WO2000053579A2

WO2000053579A2 - Solid phase synthesis of 4-aminoproline derivatives and/or combinatorial libraries thereof

Info

Publication number: WO2000053579A2
Application number: PCT/US2000/006021
Authority: WO
Inventors: Michael Douglas Hocker; Matthew Plunkett
Original assignee: Axys Pharmaceuticals, Inc.
Priority date: 1999-03-10
Filing date: 2000-03-08
Publication date: 2000-09-14
Also published as: AU3517400A; WO2000053579A3

Abstract

The present invention relates to solid phase synthesis of proline based compounds of Formula (1): R4R5NC(O)-Q-N(R1)-Y-R2 wherein R1 is an optionally substituted alkyl; R2 is alkyl, or optionally substituted aryl; R?4 and R5¿ are independently selected from cycloalkyl, or optionally substituted alkyl; R?4 and R5¿ taken together represent a five to seven membered saturated or unsaturated heterocyclic ring; Q represents (a) or (b), Y represents -S(O)¿n?-, -C(O)-, or -C(O)-NH-. Compounds of Formula (1) are prepared by a process which enables individual, parallel, and simultaneous synthesis of a plurality of compounds represented by Formula (1), starting from a compound of Formula (A): LG-C(O)-Z wherein LG represents a leaving group, Z represents (c), PG represents a protecting group.

Description

PROLINE DERIVATIVES AND SYNTHESIS THEREOF

FIELD OF INVENTION

The present invention relates to solid phase synthesis of proline based compounds of Formula 1.

BACKGROUND OF THE INVENTION

There has been much interest in synthesizing compounds which can be evaluated to determine their biological activity. Compounds have been traditionally synthesized on an individual basis. This individual synthesis of compounds is time consuming, and hence new methods for rapid synthesis of compounds are constantly sought.

Techniques have been developed in which one sequentially adds individual units as part of a synthetic process to produce a substantial number of compounds. One such technique is known as split synthesis which comprises dividing a first pool of materials into sub-pools, treating these sub- pools so as to effect a change and then again dividing the changed material into a new set of sub-pools for further treatment. This process is repeated until the desired end products are obtained.

In spite of the different synthetic techniques known in the art, the pharmaceutical industry, for example, is in search of new processes that will enable synthesis of a large number of compounds i.e., a library of compounds, at a relatively rapid pace. There is thus a need for a process for rapid parallel synthesis of multiple compounds. SUMMARY OF THE INVENTION

Keeping the above discussed needs in mind, the present invention provides a process for synthesis of a compound of Formula 1 :

R⁴R⁵NC(0)-Q-N(R )-Y-R² Formula 1 wherein

R¹ represents a C,-C₄ straight chain or a C₄-C₈ branched alkyl radical, substituted with one or more substituents selected from a group consisting of

H, tertiary amine, optionally substituted aryl, optionally substituted hetero cycloalkyl and optionally substituted heteroaryl;

R² represents C,-C₄ alkyl, optionally substituted aryl, or optionally substituted heteroaryl; R⁴ and R⁵ can be the same or different, and are independently selected from a group consisting of C₅-C₈ cycloalkyl, C,-C₈ straight chain alkyl, and C_4.8 branched alkyl, said alkyl groups substituted with (CH ^-O^CH ^-OH,

(CH₂),_.4-OH, R⁶, or optionally substituted heteroaryl; alternatively

R⁴ and R⁵ along with the nitrogen atom that they are attached to can be taken together to represent a five to seven membered saturated or unsaturated heterocyclic ring optionally substituted with R⁶;

Q represents

Y represents -S(0)_n-, -C(O)- , or -C(0)-NH- R⁶ represents H, C,-C₄ alkyl, C₄-C₈ branched alkyl, N[(CH₂)_1.4-CH₃]₂, (CH₂)_M-

OH, OC,.₄ alkyl, C_6.10 aryl, optionally substituted-C_5.10 saturated or unsaturated heterocyclyl, C_5.10 ara-C,_.4 alkyl, C_5.10 heteroaryl, or C₅-C₁₀ heteroara-C^ , alkyl; R¹⁰ and R¹¹ independently at each occurrence represent H, C₄-C₈ branched alkyl, (CH₂)₁.₄-0-C₁-C₄-alkyl, C C₄-alkyl substituted with R¹², C₆-C₁₀- substituted or unsubstituted aryl or heteroaryl, C₆-C₁₀ ara-C C₄-alkyl, or C₆-C₁₀- heteroara-C,-C₄-alkyl;

R¹² represents R⁶, O-C^C.-alkyl, O-C_6.10-optionally substituted aryl, or 0-C₆- C^-ara-C^ , alkyl;

R¹⁴ represents C_5.10-heteroaryl, C,_.4-alkyl, said alkyl substituted with H, substituted or unsubstituted C_6.10-aryl, and substituted or unsubstituted C_5.10- heteroaryl; and n represents an integer from 1-2; said process comprising the steps of:

(a) reacting a compound of Formula A

LG-C(0)-Z Formula A

wherein LG represents a leaving group, Z represents

PG

N

^N> D1 1

O

PG represents a protecting group, and R¹¹ is as defined above, with a linker molecule comprising an amine group represented by

SS-LM-NH, wherein SS is a solid support and LM is a linker molecule, to yield a product represented by Formula B

SS-LM-NHC(0)-Z Formula B

wherein SS, LM, and Z are as defined above;

(b) reacting the product represented by Formula B with a compound of

Formula C

R¹-NH₂ Formula e

to yield a product of Formula D,

SS-LM-NH-C(0)-QP-NHR¹ Formula D

wherein SS, LM, and R¹ are as defined above, QP represents

PG

PG and R¹¹ is as defined above; (c) reacting a product of Formula D with a compound of Formula E

X-Y-R² Formula E wherein Y is as defined above, and X represents Cl, Br, F, or I, to yield a product of Formula F

SS-LM-NH-C(0)-QP-N(R¹)-Y-R²; Formula F,

wherein SS, LM, QP, Y, R¹, R² are as defined above; (d) sequentially reacting a product of Formula F with: (i) a protonating agent; (ii) an epoxide represented by Formula G

A

R 10

wherein R¹⁰ is as defined above; or an acid represented by Formula H

R¹⁴-COOH Formula H where R¹⁴ is as defined above;

(iii) a protonating agent;

(iv) trimethyl silyldiazomethane; and (v) a compound of Formula R⁴R⁵NH, to yield a compound of Formula 1

DETAILED DESCRIPTION

One embodiment of the present invention provides a process wherein step (a) is carried out at a temperature in the range of from about -78 °C to about 25 °C, with the temperature range of from about -50 °C to about 0 °C being preferred. A particularly preferred temperature for the process of the present invention ranges from about -40 °C to about -10°C.

Another embodiment of the present invention provides a process wherein step (d) (ii) comprises an epoxide represented by Formula G:

wherein R¹⁰ represents H, C₄-C₈ branched alkyl, (CH₂)_1.4-0-C,-C₄ alkyl, C,-C₄ alkyl substituted with R¹², C₆-C₁₀ substituted or unsubstituted aryl or heteroaryl, C₆-C₁₀ ara-C^C,, alkyl, or C₆-C₁₀ heteroara-C,-C₄ alkyl.

Yet another embodiment of the present invention provides a process wherein step (d) (ii) comprises an acid represented by Formula H:

R¹⁴-COOH Formula H

wherein R¹⁴ is as defined above. Preferred embodiments of the present invention provide a process wherein P.¹ represents a C,-C₄ straight chain alkyl radical substituted with H, optionally substituted hetero cycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; R² represents C,_.2 alkyl, or substituted aryl; R⁴ and R⁵ can be the same or different, and are independently selected from C ₄ straight chain alkyl, C₄.₈ branched alkyl, and H, said alkyl groups substituted with R⁶; or R⁴ and R⁵ when taken together along with the nitrogen atom that they are attached to represent a five to six membered heterocyclic ring; R^δ represents H, C_M alkyl, N[(CH₂)_1.4CH₃]₂, (CH₂)_0.4OH, C_6.10 aryl, optionally substituted-C_5.6 saturated or unsaturated heterocyclyl, or C_6.1C aryl; R¹⁰ and R' independently at each occurance represent H, or C,_.4 alkyl substituted with R⁶; R¹² represents R⁶, or phenyl substituted with one or more of halogen, C,_.4 alkyl, and C_1.4alkoxy; and R¹⁴ represents C_5.10 heteroaryl, C,.., alkyl, said alkyl substituted with H, Ph, or C_5.10 unsubstituted heteroaryl.

Another preferred embodiment of the present invention provides a process wherein R² represents a -C C₂ alkyl,

R⁸ and R⁹ independently at each occurrence represent R⁶ or C(0)-C_1.4 alkyl. Preferred R⁸ and R⁹ groups are H, OC,-C₂ alkyl, C.-C₂ alkyl, and C(0)-CH₃.

DEFINITIONS

As used in the present invention the following terms and abbreviations have the following meaning, unless otherwise indicated.

Parallel synthesis: This term indicates simultaneous synthesis of independent (individual) compounds.

Library of compounds: This term indicates a collection of independent (individual) compounds. Generally the term library of compounds indicates a collection of individual compounds distinct from one another. Also included in the library of compounds is a mixture of the individual compounds.

"Alkyl", or "alkyl radical" is meant to indicate a hydrocarbon moiety of up to 8 carbon atoms. This hydrocarbon is generally attached to at least one other atom, and can be straight chain, or branched, or cyclic. The term "alkylene" represents a divalent hydrocarbon having from 1 to 10 carbon atoms. Illustrative examples are methylene (-CH₂-), ethylene (-CH₂-CH₂-), and propylene (-CH₂-CH₂-CH₂-).

The term "alkelene" represents an alkyl group, as defined above, except that it has at least one center of unsaturation, i.e., a double bond. Illustrative examples are butene, cyclo butadiene, propene, and pentene.

The term "cycloalkyl", "cycloalkyl ring", or "cycloalkyl radical" indicates a saturated or partially unsaturated three to ten carbon monocyclic or bicyclic hydrocarbon moiety which is optionally substituted with an alkyl group. The term "straight chain alkyl" is meant to represent an unbranched hydrocarbon moiety of up to 8 carbon atoms. An example of a straight chain alkyl is a n- pentyl group.

The term "hetero cycloalkyl" or "hetero cycloalkyl radical" means cycloalkyl, as defined above, except one or more of the carbon atoms indicated are replaced by a hetero atom chosen from N, NR²⁰, O , S(O), S(0)₂ and S, wherein R²⁰ is (C_1.6)alkyl, hetero(C_2.6)alkyl or hydrogen. Illustrative examples of the term heterocyclo(C_5.14)alkyl are morpholinyl, indolinyl, piperidyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, quinuclidinyl, morpholinyl, etc.).

As used in the present invention, the illustration:

generally indicates a point of attachment of the group, comprising the illustration, to another group or atom.

The term "Ph" represents an optionally substituted phenyl radical or group. The term "aryl" means an aromatic monocyclic, bicyclic, or a fused polycyclic hydrocarbon radical containing from 6 to 14 carbon atoms indicated.

Thus a C₆-C₁₄ aryl group includes phenyl, naphthyl, anthracenyl, etc. The term "heteroaryl" means aryl, as defined above, wherein one or more of the carbon atoms is replaced by a hetero atom chosen from N, O, and S. The hetero atoms can exist in their chemically allowed oxidation states. Thus Sulfur (S) can exist as a sulfide, sulfoxide, or sulfone. Each heteroaryl ring comprises from five (5) to fourteen (14) atoms. Illustrative examples of heteroaryl groups are thienyl, furyl, pyrrolyl, indolyl, pyrimidinyl, isoxazolyl, purinyl, imidazolyl, pyridyl, pyrazolyl, quinolyl, and pyrazinyl.

"Optional substituents" for aryl, hetero aryl, and Ph groups, unless otherwise indicated, are one or more substituents independently selected from a group consisting of H, C,.₄ alkyl, NH₂, halogen, 0-C,.₄ alkyl, NHC,-C₄ alkyl, N(C,-C₄)₂ alkyl, and CF₃. The term "tertiary amine", as used herein, represents a group containing a nitrogen atom substituted with a group other than a hydrogen atom. Illustrative examples of such groups are alkyl, cycloalkyl, and ketones. "Optional" "or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, the phrase "optionally is substituted with one to three substituents" means that the group referred to may or may not be substituted in order to fall within the scope of the invention.

The term "halogen" represents at least one of chlorine, bromine, iodine, and fluorine radicals. The term "linker molecule" "LM" as used in the present invention, represents a covalent moiety commonly known to one skilled in the art as tether for attaching the molecules of interest to a solid support. The term solid support (SS), as used in the present invention, signifies polymeric material for supported synthesis. A detailed description of the terms linker molecule, and solid support can be found in The Combinatorial Index, B. A. Bunin, 1998, which is incorporated herein by reference.

As used in the present invention, the term "LG" or "leaving group" is as understood by one skilled in the art, and is intended to represent a group that will be replaced by another group which generally acts as a nucleophile. A detailed description of leaving groups can be found in Organic Chemistry, K. P. C Vollhardt, 1987. Illustrative examples of leaving groups are halogen (Cl, I, Br, and F), tosylate, mesylate, and triflate. "Protecting group" or "PG", as used in the present invention, is a group that is attached to, or placed on, an atom so the protected atom does not react with reactants, thereby temporarily rendering the protected atom inactive. Illustrative examples of protecting groups are tetrahydropyran (THP), tert-butyl- oxy carbonyl (BOC), and fluoromethyloxy carbonyl (FMOC). A comprehensive list and description of protecting groups can be found in Protective groups in Organic Synthesis, second edition, T.W. Greene and P.G.M. Wuts, 1991 , which are incorporated herein by reference.

"Inert solvent" as used herein represents solvents which do not react with the reagents dissolved therein. Illustrative examples of inert solvents are tetrahydrofuran (THF), methylene chloride, dichloro methane (DCM), ethyl acetate (EtOAc), dimethyl formamide (DMF), diaoxane, chloroform, and dimethyl sulfoxide (DMSO). The term "protic solvent" is intended to mean a solvent which has an acidic proton and generally the solvent is polar, as understood by one skilled in the art. Illustrative examples of protic solvents are methanol, ethanol, propanol, and water. A detailed description of the term protic solvent can be found in Organic Chemistry, K. P. C Vollhardt, 1987, and is incorporated herein by reference.

The term "protonating agent", as used herein, signifies an agent which provides a proton to electron rich atoms such as a nitrogen atom. Illustrative examples of protonating agents are hydrochloric acid, acetic acid, and trifluoroacetic acid. The term protonating agent is known to one skilled in the art, and a detailed description of the same is available in Protective Groups in Organic Synthesis, 2nd edition, T. W. Greene and P. G. M. Wuts, 1991 , which is incorporated herein by reference. The following abbreviations used in describing the process of the present invention have the following meaning: AcOH: acetic acid

DCM: dichloromethane

DIEA: diisopropylethylamine DIC: diisopropylcarbodiimide. DMSO: dimethylsulfoxide

ETOAc: ethyl acetate

HoBt: hydroxybenzo triazole

MeOH: methanol

MeCN: acetonitrile

SLE: liquid-liquid solid supported extraction, or solid-liquid extraction

TEA: triethylamine

TLC: thin layer chromatography

THF: tetrahydrofuran

TFA: trifluoroacetic acid

EXPERIMENTAL DETAILS

The following procedures will further illustrate the process of the present invention. The general procedures of the present invention are outlined below.

Preparing compound SS-LM-NH 2

The Compound represented by SS-LM-NH₂, can be purchased from Nova Biochem, or prepared using the following general procedure: An aminomethylpolystyrene resin was mixed with 2 to 4 mole equivalents each of a carboxysulfonamide (in particular carboxyarylsulfonamide), hydroxybenzo triazole (HOBt), and diisopropylcarbodiimide (DIC). An inert solvent, preferably dimethyl formamide (DMF) was then added to this mixture until the resin swelled and this mixture was then stirred from 12 to 24 hours, to yield a compound represented by SS- LM-NH₂.

Preparation of SS-LM-NHC(0)-Z (Formula B)

In a round bottom flask was mixed from 1 to 3 equivalents of LG-C(0)-Z, a compound of Formula A, from 1 to 5 equivalents of a base, preferentially DIEA, and from 1 to 2 equivalents of SS-LM-NH₂, in an inert solvent, preferably dichloromethane (DCM). This mixture was stirred at a temperature ranging from about 25°C to about -70°C, followed by addition of 0.5 to 2 equivalents of benzotriazole-1 -yl-oxy-tris-pyrrolidino-phosphonium hexafluoro-phosphate

(PyBOP) with stirring. The resulting resin was washed at room temperature using a protic solvent followed by an inert solvent to yield a product of Formula B. Preparation of SS-LM-NH-C(0)-NHR¹ (Formula D)

A solution of a compound of Formula B in an inert solvent, preferably dichloromethane, was mixed in rapid succession with trimethyl orthoformate, a protic acid, preferably acetic acid, and R¹-NH₂ (a compound of Formula C). To this stirring mixture was added a reducing agent, preferably sodium triεcetoxyborohydride. The resulting mixture was stirred for up to an additional four hours. The resin thus formed was washed with a protic solvent, followed by treatment with an acid solution. A final wash with a protic solvent, preferably methanol followed by vacuum drying gave a compound of Formula D.

Preparation of SS-LM-NH-C(0)-QP-NR¹-Y-R² (Formula F)

A compound of Formula D was mixed with an inert solvent, and X-Y-R²

(a compound of Formula E), optionally in the presence of a base. The resulting mixture was agitated for 0.5 to five hours followed by rinsing with an inert solvent to yield a compound of Formula F.

Preparation of a Compound of Formula 1

A product of Formula F, in an inert solvent, was sequentially treated with an acid, followed by rinsing with methanol and drying under vacuum. This acid treated compound was further reacted in one of the ways, as discussed below. This acid treated compound was then either treated with a solution of an epoxide of Formula G, (when step (d) (ii) comprises an epoxide) followed by treatment with an amine, preferably a tertiary amine such as triethyl amine. (The epoxide solution was freshly prepared just before use. A preferred solvent system was an aqueous mixture of dimethyl formamide (DMF). An epoxide solution in a solvent system which is a mixture of a protic and non protic polar or non polar solvent system was preferred.) The reaction vessel was then sealed and the mixture therein agitated for 8 to 16 hours.

The reaction vessel was sealed and agitation of the reaction mixture started within one hour of the preparation of the epoxide solution. Following the 8-16 h agitation, the resulting resin was rinsed with a polar solvent, preferably DMF, followed by methylene chloride. This rinsing was then followed by vacuum drying.

Alternatively, the acid treated compound was reacted with a DMF solution of a carboxylic acid, (when step (d) (ii) comprises an acid represented by Formula H) followed by the addition of diisopropylethyl amine (DIEA). The solution was then mixed with PyBOP. This mixture was allowed to react for up to three hours, and the reaction plates were rinsed with DCM. The DCM was removed by conventional methods.

The next step in the sequence involved treating the respective compounds with an acid, preferably trifluoroacetic acid (TFA), rinsing with an inert solvent, and followed by treatment with a solution of an alkylating agent, preferably diazomethane or trimethyl siiyl diazomethane (TMS-diazomethane), in an inert solvent. The alkylating agent was quickly washed off using an inert solvent, thereby avoiding side reactions. The final step in the sequence involved the overnight reacting of the above diazomethane treated compound with an amine, represented by R⁴R⁵NH, in an inert solvent, preferably THF, to yield a compound of Formula 1.

Detailed Procedure and Examples

The following detailed procedures and examples will further illustrate the process of the present invention. Preparing compound SS-LM-NH₂

Aminomethylpolystyrene resin (MidWest Biotech, ~1 mmol/g) was added to a laboratory bottle followed by addition of 4- carboxybenzenesulfonamide (obtained from Aldrich Chemicals, MW = 201 , 2.0 mole equiv.) and HOBt (MW = 153, 2.0 mole equivε'ents). DMF was added until the minimum volume needed to swell the resin was reached (approx. 8 mL / g resin). DIC (MW = 126, d = 0.806, 2.0 mole equiv.) and the reaction mixture was then agitated for 8-16 h. The next day the resin was transferred to a fritted glass funnel and rinsed (with 30 sec. stirring between rinses) with DCM (2x) and MeOH (1x). The rinse cycle was repeated until no Ultra violet (UV) active material was visible on a TLC plate in the wash solvents. The resin was dried under vacuum to a constant mass, to yield the compound SS-LM-NH₂.

Oxidation of Boc-Hydroxyproline-OH (Boc-Hyp) to give Boc-Ketoproline

Dimethyl sulfoxide (DMSO) (83.9 g) was added to DCM (500 mL) in a 2 L round bottomed flask equipped with a mechanical stirrer and under a nitrogen atmosphere. The round bottom flask was placed in a dry ice / acetone bath and allowed to cool to about -78°C. After 20 min 100 g of oxalyl chloride was added drop wise to the stirring solution (1 drop/sec). The reaction mixture was stirred for 30 minutes. In a separate round bottom flask, 166 g of BocHyp was dissolved in 200 mL of anhydrous THF. The flask was sealed under nitrogen and placed into a dry ice / acetone bath and the reaction mixture was stirred until it reached a temperature of about -72°C. The BocHyp solution was then transferred with a large bore cannula into the stirring solution. After all of the Boc-Hyp solution was transferred the reaction was stirred for one hour. The temperature was maintained at -72°C with the addition of dry ice, as needed. Triethyl amine (TEA) (400 mL) was added drop wise to the solution over 30 min. The reaction mixture was removed from the dry ice / acetone bath and was allowed to warm to 0°C. The flask was then placed into an ice bath with stirring for 1 h. Aqueous sodium carbonate (84 g, 1.1 eq, 1 L ) and was added to the reaction mixture. The flask was removed from the ice bath and was stirred for an additional 1 h. The mixture was then poured into a 3 L separatory funnel, DCM (1 L) added, shaken, and the aqueous layer was separated. The remaining organic solution was then extracted with additional Na₂C0₃ (0.5 N, 500 mL) solution, which was combined with the aqueous solution. This combined aqueous solution was then extracted with ethyl acetate (EtOAc) (1 L). The aqueous phase was separated and concentrated HCI was added in drop wise portions (about 0.5 ml per minute) with stirring until pH of the aqueous layer was 3. This aqueous phase was then extracted twice with EtOAc (1 L each), the organic layers combined, 250 g NaCl was added to the aqueous phase, and a final extraction of EtOAc (1 L) was performed. This was combined with the previous EtOAc layers. TLC was performed (silica plates; 50:25:1 EtOAc: hexanes: AcOH; ninhydrin stain) to confirm that the product was completely removed from the aqueous layer. Na₂S0₄ (200 g) was added to the EtOAc and the solution was allowed to sit for 30 min. This solution was filtered through a sintered glass funnel and the filtrate was concentrated and dried under vacuum to yield a solid. MeCN (250 mL) was added to the solid, which was broken up with a spatula. The mixture was heated to 40°C and stirred magnetically in a vigorous manner for 30 min. The product was then cooled gradually to -5°C, and the resulting crystals were collected by filtration to yield Boc-ketoproline. Product identity and purity were evaluated by HPLC, TLC, and NMR. (Product purity was improved, as needed, by repeating the MeCN crystallization.) The solid was dried under vacuum. Acylation of the resin

SS-LM-NH, + LG-C(0)-Z ► SS-LM-NHC(0)-Z

Formula B LG (leaving group) = oxybenzotriazole (OBT)

Z =

R¹¹ = H. A round-bottom flask was charged with DCM (5 mL / g resin), followed by Boc-Ketoproline (2 equiv), DIEA (4 equiv), and sulfonamide resin (1 equiv). The mixture was cooled to -25 °C in a dry ice / MeCN bath and PyBOP (2 equiv) was added with vigorous stirring. The mixture was allowed to react for 30 min, at which point the dry ice bath was removed and the flask was allowed to warm to room temperature. The resin was rinsed with MeOH and DCM several times to yield the desired compound of Formula B.

Reductive amination:

SS-LM-NHC^Z + R'-NHs ► SS-LM-NHC(O)-QP-NHR'

Formula B Formula D

Ketoproline-derived resin (a compound of Formula B, ~1 mmol) and

DCM (5 mL / g resin) were added to an appropriately sized round bottom flask.

Trimethylorthoformate (0.80 mL / g resin), acetic acid (0.60 mL / g resin), and the appropriate reducing amine (R¹-NH₂, 4 equivalents) were added in rapid succession. Sodium triacetoxyborohydride (2.5 equiv., 0.53 g / g resin) was added and the mixture was stirred for 2 h. The reaction mixture was transferred to a fritted glass reaction vessel (LAMPS vessel), and the resin was rinsed with DCM (2 x) and MeOH (2 x). The resin was treated with a 90:10:1 solution of DCM : MeOH : TFA for 5 min, then rinsed with MeOH (4x). The resulting resin was dried under vacuum to yield the desired compound of Formula D.

The appropriate reducing amines used in this reaction step can be selected from tetrahydrofurfurylamine, tryptamine, N,N-diethylethylenediamine, butylamine, 4-(3-aminopropyl)imidazole, 1 -(3-aminopropyl)-4-methyl piperazine, 1 ,3-aminopropylpyrrolidinone, 4-(2-aminoethyl)morpholine, 2- fluorophenethylamine, 3-butoxypropyl-amine, 4-methoxybenzyl amine, and 3,4-dimethoxyphenethylamine.

Distal acylation:

SS-LM-NHC(O)-QP-NHR¹ + X-Y-R²

Formula D

SS-LM-NHC(0)-QP-N(R')-Y-R² Formula F

Three different classes of reagents used to acyiate the unreactive 4- aminoalkyl substituent were sulfonyl chloride, isocyanate, and acetic anhydride. Conditions for each acylation varied. The dried resin from the previous reductive amination step was partitioned into 6 reaction vessels, 26 g each. DCM (5 mL / g resin), DIEA (6 equiv.,) and the appropriate acylating agent (3 equiv.) were combined, agitated, and allowed to react for the specified amount of time. Stirring time for sulfonyl chlorides was 2h, isocyanates was 4 h, and for acetic anhydride was 4 h. The resin was rinsed with DCM (5 x) and taken on to the next step. Illustrative examples of acylating reagents that can be used in this step are acetic anhydride, 2,5-dimethylphenyl isocyanate, methyl isocyanate, tosyl chloride, 3,4-dimethoxybenzene sulfonyl chloride, 4-acetylphenyl isocyanate, and 8-quinoline sulfonyl chloride.

Preparation Of A Compound of Formula 1

SS-LM-NHC(O)-QP-N(R¹)-Y-R²

Formula F

SS-LM-NHC(O)-QH-N(R¹ )-Y-R²

The resin (swelled from the previous step in DCM) was treated with TFA

(5-10 mL / g resin) for 20 min. The resin was rinsed with MeOH until the effluent had a pH of less than 3. The resin was dried under vacuum until it was of constant mass.

Epoxide ring opening:

SS-LM-NHC(0)-QH-N(R¹)-Y-R²

SS-LM-NHC(0)-Q-N(R¹ )-Y-R² wherein Q is

The resin was partitioned into Polyfiltronics plates (-0.14 g/well, 1 plate per reducing amine/distal acylator combination). Each plate was placed into an open clamp (with a sheet of Teflon at the bottom) and sealed. Separately, epoxide solutions were prepared as described below. Each epoxide 90.8 mmol/well, 5 equiv., 80 mmol) was weighed and diluted to 100 ml with a solution of 20:80 water : DMF mixture. The solutions were mixed and added to the appropriate wells (1.0 ml /well). Finally, triethyl amine (TEA) (0.84 mmol/well, 5 equiv., 114 μL) was added to each well. It was very important that the reaction plates be sealed within one hour from the first dilution of the epoxides.

Plates were sealed at the top with a Beckman polyethylene square well cap. The cap was completely sealed, then a top clamp was placed over the polyethylene cap and tightened firmly with wingnuts. The plate was placed on its side and shaken for 8-16 h. The plate was removed from the clamp, filtered, and the resin was rinsed four (4) times with DMF, and then three (3) times with DCM. Each rinse solvent was allowed to gravity filter out the bottom of the Polyfiltronics plate. Residual solvent was removed by placing each plate on a vacuum box. This rinsing step was used for every resin rinsing step. It was important that the solvent used for rinsing not be added while the plates were on the vacuum box.

Illustrative examples of epoxides that can be used in this step are 4- (2,3-epoxypropyl) morpholine, glycidyl isopropyl ether, glycidyl 2-methylphenyl ether, glycidyl 4-methoxyphenyl ether, glycidol, propylene oxide, and N-(2,3- epoxypropyl)phthalimide.

Carboxylic acid acylation:

SS-LM-NHC(0)-QH-N(R¹)-Y-R^{2 ►} SS-LM-NHC(0)-Q-N(R¹)-Y-R² wherein Q is

The resin was partitioned into Polyfiltronics plates (-0.15 g / well, 2 plates per reducing amine / distal acylator combination). Each plate was placed into an open clamp and sealed. Separately, activated solutions of the 11 carboxylic acids were prepared as described below. Each carboxylic acid (0.5 mmol / well, 3 equiv., 48 mmol) was weighed into a separate reaction vessel. DMF (0.8 mL / well, 77 mL) and DIEA (1.5 mmol / well, 9 equiv., 144 mmol, 25.0 mL) were added. The solutions were mixed and PyBOP (3 equiv., 48 mmol, 25.0 g) was added. The solutions were again mixed and added to the appropriate wells (1.0 mL / well). The mixture was allowed to react for 1 h, the plate was taken from the clamp, filtered, and the resin was rinsed 3 times with DCM. After the DCM from each rinse solvent addition had been allowed to gravity filter out from the bottom of the Polyfiltronics plate, each plate was placed on the vacuum box to remove any remaining solvent. This type of cycle was used for every resin rinsing step.

Illustrative examples of carboxylic acids that can be used in this step are 3-benzoylpropionic acid, 4-methoxy-2-quinolinecarboxylic acid, mefenamic acid, benzoic acid, 2,6-dimethoxynicotinic acid, 2,5-dimethoxybenzoic acid, 5-methoxyindole-2-carboxylic acid, 2-(carboxymethylthio)-4-methylpyrimidine, 2-pyrazinecarboxylic acid, acetic acid, and 3,4-methylenedioxyphenyl acetic acid. Linker activation and amine cleavage:

SS-LM-NHC(0)-Q-N(R )-Y-R² ► R⁴(R⁵)NC(O)-Q-N(R¹)-Y-R²

Formula 1

Each well, from the preceding step, was treated with 1 % TFA in DCM (1 mL) and the solvent was allowed to filter by gravity. The plate was rinsed with DCM until the pH was less than pH 4. The plate was then reclamped and 1 mL of a 20% solution of commercial TMS-diazomethane (Aldrich, 2 M in hexanes) in DCM was added to each well. About 3 minutes later the resin was rinsed with DCM until no yellow color was seen in the effluent (generally 3-4 rinses).

The plates were placed into open clamps with Teflon sheeting to seal at the bottom, and the plates were clamped shut. A solution of the appropriate cleaving amine (0.5 M, 1.0 mL) in anhydrous, inhibitor-free THF was added to each well in the Polyfiltronic plate. The plate was placed into a sealed container containing enough THF to produce a saturated atmosphere of THF, and the reaction was allowed to proceed for 8-16 h at 25 °C. The resin was then rinsed with THF (2 x 0.5 mL) and the elute was collected into a 2 mL Beckman deepwell microtiter plate. The plates were concentrated on the Savant to dryness. Illustrative examples of cleaving amines used in this reaction step are dimethylamine, piperidine, 2-amino-5-diethylaminopentane, N,N- diethylethylene diamine, 2-aminoethoxyethanol, 4-(2-aminoethyl) morpholine, 1 -(3-aminopropyl)-4-methylpiperazine, tetrahydro- furfurylamine, butylamine, aminoethanol, ammonia, tryptamine, 1 -aminoindan, 1 -(4-fluorophenyl) ethylamine, 1 ,3-aminopropyl-pyrrolidinone, 3,4-dimethoxyphenethylamine, 2- amino-6-fluorobenzylamine, 4-hydroxypiperadine, 4-amino-1 - benzylpiperadine, and 4-(3-aminopropyl)-4-methylpiperazine.

11 Solid-liquid Extraction (SLE): Used to remove excess cleaving amines.

Cleaving amines generally fall into hydrophobic and water soluble groups. The SLE conditions for these two groups were different. For the hydrophobic set, a 2.7 micron Polyfiltronics plate was filled to within 5 mm of the top with Varian SLE packing material. To each well was added 0.4 mL of 2 M aqueous HCI. The residue was taken up in 0.5 mL of 1 :4 (v/v) THF / DCM and transferred to the top of the SLE plate (Matrix). The SLE plate was rinsed three more times with 0.5 mL of the 20% THF / DCM solution (Matrix). The purified products were collected in another Beckman plate. Concentration on the Savant and final drying under high vacuum gave the purified products.

For the water soluble amines, the SLE material was activated with deionized water, and the extractions were performed in 100% DCM. The following compounds (Standards 1 -12) were prepared using the process of the present invention.

Standard 1

Calculated MW = 618.39

Standard 2 Calculated MW =680.43

10 Standard 3 Calculated MW = 681.25

Standard 4 Calculated MW = 532.33

10

Standard 5 Calculated MW = 537.35

WO 00/53579 PCTVUSOO/06021

Standard 6: MDH21-6 Calculated MW = 765.39

Standard 7

Calculated MW = 653.34

10 Mass observed (M+1) MW: 654.5

Standard 8

Calculated MW = 754.32

15 Mass observed (M+ 1): 755.5

Standard 9

Calculated MW = 607.25

Mass observed (M+1): 608.3

Standard 10

Calculated MW = 697.33

Mass observed (M+1): 699.5

Standard 11

Calculated MW = 601.25

Mass observed (M+1): 602.3

Standard 12

Calculated MW = 701.34

Mass observed (M+1): 702.5

Claims

1. A process for synthesis of a compound of Formula 1 :

R⁴R⁵NC(0)-Q-N(R¹)-Y-R²

Formula 1 wherein

R¹ represents a C C₄ straight chain or a C₄-C₈ branched alkyl radical, substituted with one or more substituents selected from a group consisting of H, tertiary amine, optionally substituted aryl, and optionally substituted heteroaryl;

R² represents -C C₄ alkyl, or optionally substituted aryl; R⁴ and R⁵ can be the same or different, and are independently selected from a group consisting of -C₅-C₈ cycloalkyl, -C C₈ straight chain alkyl, and C₄.₈ branched alkyl, said alkyl groups substituted with -(CH₂),_.4-0-(CH₂)_1.4-OH, - (CH₂),_.4-OH, R⁶, or optionally substituted heteroaryl; alternatively R" and R⁵ along with the nitrogen atom to which they are attached can be taken together to represent a five to seven membered saturated or unsaturated heterocyclic ring optionally substituted with R⁶; Q represents

Y represents -S(0)_n-, -C(O)- , or -C(0)-NH- ;

R⁶ represents H, -C,-C₄ alkyl, C₄-C_B branched alkyl, -N[(CH₂)_1.4-CH₃]₂, -(CH_a)_M- OH, -OC,.₄ alkyl, -C₆.₁₀ aryl, -C₅.₁₀ saturated or unsaturated heterocyclyl, -C_5.10 ara-C,.,, alkyl, -C₅.₁₀ heteroaryl, or -C₅-C₁₀ heteroara-C,-C₄ alkyl; R¹⁰ and R ¹ independently at each occurrence represent H, -C₄-C₈ branched alkyl, -(CH₂)_M-0-C,-C₄ alkyl, -C,-C₄ alkyl substituted with R¹², -C_β-C₁₀ substituted or unsubstituted aryl or heteroaryl, -C₆-C₁₀ ara-C^C,, alkyl, or -C₆-C,₀ heteroara-C,-C₄ alkyl;

R¹² represents R⁶, -0-C,-C₄ alkyl, or -O-C₆-C₁₀ ara-C C₄ alkyl; R¹⁴ represents -C_0.4 alkyl, said alkyl substituted with H, substituted or unsubstituted C₆.₁₀ aryl, and substituted or unsubstituted C_5.10 heteroaryl; and n represents an integer from 1 -2; said process comprising the steps of: (a) reacting a compound of Formula A

LG-C(0)-Z Formula A

wherein LG represents a leaving group, Z represents

PG represents a protecting group, and R¹¹ is as defined above, with an amine group containing linker molecule attached to a solid support, represented by

SS-LM-NH₂

wherein SS is a solid support, and LM is a linker molecule, to yield a product represented by Formula B

SS-LM-NHC(0)-Z Formula B wherein SS, LM, and Z are as defined above;

(b) reacting a product represented by Formula B with a compound of

Formula C

R¹-NH₂ Formula e

to yield a product of Formula D,

SS-LM-NH-C(0)-QP-NHR¹ Formula D

wherein SS, LM, and R¹ are as defined above, QP represents

PG

PG represents a protecting group, and R¹ is as defined above; (c) reacting a product of Formula D with a compound of Formula E

X-Y-R² Formula E

wherein Y is as defined above, and X represents C1 , Br, F, or I, to yield a product of Formula F

SS-LM-NH-C(0)-QP-N(R')-Y-R²; Formula F,

wherein SS, LM, QP, Y, R\ R² are as defined above; (d) sequentially reacting a product of Formula F with: (i) a protonating agent;

(ii) an epoxide represented by Formula G

wherein R¹⁰ is as defined above; or an acid represented by Formula H

R¹⁴-COOH Formula H where R¹⁴ is as defined above; (iii) a protonating agent; (iv) trimethyl silyldiazomethane; and (v) a compound of Formula R⁴R⁵NH, to yield a compound of Formula 1.

2. A process of claim 1 wherein step (a) is carried out at a temperature in the range of from about -78 °C to about 25 °C.

3 A process of claim 2 wherein the temperature ranges from about -50°C to about 0°C.

4. A process of claim 3 wherein the temperature ranges from about -40 °C to about -10°C.

5. A process of claim 3 wherein step (d) (ii) comprises an epoxide represented by Formula G:

6. A process of claim 3 wherein step (d) (ii) comprises an acid represented by Formula H:

R¹⁴-COOH.

7. A process of claim 5 wherein R¹ represents a -C C₄ straight chain alkyl radical substituted with H, optionally substituted aryl, or optionally substituted heteroaryl;

R² represents -C,_.. alkyl, or substituted aryl; R⁴ and R⁵ can be the same or different, and are independently selected from

-C,_.4 straight chain alkyl, -C_4.8 branched alkyl, and H, said alkyl groups substituted with R⁶; alternatively

R⁴ and R^s when taken together along with the nitrogen atom that they are attached to represent a five to six membered heterocyclic ring; R⁶ represents H, -C alkyl, -N[(CH₂)_1.4CH₃]₂, -(CH₂)_0.4OH, -C₆,₀ aryl, -C_5.6 saturated or unsaturated heterocyclyl, or -C_6.10 aryl;

R¹⁰ and R¹¹ independently at each occurance represent H, or -C,_.4 alkyl substituted with R⁶;

R¹² represents H, R⁶, or phenyl substituted with one or more of halogen, -C,_.,, alkyl, or -C^alkoxy; and R¹⁴ represents -C_5.10 heteroaryl, -C,_.. alkyl, said alkyl substituted with H, Ph, or -C₅.₁₀ heteroaryl.

8. A process of claim 7 wherein R² represents a -0,-0, alkyl radical,

R⁸ and R⁹ independently at each occurrence represent H, -OC C₂ alkyl, -C,-C₂ alkyl radical, or -C(0)-CH₃.

9. A process of claim 6 wherein

R¹ represents a -C.-C₄ straight chain alkyl radical substituted with H, optionally substituted aryl, or optionally substituted heteroaryl;

R² represents -C,.., alkyl, or substituted aryl;

R⁴ and R⁵ can be the same or different, and are independently selected from

-C^ straight chain alkyl, -C_4.8 branched alkyl, and H, said alkyl groups substituted with R⁶; alternatively

R⁴ and R⁵ when taken together along with the nitrogen atom that they are attached to represent a five to six membered heterocyclic ring;

R⁶ represents H, -C_M alkyl, -N[(CH₂)_1.4CH₃]₂, -(CH₂)_0.4OH, -C₆,₀ aryl, -C_5.6 saturated or unsaturated heterocyclyl, or -C_6.10 aryl;

R¹⁰ and R¹¹ independently at each occurance represent H, or -C,^ alkyl substituted with R⁶;

R¹² represents H, R⁶, or phenyl substituted with one or more of halogen, -C,_.4 alkyl, and -C,_.4alkoxy; and

R¹⁴ represents -C_5.10 heteroaryl, or -C,_.. alkyl, said alkyl substituted with H, Ph, or -C_5.10 heteroaryl.

10. A process of claim 9 wherein R² represents a -C,-C₂ alkyl radical,

R⁸ and R⁹ independently at each occurrence represent H, -OC,-C₂ alkyl, -C,-C₂ alkyl radical, or -C(0)-CH₃.