Fluorohydroxyproline derivatives useful in the preparation of proteolysis targeted chimeras

Abstract

There is provided novel small molecule E3 ubiquitin ligase protein binding ligand compounds, and to their utility in PROteolysis Targeted Chimeras (PROTACs), as well as processes for their preparation thereof, and use in medicine. There is particularly provided novel small molecule E3 ubiquitin ligase protein binding inhibitor compounds based on a fluorohydroxyproline scaffold, to their utility as ligands in synthesizing novel PROTACs, and to synthetic methods therefor.

Claims

1. A compound having the structure:
A-L-B, or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof, wherein A is an E3 ubiquitin ligase protein binding ligand compound of formula I: ##STR00132## wherein L is a group which is directly bonded to the compound of formula I and wherein L is —(CH.sub.2).sub.nL.sup.1(CH.sub.2O).sub.p—, wherein L.sup.1 is a covalent bond, a 5 or 6 membered heterocyclic or heteroaromatic ring containing 1, 2 or 3 nitrogen atoms, phenyl, —(C.sub.2-C.sub.4)alkyne, —SO.sub.2—, or —NH—, or wherein L is a —(CH.sub.2CH.sub.2O).sub.b— group wherein b is 1 to 10 and wherein L is directly bonded to the compound of formula I, wherein n and p are independently 0 to 10, wherein X is C or N, wherein R.sub.1 is a covalent C-linked bond to L, a —(CH.sub.2).sub.mQ.sub.v group with a covalent C-linked bond to L, a (C.sub.1-C.sub.4) alkyl group, or a C-linked (C.sub.3-C.sub.4) heterocyclic group, wherein m is 0, 1 or 2 and v is 0 or 1, wherein Q is a (C.sub.3-C.sub.4)cyclic or (C.sub.3-C.sub.4)—C—linked nitrogen containing heterocyclic group, wherein one of the ring atoms in the Q group is optionally substituted with a —NHC(O) group or a —C(O) group, wherein said R.sub.1 groups may be optionally substituted by one or more groups independently selected from F, CN, C(O) or C(O)(C.sub.1-C.sub.3)alkyl, wherein R.sub.2a is OH, —CHF.sub.2, —CF.sub.3, or NH.sub.2, wherein R.sub.2b is H, .sup.2H, .sup.3H, a —(C.sub.1-C.sub.3) alkyl group, an aryl group, a heteroaryl group, —CF.sub.3, —CF.sub.2H, or a —CF.sub.2—(C.sub.1-C.sub.2) alkyl group, wherein R.sub.x is H, OH, —CHF.sub.2, —CF.sub.3, NH.sub.2 or F, wherein R.sub.3 and R.sub.4 are independently selected from H, a covalent C-linked, a covalent O-linked, or a covalent C(O)-linked bond to L, R.sub.5 is a —(C.sub.1-C.sub.3) alkyl group or a covalent C-linked bond to L, wherein Y is ##STR00133## wherein Z is CR.sub.6R.sub.7R.sub.8 or SR.sub.6R.sub.7R.sub.8R.sub.9R.sub.10, wherein R.sub.11 is a covalent C-linked bond or a ##STR00134## group, wherein R.sub.12 is —C(O)— or a -C(=)-R.sub.13 group, wherein when Z is CR.sub.6R.sub.7R.sub.8, R.sub.6 and R.sub.7 are each independently —(C.sub.1-C.sub.3) alkyl groups, or wherein R.sub.6 and R.sub.7 together with the C-atom to which they are attached form a —(C.sub.3-C.sub.4) cycloalkyl group, wherein when Z is CR.sub.6R.sub.7R.sub.8, R.sub.8 is a —(C.sub.1-C.sub.3) alkyl group, a —(CH.sub.2).sub.qR.sub.8* group wherein q is 0, 1 or 2, a —C(O)—R.sub.8* group, or a —N(H)—R.sub.8* group, and wherein R.sub.8* is a covalent C-, S-, or N-linked bond to L, or H, or wherein when Z is SR.sub.6R.sub.7R.sub.8R.sub.9R.sub.10, R.sub.6, R.sub.7, R.sub.8, R.sub.9 and R.sub.10 are each independently selected from: F; or —(C.sub.1-C.sub.3) alkyl groups, wherein R.sub.13 is H, F or a—(C.sub.1-C.sub.3) alkyl group, wherein the —(C.sub.1-C.sub.3) alkyl groups, or —(C.sub.3-C.sub.4) cycloalkyl groups where present in a Y group are optionally substituted by one or more substituents independently selected from: methyl; OH; or F, wherein B is a ligand which binds to a target protein or polypeptide which is to be degraded by ubiquitin ligase and is linked to A through a —C- linkage to the L group and B is independently selected from: ##STR00135##

2. The compound according to claim 1 having the structure A-L-B wherein A is an E3 ubiquitin ligase protein binding ligand compound of formula IA: ##STR00136## wherein X is N; wherein L is a —(CH.sub.2CH.sub.2O).sub.b− group wherein b is 1 to 10 and wherein L is directly bonded to the compound of formula IA, wherein R.sub.1 is a covalent C-linked bond to L, a —(CH.sub.2).sub.mQ.sub.v group with a covalent C-linked bond to L, a (C.sub.1-C.sub.4) alkyl group, or a C-linked (C.sub.3-C.sub.4) heterocyclic group, wherein m is 0, 1 or 2 and v is 0 or 1,and wherein when m is 0, v is 1; wherein Q is a (C.sub.3-C.sub.4)cyclic or (C.sub.3-C.sub.4)-C-linked nitrogen containing heterocyclic group, wherein one of the ring atoms in the Q group is optionally substituted with a —NHC(O) group or a —C(O) group, wherein said R.sub.1 groups may be optionally substituted by one or more groups independently selected from F, CN, C(O) or C(O)CH.sub.3, wherein R.sub.2a is OH, wherein R.sub.2b is H, wherein R.sub.3 and R.sub.4 are independently selected from H, a covalent C-linked, a covalent O-linked, or a covalent C(O)-linked bond to L, R.sub.5 is a —(C.sub.1-C.sub.3) alkyl group or a covalent C-linked bond to L, wherein Y is ##STR00137## wherein W is O, wherein R.sub.6 and R.sub.7 are each independently —(C.sub.1-C.sub.3) alkyl groups, or wherein R.sub.6 and R.sub.7 together with the C-atom to which they are attached form a —(C.sub.3-C.sub.4) cycloalkyl group, wherein R.sub.8 is a —(C.sub.1-C.sub.3) alkyl group, a —(CH.sub.2).sub.qR.sub.8* group wherein q is 0, 1 or 2, a —C(O)—R.sub.8* group, or a —N(H)—R.sub.8* group, and wherein R.sub.8* is a covalent C-, S-, or N-linked bond to L, or H, wherein the —(C.sub.1-C.sub.3) alkyl groups, or —(C.sub.3-C.sub.4) cycloalkyl groups where present in a Y group are optionally substituted by one or more substituents independently selected from: methyl; OH; or F, and wherein B is linked to A though a -C-linkage to the L group; and B is a ligand which binds to a target protein or polypeptide which is to be degraded by ubiquitin ligase and is linked to A though a -C-linkage to the L group and B is independently selected from: ##STR00138## or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph of the compound.

3. The compound according to claim 2 having the structure A-L-B wherein A is an E3 ubiquitin ligase protein binding ligand compound of formula IA wherein R.sub.1, R.sub.3, R.sub.4, and R.sub.5, are as indicated in formula IA: wherein L is directly bonded to the compound of formula IA at the R.sub.1 position, wherein R.sub.1 is an optionally substituted cyclopropyl group with a covalent C-linked bond to L or R.sub.1 is a covalent C-Linked bond to L, wherein X is N, wherein R.sub.2a is OH, wherein R.sub.2b, R.sub.x, R.sub.3 and R.sub.4 are all H, wherein R.sub.5 is a —CH.sub.3 group, wherein Y is ##STR00139## wherein W is O, and wherein B is independently selected from: ##STR00140## or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof.

4. The compound according to claim 2 having the structure A-L-B wherein one of: (i) A is a compound of formula IA wherein L is directly bonded to the compound of formula IA at the R.sub.3 position, wherein L is a —(CH.sub.2CH.sub.2O).sub.b— group wherein b is 1 to 10, wherein R.sub.2a is OH, wherein R.sub.2b, R.sub.x, and R.sub.4 are all H, wherein R.sub.3 is a covalent C-linked, a covalent O-linked, or a covalent —C(O)-linked bond to L, wherein R.sub.5 is a —CH.sub.3 group, wherein Y is ##STR00141## wherein W is O, and or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof; or (ii) A is a compound of formula IA wherein L is directly bonded to the compound of formula IA at the R.sub.4 position, wherein L is a —(CH.sub.2CH.sub.2O).sub.b— group wherein b is 1 to 10, wherein R.sub.2a is OH, wherein R.sub.2b, R.sub.x, and R.sub.3 are all H, wherein R.sub.4 is a covalent C-linked, a covalent O-linked, or a covalent —C(O)-linked bond to L, wherein R.sub.5 is a —CH.sub.3 group, wherein Y is ##STR00142## wherein W is O, and wherein B is independently selected from: ##STR00143## or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof; or (iii) A is a compound of formula IA wherein L is directly bonded to the compound of IA at the R.sub.5 -position, wherein R.sub.2a is OH, wherein R.sub.2b, R.sub.x, R.sub.3 and R.sub.4 are all H, wherein R.sub.5 is a covalent C-linked bond to L, wherein L is a —(CH.sub.2CH.sub.2O).sub.b— group wherein b is 1 to 10, wherein Y is ##STR00144## wherein W is O, and wherein B is independently selected from: ##STR00145## or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof; or (iv) A is a compound of formula IA wherein L is directly bonded to the compound of formula IA at the R.sub.8* position, wherein L is a —(CH.sub.2CH.sub.2O).sub.b— group wherein b is 1 to 10, wherein R.sub.2a is OH, wherein R.sub.2b, R.sub.3, and R.sub.4 are all H, wherein R.sub.5 is a —CH.sub.3 group, wherein Y is ##STR00146## wherein W is O, wherein R.sub.8 is a —(CH.sub.2).sub.qR.sub.8* group wherein q is 0, 1 or 2, and wherein R.sub.8* is a covalent C-, S-, or N-linked bond to L, and wherein B is independently selected from: ##STR00147## or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof.

5. The compound according to claim 1 independently selected from: (2R,3R,4S)-1-((S)-2-(tert-butyl)-17-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-4,16-dioxo-6,9,12-trioxa-3,15-diazaheptadecanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide; (2R,3S,4S)-1-((S)-2-(tert-butyl)-17-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-4,16-dioxo-6,9,12-trioxa-3,15-diazaheptadecanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide; (2R,3R,4S)-1-((S)-1-(4-((2S,4R)-1-acetyl-4-(4-chlorobenzyl)-2-methyl-1,2,3,4-tetrahydroquinolin-6-yl)phenyl)-12-(tert-butyl)-1, 10-dioxo-5,8-dioxa-2, 11-diazatridecan-13-oyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide; (2R,3S,4S)-1-((S)-1-(4-((2S,4R)-1-acetyl-4-(4-chlorobenzyl)-2-methyl-1,2,3,4-tetrahydroquinolin-6-yl)phenyl)-12-(tert-butyl)-1, 10-dioxo-5,8-dioxa-2, 11-diazatridecan-13-oyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide; (2R,3R,4S)-1(R)-3(6-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)hexyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-m ethylbutanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide; (2R,3R,4S)-1-((S)-2-acetamido-3,3-dimethylbutanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide; (2R,3S,4S)-1-((S)-2-acetamido-3,3-dimethylbutanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide; (2R,3R,4S)-3-fluoro-1(S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide; (2R,3S,4S)-3-fluoro-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide; (2R,3R,4 S)-1-((S)-2-(1-cyanocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide; and (2R,3S,4S)-1-((S)-2-(1-cyanocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide; or a pharmaceutically acceptable salt, enantiomer, stereoisomer, hydrate, solvate, or polymorph thereof.

6. A pharmaceutical composition comprising an effective amount of a compound according to claim 1 in combination with a pharmaceutically acceptable carrier, additive or excipient.

7. A method for the prophylaxis or treatment of a disease or condition associated with deregulation of protein activity of one or more proteins within the Bromo- and Extra-terminal (BET) family of proteins BRD2, BRD3 and BRD4, the method comprising the administration of the compound of structure A-L-B of claim 1 to a subject suffering from or likely to be exposed to said disease or condition; wherein: the disease or condition is selected from a disease or condition associated with selective degradation of the BRD4 protein within the bromodomain of the BET family of proteins; or the disease or condition is independently selected from: cancer; benign proliferative disorders; infection or non-infectious inflammatory events; autoimmune diseases; inflammatory diseases; systemic inflammatory response syndromes; viral infections and diseases; ophthalmological conditions.

8. A PROTAC compound having the structure A-L-B according to the compound of A-L-B underlaying the pharmaceutical composition according to claim 6, wherein said target protein is selected from one of: the group consisting of structural proteins, receptors, enzymes, cell surface proteins, proteins pertinent to the integrated function of a cell, proteins involved in catalytic activity, aromatase activity, motor activity, helicase activity, metabolic processes, antioxidant activity, proteolysis, biosynthesis, proteins with kinase activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, ligase activity, enzyme regulator activity, signal transducer activity, structural molecule activity, binding activity, receptor activity, cell motility, membrane fusion, cell communication, regulation of biological processes, development, cell differentiation, response to stimulus, behavioral proteins, cell adhesion proteins, proteins involved in cell death, proteins involved in protein transporter activity, proteins involved in nuclear transport, proteins involved in ion transporter activity, proteins involved in channel transporter activity, proteins involved in carrier activity, proteins involved in permease activity, proteins involved in secretion activity, proteins involved in electron transporter activity, proteins involved in pathogenesis, proteins involved in chaperone regulator activity, proteins involved in nucleic acid binding activity, proteins involved in transcription regulator activity, proteins involved in extracellular organization and proteins involved in biogenesis activity and translation regulator activity; or the group consisting of B7.1 and B7, TI FRIm, TNFR2, NADPH oxidase, BcIIBax and other partners in the apotosis pathway, C5a receptor, HMG-CoA reductase, PDE V phosphodiesterase type, PDE IV phosphodiesterase type 4, PDE I, PDEII, PDEIII, squalene cyclase inhibitor, CXCRI, CXCR2, nitric oxide synthase, cyclo-oxygenase 1, cyclo-oxygenase 2, 5HT receptors, dopamine receptors, G Proteins, histamine receptors, 5-lipoxygenase, tryptase serine protease, thymidylate synthase, purine nucleoside phosphorylase, GAPDH trypanosomal, glycogen phosphorylase, Carbonic anhydrase, chemokine receptors, JAW STAT, Retinoid X Receptor, HIV 1 protease, HIV 1 integrase, influenza, neuraminidase, hepatitis B reverse transcriptase, sodium channel, multi drug resistance, protein P-glycoprotein and MRP, tyrosine kinases, CD23, CD 124, tyrosine kinase p56 Ick, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF-alphaR, ICAM1, Cat+channels, VCAM, VLA-4 integrin, selectins, CD40/CD40L, newokinins and receptors, inosine monophosphate dehydrogenase, p38 MAP Kinase, RasIRafIMEWERK pathway, interleukin-1 converting enzyme, caspase, HCV, NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotide formyl transferase, rhinovirus, 3C protease, herpes simplex virus- 1, protease, cytomegalovirus protease, poly (ADP-ribose) polymerase, cyclin dependent kinases, vascular endothelial growth factor, oxytocin receptor, microsomal transfer protein inhibitor, bile acid transport inhibitor, 5 alpha reductase inhibitors, angiotensin 11, glycine receptor, noradrenaline reuptake receptor, endothelin receptors, neuropeptide Y and receptor, adenosine receptors, adenosine kinase and AMP deaminase, purinergic receptors P2Y1, P2Y2, P2Y4, P2Y6, and P2X1-7, farnesyltransferases, geranylgeranyl transferase, TrkA a receptor for NGF, beta-amyloid, tyrosine kinase FIk-IIKDR, vitronectin receptor, integrin receptor, Her-21 neu, telomerase inhibition, cytosolic phospholipaseA2 and EGF receptor tyrosine kinase, ecdysone 20-monooxygenase, ion channel of the GABA gated chloride channel, acetylcholinesterase, voltage-sensitive sodium channel protein, calcium release channel, and chloride channels; acetyl-CoA carboxylase, adenylosuccinate synthetase, protoporphyrinogen oxidase, and enolpyruvylshikimate-phosphate synthase.

9. A PROTAC compound having the structure A-L-B according to claim 1 wherein one of: B is an Hsp90 inhibitor; a kinase inhibitor, a phosphatase inhibitor, an MDM2 inhibitor, a compound which targets human BET Bromodomain-containing proteins, an HDAC inhibitor, a human lysine methyltransferase inhibitor, a compound targeting RAF receptor, a compound targeting FKBP, an angiogenesis inhibitor, an immunosuppressive compound, a compound targeting an aryl hydrocarbon receptor, a compound targeting an androgen receptor, a compound targeting an estrogen receptor, a compound targeting a thyroid hormone receptor, a compound targeting HIV protease, a compound targeting HIV integrase, a compound targeting HCV protease or a compound targeting acyl protein thioesterase 1 and/or 2.

10. A method of regulating protein activity of a target protein in a patient in need comprising administering to said patient an amount of a compound according to claim 1.

11. The compound of formula I, suitable for use in a PROTAC compound of structure A-L-B according to claim 1 wherein the compound of formula I, can be modified for covalent bonding to a B group as defined in claim 1, via a linker group L, or a pharmaceutically acceptable salt, stereoisomer, solvate or polymorph thereof.

12. A composition comprising a pharmaceutical intermediate compound of general formula II: ##STR00148## wherein said compound comprises one of the following embodiments: (i) R.sub.2a is OH, R.sub.2b is H, and wherein LHS is a 9-phenyl-9-fluorenyl group, a fluoroenylmethyloxycarbonyl group, a tert-butyloxycarbonyl protecting group (BOC group), or an acetamide group and wherein RHS is, —CO.sub.2CH.sub.3 or —CO.sub.2Bn; or (ii) R.sub.2a is OH, R.sub.2b is H, LHS is a BOC group and RHS is a —CO.sub.2Bn group having the structural formulae II-A, II-B and/or II-C: ##STR00149## (iii) R.sub.2a is OH, R.sub.2b is H, LHS is a BOC group and RHS is a —CO.sub.2H group having the structural formulae II-E, II-F, II-G and/or II-H: ##STR00150## (iv) R.sub.2a is OH, R.sub.2b is H, wherein LHS is an acetamide group and RHS is a —CO.sub.2Me group having the structural formulae II-I, II-J, II-K and/or II-L: ##STR00151## (v) R.sub.2a is OH, R.sub.2b is H, LHS is a 9-phenyl-9-fluorenyl group and RHS is —CO.sub.2H, —CO.sub.2CH.sub.3 or —CO.sub.2D, where D is an alternative alkyl ester or a benzyl ester, having the structural formulae II-M, II-N, II-P and/or II-O: ##STR00152##

13. A process for the preparation of compounds of general formula II: ##STR00153## wherein R.sub.2a is OH, —CHF.sub.2, —CF.sub.3, or NH.sub.2, wherein R.sub.2b is H, .sup.2H, .sup.3H, a —(C.sub.1-C.sub.3) alkyl group, an aryl group, a heteroaryl group, —CF.sub.3, —CF.sub.2H, or a —CF.sub.2—(C.sub.1-C.sub.2) alkyl group, wherein LHS is: H; an amine protecting group selected from: a 9-phenyl-9-fluorenyl, a fluoroenylmethyloxycarbonyl protecting group (Fmoc), a tert-butyloxycarbonyl protecting group (BOC group), or an acetamide group; or a suitable alternative amine protecting group for the ring-N, wherein RHS is —C(O)OH, —C(O)OCH.sub.3 or —C(O)OD where D is an alternative alkyl ester or a benzyl ester, wherein the compound of formula II is not ##STR00154## from intermediate compounds of general formula III, wherein the LHS is H; an amine protecting group selected from: a 9-phenyl-9-fluorenyl group, a fluoroenylmethyloxycarbonyl group, a BOC group, or an acetamide group; or a suitable alternative amine protecting group for the ring-N: ##STR00155## and wherein said compounds of general formula III are converted to compounds of general formula II via treatment with a suitable reducing agent.

14. A process for the preparation of intermediate compounds of structural formulae II-A II-B, and II-C as defined in claim 12 from intermediate compounds of structural formulae III-A or III-B: ##STR00156## wherein said intermediate compounds of structural formulae III-A or III-B are converted into structural formulae II-A, II-B, and II-C by treatment with a suitable reducing agent.

15. A process for the preparation of intermediate compounds of general formula III, via conversion of starting compounds of formula V to intermediate compounds of general formula IV with subsequent transformation into compounds of general formula III: ##STR00157## wherein said compounds of general formula V are converted to compounds of general formula IV via a 2-step approach with protection using a TMSO- group followed by fluorination of the protected intermediate compound IV to provide the desired 3-fluoro-compounds, wherein RHS is: —C(O)OH, —C(O)OCH.sub.3 or —C(O)OD where D is an alternative alkyl ester or a benzyl ester, and wherein LHS is: H; an amine protecting group selected from: a 9-phenyl-9-fluorenyl, a fluoroenylmethyloxycarbonyl protecting group (Fmoc), a tert-butyloxycarbonyl protecting group (BOC group), or an acetamide group; or a suitable alternative amine protecting group for the ring-N.

16. A process for the preparation of intermediate compounds of structural formula III-A and III-B, via conversion of compound of structural formula V to an intermediate compound of structural formula IV with subsequent transformation into compounds of formulae III-A and III-B: ##STR00158## wherein compound formula V is converted to compounds III-A and III-B via a 2-step approach, with protection using a TMSO- group followed by fluorination of the protected intermediate compound IV to provide the desired 3-fluoro-compounds of formulae III-A and III-B.

17. The compound of claim 1, of structure A-L-B wherein A is a compound of formula I, wherein R.sub.2a is OH, R.sub.2b is H, and R.sub.x is H and wherein L is a group which is directly bonded to the compound of formula I and wherein L is —(CH.sub.2).sub.nL.sup.1(CH.sub.2O).sub.p—, wherein L.sup.1 is a covalent bond, a 5 or 6 membered heterocyclic or heteroaromatic ring containing 1, 2 or 3 nitrogen atoms, phenyl, —(C.sub.2-C.sub.4)alkyne, —SO.sub.2—, or —NH—.

18. The compounds of general formula II according to claim 12 wherein LHS is a 9-phenyl-9-fluorenyl group and RHS is —C(O)OCH.sub.3 having the structural formulae II-Q, II-R, II-S and II-T: ##STR00159##

19. The pharmaceutical composition of claim 6, further comprising an additional bioactive agent independently selected from one or more agents for the treatment of: cancer; benign proliferative disorders; infection or non-infectious inflammatory events; autoimmune diseases; inflammatory diseases; systemic inflammatory response syndromes; viral infections and diseases; and ophthalmological conditions.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1: illustrates the structures of compounds JQ1, VHL-1, I-BET 762 and VHL-2.

(2) FIG. 2 illustrates the binding affinities and ΔH against first and second bromodomains and VBC obtained in isothermal titration calorimetry (ITC) experiments for previously reported non-fluorinated PROTACs MZ1, MZ2 and MZ3, bromodomain inhibitor JQ1 and previously reported VHL ligand VHL-1.

(3) FIG. 3: illustrates the binding affinities and ΔH against VBC obtained in isothermal titration calorimetry (ITC) experiments for VHL ligands 14b (Kd=0.235 μM, ΔH=−6566 cal/mol, ΔS=8.30 cal/mol/deg) and 14d (Kd==0.235 μM, ΔH=−6566 cal/mol, ΔS==8.30 cal/mol/deg).

(4) FIG. 4A: is a representation of the protein-ligand crystal structure of VHL-1 where the scaffold of VHL-1 is seen in 3D-format.

(5) FIG. 4B: is a representation of the protein-ligand crystal structure of VHL-2 where the scaffold of VHL-2 is seen in 3D-format.

(6) FIG. 4C: is a representation of the protein-ligand crystal structure of JQ1 where the scaffold of JQ1 is seen in 3D-format.

(7) FIG. 5: illustrates the Time dependent treatment over 36 h of HeLa cells with (a) 0.01% DMSO, (b) 1 μM JQ1.

(8) FIG. 6: illustrates the compound dose-dependent intracellular activities results obtained when HeLa cells were treated with various concentrations of PROTAC 18d against BRD2 and BRD4 (long and short isoforms) with an actin control. Protein levels were visualised by immunoblot (middle panel) and quantified relative to DMSO control (bottom panel).

(9) FIG. 7: illustrates the structures of cisMZ1, (2S,4S)-1-((S)-2-(tert-butyl)-17-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-4,16-dioxo-6,9,12-trioxa-3,15-diazaheptadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide and MZ1, (2S,4R)-1-((S)-2-(tert-butyl)-17-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-4,16-dioxo-6,9,12-trioxa-3,15-diazaheptadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide.

(10) FIG. 8 illustrates the structure of (2R,3R,4S)-1-((S)-2-(tert-butyl)-17-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-4,16-dioxo-6,9,12-trioxa-3,15-diazaheptadecanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide 18b and further illustrates the compound dose-dependent intracellular activities results obtained when HeLa cells were treated with various concentrations of PROTAC 18b against BRD2, BRD3 and BRD4 (long and short isoforms). Protein levels were visualized by immunoblot and quantified (bottom graph).

(11) FIG. 9 illustrates the structure of (2S,4R)-1-((R)-2-acetamido-3-((6-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)hexyl)thio)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide 35 and further illustrates the compound dose-dependent intracellular activities results obtained when HeLa cells were treated with various concentrations of PROTAC 35 against BRD2, BRD3 and BRD4 (long and short isoforms). Protein levels were visualized by immunoblot (left panel) and quantified (right graph).

BIOLOGICAL METHODS

(12) ITC Data on the Binding of VHL Ligands 14b and 14d

(13) Titrations were performed on an ITC200 micro-calorimeter (GE Healthcare). Compounds 14b and 14d were diluted from a DMSO stock solution to 0.6 mM in a 20 mM HEPES, 150 mM NaCl, 1 mM TCEP (pH 7) buffer. The compound was titrated against 60 μM VBC complex, equilibrated in the same buffer. Total amount of DMSO in the buffer was 3%. The data were fitted to a single binding site model to obtain the stoichiometry n, the dissociation constant K.sub.d and the enthalpy of binding ΔH using the Microcal LLC ITC200 Origin software provided by the manufacturer.

(14) Tissue Culture

(15) HeLa cells were cultured in DMEM supplemented with 10% FBS, 1% L-glutamine and 100 U/ml of penicillin/streptomycin. Cells were maintained for no more than 30 passages at 37° C. and 5% CO.sub.2.

(16) Western Blotting

(17) For protein extracts the dishes were placed on ice. The media was aspirated and the tissue layer washed twice with ice cold PBS. 120 μl of RIPA-buffer containing Protease inhibitor was added and the cells detached from the surface with a cell scraper. After removal of the insoluble fraction by centrifugation the protein concentration of the supernatant was determined by a Pierce™ BCA Protein Assay Kit. Protein extracts were fractionated by SDS-PAGE on 3-8% Tris-Acetate NuPage® Novex® (Life Technologies) polyacrylamide gels and transferred to a nitrocellulose membrane using i-Blot® 2 from Life Technologies. The membrane was then blocked with 3.5% Bovine Serum albumin (BSA) in Tris-buffered saline (TBS) with 0.1% Tween-20. For detecting proteins the following primary antibodies in the given concentrations were used: anti-BRD2 (Abcam, ab139690, EPR7642) 1:2000, anti-BRD4 (Abcam, ab128874, EPR5150(2)) 1:1000, anti-β-Actin (Cell Signaling Technology, 4970S, 13E5) 1:2000. For visualisation a Li-Cor Biosciences Odyssey system with the following secondary fluorescent Antibodies from Li-Cor Biosciences was used: IRDye800CW Goat Anti-Mouse (926-32210), IRDye800CW Donkey Anti-Rabbit (926-32213), both in concentrations of 1:10 000. Membranes were incubated with the corresponding antibodies either at 4° C. for 12 h or at 25° C. for 4 h. Between incubation with the different antibodies membranes were stripped with 0.25 M solution of Glycine.HCl at pH 2.

(18) Pharmaceutical Compositions

(19) PROTAC compounds of the invention can be administered as pharmaceutical compositions by any conventional route, in particular enterally, e.g., orally, e.g., in the form of tablets or capsules, or parenterally, e.g., in the form of injectable solutions or suspensions, topically, e.g., in the form of lotions, gels, ointments or creams, or in a nasal or suppository form. Pharmaceutical compositions comprising a PROTAC compound of the present invention in free form or in a pharmaceutically acceptable salt form in association with at least one pharmaceutically acceptable carrier or diluent can be manufactured in a conventional manner by mixing, granulating or coating methods. For example, oral compositions can be tablets or gelatin capsules comprising the active ingredient together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners. Injectable compositions can be aqueous isotonic solutions or suspensions, and suppositories can be prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers.

(20) In addition, they may also contain other therapeutically valuable substances. Suitable formulations for transdermal applications include an effective amount of a PROTAC compound of the present invention with a carrier. A carrier can include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. For example, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin. Matrix transdermal formulations may also be used. Suitable formulations for topical application, e.g., to the skin and eyes, are preferably aqueous solutions, ointments, creams or gels well-known in the art. Such may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

(21) The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a PROTAC compound of the present invention formulated together with one or more pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

(22) The pharmaceutical compositions of this invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), buccally, or as an oral or nasal spray.

(23) Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

(24) Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S. P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

(25) Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the PROTAC compounds of the invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatine capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

(26) The PROTAC compounds can also be provided in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

(27) Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, eye ointments, powders and solutions are also contemplated as being within the scope of this invention. The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

(28) Powders and sprays can contain, in addition to the PROTAC compounds of this invention, excipients such as lactose, talc, silicic acid, aluminium hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons. Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

(29) According to the methods of treatment of the present invention, diseases, conditions, or disorders are treated or prevented in a subject, such as a human or other animal, by administering to the subject a therapeutically effective amount of a PROTAC compound of the invention, in such amounts and for such time as is necessary to achieve the desired result. The term “therapeutically effective amount” of a compound of the invention, as used herein, means a sufficient amount of the compound so as to decrease the symptoms of a disorder in a subject. As is well understood in the medical arts a therapeutically effective amount of a PROTAC compound of this invention will be at a reasonable benefit/risk ratio applicable to any medical treatment.

(30) The dosage for the instant compounds can vary according to many factors, including the type of disease, the age and general condition of the patient, the particular compound administered, and the presence or level of toxicity or adverse effects experienced with the drug. A representative example of a suitable dosage range is from as low as about 0.025 mg to about 1000 mg. However, the dosage administered is generally left to the discretion of the physician.

(31) A wide variety of pharmaceutical dosage forms for mammalian patients can be employed. If a solid dosage is used for oral administration, the preparation can be in the form of a tablet, hard gelatin capsule, troche or lozenge. The amount of solid carrier will vary widely, but generally the amount of the PROTAC compound will be from about 0.025 mg to about 1 g, with the amount of solid carrier making up the difference to the desired tablet, hard gelatin capsule, troche or lozenge size. Thus, the tablet, hard gelatin capsule, troche or lozenge conveniently would have, for example, 0.025 mg, 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 25 mg, 100 mg, 250 mg, 500 mg, or 1000 mg of the present compound. The tablet, hard gelatin capsule, troche or lozenge is given conveniently once, twice or three times daily.

(32) In general, PROTAC compounds of the invention will be administered in therapeutically effective amounts via any of the usual and acceptable modes known in the art, either singly or in combination with one or more therapeutic agents. A therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors.

(33) In certain embodiments, a therapeutic amount or dose of the compounds of the present invention may range from about 0.1 mg/Kg to about 500 mg/Kg, alternatively from about 1 to about 50 mg/Kg. In general, treatment regimens according to the present invention comprise administration to a patient in need of such treatment from about 10 mg to about 1000 mg of the compound(s) of this invention per day in single or multiple doses. Therapeutic amounts or doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents. Upon improvement of a subject's condition, a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level, treatment should cease. The subject may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific inhibitory dose for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

(34) The invention also provides for pharmaceutical combinations, e.g. a kit, comprising a) a first agent which is a PROTAC compound of the invention as disclosed herein, in free form or in pharmaceutically acceptable salt form, and b) at least one co-agent. The kit can comprise instructions for its administration. The terms “co-administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term “fixed combination” means that the active ingredients, e.g. a PROTAC compound of the invention and a co-agent, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the active ingredients, e.g. a PROTAC compound of the invention and a co-agent, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g. the administration of three or more active ingredients. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, wool fat, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes, oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminium hydroxide; alginic acid; pyrogen-free water, isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulphate and magnesium stearate, as well as colouring agents, releasing agents, coating agents, sweetening, flavouring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

(35) Chemistry—Materials and Methods

(36) All chemicals, unless otherwise stated were commercially available and used without further purification. Solvents were anhydrous and reactions preformed under positive pressure of nitrogen or argon. Enantiopure (+)-JQ-1 and I-BET726 were purchased from Medchemexpress LLC, Princeton, USA. Flash column chromatography (FCC) was performed using a Teledyne Isco Combiflash Rf or Rf200i. As prepacked columns RediSep Rf Normal Phase Disposable Columns were used.

(37) NMR spectra were recorded on a Bruker 500 Ultrashield or a Bruker Ascend 400. Chemical shifts are quoted in ppm and referenced to the residual solvent signals: .sup.1H δ=7.26 (CDCl.sub.3), .sup.13C δ=77.16 (CDCl.sub.3), .sup.1H δ=3.31 (MeOD), .sup.13C δ=49.15 (MeOD), .sup.1H δ=4.79 (D.sub.2O). Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), broad (br) or a combination thereof. Coupling constants (J) are measured in Hz. Symbol “*” labels the signals of the minor rotamer when clearly distinguishable from the major one.

(38) Low resolution MS and analytical HPLC traces were recorded on an Agilent Technologies 1200 series HPLC connected to an Agilent Technologies 6130 quadrupole LC/MS, connected to an Agilent diode array detector. The column used was a Waters XBridge column (50 mm×2.1 mm, 3.5 μm particle size) and the compounds were eluted with a gradient of 5-95% acetonitrile/water+0.1% formic acid over 3 minutes (METHOD 1) or over 7 minutes (METHOD 2).

(39) Preparative HPLC was performed on a Gilson Preparative HPLC System with a Waters X-Bridge C18 column (100 mm×19 mm; 5 μm particle size) and a gradient of 5% to 95% acetonitrile in water over 10 minutes, flow 25 mL/min, with 0.1% ammonia in the aqueous phase.

(40) Abbreviations used: ACN for acetonitrile, DCM for dichloromethane, EtOAc for ethyl acetate, Et.sub.2O for diethyl ether, DMSO for dimethyl sulfoxide, DIPEA for N,N-diisopropylethylamine, MeOH for methanol, TEA for triethylamine, DMF for N,N-dimethylformamide, HATU for 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate, HOAT for 1-hydroxy-7-azabenzotriazole, TMSOTf for trimethylsilyltriflate, TFA for trifluoroacetic acid.

(41) Preparatory Compounds for the Synthesis of F-HYP in Accordance with Scheme 1 Process

Preparative Compound 1: 2-benzyl 1-(tert-butyl) (S)-4-oxopyrrolidine-1,2-dicarboxylate

(42) Preparative compound 1 was prepared from commercially available 2-benzyl 1-(tert-butyl) (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate in accordance with the method reported by Qui, Journal of Organic Chemistry, 67(20), 7162-7164. CrO.sub.3 (17.0 g, 0.17 mol) was added slowly with stirring over 30 min to a solution of pyridine (30 mL) in DCM (80 mL) at 0° C. The mixture was warmed to room temperature and 2-benzyl 1-(tert-butyl) (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (6.0 g, 18.69 mmol) in DCM (60 mL) was added under stirring. The reaction was stirred vigorously for 4 h at room temperature. The formed dark solid was decanted and washed with DCM (3×100 mL). The organic phases were washed with saturated aqueous NaHCO.sub.3, 10% aqueous critic acid, and brine and dried over anhydrous MgSO.sub.4. The solvent was removed in vacuo to yield an oily residue, which was purified by flash chromatography (heptane/ethyl acetate, 3:1) to give 4 (5.0 g, 84%) as a clear oil. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 7.35 (m, 5H), 5.28-5.09 (m, 2H), 4.88-4.72 (dd, J=10.2, 9.3 Hz, 1H), 3.92-3.87 (d, J=13.5 Hz, 2H), 2.97-2.91 (m, 1H), 2.64-2.54 (m, 1H), 1.47-1.37 (m, 9H).

Preparative Compound 2: 2-benzyl 1-(tert-butyl) (2R)-3-fluoro-4-oxopyrrolidine-1,2-dicarboxylate

(43) ##STR00081##

(44) A solution of 2-benzyl 1-(tert-butyl) (S)-4-oxopyrrolidine-1,2-dicarboxylate (1) (1.255 g, 3.929 mmol) in THF (5 mL) was added dropwise to a stirred solution of LHMDS (1M in THF, 4.35 mL) in THF (4.35 mL) at −78° C. The mixture was stirred at −78° C. for 2 hr, then TMSCl (1.25 mL, 10 mmol) was added dropwise. After 30 minutes of continued stirring at −78° C., the cooling bath was removed and the reaction mixture was allowed to warm to r.t. and stirred for additional 3 hours. The reaction mixture was then concentrated under vacuum to small volume, pentane (50 mL) was added and the mixture was poured into a saturated solution of NaHCO.sub.3 (100 mL). The mixture was vigorously shaken and the organic layer was separated, washed with brine, then dried over anhydrous MgSO.sub.4. The volatiles were removed under reduced pressure to obtain a light yellow oil corresponding to the trimethylsilylenolate which was dissolved in acetonitrile (60 mL) and cooled to 0° C. Selectfluor (2.040 g, 5.750 mmol) was added in one portion and the mixture was carefully allowed to reach 10° C. over a 3 hr period. TLC analysis (EtOAc/Heptane 3:7) showed complete conversion of the starting material A saturated solution of NH.sub.4Cl was added (30 ML), the resulting mixture was vigorously shaken and the organic layer was separated. The aqueous layer was extracted with EtOAc (2×30 mL) and the organic phase was washed with brine, then dried over MgSO.sub.4. Volatiles were removed under reduced pressure to obtain a yellow oil which was quickly passed through a short silica column eluted with a mixture of EtOAc/Heptane 4:6 to obtain the desired crude fluoroketone (2) (662 mg 50% yield) as a mixture of diasteroisomers. This crude mixture proved to be unstable in solution and therefore was used immediately in the next step. .sup.1H-NMR (mixture of diasteroisomers and their cis/trans rotamer, CDCl.sub.3) δ: 7.40-7.34 (m, 5H), 5.38-4.81 (m, 4H), 4.12-3.95 (m, 2H), 1.49-1.39 (m, 9H). .sup.19F NMR δ −186.91, −187.57 (a isomer) −205.18, −205.92 (β isomer).

(45) Alternatively, 2-Benzyl 1-(tert-butyl) (S)-4-((trimethylsilyl)oxy)-2,5-dihydro-1H-pyrrole-1,2-dicarboxylate and Selectfluor™ were dissolved in anhydrous acetonitrile (0.67 M and 0.1 M respectively) and stored under inert atmosphere. The solutions of silyl enol ether and Slectfluor were pumped at a rate of 0.77 mL/min in a 10 mL flow reactor (residence time 6.5 min) heated at 50° C. The crude product treated with a saturated solution of NH.sub.4Cl and extracted with ethyl acetate and the organic phase was dried over MgSO4. Solvents were evaporated to obtain the crude product which can be further purified by column chromatography on silica accordingly to the conditions described for the batch synthesis above. This flow process provided a reliable and constant yield of fluorinated products while providing a ratio of the diastereoisomers as disclosed for the batch process.

Reduction of 2-benzyl 1-(tert-butyl) (2R)-3-fluoro-4-oxopyrrolidine-1,2-dicarboxylate with NaBH.SUB.4 .to Prepare Preparative Compounds 3a, 3b, and 3c

(46) Fluoroketone 2 (500 mg, 1.482 mmol) was dissolved in a mixture of THF/EtOH 1:1 (10 mL) and cooled to 0° C. NaBH.sub.4 (56 mg, 1.482 mmol) was added portion-wise and the reaction mixture was stirred for 1 hour at 0 C. TLC analysis (EtOAc/Heptane 1:1) showed complete conversion of the starting material. The reaction mixture was concentrated under vacuum, EtOAc was added (15 mL) and a solution of NaHSO.sub.4 (5%) was added dropwise until a pH of 3 to 4 was obtained. The acidified mixture was then washed with brine (10 mL), and the organic phase was dried over anhydrous MgSO.sub.4. Volatiles were removed under reduced pressure to obtain yellow oil which was subjected to FCC (EtOAc/Heptane 3:7 to 1:1) to separate the 3 diasteroisomers (described herein in order of elution). Alternatively, the mixture was dissolved in methanol (8 mL) and purified by preparative HPLC (5-90% ACN in H.sub.2O over 15 min).

Preparative Compound 3c: 2-benzyl 1-(tert-butyl) (2R,3R,4R)-3-fluoro-4-hydroxypyrrolidine-1,2-dicarboxylate

(47) ##STR00082##

(48) (11% by .sup.19F-NMR, white solid, 8% isolated yield) .sup.1H-NMR (CDCl.sub.3) δ: 7.37-7.36 (m, 5H), 5.33-5.15 (m, 2H), 4.99 (d, J.sub.H—F=49.1 Hz, 1H), 4.95* (d, J.sub.H—F=49.0 Hz, 1H), 4.60* (d, J.sub.H—F=24.8 Hz, 1H), 4.48 (d, J.sub.H—F=24.8 Hz, 1H), 4.33-4.29 (m, 1H), 3.74-3.64 (m, 1H), 2.81* (d, J.sub.H—H=9.3 Hz, 1H), 2.72 (d, J.sub.H—H=9.5 Hz, 1H), 1.48* (s, 9H), 1.34 (s, 9H). .sup.19F-NMR −179.71*, −180.11. .sup.13C NMR: 170.5 (d, J.sub.C—F=15.9 Hz), 154.3*, 153.6, 134.89*, 134.72, 128.83, 128.75, 128.71, 128.64, 128.56, 128.27, 96.96 (d, J.sub.C—F 191.1 Hz), 95.91* (d, J.sub.C—F 191.2 Hz), 80.98, 80.94*, 73.64* (d, J.sub.C—F=27.8 Hz), 72.71 (d, J.sub.C—F=29.0 Hz), 67.97, 64.67 (d, J.sub.C—F=24.7 Hz), 64.41* (d, J.sub.C—F=24.4 Hz), 53.31*, 52.87, 28.34*, 28.112. C.sub.17H.sub.22FNO.sub.5, expected 339.2, found m/z=240.1, [M-Boc+H].sup.+.

Preparative Compound 3b: 2-benzyl 1-(tert-butyl) (2R,3R,4S)-3-fluoro-4-hydroxypyrrolidine-1,2-dicarboxylate

(49) ##STR00083##

(50) (30% by .sup.19F-NMR, white solid, 25% isolated yield) .sup.1H-NMR (CDCl.sub.3) δ: 7.38-7.35 (m, 5H), 5.29-5.11 (m, 2H), 4.99-4.88 (m, 1H), 4.64-4.47 (m, 1H), 4.41-4.36 (m, 1H), 3.94-3.87 (m, 1H), 3.37-3.27 (m, 1H), 2.09 (d, J.sub.H—H 7.45 Hz), 1.47* (9H), 1.33 (9H). .sup.19F-NMR δ: −199.74, −200.29*. .sup.13C-NMR δ: 169.1 (d, J.sub.C—F=13.1 Hz), 168.7* (d, J.sub.C—F=12.9 Hz), 154.07*, 153.21, 135.10*, 134.9, 128.8, 128.6, 128.5, 128.2, 94.0 (d, J.sub.C—F=190.0 Hz), 93.2* (d, J.sub.C—F=189.6 Hz), 80.9, 70.3* (d, J.sub.C—F=18.2 Hz), 69.6 (d, J.sub.C—F=17.0 Hz), 67.6, 63.7 (d, J.sub.C—F=23.9 Hz), 63.5* (d, J.sub.C—F=23.8 Hz), 49.8*, 49.3, 23.8*, 28.1. C.sub.17H.sub.22FNO.sub.5, expected 339.2, found m/z=240.1, [M-Boc+H].sup.+.

Preparative Compound 3a: 2-benzyl 1-(tert-butyl) (2R,3S,4R)-3-fluoro-4-hydroxypyrrolidine-1,2-dicarboxylate

(51) ##STR00084##

(52) (58% by .sup.19F-NMR, white solid, 50% isolated yield). .sup.1H-NMR (CDCl.sub.3) δ: 7.37-7.32 (m, 5H), 5.35-5.10 (m, 3H), 4.64* (dd, J.sub.H—F=21.0 Hz, J.sub.H—H 5.9 Hz, 1H), 4.54 (dd, J-H—F=21.7 Hz, J.sub.H—H 5.9 Hz, 1H), 4.28 (br s, 1H), 3.87 (dd, J.sub.H—H=11.2 Hz, J.sub.H—H=6.6 Hz, 1H), 3.82* (dd, J.sub.H—H=11.1 Hz, J.sub.H—H=6.6 Hz, 1H), 3.48-3.41 (m, 1H), 2.75 (br s, 1H), 1.46* (s, 9H), 1.32 (s, 9H). .sup.19F-NMR δ: −207.06, −207.67*. .sup.13C-NMR δ: 168.3 (d, J.sub.C—F=7.0 Hz), 167.9* (d, J.sub.C—F=7.2 Hz), 153.9*, 153.2, 135.2*, 135.0, 128.6, 128.5, 128.4, 128.3, 128.2, 91.5 (d, J.sub.C—F=189.6 Hz), 90.8* (d, J.sub.C—F=188.6 Hz), 81.1, 70.6* (d, J.sub.C—F=17.7 Hz), 70.1 (d, J.sub.C—F=17.6 Hz), 67.6, 61.6 (d, J.sub.C—F=21.9 Hz), 61.2* (d, J.sub.C—F=22.0 Hz), 50.6*, 50.0, 28.3*, 28.1. C.sub.17H.sub.22FNO.sub.5, expected 339.2, found m/z=240.1, [M-Boc+H].sup.+.

(53) Mitsunobu Inversion, General Procedure in Accordance with Stage (iii) in Scheme 1 Process.

(54) To a solution of fluoro-hydroxyproline (0.435 mmol), triphenylphosphine (342.0 mg, 1.306 mmol) and 4-nitrobenzoic acid (218.2 mg, 1.306 mmol) in THF (2 mL), diisopropyl azodicarboxylate (DIAD) was added dropwise at 0° C. The flask was left at 0° C. for 4 hr, then the ice bath was removed and the mixture was stirred at r.t. for 24 hr. Et.sub.2O was added (10 mL) and the mixture was washed with saturated NaHCO.sub.3 (5 mL). The mixture was washed with brine (10 mL), the organic phase was dried over anhydrous MgSO.sub.4. Volatiles were removed under reduced pressure to obtain yellow oil which was subjected to FCC (EtOAc/Heptane 1:9 to 3:7) to isolate the desired product.

Preparative Compound 4a: 2-benzyl 1-(tert-butyl) (2R,3S,4S)-3-fluoro-4-((4-nitrobenzoyl)oxy) pyrrolidine-1,2-dicarboxylate

(55) ##STR00085##

(56) Preparative compound 4a was obtained from preparative compound 3a according to the general procedure for the Mitsunobu reaction as detailed hereinbefore (187 mg, 88% isolated yield), as a pale yellow wax. .sup.1H-NMR (CDCl.sub.3) δ: 8.31-8.29 (m, 2H), 8.17-8.15 (m, 2H), 7.38-7.35 (m, 5H), 5.57-5.54 (m, 1H), 5.45-5.33 (m, 1H), 5.31-5.16 (m, 2H), 4.84* (dd, J.sub.H—F=23.3 Hz, J.sub.H—H=5.7 Hz, 1H), 4.72 (dd, J.sub.H—F=24.32 Hz, J.sub.H—H=5.5 Hz, 1H), 4.05-4.00 (m, 1H), 3.90 (d, J.sub.H—H=12.8 Hz, 1H), 3.76* (d, J.sub.H—H=12.7 Hz), 1.46* (s, 9H), 1.36 (s, 9H). .sup.19F-NMR δ: −192.43, −193.28*. C.sub.24H.sub.25FN.sub.2O.sub.8, rt=1.962 min, expected 488.1, found m/z=389.1, [M-Boc+H].sup.+.

Preparative Compound 4b: 2-benzyl 1-(tert-butyl) (2R,3R,4R)-3-fluoro-4-((4-nitrobenzoyl)oxy) pyrrolidine-1,2-dicarboxylate

(57) ##STR00086##

(58) Preparative compound 4b was obtained from preparative compound 3b according to the general procedure for the Mitsunobu reaction as detailed hereinbefore (30 mg, 95% yield, light yellow wax). .sup.1H NMR (500 MHz, CDCl.sub.3) 8.22-8.18 (m, 2H), 8.02-7.95 (m, 2H), 7.26-7.22 (m, 5H), 5.57-5.54 (m, 1H), 5.34-5.06 (m, 3H), 4.89-4.67 (m, 1H), 4.03-3.96 (m, 1H), 3.88-3.74 (m, 1H), 1.50* (s, 9H), 1.40 (s. 9H). .sup.19F-NMR δ: −182.16, −183.06*. C.sub.24H.sub.25FN.sub.2O.sub.8, rt=1.975 min, expected 488.1, found m/z=389.1, [M-Boc+H].sup.+.

Preparative Compound 3d: 2-benzyl 1-(tert-butyl) (2R,3S,4S)-3-fluoro-4-hydroxypyrrolidine-1,2-dicarboxylate

(59) ##STR00087##

(60) To a solution of the 4-nitrobenzonate ester, preparative compound 4a (24.4 mg, 0.05 mmol) in methanol (1.0 mL), sodium azide (10 mg, 0.15 mmol) was added under stirring. The mixture was heated and stirred to 50° C. until TLC analysis (EtOAc/Heptane 4:6) showed complete conversion of the starting material (15-30 min). The mixture was cooled to 0° C., brine was added (1 mL) and extracted with EtOAc (3×5 mL). The organic phase was dried over anhydrous MgSO.sub.4, the volatile components (solvent) were removed under reduced pressure, to obtain a yellow oil which was subjected to FCC (EtOAc/Heptane 1:1) to isolate the desired product (13.5 mg, 80% isolated yield) as a pale yellow wax. .sup.1H-NMR (CDCl.sub.3) δ: 7.36-7.35 (m, 5H), 5.31-5.05 (m, 3H), 4.74* (dd, J.sub.H—F=24.3 Hz, J.sub.H—H 5.1 Hz, 1H), 4.65 (dd, J.sub.H—F=25.5 Hz, J.sub.H—H 5.2 Hz, 1H), 4.45-4.42 (m, 1H), 3.77-3.74 (m, 1H), 3.68-3.56 (m, 1H), 2.33 (br s, 1H), 1.46* (s, 9H), 1.33 (s, 9H). .sup.19F-NMR δ: −192.30, −192.41*. .sup.13C-NMR δ: 167.5 (d, J.sub.C—F=8.3 Hz), 167.3* (d, J.sub.C—F=10.8 Hz), 154.4*, 153.9, 135.4*, 135.3, 130.7, 128.6, 128.5, 123.2, 123.6, 95.5 (d, J.sub.C—F=187.7 Hz), 94.8* (d, J.sub.C—F=187.3 Hz), 80.9, 80.8*, 72.8* (d, J.sub.C—F=28.0 Hz), 72.1 (d, J.sub.C—F=27.0 Hz), 67.2, 62.4 (d, J.sub.C—F=21.2 Hz), 61.9* (d, J.sub.C—F=21.0 Hz), 52.1*, 51.8, 28.4*, 28.1. C.sub.17H.sub.22FNO.sub.5, expected 339.2, found m/z=240.1, [M-Boc+H].sup.+.

Preparative Compound 3c: 2-benzyl 1-(tert-butyl) (2R,3R,4R)-3-fluoro-4-hydroxypyrrolidine-1,2-dicarboxylate

(61) ##STR00088##

(62) Preparative compound 3c, was obtained following the procedure as reported hereinbefore for preparative compound 3d. The desired product was obtained in 83% isolated yield as a transparent oil, analytical data matches with the product obtained from the direct reduction of the fluoroketone 3c.

(63) General Procedure for the De-Benzylation of Benzylester Intermediate Preparative Compounds in Accordance with Stage (v) of Scheme 1 Process

(64) The selected benzyl ester 3a, 3b, 3c or 3d (85 mg, 0.250 mmol) was dissolved in a mixture of MeOH/THF 1:2 (15 mL), a catalytic amount of palladium over carbon (10%, dry) was added and the mixture was stirred under hydrogen atmosphere until TLC analysis (EtOAc/Heptane 1:1) showed complete conversion of the starting material. The mixture was then filtered on a celite pad and the solvents were removed in vacuum to obtain the de-benzylated product which was used directly in the next stage without further purification.

Preparative Compound 5d: (2R,3S,4S)-1-(tert-butoxycarbonyl)-3-fluoro-4-hydroxypyrrolidine-2-carboxylic Acid

(65) ##STR00089##

(66) Starting from the corresponding preparative benzyl ester, 4d using the de-benzylation procedure detailed hereinbefore preparative compound 5d was obtained as a white solid (62 mg) in 99% isolated yield. .sup.1H-NMR (CD.sub.3OD) δ: 5.13-4.99 (m, 1H), 4.61-4.50 (m, 1H), 4.30-7.27 (m, 1H), 3.61-3.54 (m, 2H), 1.476* (s, 9H), 1.44 (s, 9H). .sup.19F-NMR δ: −191.59, −191.73*. .sup.13C-NMR δ: 169.6 (d, J.sub.C—F=8.5 Hz), 169.2* (d, J.sub.C—F=8.4 Hz), 155.0*, 154.6, 95.5 (d, J.sub.C—F=186.0 Hz), 95.2* (d, J.sub.C—F=128.5 Hz), 80.6, 80.4*, 71.9* (d, J.sub.C—F=26.6 Hz), 71.3 (d, J.sub.C—F=26.7 Hz), 62.4 (d, J.sub.C—F=21.3 Hz), 62.0* (d, J.sub.C—F=21.6 Hz), 52.2*, 51.3, 27.3*, 27.0. C.sub.10H.sub.16FNO.sub.5, expected 249.1, found m/z=150.1, [M-Boc+H].sup.+.

Preparative Compound 5b: (2R,3R,4S)-1-(tert-butoxycarbonyl)-3-fluoro-4-hydroxypyrrolidine-2-carboxylic Acid

(67) ##STR00090##

(68) Starting from the corresponding preparative benzyl ester, 4b using the de-benzylation procedure detailed hereinbefore preparative compound 5b was obtained as a white solid (62 mg) in 99% isolated yield. .sup.1H-NMR (CD3OD) δ: 5.01-4.93 (m, 1H), 4.43-4.27 (m, 2H), 3.78-3.74 (m, 1H), 3.30-3.28 (m, 1H), 1.49* (s, 9H), 1.44 (s, 9H). .sup.19F-NMR δ: −199.02, −199.10*. C.sub.10H.sub.16FNO.sub.5, expected 249.1, found m/z=150.1, [M-Boc+H].sup.+.

Preparative Compound 5e: (2R,3S,4S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-3-fluoro-4-hydroxypyrrolidine-2-carboxylic Acid

(69) ##STR00091##

(70) Compound 5d ((2R,3S,4S)-1-(tert-butoxycarbonyl)-3-fluoro-4-hydroxypyrrolidine-2-carboxylic acid) (67 mg, 0.36 mmol) was BOC deprotected using a solution of 4 N HCl in Dioxane (2 mL) in DCM (2 mL). Solvents were evaporated and the crude product was dissolved in a mixture of water/dioxane (1:1, 4 mL). Sodium hydrogencarbonate (94 mg, 1.1.6 mmol) was added and the mixture was stirred at room temperature for 10 minutes. 9-Fluorenylmethyl N-succinimidyl carbonate (121 mg, 0.36 mmol) was added in small portions and the mixture was stirred at room temperature overnight). The reaction was cooled to 0° C. and treated with KHSO.sub.4 (5%) to pH=3-4. The desired product was extracted with ethyl acetate (3×15 mL) and the organic phase was dried over MgSO4. Solvents were evaporated to obtain the crude product which can be further purified by column chromatography on silica using a gradient from 5% to 20% of MeOH in DCM. (Obtained 106 mg, 79% yield). MS analysis: C20H18FNO, expected 371.4, found 372.4 [M+H.sup.+]

(71) .sup.1H NMR (500 MHz, CDCl3/MeOD 8:2) mixture of rotamers, δ: 7.73-7.69 (m, 2H), 7.59-7.52 (m, 2H), 7.37-7.31 (m, 2H), 7.29-7.24 (m, 2H), 5.18-5.02 (m, 1H), 4.75-4.61 (m, 1H), 4.38-4.14 (m, 4H), 3.79-3.57 (m, 4H). .sup.19F-NMR δ: −188.40, −188.58. 13C NMR δ: 172.6, 155.3, 155.2, 143.7, 143.6, 143.5, 143.4, 141.0, 141.0, 140.9, 127.5, 127.5, 126.9, 124.9, 124.9, 119.7, 119.7, 96.2, 95.3, 94.7, 93.8, 71.9, 71.7, 71.2, 71.0, 68.0, 67.7, 66.8, 62.3, 62.1, 62.1, 57.5, 52.4, 52.1,

Preparative Compound 5f: (2R,3R,4S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-3-fluoro-4-hydroxypyrrolidine-2-carboxylic Acid

(72) ##STR00092##

(73) Compound 5b ((2R,3R,4S)-1-(tert-butoxycarbonyl)-3-fluoro-4-hydroxypyrrolidine-2-carboxylic acid) (67 mg, 0.36 mmol) was BOC deprotected using a solution of 4 N HCl in Dioxane (2 mL) in DCM (2 mL). Solvents were evaporated and the crude product was dissolved in a mixture of water/dioxane (1:1, 4 mL). Sodium hydrogencarbonate (94 mg, 1.1.6 mmol) was added and the mixture was stirred at room temperature for 10 minutes. 9-Fluorenylmethyl N-succinimidyl carbonate (121 mg, 0.36 mmol) was added in small portions and the mixture was stirred at room temperature overnight). The reaction was cooled to 0° C. and treated with KHSO.sub.4 (5%) to pH=3-4. The desired product was extracted with ethyl acetate (3×15 mL) and the organic phase was dried over MgSO4. Solvents were evaporated to obtain the crude product which can be further purified by column chromatography on silica using a gradient from 5% to 20% of MeOH in DCM. (Obtained 100 mg, 82% yield). MS analysis: C20H18FNO, expected 371.4, found 372.4 [M+H.sup.+]

(74) .sup.1H NMR (500 MHz, CDCl3) (mixture of rotamers) δ: 7.76-7.69 (m, 2H), 7.56-7.49 (m, 2H), 7.41-7.27 (m, 4H), 5.21-4.93 (m, 2H), 4.61 (d, J=18.6 Hz, 1H), 4.46-4.37 (m, 3H), 4.25-4.10 (m, 1H), 3.91-3.84 (m, 1H), 3.40 (t, J=9.3 Hz, 1H). 19F NMR δ: −199.35, −201.73. 13C NMR δ: 172.0, 171.9, 170.9, 170.8, 156.4, 154.6, 143.7, 143.6, 141.6, 141.6, 128.2, 127.4, 125.2, 125.2, 120.3, 93.8, 92.3, 70.2, 70.1, 69.7, 69.5, 68.8, 68.2, 64.0, 63.8, 63.6, 63.4, 49.6, 47.2,

(75) Preparatory Compounds for the Preparation of Stereoselective F-HYP in Accordance with Scheme 2 Starting Material, Ketone 6:

(76) Starting ketone 6 was prepared in accordance with the method as described in Zanato et al, Org. Biomol. Chem., 2014, 12, 9638.

(77) A solution of methyl (2S, 4R)-4-hydroxypyrrolidine-2-carboxylate hydrochloride (3.0 g, 16.5 mmol) and chlorotrimethylsilane (5.2 mL, 41.3 mmol) in DCM (40 mL) at 0° C. was treated with TEA under stirring (8.0 mL, 57.8 mmol) and allowed to reach r.t. The mixture was stirred at reflux for 1 h, cooled to 0° C., treated with MeOH (1.0 mL) in DCM (4.5 mL), allowed to warm to r.t. for 1 h and then treated with 9-bromo-9-phenylfluorene (6.9 g, 21.45 mmol) and Pb(NO3)2 (4.9 g, 14.9 mmol). The mixture was stirred at r.t. for 96 h, filtered and evaporated. The remaining solid was dissolved in MeOH (54 mL) and citric acid (5.5 g) was added. The solution was vigorously stirred for 1 additional hour, solvent was evaporated under reduce pressure. EtOAc (50 mL) was added and the mixture was carefully washed with saturated NaHCO.sub.3 solution. The organic phase was dried over anhydrous MgSO.sub.4, then solvent was removed under reduced pressure. The crude product was purified by flash chromatography (Heptane/EtOAc 1:1) to give the Pf protected compound (5.7 g, 90%) as a white foam. .sup.1H NMR (400 MHz, CDCl3) δ: 1.79 (ddd, 1H, J=5.6, 8.9, 13.0 Hz), 1.98 (dt, 1H, J=5.6, 12.6 Hz), 2.92 (dd, 1H, J=4.8, 9.9 Hz), 3.24 (s, 3H), 3.29 (dd, 1H, J=5.3, 8.8 Hz), 3.57 (dd, 1H, J=5.3, 10.0 Hz), 4.43-4.58 (m, 1H), 7.16 (td, 1H, J=1.1, 7.5 Hz), 7.21-7.35 (m, 6H), 7.43 (td, 1H, J=1.1, 7.5 Hz), 7.50-7.59 (m, 3H), 7.65 (dd, 1H, J=0.7, 6.8 Hz), 7.74 (dd, 1H, J=0.8, 6.8 Hz); 13C NMR (100 MHz, CDCl3) δ: 40.0, 51.3, 56.8, 59.3, 70.4, 76.1, 119.8, 120.1, 126.4, 127.1, 127.2, 127.3 (×2), 127.4, 127.6, 128.3 (×2), 128.4, 128.8, 139.9, 141.5, 142.7, 146.1, 147.2, 175.8.

Preparative Compound 7: Methyl (2R,3S)-3-fluoro-4-oxo-1-(9-phenyl-9H-fluoren-9-yl)pyrrolidine-2-carboxylate

(78) ##STR00093##

(79) A solution of starting ketone 6 (600 mg, 1.57 mmol) in DCM (15 mL) was cooled to −30° C., and TEA (700 μL, 4.70 mmol) was added dropwise. Trimethylsilyltriflate (TMSOTf) (570 μL, 3.13 mmol) was then added dropwise at −30° C. The mixture was stirred for 2 hr and kept below −10° C. The solvent was then removed under vacuum and pentane (25 mL) was added. The mixture was poured into a saturated solution of NaHCO.sub.3 (50 mL) and was vigorously shaken. The organic layer was separated, washed with brine, then dried over anhydrous MgSO.sub.4. The solvent (volatiles) was removed under reduced pressure to obtain a light yellow oil, corresponding to a non-isolated intermediate trimethylsilylenolate, which was dissolved in acetonitrile (30 mL) and cooled to −40° C. Selectfluor (834 mg, 2.35 mmol) was added in one portion and the mixture was allowed to reach 10° C. overnight. TLC analysis (EtOAc/Heptane 2:8) showed complete conversion of the starting material. A saturated solution of NH.sub.4Cl was added (30 ML), the resulting mixture was vigorously shaken and the organic layer was then separated. The aqueous layer was extracted with EtOAc (2×30 mL) and the organic phase was washed with brine, then dried over MgSO.sub.4. Volatiles were removed under reduced pressure to obtain the crude product as yellow oil, which was purified by FCC (EtOAc/Heptane 1:9) to obtain the desired fluoroketone (346 mg, 55% yield) as a white solid. MS analysis do not show any informative result as the only ion detected corresponded to the 9-phenylfluorenyl carbocation m/z=241.1.

(80) .sup.1H-NMR (CDCl.sub.3) δ: 7.76 (d, J.sub.H—H=7.6 Hz, 1H), 7.71 (d, J.sub.H—H=7.6 Hz, 1H), 7.46-7.325 (m, 11H), 5.07 (dd, J=H—F=51.0 Hz, J.sub.H—H=7.9 Hz, 1H), 4.03 (d, J=8.1 Hz, 1H), 3.98 (d, J.sub.H—H=17.9 Hz, 1H), 3.63 (d, J.sub.H—H=17.9 Hz, 1H), 3.19 (s, 3H). 19F-NMR −206.52. .sup.13C-NMR δ: 205.4 (d, J.sub.C—F=12.7 Hz), 169.3, 124.2, 144.7, 141.2, 140.6, 139.9, 129.3, 129.2, 128.8, 123.3, 128.0, 127.7, 126.8, 126.6, 125.1, 120.5, 120.4, 89.1 (d, J.sub.C—F=205.4 Hz), 75.1, 60.0 (d, J.sub.C—F=20.3. Hz), 51.8 (d, J.sub.C—F=22.8 Hz).

Preparative Compound 8: Methyl (2R,3S,4R)-3-fluoro-4-hydroxy-1-(9-phenyl-9H-fluoren-9-yl)pyrrolidine-2-carboxylate

(81) ##STR00094##

(82) Preparative fluoroketone compound 7 (638 mg, 1.59 mmol) was dissolved in THF/EtOH 1:1 (20 mL) at 0° C. NaBH.sub.4 (63 mg, 1.67 mmol) was added portion-wise and the reaction mixture was stirred for 1 hour at 0 C. TLC analysis (EtOAc/Heptane 3:7) showed complete conversion of the starting material. The reaction mixture was concentrated under vacuum, EtOAc was added (30 mL) and a solution of NaHSO.sub.4 (5%) was added dropwise until a pH of 3 to 4 was obtained. The acidified mixture was washed with brine (10 mL), and the organic phase was dried over anhydrous MgSO.sub.4. Volatiles were removed under reduced pressure to obtain the title compound as a white solid in quantitative yield.

(83) .sup.1H-NMR (CDCl.sub.3) δ: 7.79 (d, J.sub.H—H=7.6 Hz, 1H), 7.65 (d, J.sub.H—H=7.5 Hz, 1H), 7.55-7.53 (m, 2H), 7.49-7.48 (m 1H), 7.44-7.42 (m, 1H), 7.36-7.33 (m, 1H), 7.30-7.26 (m, 5H), 7.15-7.12 (m, 1H), 4.76-4.64 (m, 1H), 4.15 (br s, 2H), 3.45 (dd, J.sub.H—H=10.6 Hz, J.sub.H—H=5.0 Hz, 1H), 3.39 (s, 3H), 3.27 (dd, J.sub.H—F=8.8 Hz, J.sub.H—H=6.3 Hz, 1H), 3.05 (d, J.sub.H—H=10.6 Hz, 1H). .sup.19F-NMR δ: −205.26. .sup.13C-NMR δ: 173.8 (d, J.sub.C—F=4.6 Hz), 147.4, 144.6, 141.9, 140.8, 139.2, 129.1, 128.7, 128.6, 127.9, 127.7, 127.4, 126.0, 126.3, 120.4, 120.1, 90.7 (d, J.sub.C—F=197.6 Hz), 75.6, 70.8 (d, J.sub.C—F=16.1 Hz), 60.8 (d, J.sub.C—F=21.8 Hz), 52.9 (d, J c.sub.F=3.5 Hz), 52.2, 29.7.

Preparative Compound 10: Methyl (2R,3S,4S)-3-fluoro-4-((4-nitrobenzoyl)oxy)-1-(9-phenyl-9H-fluoren-9-yl)pyrrolidine-2-carboxylate

(84) ##STR00095##

(85) Preparative compound 10 was prepared from preparative compound 8 according to the general procedure for the Mitsunobu inversion as detailed hereinbefore. Compound 10 was obtained in 87% yield.

(86) .sup.1H-NMR (CDCl.sub.3) δ: 8.30-8.28 (m, 2H), 8.14-8.12 (m, 2H), 7.74-7.73 (m, 1H), 7.69-7.68 (m, 1H), 7.52-7.50 (m, 2H), 7.40-7.38 (m, 4H), 7.28-7.18 (m, 5H), 5.75-5.68 (m, 1H), 5.06 (ddd, J H-F+53.5 Hz, J.sub.H—H=8.0 Hz, J.sub.H—H=5.5 Hz, 1H), 4.04 (dd, J.sub.H—F=10.2 Hz, J.sub.H—H=6.8 Hz, 1H), 3.67 (dd, J.sub.H—H=7.9 Hz, J.sub.H—H=6.7 Hz, 1H), 3.36 (s, 3H), 3.08 (dd, J.sub.H—H=10.5 Hz, J.sub.H—H=5.6 Hz, 1H). .sup.19F-NMR δ: −196.44. .sup.13C-NMR δ: 170.3 (d, J.sub.C—F=4.5 Hz), 163.9, 150.8, 146.1, 145.6, 141.1, 140.1, 134.7, 130.9, 129.0, 128.6, 127.9, 127.8, 127.4, 127.1, 126.0, 123.6, 120.3, 120.1, 93.3 (d, J.sub.C—F=195.3 Hz), 75.4, 61.9 (d, J.sub.C—F=21.7 Hz), 51.7, 49.7 (d, J.sub.C—F=5.6 Hz).

Preparative Compound 11: Methyl (2R,3S,4S)-3-fluoro-4-hydroxy-1-(9-phenyl-9H-fluoren-9-yl)pyrrolidine-2-carboxylate

(87) ##STR00096##

(88) To a stirred solution of the 4-nitrobenzoate ester of preparative compound 10 (217 mg, 0.391 mmol) in THF (8 mL) at 0° C., LiOH (19 mg, 0.469 mmol) dissolved in water (2 mL) was added dropwise. The mixture was stirred at 0° C. for 2 hours. TLC analysis (EtOAc/Heptane 3:7) showed complete conversion of the starting material. The reaction mixture was concentrated under vacuum, water (5 mL) was added and the mixture was extracted with EtOAc (3×15 mL). The organic phase was washed with brine (5 mL), dried over anhydrous MgSO.sub.4 and the volatiles were removed under reduced pressure. The crude product was purified by FCC (EtOAc/Heptane 1:9) to obtain the title compound as white foam in quantitative yield.

(89) .sup.1H-NMR (CDCl.sub.3) δ: 7.75 (d, J.sub.H—H=7.5 Hz, 1H), 7.65 (d, J.sub.H—H=7.5 Hz, 1H), 7.54-7.52 (m, 2H), 7.49 (d, J.sub.H—H=7.6 Hz, 1H), 7.46-7.43 (m, 1H), 7.35-7.23 (m, 6H), 7.18-7.15 (m, 1H), 4.71-4.53 (m, 2H), 3.61-3.58 (m, 1H), 3.47-3.44 (m, 1H), 3.38 (s, 3H), 2.29-2.84 (m, 1H). .sup.19F-NMR δ: −197.63.

Preparative Compound 9a: (2R,3S,4R)-3-fluoro-4-hydroxy-2-(methoxycarbonyl)pyrrolidin-1-ium 2,2,2-trifluoroacetate

(90) ##STR00097##

(91) Preparative compound 8 (100 mg, 0.248 mmol) was dissolved in DCM (10 mL). Triisopropylsilane (TIPS) (760 μL) and trifluoroacetic acid (TFA) (500 μL) were added, and the resultant yellow mixture was left to react at room temperature for 2 h. Water 5 mL was added, the mixture was vigorously shaken and the aqueous phase was separated, washed with 2 mL of Et.sub.2O and freeze-dried to afford the title compound in 80% yield.

(92) .sup.1H-NMR (D.sub.2O) δ: 5.46-5.34 (m, 1H), 4.83 (dd, J H—F=32.1 Hz, J.sub.H—H=2.9 Hz, 1H), 4.70-4.61 (m, 1H), 3.85 (s, 3H), 3.76-3.72 (m, 1H), 3.32-3.28 (m, 1H). .sup.19F-NMR δ: −75.56 (3F), −208.8 (1F).

Preparative Compound 9b: (2R,3S,4S)-3-fluoro-4-hydroxy-2-(methoxycarbonyl)pyrrolidin-1-ium 2,2,2-trifluoroacetate

(93) ##STR00098##

(94) Prepared as described for compound 9a from preparative compound 11 in 82% yield. 1H-NMR (D2O) δ: 5.38 (d, J H—F=48.9 Hz, 1H), 4.95 (dd, J H—F=33.3 Hz, J.sub.H—H=2.3 Hz, 1H), 4.67 (br s, 1H), 3.86 (s, 3H), 3.75-3.72 (m, 1H), 3.47 (d, J.sub.H—H=1301 Hz, 1H). .sup.19F-NMR δ: −75.56 (3F), −192.27 (1F). .sup.13C-NMR δ: 165.9 (d, J.sub.C—F=5.6 Hz), 162.3 (d, J.sub.C—F=35.5 Hz), 116.3 (d, J.sub.C—F=291.7 Hz), 94.4 (d, J.sub.C—F=185.6 Hz), 71.6 (d, J.sub.C—F=27.2 Hz), 62.7 (d, J.sub.C—F=21.3 Hz), 54.1, 51.4.

(95) Scheme 3—General Procedure for Synthesis of Compounds of Formula I, IA or IB (A-Groups)

(96) Any compounds of formula I as defined herein can be prepared in accordance with the general methodology in Scheme 3 by selection of the appropriate reagents at the following stages: choice of C- or N-5 membered ring and selection of the desired R.sup.2a, R.sup.2b, R.sup.x, substituents thereon as the start-point; selection of the desired R.sup.3, R.sup.4 and R.sup.5 groups to determine the appropriate reagent for utility in stage (i); selection of the desired Y group (Y.sub.A, Y.sub.B, Y.sub.C) to determine the appropriate reagents at stages (ii) and (iii); and finally selection of the desired R.sup.1 group in the final stages to provide the compound of formula I, IA or IB.

(97) ##STR00099##

(98) As will be appreciated by the skilled chemist, using the general procedures indicated in Scheme 3, and via selection of the appropriate starting materials (5a-d) and using the methodology provided for the preparation of Boc-protected compounds (13a-d) and compounds of formula IA (14a-e) any Boc-protected amine suitable for use in the preparation of compounds of formula I, or any compound of formula IA or IB as defined herein can be prepared.

(99) In particular in step (i) the use of alternative starting materials, provides alternative final compounds having different R.sup.2a and/or R.sup.2b groups, to that provided in the above examples, or compounds where X=C, as opposed to N as illustrated in the above examples. Exemplary commercially available materials suitable for such use include: 2-trifluoromethyl; 2-fluoroproline; 2,2-difluoroproline; and 2-aminoproline. As will also be appreciated further starting reagents suitable for use in step (i) can be readily prepared from hydroxyproline, or from one or other commercially available alternatives, for example, 2-aminoproline (where R.sup.2a is —NH.sub.2 and R.sup.2b is H) in the final compound (where X=N) can be made starting from hydroxyproline via suitable modification of the synthetic routes provided herein for synthesis of F-HYP starting materials.

(100) A general method for Boc-deprotection is provided in Scheme 7 herein.

(101) Preparation of Compounds of General Formula I, IA or IB in Accordance with Scheme 3

Preparative Compound 12b: tert-butyl (2R,3R,4S)-3-fluoro-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylate

(102) ##STR00100##

(103) To a solution of (4-(4-methylthiazol-5-yl)phenyl)methanamine (32.7 mg, 0.160 mmol) in DMF (1 mL), a mixture of N-Boc-fluoro-hydroxyproline 5b (40.0 mg, 0.160 mmol), HATU (61.0 mg, 0.106 mmol), 1-hydroxy-7-azabenzotriazole (HOAT) (22.0 mg, 0.160 mmol) and N,N-Diisopropylethylamine, also called Hünig's base (DIPEA) (85 μL, 0.480 mmol) in DMF (0.5 mL) was added dropwise under stirring at r.t. After one hour, HPLC analysis showed complete conversion of the starting material and formation of the desired product (METHOD 1, rt=1.451 min, m/z=436.2, [M+H].sup.+). The reaction was diluted with DCM (10 mL), washed with water (2 mL), the organic phase was extracted and then dried over anhydrous MgSO.sub.4 and the solvents were then removed under vacuum. Purification by preparative HPLC (acidic method) afforded the title compound as a white solid (60 mg) in 86% yield.

(104) .sup.1H-NMR (MeOD) δ: 9.10-9.07 (m, 1H), 7.47-7.46 (m, 4H), 4.97-4.85 (overlapped with signal of residual water in the solvent, m, 1H), 4.52-4.33 (m, 4H), 3.81-3.76 (m, 1H), 2.51 (s, 3H), 1.49* (s, 9H), 1.33 (s, 9H). .sup.19F-NMR δ: −198.50, −199.21*.

(105) The starting material, 4-(4-methylthiazol-5-yl)phenyl)methanamine was prepared in accordance with the methodology in unpublished international patent application PCT/GB2016/050691, preparative compound (2).

Preparative Compound 12d: tert-butyl (2R,3S,4S)-3-fluoro-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylate

(106) ##STR00101##

(107) Preparative compound 12d, was obtained in accordance with the methodology as described for preparative compound 12b, and starting from compound 5d, HPLC analysis (METHOD 2) rt=3.007 min, m/z=436.2, [M+H].sup.+. (82% yield, white solid). .sup.1H-NMR (CD3OD) δ: 8.79 (s, 1H), 7.48-7.44 (m, 4H), 5.12-5.01 (m, 1H), 4.63-4.39 (m, 3H), 4.34-4.32 (m, 1H), 3.76-3.62 (m, 2H), 2.49 (s, 3H), 1.50* (s, 9H), 1.34 (s, 9H). .sup.19F-NMR δ: −193.81, −194.03*

Preparative Compound 13b: tert-butyl ((S)-1-((2R, 3R, 4S)-3-fluoro-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate

(108) ##STR00102##

(109) To a solution of preparative compound 12b (22.0 mg, 0.05056 mmol) in DCM/MeOH 9:1 (1.0 mL), HCl in dioxane (4M, 1.0 mL) was added and mixture was stirred for 4 hours at room temperature. The volatiles were removed under vacuum, and the residue was dissolved in water and freeze-dried to afford the deprotected amine as the corresponding hydrochloride salt form in quantitative yield as a light yellow powder. This salt was used without any further purification, and was suspended in DMF (1 mL) and DIPEA (22 μL, 0.126 mmol) was added. To the resulting solution, a mixture of (S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoic acid (11.7 mg, 0.0506 mmol), HATU (19.0 mg, 0.0506 mmol), HOAT (6.9 mg, 0.05056 mmol) and DIPEA (22 μL, 0.126 mmol) in DMF (0.5 mL) was added under stirring at r.t. After 3 hours, HPLC analysis (METHOD 1, rt=1.646 min, m/z=493.2, [M-tBu+H].sup.+) showed complete conversion of the starting material and the formation of the desired product. DCM (10 ml) was added and the mixture was washed with water (1 mL) and NaHSO.sub.4 solution (5%, 1 mL). The organic layer was dried over anhydrous MgSO.sub.4 and the solvents were removed under reduced pressure. The crude product was obtained as a yellow wax (25 mg, 90% yield) and was used directly in the next step without any further purification.

(110) .sup.1H NMR (500 MHz, MeOD) δ: 8.94 (s, 1H), 7.48-7.42 (m, 4H), 5.05-4.92 (m, 1H), 4.71-4.63 (m, 1H), 4.59-4.47 (m, 2H), 4.41-4.35 (m, 1H), 4.30 (s, 1H), 4.08-4.03 (m, 1H), 3.79-3.72 (m, 1H), 2.49 (s, 3H), 1.46* (s, 9H), 1.02 (s, 9H). .sup.19F-NMR δ: −200.36.

Preparative Compound 13d: tert-butyl ((S)-1-((2R,3S,4S)-3-fluoro-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate

(111) ##STR00103##

(112) Preparative compound 13d, was obtained in accordance with the methodology as described for preparative compound 13b, and starting from preparative compound 12d. The crude product was obtained as a yellow wax in 88% yield. HPLC analysis (METHOD 1): rt=1.639 min, m/z=493.2, [M M-tBu+H].sup.+. .sup.1H-NMR (MeOD) δ: 8.93 (s, 1H), 8.63-8.61 (m, 1H), 7.47-7.42 (m, 4H), 5.14-5.02 (m, 1H), 4.79 (dd, J H—F=26.0 Hz, J H—H=5.3 Hz, 1H), 4.54-4.43 (m, 3H), 4.30 (s, 1H), 3.97-3.91 (m, 2H), 2.49 (s, 3H), 1.44 (cis BOC rotmer, s, 9H), 1.06 (s, 9H). .sup.19F-NMR δ: −193.39.

Preparative Compound 14b: tert-butyl ((S)-1-((2R,3R,4S)-3-fluoro-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate

(113) ##STR00104##

(114) To a solution of preparative compound 13b (25.0 mg, 0.0455 mmol) in DCM/MeOH 9:1 (1.0 mL), HCl in dioxane (4M, 1.0 mL) was added and the resultant mixture was stirred for 4 hours at room temperature. The volatiles were removed under vacuum, the residue was dissolved in water and freeze-dried to afford the deprotected amine as a light yellow powder in quantitative yield in the form of the corresponding hydrochloride salt. This salt was used directly without any further purification and was suspended in DCM (1 mL), TEA (12.7 μL, 0.091 mmol) was added, followed by acetic anhydride (4.3 μL, 0.0455 mmol) was added at 0° C., and the resultant mixture was stirred at 0° C. for two hours. HPLC analysis showed complete conversion of the starting material and the formation of the desired product (METHOD 2 rt=2.819 min, m/z=491.2, [M+H].sup.+). The volatile solvents were removed and the crude was purified by preparative HPLC to obtain the title compound as a white solid (17.0 mg, 75% yield).

(115) .sup.1H NMR (500 MHz, MeOD) δ: 8.89 (s, 1H), 7.47-7.41 (m, 4H), 5.03-4.91 (m, 1H), 4.65 (dd, J H—F=21.4 Hz, J H—H=2.8 Hz, 1H), 4.60 (s, 1H), 4.58-4.35 (m, 3H), 4.06-4.03 (m, 1H), 3.79-3.75 (m, 1H), 2.48 (s, 3H), 2.01 (s, 3H), 1.04 (s, 9H). .sup.19F-NMR δ: −199.02 (cis rotmer) −200.31.

Preparative Compound 14d: tert-butyl ((S)-1-((2R,3S,4S)-3-fluoro-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate

(116) ##STR00105##

(117) Preparative compound 14d, was obtained in accordance with the methodology as described for preparative compound 14b, and starting from preparative compound 13d. The product was obtained in 76% yield as a white solid. HPLC analysis (METHOD 2) rt=2.901 min, m/z=491.2, [M+H].sup.+. .sup.1H NMR (500 MHz, MeOD) δ: 8.88 (s, 1H), 7.47-7.41 (m, 4H), 5.14-5.00 (m, 1H), 4.77 (dd, J H—F=26.15 Hz, J H—H=5.2 Hz, 1H), 4.63 (s, 1H), 4.55-4.32 (m, 3H), 4.00-3.91 (m, 2H), 2.48 (s, 3H), 2.01 (s, 3H), 1.08 (s, 9H). .sup.19F-NMR δ: −193.12.

Preparative Compound 14e: 2R,3R,4S)-1-((S)-2-(1-cyanocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide

(118) ##STR00106##

(119) To a solution of preparative compound 13d (10.0 mg, 0.0206 mmol) in DMF (0.3 mL), DIPEA (8.8 μL, 0.0515 mmol) was added under stirring at r.t. To the resulting solution, a mixture of 1-cyanocyclopropane-1-carboxylic acid (2.3 mg, 0.0206 mmol), HATU (8.0 mg, 0.0206 mmol), HOAT (2.8 mg, 0.0206 mmol) and DIPEA (8.8 μL, 0.0515 mmol) in DMF (0.3 mL) was added dropwise under stirring at r.t. After 2 hours, HPLC analysis showed complete conversion of the starting material and the formation of the expected product (METHOD 1, rt=1.532 min, m/z=542.2, [M+H].sup.+). DCM (10 ml) was added and the mixture was washed with water (1 mL). The organic layer was dried over anhydrous MgSO.sub.4 and the solvents were removed under reduced pressure. The crude was purified by preparative HPLC to obtain the title compound as a white solid (8 mg, 72% yield). .sup.1H NMR (400 MHz, MeOD) δ: 8.90 (s, 1H), 7.49-7.44 (m, 4H), 5.08-4.93 (m, 1H), 4.67 (dd, J.sub.H—F=20.9, dd, J.sub.H—F=3.2 Hz, 1H), 4.66 (s, 1H), 4.61-4.35 (m, 3H), 4.04 (dd, J=6.0, 10.2 Hz, 1H), 3.75-3.69 (m, 1H), 2.50 (s, 3H), 1.70-1.55 (m, 4H), 1.07 (s, 9H). .sup.19F-NMR δ: −198.8*, −200.71.

(120) Scheme 4—General Process for Coupling A to Az-L to Provide Azide of Structure Az-L-A

(121) Scheme 4 illustrates the synthetic methodology for the provision of azide intermediate compounds of structure Az-L-A suitable for use in the preparation of F-HYP containing PROTACs of structure A-L-B. As outlined in Scheme 4, the starting protected amines, for example preparative compounds (13a-d), are firstly de-protected to furnish amine intermediates, for example preparative compounds (15a-d) by using HCl in dioxane, in stage (i), with subsequent treatment with the desired linker, for example Az-PEG-linkers (A6) or (A7) in the presence of HATU and HOAT and the pH being adjusted to >9 by addition of DIPEA. After stirring for 4 h at 25° C. the reaction mixture was extracted with water. The organic phase was dried over Magnesium sulfate and evaporated to dryness. The crude product was purified by flash column chromatography using a gradient of 0%-6% of methanol in dichloromethane to furnish the desired intermediate azide of structure Az-L-A.

(122) ##STR00107##

(123) Preparatory Compounds for Preparing Intermediate Azides Az-L-A in Accordance with Scheme 4

Preparative Compound 16d: (2R,3S,4S)-1-((S)-14-azido-2-(tert-butyl)-4-oxo-6,9,12-trioxa-3-azatetradecanol)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide

(124) ##STR00108##

(125) To a solution of preparative compound 13d (15 mg, 0.034 mmol) in DCM (0.5 mL), HCl in dioxane (4M, 0.5 mL) was added. Methanol (0.2 mL) was added to solubilize the precipitate which formed after few minutes. After 2 hours, HPLC analysis showed complete conversion of the starting material and formation of the desired free amine 15d (METHOD 2 rt=2.403 min, m/z=449.2, [M+H].sup.+). The volatile components were removed under reduced pressure and the crude product was suspended in DMF (1 mL). DIPEA (10 μL, 0.051 mmol) was added, followed by a mixture of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)acetic acid (A6) (8.0 mg, 0.034 mmol), HATU (13.0 mg, 0.034 mmol), HOAT (4.6 mg, 0.034 mmol) and DIPEA (10 μL, 0.051 mmol). The resulting mixture was stirred at room temperature for one hour. HPLC analysis showed complete conversion of the starting material and formation of the desired product (METHOD 2 rt=3.241 min, m/z=664.3, [M+H].sup.+). The crude mixture was diluted with EtOAc (10 mL), washed with brine (2 mL) and solvents were removed under reduced pressure. The crude product was purified by preparative HPLC to obtain the desired compound as transparent oil (11.2 mg, 50% yield).

Preparative Compound 16e: (2R,3S,4S)-1-((S)-2-(2-(2-(2-azidoethoxy)ethoxy)acetamido)-3,3-dimethylbutanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide

(126) ##STR00109##

(127) Preparative compound 16e, was obtained in accordance with the methodology as described for preparative compound 16d, and starting from preparative compound 15e. The product was obtained in 55% yield, transparent oil. HPLC analysis (METHOD 2) rt=3.226 min, m/z=620.3, [M+H].sup.+.

(128) Scheme 5—General Process for Making A-˜L-B PROTACs from De-Protected Intermediates

(129) Scheme 5 illustrates the process for the preparation of PROTACs of structure A-L-B from preparative intermediate azide compounds of structure Az-L-A. The starting azide, of structure Az-L-A, such as for example preparatory intermediate compound (16a) (40 μmol) was dissolved in methanol (5 ml). A catalytic amount of palladium on charcoal (10 wt %, dry) was added and the reaction mixture was then stirred under an atmosphere of hydrogen gas for about 1 h at 25° C. The reaction mixture was then filtered through a syringe filter and the resulting solution evaporated to dryness to obtain the desired intermediate amine, corresponding to the starting azide was then linked to the desired B-group without further purification. The intermediate amine, structure NH.sub.2-L-A in this general example, the amine equivalent of intermediate compound (17a) and the desired B-group, in this general example suitable B-groups include the: free acid of JQ1 (11.4 mg, 25 μmol, 1 eq.), or I-BET726 (10.9 mg, 25 μmol, 1 eq.), or free acid of I-BET762 (9.92 mg, 25 μmol, 1 eq.), were then dissolved in DMF (0.5 ml) CM (2 ml). HATU (14.3 mg, 37.5 μmol, 1.5 eq.) was then added and the pH of the resultant mixture was adjusted to >9 by adding DIPEA (17.5 μl, 100 μmol, 4 eq.). After stirring the reaction mixture at 25° C. for 3 h the solvent was removed in vacuum. Purification of the crude product was achieved by preparative HPLC as described in the general information in order to furnish the desired PROTAC.

(130) For the avoidance of doubt, such intermediate amines are prepared from the corresponding de-protected azides by any suitable methods, and in particular, via reduction over palladium, with the resultant amines being utilized directly without further purification.

(131) Any PROTAC compound of structure A-L-B can be prepared in accordance with the general procedure outlined starting from intermediate compounds (13), by use of the appropriate starting Az-L-A compound and the desired B-group, as outlined in Scheme 2.

(132) The PROTAC compounds of Examples 18d and 18e as detailed herein after were prepared in accordance with the above general methodology from the appropriate starting preparatory azide and B-group.

(133) ##STR00110##

(134) Exemplary PROTACs Prepared in Accordance with the Process of Scheme 5

Example Compound 18d: 2R,3S,4S)-1-((S)-2-(tert-butyl)-17-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-4,16-dioxo-6,9,12-trioxa-3,15-diazaheptadecanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide

(135) ##STR00111##

(136) To a solution of preparative compound 16d (7.0 mg, 0.0105 mmol) in methanol (2.0 mL), a catalytic amount of Pd/C (10% dry) was added. The mixture was stirred under hydrogen atmosphere for one hour. HPLC analysis showed complete conversion of the starting material and formation of the desired intermediate amine 17d ((METHOD 2, rt=2.674 min, m/z=638.3, [M+H].sup.+). The mixture was filtered through a syringe filter and the solvent was removed. The crude amine intermediate was then dissolved in DMF (0.5 mL), and added to a solution of JQ1-COOH (4.2 mg, 0.0105 mmol), HATU (4.0 mg, 0.0105 mmol), HOAT (1.5 mg, 0.0105 mmol) and DIEPA (5.4 μL, 0.0315 mmol) in DMF (0.05 mL). The resultant mixture was stirred at room temperature for 3 hr. HPLC analysis (METHOD 2 rt=3.532 min, m/z=1020.3, [M+H].sup.+) showed complete conversion of the starting material and formation of the desired PROTAC product. Purification by preparative HPLC afforded the pure product, 7.5 mg, 70% yield, as white solid.

(137) .sup.1H NMR (400 MHz, MeOD) δ: 8.93 (s, 1H), 7.48-7.39 (m, 8H), 5.15-4.99 (m, 1H), 4.83-4.72 (m, 2H), 4.67-4.63 (m, 1H), 4.51-4.41 (m, 3H), 4.12-4.03 (m, 2H), 3.96-3.93 (m, 2H), 3.74-3.59 (m, 11H), 3.49-3.43 (m, 3H), 2.71 (s, 3H), 2.48 (s, 6H), 2.46 (s, 6H), 1.71 (s, 3H), 1.08 (s, 9H). .sup.19F-NMR δ: −195.32

Example Compound 18b: (2R,3R,4S)-1-((S)-2-(tert-butyl)-17-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-4,16-dioxo-6,9,12-trioxa-3,15-diazaheptadecanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide

(138) ##STR00112##

(139) Prepared as described for preparative compound 18d, 72% yield, white solid. HPLC analysis (METHOD 2 rt=3.554 min, m/z=1020.3, [M+H].sup.+)

(140) .sup.1H NMR (400 MHz, MeOD) δ: 8.88 (s, 31367398H), 7.48-7.39 (m, 9H), 5.06-4.90 (m, 1H), 4.71-4.68 (m, 1H), 4.66-4.48 (m, 4H), 4.36 (d, J=16.1 Hz, 1H), 4.10-4.05 (m, 3H), 3.77-3.66 (m, 10H), 3.61 (t, J=5.8 Hz, 2H), 3.49-3.43 (m, 3H), 2.70 (s, 3H), 2.48 (s, 3H), 2.46 (s, 3H), 1.71 (s, 3H), 1.06 (s, 9H). .sup.19F-NMR δ: −200.23.

Example Compound 18e: (2R,3S,4S)-1-((S)-1-(4-((2S,4R)-1-acetyl-4-(4-chlorobenzyl)-2-methyl-1,2,3,4-tetrahydroquinolin-6-yl)phenyl)-12-(tert-buytl)-1,10-dioxo-5,8-dioxa-2,11-diazatridecan-13-oyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide

(141) ##STR00113##

(142) Example compound 18e, was obtained in accordance with the methodology as described for preparative compound 18d, and starting from preparative compound 16e. The product was obtained in 75% yield as a white solid. HPLC analysis (METHOD 2) rt=3.662 min, m/z=883.4, [M-para-chloroaniline].sup.+.

(143) .sup.1H NMR (400 MHz, MeOD) δ: 8.87 (s, 1H), 8.60 (t, J=5.7 Hz, 1H), 7.90-7.75 (m, 3H), 7.64-7.56 (m, 4H), 7.44-7.36 (m, 5H), 7.12-7.09 (m, 2H), 6.73-6.68 (m, 2H), 5.15-5.00 (m, 1H), 4.84-4.75 (m, 2H), 4.46-4.41 (m, 3H), 4.26 (dd, J=4.8, 12.6 Hz, 1H), 4.10-3.94 (m, 4H), 3.76-3.60 (m, 8H), 2.74-2.66 (m, 1H), 2.44 (s, 3H), 2.25 (s, 3H), 1.37-1.29 (m, 1H), 1.19 (d, J=6.5 Hz, 3H), 1.05 (s, 9H). .sup.19F-NMR δ: −192.18

(144) Scheme 6—Synthesis of Azide-Linker Groups (Az-L)

(145) ##STR00114##

(146) Any azide-linker suitable for use in the preparation of an intermediate compound of structure Az-L-A as defined herein can be prepared in accordance with the general methodology outlined in Scheme 6 for the preparation of azide-linker compounds (A6) and (A7) by selection of the appropriate starting material.

(147) Compound (A6) was Synthesized Starting from Triethylene Glycol.

(148) Tri-ethylene glycol (120 mmol, 3 eq.) was dissolved in anhydrous THF (80 ml) and triethylamine (80 mmol, 2 eq.) was added. At 0° C. p-toluenesulfonyl chloride (40 mmol, 1 eq.) in anhydrous THF (10 ml) was added dropwise over 45 min. The reaction mixture was allowed to warm to room temperature and stirred overnight. The solvent was then removed in vacuo and the crude mixture purified by flash column chromatography using a gradient from 30%-90% Ethyl acetate in heptane.

(149) The tosylates (20 mmol, 1 eq.) were dissolved in ethanol, sodium azide (40 mmol, 2 eq.) was added and the reaction mixture heated to reflux for 18 h. After cooling to room temperature the solvent was removed in vacuo and the residue dissolved in water. The aqueous phase was extracted three times with dichloromethane. The organic phase was then dried over magnesium sulphate and then the solvent removed in vacuo.

(150) At 0° C. to the solution of the azides (10 mmol, 1 eq.) dissolved in anhydrous THF (25 ml) sodium hydride (20 mmol, 2 eq.) was added and the reaction mixture stirred for 45 min. Bromoacetic acid (10 mmol, 1 eq.) in anhydrous THF (25 ml) was then added and the reaction mixture allowed to warm to room temperature and stirred for 18 h. The solvent was removed in vacuo, the residue acidified to pH 2 with 1 M hydrochloric acid and the aqueous phase extracted for three times with dichloromethane. The combined organic layers were dried over magnesium sulphate and then purified by flash column chromatography using 10% methanol in dichloromethane to obtain the title compound (A6).

(151) Compound (A7) was synthesized in accordance with the method provided for compound (A6) and starting from tetraethylene glycol.

(S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic Acid (19)

(152) ##STR00115##

(153) (+)-JQ-1 (50 mg, 109 μmol) was dissolved in formic acid (3 ml) and stirred for 18 h at 25° C. After addition of water the reaction mixture was extracted three times with dichloromethane. The combined organic layers were dried over magnesium sulfate and evaporated to dryness to obtain the title compound which was directly used for the next reaction step. HPLC analysis (METHOD 2): rt=3.314 min, m/z=401.0, [M+H].sup.+. Yield 42.1 mg (96%).

(154) Preparation of Compounds of General Formula I, IA or IB in Accordance with General Scheme C

(2R,3R,4S)-1-((R)-2-amino-3-methyl-3-(tritylthio)butanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (20)

(155) ##STR00116##

(156) To a solution of 12b (100 mg, 0.230 mmol) in DCM/MeOH 9:1 (1 mL), 4N HCl in dioxane (1 mL) was added and mixture was stirred for 4 hours at room temperature. Volatiles were removed under vacuum, the residue was dissolved in water and freeze-dried to afford (2R,3R,4S)-3-fluoro-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-ium chloride (85 mg, 99%) as a light yellow powder which was used without any further purification.

(157) To a solution of Fmoc-S-trityl-L-penicillamine (141.8 mg, 0.230 mmol) in DMF (1 mL), HATU (87.5 mg, 0.230 mmol) and HOAT (31 mg, 0.230 mmol) were added, followed by DIPEA (99 μL, 0.575 mmol). The bright yellow solution was then added to a mixture of (2R,3R,4S)-3-fluoro-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-ium chloride (85 mg, 0.230 mmol) and DIPEA (99 μL, 0.575 mmol) in DMF (1.5 mL). After 2 hours complete conversion of the starting materials was observed by HPLC-MS (basic method), water was added (10 mL) and the mixture was extracted with AcOEt (3×25 mL). The organic phase was washed with brine (10 mL) and dried over anhydrous MgSO.sub.4. Solvents were removed under vacuum to afford compound 11 which was dissolved in DCM (2 mL). Piperidine (400 μL˜4 mmol) was added and the reaction mixture was stirred for 1 hour. Volatiles were removed under vacuum and the crude was purified by FCC (from 5 to 15% of 0.7 M NH.sub.3 in MeOH in DCM) to afford the title compound 12 as a white solid (122 mg, 75% yield). MS analysis: C40H41FN4O3S2 expected 708.3, found 709.3 [M+H.sup.+].

(158) .sup.1H NMR (500 MHz, CDCl.sub.3) δ: 8.63 (s, 1H), 7.85 (t, J=5.3 Hz, 1H), 7.27-7.11 (m, 19H), 5.50 (d, J=2.9 Hz, 1H), 5.04-4.90 (m, 1H), 4.57 (dd, J=7.0, 15.1 Hz, 1H), 4.53-4.44 (m, 2H), 4.35-4.27 (m, 1H), 3.30 (d, J=12.0 Hz, 1H), 2.99-2.93 (m, 1H), 2.44 (s, 3H), 1.11 (s, 3H), 1.08 (s, 3H). 19F NMR δ: −204.48.

(2R,3R,4S)-1-((R)-2-acetamido-3-methyl-3-(tritylthio)butanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (21)

(159) ##STR00117##

(160) To a solution of (2R,3R,4S)-1-((R)-2-amino-3-methyl-3-(tritylthio)butanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (30 mg, 0.042 mmol) in DCM (0.5 mL) at 0° C., TEA (7 μL, 0.050 mmol) and acetic anhydride (5 μL, 0.050 mmol) were added. The mixture was let to react at room temperature for 2 hours. The mixture was diluted with DCM (1 mL), washed with water (1 mL) and brine (1 mL), dried over MgSO.sub.4 and the solvent was removed under reduced pressure to afford the title compound (31 mg, 98% yield) which was used without further purification. MS analysis: C42H43FN4O4S2, expected 750.3 found 751.3 [M+H.sup.+].

(2R,3R,4S)-3-fluoro-1-((R)-2-(1-fluorocyclopropane-1-carboxamido)-3-methyl-3-(tritylthio)butanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (22)

(161) ##STR00118##

(162) To a solution of compound (2R,3R,4S)-1-((R)-2-amino-3-methyl-3-(tritylthio)butanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (30 mg, 0.042 mmol), HATU (16 mg, 0.042 mmol), HOAT (5.71 mg, 0.042 mmol) 1-fluorocyclopropane-1-carboxylic acid (4.3 mg, 0.042 mmol) in DMF (1 mL), DIPEA (25 μL, 0.141 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours, then water (1 mL) was added and the resulting mixture was extracted with DCM (3×5 mL). After drying the organic phase over MgSO.sub.4 and the solvent was removed under reduced pressure to afford the title compound (28.3 mg, 85% yield) which was used without further purification.

(2R,3R,4S)-1-((R)-2-acetamido-3-mercapto-3-methylbutanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (23)

(163) ##STR00119##

(164) Compound 22 (30 mg, 0.04 mmol) was dissolved in 1.8 mL of DCM. TIPS (0.1 mL) and TFA (0.1 mL) were added, and the yellow mixture was let to react at room temperature for 2 h. HPLC analysis (acidic method) showed complete conversion of the starting material. Volatiles were removed and the crude was dissolved in MeOH, filtered and purified by preparative HPLC and freeze-dried to give pure deprotected compound as white solid (16 mg, 79% yield). MS analysis: C23H29FN4O4S2 expected 508.2, found 509.2 [M+H.sup.+].

(165) .sup.1H NMR (500 MHz, MeOD) δ: 8.88 (s, 1H), 8.84 (t, J=6.0 Hz, 0H), 8.14 (d, J=8.9 Hz, 1H), 7.47-7.42 (m, 4H), 5.05-4.92 (m, 1H), 4.91 (d, J=8.8 Hz, 1H), 4.65 (dd, J=2.5, 21.5 Hz, 1H), 4.57-4.37 (m, 3H), 4.29 (dd, J=6.4, 10.3 Hz, 1H), 3.75-3.70 (m, 1H), 2.49 (s, 3H), 2.03 (s, 3H), 1.43 (s, 3H), 1.40 (s, 3H). 19F NMR δ: −199.94 13C NMR: 173.3, 171.5, 170.5, 153.0, 149.2, 140.0, 133.4, 131.8, 130.5, 129.1, 95.3, 93.8, 71.1, 70.9, 66.2, 66.0, 59.4, 52.2, 46.8, 43.9, 29.8, 28.8.

(2R,3R,4S)-3-fluoro-1-((R)-2-(1-fluorocyclopropane-1-carboxamido)-3-mercapto-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (24)

(166) ##STR00120##

(167) Prepared as described for compound 23, obtained 14 mg, 74% yield.

(168) .sup.1H NMR (500 MHz, MeOD) δ: 8.88 (s, 1H), 7.48-7.43 (m, 4H), 5.06-4.92 (m, 2H), 4.68 (dd, J=2.9, 21.2 Hz, 1H), 4.59-4.37 (m, 3H), 4.27 (dd, J=6.4, 10.0 Hz, 1H), 3.77-3.72 (m, 1H), 2.49 (s, 3H), 1.46 (s, 3H), 1.42-1.32 (m, 7H). 19F NMR δ: −200.07, −198.33. 13C NMR: 171.8, 171.7, 171.0, 170.4, 170.4, 152.9, 149.2, 140.0, 133.4, 131.8, 130.5, 129.6, 129.0, 95.3, 93.8, 80.1, 78.3, 71.1, 70.9, 66.1, 66.0, 58.4, 52.3, 47.4, 44.0, 30.1, 29.0, 15.9, 14.3, 14.2.

(2R,3R,4S)-1-((R)-2-acetamido-3-((6-aminohexyl)thio)-3-methylbutanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (25)

(169) ##STR00121##

(170) Under nitrogen and at 0° C., a solution of compound 23 (10 mg, 0.020 mmol) in DMF (0.5 mL) was treated with DBU (3.3 μL, 0.022 mmol) followed by N-(4-Bromohexyl)phthalimide (6.6 mg, 0.022 mmol). The reaction mixture was let to react at room temperature until complete conversion of the starting material was observed by HPLC (acidic method, 1-3 h). The reaction was cooled to 0° C. and treated with few drops of KHSO.sub.4 (5%) to pH=3-4. The solvent was removed under vacuum and the crude was dissolved in MeOH, filtered and purified by preparative HPLC to obtain 11.5 mg (80% yield) of alkylated product. MS analysis: C37H44FN5O6S2 expected 737.3, found 738.3 [M+H.sup.+]. The alkylated product was then dissolved in ethanol (0.5 mL) and treated with hydrazine monohydrate (24 μL, 0.32 mmol) at 60° C. for two hours. The reaction mixture was cooled to room temperature, filtered and purified by preparative HPLC to give the expected amine MS analysis: C29H42FN5O4S2 expected 607.3, found 608.3[M+H.sup.+], which was used as crude.

(2R,3R,4S)-1-((R)-3-((6-aminohexyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (26)

(171) ##STR00122##

(172) Prepared according to the procedure described for compound 25, used as crude.

(2R,3R,4S)-1-((R)-2-acetamido-3-((6-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)hexyl)thio)-3-methylbutanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (27)

(173) ##STR00123##

(174) Crude (2R,3R,4S)-1-((R)-2-acetamido-3-((6-aminohexyl)thio)-3-methylbutanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (assumed 0.0108 mmol) was dissolved in DMF (0.25 mL) and added to a solution of (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid (+)-JQ1-COOH (4.32 mg, 0.0108 mmol), HATU (4.1 mg, 0.0108 mmol), HOAT (1.5 mg, 0.0108 mmol) and DIPEA (5.5 μl, 0.0324 mmol) in DMF (0.5 mL). After stirring at room temperature for 1 h, the reaction was quenched with water (0.1 mL) and the mixture of water/DMF was removed under high vacuum at room temperature (overnight). The crude mixture was dissolved in MeOH, filtered and purified by preparative HPLC to give the title compound. Obtained 5 mg, 45% yield. MS analysis: C48H57ClFN9O5S3 expected 989.3, found 990.3 [M+H.sup.+].

(175) .sup.1H NMR (500 MHz, MeOD) δ 8.91 (s, 1H), 8.62 (t, J=5.9 Hz, 1H), 8.19 (d, J=8.8 Hz, 1H), 7.47-7.41 (m, 9H), 5.07-4.94 (m, 1H), 4.92-4.90 (m, 1H), 4.70-4.63 (m, 2H), 4.55-4.39 (m, 3H), 4.13 (dd, J=6.8, 9.9 Hz, 1H), 3.79-3.75 (m, 1H), 3.45-3.39 (m, 1H), 3.31-3.18 (m, 4H), 2.71 (s, 3H), 2.56 (t, J=7.4 Hz, 2H), 2.48 (s, 3H), 2.45 (s, 3H), 2.02 (s, 3H), 1.70 (s, 3H), 1.56-1.35 (m, 14H). 19F NMR=−200.10. 13C NMR: 171.7, 171.1, 164.9, 155.6, 151.6, 150.8, 147.5, 138.5, 136.7, 136.6, 132.1, 132.0, 132.0, 130.7, 130.2, 129.9, 129.0, 128.4, 127.6, 93.8, 69.6, 69.5, 55.5, 53.8, 50.6, 38.9, 37.3, 29.1, 28.9, 28.4, 27.8, 26.2, 24.5, 24.2, 20.8, 14.3, 13.0, 11.5, 10.1.

(2R,3R,4S)-1-((R)-3-((6-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)hexyl)thio)-2-(1-fluorocyclopropane-1-carboxamido)-3-methylbutanoyl)-3-fluoro-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (28)

(176) ##STR00124##

(177) Prepared according to the procedure described for compound 28, 4 mg, 38% yield. MS analysis: C50H58ClF2N9O5S3 expected 1033.3, found 1034.3 [M+H.sup.+].

(178) .sup.1H NMR (500 MHz, MeOD) δ: 8.88 (s, 1H), 8.31 (t, J=5.7 Hz, 1H), 7.76-7.73 (m, 1H), 7.47-7.40 (m, 9H), 5.07-4.95 (m, 1H), 4.92-4.90 (m, 1H), 4.69 (dd, J=2.8, 21.5 Hz, 1H), 4.65-4.61 (m, 1H), 4.59-4.37 (m, 3H), 4.14 (dd, J=6.4, 10.1 Hz, 1H), 3.78-3.73 (m, 1H), 3.44-3.39 (m, 1H), 3.30-3.18 (m, 3H), 2.70 (s, 3H), 2.61-2.53 (m, 2H), 2.49 (s, 3H), 2.45 (s, 3H), 1.55-1.31 (m, 18H). 19F NMR: −200.22, −198.08.

(179) Comparative Compounds with Pyrrolidine Ring not Comprising the Fluor Substituent

(2S,4R)-1-((R)-2-amino-3-methyl-3-(tritylthio)butanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (29)

(180) ##STR00125##

(181) Prepared according to the procedure described for compound 20.

(182) .sup.1H-NMR (400 MHz, CD.sub.3OD, 25° C.) δ: 8.90 (s, 1H), 7.63-7.60 (m, 6H), 7.40-7.33 (m, 4H), 7.31-7.29 (m, 6H), 7.24-7.19 (m, 3H), 4.46 (t, J=8.2 Hz, 1H), 4.37 (br s, 1H), 4.31 (m, 2H), 3.35 (s, 1H), 3.24 (dd, J=11.1 Hz, J=4.1 Hz, 1H), 3.07-3.04 (m, 1H), 2.70 (s, 1H), 2.47 (s, 3H), 2.16-2.11 (m, 1H), 1.99-1.92 (m, 1H), 1.26 (s, 3H), 1.19 (s, 3H).

(183) .sup.13C-NMR (101 MHz, CD.sub.3OD, 25° C.) δ: 172.9, 171.4, 151.5, 147.7, 144.9, 138.7, 132.0, 129.7, 129.0, 127.5, 127.4, 126.5, 69.3, 67.7, 59.4, 57.6, 57.3, 56.7, 42.1, 37.4, 24.6, 24.1, 14.4.

(2S,4R)-1-((R)-2-acetamido-3-methyl-3-(tritylthio)butanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (30)

(184) ##STR00126##

(185) Prepared according to the procedure described for compound 21.

(186) MS analysis: C.sub.42H.sub.44N.sub.4O.sub.4S.sub.2 expected 732.3, found 733.3 [M+H.sup.+].

(187) .sup.1H-NMR (400 MHz, CDCl.sub.3, 25° C.) δ: 8.71 (s, 1H), 7.52-7.49 (m, 6H), 7.39-7.31 (m, 3H), 7.25-7.20 (m, 11H), 6.25 (d, J=5.2 Hz, 1H), 4.64 (t, J=8.1 Hz, 1H), 4.37 (br s, 1H), 4.31-4.18 (m, 2H), 3.61 (d, J=5.3 Hz, 1H), 3.52-3.49 (m, 1H), 3.30-3.29 (m, 1H), 3.22 (dd, J=11.5 Hz, J=3.6 Hz, 1H), 2.52 (s, 3H), 2.36-2.30 (m, 1H), 2.16-2.11 (m, 1H), 1.95 (s, 3H), 1.76 (br s, 1H), 1.18 (s, 3H), 0.97 (s, 3H).

(188) .sup.13C-NMR (101 MHz, CDCl.sub.3, 25° C.) δ: 170.7, 170.5, 170.3, 150.3, 148.5, 144.2, 138.2, 131.7, 130.8, 129.7, 129.6, 129.4, 127.9, 127.0, 77.2, 70.1, 68.5, 58.5, 57.1, 56.6, 53.6, 42.8, 36.4, 26.1, 25.4, 22.9, 16.2.

(2S,4R)-1-((R)-2-(1-fluorocyclopropane-1-carboxamido)-3-methyl-3-(tritylthio)butanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (31)

(189) ##STR00127##

(190) Prepared according to the procedure described for compound 22.

(191) MS analysis: C44H45FN4O4S2 expected 776.3 found 777.3 [M+H.sup.+].

(192) .sup.1H NMR (400 MHz, CDCl3) δ: 8.71 (s, 1H), 7.54-7.51 (m, 6H), 7.34-7.31 (m, 3H), 7.25-7.19 (m, 12H), 4.69-4.64 (m, 1H), 4.38-4.36 (m, 1H), 4.32-4.19 (m, 2H), 3.66 (d, J=4.2 Hz, 1H), 3.50 (d, J=11.6 Hz, 1H), 3.26 (dd, J=3.9, 11.6 Hz, 1H), 3.09 (d, J=5.9 Hz, 1H), 2.41-2.33 (m, 1H), 2.14-2.07 (m, 1H), 1.38-1.21 (m, 7H), 0.97 (s, 3H). 19F NMR: −197.41.

(2S,4R)-1-((R)-2-(1-cyanocyclopropane-1-carboxamido)-3-methyl-3-(tritylthio)butanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (32)

(193) ##STR00128##

(194) Prepared as described for compound 22. 72% yield.

(195) MS analysis: C45H45N5O4S2, 784.0, found 785.0.

(196) .sup.1H NMR (400 MHz, CDCl3) d 8.71 (s, 1H), 7.57-7.53 (m, 6H), 7.34-7.32 (m, 2H), 7.25-7.16 (m, 13H), 4.64 (t, J=8.1 Hz, 1H), 4.38 (t, J=3.5 Hz, 1H), 4.29 (d, J=5.6 Hz, 2H), 3.66 (d, J=5.1 Hz, 1H), 3.47 (d, J=11.8 Hz, 1H), 3.24 (dd, J=3.6, 11.7 Hz, 1H), 2.79 (d, J=6.1 Hz, 1H), 2.53 (s, 3H), 2.50-2.36 (m, 1H), 2.12-2.06 (m, 1H), 1.69-1.62 (m, 2H), 1.58-1.46 (m, 3H), 1.20 (s, 3H).

(2S,4R)-1-((R)-2-acetamido-3-mercapto-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (33)

(197) ##STR00129##

(198) Prepared according to the procedure described for compound 23. (62 mg, 79% yield). MS analysis: C.sub.23H.sub.30N.sub.4O.sub.4S.sub.2 expected 490.2, found 491.1 [M+H.sup.+].

(199) .sup.1H-NMR (400 MHz, CDCl.sub.3, 25° C.) δ: 8.68 (s, 1H), 7.39-7.33 (m, 4H), 7.20-7.17 (m, 1H), 6.55 (d, 1H), 4.68 (t, J=8.0 Hz, 1H), 4.59-4.52 (m, 3H), 4.35 (dd, J=14.9 Hz, J=5.2 Hz, 1H), 4.19-4.16 (m, 1H), 3.70 (dd, J=11.2 Hz, J=3.6 Hz, 1H), 3.15 (br s, 1H), 2.29 (br s, 1H), 2.52 (s, 3H), 2.48-2.42 (m, 1H), 2.20-2.15 (m, 1H), 2.01 (s, 3H), 1.36 (s, 3H), 1.31 (s, 3H).

(200) .sup.13C-NMR (101 MHz, CDCl.sub.3, 25° C.) δ: 170.9, 170.7, 170.6, 150.4, 148.6, 137.9, 131.5, 131.1, 129.6, 128.1, 70.1, 58.9, 57.5, 56.6, 46.1, 43.3, 36.5, 30.7, 28.7, 23.0, 16.1.

(2S,4R)-1-((R)-2-acetamido-3-((6-aminohexyl)thio)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (34)

(201) ##STR00130##

(202) Prepared according to the procedure described for compound 25.

(203) (6.4 mg, 68% yield). MS analysis: C.sub.29H.sub.43N.sub.5O.sub.4S.sub.2 expected 589.3, found 590.2 [M+H.sup.+].

(204) .sup.1H NMR (400 MHz, CD.sub.3OD, 25° C.) δ: 8.88 (s, 1H), 7.46-7.41 (m, 4H), 4.92 (s, 1H), 4.58 (t, J=8.3 Hz 1H), 4.52-4.38 (m, 3H), 3.93-3.84 (m, 1H), 3.85 (dd, J=10.8 Hz, J=4.0 Hz, 1H), 2.64 (t, J=7.3 Hz, 2H), 2.56 (t, J=7.4 Hz, 2H) 2.48 (s, 3H), 226-2.23 (m, 1H), 2.14-2.10 (m, 1H), 2.00 (s, 3H), 1.49-1.28 (m, 16H).

(205) .sup.13C-NMR (101 MHz, CD.sub.3OD, 25° C.) δ: 174.4, 173.2, 171.6, 153.0, 149.2, 140.3, 133.5, 131.8, 130.6, 129.2, 71.1, 61.1, 58.0, 57.3, 43.7, 42.3, 39.2, 32.8, 30.7, 30.1, 29.3, 27.6, 26.3, 25.7, 22.5, 16.0.

(2S,4R)-1-((R)-2-acetamido-3-((6-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazol[4,3-a][1,4]diazepin-6-yl)acetamido)hexyl)thio)-3-methylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (35)

(206) ##STR00131##

(207) Prepared accordingly to procedure 27. Obtained 7.3 mg, 70% yield. MS analysis: C.sub.48H.sub.58N.sub.9ClO.sub.5S.sub.3 expected 971.3, found 972.3 [M+H.sup.+].

(208) .sup.1H-NMR (400 MHz, CD.sub.3OD, 25° C.) δ: 8.99 (s, 1H), 8.43 (t, J=5.9 Hz, 1H exch.), 8.12 (d, J=9.4 Hz, 1H exch.), 8.07 (s, 1H exch.), 7.47-7.40 (m, 8H), 4.93-4.91 (m, 1H), 4.69-4.65 (m, 1H), 4.58 (t, J=8.3 Hz, 1H), 4.52-4.38 (m, 3H), 3.93-3.91 (m, 1H), 3.85 (dd, J=10.8 Hz, J=3.96 Hz, 1H), 3.45-3.39 (m, 1H), 3.27-3.16 (m, 3H), 2.72 (s, 3H), 2.26 (t, J=7.0 Hz, 2H), 2.49 (s, 3H), 2.45 (s, 3H), 2.25-2.22 (m, 1H), 2.14-2.07 (m, 1H), 2.00 (s, 3H), 1.70 (s, 3H), 1.54-1.36 (m, 14H).

(209) .sup.13C-NMR (101 MHz, CD.sub.3OD, 25° C.) δ: 174.4, 173.2, 172.6, 171.6, 166.7, 157.1, 153.4, 152.6, 148.5, 140.5, 138.4, 137.9, 133.9, 133.6, 132.3, 132.2, 131.6, 131.4, 130.6, 130.0, 129.7, 129.3, 71.0, 61.2, 58.1, 57.4, 55.2, 43.7, 40.5, 39.2, 38.7, 30.7, 30.5, 30.0, 29.3, 27.7, 26.2, 25.9, 22.5, 15.7, 14.6, 13.1, 11.7.