2-FLAG-Gro/TLE1.

Materials and Methods 4:Compound Screening
A chemical library of 1800 small organic molecules containing indole moieties (Tripos Receptor Research) was screened in HEK293 cells expressing pHes1-Ub-Luciferase with or without pC1-Hes1following a 2.5 μM treatment for 48 h. Luminescence was amplified using the Luciferase Assay System (Promega) and measured with a microplate reader (MTP-880 Corona).To validate hit compounds, HEK293 cells expressing pHes1-EGFPd2 together with pCMV-Hes1-DsRed were treated with 10 μM of compounds for 48 h, prior to confocal microscopy imaging with CellVoyager^TM CV1000 (Yokogawa). Image data corresponding to fluorescence intensities were quantified using ImageJ software. The fluorescence intensity of the EGFPd2 signal was divided by the intensity of the DsRed signal for normalization.

Materials and Methods 5:Cell Proliferation
Cell proliferation was evaluated using WST-8 assay kit (Dojindo Molecular Technologies) or CellTiter 96 (registered trademark) Aqueous One Solution (Promega) for HEK293 and pancreatic cancer cells, respectively. Absorbance was measured using a microplate reader. For mitotic index determination, DNA was stained with 20 μM Hoechst 33342 (Thermo Fisher Scientific) for 10 min at RT prior to confocal microscopy imaging. The percentage of cells displaying condensed chromatin was determined following cell counting using the cell counter plugin in ImageJ. To evaluate cell survival, cells were incubated with a 0.2% Trypan Blue (Thermo Fisher Scientific) prior to cell counting with a Countess Automated Cell Counter (Invitrogen).

Materials and Methods 6:Cell Viability and Apoptosis
Early apoptotic and necrotic cells were stained with the Annexin V-Early Apoptosis Detection Kit (Cell Signaling Technology) and imaged by confocal microscopy. Cells that were positive for Alexa Fluor 488 Annexin V but negative for propidium iodide were considered early apoptotic while those that were both Annexin V and PI positive were considered necrotic. Cells that were both Annexin V and PI negative were considered live.

Materials and Methods 7:Fluorescence Imaging
For immunocytochemistry, cells were fixed with a 4% paraformaldehyde solution (PFA; Muto Pure Chemicals Co. Ltd.), prior to blocking with 5% Blocking one-P (Nacalai Tesque Inc.) and 0.1% Triton X-100 (Sigma Aldrich) in phosphate buffered saline (PBS; Life Technologies). Incubation with primary antibodies was carried out in antibody dilution buffer (2.5% Blocking One-P and 0.05% Triton X-100 in PBS) overnight at 4°C. Cells were then incubated with Alexa fluor 488- and Alexa fluor 568-cojugated secondary antibodies (Thermo Fisher Scientific) for 1 h at room temperature (RT) and imaged by confocal microscopy. To evaluate colocalization between PHB2 and Hes1, Pearson correlation coefficient was determined using the JACoP plugin (Bolte and Cordelieres, 2006, Journal of Microscopy-Oxford 224, 213-232.) from ImageJ. For JITV10 and mitochondrial imaging, cells were incubated with JITV10 (20 μM) together with 200 nM of MitoTracker Green (Thermo Fisher Scientific) for 45 min at 37°C. Cells were fixed with 4% PFA and incubated with a Streptavidin- Alexa Fluor 568 conjugate (Thermo Fisher Scientific) for 1 h at RT prior to confocal microscopy imaging. The production of superoxide was visualized by fluorescence microscopy following a 15 min incubation at 37°C with 4 μM MitoSOX Red indicator (Thermo Fisher Scientific).

Materials and Methods 8:Target Identification
Cells lysates were prepared in PBS with protease inhibitors (Complete Mini; Roche Life Sciences) by sonicating HEK293 cells with an ultrasonic processor (Astrason W-385; Heat System-ultrasonics). Resulting lysates were incubated with compounds together with Dynabeads M-280 streptavidin (Thermo Fisher Scientific) overnight at 4°C. Samples were boiled at 100°C for 5 min prior to separation onto mini II polyacrylamide 4/20 gels (Cosmo Bio Co., Ltd.), followed by silver staining (Silver Stain MS Kit; Wako Pure Chemical Industries, Ltd.). Gel pieces were excised and destained according to the manufacturer’s instructions. Samples were digested with trypsin, and resulting peptides were analyzed by nanoscale liquid chromatography- tandem mass spectroscopy (nano LC-MS/MS). Target proteins were identified following MS/MS ion search using Mascot server software from Matrix Science.

Materials and Methods 9:Target Validation
HEK293 cells expressing pCMV2-FLAG-Gro/TLE1 or pCMV3-FLAG-PHB2 were lysed in 150 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1% Nonidet P-40 substitute (Amresco) with protease inhibitors prior to centrifugation. Cell lysates were incubated with the indicated compounds for 1 h at 4°C and exposed to UV light for 30 min at 4°C (Bio-link Crosslinker BLX-365; Cosmo Bio). Lysates were then incubated with NeutrAvidin agarose beads (Thermo Fisher Scientific) for 2 h at 4°C. A fraction of each lysate was collected prior to the washing step (i.e., input fraction). Resin-bound complexes were washed 3 times with lysis buffer and boiled at 100°C for 10 min in reducing 2x Laemmli sample buffer (Bio-Rad) for elution. Human recombinant PHB2 protein (Abnova) was incubated with increasing concentrations of JITV10 overnight at 4°C and exposed to UV light for 30 min at 4°C. Samples were then boiled at 100°C for 10 min in reducing 2x Laemmli sample buffer and resolved by SDS-polyacrylamide gel electrophoresis. Biotinylated compounds were detected with a high sensitivity streptavidin-HRP conjugate (Thermo Fisher Scientific).

Materials and Methods 10:Western Blotting
Samples were resolved on 10% mini-protean TGX gels (Bio-Rad), prior to transfer onto Nitrocellulose blotting membranes (Ge Healthcare Life Sciences). Membranes were incubated overnight with primary antibodies, followed by a 1 h incubation at RT with ECL-peroxidase- labelled anti-mouse (Ge Healthcare Life Sciences) or horseradish peroxidase (HRP)-linked anti-rabbit (Cell Signaling Technology) in immunoreaction enhancer solution (Can Get Signal; Toyobo Co. Ltd.). Immunoreactive proteins were visualized using Amersham ECL Prime Western Blotting Detection Reagent (Ge Healthcare Life Sciences) and an ImageQuant LAS 500 imaging system (Ge Healthcare Life Sciences).

Materials and Methods 11:Co-immunoprecipitation Studies
HEK293 cells expressing Flag-PHB2 alone or together with Hes1 were lysed in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1% Nonidet P-40 substitute with protease inhibitors. Cell lysates were precleared with 2 mg of protein A-Sepharose (Sigma) for 45 min at 4°C and incubated for 48 h at 4°C with FLAG M2 mouse monoclonal antibody, together with JI051 at the concentration indicated. Labelled proteins were immunoprecipitated with 6 mg of protein A-Sepharose for 2 h at 4°C. Complexes were washed 3 times with lysis buffer and dissolved in reducing 2x Laemmli sample buffer prior to a 10 min incubation at 100°C.

Materials and Methods 12:siRNA experiments
siRNA oligonucleotide duplexes targeting human PHB2 were purchased from OriGene. The targeting sequences were as follows: PHB2B 5’-GUGAUUUCCUACAGUGUUGUUCCCT-3’ (Sequence Number 5, nucleotides 1140-1164) and PHB2C 5’-UCUAUCUCACAGCUGACAACCUUGT-3’ (Sequence Number 6, nucleotides 812-836). HEK293 cells were transfected with 50 or 75 nM siRNA using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions. Western blotting analysis and confocal imaging were performed 72 h after transfection.

Materials and Methods 13:MEF Hes1 KO Cell Line
Immortalized mouse embryonic fibroblasts (MEF) from the Hes1-KO mice (Ishibashi et al., 1995, Gene Dev. 9, 3136-3148.) were generated as below. Primary cultures of the E14.5 Hes1-KO and its littermate wild-type control embryo were maintained for 1 month with DMEM + 10% FBS + penicillin/streptomycin. Spontaneously immortalized colonies were picked up and expanded. Genotyping of the Hes1 locus was confirmed by PCR using the genomic DNA of MEFs.

Materials and Methods 14:Aqueous Solubility
Aqueous solubility was evaluated using a high throughput turbidimetric assay. Compounds were diluted in PBS (pH 7.4) and 1% DMSO at the following concentrations: 1, 3, 10, 30, and 100 μM, and incubated for 2 h at 37°C, prior to absorbance measurements at 620 nm.

Materials and Methods 15:Quantification and Statistical Analysis
Indicated sample sizes were chosen based on preliminary experiments to detect significant differences in mean values. With large enough sample sizes, Skewness and Kurtosis were calculated to verify that data were normally distributed. Two-tailed Student’s t-test was used for comparing means of numerical values when indicated. A difference in means was considered statistically significant at p < 0.05 or p < 0.01. Variation within each group of data is shown as error bars representing standard deviation or standard error of the mean as indicated.

Experimental Example 1:High-throughput screening for Hes1 modulators
The tetrapeptide WRPW (Trp-Arg-Pro-Trp) motif of Hes1 is a functional transcriptional repression domain that is sufficient to recruit TLE1 to Hes1-responsive promoters (Fisher et al., 1996, Mol. Cell. Biol. 16, 2670-2677.). Since the protein-protein interaction domain is composed of two tryptophan residues, each containing an indole ring, we searched a chemical library enriched with indole-like π-electron rich pharmacophores for a small molecule that relieves the Hes1-mediated transcriptional repression. A total of 1,800 compounds were screened with a luciferase reporter assay to study the activity of Hes1 promoter (Figure 1A). Exogenous Hes1 expression decreased the expression of the reporter gene to 56.5% ± 4.1 (DMSO; vehicle control), due to negative feedback regulation on Hes1 promoter (data not shown). The ability of each small molecule to revert Hes1-mediated luciferase gene repression was reported as an increase in the luminescence signal. Out of the compounds tested, 17 reduced the effect of Hes1 on the reporter gene by at least 60% compared to DMSO.

Figure 1A through D show the isolation of Hes1 modulators. The ability of the selected small molecules to block Hes1-mediated repression was further examined by confocal microscopy, using a Hes1 promoter-driven EGFPd2 reporter assay (Figure 1A). Out of the 17 compounds, 3 of them (D8C, L4F, and T10E) produced a fluorescence signal at least 3-fold higher compared to DMSO in cells overexpressing Hes1 (data not shown). Among these compounds, D8C, an indolylacrylamide molecule, induced the greatest response, with a 5.2- fold increase in fluorescence signal. Based on these results, D8C was chosen as a lead compound for further analysis. Figures 1B and 1C highlight the effects of the validated compound on Hes1- mediated transcriptional repression on luciferase and EGFPd2 reporter gene, respectively. The chemical structures of the validated compounds are shown in Figure 1D.

Experimental Example 2:Effect of D8C derivatives of cell proliferation
Besides its role in cell differentiation, Hes1 is also important for cell proliferation as progenitor cells with inactivated Hes1 are no longer maintained in a proliferative state, choosing instead to exit the cell cycle with upregulation of the cyclin kinase inhibitors p27 and p57 (Georgia et al., 2006, Dev. Biol. 298, 22-31.; Monahan et al., 2009, Endocrinology 150, 4386-4394.). In order to examine whether D8C influences cell growth, a colorimetric cell proliferation assay was carried out using the water-soluble tetrazolium salt WST-8. Figure 2A through D show that D8C inhibits cell proliferation. As shown in Figure 2A, incubation with D8C led to a time-dependent decrease in proliferation, reaching a plateau of 38% (as compared to cells treated with DMSO) after 72 h. Although the cell proliferation was significantly decreased, assessment of cell viability according to Trypan Blue exclusion indicated no significant difference in cell survival rate between cells treated with D8C or DMSO, indicating that the remaining cells are viable (Figure 2B).

To improve the potency of D8C, we synthesized a series of 130 compounds derived from its chemical structure, including modifications of the ethoxyphenetyl, amide, alkene, and indole moieties (Table 1-6). Structure-activity relationship (SAR) studies revealed that the ethoxy group needs to be located in the ortho position of the phenyl ring; meta and para positions resulted in a significant loss in activity. Introduction of longer alkyl groups or substitution with halogen atoms also resulted in loss of activity. Other modifications around the amide bond were not tolerated for activity, and deletion of the double bond or the indole ring resulted in activity loss. In contrast, modifications including the introduction of a methoxy group (JI051) or a methyl group (JI021) at position 7 of the indole ring resulted in EC50 values lower than that of D8C: JI051 showed a 6- fold lower EC50 value than D8C (Figure 2C). Similar trends were also observed with the EGFPd2 reporter assay (data not shown). Chemical structures of D8C, JI051, and JI046 with their respective EC50 values are summarized in Figure 2D. Further investigation of cell survival with Alexa Fluor 488 annexin V conjugate and propidium iodide indicated that JI051 is likely interfering with cell proliferation without increasing apoptosis or necrosis (data not shown), suggesting that the JI051 effect on cell growth is not due to compound toxicity.

To determine whether the decrease in proliferation could be attributed to cell cycle arrest, HEK293 cells were transfected with the fluorescent ubiquitination-based cell-cycle indicators (Fucci) (Sakaue-Sawano et al., 2008, Cell 132, 487-498.). These probes exploit the cell cycle-dependent proteolysis of Cdt1 and geminin by E3 ligases SCFSkp2 and APCCdh1, enabling the visualization of G1 and G2/M phases in red and green, respectively. Figure 3A and B show that JI051 induces cell cycle arrest in G2/M. As shown in Figure 3A, treatment with JI051 induced a dose-dependent increase in Geminin/Cdt1 fluorescence ratio, suggesting an increase in the cell population in G2/M phase of the cell cycle. A similar increase was also observed with the microtubule-depolarizing agent Nocodazole, which is known to arrest cell cycle progression in G2/M (Choi et al., 2011, PloS One 6.). As the Fucci probes do not enable the distinction between G2 and M phases of the cell cycle, we examined the effect of JI051 on chromatin condensation to evaluate the proportion of the cells undergoing mitosis. As shown in Figure 3B, the proportion of the cells with visible chromosomes increased from 3.6% to 37.9% following treatment with JI051. Fluorescence microscopy analysis also showed incomplete chromosome congression at the metaphase plate, suggesting that the cells are blocked in mitotic prometaphase.

Experimental Example 3:Target identification for JI051
As our initial strategy was to mimic the WRPW motif of Hes1 for blocking the Hes1-TLE1 interaction, we attempted to determine whether JI051 interacts with TLE1. We synthesized a JI051 photoreactive probe (JITV10) equipped with a diazirine group for covalent binding upon UV irradiation, and a biotin moiety for pulldown with NeutrAvidin agarose beads (data not shown). However, a 5-fold excess of JI051 failed to compete with JITV10 and comparable labeling was obtained with JIN04 (a negative control lacking the JI051 moiety), showing that TLE1 labeling is not specific for JI051 (data not shown). Furthermore, confocal microscopy studies with a fluorescent streptavidin conjugate (data not shown) revealed that JITV10 failed to colocalize with EGFP-TLE1 in the nucleus. Colocalization with MitoTracker green indicated that JITV10 was localized in the mitochondria (data not shown). Taken together, these data indicate that TLE1 is not the genuine target for JI051.

To identify the true protein target for JI051, we carried out immunomagnetic isolation of target protein(s) with streptavidin-coupled Dynabeads for optimal washing efficiency. Figure 4A through C show that prohibitin2 is JI051 target. We also used a biotinylated version of JI051 (JITV14) without the diazirine moiety to reduce unspecific binding. Analysis of bound proteins by SDS-PAGE (Figure 4A), showed a distinct band pattern for JITV14 compared to JIN05 (negative control without the JI051 moiety). The protein bands that were efficiently displaced by a 10-fold excess of JI130 (a JI051 derivative with improved solubility; Table 1-6) were isolated and digested with trypsin, and the resulting peptides were analysed by nanoscale liquid chromatography/tandem mass spectrometry (nano LC-MS/MS). Tandem mass spectroscopy (MS/MS) ion search using the Mascot server (Matrix Science) for protein identification identified four potential targets with a probability-based Mowse score above 100 (Perkins et al., 1999, Electrophoresis 20, 3551-3567): prohibitin 2 (PHB2), NAD(P)H quinone dehydrogenase 2 (NQO2), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and beta-actin (wherein ion scores greater than 39 were considered significant, with p<0.05). The mitochondrial protein, PHB2, showed the highest protein score (207) with 12 matching peptides covering 51% of the protein sequence (data not shown). Beta actin (ACTB) and GAPDH were discarded as potential targets as they are abundantly expressed and appear regularly as protein candidates in our target identification studies. In addition, ACTB and GAPDH were only partially displaced upon competition with a 10-fold excess of JI130, suggesting low specificity in the probe binding. Quinone reductase 2 (NQO2) has previously been reported as an off-target for acetaminophen and to be responsible for acetaminophen-induced superoxide production (Miettinen et al., 2014, Mol. Pharm. 12, 4395-43404.). To find out whether JI051 may be activating NQO2 as in the case of acetaminophen, we examined the effect of JI051 on superoxide production and found that JI051 does not induce a significant increase in superoxide levels (data not shown). Based on these results, we focused on PHB2 as a target candidate for JI051.

We performed a series of pulldown studies in cells overexpressing Flag-PHB2 to confirm that PHB2 is a target for JI051. Western blotting analysis revealed that PHB2 was photoreacted and pulled down (i.e., bound fraction) with JITV10 but not with JIN04 and displaced with a 2.5-fold excess of JI051 (Figure 4B). To determine whether JITV10 binds directly to PHB2 or needs additional proteins that are found in cell lysates, a series of photoaffinity studies with human recombinant PHB2 protein was carried out, using streptavidin conjugated with horseradish peroxidase (HRP) for detection. Analysis of PHB2 recombinant protein incubated with JITV10 revealed dose-dependent biotinylation of PHB2 upon UV irradiation (K_D = 3.7 μM; Figure 4C), indicating that JITV10 binds directly to PHB2 (data not shown). We used immunocytochemical studies to examine whether JITV10 and PHB2 were colocalized in cells (data not shown). Confocal imaging revealed that labeling of JITV10 with a streptavidin-Alexa 568 conjugate displayed a strong overlap with PHB2 immunostaining. Taken together, these results suggest that PHB2 is a bona fide target for JI051.

Experimental Example 4:Effects of JI051 and PHB2 on subcellular localization of Hes1
We then tested the possibility that PHB2 regulates Hes1 signaling by direct interaction through a series of co-immunoprecipitation studies in HEK293 cells transfected with Flag-PHB2 alone or together with Hes1. Figure 5A and B show that PHB2 is interacting with Hes1. Western blot analysis with a Hes1 antibody revealed that Hes1 is specifically co-immunoprecipitated with PHB2 in cells co-expressing Flag-PHB2 and Hes1 (Figure 5A), suggesting that the two proteins interact with each other. The amount of Hes1 co-immunoprecipitated with PHB2 strongly increased upon incubation with JI051, reaching saturation around 1 μM. These results not only indicate that Hes1 interacts directly with PHB2, but also suggest that JI051 serves as a stabilizer for the Hes1-PHB2 interaction. To determine whether the compound could increase the colocalization of Hes1 and PHB2 in intact cells, double immunostaining was carried out in HEK293 cells. As shown in Figure 5B, treatment with JI051 induced a 3-fold increase in Pearson’s correlation coefficient, indicating that the compound increases spatial overlap between Hes1 and PHB2. Colocalization was mainly observed outside the nucleus, suggesting that PHB2 may also influence Hes1 subcellular localization upon treatment with JI051 as compared to cells treated with DMSO wherein Hes1 is mainly nuclear.

Experimental Example 5:Effect of PHB2 and Hes1 on cell cycle arrest
We next carried out siRNA knockdown experiments to determine whether PHB2 is involved in JI051-mediated chromatin condensation. PHB2 siRNAs induced a substantial decrease in cell proliferation as compared to cells treated with scrambled siRNAs (26.6% ± 0.6 and 77.4% ± 4.7, respectively; data not shown). This reduction in cell growth was associated with a stretched cell morphology. These effects are both consistent with previous studies in PHB2-depleted cells (Sievers et al., 2010, Plos One 5.; Merkwirth et al., 2008, Gene Dev. 22, 476-488.). However, condensed chromatin was not observed in cells treated with PHB2 siRNAs alone (data not shown) as opposed to previous studies in HeLa cells (Takada et al., 2007, Curr Biol 17, 1356-1361.), suggesting that PHB2-depleted cells are not arrested in mitosis but rather at a different phase of the cell cycle in our experimental conditions. Mitotic arrest may be dependent on cell type or may require higher PHB2 depletion (~50%, data not shown; 60%, data not shown). Although PHB2 siRNAs did not have a direct effect on mitotic index, they caused a 62.4% decrease in the JI051-induced chromatin condensation compared to scrambled siRNA control (10.3% ± 3.2 and 27.4% ± 6.5, respectively), indicating that PHB2 is required for JI051 response. In addition to PHB2, we also examined the effect of Hes1 gene knockout (Hes1 KO) on JI051 response. JI051 only induced a minor decrease in cell proliferation in Hes1-depleted cells as compared to control cells (92.5% ± 1.9 and 64.5% ± 0.9, respectively), indicating that Hes1 is important for JI051 response (data not shown).

We then tested the effect of Hes1-DsRed overexpression (i.e., exogenous Hes1) on chromatin appearance. Cells were divided into 2 population according to presence (Hes1-DsRed (+)) or absence (Hes1-DsRed (-)) of red labeling prior to mitotic index analysis. Only a small proportion of the cells overexpressing Hes1 exhibited condensed chromatin as compared to untransfected cells (3.3% ± 2.1 and 27.3% ± 2.0, respectively), indicating that exogenous Hes1 can revert JI051 effect on chromatin appearance (data not shown). The effect of Hes1 overexpression on G2/M cell cycle arrest was also investigated wherein HEK293 cells were transfected with increasing amounts of pC1-Hes1 cDNA. Hes1 blocked JI051-mediated G2/M cell cycle arrest in a dose-dependent manner, reaching levels similar to those observed in the absence of the compound (data not shown). Taken together, these results indicate that both PHB2 and Hes1 play a role in JI051-mediated cell cycle arrest.

Experimental Example 6:JI051 is acting downstream of Notch signaling
To shed further light onto JI051 mechanism, we compared JI051 response on cell proliferation to that of the widely employed γ-secretase inhibitor DAPT (N-[N-(3,5-Difluorophenylacetyl-L- alanyl)]-S-phenylglycine t-Butyl ester). DAPT induced a dose- dependent decrease in cell growth as reported previously (data not shown) (Grottkau et al., 2009, Int J Oral Sci 1, 81-89.; Wu et al., 2014, Oncol Lett 8, 55-61.). Addition of JI051 did not block DAPT response but rather showed an additive effect until 100 μM DAPT (data not shown), suggesting that DAPT and JI051 act through a different mechanism. This hypothesis was then confirmed by siRNA experiments wherein we showed that PHB2 gene knockdown does not affect DAPT response on cell proliferation while it did reverse that of JI051 (data not shown). Based on these data, we conclude that the DAPT effect on cell proliferation is PHB2 independent. Additionally, Hes1 gene depletion (data not shown) did not inhibit DAPT- mediated decrease in cell growth while JI051 response was blocked (data not shown). Altogether, these data suggest that JI051 is acting through a different mechanism than γ-secretase inhibitors by inhibiting Notch downstream effector Hes1 via PHB2. Moreover, upregulation of Hes1 transcription through Notch intracellular domain (NICD) overexpression (Ohtsuka et al., 1999, Embo J 18, 2196-2207.) was not inhibited by JI051, confirming that our Hes1 inhibitor is acting downstream of Notch- RBPJ signaling and not upstream (data not shown).

Experimental Example 7:Effect of JI051 and its derivative on pancreatic cancer cells
As Notch signaling pathway appears to be activated in human pancreatic cancer, we tested the effects of our Hes1 inhibitors on a pancreatic ductal adenocarcinoma cell line. Figure 6A through D show that JI130 inhibits MIA PaCa-2 cell growth. As shown in Figure 6A, treatment of MIA PaCa-2 cells with JI051 and JI130 (a JI051 derivative with improved solubility; Table 1-6 and Figure 6C) resulted in a dose-dependent decrease in cell growth as compared to cells treated with DMSO. Dose-response studies revealed an EC₅₀ of 49 nM for JI130, comparable to 59 nM for the pyrimidine antimetabolite Gemcitabine, a chemotherapy drug recommended as a first-line treatment in pancreatic cancer in combination with nab-Paclitaxel (Von Hoff et al., 2013, N. Engl. J. Med. 369, 1691-1703.) (Figure 6B). Fluorescence imaging following staining with Hoechst 33342 not only indicated the presence of condensed chromatin but also additional morphological alterations, including micronucleation and multinucleation (Figure 6D). In addition to MIA PaCa-2 cells, JI130 was able to suppress cell growth in several pancreatic cancer cell lines including CFPAC-1, PK9 and KP4-1 (data not shown).

Finally, we tested the effects of JI130 on tumor growth in a pancreatic tumor xenograft model created with implanted MIA PaCa-2 cells. Figure 7A through E show that JI130 inhibits tumor growth in xenograft model. Mice were treated according to the protocol detailed in Figure 7A. As shown in Figure 7B, treatment with JI130 induced a significant decrease in the tumor volume as compared to cells treated with the vehicle (DMSO). A decrease in the tumor weight was also observed after treatment with JI130 (Figures 7C and 7D) without any noticeable change in body weight (Figure 7E).

To examine whether our Hes1 inhibitor also induced cell cycle arrest in vivo, immunohistochemical staining on MIA PaCa-2 xenograft tumors with the proliferation marker protein Ki-67 was carried out. Treatment with JI130 induced a significant decrease in the proportion of Ki-67 positive cells compared to DMSO (data not shown) (26.8% ± 3.3 and 51.2% ± 4.5, respectively), suggesting that our Hes1 inhibitor is decreasing tumor volume/weight by interfering with cell proliferation. Further animal studies are required to better understand the mode of action in vivo.

Claims

A cell cycle progression inhibitor comprising a Hes1 protein-PHB2 protein binding enhancer.
The cell cycle progression inhibitor according to claim 1, which induces G2/M phase cell cycle arrest.
The cell cycle progression inhibitor according to claim 1 or 2, wherein the binding enhancer is at least one member selected from the group consisting of a compound represented by Formula (1):

wherein R¹¹ and R¹² are identical or different, and each represents -(O)_n-R¹⁰ wherein R¹⁰ represents alkyl, alkenyl, or alkynyl, and n is 0 or 1,
m is 0 or an integer of 1 to 3,
R² represents a single bond or a linker,
R¹¹ and R², taken together with the carbon atoms to which they are attached, may form a ring,
R³ represents -NH-CO- or -CO-NH-,
R⁴ represents a single bond or a linker, and
R⁵ represents substituted or unsubstituted indolyl, and
salt, hydrate, and solvate thereof.
The cell cycle progression inhibitor according to claim 3, wherein the compound is a compound represented by Formula (1A):

wherein R¹¹, R¹², m, R², and R⁴ are as defined above;
R⁵¹, R⁵², R⁵³, R⁵⁴, and R⁵⁵ are identical or different, and each represents hydrogen, hydroxyl, halogen, or -(O)_p-R^50a wherein R^50a represents substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, or substituted or unsubstituted aryl, and p is 0 or 1; and
R⁵⁶ represents hydrogen or -(R^50b)_q-R^50c wherein R^50b represents -C(=O)-, -C(=O)-O-, -S(O)-, or -S(O)₂-, and R^50c represents substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, or substituted or unsubstituted aryl, and q is 0 or 1.
A cytostatic agent comprising a Hes1 protein-PHB2 protein binding enhancer.
An anticancer agent comprising a Hes1 protein-PHB2 protein binding enhancer.
A compound represented by Formula (1):

wherein R¹¹ and R¹² are identical or different, and each represents -(O)_n-R¹⁰ wherein R¹⁰ represents alkyl, alkenyl, or alkynyl, and n is 0 or 1,
m is 0 or an integer of 1 to 3,
R² represents a single bond or a linker,
R¹¹ and R², taken together with the carbon atoms to which they are attached, may form a ring,
R³ represents -NH-CO- or -CO-NH-,
R⁴ represents a single bond or a linker, and
R⁵ represents substituted or unsubstituted indolyl.