AU2002322344A1 - Fluoropyrrolidines as dipeptidyl peptidase inhibitors - Google Patents

Description

FLU0R0PYRR0L1D1NES AS DIPEPTIDYL PEPT1DASE INHIBITORS

FIELD OF INVENTION

The present invention relates to compounds inhibiting dipeptidyl peptidases, such

as II (DPP-II) and IV (DPP-IV), to methods for their production, and to their therapeutic

utility.

BACKGROUND OF THE INVENTION

Dipeptidyl peptidase IV (DPP-IV) is a post-proline/alanine cleaving serine protease

found in various tissues of the body including kidney, liver, and intestine. DPP-IV is

thought to regulate the activity of multiple physiogically important peptides, including,

but not limited to, GLPl, GIP, GLP2, GRP, vasoactive intestinal peptide, peptide histidine

methionine, PYY, substance P, beta-casomorphine, NPY, PACAP38, prolactin, chorionic

gonadotropin, aprotinin, corticotropin-like intermediate lobe peptide, pituitary adenylyl

cyclase-activating peptide, (Tyr)melanostatin, LD78beta(3-70), RANTES, eotaxin

procolipase, enterostatin, vasostatin 1 , endomorphin, morphiceptin, stromal cell derived

factor, macrophage-derived chemokine, granulocyte chemotactic protein-2, and GHRH/GRF. As examples of the therapeutic value of DPP-IV, DPP-IV is believed to be

involved in a variety of metabolic, gastrointestinal, viral, and inflammatory diseases,

including, but not limited to, diabetes, obesity, hyperlipidemia, dermatological or

mucous membrane disorders, psoriasis, intestinal distress, constipation, autoimmune

disorders such as encephalomyelitis, complement mediated disorders such as

glomerulonepritis, lipodystrophy, and tissue tissue damage, psychosomatic, depressive,

and neuropsychiatric disease such as anxiety, depression, insomnia, schizophrenia,

epilepsy, spasm, and chronic pain, HIV infection, allergies, inflammation, arthritis,

transplant rejection, high blood pressure, congestive heart failure, tumors, and stress- induced abortions, for example cytokine-mediated murine abortions. For example, DPP-

IV, also known as CD26, mediates T-cell activation and HIV infection (Ohtsuki et al.,

2000). T-cells expressing DPP-IV/CD26 are preferentially infected and depleted in HIV-

infected individuals (Ohtsuki et al., 2000). DPP-IV inhibitors have demonstrated anti-

inflammatory effects in animal models of arthritis (Tanaka et al, 1997). Additionally,

DPP-IV inhibition has been shown to prolong cardiac transplant survival (Korom et al.,

1997). In vitro studies suggest that DPP-IV/CD26 expression correlate with tumor

progression of malignant melanomas of the skin (Van den Oord, 1998). Furthermore, DPP-IV is thought to regulate metabolism by cleaving the penultimate proline/alanine at

the amino-terminus of polypeptides (Mentlein, 1999), such as glucagon-like peptides

(GLP) and neuropeptide Y (NPY).

More specifically, GLPs help metabolize glucose and, thus, regulation of GLPs

likely should be beneficial in the treatment of metabolic disorders such as diabetes.

Diabetes, for example type 2 (also called noninsulin-dependent diabetes mellitus

(NIDDM) or maturity-onset) diabetes, results in elevated blood sugar levels due to

absolute or relative insufficiencies of insulin. Type 2 diabetes is the more common form

of diabetes, accounting for 90% of cases, or about 16 million Americans. Most type 2

diabetics produce variable, sometimes normal, amounts of insulin, but they have

abnormalities in liver and muscle cells that resist its actions. Insulin attaches to the

receptors of cells, but glucose does not get inside, a condition known as insulin

resistance. Many type 2 diabetics seem to be incapable of secreting enough insulin to

overcome insulin resistance. GLP-1 enhances insulin secretion. Thus, regulation of GLP-

1 correlates to a regulation of insulin secretion. Moreover, GLP-1 decreases hepatic

glucose production, gastric emptying, and food intake (Deacon et al., 1995). Further, GLP-2 maintains the integrity of the intestinal mucosal epithelium via effects on gastric

motility, nutrient absorption, crypt cell proliferation and apoptosis, and intestinal

permeability (Drucker, 2001).

DPP-IV inhibitors preserve GLP-1 function for a longer time (Balka, 1999). Thus,

DPP-IV inhibitors may promote satiety, weight loss, and the antidiabetic effects of GLP-1

(Deacon et al., 1995; Hoist and Deacon, 1998). For example, inhibition of DPP-IV with the known compound NVP-DPP728 increases plasma GLP-1 (2-36 amide) concentrations

and improves oral glucose tolerance in obese Zucker rats. See, Diabetologia 42: 1324-

1331. Both subcutaneously and intravenously administered GLP-1 is rapidly degraded

from the NH2-terminus in type II diabetic patients and in healthy subjects. See, Diabetes

44:1126, 1995.

Moreover, DPP-IV inhibitors preserve GLP-2 for longer periods of time and, thus,

may be useful for treating intestinal insufficiencies and mucous membrane disorders

(Hartmann B et al., 2000).

While DPP-IV is the predominate protease regulating GLP turnover, similar

substrate or inhibitor specificity may be observed for related proteases. Related serine proteases include, but are not limited to, dipeptidyl peptidase-ll (DPP-II), dipeptidyl

peptidase IV beta, dipeptidyl peptidase 8, dipeptidyl peptidase 9, aminopeptidase P,

fibroblast activating protein alpha (seprase), prolyl tripeptidyl peptidase, prolyl

oligopeptidase (endoproteinase Pro-C), attractin (soluble dipeptidyl-aminopeptidase),

acylaminoacyl-peptidase (N-acylpeptide hydrolase; fMet aminopeptidase) and lysosomal

Pro-X carboxypeptidase (angiotensinase C, prolyl carboxypeptidase). Proline-cleaving

metallopeptidases that may share similar substrate or inhibitor specificity to DPP-IV

include membrane Pro-X carboxypeptidase (carboxypeptidase P), angiotensin-converting enzyme (Peptidyl-dipeptidase A multipeptidase], collagenase 1 (interstitial collagenase;

matrix metalloproteinase 1 ; MMP-1 ; Mcol-A), ADAM 10 (alpha-secretase, myelin-

associated disintegrin metalloproteinase), neprilysin (atriopeptidase; CALLA; CD10;

endopeptidase 24.1 1 ; enkephalinase), Macrophage elastase (metalloelastase; matrix metalloproteinase 12; MMP-12], Mat ilysin (matrix metalloproteinase 7; MMP-7), and

neurolysin (endopeptidase 24.16; microsomal endopeptidase; mitochondrial

oligopeptidase). See http://merops.iapc.bbsrc.ac.uk/.

Furthermore, beyond mammalian serine peptidases and proline-cleaving

metallopeptidases, other non-mammalian proteases may share similar substrate or inhibitor specificity to DPP-IV. Non-limiting examples of such non-mammalian serine

proteases include prolyl aminopeptidase (prolyl iminopeptidase), IgAl -specific serine

type prolyl endopeptidase (IgA protease, Neisseria, Haemophilus), dipeptidyl aminopeptidase A (STE13) (Saccharomyces cerevisiae), dipeptidyl aminopeptidase B

(fungus), prolyl oligopeptidase homologue (Pyrococcus sp.), oligopeptidase B (Eseherichia

coli alkaline proteinase II; protease II), dipeptidyl aminopeptidase Bl (Pseudomonas sp.),

dipeptidyl-peptidase IV (bacteria), dipeptidyl aminopeptidase (Aureobacterium),

dipeptidyl-peptidase IV (insect), dipeptidyl-peptidase V, allergen Tri 1 4 (Trichophyton

tonsurans), secreted alanyl DPP (Aspergillus oryzae), peptidase ll-mes (Prosopis velutina),

and bamboo serine proteinase (Pleioblastus hindsii). Non-limiting examples of such

non-mammalian proline-cleaving metallopeptidases include penicillolysin (fungal acid

metalloendopeptidase), proline-specific peptidyl-dipeptidase (Streptomyces), coccolysin

(gelatinase, Enterococcus faecalis), aminopeptidase Ey, (hen egg yolk) (apdE g.p.; Gallus

gallus domesticus), gametolysin (Chlamydomonas cell wall degrading protease), and snake venom proline-cleaving metalloproteases as well. See

http://merops.iapc.bbsrc.ac.uk/ for further reference.

Dipeptidyl peptidase II (DPP II) is a serine protease localized to lysosomes in cells

and believed to be involved in lysosomal degradation and protein turnover. The order of

expression of DPP-II is kidney » testis > or = heart > brain > or = lung > spleen >

skeletal muscle > or = liver (Araki H et al., J Biochem (Tokyo) 2001, 129:279-88). This

expression suggests possible utility in kidney or lysosomal-related disorders. Substrate

specificity studies indicated that purified DPP-II hydrolyzes specifically alanine or proline

residues at acidic pH (4.5-5.5). DPP-II has significant sequence homology and substrate

specificity to quiescent cell proline dipeptidase and prolyl carboxypeptidase, suggesting

possible overlapping functions between these proteases (Araki H et al., J Biochem (Tokyo)

2001, 129:279-88).

The present invention includes novel DPP-II and/or DPP-IV inhibitors, as well as

methods of their therapeutic use and methods of their production. While not being

limited thereby, the compounds of the present invention are believed useful for the

treatment of a variety of metabolic, gastrointestinal, viral, and inflammatory diseases,

including, but not limited to, diabetes, obesity, hyperlipidemia, dermatological or

mucous membrane disorders, psoriasis, intestinal distress, constipation, autoimmune

disorders such as encephalomyelitis, complement mediated disorders such as

glomerulonepritis, lipodystrophy, and tissue damage, psychosomatic, depressive, and

neuropsychiatric disease such as anxiety, depression, insomnia, schizophrenia, epilepsy, spasm, and chronic pain, HIV infection, allergies, inflammation, arthritis, transplant

rejection, high blood pressure, congestive heart failure, tumors, and stress-induced

abortions, for example cytokine-mediated murine abortions. As compared to other di peptidyl peptidase inhibitors, the compounds of the

present invention provide improved stability, potency, duration of action, and/or

safety/toxicity profiles.

SUMMARY OF THE INVENTION

The present invention includes compounds of formula (I):

including salts, solvates, and pharmaceutically functional derivatives thereof, wherein

X is F or H; R¹ and R² each are either H; alkyl; optionally substituted aryl or heteroaryl; or

combine to form a 3 to 14 membered ring sytem, optionally containing one or more

heteroatoms, and optionally containing one or more degrees of unsaturation. When R¹ and R² are optionally substituted aryl or heteroaryl, then R³ is H or alkyl. Otherwise, R³ is

where n is 0-5; m is 0-12; Y is S(0)_P, O, alkylene, alkenylene, alkynylene, or a bond; p is

0-2; R⁴ is R⁵ when Y is S, 0, alkylene, alkenylene, alkynylene, or a bond, where R⁵ is

optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, or heterocyclyl;

and R⁴ is R⁶ when Y is S(0) or S(0)₂, where R⁶ is optionally substituted alkyl, aryl,

cycloalkyl, heteroaryl, amino, alkylamino, arylamino, heteroarylamino, cycloalkylamino,

or hydroxy. Although not limiting, preferred optional substitutents include one or more of

alkyl, alkoxy, aryloxy, halogen, haloalkyl, cyano, alkylsulfonyl, or aryl. More preferably,

optional substituents includes one or more of G-Cs alkyl, G-Cs alkoxy, phenoxy,

benzoxy, fluorine, chlorine, G-Ce haloalkyl, cyano, G-Gs alkylsulfonyl, phenyl, or benzyl.

In one embodiment, R¹ and R² are each aryl, and R³ is H. Preferably, each aryl is

phenyl. More preferably each phenyl is substituted with halogen. More preferably each halogen is fluorine, more preferabyl 4-fluorine.

In another embodiment, R¹ and R² are each alkyl and R³ is

Preferably each of alkyl is Ci-Ce alkyl. More preferably each alkyl is methyl. Preferably n

is 0, m is 1 , and Y is S(0)_P. In one preferred embodiment, p is 0, R⁴ is R⁵, and R⁵ is

optionally substituted aryl. Preferably R⁵ is phenyl substituted with alkoxy. More preferably the alkoxy is methoxy. In another preferred embodiment, p is 1 or 2, R⁴ is R⁶,

and R⁶ is optionally substitued aryl. Preferably R⁶ is phenyl substituted with alkoxy.

More preferably the alkoxy is methoxy. More preferably p is 2.

In one embodiment, preferably the depicted NH2 group is cis to the depicted

nitrile warhead. In another embodiment preferably the depicted NH2 group is trans to

the depicted nitrile warhead.

Preferably A is H. Preferably the depicted F is cis to the depicted nitrile.

Particularly preferred compounds of the present invention include compounds of

formula (I):

including salts, solvates, and pharmaceutically functional derivatives thereof, wherein

X is F or H; R¹ and R² each are optionally substituted aryl or heteroaryl; and R³ is H or alkyl.

Particularly preferred compounds of the present invention include compounds of

formula (I):

including salts, solvates, and pharmaceutically functional derivatives thereof, wherein

X is F or H; R¹ and R² each are either

(i) alkyl; or

(ii) combine to form a 3 to 14 membered ring sytem, optionally containing

one or more heteroatoms, and optionally containing one or more degrees

of unsaturation;

R³ is

where n is 0-5; m is 0-12; Y is S(0)_P, 0, alkylene, alkenylene, or alkynylene; p is 0-2; R⁴ is

R⁵ when Y is S, 0, alkylene, alkenylene, or alkynylene, where R⁵ is optionally substituted

alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, or heterocyclyl; and R⁴ is R⁶ when Y is

S(0) or S(0)₂, where R⁶ is optionally substituted alkyl, aryl, cycloalkyl, heteroaryl, amino,

alkylamino, arylamino, heteroarylamino, cycloalkylamino, or hydroxy.

Preferably p is 1 or 2. More preferably p is 2. Preferably R¹ and R² each are alkyl.

More preferably R¹ and R² are each G-Ce alkyl. More preferably R¹ and R² are each

methyl.

Particularly preferred compounds of the present invention include:

Particularly preferred compounds are selected from: (2S,4S)-1-[(2S)-2-Amino-3,3-diphenylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile

hydrochloride; (2S,4S)-1 -[(2S)-2-Amino-3,3-bis(4-fluorophenyl)propanoyl]-4-fluoropyrrolidine-2-

carbonitrile hydrochloride;

(2S,4S)-1-[(2S)-2-Amino-5-(4-fluorophenyl)-3,3-dimethylpentanoyl]-4-fluoro-2-

pyrrolidinecarbonitrile hydrochloride; (2S,4R)-1-[(2S)-2-Amino-4-(4-fluorophenyl)-3,3-dimethylbutanoyl]-4-

fluoropyrrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-{(2R)-2-Amino-3-[(4-methoxybenzyl)thio]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-{(2R)-2-Amino-3-methyl-3-[(3-phenylpropyl)thio]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-{(2R)-2-Amino-3-methyl-3-[(2-phenylethyl)thio]butanoyl}-4-

fluoropyrrolidine-2-carbonitriIe hydrochloride;

(2S,4S)-1-((2R)-2-Amino-3-{[3-(4-fluorophenyl)propyl]thio}-3-methylbutanoyl)-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-{(2R)-2-Amino-3-[(4-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-{(2R)-2-Amino-3-[(4-methoxybenzyl)sulfinyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride; (2S,4S)-1-((2R)-2-Amino-3-methyl-3-{[4-(trifluoromethyl)benzyl]thio}butanoyl)-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-[(2S)-2-Amino-2-(1-vinylcyclopentyl)ethanoyl]-4-fluoropyrrolidine-2-

carbonitrile hydrochloride;

(2S,4S)-1-[(2S)-2-Amino-5-(4-methoxyphenyl)-3,3-dimethylpentanoyl]-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-{(2R)-2-Amino-3-methyl-3-[(3-phenylpropyl)sulfonyl]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile; (2S,4S)-1 -{(2R)-2-Amino-3-methyl-3-[(4-methylbenzyl)sulfonyl]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile; (2S,4S)-1-((2R)-2-amino-3-{[4-(benzyloxy)benzyl]suIfonyl}-3-methylbutanoyl)-4-

fluoropyrrolidine-2-carbonitrile;

(2S,4S)-1 -{(2R)-2-amino-3-[(4-cyanobenzyl)sulfonyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile; (2S,4S)-1-((2R)-2-amino-3-methyl-3-{[4-(methylsulfonyl)benzyl]sulfonyl}butanoyl)-4-

fluoropyrrolidine-2-carbonitrile;

(2S,4S)-1-{(2S)-2-Amino-2-[1 -(4-fluorobenzyl)cyclopentyl]ethanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-((2S)-2-Amino-2-{ l -[4-(trifluoromethyl)phenyl]cyclopentyl}ethanoyl)-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-{(2S)-2-Amino-2-[1-(4-fluorobenzyl)cyclopropyl]ethanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-[(2R)-2-Amino-3- (benzylsulfonyl)-3-methylbutanoyl]-4-fluoropyrrolidine-2-

carbonitrile hydrochloride; (2S,4S)-1-{(2R)-2-amino-3-[(3-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-{(2R)-2-Amino-3-[(1 ,1 '-biphenyl-4-ylmethyl)sulfonyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-{(2R)-2-Amino-3-[(2-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-{(2R)-2-Amino-3-methyl-3-[(pyridin-3-ylmethyl)thio]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile;

(2S,4S)-1-{(2R)-2-Amino-3-methyl-3-[(pyridin-2-ylmethyl)thio]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride; (2S,4S)-1 -{(2R)-2-amino-3-methyl-3-[(pyridin-4-ylmethyl)thio]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1 -{(2R)-2-amino-3-[(4-fluorobenzyl)sulfonyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride; (2S,4S)-1 -{(2R)-2-amino-3-methyl-3-[(3-phenoxybenzyl)sulfonyl]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-{(2R)-2-amino-3-methyl-3-[(3-phenoxybenzyl)thio]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-((2R)-2-Amino-3-{[(5-chloro-1 ,1-dioxido-1 -benzothien-3-

yl)methyl]sulfonyl}-3-methylbutanoyl)-4-fluoropyrrolidine-2-carbonitrile

hydrochloride;

(2S,4S)-1-{(2R)-2-Amino-3-[(2,1,3-benzoxadiazol-5-ylmethyl)thio]-3-methylbutanoyl}-

4-fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1 -{(2R)-2-Amino-3-methyl-3-[(pyridin-4-ylmethyl)sulfonyl]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-[(2S)-2-Amino-3-pyridin-4-ylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-[(2S)-2-Amino-3-pyridin-3-ylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile

hydrochloride; (2S,4S)-1 -[(2S)-2-Amino-3-piperidin-4-ylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile

dihydrochloride;

(25,45)-1-[(2S)-2-Amino-3-piperidin-3-ylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile

dihydrochloride; (2S4S)-1-[(2S)-2-Amino-3-piperidin-2-ylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile dihydrochloride;

(2S,4S)-1-{(2S)-2-Amino-3-[1-(isopropylsulfonyl)piperidin-4-yl]propanoyl}-4-

fluoropyrrolidine-2-carbonitrilehydrochloride; (2S,4S)-1-{(25)-2-Amino-3-[1-(4-methylphenylsulfonyl)piperidin-4-yl]propanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-{(2S)-2-Amino-3-[l-(isopropylsulfonyl)piperidin-3-yl]propanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S4S)-1-{(2S)-2-Amino-3-[1-(4-methylphenylsulfonyl)piperidin-3-γl]propanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S45)-1 -[(2S)-2-Amino-3-(l-benzothien-3-yl)propanoyl]-4-fluoropyrrolidine-2-

carbonitrile hydrochloride;

(2S,4S)-1-{(2S)-2-Amino-3-methyl-3-[4-(trifluoromethyl)phenyl]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride; and (3R)-3-Amino-4-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]-2-methyl-4-oxobutane-2-

sulfonic acid.

Another aspect of the present invention includes pharmaceutical formulations

that include a compound of the present invention. Preferably the pharmaceutical

formulation further includes a pharmaceutically acceptable carrier.

Another aspect of the present invention includes a method of inhibiting a post

proline/analine cleaving protease through administration of a compound of the present

invention. Preferably the post proline/analine cleaving protease is a serine protease.

More preferably the serine protease is a dipeptidyl peptidase. More preferably the

dipeptidyl peptidase is DPP-II or DPP-IV. Another aspect of the present invention includes a method for the treatment or

prophylaxis of metabolic disorders, gastrointestinal disorders, viral disorders,

inflammatory disorders, diabetes, obesity, hyperlipidemia, dermatological or mucous

membrane disorders, psoriasis, intestinal distress, constipation, autoimmune disorders,

encephalomyelitis, complement mediated disorders, glomerulonepritis, lipodystrophy,

tissue damage, psychosomatic, depressive, and neuropsychiatric disorders, HIV infection,

allergies, inflammation, arthritis, transplant rejection, high blood pressure, congestive

heart failure, tumors, and stress-induced abortions through the administration of a

compound of the present invention. Preferably the administration is for the treatment

or prophylaxis of diabetes.

Another aspect of the present invention includes the use of a compound of the

present invention in the manufacture of a medicament for the inhibition of a post

proline/analine cleaving protease. Preferably the post proline/analine cleaving protease

is a serine protease. More preferably the serine protease is a dipeptidyl peptidase. More

preferably the dipeptidyl peptidase is DPP-II or DPP-IV.

Another aspect of the present invention includes the use of a compound of the

present invention in the manufacture of a medicament for the treatment or prophylaxis

of metabolic disorders, gastrointestinal disorders, viral disorders, inflammatory disorders,

diabetes, obesity, hyperlipidemia, dermatological or mucous membrane disorders,

psoriasis, intestinal distress, constipation, autoimmune disorders, encephalomyelitis,

complement mediated disorders, glomerulonepritis, lipodystrophy, tissue damage,

psychosomatic, depressive, and neuropsychiatric disorders, HIV infection, allergies,

inflammation, arthritis, transplant rejection, high blood pressure, congestive heart

failure, tumors, and stress-induced abortions. Another aspect of the present invention includes a compound of the present

invention for use as an active therapeutic substance. In addtion a compound of the

present invention may be used in the manufacture of a medicament for the inhibition of

serine protease. Preferably such use is for the manufacture of a medicament for the

treatment or prophylaxis of metabolic disorders, gastrointestinal disorders, viral

disorders, inflammatory disorders, diabetes, obesity, hyperlipidemia, dermatological or

mucous membrane disorders, psoriasis, intestinal distress, constipation, autoimmune disorders, encephalomyelitis, complement mediated disorders, glomerulonepritis,

lipodystrophy, tissue damage, psychosomatic, depressive, and neuropsychiatric disorders,

HIV infection, allergies, inflammation, arthritis, transplant rejection, high blood pressure,

congestive heart failure, tumors, and stress-induced abortions.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The term "alkyl" refers to a straight or branched chain saturated aliphatic

hydrocarbon that may be optionally substituted, with multiple degrees of substitution

being allowed. Examples of "alkyl" include, but are not limited to methyl, ethyl, n-

propyl, isopropyl, n-butyl, t-butyl, n-pentyl, isobutyl, and the like.

As used throughout this specification, the preferred number of carbon atoms will

be represented by, for example, the phrase "Cx-C_y alkyl" which refers to an alkyl group, as

herein defined, containing the specified number of carbon atoms. Similar terminology

will apply for other preferred ranges as well.

The term "alkylene" refers to a divalent straight or branched chain aliphatic

hydrocarbon radical that may be optionally substituted, with multiple degrees of

substitution being allowed. An example of "alkylene" includes, without limitation,

methylene, namely -CH2-. The term "alkenyl" refers to a straight or branched chain aliphatic hydrocarbon,

containing one or more carbon-to-carbon double bonds that may be optionally

substituted, with multiple degrees of substitution being allowed. Examples include, but are not limited to, vinyl and the like.

As used herein the term "alkenylene" refers to a divalent straight or branched

chain aliphatic hydrocarbon radical, containing one or more carbon-to-carbon double

bonds, which may be optionally substituted, with multiple degrees of substitution being

allowed. An example of "alkenylene" includes, without limitation, vinylene, namely, - CH=CH-.

As used herein the term "alkynyl" refers to a straight or branched aliphatic

hydrocarbon containing one or more triple bond, which may optionally be substituted,

with multuiple degrees of substitution being allowed. Examples of "alkynyl" as used

herein include, but are not limited to, ethynyl and the like.

As used herein the term "alkynylene" refers to a divalent straight or branched

chain aliphatic hydrocarbon radical, containing at least one carbon-to-carbon triple

bond, that may be further substituted, with multiple degrees of substitution being allowed. An example of "alkynylene" includes, without limitation, ethynylene, namely -

C≡C-.

The term "aryl" refers to an aromatic ring system, such as an optionally

substituted benzene ring system, such as phenyl. The term encompasses fused systems

where one or more optionally substituted benzene rings form, for example, anthracene,

phenanthrene, or naphthalene ring systems. The term includes ring(s) optionally

substituted, with multiple degrees of substitution being allowed, and also includes an

optional alkylene linker, such as G-Ce alkylene, through which the aryl group may be attached. Examples of "aryl" groups include, but are not limited to phenyl, benzyl, 2-

naphthyl, 1-naphthyl, biphenyl, as well as substituted derivatives thereof.

The term "heteroaryl" refers to a monocyclic aromatic ring system, or to a fused

bicyclic aromatic ring system comprising two or more aromatic rings. These heteroaryl

rings contain one or more nitrogen, sulfur, and/or oxygen atoms, where N-oxides, sulfur

oxides, and dioxides are permissible heteroatom substitutions and the rings may be

optionally substituted, with multiple degrees of substitution being allowed. The term

includes ring(s) optionally substituted, with multiple degrees of substitution being

allowed, and also includes an optional alkylene linker, such as G-Ce alkylene, through

which the heteroaryl group may be attached. Examples of "heteroaryl" groups used

herein include furan, thiophene, pyrrole, imidazole, pyrazole, triazole, tetrazole, thiazole,

oxazole, isoxazole, oxadiazole, thiadiazole, isothiazole, pyridine, pyridazine, pyrazine,

pyrimidine, quinoline, isoquinoline, benzofuran, benzothiophene, indole, indazole, and

substituted versions thereof.

As used herein, the term "cycloalkyl" refers to a mono- or bi-cyclic hydrocarbon

ring system, which may be further substituted with multiple degrees of substitution

being allowed, and which optionally includes an alkylene linker through which the

cycloalkyl may be attached. Exemplary "cycloalkyl" groups include, but are not limited

to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. When substituted,

one preferred substituent location for cycloalkyl groups of the present invention is at

the "1 -position." To illustrate, without limitation, a preferred location for a substituent

is represented below with the substituent referred to as "R": or more generically,

The term "cycloalkyl" includes bridged or fused ring systems, as well, such as

hydrindane, decalin, or adamantyl. For ease of reference, also included within the term

are cycloalkyl/aryl fused systems where, for example, a cycloalkyl, such as cyclohexyl, is

fused with an aromatic ring, such as a benzene ring, to form groups such as

As used herein, the term "heterocyclic" or the term "heterocyclyl" refers to a heterocyclic ring, preferably three to twelve-membered, that is either saturated or has

one or more degrees of unsaturation. These heterocyclic rings contain one or more

heteroatom, such as nitrogen, sulfur, and/or oxygen atoms, where N-oxides, sulfur

oxides, and dioxides are permissible heteroatom substitutions. As used herein

heterocyclic groups optionally may be substituted, with multiple degrees of substitution being allowed, and also includes an optional alkylene linker, such as G-Cε alkylene,

through which the heterocyclyl group may be attached. Such a ring may be optionally

fused to one or more of another "heterocyclic" ring(s), aryl ring(s), or cycloalkyl ring(s).

Examples of "heterocyclic" include, but are not limited to, tetrahydrofϋran, pyran, 1 ,4-

dioxane, 1 ,3-dioxane, piperidine, pyrrolidine, morpholine, tetrahydrothiopyran,

tetrahydrothiophene, and the like.

The term "halogen" refers to fluorine, chlorine, bromine, or iodine. The term "haloalkyl" refers to an alkyl group, as defined herein that is substituted

with at least one halogen. Non-limiting examples of "haloalkyl" groups include methyl,

ethyl, propyl, isopropyl, n-butyl, and t-butyl substituted independently with one or more

halogens, e.g., fluoro, chloro, bromo, and/or iodo. The term "haloalkyl" should be

interpreted to include such substituents as perfluoroalkyl, for example, trifluoromethyl, CF₃, and the like.

As used herein, the term "haloalkoxy" refers to the group -ORa, where Ra is

haloalkyl as herein defined.

As used herein, the term "alkoxy" refers to the group -ORa, where Ra is alkyl as herein defined.

As used herein, the term "aryloxy" refers to the group -ORb, where Rb is aryl as

herein defined.

As an example, and to be applied throughout the specification, since the term

"aryl" includes optionally substituted aryl groups, the term "aryloxy" includes optionally

substituted aryloxy groups. The optional substitution applies for all applicable terms

herein defined. Further, as defined above, the term "aryl" includes alkylene-linked aryl groups. Thus, terms such as "aryloxy" and the like should be considered to include

alkylene-linked aryl groups. As an example and not meant as limiting, one aryloxy group

may be -ORb, where Rb is benzyl. As used herein, the term "heteroaryloxy" refers to the group -ORb, where Rb is

heteroaryl as herein defined.

As used herein, the term "alkoxycarbonyl" refers to the group -C(0)0R_a, where Ra

is alkyl as herein defined. As used herein, the term "aryloxycarbonyl" refers to the group -C(0)0Ra, where

Ra is aryl as herein defined.

As used herein, the term "heteroaryloxycarbonyl" refers to the group -C(0)0Ra,

where Ra is heteroaryl as herein defined.

As used herein, the term "alkoxythiocarbonyl" refers to the group -C(S)OR_a,

where Ra is alkyl as herein defined.

As used herein, the term "aryloxythiocarbonyl" refers to the group -C(S)ORa,

where Ra is aryl as herein defined.

As used herein, the term "heteroaryloxythiocarbonyl" refers to the group -

C(S)ORa, where Ra is heteroaryl as herein defined.

As used herein, the term "oxo" refers to the group =0. As used herein, the term "mercapto" refers to the group -SH.

As used herein, the term "thio" shall refer to the group -S-.

As used herein, the term "sulfinyl" shall refer to the group ~S(0)-.

As used herein, the term "sulfonyl" shall refer to the group -S(0)2-.

As used herein, the term "alkylthio" refers to the group -SRa, where Ra is alkyl as

herein defined.

As used herein, the term "arylthio" refers to the group -SRb, where Rb is aryl as

herein defined. As used herein, the term "heteroarylthio" refers to the group -SRb, where Rb is

heteroaryl as herein defined.

As used herein, the term "alkylsulfinyl" refers to the group -S(0)Ra, where R_a is

alkyl as herein defined. As used herein, the term "arylsulfinyl" refers to the group -S(0)Rb, where Rb is aryl as herein defined.

As used herein, the term "heteroarylsulfinyl" refers to the group -S(0)Rb, where Rb is heteroaryl as herein defined.

As used herein, the term "alkylsulfonyl" refers to the group -S(0)2Ra, where Ra is alkyl as herein defined.

As used herein, the term "cycloalkylsulfonyl" refers to the group -S(0)2Ra, where

Ra is cycloalkyl as herein defined.

As used herein the term "arylsulfonyl" refers to the group -S(0)2Rb, where Rb is aryl as herein defined.

As used herein the term "heteroarylsulfonyl" refers to the group -S(0)2Rb, where

Rb is heteroaryl as herein defined.

As used herein, the term "aminosulfonyl" refers to the group -S(0)2NH2.

As used herein, the term "cyano" refers to the group -CN.

As used herein the term "cyanoalkyl" refers to the group -RaCN wherein Ra is an

alkylene as herein defined.

As used herein, the term "carboxy" refers to the group -COOH.

As used herein, the term "carbamoyl" refers to the group -C(0)NH2.

As used herein, the term "alkylcarbamoyl" refers to the group -C(0)N(Ra)2, where

one Ra is alkyl and the other Ra is independently H or alkyl.

As used herein, the term "arylcarbamoyl" refers to the group -C(0)N(Ra)2, where

one Ra is aryl and the other Ra is independently H or aryl, as herein defined. As used herein, the term "heteroarylcarbamoyl" refers to the group -C(0)N(Ra)2,

where one Ra is heteroaryl and the other Ra is independently H or heteroaryl, as herein defined.

As used herein, the term "thiocarbamoyl" refers to the group -C(S)NH2.

As used herein, the term "alkylthiocarbamoyl" refers to the group -C(S)N(Ra)2,

where one Ra is alkyl and the other Ra is independently H or alkyl.

As used herein, the term "arylthiocarbamoyl" refers to the group -C(S)N(Ra)2,

where one Ra is aryl and the other Ra is independently H or aryl, as herein defined.

As used herein, the term "heteroarylthiocarbamoyl" refers to the group

-C(S)N(Ra)2, where one Ra is heteroaryl and the other Ra is independently H or heteroaryl,

as herein defined.

As used herein, the term "amino" refers to the group -NH2. As used herein, the term "alkylamino" refers to the group -N(Ra)2, where one Ra is

alkyl and the other Ra independently is H or alkyl, as herein defined.

As used herein, the term "cycloalkylamino" refers to the group -N(Ra)2, where one

Ra is cycloalkyl and the other Ra independently is H or cycloalkyl, as herein defined.

As used herein, the term "arylamino" refers to the group -N(Ra)2, where one Ra is

aryl and the other Ra independently is H or aryl, as herein defined.

As used herein, the term "heteroarylamino" refers to the group -N(Ra)2, where

one Ra is heteroaryl and the other Ra independently is H or heteroaryl, as herein defined.

As used herein, the term "acyl" refers to the group -C(0)Ra, where Ra is alkyl, aryl,

heteroaryl, cycloalkyl, or heterocyclyl, each as herein defined.

As used herein, the term "thioacyl" refers to the group -C(S)Ra, where R_a is alkyl,

aryl, heteroaryl, cycloalkyl, or heterocyclyl, each as herein defined. As used herein, the term "hydroxy" refers to the group -OH.

As used herein the term "hydroxyalkyl" refers to the group -RaOH wherein Ra is an alkylene as herein defined.

Also, as used herein throughout the present specification, the phrase "optionally

substituted" denotes an optional substitution, one or more times, with, as the terms are

herein defined, acyl; alkyl; alkenyl; alkynyl; alkylsulfonyl; alkoxy; cyano; halogen;

haloalkyl; hydroxy; nitro; aryl, which may be further substituted with acyl, alkoxy, alkyl,

alkenyl, alkynyl, alkylsulfonyl, cyano, halogen, haloalkyl, hydroxy, or nitro; heteroaryl,

which may be further substituted with acyl, alkoxy, alkyl, alkenyl, alkynyl, alkylsulfonyl,

cyano, halogen, haloalkyl, hydroxy, or nitro; arylsulfonyl, which may be further

substituted with acyl, alkoxy, alkyl, alkenyl, alkynyl, alkylsulfonyl, cyano, halogen,

haloalkyl, hydroxy, or nitro; heteroarylsulfonyl, which may be further substituted with

acyl, alkoxy, alkyl, alkenyl, alkynyl, alkylsulfonyl, cyano, halogen, haloalkyl, hydroxy, or

nitro; aryloxy, which may be further substituted with acyl, alkoxy, alkyl, alkenyl, alkynyl,

alkylsulfonyl, cyano, halogen, haloalkyl, hydroxy, or nitro; heteroaryloxy, which may be

further substituted with acyl, alkoxy, alkyl, alkenyl, alkynyl, alkylsulfonyl, cyano, halogen,

haloalkyl, hydroxy, or nitro; -R'OR'R⁴; or -NR⁴R⁵; where for each occurrence R' is

alkylene, alkenylene, or alkynylene, and R⁴ and R⁵ are each independently selected from

H, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylsulfonyl,

arylsulfonyl, or heteroarylsulfonyl, where each occurrence of such aryl or heteroaryl may

be substituted with one or more acyl, alkoxy, alkyl, alkenyl, alkylsulfonyl, cyano, halogen,

haloalkyl, hydroxy, or nitro, or R⁴ and R⁵ may combine to form a ring, optionally having

additional heteroatoms, optionally having one or more degrees of unsaturation, and optionally being further substituted with acyl, alkoxy, alkyl, alkenyl, alkynyl,

alkylsulfonyl, cyano, halogen, haloalkyl, hydroxy, or nitro.

The compounds of the present invention may have the ability to crystallize in more than one form, a characteristic known as polymorphism. All polymorphic forms

("polymorphs") are within the scope of the present invention. Polymorphism generally

can occur as a response to changes in temperature or pressure, or both, and can also result from variations in the crystallization process. Polymorphs can be distinguished by

various physical characteristics that are known in the art such as x-ray diffraction

patterns, solubility, and melting point.

Certain of the compounds described herein contain one or more chiral centers, or

may otherwise be capable of existing as multiple stereoisomers. The scope of the

present invention includes pure stereoisomers as well as mixtures of stereoisomers, such

as purified enantiomers/diasteromers or enantiomerically/diastereomerically enriched

mixtures. Also included within the scope of the invention are the individual isomers of

the compounds per se, as well as any wholly or partially equilibrated mixtures thereof.

The present invention covers the individual isomers of the compounds represented by

the formulas above as mixtures with isomers thereof in which one or more chiral centers are inverted.

As noted above, the present invention includes salts, solvates, and

pharmaceutically functional derivatives of the compounds of the present invention.

Salts include addition salts, metal salts, or optionally alkylated ammonium salts.

Examples of such salts include hydrochloric, hydrobromic, hydroiodic, phosphoric,

sulfuric, trifluoroacetic, trichloroacetic, oxalic, maleic, pyruvic, malonic, succinic, citric,

mandelic, benzoic, cinnamic, methane sulphonic, ethane sulphonic, picric, and the like. Further salts include lithium, sodium, potassium, magnesium, and the like. Still further

salts include acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate,

borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate,

dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate,

glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydroxynaphthoate,

isethionate, lactate, lactobionate, laurate, malate, mandelate, mesylate, methylbromide,

methylnitrate, methylsulfate, monopotassium maleate, mucate, napsylate, nitrate, N-

methylglucamine, pamoate (embonate), palmitate, pantothenate,

phosphate/diphosphate, polygalacturonate, potassium, salicylate, sodium, stearate,

subacetate, tannate, tartrate, teoclate, tosylate, triethiodide, trimethylammonium, and

valerate salts. Reference is also made to Journal of Pharmaceutical Science, 1997, 66, 2,

incorporated herein by reference, as relevant to salts.

As used herein, the term "solvate" refers to a complex of variable stoichiometry

formed by a solute or a salt or pharmaceutically functional derivative thereof and a

solvent. Such solvents for the purpose of the invention should not interfere with the

biological activity of the solute. Examples of solvents include, but are not limited to

water, methanol, ethanol, and acetic acid. Preferably the solvent used is a

pharmaceutically acceptable solvent. Examples of pharmaceutically acceptable solvents

include water, ethanol, and acetic acid.

The term "pharmaceutically functional derivative" refers to any pharmaceutically

acceptable derivative of a compound of the present invention, for example, an ester or an amide, which upon administration to a mammal is capable of providing (directly or

indirectly) a compound of the present invention or an active metabolite thereof. Such

derivatives are recognizable to those skilled in the art, without undue experimentation. Nevertheless reference is made to the teaching of Burger's Medicinal Chemistry and

Drug Discovery, 5^th Edition, Vol 1 : Principles and Practice, which is incorporated herein

by reference to the extent of teaching pharmaceutically functional derivatives.

While compounds of the present invention may be administered as the raw chemical,

preferably the compounds of the present invention are presented as an active ingredient

within a pharmaceutical formulation as known in the art. Accordingly, the present

invention further includes a pharmaceutical formulation comprising a compound of the

present invention, or salt, solvate, or pharmaceutically functional derivative thereof

together with one or more pharmaceutically acceptable carriers. Optionally, other

therapeutic and/or prophylactic ("active") ingredients may be included in the

pharmaceutical formulation as well. For example, the compounds of the -present invention may be combined with other anti-diabetic agents, such as one or more of the

following agents: insulin, α-glucosidase inhibitors, biguanides, insulin secretagogue, or

insulin sensitizers. Non-limiting examples of α-glucosidase inhibitors include acarbose,

emiglitate, miglitol, and voglibose. Non-limiting examples of biguanides include

metformin, buformin, and phenformin. Non-limiting examples of insulin secretagogues

include sulphonylureas. Non-limiting examples of insulin sensitizers include peroxisome

proliferator activated receptor (PPAR) ligands, such as PPAR-γ agonists, for example

Actos™ and Avandia™. Formulations of the present invention include those especially formulated for

oral, buccal, parental, transdermal, inhalation, intranasal, transmucosal, implant, or

rectal administration. Among the variety of administrations, oral administration

typically is preferred. For oral administration tablets, capsules, and caplets may contain

conventional excipients such as binding agents, fillers, lubricants, disinteg rants, and/or wetting agents. Non-limiting examples of binding agents include syrup, acacia, gelatin,

sorbitol, tragacanth, mucilage of starch, or polyvinylpyrrolidone (PVP). Non-limiting

examples of fillers include, for example, lactose, sugar, microcrystalline cellulose, maize-

starch, calcium phosphate or sorbitol. Non-limiting examples of lubricants include, for

example, magnesium sterate, stearic acid, talc, polyethylene glycol or silica. Non-

limiting examples of disintegrants include, for example, potato starch or sodium starch

glycollate. A non-limiting example of a wetting agent includes sodium lauryl sulfate.

The tablets additionally may be coated according to methods known in the art.

Alternatively, the compounds of the present invention may be incorporated into

oral liquid preparations such as aqueous or oily suspensions, solutions, emulsions, syrups,

or elixirs. Moreover, formulations containing these compounds may be presented as a

dry product for constitution with water or other suitable vehicle before use. Liquid

preparations may contain conventional additives. Non-limiting examples of such

additives include suspending agents such as sorbitol syrup, methyl cellulose,

glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum

sterate gel or hydrogenated edible fats. Additionally, emulsifying agents such as

lecithin, sorbitan mono-oleate or acacia; non-aqueous vehicles (which may include

edible oils) such as almond oil, fractionated coconut oil, oily esters, propylene glycol or

ethyl alcohol my be included. Further, preservatives such as methyl or propyl p-

hydroxybenzoates or sorbic acid, may be incorporated into the preparation. Such

preparations may also be formulated as suppositories, for example, containing

conventional suppository bases such as cocoa butter or other glycerides.

Additionally, formulations of the present invention may be formulated for

parenteral administration by injection or continuous infusion. Formulations for injection may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles,

and may contain formulatory agents such as suspending, stabilizing and/or dispersing

agents. Alternatively, the active ingredient may be in powder form for constitution with

a suitable vehicle, for example, sterile, pyrogen-free water, before use.

The formulations according to the invention may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation, for

example, subcutaneously or intramuscularly, or by intramuscular injection. Accordingly,

the compounds of the invention may be formulated with suitable polymeric or

hydrophobic materials, such as an emulsion in an acceptable oil, ion exchange resins, or

as sparingly soluble derivatives, such as a sparingly soluble salt.

Pharmaceutical formulations may be presented in unit dose forms containing a

predetermined amount of active ingredient per unit dose. Such a unit may contain certain amounts of a compound of the present invention depending on the condition

being treated, the route of administration, and the age, weight and condition of the

patient. Examples of such amounts include the formulation containing about 0.1 to

about 99.9% active ingredient. Preferred unit dosage formulations are those containing

a predetermined dose, such as a daily dose, or an appropriate fraction thereof, of an

active ingredient. Such pharmaceutical formulations may be prepared by any of the

methods well known in the pharmacy art.

As used herein, the term "effective amount" means that amount of a drug or

pharmaceutical agent that will elicit the biological or medical response of a tissue,

system, animal, or human that is being sought, for instance, by a researcher or clinician.

Furthermore, the term "therapeutically effective amount" means any amount which, as

compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side

effect, or a decrease in the rate of advancement of a disease or disorder. The term also

includes within its scope amounts effective to enhance normal physiological function.

The therapeutically effective amount of a compound of the present invention

will depend upon a number of factors including, for example, the age and weight of the

animal, the precise condition requiring treatment and its severity, the nature of the

formulation, and the route of administration. Therapeutic effectiveness ultimately will be at the discretion of the attendant physician or veterinarian. An effective amount of a

salt or solvate, or pharmaceutically functional derivative thereof, may be determined as

a proportion of the effective amount of a compound of the present invention per se.

Dosages may vary, depending upon the appropriate inhibition of DPP-IV for purposes of

treatment or prophylaxis of a variety of metabolic, gastrointestinal, viral, and inflammatory diseases, including, but not limited to, diabetes, obesity, hyperlipidemia,

dermatological or mucous membrane disorders, psoriasis, intestinal distress, constipation,

autoimmune disorders such as encephalomyelitis, complement mediated disorders such

as glomerulonepritis, lipodystrophy, and tissue damage, psychosomatic, depressive, and

neuropsychiatric disorders, HIV infection, allergies, inflammation, arthritis, transplant

rejection, high blood pressure, congestive heart failure, tumors, and stress-induced

abortions, for example cytokine-mediated murine abortions.

No toxicological effects are indicated/expected when a compound of the present

invention is administered in the above mentioned dosage range.

The following examples illustrate aspects of this invention, but should not be

construed as limitations. As used herein the symbols and conventions used in these

processes, schemes and examples are consistent with those used in the contemporary scientific literature, for example, the Journal of the American Chemical Society or the

Journal of Biological Chemistry. Unless otherwise noted, all starting materials were

obtained from commercial suppliers and used without further purification.

¹H NMR spectra were recorded on a Varian VXR-300, a Varian Unity-300, a Varian

Unity-400 instrument, or a General Electric QE-300. Chemical shifts are expressed in

parts per million (ppm, δ units). Coupling constants are in units of hertz (Hz). Splitting

patterns describe apparent multiplicities and are designated as s (singlet), d (doublet), t

(triplet), q (quartet), m (multiplet), br (broad).

Low-resolution mass spectra (MS) were recorded on a JOEL JMS-AX505HA, JOEL

SX-102, or a SCIEX-APIiii spectrometer; high resolution MS were obtained using a JOEL

SX-102A spectrometer. All mass spectra were taken under electrospray ionization (ESI),

chemical ionization (Cl), electron impact (El) or by fast atom bombardment (FAB) methods. Infrared (IR) spectra were obtained on a Nicolet 510 FT-IR spectrometer using

a 1-mm NaCl cell. All reactions were monitored by thin-layer chromatography on 0.25

mm E. Merck silica gel plates (60F-254), visualized with UV light, 5% ethanolic

phosphomolybdic acid or p-anisaldehyde solution. Flash column chromatography was

performed on silica gel (230-400 mesh, Merck). Optical rotations were obtained using a

Perkin Elmer Model 241 Polarimeter. Melting points were determined using a Mel-Temp

II apparatus and are uncorrected. IUPAC names are included to further identify particular compounds of the

present invention. The IUPAC names stated herein should in no way limit the scope of

the present invention. EXPERI MENTALS

In accordance with the present invention and as below, one embodiment of the

compounds of the present invention can be prepared by reacting a compound of

formula II with an α- or β-amino carboxylate or with an α- or β-amino activated

carboxylate, both designated herein generally as aminocarboxylates, under standard

coupling conditions, for example, with HATU, DMF, Hunigs base.

formula II formula I

More specifically, a compound of formula II may be reacted with an amino

carboxylate, where the amino carboxylate is suitably protected, for example on the α-

nitrogen, with an appropriate protecting group such as, for example, a t-butyl carboxy protecting group.

In an alternate embodiment, a compound of formula II may be reacted with an

amino activated carboxylate, such as, for example, N-hydroxysuccinimide ester or acid

chloride, where the amino activated carboxylate is suitably protected, for example, on

the α-nitrogen with an appropriate protecting group such as, for example, a t-butyl

carboxy protecting group. Removal of the protecting group under suitable conditions,

such as, for example, trifluoroacetic acid for the removal of the t-butyl carboxy, then

generates compounds of formula (I). For further detail regarding the preparation of amino carboxylates for use in

preparing the compounds of the present invention, reference may be had to WO

95/15309 and WO 98/19998, each herein incorporated by reference as related to the

preparation of such reactants.

Intermediate Example 1

(2S, 4S)-4-Fluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate

A. Methyl (2S, 4R)-4-hydroxy-2-pyrrolidinecarboxylate hydrochloride

To a MeOH solution (420 mL) containing L-hydroxyproline (62.67 g, 478 mmol) cooled in

an ice water bath was added thionyl chloride (58.6 g, 492.3 mmol) dropwise. Upon

complete addition the suspension was stirred at RT for 2 h. The mixture was then heated

to reflux for 6 h at which time it was cooled to RT and the solvent removed in vacuo.

The residual solid was pumped on under high vacuum yielding 86.03 g (474 mmol, 99%

yield) of compound A as a white solid.

¹H NMR (d₄-MeOH) 400 MHz δ 4.62-4.57 (m, 2H), 3.85 (s, 3H), 3.43 (dd, I H, J = 12.2, 3.7

Hz), 3.30 (m, 1 H), 2.41 (dd, 1 H, J = 13.6, 7.6 Hz), 2.19 (m, l H) ppm.

B. 1-Tert-butyl-2-methyl (2S, 4R)-4-hydroxy-1 ,2-pyrrolidinedicarboxylate.

To a CH2CI2 solution (1.4 L) containing compound A (88.67 g, 0.49 mol) and di-t-

butyldicarbonate (109.8 g, 0.50 mol) was added, at 0°C, triethylamine (123.6 g, 1.22 mol)

dropwise over 1.5 h. The resulting solution was then slowly allowed to warm to RT overnight. The solvent was then removed in vacuo and Et2θ was added to the residual

solid. The solid was collected via vacuum filtration and washed thoroughly with Et∑O.

The filtrate then had the solvent removed in vacuo and was dissolved in CH2CI2. The

organics were washed with sat. NaCl and sat. NaHC0₃ followed by drying over MgS0₄.

Filtration and removal of the solvent in vacuo yielded a light yellow oil which, after

pumping on under high vacuum for ~ 15 min., solidified. The resulting solid had 500 mL

of hexanes added to it and then stirred overnight. The solid was collected via vacuum

filtration and pumped on under high vacuum yielding 104.5g (0.43 mol, 87% yield) compound B as a white solid.

¹H NMR (d₄-MeOH) 400 MHz δ 4.37-4.32 (m, 2H), 3.72-3.71 (m, 3H), 3.51 (m, 1 H), 3.43

(m, 1 H), 2.23 (m, 1 H), 2.02 (m, I H), 1.42 (m, 9H) ppm.

C. 1-Tert-butyl 2-methyl (2S, 4S)-4-fluoro-1 ,2-pyrrolidinedicarboxylate.

To a 2 L flask containing compound B (124.25 g, 0.51 mol), in 1.25 L of 1 ,2-

dichloroethane cooled to -30 °C, was added DAST neat (125 g, 0.78 mol). The reaction

slowly warmed to -10 °C over 1 hr at which time the cold bath was removed. Stirring

continued at RT for 24 hr when the dark solution was poured into two 2 L flasks that

contained crushed ice and solid NaHC0₃. The flasks were periodically swirled and stirred

until no CO2 evolution was observed (note: additional solid NaHC0₃ was periodically

added). The organic layer was separated and the aqueous layer extracted with CH2CI2.

After drying over MgS0₄ the solvent was removed in vacuo and the residual dark oil was

dissolved in 200 mL of EtOAc and then 800 mL of hexanes was added. To this solution

was added 100 g of SiO₂. After stirring for 30 min the solution was filtered with the

Siθ2 being washed with hexanes/EtOAc (4:1 , ~ 500 mL). Removal of the solvent in vacuo and pumping on under high vacuum overnight yielded 121.81 g (0.40 mol, 97% yield) of

compound C as a dark oil.

'H NMR (CDCb) 400 MHz δ 5.18 (d(br), l H, _/ = 53 Hz), 4.53 (d, 1/2H, J = 9.7 Hz), 4.40 (d,

1/2H, J = 9.4 Hz), 3.87-3.59 (m, 2H), 3.73 (s, 3H), 2.51-2.28 (m, 2H), 1.46 (s, 3H, rotomer), 1.41 (s, 6H, rotomer) ppm.

D. (2S, 4S)-1-(Tert-butoxycarbonyl)-4-fluoro-2-pyrrolidinecarboxylic acid.

To a 2 L flask containing compound C (121.8 g, 0.49 mol), in 1.1 L of dioxane, was added

380 mL of H2O followed by lithium hydroxide hydrate (103.6 g, 2.46 mol) at RT. The

resulting solution stirred for 23 hr (note: by TLC the reaction appeared to be done after

5 hr) at which time the bulk of the dioxane was removed in vacuo. The residual material

was dissolved in additional H2O and then charcoal was added. After stirring for 15 min.,

the solution was filtered through a bed of celite. The filtrate had solid NaCl added to it

until it didn't dissolve any further. It was then cooled in an ice water bath and was acidified with concentrated HCl to pH 3 whilst maintaining the solution temperature

between 5 - 10 °C. The product began to precipitate out at pH 4 and upon reaching pH

3 the tan solid was collected via vacuum filtration. After pumping on under high

vacuum overnight the solid was dissolved in CH₃CN (1.5 L) and dried over MgS0₄.

Removal of the solvent in vacuo and drying under high vacuum yielded 92.7 g (0.40 mol,

81% yield) of compound as a tan solid.

¹H NMR (de-DMSO) 400 MHz δ 12.5 (s(br), 1 H), 5.22 (d(br), 1 H, J = 54 Hz), 4.25 (m, 1 H),

3.60-3.43 (m, 2H), 2.45 (m, 1 H), 2.20 (m, 1 H), 1.33 (m, 9H) ppm. E. Tert-butyl (2S, 4S)-2-(aminocarbonyl)-4-fluoro-1-pyrrolidinecarboxylate.

To a 2 L, 3-neck flask equipped with an air-driven stirrer was added compound D (92.7 g,

0.40 mol), CHsCN (1.1 L), di-t-butyldicarbonate (130 g, 0.60 mol), and pyridine (32.4 g,

0.41 mol) at RT. After stirring for 20 min., ammonium hydrogen carbonate (47.2 g, 0.60

mol) was added. The reaction stirred for 23 hr at which time the bulk of the CH₃CN was

removed in vacuo. The residue was then dissolved in CH2CI2 and washed with a 1 :1 1 M HCI/sat. NaCl solution. The aqueous layer was then extracted 2X with CH2CI2. The

combined organic layers were dried (MgS0₄) and the solvent removed in vacuo. The tan

solid was triturated with hexanes (~ 0.5 L) and collected via vacuum filtration. After

pumping under high vacuum, 68.75 g (0.30 mol, 74% yield) compound E was obtained as

a light tan solid. The filtrate after removal of the solvent in vacuo gave a dark oil that

also appeared to contain additional product by ¹H NMR.

Η NMR (de-DMSO) 400 MHz δ 7.21 (s(br), 1/2H), 7.14 (s(br), 1/2H), 6.94 (s(br), 1 H), 5.19

(d(br), 1 H, J = 54 HZ), 4.1 1 (m, I H), 3.63-3.47 (m, 2H), 2.38 (m, 1 H), 2.1 1 (m, 1 H), 1.39 (s,

3H, rotomer), 1.34 (s, 6H, rotomer) ppm.

F. Tert-butyl (2S, 4S)-2-cyano-4-fluoro-1 -pyrrolidinecarboxylate.

To a flask containing imidazole (2.93 g, 43.1 mmol), was added pyridine (75 g, 0.9 mol,

15 volumes by weight to the amide). The solution was then cooled to 0 °C and after

stirring for 10 min., BOC-4-fluoroproline carboxamide (5.0 g, 21.6 mmol) was added in

one portion. The solution was then cooled to -30 °C (note: going below this

temperature may lead to a heterogeneous solution) and POCb (13.2 g, 86.4 mmol) was

added dropwise over 5 minutes. Upon complete addition the dry-ice acetone bath was

replaced with an ice water bath and stirring continued at 0 °C for 1 hr at which time it was poured into a crushed ice, solid NaCl and EtOAc mixture. The organic layer was

separated and the aqueous layer was extracted with EtOAc (2X). The solvent was

removed in vacuo (note: keep rotovap bath < 35°C) and the residue dissolved in EtOAc.

The organics were washed with sat. NaCl and 1 M HCl (2X). After drying over MgS0₄ the

solvent was removed in vacuo yielding 4.0 g (18.6 mmol, 86% yield) of compound F as a light tan solid.

'H NMR (d₄-MeOH) 400 MHz δ 5.32 (d(br), 1 H, 1 = 52 Hz), 4.78 (m, 1 H), 3.74-3.48 (m,

2H), 2.55-2.40 (m, 2H), 1.52-1.43 (m, 9H) ppm.

G. (2S, 4S)-4-Fluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate.

To a CH₃CN solution (1 L) containing compound F (56.62 g, 0.26 mol), was added p-

toluenesulfonic acid hydrate (75.4 g, 0.40 mol) at RT. After 24 hr the CH₃CN was

removed in vacuo and then the residual brown oil was dissolved in 500 mL of EtOAc.

Within 1 min a solid precipitated out and the solution was cooled in an ice-water bath

and after stirring for 1 hr the solid was collected via vacuum filtration. The collected

solid was rinsed with cold (-20 °C) EtOAc (~ 500 mL) and then pumped on under high

vacuum overnight yielding 60.94 g (0.21 mol, 82% yield) of compound G as a light tan

solid.

¹H NMR (d₄-MeOH) 400 MHz δ 7.69 (d, 2H, J = 8.1 Hz), 7.23 (d, 2H, J = 8.0 Hz), 5.52 (dd,

1 H, J = 51 , 3.4 Hz), 4.96 (dd, 1 H, J = 9.8, 3.6 Hz), 3.78 (m, 1 H), 3.55 (m, 1 H), 2.84-2.63 (m,

2H), 2.36 (s, 3H) ppm. Alternative Route for Intermediate Example 1

(2S, 4S)-4-Fluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate.

Tert-butyl (2S,4R)-2-(aminocarbonyl)-4-hydroxypyrrolidine-1 -carboxylate

To a THF solution (420 mL) containing BOC-L-hydroxyproline (30.0 g, 129 mmol) and

triethylamine (14.4 g, 141.9 mmol) cooled to -15 °C was added ethyl chloroformate

(15.4 g, 141.9 mmol) dropwise. The resulting solution stirred for 10 min when 80 mL of

28% H4OH was added. The reaction was allowed to slowly warm to 5 °C over 2 hr at which time sat. NH4CI was added until the entire white solid had dissolved. The THF was

separated and the aqueous layer extracted with THF. The combined organic layers were

dried (MgS0₄) and the solvent removed in vacuo. The residual oil was treated with Et2U

and a small amount of CH2CI2 and MeOH. After storing in the freezer for 1 hr the

resulting white solid was collected via vacuum filtration yielding 22.0 g (95.6 mmol, 74%

yield) of compound A.

'H NMR (de-DMSO) 400 MHz δ 7.33-7.29 (d(br), 1 H, rotomers), 6.88-6.80 (d(br),

1 H, rotomers), 4.94 (s(br), 1 H), 4.18 (s(br), 1 H), 4.04 (m, 1 H), 3.35 (m, 1 H), 3.21 (m, 1 H),

1.99 (m, 1H), 1.77 (m, 1 H), 1.36-1.31 (d, 9H, rotomers) ppm.

B. Tert-butyl (2S,4R)-2-cyano-4-hydroxypyrrolidine-1 -carboxylate.

To a pyridine solution (180 mL) containing compound A (17.89 g, 77.8 mmol) cooled to -

20 °C was added trifluoroacetic anhydride (40.8 g, 194.4 mmol) dropwise. Upon complete addition the reaction was allowed to warm to RT. After 6 hr the reaction was

quenched with H2O and then poured into EtOAc (ca 500 mL). The organics were washed

with sat. NaCl, 1.0 M HCl and 2.0 M NaOH followed by drying over MgS0₄. The filtrate

had charcoal added to it and after stirring for 10 min the solution was filtered through a

bed of celite. The solvent was removed in vacuo (the rotovap temperature was at 34 °C)

yielding 13.21 g (62.3 mmol, 80% yield) of compound B as an orange oil.

'H NMR (d₄-MeOH) 400 MHz δ 4.60 (m, 1 H), 4.40 (s(br), 1 H), 3.49 - 3.41 (m, 2H),

2.36 - 2.34 (m, 2H), 1.51-1.48 (m, 9H, rotomers)

C. Tert-butyl (2S, 4S)-2-cyano-4-fluoro-1 -pyrrolidinecarboxylate.

To a 1,2-dichloroethane solution (300 mL) containing compound B (13.21 g, 62.3

mmol) cooled to -30 °C was added DAST (15.1 g, 93.4 mmol). After 30 min the cold bath

was removed and stirring continued for 24 hr at which time the reaction was quenched

carefully with sat. NaHC0₃. The solution was then poured onto crushed ice and the

organics extracted with CH2CI2 (2X). After a final washing with sat. NaHS0₄ the organics

were dried (MgS0₄) and the solvent removed in vacuo yielding 10.86 g (50.7 mmol, 81%

yield) of compound C as a brown semi-solid.

¹H NMR (d₄-MeOH) 400 MHz δ 5.32 (d(br), 1 H, J = 52 Hz), 4.78 (m, 1 H), 3.74-3.48 (m,

2H), 2.55-2.40 (m, 2H), 1.52-1.43 (m, 9H) ppm.

D. (2S, 4S)-4-Fluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate.

To an CH3CN solution (200 mL) containing compound C (10.86 g, 50. 7 mmol)

was added p-toluenesulfonic acid (14.8 g, 78 mmol) at RT. The resulting solution stirred

for 24 hr at which time the CH3CN was removed in vacuo. The residual brown oil was dissolved in EtOAc (300 mL) and within 1 min a solid precipitated out. The solution was

cooled in an ice-bath for 2 hr and then the solid collected via vacuum filtration. It was

then washed with 300 mL of cold (-20 °C) EtOAc yielding 10.07 g (35.2 mmol, 69% yield) of compound D.

¹H NMR (d₄-MeOH) 400 MHz δ 7.69 (d, 2H, J = 8.1 Hz), 7.23 (d, 2H, J = 8.0 Hz), 5.52 (dd,

1 H, J = 51 , 3.4 Hz), 4.96 (dd, 1 H, J = 9.8, 3.6 Hz), 3.78 (m, 1 H), 3.55 (m, 1 H), 2.84-2.63 (m, 2H), 2.36 (s, 3H) ppm.

Intermediate Example 2

(2S)-4,4-Difluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate.

A. Tert-butyl (2S, 4S)-2-(aminocarbonyl)-4-hydroxy-1-pyrrolidinecarboxylate.

To a THF solution (420 mL) containing BOC-L-hydroxyproline (30.0 g, 129 mmol) and

EtaN (14.4 g, 141.9 mmol) cooled to -15 °C was added ethyl chloroformate (15.4 g, 141.9

mmol) dropwise. The resulting white solution stirred at -15 °C for 30 min when 80 mL

of a 28% NH4OH solution was added via syringe. Upon complete addition the cold bath

was removed and stirring continued for 19 hr. The homogeneous solution was poured

into sat. NH4CI and the organic layer separated. The aqueous layer was extracted with

THF and then the combined organic layers dried (MgS0₄). The solvent was removed in

vacuo and the semisolid pumped under high vacuum for 2 hr. The resulting white solid was collected via vacuum filtration with Et2θ yielding 15.86 g (68.9 mmol, 53% yield) of compound A.

Η NMR (de-DMSO) 400 MHz δ 7.34 (s(br), 1 H, rotomer), 7.31 (s(br), 1 H, rotomer), 6.90

(s(br), 1 H, rotomer), 6.82 (s(br), 1 H, rotomer), 4.95 (d, 1 H, J = 3.1 Hz), 4.05 (m, 1 H), 3.36

(m, 1 H), 3.22 (m, 1 H), 2.03 (m, 1 H), 1.78 (m, 1 H), 1.37 (s, 3H, rotomer), 1.32 (s, 6H, rotomer) ppm.

B. Tert-butyl (2S)-2-(aminocarbonyl)-4-oxo-1-pyrrolidinecarboxylate.

To a CH2CI2 solution (12 mL) containing oxalyl chloride (607 mg, 4.78 mmol) cooled to -

78 °C was added a CH2CI2 solution (3 mL) containing DMSO. After 5 min tert-butyl (2S,

4S)-2-(aminocarbonyl)-4-hydroxy-1 -pyrrolidinecarboxylate (1.0 g, 4.35 mmol, as

described in step E above) in a CH2CI2/THF solution (20 mL/15 mL) was added dropwise.

Upon complete addition the reaction stirred for 20 min when Et₃N (2.20 g, 21.7 mmol)

was added. After 10 min the cold bath was removed and stirring continued for 1 hr. The solution was poured into sat. NaHC0₃ and the organics extracted with CH2CI2. After

drying over MgSU4 the solvent was removed in vacuo and the residual yellow oil purified

via column chromatography (CH∑Cb/MeOH (15:1)) yielding 560 mg (2.45 mmol, 56%

yield) of compound B as a white solid.

¹H NMR (de-DMSO) 400 MHz δ 7.58 (s(br), 1 H), 7.15 (s(br), 1 H, rotomer), 7.09 (s(br), 1 H,

rotomer), 4.51 (d, 1 H, J = 9.7 Hz, rotomer), 4.46 (d, 1 H, J = 8.8 Hz, rotomer), 3.76-3.64

(m, 2H), 3.02 (m, 1 H), 2.28 (m, 1 H), 1.39 (s, 3H, rotomer), 1.37 (s, 6H, rotomer) ppm. C. Tert-butyl (2S)-2-(aminocarbonyl)-4,4-difluoro-1-pyrrolidinecarboxylate.

To a CH2CI2 solution (10 mL) containing compound B (423 mg, 1.85 mmol) cooled to -70

°C was added DAST (889 mg, 5.50 mmol). The resulting solution stirred at -70 °C for 30

min and then at RT for 2 hr. The reaction was quenched with sat. NaHC0₃ and the

organics extracted with CH2CI2. After drying over MgSθ4 the residual yellow solid was

purified via column chromatography (CH∑Cb/MeOH (15:1)) yielding 211 mg (0.84 mmol, 46% yield) of compound C.

'H NMR (d₄-MeOH) 400 MHz δ 4.38 (m, 1 H), 3.84-3.75 (m, 2H), 2.76 (m, 1 H), 2.41 (m,

1 H), 1.44 (s(br), 9H) ppm.

D. Tert-butyl (2S)-2-cyano-4,4-difluoro-1 -pyrrolidinecarboxylate.

To a pyridine solution (20 mL) containing compound C (658 mg, 2.63 mmol) and

imidazole (358 mg, 5.26 mmol) cooled to -35 °C was added POCb (1.61 g, 10.5 mmol).

The resulting slurry stirred for 1.5 hr at which time it had warmed to 10 °C. The solution

was diluted with EtOAc and them washed 3X with 1 M HCl. After drying over MgS0₄ the

solvent was removed in vacuo yielding 610 mg (2.63 mmol, 100% yield) of compound D

which was taken directly on to E (below).

'H NMR (d4-MeOH) 400 MHz δ 4.88 (s(br), 1 H), 3.79-3.72 (m, 2H), 2.87 (m, 1 H), 2.69 (m,

1 H), 1.50 (s, 9H) ppm.

E. (2S)-4,4-Difluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate.

To a CH3CN solution (15 mL) containing compound D (512 mg, 2.21 mmol) was added p-

toluenesulfonic acid hydrate (839 mg, 4.42 mmol) at RT. The resulting solution stirred for

2 hr at which time the CH3CN was removed in vacuo. To the residual oil was added EtOAc (~ 10 mL) and this was then removed in vacuo. The resulting solid was triterated

with Et∑O followed by addition of EtOAc. The solid was collected via vacuum filtration

yielding 375 mg (1.23 mmol, 56% yield) of compound E as a white solid.

¹H NMR (d₄-MeOH) δ 400 MHz δ 7.70 (d, 2H, J = 8.2 Hz), 7.23 (d, 2H, J = 7.9 Hz), 5.12 (t,

1 H, 1 = 7.9 Hz), 3.91 -3.78 (m, 2H), 3.08-2.89 (m, 2H), 2.89 (s, 3H), 2.36 (s, 9H) ppm.

EXAMPLES

Example 1

(2S,4S)-1-[(2S)-2-Amino-3,3-diphenylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile

hydrochloride

A. (2S,4S)-1-[(2S)-2-Amino-3,3-diphenylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile

hydrochloride. To dry DMF (25 mL) was added (2S)-2-[(tert-butoxycarbonyl)amino]-3,3-

diphenylpropanoic acid (1.96 g, 6.0 mmol), HATU (2.28 g, 6.0 mmol) and N,N-

diisopropylethylamine (1.05 mL, 6.0 mmol). After stirring at RT for 30 min (2S, 4S)-4-

fluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate (1.31 g, 4.5 mmol) and

additional N,N-diisopropylethylamine (0.783 mL, 4.5 mmol) were added. This solution

was allowed to stir at RT for 12 hr and then saturated sodium bicarbonate (1 10 mL) was

added. The mixture was extracted with ethyl acetate (2 X 100 mL) and the organics were washed with saturated NaCl (100 mL), dried over MgS0₄ and concentrated to dryness to

give a crude solid. The solid was chromatographed (silica gel, hexanes/EtOAc 4:1) to

provide a solid which was stirred in a dioxane-HCI (4.0 M, 20 mL) solution for 2 hrs

followed by the addition of diethyl ether (100 mL). The resulting precipitate was

collected by filtration and dried under high vacuum yielding 1.36 g (3.64 mmol, 81 %

yield) of compound A as a white solid.

'H NMR (de-DMSO) 400 MHz δ 8.45-8.41 (s(br), 3H), 7.70 (m, 2H), 7.38-7.36 (m, 2H),

7.29-7.26 (m, 3H), 7.24-7.18 (m, 3H), 5.39 (d, 1 H, J= 51 Hz), 4.89-4.85 (m, 2H), 4.38 (d,

1 H, J = 1 1.3 Hz), 3.98 (ddd, 1 H, J = 38.8, 12.4 Et 3.1 Hz), 3.39-3.33 (m, 1 H), 2.30-2.18 (m,

2H) ppm.

(2S,4S)-1-[(2S)-2-Amino-3,3-bis(4-fluorophenyl)propanoyl]-4-flυoropyrrolidine-2-

carbonitrile hydrochloride

A. 3,3-Bis(4-fluorophenyl)-3-hydroxypropanoie acid.

To an anhydrous THF (80 mL) solution of n-butyl lithium (46 mL of 2.5 M, 115 mmol) at

0 °C was added dropwise diisopropylamine (1 1.13 g, 1 15 mmol) and the solution stirred

for 10 minutes. Keeping the solution at 0 °C, acetic acid (2.64 g, 44 mmol) was added

dropwise and the mixture stirred for 10 min and it was then heated 50 °C. After 30 min a heavy precipitate had formed and the solution was allowed to cool. A solution of 4,4'-

diflurobenzophenone (9.6 g, 0.044 mol) in THF (50 mL, anhydrous) was added at 0 °C,

and the solution stirred at room temperature overnight. Water (100 mL) and diethyl

ether (100 mL) were added and the aqueous layer was separated and acidified with 1 M

HCl to pH 3. The organics were extracted with ethyl acetate (3 X 200 mL) followed by

drying over MgS0₄. Filtration and removal of the solvent in vacuo yielded a crude white

solid that could be washed with cold CHCb to remove trace amounts of the

benzophenone. The solid was dried under high vacuum yielding 5.63 g (20.2 mmol, 46%

yield) of compound A as a white solid.

¹H NMR (de-DMSO) 400 MHz δ 12.4 (s(br), l H), 7.48-7.39 (m, 4H), 7.19-7.02 (m, 4H),

5.91 (s(br), 1 H), 3.25 (s, 2H) ppm.

B. 3,3-Bis(4-fluorophenyl)acrylic acid.

To a 20% solution of sulfuric acid in acetic acid (50 mL, V/V) was compound A (5.6 g,

20.2 mmol) and the mixture stirred for 30 minutes at RT. To this solution was added H2O

(500 mL) and the organics were extracted with ethyl acetate (3 X 150 mL) followed by

drying over MgSU4. Filtration and removal of the solvent in vacuo yielded a white solid.

The solid was dried under high vacuum yielding 4.97g (19.1 mmol, 95% yield) of

compound B as a white solid.

¹H NMR (CDCb) 400 MHz δ 7.27-7.21 (m, 2H), 7.19-7.13 (m, 2H), 7.10-6.95 (m, 4H), 6.26

(s, ! H) ppm. C. 3,3-Bis(4-fluorophenyl)propanoic acid.

To a solution of compound B (2.5 g, 9.61 mmol) in ethyl acetate (250 mL) was added 10%

palladium on carbon (50% w/w) and hydrogenated at 1 atmosphere of hydrogen for 12

hours. The heterogeneous solution was filtered through celite and concentrated in vacuo

to provide a yellow oil. The oil was dried under high vacuum yielding 2.40 g (9.16 mmol,

95 % yield) of compound C as a yellow oil.

'H NMR (de-DMSO) 400 MHz δ 12.08 (brs, 1 H), 7.40-7.30 (m, 4H), 7.15-7.05 (m, 4H), 4.45

(t, 1 H, J = 8.1 Hz), 3.05(d, 2H, J = 8.1 Hz) ppm.

D. (4S,5R)-3-[3,3-Bis(4-fluorophenyl)propanoyl]-4-methyl-5-phenyl-1 ,3-oxazolidin-2- one.

To a THF (50 mL, anhydrous) containing compound C (2.0 g, 7.63 mmol) was added N,N-

diisopropylethylamine (1.18 g, 9.16 mmol) and then the solution cooled to -78 °C. To

this solution was added trimethylacetyl chloride (0.97 g, 8.01 mmol) and the solution

warmed to 0 °C over 1 hour. The cloudy mixture was filtered and the filtrate added

slowly over 10 min to a solution of the lithiated (4S,5R)-(-)-4-methyl-5-phenyl-2-

oxazolidinone at -78 °C, which was prepared by the dropwise addition of n-butyl lithium

(3.0 mL of 2.5 M, 7.63 mmol) to a THF (50 mL) solution of (4S,5R)-(-)-4-methyl-5-

phenyl-2-oxazolidinone (1.35 g, 7.63 mmol) at -78 °C which had stirred for 10 min to provide the lithiated (4S,5R)-(-)-4-methyl-5-phenyl-2-oxazolidinone. The yellow

mixture was warmed to 0 °C and quenched with H2O (50 mL) and extracted with diethyl

ether (3 X 250 mL) followed by drying over MgS0₄. Filtration and removal of the solvent

in vacuo yielded a solid. Flash chromatography (silica gel, 20 % ethyl acetate/ hexanes) provided compound D. The white solid was dried under high vacuum yielding 2.31g

(5.49 mmol, 72% yield) as a white solid.

¹H NMR (de-DMSO) 400 MHz δ 7.40-7.25 (m, 9H), 7.18-7.02 (m, 4H), 5.76 (d, 1 H, J = 7.6

Hz), 4.65 (m, 1 H), 4.58 (t, 1 H, J = 7.6 Hz), 3.72 (dd, 1 H, J = 16.8, 7.0 Hz) 3.57 (dd, 1 H, J =

16.8, 7.0 Hz), 0.58 (d, 3H, J = 6.7 Hz) ppm.

E. (4S,5R)-3-[(2S)-2-Azido-3,3- bis(4-fluorophenyl) propanoyl]-4-methyl-5-[(l E,3Z)-1-

methylhexa-1 ,3,5-trienyl]-1 ,3-oxazolidin-2-one.

To a THF (50 mL, anhydrous) solution containing compound D (2.0 g, 4.75 mmol) at -78

°C was added dropwise potassium bis(trimethylsilyl)amide (10.0 mL of 0.5 M toluene

solution, 4.98 mmol). After stirring for 10 min 2,4,6-triisopropylbenzenesulfonyl azide

(trisyl azide) (1.84 g, 5.94 mmol) in THF (10 mL, anhydrous) was added in one portion.

After 3 minutes acetic acid was added (1.31 g, 21.8 mmol) at -78 °C and then the reaction quickly warmed to 30 °C and stirred for 1 hr at that temperature generating a

light yellow solution. To this solution was added H2O (100 mL) and the organics were

extracted with ethyl acetate (500 mL). After washing with sat. NaHCO₃ (l00 mL) and drying over MgS0₄the solvent was reomved in vacuo yielding a yellow oil. Column

chromatography (ethyl acetate/hexanes 1 :9) provided compound E as a white solid.

HPLC showed a single diastereoisomer. The white solid was dried under high vacuum

yielding 1.7 Ig (3.70 mmol, 78% yield) as a white solid.

'H NMR (CDCb) 400 MHz δ 7.42-7.35 (m, H), 7.25-7.18 (m, H), 7.10-7.06 (m, 2H), 7.05-

6.92 (m, 2H), 5.95 (d, 1 H, J = 10.8 Hz), 5.05 (d, 1 H, J = 7.1 Hz), 4.60 (d, 1 H, J = 10.8 Hz),

4.38 (m, 1 H), 0.95 (d, 3H, J = 6.8 Hz) ppm. F. (2S)-2-Azido-3,3-bis(4-fluorophenyl)propanoic acid.

To a THF/H2O (4:1, 50 mL) solution of compound E (1.5 g, 3.25 mmol) at 0 °C was added a

solution of lithium hydroxide (0.272 g, 6.49 mmol) in hydrogen peroxide (1.50 mL of

30% soln in H20, 48.75 mmol). The mixture was stirred at 0 °C for 1 hr and then

quenched with Na2Sθ4 (6.3 g, 50 mL of 1.0 M solution in H2O). The THF was removed in

vacuo and the solution acidified to pH 1 with 6.0 M HCl at O^'C. The organics were

extracted with ethyl acetate (2 X 200 mL) followed by drying over MgS04. Filtration and

removal of the solvent in vacuo yielded a clear oil. Column chromatography

(EtOAc/hexanes/acetic acid 50:50:1) provided compound F as a white solid. The solid

was dried under high vacuum yielding 0.78 g (2.60 mmol, 80% yield) as a white solid.

'H NMR (CDCb) 400 MHz δ 9.60(s(br), 1 H), 7.25-7.10 (m, 4H), 7.10-6.95 (m, 4H), 4.50 (d,

2H, _/ = 8.6 Hz) ppm.

G. (2S)-2-Amino-3,3-bis(4-fluorophenyl)propanoic acid.

To an ethyl acetate (250 mL) solution of compound F (1.5 g, 4.95 mmol) was added 10% palladium on carbon (10% w/w) and hydrogenated at 1 atmosphere of hydrogen for 12

hr. The heterogeneous solution was filtered through celite (1 g) and the filtrate

concentrated in vacuo to provide a clear oil. The oil was dried under high vacuum

yielding 1.30 g (4.70 mmol, 95% yield) of compound G as a white solid.

'H NMR (de-DMSO) 400 MHz δ 10.2(s(br), 1 H), 7.38-7.27(m, 4H), 7.08-6.98 (m, 4H), 4.25

(d, 1 H, J = 8.3 Hz), 3.95 (d, 1 H, J = 8.3 Hz) ppm. H. (2S)-2-[(tert-Butoxycarbonyl)amino]-3,3-bis(4-fluorophenyl)propanoic acid.

To a CH2CI2 (150 mL) solution containing compound G (1.30 g, 4.69 mmol) was added

triethylamine (2.37 g, 23.4 mmol) and di-tert-butyl dicarbonate (1.23 g, 5.63 mmol).

After stirring for 12 hr H2O (50 mL) and CH2CI2 (300 mL) were added and the solution

acidified to pH 3 with 1.0 M HCl. Separation of the ethyl acetate layer followed by

drying over MgSθ4 and removal of the solvent in vacuo yielded a clear oil. The oil was

dried under high vacuum yielding 1.68 g (4.4 mmol, 95% yield) of compound H as a

white solid.

H NMR (de-DMSO) 400 MHz δ 12.4 (s(br), 1 H), 7.35-7.22 (m, 4H), 7.15-6.95 (m, 4H),

4.78 (t, 1 H, J = 8.9 HZ), 4.25 (d, 1 H, J = 8.9 Hz), 3.05 (m, 1 H), 1.20 (s, 3H), 1.15 (s, 6H) ppm.

I. (2S,4S)-1-[(2S)-2-(tert-Butoxycarbonyl)amino-3,3-bis(4-fluorophenyl)propanoyl]-4-

fluoropyrrolidine-2-carbonitrile.

To a DMF solution (25 mL, anhydrous) was compound H (1.0 g, 2.65 mmol) and HATU

(1.0 g, 2.65 mmol). To this solution was added N,N-diisopropylethylamine (0.462 mL,

2.65 mmol) and after 30 min (2S, 4S)-4-fluoro-2-pyrrolidinecarbonitrile 4-

methylbenzenesulfonate (0.619 g, 2.12 mmol) and additional N,N-diisopropylethylamine

(0.37 mL, 2.12 mmol) were added. This solution was allowed to stir at RT for 12 hr and

then saturated sodium bicarbonate (100 mL) was added. The resulting gummy mixture

was extracted with ethyl acetate (3 X 100 mL) and the organics were washed with

saturated NaCl (50 mL) followed by drying over MgS0₄. Filtration and removal of the

solvent in vacuo yielded a clear oil. The oil was chromatographed on silica gel (hexanes/EtOAc 4:1) to provide a white solid. The solid was dried under high vacuum

yielding 815 mg (1.72 mmol, 65% yield) of compound I as a white solid.

¹H NMR (CDCb) 400 MHz δ 7.38-7.32 (m, 2H), 7.21-7.15 (m, 2H), 7.12-6.98(m, 4H), 5.15

(d, 1 H, J= 51 Hz), 5.03 (d, 1 H, J = 8.9 Hz, 4.89 (d, 1 H, J = 1 1.2 Hz), 4.86 (d, 1 H, I = 8.9 Hz),

4.40 (d, 1 H, J = 1 1.2 Hz), 3.83 (ddd, 1 H, J = 36.8, 12.1 , 3.7 Hz), 3.05 (d, 1 H, J = 12.2 Hz),

2.62 (t, 1 H, I = 15.3 Hz), 2.25 (m, 1 H), 1.38 (s, 9H) ppm.

J. (2S,4S)-1-[(2S)-2-Amino-3,3-bis(4-fluorophenyl)propanoyl]-4-fluoropyrrolidine-2-

carbonitrile hydrochloride. To compound I (0.5 g, 1.05 mmol) was added 4.0 N HCl in 1 ,4-dioxane (10 L, 40 mmol)

and after 3 hr diethyl ether (100 mL) was added. The resulting precipitate was collected

by filtration and after drying under high vacuum 0.41 g (I .Ommol, 95% yield) of

compound A was obtained as a white solid.

¹H NMR (de-DMSO) 400 MHz δ 8.42 (s(br), 3H), 7.72-7.66 (m, 2H), 7.38-7.32 (m, 2H),

7.25-7.19 (m, 2H), 7.06-7.0 (m, 2H), 5.38 (d, 1 H, = 51 Hz), 4.91 (d, 2H, J = 8.8 Hz), 4.82

(d, 1 H, J = 1 1.3 Hz), 4.41 (d, 1 H, J = 1 1.3 Hz), 3.86 (ddd, 1 H, J = 39.2, 12.4, 3.1 Hz), 3.45

(q, 1 H, J = 12.4 Hz), 2.38-2.20 (m, 2H) ppm.

(2S,4S)-1 -[(2S)-2-Amino-5-(4-fluorophenyl)-3,3-dimethylpentanoyl]-4-fluoro-2-

pyrrolidinecarbonitrile hydrochloride

A. 4-(4-Fluorophenyl)-2,2-dimethylbutanal

To a flask containing 4-(4-fluorophenyl)-2,2-dimethylbutanenitrile (see: Org. Lett. 2000,

2, 3285; Knochel, P. et al. for preparation of this compound) (13.4 g, 70.2 mmol) in 300

mL of toluene cooled to -78 °C was added 70.2 mL of a 1.5 M toluene solution of DIBAL

(105.3 mmol). After 2.5 hr a H2O/THF (250 mL/50 mL) solution containing 17.9 g of

sodium acetate and 17.9 mL of acetic acid was gradually added. Upon complete addition

celite and Et2θ were added and the flask was allowed to warm to RT. After stirring for

1 hr the heterogeneous solution was filtered through a bed of celite. The celite was

rinsed with Et2θ and then the filtrate was washed with H2O. After drying over MgS0₄ the

solvent was removed in vacuo and the residual oil was purified via column

chromatography (5% EtOAc/hexanes) yielding 1 1.3 g (58.2 mmol, 83% yield) of

compound A.

'H NMR (CDCb) 400 MHz δ 9.47 (s, IH), 7.12 - 7.10 (m, 2H), 7.09 - 6.93 (m, 2H), 2.51 -

2.47 (m, 2H), 1.77 - 1.72 (m, 2H), 1.12 (s, 6H) ppm.

B. 5-(4-Fluorophenyl)-3,3-dimethyl-2-({(1 R)-1 -phenyl-2-[(trimethylsilyl)oxy]ethyl}

amino)pentanenitrile

To a CH2CI2 solution (250 mL) containing compound A (9.07 g, 46.8 mmol) was added

(R)-phenylglycinol (6.41 g, 46.8 mmol) at RT. The resulting solution stirred for 1.5 hr at

which time it was cooled to 0 °C and trimethylsilyl cyanide (9.28 g, 93.5 mmol) was

added. The yellow solution was slowly allowed to warm to RT overnight. It was then

quenched with sat. NaHSU4 and the organic layer separated. The organics were washed with H2O dried (MgS0₄) and decolorized with charcoal. After filtering through celite the

yellow oil was purified via column chromatography (hexanes/EtOAc (9:1)) yielding 1 1.9 g

(28.9 mmol, 62% yield) of compound B as 6:1 mixture of diastereomers.

Major diastereomer: ¹H NMR (CDCb) 400 MHz δ 7.38 - 7.29 (m, 5H), 7.1 1 - 7.07 (m, 2H),

6.98 - 6.93 (m, 2H), 4.03 (dd, 1 H, J = 9.95, 3.8 Hz), 3.68 (m, 1 H), 3.52 (t, 1 H, J = 10.2 Hz),

3.06 (d, I H), J = 13.4 Hz), 2.44 - 2.34 (m, 3H), 1.73 - 1.65 (m, 2H), 1.1 1 (s, 3H), 1.09 (s, 3H), 0.14 (s, 9H) ppm.

C. 5-(4-Fluorophenyl)-2-{[(l R)-2-hydroxy-1-phenylethyl]amino}-3,3-

dimethylpentanenitrile.

To a MeOH solution (300 mL) containing compound B (1 1.85 g, 28.8 mmol) was added

potassium fluoride (16.7 g, 287.6 mmol) at RT. The solution stirred for 1.5 hr at which

time the bulk of the MeOH was removed in vacuo. CH2CI2 and H2O were added and the

organic layer separated. After drying over MgS0₄ the solvent was removed in vacuo and

the resulting oil purified via column chromatography (hexanes/EtOAc (4:1)) yielding 8.86

g (26.1 mmol, 90% yield) of compound C as a 6:1 mixture of diastereomers.

Major diastereomer: 'H NMR (CDCb) 400 MHz δ 7.38 - 7.30 (m, 5H), 7.10 - 7.06 (m, 2H),

6.97 - 6.92 (m, 2H), 4.06 (dd, 1 H, J = 9.5, 4.0 Hz), 3.79 (m, 1 H), 3.58 (m, 1 H), 3.07 (d, 1 H, J

= 12.5 Hz), 2.43 - 2.30 (m, 3H), 1.95 (dd, 1 H, J = 7.8, 4.1 Hz), 1.74 - 1.64 (m, 2H), 1.11 (s,

3H), 1.09 (s, 3H) ppm.

D. 5-(4-Fluorophenyl)-3,3-dimethyl-2-[[(E)-phenylmethylidene]amino}pentanenitrile.

To a CH2CI2 (10 mL)/MeOH (4 mL) solution containing compound C (494 mg, 1.45 mmol)

cooled to 0 °C was added lead tetraacetate (838 mg, 1.89 mmol). The resulting solution stirred overnight at which time it had warmed to ambient temperature. To this solution

was added 20 mL of a pH 7.2 phosphate buffer. After 30 min the cloudy solution was

filtered through a bed of celite with the celite being rinsed thoroughly with CH2CI2. The

filtrate had H2O added to it and the organic layer was separated. The aqueous layer was

extracted with CH2CI2 and then the combined organic layers were dried^' (MgSθ4). The

solvent was removed in vacuo and the oil purified via column chromatography (hexanes/EtOAc (9:1)) yielding 270 mg (0.88 mmol, 60% yield) of compound D.

¹H NMR (CDCb) 400 MHz δ 8.50 (d, I H. J = 1.4 Hz), 7.81 - 7.80 (d, 2H, J = 6.6 Hz), 7.54 -

7.43 (m, 3H), 7.17 - 7.13 (m, 3H), 6.99 - 6.94 (m, 2H), 4.48 (s, 1 H), 2.68 - 2.64 (m, 2H),

1.81 - 1.74 (m, 2H), 1.19 (s, 3H), 1.15 (s, 3H) ppm.

E. 5-(4-Fluorophenyl)-3,3-dimethylnorvailine hydrochloride.

To a flask containing compound D (268 mg, 0.87 mmol) was added 7 mL of concentrated

HCl. The solution was heated to reflux for 18 hr at which time it was cooled and

extracted with Et₂0. The aqueous layer had the solvent removed in vacuo and the

residual oily solid was dissolved in Et2θ/MeOH and was decolorized with charcoal. After

filtration and removal of the solvent in vacuo the residue was triterated with Et₂0 and

the solvent removed in vacuo yielding 180 mg (0.65 mmol, 75% yield) of compound E as

an oily solid which was taken on crude.

¹H NMR (d₄-Me0H) 400 MHz δ 7.25 - 7.20 (m, 2H), 7.01 - 6.95 (m, 2H), 4.45 (s(br), 1 H),

2.67 - 2.63 (m, 2H), 1.76 - 1.72 (m, 2H), 1.22 - 1.13 (m, 6H).

F. N. Tert-butoxycarbonyl)-5-(4-fluorophenyl)-3,3-dimethylnorvaline. To a dioxane solution (5 mL) containing compound E (180 mg, 0.65 mmol) was added 2.5

mL of a 2 M NaOH solution at RT. The resulting solution then had di-t-butyldicarbonate

(284 mg, 1.34 mmol) added to it. After stirring overnight the solution was acidified with

1.0 M HCl. The organics were extracted with EtOAc (2X), dried (MgSθ4) and the solvent

removed in vacuo. The residual oil was purified via column chroma-tography (5%

MeOH/CH∑Cb) yielding 76 mg (0.22 mmol, 34% yield) of compound F.

Η NMR (d₄-MeOH) 400 MHz δ 7.18 - 7.15 (m, 2H), 6.96 - 6.92 (m, 2H), 6.62 (d, 1 H, J =

9.3 Hz), 4.18 (d, 1 H, J = 9.5 Hz), 2.65 - 2.58 (m, 2H), 1.62 - 1.53 (m, 2H), 1.44 (s, 9H), 1.05

(s, 3H), 1.01 (s, 3H) ppm.

G. Tert-butyl (lS)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidinyl]carbonyl}-4-(4-

fluorophenyl)-2,2-dimethylbutylcarbamate.

To a DMF solution (4 mL) containing compound F (172 mg, 0.51 mmol) was added

diisopropylethyl amine (98 mg, 0.76 mmol) followed by HATU (213 mg, 0.56 mmol) at RT.

After 15 min (2S,4S)-4-fluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate (153

mg, 0.53 mmol) and diisopropylethyl amine (68 mg, 0.53 mmol) in 2 mL of DMF was

added. The reaction stirred overnight at which time it was poured into EtOAc. The

organics were washed with H2O (2X), sat. NaHC0₃ and sat. NaHS0₄. After drying over

MgSU4 the solvent was removed in vacuo and the residual yellow oil purified via column

chromatography (2% MeOH/CH2Cb) yielding 147 mg of compound G which underwent

further purification via semi-prep HPLC yielding 80 mg of compound G.

'H NMR (CDCb) 400 MHz δ 7.15 - 7.12 (m, 2H), 6.96 - 6.92 (m, 2H), 5.40 (d(br), 1 H, J =

52.0 Hz), 5.18 (d, 1 H, J = 9.7 Hz), 5.04 (d, 1 H, J = 9.3 Hz), 4.31 (d, 1 H, J = 9.9 Hz), 4.1 1 (s, 1 H), 4.03 (m, 1 H), 2.70 - 2.52 (m, 3H), 2.33 (m, 1 H), 1.64 - 1.60 (m, 2H), 1.41 (s, 9H), 1.11 (s, 3H), 1.09 (s, 3 H) ppm.

H. (2S,4S)-1-[(2S)-2-Amino-5-(4-fluorophenyl)-3,3-dimethylpentanoyl]-4-fluoro-2-

pyrrolidinecarbonitrile hydrochloride.

To a CH2CI2 solution (4 mL) containing compound G (80 mg, 0.18 mmol) was added 71 μL

of TFA (5 eq). After 30 min another 5 eq of TFA was added followed by an additional 10

eq after an hour. Upon stirring for a total of 3.5 hr the solvent was removed in vacuo

and the residue dissolved in EtOAc. The organics were washed with sat. NaHC0₃, dried

(MgS04) and the solvent removed in vacuo. The residual oil had 1.5 mL of dioxane added

to it followed by 2.0 mL of a 4.0 M dioxane solution of HCl. The solvent was removed in vacuo and Et∑O added. The precipitated solid was collected via vacuum filtration yielding

40 mg (0.1 1 mmol, 60% yield) of compound H.

¹H NMR (d₄-MeOH) 400 MHz δ 7.25 - 7.22 (m, 2H), 7.00 - 6.95 (m, 2H), 5.46 (d(br), 1 H, J

= 51.1 Hz), 5.09 (d, 1 H, J = 9.4 Hz), 4.12 (m, 1 H), 4.03 (s, 1 H), 3.90 (ddd, 1 H, J = 37.6,

12.7, 3.0 Hz), 2.68 - 2.44 (m, 4H), 1.78 - 1.73 (m, 2H), 1.21 (s, 6H) ppm.

Example 4

(2S,4R)-1-[(2S)-2-Amino-4-(4-fluorophenyl)-3,3-dimethylbutanoyl]-4-

fluoropyrrrolidine-2-carbonitrile hydrochloride A. 3-(4-Fluorophenyl)-2,2-dimethylpropanenitrile.

To a toluene solution (60 mL) containing isobutyronitrile (5.0 g, 72.3 mmol) cooled to 0

°C was added 152 mL of a 0.5 M toluene solution of KHMDS (76.0 mmol). After 15 min

4-fluorobenzylbromide (14.4 g, 76.0 mmol) was added. The solution slowly warmed to

ambient temperature overnight at which time it was quenched with H2O. The organics

were extracted with EtOAc and washed with sat. NaHSU4. After drying over MgSU4 the

solvent was removed in vacuo and the residual yellow oil purified via column

chromatography (5%EtOAc/hexanes) yielding 10.05 g (56.8 mmol, 79% yield) of compound A.

Η NMR (CDCb) 400 MHz δ 7.25 - 7.22 (m, 2H), 7.05 - 7.00 (m, 2H), 2.78 (s, 2H), 1.34 (s,

6H) ppm.

B. 3-(4-Fluorophenyl)-2,2-dimethylpropanol.

To a toluene solution (100 mL) containing compound A (10.0 g, 56.5 mmol) cooled to -

78 °C was added 56.5 mL of a 1.5 M toluene solution of DIBAL (84.7 mmol). After 2.5 hr

a H2O/THF solution (160 mL/40 mL) containing sodium acetate (14.3 g) and acetic acid

(14.3 mL) was added followed by celite. The solution stirred for 1 hr and then Et∑O was

added. The solution was filtered through celite with the celite being rinsed thoroughly

with Et2θ. The filtrate was washed with H2O (2X) and then with sat. NaHC0₃. After drying

over MgSθ4 the solvent was removed in vacuo and the residual oil purified via column

chromatography (5%EtOAc/hexanes) yielding 9.23 g (51.3 mmol, 91% yield) of

compound B.

¹H NMR (CDCb) 400 MHz δ 9.56 (s, 1 H), 7.06 - 7.03 (m, 2H), 6.97 - 6.93 (m, 2H), 2.75 (s,

2H), 1.04 (s, 6H) ppm. C 2-Amino-4-(4-Fluorophenyl)-3,3-dimethylbutanenitrile.

To a MeOH solution (32 mL) containing compound B (5.0 g, 27.8 mmol) was added 3.8

mL of 30% NH40H, potassium cyanide (1.9 g, 29.2 mmol) and 15 mL of H2O. To this

solution was added ammonium chloride (1.64 g, 30.6 mmol) at RT. The resulting solution

stirred overnight at which time it was heated to 70 °C. After 5 hr the solution was cooled, diluted with EtOAc and then washed with H2O (2X) followed by sat. NaHC0₃.

After drying over MgS0₄ the solvent was removed in vacuo and the remaining yellow oil

purified via column chromatography (hexanes/EtOAc (1 :1)) yielding 3.69 g (17.9 mmol,

64% yield) of compound C.

'H NMR (CDCb) 400 MHz δ 7.17 - 7.13 (m, 2H), 7.00 - 6.95 (m, 2H), 3.33 (t, 1 H, J = 8.5

Hz), 2.80 (d, 1 H, J = 13.4 Hz), 2.61 (d, 1 H, J = 13.4 Hz), 1.58 - 1.57 (m, 2H), 1.03 (s, 3H),

1.02 (s, 3H) ppm.

D. 2-Amino-4-(4-Fluorophenyl)-3,3-dimethylbutanoic acid.

To a flask containing compound C (3.69 g, 17.9 mmol) was added 80 mL of concentrated

HCl. The solution was then heated to reflux for 24 hr. The heterogeneous solution was

then cooled in an ice bath and the white solid collected via vacuum filtration. After

pumping on under high vacuum 4.06 g of a white powder was obtained. This material

was taken on crude.

E. 2-[(Tert-butoxycarbonyl)amino]-4-(4-fluorophenyl)-3,3-dimethylbutanoic acid.

To a dioxane solution (20 mL) containing compound D (2.0 g, 7.66 mmol) was added 5

mL of H2O and 9.6 mL of a 2.0 M aqueous solution of NaOH. To this solution was added di-t-butyl dicarbonate (2.34 g, 10.7 mmol) at RT. After stirring overnight the

heterogeneous solution was poured into sat. NaHC0₃ and washed with Et2θ. The Et2θ

layer was washed with sat. NaHC0₃ and then the combined aqueous layers were acidified

with 1.0 M HCl and extracted with EtOAc (2X). After drying over MgSθ4 the solvent was

removed in vacuo yielding 928 mg (2.86 mmol, 37% yield) of compound E.

Η NMR (d₄-MeOH) 400 MHz δ 7.21 - 7.18 (m, 2H), 7.00 - 6.95 (m, 2H) 6.74 (d, 1 H, J =

9.0 Hz), 3.98 (d, 1 H, J = 9.0 Hz), 2.68 (d, 1 H, J = 13.4 Hz), 2.62 (d, 1 H, J = 13.3 Hz), 1.45

(s(br), 9H), 0.93 (s, 3H), 0.92 (s, 3H) ppm.

F. Tert-butyl (lS)-1 -{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1 -yl]carbonyl}-3-(4-

fluorophenyl)-2,2-dimethylpropyl carbamate.

To a DMF solution (20 mL) containing compound E (928 mg, 2.86 mmol) was added

diisopropylethylamine (406 mg, 3.14 mmol) followed by HATU (1.14 g, 3.00 mmol) at RT.

After 30 min (2S,4S)-4-fiuoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate (857

mg, 3.00 mmol) and diisopropylethylamine (388 mg, 3.00 mmol) in 13 mL DMF was

added. After stirring overnight the solution was poured into EtOAc and washed with H2O

(2X), sat. NaHC0₃ and 1.0 M HCl. After drying over MgSθ4 the solvent was removed in

vacuo. The residual oil was purified via column chromatography (hexanes/EtOAc/CH∑C

(5:4:1)) yielding 273 mg (0.65 mmol, low Rf material) of the (S)-diastereromer as

determined by vibrational circular dichroism and 226 mg of a mixture of the (R)-

diastereomer plus an unknown impurity. The (S)-diastereomer was taken on.

'H NMR (CDCb) 400 MHz δ 7.15 - 7.1 1 (m, 2H), 7.01 - 6.96 (m, 2H), 5.32 (dt, 1 H, J =

51.3, 3.3 Hz), 5.20 (d, 1 H, J = 9.7 Hz), 5.01 (d, 1 H, J = 9.3 Hz), 4.16 (d, 1 H, J = 10.1 Hz), 3.91 (m, 1 H), 3.68 (dd, 1 H, J = 23.6, 12.1 Hz), 2.72 - 2.56 (m, 2H), 2.30 (m, 1 H), 1.43 (s,

9H), 1.08 (s, 3H), 0.95 (s, 3H) ppm.

G. (2S,4R)-1 -[(2S)-2-Amino-4-(4-fluorophenyl)-3,3-dimethylbutanoyl]-4-

fluoropyrrrolidine-2-carbonitrile hydrochloride.

To a CH2CI2 solution (7 mL) containing compound F (242 mg, 0.58 mmol) was added 443

μL (10 eq) of TFA at RT. After 30 min and additional 10 eq of TFA was added and after a

total of 3 hr the reaction mixture was poured into sat. NaHC0₃. The organics were

extracted with EtOAc, dried (MgSU4) and the solvent removed in vacuo. The residual oil

was dissolved in dioxane (4 mL) and then 4.0 L of a 4.0 M HCl solution in dioxane was

added. The solvent was removed in vacuo and the residue triturated with Et∑O. The resulting solid was collected via vacuum filtration affording 175 mg (0.46 mmol, 79%

yield) of salt G.

¹H NMR (d₄-MeOH) 400 MHz δ 7.22 - 7.19 (m, 2H), 7.08 - 7.04 (m, 2H), 5.43 (d(br), 1 H, J

= 51.1 Hz), 5.09 (d, 1 H, J = 9.5 Hz), 4.05 (s, 1 H), 3.97 - 3.74 ( , 2H), 2.82 (d, 1H, J = 13.4

Hz), 2.72 (d, I H, J = 13.2 Hz), 2.64 - 2.39 (m, 2H), 1.07 (s, 3H), 1.00 (s, 3H) ppm.

Example 5

(2S,4S)-1-{(2R)-2-Amino-3-[(4-methoxybenzyl)thio]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride A. Tert-butyl (l R)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]carbonyl}-2-[(4-

methoxybenzyl)thio]-2-methylpropylcarbamate.

To a DMF solution (35 mL) containing (2R)-2-[(tert-butoxycarbonyl)amino]-3-[(4-

methoxybenzyl)thio]-3-methylbutanoic acid (2.0 g, 5.41 mmol, 1.2 eq) was added N,N-

diisopropylethylamine (1.06 mL, 6.09 mmol, 1.35 eq) followed by HATU (2.9 g, 7.67

mmol, 1.7 eq). The resulting amber solution was stirred at RT for 20 min. To this stirring

solution was added a solution of (2S, 4S)-4-fluoro-2-pyrrolidinecarbonitrile 4-

methylbenzenesulfonate (1.29 g, 4.51 mmol, 1.0 eq) and N,N-diisopropylethylamine (864

μL, 4.96 mmol, 1.1 eq) in DMF (12 mL). The resulting solution was stirred at RT for 18 hr

and was then quenched with a sat. NaHC0₃ solution (ca 50 mL). The cloudy solution was

then poured into H2O (ca 100 mL) and extracted with EtOAc (3 x 50 mL). The combined

organic extracts were washed with H2O (2X) and brine (1X); dried over MgSU4 and concentrated in vacuo. The resulting amber oil was purified via flash chromatography

using 1 :1 hexanes/EtOAc as the mobile phase. Isolated 1.96 g (4.23 mmol, 78% yield) of

compound A as a white foam.

'H NMR (CDCb) 400 MHz δ 7.29 (d, 2H, J = 8.8 Hz), 6.83 (d, 2H, J = 8.6 Hz), 5.48 (d, 1 H, J

= 51.4 Hz), 5.43 (d, 1 H, J = 9.2 Hz), 5.04 (d, 1 H, J = 9.3 Hz), 4.38 (d, 1 H, J = 8.9 Hz), 4.27

(m, 1 H), 4.04 (m, 1 H), 3.80 (s, 2H), 3.77 (s, 3H), 2.72 (t, 1 H, 15.5 Hz), 2.37 (m, 1 H), 1.47-

1.41 (m, 14H) ppm.

B. (2S,4S)-1-{(2R)-2-Amino-3-[(4-methoxybenzyl)thio]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride

To a CH2CI2 (60 mL) solution containing trifluoroacetic acid (8 mL) was added compound

A (1.2 g, 2.58 mmol, 1.0 eq). The resulting pale yellow solution was stirred at RT for 2 hr at which time the solvent was removed in vacuo. The resulting TFA salt was converted

to the free base by addition of a sat. NaHC0₃ solution and extracting the aqueous layer

(2X) with EtOAc. The combined organic extracts were dried over Na2S0₄ and

concentrated in vacuo. The resulting off-white foam was purified via flash chromatography using 5% MeOH (with 0.1% NH₃) in CH2CI2 as the mobile phase

affording 897 mg (2.45 mmol, 95% yield) of a white foam. To form the HCl salt, the free

base was taken up in Et₂0 and acetone added until all solids were in solution. 2.0 M HCl

in ether was added dropwise until no more precipitate formed. The precipitate was then

filtered and washed several times with Et2θ. The resulting salt was dried under high

vacuum.

¹H NMR (D2O) 400 MHz δ 7.31 (d, 2H, J = 8.6 Hz), 6.88 (d, 2H, J = 8.5 Hz), 5.49 (d, 1 H, J =

50.5 Hz), 5.00 (d, 1 H, J = 9.8 Hz), 4.02-3.52 (m, 8H), 2.65 (t, 1 H, J = 15.7 Hz), 2.40 (m, 1 H),

1.44 (s, 3H), 1.31 (s, 3H) ppm.

Example 6

(2S,4S)-1-{(2R)-2-Amino-3-methyl-3-[(3-phenylpropyl)thio]butanoyl)-4-

fluoropyrrolidine-2-carbonitrile hydrochloride A. Methyl (2R)-2-amino-3-mercapto-3-methylbutanoate

To a methanol (18 mL, 1.14 M) solution containing L-penicillamine (3.0 g, 20.11 mmol,

1.0 eq) was added thionyl chloride (1.5 mL, 20.7 mmol, 1.03 eq). The resulting mixture

was heated to reflux for 18 hr and the solvent removed in vacuo. The resulting clear semi-solid was used in the next step without further purification. Isolated 3.28 g (20.1

mmol, 100% yield).

B. Methyl (2R)-2-[(tert-butoxycarbonyl)amino]-3-mercapto-3-methylbutanoate

To a CH2CI2 (60 mL) containing compound A (3.28 g, 20.1 mmol, 1.0 eq) was added di-

tert-butyldicarbonate (4.48 g, 20.5 mmol, 1.02 eq) and triethylamine (7.0 mL, 50.3 mmol,

2.5 eq). The resulting mixture was allowed to stir at RT for 18 hr. The solvent was

removed in vacuo and the residue taken up it Et2θ. The insoluble white solid was filtered

and the filtrate washed with brine, dried over MgS0₄, and concentrated in vacuo.

Resulting white solid was used in next step without further purification. Isolated 5.01 g

(19.02 mmol, 95% yield).

¹H NMR (d₄-MeOH) 400 MHz δ 4.26 (m, 1 H), 3.72 (s, 3H), 1.44-1.42 (m, 15H) ppm.

C. Methyl (2R)-2-[(tert-butoxycarbonyl)amino]-3-methyl-3-[(3-

phenylpropyl)thio]butanoate

To a CHsCN (20 mL) solution containing compound B (346 mg, 1.31 mmol, 1.0 eq) was

added sodium tert-butoxide (138 mg, 1.44 mmol, 1.1 eq). The resulting pale yellow

mixture was allowed to stir at RT for 10 min at which time 1-bromo-3-phenylpropane

(219 μL, 1.44 mmol, 1.1 eq) was added. The resulting solution was allowed to stir at RT

for 18 hr. The reaction was then quenched with a sat NH4CI solution, poured into H2O,

and extracted (2X) with EtOAc. The combined extracts were dried over MgS0₄ and

concentrated in vacuo. Purified via flash chromatography using 2:1 Hexane/EtOAc as

the mobile phase. Isolated 376 mg (0.988 mmol, 76% yield). Η NMR (CDCb) 400 MHz δ 7.30-7.16 (m, 5H), 5.38 (d, 1 H, J = 8.8 Hz), 4.29 (d, 1 H, J = 9.0

Hz), 3.71 (s, 3H), 2.72 (t, 2H, J = 7.4 Hz), 2.58-2.51 (m, 2H), 1.89-1.82 (m, 2H), 1.44 (s, 9H),

1.36 (d, 6H, J = 8.8 Hz) ppm.

D. (2R)-2-[(tert-Butoxycarbonyl)amino]-3-methyl-3-[(3-phenylpropyl)thio]butanoic

acid.

To a dioxane (7.5 mL) / H2O (2.5 mL) solution containing compound C (376 mg, 0.988

mmol, 1.0 eq) was added lithium hydroxide monohydrate (207 mg, 4.94 mmol, 5.0 eq).

The resulting heterogeneous mixture was allowed to stir at RT for 18 hr. The reaction

was then quenched with a sat. NH4CI solution, poured into H2O, and extracted (2X) with

EtOAc. The combined extracts were dried over MgS0₄ and concentrated in vacuo.

Purified via flash chromatography using 1 :1 hexanes/EtOAc (with 0.1% AcOH) as the

mobile phase. Isolated 230 mg (0.626 mmol, 64% yield).

Η NMR (CDCb) 400 MHz δ 7.31-7.16 (m, 5H), 5.51 (d, 1 H, J = 9.5 Hz), 4.33 (d, 1 H, J = 9.5

Hz), 3.41 (t, 2H, .7 = 6.8 Hz), 2.79 (t, 2H, - = 7.3 Hz), 2.20-2.13 (m, 2H), 1.47-1.44 (m, 15H)

ppm.

E. Tert-butyl (l R)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]carbonyl}-2-methyl-2-

[(3-phenylpropyl)thio]propylcarbamate. To a DMF solution (5 mL) containing compound D (230 mg, 0.626 mmol, 1.2 eq)

was added N,N-diisopropylethylamine (123 μL, 0.705 mmol, 1.35 eq) followed by HATU

(337 mg, 0.887 mmol, 1.7 eq). The resulting amber solution was stirred at RT for 20 min.

To this stirring solution was added a solution of (2S, 4S)-4-fluoro-2-

pyrrolidinecarbonitrile 4-methylbenzenesulfonate (149 mg, 0.522 mmol, 1.0 eq) and N,N-diisopropylethylamine (100 μL, 0.574 mmol, 1.1 eq) in DMF (2 mL). The resulting

solution was stirred at RT for 18 hr and quenched with a sat. NaHC0₃ solution (ca 5 mL).

The cloudy solution was then poured into H2O (ca 20 mL) and extracted with EtOAc (3 X

10 mL). The combined organic extracts were washed with H2O (2X) and brine (1X); dried

over MgS0₄ and concentrated in vacuo. The resulting amber oil was purified via flash

chromatography using 1 :1 hexanes/EtOAc as the mobile phase. Isolated 190 mg (0.411 mmol, 79% yield) of compound E as a white foam.

Η NMR (CDCb) 400 MHz δ 7.28-7.17 (m, 5H), 5.46 (d, 1 H, J = 51.4 Hz), 5.37 (d, 1 H, J =

9.0 Hz), 5.00 (d, 1 H, J = 9.3 Hz), 4.38-4.29 (m, 2H), 4.01 (m, 1 H), 2.77-2.58 (m, 5H), 2.35

(m, 1 H), 1.92-1.80 (m, 2H), 1.44-1.34 (m, 15H) ppm.

F. (2S,4S)-1-{(2R)-2-Amino-3-methyl-3-[(3-phenylpropyl)thio]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile

To a CH2CI2 (10 mL) solution containing trifluoroacetic acid (1 mL) was added compound

E (190 mg, 0.41 1 mmol, 1.0 eq). The resulting pale yellow solution was stirred at RT for 2

hours at which time the solvent was removed in vacuo. The resulting TFA salt was

converted to the free base by addition of a sat. NaHC0₃ solution and extracting the

aqueous layer (2X) with EtOAc. The combined organic extracts were dried over Na2SU

and concentrated in vacuo. The resulting off-white foam was purified via flash

chromatography using 5% MeOH (with 0.1% NH₃) in CH2CI2 as the mobile phase

affording 134 mg (0.389 mmol, 94% yield) of a white foam. To form the HCl salt, the

free base was taken up in Et∑O and acetone added until all solids were in solution. 2.0 M

HCl in ether was added dropwise until no more precipitate formed. The precipitate was then filtered and washed several times with Et2θ. The resulting salt was dried under

high vacuum.

'H NMR (D2O) 400 MHz δ 7.23-7.1 1 (m, 5H), 5.46 (d, 1 H, J = 50.4 Hz), 4.98 (d, 1 H, J = 9.1

Hz), 4.04-3.96 (m, 2H), 3.68 (m, 1 H), 2.62-2.30 (m, 6H), 1.78-1.71 (m, 2H), 1.29 (d, 6H, J = 9.7 Hz) ppm.

Example 7

(2S,4S)-1-{(2R)-2-Amino-3-methyl-3-[(2-phenylethyl)thio]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride A. Methyl (2R)-2-[(tert-

butoxycarbonyl)amino]-3-methyl-3-[(2-phenylethyl)thio]butanoate. To a CH₃CN (50 mL) solution containing methyl (2R)-2-[(tert-butoxycarbonyl)amino]-3-

mercapto-3-methylbutanoate (as described above) (1.26 g, 4.78 mmol, 1.0 eq) was added

sodium tert-butoxide (597 mg, 6.21 mmol, 1.3 eq). The resulting pale yellow mixture

was allowed to stir at RT for 10 min at which time (2-bromoethyl) benzene (718 μL, 5.26

mmol, 1.1 eq) was added. The resulting solution was allowed to stir at RT for 18 hr. The

reaction was then quenched with a sat NH4CI solution, poured into H2O, and extracted

(2X) with EtOAc. Combined extracts were dried over MgS04 and concentrated in vacuo.

Purified via flash chromatography using 5:1 hexanes/EtOAc as the mobile phase.

Isolated 761 mg (2.07 mmol, 45% yield).

¹H NMR (CDCb) 400 MHz δ 7.31-7.19 (m, 5H), 5.36 (d, 1 H, J = 9.0 Hz), 4.33 (d, 1 H, J = 9.1

Hz), 3.71 (s, 3H), 2.83-2.78 (m, 4H), 1.44 (s, 9H), 1.36 (d, 6H, J = 7.5 Hz) ppm. B. (2R)-2-[(Tert-butoxycarbonyl)amino]-3-methyl-3-[(2-phenylethyl)thio]butanoic acid.

To a dioxane (20 mL) / H2O (7 mL) solution containing compound A (761 mg, 2.07 mmol,

1.0 eq) was added lithium hydroxide monohydrate (434 mg, 10.35 mmol, 5.0 eq). The

resulting heterogeneous mixture was allowed to stir at RT for 18 hr. The reaction was

then quenched with a sat NH4CI solution, poured into H2O, and extracted (2X) with EtOAc. The combined extracts were dried over MgS0₄ and concentrated in vacuo.

Purified via flash chromatography using 1 :1 hexanes/EtOac (with 0.1% AcOH) as the

mobile phase. Isolated 502 mg (1.42 mmol, 69% yield).

¹H NMR (CDCb) 400 MHz δ 7.31-7.18 (m, 5H), 5.42 (d, 1 H, J = 8.3 Hz), 4.32 (s(br), 1 H),

2.87-2.81 (m, 4H), 1.44 (s, 9H), 1.40 (s, 3H), 1.36 (s, 3H) ppm.

C. Tert-butyl (l R)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]carbonyl}-2-methyl-2-

[(2-phenylethyl)thio]propylcarbamate.

To a DMF solution (10 mL) containing compound B (502 mg, 1.42 mmol, 1.2 eq) was

added N,N-diisopropylethylamine (278 μL, 1.59 mmol, 1.35 eq) followed by HATU (764

mg, 2.01 mmol, 1.7 eq). The resulting amber solution was stirred at RT for 20 min. To

this stirring solution was added a solution of (2S, 4S)-4-fluoro-2-pyrrolidinecarbonitrile

4-methylbenzenesulfonate (338 mg, 1.18 mmol, 1.0 eq) and N,N-diisopropylethylamine

(226 μL, 1.3 mmol, 1.1 eq) in DMF (5 mL). The resulting solution was stirred at RT for 18

h and quenched with a sat. NaHCOs solution (ca 5 mL). The cloudy solution was then

poured into H2O (ca 20 mL) and extracted with EtOAc (3 X 10 mL). The combined

organic extracts were washed with H2O (2X) and brine (1X); dried over MgSCU and

concentrated in vacuo. The resulting amber oil was purified via flash chromatography using 1 :1 hexanes/EtOAc as the mobile phase. Isolated 361 mg (0.803 mmol, 80% yield) of compound C as a white foam.

'H NMR (CDCb) 400 MHz δ 7.30-7.20 (m, 5H), 5.46 (d, 1 H, J = 51.0 Hz), 5.38 (d, 1 H, J =

9.2 Hz), 5.01 (d, 1 H, J = 9.4 Hz), 4.36-4.25 (m, 2H), 4.06 (m, 1 H), 2.87-2.84 (m, 4H), 2.70

(t, 1 H, J = 15.0 Hz), 2.35 (m, 1 H), 1.41-1.37 (m, 15H) ppm.

D. (2S,4S)-1-{(2R)-2-Amino-3-methyl-3-[(2-phenylethyl)thio]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile.

To a CH2CI2 (20 mL) solution containing trifluoroacetic acid (2 mL) was added compound

C (361 mg, 0.803 mmol, 1.0 eq). The resulting pale yellow solution was stirred at RT for

2 h at which time the solvent was removed in vacuo. The resulting TFA salt was

converted to the free base by addition of a sat. NaHC0₃ solution and extracting the

aqueous layer (2X) with EtOAc. The combined organic extracts were dried over Na2S0₄

and concentrated in vacuo. The resulting off-white foam was purified via flash

chromatography using 5% MeOH (with 0.1% NH3) in CH2CI2 as the mobile phase

affording 223 mg (0.638 mmol, 80% yield) of a white foam. To form the HCl salt, the

free base was taken up in Et2θ and acetone added until all solids were in solution. 2.0 M

HCl in ether was added dropwise until no more precipitate formed. The precipitate was

then filtered and washed several times with Et∑O. The resulting salt was dried under

high vacuum.

¹H NMR (D2O) 400 MHz δ 7.20-7.09 (m, 5H), 5.43 (d, 1 H, J = 50.5 Hz), 4.94 (d, 1 H, J = 9.5

Hz), 4.13 (m, 1 H), 3.89 (s, 1 H), 3.71 (m, 1 H), 2.84-2.70 (m, 4H), 2.59 (t, 1 H J = 15.7 Hz),

2.37 (m, 1 H), 1.28 (s, 3H), 1.22 (s, 3H) ppm. Example 8

(2S,4S)-1-((2R)-2-Amino-3-{[3-(4-fluorophenyl)propyl]thio}-3-methylbutanoyl)-4-

fluoropyrrolidine-2-carbonitrile hydrochloride

A. 3-(4-Fluorophenyl)propan-1-ol.

To a THF (200 mL) solution containing 3-(4-fluorophenyl)propanoic acid (33.6 g, 0.2 mol)

was added a 1.0 M solution of LiAIH4 in Et2U (200 mL, 0.2 mol) over a period of 0.5 h.

The resulting mixture was allowed to stir at RT for 18 h. The reaction was slowly

quenched with H2O and the aqueous layer extracted (1x) with Et2θ. The combined

organic extracts were dried over MgS04 and concentrated in vacuo. The resulting oil was used in the next step without further purification. Isolated 31.4 g (0.2 mol, 100%

yield).

B. 1 -(3-Chloropropyl)-4-fluorobenzene. To a CH2CI2 (300 mL) solution containing compound A (31.4 g, 0.2 mol) was added

dichlorotriphenylphosphorane (110 g, 0.33 mol). The resulting mixture was stirred at RT

for 20 min and then concentrated in vacuo. Hexanes (ca 200 mL) was added to the

residue and the resulting heterogeneous mixture was stirred at RT for 30 min. The

insoluble solids were filtered and washed with hexanes. The filtrate was concentrated in

vacuo and the resulting oil was used in the next step without further purification.

Isolated 33.3 g (0.193 mol, 96% yield). C. Methyl (2R)-2-[(tert-butoxycarbonyl)amino]-3-{[3-(4-fluorophenyl)propyl]thio}-3- methylbutanoate.

To a CH3CN (60 mL) solution containing methyl (2R)-2-[(tert-butoxycarbonyl)amino]-3-

mercapto-3-methylbutanoate (as described above) (1.51 g, 5.73 mmol, 1.0 eq) was added

sodium tert-butoxide (716 mg, 7.45 mmol, 1.3 eq). The resulting pale yellow mixture was allowed to stir at RT for 10 min at which time 1-(3-chloropropyl)-4-fluorobenzene

(as described above) (1.09 g, 6.3 mmol, 1.1 eq) was added. The resulting solution was

allowed to stir at RT for 18 hr. The reaction was then quenched with a sat. NH4CI

solution, poured into H2O, and extracted (2X) with EtOAc. The combined extracts were

dried over MgSU4 and concentrated in vacuo and then purified via flash

chromatography using 5:1 hexanes/EtOAc as the mobile phase. Isolated 700 mg (1.75

mmol, 31% yield).

'H NMR (CDCb) 400 MHz δ 7.14-7.10 (m, 2H), 6.98-6.93 (m, 2H), 5.36 (d, 1 H, _/ = 9.1 Hz),

4.29 (d, 1 H, J = 9.2 Hz), 3.71 (s, 3H), 2.68 (t, 2H, J = 7.4 Hz), 2.56-2.49 (m, 2H), 1.86-1.79

(m, 2H), 1.43 (s, 9H), 1.35 (d, 6H, _/ = 6.8 Hz) ppm.

D. (2R)-2-[(Tert-butoxycarbonyl)amino]-3-{[3-(4-fluorophenyl)propyl]thio}-3-

methylbutanoic acid.

To a dioxane (20 mL) / H2O (7 mL) solution containing compound A (700 mg, 1.75 mmol,

1.0 eq) was added lithium hydroxide monohydrate (367 mg, 8.75 mmol, 5.0 eq). The

resulting heterogeneous mixture was allowed to stir at RT for 18 hr. The reaction was

then quenched with a sat NH4CI solution, poured into H2O, and extracted (2X) with

EtOAc. The combined extracts were dried over MgS04 and concentrated in vacuo. Purified via flash chromatography using 1 :1 hexanes/EtOAc (with 0.1% AcOH) as the

mobile phase. Isolated 512 mg (1.33 mmol, 76% yield).

¹H NMR (CDCb) 400 MHz δ 7.13-7.10 (m, 2H), 6.99-6.93 (m, 2H), 5.41 (d, 1 H, J = 8.1 Hz),

4.29 (s(br), 1 H), 2.68-2.55 (m, 4H), 1.88-1.80 (m, 2H), 1.43 (s, 9H), 1.39 (s, 3H), 1.36 (s, 3H) ppm.

E. Tert-butyl (l R)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]carbonyl}-2-{[3-(4-

fluorophenyl)propyl]thio}-2-methylpropylcarbamate

To a DMF solution (15 mL) containing compound B (512 mg, 1.33 mmol, 1.2 eq) was

added N,N-diisopropylethylamine (261 μL, 1.5 mmol, 1.35 eq) followed by HATU (717

mg, 1.89 mmol, 1.7 eq). The resulting amber solution was stirred at RT for 20 min. To

this stirring solution was added a solution of (2S, 4S)-4-fluoro-2-pyrrolidinecarbonitrile

4-methylbenzenesulfonate (317 mg, 1.1 1 mmol, 1.0 eq) and N,N-diisopropylethylamine

(213 μL, 1.22 mmol, 1.1 eq) in DMF (10 mL). The resulting solution was stirred at RT for

18 hr and quenched with a sat. NaHC0₃ solution (ca 5 mL). The cloudy solution was then

poured into H2O (ca 20 mL) and extracted with EtOAc (3 X 10 mL). The combined

organic extracts were washed with H2O (2X) and brine (1X); dried over MgS0₄ and

concentrated in vacuo. The resulting amber oil was purified via flash chromatography

using 1 :1 Hexane/EtOAc as the mobile phase. Isolated 420 mg (0.872 mmol, 79% yield)

of compound C as a white foam.

¹H NMR (CDCb) 400 MHz δ 7.14-7.1 1 (m, 2H), 6.97-6.92 (m, 2H), 5.47 (d, 1 H, J = 50.9

Hz), 5.36 (d, 1 H, J = 8.4 Hz), 5.00 (d, 1 H, J = 9.3 Hz), 4.42-4.32 (m, 2H), 4.01 (m, 1 H),

2.74-2.57 (m, 5H), 2.36 (m, 1 H), 1.89-1.76 (m, 2H), 1.41-1.37 (m, 15H) ppm. F. (2S,4S)-1-((2R)-2-Amino-3-{[3-(4-fluorophenyl)propyl]thio}-3-methylbutanoyl)-4-

fluoropyrrolidine-2-carbonitrile.

To a CH2CI2 (30 mL) solution containing trifluoroacetic acid (3 mL) was added compound

C (420 mg, 0.872 mmol, 1.0 eq). The resulting pale yellow solution was stirred at RT for

2 hr at which time the solvent was removed in vacuo. The resulting TFA salt was

converted to the free base by addition of a sat. NaHC0₃ solution and extracting the aqueous layer (2X) with EtOAc. The combined organic extracts were dried over Na2S0₄

and concentrated in vacuo. The resulting off-white foam was purified via flash

chromatography using 5% MeOH (with 0.1% NH₃) in CH2CI2 as the mobile phase

affording 275 mg (0.721 mmol, 83% yield) of a white foam. To form the HCl salt, the

free base was taken up in Et2θ and acetone added until all solids were in solution. 2.0 M

HCl in ether was added dropwise until no more precipitate formed. The precipitate was

then filtered and washed several times with Et∑O. The resulting salt was dried under

high vacuum.

Η NMR (D2O) 400 MHz δ 7.15-7.10 (m, 2H), 6.96-6.90 (m, 2H), 5.48 (d, 1 H, J= 50.9 Hz),

4.99 (d, 1 H, J = 9.6 Hz), 4.07-3.98 (m, 2H), 3.74 (m, 1 H), 2.64-2.32 (m, 6H), 1.80-1.68 (m,

2H), 1.30 (d, 6H, .7 = 6.6 Hz) ppm.

Example 9

(2S,4S)-1-{(2R)-2-Amino-3-[(4-methoxybenzyl)sulfonyl1-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride A. Tert-butyl (l R)-1 -{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1 -yl]carbonyl}-2-[(4-

methoxybenzyl)sulfonyl]-2-methylpropylcarbamate.

To a CHCb (25 mL) solution containing tert-butyl (1 R)-1-{[(2S,4S)-2-cyano-4- fluoropyrrolidin-1-yl]carbonyl}-2-[(4-methoxybenzyl)thio]-2-methylpropylcarbamate

(as described above) (473 mg, 1.017 mmol, 1.0 eq) was added 3-chloroperoxybenzoic

acid (1.76 g, 10.18 mmol, 10 eq). The resulting mixture was stirred at RT for 18 hr. The

reaction was then quenched with 1.0 N NaOH (ca 5mL) and the layers separated. The

organic layer was dried over MgS0₄ and concentrated in vacuo. The resulting white

fluffy solid was used in the next step without further purification. Isolated 400 mg (0.805 mmol, 80% yield).

¹H NMR (CDCb) 400 MHz δ 7.35 (d, 2H, J = 8.8 Hz), 6.92 (d, 2H, J = 8.8 Hz), 5.48 (d, 1 H, J

= 51.0 Hz), 5.04 (d, 1 H, J = 9.1 Hz), 4.99 (d, 1 H, J = 9.4 Hz), 4.34-3.98 (m, 4H), 3.81 (s,

3H), 2.73 (t, 1 H, J = 15.0 Hz), 2.39 (m, 1 H), 1.58 (s, 3H), 1.53 (s, 3H), 1.42 (s, 9H) ppm.

B. (2S,4S)-1-{(2R)-2-Amino-3-[(4-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile.

To a CH2CI2 (40 mL) solution containing trifluoroacetic acid (4 mL) was added compound

A (400 mg, 0.805 mmol, 1.0 eq). The resulting pale yellow solution was stirred at RT for

2 hr at which time the solvent was removed in vacuo. The resulting TFA salt was

converted to the free base by addition of a sat. NaHCOs solution and extracting the

aqueous layer (2X) with EtOAc. The combined organic extracts were dried over a∑S04

and concentrated in vacuo. The resulting off-white foam was purified via flash

chromatography using 5% MeOH (with 0.1% NH₃) in CH2CI2 as the mobile phase affording 296 mg (0.745 mmol, 93% yield) of a white foam. To form the HCl salt, the

free base was taken up in Et2θ and acetone added until all solids were in solution. 2.0 M

HCl in ether was added dropwise until no more precipitate formed. The precipitate was

then filtered and washed several times with Et2θ. The resulting salt was dried under high vacuum.

'H NMR (D2O) 400 MHz δ 7.28 (d, 2H, I = 8.8 Hz), 6.92 (d, 2H, J = 8.8 Hz), 5.47 (d, 1 H, J =

50.6 Hz), 5.00 (d, 1 H, J = 10.0 Hz), 4.66 (s, 1 H), 4.51 (s, 2H), 4.05 (m, I H), 3.81 (m, 1 H),

3.69 (s, 3H), 2.64 (t, I H, J = 15.7 Hz), 2.42 (m, 1 H), 1.62 (s, 3H), 1.48 (s, 3H) ppm.

Example 10

(2S,4S)-1-{(2R)-2-Amino-3-[(4-methoxybenzyl)sulfinyl]-3-methylbutanoyll-4-

fluoropyrrolidine-2-carbonitrile hydrochloride

A. Tert-butyl (l R)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1 -yl]carbonyl}-2-[(4-

methoxybenzyl)sulfinyl]-2-methylpropylcarbamate

To a methanol (10 mL) solution containing tert-butyl (1 R)-1-{[(2S,4S)-2-cyano-4-

fluoropyrrolidin-1-yl]carbonyl}-2-[(4-methoxybenzyl)thio]-2-methylpropylcarbamate

(as described above) (660 mg, 1.42 mmol, 1.0 eq) was added a solution of Nal0₄ (334 mg,

1.56 mmol, 1.1 eq) in H2O (10 mL). The resulting mixture was stirred at RT for 18 hr. The

resulting heterogeneous mixture was partitioned between CH2CI2 and H2O. The organic

layer was dried over MgS0₄ and concentrated in vacuo. Resulting white fluffy solid was used in the next step without further purification. Isolated 600 mg (1.25 mmol, 87%

yield) as a mixture of diastereomers.

¹H NMR (CDCb) 400 MHz δ 7.27-7.25 (m, 2H), 6.90-6.88 (m, 2H), 5.87 (s(br), 1 H), 5.40 (m,

1 H), 5.03-4.95 (m, 2H), 4.19-3.66 (m, 7H), 2.65 (m, 1 H), 2.36 (m, 1 H), 1.49-1.38 (m, 15H) ppm.

B. (2S,4S)-1-{(2R)-2-Amino-3-[(4-methoxybenzyl)sulfinyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile.

To a CH2CI2 (50 mL) solution containing trifluoroacetic acid (5 mL) was added compound A (600 mg, 1.25 mmol, 1.0 eq). The resulting pale yellow solution was stirred at RT for 2

hr at which time the solvent was removed in vacuo. The resulting TFA salt was

converted to the free base by addition of a sat. NaHC0₃ solution and extracting the

aqueous layer (2X) with EtOAc. The combined organic extracts were dried over a2SU4

and concentrated in vacuo. The resulting off-white foam was purified via flash

chromatography using 5% MeOH (with 0.1% NH₃) in CH2CI2 as the mobile phase

affording 368 mg (0.965 mmol, 77% yield) of a white foam. To form the HCl salt, the

free base was taken up in Et∑O and acetone added until all solids were in solution. 2.0 M

HCl in ether was added dropwise until no more precipitate formed. The precipitate was

then filtered and washed several times with Et2θ. The resulting salt was dried under

high vacuum.

¹H NMR (D2O) 400 MHz δ 7.25-7.21 (m, 2H), 6.93-6.90 m, 2H), 5.42 (m, 1 H), 5.02 (m, 1 H),

4.48 (m, 1 H), 4.24-3.70 (m, 7H), 2.68-2.31 (m, 2H), 1.52-1.32 (m, 6H) ppm. Example 1 1

(2S,4S)-1-((2R)-2-Amino-3-methyl-3-{[4-(trifluoromethyl)benzyl]thio}butanoyl)-4-

fluoropyrrolidine-2-carbonitrile hydrochloride A. (2R)-2-[(Tert-butoxycarbonyl)amino]-3-mercapto-3-methylbutanoic acid.

To a THF (300 mL) solution containing L-penicillamine (5.0 g, 33.51 mmol, 1.0 eq) was added potassium hydroxide (1.88 g, 33.51 mmol, 1.0 eq). The mixture was then cooled

to 0 °C and di-tert-butyldicarbonate (7.31 g, 33.51 mmol, 1.0 eq) was added. The

resulting stirred mixture was allowed to warm to RT overnight. The reaction was then

poured into H2O (ca 300 mL) and extracted (2X) with EtOAc. The combined organic

extracts were dried over MgSU4 and concentrated in vacuo. The resulting white solid

was used in the next step without further purification. Isolated 7.68 g (30.80 mmol,

92⁰/0 yield).

'H NMR (CDCb) 400 MHz δ 5.51 (d, 1 H, J = 9.6 Hz), 4.33 (d, 1 H, J = 9.6 Hz), 1.55 (s, 3H),

1.45 (s, 9H), 1.41 (s, 3H) ppm.

B. (2R)-2-[(Tert-butoxycarbonyl)amino]-3-methyl-3-{[4-

(trifluoromethyl)benzyl]thio}butanoic acid.

To a CHaCN (40 mL) solution containing compound A (1.0 g, 4.01 1 mmol, 1.0 eq) was

added sodium tert-butoxide (810 mg, 8.42 mmol, 2.1 eq). The resulting pale yellow

solution was allowed to stir for 10 min at which time 4-(trifluoromethyl)benzylbromide (

1.05 g, 4.41 mmol, 1.1 eq). The resulting mixture was heated to 50 °C and allowed to stir for 18 hr. The reaction was then poured into H2O (ca 50 mL) and extracted (2X) with

EtOAc. The combined extracts were dried over MgSU4 and concentrated in vacuo. The

resulting solid was used in the next step without further purification. Isolated 1.45 g

(3.55 mmol, 89% yield).

'H NMR (CDCb) 400 MHz δ 7.55 (d, 2H, J = 8.1 Hz), 7.44 (d, 2H, J = 8.1 Hz), 5.39 (s(br),

1 H), 4.44 (s(br), 1 H), 3.85 (s, 2H), 1.45-1.43 (m, 15H) ppm.

C. Tert-butyl (l R)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1 -yl]carbonyl}-2-methyl-2-

{[4-(trifluoromethyl)benzyl]thio}propylcarbamate. To a DMF solution (30 mL) containing compound B (1.45 mg, 3.55 mmol, 1.2 eq) was

added N,N-diisopropylethylamine (698 μL, 4.01 mmol, 1.35 eq) followed by HATU (1.92

g, 5.05 mmol, 1.7 eq). The resulting amber solution was stirred at RT for 20 min. To this

stirring solution was added a solution of (2S, 4S)-4-fluoro-2-pyrrolidinecarbonitrile 4-

methylbenzenesulfonate (849 mg, 2.97 mmol, 1.0 eq) and N,N-diisopropylethylamine

(569 μL, 3.27 mmol, 1.1 eq) in DMF (25 mL). The resulting solution was stirred at RT for

18 hr and then quenched with a sat. NaHC0₃ solution (ca 5 mL). The cloudy solution was

then poured into H2O (ca 20 mL) and extracted with EtOAc (3 X 10 mL). The combined

organic extracts were washed with H2O (2X) and brine (1X); dried over MgS0₄ and

concentrated in vacuo. The resulting amber oil was purified via flash chromatography

using 1 :1 hexanes/EtOAc as the mobile phase. Isolated 682 mg (1.35 mmol, 45% yield)

of compound C as a white foam.

'H NMR (CDCb) 400 MHz δ 7.55 (d, 2H, J = 8.1 Hz), 7.50 (d, 2H, J = 8.8 Hz), 5.49 (d, 1 H, J

= 50.8 Hz), 5.42 (d, 1 H, J = 9.9 Hz), 5.03 (d, 1 H, J = 10.0 Hz), 4.40 (d, 1 H, J = 9.5 Hz), 4.32 (m, 1 H), 4.04 (m, 1 H), 3.88 (s, 2H), 2.74 (t, 1 H, J = 15.1 Hz), 2.38 (m, 1 H), 1.47 (s, 3H), 1.42

(s, 9H), 1.41 (s, 3H) ppm.

D. (2S,4S)-1 -((2R)-2-Amino-3-methyl-3-{[4-(trifluoromethyl)benzyl]thio}butanoyl)-4-

fluoropyrrolidine-2-carbonitrile.

To a CH2CI2 (70 mL) solution containing trifluoroacetic acid (7 mL) was added compound

A (682 mg, 1.35 mmol, 1.0 eq). The resulting pale yellow solution was stirred at RT for 2

hr at which time the solvent was removed in vacuo. The resulting TFA salt was

converted to the free base by addition of a sat. NaHC0₃ solution and extracting the

aqueous layer (2X) with EtOAc. The combined organic extracts were dried over Na₂S0₄

and concentrated in vacuo. The resulting off-white foam was purified via flash

chromatography using 5% MeOH (with 0.1% NH₃) in CH2CI2 as the mobile phase

affording 390 mg (0.967 mmol, 72% yield) of a white foam. To form the HCl salt, the

free base was taken up in Et₂0 and acetone added until all solids were in solution. 2.0 M

HCl in ether was added dropwise until no more precipitate formed. The precipitate was

then filtered and washed several times with Et2U. The resulting salt was dried under

high vacuum.

'H NMR (D2O) 400 MHz δ 7.57 (d, 2H, J = 8.1 Hz), 7.49 (d, 2H, J = 8.5 Hz), 5.46 (d, 1 H, J =

51.4 Hz), 4.98 (d, 1 H, J = 9.5 Hz), 4.05-3.53 (m, 5H), 2.62-2.28 (m, 2H), 1.40 (s, 3H), 1.31

(s, 3H) ppm. Example 12

(2S,4S)-1-[(2S)-2-Amino-2-(l-vinylcyclopentyl)ethanoyl]-4-fluoropyrrolidine-2-

carbonitrile hydrochloride A. Tert-butyl (1 S)-2-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]-2-oxo-1-(1-

vinylcyclopentyl)ethylcarbamate.

To a DMF solution (25 mL) containing racemic [(tert-butoxycarbony)lamino](1-

vinylcyclopentyQacetic acid (as described in Robl, Jeffrey A.; Sulsky, Richard B.; Augeri, David J.; Magnin, David R.; Hamann, Lawrence G.; Betebenner, David A. Preparation of

fused cyclopropylpyrrolidine-based inhibitors of dipeptidyl peptidase IV , WO

200168603A2) (1.07 g, 3.97 mmol) was added Hunig's Base (0.835 mL, 4.37 mmol) and

HATU (1.66 g, 4.37 mmol). The resulting solution was stirred for 15 minutes at RT, an

then a solution of (2S,4S)-4-fluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate

(1.14 g, 3.97 mmol) and Hunig's base (0.835 mL, 4.37 mmol) in 10 mL of DMF was added

dropwise over 2-3 minutes. The resulting solution was stirred at RT for 18 hr then

quenched with H2O. The solution was poured into H2O and the organics extracted with

EtOAc (2X). After washing with sat. NaCl and 1.0 N HCl the organics were dried (MgS0₄)

and the solvent removed in vacuo to provide a dark oil, which was purified on silica gel

using a Biotage FlashElute System, eluting in gradient fashion with hexanes / EtOAc 3:1

to 1 :1 to afford 2 products, diastereomeric at the alpha-amino position (C1), in approximately a 1 :1 ratio (582 mg of diastereomer 1 , 628 mg of diastereomer 2, 83%

overall yield). The slower-eluting diastereomer was determined to have the (S) absolute

stereochemistry on the basis of vibrational circular dichroism analysis of the final product.

¹H NMR (CDCb) 400 MHz δ 5.92 (dd l H, _/ = 10.8, 17.3 Hz), 5.38 (m, 1 H), 5.22 (dd, 2 H, J =

10.8, 27.4 Hz), 5.02 (d, 1 H, J = 10.3 Hz), 4.23 (d, 1 H, J = 9.2 Hz), 4.06 (m, 2H), 2.63 (t, 1 H,

1 = 15.5 Hz), 2.40-2.23 (m, 2H), 1.85 (m, 1 H), 1.75-1.52 (m, 6H), 1.43 (m, 1 H), 1.37 (s, 9H)

ppm.

B. (2S,4S)-1-[(2S)-2-amino-2-(1 -vinylcyclopentyl)ethanoyl]-4-fluoropyrrolidine-2-

carbonitrile hydrochloride.

To a flask containing tert-butyl (l S)-2-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]-2-oxo-

1 -(l-vinylcyclopentyl)ethylcarbamate (70 mg, 0.19 mmol) in 1 mL of 1 ,4-dioxane was

added 2 mL of 4 N HCl in 1 ,4-dioxane followed by 1 mL of 2 N HCl in Et2θ. The resulting

solution was stirred at RT for 3 hr at which time starting material had been consumed as

judged by TLC. 3 mL of Et∑O was added to the reaction mixture followed by 3 mL of

hexanes. The resulting light tan precipitate was filtered, triturated with Et2θ, collected

via vacuum filtration and dried under vacuum to afford compound B as a cream-colored

solid (35 mg, 70% yield).

Η NMR (d₄-MeOH) 400 MHz δ 5.83 (dd, 1 H, J = 10.7, 17.6 Hz), 5.36 (dd, 2H, J = 10.7,

27.7 Hz), 5.07 (d, 1 H, J = 9.7 Hz), 4.16 (m, 2H), 3.79 (m, 1 H), 2.63 (t, 1 H, J = 16.2 Hz), 2.45

(m, 1 H), 1.86-1.58 (m, 7H), 1.36'(M, 3H) ppm. Example 13

(2S,4S)-1 -[(2S)-2-Amino-5-(4-methoxyphenyl)-3,3-dimethylpentanoyl]-4-

fluoropyrrolidine-2-carbonitrile hydrochloride.

A. 4-(4-Methoxyphenyl)-2,2-dimethylbutanal.

To a toluene solution (80 mL) containing 4-(4-methoxyphenyl)-2,2-

dimethylbutanenitrile (3.65 g, 18.0 mmol; see: Knochel, P. et al. Org. Lett. 2000, 2, 3285

for the preparation of this compound) cooled to -78 "C was added 18.0 mL of a 1.5 M toluene solution of DIBAL (27.0 mmol). After 3 hr at -78 °C the reaction was quenched

with a H∑O/THF solution (75 mL/15 mL) containing sodium acetate (4.6 g) and acetic acid

(4.6 mL) at -78 °C. To this solution was added celite followed by Et2θ. The solution was

allowed to warm to RT at which time it was filtered through a bed of celite. The celite

was rinsed thoroughly with Et2θ and then the filtrate washed with H2O. After drying

over MgS0₄ the solvent was removed in vacuo and the residue purified via column

chromatography (hexanes/EtOAc (9:1)) yielding 3.55 g (17.2 mmol, 96% yield) of

compound A.

¹H NMR (CDCb) 400 MHz δ 9.47 (s, 1 H), 7.07 (d, 2H, I = 8.6 Hz), 6.81 (d, 2H, J = 8.4 Hz),

3.78 (s, 3H), 2.48 - 2.44 (m, 2H), 1.77 - 1.73 (m, 2H), 1.1 1 (s, 6H) ppm. B. 1,1,1-Trichloro-5-(4-methoxyphenyl)-3,3-dimethylpentan-2-ol.

To a THF solution (10 mL) containing compound A (51 1 mg, 2.48 mmol) and anhydrous

chloroform (31 1 mg, 2.60 mmol) cooled to -78 °C was added 2.6 mL of a 1.0 M THF

solution of LiHMDS (2.60 mmol) dropwise over 5 min. The resulting solution stirred at -

78 °C for 30 min at which time the reaction was quenched with H2O. Upon warming the

organics were extracted with EtOAc and after drying over MgS0₄ the solvent was removed in vacuo. The residual yellow oil was purified via column chromatography

(10%Et0Ac/hexanes) yielding 588 mg (1.81 mmol, 73% yield) of compound B as a white solid.

Η NMR (CDCb) 400 MHz δ 7.10 (d, 2H, J = 8.7 Hz), 6.82 (d, 2H, J = 8.4 Hz), 4.05 (d, 1 H, J

= 5.7 Hz), 3.78 (s, 3H), 2.95 (d, 1 H, _/ = 5.7 Hz), 2.64 - 2.59 (m, 2H), 1.95 (m, 1 H), 1.73 (m,

1 H), 1.32 (s, 3H), 1.25 (s, 3H) ppm.

C. 2-Azido-5-(4-methoxyphenyl)-3,3-dimethylpentanoic acid. To a DME solution (4 mL) containing compound B (581 mg, 1.79 mmol) was added 7 mL

of an aqueous solution containing sodium azide (232 mg, 3.58 mmol) and sodium

hydroxide (286 mg, 7.15 mmol) at RT. After stirring overnight the solution was poured

into 2.0 M NaOH and the organics extracted with Et₂0. The aqueous layer was acidifed

with sat. NaHSU4 and the organics extracted with EtOAc (2X). After drying over MgS0₄

the solvent was removed in vacuo and the residual oil purified via column

chromatography (5%MeOH/CH2C ) yielding 186 mg (0.67 mmol, 37% yield) of

compound C.

'H NMR (CDCb) 400 MHz δ 7.09 (d, 2H, J = 8.4 Hz), 6.82 (d, 2H, J = 8.6 Hz), 3.91 (s, IH),

3.78 (s, 3H), 2.58 - 2.52 (m, 2H), 1.71 - 1.61 (m, 2H), 1.10 (s, 6H) ppm. D. (2S,4S)-1 -[2-Azido-5-(4-methoxyphenyl)-3,3-dimethylpentanoyl]-4- fluoropyrrolidine-2-carbonitrile.

To a DMF solution (15 mL) containing compound C (696 mg, 2.52 mmol) and N,N-

diisopropylethylamine (358 mg, 2.77 mmol) was added HATU (1.05 g, 2.77 mmol) at RT.

After 20 min a DMF solution (8 mL) containing (2S,4S)-4-fluoro-2-

pyrrolidinecarbonitrile 4-methylbenzenesulfonate (755 mg, 2.64 mmol) and N,N-

diisopropylethylamine (341 mg, 2.64 mmol) was added. The solution stirred overnight at

which time it was diluted with EtOAc and washed with H2O (3X), sat. NaHCOsand 2.0 M

HCl. After drying over MgSU4 the solvent was removed in vacuo. The residual yellow oil

was purified via column chromatography (initially hexanes/EtOAc/CH∑C (6:3:1) then

hexanes/EtOAc/CH2Cb (3:1 :1)) yielding 605 mg (1.62 mmol, 64% yield) of compound D as

a white solid that by ¹H NMR was a 3:2 mixture of diastereomers. The diastereomeric

mixture was taken on.

E. (2S,4S)-1-[(2S)-2-Amino-5-(4-methoxyphenyl)-3,3dimethylpentanoyl]-4-

fluoropyrrolidine-2-carbonitrile hydrochloride.

To an EtOH solution (8 mL) containing 10% Pd/C (200 mg) was added a 1 :1 THF/EtOH

solution (10 mL) containing compound D (593 mg, 1.59 mmol). The solution was

degassed 4X and then placed under a balloon of hydrogen. After 6.5 hr the solution was

filtered through a bed of celite with the celite being rinsed thoroughly with CH2CI2. The

solvent was removed in vacuo and the residual solid purified via column

chromatography (5%methanolic NH3/CH2CI2) yielding 251 mg (0.72 mmol) of compound

E as the freebase. This material was then dissolved in 2 mL of CH2CI2 and 3.0 mL of a 4.0 M dioxane solution of HCl was added. The solvent was then immediately removed in

vacuo and the residual oil triterated with Et₂0. The resulting soild was collected via

vacuum filtration yielding 231 mg (0.60 mmol) of compound E as the hydrochloride salt.

H NMR (d₄-MeOH) 400 MHz δ 7.13 (d, 2H, J = 8.6 Hz), 6.81 (d, 2H, J = 8.7 Hz), 5.31

(d(br), 1 H, J « 51.1 Hz), 5.09 (d, 1 H, J = 9.5 Hz), 4.10 (m, I H), 4.01 (s, 1 H), 3.86 (m, 1 H),

3.74 (s, 3H), 2.65 - 2.42 (m, 4H), 1.76 - 1.72 (m, 2H), 1.20 (s, 6H) ppm.

Example 14

(2S,4S)-1-{(2R)-2-Amino-3-methyl-3-[(3-phenylpropyl)sulfonyl]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile

A. Tert-butyl (l R)-1 -{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]carbonyl}-2-methyl-2-

[(3-phenylpropyl)sulfonyl]propylcarbamate.

To a CHCb (50 mL) solution containing tert-butyl (l R)-1-{[(2S,4S)-2-cyano-4- fluoropyrrolidin-1-yl]carbonyl}-2-methyl-2-[(3-phenylpropyl)thio]propylcarbamate (as

described above) (830 mg, 1.79 mmol, 1.0 eq) was added 3-chloroperoxybenzoic acid

(3.09 g, 17.9 mmol, 10 eq). The resulting mixture was stirred at RT for 18 h. The

reaction was then quenched with 1.0 N NaOH (ca 10 mL) and the layers separated. The

organic layer was dried over MgSU4 and concentrated in vacuo. The resulting white

fluffy solid was used in the next step without further purification. Isolated 800 mg of

compound A (1.62 mmol, 91% yield). B. (2S,4S)-1-{(2R)-2-amino-3-methyl-3-[(3-phenylpropyl)sulfonyl]butanoyl}-4- fluoropyrrolidine-2-carbonitrile

To a CH2CI2 (50 mL) solution containing trifluoroacetic acid (5 mL) was added compound

A (800 mg, 1.62 mmol, 1.0 eq). The resulting pale yellow solution was stirred at RT for 2

h at which time the solvent was removed in vacuo. The resulting TFA salt was converted

to the free base by addition of a sat. NaHC0₃ solution and extracting the aqueous layer (2X) with EtOAc. The combined organic extracts were dried over Na₂Sθ4 and

concentrated in vacuo. The resulting off-white foam was purified via flash

chromatography using 5% MeOH (with 0.1% NH₃) in CH2CI2 as the mobile phase

affording 532 mg of compound B as the freebase (1.35 mmol, 83% yield). To form the

HCl salt, the free base was taken up in Et2θ and acetone added until all solids were in

solution. 2.0 M HCl in ether was added dropwise until no more precipitate formed. The

precipitate was then filtered and washed several times with Et∑O. The resulting salt was

dried under high vacuum.

'H NMR (D2O) 400 MHz δ 7.26-7.13 (m, 5H), 5.48 (d, 1 H, J = 50.7 Hz), 5.00 (d, 1 H, J = 9.7

Hz), 4.63 (s, 1 H), 4.02 (m, 1 H), 3.81 (m, 1 H), 3.20-3.15 (m, 2H), 2.71 (t, 2H, J = 7.5 Hz),

2.65-2.33 (m, 2H), 2.09-2.01 (m, 2H), 1.50 (s, 3H), 1.35 (s, 3H) ppm.

Example 15

(2S,4S)-1-{(2R)-2-Amino-3-methyl-3-[(4-methylbenzyl)sulfonyl]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile. A. (2R)-2-[(Tert-butoxycarbonyl)amino]-3-methyl-3-[(4-methylbenzyl)thio]butanoic acid

To a 1.0 N NaOH (50 mL) solution containing (2R)-2-[(tert-

butoxycarbonyl)amino]-3-mercapto-3-methylbutanoic acid (as described above) (1.0 g,

4.01 mmol, 1.0 eq) was added 4-methylbenzyl chloride (584 μL, 4.41 mmol, 1.1 eq). The

resulting mixture was allowed to stir at RT for 18 h. The reaction mixture was then

poured into a seperatory funnel and washed with Et2θ (1X) to remove any unreacted

chloride. Concentrated HCl was added to the aqueous layer until the pH reached 2-3. The aqueous layer was then extracted with Et2U (2X). The combined organic extracts

were dried over MgSU4and concentrated in vacuo. The resulting white fluffy solid was

used in the next step without further purification. Isolated 1.29 g of compound A (3.65

mmol, 91% yield).

'H NMR (CDCb) 400 MHz δ 7.21 (d, 2H, 1 = 7.9 Hz), 7.11 (d, 2H, J = 8.3 Hz), 5.46

(d (br), 1 H, = 8.5 Hz), 4.40 (s (br), 1 H), 3.83 (q, 2H, 1 = 11.7 Hz), 2.31 (s, 3H), 1.48-1.42

(m, 15H) ppm.

B. Tert-butyl (1 R)-1 -{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]carbonyl}-2-methyl-2-

[(4-methylbenzyl)thio]propylcarbamate.

To a DMF solution (25 mL) containing compound A (1.29 g, 3.65 mmol, 1.2 eq) was

added N,N-diisopropylethylamine (715 μL, 4.1 mmol, 1.35 eq) followed by HATU (1.96 g,

5.17 mmol, 1.7 eq). The resulting amber solution was stirred at RT for 20 min. To this

stirring solution was added a solution of (2S, 4S)-4-fluoro-2-pyrrolidinecarbonitrile 4-

methylbenzenesulfonate (869 mg, 3.04 mmol, 1.0 eq) and N,N-diisopropylethylamine

(582 μL, 3.34 mmol, 1.1 eq) in DMF (15 mL). The resulting solution was stirred at RT for

18 h and quenched with a sat NaHCOs solution (ca 5 ml). The cloudy solution was then poured into H2O (ca 20 mL) and extracted with EtOAc (3 x 10 ml). The combined organic

extracts were washed with H2O (2X) and brine (1X); dried over MgSU4 and concentrated

in vacuo. The resulting amber oil was purified via flash chromatography using 1 :1

hexanes/EtOAc as the mobile phase. Isolated 1.14 g (2.54 mmol, 84% yield) of

compound B as a white foam.

¹H NMR (CDCb) 400 MHz δ 7.26 (d, 2H, J = 8.4 Hz), 7.10 (d, 2H, J = 7.8 Hz), 5.48 (d, 1 H, J

= 51.9 Hz), 5.44 (d, 1 H, J = 9.5 Hz), 5.04 (d, 1 H, I = 9.2 Hz), 4.37 (d, 1 H, J = 9.0 Hz), 4.25

(m, 1 H), 4.04 (m, 1 H), 3.81 (s, 2H), 2.72 (t, l H, _/ = 15.2 Hz), 2.45-2.28 (m, 4H), 1.47-1.41

(m, 15H) ppm.

C. tert-butyl (1 R)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]carbonyl}-2-methyl-2-

[(4-methylbenzyl)sulfonyl]propylcarbamate.

To a CHCb (75 mL) solution containing compound B (1.14 g, 2.54 mmol, 1.0 eq) was

added 3-chloroperoxybenzoic acid (3.09 g, 17.9 mmol, 10 eq). The resulting mixture was

stirred at RT for 18 h. The reaction was then quenched with 1.0 N NaOH (ca 15 mL) and

the layers separated. The organic layer was dried over MgSU4 and concentrated in

vacuo. The resulting white fluffy solid was used in the next step without further

purification. Isolated 1.03 g of compound C (2.14 mmol, 84% yield).

¹H NMR (CDCb) 400 MHz δ 7.32 (d, 2H, J = 8.0 Hz), 7.19 (d, 2H, J = 7.9 Hz), 5.51 (m, 1 H),

5.48 (d, 1 H, J = 50.7 Hz), 5.04 (d, 1 H, J = 9.2 Hz), 4.99 (d, 1 H, J = 9.7 Hz), 4.32-3.97 (m,

4H), 2.72 (t, 1 H, J = 15.7 Hz), 2.46-2.29 (m, 4H), 1.58 (s, 3H), 1.52 (s, 3H), 1.41 (s, 9H)

ppm. D. (2S,4S)-1-{(2R)-2-amino-3-methyl-3-[(4-methylbenzyl)sulfonyl]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile

To a CH2CI2 (100 mL) solution containing trifluoroacetic acid (10 mL) was added

compound C (1.03 g, 2.14 mmol, 1.0 eq). The resulting pale yellow solution was stirred

at RT for 2 h at which time the solvent was removed in vacuo. The resulting TFA salt was

converted to the free base by addition of a sat. NaHC0₃ solution and extracting the aqueous layer (2X) with EtOAc. The combined organic extracts were dried over Na2S0₄

and concentrated in vacuo. The resulting off-white foam was purified via flash

chromatography using 5% MeOH (with 0.1% NH₃) in CH2CI2 as the mobile phase

affording 577 mg (1.51 mmol, 71% yield) of compound D freebase as a white foam. To

form the HCl salt, the free base was taken up in Et2θ and acetone added until all solids

were in solution. 2.0 M HCl in ether was added dropwise until no more precipitate

formed. The precipitate was then filtered and washed several times with Et∑O. The

resulting salt was dried under high vacuum.

'H NMR (d₄-MeOH) 400 MHz δ 7.38 (d, 2H, J = 8.1 Hz), 7.24 (d, 2H, J = 7.7 Hz), 5.56 (d,

1 H, J = 50.7 Hz), 5.12 (d, 1 H, J = 9.7 Hz), 4.74 (s, 1 H), 4.64 (ABq, 2H, J = 19.7, 13.3 Hz), 4.12 (m, 1 H), 3.94 (m, 1 H), 2.70-2.43 (m, 2H), 2.36 (s, 3H), 1.76 (s, 3H), 1.58 (s, 3H) ppm.

Example 16

(2S,4S)-1-((2R)-2-amino-3-{[4-(benzyloxy)benzyl]sulfonyl}-3-methylbutanoyl)-4-

fluoropyrrolidine-2-carbonitrile.

A. (2R)-3-{[4-(benzyloxy)benzyl]thio}-2-[(tert-butoxycarbonyl)amino]-3-

methylbutanoic acid

To a 1.0 N NaOH (25 mL) solution containing (2R)-2-[(tert-

butoxycarbonyl)amino]-3-mercapto-3-methylbutanoic acid (as described above) (500

mg, 2.01 mmol, 1.0 eq) was added 4-benzyloxybenzyl chloride (515 mg, 2.21 mmol, 1.1

eq). The resulting mixture was allowed to stir at RT for 18 h. The reaction mixture was

then poured into a seperatory funnel and washed with Et2θ (1X) to remove any

unreacted chloride. Concentrated HCl was added to the aqueous layer until the pH

reached 2-3. The aqueous layer was then extracted with Et2θ (2X). The combined organic extracts were dried over MgS0₄and concentrated in vacuo. The resulting white

fluffy solid was used in the next step without further purification. Isolated 700 mg of

compound A (1.57 mmol, 78% yield).

'H NMR (CDCb) 400 MHz δ 7.42-7.31 (m, 5H), 7.24 (d, 2H, J = 8.6 Hz), 6.90 (d, 2H,

J = 8.6 Hz), 5.46 (s(br), 1 H), 5.03 (s, 2H), 4.39 (s(br), 1 H), 3.81 (q, 2H, J = 11.5 Hz), 1.47-

1.41 (m, 15H) ppm.

B. Tert-butyl (l R)-2-{[4-(benzyloxy)benzyl]thio}-1 -{[(2S,4S)-2-cyano-4-

fluoropyrrolidin-1 -yl]carbonyl}-2-methylpropylcarbamate.

To a DMF solution (20 mL) containing compound A (700 mg, 1.57 mmol, 1.2 eq) was

added N,N-diisopropylethylamine (308 μL, 1.77 mmol, 1.35 eq) followed by HATU (847

mg, 2.23 mmol, 1.7 eq). The resulting amber solution was stirred at RT for 20 min. To

this stirring solution was added a solution of (2S, 4S)-4-fluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate (375 mg, 1.31 mmol, 1.0 eq) and N,N-diisopropylethylamine

(251 μL, 1.44 mmol, 1.1 eq) in DMF (10 mL). The resulting solution was stirred at RT for

18 h and quenched with a sat. NaHC0₃ solution (ca 5 mL). The cloudy solution was then

poured into H2O (ca 20 mL) and extracted with EtOAc (3 x 10 mL). The combined

organic extracts were washed with H2O (2X) and brine (IX); dried over MgS0₄ and

concentrated in vacuo. The resulting amber oil was purified via flash chromatography

using 1 :1 hexanes/EtOAc as the mobile phase. Isolated 340 mg (0.628 mmol, 48% yield) of compound B as a white foam.

¹H NMR (CDCb) 400 MHz δ 7.42-7.27 (m, 7H), 6.91 (d, 2H, I = 8.8 Hz), 5.46 (d, 1 H, J =

50.9 Hz), 5.43 (d, 1 H, J = 9.3 Hz), 5.05-5.01 (m, 3H), 4.37 (d, 1 H, J = 9.0 Hz), 4.26 (m, 1H),

4.03 (m, 1 H), 3.80 (s, 2H), 2.71 (t, 1 H, J = 15.2 Hz), 2.36 (m, 1 H), 1.47-1.41 (m, 15H) ppm.

C. Tert-butyl (l R)-2-{[4-(benzyloxy)benzyl]sulfonyl}-1-{[(2S,4S)-2-cyano-4-

fluoropyrrolidin-1 -yl]carbonyl}-2-methylpropylcarbamate.

To a CHCb (30 mL) solution containing compound B (340 mg, 0.628 mmol, 1.0 eq) was

added 3-chloroperoxybenzoic acid (1.08 g, 6.28 mmol, 10 eq). The resulting mixture was

stirred at RT for 18 h. The reaction was then quenched with 1.0 N NaOH (ca 5 mL) and

the layers separated. The organic layer was dried over MgS0₄ and concentrated in

vacuo. The resulting white fluffy solid was used in the next step without further

purification. Isolated 280 mg of compound C (0.488 mmol, 78% yield).

^ NMR (CDCb) 400 MHz δ 7.43-7.32 (m, 7H), 7.00 (d, 2H, J = 8.8 Hz), 5.51 (s(br), 1 H),

5.48 (d, 1 H, J = 50.7 Hz), 5.06-4.97 (m, 4H), 4.34-3.99 (m, 4H), 2.73 (t, 1 H, J = 15.2 Hz),

2.38 (m, 1 H), 1.59 (s, 3H), 1.53 (s, 3H), 1.42 (s, 9H) ppm. D. (2S,4S)-1-((2R)-2-Amino-3-{[4-(benzyloxy)benzyl]sulfonyl}-3-methylbutanoyl)-4-

fluoropyrrolidine-2-carbonitrile.

To a CH2CI2 (27 mL) solution containing trifluoroacetic acid (3 mL) was added compound

C (280 mg, 0.488 mmol, 1.0 eq). The resulting pale yellow solution was stirred at RT for

2 h at which time the solvent was removed in vacuo. The resulting TFA salt was

converted to the free base by addition of a sat. NaHC0₃ solution and extracting the

aqueous layer (2X) with EtOAc. The combined organic extracts were dried over Na2S0₄

and concentrated in vacuo. The resulting off-white foam was purified via flash chromatography using 5% MeOH (with 0.1% NH3) in CH2CI2 as the mobile phase

affording 182 mg (0.385 mmol, 79% yield) of compound D freebase as a white foam. To

form the HCl salt, the free base was taken up in Et∑O and acetone added until all solids

were in solution. 2.0 M HCl in ether was added dropwise until no more precipitate

formed. The precipitate was then filtered and washed several times with Et∑O. The

resulting salt was dried under high vacuum.

¹H NMR (d₄-MeOH) 400 MHz δ 7.44-7.29 (m, 7H), 7.05 (d, 2H, J = 8.8 Hz), 5.56 (d, 1 H, J =

50.0 Hz), 5.1 1-5.09 (m, 3H), 4.72 (s, 1 H), 4.61 (ABα, 2H, J = 18.1 , 13.5 Hz), 4.13 (m, 1 H),

3.91 (m, 1 H), 2.70-2.42 ( , 2H), 1.75 (s, 3H), 1.58 (s, 3H) ppm.

Example 17

(2S,4S)-1-{(2R)-2-amino-3-[(4-cyanobenzyl)sulfonyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile. A. (2R)-2-[(tert-butoxycarbonyI)amino]-3-[(4-cyanobenzyl)thio]-3-methylbutanoic acid

To a 1.0 N NaOH (25 mL) solution containing (2R)-2-[(tert-

butoxycarbonyl)amino]-3-mercapto-3-methylbutanoic acid (as described above) (500

mg, 2.01 mmol, 1.0 eq) was added 4-cyanobenzyl bromide (433 mg, 2.21 mmol, 1.1 eq).

The resulting mixture was allowed to stir at RT for 18 h. The reaction mixture was then

poured into a seperatory funnel and washed with Et₂0 (1X) to remove any unreacted

chloride. Concentrated HCl was added to the aqueous layer until the pH reached 2-3. The aqueous layer was then extracted with Et.20 (2X). The combined organic extracts

were dried over MgSU4and concentrated in vacuo. The resulting white fluffy solid was

used in the next step without further purification. Isolated 792 mg of compound A

(1.72 mmol, 86% yield).

B. Tert-butyl (l R)-2-[(4-cyanobenzyl)thio]-1 -{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-

yl]carbonyl}-2-methylpropylcarbamate. To a DMF solution (25 mL) containing compound A (792 mg, 1.72 mmol, 1.2 eq) was

added N,N-diisopropylethylamine (337 μL, 1.93 mmol, 1.35 eq) followed by HATU (924

mg, 2.43 mmol, 1.7 eq). The resulting amber solution was stirred at RT for 20 min. To

this stirring solution was added a solution of (2S, 4S)-4-fluoro-2-pyrrolidinecarbonitrile

4-methylbenzenesulfonate (409 mg, 1.43 mmol, 1.0 eq) and N,N-diisopropylethylamine

(274 μL, 1.57 mmol, 1.1 eq) in DMF (10 mL). The resulting solution was stirred at RT for

18 h and quenched with a sat. NaHC0₃ solution (ca 5 mL). The cloudy solution was then

poured into H2O (ca 20 mL) and extracted with EtOAc (3 x 10 ml). The combined organic

extracts were washed with H2O (2X) and brine (1X); dried over MgSU4 and concentrated

in vacuo. The resulting amber oil was purified via flash chromatography using 1 :1 hexanes/EtOAc as the mobile phase. Isolated 572 mg (1.24 mmol, 87% yield) of compound B as a white foam.

Η NMR (CDCb) 400 MHz δ 7.59 (d, 2H, I = 8.4 Hz), 7.50 (d, 2H, J = 8.4 Hz), 5.50 (d, 1 H, J

= 50.5 Hz), 5.42 (d, 1 H, J = 9.7 Hz), 5.03 (d, 1 H, J = 9.5 Hz), 4.41 -4.30 (m, 2H), 4.04 (m,

1 H), 3.88 (s, 2H), 2.75 (t, 1 H, I = 15.7 Hz), 2.39 (m, 1 H), 1.46-1.41 (m, 15H) ppm.

C. Tert-butyl (l R)-2-[(4-cyanobenzyl)sulfonyl]-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-

1-yl]carbonyl}-2-methylpropylcarbamate.

To a CHCb (50 mL) solution containing compound B (572 mg, 1.24 mmol, 1.0 eq) was

added 3-chloroperoxybenzoic acid (2.14 g, 12.4 mmol, 10 eq). The resulting mixture was stirred at RT for 18 h. The reaction was then quenched with 1.0 N NaOH (ca 5 mL) and

the layers separated. The organic layer was dried over MgSU4 and concentrated in

vacuo. The resulting white fluffy solid was used in the next step without further

purification. Isolated 487 mg of compound C (0.989 mmol, 80% yield).

'H NMR (CDCb) 400 MHz δ 7.70 (d, 2H, J = 8.5 Hz), 7.57 (d, 2H, J = 8.4 Hz), 5.53 (s(br),

1 H), 5.51 (d, 1 H, J = 50.7 Hz), 5.04 (d, 1 H, J = 9.9 Hz), 4.99 (d, 1 H, J = 9.8 Hz), 4.43-4.29 (m, 3H), 4.04 (m, 1 H), 2.77 (t, 1 H, J = 15.0 Hz), 2.42 (m, 1 H), 1.61 (d, 6H, J = 5.8 Hz), 1.44

(s, 9H) ppm.

D. (2S,4S)-1 -{(2R)-2-Amino-3-[(4-cyanobenzyl)sulfonyl]-3-methylbutanoγl}-4-

fluoropyrrolidine-2-carbonitrile.

To a CH2CI2 (54 mL) solution containing trifluoroacetic acid (6 mL) was added compound

C (487 mg, 0.989 mmol, 1.0 eq). The resulting pale yellow solution was stirred at RT for

2 h at which time the solvent was removed in vacuo. The resulting TFA salt was converted to the free base by addition of a sat. NaHCOa solution and extracting the

aqueous layer (2X) with EtOAc. The combined organic extracts were dried over Na₂S0₄

and concentrated in vacuo. The resulting off-white foam was purified via flash

chromatography using 5% MeOH (with 0.1% NH₃) in CH2CI2 as the mobile phase

affording 285 mg (0.726 mmol, 73% yield) of compound D freebase as a white foam. To

form the HCl salt, the free base was taken up in Et2θ and acetone added until all solids

were in solution. 2.0 M HCl in ether was added dropwise until no more precipitate

formed. The precipitate was then filtered and washed several times with Et∑O. The resulting salt was dried under high vacuum.

¹H NMR (D2O) 400 MHz δ 7.72 (d, 2H, J = 8.6 Hz), 7.51 (d, 2H, J = 8.5 Hz), 5.49 (d, 1 H, J =

50.6 Hz), 5.02 (d, 1 H, J = 9.7 Hz), 4.72-4.67 (m, 3H), 4.08 (m, 1 H), 3.85 (m, 1 H), 2.66-2.35

(m, 2H), 1.66 (s, 3H), 1.51 (s, 3H) ppm.

Example 18

(2S,4S)-1-((2R)-2-amino-3-methyl-3-{[4-(methylsulfonyl)benzyl]sulfonyl}butanoyl)-4-

fluoropyrrolidine-2-carbonitrile.

A. (2R)-2-[(tert-butoxycarbonyl)amino]-3-methyl-3-{[4-

(methylsulfonyl)benzyl]thio}butanoic acid

To a 1.0 N NaOH (25 mL) solution containing (2R)-2-[(tert-

butoxycarbonyl)amino]-3-mercapto-3-methylbutanoic acid (as described above) (500

mg, 2.01 mmol, 1.0 eq) was added 4-methylsulfonylbenzyl chloride (452 mg, 2.21 mmol, 1.1 eq). The resulting mixture was allowed to stir at RT for 18 h. The reaction mixture

was then poured into a seperatory funnel and washed with Et∑O (1X) to remove any

unreacted chloride. Concentrated HCl was added to the aqueous layer until the pH

reached 2-3. The aqueous layer was then extracted with Et2θ (2X). The combined

organic extracts were dried over MgSθ4and concentrated in vacuo. The resulting white

fluffy solid was used in the next step without further purification. Isolated 768 mg of

compound A (1.84 mmol, 92% yield).

Η NMR (CDCb) 400 MHz δ 7.87 (d, 2H, I = 8.5 Hz), 7.54 (d, 2H, J = 8.5 Hz), 5.39

(s(br), 1 H), 4.44 (s(br), 1 H), 3.88 (s, 2H), 3.03 (s, 3H), 1.46-1.43 (m, 15H) ppm.

B. Tert-butyl (l R)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]carbonyl}-2-methyl-2-

{[4-(methylsulfonyl)benzyl]thio}propylcarbamate.

To a DMF solution (25 mL) containing compound A (768 mg, 1.84 mmol, 1.2 eq) was

added N.N-diisopropylethylamine (361 μL, 2.07 mmol, 1.35 eq) followed by HATU (989

mg, 2.60 mmol, 1.7 eq). The resulting amber solution was stirred at RT for 20 min. To

this stirring solution was added a solution of (2S, 4S)-4-fluoro-2-pyrrolidinecarbonitrile

4-methylbenzenesulfonate (438 mg, 1.53 mmol, 1.0 eq) and N.N-diisopropylethylamine

(293 μL, 1.68 mmol, 1.1 eq) in DMF (10 mL). The resulting solution was stirred at RT for

18 h and quenched with a sat. NaHCOs solution (ca 5 mL). The cloudy solution was then

poured into H2O (ca 20 mL) and extracted with EtOAc (3 x 10 mL). The combined

organic extracts were washed with H2O (2X) and brine (1X); dried over MgS04 and

concentrated in vacuo. The resulting amber oil was purified via flash chromatography

using 1 :1 hexanes/EtOAc as the mobile phase. Isolated 683 mg (1.33 mmol, 87% yield)

of compound B as a white foam. 'H NMR (CDCb) 400 MHz δ 7.86 (d, 2H, J = 8.5 Hz), 7.58 (d, 2H, J = 8.3 Hz), 5.50 (d, 1 H, J

= 50.9 Hz), 5.43 (d, 1 H, J = 9.9 Hz), 4.43-4.30 (m, 2H), 4.03 (m, I H), 3.90 (s, 2H), 3.02 (s,

3H), 2.74 (t, 1 H, J = 15.6 Hz), 2.39 (m, 1 H), 1.47-1.41 (m, 15H) ppm.

C. Tert-butyl (l R)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1 -yl]carbonyl}-2-methyl-2-

{[4-(methylsulfonyl)benzyl]sulfonyl}propylcarbamate.

To a CHCb (50 mL) solution containing compound B (683 mg, 1.33 mmol, 1.0 eq) was

added 3-chloroperoxybenzoic acid (2.30 g, 13.3 mmol, 10 eq). The resulting mixture was

stirred at RT for 18 h. The reaction was then quenched with 1.0 N NaOH (ca 5 mL) and the layers separated. The organic layer was dried over MgS0₄ and concentrated in

vacuo. The resulting white fluffy solid was used in the next step without further

purification. Isolated 581 mg of compound C (1.06 mmol, 80% yield).

'H NMR (CDCb) 400 MHz δ 7.98 (d, 2H, J = 8.6 Hz), 7.66 (d, 2H, 1 = 8.5 Hz), 5.53 (s, 1 H),

5.51 (d, 1 H, J = 50.5 Hz), 5.08 (d, 1 H, J = 10.0 Hz), 4.99 (d, 1 H, J = 9.5 Hz), 4.44-4.32 (m,

2H), 4.03 (m, 1 H), 3.06 (s, 3H), 2.77 (t, 1 H, J = 16.0 Hz), 2.41 (m, 1 H), 1.63 (s, 3H), 1.60 (s,

3H), 1.43 (s, 9H) ppm.

D. (2S,4S)-1-((2R)-2-Amino-3-methyl-3-{[4-(methylsulfonyl)benzyl]sulfonyl}butanoyl)-

4-fluoropyrrolidine-2-carbonitrile.

To a CH2CI2 (63 mL) solution containing trifluoroacetic acid (7 mL) was added compound

C (581 mg, 1.06 mmol, 1.0 eq). The resulting pale yellow solution was stirred at RT for 2

h at which time the solvent was removed in vacuo. The resulting TFA salt was converted

to the free base by addition of a sat. NaHCOs solution and extracting the aqueous layer

(2X) with EtOAc. The combined organic extracts were dried over Na2Sθ and concentrated in vacuo. The resulting off-white foam was purified via flash

chromatography using 5% MeOH (with 0.1% NH₃) in CH2CI2 as the mobile phase affording 296 mg (0.664 mmol, 63% yield) of compound D freebase as a white foam. To

form the HCl salt, the free base was taken up in Et2θ and acetone added until all solids

were in solution. 2.0 M HCl in ether was added dropwise until no more precipitate

formed. The precipitate was then filtered and washed several times with Et2θ. The resulting salt was dried under high vacuum.

'H NMR (D2O) 400 MHz δ 7.91 (d, 2H, J = 8.1 Hz), 7.63 (d, 2H, J = 8.2 Hz), 5.49 (d, 1 H, J =

50.3 Hz), 5.02 (d, 1 H, J = 9.7 Hz), 4.77-4.68 (m, 3H), 4.09 (m, 1 H), 3.85 (m, 1 H), 3.14 (s,

3H), 2.66-2.36 (m, 2H), 1.67 (s, 3H), 1.53 (s, 3H) ppm.

Example 19

(2S,4S)-1-{(2S)-2-Amino-2-[l-(4-fluorobenzyl)cyclopentyl]ethanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride.

A. 1 -(4-Fluorobenzyl)cyclopentanecarbonitrile.

To a stirred solution of 4-fluorobenzyl bromide (10.0 g, 52.9 mmol) in toluene (100 mL)

was added cyclopentanecarbonitrile (6.1 mL, 58.2 mmol). The mixture was stirred

thoroughly and cooled to 0 °C. A solution of 0.5 M KHMDS in toluene (159 mL, 79.35

mmol) was added slowly via an addition funnel, and the reaction mixture was then

warmed to RT and stirred for 14 hours. The reaction mixture was then quenched with 1.0 M HCl until pH <7. The layers were separated and aqueous layer was re-extracted

with EtOAc. The combined organic layers were washed with saturated NaHC0₃, water,

brine, dried over MgS04, filtered, and concentrated in vacuo. Purification via silica gel

chromatography (10:1 hexanes: EtOAc) afforded 8.61 g (80% yield) of compound A as a yellow liquid.

'H NMR (CDCb) 400 MHz δ 7.28-7.24 (m, 2H), 7.01 (t, J=8.7 Hz, 2H), 2.84 (s, 2H), 2.07-

2.01 (m, 2H), 1.89-1.66 (m, 6H) ppm.

B. 1-(4-Fluorobenzyl)cyclopentanecarbaldehyde.

A stirred solution of compound A (8.61 g, 42.36 mmol) in toluene (150 mL) was

cooled to -78 °C. Neat diisobutylaluminum hydride (DIBAL, 1 1.3 mL, 63.54 mmol) was

added dropwise via syringe and the reaction mixture was stirred at -78°C for 2 hours. A

solution of THF (100 mL), water (30 mL), acetic acid (8 mL), and sodium acetate (7 g) was

carefully added and the reaction mixture was warmed to RT and stirred one additional

hour. After adding Et2θ, the biphasic mixture was vacuum filtered through a pad of

Celite, poured into a separatory funnel, and the aqueous layer drawn off. The organic

layer was washed with saturated NaHC0₃, water, brine, dried over MgSθ4, filtered, and

concentrated in vacuo. Purification via silica gel chromatography (10:1 hexanes: EtOAc)

afforded 6.55 g (75% yield) of compound B as a colorless liquid.

'H NMR (CDCb) 400 MHz δ 9.53 (s, 1 H), 7.07 (dd, J=8.4, 5.5 Hz, 2H), 6.93 (t, .7=8.7

Hz, 2H), 2.88 (s, 2H), 1.93-1.87 (m, 2H), 1.67-1.48 (m, 6H) ppm. C. Amino[1 -(4-fluorobenzyl)cyclopentyl]acetonitrile.

To a stirred solution of compound B (6.55 g, 31.76 mmol) in methanol (30 mL) and water (18 mL) was added NH4OH (-30% ammonia content, 4.4 mL, 33.35 mmol),

KCN (2.17 g, 33.35 mmol), and NH₄CI (1.87 g, 34.94 mmol). The cloudy reaction mixture

was heated to 70°C for 14 hours. After cooling to RT, the reaction mixture was diluted

with EtOAc and washed with saturated NaHC0₃, water, brine, dried over MgSθ4, filtered,

and concentrated in vacuo. Flash chromatography over silica gel (4:1 hexanes: EtOAc)

afforded 5.99 g (81% yield) of compound C as a colorless, viscous oil.

Η NMR (CDCb) 400 MHz δ 7.23-7.19 (m, 2H), 6.98 (t, .7=8.7 Hz, 2H), 3.54 (d,

.7=3.5 Hz, 1 H), 2.97 (d, .7=13.6 Hz, 1 H), 2.63 (d, J=l 3.7 Hz, 1 H), 2.08 (br s, 2H), 1.75-1.57 (m, 8H) ppm.

D. Amino[l-(4-fluorobenzyl)cyclopentyl]acetic acid hydrochloride.

To a stirred solution of compound C (5.99 g, 25.79 mmol) in glacial acetic acid

(20 mL) was added concentrated HCl (100 mL). The reaction mixture was heated to a

gentle reflux (130 °C) for 16 hours. After cooling to RT, the reaction mixture was

concentrated in vacuo to dryness producing a white solid. The solid was washed with

Et2U on a glass frit via vacuum filtration and dried under high vacuum to produce 6.33 g

(85% yield) of compound D as a white solid.

'H NMR (CD₃0D) 400 MHz δ 7.30 (dd, .7=8.7, 5.4 Hz, 2H), 7.04 (t, J=8.8 Hz, 2H),

3.89 (s, 1 H), 2.85-2.76 (m, 2H), 1.79-1.43 (m, 8H) ppm. E. [(tert-Butoxycarbonyl)amino][1-(4-fluorobenzyl)cyclopentyl]acetic acid.

To a stirred solution of compound D (6.33 g, 21.99 mmol) in 1 ,4-dioxane (100

mL) was added a solution of 1.0 M NaOH in water (76 mL, 75.57 mmol) followed by

water (24 mL). The reaction mixture was stirred at RT for several minutes until all of the

starting material was completely dissolved. Solid di-tert-butyldicarbonate (BOC2O, 8.25

g, 37.78 mmol) was added and the reaction mixture was stirred at RT for 14 hours.

Concentrated HCl was then added slowly until pH < 7 followed by dilution with EtOAc.

The aqueous layer was separated and then re-extracted with two portions of EtOAc. The

combined organic layers were dried over MgSθ4, filtered, and concentrated in vacuo to

produce 7.95 g (90% yield) of compound E as a white foam.

Η NMR (CDCb) 400 MHz δ 7.20 (dd, .7=8.6, 5.5 Hz, 2H), 6.96 (t, J=8.7 Hz, 2H),

5.00 (d, .7=9.1 Hz, I H), 4.28 (d, J=9.0 Hz, I H), 2.72-2.64 (m, 2H), 1.63-1.35 (m, 8H), 1.45

(s, 9H) ppm.

F. tert-Butyl (lS)-2-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]-1 -[1-(4-

fluorobenzyl)cyclopentyl]-2-oxoethylcarbamate.

To a stirred solution of compound E (1.90 g, 5.41 mmol) in DMF (54 mL) was added (2S,4S)-4-fluoropyrrolidine-2-carbonitrile 4-methylbenzenesulfonate (1.55 g, 5.41

mmol), HATU (2.06 g, 5.41 mmol), and diisopropylethylamine (2.83 mL, 16.23 mmol). The

reaction mixture was stirred at RT for 16 hours. After adding water (50 mL), the reaction

mixture was extracted with five portions of EtOAc. The combined extracts were washed

with water, saturated NH4CI, saturated NaHC0₃, brine, dried over MgSOvfiltered, and

concentrated in vacuo. The two diastereomers were separated via flash chromatography

over silica gel (2:1 hexanes: EtOAc) to afford 586 mg (25% yield of the desired diastereomer) of compound F as a colorless oil. Compound F was the more polar (lower

Rf) of the two diastereomers.

'H NMR (CDCb) 400 MHz δ 7.20-7.16 (m, 2H), 7.00 (t, .7=8.7, 2H), 5.22 (br d,

.7=51.1 Hz, 1 H), 5.15 (d, .7=5.9 Hz, 1 HJ. 4.93 (d, J=9.3 Hz, 1 H), 4.26 (d, .7=9.7 Hz, 1 H), 3.77-

3.63 (m, 1 H), 3.28-3.19 (m, 1 H), 2.78-2.52 (m, 4H), 2.32-2.1 1 (m, 2H), 1.73-1.52 (m, 6H), 1.42 (s, 9H) ppm.

G. (2S,4S)-1-{(2S)-2-Amino-2-[1-(4-fluorobenzyl)cyclopentyl]ethanoyl}-4-

fluoropyrrolidine-2-carbonitrile.

To a stirred solution of compound F (586 mg, 1.31 mmol) in CH2CI2 (13 mL) was added TFA (1.00 mL, 13.1 mmol). The reaction mixture was stirred at RT for 14 hours.

After concentration in vacuo, the reaction mixture was re-dissolved in EtOAc and

washed with saturated NaHC0₃, dried over MgSU4, filtered, and concentrated in vacuo.

Purification via silica gel flash chromatography (5% MeOH (with 2% NH₃) in CH2CI2)

afforded 160 mg (35% yield) of compound G as a white solid.

'H NMR (CDCb) 400 MHz δ 7.21-7.17 (m, 2H), 6.99 (t, J=8.6 Hz, 2H), 5.24 (br d,

.7=51.1 Hz, 1 H), 4.93 (d, .7=9.3 Hz, 1 H), 3.53-3.40 (m, 1 H), 3.34 (s, 1 H), 3.29-3.20 (m, 1 H),

2.92 (d, .7=13.6 Hz, 1 H), 2.66 (d, .7=13.7 Hz, 1 H), 2.63-2.49 (m, 2H), 2.31-1.32 (m, 8H) ppm.

H. (2S,4S)-1 -{(2S)-2-Amino-2-[1-(4-fluorobenzyl)cyclopentyl]ethanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride.

To a stirred solution of compound G (160 mg, 0.460 mmol) in Et2θ (5 mL) was added a

solution of 2.0 M HCl in Et∑O (1.0 mL). The reaction mixture was stirred at RT for 5

minutes, during which time a white solid precipitated. The solid was collected via vacuum filtration on a glass frit and dried overnight under high vacuum to give 139 mg

(79% yield) of compound H as a white solid.

'H NMR (CD₃0D) 400 MHz δ 7.31 -7.27 (m, 2H), 7.13 (t, .7=8.8 Hz, 2H), 5.30 (br d,

.7=51.1 , 1 H), 5.01 (d, .7=9.5 Hz, 1 H), 4.04 (s, 1 H), 3.56-3.43 (m, 1 H), 3.20-3.1 1 (m, 1 H), 2.90

(d, J=13.9 Hz, 1 H), 2.73 (d, .7=14.0 Hz, 1 H), 2.60-2.30 (m, 2H), 2.14-1.37 (m, 8H) ppm.

(2S,4S)-1-((2S)-2-Amino-2-{l-[4-(trifluoromethyl)phenyl]cyclopentyl}ethanoyl)-4-

fluoropyrrolidine-2-carbonitrile hydrochloride.

A. 1 -[4-(Trifluoromethyl)phenyl]cyclopentanecarbonitrile.

To a stirred solution of 4-fluorobenzotrifluoride (5.0 g, 30.47 mmol) in toluene (40 mL)

was added cyclopentanecarbonitrile (10.5 mL, 100.55 mmol) followed by a solution of

0.5 M KHMDS in toluene (92 mL, 45.71 mmol). The reaction mixture was heated to 70

°C and stirred for 14 hours. After cooling to RT, the reaction mixture was quenched

with 1 M HCl until pH < 7. The layers were separated and the organic layer was washed

with saturated NaHCOs, water, brine, dried over MgS04, filtered, and concentrated in

vacuo. Purification via silica gel chromatography (6:1 hexanes: EtOAc) afforded 7.53 g of

a mixture of compound A and residual cyclopentanecarbonitrile. Because these two compounds were not easily separable by chromatography, this mixture was carried directly to the next step.

B. 1-[4-(Trifluoromethyl)phenyl]cyclopentanecarbaldehyde.

A stirred solution of compound A (7.53 g as described above) in toluene (100 mL)

was cooled to -78°C. A solution of 1.5 M diisobutylaluminum hydride in toluene (DIBAL,

32.0 mL, 47.21 mmol) was added slowly via syringe and the reaction mixture was stirred

at -78 °C for 2 hours. A solution of THF (100 mL), water (20 mL), acetic acid (6 mL), and

sodium acetate (6.6 g) was carefully added and the reaction mixture was warmed to RT

and stirred one additional hour. After adding Et2θ, the biphasic mixture was vacuum

filtered through a pad of Celite, poured into a separatory funnel, and the aqueous layer

drawn off. The organic layer was washed with saturated NaHC0₃, water, brine, dried

over MgS04, filtered, and concentrated in vacuo. Purification via silica gel

chromatography (10:1 hexanes: EtOAc) afforded 3.91 g (53% yield over the last two

steps) of compound B as a colorless liquid.

¹H NMR (CDCb) 400 MHz δ 9.41 (s, 1 H), 7.61 (d, J=8.1 Hz, 2H), 7.38 (d, 7=8.2 Hz,

2H), 2.58-2.52 (m, 2H), 1.93-1.65 (m, 6H) ppm.

C. Amino{1-[4-(trifluoromethyl)phenyl]cyclopentyl}acetonitrile.

To a stirred solution of compound B (3.91 g, 16.14 mmol) in methanol (20 mL)

and water (12 mL) was added NH₄0H (~30% ammonia content, 2.20 mL, 16.95 mmol),

KCN (1.1 1 g, 16.95 mmol), and NH₄CI (950 mg, 17.76 mmol). The cloudy reaction

mixture was heated to 70 °C for 6 hours. After cooling to RT, the reaction mixture was

diluted with EtOAc and washed with saturated NaHCOs, water, brine, dried over MgS0₄, filtered, and concentrated in vacuo. Flash chromatography over silica gel (2:1

hexanes:EtOAc) afforded 2.16 g (50% yield) of compound C as a white solid.

¹H NMR (CDCb) 400 MHz δ 7.63 (d, 7=8.4 Hz, 2H), 7.54 (d, .7=8.4 Hz, 2H), 3.74 (s,

1 H), 2.43-2.36 (m, 1 H), 2.32-2.25 (m, 1 H), 2.15-2.08 (m, 1 H), 2.01-1.93 (m, 1 H), 1.85-1.72 (m, 4H), 1.54 (br s, 2H) ppm.

D. Amino{1-[4-(trifluoromethyl)phenyl]cyclopentyl}acetic acid hydrochloride.

To a stirred solution of compound C (2.16 g, 8.05 mmol) in glacial acetic acid (10

mL) was added concentrated HCl (50 mL). The reaction mixture was heated to a gentle

reflux (130 °C) for 16 hours. After cooling to RT, the reaction mixture was concentrated

in vacuo to dryness producing a white solid. The solid was dried under high vacuum to

produce 2.61 g (100% yield) of compound D as a white solid.

¹H NMR (d₄-MeOH) 400 MHz δ 7.69 (d, .7=8.4 Hz, 2H), 7.60 (d, 7=8.4 Hz, 2H), 4.20

(d, 7=2.4 Hz, 1 H), 2.42-2.34 (m, 2H), 2.27-2.19 (m, 1 H), 2.14-2.07 (m, 1 H), 1.86-1.79 (m,

2H), 1.61-1.51 (m, 2H) ppm.

E. [(tert-Butoxycarbonyl)amino]{1-[4-(trifluoromethyl)phenyl]cyclopentyl}acetic acid.

To a stirred solution of compound D (2.61 g, 8.05 mmol) in 1 ,4-dioxane (20 mL)

and water (20 mL) was added solid NaOH (1.08 g, 27.06 mmol). After stirring at RT for

several minutes, solid di-tert-butyldicarbonate (BOC2O, 3.94 g, 18.04 mmol) was added

and the reaction mixture was stirred at RT for 14 hours. Concentrated HCl was then

added slowly until pH < 7 followed by dilution with EtOAc. The aqueous layer was

separated, saturated with NaCl, and then re-extracted with two portions of EtOAc. The combined organic layers were dried over MgSU4, filtered, and concentrated in vacuo to

produce 3.01 g (86% yield) of compound E as a colorless, viscous oil.

Η NMR (CDCb) 400 MHz δ 7.55 (d, 7=7.8 Hz, 2H), 7.40 (d, 7=8.2 Hz, 2H), 4.85 (d,

7=8.5 Hz, 1 H), 4.55 (d, 7=8.5 Hz, 1 H), 2.43-1.59 (m, 8H), 1.42 (s, 9H) ppm.

F. tert-Butyl (lS)-2-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1 -yl]-2-oxo-1-{l-[4-

(trifluoromethyl)phenyl]cyclopentyl}ethylcarbamate.

To a stirred solution of compound E (1.01 g, 2.61 mmol) in DMF (26 mL) was

added (2S,4S)-4-fluoropyrrolidine-2-carbonitrile 4-methylbenzenesulfonate (747 mg,

2.61 mmol), HATU (993 mg, 2.61 mmol), and diisopropylethylamine (1.40 mL, 7.83

mmol). The reaction mixture was stirred at RT for 16 hours. After adding water (20 mL),

the reaction mixture was extracted with five portions of EtOAc. The combined extracts were washed with water, 1.0 M NaHS0₄, saturated NaHC0₃, brine, dried over MgSθ4,

filtered, and concentrated in vacuo. Purification via silica gel chromatography (1 :1

hexanes: EtOAc) produced 442 mg (35% yield) of the product as a mixture of

diastereomers. Further silica gel chromatography (2:1 hexanes: EtOAc) separated the

diastereomers and afforded 145 mg of compound F as a white foam. Compound F was

the more polar (lower Rf) of the two diastereomers.

'H NMR (CDCb) 400 MHz δ 7.63 (d, 7=8.4 Hz, 2H), 7.56 (d, 7=8.6 Hz, 2H), 5.27 (d,

7=9.7 Hz, 1 H), 5.08 (br d, 7=50.9 Hz, 1 H), 4.84 (d, 7=9.5 Hz, 1 H), 4.49 (d, 7=9.9 Hz, 1 H),

3.63-3.50 (m, 1 H), 2.77 (dd, 7=23.6, 12.0 Hz, 1 H), 2.48 (t, 7=15.5 Hz, 1 H), 2.25-2.20 (m,

2H), 2.10-2.03 (m, 2H), 1.77-1.71 (m, 2H), 1.52-1.48 (m, 2H), 1.41 (s, 9H) ppm. G. (2S,4S)-1 -((2S)-2-Amino-2-{l-[4-(trifluoromethyl)phenyl]cyclopentyl}ethanoyl)-4-

fluoropyrrolidine-2-carbonitrile.

To a stirred solution of compound F (145 mg, 0.30 mmol) in CH2CI2 (3 mL) was

added TFA (0.1 16 mL, 1.50 mmol). The reaction mixture was stirred at RT for 14 hours.

After concentration in vacuo, the reaction mixture was re-dissolved in EtOAc and

washed with saturated NaHC0₃, dried over MgS04, filtered, and concentrated in vacuo.

Purification via silica gel flash chromatography (5% MeOH (with 2% NH3) in CH2CI2)

afforded 62 mg (54% yield) of compound G as a white solid.

'H NMR (CDCb) 400 MHz δ 7.62 (d, 7=8.2 Hz, 2H), 7.54 (d, 7=8.6 Hz, 2H), 5.12 (br

d, 7=51.1 Hz, 1 H), 4.86 (d, 7=9.5 Hz, 1 H), 3.57 (s, 1 H), 3.55-3.42 (m, 1 H), 2.97-2.88 (m,

1H), 2.50 (t, 7=15.2 Hz, I H), 2.24-2.10 (m, 5H), 1.99 (br s, 2H), 1.76-1.71 (m, 2H), 1.54- 1.41 (m, 2H) ppm.

H. (2S,4S)-1 -((2S)-2-Amino-2-{l -[4-(trifluoromethyl)phenyl]cyclopentyl}ethanoyl)-4-

fluoropyrrolidine-2-carbonitrile hydrochloride.

Diethyl ether (3 mL) was added to a flask containing compound G (51 mg, 0.133 mmol).

A few drops of acetone were added to allow the solution to become homogeneous. A

solution of 2.0 M HCl in Et∑O (1.0 mL) was added and the reaction mixture was stirred at

RT for 5 minutes. The solvent was removed in vacuo and the resulting solid was dried

overnight under high vacuum to afford 55 mg (98% yield) of compound H as a white

solid.

'H NMR (D2O) 400 MHz δ 7.62 (d, 7=8.8 Hz, 2H), 7.58 (d, 7=9.0 Hz, 2H), 5.13 (br d,

7=51.1 Hz, 1 H), 4.86 (d, 7=9.5 Hz, 1H), 4.37 (s, 1 H), 3.54-3.40 (m, 1 H), 2.95-2.86 (m, IH),

2.44-2.18 (m, 4H), 1.92-1.84 (m, 2H), 1.64-1.58 (m, 2H), 1.33-1.25 (m, 2H) ppm.

(2S,4S)-1-{(2S)-2-Amino-2-[1-(4-fluorobenzyl)cyclopropyl]ethanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride

A. 1-(4-Fluorobenzyl)cyclopropanecarbonitrile.

A stirred solution of cyclopropanecarbonitrile (4.3 mL, 58.2 mmol) in toluene (100 mL)

was cooled to 0 °C. A solution of 0.5 M KHMDS in toluene (159 mL, 79.35 mmol) was

added slowly via an addition funnel and the reaction mixture was stirred at 0 °C for 30

minutes. A solution of 4-fluorobenzyl bromide (10.0 g, 52.9 mmol) in toluene (20 mL)

was then added dropwise at 0 °C. The reaction mixture was warmed to RT and stirred

for 3 hours. The reaction mixture was then quenched with 1.0 M HCl until pH < 7. The

layers were separated and aqueous layer was re-extracted with EtOAc. The combined

organic layers were washed with saturated NaHCCb, water, brine, dried over MgS0₄,

filtered, and concentrated in vacuo. Purification via silica gel chromatography (5%

EtOAc in hexanes) afforded 7.76 g (84% yield) of compound A as a yellow liquid.

¹H NMR (CDCb) 400 MHz δ 7.25-7.22 (m, 2H), 7.03 (t, 7=8.7 Hz, 2H), 2.77 (s, 2H),

1.28 (dd, 7=7.1 , 5.1 Hz, 2H), 0.94 (dd, 7=7.1 , 5.1 Hz, 2H) ppm. B . 1 -(4-Fluorobenzyl)cyclopropanecarbaldehyde.

A stirred solution of compound A (7.76 g, 44.29 mmol) in toluene (150 mL) was

cooled to -78 °C. Neat diisobutylaluminum hydride (DIBAL, 1 1.8 mL, 66.43 mmol) was

added dropwise via syringe and the reaction mixture was stirred at -78 °C for 2 hours.

A solution of THF (100 mL), water (40 mL), acetic acid (8 mL), and sodium acetate (7.5 g)

was carefully added and the reaction mixture was warmed to RT and stirred one

additional hour. After adding Et∑O, the biphasic mixture was vacuum filtered through a

pad of celite, poured into a separatory funnel, and the aqueous layer drawn off. The

organic layer was washed with saturated NaHC0₃, water, brine, dried over MgSθ4,

filtered, and concentrated in vacuo. Purification via silica gel chromatography (5%

EtOAc in hexanes) afforded 7.20 g (91% yield) of compound B as a colorless liquid.

'H NMR (CDCb) 400 MHz δ 8.71 (s, 1 H), 7.16-7.13 (m, 2H), 6.94 (t, 7=8.8 Hz, 2H),

2.96 (s, 2H), 1.20-1.17 (m, 2H), 1.00-0.97 (m, 2H) ppm.

C. Amino[l -(4-fluorobenzyl)cyclopropyl]acetonitrile.

To a stirred solution of compound B (7.20 g, 40.40 mmol) in methanol (40 mL)

and water (25 mL) was added NH4OH (~30% ammonia content, 5.51 mL, 42.42 mmol),

KCN (2.76 g, 42.42 mmol), and NH4CI (2.38 g, 44.44 mmol). The cloudy reaction mixture

was heated to 70 °C for 14 hours. After cooling to RT, the reaction mixture was diluted

with EtOAc and washed with saturated NaHC0₃, water, brine, dried over MgSU4, filtered,

and concentrated in vacuo. Flash chromatography over silica gel (20% EtOAc in

hexanes) afforded 2.21 g (27% yield) of compound C as a colorless, viscous oil. ¹H NMR (CDCb) 400 MHz δ 7.22-7.19 (m, 2H), 6.98 (t, 7=8.6 Hz, 2H), 3.62 (d,

7=3.3 Hz, 1 H), 3.12 (d, 7=14.3 Hz, 1 H), 2.56 (d, 7=14.3 Hz, 1 H), 1.93 (br s, I H). 1.82 (br s. 1 H), 0.78-0.76 (m, 2H), 0.67-0.64 (m, 1 H), 0.54-0.51 (m, 1 H) ppm.

^* D. Amino[l-(4-fluorobenzyl)cyclopropyl]acetic acid hydrochloride.

To a stirred solution of compound C (2.21 g, 10.82 mmol) in glacial acetic acid

(10 mL) was added concentrated HCl (50 mL). The reaction mixture was heated to a

gentle reflux (130 °C) for 16 hours. After cooling to RT, the reaction mixture was

concentrated in vacuo to dryness producing a white solid. The solid was washed with

Et2θ on a glass frit via vacuum filtration and dried under high vacuum to produce 2.81 g

(100% yield) of compound D as a white solid.

' NMR (d₄-MeOH) 400 MHz δ 7.18 (dd, 7=8.8, 5.5 Hz, 2H), 7.01 (t, 7=8.8 Hz, 2H),

3.54 (s, 1 H), 2.84 (d, 7=4.9 Hz, 2H), 0.89-0.84 (m, 1 H), 0.70-0.59 (m, 2H), 0.51-0.47 (m,

l H) ppm.

E. [(tert-Butoxycarbonyl)amino][l-(4-fluorobenzyl)cyclopropyl]acetic acid.

To a stirred solution of compound D (2.81 g, 10.85 mmol) in 1 ,4-dioxane (50 mL)

was added a solution of 1.0 M NaOH in water (33 mL, 33.03 mmol) followed by water

(17 mL). The reaction mixture was stirred at RT for several minutes until all of the

starting material was completely dissolved. Solid di-tert-butyldicarbonate (BOC20, 3.60

g, 16.52 mmol) was added and the reaction mixture was stirred at RT for 14 hours.

Concentrated HCl was then added slowly until pH < 7 followed by dilution with EtOAc.

The aqueous layer was separated and then re-extracted with two portions of EtOAc. The combined organic layers were dried over MgSU4, filtered, and concentrated in vacuo to

produce 4.06 g (1 12% yield) of compound E as a viscous, light yellow oil.

¹H NMR (CDCb) 400 MHz δ 7.19-7.15 (m, 2H), 6.95 (t, 7=8.7 Hz, 2H), 4.98 (d,

7=7.0 Hz, 1 H), 3.98 (d, 7=7.5 Hz, I H), 2.85 (d, 7=14.5 Hz, 1 H), 2.60 (d, 7=14.2 Hz, I H), 1.45

(s, 9H), 0.79-0.72 (m, 2H), 0.51 -0.47 (m, 2H) ppm.

F. tert-Butyl (lS)-2-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]-1-[l -(4-

fluorobenzyl)cyclopropyl]-2-oxoethylcarbamate.

To a stirred solution of compound E (4.06 g, 12.56 mmol) in DMF (125 mL) was

added (2S,4S)-4-fluoropyrrolidine-2-carbonitrile 4-methylbenzenesulfonate (3.60 g,

12.56 mmol), HATU (4.78 g, 12.56 mmol), and diisopropylethylamine (6.60 mL, 37.68

mmol). The reaction mixture was stirred at RT for 16 hours. After adding water (125

mL), the reaction mixture was extracted with four portions of EtOAc. The combined extracts were washed with saturated NH4CI, saturated NaHC0₃, water, brine, dried over

MgSU4, filtered, and concentrated in vacuo. Purification via flash chromatography over

silica gel (1 :1 hexanes: EtOAc) afforded^' 1.29 g (25% yield) of the product as a mixture of diastereomers. Additional silica gel flash chromatography (2:1 hexanes: EtOAc) separated

the diastereomers and afforded 853 mg of compound F as a white foam. Compound F

was the more polar (lower Rf) of the two diastereomers.

'H NMR (CDCb) 400 MHz δ 7.29 (dd, 7=8.6, 5.5 Hz, 2H), 7.03 (t, 7=8.7 Hz, 2H),

5.15 (d, 7=9.3 Hz, 1 H), 5.14 (br d, 7=51.1 Hz, 1 H), 4.76 (d, 7=9.3 Hz, 1 H), 4.42 (d, 7=9.3 Hz,

1 H), 3.29-3.16 (m, 1 H), 3.15-3.1 1 (m, 1 H), 3.07-2.98 (m, 1 H), 2.52 (t, 7=15.2 Hz, 1 H),

2.24-2.17 (m, 1 H), 2.14-2.07 (m, 1 H), 1.44 (s, 9H), 0.86-0.76 (m, 2H), 0.56-0.45 (m, 2H)

ppm. G. (2S,4S)-1-{(2S)-2-Amino-2-[l -(4-fluorobenzyl)cyclopropyl]ethanoyl}-4-

fluoropyrrolidine-2-carbonitrile.

To a stirred solution of compound F (853 mg, 2.03 mmol) in CH2CI2 (20 mL) was

added TFA (1.56 mL, 20.4 mmol). The reaction mixture was stirred at RT for 18 hours.

After concentration in vacuo, the reaction mixture was re-dissolved in EtOAc and washed with saturated NaHC0₃, dried over MgSθ4, filtered, and concentrated in vacuo.

Purification via silica gel flash chromatography (5% MeOH (with 2% NH₃) in CH2CI2)

afforded 401 mg (62% yield) of compound G as a white solid.

'H NMR (CDCb) 400 MHz δ 7.27-7.24 (m, 2H), 7.00 (t, 7=8.6 Hz, 2H), 5.17 (br d,

7=51.3 Hz, 1 H), 4.76 (d, 7=9.3 Hz, 1 H), 3.48 (s, 1 H), 3.32-3.01 (m, 3H), 2.54 (t, 7=15.0 Hz,

1 H), 2.22-2.05 (m, 2H), 1.74 (br s, 2H), 0.79-0.71 (m, 2H), 0.53-0.39 (m, 2H) ppm.

H. (2S,4S)-1-{(2S)-2-Amino-2-[1-(4-fluorobenzyl)cyclopropyl]ethanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride.

Diethyl ether (8 mL) was added to a flask containing compound G (401 mg, 1.26 mmol).

Approximately 1 mL of acetone was added to make the solution homogeneous. A

solution of 2.0 M HCl in Et2U (6.0 mL) was added and the reaction mixture was stirred

for 5 minutes. Removal of the solvent in vacuo produced a white solid that was

triturated with a portion of the HCl/Et₂O solution. The solid was collected via vacuum

filtration on a glass frit, washed with several portions of Et∑O, and dried overnight under

high vacuum to give 376 mg (84% yield) of compound H as a white solid. 'H NMR (D2O) 400 MHz δ 7.29-7.23 (m, 2H), 7.07-7.01 (m, 2H), 5.22 (br d, 7=50.6

Hz, 1 H), 4.82 (d, 7=9.1 Hz, 1 H), 4.10 (s, 1 H), 3.25-2.92 (m, 3H), 2.28 (t, 7=16.0 Hz, 1 H),

2.32-2.21 (m, 2H), 1.16-0.43 (m, 4H) ppm.

Example 22

(2S,4S)-1-[(2R)-2-Amino-3- (benzylsulfonyl)-3-methylbutanoyl]-4-fluoropyrrolidine-2-

carbonitrile hydrochloride

A. (2R)-3-(benzylthio)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoic acid To a 1.0 N KOH solution (50 mL) containing (2R)-2-[(tert-butoxycarbonyl)amino]-3-

mercapto-3-methylbutanoic acid (1 g, 0.00401 mole) was added benzyl bromide (755

mg, 0.00441 mole) at RT. After 17.0 hours the reaction mixture was diluted with water

and washed with diethyl ether. The organics were discarded and the aqueous layer was

cooled to 0-5 °C. The aqueous solution was made acidic (pH 3.5) using concentrated HCl

and the product was extracted into EtOAc. The organics were dried over gSθ4 and

concentrated to dryness to yield a total of 1.1 g (81%) of compound A as a pure oily

solid.

'H NMR (CDCb) 400 MHz δ 7.32-7.20 (m, 5H), 5.44 (d (br), 1 H, 7 = 7.2 Hz), 4.38 (d (br),

1 H, 7 = 8.0 Hz), 3.80 (m, 2H), 1.45- 1.43 (m, 15H) ppm. B. Tert-butyl (l R)-2-(benzylthio)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-

yl]carbonyl}-2-methylpropylcarbamate.

To a DMF solution (20 mL) containing compound A (l .1 g, 0.0032 mole) and N,N-

diisopropylethylamine (1.4 g, 0.0107 mole) was added HATU (1.85 g, 0.0049 mole) at RT.

After 30 minutes the reaction mixture was mixed with (2S, 4S)-4-fluoro-2-

pyrrolidinecarbonitrile 4-methylbenzenesulfonate (1.0g, 0.0035 mole). After stirring

overnight the reaction was quenched with NaHC0₃ and the organics extracted with

EtOAc (2X). The organics were washed with sat. NaCl and dried over MgSθ4. Removal of

the solvent in vacuo and purification via column chromatography (1/1 hexanes/EtOAc)

yielded 445 mg (32%) of compound B.

'H NMR (CDCb) 400 MHz δ 7.36-7.18 (m, 5H), 5.37 (d (br), 1 H), 5.45 (d (br), 1 H, 7=51.6

Hz), 5.00 (d, 1 H, 7 = 9.2 Hz), 4.35 (d, 1 H, 7=8.8), 4.27-3.92 (m, 4H), 2.67-2.59 (m, 1 H),

2.40-2.23 (m, 1 H), 1.46-1.36 (m, 15H) ppm.

C. Tert-butyl (l )-2-(benzylsulfonyl)-1 -{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-

yl]carbonyl}-2-methylpropylcarbamate.

To a CH2CI2 solution (75 mL) containing compound B (445 mg, 0.00102 mole) was added

3-chloroperoxybenzoic acid (57-86%) (1.8g 0.0102 mole). The reaction mixture was

allowed to stir at RT for a period of 16.0 hours followed by neutralization with 2.0 N

KOH. The organics were separated, dried over MgS0₄ and concentrated to dryness to

afford 421 mg of crude compound C (88%). The resulting tan solid was used without

further purification. D. (2S,4S)-1-[(2R)-2-Amino-3- (benzylsulfonyl)-3-methylbutanoyl]-4-fluoropyrrolidine- 2-carbonitrile hydrochloride.

To a CH2CI2 solution (45mL) containing compound C (421 mg, 0.00102 mole) was added

TFA (5 mL). The reaction mixture was allowed to stir at RT for a period of 16.0 hours

followed by neutralization with sat. NaHCOa. The organics were dried over MgS0₄ and

concentrated to dryness to afford a crude solid. The resulting solid was purified using silica gel chromatography (99% CH2CI2/1⁰/0 MeOH with 2.0 M NHs). The pure tan solid

was dissolved into an acetone/ether mixture (1/1) and precipitated using 2.0 N HCl in

ether. The resulting solid was filtered in vacuo and washed with ether. After drying under high vacuum the reaction yielded a total of 96 mg of compound D.

¹H NMR (d₄-MeOH) 400 MHz δ 7.49-7.35 (m, 5H), 5.47 (d (br), I H, 7=50.8 Hz), 5.10 (d,

1 H, 7 = 8.8 Hz), 4.92-4.59 (m, 3H), 4.15-3.91 (m, 2H). 2.71 -2.43 (m, 2H), 1.77 (s, 3H), 1.60 (s, 3H) ppm.

(2S,4S)-1-{(2R)-2-amino-3-[(3-methoxybenzyl)sυlfonyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride.

A. (2R)-2-[(tert-butoxycarbonyl)amino]-3-[(3-methoxybenzyl)thio]-3-methylbutanoic

acid. To a 1.0 N KOH solution (50 mL) containing (2R)-2-[(tert-butoxycarbonyl)amino]-3-

mercapto-3-methylbutanoic acid (1 g, 0.00401 mole) was added 3-methoxybenzyl

chloride (691 mg, 0.00441 mole) at RT. After 17.0 hours the reaction mixture was

diluted with water (50 mL) and washed with diethyl ether. The organics were discarded

and the aqueous layer was cooled to 0-5 °C. The aqueous layer was made acidic (pH 3.5)

using concentrated HCl and extracted into EtOAc. The organics were dried over MgSU4

and concentrated to dryness to yield a total of 1.35 g (91%) of compound A.

^]H NMR (CDCb) 400 MHz δ 7.19 (dd, I H, 7=7.8 Hz), 6.90 (d, I H, 7=7.2 Hz), 6.86 (s, 1 H),

6.76 (dd, 1 H, 7=8.4 Hz), 5.43 (d (br), 1 H, 7 = 6.4 Hz), 4.39 (d (br), 1 H, 7 = 6.8 Hz), 3.82-3.74

(m, 5H), 1.45-1.35 (m, 15H) ppm.

B. Tert-butyl (l R)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]carbonyl}-2-[(3-

methoxybenzyl)thio]-2-methylpropylcarbamate.

To a DMF solution (25 ml) containing compound A (1.3 g, 0.0035 mole) and N,N-

diisopropylethylamine (1.36 g, 0.0106 mole) was added HATU (2.0 g, 0.0053 mole) at RT. After 30 minutes the reaction mixture was mixed with (2S, 4S)-4-fluoro-2-

pyrrolidinecarbonitrile 4-methylbenzenesulfonate (1.1 g, 0.0038 mole). After stirring

overnight the reaction was quenched with NaHC0₃ and the organics extracted with

EtOAc (2X). The organics were washed with sat. NaCl and dried over MgSU4. Removal of

the solvent in vacuo and purification via column chromatography (1/1 hexanes/EtOAc)

yielded 617 mg (38%) of compound B.

¹H NMR (CDCb) 400 MHz δ 7.18 (dd, 1 H, 7=7.8 Hz), 6.95 (s, 1 H,), 6.92 (d (br), 1 H, 7=2.8),

6.74 (dd, 1 H, 7=8.2 Hz), 5.41 (d (br), 1 H, 7 = 8.8 Hz), 5.39 (d (br), 1 H, 7=51.2), 5.00 (d, 1 H, 7=9.6 Hz), 4.35 (d, 1 H, 7 = 8.8 Hz), 4.28-3.78 (m, 7H), 2.71-2.52 (m, 1 H), 2.46-2.27 (m, 1 H), 1.62-1.39 (m, 15H) ppm.

C. Tert-butyl (l R)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1 -yl]carbonyl}-2-[(3- methoxybenzyl)sulfonyI]-2-methylpropylcarbamate.

To a CH2CI2 solution (100 mL) containing compound B (617 mg, 0.0013 mole) was added

3-chloroperoxybenzoic acid (57-86%) (2.3g 0.013 mole). The reaction mixture was

allowed to stir at RT for a period of 16.0 hours followed by neutralization with 2.0 N

KOH. The organics were dried over MgSθ4 and concentrated to dryness to afford 647mg

of crude solid (98%). The resulting off white solid was used without further purification.

Η NMR (CDCb) 400 MHz δ 7.30-7.25 (m, 1 H), 7.01-6.98 (m, 2H,), 6.92-6.88 (m, 1 H), 5.49

(d (br), 1 H, 7=8.4 Hz), 5.39 (d (br), 1 H, 7 = 50.8 Hz), 5.03 (d (br), 1 H, 7=9.2), 4.96 (d, 1 H, 7 =

9.2 Hz), 4.32-3.80 (m, 4H), 3.79 (s, 3H), 2.71 -2.54 (m, 1 H), 2.46-2.24 (m, 1 H), 1.59-1.39 (m, 15H) ppm.

D. (2S,4S)-1 -{(2R)-2-Amino-3-[(3-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride.

To a CH2CI2 solution (45 mL) containing compound C (647 mg, 0.0013 mole) was added

TFA (5mL). The reaction mixture was allowed to stir at RT for a period of 16.0 hours

followed by neutralization with sat. NaHC0₃. The organics were separated, dried over

MgSU4 and concentrated to dryness to afford a crude solid. The resulting solid was

purified using silica gel chromatography (99% CH2Cb/1% MeOH with 2.0M NH₃). The

pure solid was dissolved into an acetone/ether mixture (1/1) and precipitated using 2.0 N HCl in ether. The resulting solid was filtered in vacuo and washed with ether. After

drying under high vacuum the reaction yielded a total of 221 mg of compound D.

'H NMR (d₄-MeOH) 400 MHz δ 7.31 (dd, 1 H, 7=8.0 Hz), 7.18-7.06 (m, 2H), 6.98 (d,

1 H, 7=8.0 Hz), 5.49 (d (br), 1 H, 7=50.8 Hz), 5.10 (d, 1 H, 7 = 9.2 Hz), 4.72-4.58 (m, 3H),

4.15-3.88 (m, 2H), 3.81 (s, 3H), 2.69-2.44 (m, 2H), 1.75 (s, 3H), 1.59 (s, 3H) ppm.

(2S,4S)-1-{(2R)-2-Amino-3-[(1 ,1 '-biphenyl-4-ylmethyl)sulfonyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride

A. 2R)-3-[(1 ,1'-Biphenyl-4-ylmethyl)thio]-2-[(tert-butoxycarbonyl)amino]-3-

methylbutanoic acid.

To a 1.0 N KOH solution (50 L) containing 1,4-dioxane (10 mL), and (2R)-2-[(tert-

butoxycarbonyl)amino]-3-mercapto-3-methylbutanoic acid (1 g, 0.00401 mole) was

added 4-phenylbenzyl chloride (894 mg, 0.00441 mole) at RT. After 17.0 hours the

reaction mixture was diluted with water and washed with diethyl ether. The organics

were discarded and the aqueous layer was cooled to 0-5 °C. The aqueous solution was

made acidic (pH 3.5) using concentrated HCl and the product was extracted into ether.

The organics were dried over MgS0₄ and concentrated to dryness to yield a total of 1.4 g

(82%) of compound A as a white solid. Η NMR (CDCb) 400 MHz δ 7.59-7.31 (m, 9H), 5.49 ( , 1 H), 4.32 (d (br), 1 H, 7 = 9.6 Hz),

3.88-3.71 (m, 2H), 1.57-1.40 (m, 15H) ppm.

B. Tert-butyl (l R)-2-[(l ,1 '-biphenyl-4-ylmethyl)thio]-1-{[(2S,4S)-2-cyano-4-

fluoropyrrolidin-1 -yl]carbonyl}-2-methylpropylcarbamate.

To a DMF solution (25 mL) containing compound A (1.4 g, 0.0034 mole) and N,N-

diisopropylethylamine (1.3 g, 0.0101 mole) was added HATU (1.9 g, 0.0051 mole) at RT.

After 30 minutes the reaction mixture was mixed with (2S, 4S)-4-fluoro-2-

pyrrolidinecarbonitrile 4-methylbenzenesulfonate (1.1 g, 0.0037 mole). After stirring

overnight the reaction was quenched with NaHC0₃ and the organics extracted with

EtOAc (2X). The organics were washed with sat. NaCl and dried over MgS0₄. Removal of

the solvent in vacuo and purification via column chromatography (1/1 hexanes/EtOAc)

yielded 828mg (48% yield) of extremely pure product.

'H NMR (CDCb) 400 MHz δ 7.57-7.30 (m, 9H), 5.47-5.33 (m, 2H), 5.03 (d(br), 1 H,

7=9.6), 4.39 (d, 1 H, 7 = 8.8 Hz), 4.31-3.97 (m, 2H), 3.89 (s, 2H), 2.68 (dd, 1 H, 7=15.6 Hz),

2.44-2.28 (m, 1 H), 1.58-1.41 (m, 15H) ppm.

C. Tert-butyl (l R)-2-[(1,1'-biphenyl-4-ylmethyl)sulfonyl]-1-{[(2S,4S)-2-cyano-4-

fluoropyrrolidin-1-yl]carbonyl}-2-methylpropylcarbamate.

To a CH2CI2 solution (100 mL) containing compound B (828 mg, 0.0016 mole) was added

3-chloroperoxybenzoic acid(57-86%) (2.8 g 0.0162 mole). The reaction mixture was

allowed to stir at RT for a period of 16.0 hours followed by neutralization with 2.0 N

KOH. The organics were dried over MgS04 and concentrated to dryness to afford 867 mg of compound C (99% yield). The resulting white solid was used without further purification.

¹H NMR (CDCb) 400 MHz δ 7.63-7.33 (m, 9H), 5.53 (d(br), 1 H, 7=8.4 Hz), 5.42 (d(br), 1 H, 7

= 50.8 Hz), 5.07 (d(br), 1 H, 7=9.2), 4.99 (d, 1 H, 7 = 9.2 Hz), 4.39-4.00 (m, 4H), 2.70 (dd,

1 H, 7=15.2 Hz), 2.47-2.34 (m, 1 H), 1.62-1.40 (m, 15H) ppm.

D. (2S,4S)-1-{(2R)-2-amino-3-[(l ,1 '-biphenyl-4-ylmethyl)sulfonyl]-3-methylbutanoyl}-

4-fluoropyrrolidine-2-carbonitrile hydrochloride.

To a CH2CI2 solution (45 mL) containing compound C (867 mg, 0.0016 mole) was added

TFA (5 mL). The reaction mixture was allowed to stir at RT for a period of 16.0 hours

followed by neutralization with sat. NaHC0₃. The organics were separated, dried over

MgSθ4 and concentrated to dryness to afford a crude solid. The resulting solid was

purified using silica gel chromatography (99% CH₂Cb/l% MeOH with 2.0M NHa). The

pure solid was dissolved into an acetone/ether mixture (1/1) and precipitated using 2.0 N

HCl in ether. The resulting solid was filtered in vacuo and washed with ether. After

drying under high vacuum the reaction yielded a total of 307mg of compound D.

¹H NMR (d₄-MeOH) 400 MHz δ 7.66-7.32 (m, 9H), 5.48 (d(br), 1 H, 7=50.4 Hz), 5.10 (d,

I H, 7=8.0), 4.86-4.64 (m, 3H), 4.19-3.86 (m, 2H), 2.70-2.41 (m, 2H), 1.78 (s, 3H), 1.63 (s,

3H) ppm. Example 25

(2S,4S)-1-{(2R)-2-Amino-3-[(2-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-

fluoropyrro ιdine-2-carbonitrile hydrochloride A. (2R)-2-[(Tert-butoxycarbonyl)amino]-3-[(2-methoxybenzyl)thio]-3-methylbutanoic

acid.

To a 1.0 N KOH solution (50 mL) containing (2R)-2-[(tert-butoxycarbonyl)amino]-3-

mercapto-3-methylbutanoic acid (1 g, 0.00401 mole) was added 2-methoxybenzyI

chloride (691 mg, 0.00441 mole) at RT. After 17.0 hours the reaction mixture was diluted

with water and washed with diethyl ether. The organics were discarded and the aqueous

layer was cooled to 0-5 °C. The aqueous solution was made acidic (pH 3.5) using

concentrated HCl and the product was extracted into EtOAc. The organics were dried

over MgSU4 and concentrated to dryness to yield a total of 1.1 g (74%) of compound A.

Η NMR (CDCb) 400 MHz δ 7.25-7.21 (m, 2H), 6.90-6.85 (m, 2H), 5.63-5.55 (m, I H), 4.31

(d(br), 1 H, 7 = 9.2 Hz), 3.88-3.79 (m, 5H), 1.58-1.33 (m, 15H) ppm.

B. Tert-butyl (1 R)-1 -{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1 -yl]carbonyl}-2-[(2-

methoxybenzyl)thio]-2-methylpropylcarbamate.

To a DMF solution (25 mL) containing compound A (1.0 g, 0.0027 mole) and N.N-

diisopropylethylamine (1.05 g, 0.0081 mole) was added HATU (1.5 g, 0.00405 mole) at RT. After 30 minutes the reaction mixture was mixed with (2S, 4S)-4-fluoro-2-

pyrrolidinecarbonitrile 4-methylbenzenesulfonate (851 mg, 0.003 mole). After stirring

overnight the reaction was quenched with NaHC0₃ and the organics extracted with

EtOAc (2X). The organics were washed with sat. NaCl and dried over MgS0₄. Removal of

the solvent in vacuo and purification via column chromatography (1/1 hexanes/EtOAc)

yielded 668 mg (53% yield) of compound B.

'H NMR (CDCb) 400 MHz δ 7.31 (dd, 1 H, 7=7.6Hz), 7.19 (dd, 1 H, 7=8.0 Hz), 6.92-

6.83 (m, 2H), 5.47-5.31 (m, 2H), 5.03 (d(br), 1 H, 7=9.2), 4.34 (d, 1 H, 7 = 8.4 Hz), 4.26-3.66

(m, 7H), 2.68 (dd, 1 H, 7=15.2 Hz), 2.43-2.26 (m, 1 H), 1.57-1.37 (m, 15H) ppm.

C. Tert-butyl (l R)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]carbonyl}-2-[(2-

methoxybenzyl)sulfonyl]-2-methylpropyl carbamate.

To a CH2CI2 solution (75 mL) containing compound B (668 mg, 0.0014 mole) was added

3-chloroperoxybenzoic acid(57-86%) (2.5 g 0.0140 mole). The reaction mixture was

allowed to stir at RT for a period of 16.0 hours followed by neutralization with 2.0 N

KOH. The organics were dried over MgSU4 and concentrated to dryness to afford 678

mg of compound C (95%). The resulting off white solid was used without further

purification.

¹H NMR (CDCb) 400 MHz δ 7.44 (dd, 1 H, 7=7.6Hz), 7.34 (m, 1 H), 6.99-6.91 (m, 2H), 5.61

5.57 (m, 1 H), 5.40 (d(br), 1 H, 7=51.2 Hz), 5.00-4.95 (m, 2H), 4.49-3.85 (m, 7H), 2.65 (dd,

1 H, 7=15.6 Hz), 2.45-2.28 (m, 1 H), 1.62-1.37 (m, 15H) ppm.

D. (2S,4S)-1-{(2R)-2-amino-3-[(2-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride. To a CH2CI2 solution (45 mL) containing compound C (678 mg, 0.00136 mole) was added

TFA (5 mL). The reaction mixture was allowed to stir at RT for a period of 16.0 hours

followed by neutralization with sat. NaHC0₃. The organics were separated, dried over

MgS04 and concentrated to dryness to afford a crude solid. The resulting solid was

purified using silica gel chromatography (99% CH₂Cb/l% MeOH with 2.0 M NH₃). The

pure solid was dissolved into an acetone/ether mixture (1/1) and precipitated using 2.0 N HCl in ether. The resulting solid was filtered in vacuo and washed with ether. After

drying under high vacuum the reaction yielded a total of 313 mg of compound D.

'H NMR (d₄-MeOH) 400 MHz δ 7.45-7.38 (m, 2H), 7.06 (d, 1 H, 7=8.4 Hz), 6.99-6.99 (dd,

1 H, 7=7.2 Hz), 5.49 (d(br), 1 H, 7=50.8 Hz), 5.09 (m, 1 H, 7=9.2), 4.13-3.84 (m, 6H), 2.67-

2.44 (m, 2H), 1.74 (s, 3H), 1.58 (s, 3H) ppm.

(2S,4S)-1-{(2R)-2-Amino-3-methyl-3-[(pyridin-3-ylmethyl)thio]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile.

A. (2R)-2-[(Tert-butoxycarbonyl)amino]-3-methyl-3-[(pyridin-3-ylmethyl)thio]butanoic

acid.

To a 1.0 N KOH solution (50 mL) containing (2R)-2-[(tert-butoxycarbonyl)amino]-3-

mercapto-3-methylbutanoic acid (1 g, 0.00401 mole) was added 3-

(bromomethyl)pyridine hydrobromide (1.1 g, 0.00441 mole) at RT. After 17.0 hours the reaction mixture was diluted with water and washed with diethyl ether. The organics

were discarded and the aqueous layer was cooled to 0-5 °C. The aqueous solution was

made acidic (pH 4.0) using concentrated HCl and the product was extracted into EtOAc.

The organics were dried over Na2Sθ4 and concentrated to dryness to yield a total of 403

mg (30%) of compound A as a pure oily solid.

'H NMR (CDCb) 400 MHz δ 8.77 (s, 1 H), 8.56 (d, 1 H, 7=5.6 Hz), 7.80 (d, 1 H, 7=8.0 Hz), 7.37

(dd, 1 H, 7=8.0 Hz), 5.47 (d(br), 1 H, 7 = 9.6 Hz), 4.53 (d(br), 1 H, 7 = 9.6 Hz), 3.95 (dd, 2H,

7=69.6 Hz), 1.60-1.35 (m, 15H) ppm.

B. Tert-butyl (l R)-1 -{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]carbonyl}-2-methyl-2-

[(pyridin-3-ylmethyl)thio]propylcarbamate.

To a DMF solution (20 mL) containing compound A (400 mg, 0.0012 mole) and N,N-

diisopropylethylamine (456 mg, 0.0035 mole) was added HATU (670 mg, 0.0018 mole) at RT. After 30 minutes the reaction mixture was mixed with (2S, 4S)-4-fluoro-2-

pyrrolidinecarbonitrile 4-methylbenzenesulfonate (370 mg, 0.0013 mole). After stirring

overnight the reaction was quenched with NaHC0 and the organics extracted with

EtOAc (2X). The organics were washed with sat. NaCl and dried over Na2Sθ4. Removal of

the solvent in vacuo and purification via column chromatography (1/1 hexanes/EtOAc)

yielded 314 mg (61%) of compound B.

Η NMR (CDCb) 400 MHz δ 8.58 (s, 1 H), 8.46 (d, 1 H, 7=4.8 Hz), 7.71 (d, 1 H, 7=8.0

Hz), 7.21 (dd, 1 H, 7=8.0 Hz), 5.45 (d(br), 1 H, 7=9.2 Hz), 5.43 (d(br), 1 H, 7=51.2 Hz), 5.02 (d,

1 H, 7=9.2 Hz), 4.41 (d, 1 H, 7 = 9.6 Hz), 4.37-3.79 (m, 4H), 2.70 (dd, 1 H, 7=15.6 Hz), 2.46-

2.29 (m, 1 H), 1.55-1.39 (m, 15H) ppm. C. (2S,4S)-1-{(2R)-2-Amino-3-methyl-3-[(pyridin-3-ylmethyl)thio]butanoyl}-4- fluoropyrrolidine-2-carbonitrile hydrochloride.

To a CH2CI2 solution (22 mL) containing compound B (314 mg, O.720mmol) was added

TFA (3 mL). The reaction mixture was allowed to stir at RT for a period of 16.0 hours

followed by neutralization with sat. NaHC0₃. The organics were separated, dried over

Na2S0₄ and concentrated to dryness to afford a crude solid. The resulting solid was purified using silica gel chromatography (99% CH2C /1% MeOH with 2.0 M NHa). The

pure solid was dissolved into an acetone/ether mixture (1/1) and precipitated using 2.0 N

HCl in ether. The resulting solid was filtered in vacuo and washed with ether. After drying under high vacuum the reaction yielded a total of 69 mg of compound C.

'H NMR (CDCb) 400 MHz δ 8.57 (s, 1 H), 8.46 (d, 1 H, 7=5.2 Hz), 7.71 (d, 1 H, 7=6.4 Hz), 7.23

(dd, 1 H, 7=8.0 Hz), 5.44 (d(br), 1 H, 7=51.2 Hz), 4.97 (d, 1 H, 7=9.6 Hz), 4.20-3.58 (m, 5H),

2.66 (dd, 1 H, 7=15.2 Hz), 2.39-2.22 (m, 1 H), 1.78 (s(br), 2H), 1.48 (s, 3H), 1.39 (s, 3H) ppm.

(2S,4S)-1-{(2R)-2-Amino-3-methyl-3-[(pyridin-2-ylmethyl)thio]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride.

A. (2R)-2-[(Tert-butoxycarbonyl)amino]-3-methyl-3-[(pyridin-2-ylmethyl)thio]butanoic

acid. To a 1.0 N KOH solution (50 mL) containing (2R)-2-[(tert-butoxycarbonyl)amino]-3-

mercapto-3-methylbutanoic acid (1 g, 0.00401 mole) was added 2-

(bromomethyl)pyridine hydrobromide (1.1 g, 0.00441 mole) at RT. After 17.0 hours the

reaction mixture was diluted with water and washed with diethyl ether. The organics

were discarded and the aqueous layer was cooled to 0-5 °C. The aqueous solution was

made acidic (pH 4.0) using concentrated HCl and the product was extracted into EtoAc.

The organics were dried over Na2Sθ4 and concentrated to dryness to yield a total of 557

mg (41%) of compound A as a white solid.

¹H NMR (CDCb) 400 MHz δ 8.53 (d, 1 H, 7=4.8 Hz), 7.75 (dd, 1 H, 7=7.6 Hz), 7.34-7.30 (m,

2H), 5.73 (d(br), 1 H, 7 = 8.8 Hz), 4.47 (d, 1 H, 7 = 8.8 Hz), 4.41 -4.01 (m, 2H) 1.55 (m, 15H)

ppm.

B. Tert-butyl (l R)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1 -yl]carbonyl}-2-methyl-2-

[(pyridin-2-ylmethyl)thio]propyl carbamate. To a DMF solution (20 mL) containing compound A (557 mg, 0.0016 mole) and N,N-

diisopropylethylamine (635 mg, 0.0049 mole) was added HATU (912 mg, 0.0024 mole) at

RT. After 30 minutes the reaction mixture was mixed with (2S, 4S)-4-fluoro-2-

pyrrolidinecarbonitrile 4-methylbenzenesulfonate (504 mg, 0.00176 mole). After stirring

overnight the reaction was quenched with NaHC0₃ and the organics extracted with

EtOAc (2X). The organics were washed with sat. NaCl and dried over Na2S0₄. Removal of

the solvent in vacuo and purification via column chromatography (1/1 hexanes/EtOAc)

yielded 466 mg (65%) of compound B.

'H NMR (de Acetone) 400 MHz δ 8.46 (d, 1 H, 7=4.4 Hz), 7.71 (dd, 1 H, 7=7.6 Hz), 7.48 (d,

1 H, 7=8.0 Hz), 7.21 (dd, 1 H, 7=7.6 Hz), 6.25 (d(br), 1 H, 7=8.4 Hz), 5.60 (d(br), 1 H, 7=50.8 Hz), 5.05 (m, 1 H), 4.61 (d, 1 H, 7 = 8.8 Hz), 4.43-3.83 (m, 4H), 2.83-2.52 (m, 2H), 1.50-1.35 (m, 15H) ppm.

C. (2S,4S)-1 -{(2R)-2-Amino-3-methyl-3-[(pyridin-2-ylmethyl)thio]butanoyl}-4- fluoropyrrolidine-2-carbonitrile hydrochloride.

To a CH2CI2 solution (45 mL) containing compound B (314 mg, 0.00107mol) was added TFA (5 mL). The reaction mixture was allowed to stir at RT for a period of 16.0 hours

followed by neutralization with sat. NaHC0₃. The organics were separated, dried over

Na2S0₄ and concentrated to dryness to afford a crude solid. The resulting solid was

purified using silica gel chromatography (99% CH2C /l MeOH with 2.0 M NHa). The

pure solid was dissolved into an acetone/ether mixture (1/1) and precipitated using 2.0 N

HCl in ether. The resulting solid was filtered in vacuo and washed with ether. After

drying under high vacuum the reaction yielded a total of 127 mg of compound C.

'H NMR (D2O) 400 MHz δ 8.54 (d, 1 H, 7=5.6 Hz), 8.34 (dd, 1 H, 7=8.0 Hz), 7.92 (d, 1 H,

7=8.4 Hz), 7.21 (dd, 1 H, 7=6.4 Hz), 5.44 (d(br), 1 H, 7=50.4 Hz), 5.02 (d, 1 H, 7=10 Hz), 4.60

(m, 5H), 2.62 (dd, 1 H, 7=15.6 Hz), 2.54-2.34 (m, 1 H), 1.40 (s, 6H) ppm.

(2S,4S)-1 -{(2R)-2-amino-3-methyl-3-[(pyridin-4-ylmethyl)thio]butanoyl)-4-

fluoropyrrolidine-2-carbonitrile hydrochloride A. (2R)-2-[(tert-butoxycarbonyl)amino]-3-methyl-3-[(pyridin-4-ylmethyl)thio]butanoic acid.

To a 1.0 N KOH solution (50 mL) containing (2R)-2-[(tert-butoxycarbonyl)amino]-3-

mercapto-3-methylbutanoic acid (1 g, 0.00401 mole) was added 4-

(bromomethyl)pyridine hydrobromide (1.1 g, 0.00441 mole) at RT. After 17.0 hours the

reaction mixture was diluted with water and washed with diethyl ether. The organics

were discarded and the aqueous layer was cooled to 0-5 °C. The aqueous solution was

made acidic (pH 4.0) using concentrated HCl and the product was extracted into EtOAc.

The organics were dried over Na2S0₄ and concentrated to dryness to yield a total of 482

mg (35%) of compound A as a white solid.

Η NMR (CDCb) 400 MHz δ 8.48 (d, 2H, 7=6.0 Hz), 7.40 (d, 2H, 7=5.6 Hz), 5.48 (d(br), 1 H, 7

= 9.2 Hz), 4.41 (d, 1 H, 7 = 9.2 Hz), 3.82 (s, 2H) 1.52-1.38 (m, 15H) ppm.

B. Tert-butyl (l R)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1 -yl]carbonyl}-2-methyl-2-

[(pyridin-4-ylmethyl)thio]propylcarbamate.

To a DMF solution (20 mL) containing compound A (482 mg, 0.00142 mole) and N.N-

diisopropylethylamine (549 mg, 0.0042 mole) was added HATU (810 mg, 0.00213 mole)

at RT. After 30 minutes the reaction mixture was mixed with (2S, 4S)-4-fluoro-2-

pyrrolidinecarbonitrile 4-methylbenzenesulfonate (447 mg, 0.00156 mole). After stirring

overnight the reaction was quenched with NaHC0₃ and the organics extracted with

EtOAc (2X). The organics were washed with sat. NaCl and dried over Na₂S04. Removal of

the solvent in vacuo and purification via column chromatography (1/1 hexanes/ EtOAc)

yielded 476 mg (77%) of compound B. Η NMR (CDCb) 400 MHz δ 8.51 (d, 2H, 7=4.4 Hz), 7.31 (d, 2H, 7=6.4 Hz), 5.43 (d(br), 1 H.

7=9.6 Hz), 5.43 (d(br), 1 H, 7=51.2 Hz), 5.01 (d, 1 H, 7=9.6 Hz), 4.39 (d, I H, 7=9.6 Hz), 4.35-

3.96 (m, 2H), 3.80 (s, 2H), 2.70 (dd, 1 H, 7=15.4), 2.46-2.29 (m, 1 H), 1.45-1.41 (m,15H) ppm.

C. (2S,4S)-1-{(2R)-2-amino-3-methyl-3-[(pyridin-4-ylmethyl)thio]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride.

To a CH2CI2 solution (45 mL) containing compound B (476 mg, 0.00109mol) was added

TFA (5 mL). The reaction mixture was allowed to stir at RT for a period of 16.0 hours

followed by neutralization with sat. NaHC0 . The organics were separated, dried over

a2SU4 and concentrated to dryness to afford a crude solid. The resulting solid was

purified using silica gel chromatography (99% CH2C /1% MeOH with 2.0 M NHa). The

pure solid was dissolved into an acetone/ether mixture (1/1) and precipitated using 2.0 N

HCl in ether. The resulting solid was filtered in vacuo and washed with ether. After

drying under high vacuum the reaction yielded a total of 107 mg of compound C.

¹H NMR (d₄-MeOH) 400 MHz δ 8.81 (d, 2H, 7=4.8 Hz), 8.08 (d, 2H, 7=6.4 Hz), 5.51 (d(br),

1 H, 7=51.2 Hz), 5.10 (d, I H, 7=9.2 Hz), 4.35-3.96 (m, 4H), 2.65-2.43 (m, 2H), 1.62-1.42

(m,6H) ppm.

Example 29

(2S,4S)-1-{(2R)-2-amino-3-[(4-fluorobenzyl)sulfonyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride .

A. (2R)-2-[(tert-butoxycarbonyl)amino]-3-[(4-fluorobenzyl)thio]-3-methylbutanoic acid.

To a 1.0 N KOH solution (50 mL) containing (2R)-2-[(tert-butoxycarbonyl)amino]-3-

mercapto-3-methylbutanoic acid (1.0 g, 0.00401 mole) was added 1 -(bromomethyl)-4-

fluorobenzene (834 mg, 0.00441 mole) at RT. After 17.0 hours the reaction mixture was

diluted with water and washed with ether. The organics were discarded and the

aqueous layer was cooled to 0-5 °C. The aqueous solution was made acidic (pH 3.5)

using concentrated HCl and the product was extracted into EtOAc. The organics were

dried over MgSU4 and concentrated to dryness to yield a total of 1.2 g (86%) of

compound A as an oil.

'H NMR (CDCb) 400 MHz δ 7.29-7.26 (m, 2H), 6.99-6.94 (m, 2H), 5.40 (d(br), 1 H, 7 = 8.0

Hz), 4.40 (d(br), 1 H, 7 = 8.0 Hz), 3.80-3.74 (m, 2H) 1.50-1.40 (m, 15H) ppm.

B. Tert-butyl (l R)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1 -yl]carbonyl}-2-[(4-

fluorobenzyl)thio]-2-methylpropylcarbamate.

To a DMF solution (25 mL) containing compound A (1.2 g, 0.0034 mole) and N,N-

diisopropylethylamine (1.3 g, 0.0101 mole) was added HATU (1.9 g, 0.0051 mole) at RT.

After 30 minutes the reaction mixture was mixed with (2S, 4S)-4-fluoro-2-

pyrrolidinecarbonitrile 4-methylbenzenesulfonate (1.1 g, 0.0037 mole). After stirring

overnight the reaction was quenched with NaHCOa and the organics extracted with

EtOAc (2X). The organics were washed with sat. NaCl and dried over a2S0₄. Removal of

the solvent in vacuo and purification via column chromatography (1/1 hexanes/EtOAc)

yielded 929mg (61% yield) of compound B. 'H NMR (CDCb) 400 MHz δ 7.33 (dd, 2H, 7=8.4 Hz), 6.96 (dd, 2H, 7=8.8 Hz), 5.42 (d(br),

1 H, 7=9.2 Hz), 5.43 (d(br), 1 H, 7=51.6 Hz), 5.02 (d, 1 H, 7=9.6 Hz), 4.38 (d, 1 H, 7=9.2 Hz),

4.34-3.97 (m, 2H), 3.82 (s, 2H), 2.69 (dd, 1 H, 7=15.4 Hz), 2.46-2.28 (m, 1 H), 1.45-1.41 (m,15H) ppm.

C. Tert-butyl (l R)-1 -{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]carbonyl}-2-[(4-

fluorobenzyl)sulfonyl]-2-methylpropylcarbamate.

To a CH2CI2 solution (100 mL) containing compound B (929 mg, 0.0019 mole) was added

3-chloroperoxybenzoic acid (57-86%) (3.3 g 0.0191 mole). The reaction mixture was

allowed to stir at RT for a period of 16.0 hours followed by neutralization with 2.0 N

KOH. The organics were dried over MgSθ4 and concentrated to dryness to afford 678

mg of compound C (97% yield). The resulting off white solid was used without further

purification.

'H NMR (CDCb) 400 MHz δ 7.40 (dd, 2H, 7=8.8 Hz), 7.07 (dd, 2H, 7=8.6 Hz), 5.52 (d(br),

1 H, 7=9.2 Hz), 5.43 (d(br), 1 H. 7=51.2 Hz), 5.03 (d, 1 H, 7=9.6 Hz), 4.97 (d, 1 H, 7=9.6 Hz),

4.36-3.74 (m, 4H), 2.70 (dd, 1 H, 7=15.2 Hz), 2.47-2.28 (m, 1 H), 1.72-1.35 (m, 15H) ppm.

D. (2S,4S)-1-{(2R)-2-Amino-3-[(4-fluorobenzyl)sulfonyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride.

To a CH2CI2 solution (45 mL) containing compound C (678 mg, 0.00140 mole) was added

TFA (5 mL). The reaction mixture was allowed to stir at RT for a period of 16.0 hours

followed by neutralization with sat. NaHC0₃. The organics were separated, dried over

MgS04 and concentrated to dryness to afford a crude solid. The resulting solid was

purified using silica gel chromatography (99% CH2CI2/I % MeOH with 2.0 M NH₃). The pure solid was dissolved into an acetone/ether mixture (1/1) and precipitated using 2.0 N

HCl in ether. The resulting solid was filtered in vacuo and washed with ether. After

drying under high vacuum the reaction yielded a total of 254 mg of compound D as a white solid.

'H NMR (CDCb) 400 MHz δ 7.39 (dd, 2H, 7=8.4 Hz), 7.08 (dd, 2H, 7=8.6 Hz), 5.41 (d(br),

1 H, 7=50.8 Hz), 4.94 (d, 1 H, 7=9.6 Hz), 4.63 (d, 1 H, 7=13.2 Hz), 4.35-3.55 (m, 4H), 2.70 (dd,

1 H, 7=15.4 Hz), 2.43-2.26 (m, 1 H), 1.80-1.39 (m, 8H) ppm.

Example 30

(2S,4S)-1-{(2R)-2-amino-3-methyl-3-[(3-phenoxybenzyl)sulfonyl]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride.

A. (2R)-2-[(Tert-butoxycarbonyl)amino]-3-methyl-3-[(3-phenoxybenzyl)thio]butanoic

acid. To a 1.0 N KOH solution (50 mL) containing (2R)-2-[(tert-butoxycarbonyl)amino]-3-

mercapto-3-methylbutanoic acid (1 g, 0.00401 mole) was added 1-(chloromethyl)-3-

phenoxybenzene (964 mg, 0.00441 mole) and 1 ,4-dioxane (15 mL) at RT. After 17.0

hours the reaction mixture was diluted with water and washed with diethyl ether. The

organics were discarded and the aqueous layer was cooled to 0-5 °C. The aqueous

solution was made acidic (pH 3.5) using concentrated HCl and the product was extracted into ether. The organics were dried over MgSU4 and concentrated to dryness to yield a

total of 1.4 g (82%) of compound A as a white solid.

¹H NMR (CDCb) 400 MHz δ 7.34-6.98 (m, 8H), 6.85 (dd, 1 H, 7=8.0 Hz), 5.44 (m, 1 H), 4.38

(m, 1 H), 3.80 (m, 2H), 1.50-1.40 (m, 15H) ppm.

B. Tert-butyl (l R)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]carbonyl}-2-methyl-2-

[(3-phenoxybenzyl)thio]propylcarbamate.

To a DMF solution (20 mL) containing compound A (1.4 g, 0.0032 mole) and N,N-

diisopropylethylamine (1.3 g, 0.0100 mole) was added HATU (1.8 g, 0.0048 mole) at RT.

After 30 minutes the reaction mixture was mixed with (2S, 4S)-4-fluoro-2-

pyrrolidinecarbonitrile 4-methylbenzenesulfonate (1.0 g, 0.0035 mole). After stirring

overnight the reaction was quenched with NaHC0₃ and the organics extracted with

EtOAc (2X). The organics were washed with sat. NaCl and dried over MgSU4. Removal of

the solvent in vacuo and purification via column chromatography (1/1 hexanes/ EtOAc)

yielded 1.1 g (65%) of compound B.

'H NMR (CDCb) 400 MHz δ 7.34-6.97 (m, 8H), 6.84 (dd, 1 H, 7=8.0 Hz), 5.44 (d(br), 1 H,

7=9.2 Hz), 5.39 (d(br), 1 H, 7=51.2 Hz), 5.01 (d, 1 H, 7=9.6 Hz), 4.36 (d, 1 H, 7=9.2 Hz), 4.29-

3.90 (m, 2H), 3.80 (s, 2H), 2.66 (dd, 1 H, 7=15.4 Hz), 2.43-2.26 (m, 1 H), 1.52-1.38 (m,15H)

ppm.

C. Tert-butyl (l R)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1 -yl]carbonyl}-2-methyl-2-

[(3-phenoxybenzyl)sulfonyl]propyl carbamate.

To a CH2CI2 solution (50 mL) containing compound B (600 mg, 0.001 1 mol) was

added 3-chloroperoxybenzoic acid (57-86%) (2.0 g 0.01 1 mole). The reaction mixture was allowed to stir at RT for a period of 16.0 hours followed by neutralization with 2.0 N

KOH. The organics were dried over MgSθ4 and concentrated to dryness to afford 594

mg of compound C (93% yield). The resulting off white solid was used without further

purification.

' NMR (CDCb) 400 MHz δ 7.34-7.00 (m, 9H), 5.40 (d(br), 1 H, 7=51.2 Hz), 5.27 (d(br), 1 H,

7=8.8 Hz), 4.93 (d, 1 H, 7=9.6 Hz), 4.62 (d, 1 H, 7=13.2 Hz), 4.46-3.55 (m, 4H), 2.69 (dd, 1H,

7=15.4 Hz), 2.42-2.25 (m, 1 H), 1.62-1.43 (m,15H) ppm.

D. (2S,4S)-1-{(2R)-2-amino-3-methyl-3-[(3-phenoxybenzyl)sulfonyl]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride.

To a CH2CI2 solution (45 mL) containing compound C (678 mg, 0.00106 mole) was added

TFA (5 mL). The reaction mixture was allowed to stir at RT for a period of 16.0 hours

followed by neutralization with sat. NaHCC The organics were separated, dried over

MgSU4 and concentrated to dryness to afford a crude solid. The resulting solid was

purified using silica gel chromatography (99% CH2C /1% MeOH with 2.0 M NH₃). The

pure solid was dissolved into an acetone/ether mixture (1/1) and precipitated using 2.0 N

HCl in ether. The resulting solid was filtered in vacuo and washed with ether. After

drying under high vacuum the reaction yielded a total of 268 mg of compound D as a

white solid.

'H NMR (de-DMSO) 400 MHz δ 7.42-6.99 (m, 9H), 5.54 (d(br), 1 H, 7=51.2 Hz), 5.04 (d, 1H,

7=9.2 Hz), 4.70 (m, 2H), 4.46 (m, 1 H), 4.16-3.79 (m, 2H), 2.55-2.30 (m, 2H), 1.55 (s, 3H),

1.47 (s, 3H) ppm. Example 31

(2S,4S)-1-{(2R)-2-amino-3-methyl-3-[(3-phenoxybenzyl)thio]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride

To a CH2CI2 solution (45mL) containing tert-butyl (1 R)-1 -{[(2S,4S)-2-cyano-4- fluoropyrrolidin-1-yl]carbonyl}-2-methyl-2-[(3-phenoxybenzyl)thio]propylcarbamate

(500 mg, 0.0010 mol) was added TFA (5 mL). The reaction mixture was allowed to stir at RT for a period of 16.0 hours followed by neutralization with sat. NaHC0₃. The organics

were separated, dried over MgSU4 and concentrated to dryness to afford a crude solid.

The resulting solid was purified using silica gel chromatography (99% CH2Cb/1% MeOH

with 2.0 M NHs). The pure solid was dissolved into an acetone/ether mixture (1/1) and

precipitated using 2.0 N HCl in ether. The resulting solid was filtered in vacuo and

washed with ether. After drying under high vacuum the reaction yielded a total of 132

mg of the title compound .

'H NMR (D2O) 400 MHz δ 7.17-6.71 (m, 9H), 5.38 (d(br), 1 H, 7=50.4 Hz), 5.00 (d, 1 H,

7=9.6 Hz), 4.16-3.60 (m, 4H), 2.62-2.30 (m, 2H), 1.38-1.29 (s, 6H) ppm.

(2S,4S)-1-((2R)-2-Amino-3-{[(5-chloro-1 ,1 -dioxido-1 -benzothien-3-

yl)methyl]sulfonyl}-3-methylbutanoyl)-4-fluoropyrrolidine-2-carbonitrile hydrochloride. A. (2R)-2-[(tert-butoxycarbonyl)amino]-3-{[(5-chloro-1 -benzothien-3-yl)methyl]thio}-

3-methylbutanoic acid.

To a 1.0 N KOH solution (50 mL) containing (2R)-2-[(tert-butoxycarbonyl)amino]-3-

mercapto-3-methylbutanoic acid (1 g, 0.00401 mole) was added 3-(bromomethyl)-5-

chloro-1-benzothiophene (1.2 g, 0.00441 mole) and 1 ,4-dioxane (10 mL) at RT. After

17.0 hours the reaction mixture was diluted with water and washed with diethyl ether.

The organics were discarded and the aqueous layer was cooled to 0-5 °C. The aqueous

solution was made acidic (pH 3.5) using concentrated HCl and the product was extracted

into ether. The organics were dried over MgSU4 and concentrated to dryness to yield a

total of 1.0 g (59%) of compound A as a tan solid.

'H NMR (CDCb) 400 MHz δ 7.84 (s, 1 H), 7.72 (d, 1 H, 7=8.4 Hz), 7.39 (s, 1 H), 7.30 (d, I H.

7=8.4 Hz), 5.44 (m, 1 H), 4.45 (m, 1 H, 7=7.6 Hz), 4.07-4.00 (m, 2H), 1.55-1.40 (m, 15H)

ppm.

B. Tert-butyl (1 R)-2-{[(5-chloro-1-benzothien-3-yl)methyl]thio}-1 -{[(2S,4S)-2-cyano-

4-fluoropyrrolidin-1 -yl]carbonyl}-2-methylpropyIcarbamate. To a DMF solution (20 mL) containing compound A (1.0 g, 0.0023 mole) and N,N-

diisopropylethylamine (902 mg, 0.007 mole) was added HATU (1.3 g, 0.00345 mole) at

RT. After 30 minutes the reaction mixture was mixed with (2S, 4S)-4-fluoro-2-

pyrrolidinecarbonitrile 4-methylbenzenesulfonate (725 mg, 0.0025 mole). After stirring

overnight the reaction was quenched with NaHC0₃ and the organics extracted with

EtOAc (2X). The organics were washed with sat. NaCl and dried over MgSθ4. Removal of

the solvent in vacuo and purification via column chromatography (1/1 hexanes/EtOAc)

yielded 738 mg (60%) of compound B.

'H NMR (CDCb) 400 MHz δ 7.91 (s, 1 H), 7.72 (d, 1 H, 7=8.8 Hz), 7.47 (s, 1 H), 7.30

(d, 1 H, 7=8.4 Hz), 5.49-5.35 (m, 2H), 5.04 (d, 1 H, 7=9.6 Hz), 4.41 (d, 1 H, 7=9.2 Hz), 4.35- 3.98 (m, 4H), 2.69 (dd, 1 H, 7=15.2 Hz), 2.46-2.29 (m, 1 H), 1.47-1.40 (m, 15H) ppm.

C. Tert-butyl (l R)-2-{[(5-chloro-1 ,1 -dioxido-1-benzothien-3-yl)methyl]sulfonyl}-1-

{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]carbonyl}-2-methylpropylcarbamate.

To a CH2CI2 solution (100 mL) containing compound B (738 mg, 0.0014 mole) was

added 3-chloroperoxybenzoic acid (57-86%) (4.8 g 0.028 mole). The reaction mixture

was allowed to stir at RT for a period of 16.0 hours followed by neutralization with 2.0 N

KOH. The organics were dried over MgSU4 and concentrated to dryness to afford 803

mg of compound C (97% yield). The resulting off white solid was used without further

purification.

Η NMR (CDCb) 400 MHz δ 8.05 (s, 1 H), 7.96 (d, 1 H, 7=7.6 Hz), 7.48 (m, 3H), 5.45 (d(br),

1 H 7=10 Hz), 5.44 (d(br), 1 H, 7=50.8 Hz), 5.02 (d, 1 H, 7=10 Hz), 4.95 (d, 1 H, 7=9.6 Hz),

4.77-3.96 (m, 4H), 2.70 (dd, 1 H, 7=14.8 Hz), 2.46-2.30 (m, 1 H), 1.70-1.37 (m, 15H) ppm. D. (2S,4S)-1-((2R)-2-amino-3-{[(5-chloro-1 ,1-dioxido-1-benzothien-3-

yl)methyl]sulfonyl}-3-methylbutanoyl)-4-fluoropyrrolidine-2-carbonitrile hydrochloride.

To a CH2CI2 solution (45 mL) containing compound C (500 mg, 0.0010 mole) was added

TFA (5 mL). The reaction mixture was allowed to stir at RT for a period of 16.0 hours

followed by neutralization with sat. NaHC0₃. The organics were separated, dried over

MgS0₄ and concentrated to dryness to afford a crude solid. The resulting solid was

purified using silica gel chromatography (99% CH∑Cb/ o MeOH with 2.0 M NHa). The

pure solid was dissolved into an acetone/ether mixture (1/1) and precipitated using 2.0 N HCl in ether. The resulting solid was filtered in vacuo and washed with ether. After

drying under high vacuum the reaction yielded a total of 132 mg of compound D.

'H NMR (D2O) 400 MHz δ 7.99 (s, 1 H), 7.81 -7.56 (m, 3H), 5.44 (d(br), 1 H, 7=50.4

Hz), 5.01 (d, 1 H, 7=9.6 Hz), 4.19-3.80 (m, 4H), 2.66-2.22 (m, 2H), 1.58 (s, 3H), 1.43 (s, 3H) ppm.

Example 33

(2S,4S)-1-{(2R)-2-Amino-3-[(2,1 ,3-benzoxadiazol-5-ylmethyl)thio]-3-methylbutanoyl)-

4-fluoropyrrolidine-2-carbonitrile hydrochloride

A. (2R)-3-[(2,1 ,3-Benzoxadiazol-5-ylmethyl)thio]-2-[(tert-butoxycarbonyl)amino]-3-

methylbutanoic acid. To a 1.0 N KOH solution (50 mL) containing (2R)-2-[(tert-butoxycarbonyl)amino]-3-

mercapto-3-methylbutanoic acid (1 g, 0.00401 mole) was added 5-(bromomethyl)-2,1,3-

benzoxadiazole (940 mg, 0.00441 mole) and 1 ,4-dioxane (5 mL) at RT. After 17.0 hours

the reaction mixture was diluted with water and washed with diethyl ether. The

organics were discarded and the aqueous layer was cooled to 0-5 °C. The aqueous

solution was made acidic (pH 4.0) using concentrated HCl and the product was extracted into EtOAc. The organics were dried over Na2SU4 and concentrated to dryness to yield a

total of 622 mg (41%) of compound A as a brown solid.

¹H NMR (CDCb) 400 MHz δ 7.76 (d, 1 H, 7=9.2 Hz), 7.72 (s, 1 H), 7.44 (d, 1 H, 7=9.2 Hz),

5.40 (d (br), 1 H, 7 = 8.0 Hz), 4.46 (d (br), 1 H, 7 =8.4 Hz), 3.89 (s, 2H) 1.47-1.40 (m, 15H) ppm.

B. Tert-butyl (l R)-2-[(2,1,3-benzoxadiazol-5-ylmethyl)thio]-1-{[(2S,4S)-2-cyano-4-

fluoropyrrolidin-1-yl]carbonyl}-2-methylpropylcarbamate.

To a DMF solution (20 mL) containing compound A (622 mg, 0.00163 mole) and N.N-

diisopropylethylamine (632 mg, 0.0049 mole) was added HATU (930 mg, 0.00244 mole)

at RT. After 30 minutes the reaction mixture was mixed with (2S, 4S)-4-fluoro-2-

pyrrolidinecarbonitrile 4-methylbenzenesulfonate (514 mg, 0.00180 mole). After stirring

overnight the reaction was quenched with NaHC0₃ and the organics extracted with

EtOAc (2X). The organics were washed with sat. NaCl and dried over a2SU4. Removal of

the solvent in vacuo and purification via column chromatography (1/1 hexanes/EtOAc)

yielded mg 444 mg (45% yield) of compound B.

'H NMR (CDCb) 400 MHz δ 7.78 (s, 1 H), 7.76 (d, 1 H, 7=9.2 Hz), 7.48 (d, I H, 7=9.2 Hz),

5.43 (d (br), 1 H, 7=9.6 Hz), 5.45 (d (br), 1 H, 7=50.8 Hz), 5.02 (d, 1 H, 7=9.6 Hz), 4.44 (d, 1 H, 7=9.6 Hz), 4.39-3.89 (m, 4H), 2.72 (dd, 1 H, 7=15.2), 2.48-2.32 (m, 1 H), 1.60-1.40 (m, 15H) ppm.

C. (2S,4S)-1 -{(2R)-2-Amino-3-[(2,1 ,3-benzoxadiazol-5-ylmethyl)thio]-3-

methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile hydrochloride.

To a CH2CI2 solution (45 mL) containing compound B (444 mg, 0.770mmol) was added

TFA (5 mL). The reaction mixture was allowed to stir at RT for a period of 16.0 hours

followed by neutralization with sat. NaHC0₃. The organics were separated, dried over

Na2SU4 and concentrated to dryness to afford a crude solid. The resulting solid was

purified using silica gel chromatography (99% CH2C /l MeOH with 2.0M NH₃). The

pure solid was dissolved into an acetone/ether mixture (1/1) and precipitated using 2.0 N HCl in ether. The resulting solid was filtered in vacuo and washed with ether. After

drying under high vacuum the reaction yielded a total of 105 mg of compound C.

^ NMR (de-DMSO) 400 MHz δ 8.02-7.99 (m, 2H), 7.63 (d, 1 H, 7=9.6 Hz), 5.55 (d (br), 1 H,

7=51.2 Hz), 5.07 (d, 1 H, 7=9.2 Hz), 4.22-3.98 (m, 5H), 2.58-2.32 (m, 2H), 1.48 (s, 3H), 1.40

(s, 3H) ppm.

(2S,4S)-1 -{(2R)-2-Amino-3-methyl-3-[(pyridin-4-ylmethyl)sulfonyl]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride To a CH2CI2 solution (90 mL) containing tert-butyl (l R)-1 -{[(2S,4S)-2-cyano-4-

fluoropyrrolidin-1-yl]carbonyl}-2-methyl-2-[(pyridin-4-ylmethyl)thio]propylcarbamate

(500 mg, 0.001 1 mol) was added TFA (10 mL). The reaction mixture was allowed to stir at RT for a period of 16.0 hours followed by the addition of 3-chloroperoxybenzoic acid

(2.0 g, 0.01 14 moles). The reaction was stirred for a period of 4.0 hours followed by

neutralization with 2.0 N NaOH. The organics were separated, dried over Na₂Sθ4 and

concentrated to dryness to afford a crude solid. The resulting solid was purified using

silica gel chromatography (99% CH∑Cfe/l MeOH with 2.0 M NHs). The pure solid was dissolved into an acetone/ether mixture (1/1) and precipitated using 2.0 N HCl in ether.

The resulting solid was filtered in vacuo and washed with ether. After drying under high

vacuum the reaction yielded a total of 22 mg of the title compound.

'H NMR (d4CDCb) 400 MHz δ 8.64 (d, 2H, 7=5.6 Hz), 7.37 (d, 2H, 7=5.6 Hz), 5.42 (d (br),

1 H, 7=51.2 Hz), 4.94 (d, 1 H, 7=9.6 Hz), 4.77 (d, 1 H, 7=13.2 Hz), 4.39-3.75 (m, 4H), 2.71 (dd,

1 H, 7=15.6 Hz), 2.43-2.29 (m, 1 H), 2.06-1.86 (s (br), 2H), 1.62 (s, 3H), 1.46 (s, 3H) ppm.

(2S4S)-1-[(2S)-2-Amino-3-pyridin-4-ylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile

hydrochloride A. (2S,4S)-1-[(25)-2-amino-3-pyridin-4-ylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile hydrochloride

To dry DMF (25 mL) was added (25)-2-[(tert-butoxycarbonyl)amino]-3-pyridin-4-

ylpropanoic acid (705 mg, 2.65 mmol), HATU (1.0 g, 2.65 mmol) and N,N-

diisopropylethylamine (0.46 mL, 2.65 mmol). After stirring at RT for 30 min (2S, 4S)-4-

fluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate (581 mg, 1.99 mmol) and

additional N.N-diisopropylethylamine (0.35 mL, 1.99 mmol) were added. This solution

was allowed to stir at RT for 12 hours. Then was added saturated sodium bicarbonate

(100 mL) and the mixture was extracted with ethyl acetate (2 X 100 mL) and the

organics were washed with saturated NaCl (100 mL) and dried over MgSU4 and

concentrated to dryness to give the crude solid. The solid was purified by column chromatography (silica gel, hexanes/EtOAc 4:1) to provide a solid which was stirred in a

dioxane solution containing HCl (4.0 M, 20 mL) for 2 hrs followed by the addition of

diethyl ether (100 mL). The precipitated solid was collected by filtration and dried under

high vacuum yielding 388 mg (1.3 mmol, 65% yield) of compound A as a white solid.

¹H NMR (de-DMSO) 400 MHz δ 8.92 (s(br), 3H), 8.84 (d, 2H, 7= 6.2 Hz), 7.96 (d, 2H, 7=

6.2 Hz), 5.45 (d, 1 H, 7 = 51 Hz), 5.00 (d, 1 H, 7 = 8.8 Hz), 4.47 (m(br), 1 H), 4.05-3.78 (m,

2H), 3.41-3.36 (m, 2H), 2.42-2.25 (m, 2H) ppm.

(25,45)-1-[(25)-2-Amino-3-pyridin-3-ylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile hydrochloride

A. (2S,4S)-1-[(2S)-2-amino-3-pyridin-3-ylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile hydrochloride

To dry DMF (25 mL) was added (2S)-2-[(£er£-butoxycarbonyl)amino]-3-pyridin-3- ylpropanoic acid (534 mg, 2.0 mmol), HATU (0.76 g, 2.0 mmol) and N,N-

diisopropylethylamine (0.35 mL, 2.0 mmol). After stirring at RT for 30 min (2S, 4S)-4-

fluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate (438 mg, 1.5 mmol) and

additional N.N-diisopropylethylamine (0.26 mL, 1.5 mmol) were added. This solution was allowed to stir at RT for a total of 12 hr and then saturated sodium bicarbonate (150 mL)

was added. The mixture was extracted with ethyl acetate (3 X 100 mL) and the organics

were washed with saturated NaCl (100 mL), dried over MgSU4 and concentrated to

dryness to give the crude solid. The solid was chromatographed (silica gel,

hexanes/EtOAc 4:1) to provide a solid which was stirred in a dioxane-HCI (4.0 M, 25 mL)

solution for 2 hrs followed by the addition of diethyl ether (200 mL). The precipitated

solid was collected by filtration and dried under high vacuum yielding 266 mg (0.89

mmol, 59% yield) of compound A as a white solid.

Η NMR (de-DMSO) 400 MHz δ 8.88 (s, 1 H), 5.51 (d, 1 H, 7= 51 Hz), 5.01 (d, 1 H, 7 = 8.8

Hz), 4.45-4.38 (m(br), 1 H), 4.00-3.86 (m, 2H), 3.38-3.27 (m, 2H), 2.42-2.30 (m, 3H) ppm. Example 37

(25,45)-1-[(2S)-2-Amino-3-piperidin-4-ylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile dihydrochloride

A. (2S)-2-[(tert-butoxycarbonyl)amino]-3-piperidin-4-ylpropanoic acid acetic acid salt. To an acetic acid (glacial, 50 mL) solution of (2S)-2-[(£ert-butoxycarbonyl)amino]-3-

pyridin-4-ylpropanoic acid (0.69 g, 2.58 mmol) was added 10% Pd/C (50 % w/w, 0.35 g) and hydrogenated at 60 psi under a hydrogen atmosphere. The solution was filtered

through celite and concentrated to provide a white solid. Then dried under high vacuum

yielding 822 mg (2.48 mmol, 96% yield) of compound A as the acetic acid salt.

'H NMR (de-DMSO) 400 MHz δ 10.5 (s(br), 1 H), 6.21 -6.19 (m(br), I H), 3.69-3.62 (m, I H),

3.19-3.06 (m, 2H), 2.75-2.65 (m, 2H), 1.92 (s, 3H), 1.80-1.45 (m, 6H), 1.35 (s, 9H), 1.21-

1.05 (m, 1 H) ppm.

B. (2S)-2-[(7^'er-'-butoxycarbonyl)amino]-3-[1-(£ert-butoxycarbonyl)piperidin-4-

yl]propanoic acid.

To a CH2CI2 (150 mL) solution of (2S)-2-[(tert-butoxycarbonyl)amino]-3-piperidin-4-

ylpropanoic acid acetic acid salt (1.0 g, 3.00 mmol) was added triethylamine (1.52 g, 15

mmol) and di- tert-butyl dicarbonate (786 mg, 3.6 mmol) and stirred for 12 hr. Then

was added H2O (50 mL) and CH2CI2 (300 mL), acidified to pH 4 with 1.0 M HCl. Separation of the ethyl acetate layer followed by drying over MgSU4 and filtration and

removal of the solvent in vacuo yielded a clear oil. The oil was dried under high vacuum

yielding 1.06 g (2.86 mmol, 95% yield) of compound B as a white solid.

¹H NMR (de-DMSO) 400 MHz δ 12.4 (s(br), 1 H), 7.08 (d, 1 H, 7 = 8.3 Hz), 3.98-3.81 (m(br),

3H), 2.78-2.59 (m(br), 2H), 1.68-1.42 (m, 6H), 1.39 (s, 16H), 1.02-0.89 (m, 2H) ppm.

C. (2S,4S)-1-[(2S)-2-Amino-3-piperidin-4-ylpropanoyl]-4-fluoropyrrolidine-2-

carbonitrile hydrochloride.

To dry DMF (25 mL) was added (2S)-2-[(tert-butoxycarbonyl)amino]-3-[1 -(tert-

butoxycarbonyl)piperidin-4-yl]propanoic acid (0.85 g, 2.28 mmol), HATU (0.87 g, 2.28

mmol) and N.N-diisopropylethylamine (0.40 mL, 2.28 mmol). After stirring at RT for 30

min (2S, 4S)-4-fluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate (500 mg, 1.71

mmol) and additional N.N-diisopropylethylamine (0.30 mL, 1.71 mmol) were added. This

solution was allowed to stir at RT for 12 hr and then saturated sodium bicarbonate (100

mL) was added. The mixture was extracted with ethyl acetate (2 X 100 mL) and the

organics were washed with saturated NaCl (50 mL) followed by drying over MgS0₄.

Evaporation in vacuo gave the crude solid which was chromatographed (silica gel,

hexanes/EtOAc 4:1) to provide a solid which was stirred in a dioxane-HCI (4.0 M, 20 mL)

solution for 2 hrs followed by the addition of diethyl ether (100 mL). The resulting

precipitate was collected by filtration and dried under high vacuum yielding 432 mg

(1.26 mmol, 74% yield) of compound C as a white solid.

¹H NMR (de-DMSO) 400 MHz δ 9.0-8.83 (s(br), 2H), 8.80-8.60 (s(br), 3H), 5.52 (d, 1 H, 7 =

51 Hz), 5.06 (d, I H, J = 8.8 Hz), 4.07-4.01 (m(br), I H). 3.94-3.81 (m, 2H), 3.23-3.20 (m, 2H), 2.87 (s, I H), 2.74-2.66 (m, 3H), 2.46-2.37 (m, 2H), 1.95-1.91 ( , 1 H), 1.77-1.58 (m, 3H), 1.42-1.31 (m, 1 H) ppm.

(2S45)-1-[(2S)-2-Amino-3-piperidin-3-ylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile

dihydrochloride

A. (2S)-2-[(£er£-butoxycarbonyl)amino]-3-piperidin-3-ylpropanoic acid.

To an acetic acid (glacial, 50 mL) solution of (2S)-2-[(tert-butoxycarbonyl)amino]-3-

pyridin-3-ylpropanoic acid (0.8 g, 3.0 mmol) was added 10% Pd/C (50 % w/w, 0.40 g)

and hydrogenated at 60 psi under a hydrogen atmosphere. The solution was filtered

through celite, concentrated and then dried under high vacuum yielding 946 mg (2.85

mmol, 95% yield) of compound A as the acetic acid salt.

'H NMR (de-DMSO) 400 MHz δ 10.2 (s(br), I H). 6.15-6.05 (m, 1 H), 3.69-2.64 (m, 1 H),

3.21-3.07 (m, 2H), 2.67-2.61 (m, 1 H), 2.39-2.31 (m(br), 1 H),1.95 (s, 3H), 1.91-1.41 (m,

6H), 1.91 (s, 9H), 1.08-1.01 (m, 1 H) ppm.

B. (2S)-2-[(7ert-butoxycarbonyl)amino]-3-[1-(tert-butoxycarbonyl)piperidin-3-

yl]propanoic acid.

To a CH2CI2 (150 mL) solution containing compound A (1.0 g, 3.00 mmol) was added

triethylamine (1.52 g, 15 mmol) and di-tert-butyl dicarbonate (786 mg, 3.6 mmol) and stirred for 12 hr. The reaction was then quenched with H2O (50 mL) and CH2CI2 (300 mL)

added. The organic layer was separated and the aqueous layer acidified to pH 4 with 1.0

M HCl followed by extraction with EtOAc. The combined organic layers were dried over

MgSU4 and the solvent removed in vacuo yielding a clear oil. The oil was dried under

high vacuum yielding 1.07 g (2.87 mmol, 95% yield) of compound B as a white solid.

¹H NMR (de-DMSO) 400 MHz δ 12.15 (s(br), 1 H), 7.17 (d, 1 H, 7 = 8.3 Hz), 3.95-3.87

(m(br), 3H), 2.61-2.45 (m(br), 2H), 1.73-1.40 (m, 6H), 1.35 (s, 16H), 1.2-0.89 (m, 2H) ppm.

C. (2S,45)-1 -[(2S)-2-Amino-3-piperidin-3-ylpropanoyl]-4-fluoropyrrolidine-2- carbonitrile dihydrochloride.

To dry DMF (25 mL) was added compound B (0.85 g, 2.28 mmol), HATU (0.87 g, 2.28

mmol) and N.N-diisopropylethylamine (0.40 mL, 2.28mmol). After stirring at RT for 30

minutes (2S, 4S)-4-fluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate (500 mg,

1.71 mmol) and additional N.N-diisopropylethylamine (0.30 mL, 1.71 mmol) were added.

This solution was allowed to stir at RT for a total of 12 hours at which time saturated

sodium bicarbonate (100 mL) was added. The mixture was extracted with ethyl acetate

(2 X 100 mL) and the organics were washed with saturated NaCl (50 mL) and dried over

MgS04. Removal of the solvent in vacuo gave the crude solid. The solid was

chromatographed (silica gel, hexanes/EtOAc 4:1) to provide a solid which was stirred in a

dioxane-HCl (4.0 M, 20 mL) solution for 2 hrs followed by the addition of diethyl ether

(100 mL). The resulting precipitate was collected by filtration and dried under high

vacuum yielding 419 mg (1.23 mmol, 72% yield) of compound C as a white solid.

^]H NMR (de-DMSO) 400 MHz δ 9.0-8.83 (s(br), 2H), 8.60-8.50 (s(br), 3H), 5.52 (d, 1 H.7

=51 Hz), 5.02 (d, 1 H, 7 = 8.8 Hz), 4.13-4.08 (m(br), 1 H). 3.99-3.87 (m, 2H), 3.37-3.31 (m, 2H), 3.05-3.01 (m, 1 H), 2.87 (m, 2H), 2.46-2.37 (m, 2H), 1.95-1.91 (m, 5H), 1.22-1.1 1 (m, 1 H) ppm.

(25,45)- 1 -[(25)-2-Amino-3-piperidin-2-ylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile

dihydrochloride

A. (25)-2-[(7ert-butoxycarbonyl)amino]-3-piperidin-2-ylpropanoic acid

To an acetic acid (glacial, 50mL) solution of (25)-2-[(fert-butoxycarbonyl)amino]-3-

pyridin-2-ylpropanoic acid (1.6 g, 6.0 mmol) was added 10% Pd/C (50 % w/w, 0.80 g)

and hydrogenated at 60 psi under a hydrogen atmosphere. The solution was filtered

through celite, concentrated and then dried under high vacuum yielding 951 mg (2.85

mmol, 95% yield) of compound A as the acetic acid salt.

NMR (de-DMSO) 400 MHz δ 10.15 (s(br), 1 H), 6.21-6.19 (m, 1 H), 3.69-2.64 (m, 1 H),

3.61-3.56 (m, 2H), 3.27-3.12 (m, 2H), 2.72-2.65 (m, 1 H), 1.89 (s, 3H), 1.81-1.63 (m, 5H),

1.52-2.42 (m, 1 H), 1.91 (s, 9H) ppm.

B. (25)-2-[(Tert-butoxycarbonyl)amino]-3-[1 -(tert-butoxycarbonyl)piperidin-2-

yl]propanoic acid. To a CH2CI2 (150 mL) solution of compound A (1.5 g, 4.50 mmol) was added

triethylamine (2.27 g, 23 mmol) and di-tert-butyl dicarbonate (1.18 g, 5.4 mmol). After

stirring for 12 hr H2O (50 mL) and CH2CI2 (300 mL) were added followed by acidification

to pH 3 with 1.0 M HCl. Separation of the organic layer followed by drying over MgS0₄

and removal of the solvent in vacuo yielded a clear oil. The oil was dried under high

vacuum yielding 1.68g (4.45 mmol, 95% yield) of compound B as a white solid.

¹H NMR (de-DMSO) 400 MHz δ 12.2 (s(br), 1 H), 7.15 (d, 1 H, 7 = 8.3 Hz), 4.25-3.99 (m(br),

3H), 3.89-3.78 (m(br), 2H), 2.68-2.58 (m, 1 H) 1.79-1.40 (m, 5H), 1.35 (s, 16H), 1.2-1.14 (m, 2H) ppm.

C. (25,45)-1-[(25)-2-Amino-3-piperidin-2-ylpropanoyl]-4-fluoropyrrolidine-2-

carbonit ile dihydrochloride.

To dry DMF (25 mL) was added compound B (850 mg, 2.28 mmol), HATU (869 mg, 2.28 mmol) and N.N-diisopropylethylamine (0.40 mL, 2.28 mmol). After stirring at RT for 30

min (2S, 4S)-4-fluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate (500 mg, 1.71

mmol) and additional N.N-diisopropylethylamine (0.30 mL, 1.71 mmol) were added. This

solution was allowed to stir at RT for 12 hr and then saturated sodium bicarbonate (110

mL) was added. The mixture was extracted with ethyl acetate (2 X 100 mL) and the

organics were washed with saturated NaCl (50 mL) and dried over MgSU4. Removal of

the solvent in vacuo gave a crude solid. The solid was chromatographed (silica gel,

hexanes/EtOAc 4:1) to provide a solid which was stirred in a dioxane-HCl (4.0 M, 20 mL)

solution for 2 hrs followed by the addition of diethyl ether (100 mL). The resulting

precipitate was collected by filtration and dried under high vacuum yielding 365 mg

(1.07mmol, 63% yield) of compound C as a white solid. 'H NMR (de-DMSO) 400 MHz δ 9.32-9.10 (s(br), 2H), 8.79-8.53 (s(br), 3H), 5.51 (d, 1 H.7

=51 Hz), 5.04 (d, 1 H, 7 = 8.8 Hz), 4.25-4.21 (m(br), 1 H), 4.19-3.88 (m, 2H), 3.58-3.46 (m,

1 H), 3.42-3.32 (m, 2H), 2.85 (s, 1 H), 2.71 -2.63 (m, 2H), 2,27-2.14 (m, 1 H), 2.05-1.97 (m,

1 H), 1.81-1.40 (m, 5H) ppm.

(25,45)-1-{(25)-2-Amino-3-[l-(isopropylsulfonyl)piperidin-4-yl]propanoyl)-4-

fluoropyrrolidine-2-carbonitrilehydrochloride A. (25)-2-[(7ert-butoxycarbonyl)amino]-3-[l -(isopropylsulfonyl)piperidin-4-yl]propanoic

acid.

To an CH₃CN (150 mL) solution of compound A from example 12 (1.0 g, 3.00 mmol) was

added triethylamine (1.52 g, 15 mmol) and isopropylsulfonyl chloride (513 mg, 3.6

mmol). After stirring for 6 hrs the acetonitrile was removed in vacuo and H2O (50 mL)

and ethyl acetate (300 mL) were added. Separation of the ethyl acetate layer followed

by drying over MgSU4 and removal of the solvent in vacuo yielded a solid which was

washed with ether and dried under high vacuum yielding 860 mg (2.26 mmol, 75%

yield) of compound A as a white solid. Η NMR (de-DMSO) 400 MHz δ 12.4 (s(br), 1 H), 7.15 (d, 1 H, 7 = 8.3 Hz), 4.37-4.29 (m,

1 H), 3.99-3.95 (m(br), 1 H), 3.85-3.80 (m, 1 H), 3.62-3.58 (m, 1 H), 2.97-2.92 (m, 1 H), 2.42-

2.37 (m, 1 H), 1.91-1.89 (m, I H) 1.87-1.46 (m, 7H), 1.37 (s, 9H), 1.19-0.89 (d, 4H) ppm.

B. (25,45)-1-{(25)-2-Amino-3-[l-(isopropylsulfonyl)piperidin-4-yl]propanoyl}-4-

fluoropyrrolidine-2-carbonitrilehydrochloride.

To dry DMF (25 mL) was added compound A (1.75 g, 4.62 mmol), HATU (1.76 g, 4.62

mmol) and N.N-diisopropylethylamine (0.80 mL, 4.62 mmol). After stirring at RT for 30

minutes (2S, 4S)-4-fluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate (1.0 g,

3.47 mmol) and additional N.N-diisopropylethylamine (0.60 mL, 3.47 mmol) were added.

This solution was allowed to stir at RT for 12 hr and then saturated sodium bicarbonate

(1 10 mL) was added. The mixture was extracted with ethyl acetate (2 X 100 mL) and the

organics were washed with saturated NaCl (50 mL) and dried over MgSU4. Removal of

the solvent in vacuo gave a crude solid. The solid was chromatographed (silica gel,

hexanes/EtOAc 4:1) to provide a solid which was stirred in a dioxane-HCl (4.0 M, 20 mL)

solution for 2 hrs followed by the addition of diethyl ether (100 mL). The precipitate was

collected by filtration and dried under high vacuum yielding 969 mg (2.35 mmol, 68 %

yield) of compound B as a white solid..

'H NMR (de-DMSO) 400 MHz δ 8.51-8.44 (s(br), 3H), 5.15 (d, 1 H, 7= 51 Hz), 5.06 (d, I H, 7

= 8.8 Hz), 4.04-4.01 (s(br), 1 H), 3.96-3.84 (m, 2H), 3.72-3.61 (m, 2H), 3.51-3.37 (m(br),

2H), 3.35-3.27 (m, 1 H), 2.79-2.76 (m, 2H), 1.82-1.69 (m, 5H), 1.17 (m, 8H) ppm.

(25,45)-1-{(25)-2-Amino-3-[l-(4-methylphenylsulfonyl)piperidin-4-yl]propanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride

A. (25)-2-[(rert-butoxycarbonyl)amino]-3-[l -(4-methylphenylsulfonyl)piperidin-4-

yljpropanoic acid.

To a CH₃CN (150 mL) solution of compound A from example 12 (1.0 g, 3.00 mmol) was

added triethylamine (1.52 g, 15 mmol) and toluenesulfonyl chloride (686 mg, 3.6 mmol).

After stirring for 6 hrs the acetonitrile was removed in vacuo and H2O (50 mL) and ethyl

acetate (300 mL) were added. Separation of the ethyl acetate layer followed by drying

over MgS0₄ and removal of the solvent in vacuo yielded a solid which was washed with

ether and dried under high vacuum yielding 1.09 g (2.56 mmol, 85% yield) of compound

A as a white solid.

Η NMR (de-DMSO) 400 MHz δ 12.4 (s(br), I H). 7.61 (d, 2H, 7 = 8.2 Hz), 7.42 (d, 2H, 7 =

8.2 Hz), 7.13 (d, 1 H, 7 = 8.3 Hz), 4.27-4.21 (m, 1 H), 3.98-3.95 (m, 1 H), 3.58-3.52 (m, 1 H),

2.98-2.91 (m, 1 H), 2.42 (s, 3H), 2.21-2.20 (m, 1 H), 1.81-1.50 (m, 6H), 1.39 (s, 9H), 1.21-

1.08 (m, 1 H) ppm.

B. (25,45)-1 -{(25)-2-Amino-3-[1-(4-methylphenylsulfonyl)piperidin-4-yl]propanoy|}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride. To dry DMF (25 mL) was added compound A (2.13 g, 5.0 mmol), HATU (1.90 g, 5.00

mmol).and N.N-diisopropylethylamine (0.87 mL, 5.0 mmol). After stirring at RT for 30

minutes (2S, 4S)-4-fluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate (1.1 g,

3.75 mmol) and additional N.N-diisopropylethylamine (0.65 mL, 3.75 mmol) were added.

This solution was allowed to stir at RT for 12 hr and then saturated sodium bicarbonate

(110 mL) was added. The mixture was extracted with ethyl acetate (2 X 100 mL) and the

organics were washed with saturated NaCl (50 mL) and dried over MgSU4. Removal of

the solvent in vacuo gave a crude solid that was chromatographed (silica gel, hexanes/EtOAc 4:1) to provide a solid which was stirred in a dioxane-HCl (4.0 M, 20 mL)

solution for 2 hrs followed by the addition of diethyl ether (100 mL). The resulting

precipitate was collected by filtration and dried under high vacuum yielding 1.10 g (2.4

mmol, 64 % yield) of compound B as a white solid.

¹H NMR (de-DMSO) 400 MHz δ 8.21-8.05 (s(br), 3H). 7.60 (d, 2H, 7 = 7.9 Hz), 7.43 (d, 2H.

7 = 7.9 Hz), 3.91-3.88 (m, 2H), 3.07-2.99 (m, 1 H), 2.43 (s, 3H), 2.09-2.03 (m, I H), 1.94-

1.29 (m, 8H), 1.12-1.02 (m, 1 H) ppm.

Example 42

(25,45)-1-{(25)-2-Amino-3-[l-(isopropylsulfonyl)piperidin-3-yπpropanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride A. (25)-2-[(£ert-butoxycarbonyl)amino]-3-[l -(isopropylsulfonyl)piperidin-3-yl]propanoic acid.

To a CH₃CN (150 mL) solution compound A from example 13 (1.0 g, 3.00 mmol) was

added triethylamine (1.52 g, 15 mmol) and isopropylsulfonyl chloride (513 mg, 3.6

mmol). After stirring for 6 hr the acetonitrile was removed in vacuo and then added H2O

(50 mL) and ethyl acetate (300 mL) were added. Separation of the ethyl acetate layer

followed by drying over MgS0₄ and removal of the solvent in vacuo yielded a solid

which was washed with ether and dried under high vacuum yielding 926 mg (2.43 mmol,

81% yield) of compound A as a white solid.

'H NMR (de-DMSO) 400 MHz δ 12.2 (s(br), 1 H), 7.18 (d, 1 H, 7 = 8.3 Hz), 3.94-3.89 (m.

1 H), 3.55-3.49 (m(br)„ 2H), 3.35-3.30 (m, 2H), 2.89-2.85 (m, 1 H), 2.62-2.57 (m, 1 H), 1.91-

1.42 (m, 5H), 1.39 (s, 9H), 1.19 (d, 6H) ppm.

B. (25,45)-1-{(25)-2-Amino-3-[1-(isopropyIsulfonyl)piperidin-3-yl]propanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride.

To dry DMF (25 mL) was added compound A (1.75g, 4.62 mmol), HATU (1.76 g, 4.62

mmol) and N.N-diisopropylethylamine (0.80 mL, 4.62 mmol). After stirring at RT for 30

minutes (2S, 4S)-4-fluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate (1.0 g,

3.47 mmol) and additional N.N-diisopropylethylamine (0.60 mL, 3.47 mmol) were added.

This solution was allowed to stir at RT for 12 hr and then saturated sodium bicarbonate

(1 10 mL) was added. The mixture was extracted with ethyl acetate (2 X 100 mL) and the

organics were washed with saturated NaCl (50 mL) and dried over MgSU4. Removal of

the solvent in vacuo gave a crude solid. This solid was chromatographed (silica gel,

hexanes/EtOAc 4:1) to provide a solid which was stirred in a dioxane-HCl (4.0 M, 20 mL) solution for 2 hrs followed by the addition of diethyl ether (100 mL). The resulting

precipitate was collected by filtration and dried under high vacuum yielding 1.01 g

(2.46 mmol, 71 % yield) of compound B as a white solid.

¹H NMR (de-DMSO) 400 MHz δ 8.42-8.39 (s(br), 3H), 5.51 (d, 1 H, 7 =51 Hz), 5.06 (d, 1 H. 7

= 8.8 Hz), 4.07-4.01 (s(br), I H). 3.98-3.84 (m, 2H), 3.52-3.49 (m, 1 H), 3.41-3.27 (m(br),

4H), 3.09-3.02 (m, l H), 2.98-2.80 (m, 1 H), 2.65-2.61 (m, 1 H), 1.77-1.63 (m, 6H), 1.18 (m, 8H) ppm.

Example 43

(25,45)-1-{(25)-2-Amino-3-[l-(4-methylphenylsulfonyl)piperidin-3-yl]propanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride

A. (25)-2-[(rert-butoxycarbonyl)amino]-3-[l -(4-methylphenylsulfonyl)piperidin-3-

yl]propanoic acid

To an CHsCN (150 mL) solution of compound A from example 13 (1.Og, 3.00mmol) was

added triethylamine (1.52 g, 15 mmol) and toluene sulfonyl chloride (686 mg, 3.6

mmol). After stirring for 6 hrs the acetonitrile was removed in vacuo and then H2O (50

mL) and ethyl acetate (300 mL) were added. Separation of the ethyl acetate layer

followed by drying over MgS0₄ and removal of the solvent in vacuo yielded a solid which was washed with ether and dried under high vacuum yielding 946 mg (2.22 mmol, 74% yield) of compound A as a white solid.

'H NMR (de-DMSO) 400 MHz δ 12.4 (s(br), 1 H), 7.61 (d, 2H, 7 = 8.2 Hz), 7.41 (d, 2H, 7 =

8.2 Hz), 7.15 (d, 1 H, 7 = 8.3 Hz), 3.98-3.91 (m, 1 H), 3.45-3.41 (m, 2H), 2.42 (s, 3H), 2.21-

2.20 (m, 1 H), 2.05-1.95 (m, I H). 1.87-1.45 (m, 6H), 1.42 (s, 9H), 1.21 -1.08 ( , 1 H) ppm.

B. (25,45)-1-{(25)-2-Amino-3-[1-(4-methylphenylsulfonyl)piperidin-3-yl]propanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride.

To a dry DMF (25 mL) was added compound A (2.13 g, 5.0 mmol), HATU ( 1.90 g, 5.00

mmol) and N.N-diisopropylethylamine (0.87 mL, 5.0 mmol). After stirring at RT for 30 minutes (2S, 4S)-4-fluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate (1.1 g,

3.75 mmol) and additional N.N-diisopropylethylamine (0.65 mL, 3.75 mmol) were added. This solution was allowed to stir at RT for a total of 12 hr and then saturated sodium

bicarbonate (1 10 mL) was added. The mixture was extracted with ethyl acetate (2 X 100

mL) and the organics were washed with saturated NaCl (50 mL) and dried over MgSθ4

and concentrated to dryness to give a crude solid. The solid was chromatographed (silica gel, hexanes/EtOAc 4:1) to provide a solid which was stirred in a dioxane-HCl (4.0 M, 20

mL) solution for 2 hrs followed by the addition of diethyl ether (100 mL). The resulting

precipitate was collected by filtration and dried under high vacuum yielding 1.17 g (2.55

mmol, 51 % yield) of compound B as a white solid..

'H NMR (de-DMSO) 400 MHz δ 8.50-8.43 (s(br), 3H), 7.61 (d, 2H, 7 = 7.9 Hz), 7.43 (d. 2H,

7 = 7.9 Hz), 5.51 (d, 1 H, 7= 51 Hz), 5.07 (d, 1 H, 7 = 8.8 Hz), 4.07-4.01 (brm, 1 H), 3.91-3.83

(m, 1 H), 3.48-3.40 (m, 2H), 3.17-3.05 (m, 2H), 2.57-2.51 (m, 1 H), 2.38 (s, 3H), 1.84-1.23

(m, 8H), 1.07-1.05 (m, 1 H) ppm. Example 44

(25,45)-1-[(25)-2-Amino-3-(l -benzothien-3-yl)propanoyl]-4-fluoropyrrolidine-2-

carbonitrile hydrochloride

A. (25,45)-1-[(25)-2-Amino-3-(1 -benzothien-3-yl)propanoyl]-4-fluoropyrrolidine-2-

carbonitrile hydrochloride.

To dry DMF (25 mL) was added (25)-3-(1 -benzothien-3-yl)-2-[(fert-

butoxycarbonyl)amino]propanoic acid (1.92 g, 6.0 mmol), HATU (2.28 g, 6.0 mmol) and

N.N-diisopropylethylamine (1.05 mL, 6.0 mmol). After stirring at RT for 30 min (2S, 4S)- 4-fluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate (1.31 g, 4.5 mmol) and

additional N.N-diisopropylethylamine (0.78 mL, 4.5 mmol) were added. This solution was

allowed to stir at RT for 12 hr and then saturated sodium bicarbonate (1 10 mL) was added. The mixture was extracted with ethyl acetate (2 X 100 mL) and the organics were

washed with saturated NaCl (50 mL), dried over MgSU4 and concentrated to dryness to

give a crude solid. The solid was chromatographed (silica gel, hexanes/EtOAc 4:1) to

provide a solid which was stirred in a dioxane-HCl (4.0 M, 20 mL) solution for 2 hrs

followed by the addition of diethyl ether (100 mL). The precipitate was collected by

filtration and dried under high vacuum yielding 1.0 g (2.84 mmol, 63 % yield) of

compound A as a white solid. ¹H NMR (de-DMSO) 400 MHz δ 8.70-8.61 (s(br), 3H), 8.05-8.00 (m, 2H), 7.45-7.36 (m,

3H), 5.21 (d, 1 H, 7= 51 Hz), 5.01 (d, 1 H, 7 = 8.8 Hz), 4.23-4.20 (m, 1 H), 3.71 (ddd, 1 H, 7 =

41.1 , 12.3, 3.2 Hz), 3.55-3.51 (m, 1 H), 3.30-3.24 (m, 1 H), 2.73 (q, 1 H, 7 = 12.1 Hz), 2.39- 2.14 (m, 2H) ppm.

Example 45

(2S,4S)-1 -{(2S)-2-Amino-3-methyl-3-[4-(trifluoromethyl)phenyl]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride . A. 2-Methyl-2-[4-(trifluoromethyl)phenyl]propanal.

To a toluene solution (90 mL) containing 2methyl-2-[4-(trifluoromethyl)phenyl]

propanenitrile (see: 7. Am. Chem. Soc. 2000, 722, 712, Caron, S. et al. for the preparation

of this compound) (5.54 g, 26.0 mmol) cooled to -78 °C was added 26.0 mL of a 1.5 M

toluene solution of DIBAL (39.0 mmol). The resulting solution stirred at -78 °C for 2.5 hr

at which time it was quenched with an aqueous THF solution (105 mL/20 mL) containing

sodium acetate (6.6 g) and acetic acid (6.6 mL). After 5 min the cold bath was removed

and stirring continued for 20 min at RT when celite and Et2θ were added. Upon stirring

an additional 30 min, the heterogeneous solution was filtered through a bed of celite.

The celite was rinsed thoroughly with Et∑O and then the aqueous layer separated. The

organics were washed with H2O, dried (MgSU4) and the solvent removed in vacuo. The residual oil was purified via column chromatography (5%EtOAc/hexanes) yielding 4.22 g

(19.5 mmol, 75% yield) of compound A as a clear oil.

'H NMR (CDCb) 400 MHz δ 9.52 (s, 1 H), 7.63 (d, 2H, 7 = 8.2 Hz), 7.39 (d, 2H, 7 = 8.2 Hz),

1.49 (s, 3H) ppm.

B. 2-Amino-3-methyl-3-[4-(trifluoromethyl)phenyl]butanenitrile.

To a MeOH solution (10 mL) containing compound A (1.87 g, 8.66 mmol) was added 1.2

mL of 30% NH₄0H, H₂0 (6 mL) and potassium cyanide (592 mg, 9.09 mmol). To this

solution was added ammonium chloride (510 mg, 9.53 mmol) at RT. After 1 hr the

solution was heated to 70 °C for 6 hr. Upon cooling, the reaction was diluted with EtOAc

and then washed with sat NaHC0₃ and H2O. After drying over MgS0₄ the solvent was

removed in vacuo and the residue purified via column chromatography (EtOAc/hexanes

(3:2)) yielding 1.52 g (6.27 mmol, 72% yield) of compound B.

Η NMR (CDCb) 400 MHz δ 7.63 (d, 2H, 7 = 8.4 Hz), 7.55 (d, 2H, 7 = 8.5 Hz), 3.80 (t, 1 H, 7

= 7.9 Hz), 1.56 (s, 3H), 1.53 (s, 3H), 1.40 (d(br), 2H, 7 = 6.9 Hz) ppm.

C. 2-Amino-3-methyl-3-[4-(trifluoromethyl)phenyl]butanoic acid hydrochloride.

To a flask containing compound B (2.96 g, 12.2 mmol) was added 10 mL of acetic acid

followed by 60 mL of concentrated HCl. The solution was then heated to reflux

overnight and upon cooling the bulk of the solvent was removed in vacuo. The resulting

white solid was triterated with an Et2θ/hexanes (2:1) solution and collected via vacuum

filtration. After pumping on under high vacuum 3.13 g (10.5 mmol, 86% yield) of

compound C was obtained as a white solid. Η NMR (d₄-MeOH) 400 MHz δ 7.69 - 7.68 (m, 4H), 4.33 (s, 1 H), 1.57 (s, 3H), 1.53 (s, 3H)

ppm.

D. 2-[(Tert-butoxycarbonyl)amino]-3-methyl-3-[4-(trifluoromethyl)phenyl]butanoic acid.

To a dioxane (30 mL)/H2θ (7 mL) solution containing compound C (3.05 g, 10.2 mmol)

was added 12.8 mL of a 2.0 M NaOH solution (25.6 mmol) followed by di-t-butyl

dicarbonate (3.13 g, 14.4 mmol) at RT. The solution stirred overnight at which time it

was poured into sat. NaHC0₃ and washed with Et2U. The Et2θ layer was washed with sat.

NaHC0₃ and then the aqueous layers combined. After acidification with 1.0 M HCl the

organics were extracted with EtOAc (2X). After drying over MgSU4 the solvent was

removed in vacuo yielding 3.58 g (9.92 mmol, 97% yield) of compound D as a white solid.

'H NMR (CDCb) 400 MHz δ 7.57 (d, 2H, 7 = 8.2 Hz), 7.49 (d, 2H, 7 = 8.3 Hz), 4.99 (d(br),

l H, 7 = 9.2 Hz), 4.63 (d(br), 1 H, 7 = 9.3 Hz), 1.45 (s, 3 H), 1.44 (s, 3H), 1.36 (s, 9H) ppm.

E. Tert-butyl (lS)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-l-yl]carbonyl}-2-methyl-2-

{4-(trifluoromethyl)phenyl]propylcarbamate.

To a DMF solution (45 mL) containing compound D (2.0 g, 5.54 mmol) was added N.N-

diisopropylethylamine (788 mg, 6.09 mmol) followed by HATU (2.21 g, 5.82 mmol) at RT.

The resulting solution stirred for 30 min at which time a DMF solution (27 mL)

containing (2S,4S)-4-fluoro-2-pyrrolidinecarbonitrile 4-methylbenzenesulfonate (1.66 g,

5.82 mmol) and N.N-diisopropylethylamine (752 mg, 5.82 mmol) was added. After

stirring overnight the solution was diluted with EtOAc and washed with H2O (3X), sat. NaHC0₃ and 1.0 M HCl. After drying over MgSU4the solvent was removed in vacuo and

the residual dark oil purified via column chromatography (hexanes/EtOAc/CH∑Cb (5:4:1)).

A low Rf material (610 mg, 1.37 mmol) was obtained which was a single stereoisomer by

'H NMR and LC plus 335 mg of a high Rf material which contained desired product, but

was contaiminated with another compound. The low Rf material turned out to be the (S)-diastereomer after in vitro testing.

Low Rfdiastereomer:

¹H NMR (CDCb) 400 MHz δ 7.65 - 7.60 (m, 2H), 7.59 - 7.55 (m, 2H), 5.29 (d, 1 H, 7 = 9.9

Hz), 5.06 (dt, 1 H, 7 = 51.1 , 3.3 Hz), 4.85 (d, 1 H, 7 = 9.4 Hz), 4.34 (d, 1 H, 7 = 9.9 Hz), 3.55

(ddd, 1 H, 7 = 3.4, 12.1 , 36.2 Hz), 2.70 (dd, 1 H, 7 = 12.3, 17.8 Hz), 2.48 (t, 1 H, 7 = 15.4 Hz).

2.15 (m, l H), 1.53 (s, 3H), 1.51 (s, 3H), 1.40 (s, 9H) ppm.

F. (2S,4S)-1 -{(2S)-2-Amino-3-methyl-3-[4-(trifluoromethyl)phenyl]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride. To a CH2CI2 solution (10 mL) containing compound E (643 mg, 1.44 mmol, low Rf isomer)

was added TFA (1.65 g, 14.4 mmol) at RT. After 1 hr an additional 5 eq of TFA was added

and after a total of 2 hr the solvent was removed in vacuo and the residual oil dissolved

in 5 mL of dioxane. To this solution was added 10 mL of a 4.0 M dioxane solution of HCl.

The solvent was removed in vacuo followed by the addition of CH2CI2 and Et2θ. This

process was repeated 3X until a consistent solid formed. The solid was collected via

vacuum filtration yielding 406 mg (1.03 mmol, 72% yield) of compound F.

H NMR (d4-MeOH) 400 MHz δ 7.74 - 7.66 (m, 4H), 5.13 (d(br), 1 H, 7 = 51.3 Hz), 4.97 (d,

1 H, 7 = 9.3 Hz), 4.25 (s, 1 H), 3.52 (m, 1 H), 2.74 (dd, 1 H, 7 = 22.0, 1 1.9 Hz), 2.47 - 2.22 (m,

2H), 1.67 (s, 3H), 1.60 (s, 3H) ppm. Example 46

(3R)-3-Amino-4-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1 -yl]-2-methyl-4-oxobutane-2- sulfonic acid

A. (4R)-2,2,5,5-Tetramethyl-1 ,3-thiazolidine-4-carboxylic acid.

To a suspension of L-penicillamine (0.300 g, 2.01 mmol) in methanol (2 mL) was

added acetone (4 mL, 54.5 mmol). The solution become clear and was stirred for ca. 3

hours after which it became cloudy again. The solution was concentrated to afford 0.377 g of compound A as a white solid (99% yield). ^'

'H-NMR (de-DMSO) 400 MHz δ 3.74 (s, 1 H), 2.48 (m, 2H), 1.54 (m, 6H), 1.43 (s,

3H), 1.18 (s, 3H) ppm.

B. (2S,4S)-4-Fluoro-1-{[(4R)-2,2,5,5-tetramethyl-1 ,3-thiazolidin-4-yl]carbonyl}

pyrrolidine-2-carbonitrile. A solution of compound A (0.256 g, 1.35 mmol), (2S,4S)-4-fluoropyrrolidine-2-

carbonitrile tosylate (0.387 g, 1.35 mmol), 1-hydroxybenzotriazole hydrate (0.182 g, 1.35

mmol) and N.N'-dicyclohexylcarbodiimide (0.279 g, 1.35 mmol) and N,N'-

diisopropylethylamine (0.280 mL, 1.63 mmol) in tetrahyd ofuran (13 mL) was stirred for

ca. 48 hours and then concentrated. The residue was taken up with ethyl acetate and

filtered. The filtrate was washed with aqueous NaHC0₃ and brine. The organic layer was dried over MgS04 and concentrated. The crude mixture was chromatographed on silica

gel (5% MeOH/95% CHCb) to afford 0.171 g of compound B as a white solid (44% yield).

'H-NMR (de-DMSO) 400 MHz δ 5.48 (d, 7= 50 Hz, 1 H), 5.02 (m, 1 H), 4.05-3.85 ( ,

3H), 3.48 (m, 1 H), 2.42 (m, 1 H), 1.54 (m, 5H), 1.47 (s, 3H), 1.22 (s, 3H) ppm.

C. (2S,4S)-1-[(2R)-2-Amino-3-mercapto-3-methylbutanoyl]-4-fluoropyrrolidine-2- carbonitrile hydrochloride.

To a solution of compound B (0.143 g, 0.501 mmol) in 50% v:v water/methanol

(20 mL) was added 1.0 N HCl (0.550 mL, 0.550 mmol). The solution was mixed at ca. 500

mbar at ca. 35 °C for ca. 1 hour. The solution was concentrated, taken up with

tetrahydrofuran, dried over MgSθ4 and concentrated again to afford 0.163 g of compound C as an off-white solid (100% yield).

'H-NMR (de-DMSO) 400 MHz δ 8.61 (s, 1 H), 5.54 (d, 7= 52 Hz, 1 H), 5.05 (d, 7 = 9

Hz, 1 H), 4.28-3.89 (m, 4H), 2.47 (m, DMSO overlap, 1 H), 1.74 (m, 3H), 1.43 (m, 6H), 1.33

(s, 3H) ppm.

D. rert-butyl-(l R)-1-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-1 -yl]carbonyl}-2-mercapto-

2-methylpropyl carbamate.

To a solution of compound C (0.141 g, 0.501 mmol) and N,N-

diisopropylethylamine (0.090 mL, 0.526 mmol) in dichloromethane (3 mL) was added di-

(tert-butyl)-dicarbonate (0.120 g, 0.551 mmol) followed by additional N,N-

diisopropylethylamine (0.100 mL, 0.584 mmol). The solution was stirred for ca. 2 hours

and then concentrated. The crude mixture was chromatographed on silica gel (2%Me0H/98% CHCb) to afford 0.088 g of compound D as an oil which crystallized into a white solid (51% yield).

¹H-NMR (de-DMSO) 400 MHz δ 6.99 (d, 7= 9 Hz, 1 H), 5.53 (d, 7= 51 Hz, 1 H), 4.97

(d, 7= 7 Hz, 1 H), 4.37-3.74 (m, 3H), 2.95 (s, 1 H), 2.40 (m, 1 H), 1.35 (m, 15H) ppm.

E. (3R)-3-Amino-4-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1 -yl]-2-methyl-4-oxobutane-2- sulfonic acid. °

To a solution of compound D (4.412 g, 12.8 mmol) in methanol (18 mL) was

added 30% aqueous hydrogen peroxide (5.9 mL). The solution was stirred for ca. 30

hours, concentrated, lyophilized and dried over P2O5 at ca 40 °C for at least 12 hours.

The crude mixture was taken up with a 20% methanol/80% chloroform/acetic acid

solution and a solid precipitated. The suspension was filtered to afford 0.607 g of

compound E as a pink solid (16% yield).

'H-NMR (de-DMSO) 400 MHz δ 8.12 (s, 3H), 5.50 (d, 7= 50 Hz, 1 H), 5.07 (d, 7= 9 Hz, 1 H),

4.27-3.30 (m, 6H), 2.30 (m, DMSO overlap, 1 H), 1.26 (s, 3H), 1.15 (s, 3H) ppm.

BIOLOGICAL DATA

Materials:

H-Ala-Pro-pNA*HCI was purchased from BACHEM Bioscience Inc. (product no. L-

1 115). A 500 mM stock solution was prepared with dimethylsulfoxide and stored at -20

°C. Gly-Pro-AMC was purchased from Enzyme System Products (product no. AMC-39)

and stored at -20 °C as a 10 mM stock solution in dimethylsulfoxide. Test compounds

were dissolved to 10 mM in dimethylsulfoxide and this was used as a stock solution for

DPP-IV titration assays. Athens Research and Technology, Inc prepared the purified human DPP-IV. The material was isolated from human prostasomes using the method of

DeMeester et al., J. Immunol. Methods 189, 99-105. (1996). DPP-IV Assay:

Two-fold serial dilutions of test compounds in 100 % dimethylsulfoxide were

performed in 96-well polystyrene flat bottom plates (Costar, #9017). The average

enzymatic activity from wells containing dimethylsulfoxide but lacking test compound was used as a control value for calculating percent inhibition. DPP-IV (20 ng/mL) was

mixed in microtiter plates with test compounds, substrate and assay buffer to yield 100

μM H-Ala-Pro-pNA«HCI in 25 mM Tris, pH 7.5, 10 mM KCI, 140 mM NaCl. The intact

peptide contains a p-nitrophenylanilide which, when hydrolyzed by DPP-IV, releases the

absorbant p-nitrophenylaniline. The absorbency was monitored in 20 minutes intervals

at a wavelength of 387 nm using a Molecular Devices SpectraMax 250 absorbency plate

reader. The enzymatic activity was determined by estimating the best linear fit to the data. Values for enzymatic activity were taken directly from the linear fit determined by

the software on the plate reader.

Data Analysis: The enzymatic activity was determined by estimating the best

linear fit to the data. Data reduction was performed using the Microsoft Excel

RoboSage.

Determination of ICBO values: The enzymatic activity was plotted against the

concentration of test compound, including [I] = 0, and the ICso determined from a fit of

equation 2 to the data.

RATE = Vmax/ (l + ([!]/ ICso )) (2)

Vmax was the best fit estimate of the maximal enzymatic activity. Determination of Ki values: Ki values were calculated from ICso values using equation 3 assuming a competitive model.

, = /C₅₀ * [l ]

^{(s + κ}*r (3)

The apparent pKi values were > 5.0 for each of the examples.

DPP-II Assay:

The intermediate plate contained 5.3 μL of test compound in 2-fold serial

dilutions across the plate. A volume of 209 μL of buffer (100 mM sodium acetate pH

5.5) containing substrate (H-Lys-Ala-pNA-2HCI; product no. L-2085; BACHEM Bioscience

Inc.:) was added to each well of the intermediate plate, then mixed. The reaction was

initiated with the transfer of 180 μL of the substrate/test compound solution to the

assay plate containing 20 μL of enzyme. Final concentrations in the assay were 100 nM

enzyme and 1000 μM substrate in 100 mM NaOAc, pH 5.5, 2.5% DMSO in a final volume

of 200 μL. The absorbance was monitored every 20 minutes for 5 hours at 387 nm using

a Molecular Devices SpectraMax 250 absorbance plate reader.

Data Analysis: The enzymatic activity was determined by estimating the best

linear fit to the data. Data reduction was performed using the Microsoft Excel

RoboSage.

Determination of IC50 values: The enzymatic activity was plotted against the

concentration of test compound, including [I] = 0, and the ICBO determined from a fit of

equation 2 to the data. RATE = Vmax/ (l + ([!]/ ICso )) (2)

V ax was the best fit estimate of the maximal enzymatic activity.

Determination of Ki values: Ki values were calculated from ICso values using equation 3

assuming a competitive model.

K_t = /C₅₀ * [l ] (S + K_m) (3)

Certain compounds of the present invention showed activity for DPP-II, for example pKi

values > 6.0, while others demonstrated selectivity for DPP-IV, discussed hereinabove.

In vivo studies:

Age and weight matched male CD1 mice were housed individually at 72° F and

50% relative humidity with a 12 h light/dark cycle. Animals were dosed by oral gavage

with 10 ml/kg vehicle (0.5% methylcellulose (HPMC) with 0.1% Tween 80) or 1 mg/kg

test compound in vehicle. The animals were anesthetized with isofluorane for blood

collection at the specified times (0-6 hours). Plasma DPP-IV activity was measured using

the fluorogenic substrate Gly-Pro-AMC (50 μM) according to the manufacturers

specification (Enzyme System Products, Livermore CA). The substrate was mixed with 50

mM Tris, pH 7.8 and 20% plasma. The samples were incubated for 20 min at 30°C and

fluorescence measured using a cytofluor spectrofluoremeter with the filters set at 360

nm excitation and 460 nm emission.

COMPARATIVE EXAMPLES

The compounds of the present invention demonstrate a number of preferred

characterisitcs. Not to be limtied thereby, but the preferred characteristics of the compounds of the present invention are believed due to the unexpected benefits gained

through manipulation of the substitution patterns of the compounds of Formula (I). The

results of comparative testing illustrate several surprising and unexpected benefits.

More specifically, the comparative tests indicate that the compounds of the present

invention demonstrate: (i) increased potency, as characterized by DPP-IV inhibition

activity measured from plasma; (ii) increased selectivity; (iii) increased duration of

action; and/or (iv) an increased safety profile.

Comparison Examples

Comparison Example 1 Comparison Example 2 Comparison Example 3

bon

Comparison Example 4

Comparison Example 5 Comparison Example 6

Comparator I - Potency/Duration

Surprisingly, with regard to inhibtion of dipeptidyl peptidases, in particular with

DPP-IV, compounds that demonstrate high affinity for the enzyme (i.e. potent) do not

necessarily possess the duration of action desired for therapeutic effectievness.

Duration of action, as used herein, includes the ability of the compound to maintain

effectiveness, preferably therapeutic effectiveness, over a sustained period of time. A

compound demonstrating the preferred level of duration of action provides therapeutic

effectiveness with once-daily dosing. A once-a-day dosage regimen, as is appreciated in

the pharmaceutical arts, may be advantageous over multiple daily dosing regimens for

reasons such as patient compliance and convenience. Thus, preferred compounds should

not only be sufficiently potent but should also demonstrate desired levels of duration of

action.

Unfortunately, as is appreciated by those skilled in the art, there is no necessary

correlation between potency and duration of action. In other words, although a

compound demonstrates high affinity for the DPP-IV enzyme, the compound does not

necessarily have appropriate duration properties to be considered for preferential once-

daily dosing. The following illustrates this lack of correlation between potency and

duration of action. The following data illustates the invention but should not be

interpreted to limit the invention.

Age and weight matched male CD rats were housed individually at 72° F and

50% relative humidity with a 12 h light/dark cycle. Animals were dosed by oral gavage

with vehicle (10 mM acetic acid) or 1 mg/kg test compound in vehicle. After 6 hrs

administration of compound, the rats were anesthetized with isofluorane for cardiac

blood collection. DPP-IV activity in serum was measured using the fluorogenic substrate Gly-Pro-AMC (50 mM) according to the manufacturers specification (Enzyme System

Products, Livermore CA). The substrate was mixed with 50 mM Tris, pH 7.8, in plasma

(20% final v/v) and the samples were incubated for 5-20 min at 30°C. DPP-IV activity

was determined by measuring fluorescence using a cytofluor spectrofluoremeter with

the filters set at 360 nm excitation and 460 nm emission. Averages and standard

deviations from each group (n=3) were calculated using students t-test and compared to

vehicle treated rats to determine percent inhibition as shown in Table I.

TABLE I

ICBO (nM) Percent

Human DPP-IV DPP-IV inhibition At 6 hrs In Rats Comparison Example 1 1.3 32 ± 3

Comparison Example 2 32 + 3

Comparison Example 4 5.6 2.8 + 0.1

Comparison Example 5 71 + 13

Example 2 22 68 + 4

Example 9 72 64 + 12

As shown, Comparison Examples 1, 2, and 4 are very potent, each having ICBO

values of <5.6 nM. The compounds illustrative of the present invention, Examples 2 and

9 demonstrate adequate potency, namely ICBO values of 22 and 72 respectively. When

the Comparison Examples 1 , 2, and 4 were tested for duration of action, however, they each demonstrated a poor level of duration. The compounds of the present invention,

although less potent, present a duration of action preferred for overall therapeutic profile.

Comparison Example 5 is included to further demonstrate the unpredictability

between potency and duration of action. More specifically, Comparison Example 5

illustrates one compound having both high binding affinity and duration of action.

Thus, the relationship of potency to duration of action can not be characterized strictly

as either a parallel or an inverse relationship. Rather the relationship is not predictable.

This lack of correlation between potency and duration of action presents a lack

of predictability among inhibitors of dipeptidyl peptidases, specifically DPP-IV. This

unpredictability has not heretofore been recognized. As shown above, binding data,

alone, may or may not be enough to ascertain the therapeutic utility of such

compounds. The compounds of the present invention demonstrate desired levels of both

potency and duration of action in addition to the other properties herein discussed.

Comparator II - β-substitution and Cyclization

As described herein, the compounds of the present invention have at least two

identical β-substitutions, namely, R¹ and R² are the same. As referenced herein the

compounds of the present invention are di-β-substituted, as illustrated with Examples 1

and 10, respectively:

β-carbon (Example 1) β-carbon (Example 10). Thus, using Example 10 as an example, one preferred embodiment is described when R¹

and R² are each alkyl, more preferably Ci-Ce alkyl, more preferably, methyl. Another

preferred embodiment is illustrated by Example 1 , namely, when R¹ and R² are each aryl,

more preferably phenyl, and, as illustrated in Example 2, more preferably phenyl

substituted with halogen, preferably fluorine. Generally, di-β-substituted compounds,

such as those of the present invention, do not tend to cyclize with the nitruile warhead

to form the cyclic amidine, and, therefore, are stable.

On the other hand, mono-β-substituted compounds, for example, Comparison

Examples 3 and 4 illustrated hereinabove, where the β carbon does not carry two

identical substituents tend to cyclize and, therefore, demonstrate relatively short

stability half-lives, as measured by 1 1/2.

In addition, generally compounds with the gem-substitution pattern on the β-

carbon demonstrate increased selectivity as well.

Stability studies for DPP IV compounds

Typically 0.2 - 0.5 mg of compound is dissolved in 1 mL of Phosphate Buffered

Saline (PBS) in a HPLC sample vial and placed in an autosampler thermostatically

controlled at 37°C. An initial time point HPLC chromatogram is obtained followed by

successive HPLC chromatograms at time points of 24, 48, 72, etc hours.

HPLC method:

Column: Phenomenex Luna C18(2), 3μ, 100 x 4.6 mm, thermostated at 40 °C

Mobile Phase A: 95:5:0.2% Water: Acetonitrile: Trifluoroacetic acid

Mobile Phase B: 5:95:0.2% Water: Acetonitrile: Trifluoroacetic acid

Flow rate: 1 mL/minute Gradient: 0 - 100 %B in 15 minutes

UV Detection wavelength: 215 nm

The area under the drug substance peak is compared to the initial area for each

chromatogram and plotted as percent remaining from initial time (to).

Comparison of the cyclization/degradation reaction half-lives (t 1/2) of

compounds show increased stability for the di-β-substituted compounds, such as

Examples 2 and 9. Increased 1 1/2 is related directly to increased stability of the compound.

TABLE 2

^*Note: For Comparison Example 3 the autosampler was not thermostatically controlled

as described hereinabove, rather the autosampler was left at ambient room temperature.

Typically an increase in 10 °C increases the reaction rate two-fold, thereby decreasing

the 1 1/2 by two-fold. As will be apprciated by those skilled in the art, a reasonably

calculated 1 1/2 for Comparison Example 3 would be one half (or less) of the measured

value at room temperature (23 °C). Thus, in addition to potency and duration of action, the compounds of the present invention demonstrate desired stability as well.

Comparator 111 - Safety Profile

The dog is a common non-rodent species routinely used to satisfy regulatory

requirements. The beagle was chosen to examine the safety of the compounds of the

present invention because of the knowledge of this strain's general pathology and

response to a wide variety of drugs. Dogs (Marshall Farms Inc., North Rose, NY, USA)

from 9 to 12 months (5 to 12 kg) were dosed by oral gavage at 10 mL/kg/day with

vehicle (10 mM acetic acid, pH 3.3) or test compound in vehicle. Each dose was followed

by a flush of approximately 10 mL of water, once daily with 24-48 hours between each

subsequent dose. If limiting gastrointestinal toxicity was not observed, the dose was

increased 3-fold no sooner than approximately 24 hours after the preceding dose. Limiting GI toxicity includes diarrhea, loose or mucoid feces, blood in stool, and/or

sloughing of the gut epithelium. Clinical observations were made at least three times

daily during treatment (once prior to dosing, approximately one hour post dose, and

once in conjunction with pm viability). DPP-IV activity in serum was measured using the

fluorogenic substrate Gly-Pro-AMC (50 mM) according to the manufacturers

specification (Enzyme System Products, Livermore CA). The substrate was mixed with 50

mM Tris, pH 7.8, in serum (20% final v/v) and the samples were incubated for 5-20 min

at 30°C. DPP-IV activity was determined by measuring fluorescence using a cytofluor

spectrofluoremeter with the filters set at 360 nm excitation and 460 nm emission.

The compound illustrated above as Comparison Example 5 contains a relatively

small P2 portion as determined by molecular weight. Thus, Comparison Example 5 and

compounds of the present invention, which are herein described as having large P2 portions, namely Examples 2 and 9 for illustration, were each tested in the described

dose escalation studies.

Based on dose escalation studies in dogs, 0.3 mg/kg of Comparison Example 5

caused loose/mucoid feces with traces of blood whereas 1 mg/kg of a compound of the

present invention, Example 9 caused no adverse affects. Upon escalation to 3 mg/kg,

however, Example 9 caused loose/mucoid feces with traces of blood.

In contrast, 12 hours after oral dosing 0.2 mg/kg of Comparison Example 5,

Comparison Example 5 inhibited DPP4 by 2%. Surprisingly, 0.2 mg/kg of Example 9

inhibited DPP4 by 76% indicating that Example 9 significantly inhibits DPP4 activity in serum.

Likewise with other compounds of the present invention, 10 mg/kg of Example 2

had no adverse affects on gastrointestinal toxicity and 0.5 mg/kg of Example 2 inhibited

DPP4 by 45% 12 hours after oral dosing.

Based upon dose escalation studies in dogs, compounds of the present invention,

as illustrated by Examples 2 and 9, that have a larger molecular weight P2 portion are

preferred.

Comparator IV - Fluorinated v. non-fluorinated

When compounds of the present invention were tested in vivo at time periods

ranging from 0 to 10 hours, the flurorinated compounds of the present invention

demonstrated a significant increase in DPP-IV inhibition over their non-fluorinated

counterparts. Thus, the compounds of the present invention provide an unexpected

potency that was not heretofore appreciated. For example, Example 2 and its non-

fluorinated version, Comparison Example 6, were compared to determine the benefit of

the 4-fluoro substituent. Surprisingly, the 4-fluoro substituent prolongs the duration of the enzyme inhibitor complex dramatically as well as providing a significant increase in

potency. These beneficial properties have not heretofore been appreciated.

TABLE 3

All research complied with the principles of laboratory animal care (NIH

publication No. 85-23, revised 1985) and GlaxoSmithKline policy on animal use.

Although specific embodiments of the present invention have been illustrated

and described in detail, the invention is not limited thereto. The above detailed

description of preferred embodiments is provided for example only and should not be

construed as constituting any limitation of the invention. Modifications will be obvious

to those skilled in the art, and all modifications that do not depart from the spirit of the

invention are intended to be included within the scope of the appended claims.

Claims (51)

What is claimed is:

1. A compound of formula (I):

including salts, solvates, and pharmaceutically functional derivatives thereof, wherein

X is F or H;

R¹ and R² each are either

(i) H;

(ii) alkyl;

(iii) optionally substituted aryl or heteroaryl; or

(iv) combine to form a 3 to 14 membered ring sytem, optionally containing

one or more heteroatoms, and optionally containing one or more degrees of

unsaturation;

when R¹ and R² are (iii), then R³ is H or alkyl;

when R¹ and R² are (i), (ii), or (iv), then R³ is

where n is 0-5;

m is 0-12;

Y is S(0)_P, 0, alkylene, alkenylene, alkynylene, or a bond;

p is 0-2; R⁴ is R⁵ when Y is S, 0, alkylene, alkenylene, alkynylene, or a bond, where R⁵ is optionally

substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, or heterocyclyl;

R⁴ is R⁶ when Y is S(0) or S(0)2, where R⁶ is optionally substituted alkyl, aryl, cycloalkyl,

heteroaryl, amino, alkylamino, arylamino, heteroarylamino, cycloalkylamino, or hydroxy.

2. The compound of claim 1 wherein the optional substitutent is one or more of alkyl,

alkoxy, aryloxy, halogen, haloalkyl, cyano, alkylsulfonyl, or aryl.

3. The compound of claim 2 wherein the optional substituent is one or more of Ci-Ce

alkyl, Ci-Ce alkoxy, phenoxy, benzoxy, fluorine, chlorine, Ci-Cβ haloalkyl, cyano, Ci-Cε

alkylsulfonyl, phenyl, or benzyl.

4. The compound of claim 1 wherein R¹ and R² are each aryl, and R³ is H.

5. The compound of claim 4 wherein each of said aryl is phenyl.

6. The compound of claim 5 wherein each of said phenyl is substituted with halogen.

7. The compound of claim 6 wherein each of said halogen is fluorine.

8. The compound of claim 1 wherein R¹ and R² are each alkyl and R³ is

9. The compound of claim 8 wherein each of said alkyl is Ci-Ce alkyl.

10. The compound of claim 9 wherein each of said alkyl is methyl.

1 1. The compound of claim 10 wherein n is 0, m is 1 , and Y is S(0)_P.

12. The compound of claim 11 wherein p is 0, R⁴ is R⁵, and R⁵ is optionally substituted

aryl.

13. The compound of claim 12 wherein R⁵ is phenyl substituted with alkoxy.

14. The compound of claim 13 wherein said alkoxy is methoxy.

15. The compound of claim 1 1 wherein p is 1 or 2, R⁴ is R⁶, and R⁶ is optionally

substitued aryl.

16. The compound of claim 15 wherein R⁶ is phenyl substituted with alkoxy.

17. The compound of claim 16 wherein said alkoxy is methoxy.

18. The compound of claim 17 wherein p is 1.

19. The compound of claim 17 wherein p is 2.

20. The compound of claim 1 wherein the depicted NH2 group is cis to the depicted

nitrile warhead.

21. The compound of claim 1 wherein the depicted NH2 group is trans to the depicted

nitrile warhead.

22. The compound of claim 1 wherein A is H.

23. The compound of claim 1 wherein the depicted F is cis to the depicted nitrile.

24. A compound of formula (I):

including salts, solvates, and pharmaceutically functional derivatives thereof, wherein

X is F or H;

R¹ and R² each are optionally substituted aryl or heteroaryl; and

R³ is H or alkyl.

25. A compound of formula (I):

including salts, solvates, and pharmaceutically functional derivatives thereof, wherein X is F or H;

R¹ and R² each are either

(iii) alkyl; or

(iv) combine to form a 3 to 14 membered ring sytem, optionally containing

one or more heteroatoms, and optionally containing one or more degrees

of unsaturation; R³ is

where n is 0-5; m is 0-12;

Y is S(0)_P, 0, alkylene, alkenylene, or alkynylene;

p is 0-2;

R⁴ is R⁵ when Y is S, 0, alkylene, alkenylene, or alkynylene, where R⁵ is optionally

substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, or heterocyclyl;

R⁴ is R⁶ when Y is S(0) or S(0)₂, where R⁶ is optionally substituted alkyl, aryl, cycloalkyl,

heteroaryl, amino, alkylamino, arylamino, heteroarylamino, cycloalkylamino, or hydroxy.

26. The compound of claim 25 wherein p is 1 or 2.

27. The compound of claim 26 wherein p is 2.

28. The compound of claim 25 wherein R¹ and R² each are alkyl.

29. The compound of claim 28 wherein R¹ and R² are each G-Ce alkyl.

30. The compound of claim 29 wherein R¹ and R² are each methyl.

31. A compound selected from:

32. A compound selected from:

33. A compound selected from: (2S,4S)-1 -[(2S)-2-Amino-3,3-diphenylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile

hydrochloride; (2S,4S)-1-[(2S)-2-Amino-3,3-bis(4-fluorophenyl)propanoyl]-4-fluoropyrrolidine-2-

carbonitrile hydrochloride; (2S,4S)-1 -[(2S)-2-Amino-5-(4-fluorophenyl)-3,3-dimethylpentanoyl]-4-fluoro-2-

pyrrolidinecarbonitrile hydrochloride; (2S,4R)-1 -[(2S)-2-Amino-4-(4-fluorophenyl)-3,3-dimethylbutanoyl]-4-

fluoropyrrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1 -{(2R)-2-Amino-3-[(4-methoxybenzyl)thio]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-{(2R)-2-Amino-3-methyl-3-[(3-phenylpropyl)thio]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1 -{(2R)-2-Amino-3-methyl-3-[(2-phenylethyl)thio]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1 -((2R)-2-Amino-3-{[3-(4-fluorophenyl)propyl]thio}-3-methylbutanoyl)-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-{(2R)-2-Amino-3-[(4-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-{(2R)-2-Amino-3-[(4-methoxybenzyl)sulfinyl]-3-methylbutanoyl}-4-

fluoropyrroIidine-2-carbonitrile hydrochloride;

(2S,4S)-1 -((2R)-2-Amino-3-methyl-3-{[4-(trifluoromethyl)benzyl]thio}butanoyl)-4-

fluoropyrrolidine-2-carbonitrile hydrochloride; (2S,4S)-1 -[(2S)-2-Amino-2-(l -vinylcyclopentyl)ethanoyl]-4-fluoropyrrolidine-2-

carbonitrile hydrochloride; (2S,4S)-1-[(2S)-2-Amino-5-(4-methoxyphenyl)-3,3-dimethylpentanoyl]-4-

fluoropyrrolidine-2-carbonitrile hydrochloride; (2S,4S)-1 -{(2R)-2-Amino-3-methyl-3-[(3-phenylpropyl)sulfonyl]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile; (2S,4S)-1-{(2R)-2-Amino-3-methyl-3-[(4-methylbenzyl)sulfonyl]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile; (2S,4S)-1 -((2R)-2-amino-3-{[4-(benzyloxy)benzyl]sulfonyl}-3-methylbutanoyl)-4-

fluoropyrrolidine-2-carbonitrile;

(2S,4S)-1 -{(2R)-2-amino-3-[(4-cyanobenzyl)sulfonyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile;

(2S,4S)-1-((2R)-2-amino-3-methyl-3-{[4-(methylsulfonyl)benzyl]sulfonyl}butanoyl)-4-

fluoropyrrolidine-2-carbonitrile;

(2S,4S)-1 -{(2S)-2-Amino-2-[1-(4-fluorobenzyl)cyclopentyl]ethanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-((2S)-2-Amino-2-{l -[4-(trifluoromethyl)phenyl]cyclopentyl}ethanoyl)-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-{(2S)-2-Amino-2-[l-(4-fluorobenzyl)cyclopropyl]ethanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1 -[(2R)-2-Amino-3- (benzylsulfonyl)-3-methylbutanoyl]-4-fluoropyrrolidine-2-

carbonitrile hydrochloride;

(2S,4S)-1-{(2R)-2-amino-3-[(3-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1 -{(2R)-2-Amino-3-[(l ,1'-biphenyl-4-ylmethyl)sulfonyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride; (2S,4S)-1-{(2R)-2-Amino-3-[(2-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride; (2S,4S)-1 -{(2R)-2-Amino-3-methyl-3-[(pyridin-3-ylmethyl)thio]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile; (2S,4S)-1 -{(2R)-2-Amino-3-methyl-3-[(pyridin-2-ylmethyl)thio]butanoγl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride; (2S,4S)-1 -{(2R)-2-amino-3-methyl-3-[(pyridin-4-ylmethyl)thio]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-{(2R)-2-amino-3-[(4-fIuorobenzyl)sulfonyl]-3-methylbutanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1 -{(2R)-2-amino-3-methyl-3-[(3-phenoxybenzyl)sulfonyl]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-{(2R)-2-amino-3-methyl-3-[(3-phenoxybenzyl)thio]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-((2R)-2-Amino-3-{[(5-chloro-1 ,1-dioxido-1 -benzothien-3- yl)methyl]sulfonyl}-3-methylbutanoyl)-4-fluoropyrrolidine-2-carbonitrile

hydrochloride;

(2S,4S)-1-{(2R)-2-Amino-3-[(2,1 ,3-benzoxadiazol-5-ylmethyl)thio]-3-methylbutanoyl}-

4-fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1-{(2R)-2-Amino-3-methyl-3-[(pyridin-4-ylmethyl)sulfonyl]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S4S)-1-[(2S)-2-Amino-3-pyridin-4-ylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile

hydrochloride;

(2S4S)-1 -[(2S)-2-Amino-3-pyridin-3-ylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile

hydrochloride; (2S,4S)-1-[(2S)-2-Amino-3-piperidin-4-ylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile

dihydrochloride; (2S,45)-1-[(25)-2-Amino-3-piperidin-3-ylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile

dihydrochloride; (25,45)- 1 -[(2S)-2-Amino-3-piperidin-2-ylpropanoyl]-4-fluoropyrrolidine-2-carbonitrile dihydrochloride;

(2S,4S)-1 -{(2S)-2-Amino-3-[l-(isopropylsulfonyl)piperidin-4-yl]propanoyl}-4-

fluoropyrrolidine-2-carbonitrilehydrochloride;

(25,45)- 1 -{(25)-2-Amino-3-[l-(4-methylphenylsulfonyl)piperidin-4-yl]propanoyl}-4- fluoropyrrolidine-2-carbonitrile hydrochloride;

(2S,4S)-1 -{(25)-2-Amino-3-[l-(isopropylsulfonyl)piperidin-3-yl]propanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(25,45)- 1-{(25)-2-Amino-3-[l-(4-methγlphenylsulfonyl)piperidin-3-yl]propanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride;

(25,45)- 1-[(25)-2-Amino-3-(l -benzothien-3-yl)propanoyl]-4-fluoropyrrolidine-2-

carbonitrile hydrochloride;

(2S,4S)-1-{(2S)-2-Amino-3-methyl-3-[4-(trifluoromethyl)phenyl]butanoyl}-4-

fluoropyrrolidine-2-carbonitrile hydrochloride; and

(3R)-3-Amino-4-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]-2-methyl-4-oxobutane-2-

sulfonic acid.

34. A pharmaceutical formulation comprising a compound of claims 1 to 33.

35. The pharmaceutical formulation of claim 34 further comprising a pharmaceutically

acceptable carrier.

36. A method of inhibiting a post proline/analine cleaving protease comprising

administering a compound of claims 1 to 33.

37. The method of claim 36 wherein the post proline/analine cleaving protease is a

serine protease.

38. The method of claim 37 wherein the serine protease is a dipeptidyl peptidase.

39. The method of claim 38 wherein the dipeptidyl peptidase is DPP-II.

40. The method of claim 38 wherein the dipeptidyl peptidase is DPP-IV.

41. A method for the treatment or prophylaxis of metabolic disorders, gastrointestinal

disorders, viral disorders, inflammatory disorders, diabetes, obesity, hyperlipidemia,

dermatological or mucous membrane disorders, psoriasis, intestinal distress,

constipation, autoimmune disorders, encephalomyelitis, complement mediated

disorders, glomerulonepritis, lipodystrophy, tissue damage, psychosomatic,

depressive, and neuropsychiatric disorders, HIV infection, allergies, inflammation,

arthritis, transplant rejection, high blood pressure, congestive heart failure, tumors,

and stress-induced abortions comprising administering a compound of claims 1 to 33.

42. The method of claim 41 wherein the compound of claims 1 to 33 is administered for

the treatment or prophylaxis of diabetes.

43. Use of a compound of claims 1 to 33 in the manufacture of a medicament for the

inhibition of a post proline/analine cleaving protease.

44. The use of claim 43 wherein the post proline/analine cleaving protease is a serine

protease.

45. The use of claim 44 wherein the serine protease is a dipeptidyl peptidase.

46. The use of claim 45 wherein the dipeptidyl peptidase is DPP-II.

47. The use of claim 45 wherein the dipeptidyl peptidase is DPP-IV.

48. Use of a compound of claims 1 to 33 in the manufacture of a medicament for the

treatment or prophylaxis of metabolic disorders, gastrointestinal disorders, viral

disorders, inflammatory disorders, diabetes, obesity, hyperlipidemia, dermatological or mucous membrane disorders, psoriasis, intestinal distress, constipation,

autoimmune disorders, encephalomyelitis, complement mediated disorders,

glomerulonepritis, lipodystrophy, tissue damage, psychosomatic, depressive, and

neuropsychiatric disorders, HIV infection, allergies, inflammation, arthritis, transplant

rejection, high blood pressure, congestive heart failure, tumors, and stress-induced

abortions.

49. A compound according to claims 1 to 33 for use as an active therapeutic substance.

50. A compound according to claims 1 to 33 for use in the manufacture of a

medicament for the inhibition of serine protease.

51. A compound according to claims 1 to 33 for use in the manufacture of a

medicament for the treatment or prophylaxis of metabolic disorders, gastrointestinal

disorders, viral disorders, inflammatory disorders, diabetes, obesity, hyperlipidemia,

dermatological or mucous membrane disorders, psoriasis, intestinal distress,

constipation, autoimmune disorders, encephalomyelitis, complement mediated

disorders, glomerulonepritis, lipodystrophy, tissue damage, psychosomatic,

depressive, and neuropsychiatric disorders, HIV infection, allergies, inflammation,

arthritis, transplant rejection, high blood pressure, congestive heart failure, tumors,

and stress-induced abortions.