Synthesis of Novel Potent Dipeptidyl Peptidase IV Inhibitors with Enhanced Chemical Stability: Interplay between the N-Terminal Amino Acid Alkyl Side Chain and the Cyclopropyl Group of α -Aminoacyl-L-cis-4,5-methanoprolinenitrile-Based Inhibitors

Article abstract of DOI:10.1021/jm049924d

A series of methanoprolinenitrile-containing dipeptide mimetics were synthesized and assayed as inhibitors of the N-terminal sequence-specific serine protease dipeptidyl peptidase IV (DPP-IV). The catalytic action of DPP-IV is the principle means of degradation of glucagon-like peptide-1, a key mediator of glucose-stimulated insulin secretion, and DPP-IV inhibition shows clinical benefit as a novel mechanism for treatment of type 2 diabetes. However, many of the reversible inhibitors to date suffer from chemical instability stemming from an amine to nitrile intramolecular cyclization. Installation of a cyclopropyl moiety at either the 3,4- or 4,5-position of traditional 2-cyanopyrrolidide proline mimetics led to compounds with potent inhibitory activity against the enzyme. Additionally, cis-4,5-methanoprolinenitriles with β-branching in the N-terminal amino acid provided enhanced chemical stability and high inhibitory potency. This class of inhibitors also exhibited the ability to suppress prandial glucose elevations after an oral glucose challenge in male Zucker rats.

Full text of DOI:10.1021/jm049924d

J . Med. Chem. 2004, 47, 2587-2598
2587
Syn th esis of Novel P oten t Dip ep tid yl P ep tid a se IV In h ibitor s w ith En h a n ced
Ch em ica l Sta bility: In ter p la y betw een th e N-Ter m in a l Am in o Acid Alk yl Sid e
Ch a in a n d th e Cyclop r op yl Gr ou p of
r-Am in oa cyl-L-cis-4,5-m eth a n op r olin en itr ile-Ba sed In h ibitor s
David R. Magnin,† J effrey A. Robl,*,† Richard B. Sulsky,† David J . Augeri,†, Yanting Huang,†
Ligaya M. Simpkins,† Prakash C. Taunk,† David A. Betebenner,†,# J ames G. Robertson,‡ Benoni E. Abboa-Offei,‡
Aiying Wang,‡ Michael Cap,‡ Li Xin,‡ Li Tao,[ Doree F. Sitkoff,∞ Mary F. Malley,O J ack Z. Gougoutas,O
Ashish Khanna,§ Qi Huang,‡ Song-Ping Han,‡ Rex A. Parker,‡ and Lawrence G. Hamann*,†
Departments of Discovery Chemistry, Metabolic Research, Exploratory Pharmaceutics, Computer-Assisted Drug Design,
Solid State Chemistry, and Pharmaceutical Candidate Optimization, Bristol-Myers Squibb, Pharmaceutical Research Institute,
P.O. Box 5400, Princeton, New J ersey 08543-5400
Received J anuary 27, 2004
A series of methanoprolinenitrile-containing dipeptide mimetics were synthesized and assayed
as inhibitors of the N-terminal sequence-specific serine protease dipeptidyl peptidase IV (DPP-
IV). The catalytic action of DPP-IV is the principle means of degradation of glucagon-like
peptide-1, a key mediator of glucose-stimulated insulin secretion, and DPP-IV inhibition shows
clinical benefit as a novel mechanism for treatment of type 2 diabetes. However, many of the
reversible inhibitors to date suffer from chemical instability stemming from an amine to nitrile
intramolecular cyclization. Installation of a cyclopropyl moiety at either the 3,4- or 4,5-position
of traditional 2-cyanopyrrolidide proline mimetics led to compounds with potent inhibitory
activity against the enzyme. Additionally, cis-4,5-methanoprolinenitriles with â-branching in
the N-terminal amino acid provided enhanced chemical stability and high inhibitory potency.
This class of inhibitors also exhibited the ability to suppress prandial glucose elevations after
an oral glucose challenge in male Zucker rats.
In tr od u ction
GLP-1 (9-36) by cleavage of the N-terminal dipeptide
residues. The truncated metabolite has antagonist
activity against the GLP-1 receptor both in vitro and
in vivo.9 The principle enzyme responsible for the
cleavage of GLP-1 (7-36) amide to GLP-1 (9-36) amide
is dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5), a
nonclassical, sequence-specific serine protease that
catalyzes the cleavage of dipeptides from the N-termi-
nus of proteins with the sequence H-X-Pro-Y or H-X-
Ala-Y (where X, Y ) any amino acid; Y * Pro).10
Inhibition of DPP-IV has been shown to be effective at
sustaining circulating levels of GLP-1 (7-36) and
therefore offers a new therapeutic approach for the
treatment of type 2 diabetes.11
With the spread of Western lifestyles, the prevalence
of type 2 diabetes in the world’s population is rising.1
Current treatment strategies include reducing insulin
resistance using glitazones,2 supplementing insulin
supplies with exogenous insulin,3 increasing insulin
secretion with sulfonylureas,4 reducing hepatic glucose
output with biguanides,5 and limiting glucose absorption
with glucosidase inhibitors.6 Promising new targets for
drug development are also emerging. Of particular
interest is the pharmacology surrounding the incretin
hormone glucagon-like peptide 1 (GLP-1).7 GLP-1 is
known to function as a mediator of glucose-stimulated
insulin secretion, and several clinical studies have
shown that administration of the peptide or its ana-
logues results in antidiabetic action in subjects with type
2 diabetes.8 Although GLP-1 is secreted as GLP-1 (7-
36) amide from the small and large intestines in
response to dietary signals, it is rapidly truncated to
Early reports of DPP-IV inhibitors included proline-
based dipeptide mimics bearing boronic acid12 (1) or
diphenyl phosphonate substituents (2).13 These com-
pounds were irreversible inhibitors of DPP-IV or were
slow to dissociate from the enzyme. Several first-
generation dipeptide surrogates have been disclosed as
reversible inhibitors of DPP-IV, including both C-
substituted (3) and N-substituted (4) glycinylprolineni-
trile dipeptide analogues.14,15 These compound classes
include many potent inhibitors of the enzyme, but all
suffer from chemical instability whereby the N-terminal
amine intramolecularly cyclizes onto the nitrile, forming
inactive cyclic imidates and/or their diketopiperazine
hydrolysis products. However, more recent publications
have disclosed a series of more hindered N-alkylamines
(5) that have much greater chemical stability.16 Thia-
* To whom correspondence should be addressed. For J .A.R.: tele-
phone, 609-818-5048; fax, 609-818-3550; e-mail, jeffrey.robl@bms.com.
For L.G.H.: telephone,609-818-5526;fax,609-818-3550;e-mail: lawrence.
hamann@bms.com.
† Department of Discovery Chemistry.
Present address: Lexicon Pharmaceuticals, 350 Carter Road,
Princeton, NJ 08540.
#
Present address: Pharmaceutical Discovery Division, Abbott
Laboratories, Abbott Park, IL 60064.
[ Department of Exploratory Pharmaceutics.
∞ Department of Computer-Assisted Drug Design.
O Department of Solid State Chemistry.
§ Department of Pharmaceutical Candidate Optimization.
‡ Department of Metabolic Research.
10.1021/jm049924d CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/13/2004

2588 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Magnin et al.
Sch em e 1a
a
(a) N-Boc-amino acid, EDAC, DMAP or PyBop, 50-90%; (b)
(for intermediates 8-10) POCl3, pyridine, imidazole, 70-90%; (c)
TFA, CH2Cl2, TFA or HCl in Et2O or EtOAc, 70-90%.
F igu r e 1. Known proline-derived dipeptidyl peptidase IV
inhibitors.
anoproline,19 and L-2,3-methanoproline20 are shown in
Scheme 1.
zolidide (6) and pyrrolidide (7) based inhibitors have also
been disclosed that lack the nitrile moiety and thus
possess greater stability, though at the cost of reduced
potency17a-c with a few very recent notable excep-
tions.17d-f These compounds are shown in Figure 1.
Standard conditions were employed to couple the
enantiomerically pure L-cis-4,5-cyclopropylprolineamide
nucleus (8) to give the corresponding Boc-protected
dipeptides in good to excellent yield.21 Dehydration14b,22
(POCl3, pyridine, imidazole) of the amide and removal23
of the N-terminal Boc protecting group (TFA or HCl)
gave the proline dipeptide (12) in high yield. Similar
protocols were used for the L-cis-3,4-methanoproline
fragment 10 and the L-4,5-trans-methanoproline com-
pound 9. The known (-)-(2S,3R)-methanoprolinenitrile
(11) was prepared according to the method of Hercout.20a
In this instance, the dehydration reaction was per-
formed prior to introduction of the isoleucine fragment.
Preparation of a library was undertaken to probe
structure-activity relationships between the N-termi-
nal amino acid residue and the cis-4,5-methanopro-
linenitriles. These inhibitors were generated in a three-
step sequence in parallel array format in a manner
similar to that described in Scheme 1. Initial reaction
of the Boc-protected amino acid with methanoprolinea-
mide, EDAC, and DMAP in dichloromethane, and
subsequent purification through an SCX ion exchange
cartridge, gave good to excellent yields of coupled
dipeptides. Dehydration and acid-promoted deprotection
(TFA in dichloromethane) yielded the inhibitors as TFA
salts. Further purification of the final products was
easily accomplished by preparative reverse-phase HPLC
or by trituration with Et2O.
Holmberg has prepared tert-alkylmalonic acid deriva-
tives through a TiCl4-mediated Knoevenagel process
and subsequent copper-assisted conjugate addition of a
Grignard reagent or conjugate reduction. Following this
method (Scheme 2), diesters can be converted to the
protected amino acids in three steps.24 This methodology
provided an approach to the cycloalkylglycine amino
acid derivatives 20. First, the malonic acid diesters 17
were subjected to conjugate addition of a methyl group
or hydride and then hydrolyzed to the corresponding
monoacids 18. Subsequent Curtius rearrangement of 18
by treatment with diphenylphosphoryl azide, followed
by trapping of the intermediate isocyanate with benzyl
alcohol, provided the Cbz adducts 19. Finally, the esters
were hydrolyzed to give the amino acids 20 in racemic
form. The racemic Boc-protected acids 20 were then
coupled to the L-cis-4,5-methanoprolineamide group.
We endeavored to synthesize novel dipeptide sur-
rogates containing a proline mimic linked to an N-
terminal amino acid that would act as reversible
inhibitors of DPP-IV with maximum potency and en-
hanced solution stability. Hanessian has reported that
installation of a 4,5-methano moiety into a proline
residue has the effect of flattening the five-membered
ring,18 and one might extend a similar conformational
argument to other cyclopropanated prolines as well. By
inference, the cyclopropane bridge may occupy the
space-filling region that would normally be given to the
methylene group in the puckered or “envelope” confor-
mation of a typical five-member ring. With this prece-
dent we sought to establish whether a methanoproline
derivative could serve as a viable proline surrogate in
a DPP-IV inhibitor. Our strategy was to prepare dipep-
tide surrogates containing a cyclopropanated prolineni-
trile derived from L-proline. The regio- and stereochem-
ical disposition of the cyclopropane bridge was varied
in order to identify a compound with maximal stability
and potency. Herein, we report the discovery of L-cis-
4,5-methanoprolinenitrile dipeptides as potent inhibi-
tors of DPP-IV with increased chemical stability and
high potency. In addition, we also present data demon-
strating that these novel DPP-IV inhibitors effectively
lower plasma glucose after a glucose challenge in rodent
models.
Ch em istr y
Dipeptides (12-15) composed of N-terminal isoleucine
appended to cyclopropylprolineamides or nitriles derived
from L-proline (8-11) were targeted to probe the
potential for this type of inhibitor scaffold. Isoleucine
was selected as the N-terminal residue because it was
the most potent natural amino acid reported in the
2-cyanopyrrolidide series.14a These inhibitors were ex-
pected to provide a dependable inhibitory benchmark
for the differing methanoproline structures. The syn-
thetic routes used to prepare the dipeptides derived from
L-cis- and L-trans-4,5-methanoproline,18b L-cis-3,4-meth-

Dipeptidyl Peptidase IV Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2589
Sch em e 2a
Ta ble 1. In Vitro Inhibition Constants for Porcine DPP-IV and
Solution Stability Half-Lives for Prolinenitrile DPP-4 Inhibitors
N-terminal
amino acida
substituted
stability,
compd
prolinenitrile Ki (nM)b
t1/2 (h)c
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
Ile
Ile
Ile
Ile
Ile
Val
Val
tert-Leu
tert-Leu
tert-Leu
Phe
Trp
N-Me-Val
Pro
Pip
Met
prolinenitrile 2 ( 0.5
5
a
trans-4,5-
trans-2,3-
cis-4,5-
cis-3,4-
cis-4,5-
cis-3,4-
prolinenitrile 8 ( 0.5
cis-4,5-
cis-3,4-
cis-4,5-
cis-4,5-
cis-4,5-
cis-4,5-
cis-4,5-
cis-4,5-
cis-4,5-
cis-4,5-
cis-4,5-
1620 ( 80
7500 ( 200
25 ( 1
(a) TiCl4, THF, CCl4, diethyl malonate, 0 °C, then pyridine, 0
°C to room temp, 68%; (b) MeMgI, CuCl, Et2O, 0 °C, 69%; (c)
NaOH, THF, EtOH, 78%; (d) (i) diphenylphosphorylazide, NEt3,
PhH, reflux; (ii) BnOH, reflux, 18 h, 100%; (e) H2, Pd(OH)2, EtOAc,
100%; (f) (Boc)2O, K2CO3, THF, H2O, 92%, two steps; (g) NaOH,
MeOH, THF, room temp, then aqueous HCl, 95%.
22
4
28
2
27
42
4
15 ( 1
29 ( 1
12 ( 1
7 ( 0.5
14 ( 1
65 ( 3
Processing as previously described (Scheme 1) gave a
mixture of diastereomers that were separated at the
nitrile stage by silica gel flash chromatography. The
later-eluting isomer in each case was consistently
identified as the desired L,L-dipeptide.25
230 ( 10
1940 ( 80
107 ( 5
10000
135 ( 10
12 ( 0.5
4 ( 0.5
15 ( 1
Cb-Gly
Cp-Gly
Ch-Gly
(1-Me-Cb-1-yl)-Gly cis-4,5-
(1-Me-Cp-1-yl)-Gly cis-4,5-
(1-Me-Ch-1-yl)-Gly cis-4,5-
Resu lts a n d Discu ssion
All compounds were tested in vitro against purified
porcine DPP-IV. Inhibition was determined against the
substrate H-Ala-Pro-pNA. Production of p-nitroaniline
(pNA) was measured at 405 nm over 15 min. A com-
parison of methanoproline-derived isoleucine N-termi-
nal dipeptides shows that the enzyme inhibitory activity
is critically dependent on both the position and the
orientation of the methano bridge (Table 1). The pres-
ence of the methano bridge on the trans side of the
prolinenitrile (e.g., 22, 23) resulted in a significant loss
of activity relative to the unsubstituted prolinenitrile
21. This result was consistent with early reports where
insertion of a methyl group at the 2-postion of 2-cyan-
opyrrolidides resulted in a 2000-fold decrease in activ-
ity.26 In contrast, when the methano bridge was oriented
cis to the nitrile at either the 4,5- or 3,4-position,
inhibitory activity was only slightly diminished. This
is clearly illustrated with cis-4,5-methano inhibitors 24
and 26 and cis-3,4-methano inhibitors 25 and 27, where
inhibitory potency resides in the 20 nM range. A more
complete exploration of N-terminal amino acids reveals
that increasing the degree of â-branching to that of tert-
Leu (e.g., 29) further increases potency in the 4,5-
methano series to that of the prolinenitrile version (28).
Replacement of these alkyl substituents with aromatic
residues such as Phe (31) or Trp (32) in the N-terminal
position significantly eroded potency. Alkyl substitution
on the terminal amine was generally ill-tolerated in this
series (e.g., 33-35), though it would appear that the
relatively more accessible N-terminus found in proline
derivative 34 is capable of restoring a modest degree of
inhibitory activity. Replacement of Leu and tert-Leu side
chains with medium-ring cycloalkyl and methylcy-
cloalkyl (37-42) generally led to inhibitors with com-
parable or slightly improved activity, though potency
dropped steadily with increasing larger ring size (data
not shown). The Cp-Gly and Me-Cp-Gly derived ana-
logues 38 and 41 were among the most potent compound
prepared in this series, exhibiting Ki values of 4 and 7
nM, respectively.
19
24
11 ( 0.5
7 ( 0.5
8 ( 0.5
a
b
Cb ) cyclobutyl; Cp ) cyclopentyl; Ch ) cyclohexyl. Values
represent the mean ( SEM and are at least triplicate determina-
tions. c Solution stability data are measured at 39.5 °C and pH
7.2 in phosphate buffer.
Sch em e 3
Solu tion Sta bility. After the discovery that the L-cis-
4,5- and L-cis-3,4-methanoprolinenitriles were potent
inhibitors of DPP-IV in vitro, a comparative study was
initiated to investigate aqueous solution stability of
these analogues. The N-terminal valine, isoleucine, and
tert-leucine dipeptide nitriles were selected in order to
make direct comparisons between methanoprolinenitrile
and unsubstituted prolinenitrile compounds with re-
spect to solution stability. Reaction rates were moni-
tored by following the disappearance of starting mate-
rial on reverse-phase HPLC at pH 7.2 and 39.5 °C in
phosphate buffer. HPLC mass spectral analysis revealed
that the two major products that formed during the
stability experiments had either an identical mass or
an M + 1 mass to the parent starting material. These
data are consistent with the formation of intramolecular
cyclization products, with the initial cyclic imidate (X
) NH) surrendering to the diketopiperazine (X ) O)
upon hydrolysis (Scheme 3).
Two interesting aspects of the solution half-life data
should be noted. The first is that there is increased
solution stability associated with the cis-4,5-methano-
prolinenitrile derivatives when compared to either the

2590 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Magnin et al.
cis-3,4-methanoprolinenitrile or the unadorned pro-
linenitrile compounds. For instance, in the case where
the N-terminal amino acid is isoleucine, the isomeric
4,5-methano-substituted compound 24 has a solution
half-life of 22 h, which is 5.5-fold longer than the
analogous 3,4-methano-substituted compound 25 and
approximately 4.5-fold longer than the unadorned pro-
linenitrile analogue 21. The second feature is that there
is a strong correlation between steric size at the â-posi-
tion of the N-terminal amino acid and relative solution
stability. Increasing the degree of branching at the
â-position of the alkylglycine substituent increases
solution stability. The most stable compounds within
their respective prolinenitrile series have the tert-Leu
N-terminal amino acid fragment in common. For ex-
ample, unadorned prolinenitrile 28 (t1/2 ) 27 h) is more
stable than its less branched isomer 21 (t1/2 ) 5 h).
Similarly, cis-4,5-methanoprolinenitrile 29 (t1/2 ) 42 h)
is more stable than the less branched dipeptidenitriles
24 and 39 (t1/2 ) 22 and 19 h, respectively), though the
magnitude of the effect of increased â-branching in this
series appears to be blunted by the inherent baseline
stability imparted by the methano bridge. This observa-
tion is in agreement with data reported by Coutes12a
and Snow27 where the rates of cyclization for Xaa-
boroproline dipeptides were shown to be Gly-BoroPro
> Ala-BoroPro > Val-BoroPro.
F igu r e 2. The upper structure depicts the local low-energy
minimum for 29 where the reactive amine is close to the nitrile
(anti conformation). The lower structure is the solid-state
conformation observed in the X-ray crystallographic structure
of the TFA salt of compound 29 (syn conformation). Two
independent cations were observed in the crystal structure,
though because these have identical conformations, only one
is shown for purposes of clarity. Carbon atoms C(2) and C(8)
are labeled.
Com p u ta tion a l An a lysis. Computational analysis
was undertaken to more fully understand the relative
stabilities of the methanoprolinenitriles. Ground-state
conformations were generated for methanoprolinenitrile
and prolinenitrile forms of the N-terminal tert-leucine
dipeptide compounds. The calculated ground-state struc-
ture for the tert-leucine dipeptidenitrile is identical to
the conformation observed through single-crystal X-ray
structural analysis28 of the TFA salt of 29 (rms ) 0.1 Å
for calculated and observed heavy atoms; see Figure 2,
lower structure). Both have the same syn conformation
around the amide bond, characterized by a small C(2)-
N-C(8)dO torsional angle (-5° and +2° for the TFA
structure). It is of additional interest that a similar
conformation has been observed in several recently
disclosed cocrystal structures of DPP-IV/inhibitor com-
plexes.29
stability due to side chain bulk (specifically â-branching)
and the increase in stability upon conversion from
prolinenitrile to cis-4,5-methanoprolinenitrile. Calcu-
lated relative energies between the ground-state con-
formation and the local low-energy minimum confor-
mation that brings the reactive amine and nitrile in
proximity are presented in Table 2 for the methanopro-
linenitrile and prolinenitrile forms of N-terminal tert-
leucine and alanine dipeptidenitriles, as well as for the
same prolinenitriles with a simple acetamide cap.
Con for m a t ion a l St a b ilit y Du e t o Sid e Ch a in
Bu lk . The values in the first column of Table 2 compare
the conformational energy differences between the
ground state and the geometry where internal cycliza-
tion could occur for the unsubstituted prolinenitrile
series. The ab initio (G98) results are expected to be
considerably more accurate than the force field values
and indicate that the energy required to assume the anti
conformation increases with side chain bulk (e.g., 0.3,
1.9, 2.8 kcal/mol for no side chain, the alanine side
chain, and the tert-leucine side chain, respectively). The
force field energy results agree qualitatively with the
ab initio values and suggest that the primary contribu-
tion is due to van der Waals interactions. Examination
of the structures reveals extremely close contacts be-
tween two of the side chain methyl groups and the
carbonyl oxygen in the anti conformation (approxi-
mately 3 Å each) that would increase the energy barrier
for internal cyclization and thus lead to greater stability.
A similar trend is observed for increasing side chain
bulk in the methano-substituted series (column 2), and
these values are in good agreement with the solution
data where increased â-branching enhances stability.
Con for m a tion a l Sta bility Du e to Meth a n o Su b-
stitu tion . As shown in the third column of Table 2, the
ab initio (G98) energy calculations indicate that the
In addition to the syn conformation, there is a
calculated local low-energy minimum where the reactive
amine and nitrile are close to each other; in this anti
conformation (Figure 2, upper structure), the C(2)-N-
C(8)dO torsional angle is 180°. Moreover, the angle
between the amine N and the C≡N group is 109° ( 1°
and the distance between the these reactive partners
is 2.95 Å. It is therefore reasonable to assume that the
observed intramolecular cyclization is initiated from this
conformation. The value of 109° between the amine
group and the nitrile is in close agreement with the
hypothetical angle of attack of at least 108° reported
by Baxter and Connor.30 It was envisioned that the
relative energetic differences between the global mini-
mum and the reactive local minimum would represent
a means to evaluate the relative stabilities of compounds
in solution.
The calculated conformations and their relative ener-
gies can be used to examine the basis for two aspects of
the experimental compound stability: the increase in

Dipeptidyl Peptidase IV Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2591
Ta ble 2. Calculated Energya Differences between the
Calculated Ground State and the Local Low-Energy Minimum
Required for Cyclization
28 (Prolinenitrile) and 29 (Methanoprolinenitrile)
(A ) tert-Leu)
method interaction
∆H(28) b ∆H(29) b ∆∆H(29 - 28)
G98 ab initio
2.8
3.4
0.6
Insight CFF force field
total nonbond
van der Waals
electrostatic
3.5
3.3
0.2
4.2
3.9
0.3
0.7
0.6
0.1
F igu r e 3. Effects of inhibitors 29 (b) and 41 (9) dosed at 3
µmol/kg po versus vehicle control (O) on plasma DPP-IV
activity in Zuckerfa/fa rats.
43 (Prolinenitrile) and 44 (Methanoprolinenitrile)
(A ) Ala)
IV inhibition, increases in plasma insulin levels, and
an improvement in glucose tolerance.31 Compounds 29
and 41 were potent inhibitors of DPP-IV in vitro and
demonstrated excellent solution stability. As such, these
inhibitors were selected to determine the effects of DPP-
IV inhibition in vivo on glucose tolerance and insulin
secretion in Zuckerfa/fa rats. An oral glucose tolerance
test (oGTT) in the Zuckerfa/fa rat is a frequently used
model of hyperglycemia in type 2 diabetes and obesity
research. Zuckerfa/fa rats are severely hyperphagic,
extremely obese, markedly insulin-resistant, and mildly
hyperglycemic because of a mutation and loss of function
of the leptin receptor gene.32,33 Fasted male Zuckerfa/fa
rats were dosed orally with water or with inhibitors 29
and 41 (3 µmol/kg), and an oGTT was conducted 0.5 h
after the dosing. Plasma DPP-IV activity, glucose, and
insulin levels were then monitored over a 2 h period.
Figures 3-5 show the ex vivo plasma DPP-IV activity,
insulin response, and glucose excursion curves in re-
sponse to an oral glucose challenge (2 g/kg). Animals in
the control group reached peak plasma glucose levels
60 min after glucose administration, at which point the
drug-treated animals exhibited a 30-35% decrease in
glucose levels compared to controls (control animals, 356
mg/dL; compound 29 treated animals, 226 mg/dL;
compound 41 treated animals, 245 mg/dL). Glucose
levels were significantly reduced in the drug-treated
animals from 30 min onward, with maximal reductions
in glucose observed at 90 min (-34% to -38%). Plasma
DPP-IV activity was maximally suppressed (60%) 30
min after dosing (Figure 3), and the effects of these
inhibitors were sustained throughout the course of the
experiment (60-35%). The insulin response to oral
glucose was also enhanced by treatment with DPP-IV
inhibitors (Figure 4), demonstrating the link between
the glucose-lowering effects and DPP-IV inhibition of
these compounds.
method interaction
∆H(43) b ∆H(44) b ∆∆H (44 - 43)
G98 ab initio
1.9
2.6
0.6
Insight CFF force field
total nonbond
van der Waals
electrostatic
3.3
2.9
0.4
3.9
3.4
0.4
0.6
0.6
0.0
45 (Prolinenitrile) and 46 (Methanoprolinenitrile)
(A ) Ac)
method interaction
∆H(45) b ∆H(46) b ∆∆H (46 - 45)
G98 ab initio
0.3
0.9
0.6
Insight CFF force field
total nonbond
van der Waals
electrostatic
-0.2
-0.4
0.2
0.0
-0.2
0.3
0.2
0.2
0.1
a
Energies are for the cis-4,5-methanoprolinenitrile and pro-
linenitrile versions of the compounds and are given in kcal/mol.
Energies are for the anti conformation (Scheme 3 and Figure 2
upper structure) relative to the global minimum conformation,
which corresponds to the syn geometry in Scheme 3 and Figure 2
lower structure.
b
addition of the methano bridge increases the energy
barrier toward adopting the conformation required for
cyclization by approximately 0.6 kcal/mol for the N-
terminal tert-leucine dipeptide compound as well as for
the alanine dipeptide and acetamide compounds. The
lack of dependence on the nature of the side chain
suggests that the observed increase in stability of the
cis-4,5-methano compounds is due to a local interaction
between the methano bridge and the N-terminal amino
acid that favors the ground state relative to the anti
conformation where cyclization is initiated. The force
field energy analysis (Insight CFF) results also agree
qualitatively, implying that van der Waals interactions
are primarily responsible for the increase in energy.
These conclusions are supported by the cyclization rate
differences between the isomeric isoleucine derivatives
24 (t1/2 ) 22 h) and 21 (t1/2 ) 5 h), where a 4.5-fold
increase in stability is observed for the methanopro-
linenitrile. This increase in stability is in fair agreement
with the calculated value of 0.6 kcal/mol. The calculated
∆∆H and observed solution half-life results support
contributions to stability from both â-branching and the
presence of the 4,5-methano bridge on the pyrrolidine
ring.
Interestingly, a significant decrease in plasma glucose
levels occurred when DPP-IV activity in plasma was
inhibited only by 35-60%. This finding suggests that
it may not be necessary to completely suppress plasma
DPP-IV activity in order to achieve antihyperglycemic
efficacy in type 2 diabetics. However, differences may
exist in the inhibition of the turnover of native sub-
strates such as GLP-1 compared with that of the
pseudosubstrate used in this assay, and potential assay
In Vivo Activity. In vivo evaluation of DPP-IV
inhibitors has supported the connection between DPP-

2592 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Magnin et al.
Exp er im en ta l Section
Gen er a l Ch em ica l P r oced u r es. All reactions were carried
out using oven-dried or flame-dried round-bottomed (rb) flasks
and glassware under a static atmosphere of argon or nitrogen,
and the mixtures were stirred magnetically unless otherwise
noted. All reagents used were of commercial quality and were
obtained from Aldrich Chemical Co., Sigma Chemical Co.,
Lancaster Chemical Co., or Acros Chemical Co. All reactions
were carried out using commercially available anhydrous
solvents from Aldrich Chemical Co. or EM Science Chemical
Co. unless otherwise noted. “Brine” refers to a saturated
aqueous solution of NaCl. Unless otherwise specified, solutions
of common inorganic salts used in workups are aqueous
solutions. 1H (500 MHz) and 13C (125 MHz) NMR spectra were
1
recorded on a J EOL J NM-ECP500 spectrometer, and H (400
MHz) and 13C (100 MHz) spectra were recorded on a J EOL
GSX400 spectrometer. Chemical shifts are given in parts per
million (ppm) downfield from internal reference tetramethyl-
silane in δ units, and coupling constants (J values) are given
in hertz (Hz). Selected data are reported in the following
manner: chemical shift; multiplicity; coupling constants;
assignment. Elemental analyses were performed by the Ana-
lytical Chemistry department at Bristol-Myers Squibb. Melting
points were taken on a Hoover Uni-melt melting point ap-
paratus and are uncorrected. Boiling points are reported
uncorrected. Ku¨gelrohr distillations were performed using a
Bu¨chi GKR-51 apparatus, and reported boiling points cor-
respond to uncorrected oven air bath temperatures. Optical
rotations were obtained on a Perkin-Elmer 241 polarimeter
using a cell 1 dm in length and are reported as follows:
F igu r e 4. Effects of inhibitors 29 (b) and 41 (9) dosed at 3
µmol/kg po versus vehicle control (O) on plasma glucose after
an oGTT in Zuckerfa/fa rats.
[R]temp
(concentration in g/100 mL, solvent). All flash
wavelength
chromatographic separations were performed using E. Merck
silica gel (particle size, 0.040-0.063 mm). Reactions were
monitored by TLC using 0.25 mm E. Merck silica gel plates
(60 F254) and were visualized using UV light and 5% phospho-
molybdic acid in 95% EtOH, or by sequential treatment with
1 N HCl in CH3OH followed by ninhydrin staining. LC/MS
data were recorded on a Shimadzu LC-10AT equipped with
an SIL-10A injector, an SPD-10AV detector normally operating
at 220 nm and interfaced to a Micromass ZMD mass spec-
trometer. LC/MS or HPLC retention times are reported using
a Phenomenex Luna C-18 4.6 mm × 50 mm column, with
elution with a 4 min gradient of 0-100% solvent B, where
solvent A is 10:90:0.1 CH3OH-H2O-TFA and solvent B is 90:
10:0.1 CH3OH-H2O-TFA (HPLC, method 1). Other HPLC
methods include the following: method 2, YMC S-5 C-18 4.6
mm × 50 mm, 0-100% B, elution with a 4 min gradient of
0-100% solvent B, where solvent A is 10:90:0.1 CH3CN-H2O-
TFA and solvent B is 90:10:0.1 CH3CN-H2O-TFA; method
3, Zorbax SB C-18 4.6 mm × 75 mm column, elution with an
8 min gradient of 0-100% solvent B, where solvent A is 10:
90:0.1 CH3OH-H2O-H3PO4 and solvent B is 90:10:0.1 CH3-
OH-H2O-H3PO4. All solvents were removed by rotary evapo-
ration under vacuum using a standard rotary evaporator
F igu r e 5. Effects of inhibitors 29 (b) and 41 (9) dosed at 3
µmol/kg po versus vehicle control (O) on plasma insulin after
an oGTT in Zuckerfa/fa rats.
artifacts relating to the kinetic aspects of certain inhibi-
tors cannot be ruled out at present.
Con clu sion
We have demonstrated that the prolinenitrile frag-
ment of previously reported DPP-IV inhibitors can be
replaced with either a cis-3,4-methano- or a cis-4,5-
methanoprolinenitrile ring system to provide novel and
highly potent DPP-IV inhibitors. Solution stability
studies demonstrate that introduction of either a steri-
cally bulky amino acid side chain on the N-terminal
amino acid or a cis-4,5-methano bridge to the prolineni-
trile moiety significantly enhances solution stability,
minimizing a known intramolecular cyclization path-
way. The greatest improvement in stability is observed
when both of these structural features are present and
at work in concert. This added stability has the potential
to beneficially impact the chemical and formulation
stability of cyanopyrrolidide-based pharmaceuticals. In
many cases, the presence of a â-branched amino acid
also provides increased inhibitory potency as well as
solution stability. This class of inhibitors has been
shown to be effective in suppression of prandial glucose
elevations after an oral glucose challenge. These initial
findings are being used as the basis for the development
of DPP-IV inhibitors with even greater stability and in
vivo efficacy for the treatment of diabetes and impaired
glucose homeostasis through potentiation of meal-
induced levels of GLP-1 (7-36) and insulin.
equipped with
a dry ice condenser. All filtrations were
performed using vacuum unless otherwise noted.
Rep r esen ta tive Exa m p le of P r ep a r a tion : Gen er a l
Meth od A. N-Boc-L-cis-4,5-m eth a n op r olin ea m id e. To a
solution of N-Boc-4,5-methanoproline18b (1.20 g, 5.28 mmol)
in THF (20 mL) at -15 °C was added 4-methylmorpholine
(0.71 mL, 6.50 mmol) and then isobutyl chloroformate (0.78
mL, 6.00 mmol) over 5 min. The reaction mixture was stirred
at -15 °C for 30 min, cooled to -30 °C, and then treated with
a solution of NH3 in dioxane (50 mL, 25 mmol). The reaction
mixture was stirred at -30 °C for 0.5 h, warmed to room
temperature, and stirred overnight. The reaction mixture was
quenched with citric acid solution (pH 4) and extracted with
Et2O (3 × 50 mL). The combined organic fractions were washed
with brine, dried (Na2SO4), and concentrated. The residue was
purified by flash column chromatography on silica gel with
EtOAc to give 1.00 g (84%) of the title compound. 1H NMR
and 13C NMR signals were very broad and poorly defined as a
result of carbamate rotamers. Anal. (C11H18N2O‚0.4H2O) C, H,
N.

Dipeptidyl Peptidase IV Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2593
L-cis-4,5-Meth a n op r olin ea m id e TF A Sa lt (8). To a solu-
tion of N-Boc-4,5-methanoprolineamide (0.90 g, 4.00 mmol) in
CH2Cl2 (3 mL) at 0 °C was added TFA (3 mL). The reaction
mixture was stirred at 0 °C for 18 h and concentrated under
reduced pressure to give 0.98 g (100%) of the title compound
as an oil that solidified upon prolonged standing. Alternatively,
the protected material can be treated with HCl(g) in EtOAc
to give a white powder. Data for HCl salt: 1H NMR (CD3OD,
400 MHz) δ 0.78 (m, 1H), 0.95 (q, J ) 8.0, 1H), 1.85 (m, 1H),
2.27 (dd, J ) 14.1, 3.0, 1H), 2.75 (m, 1H), 3.42 (m, 1H), 4.57
(dd, J ) 11.1, 3.0, 1H); 13C NMR (CD3OD, 100 MHz) δ 10.7,
18.8, 33.3, 38.3, 62.2, 170.3. Anal. (C6H10N2O‚1.03HCl‚
0.10H2O) C, H, N, Cl.
(dd, J ) 11.5, 2.5, 1H), 2.65 (m, 1H), 3.95 (m, 1H), 4.51 (s,
1H), 5.09 (dd, J ) 11.2, 2.2, 1H).
An oven-dried 15 mL test tube was charged with the N-Boc-
tert-leucine-4,5-methanoprolinenitrile (45 mg, 0.14 mmol), CH2-
Cl2 (1 mL), and TFA (1 mL). The reaction mixture was vortexed
for 40 min at room temperature, diluted with toluene (4 mL),
and concentrated under reduced pressure to a thick oil. The
product was purified by reverse-phase preparative HPLC on
a YMC S5 ODS 20 mm × 250 mm column to give 14 mg (35%)
of the title compound. Purification conditions were the follow-
ing: gradient elution from 10:90:0.1 CH3OH-H2O-TFA to 90:
10:0.1 CH3OH-H2O-TFA over 18 min; flow rate, 20 mL/min;
detection wavelength, 220 nm; retention time, 10 min. 1H NMR
(DMSO-d6, 400 MHz) δ 0.73 (m, 1H), 1.05 (s + m, 10H), 1.99
(m, 1H), 2.26 (dd, J ) 13.5, 2.2, 1H), 2.50 (m, 1H), 4.11 (m,
Am id es P r ep a r ed fr om Kn ow n Ca r boxylic Acid s by
Met h od A. l-tr a n s-4,5-Met h a n op r olin ea m id e TF A Sa lt
1H), 4.37 (s, 1H), 5.26 (dd, J ) 10.5, 2.2, 1H), 8.20 (s, 2H); 13
C
1
(9). H NMR (CD3OD, 400 MHz) δ 0.89 (q, J ) 6.0, 1H), 0.95
NMR (DMSO-d6, 100 MHz) δ 12.7, 17.3, 25.9, 29.8, 34.2, 37.6,
45.0, 57.6, 118.0 (q, J ) 280), 119.5, 157.5 (q, J ) 38), 166.7.
L-ter t-Leu cin yl-L-cis-4,5-m eth an opr olin en itr ile HCl Salt
(29). An oven-dried 15 mL test tube was charged with N-Boc-
tert-leucine-4,5-methanoprolinenitrile (0.45 mg, 0.14 mmol)
and Et2O (1 mL) and was treated with a 2 N HCl solution in
Et2O (1 mL). The reaction mixture was vortexed for 15 min at
room temperature and concentrated under reduced pressure
to a white powder. The product was purified by trituration
with Et2O, collected, and dried under vacuum to give the title
(m, 1H), 1.90 (m, 1H), 2.18 (td, J ) 5.0, 11.4, 1H), 2.59 (dd, J
) 8.1, 13.2, 1H), 3.47 (td, J ) 2.5, 6.6, 1H), 4.01 (dd, J ) 8.1,
10.8, 1H); 13C NMR (CD3OD, 100 MHz) δ 7.1, 17.0, 32.0, 36.1,
58.3, 118.6 (q, J ) 263.5), 163.0 (q, J ) 35.4), 172.0. Anal.
(C6H10N2O‚1.00TFA) C H, N, F.
L-cis-3,4-Meth a n op r olin ea m id e TF A Sa lt (10). 1H NMR
(CD3OD, 400 MHz) δ 0.60 (m, 1H), 0.75 (q, J ) 7.5, 1H), 1.85
(m, 1H), 2.10 (m, 1H), 3.45 (d, J ) 10.9, 1H), 3.49 (dd, J )
11.0, 3.5, 1H), 4.44 (d, J ) 4.3, 1H); 13C NMR (CD3OD, 100
MHz) δ 5.3, 17.5, 20.4, 62.5, 170.4 (TFA not observed). Anal.
(C6H10N2O‚1.30TFA) C, H, N, F.
L-2,3-Meth a n op r olin en itr ile HCl Sa lt (11). 1H NMR
(CD3OD, 400 MHz) δ 1.55 (m, 1H), 1.70 (m, 1H), 2.10 (m, 1H),
2.25 (m, 1H), 2.37 (m, 1H), 2.67 (m, 1H), 3.15 (m, 1H).
Rep r esen ta tive Exa m p le of P r ep a r a tion : Gen er a l
Meth od B. l-ter t-Leu cin yl-L-cis-4,5-m eth a n op r olin en i-
tr ile TF A Sa lt (29). An oven-dried 15 mL test tube was
charged with the L-cis-4,5-methanoprolineamide TFA salt (56
mg, 0.22 mmol), N-tert-butyloxycarbonyl-L-tert-leucine (53 mg,
0.23 mmol), 4-(dimethylamino)pyridine (0.11 g, 0.88 mmol),
and CH2Cl2 (4 mL). The mixture was treated with 1-[(3-
(dimethyl)amino)propyl]-3-ethylcarbodiimide (84 mg, 0.44
mmol), and the tube was sealed under a nitrogen atmosphere.
The tube was placed in a shaker and shaken overnight. The
product was purified by solid-phase extraction using a United
Technology SCX column (2 g of sorbent in a 6 mL column) by
loading the material on an SCX ion exchange column and
successively washing with CH2Cl2 (5 mL), 1:3 CH3OH-CH2-
Cl2 (5 mL), 1:2 CH3OH-CH2Cl2 (5 mL), and 1:1 CH3OH-CH2-
Cl2 (10 mL). The product-containing fractions were concen-
trated under reduced pressure to give the desired amide.
Further purification by reverse-phase preparative HPLC on
a YMC S5 ODS 20 mm × 250 mm column gave the desired
amide (50 mg, 68% yield). Purification conditions were the
following: gradient elution from 30:70:0.1 CH3OH-H2O-TFA
to 90:10:0.1 CH3OH-H2O-TFA over 15 min; flow rate, 20 mL/
min; detection wavelength, 220 nm; retention time, 14 min.
1H NMR (CD3OD, 500 MHz) δ 0.87 (m, 1H), 1.08 (s + m, 10H),
1.44 (s, 9H), 1.75 (m, 1H), 2.01 (dd, J ) 13.7, 3.5, 1H), 2.58
(m, 1H), 3.87 (m, 1H), 4.53 (s, 1H), 4.77 (dd, J ) 11.8, 3.2,
1H); 13C NMR (CD3OD, 125 MHz) δ 14.6, 18.4, 27.1, 28.7, 31.4,
36.4, 39.9, 60.4, 62.7, 80.7, 157.9, 172.4, 176.7.
1
compound (25 mg, 85%). H NMR (CD3OD, 400 MHz) δ 0.91
(m, 1H), 1.13 (s + m, 10H), 2.00 (m, 1H), 2.32 (dd, J ) 14.0,
2.2, 1H), 2.65 (m, 1H), 3.97 (m, 1H), 4.37 (s, 1H), 5.26 (dd, J )
10.5, 2.2, 1H); 13C NMR (CD3OD, 100 MHz) δ 14.1, 19.1, 26.7,
31.4, 35.7, 39.4, 46.9, 60.1, 120.4, 168.1. Anal. (C12H20ClN3O‚
0.25H2O) C, H, N.
Dip ep tid e Nitr iles P r ep a r ed by Gen er a l Meth od B.
1
L-Isoleu cin e-L-p r olin en itr ile HCl Sa lt (21). H NMR (400
MHz, CD3OD) δ 0.99 (t, J ) 7.2, 3H), 1.10 (d, J ) 6.6, 3H),
1.20 (m, 2H), 1.70 (m, 1H), 1.90-2.45 (m, 4H), 3.70 (m, 2H),
4.11 (s, 1H), 4.81 (d, J ) 8.0, 1H); 13C NMR (CD3OD, 100 MHz)
δ 11.7, 15.3, 25.1, 26.4, 30.9, 37.8, 48.0, 57.4, 119.2, 169.1. Anal.
(C11H19N3O‚1.00HCl‚0.5H2O) C, H, N.
L-Isoleu cin yl-L-cis-4,5-m eth a n op r olin en itr ile TF A Sa lt
(24). 1H NMR (D2O, 500 MHz) δ 0.90 (m, 1H), 0.95 (t, J ) 7.3,
3H), 1.12 (d, J ) 7.3, 3H), 1.17 (m, 1H), 1.25 (m, 1H), 1.47 (m,
1H), 2.05 (m, 1H), 2.32 (m, 1H), 2.55 (dd, J ) 13.8, 2.8, 1H),
2.78 (m, 1H), 3.73 (td, J ) 6.4, 2.8, 1H), 4.46 (d, J ) 9.1, 1H),
5.32 (dd, J ) 11, 2.8, 1H); 13C NMR (D2O, 125 MHz) δ 10.9,
13.7, 14.9, 18.5, 23.6, 30.1, 35.5, 38.0, 47.0, 56.9, 116.6 (q, J )
290), 119.8, 163.2 (q, J ) 35), 165.4. HPLC: method 3; tR
)
2.49 3.03 min. HRMS (FAB) m/z: [M + H]+ calcd for C12H20N3O,
222.1606; found, 222.1606.
L-Valin yl-L-cis-4,5-m eth an opr olin en itr ile TFA Salt (26).
1H NMR (CD3OD, 500 MHz) δ 0.93 (m, 1H), 1.07 (d, J ) 7.1,
3H), 1.09 (m, 1H), 1.17 (d, J ) 7.1, 3H), 2.01 (m, 1H), 2.35
(dd, J ) 13.8, 3.0, 1H), 2.50 (m, 1H), 2.65 (m, 1H), 3.82 (td, J
) 6.0, 2.5, 1H), 4.39 (d, J ) 5.0, 1H), 5.16 (dd, J ) 11.0, 2.2,
1H); 13C NMR (CD3OD, 100 MHz) δ 14.3, 17.4, 19.4, 30.9, 31.8,
38.7, 47.6, 58.5, 120.8, 168.9. HPLC: method 2; tR ) 0.89 min.
HRMS (FAB) m/z: [M + H]+ calcd for C11H17N3O, 208.1450;
found, 208.1447.
L-ter t-Leu cin yl-L-p r olin en itr ile TF A Sa lt (28). 1H NMR
(CD3OD, 400 MHz) δ 1.13 (s, 9H), 1.90-2.45 (m, 4H), 3.72 (t,
J ) 6.3, 1H), 4.00 (s, 1H), 4.81 (d, J ) 8.0, 1H); 13C NMR (CD3-
OD, 100 MHz) 26.7, 26.8, 31.2, 35.9, 48.2, 60.5, 119.4 (q, J )
248), 119.5, 163.2 (q, J ) 46), 169.1. Anal. (C11H19N3O‚
1.00TFA) C, H, N, F.
L-P h en yla la n in yl-L-cis-4,5-m eth a n op r olin en itr ile TF A
Sa lt (31). 1H NMR (CD3OD, 500 MHz) δ 0.31 (m, 1H), 0.66
(dd, J ) 8.5, 6.5, 1H), 1.84 (m, 1H), 2.25 (dd, J ) 13.8, 2.5,
1H), 2.57 (m, 1H), 3.25 (m, 1H), 3.30 (s, 2H), 3.65 (m, 1H),
4.73 (t, J ) 6.6, 1H), 5.10 (dd, J ) 10.2, 2.2, 1H), 7.32 (m, 5H);
13C NMR (CD3OD, 100 MHz) δ 13.7, 18.7, 31.9, 38.4, 38.7, 47.6,
54.3, 118.0 (q, J ) 280), 120.0, 129.4, 130.7, 131.3, 135.1, 164.0
(q, J ) 43), 168.0. HPLC: method 2; tR ) 1.33 min. HRMS
(FAB) m/z: [M + H]+ calcd for C15H17N3O, 256.1450; found,
256.1445.
An oven-dried 15 mL test tube was charged with the N-Boc-
tert-leucine-cis-4,5-methanoprolineamide (50 mg, 0.15 mmol),
imidazole (31 mg, 0.46 mmol), and pyridine (1 mL). The tube
was sealed under nitrogen atmosphere and cooled to -30 °C.
Slow addition of POCl3 (141 mg, 88 µL, 0.92 mmol) gave after
mixing a thick slurry. The tube was mixed at -30 °C for 1 h,
and the volatiles were evaporated. The product was purified
by solid-phase extraction using a United Technology silica
extraction column (2 g of sorbent in a 6 mL column) by loading
the material on a silica column and successively washing with
CH2Cl2 (5 mL), 5:95 CH3OH-CH2Cl2 (5 mL), 7:93 CH3OH-
CH2Cl2 (5 mL), and 12:88 CH3OH-CH2Cl2 (10 mL). The
product-containing fractions were pooled and concentrated
under reduced pressure to give the N-Boc-protected nitrile
1
compound (46 mg, 96%). H NMR (CD3OD, 500 MHz) δ 0.88
(m, 1H), 1.08 (s + m, 10H), 1.43 (s, 9H), 1.94 (m, 1H), 2.32

2594 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Magnin et al.
L-Tr yp t op h a n yl-L-cis-4,5-m et h a n op r olin en it r ile TF A
Sa lt (32). 1H NMR (CD3OD, 500 MHz) δ 0.00 (m, 2H), 1.64
(m, 1H), 2.11 (dd, J ) 13.8, 2.2 Hz, 1H), 2.44 (m, 1H), 3.23 (s,
2H), 3.35 (m, 1H), 4.71 (t, J ) 6.6, 1H), 5.00 (dd, J ) 10.5, 2.2,
1H), 7.00 (t, J ) 7.2, 1H), 7.07 (t, J ) 7.7, 1H), 7.10 (s, 1H),
7.32 (d, J ) 8.2, 1H), 7.56 (d, J ) 7.7, 1H); 13C NMR (CD3OD,
100 MHz) δ 13.4, 18.6, 28.8, 31.8, 38.9, 47.4, 53.4, 107.7, 113.1,
119.2, 120.5, 120.9, 123.3, 126.2, 128.9, 138.9, 169.5. HPLC:
method 2; tR ) 1.39 min. HRMS (FAB) m/z: [M + H]+ calcd
for C17H18N4O, 295.1559; found, 295.1558.
L-N-Meth ylvalin yl-L-cis-4,5-m eth an opr olin en itr ile TFA
Sa lt (33). 1H NMR (CD3OD, 500 MHz) δ 0.94 (m, 1H), 1.11
(m + d, J ) 7.2, 4H), 1.18 (d, J ) 7.2, 3H), 2.02 (m, 1H), 2.35
(dd, J ) 13.8, 2.3, 1H), 2.50 (m, 1H), 2.65 (m, 1H), 2.70 (s,
3H), 3.85 (td, J ) 6.0, 2.7, 1H), 4.42 (d, J ) 5.0, 1H), 5.20 (dd,
J ) 10.5, 2.2, 1H); 13C NMR (CD3OD, 100 MHz) δ 14.4, 18.3,
19.0, 19.5, 31.4, 31.8, 33.8, 38.8, 47.6, 66.8, 120.7, 167.5.
HPLC: method 2; tR ) 0.87 min. HRMS (FAB) m/z: [M + H]+
calcd for C12H19N3O, 222.1606; found, 222.1604.
0.99 (m, 1H), 1.19 (q, J ) 8.8, 6.6, 1H), 1.37 (s, 3H), 1.89 (m,
3H), 2.12 (m, 2H), 2.24 (m, 1H), 2.47 (dd, J ) 13.6, 2.6, 1H),
2.54 (m, 1H), 2.72 (m, 1H), 3.90 (m, 1H), 4.82 (s, 1H), 5.27
(dd, J ) 10.8, 2.4, 1H); 13C NMR (D2O, 400 MHz) δ 14.1, 15.1,
18.5, 21.1, 30.0, 30.9, 31.0, 38.6, 40.1, 46.8, 59.0, 120.0, 167.8.
HRMS (ESI) m/z: [M + H]+ calcd for C13H19N3O, 234.1607;
found, 234.1592. HPLC: method 3; tR ) 3.55 min. Anal.
(C13H19N3O‚1.00TFA) C, H, N, F.
(S)-1-Meth ylcyclop en t-1-ylglycin yl-L-cis-4,5-m eth a n o-
p r olin en itr ile TF A Sa lt (41). 1H NMR (D2O, 400 MHz) δ
0.82 (m, 1H), 0.95 (s, 3H), 1.00 (q, J ) 7.1, 1H), 1.25-1.75 (m,
8H), 1.90 (m, 1H), 2.28 (dd, J ) 14.0, 2.7, 1H), 2.54 (m, 1H),
3.70 (td, J ) 6.1, 3.1, 1H), 4.35 (s, 1H), 5.08 (dd, J ) 8.5, 2.6,
1H); 13C NMR (D2O, 100 MHz) δ 13.8, 14.5, 21.3, 23.2, 23.4,
30.0, 36.2, 36.7, 45.7, 46.7, 58.8, 119.8, 168.2. HRMS (FAB)
m/z: [M + H]+ calcd for C14H20N3O, 248.1756; found, 248.1749.
Anal. (C14H21N3O‚1.00TFA‚0.89H2O) C, H, N.
(S)-1-Met h ylcycloh ex-1-ylglycin yl-L-cis-4,5-m et h a n o-
p r olin en itr ile TF A Sa lt (42). 1H NMR (D2O, 400 MHz) δ
0.86 (m, 1H), 1.03 (s, 3H), 1.07 (m, 2H), 1.39 (m, 9H), 1.95 (m,
1H), 2.31 (dd, J ) 13.6, 2.6, 1H), 2.58 (m, 1H), 3.76 (m, 1H),
4.26 (s, 1H), 5.12 (dd, J ) 11, 2.6, 1H); 13C NMR (D2O, 400
MHz) δ 13.8, 18.2, 18.5, 20.9, 21.0, 25.3, 30.0, 33.5, 34.1, 37.9,
38.9, 46.7, 60,0, 119.8, 168.0. HRMS (ESI) m/z: [M + H]+ calcd
for C15H23N3O, 262.1920; found, 262.1904. HPLC: method 3;
tR ) 4.77 min. Anal. (C15,H23N3O‚1.00TFA‚0.50H2O) C, H, N,
F.
L-P r olin yl-L-cis-4,5-m et h a n op r olin en it r ile TF A Sa lt
(34). 1H NMR (CD3OD, 400 MHz) δ 0.93 (m, 1H), 1.10 (m, 1H),
2.00 (m, 1H), 2.11 (m, 3H), 2.38 (dd, J ) 13.8, 2.2, 1H), 2.66
(m, 2H), 3.43 (m, 2H), 3.75 (td, J ) 6.0, 2.7, 1H), 4.85 (t, J )
8.8, 1H), 5.11 (dd, J ) 12.6, 2.2, 1H); 13C NMR (CD3OD, 100
MHz) δ 14.1, 19.2, 25.4, 29.8, 30.0, 32.0, 38.3, 47.8, 61.1, 120.7,
168.4. HPLC: method 2; tR ) 0.48 min. HRMS (FAB) m/z: [M
+ H]+ calcd for C11H15N3O, 206.1296; found, 206.1293.
L-P ip ecolin yl-L-cis-4,5-m eth a n op r olin en itr ile TF A Sa lt
Rep r esen ta tive Exa m p le of P r ep a r a tion : Gen er a l
Meth od C. l-ter t-Leu cin yl-L-cis-3,4-m eth a n op r olin en i-
tr ile TF A Sa lt (30). An oven-dried rb flask was charged with
cis-3,4-methanoprolineamide TFA salt (50 mg, 0.21 mmol),
N-tert-butyloxycarbonyl-L-tert-leucine (48 mg, 0.21 mmol),
benzotriazol-1-yloxytripyrrolidinophosphonium hexafluoro-
phosphate (164 mg, 0.32 mmol), 4-methylmorpholine (70 µL,
0.63 mmol), and CH2Cl2 (4 mL). The flask was sealed under
nitrogen atmosphere and stirred overnight. The product was
purified by solid-phase extraction using a United Technology
silica extraction column (2 g of sorbent in a 6 mL column) by
loading the material on a silica column and successively
washing with CH2Cl2 (5 mL), 5:95 CH3OH-CH2Cl2 (5 mL),
7:93 CH3OH-CH2Cl2 (5 mL), and 12:88 CH3OH-CH2Cl2 (10
mL). The product-containing fractions were pooled and con-
centrated under reduced pressure to give the desired amide
1
(35). H NMR (CD3OD, 500 MHz) δ 0.93 (m, 1H), 1.12 (q, J )
6.5, 1H), 1.20 (m, 1H), 1.60-2.10 (m, 3H), 2.35 (dd, J ) 14.0,
2.5, 1H), 2.65 (m, 1H), 3.10 (m, 1H), 2.66 (m, 1H), 3.45 (d, J )
11.0, 1H), 3.56 (t, J ) 5.0, 1H), 3.68 (t, J ) 4.5, 1H), 3.76 (m,-
1H), 4.48 (dd, J ) 12.5, 3.5, 1H), 5.06 (dd, J ) 10.5, 2.5, 1H);
13C NMR (CD3OD, 125 MHz) δ 13.5, 16.5, 21.0, 21.7, 25.5, 29.1,
34.2, 40.5, 47.8, 61.4, 118.5 (q, J ) 289), 120.7, 166.3 (q, J )
36), 171.1. HPLC: method 1; tR ) 0.85 min. MS m/z 220 [M +
H]+.
L-Meth ion in yl-L-cis-4,5-m eth an opr olin en itr ile TFA Salt
1
(36). H NMR (CD3OD, 500 MHz) δ 0.93 (m, 1H), 1.13 (q, J )
7.1, 1H), 2.03 (m, 1H), 2.25 (s, 3H), 2.29 (m, 2H), 2.36 (dd, J )
13.8, 2.0, 1H), 2.66 (m, 3H), 3.79 (td, J ) 6.0, 3.0, 1H), 4.68
(dd, J ) 7.0, 4.5, 1H), 5.15 (dd, J ) 10.5, 2.0, 1H); 13C NMR
(CD3OD, 100 MHz) δ 13.9, 15.2, 19.0, 29.6, 30.3, 31.4, 38.1,
47.4, 52.4, 120.4, 168.4. HPLC: method 1; tR ) 1.18 min. MS
m/z 240 [M + H]+.
1
compound (64 mg, 90%). H NMR (CD3OD, 400 MHz) δ 0.69
(m, 1H), 0.78 (q, J ) 4.7, 1H), 1.00 (s, 9H), 1.42 (s, 9H), 1.74
(m, 1H), 1.93 (m, 1H), 3.82 (d, J ) 9.2, 1H), 3.98 (m, 1H), 4.16
(s, 1H), 4.61 (d, J ) 5.2, 1H); 13C NMR (CD3OD, 100 MHz) δ
8.9, 17.7, 21.0, 26.8, 28.7, 36.1, 52.5, 60.2, 62.5, 80.7, 156.0,
174.0, 175.0. MS m/z 340 [M + H]+.
(S )-Cyclob u t ylglycin yl-L-cis-4,5-m e t h a n op r olin e n i-
tr ile TF A Sa lt (37). 1H NMR (D2O, 400 MHz) δ 0.85 (m, 1H),
1.05 (m, 1H), 1.73 (m, 1H), 1.92 (m, 7H), 2.30 (dd, J ) 14.1,
2.2, 1H), 2.56 (m, 1H), 2.89 (m, 1H), 3.70 (m, 1H), 4.45 (d, J )
8.4, 1H), 5.06 (dd, J ) 10.8, 2.4, 1H); 13C NMR (D2O, 400 MHz)
δ 13.8, 17.8, 18.3, 24.5, 24.7, 30.1, 38.0, 46.8, 55.1, 119.9, 168.3.
HRMS (ESI) m/z: [M + H]+ calcd for C12H17N3O, 220.145;
found, 220.144. HPLC: method 1: tR ) 1.26 min. Anal.
(C12H17N3O‚1.00TFA‚0.67H2O) C, H, N, F.
(S)-Cyclop en t ylglycin yl-L-cis-4,5-m et h a n op r olin en i-
tr ile TF A Sa lt (38). 1H NMR (D2O, 400 MHz) δ 0.86 (m, 1H),
1.04 (q, J ) 7.1, 1H), 1.25-1.80 (m, 8H), 1.93 (m, 1H), 2.32
(dd, J ) 11.5, 2.6, 1H), 2.40 (m, 1H), 2.59 (m, 1H), 3.70 (td, J
) 6.1, 2.6, 1H), 4.43 (d, J ) 7.5, 1H), 5.09 (dd, J ) 11.0, 2.6,
1H); 13C NMR (D2O, 100 MHz) δ 13.5, 18.2, 24.3, 24.7, 27.9,
28.4, 30.2, 37.9, 41.3, 46.8, 55.1, 119.6, 169.1. HRMS (FAB)
m/z: [M + H]+ calcd for C13H19N3O, 234.1602; found, 234.1606.
Anal. (C13H19N3O‚1.23TFA) C, H, N.
An oven-dried flask was charged with N-Boc-tert-leucinyl-
cis-4,5-methanoprolineamide (65 mg, 0.19 mmol), imidazole
(31 mg, 0.46 mmol), and pyridine (2 mL). The flask was sealed
under nitrogen atmosphere and cooled to -30 °C. Slow
addition of POCl3 (132 mg, 81 µL, 0.86 mmol) gave, after
mixing, a thick slurry that was mixed at -30 °C for 1 h and
concentrated. The product was purified by silica gel column
chromatography using 1:5 EtOAc-CH2Cl2. The product-
containing fractions were pooled and concentrated under
reduced pressure to give the desired N-Boc-protected title
compound (50 mg, 80%). 1H NMR (CDCl3, 400 MHz) δ 0.65 (q,
J ) 4.7, 1H), 0.99 (s, 9H), 1.00 (m, 1H), 1.42 (s, 9H), 1.89 (m,
1H), 2.00 (m, 1H), 3.80 (d, J ) 9.2, 1H), 3.99 (m, 1H), 4.15 (d,
J ) 9.2, 1H), 4.72 (d, J ) 4.9, 1H), 5.18 (d, J ) 9.6, 1H); 13C
NMR (CDCl3, 100 MHz) δ 11.1, 18.5, 19.1, 26.2, 28.3, 35.3,
50.0, 50.7, 58.6, 79.9, 116.9, 156.0, 173.0. MS m/z 322 [M +
H]+.
An oven-dried flask was charged with N-Boc-tert-leucinyl-
3,4-methanoprolinenitrile (0.45 mg, 0.14 mmol), CH2Cl2 (1
mL), and TFA (1 mL). The reaction mixture was stirred for 1
h at room temperature, diluted with toluene (4 mL) and
concentrated under reduced pressure. The product was puri-
fied by reverse-phase preparative HPLC on a YMC S5 ODS
20 mm × 250 mm column to give the title compound (20 mg,
45%). Purification conditions were the following: gradient
(S)-Cycloh e xylglycin yl-L-cis-4,5-m e t h a n op r olin e n i-
1
tr ile TF A Sa lt (39). H NMR (400 MHz, CD3OD) δ 0.90 (m,
1H), 1.05-1.35 (m, 6H), 1.70-1.90 (m, 5H), 2.00 (m, 1H), 2.15
(m, 1H), 2.37 (dd, J ) 13.6, 1.8, 1H), 2.63 (m, 1H), 3.83 (m,
1H), 4.35 (d, J ) 5.7, 1H), 5.17 (dd, J ) 10.6, 2.2, 1H); 13C
NMR (D2O, 100 MHz) δ 13.9, 19.1, 26.7, 26.9, 27.1, 28.5, 30.0,
31.4, 38.4, 40.3, 47.1, 57.2, 120.5, 168.5. HRMS (FAB) m/z: [M
+ H]+ calcd for C14H21N3O, 248.1768; found, 248.1763. Anal.
(C14H21N3O‚1.00TFA) C, H, N.
(S)-1-Met h ylcyclob u t -1-ylglycin yl-L-cis-4,5-m et h a n o-
p r olin en itr ile TF A Sa lt (40). 1H NMR (D2O, 400 MHz) δ

Dipeptidyl Peptidase IV Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2595
elution from 10:90:0.1 CH3OH-H2O-TFA to 90:10:0.1 CH3-
OH-H2O-TFA over 18 min; flow rate, 20 mL/min; detection
purified by flash column chromatography on silica gel (5:95
Et2O-hexanes) to give the diethyl ester compound (1.09 g,
68.7%). 1H NMR (CDCl3, 400 MHz) δ 1.15 (s, 3H), 1.26 (t, J )
7.0, 6H), 1.57 (m, 2H), 1.55-1.70 (m, 6H), 3.34 (s, 1H), 4.20
(q, J ) 7.0, 4H); 13C NMR (CDCl3, 100 MHz) δ 14.1, 23.7, 24.0,
38.6, 43.9, 60.7, 60.8, 168.7. LC/MS m/z: 243 [M + H]+. A
solution of 2-(1-methylcyclopentyl)malonic acid diethyl ester
(736 mg, 3.04 mmol) in a mixture of EtOH (12 mL) and THF
(6 mL) was treated with 1.0 M NaOH (3.04 mL, 3.04 mmol)
and stirred at room temperature for 24 h. The reaction mixture
was evaporated to a syrup, dissolved in water (10 mL), and
extracted with Et2O (15 mL). The aqueous phase was acidified
with 1 M HCl to pH 2.0 and extracted with EtOAc (3 × 25
mL). The combined organic extracts were washed with brine,
dried (MgSO4), and evaporated to give the title compound (0.87
g, 95%). Rf ) 0.56 (CH2Cl2-CH3OH, 9:1; PMA). 1H NMR
(CDCl3, 400 MHz) δ 1.15 (s, 3H), 1.28 (t, J ) 7.0, 3H), 1.55-
1.70 (m, 8H), 3.75 (s, 1H), 4.20 (q, J ) 7.0, 2H); 13C NMR
(CDCl3, 100 MHz) δ 14.0, 23.2, 23.9, 38.5, 44.5, 60.4, 61.4,
169.7, 173.5. LC/MS m/z: 215 [M + H]+.
1
wavelength, 220 nm; tR, 10 min. H NMR (CD3OD, 400 MHz)
δ 0.55 (q, J ) 5.7, 1H), 1.10 (s + m, 10H), 1.95 (m, 1H), 2.13
(m, 1H), 3.80 (m, 2H), 3.93 (s, 1H), 4.84 (d, J ) 5.0, 1H); 13C
NMR (CD3OD, 100 MHz) δ 11.3, 19.7, 20.1, 26.8, 35.8, 52.1,
52.3, 60.5, 117.3 (q, J ) 289), 119.2, 161.6 (q, J ) 38), 170.5.
Anal. (C12H19N3O‚1.00TFA) C, H, N, F.
Dip ep t id e Nit r iles P r ep a r ed b y Gen er a l Met h od C.
l-Isoleu cin yl-L-tr a n s-4,5-m eth a n op r olin en itr ile TF A Sa lt
(22). 1H NMR (D2O, 500 MHz) δ 0.72 (m, 1H), 0.92 (t, J ) 7.3,
3H), 1.07 (d+m, J ) 7.3, 4H), 1.25 (m, 1H), 1.55 (m, 1H), 2.19
(m, 2H), 2.43 (m, 1H), 2.60 (m, 1H), 3.63 (m, 1H), 4.39 (d, J )
5.5, 1H), 4.82 (q, J ) 9.1, 1H); 13C NMR (D2O, 125 MHz) δ
10.7, 14.6, 18.2, 18.8, 23.7, 31.6, 36.4, 37.1, 50.9, 56.6, 116.6
(q, J ) 290), 118.5, 163.2 (q, J ) 35), 170.2; HPLC: method 3;
tR ) 2.89 min. HRMS (FAB) m/z: [M + H]+ calcd for
C
12H19N3O, 222.1606; found, 222.1601.
L-Isoleu cin yl-L-tr a n s-2,3-m eth a n op r olin en itr ile TF A
Sa lt (23). 1H NMR (D2O, 500 MHz) δ 0.84 (t, J ) 7.3, 3H),
1.05 (d, J ) 7.3, 3H), 1.14-1.30 (m 2H), 1.64 (m, 1H), 1.76 (m,
1H), 2.10 (m, 2H), 2.27 (m, 1H), 2.75 (m, 1H), 3.08 (m, 1H),
3.90 (m, 1H), 4.30 (d, J ) 2.3, 1H); 13C NMR (D2O, 125 MHz)
δ 11.5, 15.0, 22.4, 23.6, 24.0, 28.1, 36.5, 42.0, 44.8, 60.7, 116.6
(q, J ) 290), 117.7, 163.2 (q, J ) 35), 165.6. HPLC: method 3;
tR ) 2.49 min. HRMS (FAB) m/z: [M + H]+ calcd for
Ben zyloxyca r bon yla m in o(1-m eth ylcyclop en t-1-yl)a ce-
tic Acid Eth yl Ester (19b). A solution of 2-(1-methylcyclo-
pentyl)malonic acid monoethyl ester (0.48 g, 2.26 mmol) in dry
benzene (2.85 mL) was treated with triethylamine (0.3 mL,
2.26 mmol) and diphenylphosphoryl azide (0.5 mL, 2.26 mmol),
refluxed for 1 h, and cooled to room temperature. The solution
was treated with benzyl alcohol (0.35 mL, 3.39 mmol), refluxed
for 17 h, cooled, then partitioned between aqueous 10% citric
acid solution (3.8 mL) and EtOAc (2 × 25 mL). The combined
organic extracts were washed with 5% NaHCO3 solution, dried
(MgSO4), and evaporated. The remainder was purified by flash
column chromatography (1:9 EtOAc-hexanes) to give the title
C
12H19N3O, 222.1606; found, 222.1611.
L-Isoleu cin yl-L-cis-3,4-m eth a n op r olin en itr ile TF A Sa lt
(25). 1H NMR (500 MHz, D2O) δ 0.53 (m, 1H), 0.90 (t, J ) 7.3,
3H), 1.00 (d, J ) 6.9, 3H), 1.08 (m, 1H), 1.18 (m, 1H), 1.48 (m,
1H), 2.00 (m, 2H), 2.20 (m, 1H), 3.83 (s, 2H), 4.10 (d, J ) 5.5,
1H), 4.89 (d, J ) 5.0, 1H); 13C NMR (D2O, 125 MHz) δ 10.1,
10.8, 14.6, 18.5, 23.8, 36.4, 50.7, 51.2, 56.4, 116.6 (q, J ) 290),
118.1, 163.2 (q, J ) 35), 170.4. HPLC: method 3; tR ) 3.15
min. HRMS (FAB) m/z: [M + H]+ calcd for C12H19N3O,
222.1606; found, 222.1605.
1
compound (1.20 g, 100%). H NMR (CDCl3, 400 MHz) δ 0.92
(s + t, J ) 7.0, 6H), 1.20-1.80 (m, 8H), 4.20 (m, 3H), 5.11 (s,
2H), 5.38 (m, 1H), 7.30-7.50 (m, 5H). LC/MS m/z: 342 [M +
Na]+.
L-Valin yl-L-cis-3,4-m eth an opr olin en itr ile TFA Salt (27).
1H NMR (CD3OD, 400 MHz) δ 0.55 (q, J ) 5.7, 1H), 1.03 (d, J
) 6.5, 3H), 1.08 (d + m, J ) 7.0, 4H), 1.95 (m, 1H), 2.10 (m,
1H), 2.20 (m, 1H), 3.80 (d, J ) 9.5, 1H), 3.86 (dd, J ) 9.5, 4.8,
1H), 4.00 (d, J ) 5.7, 1H), 4.84 (d, J ) 4.8, 1H); 13C NMR (CD3-
OD, 100 MHz) δ 11.5, 17.8, 19.2, 19.8, 20.2, 31.7, 51.6, 52.1,
58.2, 118.4, 118.8 (q, J ) 289), 162.0 (q, J ) 37), 171.0. Anal.
(C11H27N3O‚1.30TFA) C, H, N, F.
Rep r esen ta tive Exa m p le of P r ep a r a tion of In ter m ed i-
a tes 19 a n d 20: Gen er a l Meth od D. 2-Cyclop en tylid en -
em a lon ic Acid Dieth yl Ester (17b). A mixture of dry THF
(200 mL) and dry CCl4 (25 mL) was cooled to 0 °C and treated
with TiCl4 (11.0 mL, 0.1 mol). The resulting yellow suspension
was stirred at 0 °C for 5 min, treated sequentially with
cyclopentanone (4.4 mL, 0.05 mol) and freshly distilled diethyl
malonate (7.6 mL, 0.05 mol), and then stirred at 0 °C for 0.5
h. The reaction mixture was then treated with a solution of
dry pyridine (16 mL, 0.20 mol) in dry THF (30 mL) and stirred
at 0 °C for 1.0 h and then at room temperature for 72 h. The
reaction mixture was quenched with water and extracted with
Et2O (2 × 100 mL). The combined organic extracts were
washed with brine and saturated NaHCO3, dried (MgSO4), and
concentrated under reduced pressure. The residue (12.1 g) was
purified by flash column chromatography (EtOAc-hexanes,
5:95) to give the title compound (5.25 g, 46%). 1H NMR (CDCl3,
400 MHz) δ 1.30 (m, 6H), 1.73 (m, 4H), 2.68 (m, 4H), 4.24 (m,
4H). LC/MS m/z: 227 [M + H]+.
2-(1-Meth ylcyclop en t-1-yl)m a lon ic Acid Mon oeth yl Es-
ter (18b). A mixture of 3.0 M methylmagnesium iodide (3.1
mL, 9.4 mmol) and cuprous chloride (10.6 mg) was stirred at
0 °C, treated with a solution of 2-cyclopentylidenemalonic acid
diethyl ester (17b) (1.41 g, 6.24 mmol) in dry Et2O (2 mL) over
5 min, and stirred at 0 °C for 1 h and then at room temperature
for 1 h. The reaction was then quenched by the dropwise
addition of ice-water (15 mL) followed by 3 M HCl (3.7 mL).
The mixture was then extracted with EtOAc (3 × 25 mL). The
combined organic extracts were washed with 1% Na2S2O3 and
brine, dried (MgSO4), and evaporated (1.57 g). The residue was
ter t-Bu tyloxyca r bon yla m in o(1-m eth ylcyclop en t-1-yl)-
a cetic Acid (20b). A solution of benzyloxycarbonylamino(1-
methylcyclopentyl)acetic acid ethyl ester (0.798 g, 2.26 mmol)
in EtOAc (35 mL) was treated with 10% palladium hydroxide
on carbon (190 mg) under an atmosphere of H2 at room
temperature for 20 h. The mixture was diluted with EtOAc
and filtered through a Celite pad, washing the pad well with
EtOAc (2 × 35 mL). The filtrate was evaporated to dryness to
give the crude amine (0.42 g, 100%). The crude amine (0.42 g,
2.26 mmol) in a mixture of THF (9 mL) and water (9 mL) was
treated with di-tert-butyl dicarbonate (0.73 g, 3.35 mmol) and
K2CO3 (0.68 g, 4.54 mmol) and stirred at room temperature
for 36 h. The reaction mixture was partitioned between water
and Et2O. The aqueous fraction was then extracted with Et2O
(3 × 30 mL), and the combined organic extracts were washed
with brine, dried (MgSO4), and evaporated. The remainder was
purified by flash column chromatography (1:9 EtOAc-hexane)
to give the desired N-Boc-protected ester (0.57 g, 92%). 1H
NMR (CDCl3, 400 MHz) δ 0.95 (s, 3H), 1.28 (t, J ) 7.0, 3H),
1.30 (m, 2H), 1.44 (s, 9H), 1.55-1.80 (m, 6H), 4.20 (q, J ) 7.0,
2H), 5.11 (d, J ) 6.0, 1H); 13C NMR (CDCl3, 100 MHz) δ 14.2,
22.5, 23.9, 24.3, 28.3, 36.8, 45.8, 60.8, 80.0, 155.7, 172.5. LC/
MS m/z: 286 [M + H]+. A solution of tert-butyloxycarbony-
lamino(1-methylcyclopentyl)acetic acid ethyl ester (0.55 g, 1.97
mmol) in MeOH (6.9 mL) and THF (6.9 mL) was treated with
1.0 M NaOH (2.9 mL, 2.9 mmol) and stirred at room temper-
ature for 24 h. The reaction mixture was evaporated, dissolved
in water (10 mL), and extracted with Et2O. The aqueous phase
was acidified to pH 2 with 1.0 M HCl (2.9 mL) and extracted
with EtOAc (3 × 30 mL). The combined organic extracts were
washed with brine, dried (MgSO4), and evaporated to give the
title compound (0.81 g, 96%): mp ) 151-152 °C. 1H NMR
(CDCl3, 400 MHz) δ 0.98 (s, 3H), 1.30 (m, 2H), 1.44 (s + m,
11H), 1.55-1.80 (m, 6H), 4.20 (d, J ) 7.0, 2H), 5.11 (d, J )
7.0, 1H); 13C NMR (CDCl3, 100 MHz) δ 22.5, 23.9, 24.3, 28.3,
36.9, 45.6, 60.8, 80.0, 155.7, 176.8. LC/MS m/z: 258 [M + H]+.

2596 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Magnin et al.
The amino acids prepared according to this method were
converted to the corresponding dipeptide nitriles using general
method B.
Refer en ces
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Diabetes, 1995-2025: Prevalence, Numerical Estimates and
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A. S.; Nuss, J . M. Current Therapies and Emerging Targets for
the Treatment of Diabetes. Curr. Pharm. Des. 2001, 7, 417-
450. (c) Harris, M. I.; Hadden, W. C.; Knowler, W. C.; Bennet,
P. H.; Prevalence of Diabetes and Impaired Glucose Tolerance
and Plasma Glucose Levels in U.S. Population Aged 20-74 yr.
Diabetes 1987, 36, 523-534.
(2) (a) Saltiel, A. R.; Olefsky, J . M. Thiazolidinediones in the
Treatment of Insulin Resistance and Type II Diabetes. Diabetes
1996, 45, 1661-1669. (b) Schoonjans, K.; Martin, G.; Staels, B.;
Auwerx, J . Peroxisome Proliferator-Activated Receptors, Or-
phans with Ligands and Functions. Curr. Opin. Lipidol. 1997,
8, 159-166. (c) Pittas, A. G.; Greenberg, A. S.; Thiazolidinediones
in the Treatment of Type 2 Diabetes. Expert Opin. Pharmaco-
ther. 2002, 3, 529-540. (d) Rami, H. K.; Smith, S. A.; Synthetic
Ligands for PPARγsReview of Patent Literature 1994-
1999. Expert Opin. Ther. Pat. 2000, 10, 623-634. (e) Van Gaal,
L.; Scheen, A. J . Are All Glitazones the Same? Diabetes Metab.
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S15.
Com p u ta tion a l Assessm en t Meth od s. Conformations
were generated for both methanoprolinenitrile and prolineni-
trile forms of the N-terminal tert-leucine dipeptide and alanine
dipeptides using Macromodel with OPLSAA parameters and
GB/SA solvation model.34 Two conformations were selected for
energetic comparisons: the global minimum, which has the
syn amide rotamer (Figure 2, lower structure), and an anti
conformation, which brings the reactive amine and nitrile close
to each other (Figure 2, upper structure). These conformations
were reminimized using ab initio quantum mechanics with the
B3LYP DFT method and the 6-31+G** basis set in Gaussian
98.0.35 To approximate van der Waals and electrostatic con-
tributions, relative energies of the conformations were also
calculated using Insight’s CFF force field.36 Ab initio energetic
minimizations and force field calculations were also performed
on the proline and methanoproline compounds with a simple
acetamide cap in both conformations.
In Vitr o Assa ys. All compounds were tested in vitro against
purified porcine DPP-IV as previously published.37 Inhibition
was determined using the substrate H-Gly-Pro-PNA. Produc-
tion of p-nitroaniline was measured at 405 nm at 9 s intervals
over 15 min. Reactions were initiated by addition of the
enzyme, and data collection was started immediately. The
reactions were run at 11 substrate concentrations (ranging
from 60 to 3000 µM) and 7 inhibitor concentrations (ranging
from 1 to 1000 nM). Enzyme reactions contained a final volume
of 100 µL, ATE buffer (100 mM Aces, 52 mM Tris, 52 mM
ethanolamine, pH 7.4), 4.5 nM porcine DPP-IV, and 1% DMSO.
In Vivo Assa y Meth od s. Male Zuckerfa/fa rats (Harlan)
weighing between 400 and 450 g were housed in a room that
was maintained on a 12 h light-dark cycle and were allowed
free access to normal rodent chow and tap water. The day
before the experiment, the rats were weighed and divided into
control and treated groups of six. Rats were fasted 17 h prior
to the start of the study. On the day of the experiment, animals
were dosed orally with vehicle (water) or DPP-IV inhibitors
(3 µmol/kg) at -30 min. Two blood samples were collected at
-30 and 0 min by tail bleed. Glucose (2 g/kg) was administered
orally at 0 min. Additional blood samples were collected at 15,
30, 60, 90, and 120 min. Blood samples were collected into
EDTA containing tubes from Starstedt. Plasma glucose was
determined by Cobas Mira (Roche Diagnostics) by the glucose
oxidation method.
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Rat plasma insulin was assayed using an ELISA kit from
Crystal Chem Inc. Properly diluted plasma samples were
added to the ELISA microplate. Insulin was then detected
using guinea pig anti-insulin serum. DPP-IV activity in rat
plasma was assayed ex vivo using Ala-Pro-AFC‚TFA, a
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reaction buffer, 50 mM Hepes, and 140 mM NaCl. The buffer
contained 0.1 mM Ala-Pro-AFC‚TFA. Fluorescence was then
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the first 20 min of the reaction, with units/mL defined as the
rate of increase of fluorescence intensity (arbitrary units) per
milliliter of plasma. All in vivo data presented are the mean
( SE (n ) 6). Data analysis was performed using ANOVA
followed by Fisher Post-hoc.
Ack n ow led gm en t. The authors thank Dr. Robert
Zahler for helpful comments in the editing of this
manuscript.
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