(2S,3S)-3-amino-4-(3,3-difluoropyrrolidin-1-yl)-N,N-dimethyl-4-oxo-2-(4-[1, 2,4]triazolo[1,5-a]-pyridin-6-ylphenyl)butanamide: A selective α-amino amide dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes

Article abstract of DOI:10.1021/jm060015t

A series of β-substituted biarylphenylalanine amides were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-4) for the treatment of type 2 diabetes. Optimization of the metabolic profile of early analogues led to the discovery of (2S

Full text of DOI:10.1021/jm060015t

3614
J. Med. Chem. 2006, 49, 3614-3627
(2S,3S)-3-Amino-4-(3,3-difluoropyrrolidin-1-yl)-N,N-dimethyl-4-oxo-2-(4-[1,2,4]triazolo[1,5-a]-
pyridin-6-ylphenyl)butanamide: A Selective r-Amino Amide Dipeptidyl Peptidase IV Inhibitor
for the Treatment of Type 2 Diabetes
Scott D. Edmondson,†,* Anthony Mastracchio,† Robert J. Mathvink,† Jiafang He,† Bart Harper,† You-Jung Park,†
Maria Beconi,|,# Jerry Di Salvo, George J. Eiermann,‡ Huaibing He,† Barbara Leiting,§ Joseph F. Leone,† Dorothy A. Levorse,†
Kathryn Lyons,† Reshma A. Patel,§ Sangita B. Patel,† Aleksandr Petrov,‡ Giovanna Scapin,† Jackie Shang,| Ranabir Sinha Roy,§
Aaron Smith,† Joseph K. Wu,§ Shiyao Xu,| Bing Zhu,| Nancy A. Thornberry,§ and Ann E. Weber†
Merck Research Laboratories, Merck & Co., Inc., Rahway, New Jersey 07065
ReceiVed January 6, 2006
A series of â-substituted biarylphenylalanine amides were synthesized and evaluated as inhibitors of dipeptidyl
peptidase IV (DPP-4) for the treatment of type 2 diabetes. Optimization of the metabolic profile of early
analogues led to the discovery of (2S,3S)-3-amino-4-(3,3-difluoropyrrolidin-1-yl)-N,N-dimethyl-4-oxo-2-
(4-[1,2,4]triazolo[1,5-a]pyridin-6-ylphenyl)butanamide (6), a potent, orally active DPP-4 inhibitor (IC50
6.3 nM) with excellent selectivity, oral bioavailability in preclinical species, and in vivo efficacy in animal
)
models. Compound 6 was selected for further characterization as a potential new treatment for type 2 diabetes.
Introduction
quently, potent small molecule DPP-4 inhibitors such as
vildagliptin (NVP-LAF237, 1),3 saxagliptin (BMS-477118, 2),4
and sitagliptin (MK-0431, 3)5 (Chart 1) have advanced into late
stage human clinical trials.
Inhibition of dipeptidyl peptidase IV (DPP-4)a has recently
emerged as a promising new approach for the treatment of type
2 diabetes mellitus.1 DPP-4 is the enzyme responsible for
inactivation of the incretin hormones glucagon-like peptide 1
(GLP-1) and glucose-dependent insulinotropic polypeptide
(GIP). These two hormones are secreted in response to nutrient
ingestion, and each enhances the glucose-dependent secretion
of insulin. Additionally, GLP-1 has been shown in mammals
to stimulate insulin biosynthesis, inhibit glucagon secretion, slow
gastric emptying, reduce appetite, and stimulate â-cell neogen-
esis and differentiation.1,2
Due to rapid processing of GLP-1 and GIP by DPP-4, the
half-lives of the active peptides in blood are extremely short.
Inhibition of DPP-4 in humans has been shown to increase
circulating GLP-1 and GIP levels which lead to decreased blood
glucose levels, hemoglobin A1c levels, and glucagon levels.2,3c,5c
DPP-4 inhibitors offer a number of potential advantages over
existing diabetes therapies including a lowered risk of hypogly-
cemia, a lowered risk of weight gain, and the potential for the
regeneration and differentiation of pancreatic â-cells. Conse-
DPP-4 is a serine protease which cleaves a dipeptide from
the N-terminus of GLP-1[7-36] to give an inactive GLP-1[9-
36] peptide. Since DPP-4 preferentially cleaves substrates with
proline at the P-1 position, it is not surprising that a common
structural motif among many DPP-4 inhibitors is a pyrrolidine-
bearing electrophile at the P-1 site linked to an R-amino acid at
the P-2 site.1c Some of these electrophiles are irreversible
inhibitors (phosphonates) while others are reversible (nitriles,
boronic acids) inhibitors.1c A potential liability of the reversible
inhibitors is poor chemical stability due to the propensity of
the free amine group to cyclize with the electrophile thus
forming a six-membered ring. Cyanopyrrolidines such as 1 and
2 are representative of this potent class of DPP-4 inhibitors,
and each of these compounds has been optimized with respect
to chemical stability and potency.3,4 An alternative option to
access potent and stable DPP-4 inhibitors would be to optimize
for increased potency at the P-2 substituent of R-amino acid
derivatives lacking an electrophile.6
Previous reports from these laboratories describe a series of
â-substituted phenylalanine derivatives which lack an electro-
phile attached to the pyrrolidine ring.7 Optimization of the
â-substituent of the phenylalanine led to â-dimethylamide 4,
an intrinsically potent DPP-4 inhibitor (IC50 ) 12 nM) with a
high serum potency shift (32-fold) due to high plasma protein
binding.7b Replacement of the 4-fluorophenyl group in the
homophenylalanine series with polar heterocycles such as a
methylpyridone (5) resulted a reduced serum shift and a good
pharmacokinetic profile in rats, dogs, and monkeys.8 Unfortu-
nately, the methylpyridone moiety is metabolically labile,
forming the free pyridone ring upon metabolism. Since this
metabolite is also a potent DPP-4 inhibitor and active at various
ion channels, further development of 5 was abandoned.
* To whom correspondence should be addressed. Scott D. Edmondson,
Merck Research Laboratories, Mail Code RY123-234 P.O. Box 2000
Rahway, NJ 07065-0900. Tel: (732) 594-0287. Fax: (732) 594-5350.
E-mail: scott_edmondson@merck.com.
† Department of Medicinal Chemistry.
§ Department of Metabolic Disorders.
| Department of Drug Metabolism.
‡ Department of Pharmacology.
Department of Immunology and Rheumatology.
# Current address: GlaxoSmithKline, RI-CEDD, 709 Swedeland Rd.,
King of Prussia, PA 19406.
a Nonstandard abbreviations: DPP-4 ) dipeptidyl peptidase 4, GLP-1
) glucacon-like peptide 1, GIP ) glucose-dependent insulinotropic
polypeptide, CBS reagent ) 2-methyl-CBS-oxazaborolidine, DPP8 )
dipeptidyl peptidase 8, DPP9 ) dipeptidyl peptidase 9, FAP ) seprase or
fibroblast activation protein, QPP ) quiescent cell proline dipeptidase, DPP-
II ) dipeptidyl peptidase II, APP ) amino peptidase P, F ) oral
bioavailability, val-pyr ) valine pyrrolidide, SAR ) structure-activity
relationships, MRT ) mean residence time, hERG ) human ether-a-go-go
which is a human cardiac potassium ion channel, OGTT ) oral glucose
tolerance test, PD ) pharmacodynamic, AMC ) aminomethylcoumarin,
Herein, we describe a series of fused heterocyclic bioisosteres
to the methylpyridone moiety that solve the liabilities associated
with 5. Further improvement of the metabolic profile of the
(S)-3-fluoropyrrolidine moiety ultimately led to the discovery
DIO ) diet-induced obese, AUC ) area under the curve, and Clp
clearance.
)
10.1021/jm060015t CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/20/2006

R-Amino Amide Dipeptidyl Peptidase IV Inhibitor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3615
Chart 1
Scheme 1a
Scheme 2a
a Reagents: (a) Me2NCR2(OMe)2, DMF, 130 °C, 12 h; (b) H2NOSO3H,
MeOH, pyr, 0 °C to room temperature; (c) t-BuOH, tol, Et3N, DPPA, rt to
100 °C, 4 h; (d) TFA/DCM (1:2), 0 °C to room temperature.
of (2S,3S)-3-amino-4-(3,3-difluoropyrrolidin-1-yl)-N,N-dimeth-
yl-4-oxo-2-(4-[1,2,4]triazolo[1,5-a]pyridin-6-ylphenyl)butana-
mide (6), a potent, selective, and orally active DPP-4 inhibitor
with an excellent pharmacokinetic profile in four species. This
compound was selected for extensive in vivo evaluation for the
treatment of type 2 diabetes.
Chemistry
a Reagents: (a) (EtO)2POCH2COCH3, DBU, THF; (b) R-CBS, cat-
echolborane, tol, -78 °C to -30 °C; (c) EDC, HOBt, Boc-Gly, DIEA,
DCM; (d) LHMDS, ZnCl2, THF, -78 °C; (e) TMSCHN2, Et2O, MeOH;
(f) 1 N LiOH, THF, MeOH; (g) EDC, RR′NH, HOBt, DIEA, DCM; (h)
KMnO4, NaIO4, K2CO3, t-BuOH, H2O; (i) EDC, HOBt, Me2NH, DIEA,
DCM; (j) bis(pinacolato)diboron, DMSO, KOAc, Pd(dppf)2Cl2, 80 °C, 4
h; (k) ArX, tol, EtOH, 2 N aqueous Na2CO3, Pd(dppf)2Cl2, 90 °C, 12 h; (l)
TFA, DCM; (m) Na2CO3, H2O; (n) HCl, DCM, rt.
Heterocyclic intermediates 8a-d and 11 were synthesized
following the routes illustrated in Scheme 1. 2-Amino-5-
bromopyridines (7) were heated with dimethylacetamide di-
methylacetal or dimethylpropionamide dimethylacetal in DMF,
followed by treatment with hydroxylamine-O-sulfonic acid to
afford the substituted 6-bromo[1,2,4]triazolo[1,5-a]pyridines
8a-d.9 A similar procedure was performed on 2-amino-4-
iodopyridine (10) to afford triazaole 11. Aminopyridine 10 was
generated via a Curtius rearrangement of acid 9 based on a
literature reported sequence.10
Inhibitors were synthesized using a variation of a route
previously described by these laboratories (Scheme 2).7b Hor-
ner-Emmons reaction of 4-bromobenzaldehyde (12) with
diethyl (2-oxopropyl)phosphonate afforded the corresponding
trans enone in excellent yield (81%). A chiral reduction of the
enone with (R)-2-methyl-CBS-oxazaborolidine (R-CBS reagent)
was next employed to afford the S-styrenol 13 in excellent yield
(90%) and enantioselectivity (96% ee).11 The assignment of an
S absolute configuration to alcohol 13 was initially supported
by CBS reagent precedent11a and later confirmed by X-ray
crystallographic analysis of analogue 23 (vide infra) which was
derived from 13. Enantiomeric excess could be enhanced to
>98% ee by recrystallization from cyclohexane. Next, the
alcohol was condensed with Boc-glycine under standard condi-
tions to afford ester 14. Exposure of 14 to Kazmaier’s enolate-
Claisen rearrangement conditions12 followed by esterification
of the resulting acid with trimethylsilyldiazomethane then
afforded N-Boc amino ester 15 in excellent yield and stereo-
selectivity. Esterification to form 15 was performed in order to
facilitate purification, and this ester was subsequently saponified
to give acid 16 which was used without purification. This
combination of a chiral CBS reduction with an enolate Claisen
rearrangement is an especially effective method of synthesizing
orthogonally functionalized, â-substituted phenylalanine deriva-
tives in high enantiomeric excess.
Next, acid 16 was coupled with 3-fluoroazetidine,13 pyrro-
lidine, (S)-3-fluoropyrrolidine,14 or 3,3-difluoropyrrolidine14
using standard coupling conditions to give amides 17a-d in
yields of 60-95%. Oxidative cleavage of the olefin to the
corresponding acids followed by standard EDC coupling of these
acids with dimethylamine then afforded dimethylamides 18a-d
in 50-90% overall yields. Treatment of bromides 18a-d with
bis(pinacolato)diboron under palladium catalysis provided bo-
ronates 19a-d in excellent yields (>80%). Suzuki couplings
of boronates 19a-d with a series of heterocyclic halides
afforded the desired coupled products, which were then depro-
tected under standard conditions to give inhibitors 6 and 20-
33 in Tables 1-3 as the trifluoroacetic acid (TFA) salts. The
TFA salt 6a was converted to a hydrochloric acid salt 6b for in
vivo evaluation.

3616 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Edmondson et al.
Table 1. Inhibitory Properties (IC50s) of Selected Biarylphenylalanine (S)-Fluoropyrrolidide Amides
a Unless otherwise noted, values reported are the mean of a minimum of two experiments with a standard deviation <25% of the mean. b Assay done in
the presence of 50% human serum. Unless otherwise noted, values reported are the mean of a minimum of two experiments with a standard deviation <40%
of the mean. c Values reported for compound 4 are taken from ref 7b. d Values reported are the results of a single experiment. e Value is 4.1 ( 3.6 nM.
Results and Discussion
DPP-4 inhibitory activities for selected (S)-3-fluoropyrroli-
dine-derived analogues are listed in Table 1. For comparison,
fluorophenyl derivative 4 is also included in the table. Although
methylpyridone 20 is similar to 4 in terms of intrinsic DPP-4
potency, 20 exhibited only a 10-fold serum potency shift
compared to a 32-fold shift observed with 4. To address the
possibility of metabolic demethylation of the N-methylpyridone
ring, a series of fused heterocycles (i.e. 21-26) was generated
which was expected to mimic the stereoelectronic effect of the
methylpyridone moiety. Heterocycles 21-26 were all comparable
to 20 in terms of intrinsic potency, but 21 and 26 suffer from
large serum potency shifts. Pharmacokinetic evaluation of 20
and 22-25 (IC50s < 10 nM) in the rat revealed that 20 and 22
suffered from low oral bioavailabilities (Frat < 3%, Table 4)
while 24 displayed a moderate oral bioavailability (Frat ) 21%).
Compounds 6a and 20-33 were evaluated for in vitro
inhibition of DPP-4.15 As a surrogate measure of plasma protein
binding, each inhibitor was also measured for DPP-4 inhibition
in the presence of 50% human serum.7b Each inhibitor was also
tested against DPP8,16 DPP9,17 FAP (seprase),18 and other
proline specific enzymes with DPP-4 like activity,19 including
QPP (DPP-II),15,20 amino peptidase P (APP), and prolidase.
Selectivity over DPP8 and DPP9 was considered particularly
important because inhibition of these enzymes has been associ-
ated with toxicity in preclinical species and their relevance to
humans is not yet known.21 Consequently, inhibition data for
each compound against DPP8 and DPP9 is presented in Table
1. With the exception of QPP (data also presented), inhibition
of the other enzymes was weak (IC50s > 40,000 nM).

R-Amino Amide Dipeptidyl Peptidase IV Inhibitor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3617
Table 2. Inhibitory Properties (IC50s) of Selected Biarylphenylalanine Amides
a Unless otherwise noted, values reported are the mean of a minimum of two experiments with a standard deviation <25% of the mean. b Assay done in
the presence of 50% human serum. Unless otherwise noted, values reported are the mean of a minimum of two experiments with a standard deviation <40%
of the mean. c Values reported are the results of a single experiment.
Table 3. Inhibitory Properties (IC50s) of Selected Biarylphenylalanine Difluoropyrrolidides
a Unless otherwise noted, values reported are the mean of a minimum of two experiments with a standard deviation <25% of the mean. b Assay done in
the presence of 50% human serum. Unless otherwise noted, values reported are the mean of a minimum of two experiments with a standard deviation <40%
of the mean.
Heterocycles 23 and 25, however, afforded the highest oral
bioavailabilities and longest half-lives in the series (Frat ) 43%
and 46%; t1/2 ) 2.0 and 4.3 h respectively).
(IC50 ) 34 nM)8 with compound 20 (IC50 ) 8.0 nM) is
illustrative of the potency enhancement afforded by the dimethyl
amide moiety. The central phenyl group appears to provide a
sufficient tether length to position the triazolopyridine hetero-
cycle proximal to Arg358. The fused triazole ring stacks against
the side chain of Phe357 and hydrogen bonds with the side chain
of Arg358. Moreover, SAR trends of analogous phenylalanine-
derived DPP-4 inhibitors illustrate the potency enhancing effect
of aromatic functionality positioned to interact with Arg358 and
Tyr547.6-8 The presence of these additional interactions with
the enzyme affords 23 a greater than 350-fold improvement in
potency compared to val-pyr.
Due to the excellent DPP-4 potency and rat pharmacokinetic
profile of 23, additional SAR surrounding this compound was
explored. Consistent with the X-ray crystal structure, the
introduction of methyl substituents on the fused triazole ring
system afforded compounds with reduced intrinsic potency (e.g.
The X-ray crystal structure determination of triazole 23 (IC50
) 4.3 nM) bound to the active site of DPP-4 is shown in Figure
1. Compound 23 (yellow) shows significant overlap with a
previously reported R-amino acid-derived DPP-4 inhibitor,
valine pyrrolidide22 (val-pyr green in Figure 1, DPP-4 IC50
)
1580 nM). The pyrrolidines of both inhibitors occupy the S1
hydrophobic pocket, and the carbonyls linked to the pyrrolidines
are within hydrogen bonding distance from the side chain of
Asn710. Furthermore, the amino group of both inhibitors
hydrogen bond to the side chains of Tyr662, Glu205, and
Glu206. In contrast to val-pyr, the larger compound 23 extends
into the S2 pocket, where the dimethyl amide carbonyl forms a
hydrogen bond with the side chain of Tyr547. A comparison
of the intrinsic potency of â-methylphenylalanine derivative 5

3618 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Table 4. Pharmacokinetic Profiles of Selected DPP-4 Inhibitorsa
Edmondson et al.
Table 5. Ion Channel Activities (IC50s) of Analogues 6a, 32, and 33
Clp
t1/2 PO AUCnorm PO Cmax
compd
Ca (µM)
hERG (µM)
Na (µM)
compd species (mL/min/kg) (h) (µM‚h/mpk)
(µM)
F (%)
6a
32
33
>100
54
>100
16
>100
>100
>100
60
20
22
23
rat
rat
rat
dog
rat
rat
rat
dog
rat
dog
rat
dog
monkey
mouse
rat
dog
rat
15
34
7.0
1.2
1.1
2.0
2.4
1.7
4.3
2.1
2.0
1.6
1.1
1.6
4.8
6.3
6.7
1.1
3.4b
2.7
3.1b
0.048
0.009
2.4
0.021
0.008
2.3
6.6
0.60
3.9
2.4
4.0
2.1
5.9
6.9
6.3
2.8
11
7.8
9.5
8.9
2
0.76
43
94
21
46
37
100
46
98
100
73
56
68
69
81
79
100
15
3.1
10.0
3.3
7.3
6.1
7.7
5.9
2.8
1.8
5.3
1.2
1.8
1.5
1.4
1.0
12
24
25
30
0.83
5.6
2.2
6.9
2.9
6.8
15
16
4.0
23
15
20
24
38
mg of protein) in both rat and human liver microsomes,
revealing a lower propensity to form reactive metabolites.
The superior metabolic profile and rat oral bioavailability of
6a relative to 23 suggested that incorporation of the difluoro-
pyrrolidine ring into analogues 24 and 25 might improve their
rat oral biovailabilities. Compounds 32 and 33 (Table 3) each
exhibit excellent intrinsic inhibition of DPP-4 (IC50s ) 6.2 nM
and 7.5 nM, respectively) with a reduced serum shift (5-fold
and 8-fold, respectively) relative to 6a (11-fold). As expected,
each these analogues also possess improved oral bioaviabilities
(Frat ) 69% for 32 and 79% for 33) in the rat relative to their
(S)-3-fluoropyrroline-derived counterparts. The dog pharmaco-
kinetic profiles of 32 and 33 revealed excellent oral bioavail-
abilities (Fdog ) 81% and 100% respectively) but mean
residence times (MRT)25 for 32 (3.4 h) and 33 (3.1 h) shorter
than the half-life and MRT for 6a (t1/2 ) 4.8 h, MRT ) 5.6 h).
Next, binding at hERG,26 human cardiac sodium channel,27
and rabbit calcium channel28 was measured as a general indicator
of ion channel activities (Table 5). Although compound 6a was
inactive at each of these ion channels (IC50s > 100 µM),
compound 32 showed activity at the potassium channel (hERG
IC50 ) 16 µM) and 33 exhibited activity at the calcium channel
(Ca2+ IC50 ) 15 µM). Additional in vitro profiling of 6a in an
extensive panel of receptor and ion channel binding and enzyme
inhibition assays showed no significant activity at 10 µM (data
not shown). Moreover, 6a also displays excellent pharmaco-
kinetic profiles in the mouse (F ) 68%, t1/2 ) 6.7 h) and rhesus
monkey (F ) 56%, t1/2 ) 6.3 h).
On the basis of an excellent off-target selectivity profile and
pharmacokinetic profile in four species, compound 6 was chosen
for further in vivo evaluation. The hydrochloride salt 6b was
assessed for its ability to improve glucose tolerance in lean mice.
Administration of single oral doses reduced the blood glucose
excursion in an oral glucose tolerance test (OGTT) in a dose-
dependent manner from 0.1 mg/kg (43% reduction) to 3.0 mg/
kg (56% reduction) when administered 60 min before an oral
dextrose challenge (5 g/kg, Figure 2). In a separate OGTT
experiment, the pharmacodynamic profile of compound 6b was
assessed. Plasma DPP-4 inhibition, compound concentration,
and active GLP-1 levels were measured 10 min after dextrose
challenge (Figure 3). At the 0.1 mg/kg dose, the plasma
concentration of 6 was 269 nM and the reduction of blood
glucose excursion corresponded to a 56% inhibition of plasma
DPP-4 activity.29 A dosage of 0.3 mg/kg corresponded to a
plasma concentration of 1390 nM and provided 83% inhibition
of DPP-4. Maximal efficacy resulted in a 2 to 3-fold increase
in active GLP-1, analogous to GLP-1 levels observed upon
glucose challenge in DPP-4 deficient mice.30 The observed
levels of DPP-4 inhibition in the PD assay do not, however,
correspond to the expected efficacy of 6b in light of the intrinsic
murine potency (mouse IC50 ) 6.0 nM). To resolve this
disconnect between in vitro potency and in vivo efficacy,
noncovalent plasma protein binding of 6 was measured (Table
6). Since compound 6 is 96% protein bound in the mouse, a
free fraction of only 4% is available in the plasma for DPP-4
inhibition. At the 0.1 mg/kg dose in the mouse, 4% of the total
plasma concentration of 6 (269 nM) corresponds to a free
fraction concentration of only 11 nM. Since the 0.1 mg/kg dose
31
6a
32
33
dog
22
a Dose: iv: 1 mpk, po: 2 mpk. b Values are for mean residence time
(MRT) since t1/2 could not be calculated due to secondary peaks in the
plasma concentration time profile.
Figure 1. Compound 23 bound to DPP-4. The overlay of compound
23 (yellow) and the substrate analogue valine-pyrrolidine (green,
1N1M.pdb) shows the similar orientation between the two compounds.
Interactions of compound 23 with DPP-4 are shown as red dotted lines.
The extensive hydrogen bond network present between the ordered
water molecules, compound 23, and protein atoms has been omitted
for clarity.
27 and 29) and increased serum shifts relative to the parent
compound 23.
Potential substitutes for the (S)-3-fluoropyrrolidine ring of
23 were next evaluated (Table 2). Fluoroazetidide 30 afforded
the best intrinsic potency (DPP-4 IC50 ) 2.7 nM) in this series
with a 15-fold serum shift. Pyrrolidide 31 (IC50 ) 5.2 nM) and
difluoropyrrolidide 6a (IC50 ) 6.3 nM) also maintained excellent
DPP-4 potency, with serum shifts of 12-fold and 11-fold,
respectively. Rat pharmacokinetic evaluation of these three
compounds revealed that 30 and 31 each presented a profile
similar to that of 23 while incorporation of the difluoropyrro-
lidine group (6a) increased the oral bioavailability substantially
(Frat ) 100%). Further pharmacokinetic evaluation of 23, 30,
31, and 6a in the dog revealed another potential advantage of
the difluoropyrrolidine. While the half-lives of 23, 30, and 31
were all less than 2.5 h, the half-life of 6a was 4.8 h.
Since the (S)-3-fluoropyrrolidine moiety in previously re-
ported DPP-4 inhibitors has been demonstrated to be a labile
site for metabolic activation, 23 was evaluated for the formation
of reactive metabolites in the presence of rat and human liver
microsomes.23,24 Compound 23 displayed a moderate potential
for metabolic activation in both species, with levels of covalent
binding due to reactive metabolites measuring 142 pmol/mg
protein and 116 pmol/mg of protein in rat and human liver
microsomes, respectively. In comparison, compound 6a showed
relatively low levels of covalent binding (less than 50 pmol/

R-Amino Amide Dipeptidyl Peptidase IV Inhibitor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3619
Figure 2. (a) Effects of compound 6 on glucose levels after an oral
glucose tolerance test in lean C57BL/6N male mice. Compound 6 or
water (vehicle) was administered 60 min prior to an oral dextrose
challenge (5 g/kg). Control animals received water only. (b) The glucose
AUC was determined from 0 to 120 min. Percent reduction values for
each treatment were generated from the AUC data normalized to the
water-challenged controls. Data are represented as mean ( SEM (n )
7).
Figure 3. Effects of compound 6 on (a) DPP-4 inhibition and (b)
GLP-1 levels after an oral glucose tolerance test in lean C57BL/6N
male mice. Compound 6 was administered 60 min prior to an oral
dextrose challenge (5 g/kg). Plasma samples were collected for analysis
10 min post-dextrose administration. Data are represented as mean (
SEM (n ) 10-14/group).
corresponds to 56% DPP-4 inhibition (from Figure 3), the
calculated free fraction concentration of 11 nM correlates well
with the measured murine DPP-4 potency (IC50 ) 6.0 nM) of
6b, thus resolving the discrepancy between the observed in vivo
efficacy and the measured in vitro potency in the mouse.31 These
results confirm the correlation between DPP-4 inhibition,
increase in GLP-1 levels, and an improvement in glucose
tolerance.
Blood glucose lowering was also demonstrated in diet-induced
obese (DIO) mice, which are hyperglycemic, hyperinsulinemic,
and show impaired glucose tolerance in response to a dextrose
challenge consistent with insulin resistance observed in type 2
diabetes mellitus. Compared to vehicle, an 84% glucose
reduction was observed at the 0.3 mg/kg oral dose of compound
6b (Figure 4), resulting in near normalization of glucose
excursion relative to lean controls.
Conclusions. A culmination of work with phenylalanine-
derived DPP-4 inhibitors has led to the discovery of compound
6, a potent DPP-4 inhibitor with a moderate serum shift that
demonstrates excellent in vitro selectivity and in vivo efficacy.
On the basis of its DPP-4 potency, selectivity, in vivo efficacy,
pharmacokinetic profile, and low potential for metabolic activa-
tion, compound 6, (2S,3S)-3-amino-4-(3,3-difluoropyrrolidin-
1-yl)-N,N-dimethyl-4-oxo-2-(4-[1,2,4]triazolo[1,5-a]pyridin-6-
ylphenyl)butanamide, was chosen for further evaluation as a
potential treatment of type 2 diabetes mellitus.
Table 6. Plasma Protein Binding of Compound 6 in Different Species
mouse
96
rat
91
dog
64
monkey
54
human
82
Liquid Chromatography-Mass Spectrometer (LC-MS), using a
Waters Xterrra MSC18 3.5um, 50 × 3.0 mm column with a binary
solvent system where solvent A ) water, 0.06% trifluoroacetic acid
(by volume) and solvent B ) acetonitrile, 0.05% trifluoroacetic
acid (by volume). The LC method used a flow rate ) 1.0 mL/mim
with the following gradient: t ) 0 min, 90% solvent A; t ) 3.75
min., 2.0% solvent A; t ) 4.75 min, 2% solvent A; t ) 4.76 min,
90% solvent A; t ) 5.5 min, 90% solvent A. High-resolution mass
spectra were acquired from a Micromass Q-TOF quadrupole-time-
of-flight mass spectrometer. All MS experiments were performed
using electrospray ionization (EI) in positive ion mode. Leucine
enkephalin was applied as a lock-mass reference for accurate mass
analysis.
Purity analyses were performed using two distinct HPLC methods
for each compound unless otherwise indicated. The above-described
LC-MS HPLC method serves as a gross analysis of purity over a
broad range, and all final compounds show a single peak (>95%
purity) using this analytical method. To evaluate purity more
accurately, a high-resolution HPLC method was used to evaluate
each final product, and the final purity using these methods are
noted with the analytical data. High-resolution HPLC purity analysis
was achieved using the following two methods. Method A is
composed of two 5 µ Intertsil ODS-2 columns including a 4.6 ×
7.5 mm precolumn and a 4.6 × 30 mm column, a flow rate ) 2.0
mL/min, temperature ) 37 °C, λ ) 210 nm, and a mobile phase )
55:45 methanol/0.04 M potassium phosphate buffer with a final
pH ) 7.3. Method B utilized a 5 µ 4.6 × 250 mm Intertsil ODS-2
column at a flow rate ) 0.7 mL/min, temperature ) 37 °C, λ )
Experimental Section
General. All commercial chemicals and solvents are reagent
grade and were used without further purification unless otherwise
specified. 1H NMR spectra were recorded on a Varian InNova 500
MHz instrument in CDCl3 or CD3OD solutions. Low-resolution
mass spectra (MS) were determined on a Micromass Platform

3620 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Edmondson et al.
6-Bromo-5-methyl[1,2,4]triazolo[1,5-a]pyridine (8b). Starting
with 6-amino-3-bromo-2-picoline, the same procedures were fol-
lowed as in the synthesis of 8a. For 8b, LC/MS m/e 197.9/199.9
1
(M + H)+; H NMR (500 MHz, CDCl3) δ 8.65 (s, 1H), 8.34 (s,
1H), 7.45 (s, 1H), 2.70 (s, 3H).
6-Bromo-8-methyl[1,2,4]triazolo[1,5-a]pyridine (8c). Starting
with 2-amino-5-bromo-3-picoline, the same procedures were fol-
lowed as in the synthesis of 8a. For 8c, LC/MS m/e 212.0/214.0
(M + H)+; 1H NMR (500 MHz, CDCl3) δ 8.41 (s, 1H), 7.74 (d, J
) 9.4 Hz, 1H), 7.65 (d, J ) 9.4 Hz, 1H), 3.00 (s, 3H).
6-Bromo-8-methyl[1,2,4]triazolo[1,5-a]pyridine (8d). Starting
with 2-amino-5-bromopyridine and dimethylacetamide dimethyl
acetal, the same procedures were followed as in the synthesis of
1
8a. For 8d, LC/MS m/e 212.1/214.1 (M + H)+; H NMR (500
MHz, CD3OD) δ 8.96 (s, 1H), 7.75 (dd, J ) 9.4, 1.8 Hz, 1H), 7.60
(d, J ) 9.6 Hz, 1H), 2.53 (s, 3H).
2-Amino-4-iodopyridine (10). To a stirred solution of 4-iodo-
picolinic acid hemi-hydroiodide hydrate (1.0 g, 3.0 mmol) in 6 mL
of tert-butyl alcohol, 6 mL of toluene, and 1.4 mL of triethylamine
was added diphenylphosphoryl azide (1.1 mL, 5.0 mmol) dropwise
over 15 min. The resultant solution was then warmed to 65 °C
and, after 1.5 h, the bath temperature was raised to 100 °C. After
12 h, the solution was cooled and concentrated under reduced
pressure, and the residue was partitioned between ethyl acetate (100
mL) and water (60 mL). The ethyl acetate layer was washed
sequentially with saturated aqueous sodium bicarbonate solution
(100 mL) and saturated aqueous brine (100 mL), dried over
magnesium sulfate, and concentrated to afford a brown solid.
Purification by flash chromatography (silica gel; 5% ethyl acetate-
hexanes eluant) afforded a pale yellow solid, which was triturated
with hexanes to afford the Boc protected amine as a white solid
(570 mg, 30% yield). MS 265.1 (M + 1 - tBu). To a solution of
the above product (1.5 g, 4.7 mmol) in 10 mL of dichloromethane
was added trifluoroacetic acid (5 mL). The resultant solution was
stirred at room temperature for 1 h, and the volatiles were removed
under reduced pressure. The residue was dissolved in water (50
mL), and the solution was neutralized by portionwise addition of
sodium bicarbonate. The mixture was extracted with ethyl acetate,
and the extract was washed with saturated aqueous brine, dried
over magnesium sulfate and concentrated to afford an off-white
solid, which was triturated with hexanes to afford 2-amino-4-
iodopyridine 10 (1.0 g, 97% yield) as a white powder. LC/MS m/e
221.1 (M + H)+; 1H NMR (500 MHz, CD3OD) δ 7.59 (d, J ) 7.2
Hz, 1H), 7.04 (s, 1H), 6.93 (d, J ) 7.2 Hz, 1H).
7-Iodo[1,2,4]triazolo[1,5-a]pyridine (11). To a stirred solution
of 10 (1.0 g, 4.5 mmol) in N,N-dimethylformamide (3.0 mL) was
added N,N-dimethylformamide dimethyl acetal (1.6 mL, 11 mmol).
The reaction mixture was heated to 130 °C in a sealed tube
overnight. After cooling to room temperature, the volatiles were
removed under reduced pressure to afford a red oil, which was
dissolved in 8.0 mL of methanol and 0.74 mL of pyridine. The
solution was cooled in an ice bath, and hydroxylamine-O-sulfonic
acid (668 mg, 5.9 mmol) was added in one portion. The reaction
mixture was allowed to warm to room temperature and was stirred
overnight. The volatiles were removed under reduced pressure, and
the residue was partitioned between saturated aqueous brine solution
and ethyl acetate. The aqueous layer was further extracted with
ethyl acetate, and the combined organic layers were washed with
saturated aqueous brine solution (100 mL), dried over magnesium
sulfate, and concentrated under reduced pressure to afford the crude
product as an orange solid. Purification by flash chromatography
(silica gel; 0 to 4% methanol/methylene chloride gradient) afforded
pure 11 (363 mg, 33% over two steps) as a pale yellow solid. LC/
MS m/e 246.1 (M + H)+; 1H NMR (500 MHz, CD3OD) δ 8.60 (d,
J ) 9.2 Hz, 1H), 8.39 (s, 1H), 8.26 (s, 1H), 7.52 (d, J ) 9.2 Hz,
1H).
Figure 4. (a) Effects of compound 6 on glucose levels after an oral
glucose tolerance test in diet-induced obese (DIO) C57BL/6N male
mice versus lean mice. Compound 6 or water (vehicle) was administered
60 min prior to an oral dextrose challenge (5 g/kg). Control animals
were lean and received vehicle followed by dextrose challenge. (b)
The glucose AUC was determined from 0 to 120 min. Percent reduction
values for each treatment were generated from the AUC data normalized
to the lean controls. Data are represented as mean ( SEM (n ) 7).
210 nm, and a mobile phase ) 55:45 methanol/0.04 M potassium
phosphate buffer with a final pH ) 7.3.
Potentiometric titration of products 4 and 6a revealed that these
compounds exist as mono-trifluoroacetic acid (TFA) salts with
0-54% excess TFA in the final forms. While amounts of excess
TFA varied widely depending on the preparation of each individual
batch, the ratios were typically closer to 1:1 parent/TFA when the
final products were lyophilized to dryness. Consequently, final
products 4, 20, 22, 23, 24, and 27-32 likely exist as the
corresponding mono-TFA salts. Final products with more basic
heterocycles such as 21, 25, 26, and 33 were not titrated.
Potentiometric titration of hydrochloric acid salt 6b revealed a ratio
of 1.06:1 ratio of parent/HCl.
6-Bromo[1,2,4]triazolo[1,5-a]pyridine (8a). To a stirred solution
of 2-amino-5-bromopyridine (25.0 g, 145 mmol) in N,N-dimethyl-
formamide (60 mL) was added N,N-dimethylformamide dimethyl
acetal (60.0 mL, 454 mmol). The reaction mixture was heated to
130 °C overnight. After cooling to room temperature, the volatiles
were removed under reduced pressure to afford the desired product
(N′-(5-bromopyridin-2-yl)-N,N-dimethylimidoformamide) as a brown
oil.
To an ice-cooled, stirred solution of the above crude product in
methanol (200 mL) and pyridine (23.0 mL, 290 mmol) was added
hydroxylamine-O-sulfonic acid (22.6 g, 200 mmol). The reaction
mixture was allowed to warm to room temperature and was stirred
overnight. The volatiles were removed under reduced pressure, and
the residue was partitioned between aqueous sodium bicarbonate
solution and ethyl acetate. The aqueous layer was further extracted
with ethyl acetate, and the combined organic layers were washed
sequentially with water (100 mL) and saturated aqueous brine
solution (100 mL), dried over magnesium sulfate, and concentrated
in vacuo to yield 8a as an orange/brown solid, which was used
without further purification (18.8 g, 66% yield over two steps).
(2S,3E)-4-(4-Bromophenyl)but-3-en-2-ol (13). To 1.92 g (48
mmol) of sodium hydride (60% dispersion in mineral oil) in 100
mL of THF at 0 °C was added 8.1 g (42 mmol) of diethyl (2-
oxopropyl)phosphonate, and the resultant mixture was stirred at 0
°C for 30 min. After the slurry became homogeneous, 7.4 g (38
1
LC/MS m/e 197.9/199.9 (M + H)+; H NMR (500 MHz, CDCl3)
δ 8.77 (d, J ) 1.4 Hz, 1H), 8.34 (s, 1H), 7.68 (d, J ) 9.4 Hz, 1H),
7.60 (dd, J ) 9.4, 1.9 Hz).

R-Amino Amide Dipeptidyl Peptidase IV Inhibitor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3621
mmol) of 4-bromobenzaldehyde was added to the mixture and the
resulting solution was stirred for 2h at 0 °C until TLC revealed
complete disappearance of starting material. Next, the reaction
mixture was diluted with 100 mL of water and the resultant mixture
was extracted with three 200-mL portions of diethyl ether. The
organic phases were then combined, washed with two 100 mL
portions of 5% hydrochloric acid solution, two 100-mL portions
of saturated aqueous sodium bicarbonate solution, two 100-mL
portions of saturated aqueous brine, dried over magnesium sulfate,
filtered, and evaporated in vacuo to yield the crude waxy solid.
The crude material was then purified by flash chromatography on
a Biotage system (silica gel, 0 to 15% ethyl acetate/hexanes
gradient) to give 6.93 g (81% yield) of 3E-4-(4-bromophenyl)but-
3-en-2-one as a pale yellow crystalline solid. LC/MS m/e 225.0/
227.0 (M + H)+; 1H NMR (500 MHz, CD3OD) δ 7.54 (d, J ) 8.5
Hz, 1H), 7.45 (d, J ) 16.5 Hz, 1H), 7.42 (d, J ) 9.5 Hz, 1H), 6.71
(d, J ) 16.4 Hz, 1H), 2.39 (s, 3H).
solution precooled to -78 °C. After stirring for 10 min at that
temperature, 55 mL of zinc chloride solution (55 mmol, 1 M in
diethyl ether) was added at -78 °C. The resultant mixture was
stirred at -78 °C for 5 h and then allowed to warm slowly to room
temperature over 3 h. After stirring an additional 2 h at room
temperature, the mixture was quenched with water and 5%
hydrochloric acid (100 mL each). The resultant mixture was then
extracted with three 300-mL portions of ethyl acetate, and the
organic phases were combined and washed sequentially with 5%
hydrochloric acid, saturated aqueous sodium bicarbonate solution,
and saturated aqueous brine (200 mL each). The organic phase was
then dried over magnesium sulfate, filtered, and evaporated in vacuo
to yield the crude acid 16 as a yellow foam. This crude material
was dissolved in 500 mL of 1:1 diethyl ether/methanol and cooled
to 0 °C. Trimethylsilyldiazomethane solution (75 mL, 150 mmol,
2 M in hexanes) was added in portions until a yellow color persisted.
After warming to room temperature, the solution was stirred an
additional 8 h, then concentrated in vacuo. The crude material was
purified by flash chromatography on a Biotage system (silica gel,
0 to 15% ethyl acetate/hexanes gradient) to give the methyl ester
15 as a colorless oil (16.5 g, 88% yield). LC/MS m/e 298.0/300.0
(M - Boc + H)+; 1H NMR (500 MHz, CDCl3) δ 7.44 (d, J ) 8.4
Hz, 2H), 7.08 (d, J ) 8.5 Hz, 2H), 5.68-5.56 (m, 2H), 4.87 (d, J
) 8.6 Hz, 1H), 4.60 (t, J ) 8.0 Hz, 1H), 3.67 (s, 4H), 1.70 (d, J
) 5.5 Hz, 3H), 1.39 (s, 9H).
To 121.5 g (540 mmol) of the above ketone dissolved in 2500
mL of toluene was added 75 mL (7.5 mmol, 1 M in toluene) of
(R)-2-methyl-CBS-oxazaborolidine catalyst, and the resultant mix-
ture was stirred with a mechanical stirrer at ambient temperature
for 20 min. The mixture was cooled to -78 °C, and 92 mL (863
mmol) of catecholborane in 400 mL of toluene was added dropwise
over 60 min. After the addition, the slurry was stirred at -78 °C
for 60 min while slowly turning homogeneous. The solution was
then stirred at -78 °C an additional 16 h (reaction time varies from
4 to 24 h) until TLC revealed complete disappearance of starting
material. Next, the reaction mixture was warmed to room temper-
ature and carefully quenched with 100 mL of water, diluted with
10 L of 1 N NaOH aqueous solution, and the resultant mixture
was extracted (in an extractor) with 8000 mL of diethyl ether. The
organic phases were then combined and washed with two 2500
mL portions of 1 N NaOH aqueous solution, 2000 mL of water,
2000 mL of 1 N aqueous hydrochloric acid, 1000 mL of saturated
aqueous brine, dried over magnesium sulfate, filtered, and evapo-
rated in vacuo to yield the crude yellow solid which was
recrystallized slowly from hexane to give alcohol 13 (110 g, 90%
yield) as pale yellow crystals. Analytical chiral HPLC (AD column,
2% ethanol/heptane) indicated that this product was 96% ee (S
enantiomer is faster eluting). An additional recrystallization in
cyclohexane afforded the S enantiomer 13 as colorless crystals in
98.2% ee (87.3 g, 71% overall yield). LC/MS m/e 209.0/211.0 (M
To a solution of 25.0 g (62.8 mmol) of methyl ester 15 in 600
mL of tetrahydrofuran (THF) were added in succession 200 mL of
methanol and 200 mL (200 mmol) of 1 N aqueous sodium
hydroxide solution. The reaction mixture was stirred at ambient
temperature for 3 h, and then the methanol and THF were removed
under reduced pressure. To the aqueous mixture was added 250
mL of 1 N hydrochloric acid, and the mixture was extracted with
ethyl acetate (3 × 300 mL). The combined organic extracts were
washed with brine (300 mL) then dried over sodium sulfate, filtered,
and concentrated in vacuo to afford the carboxylic acid 16 (24.1 g,
100% yield) which was used without further purification. LC/MS
1
m/e 384.1.386.1 (M + H)+; H NMR (500 MHz, CDCl3) δ 7.45
(d, J ) 8.5 Hz, 2H), 7.12 (d, J ) 8.5 Hz, 2H), 5.72-5.60 (m, 2H),
4.92 (d, J ) 8.9 Hz, 1H), 4.64 (t, J ) 7.8 Hz, 1H), 3.76 (t, J ) 7.0
Hz, 1H), 1.71 (d, J ) 5.7 Hz, 3H), 1.41 (s, 9H).
General Procedure for the Synthesis of Bromides 18a-c. Step
A. Synthesis of 17a-c. To a solution of acid 16 and cyclic amine
R2NH (3-fluoroazetidine,13 pyrrolidine, or (S)-3-fluoropyrrolidine14)
in DMF were added HOBt, N,N-diisopropylethylamine (DIPEA),
and EDC. The solution was stirred at ambient temperature under
nitrogen for 12-18 h, then ethyl acetate was added and the mixture
was washed sequentially with saturated aqueous sodium bicarbonate
solution, 1 N hydrochloric acid, and brine. Next, the solution was
dried over sodium or magnesium sulfate, filtered, concentrated in
vacuo, and purified by flash chromatography using a Biotage
Horizon system (silica gel, 100% hexanes to 100% ethyl acetate
gradient) to afford amides 17a-c.
Step B. Synthesis of Dimethyl Amides (18a-c). A round-
bottom flask with water and sodium periodate was stirred until
homogeneous then potassium permanganate was added to the
mixture. To this dark purple solution were added potassium
carbonate powder and olefins 17a-c in a tert-butyl alcohol solution.
The reaction mixture was stirred at ambient temperature for 12-
24 h, then treated with saturated aqueous sodium sulfite solution,
acidified with 1 N aqueous hydrochloric acid, and extracted with
ethyl acetate. The combined organic extracts were washed with
brine, and the resultant clear solution was dried over sodium or
magnesium sulfate, filtered, and concentrated in vacuo to afford
the crude acids which were used without further purification.
To a solution of the above acids in DMF was added HOBt,
DIPEA, 2 N dimethylamine (in THF), and EDC. The reaction
mixture was then stirred at ambient temperature for 12-18 h. Ethyl
acetate was then added and the mixture was washed with saturated
aqueous sodium bicarbonate solution, 1 N aqueous hydrochloric
acid, and brine, dried over sodium or magnesium sulfate, filtered
and concentrated in vacuo. Purification by flash chromatography
1
- H2O + 1)+; H NMR (500 MHz, CD3OD) δ 7.46 (d, J ) 8.5
Hz, 2H), 7.26 (d, J ) 8.5 Hz, 2H), 6.53 (d, J ) 15.8 Hz, 1H), 6.28
(dd, J ) 16.0, 6.2 Hz, 1H), 4.51 (m, 1H), 1.72 (s, 1H), 1.40 (d, J
) 6.4 Hz, 3H).
(1S,2E)-3-(4-Bromophenyl)-1-methylprop-2-en-1-yl N-(tert-
Butoxycarbonyl)glycinate (14). To 12.6 g (55 mmol) of alcohol
13 dissolved in anhydrous dichloromethane (300 mL) were added
EDC (23 g, 120 mmol), HOBt (16 g, 120 mmol), N-(tert-
butoxycarbonyl)glycine (21 g, 120 mmol), and N,N′-diisopropyl-
ethylamine (19 mL, 120 mmol). After 5 h, the mixture was
concentrated and diluted with 200 mL of 10% aqueous hydrochloric
acid. The resultant mixture was then extracted with three 300-mL
portions of diethyl ether, and the organic phases were combined
and washed sequentially with 5% hydrochloric acid, saturated
aqueous sodium bicarbonate solution, and saturated aqueous brine
(100 mL each). The organic phase was then dried over magnesium
sulfate, filtered, and evaporated in vacuo to yield the crude material
as a viscous oil. The crude material was purified by flash
chromatography on a Biotage system (silica gel, 0 to 20% ethyl
acetate/hexanes gradient) to give ester 14 (18.1 g, 86% yield) as a
colorless crystalline solid. LC/MS m/e 328.1/330.1 (M - tBu +
1
1)+; H NMR (500 MHz, CD3OD) δ 7.43 (d, J ) 8.2 Hz, 2H),
7.23 (d, J ) 8.4 Hz, 2H), 6.55 (d, J ) 15.8 Hz, 1H), 16.16 (dd, J
) 15.9, 6.7 Hz, 1H), 5.57 (m, 1H), 5.12 (s, 1H), 3.97-3.88 (m,
2H), 1.45 (s, 9H), 1.43 (d, J ) 6.4 Hz, 2H).
(âS)-4-Bromo-N-(tert-butoxycarbonyl)-â-[(1E)-prop-1-en-1-
yl]-L-phenylalanine (16). Ester 14 (18.1 g, 47 mmol) in anhydrous
tetrahydrofuran (50 mL) was added via cannula to 105 mL (105
mmol, 1 M in tetrahydrofuran) of lithium hexamethyldisilazide

3622 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Edmondson et al.
using a Biotage Horizon system (silica gel, 1:1 ethyl acetate/hexanes
to 100% ethyl acetate to 10% methanol/ethyl acetate gradient)
afforded the products 18a-c.
for C22H27FN4O3 (M + H)+ m/e 415.2145, found m/e 415.2150.
1H NMR (500 MHz, CD3OD) δ 8.03 (d, J ) 2.6 Hz, 1H), 7.91
(dd, J ) 9.4, 2.8 Hz), 7.66 (d, J ) 8 Hz), 7.48 (m, 1H), 6.66 (d,
J ) 9.3 Hz, 1H), 5.40 (m, 1H), 4.66 (dd, J ) 24.5, 8 Hz, 1H), 4.54
(dd, J ) 14.2, 8 Hz, 1H), 4.38 (dt, J ) 31.8, 11.5, 9.0 Hz, 1 H),
3.85 (m, 2H), 3.66 (s, 3H), 3.55, (m, 1H), 2.93 (dd, J ) 12.3, 4.7
Hz, 6H), 2.25 (m, 2H).
(2S,3S)-3-Amino-4-[(3S)-3-fluoropyrrolidin-1-yl]-2-(4-imidazo-
[1,2-a]pyridin-6-ylphenyl)-N,N-dimethyl-4-oxobutanamide, TFA
Salt (21). Starting from bromide 18c and 6-iodoimidazo[1,2-a]-
pyridine, the procedures summarized above provided compound
21. 98.8% purity by HPLC (Method B, tr ) 5.90 min); LC/MS
m/e 424.2 (M + H)+; HRMS (ES+) calcd for C23H26FN5O2 (M +
H)+ m/e 424.2149, found m/e 424.2169. 1H NMR (500 MHz, CD3-
OD) δ 9.14 (s, 1H), 8.29 (d, J ) 1.4 Hz, 1H), 8.28 (d, J ) 2.1 Hz,
1H), 8.09 (d, J ) 2.1 Hz, 1H), 8.04 (d, J ) 9.4 Hz, 1H), 7.85 (d,
J ) 8.2 Hz, 2H), 7.62 (d, J ) 8.2 Hz, 2H), 5.32 (dd, J ) 52.7,
33.7 Hz, 1H), 4.72 (dd, J ) 25.6, 8.4 Hz, 1H), 4.62 (dd, J ) 13.6,
8.4 Hz, 1H), 4.44-4.33 (m, 1H), 3.94-3.79 (m, 2H), 3.67-3.49
(m, 1H), 2.97 (d, J ) 7.1 Hz, 3H), 2.92 (d, J ) 3.2 Hz, 3H), 2.42-
2.16 (m, 2H).
(2S,3S)-3-N-(tert-butoxycarbonyl)amino-2-(4-bromophenyl)-
4-(3-fluoroazetidin-1-yl)-N,N-dimethyl-4-oxobutanamide (18a).
Starting from 3-fluoroazetidine12 and acid 16, the procedures above
provided compound 18a. LC/MS m/e 372.1/374.1 (M - Boc +
1
H)+; H NMR (500 MHz, CDCl3) δ 7.47 (d, J ) 8.2 Hz, 2H),
7.26-7.22 (m, 2H), 5.42-5.30 (br d, J ) 54.2 Hz, 1H), 4.93-
4.60 (m, 3H), 4.47-4.07 (m, 4H), 2.92-2.90 (m, 6H), 1.24 (s,
9H).
(2S,3S)-3-N-(tert-butoxycarbonyl)amino-2-(4-bromophenyl)-
4-N,N-dimethyl-4-oxo-4-pyrrolidin-1-ylbutanamide (18b). Start-
ing from pyrrolidine and acid 16, the procedures above provided
compound 18b. LC/MS m/e 468.1/470.1 (M + H)+; 1H NMR (500
MHz, CDCl3) δ 7.48 (d, J ) 8.2 Hz, 2H), 7.27 (d, J ) 8.4 Hz,
2H), 5.14 (t, J ) 10.5 Hz, 1H), 4.98 (d, J ) 10.5 Hz, 1H), 4.23 (d,
J ) 10.3 Hz, 1H), 4.02-3.99 (m, 1H), 3.74-3.70 (m, 1H), 3.53-
3.45 (m, 2H), 2.92 (s, 3H), 2.90 (s, 3H), 2.08-1.88 (m, 2H), 1.22
(s, 9H).
(2S,3S)-3-N-(tert-Butoxycarbonyl)amino-2-(4-bromophenyl)-
4-[(3S)-3-fluoropyrrolidin-1-yl]-N,N-dimethyl-4-oxobutana-
mide (18c). Starting from (S)-3-fluoropyrrolidine13 and acid 16,
the procedures above provided compound 18c. LC/MS m/e 486.2/
(2S,3S)-3-Amino-4-[(3S)-3-fluoropyrrolidin-1-yl]-N,N-dimeth-
yl-4-oxo-2-(4-[1,2,4]triazolo[4,3-a]pyridin-6-ylphenyl)butana-
mide, TFA Salt (22). Starting from bromide 18c and 6-bromo-
[1,2,4]triazolo[4,3-a]pyridine,33 the procedures summarized above
provided compound 22. 99.2% purity by HPLC (Method B, tr )
4.35 min); LC/MS m/e 425.2 (M + H)+; HRMS (ES+) calcd for
1
488.2 (M + H)+; H NMR (500 MHz, CDCl3) δ 7.43 (d, J ) 7.5
Hz, 2H), 7.29-7.24 (m, 2H), 5.34-4.85 (m, 3H), 4.36-4.09 (m,
2H), 3.89-3.45 (m, 3H), 2.88 (d, J ) 13.0 Hz, 3H), 2.86 (s, 3H),
2.34-2.03 (m, 2H), 1.18 (s, 9H).
1
C22H26FN6O2 (M + H)+ m/e 425.2101, found m/e 425.2114. H
NMR (500 MHz, CD3OD) δ 9.45 (s, 1H), 9.06 (s, 1H), 8.26 (d, J
) 9.2 Hz, 1H), 8.08 (d, J ) 9.2 Hz, 1H), 7.83 (d, J ) 8.0 Hz, 2H),
7.62 (d, J ) 8.0 Hz, 2H), 5.39 (dd, J ) 53.5, 34.6 Hz, 1H), 4.73
(dd, J ) 26.3, 8.3 Hz, 1H), 4.62 (dd, J ) 13.2, 8.3 Hz, 1H), 4.41-
4.34 (m, 1H), 3.96-3.80 (m, 2H), 3.67-3.48 (m, 1H), 2.97 (d, J
) 7.8 Hz, 3H), 2.92 (d, J ) 2.3 Hz, 3H), 2.41-2.08 (m, 2H).
(2S,3S)-3-Amino-4-[(3S)-3-fluoropyrrolidin-1-yl]-N,N-dimeth-
yl-4-oxo-2-(4-[1,2,4]triazolo[1,5-a]pyridin-6-ylphenyl)butana-
mide, TFA Salt (23). Starting from bromide 18c and intermediate
8a, the procedures summarized above provided compound 23.
97.7% purity by HPLC (Method B, tr ) 5.86 min); LC/MS m/e
425.3 (M + H)+; HRMS (ES+) calcd for C22H26FN6O2 (M + H)+
m/e 425.2101, found m/e 425.2106. 1H NMR (500 MHz, CD3OD)
δ 9.10 (s, 1H), 8.48 (s, 1H), 8.03 (dt, J ) 9.2, 2.1 Hz, 1H), 7.88
(d, J ) 9.3 Hz, 1H), 7.82 (dd, J ) 8.2, 2.7 Hz, 2H), 7.57 (dd, J )
8.5, 3.7 Hz, 2H), 5.40 (dd, J ) 53.2, 37.8 Hz, 1H), 4.71 (dd, J )
24.3, 8.3 Hz, 1H), 4.61 (dd, J ) 14.4, 8.2 Hz, 1H), 4.44-4.34 (m,
1H), 3.95-3.79 (m, 2H), 3.67-3.50 (m, 1H), 2.97 (d, J ) 5.3 Hz,
3H), 2.93 (d, J ) 3.4 Hz, 3H), 2.45-2.15 (m, 2H).
(2S,3S)-3-Amino-4-[(3S)-3-fluoropyrrolidin-1-yl]-N,N-dimeth-
yl-4-oxo-2-(4-[1,2,4]triazolo[1,5-a]pyridin-7-ylphenyl)butana-
mide, TFA Salt (24). Starting from bromide 18c and intermediate
11, the procedures summarized above provided compound 24.
99.7% purity by HPLC (Method B, tr ) 5.86 min); LC/MS m/e
425.3 (M + H)+; HRMS (ES+) calcd for C22H26FN6O2 (M + H)+
m/e 425.2101, found m/e 425.2117. 1H NMR (500 MHz, CD3OD)
δ 8.92 (d, J ) 7.3 Hz, 1H), 8.57 (s, 1H), 8.08 (s, 1H), 7.92 (d, J
) 8.2 Hz, 2H), 7.63 (d, J ) 7.3 Hz, 1H), 7.60 (dd, J ) 8.4, 2.0
Hz, 2H), 5.40 (dd, J ) 53.4, 35.1 Hz, 1H), 4.75-4.33 (m, 3H),
3.93-3.31 (m, 3H), 2.97 (d, J ) 6.2 Hz, 3H), 2.93 (d, J ) 3.6 Hz,
3H), 2.45-2.07 (m, 2H).
General Procedure for the Synthesis of Boronates 19a-c. To
bromides 18a-c in dimethyl sulfoxide (DMSO) was added bis-
(pinacolato)diboron,
[1,1′-bis(diphenylphosphino)ferrocene]-
dichloropalladium(II) (complex with dichloromethane (1:1)), and
potassium acetate. Nitrogen was then bubbled through the mixture
for 3 min, then the mixture was stirred at 80 °C under nitrogen for
4-12h. The mixture was cooled to ambient temperature, filtered
through a silica gel pad, and rinsed with excess ethyl acetate. The
solution was washed with two portions of brine, dried over sodium
or magnesium sulfate, filtered, and concentrated in vacuo. Purifica-
tion by flash chromatography on a Biotage Horizon system (silica
gel, 40% ethyl acetate/hexanes to 100% ethyl acetate to 20%
methanol/ethyl acetate gradient) afforded the boronates 19a-c as
dark yellow/brown foamy solids.
General Procedure for the Synthesis of Compounds 20-33.
Step A. Suzuki Coupling of Boronates 18a-c. To boronates
19a-c in ethanol/toluene (1:1) were added ArX (5-bromo-1-
methylpyridin-2(1H)-one,32 6-iodoimidazo[1,2-a]pyridine, 6-bromo-
[1,2,4]triazolo[4,3-a]pyridine,33 8a-d, 11, 5-chloropyrazolo[1,5-
a]pyrimidine,34 or 6-chloroimidazo[1,2-b]pyridizine35), [1,1′-
bis(diphenylphosphino) ferrocene]dichloro-palladium(II) (complex
with dichloromethane,1:1), and 2 N aqueous sodium carbonate
solution. The reaction mixture was stirred at 90 °C under nitrogen
for 8-14 h. After cooling to ambient temperature, ethyl acetate
was added to the mixture and the organic phase was washed
sequentially with 0.5 N aqueous sodium bicarbonate solution and
brine, dried over sodium or magnesium sulfate, filtered, and con-
centrated in vacuo. The crude material was purified by reverse phase
HPLC (YMC Pro-C18 column, gradient elution, 10 to 90% aceto-
nitrile/water with 0.1% TFA) to afford the pure coupled products.
Step B. General Procedure for tert-Butyloxycarbonyl (Boc)
Deprotection. The above coupled products were dissolved in a 1:1
mixture of dichloromethane and TFA, stirred for 30-90 min at
room temperature, then concentrated in vacuo. Purification by
reverse phase HPLC (YMC Pro-C18 column, gradient elution, 10
to 90% acetonitrile/water with 0.1% TFA) then afforded the pure
final products 20-33.
(2S,3S)-3-Amino-4-[(3S)-3-fluoropyrrolidin-1-yl]-N,N-dimeth-
yl-4-oxo-2-(4-pyrazolo[1,5-a]pyrimidin-5-ylphenyl)butana-
mide, TFA Salt (25). Starting from bromide 18c and 5-chloropyr-
azolo[1,5-a]pyrimidine,34 the procedures summarized above pro-
vided compound 25. 98.8% purity by HPLC (Method B, tr ) 7.13
min); LC/MS m/e 425.3 (M + H)+; HRMS (ES+) calcd for C22H26-
1
(2S,3S)-3-Amino-4-[(3S)-3-fluoropyrrolidin-1-yl]-N,N-dimeth-
yl-2-[4-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-4-oxo-
butanamide, TFA Salt (20). Starting from bromide 18c and
5-bromo-1-methylpyridin-2(1H)-one,32 the procedures summarized
above provided compound 20. 100% purity by HPLC (Method B,
tr ) 4.85 min); LC/MS m/e 415.6 (M + H)+; HRMS (ES+) calcd
FN6O2 (M + H)+ m/e 425.2101, found m/e 425.2114. H NMR
(500 MHz, CD3OD) δ 8.91 (d, J ) 7.6 Hz, 1H), 8.21-8.18 (m,
3H), 7.58 (d, J ) 7.7 Hz, 2H), 7.51 (d, J ) 7.3 Hz, 1H), 6.72 (d,
J ) 2.1 Hz, 1H), 5.41 (dd, J ) 52.2, 34.1 Hz, 1H), 4.74 (dd, J )
26.8, 8.0 Hz, 1H), 4.63 (dd, J ) 16.0, 8.1 Hz, 1H), 4.45-4.34 (m,
1H), 3.97-3.51 (m, 3H), 2.97-2.93 (m, 6H), 2.43-2.30 (m, 2H).

R-Amino Amide Dipeptidyl Peptidase IV Inhibitor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3623
(2S,3S)-3-Amino-4-[(3S)-3-fluoropyrrolidin-1-yl]-2-(4-imidazo-
[1,2-b]pyridazin-6-ylphenyl)-N,N-dimethyl-4-oxobutanamide, TFA
Salt (26). Starting from bromide 18c and 6-chloroimidazo[1,2-b]-
pyridizine,35 the procedures summarized above provided compound
26. LC/MS m/e 425.4 (M + H)+; HRMS (ES+) calcd for C22H26-
1H), 4.61 (d, J ) 7.7 Hz, 1 H), 4.03-3.98 (m, 1H), 3.76-3.71 (m,
1H), 3.56-3.46 (m, 2H), 2.97 (s, 3H), 2.94 (s, 3H), 2.15-1.96
(m, 4H).
(2S,3S)-3-Amino-4-(3,3-difluoropyrrolidin-1-yl)-N,N-dimeth-
yl-4-oxo-2-(4-[1,2,4]triazolo[1,5-a]pyridin-7-ylphenyl)butana-
mide, TFA Salt (32). Starting from bromide 18d and intermediate
11, the procedures summarized above provided compound 32.
95.2% purity by HPLC (Method B, tr ) 6.51 min); LC/MS m/e
443.2 (M + H)+; HRMS (ES+) calcd for C22H25F2N6O2 (M + H)+
m/e 443.2002, found m/e 443.2031. 1H NMR (500 MHz, CD3OD)
δ 9.02 (d, J ) 7.0 Hz, 1H), 8.80 (s, 1H), 8.19 (s, 1H), 7.98 (d, J
) 8.3 Hz, 2H), 7.78 (d, J ) 6.9 Hz, 1H), 7.63 (dd, J ) 8.1, 2.0
Hz, 2H), 4.78-3.72 (series of m, 6H), 2.96 (s, 3H), 2.94 (d, J )
1.5 Hz, 3H), 2.66-2.45 (m, 2H).
(2S,3S)-3-Amino-4-(3,3-difluoropyrrolidin-1-yl)-N,N- -dimeth-
yl-4-oxo-2-(4-pyrazolo[1,5-a]pyrimidin-5-ylphenyl)butana-
mide, TFA Salt (33). Starting from bromide 18d and 6-chloroim-
idazo[1,2-b]pyridizine,35 the procedures summarized above provided
compound 33. High-resolution HPLC purity was not evaluated, but
the sample was >95% pure by LC/MS HPLC method). LC/MS
m/e 443.3 (M + H)+. 1H NMR (500 MHz, CD3OD) δ 8.95 (d, J )
7.4 Hz, 1H), 8.26 (d, J ) 8.2 Hz, 2H), 8.19 (d, J ) 2.3 Hz, 1H),
7.59 (d, J ) 8.3 Hz, 2H), 7.57 (d, J ) 7.3 Hz, 1H), 6.73 (t, J ) 1.7
Hz, 1H), 4.75 (d, J ) 8.3 Hz, 1H), 4.64-4.57 (m, 2H), 4.55-3.73
(series of m, 4H), 3.32-3.31 (m, 3H), 2.94 (d, J ) 10.7 Hz, 3H),
2.67-2.43 (m, 2H).
(2S,3S,4E)-3-(4-Bromophenyl)-1-(3,3-difluoropyrrolidin-1-yl)-
1-oxohex-4-en-2-N-(tert-butoxycarbonyl)amine (17d). Acid 16
(15 g, 39 mmol) was mixed with 11.2 g (78 mmol) of 3,3-
difluoropyrrolidine,13 10.5 g (78 mmol) of HOBt, 13.7 mL (78
mmol) of N,N-diisopropylethylamine, and 200 mL of DMF. Next,
15 g (78 mmol) of EDC was added, and the solution was stirred at
ambient temperature under nitrogen for 12 h. Ethyl acetate (1.0 L)
was added, and the mixture was washed with 0.5 N aqueous sodium
bicarbonate solution (3 × 400 mL), 1 N hydrochloric acid (2 ×
400 mL), and brine (400 mL), dried over sodium sulfate, filtered,
and concentrated in vacuo to afford 17d (17.7 g, 96% yield), which
was sufficiently pure for use in subsequent steps. LC/MS m/e 375.1
(M - Boc + H)+; 1H NMR (500 MHz, CDCl3) δ 7.48 (d, J ) 8.4
Hz, 2H), 7.15 (d, J ) 8.3 Hz, 2H), 5.72-5.59 (m, 2H), 5.10-5.08
(m, 1H), 4.72-4.53 (m, 1H), 4.03-3.32 (m, 5H), 2.41-2.14 (m,
2H), 1.71 (d, J ) 6.2 Hz, 3H), 1.37 (s, 9H).
1
FN6O2 (M + H)+ m/e 425.2101, found m/e 425.2090. H NMR
(500 MHz, CD3OD) δ 8.46 (s, 1H), 8.38 (d, J ) 9.8 Hz, 1H), 8.24-
8.20 (m, 3H), 8.11 (s, 1H), 7.65 (d, J ) 8.0 Hz, 1H), 5.41 (dd, J
) 52.7, 34.4, 1H), 4.73 (dd, J ) 24.7, 8.2 Hz, 1H), 4.64 (dd, J )
14.1, 8.2 Hz, 1H), 4.45-4.34 (m, 1H), 3.93-3.79 (m, 2H), 3.68-
3.51 (m, 1H), 3.34-3.32 (m, 3H), 2.98-2.93 (m 3H), 2.46-2.04
(m, 2H).
(2S,3S)-3-Amino-4-[(3S)-3-fluoropyrrolidin-1-yl]-N,N-dimeth-
yl-2-[4-(2-methyl[1,2,4]triazolo[1,5-a]pyridin-6-yl)phenyl]-4-oxo-
butanamide, TFA Salt (27). Starting from bromide 18c and
intermediate 8d, the procedures summarized above provided
compound 27. 99.6% purity by HPLC (Method A, tr ) 0.376 min);
LC/MS m/e 439.2 (M + H)+; HRMS (ES+) calcd for C23H28FN6O2
(M + H)+ m/e 439.2252, found m/e 439.2257. 1H NMR (500 MHz,
CD3OD) δ 9.04 (d, J ) 0.7 Hz, 1H), 8.07 (dt, J ) 9.2, 1.6 Hz,
1H), 7.84-7.81 (m, 3H), 7.58-7.55 (m, 2H), 5.40 (dd, J ) 52.6,
34.5, 1H), 4.70 (dd, J ) 24.5, 8.2 Hz, 1H), 4.59 (dd, J ) 14.4, 8.0
Hz, 1H), 4.44-4.33 (m, 1H), 3.92-3.78 (m, 2H), 3.67-3.50 (m,
1H), 2.96 (d, J ) 5.2 Hz, 3H), 2.93 (d, J ) 5.1 Hz, 3H), 2.61 (s,
3H), 2.43-2.06 (m, 2H).
(2S,3S)-3-Amino-4-[(3S)-3-fluoropyrrolidin-1-yl]-N,N-dimeth-
yl-2-[4-(5-methyl[1,2,4]triazolo[1,5-a]pyridin-6-yl)phenyl]-4-oxo-
butanamide, TFA Salt (28). Starting from bromide 18c and
intermediate 8b, the procedures summarized above provided
compound 28. 99.8% purity by HPLC (Method B, tr ) 7.04 min);
LC/MS m/e 439.3 (M + H)+; HRMS (ES+) calcd for C23H28FN6O2
(M + H)+ m/e 439.2258, found m/e 439.2266. 1H NMR (500 MHz,
CD3OD) δ 8.58 (d, J ) 1.4 Hz, 1H), 7.80 (d, J ) 8.9 Hz, 1H),
7.74 (d, 9.1 Hz, 1H), 7.59 (d, J ) 1.8 Hz, 4H), 5.42 (dd, J ) 52.6,
39.9 Hz, 1H), 4.73 (dd, J ) 24.4, 8.1 Hz, 1H), 4.63 (dd, J ) 13.9,
8.1 Hz, 1H), 4.46-4.35 (m, 1H), 3.95-3.81 (m, 2H), 3.69-3.52
(m, 1H), 3.01 (d, J ) 5.3 Hz, 3H), 2.96 (d, 3.9 Hz, 3H), 2.83 (s,
3H), 2.44-2.28 (m, 2 H).
(2S,3S)-3-Amino-4-[(3S)-3-fluoropyrrolidin-1-yl]-N,N-dimeth-
yl-2-[4-(8-methyl[1,2,4]triazolo[1,5-a]pyridin-6-yl)phenyl]-4-oxo-
butanamide, TFA Salt (29). Starting from bromide 18c and
intermediate 8c, the procedures summarized above provided
compound 29. 98.5% purity by HPLC (Method A, tr ) 0.441 min);
LC/MS m/e 439.2 (M + H)+; HRMS (ES+) calcd for C23H28FN6O2
(M + H)+m/e 439.2252, found m/e 439.2260. 1H NMR (500 MHz,
CD3OD) δ 8.97 (s, 1H), 8.49 (s, 1H), 7.87-7.84 (m, 4H), 7.59-
7.56 (m, 3H), 5.42 (dd, J ) 52.6, 34.6 Hz, 1H), 4.71 (dd, J )
24.2, 8.2 Hz, 1H), 4.60 (dd, J ) 14.6, 8.0 Hz, 1H) 4.42-4.35 (m,
1H), 3.94-3.80 (m, 2H), 3.69-3.55 (m, 1H), 2.98 (d, J ) 5 Hz,
3H), 2.95 (d, J ) 4.4 Hz, 3H), 2.72 (s, 3H), 2.44-2.31 (m, 2H).
(2S,3S)-3-Amino-4-(3-fluoroazetidin-1-yl)-N,N-dimethyl-4-
oxo-2-(4-[1,2,4]triazolo[1,5-a]pyridin-6-ylphenyl)butanamide, TFA
Salt (30). Starting from bromide 18a and intermediate 8a, the
procedures summarized above provided compound 30. 100% purity
by HPLC (Method B, tr ) 5.63 min); LC/MS m/e 411.2 (M +
H)+; HRMS (ES+) calcd for C21H24FN6O2 (M + H)+ m/e 411.1939,
found m/e 411.1954. 1H NMR (500 MHz, CD3OD) δ 9.13 (s, 1H),
8.50 (s, 1H) 8.07 (dd, J ) 9.3, 1.8 Hz, 1H), 7.91 (dd, J ) 9.3, 0.8
Hz, 1H), 7.86 (d, J ) 8.2 Hz, 2H), 7.60-7.58 (m, 2H), 5.47 (bd,
J ) 57 Hz, 1H), 5.30-4.95 (m, 1H), 4.81-4.74 (m, 1H), 4.57-
4.37 (m, 3H), 4.21-4.14 (m, 1H), 2.98 (d, J ) 5.2 Hz, 3H), 2.96
(d, J ) 6.9 Hz, 3H).
(2S,3S)-N-(tert-Butoxycarbonyl)amino-2-(4-bromophenyl)-4-
(3,3-difluoropyrrolidin-1-yl)-N,N-dimethyl-4-oxobutanamide (18d).
A round-bottom flask was charged with 1.5 L of water, and 80 g
(374 mmol) of sodium periodate was added. The mixture was stirred
until homogeneous then 1.2 g (7.5 mmol) of potassium perman-
ganate was added to the mixture. To this dark purple solution were
added 5.7 g (41.1 mmol) of potassium carbonate powder (∼325
mesh) and 17.7 g (37.4 mmol) of 17d as a 500 mL tert-butyl alcohol
solution. The reaction mixture was stirred at ambient temperature
for 24 h, then treated with 50 mL of saturated aqueous sodium
sulfite solution, acidified with 1 N aqueous hydrochloric acid (400
mL), and extracted with ethyl acetate (3 × 400 mL). The combined
organic extracts were washed with brine (3 × 400 mL), and the
resultant clear solution was dried over sodium sulfate, filtered, and
concentrated in vacuo to afford the crude acid (20.38 g, 100%) as
a colorless crystalline solid which was used without further
1
purification. LC/MS m/e 377.0/379.0 (M + H)+; H NMR (500
MHz, CD3OD) δ 7.48-7.46 (m, 2H), 7.26-7.22 (m, 2H), 7.12 (br
s, 1H), 5.35 (dd, J ) 21.0, 10.4 Hz, 1H), 5.12-4.94 (m, 1H), 4.21-
4.00 (m, 3H), 3.91-3.65 (m, 2H), 2.50-2.33 (m, 2H), 1.24 (d, J
) 4.6 Hz, 9H).
(2S,3S)-3-Amino-N,N-dimethyl-4-oxo-4-pyrrolidin-1-yl-2-(4-
[1,2,4]triazolo[1,5-a]pyridin-6-ylphenyl)butanamide, TFA Salt
(31). Starting from bromide 18b and intermediate 8a, the procedures
summarized above provided compound 31. 96.8% purity by HPLC
(Method A, tr ) 0.423 min); LC/MS m/e 407.3 (M + H)+; HRMS
(ES+) calcd for C22H27N6O2 (M + H)+ m/e 407.2190, found m/e
The above acid (20.38 g, 37.4 mmol) was mixed with 10.1 g
(74.8 mmol) of HOBt and 300 mL of DMF. Next, 13 mL (74.8
mmol) of N,N-diisopropylethylamine, 37.4 mL (74.8 mmol) of 2
N dimethylamine in THF, and 14.3 g (74.8 mmol) of EDC were
then added sequentially to the solution. The reaction mixture was
then stirred at ambient temperature for 12 h. Ethyl acetate (1.2 L)
was then added, and the mixture was washed with 0.5 N aqueous
sodium bicarbonate solution (3 × 400 mL), 1 N aqueous hydro-
1
407.2196. H NMR (500 MHz, CD3OD) δ 9.10 (s, 1H), 8.50 (s,
1H), 8.05 (d, J ) 9.4 Hz, 1H), 7.89 (d, J ) 9.4 Hz, 1H), 7.82 (d,
J ) 8.3 Hz, 2H), 7.56 (d, J ) 8.1 Hz, 2H), 4.71 (d, J ) 7.8 Hz,

3624 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Edmondson et al.
chloric acid (2 × 300 mL), and brine (400 mL), dried over sodium
sulfate, filtered, and concentrated in vacuo. Purification by flash
chromatography using a Biotage Horizon system (silica gel, 1:1
ethyl acetate/hexanes to 100% ethyl acetate to 10% methanol/ethyl
acetate gradient) afforded dimethyl amide 18d as a pale yellow
foamy solid (11.53 g, 59% yield). LC/MS m/e 448.2/450.2 (M -
12.7 mmol) was dissolved in a 1:1 mixture of dichloromethane and
TFA, stirred for 30 min at room temperature, then concentrated in
vacuo. The TFA salt was then dissolved in water, and the aqueous
solution adjusted to pH 9 via addition of 2 N aqueous sodium
carbonate solution. After extracting the aqueous mixture with 3:1
chloroform:2-propanol (5 × 200 mL), the combined organic layers
were washed once with brine, then dried over sodium sulfate,
filtered, and concentrated in vacuo. The resultant free base was
then dissolved in dichloromethane, and 30 mL of 2 N hydrogen
chloride in ether was added to the solution. After stirring for 60
min, the solution was evaporated to afford the title compound as a
white hydrochloride salt. The compound was further purified by
recrystallization from ethanol/ether. Lyophilization of the recrystal-
lized compound from water/acetonitrile (40:60, 100 mL) then
afforded 6b as a fluffy white crystalline solid (3.7 g, 61% yield).
100% purity by HPLC (Method B, tr ) 7.25 min); LC/MS m/e
443.2 (M + H)+; HRMS (ES+) calcd for C22H25F2N6O2 (M + H)+
m/e 443.2007, found m/e 443.2003. 1H NMR (500 MHz, CD3OD)
δ 9.12 (s, 1H), 8.48 (s, 1H), 8.06 (dd, J ) 9.4, 1.6 Hz, 1H), 7.87
(d, J ) 9.3 Hz, 1H), 7.85 (d, J ) 8.0 Hz, 2H), 7.58 (dd, J ) 8.2,
1.6 Hz, 2H),. 4.69 (dd, J ) 57.4, 8.4 Hz, 1H), 4.58 (dd, J ) 13.8,
8.2 Hz, 1H), 4.52-3.74 (m, 6H), 2.96 (s, 3H), 2.94 (d, J ) 1.1 Hz,
3H), 2.69-45 (m, 2H).
X-ray Crystallographic Analysis. DPP-4 (residues 39-766)
was crystallized in the presence of compound 23 following the
reported conditions.22 A 94.8% complete, 5.5-fold redundant X-ray
diffraction data set to 2.4 Å was collected from a single-crystal
cooled to 100 K, using the beamline 17-BM at the Advanced Photon
Source (Argonne, IL). The structure was solved using Molecular
Replacement procedures (MOLREP36) and the 1N1M.pdb coordi-
nate file, without water, ligand, and sugar molecules. The structure
was refined against all available data to 2.4 Å. using CNX
(Accelrys37) to a crystallographic R-factor of 19.4% and an Rfree of
24.0%. The root-mean-square deviation between the model and ideal
bond distances and bond angles are 0.010 Å and 1.46°, respectively.
Coordinates have been deposited with the Protein Data Bank,
accession code 2FJP. Data collection and refinement statistics and
further refinement details are available as Supporting Information.
Oral Glucose Tolerance Test in Lean Mice. Male C57BL/6N
mice (7-12 weeks of age) from Taconic Farms, Germantown, NY
were housed 10 per cage and given access to normal diet rodent
chow (Teklad 7012) and water ad libitum. Mice (n ) 7/group) were
randomly assigned to treatment groups and fasted overnight (∼18-
21 h). Baseline (t ) -60 min) blood glucose concentration was
determined by glucometer from tail nick blood. Animals were then
treated orally with vehicle (0.25% methylcellulose, 5 mL/kg) or
compound 6b (3, 1, 0.3 and 0.1 mg/kg; 5 mL/kg). Blood glucose
concentration was measured 1 h after treatment (t ) 0 min), and
mice were then orally challenged with dextrose (5 g/kg, 10 mL/
kg). One group of vehicle-treated mice was challenged with water
as a negative control. Blood glucose levels were determined from
tail bleeds taken 20, 40, 60, and 120 min after dextrose challenge.
The blood glucose excursion profile from t ) 0 to t ) 120 min
was used to integrate an area under the curve (AUC) for each
treatment. Percent reduction values for each treatment were
generated from the AUC data normalized to the water-challenged
controls.
1
tBu + H)+; H NMR (500 MHz, CDCl3) δ 7.47 (d, J ) 8.3 Hz,
2H), 7.26-7.24 (m, 2H), 5.16-4.94 (m, 1H), 4.79-4.74 (t, J )
10.8 Hz, 1H), 4.36-4.13 (m, 2H), 3.97-3.64 (m, 3H), 2.91 (s,
3H), 2.90 (s, 3H), 2.53-2.36 (m, 2H), 1.22 (d, J ) 3.3 Hz, 9H).
(2S,3S)-3-N-(tert-Butoxycarbonyl)amino-4-(3,3-difluoropyr-
rolidin-1-yl)-N,N-dimethyl-4-oxo-2-[4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenyl]butanamide (19d). To 11.5 g (22.8
mmol) of 18d were added 11.5 (45.6 mmol) of bis(pinacolato)-
diboron, 3.7 g (4.6 mmol) of [1,1′-bis(diphenylphosphino)ferrocene]-
dichloropalladium(II) (complex with dichloromethane (1:1)), 11.2
g (114 mmol) of potassium acetate, and 70 mL of dimethyl
sulfoxide (DMSO). Nitrogen was then bubbled through the mixture
for 3 min, then the mixture was stirred at 80 °C under nitrogen for
4 h. The mixture was cooled to ambient temperature, then filtered
through a silica gel pad and rinsed with excess ethyl acetate. The
solution was washed with two portions of brine, dried over sodium
sulfate, filtered, and concentrated in vacuo. Purification by flash
chromatography on a Biotage Horizon system (silica gel, 40% ethyl
acetate/hexanes to 100% ethyl acetate to 20% methanol/ethyl acetate
gradient) afforded 19d as a yellow foamy solid (10.5 g, 84%). LC/
MS m/e 552.6 (M + H)+; 1H NMR (500 MHz, CDCl3) δ 7.71 (d,
J ) 7.7 Hz, 2H), 7.37 (dd, J ) 7.9, 1.7 Hz, 2H), 5.02 (t, J ) 10.4
Hz, 1H), 4.38 (d, J ) 10.6 Hz, 1H), 4.19-3.58 (m, 4H), 2.95 (s,
3H), 2.90 (s, 3H), 1.35 (s, 12H), 1.18 (d, J ) 5.5 Hz, 9H).
(2S,3S)-3-Amino-4-(3,3-difluoropyrrolidin-1-yl)-N,N-dimeth-
yl-4-oxo-2-(4-[1,2,4]triazolo[1,5,a]pyridin-6-ylphenyl)butana-
mide, TFA Salt (6a). To 9.27 g (16.8 mmol) of 19d in 200 mL of
ethanol/toluene (1:1) were added 6.7 g (33.6 mmol) of 6-bromo-
[1,2,4]triazolo[1,5-a]pyridine 8a, 2.9 g (3.6 mmol) of [1,1′-bis-
(diphenylphosphino) ferrocene]dichloropalladium(II) (complex with
dichloromethane,1:1), and 42 mL (84 mmol) of 2 N aqueous sodium
carbonate solution. The reaction mixture was stirred at 90 °C under
nitrogen for 12 h. After cooling to ambient temperature, 600 mL
of ethyl acetate was added to the mixture and the organic phase
was washed sequentially with 0.5 N aqueous sodium bicarbonate
solution and brine, dried over sodium sulfate, filtered, and
concentrated in vacuo. The crude material was purified by reverse
phase HPLC on a Kiloprep 100 G system (Kromasil C8 16 micron,
isocratic elution, 40% acetonitrile/water with 0.1% TFA) to afford
the coupled product as a colorless solid (6.9 g, 76% yield). LC/
1
MS m/e 543.27 (M + H)+; H NMR (500 MHz, CD3OD) δ 9.05
(s, 1H), 8.46 (s, 1H), 8.01 (d, J ) 9.1 Hz, 1H), 7.87 (d, J ) 9.1
Hz, 1H), 7.73 (d, J ) 7.5 Hz, 2H), 7.55 (d, J ) 7.5 Hz, 2H), 5.03
(dd, J ) 76.7, 10.3 Hz, 1H), 4.46 (d, J ) 10.5 Hz, 1H), 4.23-3.61
(m, 4H), 3.02 (s, 3H), 2.92 (s, 3H), 2.58-2.42 (m, 2H), 1.20 (d, J
) 5.0 Hz, 9H).
The coupled product was then dissolved in a 1:1 mixture of
dichloromethane and TFA, stirred for 30 min at room temperature,
then concentrated in vacuo. The product was purified by reverse
phase HPLC on a Kiloprep 100 G system (Kromasil C8 16 micron,
gradient elution, 0% to 65% acetonitrile/water with 0.1% TFA) to
afford the product 6a as a colorless crystalline TFA salt (3.7 g,
60% yield). 100% purity by HPLC (Method A, tr ) 0.462 min);
LC/MS m/e 443.5 (M + H)+; HRMS (ES+) calcd for C22H25F2N6O2
(M + H)+ m/e 443.2007, found m/e 443.2027. 1H NMR (500 MHz,
CD3OD) δ 9.12 (s, 1H), 8.48 (s, 1H), 8.06 (dd, J ) 9.4, 1.5 Hz,
1H), 7.89 (d, J ) 9.3 Hz, 1H), 7.85 (d, J ) 8.0 Hz, 2H), 7.58 (dd,
J ) 8.2, 1.6 Hz, 2H), 4.69 (dd, J ) 57.4, 8.4 Hz, 1H), 4.58 (dd, J
) 13.8 Hz, 8.4 Hz, 1H), 4.52-3.74 (m, 6H), 2.96 (s, 3H), 2.94 (d,
J ) 1.1 Hz, 3H), 2.82-2.44 (m, 2H).
Lean Mouse Pharmacodynamic Assay. Male C57BL/6N mice
(7-12 weeks of age, 19-26 g) from Taconic Farms, Germantown,
NY, were housed 10 per cage and given access to normal diet rodent
chow (Teklad 7012) and water ad libitum. Mice (n ) 20-28/group)
were randomly assigned to treatment groups and fasted overnight
(∼18-21 h). Baseline (t ) -60 min) blood glucose concentration
was determined by glucometer from tail nick blood. Animals were
then treated orally with vehicle (0.25% methylcellulose, 5 mL/kg)
or compound 6b (3, 1, 0.3, and 0.1 mg/kg; 5 mL/kg) and the blood
glucose concentration determined 1 h after treatment (t ) 0 min).
After the blood glucose determination at t ) 0, mice were orally
challenged with dextrose (5 g/kg, 10 mL/kg). One group of vehicle-
treated mice was challenged with water as a negative control. Blood
glucose levels were determined from tail bleeds taken 20 min after
(2S,3S)-3-Amino-4-(3,3-difluoropyrrolidin-1-yl)-N,N-dimeth-
yl-4-oxo-2-(4-[1,2,4]triazolo[1,5,a]pyridin-6-ylphenyl)butana-
mide Hydrochloride (6b). The tert-butyl [(1S,2S)-1-[(3,3-difluo-
ropyrrolidine-1-yl)carbonyl]-3-(dimethylamino)-3-oxo-2-(4-
[1,2,4]triazolo[1,5-a]pyridine-6-phenyl)propyl]carbamate (6.9 g,

R-Amino Amide Dipeptidyl Peptidase IV Inhibitor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3625
dextrose challenge. Mice were then immediately euthanized and
terminal blood samples collected by cardiac puncture. Blood
samples were collected into EDTA, and the plasma was harvested
by centrifugation. Aliquots of plasma samples were stored at -70
°C until analysis.
disturbance of samples. Four aliquots (200 or 100 µL) were taken
sequentially from the top of the vial using narrow pipet tips and
assayed for total radioactivity. The protein pellet (bottom layer)
was digested by incubating with 8 M urea or 0.01 N NaOH and
analyzed for total radioactivity by liquid scintillation counting. The
unbound fraction was determined from the ratio of radioactivity in
the protein free fraction to that in plasma prior to centrifugation.
Recoveries were calculated by adding the total radioactivity
measured in the different layers, including the protein pellet, and
comparing that value to the radioactivity present in plasma before
incubation.
Measurement of Plasma DPP-4 Activity.29 Plasma DPP-4
activity was measured using a continuous fluorometric assay with
the substrate Gly-Pro-AMC, which is cleaved by DPP-4 to release
the fluorescent AMC leaving group. A typical reaction contains
50% plasma, 50 µM Gly-Pro-AMC, and buffer (100 mM HEPES,
pH 7.5, 0.1 mg/mL BSA) in a total reaction volume of 50 to 70
µL (depending on the availability of sample). Liberation of AMC
was monitored continuously in a 96-well plate fluorometer (Spec-
tramMAX Gemini, Molecular Devices), using an excitation wave-
length of 360 nm and an emission wavelength of 460 nm. Under
these conditions, approximately 5 µM AMC is produced in 5 min
at 37 °C. The plate reader used was a SpectramMAX Gemini
(Molecular Devices). The assay exhibits linear rates only for about
5 min due to the rapid substrate depletion. Therefore, it was
important to preincubate all assay components to the assay
temperature prior to the assay. The data are reported as % inhibition
calculated as follows: %Inhibition ) 100 (1 - (Vt/Vc)), where Vt
is the rate of reaction of treated sample and Vc is the rate of reaction
of control sample.
Measurement of Plasma active (intact) GLP-1. Plasma intact
GLP-1 was measured using a 96-well ELISA kit for active hormone,
purchased from Linco Research Inc (St. Charles, MO, cat. # EGLP-
35K). The assay has a detection limit of 2 pM and is selective for
active GLP-1 (GLP-1[7-36] amide and GLP-1[7-37]). The DPP-4
inhibitor valine thiazolidide (100 µM) was added to plasma aliquots
for active GLP-1 measurements to prevent degradation of the
hormone.
Diltiazem/Ca+2 L-type Channel Binding Assay. L-type Ca2+
binding was performed following a published procedure27 using
membranes prepared from rabbit skeletal muscle. Compounds were
3
serially diluted in DMSO then mixed with H-diltiazem (20 nM
final concentration, NEN) and 40-70 µg membranes in assay buffer
(50 mM Tris, pH 7.4). The assay was incubated for 60 min at room
temperature with shaking. Following incubation, the membranes
were harvested using a Packard Filtermate Harvester and GF/C filter
plates that had been presoaked in 0.3% polyethylenimine (PEI).
The plates were then washed three times with ice-cold assay buffer.
Plates were then air-dried and counted in a Packard TopCounter.
Total binding was determined in wells with DMSO only, and
nonspecific binding was determined in the presence of 20 µM
unlabeled diltiazem. Data analysis was performed using Micorsoft
Excel and GraphPad Prism software.
Cardiac Type-2 Na+2 Channel Binding Assay. Inhibition of
binding to the human cardiac Na+ Channel (hNav 1.5, hH1a) was
performed using membranes made from HEK293 cells stably
expressing the channel as described in the literature.28 Compounds
were serially diluted in DMSO and added to ligand (3H-BPBTS,
1 nM final concentration, MRL Compound Synthesis) and 40-80
µg of membranes in assay buffer (20 mM Tris-HCl, pH 7.4; 0.01
mg/mL Bacitracin). The assay was then incubated overnight (16
h) at room temperature with shaking. Following the incubation, a
Packard Filtermate Harvester and GF/B filter plates (presoaked in
0.3% PEI) were used to harvest the membranes. The plates washed
three times with ice-cold wash buffer (20 mM Tris, pH 7.4; 150
mM NaCl; 0.05% Triton X-100). The filter plates were then air-
dried and counted in a Packard TopCount scintillation counter. Total
binding was determined in wells with DMSO only, and nonspecific
binding was determined in the presence of 5 µM unlabeled ligand.
Data analysis was performed using Micorsoft Excel and GraphPad
Prism software.
Determination of Plasma Concentration of Compound 6.
Plasma concentrations of compound 6 were determined 20 min after
dextrose challenge and 80 min postcompound administration by
liquid chromatography/tandem mass spectrometry. Plasma was
prepared for MS analysis by solid-phase extraction using OASIS-
HLB 96-well extraction plates. The typical limit of quantitation
was 10 nM.
Oral Glucose Tolerance Test in Diet Induced Obese (DIO)
Mice. Male C57BL/6N mice were purchased from Taconic Farms,
Germantown, NY. At 5 weeks of age they were placed on a high
fat diet F-3282 (35% fat by weight) supplied by BioServ, NJ, for
27 weeks. An age-matched cohort of mice was fed a normal diet
rodent chow (Teklad 7012) to provide a lean control group. All
mice were given access to food and water ad libitum. At
approximately 7 months of age, mice were used for the following
OGTT. DIO animals were randomly assigned to treatment groups.
The DIO (45-55 g) and lean mice (28-32 g) (n ) 7-8/group)
were fasted overnight (18-21 h), and baseline (t ) -60 min) blood
glucose concentration was determined by glucometer from tail nick
blood. DIO animals were then treated orally with vehicle (0.25%
methylcellulose, 5 mL/kg) or compound 6b (30, 3, 0.3 mg/kg; 5
mL/kg). Lean control mice received only vehicle. Blood glucose
concentration was measured 1 h after treatment (t ) 0 min), and
mice were then orally challenged with dextrose (2 g/kg, 10 mL/
kg). Blood glucose levels were determined from tail bleeds taken
20, 40, 60, and 120 min after dextrose challenge. The blood glucose
excursion profile from t ) 0 to t ) 120 min was used to integrate
an area under the curve (AUC) for each treatment. Percent reduction
values for each treatment were generated from the AUC data
normalized to the dextrose-challenged lean controls.
Acknowledgment. The authors thank Dr. Philip Eskola,
Daniel Kim, and Regina Black of Synthetic Services Group for
large scale synthetic support. We also thank Dr. Bernard Choi
and Falguni Patel for providing high-resolution mass spectral
analyses. We thank W. P. Feeney, J. E. Fenyk-Melody, J. C.
Hausamann, S. A. Iliff, C. N. Nunes, A. S. Parlapiano, G. M.
Seeburger, X. Shen, and K. G. Vakerich for dosing the animals
used in pharmacokinetic studies. Use of the beamline 17-BM
with beamline management and support provided by staff from
IMCA-CAT at the Advanced Photon Source was supported by
the companies of the Industrial Macromolecular Crystallography
Association through a contract with Illinois Institute of Technol-
ogy. Use of the Advanced Photon Source was supported by the
U.S. Department of Energy, Office of Science, Office of Basic
Energy Sciences, under Contract No. W-31-109-Eng-38.
Determination of Plasma Protein Binding of Compound 6a.
[3H]6a (0.5 and 20 µM was incubated with a fresh pooled source
of plasma from male CD-1 mouse, C57BL mouse, Sprague-
Dawley rat, beagle dog, rhesus monkey, and human. Following
incubations at 37 °C for 30 min, an aliquot was counted for
radioactivity, and duplicate 1 or 0.5 mL aliquots of plasma were
transferred into centrifuge tubes and centrifuged at 100 000 rpm
(Beckman centrifuge model TL-100, rotor TLA 100.2) at 37 °C
for 3 h. The centrifuge was stopped without braking to minimize
Supporting Information Available: The X-ray crystallographic
data of compound 23 bound to DPP-4 are available. This material
is available free of charge via the Internet at http://pubs.acs.org.
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R-Amino Amide Dipeptidyl Peptidase IV Inhibitor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3627
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