The highly potent and selective dipeptidyl peptidase IV inhibitors bearing a thienopyrimidine scaffold effectively treat type 2 diabetes

Article abstract of DOI:10.1016/j.ejmech.2010.10.016

New dipeptidyl peptidase IV inhibitors were designed based on Alogliptin using a scaffold-hopping strategy. All of the compounds constructed on a thienopyrimidine scaffold demonstrated good inhibition and selectivity for DPP-IV. Compound 10d exhibited subnanomolar (IC₅₀ = 0.33 nM) DPP-IV inhibitory activity, good in vivo efficacy and an acceptable pharmacokinetic profile. A pharmacokinetic-driven optimization of 10d may lead to a new class of clinical candidate DPP-IV inhibitors.

Full text of DOI:10.1016/j.ejmech.2010.10.016

European Journal of Medicinal Chemistry 46 (2011) 71e76
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
The highly potent and selective dipeptidyl peptidase IV inhibitors bearing
a thienopyrimidine scaffold effectively treat type 2 diabetes
,
,
Jifeng Deng a 1, Li Peng a 1, Guicheng Zhang a, Xiaobing Lan a, Chufang Li a, Fuxin Chen a, Yayao Zhou a,
Zuoxian Lin a, Ling Chen a, Renke Dai a, Hongjiang Xu b, Ling Yang b, Xiquan Zhang b, Wenhui Hu a
,
*
a Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kai Yuan Avenue, Guangzhou Science Park, Guangzhou 510530, China
b Jiangsu Chia-Tai Tianqing Pharmaceutical Co. Ltd, No. 8 Julong North Rd., Xinpu Lianyungang Jiangsu 222006, China
a r t i c l e i n f o
a b s t r a c t
Article history:
New dipeptidyl peptidase IV inhibitors were designed based on Alogliptin using a scaffold-hopping
strategy. All of the compounds constructed on a thienopyrimidine scaffold demonstrated good inhibition
and selectivity for DPP-IV. Compound 10d exhibited subnanomolar (IC50 ¼ 0.33 nM) DPP-IV inhibitory
activity, good in vivo efficacy and an acceptable pharmacokinetic profile. A pharmacokinetic-driven
optimization of 10d may lead to a new class of clinical candidate DPP-IV inhibitors.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Received 19 June 2010
Received in revised form
15 October 2010
Accepted 16 October 2010
Available online 26 October 2010
Keywords:
DPP-IV inhibitor
Type 2 diabetes
Scaffold hopping
1. Introduction
delay or prevent the loss of functional
slow disease progression [9].
b-cell mass and, therefore,
The high prevalence of type 2 diabetes (T2D) is a major global
public health concern and affects approximately 180 million
patients worldwide, with yet more people still undiagnosed or
at the pre-diabetes stage. T2D, known as non-insulin-dependent
diabetes mellitus, is a chronic, severe and increasingly prevalent
disease [1,2]. Although many standard therapies for T2D are avail-
able in the market, none of them can stop disease progression.
Therefore, new therapeutic agents are still needed to combat dia-
betes. Dipeptidyl peptidase IV (DPP-IV) inhibitors have emerged as
a major breakthrough in anti-diabetic drug discovery. By blocking
the DPP-IV enzyme, these inhibitors can prolong the half-life of
active glucagons like peptide-1 (GLP-1) and glucose-dependent
insulinotropic peptide (GIP) and thus stimulate insulin biosynthesis
and secretion. These inhibitors finally represent a way to effectively
and safely control blood glucose in diabetic patients [3e5].
Based on these observations, the inhibition of DPP-IV is an
attractive strategy for the treatment of diabetes, and three DPP-IV
inhibitors have been launched in the US or EU: Sitagliptin 1 [10,11],
Vildagliptin 2 [12e14] and Saxagliptin 3 [15] (Fig. 1). Alogliptin 4
[16] has twice the potency of Sitagliptin as a DPP-IV inhibitor and is
still in the FDA review process due to new guidelines for cardiac
safety data [17e19].
For a chronic disease, a drug that can produce similar beneficial
effects with less toxicity at low doses is always welcome. Though
there are several new drugs been marketed such as Sitagliptin,
Vildagliptin and Saxagliptin, and highly potent DPP-IV inhibitors
are still in great need. While many novel inhibitors have been
reported based on 1, 2 and 3, fewer analogs of Alogliptin have been
disclosed, leaving a large amount of chemical space unexplored.
Thus, our objective was to discover more inhibitors by modifying
this drug candidate using a scaffold-hopping strategy [20]. Alog-
liptin was previously obtained by replacing the scaffold of quina-
zolinone compound with a pyrimidinedione. Herein, we report the
discovery of highly potent DPP-IV inhibitors by incorporating
a thienyl ring into Alogliptin’s structure.
DPP-IV inhibitors possess several advantages over traditional
anti-diabetic drugs, such as not leading to increased bodyweight or
causing hypoglycemia, and are generally well-tolerated [6e8]. DPP-
IV inhibitors may be disease-modifying therapies because they can
2. Chemistry
* Corresponding author. Tel.: þ86 20 32015211; fax: þ86 20 32015299.
E-mail address: hu_wenhui@gibh.ac.cn (W. Hu).
Joint first authors: these two authors contribute equally to this work.
The synthesis of compounds 10aee is outlined in Scheme 1
which is similar to the synthetic protocol of Alogliptin [16].
1
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.10.016

72
J. Deng et al. / European Journal of Medicinal Chemistry 46 (2011) 71e76
Fig. 1. Important DPP-IV inhibitors.
Briefly, the commercially available starting materials 5aec were
either heated with urea [9] between 190 ꢀC and 200 ꢀC, or reacted
with chlorosulfonyl isocyanate at ꢁ60 ꢀC to generate the corre-
sponding thienopyrimidines [21] 6aee. Chlorination of 6aee with
phosphoryl trichloride yielded 7aee, which were hydrolyzed with
aqueous sodium hydroxide to give key intermediates 8aee. Selec-
tive N-alkylation was performed using a previously published
method [22] to produce compounds 9aee. The final compounds,
10aee, were obtained in high yields by the amination of the chloro
precursors 9aee with 3-(R)-aminopiperidine.
Remarkably, all the compounds except 10c were more active
than Alogliptin in vitro. Among them, compound 10d was 10 times
more active than Alogliptin. Additionally, all of the compounds in
the series demonstrated good selectivity, while none of them
inhibited DPP-8 or DPP-9. These compounds did not inhibit the
hERG ion channel and may have less risk for cardiac side effect,
which is a major concern for anti-diabetic drugs.
Compound 10a, which has no substituents on the scaffold,
showed good subnanomolar inhibition (0.87 nM, in vitro). The
addition of a methyl to the 3-position (10b) decreased the activity
slightly (1.32 nM). However, simply switching the methyl group
(10c) to a trifluoromethyl group greatly decreased the activity
(88.82 nM). Rotating the thiophenyl ring on the scaffold of 10a led
to compound 10d, which had a 2-fold increased activity (0.33 nM)
and was the most active compound in this study. Adding a 1-methyl
group onto compound 10d resulted in compound 10e, which was
still more active than Alogliptin in vitro. This simple investigation
demonstrates that the orientation of the thiophenyl moiety is not
important for inhibition and that functional groups on the ring do
3. Results and discussion
All the compounds listed in Table 1 were evaluated for activity
against the DPP-IV enzyme and for selectivity against DPP-8 and
DPP-9 [10,23]. We also conducted the human Ether-à-go-go Related
Gene (hERG) study in accordance with the new FDA guidelines for
evaluating the cardiovascular risk of new therapies for the treat-
ment of T2D [24,25].
Scheme 1. Synthesis of compounds 10aee. Reagents: (a) urea, 190e200 ꢀC; (b) ClSO2NCO, CH2Cl2, ꢁ60 ꢀC; (c) POCl3, DIEA, reflux; (d) 1 N NaOH; (e) 2-CNPhCH2Br, NaH, LiBr;
(f) 3-(R)-aminopiperidine, NaHCO3, 150 ꢀC.

J. Deng et al. / European Journal of Medicinal Chemistry 46 (2011) 71e76
73
Table 1
Inhibitory properties of compounds 10aee.
No.
X
Y
DPP-IV
DPP-8
DPP-9
hERG (mM)
IC50 (nM) IC50 (nM) IC50 (nM)
10a
10b
10c
10d
10e
Alogliptin
S
S
S
CH
CCH3
CCF3
S
0.87
1.32
88.82
0.33
1.72
3.4
>25,000
>25,000
>25,000
>25,000
>25,000
>25,000
>25,000
>25,000
>25,000
>25,000
>25,000
>25,000
484.6
376.3
NTa
517.4
203.5
>30b
CH
CCH3
S
a
NT ¼ not tested.
b
Reported data, see Ref. [26].
exert some influence on activity. The IC50 value for Alogliptin under
our experimental conditions was 3.4 nM, which is in agreement
with the literature value (<10 nM) [26].
The most active compound in Table 1, 10d, was chosen for in vivo
evaluation (Figs. 2 and 3). Oral administration of 10d in rats
significantly reduced both blood glucose levels in an oral glucose
tolerance test (OGTT) and serum DPP-IV activity in ICR male mice.
The initial results showed that the in vivo efficacy of compound 10d
is as good as Alogliptin.
Fig. 3. Effect of compounds on serum DPP-IV activity in ICR male mice. Specific activity
of serum DPP-IV tested in male C57BL/6J mice fed a high-fat diet. Compound 10d
(3 mg/kg), Alogliptin (3 mg/kg), or vehicle was orally administered. Data are mean -
ꢂ S.E.M (n ¼ 5/group).
Table 2 shows the pharmacokinetic profile of compound 10d in
Sprague Dawley Rats and Beagle Dogs. Both the T1/2 and the oral
bioavailability are acceptable. In our experiment, the oral
bioavailability (F) in Sprague Dawley Rats of 10d was 23.7% while in
beagle dogs was 42.3%, which was less than the reported value
(F ¼ 43% in rats, F ¼ 68% in dogs) for Alogliptin [16]. Thus,
compound 10d afforded desirable pharmacokinetic (PK) and
pharmacodynamic (PD) profiles and is worthy of further evaluation
as a drug candidate.
Even though compound 10d could be considered as a potential
drug candidate, there are still significant opportunities to improve
its drug-like properties. For example, there is a disconnect between
the in vitro and in vivo activity: compound 10d was more than 10
times more active than Alogliptin in vitro, but its in vivo activity was
a little lower than that of Alogliptin. The unexpectedly low in vivo
activity could be the result of moderate PK properties. Thus, a PK-
driven optimization to improve oral bioavailability of compounds
in this series will be required to generate new drug candidates in
the DPP-IV inhibitor class of therapeutics.
4. Conclusions
In summary, we have successfully generated highly potent DPP-
IV inhibitors 10a and 10d with IC50 values in the subnanomolar
range by using a scaffold replacement technique and by using
Alogliptin as a starting point. All of the compounds investigated in
the study showed good selectivity for DPP-IV over DPP-8 and DPP-9
and did not block the hERG ion channel. Compound 10d met some
of the requirements for being a drug candidate in terms of efficacy,
selectivity and acceptable pharmacokinetic profile. Most impor-
tantly, we provided a novel series of compounds with the potential
for PK-driven optimization to facilitate the discovery of anti-dia-
betic drugs.
5. Experimental
Fig. 2. Effect of compounds in the oral glucose tolerance test. Changes of blood glucose
levels (A) and AUC values of delta blood glucose between 0 and 120 min (B) in an oral
glucose tolerance test in male C57BL/6J mice fed a high-fat diet. Compound 10d (3 mg/
kg), Alogliptin (3 mg/kg), or vehicle was orally administered 30 min prior to an oral
glucose challenge (2.5 g/kg). Data are mean ꢂ S.E.M (n ¼ 5/group).
5.1. General chemistry procedures
1H NMR spectra were recorded on a Bruker Avance 400 and 13
NMR spectra on a Bruker Avance 500. Chemical shifts are expressed
C

74
J. Deng et al. / European Journal of Medicinal Chemistry 46 (2011) 71e76
Table 2
Selected PK parameters for compound 10d in rats and dogs.
Species
No.
T1/2 (h)
AUC0-t
(
m
g h mLꢁ1
)
Tmax (h)
Cmax
(
m
g mLꢁ1
)
MRT (h)
CLp (mL minꢁ1 kgꢁ1
)
F (%)
Sprague Dawley rats
Sprague Dawley rats
Beagle dogs
10d i.v. (10 mg/kg)
10d p.o. (20 mg/kg)
10d i.v. (10 mg/kg)
10d p.o. (20 mg/kg)
5.9 ꢂ 3.2
1.48 ꢂ 0.41
0.69 ꢂ 0.19
13.26 ꢂ 3.18
11.21 ꢂ 1.93
0.04 ꢂ 0.02
0.59 ꢂ 0.71
0.10 ꢂ 0.04
0.91 ꢂ 0.20
1.23 ꢂ 0.35
0.17 ꢂ 0.08
5.75 ꢂ 1.14
2.07 ꢂ 0.85
2.7 ꢂ 0.32
6.7 ꢂ 1.0
6.0 ꢂ 1.1
9.0 ꢂ 2.1
6.97 ꢂ 1.58
6.40 ꢂ 2.98
22.18 ꢂ 8.83
11.5 ꢂ 3.17
7.0 ꢂ 3.9
10.14 ꢂ 5.29
11.29 ꢂ 5.53
23.7
42.3
Beagle dogs
i.v., intravenous injection; p.o., oral administration.
in parts per million (ppm), and coupling constants are expressed in
Hertz (Hz). Splitting patterns describe apparent multiplicities and
are designated as s (singlet), d (doublet), t (triplet), q (quartet),
m (multiplet) or br (broad). Low-resolution mass spectra (MS) and
compound purity data were acquired on a Waters ZQ LC/MS single
quadrupole system equipped with an electrospray ionization (ESI)
source, a UV detector (220 nm and 254 nm), and an evaporative
light scattering detector (ELSD). Preparative HPLC was conducted
on the same system using mixtures of TFA (0.05%) buffered water
and acetonitrile. Thin-layer chromatography was performed on
0.25 mm Merck silica gel plates (60F-254) and visualized with UV
light, 5% ethanolic phosphomolybdic acid, ninhydrin or p-ani-
saldehyde solution. Flash column chromatography was performed
on silica gel (230e400 mesh, Merck).
5.3.3. 5-(Trifluoromethyl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-
dione (6c)
The title compound was prepared according to the reported
procedure [29] and was isolated as a white solid (4.7 g, yield 99.0%).
1H NMR (400 MHz, DMSO-d6):
d 12.17 (s, 1H), 11.28 (s, 1H), 7.75 (s,
1H); MS (ESI) C7H3N2O2SF3 [M ꢁ H]ꢁ 234.9.
5.3.4. Thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione (6d)
Yield: 59%. 1H NMR (400 MHz, DMSO-d6):
d 11.60e11.10 (br, s,
2H), 8.10 (d, J ¼ 5.2 Hz, 1H), 6.9 (d, J ¼ 5.2 Hz, 1H); MS: (ESI)
C6H4N2O2S [M þ H]þ 169.1.
5.3.5. 7-Methylthieno[3,2-d]pyrimidine-2,4(1H,3H)-dione (6e)
Yield: 50.0%. 1H NMR (400 MHz, DMSO-d6):
d 11.42 (s, 1H), 11.22
(s,1H), 7.69 (s,1H), 2.20 (s, 3H); MS (ESI) C7H6N2O2S [M þ H]þ 182.9.
5.4. Synthesis of compounds 7aed
5.2. Synthesis of compounds 5aec
5.2.1. Methyl 2-aminothiophene-3-carboxylate (5a)
The title compound was prepared according to the previously
reported procedure [27]. A total of 132 g of crude product was
obtained and used for the next reaction without further
purification.
5.4.1. 2,4-Dichlorothieno[2,3-d]pyrimidine (7a)
A total of 0.8 mL N,N-dimethylaniline was added to 3.0 g of
thieno[2,3-d]pyrimidine-2,4-(1H,3H)-dione 6a in 20 mL POCl3. The
mixture was then heated under reflux for 16 h. Excess POCl3 was
removed in vacuo, and the resulting residue was treated with ice
water to yield a precipitate. The solid was collected by filtration,
washed with water and dried over a funnel to afford solid 7a (1.3 g,
5.2.2. Methyl-2-amino-4-methylthiophene-3-carboxylate (5b)
The title compound was prepared according to the previously
reported procedure [28]. A total of 21.0 g of 5b was obtained in
yield 35.5%). 1H NMR (400 MHz, CDCl3):
7.43 (d, J ¼ 6.4 Hz, 1H).
d
7.62 (d, J ¼ 6.4 Hz, 1H),
60.5% yield. 1H NMR (400 MHz, CDCl3):
3.81 (s, 1H), 2.26 (s, 3H).
d 6.06 (s, 2H), 5.81 (s, 1H),
5.4.2. 2,4-Dichloro-5-methylthieno[2,3-d]pyrimidine (7b)
Yield: 31.0%. 1H NMR (400 MHz, CDCl3):
(s, 1H), 2.66 (s, 3H).
d 7.20 (s, 1H), 2.93
5.2.3. Methyl-2-amino-4-(trifluoromethyl)thiophene-3-carboxylate
(5c)
The title compound was prepared according to the reported
procedure [29] and was isolated as a brown solid (6.0 g, yield
5.4.3. 2,4-Dichloro-5-(trifluoromethyl)thieno[2,3-d]pyrimidine (7c)
Yield: 53.0%. 1H NMR (400 MHz, DMSO-d6):
d 8.88 (s, 1H).
30.0%). 1H NMR (400 MHz, CDCl3):
d 6.75 (s, 1H), 6.26 (s, 2H), 3.84
(s, 3H); MS (ESI) C7H6NO2F3 [M ꢁ H]ꢁ 224.0.
5.4.4. 2,4-Dichlorothieno[3,2-d]pyrimidine (7d)
Yield: 67%. 1H NMR (400 MHz, CDCl3):
d
8.13 (d, J ¼ 5.5 Hz, 1H),
7.55 (d, J ¼ 5.5 Hz, 1H); MS: (ESI) C6H2Cl2N2S [M þ H]þ 206.9.
5.3. Synthesis of compounds 6aee
5.4.5. 2,4-Dichloro-7-methylthieno[3,2-d]pyrimidine (7e)
5.3.1. Thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (6a)
Yield: 43.4%. 1H NMR (400 MHz, CDCl3):
d 7.74 (s, 1H), 2.49
A
mixture of methyl 2-aminothiophene-3-carboxylate 5a
(s, 3H); MS (ESI) C7H4N2Cl2S [M þ H]þ 218.9.
(20.0 g, 0.13 mol) and urea (60 g, 1.0 mol) was heated at 200 ꢀC for
2 h. The reaction mixture was cooled and poured into a sodium
hydroxide solution, and any insoluble material was removed by
filtration. The filtrate was then acidified with 2 N HCl to give a white
precipitate, which was collected by filtration, washed with water
and dried on a funnel to provide the title compound 6a (13.7 g,
yield 64%). MS (ESI): C6H4N2O2S [M ꢁ H]ꢁ 167.1.
5.5. Synthesis of compounds 8aed
5.5.1. 2-Chlorothieno-[2,3-d]-pyrimidin-4(3H)-one (8a)
A mixture of 10.2 g of 2,4-dichlorothieno-[2,3-d]-pyrimidine 7a,
120 mL 1 N NaOH and 20 mL of THF was stirred at room temper-
ature under N2 for 4 h. The solution was then chilled and adjusted
to pH 5 with AcOH. The resulting precipitate was collected, washed
with water and dried to afford 8a as a solid (8.5 g, yield 92%). MS
(ESI) C6H3ClN2OS [M ꢁ H]ꢁ 185.1.
5.3.2. 5-Methylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (6b)
The title compound was prepared from methyl-2-amino-
4-methylthiophene-3-carboxylate 5b according to the previously
reported procedure [28] in 57.0% yield. 1H NMR (400 MHz, DMSO-
d6):
d
6.07 (s, 2H), 5.82 (s, 1H), 3.82 (s, 1H), 2.26 (s, 3H); MS (ESI)
5.5.2. 2-Chloro-5-methylthieno[2,3-d]pyrimidin-4(3H)-one (8b)
Yield: 90.3%. MS (ESI) C7H5ClN2OS [M ꢁ H]ꢁ 201.1.
C7H6N2O2S [M ꢁ H]ꢁ 181.1.

J. Deng et al. / European Journal of Medicinal Chemistry 46 (2011) 71e76
75
5.5.3. 2-Chloro-5-(trifluoromethyl)thieno[2,3-d]pyrimidin-4(3H)-
one (8c)
vacuo and then purified by flash chromatography to give the title
compound 10a (12.35 g, yield 77.8%). 1H NMR (400 MHz, CDCl3):
d 7.71e7.65 (m, 1H), 7.51e7.47 (m, 1H), 7.36e7.32 (m, 2H), 7.08e7.04
(m, 2H), 5.56e5.47 (m, 2H), 3.27e3.24 (m, 1H), 3.14e3.11 (m, 1H),
2.97e2.91 (m, 1H), 2.85e2.78 (m, 1H), 2.64e2.59 (m, 1H), 1.96e1.91
(m, 1H), 1.78e1.73 (m, 1H), 1.68e1.61 (m, 1H), 1.25e1.21 (m, 1H); 13C
Yield: 96.0%. 1H NMR (400 MHz, DMSO-d6):
d
8.29 (s, 1H); MS
(ESI) C7H2N2 F3ClOS [M ꢁ H]ꢁ 253.0.
5.5.4. 2-Chlorothieno[3,2-d]pyrimidin-4(3H)-one (8d)
Yield: 90%. 1H NMR (400 MHz, DMSO-d6):
13.47 (s, 1H), 8.2
d
NMR (125 MHz, DMSO-d6) d 163.87, 159.06, 157.22, 141.25, 133.88,
(d, J ¼ 5.5 Hz, 1H), 7.55 (d, J ¼ 5.5 Hz, 1H); MS: (ESI) C6H3ClN2OS
133.44, 128.32, 127.56, 122.19, 122.14, 120.22, 117.58, 110.51, 59.07,
51.01, 47.69, 46.74, 33.60, 23.51; MS (ESI) C19H19N5OS [M þ H]þ
366.1.
[M þ H]þ 187.0.
5.5.5. 2-Chloro-7-methylthieno[3,2-d]pyrimidin-4(3H)-one (8e)
Yield: 57%. 1H NMR (400 MHz, DMSO-d6):
d
7.83 (s, 1H), 2.25
5.7.2. (R)-2-((2-(3-Aminopiperidin-1-yl)-5-methyl-4-oxothieno[2,3-
(s, 3H); MS (ESI) C7H5N2ClOS [M ꢁ H]ꢁ 199.1.
d]pyrimidin-3(4H)yl)methyl) benzonitrile (10b)
Yield: 45%. 1H NMR (400 MHz, CDCl3):
d 7.5e7.564 (m, 2H),
7.50e7.48 (m, 1H), 7.44e7.40 (m, 1H), 6.66 (s, 1H), 5.31 (s, 2H),
3.26e3.23 (m, 1H), 3.15e3.11 (m, 1H), 2.99e2.93 (m, 1H), 286e2.79
(m, 1H), 2.66e2.61 (m, 1H), 2.51 (s, 3H), 1.99e1.95 (m, 1H),
1.82e1.76 (m, 1H), 1.68e1.57 (m, 1H), 1.42e1.26 (m, 1H); 13C NMR
5.6. Synthesis of compounds 9aed
5.6.1. 2-((2-Chloro-4-oxothieno-[2,3-d]pyrimidin-3-(4H)-yl)-
methyl)benzonitrile (9a)
NaH (2.1 g, 51.6 mmol) was added to a stirred solution of 8a
(8.4 g, 44.9 mmol) in DME (120 mL) and DMF (30 mL) at 0 ꢀC.
Twenty minutes later, LiBr (7.9 g, 89.7 mmol) was added, and the
mixture was allowed to warm to room temperature. After 15 min,
(125 MHz, DMSO-d6)
d 164.24, 159.54, 156.99, 141.45, 135.82,
133.95, 133.89, 133.43, 127.61, 118.87, 117.67, 116.84, 110.53, 55.85,
51.29, 46.99, 46.46, 30.77, 22.84, 16.39; MS (ESI) C20H21N5OS,
[M ꢁ H]ꢁ 380.1.
a
-bromo-o-tolunitrile (10.15 g, 51.6 mmol) was then added, and the
5.7.3. (R)-2-((2-(3-Aminopiperidin-1-yl)-4-oxo-5-(trifluoromethyl)
thieno[2,3-d]pyrimidin-3(4H)-yl)methyl)benzonitrile (10c)
mixture was heated at 65 ꢀC overnight. After cooling, the mixture
was poured into water (1000 mL) with stirring to yield a precipi-
tate. This solid was filtered and dried to give the title compound 9a
Yield: 82.0%. 1H NMR (400 MHz, CDCl3):
d 7.68e7.65 (m, 1H),
7.55e7.51 (m, 2H), 7.36 (t, J ¼ 7.6 Hz,1H), 7.14 (d, J ¼ 8.0 Hz,1H), 5.50
(s, 2H), 3.35e3.31 (m, 1H), 3.22e3.19 (m, 1H), 3.02e2.95 (m, 1H),
2.90e2.83 (m, 1H), 2.67e2.62 (m, 1H), 1.98e1.94 (m, 1H), 1.80e1.75
(m, 1H), 1.70e1.64 (m, 1H), 1.31e1.20 (m, 1H); 13C NMR (125 MHz,
(11 g, yield 81%). 1H NMR (400 MHz, CDCl3):
d 7.78e7.70 (m, 1H),
7.58e7.49 (m, 2H), 7.45e7.40 (m, 1H), 7.33 (d, J ¼ 6.4 Hz, 1H), 6.87
(d, J ¼ 6.4 Hz, 1H), 5.75 (s, 2H).
DMSO-d6)
127.70,
d
166.14, 158.25, 157.48, 140.95, 133.89, 133.52, 128.36,
122.65,
5.6.2. 2-((2-Chloro-5-methyl-4-oxothieno[2,3-d]pyrimidin-3(4H)-
125.10(d,JC-F ¼ 7.0 Hz),124.45(q,JC-F ¼ 37.5 Hz),
yl)methyl)benzonitrile (9b)
120.50, 117.56, 114.98, 110.25, 58.55, 50.74, 47.57, 33.41, 23.38; MS
Yield: 68.7%. 1H NMR (400 MHz, CDCl3):
d 7.64 (s, 1H), 7.61e7.59
(ESI) C20H18F3N5OS [M þ H]þ 434.0.
(m, 2H), 7.48e7.44 (m,1H), 6.86 (s,1H), 5.48 (s, 2H), 2.56 (s, 3H); MS
(ESI) C15H10ClN3OS [M þ H]þ 316.0.
5.7.4. (R)-2-((2-(3-Aminopiperidin-1-yl)-4-oxothieno[3,2-d]
pyrimidin-3(4H)-yl)methyl)benzonitrile (10d)
The title compound was prepared from 2-((2-chloro-4-oxo-
thieno[3,2-d]pyrimidin-3(4H)-yl) methyl)benzonitrile (9d) in 77.8%
yield by a method analogous to that used to make 10a. 1H NMR
5.6.3. 2-((2-Chloro-4-oxo-5-(trifluoromethyl)thieno[2,3-d]
pyrimidin-3(4H)-yl)methyl)benzonitrile (9c)
Yield: 70.0%. 1H NMR (400 MHz, CDCl3):
d 7.78 (s, 1H), 7.74e7.72
(m, 2H), 7.63e7.54 (m, 1H), 7.46e7.41 (m, 1H), 7.15 (d, J ¼ 8 Hz, 1H),
(400 MHz, CD3OD):
d
7.98 (dd, J ¼ 2 Hz, 2 Hz, 1H), 7.73 (d, J ¼ 8 Hz,
5.75 (s, 2H); MS (ESI) C15H7N3F3ClOS [M þ H]þ 370.0.
1H), 7.57 (t, J ¼ 7.6 Hz, 1H), 7.42 (t, J ¼ 7.6 Hz, 1H), 7.25 (d, J ¼ 4.8 Hz,
1H), 7.12 (d, J ¼ 8 Hz, 1H), 5.56 (s, 2H), 3.39 (m, 1H), 3.21 (m, 1H),
2.88 (m, 2H), 2.67 (m, 1H), 1.96 (m, 1H), 1.76 (m, 1H), 1.67 (m, 1H),
5.6.4. 2-((2-Chloro-4-oxothieno[3,2-d]pyrimidin-3(4H)-yl)methyl)
benzonitrile (9d)
1.25 (m, 1H); 13C NMR (125 MHz, DMSO-d6)
d 158.52, 158.08,
The title compound was prepared from 2-chlorothieno[3,2-d]
pyrimidin-4(3H)-one 8d in 96.8% yield by a method analogous to
155.97, 140.86, 135.58, 133.50, 133.03, 127.90, 126.98, 124.70, 118.10,
117.16, 110.06, 58.77, 50.77, 47.29, 46.04, 33.12, 23.11; MS (ESI)
C19H19N5OS [M þ H]þ 366.1.
that used to make 9a. 1H NMR (400 MHz, DMSO-d6):
d 7.88
(d, J ¼ 5.2 Hz, 1H), 7.73 (d, J ¼ 7.6 Hz, 1H), 7.55 (t, J ¼ 7.6 Hz, 1H), 7.43
(t, J ¼ 7.6 Hz, 1H), 7.32 (d, J ¼ 5.2 Hz, 1H), 7.16 (d, J ¼ 8 Hz, 1H),
5.77 (s, 2H); MS (ESI) C14H8ClN3OS [M þ H]þ 302.0.
5.7.5. (R)-2-((2-(3-Aminopiperidin-1-yl)-7-methyl-4-oxothieno[3,2-
d]pyrimidin-3(4H)-yl)methyl) benzonitrile (10e)
Yield: 55.5%. 1H NMR (400 MHz, CDCl3):
d
7.69 (d, J ¼ 12.8 Hz,
5.6.5. 2-((2-Chloro-7-methyl-4-oxothieno[3,2-d]pyrimidin-3(4H)-
yl)methyl)benzonitrile (9e)
1H), 7.59e7.53 (m, 2H), 7.40 (t, J ¼ 7.6 Hz,1H), 7.08(d, J ¼ 8.0 Hz,1H),
5.55 (s, 2H), 3.45e3.39 (m, 1H), 3.15 (d, J ¼ 12.4 Hz, 1H), 2.95e2.81
(m, 2H), 2.74e2.69 (m, 1H), 2.34 (s, 3H), 1.97e1.94 (m, 1H),
1.78e1.74 (m, 2H), 1.35e1.26 (m, 1H); 13C NMR (125 MHz, DMSO-
Yield: 83%. MS (ESI) C15H10N3ClOS [M þ H]þ 316.0.
5.7. Synthesis of compounds 10aee
d6) d 158.47, 157.63, 154.47, 140.95, 136.04, 133.43, 133.01, 127.88,
127.71, 127.28, 118.32, 117.27, 110.30, 54.27, 51.17, 46.33, 46.07, 29.22,
5.7.1. (R)-2-((2-(3-Aminopiperidin-1-yl)-4-oxothieno-[2,3-d]-
pyrimidin-3(4H)-yl)methyl) benzonitrile (10a)
22.12, 12.37; MS (ESI) C20H21N5OS [M þ H]þ 380.1.
A mixture of 9a (13.1 g, 43.4 mmol), 3-(R)-aminopiperidine
dihydrochloride (11.5 g, 66.0 mmol) and NaHCO3 (17.4 g,
173.6 mmol) in 300 mL of ethanol in a sealed tube was heated at
150 ꢀC for 6 h. The reaction mixture was then cooled to room
temperature and filtered. The resulting filtrate was concentrated in
5.8. Biological assays for DPP-IV, DPP-8 and DPP-9 in vitro
DPP inhibition assay was performed according to a slightly
modified method of Kim et al. [10]. An enzyme and chemicals
(diluted in an assay buffer: 50 mM Tris, pH 7.5 and 0.1% bovine

76
J. Deng et al. / European Journal of Medicinal Chemistry 46 (2011) 71e76
serum albumin, pH 7.4) were mixed in a black 96-well plate, and
then the plate was incubated for 10 min at room temperature to
allow inhibitor/enzyme interaction. The enzyme reaction was
group. 5
bated at room temperature for 5 min. 10
and 20 L buffer were then added and mixed. Fluorescence at
m
L rat serum mixing with 35
m
L 80 mM MgCl2 was incu-
mL 0.1 mM Gly-Pro-AMC
m
started by the addition of 100
and incubated for 20 min at room temperature. The final concen-
tration of Gly-Pro-AMC was 50 mol/L. Gly-Pro-AMC is cleaved by
m
mol/L Gly-Pro-AMC (in assay buffer)
460 nm with excitation of 380 nm was measured over 18 min with
3-min interval. These measurements were used to calculate the
specific activity of serum DPP-IV.
m
the enzyme to release the fluorescent aminomethylcoumarin
(AMC). Liberation of AMC was monitored at an excitation wave-
length of 360 nm and an emission wavelength of 460 nm with
a microplate reader (synergy HT, BIO-TEK, USA). Three separate
experiments were performed and means of IC50 values were
calculated by a curve-fitting program (GraphPad Software Inc., San
Diego, CA) [10,23].
Acknowledgments
This research was supported by the Bureau of Science and
Technology of Guangzhou Municipality, China, by Grant No.
2009Z1-E871. We thank Hui Xie for helpful PK discussions.
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198
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