2-Cyano-4-fluoro-1-thiovalylpyrrolidine analogues as potent inhibitors of DPP-IV

Article abstract of DOI:10.1016/j.bmcl.2005.08.050

We report the synthesis and biological activity of a series of 2-cyano-4-fluoro-1-thiovalylpyrrolidine inhibitors of DPP-IV. Within this series, compound 19 provided a potent, selective, and orally active DPP-IV inhibitor which demonstrated a very long duration of action in both rat and dog.

Full text of DOI:10.1016/j.bmcl.2005.08.050

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5257–5261
2-Cyano-4-fluoro-1-thiovalylpyrrolidine analogues as
potent inhibitors of DPP-IV
Curt D. Haffner,a,* Darryl L. McDougald,a Steven M. Reister,a Brian D. Thompson,a
Christopher Conlee,a Jing Fang,a Jonathan Bass,a James M. Lenhard,b, Dallas Croom,b
Melissa B. Secosky-Chang,c Thaddeus Tomaszek,c Donavon McConn,d
Kevin Wells-Knechtd,à and Paul R. Johnsone
aDepartment of Medicinal Chemistry, GlaxoSmithKline Research and Development, 5 Moore Drive,
Research Triangle Park, NC 27709, USA
bDepartment of Metabolic Diseases, GlaxoSmithKline Research and Development, 5 Moore Drive,
Research Triangle Park, NC 27709, USA
cScreening and Compound Profiling, GlaxoSmithKline, 1250 S. Collegeville Rd., Collegeville, PA 19426, USA
dDrug Metabolism and Pharmacokinetics, GlaxoSmithKline Research and Development, 5 Moore Drive,
Research Triangle Park, NC 27709, USA
ePharmaceutical Development/Physical Properties and Developability, GlaxoSmithKline Research and Development,
5 Moore Drive, Research Triangle Park, NC 27709, USA
Received 25 July 2005; revised 12 August 2005; accepted 15 August 2005
Available online 15 September 2005
Abstract—We report the synthesis and biological activity of a series of 2-cyano-4-fluoro-1-thiovalylpyrrolidine inhibitors of DPP-
IV. Within this series, compound 19 provided a potent, selective, and orally active DPP-IV inhibitor which demonstrated a very long
duration of action in both rat and dog.
Ó 2005 Elsevier Ltd. All rights reserved.
Dipeptidyl peptidase IV (DPP-IV), also known as T-cell
antigen CD26, first identified in 1966,1 is a widely ex-
pressed serine exopeptidase. It has been shown to have
several functions in humans. First, it contributes to
extracellular matrix binding.2 Second, it functions as
an adenosine deaminase (ADA)-binding protein3 and
third, it exhibits post-proline or alanine cleaving proper-
ties from oligo or polypeptides at the N-terminus.4 It has
also been shown to inactivate two important incretins,
glucagon-like peptide 1 (GLP-1) and glucose-dependent
inhibitory polypeptide (GIP).5 Both of these incretins
act as glucose-dependent secretagogues upon meal
ingestion. However, in the presence of DPP-IV both of
them are rapidly inactivated.5 Previous studies have
shown that in patients with type 2 diabetes, GLP-1 re-
mains strongly insulinotropic even though its secretion
levels are reduced relative to non-diabetic subjects.6
However, GIPÕs insulinotropic effect is less if not absent
even though its secretion levels are normal or slightly be-
low normal.7 GLP-1Õs strong insulinotropic effect, but
poor pharmacokinetic profile, has led several companies
to identify GLP-1 agonists that are resistant to degrada-
tion by DPP-IV. Several of these have now been shown
to be clinically efficacious.8 However, all of these ago-
nists need to be dosed either s.c. or i.v. Some adverse
events have also been noted (e.g., nausea).
Keywords: DPP-IV; Glucagon-like peptide 1; Glucose-dependent
inhibitory polypeptide; Dipeptidyl peptidase IV.
An indirect approach to harnessing the antihyperglyce-
mic properties of GLP-1 is to inhibit DPP-IV, thus
increasing the t1/2 of native protein in vivo. Indeed,
numerous small molecule DPP-IV inhibitors have now
been reported9 and more importantly several of them
have achieved a positive proof-of-concept within a
*
Corresponding author. Tel.: +1 919 483 6247; fax: +1 919 315
043; e-mail: curt.d.haffner@gsk.com
Present address: Johnson & Johnson, Welsh & McKean Rds., Spring
House, PA 19477, USA.
à
Present address: Schering-Plough Research Institute, 2015 Galloping
Hill Rd., Kenilworth, NJ 07033, USA.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.08.050

5258
C. D. Haffner et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5257–5261
clinical setting. Among these are P32/98 from Probio-
drug,10 LAF-237 from Novartis,11 BMS-477118 from
Bristol-Myers Squibb,12 and MK-0431 from Merck &
Co. (Fig. 1).13
X
X
a, b
CO2Me
CONH2
N
N
BOC
BOC
X = H; 1
X = F; 2
X = H; 3
X = F; 4
The C2 nitrile moiety found on the P1 pyrrolidine ring
on a number of reported DPP-IV inhibitors not only
forms a key interaction with the catalytic serine 630,
but also in combination with the P2 basic amine can
undergo an intramolecular cyclization reaction to form
a cyclic amidine which can subsequently get hydrolyzed
to form a diketopiperazine.14 In this same report, it was
shown that steric bulk appropriately placed on the P2
fragment can slow this cyclization. Another report has
shown that a cyclopropyl ring fused on the P1 pyrroli-
dine ring can also slow this cyclization.15 An obvious
solution to this problem would be to remove the nitrile
moiety. Unfortunately in so doing one usually loses a
significant amount of potency versus DPP-IV,16
although there have been several reports recently of P1
pyrrolidine containing DPP-IV inhibitors that lack the
nitrile yet retain good potency versus DPP-IV.17 We re-
port herein a series of 2-cyano-4-fluoro-1-thiovalylpyrr-
olidine inhibitors of DPP-IV. The main focus of this
work centered on the P2 fragment and blocking groups
to slow the cyclization as noted above. However, some
interesting properties were also noted by incorporation
of a C4 fluoro substituent on the pyrrolidine ring (see
Table 3 for additional information).
X
·pTsOH
CN
c, d
N
H
X = H; 5
X = F; 6
Scheme 1. Reagents and conditions: (a) dioxane, H2O, LiOHH2O;
(b) CH3CN, BOC2O, NH4HCO3; (c) pyridine, imidazole, POCl3, 0 °C;
(d) CH3CN, pTsOH.
O
O
O
R
a
b
HO
S
HO
d
SH
BOC
HO
SH
N
HN
HN
BOC
NH2
9
8
7
X
X
c
CN
CN
or e
N
R
R
O
S
O
S
NH2 (O)n
11 - 39
HN
BOC
10
Scheme 2. Reagents and conditions: (a) THF, KOH, BOC2O; (b) 1 N
NaOH, H2O, RBr or RCl; (c) DMF, HATU, i-Pr2NEt, 5 or 6; (d)
CH2Cl2, TFA or HCl, dioxane; (e) MeOH, NaIO4 or mCPBA,
CH2Cl2; step d.
The synthesis into these compounds started with either
the known 4-fluoropyrrolidine carboxylate 218 or a mod-
ified procedure to make the known 2-cyanopyrrolidine
5.16 Hydrolysis of esters 1 and 2 generated the corre-
sponding acids, which were then converted to the pri-
mary carboxamides 3 and 4 by first generating the
mixed anhydride via di-t-butyldicarbonate (BOC2O)
and then reacting with ammonium bicarbonate. The
carboxamides 3 and 4 were then dehydrated with POCl3
followed by removal of the BOC-protecting group
with p-toluenesulfonic acid to give amines 5 and 6
(Scheme 1).
amino acid was protected as the t-butyl carbamate with
BOC2O under basic conditions to generate acid 8. The
acid was then alkylated again under basic conditions
with either the requisite alkyl bromide or chloride to
give sulfide 9. The acid was then coupled to either amine
5 or 6 utilizing HATU as the coupling agent in DMF to
afford the amide 10. The sulfide was then directly depro-
tected with TFA or HCl to yield final amine or alterna-
tively, the sulfur was oxidized to give either the sulfoxide
(sodium periodate, MeOH) or the sulfone (mCPBA,
CH2Cl2) followed by deprotection as before, yielding
final products 11–39 (Scheme 2).
The amino acid portion of the molecule was generated
by starting with the commercially available D-penicilla-
mine 7. A general synthetic route to afford the final de-
sired compounds is described in Scheme 2. Initially the
The data shown in Table 1 compare the in vitro selectiv-
ity of these inhibitors versus DPP-IV, DPP-II, and
seprase.19 Most of the compounds showed very weak
inhibitory activity against both DPP-II and seprase.
The selectivity versus seprase is especially noteworthy,
given the fact that seprase has been shown to have the
highest degree of identity to DPP-IV.20 Initially benzyl
derivatives at either the sulfide or sulfone oxidation state
in the P2 moiety were examined. In general, the func-
tionality placed on the phenyl ring produced very little
variation in the potency against DPP-IV. However,
compounds 13, 18–19, 24, and 26 did demonstrate sub
100 nM potency versus DPP-IV. Comparison of com-
pounds 17, 19, and 23 to 34, 30, and 27, respectively,
demonstrated a drop in potency against DPP-IV upon
oxidation of the sulfide to the sulfone. Extending the
S
CN
N
N
H
N
O
F
O
N
OH
NH2
LAF-237
P32/98
F
CN
NH2
O
OH
N
N
O
N
F
N
NH2
CF3
BMS-477118
MK-0431
Figure 1.

C. D. Haffner et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5257–5261
Table 1. Inhibition and selectivity properties for compounds 11–39
5259
F
CN
N
R
O
S
NH2 (O)n
Compound
n
R
Ki (nM)a
DPP-IV
DPP-II
Seprase
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
0
0
0
0
1
0
0
0
0
0
0
0
2
CH2Ph
CH2-4-F-Ph
324
281
68
>22385
>22385
>22385
>22385
>22385
>22385
>22385
17258
>23440
>23440
9450
CH2-4-CN-Ph
CH2-4-Me-Ph
CH2-4-Ph-Ph
CH2-4-OBn-Ph
CH2-3-OPh-Ph
CH2-4-SO2Me-Ph
CH2-4-OMe-Ph
CH2-3-OMe-Ph
CH2-2-OMe-Ph
CH2-4-NHCOMe-Ph
CH2-4-Pyridyl
CH2-4-Thiadiazole-Ph
OH
530
579
428
310
76
>23440
9660
20395
6863
>23440
>23440
16980
12380
7925
53
>22385
>22385
>22385
>22385
>22385
>22385
>22385
>22385
>22385
>22385
>22385
3949
316
309
225
1278
74
>23440
3656
154
40
14639
5158
9660
CH2-5-Cl-3-Benzothiophenesulfone
CH2-4-Pyridyl
CH2-2-Pyridyl
33
12
19076
>23440
>23440
>23440
>23440
1276
CH2-3-Pyridyl
18
29
CH2-4-OMe-Ph
CH2-4-OMe-Phb
CH2-4-CF3-Ph
CH2NHCOMec
CH2-3-OPh-Ph
CH2-2,3-Benzoxadiazole
(CH2)2Ph
112
63
>22385
1127
46
65
>22385
1066
12845
>23440
>23440
12176
15776
>23440
40
56
>22385
14421
523
(CH2)3Ph
(CH2)3-4-F-Ph
(CH2)3Ph
24
45
701
>22385
302
a The Ki was calculated from the equation Ki = IC50/(1+S/Km), where S is the substrate concentration.
b Mixture of diastereomers.
c The BOC protected amino acid used to synthesize this compound was commercially available.
Table 2. In vivo properties for select compounds
chain length resulted in no change in DPP-IV potency
(11 vs. 39). However, as was noted with other com-
pounds, oxidation of the chain extended analogue 37
to give 39 lowered potency by approximately 12-fold
against DPP-IV. Most of the sulfone analogues showed
very poor or no inhibition versus DPP-II. However,
DPP-II activity was noted for some, but not all, of the
sulfide compounds (e.g., 30, 32, and 34).
F
CN
N
O
S
O
O
H2N
R
Compound
R
% I in rat at
6 h at 1 mpk
Hours above IC50
at 0.2 mpk p.o. dog
The sulfonic acid 25 was also made and showed moder-
ate potency versus DPP-IV, but good to moderate selec-
tivity, respectively, against both DPP-II and seprase.
In vivo pharmacokinetic experiments revealed that the
compounds containing the sulfide moiety were rapidly
oxidized to a mixture of sulfoxide and sulfone (data
not shown). Therefore, further compound progression
focused on several of the sulfone compounds. Com-
pounds 13, 18, and 19 were orally dosed in both rat
and dog to determine their duration of action. As seen
in Table 2, compound 19 turned out to have the longest
duration in both rat and dog (84% DPP-IV inhibition at
13
18
19
CN
SO2Me
OMe
69
68b
84
10a
7.8
>12
a GI toxicity was seen in the dog when orally dosed at 10 mpk.
b Poor %F (<5%) was seen upon oral dosing in the rat.
1 mpk in rat at 6 h and >12 h above the IC50 at 0.2 mpk
in dog). Interestingly, we also found that incorporation
of a fluorine atom at C4 on the pyrrolidine ring impart-
ed unexpected differences in the pharmacological

5260
C. D. Haffner et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5257–5261
Table 3. Pharmacological differences between compounds 19 and 40
5. Mentlein, R.; Gallwitz, B.; Schmidt, W. E. Eur. J.
Biochem. 1993, 214, 829.
X
6. (a) Nauck, M. A.; Kleine, N.; Orskov, C.; Holst, J. J.;
Willms, B.; Creutzfeldt, W. Diabetologia 1993, 36, 741;
(b) Toft-Nielson, M.; Madsbad, S.; Holst, J. J. Diabetes
1996, 45, 552; (c) Toft-Nielson, M.; Madsbad, S.; Holst, J. J.
J. Clin. Endocrinol. Metab. 2001, 86, 3853; (d) Vilsboll, T.;
Krarup, T.; Deacon, C. F.; Madsbad, S.; Holst, J. J.
Diabetes 2001, 50, 609.
CN
N
O
S
O
O
H2N
OMe
19, X = F
40, X = H
7. (a) Elahi, D.; McAloon-Dyke, M.; Fukagawa, N. K.;
Meneilly, G. S.; Sclater, A. L.; Minaker, K. L.; Habener,
J. F.; Andersen, D. K. Regul. Pept. 1994, 51, 63; (b)
Vilsboll, T.; Knop, F. K.; Krarup, T.; Johansen, A.;
Madsbad, S.; Larsen, S.; Hansen, T.; Pedersen, O.; Holst,
J. J. J. Clin. Endocrinol. Metab. 2003, 88, 4897.
Compound 19 Compound 40
Ki (nM)
53
84%
115
62%
DPP-IV inhibition at 6 h 1
mpk rat
Hours above IC50 at 0.2 mpk dog >12
7
8. (a) Edward, C. M. B.; Stanley, S. A.; Davis, R.; Brynes, A.
E.; Frost, G. S.; Seal, L. J.; Ghatei, M. A.; Bloom, S. R.
Am. J. Physiol. Endocrinol. Metab. 2001, 281, E155; (b)
Egan, J. M.; Clocquet, A. R.; Elahi, D. J. Clin. Endocrinol.
Metab. 2002, 87, 1282; (c) Juhl, C. B.; Hollindal, M.;
Sturis, J.; Jakobsen, G.; Agerso, H.; Veldhuis, J.; Porksen,
N.; Schmitz, O. Diabetes 2002, 51, 424; (d) Kolterman, O.
G.; Buse, J. B.; Fineman, M. S.; Gaines, E.; Heintz, S.;
Bicsak, T. A.; Taylor, K.; Kim, D.; Aisporna, M.; Wang,
Y.; Baron, A. D. J. Clin. Endocrinol. Metab. 2003, 88,
3082; (e) Fineman, M. S.; Bicsak, T. A.; Shen, L. Z.;
Taylor, K.; Gaines, E.; Varns, A.; Kim, D.; Baron, A. D.
Diabetes Care 2003, 26, 2370; (f) Chang, A. M.; Jakobsen,
G.; Sturis, J.; Smith, M. J.; Bloem, C. J.; An, B.; Galecki,
A.; Halter, J. B. Diabetes 2003, 52, 1786; (g) Buse, J. B.;
Henry, R. R.; Han, J.; Kim, D. D.; Fineman, M. S.;
Baron, A. D. Diabetes Care 2004, 27, 2628; (h) Degn, K.
B.; Brock, B.; Juhl, C. B.; Djurhuus, C. B.; Grubert, J.;
Kim, D. D.; Han, J.; Taylor, K.; Fineman, M.; Schmitz,
O. Diabetes 2004, 53, 2397; (i) Fineman, M. S.; Shen, L.
Z.; Taylor, K.; Kim, D. D.; Baron, A. D. Diabetes Metab.
Rev. 2004, 20, 411; (j) Degn, K. B.; Juhl, C. B.; Sturis, J.;
Jakobsen, G.; Brock, B.; Chandramouli, V.; Rungby, J.;
Landau, B. R.; Schmitz, O. Diabetes 2004, 53, 1187; (k)
Harder, H.; Nielsen, L.; Thi, T. D. T.; Astrup, A. Diabetes
Care 2004, 27, 1915; (l) Madsbad, S.; Schmitz, O.;
Ranstam, J.; Jakobsen, G.; Matthews, D. R. Diabetes
Care 2004, 27, 1335; (m) Kolterman, O. G.; Kim, D. D.;
Shen, L.; Ruggles, J. A.; Nielsen, L. L.; Fineman, M. S.;
Baron, A. D. Am. J. Health Syst. Pharm. 2005, 62, 173.
9. (a) Evans, D. M. IDrugs 2002, 5, 577; (b) Wiedeman, P. E.;
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IC50 vs P450Õs
>15 lM
>422
>442
>15 lM
>195
>204
Selectivity ratio versus DPP-II
Selectivity ratio versus seprase
t1/2 (h)
2.3 (r), 3.9 (d) 4.2 (r), 3.0 (d)
% F
t1/2 offset
32 (r), 80 (d)
87 min
1733 h
4.0 (r), 100 (d)
41 min
360 h
t1/2 cyclization at 37 °C pH 7.2
properties of compound 19. A recent report has also
noted differences in potency and in vivo drug levels
when comparing a 2-cyano-4-fluoropyrrolidine deriva-
tive to its des-fluoro counterpart. However, no other
differences were noted in this report.21 Table 3 demon-
strates some of the observed differences in compound
19 versus the des-fluoro analogue, compound 40.
As can be seen in Table 3, compound 19 was more po-
tent than compound 40. It had a longer duration of ac-
tion in both rat and dog, which in part might be a result
of the differences seen in the t1/2 offset values (87 min vs.
41 min). Its pharmacokinetic properties were better, as
was its stability toward intramolecular cyclization. Both
compounds were very selective against DPP-II, seprase,
and P450Õs. Addition of the gem-dimethyl group signif-
icantly added to the stability of compound 19 toward
cyclization as the compound lacking this moiety had a
t1/2 cyclization of 80 h (compound not shown). In con-
clusion, we have identified a series of 2-cyano-4-fluoro-
1-thiovalylpyrrolidine inhibitors of DPP-IV. The most
promising compound within this series was compound
19 which demonstrated a good inhibitory, selectivity,
and pharmacokinetic profile toward DPP-IV.
10. (a) Demuth, H.-U.; Glund, K.; Banke-Bochita, J.; Rost,
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