Discovery of potent and selective β-homophenylalanine based dipeptidyl peptidase IV inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.06.099

Modification of in-house screening lead β-aminoacyl proline 8 gave an equipotent thiazolidide 9. Extensive SAR studies on the phenyl ring of 9 led to the discovery of a novel series of potent and selective DP-IV inhibitors. Introduction of a fluorine at the 2-position proved to be crucial for the potency of this series. The 2,5-difluoro (22q) and 2,4,5-trifluoro (22t) analogues were potent inhibitors of DP-IV (IC₅₀ = 270, 119 nM, respectively).

Full text of DOI:10.1016/j.bmcl.2004.06.099

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4759–4762
Discovery of potent and selective b-homophenylalanine based
dipeptidyl peptidase IV inhibitors
a
a
a
Jinyou Xu,a, Hyun O. Ok, Edward J. Gonzalez, Lawrence F. Colwell, Jr.,
*
Bahanu Habulihaz,a Huaibing He,a Barbara Leiting,b Kathryn A. Lyons,a
Frank Marsilio,b Reshma A. Patel,b Joseph K. Wu,b Nancy A. Thornberry,b
Ann E. Webera and Emma R. Parmeea
aDepartment of Medicinal Chemistry, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
bDepartment of Metabolic Disorders, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
Received 25 May 2004; revised 21 June 2004; accepted 21 June 2004
Available online 31 July 2004
Abstract—Modification of in-house screening lead b-aminoacyl proline 8 gave an equipotent thiazolidide 9. Extensive SAR studies
on the phenyl ring of 9 led to the discovery of a novel series of potent and selective DP-IV inhibitors. Introduction of a fluorine at
the 2-position proved to be crucial for the potency of this series. The 2,5-difluoro (22q) and 2,4,5-trifluoro (22t) analogues were
potent inhibitors of DP-IV (IC50 =270, 119nM, respectively).
Ó 2004 Elsevier Ltd. All rights reserved.
OH
Glucagon-like peptide-1 (GLP-1) is an incretin hormone
that is released from the gut during meals and serves as
an enhancer of glucose stimulated insulin release from
pancreatic b-cells. Chronic infusion of GLP-1 to
patients with type 2 diabetes resulted in significant
decreases in both blood glucose and hemoglobin A1c
levels,1 however, GLP-1 is rapidly degraded in plasma
by the serine protease dipeptidyl peptidase IV (DP-
IV). Inhibition of DP-IV increases the levels of endo-
genous intact circulating GLP-1. Thus, inhibition of
DP-IV is rapidly emerging as a novel therapeutic ap-
proach to the treatment of type 2 diabetes.2
NC
O
CN
O
H
N
CN
H
N
N
N
H
N
N
S
1
2
O
O
N
N
NH2
S
H
NH2
4
3
NH2 NH2
F
H
N
N
S
O O
O
MeO
N
F
A range of DP-IV inhibitors have been reported, some
of which are illustrated in Figure 1. The (S)-2-cyano-
pyrrolidine derivatives 1 and 2 are potent inhibitors of
DP-IV, but both compounds contain an electrophilic
nitrile group.3 A systemic study to improve the chemical
stability of such nitrile-containing compounds has been
reported recently.4 Inhibitors lacking an electrophile,
such as 3 and 4, are generally of only modest intrinsic
potency.5 Earlier reports from our laboratories de-
scribed 4-amino cyclohexylglycine analogues 5 and 6
N
Cl
Cl
NH2
X
OMe
5 (X = S, CH2)
7
6 (X = CHF, CF2)
Figure 1. Published DP-IV inhibitors.
as potent DP-IV inhibitors lacking an electrophile.6,7
More recently, 4-aminomethylpyrimidine 7 was re-
ported as a very potent DP-IV inhibitor (IC50
=
0.1nM).8 In an effort to find novel structural classes,
we have identified b-aminoacyl proline 8 (Fig. 2) from
the Merck sample collection as a novel lead. It was soon
discovered that the right-hand side of 8 could be
Keywords: DP-IV.
*
Corresponding author. Tel.: +1-732-594-2217; fax: +1-732-594-
5790; e-mail: jinyou_xu@merck.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.099

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J. Xu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4759–4762
Cl
With the goal of finding analogues of thiazolidide 9 (Fig.
2) with increased potency, initially we looked at replace-
ment of the phenyl group in 9 to see if it was essential for
the activity in this series. Table 1 summarizes the DP-IV
inhibitory properties of these b-amino thiazolidides.
Lengthening or shortening the distance between the phe-
nyl group and the amino group was not tolerated (10,
11). Replacement of the phenyl group with a heterocycle
usually resulted in a decrease in potency. However, the
bioisosteric thiophene 15 was only 2-fold less potent.
Replacement of the phenyl group with a cyclopentyl
or cyclohexyl group resulted in 10-fold decrease in po-
tency. We also prepared the enantiomer of 9, which
was inactive (DP-IV IC50 >100,000nM). At that point,
we decided to examine substitution on the phenyl ring
in 9.
H
O
N
NH2
O
NH2
O
O
O
N
N
S
NH
7
8
IC 50 = 1900 nM
Figure 2. Initial leads.
IC 50 = 3000 nM
replaced by a thiazolidine without a significant loss in
potency while reducing the molecule weight of the initial
lead by half. The work described here summarizes our
initial efforts at optimizing the left-hand side of this
novel series of b-homophenylalanine based DP-IV
inhibitors.
Table 2 summarizes the SAR of these derivatives. In
most cases, mono-substitution on the phenyl ring had
little effect or was detrimental to activity. However,
addition of a fluorine or methyl group at the 2-position
of the phenyl ring increased potency by 3-fold (22a,
22b). The 3,4-difluoro analogue 22n was also very inter-
esting because it was more potent than 3-fluoro ana-
logue 22e and 4-fluoro analogue 22h. We observed the
beneficial additive effect of substitution at both the
3- and 4-position on the phenyl ring. Therefore, we
The b-amino acid derived inhibitors were prepared by
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-
mediated coupling of b-amino acids with thiazolidine.
Noncommercially available b-amino acids were readily
accessible via the Arndt–Eistert homologation of a-amino
acids (Scheme 1), which were either commercially availa-
ble or prepared using EvansÕ asymmetric azidation9 or
Scho¨llkopfÕs bis-lactim methodology.10 A representative
example of the synthesis of these inhibitors is shown in
Scheme 1. Treatment of a-amino acid 19 with isopropyl
chloroformate followed by reaction with an excess of dia-
zomethane afforded diazo ketone 20. Sonication of the
diazo ketone in 1,4-dioxane in the presence of silver ben-
zoate resulted cleanly in the formation of b-amino acid
21.11 An EDC-mediated coupling of 21 with thiazolidine
followed by deprotection of the tert-butylcarbamate pro-
vided the desired compound 22t.12
Table 1. Inhibitory property of b-aminoacid thiazolidides
NH2
O
R
N
Compd
R
DP-IV IC50, nM
3000
9
Inhibitors were tested for their selectivity profiles against
a variety of DP-IV homologues and proline-specific en-
zymes including quiescent cell proline dipeptidase (QPP/
DPP-II), prolyl endopeptidase (PEP), amino peptidase P
(APP), and prolidase. Since significant off-target activity
was only observed with QPP (<100,000nM), inhibition
data are presented for DP-IV and QPP only.13
10
11
12
>100,000
16,480
39,640
N
F
F
O
13
14
15
16
17
18
44,860
51,000
5700
F
COOH
N2
N
N
F
a,b
NHBOC
NHBOC
F
F
F
19
20
S
F
F
S
N
c
d,e
F
O
COOH
46,910
27,470
33,000
NH2
22t
O
NHBOC
F
F
21
Scheme 1. Reagents: (a) Et3N, i-PrO(CO)Cl, THF; (b) CH2N2, ether;
(c) C6H5CO2–Ag+, H2O, dioxane, sonication, rt; (d) thiazolidine,
EDC, HOBT, DIEA, DMF; (e) trifluoroacetic acid, CH2Cl2, 1h.

J. Xu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4759–4762
Table 2. Inhibitory properties of b-aminoacid thiazolidides
NH2
4761
ery of a novel series of potent and selective DP-IV inhib-
itors. Introduction of a fluorine at the 2-position of the
phenyl ring proved to be crucial for the potency of these
compounds. The 2,5-difluoro (22q) and 2,4,5-trifluoro
(22t) analogues were the most potent DP-IV inhibitors,
exhibiting DP-IV IC50s of 270 and 119nM, respectively.
Incorporation of the fluorine containing b-homophenyl-
alanine derivatives described herein into a novel series of
piperazine containing DP-IV inhibitors will be described
in the following paper.14
O
R
N
Compds
R
DP-IV IC50, nM
QPP IC50, nM
9
2-H
2-F
3000
931
>100,000
49,700
92,060
>100,000
82,120
48,510
29,750
24,570
67,630
36,190
5775
22a
22b
22c
22d
22e
22f
22g
22h
22i
2-Me
2-CN
2-CF3
3-F
981
2519
2263
2026
8951
3611
3279
3028
10,700
7688
14,860
1552
1700
9400
589
3-CN
3-CF3
4-F
References and notes
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4-I
22j
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22k
22l
4-CF3
4-CN
4-NO2
ND
ND
22m
22n
22o
22p
22q
22r
22s
22t
24,330
33,890
ND
3-F, 4-F
2-F, 3-F (rac)
2-F, 4-F
2-F, 5-F
56,050
77,560
ND
270
2-F, 6-F (rac)
3-Cl, 4-Cl
2-F, 4-F, 5-F
8476
2026
119
48,510
25,380
decided to look at further effects of disubstitution of flu-
orine on the phenyl ring. A combination of 2-fluoro and
4-fluoro on the phenyl ring increased potency by 5-fold
(22p). A 10-fold increase in potency was observed with
2,5-difluoro analogue 22q. Given the additive beneficial
effects of substitution of fluorine at the 3- and 4-position
on the phenyl ring, we prepared the corresponding 2,4,5-
trifluoro analogue 22t. A dramatic 25-fold increase was
observed with 22t (IC50 =119nM). Both 22q and 22t
exhibited >200-fold selectivity over QPP.
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The pharmacokinetic properties of compounds 22q and
22t were determined in rats. These compounds suffered
from high clearance, short half-life and poor oral bio-
availability, which precluded further development of
this series (Table 3).
In summary, modification of an in-house screening lead
b-aminoacyl proline 8, gave an equipotent thiazolidide
9. SAR studies on the phenyl ring of 9 led to the discov-
Table 3. Pharmacokinetic properties (1mpk IV, 2mpk PO) of selected
DP-IV inhibitors in rats
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F
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R
NH2
O
N
1
12. Final compounds were characterized by H NMR, mass
F
S
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Compds
R
Clp (mL/min/kg)
t1/2 (h)
F (%)
22q
22t
H
F
110
120
0.5
1.1
2
3
13. (a) For assay conditions for DP-IV and QPP inhibition,
see: Leiting, B.; Pryor, K. D.; Wu, J. K.; Marsilio, F.;

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J. Xu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4759–4762
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were within 1.3-fold of the reported average.