Pyrrolidine-constrained phenethylamines: The design of potent, selective, and pharmacologically efficacious dipeptidyl peptidase IV (DPP4) inhibitors from a lead-like screening hit

Article abstract of DOI:10.1016/j.bmcl.2007.01.026

A novel series of pyrrolidine-constrained phenethylamines were developed as dipeptidyl peptidase IV (DPP4) inhibitors for the treatment of type 2 diabetes. The cyclohexene ring of lead-like screening hit 5 was replaced with a pyrrolidine to enable paralle

Full text of DOI:10.1016/j.bmcl.2007.01.026

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2005–2012
Pyrrolidine-constrained phenethylamines: The design of potent,
selective, and pharmacologically efficacious dipeptidyl peptidase IV
(DPP4) inhibitors from a lead-like screening hit
Bradley J. Backes,a,* Kenton Longenecker,b Gregory L. Hamilton,a Kent Stewart,b
Chunqiu Lai,a Hana Kopecka,a Thomas W. von Geldern,a David J. Madar,a
Zhonghua Pei,a Thomas H. Lubben,a Bradley A. Zinker,a Zhenping Tian,c
Stephen J. Ballaron,a Michael A. Stashko,a Amanda K. Mika,a
David W. A. Beno,d Anita J. Kempf-Grote,d Candace Black-Schaefer,b
Hing L. Shama and James M. Trevillyana
aMetabolic Disease Research, Abbott Laboratories, Abbott Park Road, Abbott Park, IL 60064-6099, USA
bAdvanced Technology, Abbott Laboratories, Abbott Park Road, Abbott Park, IL 60064-6099, USA
cProcess Research and Development, Abbott Laboratories, Abbott Park Road, Abbott Park, IL 60064-6099, USA
dDepartments of Exploratory Pharmacokinetics and Pharmaceutics, Global Pharmaceutical Research and Development,
Abbott Laboratories, Abbott Park Road, Abbott Park, IL 60064-6099, USA
Received 13 November 2006; revised 18 December 2006; accepted 8 January 2007
Available online 19 January 2007
Abstract—A novel series of pyrrolidine-constrained phenethylamines were developed as dipeptidyl peptidase IV (DPP4) inhibitors
for the treatment of type 2 diabetes. The cyclohexene ring of lead-like screening hit 5 was replaced with a pyrrolidine to enable par-
allel chemistry, and protein co-crystal structural data guided the optimization of N-substituents. Employing this strategy, a >400·
improvement in potency over the initial hit was realized in rapid fashion. Optimized compounds are potent and selective inhibitors
with excellent pharmacokinetic profiles. Compound 30 was efficacious in vivo, lowering blood glucose in ZDF rats that were allowed
to feed freely on a mixed meal.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Glucagon-like peptide-1 (GLP-1) therapy has emerged
as a promising treatment for type 2 diabetes.1 GLP-1,
as an incretin hormone, stimulates insulin biosynthesis
and secretion in a glucose-dependent manner while sup-
pressing glucagon release.2 Thus, blood glucose levels
may be regulated by GLP-1 with little risk of hypoglyce-
mia. Additional benefits to patients include the slowing
of gastric emptying3 and a reduction in appetite.4 Signif-
icantly, recent studies in rat suggest that GLP-1 treat-
ment may have the potential to stabilize or even
reverse disease progression by increasing b-cell mass
and function.5 Serine protease dipeptidyl peptidase IV
(DPP4) inactivates GLP-1(7–36) to give GLP-1(9–36)
by cleaving two N-terminal amino acid residues that
are required for receptor binding.6 Accordingly, inhibi-
tors of DPP4 may increase the half-life of active
GLP-1, prolonging its beneficial effects. Several clinical
trials indicate that orally administered small molecule
inhibitors of DPP4 are well-tolerated, lower blood glu-
cose and/or HbA1c levels, and increase glucose
tolerance.7
Chart 1 lists three notable DPP4 inhibitors that have en-
tered the clinic, vildagliptin 1 (LAF-237),8 sitagliptin 2
(MK-0431)9 and saxagliptin 3 (BMS-477118).10 Our
program has produced clinical compound 4 that
features an alkyne-substituted cyanopyrrolidine and
imparts the inhibitor with a superior selectivity profile.11
Lead-like12 cyclohexenyl hit 5 was identified in a
high throughput screen of our corporate compound
collection as a relatively weak inhibitor of DPP4
Keywords: DPPIV inhibitors; Diabetes; Structure-based design.
*
Corresponding author. Tel.: +1 847 938 3521; fax: +1 847 938
1674; e-mail: bradley.backes@abbott.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.026

2006
B. J. Backes et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2005–2012
HO
N-Substituted pyrrolidines were synthesized according
CN
O
H
F
to Scheme 1. Nitrostyrene 7 was treated with the azome-
thine ylide generated from the action of AgF upon
N-benzyl-N-cyanomethyl-N-trimethylsilylamine to pro-
vide pyrrolidine 8 ( ).15 Reduction of 8 ( ) with zinc
dust gave inhibitor 9 ( ). Intermediate 8 ( ) was deben-
zylated with triphosgene16 and the resulting carbamoyl
chloride was intercepted with secondary amines to pro-
vide tetra-substituted urea inhibitors 10a and b ( ) after
reduction of the nitro group, as before. When palladi-
um-mediated methods were employed to debenzylate
the pyrrolidine, competitive hydrogenolysis of the aryl
chlorides occurred. Therefore, 8 ( ) was debenzylated17
with b-trimethylsilylethyl chloroformate18 to give the
Teoc-protected pyrrolidine. Nitro reduction, treatment
with Boc-anhydride, and Teoc-removal with TASF
(tris(dimethylamino)sulfonium difluorotrimethylsilicate)
gave key intermediate 12 ( ). Acylation of 12 ( ) with
acetic anhydride, p-tosyl chloride, and isocyanates fol-
lowed by Boc-deprotection with HCl in dioxane gave
amide 13 ( ), sulfonamide 14 ( ), and urea 15a and b
( ) inhibitors, respectively. To provide carbamate inhib-
itors 16a and b ( ), alcohols were treated with DSC
(N,N0-disuccinimidyl carbonate) and the mixed carbon-
ates were intercepted in situ with 12 ( )19 followed by
Boc-removal performed as before. Palladium-mediated
N-arylation of 12 ( ) or nucleophilic aromatic substitu-
tion of heteroaryl chlorides under microwave condi-
tions20 followed by Boc-deprotection was employed
to give inhibitors 17 ( ) and 18a–d ( ). Treatment of
12 ( ) with 2,4-dichloropyrimidine gave a mixture of
19a ( ) and 19b ( ), Suzuki cross-coupling reaction of
19a–c ( ) with 3-thiophene boronic acid and removal
of the Boc-protecting group provided final compounds
20a–c ( ).
N
N
F
O
N
N
N
F
N
N
1
NH2
F3C
2
HO
CN
O
CN
O
H
N
N
N
N
NH2
4
3
HO
O
Cl
Ar
3
4
"Lead Hopping"
N
NH2
R
1
NH2
6
5 Screening Hit
Chart 1. DPP4 inhibitors.
(Ki = 0.8lM). However, the high binding efficiency13
and novel structure of 5 were compelling.14 We pro-
posed that ‘lead-hopping’ to 6 would: (1) display the
key phenethylamine pharmacophore of 5 in the 3- and
4-position of the pyrrolidine 6, (2) allow the extended
DPP4 binding pocket to be probed with nitrogen
substitution and (3) provide a structurally novel series
of DPP4 inhibitors. Given the synthetic accessibility of
substituted pyrrolidines and the ready availability
of protein co-crystal structures of inhibited complexes,
this strategy provides an iterative and rapid approach
for inhibitor optimization. Here we report the design,
development, and pharmacological evaluation of a
novel series of pyrrolidine-constrained phenethylamine
inhibitors of DPP4.
Azomethine ylide cyclo-addition chemistry employing
nitrostyrenes 21a–c, as before, gave 22a–c ( )
(Scheme 2). Nitro reduction and amine protection with
Cl
Cl
Step cmpnd R
Cl
Cl
g 13 (+) R = CH3CO-
Cl
N
h 14 (+) R = p-tolylSO2-
Cl
i
j
15a-b (+) R = R1NHCO-
16a-b (+) R = R1OCO-
O
N
NH2
k 17 (+) R = Ph
18a-b (+) R = Het
NH2
R1
c, b
Cl
N
R
N
10a-b (+)
NH2
b
Bn
R2
l
18c-d (+) R = Het
9 (+)
Step (see Table)
then m
Cl
Cl
Cl
Cl
Cl
N
Cl
Cl
Cl
a
Cl
N
Cl
Cl
o,m
n
f
d,b,e
Y
Y
NH2
N
NHBoc
X
NO2
N
Z
Bn
X
NHBoc
11 (+)
O2N
N
Z
7
Teoc
NHBoc
H
8 (+)
20a-c (+)
12 (+)
19a (+) (X = C; Y = N; Z = N)
19b (+) (X = N; Y = C; Z = N)
19c (+) (X = N; Y = N; Z = C)
Cl
S
Scheme 1. Reagents and conditions: (a) N-benzyl-N-cyanomethyl-N-trimethylsilylamine, AgF, CH3CN; (b) Zn, HCl or HOAc, MeOH;
(c) triphosgene, CH2Cl2 then R1R2NH, Et3N; (d) (c) ClCO2CH2CH2SiMe3, THF; (e) Boc2O, i-Pr2EtN, THF; (f) TASF, DMF; (g) Ac2O, i-Pr2EtN,
THF; (h) p-tosyl chloride, i-Pr2EtN, THF; (i) R1NCO, i-Pr2EtN, THF; (j) R1OH, DSC, Et3N, CH3CN then 12 ( ); (k) RX, Binap, Pd2(dba)3–
CHCl3, t-BuONa toluene, 100ꢁC; (l) RX, i-Pr2EtN, i-PrOH, 130 ꢁC, 10 min (microwave); (m) HCl in dioxane; (n) dichloroheterocycle, i-Pr2EtN,
i-PrOH, 130 ꢁC, 10 min (microwave); (o) 3-thiophene boronic acid, PdCl2(PPh3)2, Na2CO3, DMF/DME/MeOH/H2O (1:1:0.9:0.3), 150 ꢁC, 20 min
(microwave).

B. J. Backes et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2005–2012
2007
Ar
NO2
Ar
pocket and positioning the amine to interact with resi-
dues Glu205 and Glu 206 (Fig. 1a). Accordingly, substi-
tution of the distal positions of the cyclohexene directs
substituents to productive binding regions. It was appar-
ent that by ‘lead-hopping’ to pyrrolidines 6, a nitrogen
handle would be introduced in a strategic position to
probe the binding pocket with a range of nitrogen sub-
stituents. In addition, substitution of the pyrrolidine
nitrogen, from a synthetic chemistry perspective, may
simplify analoging by allowing for the use of parallel
chemistry schemes. A number of N-substituted pyrroli-
dine inhibitors were tested as racemates against DPP4
(Table 1). It was typical that alkyl, acyl, and sulfonyl-
substituted pyrrolidines were poor inhibitors of DPP4,
and compounds 9 ( ), 13 ( ), and 14 ( ), respectively,
(Ki > 2.7 lM) are representative. However, simple urea
15a ( ) (Ki = 280 nM) and carbamate 16a ( )
(Ki = 490 nM) substitutions increased potency. Diverse
libraries of carbamates, tri- and tetra-substituted ureas
were prepared to explore the possibility of further gains.
However, only modest improvements were observed for
our best compounds (urea 15b ( ) Ki = 190 nM, carba-
mate 16b ( ) Ki = 240 nM). N-Substitution with aryl
and heteroaryl groups revealed a clear trend (pryrimid-
inyl-18b ( ) (Ki = 210 nM) > pyridyl-18a ( ) (Ki = 790
nM) > Phe-17 ( ) (Ki = 2800 nM)). A co-crystal struc-
ture of 18b with human DPP4 (Fig. 1b) was analogous
to that observed for 5 above in positioning the dichlor-
ophenyl group within the S1 pocket and the primary
amine between Glu 205 and Glu 206. The N-pyrimidinyl
group is observed to stack against the phenyl ring of Phe
357. The potency of 18b, relative to the other analogs, is
likely due to some combination of (1) minimal steric
clash between the ortho pyrimidine nitrogens with the
pyrrolidine ring system, and/or (2) pi-stacking of the
electron-deficient pyrimidine system with Phe 357.
Ar
b, c
a
N
N
NHBoc
O2N
Bn
Bn
21a (+) (Ar = 2-Cl,4-Ph)
(+ ) (Ar = 2-Cl-4,5-F-Ph)
21c (+) (Ar = 2,4,5-F-Ph)
21b
23a-c (+)
22a-c (+)
Ar
Ar
N
N
NH2
N
N
e,f
d
NHBoc
24a-c (+)
N
N
g
25a-c
(+)
Cl
S
F
F
HO
F
F
F
F
O
h,i
HO
22c
(+)
.
NO2
26
OH
OH
N
HN
27
O
NHBoc
Bn
F
F
F
F
F
F
j
e or k,
then f
N
N
N
N
NH2
NHBoc
N
X
N
X
29a-h X = C
30
28a X = C
28b X = N
X = N
Cl
R
Scheme 2. Reagents and conditions: (a) N-benzyl-N-cyanomethyl-N-
trimethylsilylamine, AgF, CH3CN; (b) Zn, HOAc, MeOH; (c) Boc2O,
i-Pr2EtN, THF; (d) 4,6-dichloropyrimidine, DME, 160 ꢁC, 30 min
(microwave); (e) boronic acid, PdCl2(PPh3)2, Na2CO3, DMF/DME/
MeOH/H2O (1:1:0.9:0.3), 150 ꢁC, 20 min (microwave); (f) HCl in
dioxane; (g) D-tartaric acid; (h) RaNi, H2, EtOH, 40 psi then Boc2O,
THF; (i) Pd(OH)2, MeOH, 50ꢁC, 60psi; (j) dichloroheterocycle; (k)
Me3SnSnMe3, Pd(PPh3)4, dioxane, 105 ꢁC, then ArX, Pd(PPh3)4,
dioxane, 135 ꢁC, 1 h (microwave).
In a separate chemical effort, we have previously report-
ed on thiazole-based peptidomimetic inhibitors of DPP4
(31 shown in Fig. 1c inset).23 In this early work, we were
able to obtain a number of crystal structures of inhibited
rat DPP4 enzyme. The binding mode shown in Figure 1c
is representative. Overlay of this structure with the
structure of 18b is shown in Figure 1c and provided a
key insight in further analoging of 18b. While 31 and
18b bind in a very different manner in the S1 pocket,
the two structures converge with the amide of 31 stack-
ing against Phe357 and projecting its thiazole substitu-
ent toward Arg358. The excellent overlay of the two
structures strongly suggested that gains in potency
may be realized by extending heterocyclic, or properly
functionalized, aromatic substituents from the meta-po-
sition of the pyrimidine of 18b. An early indication of
direction dependence was observed in the relative poten-
cies of meta-substituted 18d ( ) (Ki = 140 nM) and para-
substituted 18c ( ) (Ki = 430 nM). A substantial gain in
potency was observed for thiophene-substituted com-
pounds with concomitant optimization of the positions
of the pyrimidine nitrogens, as evidenced by compound
Boc-anhydride provided 23a–c ( ). Quaternization of
the N-benzyl pyrrolidine of 23a–c ( ) under microwave
conditions using 4,6-dichloropyrimidine was accompa-
nied by debenzylation,21 and gave a convenient and time
saving synthesis of 24a–c ( ). Suzuki coupling with 3-
thiophene boronic acid was employed to provide inhib-
itors 25a–c ( ) after Boc-deprotection, as before. To
provide enantiopure material, D-tartaric acid was used
to resolve 22c ( ).22 The salt 26 was free-based and
the nitro group was reduced with Raney nickel to give
the free amine. Boc-anhydride protection was followed
by debenzylation to give 27. Substitution of 4,6-dichlo-
ropyrimidine or 2,4-dichlorotriazine with 27 gave 28a
or 28b, respectively. Suzuki cross-coupling with aryl or
heteroaryl boronic acids was employed to provide inhib-
itors 29a–e, g and 30, after Boc-removal. Treatment of
28a with (Me3Sn)2 provided a suitable intermediate for
Stille cross couplings. In situ coupling with aryl bro-
mides and Boc-deprotection gave final compounds
29f,h in a convenient one-pot procedure.
20c ( ) (Ki = 15 nM) that represents
a >50-fold
The cyclohexenyl ring of screening hit 5 ( ) serves to
efficiently constrain the aryl and amine substitutents,
directing the aryl group to the hydrophobic S1 binding
improvement over the initial screening lead.24 The co-
crystal structure of 20c confirms our design hypothesis
(Fig. 1d) with the thiophene projecting into a mainly

2008
B. J. Backes et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2005–2012
Figure 1. DPP4 co-crystal structures with inhibitors. The source of the DPP4 (human or rat) is indicated. (1a) Compound 5 (brown) in active site of
˚
rat DPP4 outlined with gray protein surface. The 2-chlorophenyl group projects into hydrophobic S1 pocket. Hydrogen bonds (N–O distances in A)
are shown as black dotted line between primary ammonium group of 5 and Glu 203-Glu 204 of rat DPP4 (Glu 205–206 of human DPP4). (1b)
Compound 18b (orange) in human DPP4. Glu 205 and Phe 357 (green) are exposed to show polar and pi-stacking interactions, respectively. (1c)
Same as Figure 1a with the rat structure of compound 31 (green, pdb code 2OAE) overlaid. The chemical structure of 31 is shown in upper left as an
inset. (1d) Compound 20c (light blue) in human DPP4 with surface removed to expose environment surrounding thiophene ring: Phe 357 C and CB,
Arg 358 CB, CG, and CD, Glu 206 O, and Ser 209 OG. (1e) Compound 29g (pink, pdb code 2OAG) in human DPP4 with polar interactions with
Arg 358 guanidinium group highlighted with orange dotted lines.
hydrophobic pocket comprised of Phe 357 C and CB,
Arg 358 CB, CG, and CD, Glu 206 O, and Ser 209
OG. The S1-pocket was probed with various halogen
substitution patterns (20c ( ), 25a–c ( )) showing
2,4-dichloro- and 2,4,5-trifluorosubstitution (25c, Ki =
17 nM) to be equipotent. Further work in this series
used the 2,4,5-trifluoro-pattern to impart compounds
with lower ClogP, lower molecular weights, and im-
proved chemical orthogonality for further analoging.25
From this point on, all 2,4,5-trifluoro analogs were pre-
pared as single enantiomers. In accord with the protein
crystal structure, compound 29b (as drawn in Scheme 2,
Ki = 6 nM, Table 2) was the active enantiomer of
25c ( ).26
At this point in the research program, a modified
enzyme assay was required to assess highly potent
compounds with low-nanomolar inhibitory potency.

B. J. Backes et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2005–2012
2009
Table 1. DPP4 inhibition constants of 9–18ba
Table 2. DPP4 inhibition constants of 18c–25ca
Ar
Cl
Cl
N
NH2
R
Compound
R
Ar
Ki (nM)
N
NH2
R
N
Cl
Cl
Cl
Compound
R
Ki (nM)
N
18c ( )
430
140
76
9 ( )
13 ( )
14 ( )
Bn–
MeCO–
3400
2700
13,000
280
Cl
Cl
Cl
MeO
p-TolylSO2–
EtNHCO–
15a ( )
15b ( )
10a ( )
10b ( )
16a ( )
(3-F)PhCH2NHCO–
EtNMeCO–
(3-MeO)PhCH2NMeCO–
EtOCO–
190
610
N
18d ( )
220
490
N
N
O
N
S
16b ( )
17 ( )
240
2800
790
N
N
20a ( )
O
Ph–
N
Cl
Cl
F
18a ( )
S
S
S
20b ( )
20c ( )
25a ( )
200
15
N
N
N
N
N
Cl
Cl
18b ( )
210
a All Ki values are an average of at least two runs.
N
N
Therefore, a second ‘tight binding’ assay was utilized
(Table 3, Ki (TB)). In addition, a third DPP4 assay in ro-
dent plasma was added to gauge the effect of compound
potency in the presence of plasma proteins (Ki TB; 10%
PL (rat plasma)). Realizing the close proximity of cat-
ionic side chain of Arg 358 to the terminal aryl of com-
pounds like 18d or 20c, anionic analogs were prepared
to exploit potential electrostatic interactions. Substitu-
tion of the pyrimidine with a 3-carboxyphenyl group
provided one of the most potent compounds in our
series (29c, Ki = 1.8 nM (TB)) and represents a 10·
improvement over aryl substitution alone (29a, Ki = 20
nM). In addition, 29c was unaffected by plasma protein
binding (Ki = 1.0 nM (TB, 10% PL)). Potency gains for
similarly positioned polar groups formed a solid trend
with small amides (29e, Ki = 7 nM), sulfonamides (29f,
Ki = 3.2 nM (TB), Ki = 1.7 nM (TB, 10%PL)), and sulf-
ones (29g, Ki = 3.4 nM (TB), Ki = 3.5 nM (TB, 10%PL))
possessing good potencies and low protein binding. Sub-
stitution of the pyrimidine with a 4-carboxyphenyl
group did not result in the same gains (29d, Ki = 85
nM), and the potency of compounds such as 29g, when
substituted with additional small groups on the aromatic
ring (5-fluoro,29h, Ki = 9 nM), was compromised. Addi-
tional increases in potency were observed when the pyri-
midinyl ring of 29g was replaced with a triazinyl ring
(30, Ki = 2.1 nM (TB)). The 2-fold gain in potency of
the triazinyl ring over the pyrimidine ring may be due
to increased pi-stacking interaction with Phe 357 of
the enzyme, although differential solvation of the two
ring systems or subtle differences in bond angles or bond
lengths can not be eliminated from consideration. The
crystal structure of 29g in DPP4 is shown in Figure 1e
34
Cl
F
F
S
S
25b ( )
35
17
N
N
N
N
C
F
F
25c ( )
F
a All Ki values are an average of at least two runs.
and exhibits the same binding site interactions identified
above with the notable addition of specific H-bonding
with the guanidine of Arg 358. Both of the sulfonyl oxy-
gens can be positioned within H-bond distance of Arg
358 (3.2 and 3.6 Ang) in a chelation-arrangement,
although we cannot eliminate mono-dentate interpreta-
tions from consideration. The most potent compounds
of our series that are substituted in the meta-position
exploit this interaction and represent a >400x improve-
ment over the initial screening hit.
Compounds 29c,f,g and 30 were evaluated (Table 4)
in vitro against the human DPP4 homologs DPP8,27
DPP9,28 prolyl oligopeptidases (POP),29 and fibroblast
activation protein a (FAPa, also called seprase).30 While
there are a number of other dipeptidyl peptidases in the

2010
B. J. Backes et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2005–2012
Table 3. DPP4 inhibition constants of 29a–30a
body, the function of most of these peptidases remains
unknown at present. There is, however, evidence indi-
cating that DPP8/9 activity may lead to profound toxic-
ity in preclinical species.31 No inhibition of POP and
FAP was observed when compounds were tested at con-
centrations up to 30lM. Inhibitors tested against DPP8
and DPP9 had little activity (Ki = 4–6lM and
Ki = 10.9–17 lM, respectively) and, therefore, high lev-
els of selectivity for DPP4.
F
F
F
N
NH2
R
Compound
29a
R
Ki (nM) Ki (10%PL,
nM)
Early assessment of the ADME characteristics of com-
pounds in the pyrrolidine series indicated that protein
binding was low since all compounds measured were
<95% protein bound (100% human plasma) and Ki val-
ue shifts were not seen for binding studies in the pres-
ence of 10% rat plasma (Table 3). In addition, all
compounds tested displayed acceptable stabilities in
rat and human liver microsome incubation studies
(t1/2 > 3h). However, inhibition of CYP2D6 was ob-
served for several racemic compounds (Table 5) including
25c ( ) (IC50 = 0.098 lM). Fortuitously, the enantiomer
with activity toward DPPIV, 29b, was >10· less potent to-
ward CYP2D6 (IC50 = 1.1 lM). In addition, inhibitors
with arene substituents that provided some of our most
potent compounds against DPP4 (29c,f and 30) lacked
CYP2D6 inhibition altogether (IC50 = >10lM).
20
6
ndc
nd
N
N
S
29b
N
N
29c
29d
1.8b
1.0
nd
HO
HO
O
O
N
N
N
85
N
The pharmacokinetics of several compounds were eval-
uated in Sprague–Dawley rats (Table 6). Both carboxyl-
ic acid-substituted 29c and sulfonamide-substituted 29f
possessed relatively high clearance rates (CLp 1.10 L/
h kg and 2.09 L/h kg, respectively) and low AUCs (po
652 ng h/kg and 515 ng h/kg, respectively). Clearance
rates for amide-substituted 29e were lower and lead to
Me2N
O
29e
29f
29g
29h
30
7
nd
1.7
3.5
nd
1.3
O
N
N
N
3.2b
N
S
O
O
N
a
higher AUC and moderate bioavailability (%
F = 42). Sulfones 29g (% F = 110) and 30 (% F = 74)
had excellent bioavailability characterized by high
AUCs in rat. Compound 30 was tested in two additional
species. In cynomolgus monkey, clearance was higher
than in rat (CLp 0.62 L/h kg vs 0.21 L/h kg, respective-
ly) and somewhat lower bioavailability was observed
(% F = 42). The bioavailability of 30 in beagle dog was
high (% F = 100) and was characterized by high AUC
and an extended half-life (t1/2 (po, h) = 14.9).
3.4b
S
O
O
N
N
F
9
S
S
O
O
N
N
N
N
2.1b
Compound 30 was chosen for in vivo evaluation due to
its excellent in vitro potency, selectivity, and pharmaco-
kinetic profile (Fig. 2). Blood glucose lowering was dem-
onstrated in female Zucker diabetic fatty (ZDF) rats of
10 weeks of age that were orally dosed with 30
(t = ꢀ240 min), and after four hours of incubation
N
O
O
a All Ki values are an average of at least two runs.
b Tight-binding assay (see text).
c nd, not determined.
Table 5. CYP Inhibitiona
Table 4. Selectivity of representative DPP4 inhibitorsa
DPP4b
DPP8
DPP9
POP
FAPa
Compound CYP2D6 (lM) CYP2C9 (lM) CYP3A4 (lM)
Compound
25c ( )
29b
29c
0.098
1.1
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
29c
29f
29g
30
1.8
3.2
3.4
2.1
>3000
4320
6440
4350
>3000
17,500
10,900
11,100
>30,000
>30,000
>30,000
>30,000
>30,000
>30,000
>30,000
>30,000
>10
>10
>10
29f
30
a Ki values (nM) for each enzyme are reported.
b Ki values for DPP4 are from tight-binding assay. For structures of the
compounds, see Table 3.
a All IC50 values are an average of at least two runs. For structures, see
Tables 2 and 3.

B. J. Backes et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2005–2012
Table 6. PK profile of selected DPP4 inhibitors
2011
Compound
Species
CLp (L/h kg)
Vss (L/kg)
Cmax (ng/mL)
t1/2 (po, h)
AUC (po) (ng h/kg)
F (%)
29c
29e
29f
29g
30
Rat
Rat
1.10
0.73
2.09
0.26
0.15
0.62
0.21
1.78
1.77
1.84
1.44
1.15
3.83
4.01
176
1270
347
6.8
3.5
652
3144
18
42
Rat
Rat
1.1
3.8
515
21
1813
1970
218
22,026
24,400
1719
110
74
Rat
Monkey
Dog
5.5
6.2
30
30
42
100
691
14.9
12,203
CLp, plasma clearance; Vss, volume of distribution at steady state; Cmax, maximal concentration when dosed orally; t1/2, terminal half-life when dosed
orally; AUC, area under curve, F, oral bioavailability.
PK was measured in rats at 5 mg/kg dose. PK was measured in dog and monkey at 2.5 mg/kg dose. For structures, see Table 3.
lowered at all three doses (0.3 mg/kg, ꢀ32%; 1 mg/kg,
ꢀ39%; 3 mg/kg ꢀ37%) in comparison to the vehicle.
The plasma concentration of 30 reached 686 48 ng/mL
(t = 0) and 373 45 ng/mL (t = 240), providing >99%
(t = 0) and >98% (t = 240 min) inhibition of DPP4 at
1 mpk, respectively.
In conclusion, a ‘lead-like’ screening hit inspired the
development of a series of pyrrolidine-constrained
DPP4 inhibitors. By lead-hopping to the synthetically
flexible pyrrolidine framework and employing struc-
ture-based design, significant gains in potency (>400·)
were realized in rapid fashion. ADME characteristics,
in vitro potency, and selectivity characteristics were
optimized in parallel to provide several compounds with
attractive profiles. Optimized compounds were very po-
tent DPP4 inhibitors with a low degree of protein bind-
ing and high selectivity against DPP4 homologs.
Compound 30 possessed attractive pharmacokinetic
profiles in multiple preclinical species. In addition, 30
was quite efficacious in vivo, lowering the blood glucose
of rats when orally dosed prior to a mixed meal.
Supplementary data
X-ray crystal structures have been deposited in the
RCSB protein data bank (www.rcsb.org) with codes
2OAE and 2OAG. Supplementary data associated with
this article can be found, in the online version, at
doi:10.1016/j.bmcl.2007.01.026.
Figure 2. A free-feeding model of female ZDF rats dosed with 30
orally. Overnight fasted female ZDF rats (n = 10/group) of 10 weeks of
age were dosed with either vehicle or DPP4 inhibitor 30. After four
References and notes
hours of pre-incubation with 30, the rats were allowed to feed (t = 0)
1. Zander, M.; Madsbad, S.; Madsen, J. L.; Holst, J. J.
Lancet 2002, 359, 824.
2. (a). Diabetologia 1992, 35, 701; For reviews see: (b) Holst,
J. J. Gastroenterology 1994, 107, 1048; (c) Drucker, D. J.
Diabetes 1998, 47, 159; (c) Deacon, C. F.; Holst, J. J.;
Carr, R. D. Drugs Today 1999, 35, 159.
freely on a highly palatable, macronutrient balanced food source (64%
CHO, 22% fat, and 14% Pro) and during which blood glucose levels
were measured. Plasma glucose change from baseline is shown on the
left panel and area under the curve (AUC) change (t = 0 to
t = 240 min) is shown on the right panel.
3. (a) Wettergren, A.; Schjoldager, B.; Mortensen, P. E.;
Myhre, J.; Christiansen, J.; Holst, J. J. Dig. Dis. Sci. 1993,
38, 665; (b) Nauck, M. A.; Niedereichholz, U.; Ettler, R.;
Holst, J. J.; Orskov, C.; Ritzel, R.; Schmiegel, W. H. Am.
J. Physiol. 1997, 273, E981.
4. Flint, A.; Raben, A.; Ersboll, A. K.; Holst, J. J.; Astrup,
A. Int. J. Obes. 2001, 25, 781.
5. Farilla, L.; Hui, H.; Bertolotto, C.; Kang, E.;
Bulotta, A.; Di Mario, U.; Perfetti, R. Endocrinology
2002, 143, 4397.
with 30, were allowed to feed freely on a balanced food
source (t = 0 min). This model is analogous to oral glu-
cose tolerance tests (OGTT) used clinically to evaluate
glycemic control, except that the ‘challenge’ is a liquid
mixed meal composed of fats, proteins, and carbohy-
drates, and thus a more realistic representation of the
nutritional makeup of normal food. Glucose excursion
as measured by the area under the curve (AUC) was

2012
B. J. Backes et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2005–2012
19. Hamilton, G. L.; Backes, B. J. Tetrahedron Lett. 2006, 47,
6. (a) Kieffer, T. J.; McIntosh, C. H. S.; Pederson, T. A.
Endocrinology 1995, 136, 3585; (b) Deacon, C. F.; Nauck,
M. A.; Toft-Nielson, M.; Pridal, L.; Willms, B.; Holst, J. J.
Diabetes 1995, 44, 1126.
967.
20. Luo, G.; Chen, L.; Poindexter, G. S. Tetrahedron Lett.
2002, 43, 5739.
´
7. (a) Ahren, B.; Simonsson, E.; Larsson, H.; Landin-Olsson,
M.; Torgeirsson, H.; Jansson, P.-A.; Sandqvist, M.;
Bavenholm, P.; Efendic, S.; Eriksson, J. W.; Dickinson,
21. Hamilton, G. L.; Backes, B. J. Tetrahedron Lett. 2006, 47,
2229.
22. A matrix of chiral acids (D-tartaric acid, D-malic acid,
(1R)-(ꢀ)10-camphorsulfonic acid, and (R)-(ꢀ)-mandelic
acid) and solvents (THF, EtOH, EtOAc and acetone) were
evaluated to resolve 21c ( ). The use of D-tartaric acid and
EtOH provided a practical solution.
´
S.; Holmes, D. Diabetes Care 2002, 25, 869; (b) Ahren, B.;
Gomis, R.; Standl, E.; Mills, D.; Schweizer, A. Diabetes
´
Care 2004, 27, 2874; (c) Ahren, B.; Landing-Olsson, L.;
Jansson, P-A.; Sevensson, M.; Holmes, D.; Schweizer, A.
´
J Clin. Endocrinol. Metab. 2004, 89, 2078; (d) Ahren, B.;
23. Shuai, Q.; Patel, J.; Zanze, I.; Dinges, J.; Wiedeman, P.;
Pei, Z.; Michmerhuizen, M. J.; Hoff, E.; Kalvin, D. M.;
von Geldern, T.; Lubben, T.; Ballaron, S.; Stashko, M.;
Zinker, B.; Djuric, S.; Beno, D.; Kempf-Grote, A. J.;
Mika, A.; Farb, T.; Perham, M. A.; Adler, A. L.;
Trevillyan, J.; Sham H. Acyl thiazolidides-novel potent
DPP-IV inhibitors, MEDI Poster 303, American Chemical
Society National Meeting, Aug 28–Sep 1, 2005; Washing-
ton, DC, USA.
24. For initial studies, thiophene substitution was evaluated
rather than thiazole substitution since the requisite start-
ing material (thiophene boronic acid) is commercially
available.
25. It is noteworthy that 2 (MK-0431) has an aryl group
with a similar 2,4,5-trifluorosubstitution pattern as 25c.
We obtained internal co-crystal structures using com-
pounds similar to 2 (Edmondson, D. D.; Parmee, E.;
Weber, A. E.; Xu, J. Dipeptidyl Peptidase Inhibitors for
the Treatment of Diabetes, Patent WO 03/000180,
January 3, 2003). The data indicated that our pyrroli-
dine-based inhibitors and the Merck inhibitors share a
similar aryl binding mode. On that basis, compounds
featuring the 2,4,5-trifluorosubstitution pattern were
included in our SAR studies.
26. An X-ray crystal structure was obtained from D-tartaric
acid salt 26. The stereochemical assignments of the
pyrrolidine substituents of 26 were in agreement with
protein crystal structure (Fig. 1d). These data further
confirm the stereochemical assignment of 29b as the active
enantiomer of 25c ( ).
Pacini, G.; Foley, J. E.; Schweizer, A. Diabetes Care 2005,
28, 1936.
8. Villhauer, E. B.; Brinkman, J. A.; Naderi, G. B.; Burkey,
B. F.; Dunning, B. E.; Prasad, K.; Mangold, B. L.;
Russell, M. E.; Hughes, T. E. J. Med. Chem. 2003, 46,
2774.
9. Kim, D.; Wang, L.; Beconi, M.; Eiermann, G. J.; Fisher,
M. H.; He, H.; Hickey, G. J.; Kowalchick, J. E.; Leiting,
B.; Lyons, K.; Marsilio, F.; McCann, M. E.; Patel, R. A.;
Petrov, A.; Scapin, G.; Patel, S. B.; Sinha Roy, R.; Wu, J.
K.; Wyvratt, M. J.; Zhang, B. B.; Zhu, L.; Thornberry, N.
A.; Weber, A. E. J. Med. Chem. 2005, 48, 141.
10. Augeri, D. J.; Robl, J. A.; Betebenner, D. A.; Magnin, D.
R.; Khanna, A.; Robertson, J. G.; Wang, A.; Simpkins, L.
M.; Taunk, P.; Huang, Q.; Han, S-P.; Abboa-Offei, B.;
Cap, M.; Xin, L.; Tao, L.; Tozzo, E.; Welzel, G. E.; Egan,
D. M.; Marcinkeviciene, J.; Chang, S. Y.; Biller, S. A.;
Kirby, M. S.; Parker, R. A.; Hamann, L. G. J. Med.
Chem. 2005, 48, 5025.
11. Madar, D. J.; Kopecka, H.; Pireh, D.; Yong, H.; Pei, Z.;
Li, X.; Wiedeman, P. E.; Djuric, S. W.; Von Geldern, T.
W.; Fickes, M. G.; Bhagavatula, L.; McDermott, T.;
Wittenberger, S.; Richards, S. J.; Longenecker, K. L.;
Stewart, K. D.; Lubben, T. H.; Ballaron, S. J.; Stashko,
M. A.; Long, M. A.; Wells, H.; Zinker, B. A.; Mika, A.
K.; Beno, D. W. A.; Kempf-Grote, A. J.; Polakowski, J.;
Segreti, J.; Reinhart, G. A.; Fryer, R. M.; Sham, H. L.;
Trevillyan, J. M. J. Med. Chem. 2006, 49, 6416.
12. Oprea, T. I.; Davis, A. M.; Teague, S. J.; Leeson, P. D.
J. Chem. Inf. Comput. Sci. 2001, 41, 1308.
27. Abbott, C. A.; Yu, D. M. T.; Woollatt, E.; Sutherland, G.
R.; McCaughan, G. W.; Gorrell, M. D. Eur. J. Biochem.
2000, 267, 6140.
13. Abad-Zapatero, C.; Metz, J. T. Drug Discovery Today
2005, 10, 464.
14. Lead-like hit 5 has been optimized to provide a clinical
candidate Pei, Z.; Li, X.; von Geldern, T. W.; Madar, D.
J.; Longenecker, K.; Yong, H.; Lubben, T. H.; Stewart, K.
D.; Zinker, B. A.; Backes, B. J.; Judd, A. S.; Mulhern, M.;
Ballaron, S. J.; Stashko, M. A.; Mika, A. K.; Beno, D. W.
A.; Reinhart, G. A.; Fryer, R. M.; Preusser, L. C.; Kempf-
Grote, A. J.; Sham, H. L.; Trevillyan, J. M. J. Med. Chem.
2006, 49, 6439.
15. Padwa, A.; Chen, Y. Y. Tetrahedron Lett. 1984, 25, 5739.
16. Jorand-Lebrun, C.; Valognes, D. Synth. Commun. 1998,
23, 1189.
17. Cambell, A. L.; Pilipauskas, D. R.; Khanna, I. K.;
Rhodes, R. A. Tetrahedron Lett. 1987, 28, 2331.
18. Shute, R. E.; Rich, D. H. Synthesis 1987, 346.
28. Ajami, K.; Abbott, C. A.; McCaughan, G. W.; Gorrell,
M. D. Biochem. Biophys. Acta 2004, 1679, 18.
29. Rosenblum, J. S.; Kozarich, J. W. Curr. Opin. Chem. Biol.
2003, 7, 496.
30. Scanlan, M. J.; Raj, B. K. M.; Calvo, B.; Garin-Chesa, P.;
Sanz-Moncasi, M. P.; Healey, J. H.; Old, L. J.; Rettig, W.
J. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5657.
31. Lankas, G. R.; Leiting, B.; Roy, R. S.; Eiermann, G. J.;
Beconi, M. G.; Biftu, T.; Chan, C.-C.; Edmondson, S.;
Feeney, W. P.; He, H.; Ippolito, D. E.; Kim, D.; Lyons, K.
A.; Ok, H. O.; Patel, R. A.; Petrov, A. N.; Pryor, K. A.;
Qian, X.; Reigle, L.; Woods, A.; Wu, J. K.; Zaller, D.;
Zhang, Z.; Zhu, L.; Weber, A. E.; Thornberry, N. A.
Diabetes 2005, 54, 2988.