Discovery of new chemotype dipeptidyl peptidase IV inhibitors having (R)-3-amino-3-methyl piperidine as a pharmacophore

Article abstract of DOI:10.1016/j.bmcl.2010.10.101

Structures containing the (R)-3-amino-3-methyl piperidine unit as a new pharmacophore moiety have been shown to possess moderate inhibitory activity for DPP-4 with good pharmacokinetics profile. One of these compounds was found to have good oral bioavailability and PK/PD profile in ZF-rat.

Full text of DOI:10.1016/j.bmcl.2010.10.101

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7246–7249
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of new chemotype dipeptidyl peptidase IV inhibitors
having (R)-3-amino-3-methyl piperidine as a pharmacophore
⇑
Yukihiro Nishio , Hidenori Kimura, Shinya Tosaki, Eiji Sugaru, Mutsuko Sakai, Masakuni Horiguchi,
Yumi Masui, Michiko Ono, Tsutomu Nakagawa, Hiroyuki Nakahira
Drug Research Division, Dainippon Sumitomo Pharma Co., Ltd, 3-1-98 Kasugade Naka, Konohana-ku, Osaka 554-0022, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 July 2010
Revised 1 October 2010
Accepted 20 October 2010
Available online 26 October 2010
Structures containing the (R)-3-amino-3-methyl piperidine unit as a new pharmacophore moiety have
been shown to possess moderate inhibitory activity for DPP-4 with good pharmacokinetics profile. One
of these compounds was found to have good oral bioavailability and PK/PD profile in ZF-rat.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Dipeptidyl peptidase 4
DPP-4
Dipeptidyl peptidase 4 inhibitors
DPP-4 inhibitors
3-Amino-piperidine
(R)-3-Amino-3-methyl piperidine
Patients suffering from type 2 diabetes are on the increase
worldwide. Although a number of therapies are available for this
condition, recent efforts have been focusing on dipeptidyl pepti-
dase IV (DPP-4) inhibitors as a new class of therapeutic agents
for type 2 diabetes.1 Indeed, a number of clinical studies have al-
ready confirmed the efficacy and good tolerance of DPP-4 inhibi-
tors. DPP-4, a serine protease distributed throughout the body,
cleaves a wide range of peptides to modulate their biological activ-
ity. One of these peptides is glucagon-like peptide-1 (GLP-1), which
plays an important role in the regulation of blood glucose level.2
GLP-1 is released after food ingestion and through its receptor
stimulates insulin biosynthesis and secretion. GLP-1 also inhibits
glucagon release, delays gastric emptying, and induces pancreatic
b-cell proliferation.3
F
F
OH
F
NH2
O
O
CN
H
N
N
N
N
N
N
1
2
CF3
CN
N
O
NH2
O
N
O
NH2
N
N
N
N
N
N
N
O
N
3
4
Figure 1. Structures of small molecule DPP-4 inhibitors.
A number of DPP-4 inhibitors, such as 1 (sitagliptin)4, 2 (vildag-
liptin),5 and 3 (alogliptin)6, have already been approved, while oth-
ers, for example, 4 (linagliptin),7 are still under development
(Fig. 1). In our search for potent DPP-4 inhibitors, we have identi-
fied a series of compounds represented here by 6, which have a
pyrolo[3,2-d]pyrimidine structure.8 The scaffold of these com-
pounds was derived from our HTS hit compound 5, which pos-
sesses a xanthine scaffold like that of 4.7 Based on 5, other series
of compounds were also identified and their SARs will be disclosed
elsewhere in the near future.
Reports describing compounds 36 and 47 indicate that the (R)-3-
amino piperidine group is essential for activity as this group inter-
acts with DPP-4 (the enzyme). This finding is based on X-ray crys-
tal analysis of the identified DPP-4 inhibitors complexed with
hDPP-4. In our study, compound 6, which has the same piperidine
unit as 3 and 4, was found to have an excellent DPP-4 inhibitory
activity, but showed modest bioavailability (B.A.) (37%). Based on
the structure of 6, we assumed that the primary amine moiety,
which has high hydrophilicity, is one of the main reasons for 6
modest B.A. From this point of view, we considered introducing
substituents at the piperidine part of 6 to increase lipophilicity,
and consequently identified (R)-3-amino-3-methyl piperidine unit
⇑
Corresponding author.
E-mail address: yukihiro-nishio@ds-pharma.co.jp (Y. Nishio).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.101

Y. Nishio et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7246–7249
7247
R2
O
R1
N
Me
NH2
N
N
Cl
CN
N
Cl
O
N
O
N
O
N
H
R3
7
NH2
N
N
N
HN
N
N
NH
O
O
R5
N
R4
N
6
5
O
Me
NH2
N
N
8
Figure 2. Synthesis of new chemotype DPP-4 inhibitors from HTS hit 5.
O
O
Me
NHBoc
Me
CO2R
Me
CO2Et
Me
NHBoc
d
c
a
O
N
O
N
HN
HN
10
11
12
13
R = Et
R = H
9
b
Scheme 1. Reagents and conditions: (a) CbzCl (1.1 equiv), Et3N (1.2 equiv), THF, rt, 10 h; (b) NaOHaq (2.0 equiv), THF, MeOH, rt, overnight; (c) DPPA (1.02 equiv), Et3N
(1.2 equiv), toluene, 100 °C, 2 h then t-BuOK (0.1 equiv), t-BuOH (10 equiv), 80 °C, 2 h, 57% from 9; (d) Pd/C (10 wt % vs 12), H2, MeOH, rt, 8 h, 95%.
as acceptable for good B.A. and appropriate DPP-4 inhibitory activ-
ity. In this Letter we describe the structure–activity relationship,
pharmacokinetics, and pharmacological evaluation of the newly
synthesized structures, which are represented here as 7 and 8
(Fig. 2).
As shown in Scheme 1, the (R)-3-amino-3-methyl piperidine 13
was synthesized in five steps from the known compound 9.9 Fol-
lowing protection of the amine by a carbobenzyloxy (Cbz), hydro-
lysis of 10 gave the carboxylic acid 11. After Curtius rearrangement
using diphenylphosphoric azid (DPPA), the solution containing the
intermediate isocyanate was first washed with water to remove
by-products, and then treated with excess amount of tert-butyl
alcohol and catalytic amount of potassium tert-butoxide to gener-
ate the tert-butoxy carbonyl (Boc) protected amine 12. The target
amine unit 13 was obtained by hydrogenation of 12.
Compounds 7a–g having a pyrolo[3,2-d]pyrimidine were syn-
thesized as shown in Scheme 2.8 Reaction of 13 with 14 without
use of any base gave 15, but the next reaction from 15 to 16 re-
quired DBU to proceed. Reaction of 16 with ethyl bromoacetate
afforded 17, which underwent cyclization in the presence of lith-
ium tert-butoxide generated in situ from lithium amide and tert-
butyl alcohol to produce the common intermediate 18. The next
intermediate 19 was obtained by methylation following reaction
of methyl isothiocyanate (MeNCS) with 18. The next reaction,
O
N
O
O
N
O
NC
HN
OtBu
Me
NC
OtBu
Me
N NHBoc
NC
NC
OtBu
Me
OtBu
c
a
b
d
NHBoc
EtO2C
N
MeS
SMe
MeS
NHBoc
Ph
Ph
Ph
14
15
16
17
Ph
N
Ph
O
N
O
Me
Me
Me
NHBoc
NHBoc
EtO2C
H2N
NHBoc
g
f
N
N
e
N
N
N
N
O
N
H
MeS
N
OtBu
OtBu
OtBu
O
O
O
20
18
19
Ph
R2
O
R1
O
O
Me
Me
H
N
h
HCl
NH2
i,j,k
NHBoc
NHBoc
Me
N
N
O
N
N
N
N
N
N
N
O
N
OtBu
21a-g
OtBu
22a-g
R3
R3
O
N
O
O
R3
7a-g
Scheme 2. Reagents and conditions: (a) 13 (1.0 equiv), toluene, 60 °C, 4 h, quant.; (b) benzylamine (1.2 equiv), DBU (2.0 equiv), CH3CN, 80 °C, 5 h, 72%; (c) ethyl bromoacetate
(1.1 equiv), K2CO3 (1.2 equiv), DMF, 50 °C, 1 h, quant.; (d) LiNH2 (2.0 equiv), t-BuOH (20 equiv), CH3CN, heptane, rt, 1 h, 72%; (e) MeNCS (2.0 equiv), K2CO3 (2.0 equiv),
pyridine, 120 °C, 6 h then MeI (1.2 equiv), K2CO3 (2.0 equiv), acetone; (f) Na2WO4Á5H2O (1.1 equiv), H2O2 aq (10 equiv), MeOH–AcOH–H2O (9/3/1), 50 °C, 5 h, 50% from 18; (g)
MeI (1.2 equiv) for 21a–c, EtI (1.2 equiv) for 21d, ClCH2CONMe2 (1.2 equiv) for 21e, Br(CH2)3OTHP (1.2 equiv) for 21f, Br(CH2)3OEt (1.2 equiv) for 21g, K2CO3 (1.5 equiv), DMF,
50 °C, 3 h; (h) Pd/C (200 wt %), HCO2NH4 (300 equiv), MeOH, reflux, 4 h, 22a–c 62%, 22d 60%, 22e 72%, 22f 58%; (i) appropriate benzyl bromide (1.1 equiv), K2CO3 (2.0 equiv),
DMF, 70 °C, 3 h; (j) PhSO3H monohydrate (2.0 equiv), 70 °C, 3 h; (k) HCl–Et2O (1.1 equiv), CHCl3, 7a 21%, 7b 18%, 7c 23%, 7d 30%, 7e 12%, 7f 21%, 7g 25% from 22a–g.

7248
Y. Nishio et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7246–7249
Ph
Ph
Ph
O
N
Me
O
N
Me
Me
NHBoc
EtO2C
H2N
NHBoc
NHBoc
c
b
N
N
a
N
N
N
N
N
N
HS
OtBu
OtBu
OtBu
O
O
O
23
18
24
R5
R4
O
N
R5
R4
Me
H
N
O
NHBoc
HCl
Me
d,e,f
e,f
N
O
N
Me
NHBoc
N
N
N
NH2
N
N
N
N
OtBu
25
N
O
OtBu
26a-d
8a-d
O
Scheme 3. Reagents and conditions: (a) MeNCS (2.0 equiv), K2CO3 (2.0 equiv), pyridine, 120 °C, 6 h; (b) Na2WO4Á5H2O (1.1 equiv), H2O2aq (10 equiv), MeOH–AcOH–H2O (9/3/
1), 50 °C, 5 h, 72% from 18; (c) Pd/C (200 wt %), HCO2NH4 (300 equiv), MeOH, reflux, 4 h, 82%; (d) appropriate benzyl bromide (1.1 equiv), K2CO3 (2.0 equiv), DMF, 70 °C, 3 h;
(e) PhSO3H monohydrate (2.0 equiv), 70 °C, 3 h; (f) HCl–Et2O (1.1 equiv), CHCl3, 8a 14%, 8b 20%, 8c 21%, 8d 15% from 26a–d.
Table 2
which included oxidation and hydrolysis of 19 was carried out un-
der the same conditions and constructed the pyrolo[3,2-d]pyrimi-
In vitro activity of the synthesized DPP-4 inhibitors
dine scaffold 20. Alkylation of 20 with appropriate alkyl halides
gave 21a–g. As removal of benzyl protection in 21 did not proceed
under usual hydrogenation conditions, that is, hydrogen atmo-
sphere with a catalytic amount of palladium/carbon (Pd/C), excess
amount of Pd/C, and ammonium formate were used under reflux.
After benzylation of 22a–g, Boc protection and substitution of
the tert-butyl ester at the 7-position of the pyrolo[3,2-d]pyrimidine
structure were cleaved by benzensulphonic acid monohydrate, and
the purified free amines were converted to 7a–g by 1 N HCl–ether
solution.
As for compounds 8a–d having a deazahypoxanthine, they were
synthesized by almost the same route as that for 7a–g (Scheme 3).
The only difference was at the first step from the common interme-
diate 18–23, which did not include methylation like in the case of
20. The rest was the same as shown in Scheme 2.
In vitro activity of the synthesized compounds listed in Table 1
was examined using human plasma DPP-4. As expected, introduc-
ing steric hindrance at the 3-position of the piperidine moiety de-
creased the activity (6: IC50 = 1.8 nM vs 7a: IC50 = 36 nM) and
increased the value of log P* (0.1 for 6 and 0.28 for 7a). However,
compound 7a retained acceptable DPP-4 inhibitory activity.
Variation of substituents at the benzyl position is reported in
Table 1, that is, 7a–c. Changes at the R1 and R2 positions did not
come to improve the inhibitory activity for DPP-4. As for R3, the
R5
R4
HCl
O
N
Me
NH2
N
N
N
8a-d
Compounds
R4
Cl
CN
Me
CN
R5
DPP-4a IC50 (nM)
8a
8b
8c
8d
F
F
F
H
103
87
23
151
a
DPP-4 inhibitory activity is given as the mean of at least three experiments.
activity was kept even in the case of a polar substituent (7e:
IC50 = 13 nM, 7f: IC50 = 23 nM), but there seems to be limitation
to the extend of the linker (7g: IC50 = 122 nM). While the series
of compounds 7a–f exhibited good DPP-4 inhibitory activity, al-
most all compounds shown in Table 2 gave less satisfactory results
(IC50 >50 nM), except for 8c (IC50 = 23 nM).
Selectivity against DPP-810 and DPP-911, both of which belong
to the same gene family as DPP-4, was also assessed. Although
the biological roles of DPP-8 and DPP-9 are still unclear, it is impor-
tant to avoid potential side effects associated with these off-target
enzymes. All compounds disclosed in this Letter showed good
selectivity (IC50 >10 lM) against DPP-8/9.
Table 1
Among the compounds listed in Tables 1 and 2, 7b–f and 8c,
which showed IC50 values <30 nM in in vitro activity assessment,
were selected for in vitro pharmacokinetics evaluation. In terms
In vitro activity of the synthesized DPP-4 inhibitors
R2
N
R1
HCl
NH2
O
Me
Table 3
N
In vitro pharmacokinetic parameter of 7b–f and 8c
N
CYP inhibitiona IC50
(l
M)
MSb (ml/min/mg protein)
O
N
R3
7a-g
R3
1A2
2D6
3A4
Human
Rat
0.046
0.013
<0.01
<0.01
0.017
0.264
7b
7c
7d
7e
7f
>40
>40
26.8
26.3
>40
>40
3.4
>40
0.4
<0.165
0.2
14.3
10.2
20.2
9.4
13.4
7.7
0.082
0.056
0.021
0.019
0.016
0.048
Compounds
R1
R2
DPP-4a IC50 (nM)
6
—
—
H
F
F
F
F
F
F
—
1.8
36
28
27
12
13
23
122
7a
7b
7c
7d
7e
7f
CN
Cl
Me
CN
Cl
Me
Me
Me
Et
CH2CONMe2
(CH2)3OH
8c
11.9
a
CYP inhibition was evaluated after incubation for 10 min with human liver
microsomes and NADPH. Initial concentration of each compound was 10 M.
l
Cl
b
MS means metabolic stability, which was evaluated after incubation for 30 min
7g
Cl
(CH2)3OEt
with hepatic microsomes and NADPH. Initial concentration of each compound was
10 lM.
a
DPP-4 inhibitory activity is given as the mean of at least three experiments.

Y. Nishio et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7246–7249
7249
ZF rats was evaluated over a time-course of 0–24 h (Fig. 3). Com-
pound 7c showed the strongest inhibition against DPP-4 at
1.0 mg/kg around 2 h after administration and remained active
for up to 10 h. In addition, AUC of plasma DPP-4 inhibition by 7c
increased in a dose dependent manner. These results indicate that
7c acts as a potent inhibitor in ZF rats.
In conclusion, we found that compounds having (R)-3-amino-3-
methyl piperidine moiety, as represented here by compound 7c,
are potent DPP-4 inhibitors with good bioavailability and PK/PD
profiles. Unfortunately, compound 7c showed modest inhibition
against hERG (IC50 = 6 lM), making further evaluation of this com-
pound was suspended. To search for the more desirable com-
pounds, additional optimization study of these series of
compounds is currently in progress.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.10.101.
References and notes
1. For recent reviews see (a) Havale, S. H.; Pal, M. Bioorg. Med. Chem. 2009, 17,
1783; (b) Mest, H.-J.; Mentlein, R. Diabetlogia 2005, 48, 616; (c) Weber, A. E. J.
Med. Chem. 2004, 47, 4135; (d) Augustyns, K.; Van der Veken, P.; Senten, K.;
Haemers, A. Expert Opin. Ther. Patents 2003, 13, 499.
2. For recent references see (a) Holst, J. J. Curr. Opin. Endocrin. Diabetes 2005, 48,
616; (b) Knudsen, L. B. J. Med. Chem. 2004, 47, 4128.
3. Farilla, L.; Hui, H.; Bertolotto, C.; Kang, E.; Bulotta, A.; Di Mario, U.; Perfetti, R.
Endocrinology 2002, 143, 4397.
4. Kim, D.; Wamg, 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.; Petrowv, A.; Scapin, G.; Patel, S. B.; SinhaRoy, R.; Wu, J. K.; Wyvratt, M. J.;
Zhang, B. B.; Zhu, L.; Thornberry, N. A.; Weber, A. E. J. Med. Chem. 2005, 48, 141.
5. 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.
6. (a) Gwaltney, S. L.; Aertgeerts, K.; Feng, J.; Kaldor, S. W.; Kassel, D. B.; Manuel,
M.; Navre, M.; Prasad, G. S.; Shi, L.; Skene, R. J.; Stafford, J. A.; Wallace, M.; Wu,
R.; Ye, S.; Zhang, Z. Abstracts of Papers, 231st, Meeting of the American Chemical
Society, Atlanta, GA, 2006; American Chemical Society: Washington, DC, 2006;
MEDI-018.; (b) Feng, J.; Zhang, Z.; Wallace, M. B.; Sttaford, J. A.; Kaldor, S. W.;
Kassel, D. B.; Navre, M.; Shi, L.; Skene, R. J.; Asakawa, T.; Takeuchi, K.; Xu, R.;
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Chem. 2007, 50, 6450.
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Figure 3. (a) Time-dependent activity of 7c in plasma after oral single adminis-
tration in ZF rats; *p <0.05, **p <0.01 versus the vehicle-treated group (Dunnett’s
multiple comparison test). (b) Dose-dependent increase in AUC of plasma DPP-4
inhibition (inhibition% Â h) in ZF rats (test of linearity (lack of fit)); **p <0.01,
compound 7c-treated Zucker-fatty rats versus vehicle-treated Zucker-fatty rats;
Dunnett multiple comparison with two-sided significance level of 5%. Data are
given as the mean SEM (n = 5).
Table 4
Pharmacokinetic parameters of 6 and 7c in rat
Dose (mg/kg)
CL (ml/min/kg)
Vdss (L/kg)
T1/2 (h)
B.A (%)
6
iv
po
iv
1
10
1
52.7
—
51.5
—
4.2
—
14.4
—
—
1.30
—
37
7c
10. (a) Abbot, C. A.; Yu, D. M.; Woollatt, E.; Sutherland, G. R.; McCaughen, G. W.;
Gorrell, M. D. Eur. J. Biochem. 2000, 267, 6140; (b) Olsen, C.; Wagtmann, N. Gene
2002, 299, 185.
po
10
4.69
69.3
11. (a) 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. K.; Zaller, D.; Zhang, X.; Zhu, L.; Weber, A. E.; Thronberry, N.
A. Diabetes 2005, 54, 2988; (b) Burkey, B. F.; Hoffmann, P. K.; Hassiepen, U.;
Trappe, J.; Juedes, M.; Foley, J. E. Diabetes Obes. Metab. 2008, 10, 1057; (c) Wu,
J.-J.; Tang, H.-K.; Yeh, T.-K.; Chen, C.-M.; Shy, H.-C.; Chu, Y.-R.; Chien, C.-H.;
Tsai, T.-Y.; Huang, Y.-C.; Huang, Y.-L.; Huang, C.-H.; Tseng, H.-Y.; Jiaang, W.-T.;
Chao, Y.-S.; Chen, X. Biochem. Pharmacol. 2009, 78, 203.
of CYP enzymes inhibition, especially 2D6, 7c and 8c were clearly
better than the other compounds (Table 3). As for metabolic stabil-
ity in rats, 7c12 was selected as the most suitable for pharmacoki-
netic (PK) study in rat.
Compound 7c showed longer half-life (T1/2) than 6 with almost
the same clearance (CL). This result is in good correlation with that
of human serum protein binding (95.1% for 7c and 71.1% for 6),
which is believed to be associated with the value of log P* (1.17
for 7c and 0.1 for 6). Compound 7c distribution volume (Vdss) in-
creased, suggesting improved B.A., and its maximum concentration
time (Tmax) was observed at 3 h after administration. These find-
ings indicate that 7c possesses favorable oral B.A. and is therefore
suitable for PK/PD evaluation in Zucker-Fatty (ZF) rat (Table 4).
To assess the potency of single oral administration of 7c (0.1–
1.0 mg/kg), the inhibitory activity of this compound for DPP-4 in
12. Analytical data of 7c as a HCl salt: 1H NMR (400 MHz, DMSO-d6) d: 1.20 (3H, s),
1.48–1.61 (2H, m), 1.65–1.90 (2H, m), 2.35 (3H, s), 2.63–2.72 (1H, m), 2.75–
2.82 (1H, m), 2.90 (1H, d, J = 11.8 Hz), 3.05 (1H, d, J = 12.1 Hz), 3.12 (3H, s), 3.39
(3H, s), 5.42 (2H, s), 5.93 (1H, dd, J = 10.2, 2.3 Hz), 6.06 (1H, s), 6.94 (1H, td,
J = 8.4, 2.7 Hz), 7.22 (1H, dd, J = 8.4, 6.0 Hz), 8.19 (3H, br s). 13C NMR (100 MHz,
CDCl3-d3) d: 18.7, 21.1, 23.0, 27.9, 31.7, 33.3, 45.8, 53.4, 54.1, 59.5, 83.9, 106.1,
112.0 (2J(C, F) = 22.9 Hz), 113.3 (2J(C, F) = 21 Hz), 130.3 (4J(C, F) = 2.7 Hz), 131.5
(3J(C, F) = 7.6 Hz), 135.6, 139.3 (3J(C, F) = 6.5 Hz), 149.8, 151.38, 154.8, 161.6
(1J(C, F) = 241 Hz). IR (ATR): 2942, 1685, 1635 cmÀ1
414.2299 (calcd m/z 414.2300 for 22H28FN5O2 + H). Anal. Calcd for
22H28FN5O2 HCl 3/2H2O: C, 55.40; H, 6.76; N, 14.68. Found: C, 55.53; H,
6.59; N, 14.59.
. HRMS (ESI+): m/z
C
C