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WO1997041093A1 - Procedes de synthese d'amines protegees par fmoc - Google Patents

Procedes de synthese d'amines protegees par fmoc Download PDF

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Publication number
WO1997041093A1
WO1997041093A1 PCT/NL1997/000235 NL9700235W WO9741093A1 WO 1997041093 A1 WO1997041093 A1 WO 1997041093A1 NL 9700235 W NL9700235 W NL 9700235W WO 9741093 A1 WO9741093 A1 WO 9741093A1
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Prior art keywords
hydrogen
alkyl
fmoc
group
aryl
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PCT/NL1997/000235
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English (en)
Inventor
Ted Allen Baer
Stephen Peter Raillard
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Affymax Technologies N.V.
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Priority to AU24111/97A priority Critical patent/AU2411197A/en
Publication of WO1997041093A1 publication Critical patent/WO1997041093A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/16—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C269/00—Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C269/04—Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups from amines with formation of carbamate groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C319/00—Preparation of thiols, sulfides, hydropolysulfides or polysulfides
    • C07C319/14—Preparation of thiols, sulfides, hydropolysulfides or polysulfides of sulfides
    • C07C319/20—Preparation of thiols, sulfides, hydropolysulfides or polysulfides of sulfides by reactions not involving the formation of sulfide groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/02—Ortho- or ortho- and peri-condensed systems
    • C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
    • C07C2603/06—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members
    • C07C2603/10—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings
    • C07C2603/12—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings only one five-membered ring
    • C07C2603/18—Fluorenes; Hydrogenated fluorenes

Definitions

  • the present invention is related to the area of chemical synthesis. More specifically, one embodiment of the present invention provides a method for the preparation of Fmoc-protected amines, and more particularly, for the preparation of Fmoc-protected amino acids.
  • Chemical protecting groups are used during synthesis reactions to temporarily protect certain functional groups on a compound against undesired reactions. When a reaction sequence is complete, and protection is no longer necessary, the protective group is removed to restore the protected functional group to its natural activity. Protective groups are removed by various procedures such as exposure to acidic or basic conditions or electromagnetic radiation (e.g., light of a prescribed wavelength).
  • a particularly preferred protecting group for amines and particularly, amino acids is the 9-fluorenylmethoxycarbonyl (Fmoc) group.
  • the Fmoc group was first introduced for the protection of an alpha-amino function of an amino acid and it was removable through the treatment of liquid ammonia in 10-12 hours at room temperature (see Carpino and Han (1970) T. Am. Chem. Sor. 92:5748); subsequently, other deprotection methods have been used. Greene et al (1991) Protective Groups in Organic Synthesis. 2nd Ed. (John Wiley & Sons, Inc. , New York). The chemistry of its deprotection centers on the acidic nature of the proton on the beta-carbon atom, and hence, upon its abstraction by base.
  • R * are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl; and q is an integer of from 1 to 10, and preferably, from 1 to 4.
  • the present invention provides new methods for the preparation of Fmoc-protected amines. These methods involve the use of mild conditions and result in the production of high yields of Fmoc-protected amines, and particularly amino acids, having high chemical and optical purity. The methods can be conducted under anhydrous conditions when necessary.
  • an amine component is treated with a silylating agent and then with an activated Fmoc-reagent to yield the Fmoc-protected amine.
  • the silylating agent is selected from the group consisting of N-methyl- N-(t ⁇ methylsilyl)trifluoracetamide (MSTFA), N,0-bis(tnmethylsilyl)acetamide (BSA), N-methyl-N-(trimethylsilyl)acetam ⁇ de (MSA), N,0-b ⁇ s (t ⁇ methylsilyl)trifluoroacetamide (BSTFA) and bistrimethyl silylurea (BSU) More preferably, N-methyl-N-(trimethylsilyl)trifluoracetamide (MSTFA) or N,0- B_s(tr_methylsilyl)acetamide (BSA) is used More preferably, at least two silylating equivalents of silylating agent are used.
  • Preferred activated Fmoc-reagents mclude the acid chloride derivative of Fmoc (Fmoc-Cl); Fmoc-N-pentafluorophenyl ester (Fmoc-OPfp) and N-(9-fluorenylmethoxycarbonyloxy)succ ⁇ n ⁇ mide (Fmoc-OSu)
  • Fmoc-OSu A particularly preferred activated Fmoc-reagent is Fmoc-OSu
  • the amine component preferably is a primary or secondary amine having the formula -NR'R", where R' and R" are independently selected from the group consisting of hydrogen alkyl, alkoxy, amino, aryl, aryloxy, heteroaryl, and arylalkyl or salts thereof, provided that either R' or R" is hydrogen. More preferably, the amine component will have the formula:
  • Rl and R ⁇ are independently selected from the groups consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, heteroaryl, carboxyl, carboxyalkyl, and arylalkyl. More preferably, the amine component will comprise an amino acid or peptide.
  • the amine component will have the formula:
  • R6 is hydrogen, C ⁇ -Cg alkyl, aryl or arylalkyl;
  • R ⁇ , R4 anc j R5 are eacn independently hydrogen, Cj-Cg alkyl, or C ⁇ -CQ alkoxy;
  • X ⁇ l and ⁇ ll are each independently selected from the group consisting of halogen, — COOH, — SH,
  • R? and R ⁇ > are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl; and q is an integer of from 1 to 10, and preferably, from 1 to 4
  • activated reagent refers to those compounds which have been modified to render the compound more reactive toward covalent bond formation with another functional group or reactive site as compared with the unmodified compound.
  • activated Fmoc-reagents include those compounds which the carboxyl group has been modified to produce an anhydride, acid chloride, or other reactive species.
  • Preferred activated Fmoc-reagents mclude the acid chloride derivative (Fmoc-Cl); Fmoc-N-pentafluorophenyl ester (Fmoc-OPfp) and N-(9-fluorenylmethoxycarbonyloxy)succin ⁇ mide (Fmoc-OSu).
  • Other activating groups are known to those of skill in the art.
  • Alkoxy refers to the group alkyl-O-.
  • Alkyl refers to a cyclic, branched, or straight chain chemical group containing only carbon and hydrogen, such as methyl, heptyl, -(CH2)2", and adamantyl. Alkyl groups can either be unsubstituted or substituted with one or more substituents, e.g., halogen, alkoxy, acyloxy, ammo, aryl, hydroxyl, mercapto, carboxy, benzyloxy, phenyl, benzyl, or other functionality which may be suitably blocked, if necessary for purposes of the invention, with a protecting group
  • substituents e.g., halogen, alkoxy, acyloxy, ammo, aryl, hydroxyl, mercapto, carboxy, benzyloxy, phenyl, benzyl, or other functionality which may be suitably blocked, if necessary for purposes of the invention, with a protecting group
  • alkyl or “alkylene” is used
  • alkyl groups as substituents are those containing 1 to 10 carbon atoms, with those containing 1 to 6 carbon atoms (i.e., lower alkyl groups) being particularly preferred
  • Preferred alkyl or alkylene groups as linking groups are those containing 1 to 20 carbon atoms, with those containing 3 to 6 carbon atoms being particularly preferred
  • Ammoni ' or "amine group” refers to the group -NR'R", where R and
  • R" are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl
  • R' and R" are hydrogen
  • m a secondary amino group, either, but not both, R' or R" is hydrogen
  • an "o -amino acid” consists of a carbon atom, called the ⁇ -carbon, to which is bonded an ammo group and a carboxyl group. Typically, this ⁇ -carbon atom is also bonded to a hydrogen atom and a distinctive group referred to as a "side chain " The hydrogen atom may also be replaced with a group such as alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, and other groups.
  • side chains of naturally occurring amino acids include, for example, hydrogen (as in glycine), alkyl [as in alanine (methyl), valine (isopropyl), leucme (sec-butyl), isoleu ⁇ ne (iso-butyl), and proline (-(CH2)3-)], substituted alkyl [as in serine (hydroxymethyl), cysteine (thiomethyl), aspartic acid (carboxymethyl), asparagine, arginme, glutamme, glutamic acid, and lysine], aryl alkyl (as in phenylalarune, histidine, and tryptophan), substituted aryl alkyl (as in tyrosine and thyroxine), and heteroaryl (as in histidine).
  • amino acids used in the present invention may possess synthetic side chains.
  • a "synthetic side chain” is any side chain not found in a naturally occurring ammo acid.
  • a synthetic side chain can be an isostere of the side chain of a naturally occurring amino acid.
  • Naturally occurring and synthetic side chains may contain reactive functionalities, such as hydroxyl, mercapto, and carboxy groups
  • reactive functionalities such as hydroxyl, mercapto, and carboxy groups
  • these groups may have to be protected to carry out the desired reaction scheme
  • the hydrogen at the ⁇ -carbon can also be replaced with other groups; those of skill in the art recognize the medicinal importance of ⁇ -methyl amino acids and other ⁇ -, ⁇ -disubs tuted amino acids
  • amino acid includes other molecules having both an amino group and a carboxyl group, such as beta-amino acids, gamma-amino acids, the photolinker described in detail below, and the like.
  • Protected amino acid refers to an ammo acid, typically an ⁇ -amino acid having either or both the amine functionality and the carboxylic acid functionality suitably protected by one of the groups described above Additionally, for those amino acids having reactive sites or functional groups on a side chain (i.e., se ⁇ ne, tyrosme, glutamic acid), the term “protected ammo acid " is meant to refer to those compounds which optionally have the side chain functionality protected as well.
  • Aryl or “Ar” refers an aromatic substituent which may be a single ring or multiple rings which are fused together, linked covalently or linked to a common group such as an ethylene or methylene moiety.
  • the aromatic rings may each contain heteroatoms, for example, phenyl, naphthyl, biphenyl, diphenylmethyl, 2,2-dipheny 1-1 -ethyl, thienyl, pyridyl and quinoxalyl.
  • the aryl moieties may also be optionally substituted with halogen atoms, or other groups such as nitro, carboxyl, alkoxy, phenoxy and the like.
  • aryl radicals may be attached to other moieties at any position on the aryl radical which would otherwise be occupied by a hydrogen atom (such as, for example, 2-pyridyl, 3- pyridyl and 4-pyridyl).
  • Aryloxy refers to the group aryl-O- or heteroaryl-O-.
  • Arylalkyl'Or “aralkyl” refers to the groups R'-Ar and R-HetAr, where
  • Ar is an aryl group
  • HetAr is a heteroaryl group
  • R' is straight-chain or branched-chain aliphatic group (for example, benzyl, phenylethyl, 3-(4- nitrophenyl)propyl, and the like).
  • Preferred aryl groups include phenyl, 1- naphthyl, 2-naphthyl, biphenyl, phenylcarboxylphenyl (i.e , derived from benzophenone), and the like.
  • Carboxy or “carboxyl” refers to the group -R'(COOH) where R' is alkyl, substituted alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heterocyclic, heteroaryl, or substituted heteroaryl.
  • Carboxyalkyl refers to the group -(CO)-OR' where R' is alkyl or substituted alkyl.
  • Carboxyaryl refers to the group -(CO)-OR' where R' is aryl, heteroaryl, or substutited aryl or heteroaryl.
  • Linker refers to a molecule or group of molecules attached to a solid support and spacing a synthesized compound from the solid support, such as for exposure /binding to a receptor.
  • Protecting group refers to a chemical group that exhibits the following characteristics: (1) reacts selectively with the desired functionality in good yield to give a derivative that is stable to the projected reactions for which protection is desired; 2) can be selectively removed from the derivatized solid support to yield the desired functionality; and 3) is removable in good yield by reagents compatible with the other functional group(s) generated in such projected reactions. Examples of protecting groups can be found in Greene et al. (1991) Protective Groups in Organic Synthesis. 2nd Ed. Qohn Wiley & Sons, Inc., New York).
  • Preferred protecting groups include photolabile protecting groups (such as methylnitropiperonyloxycarbonyl (Menpoc), methylnitropiperonyl (Menp), nitroveratryl (Nv), nitroveratryloxycarbonyl (Nvoc), or nitroveratryloxymethyl ether (Nvom)); acid-labile protecting group (such as Boc or DMT); base-labile protecting groups (such as Fmoc, F , phosphonioethoxycarbonyl (Peoc, see Kunz (1976) Chem. Ber.
  • 1Q9_:2670 groups which may be removed under neutral conditions (e.g., metal ion-assisted hydrolysis ), such as DBMB (see Chattopadhyaya et al. (1979) T.C.S. Chem. Comm. 987-990), allyl or alloc (see, e.g., Greene and Wuts, "Protective Groups in Organic Synthesis", 2nd Ed., John Wiley & Sons, Inc., New York, NY (1991), 2-haloethyl (see Kunz and Buchholz (1981) Angew. Chem. Int F.H Engl.
  • neutral conditions e.g., metal ion-assisted hydrolysis
  • DBMB see Chattopadhyaya et al. (1979) T.C.S. Chem. Comm. 987-990
  • allyl or alloc see, e.g., Greene and Wuts, "Protective Groups in Organic Synthesis
  • Solid support refers to a material or group of materials having a rigid or semi-rigid surface or surfaces.
  • at least one surface of the solid support will be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different compounds with, for example, wells, raised regions, pins, etched trenches, or the like.
  • the solid support(s) will take the form of beads, resins, gels, microspheres, or other geometric configurations.
  • a preferred solid support is a bead havmg a size is in the range of 1 nm to 100 ⁇ m, but a more massive solid support of up to 1 mm in size may sometimes be used.
  • Particularly preferred resins include Sasrin resin (a polystyrene resin available from Bachem Bioscience, Switzerland); and TentaGel S AC, TentaGel PHB, or TentaGel S NH2 resm (polystyrene-polyethylene glycol copolymer resins available from Rappe Polymere, Tubingen, Germany).
  • Stepospecific reaction refers to a reaction m which bonds are broken and made at a single asymmetric atom (usually, but not necessarily carbon), and which lead largely to a single stereoisomer. If the configuration of the asymmetric carbon is altered m the process, the reaction is said to involve an inversion of configuration. If the configuration of the asymmetric carbon remains the same, the transformation occurs with retention of configuration.
  • Isolation and purification of the compounds and intermediates described herem can be effected, if desired, by any suitable separation or purification procedure such as, for example, filtration, extraction, crystallization, column chromatography, thin-layer chromatography, thick-layer (preparative) chromatography, distillation, or a combination of these procedures
  • suitable separation and isolation procedures can be had by references to the examples hereinbelow. However, other equivalent separation or isolation procedures can, of course, also be used
  • an amine component is silylated and then treated with an activated Fmoc-reagent to yield a Fmoc protected amine.
  • the amine component can be utilized in a soluble format or can be attached to a solid support.
  • the amine component will include a functionality which can covalently bind the molecule to the solid support (e.g., an activated carbonyl, acyl halide, or activated hydroxyl) as well as the amino group or a protected derivative thereof.
  • the amine component will comprise a primary or secondary amme having the formula: -NR'R", where R' and R" are independently selected from the group consisting of hydrogen alkyl, alkoxy, amino, aryl, aryloxy, heteroaryl, and arylalkyl or salts thereof, provided that either R' or R" is hydrogen. More preferably, the amine component will have the formula:
  • Rl and R ⁇ are independently selected from the groups consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, heteroaryl, carboxyl, carboxyalkyl, carboxyaryl, and arylalkyl.
  • the amine component if not commercially available, can be prepared by standard chemical procedures.
  • the amine component will comprise an amino acid, and more preferably, an amino acid bearing a substituent on the alpha carbon
  • the amino acids finding utility in the present invention include the twenty naturally occurring ⁇ -ammo acids, in either their D- or L-enantiome ⁇ c forms.
  • Unnatural amino acids such as ⁇ , ⁇ -disubstituted amino acids, N-al yl amino acids, lactic acid, and other unconventional amino acids are also suitable components.
  • unconventional amino acids include, but are no: limited to: 4-hydroxyproline, O-phosphose ⁇ ne, 3-methylhistidine, 5- hydroxylysine, and other similar amino acids. Since peptides are composed of amino acid subunits, one of skill in the art will appreciate that peptides can also serve as amine components.
  • the amine component is a photochemically cleavable linking group which can be represented by the formula:
  • R > is hydrogen, C -Cg alkyl, aryl or arylalkyl
  • R3, R4 and R5 are each independently hydrogen, C -Cg alkyl, or Cj-Cg alkoxy
  • ⁇ ll and Y l are each independently selected from the group consisting of halogen, — COOH, — SK,
  • R? and R ⁇ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl; and q is ar. integer of from 1 to 10, and preferably, from 1 to 4.
  • the photocleavable linking groups have the formula:
  • R6 is hydrogen, Cj-Cg alkyl; R 4 and R ⁇ are each independently hydrogen, C ⁇ -Cg alkyl or -Cg alkoxy; R ⁇ is Cg alkoxy; X 11 and Y*l are each independently selected from the group consisting of — Br, — Cl, — OH, — OP, — NH2, — NHP, in which P is a suitable protecting or activating group, and —
  • R 7 and R ⁇ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl; and q is an integer of from 1 to 4.
  • R6 is hydrogen or methyl
  • R° and R ⁇ are both hydrogen
  • R3 is methoxy
  • X 11 is — NH2 or — NR 7 R8 wherein R 7 is hydrogen and R 8 is lower alkyl, more preferably, methyl.
  • the photocleavable linking groups have the formula:
  • R° is hydrogen, Cj-Cg alkyl; R ⁇ and R are each independently hydrogen,
  • R ⁇ is C ⁇ -Cg alkoxy
  • X ⁇ and ⁇ ll are each independently selected from the group consisting of — Br, — Cl, — OH, — NH2, —
  • R ⁇ is hydrogen or methyl
  • R ⁇ and R4 are both hydrogen
  • R3 is methoxy
  • X 11 is — H2 or — NR 7 R 8 wherein R 7 is hydrogen and R 8 is lower alkyl, more preferably, methyl.
  • Particularly preferred photocleavable protecting groups are: wherein R6, R5 and R 4 are hydrogen, R3 is methoxy, Y 11 is — OH, and X 11 is — NH2, and q is 1; wherein R ⁇ , R5 and R4 are hydrogen, R ⁇ is methoxy, YU is — OH, and ⁇ ll is — NH2, and q is 3; wherein R6, R5 and R 4 are hydrogen, R ⁇ is methoxy, Y*l is — OH, and X 11 is — NHMe, and q is 1; wherein R6, R5 and R 4 are hydrogen, R ⁇ is methoxy, Y l is — OH, and X 11 is — NHMe, and q is 3; wherein is methyl, R ⁇ and R4 are hydrogen, R ⁇ is methoxy, YU is — OH, and X 11 is — NH2, and q is 1; wherein R ⁇ is methyl, R ⁇ and R4 are hydrogen
  • Suitable protecting groups include acid-labile, base-labile, photoremovable, or removable under neutral conditions. See, e.g., Green, Protecting Groups in Organic Synthesis. Wiley 1985, pp. 218-288, which is incorporated herein by reference. The choice of a particular protecting group will be determined generally by the reaction conditions and by the types of protecting groups present on other components to be used in synthesis. In a most preferred embodiment, the protecting groups are photoremovable and their removal is accomplished by exposing the surface or selected regions thereof to light (e.g., from a light source through a mask) or removable under neutral conditions. Such protecting groups and techniques are described in U.S. Patent No. 5,148,854 and co-pending U.S. Patent Applications Serial No. 07/624,120, filed December 6, 1990, and 07/971,181, filed November 2, 1992.
  • the compound to be protected is first silylated through treatment with a mono- or disilyl acetamide or other agents capable of silylating an amine group without generating acid by-products.
  • a mono- or disilyl acetamide or other agents capable of silylating an amine group without generating acid by-products Preferably, N-methyl-N- (trimethylsilyl)trifluoracetamide (MSTFA), N,0-bis(trimethylsilyl)acetamide (BSA), N-methyl-N-(trimethylsilyl)acetamide (MSA), N,0-bis (tr ⁇ methylsilyl)trifluoroacetamide (BSTFA) or bistrimethyl silylurea (BSU) is used (each available from Aldrich Chemical Co., Milwaukee, WI; or Fluka, Buchs
  • an excess of the silylating agent preferably, two or more silylating equivalents, and more preferably, between about two and five equivalents, is used.
  • one additional silylating equivalent of silylating agent is used.
  • the silylation reaction is conducted in an aprotic solvent, such as methylene chloride, and at a temperature between about room temperature and 100°C, preferably, between about room temperature and about 50°C, and most preferably, at about the temperature of the refluxing solvent.
  • an aprotic solvent such as methylene chloride
  • an activated Fmoc-reagent preferably, Fmoc-Cl; Fmoc-OPfp or Fmoc-OSu, most preferably, Fmoc-OSu (each available from Aldrich Chemical Co., Milwaukee, WI; Fluka, Buchs Switzerland or Ronkonkoma, NY, or Chemlmpex).
  • 1.0 and 3.0 equivalents Preferably, between about 1.0 and 2.0 equivalents of activated Fmoc-reagent is used. Most preferably, about 1.0 equivalents of Fmoc-OSu is used. 17
  • the reaction is typically conducted at a temperature between about 0°C and 100°C, preferably, between about 0°C and 50°C, and most preferably at about room temperature. The reaction generally will go to completion within about 24 hours.
  • the reaction can be worked up under anhydrous conditions, typically, by first adding a polar, protic solvent, such as a lower alkanol, such as methanol. This leads to the conversion of the trimethylsilyl ester into the free acid and methoxytrimethylsilane.
  • the free acid may be a crystalline product that is insoluble in methanol and thus, can be further purified by washing it with methanol to obtain, after drying, a pure product. This is the method of choice for the preparation of the Fmoc-photolinker. If, however, the free acid is not insoluble in methanol, the reaction can be worked-up as shown below and described in detail in the examples:
  • the present methods provide for a high yield of Fmoc-protected amines with a high degree of purity.
  • the yield will be greater than 80%, preferably, greater than 90%, and more preferably, greater than 95%, as detailed in the experimental section below.
  • Fmoc dipeptides can occasionally be found as side-products in preparations of Fmoc amino acids, (see, Sigler supra.), the presence of possible Fmoc dipeptide impurities was investigated.
  • Fmoc (Gly-Gly) and Fmoc (Phe-Phe) were prepared as reference compounds with the newly developed procedure. The results are shown in the next table:
  • the reaction sequence described herein therefore can be characterized as being highly stereoselective and may be stereospecific.
  • the reaction sequence described herein will exhibit at least about a 60% percent enantiomeric excess overall; preferably, at least about a 70% percent enantiomeric excess; more preferably, at least about a 80% percent enantiomeric excess; and even more preferably, at least about an 90% percent enantiomeric excess, where a perfectly stereospecific reaction sequence would have a 100% percent enantiomeric excess.
  • the mixture was extracted three times with ethyl acetate.
  • the organic phase was washed with water and brine and then dried over sodium sulfate.
  • the solvent was removed by rotary evaporation and the product was dried at 50°C under vacuum.
  • This example illustrates the use of MTSFA to produc Fmoc phenylalanine.
  • the product was dried at 70°C at 50 mm Hg to obtain 88.2 g photolinker, melting point 199-202°C.
  • HPLC the product was 100% pure.
  • the mother liquors and washes were combined and stripped to dryness. Trituration and thorough washing with methanol yielded, after drying, an additional 6.5 g product, melting point 197-200°C.
  • the overall yield was 91.1%.
  • a method of making Fmoc-protected ammo acids comprising the steps of: (a) treating an amino acid with a silylating agent to form a silylated intermediate; and
  • silylating agen is selected from the group consisting of N-methyl-N-(trimethylsilyl)trifIuoracetam ⁇ de, N,0- bis(trimethylsilyl)acetamide, N-methyl-N-(trimethylsilvt)acetamice, N,0-bis (trimethylsilyl)trifluoroacetamide, and bistrimethylsilylurea.

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Abstract

Procédés de synthèse d'amines protégées par 9-fluorénylméthoxycarbonyle (Fmoc), consistant à traiter un constituant amine au moyen d'un agent de silylation puis d'un réactif Fmoc activé. Ces procédés peuvent être mis en oeuvre dans des conditions anhydres.
PCT/NL1997/000235 1996-04-30 1997-04-29 Procedes de synthese d'amines protegees par fmoc WO1997041093A1 (fr)

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US7790765B2 (en) 2007-04-30 2010-09-07 Arqule, Inc. Hydroxy sulfonate of quinone compounds and their uses
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US6887701B2 (en) 1999-07-30 2005-05-03 Large Scale Proteomics Corporation Microarrays and their manufacture
US7179638B2 (en) 1999-07-30 2007-02-20 Large Scale Biology Corporation Microarrays and their manufacture by slicing
US7812051B2 (en) * 2004-08-11 2010-10-12 Arqule, Inc. Pharmaceutical compositions of β-lapachone and β-lapachone analogs with improved tumor targeting potential
US8614228B2 (en) 2004-08-11 2013-12-24 Arqule, Inc. Quinone prodrug compositions and methods of use
US7790765B2 (en) 2007-04-30 2010-09-07 Arqule, Inc. Hydroxy sulfonate of quinone compounds and their uses
US20140303344A1 (en) * 2007-11-19 2014-10-09 Bcn Péptides, S.A. Method for the stereoselective preparation of amino acid derivatives

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