(3R)-4-[(3R)-3-Amino-4-(2,4,5-trifluorophenyl)butanoyl]-3-(2,2,2-trifluoroethyl)-1,4-diazepan-2-one, a selective dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes

Article abstract of DOI:10.1016/j.bmcl.2006.09.099

Replacement of the triazolopiperazine ring of sitagliptin (DPP-4 IC₅₀ = 18 nM) with 3-(2,2,2-trifluoroethyl)-1,4-diazepan-2-one gave dipeptidyl peptidase IV (DPP-4) inhibitor 1 which is potent (DPP-4 IC₅₀ = 2.6 nM), selective, and ef

Full text of DOI:10.1016/j.bmcl.2006.09.099

Bioorganic & Medicinal Chemistry Letters 17 (2007) 49–52
(3R)-4-[(3R)-3-Amino-4-(2,4,5-trifluorophenyl)butanoyl]-3-(2,2,2-
trifluoroethyl)-1,4-diazepan-2-one, a selective dipeptidyl peptidase
IV inhibitor for the treatment of type 2 diabetes
Tesfaye Biftu,a,* Dennis Feng,a Xiaoxia Qian,a Gui-Bai Liang,a Gerard Kieczykowski,a
George Eiermann,b Huaibing He,a Barbara Leiting,c Kathy Lyons,a Aleksandr Petrov,b
Ranabir Sinha-Roy,c Bei Zhang,c Giovanna Scapin,a Sangita Patel,a Ying-Duo Gao,a
Suresh Singh,a Joseph Wu,c Xiaoping Zhang,c Nancy A. Thornberryc and Ann E. Webera
aDepartment of Medicinal Chemistry, Merck Research Laboratories, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
bDepartment of Pharmacology, Merck Research Laboratories, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
cDepartment of Metabolic Disorders, Merck Research Laboratories, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
Received 11 September 2006; revised 28 September 2006; accepted 29 September 2006
Available online 5 October 2006
Abstract—Replacement of the triazolopiperazine ring of sitagliptin (DPP-4 IC50 = 18 nM) with 3-(2,2,2-trifluoroethyl)-1,4-diaze-
pan-2-one gave dipeptidyl peptidase IV (DPP-4) inhibitor 1 which is potent (DPP-4 IC50 = 2.6 nM), selective, and efficacious in
an oral glucose tolerance test in mice. It was selected for extensive preclinical development as a potential back-up candidate to
sitagliptin.
Ó 2006 Elsevier Ltd. All rights reserved.
The incretin hormone glucagon-like peptide 1 (GLP-1)
stimulates insulin biosynthesis and secretion, and inhib-
its glucagon release in a glucose-dependent manner.1,2
GLP-1 and its analogs have been the subject of intense
research related to the treatment of type 2 diabetes.3,4
However, active GLP-1 (GLP-1[7–36]amide) is rapidly
degraded in vivo through the action of dipeptidyl pepti-
dase IV (DPP-4), a serine protease which cleaves a
dipeptide from the N-terminus to give the inactive
GLP-1[9–36]amide.5,6 Small-molecule inhibitors7,8 of
DPP-4 have been shown to prolong the beneficial effects
of this incretin hormone, as well as stabilize other incre-
tin hormones such as glucose-dependent insulinotropic
polypeptide (GIP).9 Indeed, improved glucose tolerance
in diabetic patients was achieved in human clinical trials
with several small-molecule DPP-4 inhibitors,10 includ-
ing sitagliptin.10f In this paper, we describe the synthesis,
SAR, and biological evaluation of (3R)-4-[(3R)-3-ami-
no-4-(2,4,5-trifluorophenyl)butanoyl]-3-(2,2,2-trifluoro-
ethyl)-1,4-diazepan-2-one (1), which was chosen for
extensive pre-clinical studies as a potential back-up to
sitagliptin.
F
F
F
CF3
F
NH2
O
NH2
O
O
N
N
N
N
NH
N
F
F
CF3
1
sitagliptin
The DPP-4 inhibitors 5 reported in this manuscript were
prepared from b-amino acid intermediates 210f,11 and
substituted heterocyclic amines 3 using standard peptide
coupling conditions followed by deprotection as shown
in Scheme 1.
The imidazolone (n = 0) and piperazinone (n = 1) het-
erocycles 3 are commercially available. The diazepanon-
es (n = 2) are prepared as shown in Scheme 2. The
hydrochloride salt of amino ester 6 is condensed with
acrylonitrile 7 and the amino group of the product
formed is protected as its tert-butoxylcarbonyl (Boc)
Keywords: DPP-IV; Diabetes; Diazepanone.
*
Corresponding author. Tel.: +1 732 594 6514; e-mail:
Tesfaye_Biftu@merck.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.099

50
T. Biftu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 49–52
R'
O
HN
F
F
F
NH
n
R
R
Boc
Boc
HN
O
HN
O
R'
3
O
OH
N
EDC, HOBt, DMF
F
NH
n
2
4
F
F
R
TFA/CH2Cl2
NH2
O
R'
O
N
NH
5
n
Scheme 1.
DPP8,13 DPP9,14 and fibroblast activation protein
(FAP, also called seprase),15 PEP, and other proline spe-
cific enzymes16 with DPP-4-like activity, including quies-
cent cell proline dipeptidase (QPP, also known as DPP-
II),12,17 APP and prolidase. Activity against PEP, FAP,
APP, and prolidase was typically P100 lM and is thus
not reported. DPP8 and DPP9 activity was similar, thus
only DPP8 data are reported. Selectivity over the latter
two enzymes was of particular concern since safety stud-
ies using a DPP8/9 selective inhibitor suggest that inhibi-
tion of DPP8 and/or DPP9 may be associated with
profound toxicity in preclinical species.18 The relevance
of this toxicity to humans has yet to be determined.
R
Boc
N
1) NaOH/H2O, 70 o
2) O(COOtBu)2
C
CO2Me
+
H2N
HCl
CO2Me
CN
NC
R
6
7
8
Boc
H2, PtO2
LiOH
THF/MeOH
H2N
N
CO2Me
CHCl3, EtOH
R
9
O
Boc
R'
N
H2N
N
CO2H
Boc
N
(CH3)3Al
CH2Cl2
R
12
DPP-4 inhibitory activities of compounds 1, 13–2619 are
listed in Tables 1–4. As shown in Table 1, the difluoro-
phenyl analog 13 of sitagliptin has an IC50 of 27 nM
in the DPP-4 assay.10f Introduction of a methyl group
gave 14, which is a more potent (IC50 = 7.2 nM) DPP-
4 inhibitor.20 Replacement of the triazolopiperazine of
14 with various ring size cyclic amides, that is, imidazo-
lone, piperazolone, and diazepanone gave analogs 15,
16, and 17 with IC50s of 160, 89, and 14 nM, respective-
ly. Similar to what was seen with sitagliptin, the trifluor-
ophenyl diazepanone 18 was more potent than the
difluorophenyl analog 17.
10
1) LDA
2) RX
3) HCl or TFA
When R = H
NaH, then
EDC/HOBt
CH2Cl2
PhCH2OCH2Cl
O
R
O
R
HCl/dioxane
or TFA/CH2Cl2
Boc
H
N
NH
N
NH
11
3 (n = 2)
Scheme 2. Synthesis of hexahydrodiazepinones.
Since diazepanone 18 is the most potent compound in
this series and has slightly better activity than sitagliptin,
we investigated the activities of the four possible stereo-
isomers of this compound. As shown in Table 2, the
decreasing order of DPP-4 inhibitory activity is: 18
(R,R) > 19 (R,S) > 20 (S,S) > 21 (S,R).
derivative to provide 8, which is reduced to primary
amine 9. Amino ester 9 was hydrolyzed to acid 10 and
cyclized using amino acid coupling reagents such as
EDC to provide intermediate 11. Alternatively, direct
cyclization of 9 to N-protected hexahydrodiazepinone
11 was also conducted using trimethylaluminum.
Deprotection of Boc derivative 11 by treatment with
hydrogen chloride in dioxane or trifluoroacetic acid in
dichloromethane provided diazepanone 3 (n = 2). Pro-
tected diazepanone 12 (R0 = BOM), made from 11,
wherein R is hydrogen, was alkylated using bases such
as lithium diisopropyl amide followed by treatment with
various alkyl halides. Deprotection gave 3 (n = 2).
The most potent (R,R) isomer 18 was then elaborated
further by modifying the methyl adjacent to the carbon-
yl group of the diazepanone ring. As shown in Table 3,
the unsubstituted analog 22 showed greatly decreased
DPP-4 inhibitory potency. Aryl-alkyl substituents give
compounds such as 24, 25, and 26, which are subnano-
molar inhibitors of DPP-4 and display high selectivity
against QPP, DPP8, and the other proline peptidases
tested. However, these compounds have poor oral bio-
availability (data not shown). While ethyl analog 23 is
less active than the methyl derivative, the trifluoroethyl
Compounds 1, 13–26 were evaluated in vitro for their
inhibition of hDPP-4 activity.12 The inhibitors were also
tested against DASH family members, including

T. Biftu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 49–52
51
Table 1. Activities of imidazolone, piperazone, and diazepanone DPP-
4 inhibitors
Table 3. Inhibitory properties of substituted diazepanone analogs
F
F
F
F
R
NH2
O
R
O
NH2
O
N
NH
X
F
Compound
R
IC50 (nM)
Compound
Sitagliptin
R
F
X
DPP-4 IC50 (nM)
DPP-4
QPP
DPP8
N
N
22
18
23
1
H
140
6.6
>100,000
59,000
47,000
59,000
17,000
89,000
75,000
82,000
46,000
13,000
28,000
21,000
53,000
80,000
N
Me
Et
N
N
18
16
2.6
CH2CF3
CH2(Ph)
CF3
N
24
25
26
0.91
0.49
0.29
N
CH2(2-pyridyl)
CH2(2-pyrazolyl)
N
13
14
15
16
17
18
H
H
H
H
H
F
27
7.2
160
89
CF3
N
Table 4. Mean pharmacokinetic parameters of 1 and 18 in nonclinical
species
N
N
Parameter
Rat
Dog
N
1
18
1
18
CF3
Dose iv/po (mg/kg)
Clp (mL/min/kg)
T1/2 (h)
1/2
94
2
1/2
88
1/2
18
9
1/2
9
N
1
26
4.3
100
8.5
O
Foral (%)
NAUC (lM h/(mg/kg))
36
0.2
95
2.2
N
0.15
H
Clp, plasma clearance; Foral, oral bioavailability; nAUC, dose-nor-
malized area under the plasma concentration versus time curve.
Pharmacokinetic parameters were obtained following a iv (1 mg/kg) or
po (2 mg/kg) dose of 1 and 18 (amorphous trifluoroacetic acid salt in
rats, hydrochloride salt in dogs) in water.
O
N
NH
O
14
N
N
The pharmacokinetic properties of the two most prom-
ising analogs 1 and 18 were then determined in male
Sprague–Dawley rats and beagle dogs (1 mg/kg iv,
2 mg/kg po). As shown in Table 4, both compounds
exhibited high plasma clearance and moderate oral bio-
availability in rats. In dogs, however, plasma clearance
was moderate and oral bioavailability was very good.
In both species, the terminal half-life of trifluoroethyl
analog 1 was longer than methyl analog 18, and thus
compound 1 was chosen for more extensive study.
NH
O
6.6
NH
Table 2. Inhibitory properties of diazepanone isomers
Trifluoroethyl analog 1 is a competitive, reversible
inhibitor of DPP-4 (IC50 = 2.6 nM), twice as active
as 18. It is highly selective over all proteases tested
(IC50s > 23 lM), including DPP8 and DPP9, and a
panel of >170 enzyme and receptor assays at Panlabs
(>10 lM). In an oral glucose tolerance test
(OGTT)10f,11 in lean mice, 1 significantly reduced
blood glucose excursion in a dose-dependent manner
from 0.1 mg/kg (16% reduction) to 3 mg/kg (46%
reduction). At the 3 mg/kg dosage, plasma DPP-4
activity was inhibited by >80%, the targeted inhibition
associated with maximal efficacy, with a corresponding
plasma concentration of 266 nM. In DPP-4-deficient
mice, no inhibition of glucose excursion was observed
following administration of 1 (10 mg/kg po), indicat-
ing that the reduction observed in wild type mice is
a mechanism-based effect.
F
F
Me
O
NH2
*
O
N
**
NH
F
Compound Stereochemistry* Stereochemistry** DPP-4
IC50 (nM)
18
19
20
21
R
R
S
S
R
S
S
R
6.6
150
2,300
67,000
substituted analog
(IC50 = 2.6 nM) and >10,000-fold selectivity over QPP
and DPP8.
1
has increased activity

52
T. Biftu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 49–52
References and notes
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Figure 1. X-ray structure of compound 1 bound to active site of
DPP-4.
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Figure 1 shows compound 1 bound to the active site
of DPP-4 as presented in the crystal structure. The
basic amine group hydrogen bonds with the side
chains of Glu205 and Glu206. The 2-F atom of the
P1 fragment is within hydrogen bonding distance from
the side chain of Asn710 and the amide carbonyl
interacts with the side-chain hydroxyl of Tyr547
through a water molecule. The 7-membered ring
group fills the hydrophobic area above the side chain
of Phe357. Comparison of the structure of 1 and 18
(not shown) indicates that the methyl group points
toward a hydrophobic pocket formed by the side
chains of Phe357, Tyr547, and Tyr666, and that the
larger trifluoroethyl substituent provides a better com-
plementarity to the binding site, thus increasing the
compound potency. The crystal structure was solved
˚
at 2.35 A resolution. Coordinates for the structure of
DPP-4 in complex with 1 and 18 have been deposited
with the Protein Data Bank, accession codes 2IIT and
2IIV, respectively.
In summary, a novel series of potent diazepanone-based
DPP-4 inhibitors have been discovered. Based on its
DPP-4 potency, selectivity, in vivo efficacy, and
pharmacokinetic profile, (3R)-4-[(3R)-3-amino-4-(2,4,5-
trifluorophenyl)butanoyl]-3-(2,2,2-trifluoroethyl)-1,4-
diazepan-2-one (1) was chosen for preclinical evaluation
as a potential back-up to sitagliptin for the treatment of
type 2 diabetes mellitus.
18. Lankas, G. R.; Leiting, B.; Sinha Roy, R.; 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.; Zaller, D.; Zhang,
X.; Zhu, L.; Weber, A. E.; Thornberry, N. A. Diabetes
2005, 54, 2988.
19. All new compounds were characterized by H NMR and
LC–MS prior to submission for biological evaluation.
20. Linda Brockunier, unpublished.
Acknowledgments
We thank Dr. Phil Eskola, Gina Black, Mark Levorse,
Joe Leone, Bob Frankshun, Amanda Makarewicz, Dan-
iel Kim, and Dr. Derek Von Langen of Synthetic Servic-
es Group for large-scale synthetic support, and Dr.
Bernard Choi and Eric Streckfuss of Analytical Support
Group for open access LC–MS service.
1