Synthesis and lipid-lowering evaluation of 3-methyl-1 H-purine-2,6-dione derivatives as potent and orally available anti-obesityagents

European Journal of Medicinal Chemistry 87 (2014) 595 e 610

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Synthesis and lipid-lowering evaluation of 3-methyl-1H-purine-2,6-

dione derivatives as potent and orally available anti-obesity agents

a

,

1

a

,

1

a

,

1

a

b

Linhong He , Heying Pei , Liang Ma , Yuzhi Pu , Jinying Chen , Zhuowei Liu ,

a

b

Yan Ran , Lei Lei , Suhong Fu , Minghai Tang , Aihua Peng , Chaofeng Long ,

a, *

Lijuan Chen

a

State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, West China Medical School,

Sichuan University, Chengdu, Sichuan 610041, PR China

Guangdong ZhongSheng Pharmaceutical Co. Ltd., Dongguan 440100, Guangdong, PR China

b

a r t i c l e i n f o

a b s t r a c t

Article history:

Received 8 August 2014

Received in revised form

Obesity accompanied with metabolic disorder is often complicated with a strong link of dyslipidemia and

insulin resistance, whose indicator is the excess accumulation of triglycerides (TG) in cells. Consideration

the idea of lipid-lowering and improving insulin resistance, 34 novel compounds by combination the

xanthine scaffold with the chain of Rosiglitazone have been synthesized. Among them, several com-

pounds showed efﬁciency on reducing TG in 3T3-L1 adipoctyes, and 11c exhibited the most optimal

capacity in lipid-lowering and improving obese clinical symptoms in DIO mice. Furthermore, the hy-

drochloride of 11c (11c$HCl) showed excellent bioavailability, 58.94%, over 2 folds than that (28.03%) of

25 September 2014

Accepted 29 September 2014

Available online 30 September 2014

Keywords:

Triglycerides

Insulin resistance

Lipid-lowering

Diet induced obesity

11c, and the anti-obesity effect of 11c$HCl at 50 mg/kg dose was better than that of Metformin at 150 mg/

kg dose in DIO mice, almost reversed HFD to a normal level. Thus, 11c$HCl might be a potent and orally

available anti-obesity agent via alleviating the obese clinical symptoms, body fat, improving serum pa-

rameters and insulin resistance and TG clearance in liver.

©

2014 Published by Elsevier Masson SAS.

1. Introduction

dramatically increasing prevalence of obesity around the world

1e4], the American Medical Association classiﬁed obesity as a

[

Obesity can be deﬁned as an excess of body fat. In clinical terms,

disease in 2013. The main treatment for obesity consists of dieting

and physical exercise, but it is very difﬁcult for people to persist in

Ref. [5]. Despite a rising worldwide epidemic of obesity, there are

currently only two available drugs for the long-term treatment of

obesity: Orilistat and Lorcaserin [6,7], which were limited use

because of their austere adverse effects [8,9]. So there is a strong

unmet medical need in reliable and efﬁcient drugs targeting anti-

obesity properties and hypolipidemic activity.

a surrogate maker for body fat content is the body mass index

2

(

BMI), which exceeds 30 kg/m called obesity [1e4]. Obesity and

obesity-related disorders, often referred to as metabolic syndrome,

have been recognized as major underlying factors of the patho-

genesis of such type 2 diabetes (T2D), hypertension, dyslipidemia,

atherosclerosis, and certain types of cancer [1e4]. With the

A major cause of obesity is the accumulation of TG in adipose

tissue [10], and increased adipose tissue mass can occur through

increase in cell size, cell number, or both [2]. Nowadays, an elevated

TG is frequently the most available laboratory marker of intracel-

lular accumulation of lipids [11]. Impaired fasting glucose often is

found together with an increase TG level, which can interrupt in-

sulin action by multiple mechanisms and lead to insulin resistance

ultimately [12]. Furthermore, an increasing TG in plasma also could

active adipocytokines to increase the risk of coronary heart disease

(CHD) [13]. Therefore, the ability to reduce intracellular TG con-

centrations has been proposed as an attractive therapeutic para-

digm for obesity and related diseases.

Abbreviations: CHD, coronary heart disease; DMF, dimethylformamide; PE, pe-

troleum ether; H&E, hematoxylin and eosin; DIO, diet-induced obesity; AICAR,

acadesine 5 -monophosphate; Met, metformin; RSG, Rosiglitazone; HFD, high fat

diet; TG, Triglycerides; FBG, fasting plasma glucose; ALT, alanine transaminase; AST,

aspartate amino-transferase; LDL, low-density lipoprotein; HDL, high-density li-

poprotein; FFA, free fatty acids; OGTT, oral glucose tolerance test; ITT, insulin

tolerance test; IR, insulin resistance; HOMA-IR, homeostasis model of assessment

for insulin resistance index; NAFLD, nonalcoholic fatty liver disease; DIAD, Diiso-

propyl azodicarboxylate; DMF-DMA, Dimethoxy-N,N-dimethylmethanamine; H&E,

hematoxylin-eosin.

0

*

Corresponding author.

E-mail address: chenlijuan125@163.com (L. Chen).

There authors contributed equally to this work.

1

http://dx.doi.org/10.1016/j.ejmech.2014.09.094

0

596

L. He et al. / European Journal of Medicinal Chemistry 87 (2014) 595e610

It has been established that insulin resistance is a major factor

ﬂuorobenzaldehyde in the presence of t-BuOK as a base in DMF.

The other diketone intermediate (9) was synthesized from the most

primitive material 1-methylurea (4) Treatment of 4 and 2-

linking obesity, particularly central adiposity [12]. The insulin

signaling system plays an important role in regulating some

physiological processes of obesity, including glucose uptake, TG

synthesis, lipolysis and adipocytokine secretion [14]. Conversely,

excess lipid accumulation is a major cause of insulin resistance in

ꢀ

cyanoacetic acid at 60 C in DMF, afforded white solid in a good

yield, then 20% NaOH solution was added to dissolve the white

solid, reﬂuxing for another 2 h, taken the solution in icy water and

acidiﬁed to pH < 7 to give 5. Primary amine 5 was protected by

DMF-DMA to provide 6 with reaction selectivity. The 6 was treated

with NaH, LiBr and various halides in ice-bath to give 7. Subse-

quently, 8 was synthesized through two methods of deprotection

employing concentrated HCl or aqueous ammonia in ethanol,

respectively. The diamino uracil 9 was obtained by its conversion to

nitroso racil and reduction with sodium dithionite. Condensation of

3 with 9 in EtOH/AcOH (4/1, v/v) afforded the corresponding amino

derivatives, which, on subsequent ring closure in reﬂuxing toluene

in the presence of diisopropyl azodicarboxylate (DIAD), provided

the corresponding xanthine derivatives 10. Finally, target com-

pounds 11aex were obtained by alkylation with various kinds of

obesity [15]. Prolonged insulin resistance may eventually result in

b

cell failure and circulating insulin levels become insufﬁcient to

control blood glucose, leading to overt hyperglycaemia, hyperlip-

idemia and relative complications [16]. That is to say, potent and

novel insulin regulators have been regarded as therapeutic poten-

tial for the treatment of obesity [17].

Xanthine is a purine base found in most human body tissues and

ﬂuids and in other organisms [18]. Lots of drugs contain xanthine,

such as Theobromine [19], Caffeine [20] and Linagliptin [21], which

could improve the blood lipid proﬁle and thereby to help obese

persons lose weight, and may account for improved insulin sensi-

tivity. Rosiglitazone (RSG), a wonderful insulin sensitizer by binding

to the peroxisome proliferators-activated receptor-g, promoting

2 3

halide and K CO , summarized in Table 1.

synthesis of glucose transporters and activating adipocyte differ-

entiation [22]. Based on the structureeactivity relationship (SAR)

data reported in the literature it appears that the acidic head group

may be one of the factors responsible for side effects of RSG [23].

Researches also conﬁrmed that replacement the acidic thiazolidi-

nedione head group of RSG with other heterocycles could obtain

signiﬁcant bioactivities like anti-hyperglycemic and anti-

dyslipidemic [23,24]. Encouraged by these results above and

based on the idea of lipid lowering agents with improving insulin

resistance, we have designed and synthesized a series of novel

compounds where the acidic thiazolidinedione head group of RSG

was replaced with xanthine scaffold as shown in Fig. 1. 34 novel

derivatives were synthesized and evaluated for the ability on

reducing TG level in 3T3-L1 adipocytes as well as alleviating obese

clinical symptoms in DIO mice.

To explore the SAR, these novel N-1 or N-7 mono-substitution

and N-1,7-disubstituted derivatives were synthesized as outlined

in Scheme 2. Compound 13 was prepared according to the same

synthetic procedures reported for 10. For the synthesis of 14aed,

controlling the equivalent ratio of halides was critical, one equiv-

alent to 14aeb while two equivalents to 14ced. The position N-7 of

2 3

13 was protected by POM-Cl in the presence of K CO as a base in

anhydrous DMF, then used halides to attack N-1 in the same way to

make 15aec. Finally, deprotection of the POM group and degreas-

ing reaction with 15bec were efﬁciently achieved by using 2 M HCl

in MeOH, thus to gain carboxylic acid derivatives 16aeb. At this

stage, all the ﬁnal products were fully analyzed and characterized

by nuclear magnetic resonance (NMR), mass spectrum (MS), and

high performance liquid chromatography (HPLC) before entering

the biological test.

2

. Result and discussion

2.2. Biological evaluations

2.1. Chemistry

2.2.1. Determination the content of TG in 3T3-L1 adipocytes

TG is commonly used as indicators of intracellular accumulation

of lipids [25,26]. 3T3-L1 mouse preadipocytes are known to

differentiate into mature adipocyte-like cells in vitro, and intracel-

lular accumulation of lipid droplets has been observed in cell dif-

ferentiation [27]. All these compounds were evaluated for the

inhibitory effects on TG accumulation of 3T3-L1 adipocytes. Aca-

The preparation of a library of 3-methyl-8-(4-(2-(methyl(pyr-

idin-2-yl)amino) ethoxy)phenyl)-1H-purine-2,6(3H,7H)-dione de-

rivatives (11) have been carried out in a tandem nine-step sequence

from two important intermediates 3 and 9 as described in Scheme

1

2

. Compound 2 was afforded by treatment 2-chloropyridine (1) and

-(methylamino)ethanol in a good yield. The pivotal aldehyde in-

termediate was synthesized via reaction of and 4-

0

desine 5 -monophosphate (AICAR), Met, well known inhibitors to

reduce the TG level, were selected as positive controls [12]. On the

3

a

2

Fig. 1. Design compounds with anti-obesity activity.

L. He et al. / European Journal of Medicinal Chemistry 87 (2014) 595e610

597

ꢀ

Scheme 1. Reagents and conditions: (a) 2-(methylamino)ethanol, 150 C, 8 h; (b) t-BuOK, DMF, 4-ﬂuorobenzaldehyde, 80 C, overnight; (c) 2-cyanoacetic acid, Ac

2

O, 60 C, 5 h, then

ꢀ

2

0% NaOH (aq.), 2 h; (d) DMF-DMA, DMF, 40 C, overnight; (e) R

1

-X, LiBr, NaH, DMF, 70 C, overnight; (f) concentrated HCl or 28% NH

, 60 C, 3 h; (h) 3, EtOH, AcOH, reﬂux, then DIAD, toluene, reﬂux, 5 h; (i) R

4

OH, EtOH, 40 C, 5 h; (g) AcOH, NaNO

2

, 0 C to

ꢀ

room temperature, 5 h; then MeOH, NH

4 2

OH, Na S

2

O

4

2

-X, K

2

CO

3

, DMF, 70 C, over 5 h.

contrary, RSG, a lipogenic enhancer, was chosen as negative control

27].

As depicted in Fig. 2, 11c, 11d, 11f, 11j, 14d and 15a signiﬁcantly

suppressed the accumulation of TG in mature 3T3-L1 cells at 10 M.

substitutes at N-7 position was better than there was substitutes; 3)

when N-1 was no substitute group, the introduction of polar group

at N-7 was necessary.

[

m

Among them, 11c and d were considered as identiﬁed hits with a

favorable TG inhibition ratio 64.47% and 51.11%, which were higher

than AICAR 50.96%. 11a, without any substituent group at N-7,

showed no biological activity. However, the introduction of an alkyl

substituent led to an increase in lowering TG, and decreased

roughly when the bulk of substituent group increased by compar-

ison with 11ben. The order of potency was 1-ethyl > 1-propyl > 1-

methyl > others, which was related to their poor solubility as well

as high hydrophobicity. In order to increase water solubility of the

highly lipophilic, polar groups, such as acidic or basic functions,

were introduced into N-7 position. As for 11ken, the exposed

carboxylic group revealed a better potency in restraining adipo-

genesis than the corresponding ethyl ester compounds. Similar

cases were observed with 14c and d, but all of them did not show

potency improvements over 11c or d. Further elaboration to small

alkanes at N-1 position (11oex) did not improve potency, neither.

Nevertheless, this unfavorable result also conﬁrmed the order of

potency was 1-ethyl > 1-propyl by contrasting 11pes or 11wex.

Quite different from the aryl group at N-1 position, the introduction

of alkyl substituent at N-7 resulted in decreasing activity while N-1

was small alkanes. By contrast, the presence of the cyano group on

the terminal phenyl of 11c and d led to reduce the hydrophobicity

and enhance of inhibitor potency. Also, there was not any appre-

ciable hit among the derivatives without substituent group at N-1,

except 15a, which was particularly beneﬁcial for potency by the

ester segment that reduced hydrophobicity. In spite of 15be16b did

not exhibit a good efﬁcacy in lowering TG, the data also conﬁrmed

that N-1,7-disubstituted was a necessary portion for this series

compounds. Besides, reducing the high lipophilicity was even

important from the N-1,7-disubstituted compounds 11u, 11x, 14c

and d. Thus, the structure activity relationship of 3-methyl-1H-

purine-2,6-dione Derivatives in suppression the accumulation of

TG in mature 3T3-L1 cells could simply summary here: 1) when N-1

was aryl group, the order of potency was 1-ethyl > 1-propyl at N-7

position; 2) when N-1 was small alkanes, the efﬁcacy of no

2.2.2. Effects of 11c and d in diet-induced obesity (DIO)

On the basis of their excellent potency of lowing TG in vitro, 11c

and d were selected for further pharmacologic evaluation in DIO

model with C57Bl/6J mice [11,28]. Oral administration of high-fat

diet (HFD, Research Diets, D12492) for 12 weeks, the mice were

successfully induced obesity as evidenced by the gain of body

weight compared to the normal group. At the same time, the male

rats were in single-dose acute toxicity assessment, the maximal

feasible single oral dose, 2.5 g/kg, showed no signiﬁcant effect on

clinical signs, or body weight and mortality were observed over a 7-

day period in sub-acute toxicity measurement (Supporting

information). According to the results of in vitro assay and previ-

ous experimental dosage, we accepted a dose of 50 mg/kg/day as a

therapeutic strategy. After oral administration of HFD þ 11c (50 mg/

kg/day), HFD þ 11d (50 mg/kg/day), and HFD þ Met (150 mg/kg/

day) for another 4 weeks, the average body weight was reduced

respectively by 15.96%, 7.64% and 19.08% without dramatically

changes of food intake compared to the HFD group. The mean loss

of liver weight was 16.80%, 12.80% and 23.20%, while the mean fat

mass loss of epididymal fat tissues was 21.65%, 4.76% and 22.94%,

respectively. As a comparison, 11c exhibited superior ability in

controlling the weight of body, liver and fat than 11d from all the

results above, which was close to Met (Fig. 3).

Obesity is not only a major contributor to the metabolic

dysfunction involving lipid, but also blood glucose and insulin [29].

In order to investigate the potential effects of two candidate com-

pounds on blood glucose and insulin homeostasis, an oral glucose

tolerance test (OGTT) and insulin tolerance test (ITT) were carried

out on DIO mice after 4-week efﬁcacy study. Such two disease

models develop obese and obesity-related features such as

impaired glucose tolerance, hyperinsulinemia and hyperlipidemia

[30]. 11c showed a signiﬁcant improvement in glucose tolerance

and insulin resistance at a 50 mg/kg dose (P < 0.05), especially in

abdominal case (Fig. 4B, 4D), which was similar to Met, and 11d was

negative. The time of reducing plasma glucose concentration to the

598

L. He et al. / European Journal of Medicinal Chemistry 87 (2014) 595e610

Table 1

Structural information of 11 for pharmacological proﬁlings.

No.

R

1

R

2

No.

R

1

R

2

11a

H

11m

1

1b

11n

11o

11p

11q

11r

11s

11t

1

1c

H

11d

1

1e

1

1f

11g

11h

H

1

1i

11u

11v

11w

11z

11j

11k

11l

initial status by 50% exceed 40 min and the rate of decline in plasma

glucose concentrations every min (KTT) was >2%/min, so there was

little risk of hypoglycemia. Accordance with the OGTT test that 11c

did not alter fasting glucose level with normal glucose homeostasis.

So, 11c that eliminated excess lipid accumulation (in liver and

muscle) could improve glucose homeostasis by reversing lipid-

induced insulin resistance and alleviate obesity [12].

HOMA-IR. These results were consistent with the ITT study that

showed above.

Subsequently, we also measured other biochemical parameters

in serum level, including alanine transaminase (ALT), aspartate

aminotransferase (AST), TG, low-density lipoprotein (LDL), high-

density lipoprotein (HDL) and free fatty acids (FFA), which were

7

primary associated with dyslipidemia and obesity. HFD mice

At the end of the feeding study, fasting plasma blood (FBG) and

insulin levels were tested, and the homeostasis model assessment

of basal insulin resistance (HOMA-IR) was calculated. Lower

HOMA-IR values indicate greater insulin sensitivity, whereas higher

HOMA-IR values show lower insulin sensitivity or insulin resistance

showed an increasing level of plasma AST, ALT, TG, LDL, HDL and

FFA, which were gradually reduced in 11c mice by 83.67%, 10.28%,

6.42%, 5.88%, 106.32% and 3.82%, respectively. The decrease TG

content may result from decreasing lipid synthesis and further

increased in insulin sensitivity in 11c mice. 11d did not show

noticeable effect in these changes. Moreover, the ALT and AST levels

of 11c treated mice were similar to that of normal and Met treated

groups. To put it simply, 11c could improve plasma lipid proﬁle and

scarcely cause any hepatotoxicity, which was correlated with lower

adipose tissue and liver mass.

[11]. As shown in Table 2, 11c signiﬁcantly reversed HFD diet

induced elevations in plasma glucose (39.48%) and insulin (42.02%)

levels compared to obese controls. HOMA-IR was found to be

higher in HFD and HFD þ 11d groups. However, insulin sensitivity

was increased by 11c supplementation as indicated by lower

L. He et al. / European Journal of Medicinal Chemistry 87 (2014) 595e610

599

ꢀ

Scheme 2. Reagents and conditions: (a) AcOH, NaNO

2

, 0 C to room temperature, 5 h, then MeOH, NH

4

OH, Na

2

S

2

O

4

, 60 C, 3 h; (b) 3, EtOH, AcOH, reﬂux, 4 h, then DIAD, toluene,

ꢀ

reﬂux, 5 h; (c) RX, K

2

CO

3

, anhydrous DMF, 70 C, over 5 h; (d) 2 M NaOH, MeOH, 80 C, overnight.

Up till now, Oil Red O staining has been commonly used to screen

the changes in lipid content. However, there is concern about the

speciﬁcity and the inaccuracy of the results obtained from the Oil

Red O staining [31]. Therefore, the liver sections were not only

processed routinely for Oil Red O assay, but also together with he-

matoxylin staining. Besides, the tissues of liver and fat also stained

with hematoxylin-eosin (H&E) thus to make the result reliable. In

agreement with a decline in the content of hepatic TG, there were

numerous lipid droplets in liver tissues from HFD-induced mice

severely ruined liver histology presented as ballooning degenera-

tion and further led to the formation of steatosis. Fortunately, the

reduction of hepatic TG by the administration of 11c, d and Met was

Fig. 5BeD, the fat accumulation from liver tissues of treated mice

(HFD þ Met, HFD þ 11c) was distinctly alleviated, and the remark-

able reduction of the number and size of lipid droplets was observed

(black arrowheads). Consistently with the results above, 11c dis-

played better capacity of controlling the differentiation and size of

adipocytes than 11d, which had minimal efﬁcacy.

2.2.3. Pharmacokinetics of 11c and 11c·HCl

With the preferable in vivo evaluation, 11c was investigated the

pharmacokinetics in SpragueeDawley rats, whose bioavailability

was only 28.03% (Table 3). However, the poor oral bioavailability of

11c may have a close relationship with such a high oral dosage in

DIO model. Thus we tried to prepare various salts of 11c, such as

7.17%, 5.15% and 10.73%, respectively. Similar results were seen in

Fig. 2. Inhibitory effects of 34 novel compounds on TG accumulation in 3T3-L1 adipocytes. RSG and compounds were at a same concentration of 10

mM, whereas Met was 1.0 mM

and AICAR was 0.5 mM. Results were recorded from three independence experiments: *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. the corresponding control.

600

L. He et al. / European Journal of Medicinal Chemistry 87 (2014) 595e610

Fig. 3. 11c and d improved the metabolic symptoms in DIO mice. (A) Body weights of DIO mice, either HFD group (n ¼ 6) or treated groups (n ¼ 6), were monitored the oral

administration of 11c (50 mg/kg/day), 11d (50 mg/kg/day) and Met (150 mg/kg/day) for 4 weeks. (B) food intake, (C) liver weight, and (D) fat weight (epididymal and abdominal fat

mass) were analyzed: #, P < 0.05 vs. normal group; *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. the corresponding HFD group.

triﬂuoroacetic acid salt, benzoate, maleate and hydrochloride. Only

hydrochloride was prepared successfully owing to the weak alka-

line of 11c. As expected, 11c$HCl had an amazing solubility of

of 11c. Despite the intravenous half-life of hydrochloride was

shorter than primary compound, the oral half-life was a slight

longer, approximately 7 h. The delta-T between 11c and 11c$HCl

was only 0.6 h, which implied 11c$HCl should be hydrolyzed to 11c

quickly and taken effect. Besides, acute and sub-acute toxicity

studies of 11c$HCl in SpragueeDawley rats in series doses up to

3

.28 mg/mL at room temperature, whereas 11c was only 1 mg/mL

Supporting information). Agree with the result of solubility, the

bioavailability of 11c$HCl was also ascended to 58.94%, over 2-fold

(

Fig. 4. Effects of 11c and d on postprandial glucose levels in OGTT and ITT. Both of these tests were performed in treated DIO mice for 4 weeks after one night of fasting. Blood was

sampled from the tail vein of mice at 0, 5, 15, 30, 60, 90 and 120 min after an oral glucose load of 2.0 g/kg or intraperitoneally insulin load of 0.5 U/kg of body weight. Top panels

show plasma glucose concentration curve as a function of time and bottom panels show areas under the curves: (A, C) blood glucose level in OGTT and (B, D) blood glucose level in

ITT. Results were the mean ± standard errors (n ¼ 6) as shown: #, P < 0.05 vs. normal group; *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. the corresponding HFD group.

L. He et al. / European Journal of Medicinal Chemistry 87 (2014) 595e610

601

Table 2

a

Parameters of biochemical markers from DIO mice.

Parameter

Normal

5.0 ± 1.30*

11.85 ± 2.6

33.00 ± 18.23**

125.00 ± 35.00

0.93 ± 009*

HFD

8.51 ± 2.19

9.40 ± 1.0

122.50 ± 38.89

133.75 ± 27.20

1.09 ± 0.11

0.17 ± 0.06

0.95 ± 0.04

23.57 ± 2.44

3.50 ± 0.71

HFD þ 11c

HFD þ 11d

HFD þ Met

FBG (mM)^b

Insulin (mU)

ALT (U/L)

AST (U/L)

TG (Mm)

LDL (Mm)

HDL (Mm)

FFA (ng/L)

HOMA-IR

5.15 ± 0.63*

13.35 ± 3.1*

20.00 ± 8.66**

120.00 ± 28.72

1.02 ± 0.16

0.11 ± 0.01*

1.96 ± 0.07*

22.67 ± 3.05

3.06 ± 0.85

7.25 ± 1.74

12.04 ± 2.5

86.00 ± 55.39

150.00 ± 23.18

1.15 ± 0.06

0.16 ± 0.03

0.96 ± 0.15

24.43 ± 5.61

3.99 ± 1.65

5.32 ± 1.14*

8.10 ± 1.2

21.67 ± 2.89*

110.00 ± 30.00

0.92 ± 0.14

0.14 ± 0.07

0.12 ± 0.03

0.60 ± 0.17**

19.55 ± 1.57

2.61 ± 0.85

0.82 ± 0.09**

21.56 ± 1.26

1.93 ± 0.54**

HOMA-IR ¼ fasting plasma glucose (mmol/L) ꢁ fasting insulin (mU)/22.5.

a

The HFD-fed group (n ¼ 6) or treated groups (n ¼ 6) were monitored during the oral administration of 11c (50 mg/kg/day), 11d (50 mg/kg/day) and Met (150 mg/kg/day)

for 4 weeks. The results as were expressed as the mean ± S.D.

b

Fasting blood glucose:*, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. the corresponding HFD group.

2

.5 g/kg exhibited no toxicity according to measurements of both

physiological and metabolic variables. Based on these advantages of

1c$HCl, it was also selected for further evaluation in DIO model.

owned the best capacity in improving obesity and no abnormal

behavioral or physiological signs were observed during the

experiment.

1

A

combination of excellent in vivo activity, dramatically

2

.2.4. Effects of 11c·HCl in DIO mice

To investigate the further effect of 11c$HCl on adipocytes con-

improved pharmacokinetic proﬁle, and minimal risk of cytotoxicity

proﬁles, 11c$HCl was assessed for its ability to improved glucose

tolerance and insulin resistance in DIO mice after 4-week treat-

ment. As expected, single oral doses of 11c$HCl robustly lowered

the blood glucose (Fig. 7A, C) and augmented insulin secretion

(Fig. 7B, D) during the tests in a dose-proportional manner from 20

to 50 mg/kg. It should be noted that 11c$HCl exhibited a signiﬁcant

blood glucose-lowering effect at two doses in comparison with

Met. Even at the high dose, 11c$HCl did not alter fasting glucose

levels in DIO mice with normal glucose homeostasis, which was in

accordance with the absence of hypoglycemic effects. Moreover,

11c$HCl displayed a much stronger capacity in glucose-lowering

after the administration of insulin at all times points than Met, a

well-known biguanide drug that increases insulin sensitivity [24].

tent in vivo, we employed the DIO model where we administered

two doses of 11c$HCl (20 and 50 mg/kg/day) for 4 weeks. During or

after the treatment period, all the phenomenon and data revealed

that the curative effect of high-dose 11c$HCl was better than that of

Met treatment group in contrast to ones of HFD (P < 0.05) without

affecting dramatic changes in accumulative food intake (Fig. 6A, B).

Among them, the average body weight was reduced respectively by

12.86%, 15.08% and 11.41%, the mean mass of liver decreased

respectively by 20.00%, 22.67% and 22.00% (Fig. 6C), along with the

fat loss was by 18.18%, 56.20% and 9.92% respectively (Fig. 6D).

Particularly, the result of fat loss in high-dose 11c$HCl group came

close to the normal group. That was to say, the high-dose of 11c$HCl

Fig. 5. (A) Effects of 11c and d on hepatic TG content from DIO mice. The values represent the mean ± S.D from 6 mice in each group: *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. the

corresponding HFD group. Histological examination of liver and fat (epididymal visceral adipose tissue) samples from DIO mice: (B) Oil Red O staining and (C) Oil Red

O þ hematoxylin staining of representative liver sections, along with H&E staining of (D) liver and (E) fat tissues (scale, 200ꢁ). Lipid droplets were marks using black arrowheads.

602

L. He et al. / European Journal of Medicinal Chemistry 87 (2014) 595e610

Table 3

decreased size of fat cells as similar seen in ones from Met group.

These data are parallel with the restricting body and liver tissue

weights as a consequence of reduction in fat and TG content of DIO

mice showed above. Obviously, 11c$HCl possessed the potential for

weight lowing effect on obese symptoms and improved the obesity

related metabolic features.

Pharmacokinetic proﬁles of 11c and 11c$HCl in Mice. Data were mean concentra-

tions in mouse plasma (n ¼ 5) following a single 5.0 mg/kg intravenous dose or

20 mg/kg oral dose.

1

1c

11c$HCl

Intravenous

C

max

(

m

g/L)

341.544

0.083

1741.815

0.083

t

max (h)

1/2 (h)

8.829

5.817

AUC0-t

(L/kg)

CLtotal (L/h/kg)

g/L)

(

m

g/L$h)

404.149

142.052

11.314

54.412

2.267

1778.411

23.626

2.825

465.159

2.792

3. Conclusion

V

d

In this study, we have developed a series of 3-methyl-1H-pu-

rine-2,6-dione derivatives as novel lowering TG agents. Our struc-

tureeactivity relationship study was clear and revealed that

compounds with appropriate polarity exhibited good efﬁcacy on

lowering the synthesis of TG in 3T3-L1 adipocytes. Of these mole-

cules, compound 11c showed TG reduction and improved the obese

clinical symptoms when studied in vivo at a dose of 50 mg/kg/day,

which was found to be almost equi-efﬁcacious to Met. Further

improvement on solubility and bioavailability resulted in the

identiﬁcation of 11c-hydrochloride (11c$HCl). Thus treatment with

Oral

C

max

(

m

t

max (h)

1/2 (h)

6.304

6.973

AUC0-t

(L/kg)

CLtotal (L/h/kg)

(

m

g/L$h)

453.074

416.501

61.512

28.03

4192.938

67.208

8.31

V

d

F (%)

58.94

In short, 11c$HCl could not only lower the concentration of plasma

glucose, but also improve insulin resistance. These encouraging

results in terms of reduced glucose excursion in addition to the

effect on insulin levels and on lowering body weight gain suggested

that 11c$HCl could provide potential beneﬁts for treating obesity

and diabetes.

Similar observation was found by comparison some important

biochemical makers in plasma level after the treatment. In contract

to HFD group, the treatment of 11c$HCl effectively recovered serum

biomarkers to appropriate ranges and decreased HOMA-IR

dramatically in a dose dependent manner. Specially, high-dose

11c$HCl at two doses of 20 and 50 mg/kg/day in DIO mice revealed

good anti-obesity effect. Particularly at high dose, 11c$HCl mark-

edly alleviated the clinical symptoms of obesity, the changes of TG,

LDL, HDL, FFA and fat mass were reduced to normal group, and

histological evaluation displayed normal condition. Besides, there

were no signiﬁcant adverse effects were observed in toxicity study

with rats. Overall, potent and safe lipid-lowering compound,

11c$HCl can be expected to provide a novel therapeutics for

obesity-related disease without any risk of hypoglycemia and

toxicity.

1

1c$HCl presented ascendant improvements of TG (28.34%), LDL

(

45.16%), HDL (112.31%), FFA (20.46%) and HOMA-IR (59.34%), bet-

ter than that in Met group, almost reversed HFD diet to a normal

level (Table 4).

4. Experimental section

The content of TG in liver, morphology of adipose tissue and

liver of mice from different diet groups is depicted in Fig. 8.

Compared to HFD mice, the reduction of hepatic TG by the

administration of 11c$HCl and Met was 16.80% (20 mg/kg/day),

4.1. General synthetic methods

Chemistry reagents of analytical grade were purchased from

Changzheng Chemical Factory, Chengdu, Sichuan, P. R. China. TLC

was performed on 0.20 mm Silica Gel 60 F254 plates (Qingdao Ocean

Chemical Factory, Shandong, China). Hydrogen nuclear magnetic

21.59% (50 mg/kg/day) and 20.98%, respectively. Remarkably, the

cytoplasm of the hepatocytes from HFD mice showed micro-

vesicular vacuolation (the presence of numerous lipid droplets), as

well as hypertrophic, clear adipocytes of fat tissues, suggesting that

the diet feeding led to an excessive fat deposition and accumula-

tion. However, 11c$HCl supplementation altered adipose tissue

morphology and reduced lipid accumulation in the liver, according

with a decline in the content of TG in liver. Especially for the high

dose, 11c$HCl revealed prominent efﬁcacy in improving the extent

of fatty change, which displayed normal morphology nearly

without obvious histology changes and site of steatosis. Low-dose

1

resonance ( H NMR) spectra were recorded at 400 MHz while

13

carbon nuclear magnetic resonance ( C NMR) spectra were

recorded at 100 MHz on a Varian spectrometer (Varian, Palo Alto,

CA) model Gemini 400 and reported in parts per million. Chemical

shifts (d) are quoted in ppm relative to tetramethylsilane (TMS) as

an internal standard, where (

d

) TMS ¼ 0.00 ppm. The multiplicity of

the signal is indicated as s, singlet; brs, broad singlet; d, doublet; t,

triplet; q, quartet; m, multiplet, deﬁned as all multipeak signals

where overlap or complex coupling of signals makes deﬁnitive

descriptions of peaks difﬁcult. Mass spectra (MS) were measured by

11c$HCl fed mice had lower lipids in hepatic tissues and

Table 4

a

Parameters of biochemical markers from DIO Mice.

Parameter

Normal

4.88 ± 0.75***

4.31 ± 0.82***

29.60 ± 8.29

128.00 ± 20.59

0.96 ± 0.14**

0.13 ± 0.02**

1.02 ± 0.13

HFD

10.09 ± 1.12

8.77 ± 2.85

62.00 ± 10.53

184.67 ± 14.12

1.31 ± 0.11

0.31 ± 0.09

0.65 ± 0.36

32.35 ± 7.19

3.91 ± 1.37

HFD þ 11c$HCl-20

HFD þ 11c$HCl-50

HFD þ Met

FBG (mM)

Insulin (mU)

ALT (U/L)

8.42 ± 1.91

7.48 ± 3.45

24.00 ± 14.29

125.71 ± 16.72

1.25 ± 0.25

0.22 ± 0.03*

1.31 ± 0.26**

26.80 ± 11.13

2.98 ± 1.53

7.76 ± 1.68*

4.76 ± 2.39**

18.86 ± 3.80

131.43 ± 16,72

0.94 ± 0.33*

0.17 ± 0.03**

1.38 ± 0.30**

25.73 ± 1.42**

1.59 ± 1.03***

7.57 ± 2.02*

5.48 ± 2.60*

46.29 ± 4.53

172.00 ± 9.42

1.07 ± 0.18*

0.17 ± 0.06**

1.38 ± 0.20***

26.05 ± 6.70*

1.73 ± 0.91***

AST (U/L)

b

TG (Mm)

LDL (Mm)

HDL (Mm)

FFA (ng/L)

HOMA-IR

25.07 ± 3.49**

0.94 ± 0.23***

HOMA-IR ¼ fasting plasma glucose (mmol/L) ꢁ fasting insulin (mU)/22.5.

a

The HFD-fed group (n ¼ 6) or treated groups (n ¼ 6) were monitored during the oral administration of 11c$HCl (20 mg/kg/day), 11c$HCl (50 mg/kg/day) and Met (150 mg/

kg/day) for 4 weeks. The results as were expressed as the mean ± S.D.

b

Fasting blood glucose: *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. the corresponding HFD group.

L. He et al. / European Journal of Medicinal Chemistry 87 (2014) 595e610

603

Fig. 6. 11c$HCl improved the metabolic symptoms in DIO mice. (A) Body weights of DIO mice, either HFD group (n ¼ 6) or treated groups (n ¼ 6), were monitored the oral

administration of 11c$HCl (20 mg/kg/day), 11c$HCl (50 mg/kg/day) and Met (150 mg/kg/day) for 4 weeks. (B) Food intake, (C) liver weight, and (D) fat weight (epididymal and

abdominal fat mass) were analyzed: #, P < 0.05 vs. normal group; *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. the corresponding HFD group.

Fig. 7. Effects of 11c$HCl on postprandial glucose levels in OGTT and ITT. Both of these tests were performed in treated DIO mice for 4 weeks after one night of fasting. Blood was

sampled from the tail vein of mice at 0, 5, 15, 30, 60, 90 and 120 min after an oral glucose load of 2.0 g/kg or intraperitoneally insulin load of 0.5 U/kg of body weight. Top panels

show plasma glucose concentration curve as a function of time and bottom panels show areas under the curves: (A, C) Blood glucose level in OGTT and (B, D) blood glucose level in

ITT. Results were the means ± standard errors (n ¼ 6) as shown: #, P < 0.05 vs. normal group; *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. the corresponding HFD group.

604

L. He et al. / European Journal of Medicinal Chemistry 87 (2014) 595e610

Fig. 8. (A) Effects of 11c$HCl on hepatic TG content from DIO mice. The values represent the mean ± S.D from 6 mice in each group: *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. the

corresponding HFD group. Histological examination of liver and fat (epididymal visceral adipose tissue) samples from DIO mice: (B) Oil Red O staining and (C) Oil Red

O þ hematoxylin staining of representative liver sections, along with H&E staining of (D) liver and (E) fat tissues (scale, 200ꢁ). Lipid droplets were marks using black arrowheads.

Q-TOF Priemier mass spectrometer (Micromass, Manchester, UK).

Room temperature (rt) is within the range 20e25 C. The purity

was analyzed by HPLC system (Waters 2695, separations module)

with a photodiode array detector (Waters 2996, Milford, MA, U.S.),

and the chromatographic column was a reversed phase C18 column

4.1.3. 6-Amino-1-methylpyrimidine-2,4(1H,3H)-dione (5)

ꢀ

1-Methylurea (15.0 g, 202.5 mmol) and 2-cyanoacetic acid

(25.8 g, 303.7 mmol) in the solvent of acetic anhydride (100 mL),

ꢀ

were stirred at 60 C for 5 h and the solid was formed. The pre-

cipitation was collected by ﬁltration and washed with water to

afford the important intermediate with a good yield about 65%.

Dissolve the white solid (18 g, 127.5 mmol) with 20% NaOH solution

(100 mL), reﬂuxing for 2 h. After the reaction was completed,

cooled down the mixture to room temperature, added the hydro-

chloric acid liquid to form solid. The white precipitate was collected

by ﬁltration, washed with water and ethanol to afford the products

5 without any puriﬁcation.

(

Waters, 150 mm ꢁ 4.6 mm, i.d. 5

m

m). All compounds were sup-

plied in HPLC degree methanol with 10

mL, which was injected on a

partial loop, with isocratic elution with 70% methanol and 30%

water at a ﬂow rate of 1 mL/min. The purity of all tested compounds

was ꢂ95% according to our analytical HPLC method.

4.1.1. 2-(Methyl(pyridin-2-yl)amino)ethanol (2)

To a solution of 2-chloropyridine (6.0 g, 52.8 mmol) and 2-

ꢀ

(

methylamino)ethanol (11.9 g, 158.5 mmol), stirred at 150

C

overnight. After the reaction was completed, water was added into

the solution. The solution was extracted with CH Cl , washed with

water and brine, and dried with anhydrous Na SO . Then the sol-

vent was removed under reduced pressure and the product

appeared to be brown oil.

4

.1.4. General procedure for synthesis of compounds 6

To a suspension of compound 5 (10.0 g, 70.8 mmol) and DMF-

2

4

ꢀ

DMA (25.3 g, 212.6 mmol) in DMF (50 mL) was stirred at 40

C

overnight. After the reaction was completed, water was added into

the solution and cooled to room temperature. The white precipitate

was collected by ﬁltration, washed with water and ethanol to afford

the products 6 without any puriﬁcation.

4.1.2. 4-(2-(Methyl(pyridin-2-yl)amino)ethoxy)benzaldehyde (3)

4

-Fluorobenzalde (15.1 g, 121.6 mmol) was added dropwise to a

stirred solution of the compound 2 (7.4 g, 48.6 mmol) in DMF (60 mL)

ꢀ

cooled at 0 C. Potassium tert-butoxide (6.5 g, 58.3 mmol) was added

ꢀ

in small portions. The temperature was raised to 80 C and stirred

4.1.5. General procedure for synthesis of compounds 7

overnight. The mixture was quenched by water, extracted with

EtOAc and combined organic phases. Then the organic phases was

acidizedto pH 3e4 with diluted hydrochloric acidsolution, extracted

with water and combined aqueous phases. The aqueous phases was

basiﬁed to pH 9e10 with aqueous NaOH, extracted with EtOAc,

To a solution of compound 6 (6.0 g, 30.6 mmol) in DMF was

stirred at 0 C for 5 min, sodium hydride (770.5 mg, 32.1 mmol) was

ꢀ

added in small portions. Then lithium bromide (5.3 g, 61.2 mmol)

was feed in the same way follow by sodium hydride. Halogenated

ꢀ

substances (32.1 mmol) was input onetime, moved to 80 C and

washed with water and brine, and dried with anhydrous Na

2

SO

4

.

stirred overnight. After the reaction was completed, poured into

ice-water. And then the pure light yellow precipitate was collected

by ﬁltration, washed with water, and dried in vacuo to afford 89.7%

of 7.

Then the solvent was removed under reduced pressure. Chroma-

tography puriﬁcation of the residue product eluting with 1/5 EtOAc/

PE (petroleum ether) gave the target compounds 3.

L. He et al. / European Journal of Medicinal Chemistry 87 (2014) 595e610

605

4

.1.6. General procedure for synthesis of compounds 8

Concentrated HCl or 28% NH OH was added dropwise to a

stirred solution of the compound 7 (6.0 g, 19.3 mmol) in EtOH

and the precipitated product was isolated by ﬁltration, wash with

water, dried.

4

(

50 mL) at ice-bath until the suspension gradually turned to clear

4.1.11. General procedure for synthesis of compounds 16aeb

ꢀ

solution. Solid was formed when heated to 40 C about 30 min,

and maintain this temperature another 5 h. The mixture was

poured into ice-water and the white solid was collected by

ﬁltration, washed with water, and dried in vacuo to afford 87.9% of

compound 8.

2 M NaOH (200

15c (0.1 mmol) in ethanol. The mixture was heated to reﬂux over-

night. After the reaction was completed, acetic acid (200 L) as

mL) was added to a solution of compound 15b or

m

added to neutralize the mixture, then the solvent was evaporated.

The residue was poured onto ice-water, and the precipitated

product was isolated by ﬁltration, wash with water, dried.

4

.1.7. General procedure for synthesis of compounds 9 and 12

Sodium nitrite (1.3 g, 19.3 mmol) was added in small portions to

stirred solution of the 5,6-diamino-1-methylpyrimidine-

,4(1H,3H)-dione derivatives (12.9 mmol) in glacial acetic acid

4

.1.12. General procedure for synthesis of compound 11c·HCl

Sulfur dichloride (356.91 mg, 3.0 mmol) was added dropwise to

a

2

a solution of compound 11c (535.60 mg, 1.0 mmol) in dichloro-

methane at ice-bath. The reaction mixture was stirred for 2 h and

then concentrated in vacuo to afford ﬂaxen ﬂaky solid, then drying

in vacuum to get target product.

(

30 mL), the color changed from light red to blue, orange or

crimson. The reaction mixture was stirred for about 5 h at ambient

temperature. The colorized sediment was collected by ﬁltration,

washed with water, and dried in vacuo to afford 73.5% of nitrosated

product. To a stirred mixture nitroso compound (2.7 g, 9.5 mmol) in

4

.2. Compound analysis

2

5 mL methanol, aqueous ammonia was added dropwise till the

ꢀ

4.2.1. 2-((3-Methyl-8-(4-(2-(methyl(pyridin-2-yl)amino)ethoxy)

phenyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)

benzonitrile (11a)

solution turn to clear. The temperature was raised to 60 C, and

sodium dithionite (3.3 g, 18.9 mmol) was added in small portion

until the color disappeared. The mixture was stirred for two addi-

tional hours in order to complete the reaction. Move out the solvent

under reduced pressure distillation, washed the solid with cold

water, and dried under high vacuum to obtain the corresponding

product.

Yield 80.2%. HPLC purity: 99.28%. White solid. ¹H NMR

(

400 MHz, DMSO-d

6

):

d

13.74 (s,1H), 8.09 (d, J ¼ 8.6 Hz, 3H), 7.84 (d,

J ¼ 7.6 Hz, 1H), 7.61 (t, J ¼ 7.6 Hz, 1H), 7.51 (t, J ¼ 7.1 Hz, 1H), 7.45 (t,

J ¼ 7.6 Hz,1H), 7.29 (d, J ¼ 7.9 Hz,1H), 7.10 (d, J ¼ 8.7 Hz, 2H), 6.66 (d,

J ¼ 8.6 Hz, 1H), 6.63e6.52 (m, 1H), 5.26 (s, 2H), 4.23 (t, J ¼ 5.7 Hz,

13

2

H), 3.94 (t, J ¼ 5.7 Hz, 2H), 3.50 (s, 3H), 3.09 (s, 3H). C NMR

4.1.8. General procedure for synthesis of compounds 10 and 13

(100 MHz, DMSO-d

6

): d 159.95,157.89,154.31,151.68, 150.84,148.17,

A mixture of compound 9 or compound 12 (4.8 mmol), com-

141.13, 137.43, 133.43, 132.82, 130.70, 127.77, 126.79, 120.41, 117.25,

pound 3 (5.3 mmol) and 1.5 mL of acetic acid was reﬂuxed for 4 h in

0 mL of ethanol. Upon cooling of the mixture, the precipitate was

1

14.72, 111.57, 110.46, 107.56, 105.86, 65.72, 48.39, 42.12, 37.08,

2

þ

33.70, 29.53. MS(ESI), m/z: 508.29 [MþH] .

ﬁltered and washed with ethanol then diethyl ether to give the

Schiff-base, which was used directly in the next step. Schiff-base

4

.2.2. 2-((3,7-Dimethyl-8-(4-(2-(methyl(pyridin-2-yl)amino)

(

3.3 mmol) was heated in 20 mL of toluene. As the mixture began

ethoxy)phenyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)

methyl)benzonitrile (11b)

to reﬂux, diisopropyl azodicarboxylate (6.5 mmol) was added

through the condenser. After another 2 h, the reaction mixture was

ﬁltered, and the precipitate was washed with ethanol and diethyl

ether to give compound 10 and 13.

Yield 78.9%. HPLC purity: 99.34%. White solid. ¹H NMR

(400 MHz, DMSO-d

6

):

d

8.10 (d, J ¼ 4.4 Hz, 1H), 7.84 (d, J ¼ 7.6 Hz,

1

2

H), 7.75 (d, J ¼ 8.2 Hz, 2H), 7.62 (t, J ¼ 7.7 Hz, 1H), 7.52 (t, J ¼ 7.5 Hz,

H), 7.45 (t, J ¼ 7.5 Hz, 1H), 7.30 (d, J ¼ 7.8 Hz, 1H), 7.14 (d, J ¼ 8.3 Hz,

H), 6.68 (d, J ¼ 8.6 Hz, 1H), 6.62e6.55 (m, 1H), 5.26 (s, 2H), 4.25 (t,

4

.1.9. General procedure for synthesis of compounds 11aex, 14c,

5bec

Halogenated reagents (0.105 mmol) was added to a solution of

compound 10, 13 or 15a (0.1 mmol) in presence of anhydrous K CO

14.5 mg, 0.105 mmol) in DMF (3 mL). Further, the mixture was

1

J ¼ 5.6 Hz, 2H), 3.98 (s, 3H), 3.95 (d, J ¼ 5.4 Hz, 2H), 3.48 (s, 3H), 3.10

13

(

1

s, 3H). C NMR (100 MHz, DMSO-d

51.68, 150.84, 148.17, 141.13, 137.43, 133.43, 132.82, 130.70, 127.77,

26.79, 120.41, 117.25, 114.72, 111.57, 110.46, 107.56, 105.86, 65.72,

6

): d 159.95, 157.89, 154.31,

2

3

(

ꢀ

stirred at 80 C about 5 h. The resultant mixture was extracted with

EtOAc, washed with water and brine, and dried with anhydrous

4

8.39, 42.12, 37.08, 33.70, 29.53, 23.71. MS(ESI), m/z: 522.25

þ

[MþH] .

Na

obtain the solid product. Nevertheless, compounds 14a, 14b and

5a were puriﬁed by column chromatography on silica gel using 1/5

EtOAc/PE as mobile phase.

2 4

SO . Then the solvent was removed under reduced pressure to

4.2.3. 2-((7-Ethyl-3-methyl-8-(4-(2-(methyl(pyridin-2-yl)amino)

1

ethoxy)phenyl)-2, 6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)

methyl)benzonitrile (11c)

Yield 76.9%. HPLC purity: 99.91%. White solid.

m

T :

ꢀ

4.1.10. General procedure for synthesis of compounds 14d

178.0e178.3 C; 11c$HCl. Yield 99.46%. HPLC purity: 99.04%. Flaxen

ꢀ

1

Ethyl bromoacetate (0.3 mmol) was added to a solution of

solid. T : 135.5e135.8 C. H NMR (400 MHz, DMSO-d ): d 8.10 (d,

m

6

compound 13 (0.15 mmol), anhydrous K

2

CO

3

(20.7 mg, 0.15 mmol)

J ¼ 4.4 Hz,1H), 7.84 (d, J ¼ 7.6 Hz,1H), 7.66 (d, J ¼ 8.4 Hz, 2H), 7.61 (t,

J ¼ 7.7 Hz, 1H), 7.51 (t, J ¼ 7.5 Hz, 1H), 7.45 (t, J ¼ 7.5 Hz, 1H), 7.31 (d,

J ¼ 7.9 Hz, 1H), 7.15 (d, J ¼ 8.7 Hz, 2H), 6.67 (d, J ¼ 8.6 Hz, 1H),

6.62e6.54 (m, 1H), 5.27 (s, 2H), 4.31 (q, J ¼ 6.9 Hz, 2H), 4.25 (t,

J ¼ 5.8 Hz, 2H), 3.96 (t, J ¼ 5.8 Hz, 2H), 3.47 (s, 3H), 3.10 (s, 3H), 1.34

ꢀ

in DMF (2 mL) at room temperature. And then heat to 80 C over-

night. After the reaction was completed, the mixture was poured

into water and extracted with EtOAc, washed with water and brine,

and dried with anhydrous Na SO . Then the solvent was removed

2 4

under reduced pressure to obtain the intermediate product. Where

after the intermediate product (0.1 mmol) was dissolved in ethanol

13

(t, J ¼ 7.0 Hz, 3H). C NMR (100 MHz, DMSO-d

6

): d 159.97, 157.98,

153.95, 151.39, 150.85, 148.54, 147.48, 141.16, 137.35, 133.44, 132.85,

130.55, 127.77, 126.83, 120.60, 117.25, 114.87, 111.57, 110.40, 106.76,

105.78, 65.73, 48.38, 42.22, 41.26, 37.07, 29.55, 16.05. MS(ESI), m/z:

with stirring, added 1 M NaOH (200

overnight. Acetic acid (200 L) was added to neutralize the mixture,

the solvent was evaporated. The residue was poured onto ice-water,

mL), and left with stirring reﬂux

m

þ

536.27 [MþH] .

606

L. He et al. / European Journal of Medicinal Chemistry 87 (2014) 595e610

4

.2.4. 2-((3-Methyl-8-(4-(2-(methyl(pyridin-2-yl)amino)ethoxy)

4.2.8. 2-((3-Methyl-8-(4-(2-(methyl(pyridin-2-yl)amino)ethoxy)

phenyl)-2,6-dioxo-7-pentyl-2,3,6,7-tetrahydro-1H-purin-1-yl)

methyl)benzonitrile (11h)

phenyl)-2,6-dioxo-7-propyl-2,3,6,7-tetrahydro-1H-purin-1-yl)

methyl)benzonitrile (11d)

Yield 78.4%. HPLC purity: 100.00%. White solid.

T

m

:

Yield 74.7%. HPLC purity: 99.13%. White solid. ¹H NMR

ꢀ

1

144.7e145.0 C. H NMR (400 MHz, DMSO-d

6

):

d

8.10 (d, J ¼ 4.2 Hz,

(400 MHz, DMSO-d

6

):

d

8.10 (d, J ¼ 4.4 Hz, 1H), 7.84 (d, J ¼ 7.6 Hz,

1

H), 7.85 (d, J ¼ 7.6 Hz, 1H), 7.65 (d, J ¼ 8.4 Hz, 2H), 7.61 (t, J ¼ 7.7 Hz,

1H), 7.65 (d, J ¼ 8.4 Hz, 2H), 7.61 (t, J ¼ 7.7 Hz, 1H), 7.51 (t, J ¼ 7.5 Hz,

1H), 7.45 (t, J ¼ 7.5 Hz, 1H), 7.29 (d, J ¼ 7.9 Hz, 1H), 7.14 (d,

J ¼ 8.5 Hz, 2H), 6.67 (d, J ¼ 8.5 Hz, 1H), 6.6e6.55 (m, 1H), 5.27 (s,

2H), 4.31 (t, J ¼ 7.1 Hz, 2H), 4.25 (t, J ¼ 5.6 Hz, 2H), 3.95 (t,

J ¼ 5.6 Hz, 2H), 3.47 (s, 3H), 3.10 (s, 3H), 1.75e1.63 (m, 2H),

H), 7.51 (t, J ¼ 7.6 Hz, 1H), 7.46 (t, J ¼ 7.6 Hz, 1H), 7.30 (d, J ¼ 7.9 Hz,

H), 7.14 (d, J ¼ 8.6 Hz, 2H), 6.67 (d, J ¼ 8.6 Hz, 1H), 6.61e6.55 (m,

H), 5.26 (s, 2H), 4.27 (t, J ¼ 4. Hz, 2H), 4.24 (d, J ¼ 5.6 Hz, 2H), 3.96

(

2

t, J ¼ 5.6 Hz, 2H), 3.48 (s, 3H), 3.10 (s, 3H), 1.71 (dd, J ¼ 14.2, 6.9 Hz,

13

H), 0.73 (t, J ¼ 7.3 Hz, 3H). C NMR (100 MHz, DMSO-d

6

):

d

159.89,

1.19e1.04 (m, 4H), 0.74 (t, J ¼ 6.9 Hz, 3H). C NMR (100 MHz,

1

57.99, 154.02, 151.78, 150.84, 148.49, 147.54, 141.14, 137.32, 133.45,

32.87, 130.60, 127.76, 126.76, 120.77, 117.24, 114.84, 111.55, 110.32,

6

DMSO-d ): d 159.90, 157.96, 154.01, 151.76, 150.81, 148.49, 147.47,

141.12, 137.34, 133.41, 132.87, 130.57, 127.75, 126.73, 120.76, 117.22,

114.83, 111.55, 110.30, 106.90, 105.76, 65.71, 48.37, 45.74, 42.21,

106.94, 105.73, 65.69, 48.36, 47.23, 42.23, 37.06, 29.57, 23.60, 10.54.

þ

MS(ESI), m/z: 550.29 [MþH] .

37.06, 29.73, 29.55, 27.73, 21.33, 13.64. MS(ESI), m/z: 578.31

þ

[

MþH] .

4.2.5. 2-((7-Allyl-3-methyl-8-(4-(2-(methyl(pyridin-2-yl)amino)

ethoxy)phenyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)

methyl)benzonitrile (11e)

4.2.9. 2-((3-Methyl-8-(4-(2-(methyl(pyridin-2-yl)amino)ethoxy)

phenyl)-7-(3-methylbut-2-en-1-yl)-2,6-dioxo-2,3,6,7-tetrahydro-

1H-purin-1-yl)methyl)benzonitrile (11i)

Yield 71.0%. HPLC purity: 99.09%. White solid. ¹H NMR

(

400 MHz, DMSO-d

6

):

d

8.09 (d, J ¼ 4.0 Hz, 1H), 7.84 (d, J ¼ 7.6 Hz,

Yield 82.1%. HPLC purity: 98.21%. White solid. ¹H NMR

1

2

5

H), 7.68 (d, J ¼ 8.5 Hz, 2H), 7.62 (t, J ¼ 7.7 Hz, 1H), 7.51 (t, J ¼ 7.7 Hz,

(400 MHz, DMSO-d

6

):

d

8.10 (d, J ¼ 4.2 Hz, 1H), 7.85 (d, J ¼ 7.6 Hz,

H), 7.45 (t, J ¼ 7.5 Hz, 1H), 7.29 (d, J ¼ 7.9 Hz, 1H), 7.13 (d, J ¼ 8.5 Hz,

H), 6.67 (d, J ¼ 8.6 Hz, 1H), 6.62e6.54 (m, 1H), 6.11e5.97 (m, 1H),

.25 (s, 2H), 5.19 (d, J ¼ 10.5 Hz, 1H), 4.97 (d, J ¼ 4.0 Hz, 2H), 4.91 (d,

1H), 7.65 (d, J ¼ 8.4 Hz, 2H), 7.61 (t, J ¼ 7.7 Hz, 1H), 7.51 (t, J ¼ 7.6 Hz,

1H), 7.46 (t, J ¼ 7.6 Hz, 1H), 7.30 (d, J ¼ 7.9 Hz, 1H), 7.14 (d, J ¼ 8.6 Hz,

2H), 6.67 (d, J ¼ 8.6 Hz, 1H), 6.61e6.55 (m, 1H), 5.26 (s, 2H), 5.24 (t,

J ¼ 7.6 Hz, 1H), 4.27 (d, J ¼ 10.4 Hz, 2H), 4.26 (t, J ¼ 5.6 Hz, 2H), 3.96

(t, J ¼ 5.6 Hz, 2H), 3.48 (s, 3H), 3.10 (s, 3H), 1.63 (s, 3H), 1.52 (s, 3H).

J ¼ 17.3 Hz, 1H), 4.24 (t, J ¼ 5.6 Hz, 2H), 3.95 (t, J ¼ 5.5 Hz, 2H), 3.49

1

3

(

1

s, 3H), 3.09 (s, 3H). C NMR (100 MHz, DMSO-d

53.89, 151.70, 150.83, 148.37, 147.53, 141.09, 137.29, 133.65, 133.40,

32.84, 130.37, 127.75, 126.78, 120.37, 117.22, 116.68, 114.78, 111.54,

6

): d 160.04, 157.98,

13

6

C NMR (100 MHz, DMSO-d ): d 159.95, 158.00, 154.11, 151.58,

150.81, 148.43, 147.53, 141.13, 137.32, 135.72, 133.43, 132.85, 130.59,

127.76, 126.81, 120.68, 119.88, 117.24, 114.82, 111.56, 110.37, 106.89,

105.72, 65.70, 48.34, 44.36, 42.19, 37.05, 29.54, 25.17, 17.80. MS(ESI),

1

10.39, 106.98, 105.70, 65.72, 48.35, 47.95, 42.15, 37.05, 29.57.

þ

MS(ESI), m/z: 548.28 [MþH] .

þ

m/z: 576.31 [MþH] .

4.2.6. 2-((7-Butyl-3-methyl-8-(4-(2-(methyl(pyridin-2-yl)amino)

ethoxy)phenyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)

methyl)benzonitrile (11f)

4.2.10. 2-((7-(2-Hydroxyethyl)-3-methyl-8-(4-(2-(methyl(pyridin-

2-yl)amino)ethoxy)phenyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-

1-yl)methyl)benzonitrile (11j)

Yield 70.0%. HPLC purity: 98.74%. White solid. ¹H NMR

(

1

400 MHz, DMSO-d

H), 7.66 (d, J ¼ 8.4 Hz, 2H), 7.62 (t, J ¼ 7.7 Hz, 1H), 7.51 (t,

6

):

d

8.10 (d, J ¼ 4.0 Hz, 1H), 7.85 (d, J ¼ 7.6 Hz,

Yield 72.8%. HPLC purity: 95.73%. White solid. ¹H NMR

(400 MHz, DMSO-d

6

):

d

8.10 (d, J ¼ 3.3 Hz, 1H), 7.83 (d,

J ¼ 7.0 Hz, 1H), 7.46 (t, J ¼ 7.5 Hz, 1H), 7.29 (d, J ¼ 7.9 Hz, 1H), 7.14

J ¼ 8.0 Hz, 1H), 7.81 (d, J ¼ 8.4 Hz, 1H), 7.62 (t, J ¼ 7.6 Hz, 1H),

7.51 (t, J ¼ 7.6 Hz, 1H), 7.46 (t, J ¼ 7.4 Hz, 1H), 7.30 (d, J ¼ 7.6 Hz,

1H), 7.12 (d, J ¼ 8.5 Hz, 2H), 6.67 (d, J ¼ 8.5 Hz, 1H), 6.61e6.55 (m,

1H), 5.27 (s, 2H), 4.48 (s, 2H), 4.24 (t, J ¼ 5.6 Hz, 2H), 3.95 (t,

J ¼ 5.5 Hz, 2H), 3.77 (d, J ¼ 5.3 Hz, 2H), 3.49 (s, 3H), 3.10 (s, 3H).

(

d, J ¼ 8.6 Hz, 2H), 6.67 (d, J ¼ 8.6 Hz, 1H), 6.61e6.55 (m, 1H), 5.27

s, 2H), 4.31 (t, J ¼ 11.2 Hz, 2H), 4.25 (t, J ¼ 5.7 Hz, 2H), 3.95 (t,

J ¼ 5.6 Hz, 2H), 3.47 (s, 3H), 3.10 (s, 3H), 1.66 (dd, J ¼ 14.2, 7.3 Hz,

1

3

2

H), 1.13 (dd, J ¼ 14.5, 7.2 Hz, 2H), 0.75 (t, J ¼ 7.3 Hz, 3H). C NMR

100 MHz, DMSO-d ): 159.89, 157.97, 154.01, 151.78, 150.83,

48.51, 147.57, 141.13, 137.35, 133.45, 132.88, 130.60, 127.77, 126.75,

13

(

1

6

d

6

C NMR (100 MHz, DMSO-d ): d 159.88, 158.00, 154.05,

152.75 150.83, 148.58, 147.54, 141.16, 137.31, 133.42, 132.83, 131.16,

127.78, 126.76, 120.84, 117.25, 114.58, 111.55, 110.48, 106.99,

105.73, 65.69, 62.77, 59.95, 48.37, 42.20, 37.06, 29.55. MS(ESI), m/

120.76, 117.23, 114.84, 111.56, 110.28, 106.91, 105.77, 65.70, 48.37,

4

5.57, 42.23, 37.07, 32.13, 29.56, 18.88, 13.18. MS(ESI), m/z: 504.31

þ

[MþH] .

z: 574.28 [MþNa] .

4

.2.7. 2-((7-Isobutyl-3-methyl-8-(4-(2-(methyl(pyridin-2-yl)

4.2.11. Ethyl-2-(1-(2-cyanobenzyl)-3-methyl-8-(4-(2-

(methyl(pyridin-2-yl)amino) ethoxy)phenyl)-2,6-dioxo-2,3-

dihydro-1H-purin-7(6H)-yl)acetate (11k)

amino)ethoxy)phenyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)

methyl)benzonitrile (11g)

Yield 71.2%. HPLC purity: 98.57%. White solid. ¹H NMR

Yield 85.0%. HPLC purity: 99.12%. White solid. ¹H NMR

(

400 MHz, DMSO-d

6

):

d

8.10 (d, J ¼ 4.1 Hz, 1H), 7.85 (d, J ¼ 7.7 Hz,

(400 MHz, DMSO-d

6

):

d

8.10 (d, J ¼ 4.0 Hz, 1H), 7.86 (d, J ¼ 7.6 Hz,

1

H), 7.67 (d, J ¼ 8.5 Hz, 2H), 7.62 (t, J ¼ 7.7 Hz, 1H), 7.51 (t, J ¼ 7.6 Hz,

1H), 7.67 (d, J ¼ 8.5 Hz, 2H), 7.61 (t, J ¼ 7.7 Hz, 1H), 7.51 (t, J ¼ 7.1 Hz,

1H), 7.46 (t, J ¼ 7.6 Hz, 1H), 7.31 (d, J ¼ 7.8 Hz, 1H), 7.13 (d, J ¼ 8.6 Hz,

2H), 6.67 (d, J ¼ 8.5 Hz, 1H), 6.61e6.55 (m, 1H), 5.23 (s, 2H), 5.18 (s,

2H), 4.24 (t, J ¼ 5.7 Hz, 2H), 4.06 (t, J ¼ 5.7 Hz, 2H), 3.95 (q, J ¼ 7.2 Hz,

H), 7.46 (t, J ¼ 7.6 Hz, 1H), 7.28 (d, J ¼ 7.9 Hz, 1H), 7.28 (d, J ¼ 7.9 Hz,

H), 7.13 (d, J ¼ 8.5 Hz, 2H), 6.67 (d, J ¼ 8.6 Hz, 1H), 6.61e6.53 (m,

H), 5.26 (s, 2H), 4.23 (d, J ¼ 6.1 Hz, 2H), 4.19 (t, J ¼ 5.6 Hz, 2H), 3.95

13

(

t, J ¼ 5.6 Hz, 2H), 3.48 (s, 3H), 3.10 (s, 3H), 1.90 (m, 1H), 0.63 (d,

2H), 3.50 (s, 3H), 3.09 (s, 3H), 1.18 (t, J ¼ 7.1 Hz, 3H). C NMR

13

J ¼ 6.5 Hz, 6H). C NMR (100 MHz, DMSO-d

54.15, 152.34, 150.83, 148.50, 147.50, 141.12, 137.3, 133.44, 132.89,

30.75, 127.75, 126.69, 121.16, 117.22, 114.76, 111.54, 110.27, 107.04,

6

):

d

159.76, 157.98,

6

(100 MHz, DMSO-d ): d 167.50, 160.22, 157.99, 154.25, 152.44,

1

150.78, 148.18, 147.53, 140.93, 137.32, 133.42, 132.85, 130.44, 127.83,

126.73, 119.88, 117.2, 115.02, 111.56, 110.48, 107.26, 105.73, 65.77,

61.55, 48.34, 47.58, 42.10, 37.06, 29.64, 13.84. MS(ESI), m/z: 594.29

05.73, 65.65, 52.54, 48.38, 42.26, 37.05, 29.59, 29.15,19.18. MS(ESI),

þ

m/z: 564.33 [MþH] .

[MþH] .

L. He et al. / European Journal of Medicinal Chemistry 87 (2014) 595e610

607

4

.2.12. 2-(1-(2-Cyanobenzyl)-3-methyl-8-(4-(2-(methyl(pyridin-2-

6.64e6.55 (m, 1H), 5.02 (s, 2H), 4.31 (q, J ¼ 7.2 Hz, 2H), 4.24 (t,

J ¼ 5.8 Hz, 2H), 3.95 (t, J ¼ 5.7 Hz, 2H), 3.71 (s, 3H), 3.45 (s, 3H), 3.10

yl)amino)ethoxy) phenyl)-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-

yl)acetic acid (11l)

13

(s, 3H), 1.34 (t, J ¼ 7.0 Hz, 3H). C NMR (100 MHz, DMSO-d

6

):

1

Yield 57.9%. HPLC purity: 97.32%. White solid. H NMR (400 MHz,

d 159.88, 158.36, 157.84, 154.00, 151.17, 150.80, 148.15, 147.24,

DMSO-d

6

):

d

8.09 (d, J ¼ 4.0 Hz, 1H), 7.85 (d, J ¼ 7.6 Hz, 1H), 7.67 (d,

137.46, 130.52, 129.68, 129.22, 120.68, 114.80, 113.59, 111.56,

106.72, 105.89, 65.69, 55.00, 48.41, 43.04, 41.17, 37.07, 29.43,

16.06.MS(ESI), m/z: 541.24 [MþH] .

J ¼ 8.5 Hz, 2H), 7.62 (t, J ¼ 7.7 Hz, 1H), 7.51 (t, J ¼ 7.1 Hz, 1H), 7.46 (t,

J ¼ 7.6 Hz,1H), 7.31 (d, J ¼ 7.8 Hz,1H), 7.13 (d, J ¼ 8.6 Hz, 2H), 6.67 (d,

J ¼ 8.5 Hz, 1H), 6.61e6.55 (m, 1H), 5.24 (s, 2H), 5.07 (s, 2H), 4.32 (t,

þ

13

J ¼ 4.8 Hz, 2H), 4.07 (t, J ¼ 4.8 Hz, 2H), 3.50 (s, 3H), 3.25 (s, 3H).

C

4.2.17. 7-Allyl-1-(4-methoxybenzyl)-3-methyl-8-(4-(2-

NMR (100 MHz, DMSO-d

50.78, 148.18, 147.52, 140.93, 137.32, 133.42, 132.85, 130.44, 127.83,

26.73, 119.88, 117.21, 115.02, 111.56, 110.48, 107.26, 105.74, 65.77,

6

): d 167.51, 160.22, 157.99, 154.25, 152.45,

(methyl(pyridin-2-yl)amino) ethoxy)phenyl)-1H-purine-

1

2

,6(3H,7H)-dione (11q)

Yield 84.1%. HPLC purity: 98.82%. White solid. 1_H NMR

þ

61.55, 48.34, 42.10, 37.07, 29.64. MS(ESI), m/z: 566.29 [MþH] .

(

400 MHz, DMSO-d

6

):

d

8.09 (d, J ¼ 4.4 Hz, 1H), 7.65 (d, J ¼ 8.4 Hz,

2

1

H), 7.51 (t, J ¼ 7.9 Hz, 1H), 7.27 (d, J ¼ 8.3 Hz, 2H), 7.11 (d, J ¼ 8.5 Hz,

H), 6.85 (d, J ¼ 8.3 Hz, 2H), 6.66 (d, J ¼ 8.6 Hz, 1H), 6.61e6.55 (m,

H), 6.09e5.98 (m, 1H), 5.18 (d, J ¼ 10.6 Hz, 1H), 4.99 (s, 2H), 4.98 (d,

4.2.13. Ethyl-4-(1-(2-cyanobenzyl)-3-methyl-8-(4-(2-

(

methyl(pyridin-2-yl)amino) ethoxy)phenyl)-2,6-dioxo-2,3-

dihydro-1H-purin-7(6H)-yl)butanoate (11m)

J ¼ 4.0 Hz, 2H), 4.89 (d, J ¼ 17.3 Hz, 1H), 4.23 (t, J ¼ 5.6 Hz, 2H), 3.94

Yield 75.0%. HPLC purity: 99.10%. White solid. ¹H NMR

13

(

1

t, J ¼ 5.7 Hz, 2H), 3.71 (s, 3H), 3.47 (s, 3H), 3.09 (s, 3H). C NMR

100 MHz, DMSO d ): 159.97, 158.37, 157.98, 153.97, 151.51, 150.79,

47.99, 147.52, 137.30, 133.76, 130.35, 129.63, 129.24, 120.46, 116.51,

14.74, 113.57, 111.54, 106.93, 105.71, 65.70, 54.99, 48.35, 47.86, 43.0,

(

400 MHz, DMSO-d

6

):

d

8.10 (d, J ¼ 4.0 Hz, 1H), 7.85 (d, J ¼ 7.6 Hz,

6

d

1

2

H), 7.67 (d, J ¼ 8.5 Hz, 2H), 7.62 (t, J ¼ 7.7 Hz, 1H), 7.51 (t, J ¼ 7.1 Hz,

H), 7.46 (t, J ¼ 7.6 Hz, 1H), 7.31 (d, J ¼ 7.8 Hz, 1H), 7.13 (d, J ¼ 8.6 Hz,

H), 6.67 (d, J ¼ 8.5 Hz, 1H), 6.61e6.55 (m, 1H), 5.27 (s, 2H), 4.37 (t,

þ

3

7.04, 29.45. MS(ESI), m/z: 553.31 [MþH] .

J ¼ 6.6 Hz, 2H), 4.24 (t, J ¼ 5.6 Hz, 2H), 3.96 (t, J ¼ 5.6 Hz, 2H), 3.88

(

1

d

q, J ¼ 7.2 Hz, 2H), 3.48 (s, 3H), 3.10 (s, 3H), 2.19 (t, J ¼ 6.8 Hz, 2H),

4.2.18. 7-Ethyl-1-(4-ﬂuorobenzyl)-3-methyl-8-(4-(2-

13

.84 (m, 2H), 1.06 (t, J ¼ 7.1 Hz, 3H). C NMR (100 MHz, DMSO-d

6

):

171.80, 159.93, 157.96, 154.06, 151.88, 150.82, 148.55, 147.47, 141.10,

37.35, 133.40, 132.85, 130.71, 127.77, 126.75, 120.57, 117.23, 114.78,

(methyl(pyridin-2-yl)amino) ethoxy)phenyl)-1H-purine-

2

,6(3H,7H)-dione (11r)

1

Yield 78.0%. HPLC purity: 98.78%. White solid. ¹H NMR

111.56, 110.35,106.96,105.77, 65.71, 59.86, 48.38, 45.12, 42.21, 37.08,

(

2

4

400 MHz, DMSO-d

6

):

d

8.09 (d, J ¼ 4.7 Hz, 1H), 7.64 (d, J ¼ 8.6 Hz,

þ

3

0.01, 29.55, 25.29, 13.95. MS(ESI), m/z: 622.27 [MþH] .

H), 7.51 (t, J ¼ 7.3 Hz, 1H), 7.41 (d, J ¼ 8.7 Hz, 2H), 7.13 (t, J ¼ 8.0 Hz,

H), 6.67 (d, J ¼ 8.5 Hz, 1H), 6.61e6.55 (m, 1H), 5.07 (s, 2H), 4.31 (q,

4.2.14. 4-(1-(2-Cyanobenzyl)-3-methyl-8-(4-(2-(methyl(pyridin-2-

J ¼ 7.2 Hz, 2H), 4.24 (t, J ¼ 5.8 Hz, 2H), 3.95 (t, J ¼ 5.7 Hz, 2H), 3.46 (s,

yl)amino)ethoxy) phenyl)-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-

yl)butanoic acid (11n)

13

3

d

1

H), 3.09 (s, 3H), 1.34 (t, J ¼ 6.8 Hz, 3H). C NMR (100 MHz, DMSO-

): 160.03, 157.99, 153.95, 151.60, 150.82, 148.11, 147.53, 137.30,

30.37, 129.77, 129.69, 120.44, 115.09, 114.88, 114.76, 111.55, 106.94,

6

d

Yield 50.6%. HPLC purity: 96.32%. White solid. ¹H NMR

(

400 MHz, DMSO-d

6

):

d

8.09 (d, J ¼ 4.0 Hz, 1H), 7.86 (d, J ¼ 7.6 Hz,

1

05.72, 65.70, 55.00, 48.35, 47.89, 42.93, 37.05, 29.49. MS(ESI), m/z:

1

2

H), 7.67 (d, J ¼ 8.5 Hz, 2H), 7.63 (t, J ¼ 7.7 Hz, 1H), 7.51 (t, J ¼ 7.1 Hz,

þ

529.25 [MþH] .

H), 7.46 (t, J ¼ 7.6 Hz, 1H), 7.32 (d, J ¼ 7.8 Hz, 1H), 7.13 (d, J ¼ 8.6 Hz,

H), 6.67 (d, J ¼ 8.5 Hz, 1H), 6.62e6.55 (m, 1H), 5.25 (s, 2H), 4.37 (t,

4.2.19. 7-Allyl-1-(4-ﬂuorobenzyl)-3-methyl-8-(4-(2-

J ¼ 5.6 Hz, 2H), 4.24 (t, J ¼ 5.6 Hz, 2H), 4.07 (t, J ¼ 6.6 Hz, 2H), 2.37 (t,

13

(methyl(pyridin-2-yl)amino)etoxy) phenyl)-1H-purine-2,6(3H,7H)-

dione (11s)

J ¼ 6.8 Hz, 2H), 2.19 (m, 2H). C NMR (100 MHz, DMSO-d

171.80, 159.93, 157.96, 154.06, 151.88, 150.82, 148.55, 147.47, 141.10,

37.35, 133.40, 132.85, 130.71, 127.77, 126.75, 120.57, 117.23, 114.78,

6

):

d

Yield 73.9%. HPLC purity: 98.34%. White solid. ¹H NMR

1

(

400 MHz, DMSO-d

6

):

d

8.09 (d, J ¼ 4.6 Hz, 1H), 7.66 (d, J ¼ 8.4 Hz,

111.56, 110.35,106.96,105.77, 65.71, 59.86, 48.38, 42.21, 37.08, 30.01,

þ

2H), 7.51 (t, J ¼ 7.9 Hz, 1H), 7.19 (d, J ¼ 8.7 Hz, 2H), 7.12 (d, J ¼ 7.9 Hz,

2

9.55, 25.29. MS(ESI), m/z: 594.29 [MþH] .

2

1

2

H), 6.84 (d, J ¼ 8.3 Hz, 2H), 6.66 (d, J ¼ 8.6 Hz, 1H), 6.60e6.55 (m,

H), 6.09e5.97 (m, 1H), 5.27 (d, J ¼ 10.6 Hz, 1H), 5.04 (d, J ¼ 7.2 Hz,

H), 4.90 (d, J ¼ 17.2 Hz,1H), 4.50 (s, 2H), 4.23 (t, J ¼ 5.7 Hz, 2H), 3.94

4.2.15. 1-(4-Methoxybenzyl)-3-methyl-8-(4-(2-(methyl(pyridin-2-

yl)amino)ethoxy) phenyl)-1H-purine-2,6(3H,7H)-dione (11o)

13

1

(t, J ¼ 5.6 Hz, 2H), 3.48 (s, 3H), 3.09 (s, 3H). C NMR (100 MHz,

Yield 89.1%. HPLC purity: 98.90%. White solid.

400 MHz, DMSO-d ):

H NMR

DMSO d

6

): d 160.03, 157.99, 153.95, 151.60, 150.82, 148.11, 147.53,

(

6

d

13.66 (s,1H), 8.08 (d, J ¼ 4.0 Hz, 2H), 7.96 (d,

137.30, 133.73, 130.37, 129.77, 129.69, 120.44, 116.56, 115.09, 114.88,

J ¼ 7.6 Hz, 1H), 7.51 (t, J ¼ 7.0 Hz, 1H), 7.27 (d, J ¼ 8.6 Hz, 2H), 7.08 (d,

J ¼ 8.8 Hz, 2H), 6.85 (d, J ¼ 8.6 Hz, 2H), 6.66 (d, J ¼ 8.0 Hz, 1H),

1

14.76, 111.55, 106.94, 105.72, 65.70, 48.35, 47.89, 42.93, 37.05,

þ

29.49. MS(ESI), m/z: 541.25 [MþH] .

6

.60e6.55 (m, 1H), 5.02 (s, 2H), 4.22 (t, J ¼ 5.8 Hz, 2H), 3.94 (t,

13

J ¼ 5.8 Hz, 2H), 3.71 (s, 3H), 3.49 (s, 3H), 3.09 (s, 3H). C NMR

100 MHz, DMSO-d ): 160.05, 158.33, 157.99, 153.82, 150.94,

50.16, 147.52, 137.29, 129.76, 129.12, 128.10, 121.20, 114.77, 113.57,

11.54, 105.70, 65.66, 54.99, 48.36, 43.06, 37.03, 29.71. MS(ESI), m/z:

(

1

6

d

4.2.20. 1-Allyl-3-methyl-8-(4-(2-(methyl(pyridin-2-yl)amino)

ethoxy)phenyl)-1H-purine-2,6(3H,7H)-dione (11t)

Yield 87.9%. HPLC purity: 98.71%. White solid. ¹H NMR

þ

513.29 [MþH] .

(400 MHz, DMSO-d

6

):

d

13.60 (s, 1H), 8.12 (d, J ¼ 4.0 Hz,1H), 8.00 (d,

J ¼ 8.4 Hz, 2H), 7.50 (d, J ¼ 6.8 Hz, 1H), 7.08 (d, J ¼ 8.8 Hz, 2H), 6.66

(d, J ¼ 8.6 Hz, 1H), 6.63e6.56 (m, 1H), 5.92e5.81 (m, 1H), 5.10 (d,

J ¼ 5.6 Hz, 1H), 5.06 (d, J ¼ 5.6 Hz, 1H), 4.50 (d, J ¼ 4.8 Hz, 2H), 4.22

4.2.16. 7-Ethyl-1-(4-methoxybenzyl)-3-methyl-8-(4-(2-

(

methyl(pyridin-2-yl)amino) ethoxy)phenyl)-1H-purine-

2

,6(3H,7H)-dione (11p)

(t, J ¼ 5.8 Hz, 2H), 3.94 (t, J ¼ 5.8 Hz, 2H), 3.50 (s, 3H), 3.09 (s, 3H).

Yield 83.6%. HPLC purity: 98.76%. White solid. ¹H NMR

13

6

C NMR (100 MHz, DMSO-d ): d 160.07, 158.00, 153.52, 150.72,

(

2

400 MHz, DMSO-d

H), 7.52 (t, J ¼ 7.8 Hz, 1H), 7.28 (d, J ¼ 8.4 Hz, 2H), 7.13 (d,

6

):

d

8.09 (d, J ¼ 4.7 Hz, 1H), 7.63 (d, J ¼ 8.6 Hz,

150.08, 148.79, 147.53, 137.32, 133.16, 128.12, 121.22, 115.98, 114.83,

111.55, 107.11, 105.73, 65.67, 48.37, 42.52, 37.04, 29.72. MS(ESI), m/z:

þ

J ¼ 8.6 Hz, 2H), 6.86 (d, J ¼ 8.5 Hz, 2H), 6.68 (d, J ¼ 8.2 Hz, 1H),

433.32 [MþH] .

608

L. He et al. / European Journal of Medicinal Chemistry 87 (2014) 595e610

4

.2.21. 1,7-Diallyl-3-methyl-8-(4-(2-(methyl(pyridin-2-yl)amino)

4.2.26. 7-Ethyl-3-methyl-8-(4-(2-(methyl(pyridin-2-yl)amino)

ethoxy)phenyl)-1H-purine-2,6(3H,7H)-dione (14a)

ethoxy)phenyl)-1H-purine-2,6(3H,7H)-dione (11u)

Yield 79.2%. HPLC purity: 95.34%. White solid. ¹H NMR

Yield 68.3%. HPLC purity: 97.32%. White solid. ¹H NMR

(

2

400 MHz, DMSO-d

H), 7.56 (t, J ¼ 7.5 Hz, 1H), 7.17 (d, J ¼ 8.5 Hz, 2H), 6.72 (d,

J ¼ 8.6 Hz, 1H), 6.66e6.58 (m, 1H), 6.15e6.03 (m, 1H), 5.91e5.81

m, 1H), 5.23 (d, J ¼ 10.4 Hz, 1H), 5.16 (d, J ¼ 10.4 Hz, 1H), 5.12 (d,

6

):

d

8.15 (d, J ¼ 4.0 Hz, 1H), 7.71 (d, J ¼ 8.4 Hz,

(400 MHz, DMSO-d

6

):

d

11.13 (s, 1H), 8.09 (d, J ¼ 4.5 Hz, 1H), 7.63 (d,

J ¼ 8.7 Hz, 2H), 7.51 (t, J ¼ 7.0 Hz,1H), 7.13 (d, J ¼ 8.7 Hz, 2H), 6.67 (d,

J ¼ 8.6 Hz, 1H), 6.60e6.55 (m, 1H), 4.36 (d, J ¼ 6.9 Hz, 2H), 4.30 (d,

J ¼ 5.9 Hz, 2H), 3.95 (t, J ¼ 5.9 Hz, 2H), 3.38 (s, 2H), 3.09 (s, 3H), 1.33

(

13

J ¼ 17.2 Hz, 1H), 5.02 (d, J ¼ 4.4 Hz, 2H), 4.94 (d, J ¼ 17.2 Hz, 1H),

(t, J ¼ 7.1 Hz, 3H). C NMR (100 MHz, DMSO-d

6

): d 159.82, 158.00,

4

2

d

.54 (d, J ¼ 4.4 Hz, 2H), 4.28 (t, J ¼ 5.6 Hz, 2H), 4.00 (t, J ¼ 5.6 Hz,

154.56, 149.92, 150.83, 149.57, 147.53, 137.30, 130.47, 120.77, 114.78,

111.55, 107.13, 105.72, 65.69, 48.36, 41.08, 37.05, 28.40, 16.03.

13

H), 3.53 (s, 3H), 3.14 (s, 3H). C NMR (100 MHz, DMSO-d

159.96, 157.98, 153.63, 151.39, 150.48, 147.99, 147.52, 137.28,

33.73, 132.97, 130.30, 120.46, 116.50, 116.24, 114.73, 111.54, 106.86,

05.70, 65.69, 48.35, 47.84, 42.42, 37.04, 29.41. MS(ESI), m/z:

6

):

þ

MS(ESI), m/z: 421.25 [MþH] .

1

4

.2.27. 3-Methyl-8-(4-(2-(methyl(pyridin-2-yl)amino)ethoxy)

phenyl)-7-(3-methylbut-2-en-1-yl)-1H-purine-2,6(3H,7H)-dione

14b)

Yield 64.9%. HPLC purity: 97.81%. White solid. ¹H NMR

(400 MHz, DMSO-d ):

þ

73.27 [MþH] .

(

4

.2.22. 3-Methyl-8-(4-(2-(methyl(pyridin-2-yl)amino)ethoxy)

phenyl)-1-propyl-1H-purine-2,6(3H,7H)-dione (11v)

6

d

11.11 (s, 1H), 8.09 (d, J ¼ 4.4 Hz, 1H), 7.62 (d,

Yield 86.9%. HPLC purity: 97.22%. White solid. ¹H NMR

J ¼ 8.6 Hz, 2H), 7.51 (t, J ¼ 7.8 Hz,1H), 7.12 (d, J ¼ 8.6 Hz, 2H), 6.66 (d,

J ¼ 8.5 Hz, 1H), 6.62e6.53 (m, 1H), 5.22 (t, J ¼ 7.6 Hz, 1H), 4.92 (d,

J ¼ 5.5 Hz, 2H), 4.23 (d, J ¼ 5.6 Hz, 2H), 3.95 (d, J ¼ 5.5 Hz, 3H), 3.38

(

400 MHz, DMSO-d

6

):

d

13.60 (s,1H), 8.09 (d, J ¼ 3.9 Hz,1H), 8.04 (d,

J ¼ 8.3 Hz, 2H), 7.51 (t, J ¼ 7.8 Hz,1H), 7.08 (d, J ¼ 8.7 Hz, 2H), 6.66 (d,

J ¼ 8.5 Hz, 1H), 6.57 (s, 1H), 4.22 (d, J ¼ 5.5 Hz, 2H), 3.94 (d,

J ¼ 5.5 Hz, 2H), 3.86 (t, J ¼ 6.7 Hz, 2H), 3.49 (s, 3H), 3.09 (s, 3H), 1.58

13

(s, 3H), 3.09 (s, 2H), 1.63 (s, 3H), 1.53 (s, 3H). C NMR (1001 MHz,

6

DMSO-d ): d 159.80, 158.00, 154.71, 151.00, 150.84, 149.45, 147.53,

137.32, 135.67, 130.51, 120.85, 119.93, 114.74, 111.56, 107.26, 105.72,

13

(

d

m, 2H), 0.88 (t, J ¼ 7.5 Hz, 3H). C NMR (100 MHz, DMSO-d

6

):

160.04, 158.00, 153.88, 150.94, 149.96, 148.64, 147.53, 137.32,

28.09, 121.25, 114.82, 111.55, 107.18, 105.73, 65.67, 48.37, 42.10,

65.67, 48.34, 44.16, 37.04, 28.40, 25.19, 17.76. MS(ESI), m/z: 461.27

þ

1

3

[MþH] .

þ

7.04, 29.66, 20.82, 11.16. MS(ESI), m/z: 435.27 [MþH] .

4.2.28. 1,7-Diethyl-3-methyl-8-(4-(2-(methyl(pyridin-2-yl)amino)

4

.2.23. 7-Ethyl-3-methyl-8-(4-(2-(methyl(pyridin-2-yl)amino)

ethoxy)phenyl)-1H-purine-2,6(3H,7H)-dione (14c)

ethoxy)phenyl)-1-propyl-1H-purine-2,6(3H,7H)-dione (11w)

Yield 82.5%. HPLC purity: 95.09%. White solid. ¹H NMR

1

Yield 82.7%. HPLC purity: 99.20%. White solid.

400 MHz, DMSO-d ):

8.16 (d, J ¼ 3.9 Hz, 1H), 7.69 (d, J ¼ 8.3 Hz,

H), 7.57 (t, J ¼ 7.7 Hz,1H), 7.19 (d, J ¼ 8.3 Hz, 2H), 6.73 (d, J ¼ 8.6 Hz,

H), 6.64 (t, J ¼ 5.7 Hz, 1H), 4.36 (q, J ¼ 7.2 Hz, 2H), 4.30 (d,

H

NMR

(400 MHz, DMSO-d

6

):

d

8.09 (d, J ¼ 3.6 Hz, 1H), 7.63 (d, J ¼ 8.6 Hz,

(

2

1

6

d

2H), 7.52 (t, J ¼ 7.2 Hz, 1H), 7.13 (d, J ¼ 8.6 Hz, 2H), 6.67 (d, J ¼ 8.6 Hz,

1H), 6.62e6.56 (m, 1H), 4.30 (q, J ¼ 6.7 Hz, 2H), 4.24 (t, J ¼ 5.6 Hz,

2H), 3.96 (t, J ¼ 5.6 Hz, 2H), 3.92 (q, J ¼ 6.7 Hz, 2H), 3.45 (s, 3H), 3.10

13

J ¼ 5.5 Hz, 2H), 4.01 (t, J ¼ 5.5 Hz, 2H), 3.92 (t, J ¼ 7.2 Hz, 2H), 3.51 (s,

H), 3.16 (s, 3H), 1.64 (m, 2H), 1.40 (t, J ¼ 6.8 Hz, 3H), 0.94 (t,

(s, 3H), 1.34 (t, J ¼ 6.9 Hz, 3H), 1.14 (t, J ¼ 6.9 Hz, 3H). C NMR

3

6

(100 MHz, DMSO-d ): d 159.86, 158.00, 153.82, 150.94, 150.52,

13

J ¼ 7.3 Hz, 3H). C NMR (100 MHz, DMSO-d

54.04, 150.96, 150.71, 148.01, 147.46, 137.34, 130.51, 120.72, 114.80,

11.55, 106.69, 105.77, 65.69, 48.37, 42.00, 41.12, 37.06, 29.34, 20.81,

6

):

d

159.86, 157.96,

148.00, 147.53, 137.30, 130.50, 120.72, 114.80, 111.55, 106.75, 105.72,

1

65.70, 48.36, 41.11, 37.05, 35.61, 29.31, 16.06, 13.11. MS(ESI), m/z:

þ

449.25 [MþH] .

þ

16.05, 11.15. MS(ESI), m/z: 463.29 [MþH] .

0

4

.2.29. 2,2 -(3-Methyl-8-(4-(2-(methyl(pyridin-2-yl)amino)

4

.2.24. 3-Methyl-8-(4-(2-(methyl(pyridin-2-yl)amino)ethoxy)

phenyl)-1,7-dipropyl-1H-purine-2,6(3H,7H)-dione (11x)

Yield 79.5%. HPLC purity: 98.35%. White solid. ¹H NMR

ethoxy)phenyl)-2,6-dioxo-2,3-dihydro-1H-purine-1,7(6H)-diyl)

diacetic acid (14d)

Yield 73.8%. HPLC purity: 96.03%. White solid. ¹H NMR

(

400 MHz, DMSO-d

6

):

d

8.15 (d, J ¼ 3.9 Hz, 1H), 7.68 (d, J ¼ 8.4 Hz,

(400 MHz, DMSO-d

6

):

d

8.07 (d, J ¼ 4.0 Hz, 1H), 7.68 (t, J ¼ 7.7 Hz,

2

1

2

H), 7.57 (t, J ¼ 7.8 Hz, 1H), 7.18 (d, J ¼ 8.4 Hz, 2H), 6.73 (d, J ¼ 8.6 Hz,

1H), 7.60 (d, J ¼ 8.1 Hz, 2H), 7.12 (d, J ¼ 7.8 Hz, 2H), 6.91 (d,

J ¼ 8.6 Hz, 1H), 6.70e6.65 (m, 1H), 5.07 (s, 2H), 4.53 (s, 2H), 4.27 (t,

H), 6.63 (t, J ¼ 5.8 Hz, 1H), 4.33 (t, J ¼ 7.2 Hz, 2H), 4.28 (t, J ¼ 5.6 Hz,

H), 4.01 (t, J ¼ 5.6 Hz, 2H), 3.92 (t, J ¼ 7.2 Hz, 2H), 3.51 (s, 3H), 3.15

1

3

J ¼ 5.6 Hz, 2H), 4.00 (t, J ¼ 5.6 Hz, 2H), 3.49 (s, 3H), 3.16 (s, 3H).

C

(

s, 3H), 1.75 (m, 2H), 1.63 (m 2H), 0.93 (t, J ¼ 7.4 Hz, 3H), 0.78 (t,

6

NMR (100 MHz, DMSO-d ): d 169.43, 168.86, 160.00, 158.00, 153.85,

13

J ¼ 7.3 Hz, 3H). C NMR (100 MHz, DMSO-d

54.12, 151.37, 147.95, 147.52, 137.29, 130.55, 120.90, 114.77, 111.54,

06.89, 105.71, 65.68, 48.37, 47.22, 41.99, 37.04, 29.34, 23.61, 20.80,

6

):

d

59.79, 157.99,

152.34, 150.53, 148.03, 147.89, 137.32, 130.40, 120.17, 114.97, 111.72,

1

107.12, 105.72, 65.58, 48.91, 47.51, 41.68, 37.43, 29.48. MS(ESI), m/z:

þ

531.13 [MþNa] .

þ

1

1.13, 10.49. S(ESI), m/z: 477.27 [MþH] .

4.2.30. (3-Methyl-8-(4-(2-(methyl(pyridin-2-yl)amino)ethoxy)

4.2.25. 3-Methyl-8-(4-(2-(methyl(pyridin-2-yl)amino)ethoxy)

phenyl)-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)methyl pivalate

phenyl)-1H-purine-2,6 (3H,7H)-dione (13)

(15a)

Yield 78.0%. HPLC purity: 96.44%. White solid. ¹H NMR

Yield 47.3%. HPLC purity: 97.21%. White solid. H NMR (400 MHz,

1

(

400 MHz, DMSO-d

6

):

d

13.59 (s, 1H), 11.08 (s, 1H), 8.09 (d,

DMSO-d

6

):

d

11.27 (s, 1H), 8.09 (d, J ¼ 4.0 Hz, 1H), 7.67 (d, J ¼ 8.7 Hz,

J ¼ 4.5 Hz, 1H), 8.05 (d, J ¼ 8.4 Hz, 2H), 7.51 (t, J ¼ 7.7 Hz, 1H),

2H), 7.51 (t, J ¼ 7.8 Hz, 1H), 7.15 (d, J ¼ 8.7 Hz, 2H), 6.66 (d, J ¼ 8.5 Hz,

1H), 6.63e6.55 (m, 1H), 6.14 (s, 2H), 4.24 (t, J ¼ 5.8 Hz, 2H), 3.95 (t,

7

1

3

1

4

.07 (d, J ¼ 8.5 Hz, 2H), 6.66 (d, J ¼ 8.6 Hz, 1H), 6.60e6.55 (m,

13

H), 4.22 (t, J ¼ 5.6 Hz, 2H), 3.94 (t, J ¼ 5.6 Hz, 2H), 3.42 (s, 3H),

.09 (s, 3H). ¹³C NMR (100 MHz, DMSO-d

): 162.28, 159.97,

57.99, 156.34, 154.33, 151.25, 151.11, 147.53, 137.31, 127.99, 121.31,

14.80, 111.54, 105.72, 75.19, 65.65, 48.37, 37.03. MS(ESI), m/z:

J ¼ 5.8 Hz, 2H), 3.40 (s, 3H), 3.09 (s, 3H), 1.08 (s, 9H). C NMR

6

d

6

(100 MHz, DMSO-d ): d 177.90, 159.90, 157.99, 154.39, 151.12,

150.25, 149.97, 147.52, 137.29, 130.36, 121.49, 114.75, 111.53, 107.81,

105.70, 65.64, 64.53, 61.47, 48.37, 41.70, 37.03, 28.72. MS(ESI), m/z:

529.29 [MþNa] .

þ

10.14 [MþNa] .

L. He et al. / European Journal of Medicinal Chemistry 87 (2014) 595e610

609

4

.2.31. (1-(2-Ethoxy-2-oxoethyl)-3-methyl-8-(4-(2-

The medium was again changed to serum-containing DMEM that

contained 10 g/mL insulin but no IBMX or DEX every two days

(

methyl(pyridin-2-yl)amino)ethoxy) phenyl)-2,6-dioxo-2,3-

m

dihydro-1H-purin-7(6H)-yl)methyl pivalate (15b)

until all the preadipocytes differentiated successfully. Generally full

differentiation needs 8e12 days. The twelfth day after differentia-

tion, the culture medium was replaced by DMEM supplemented

Yield 60.2%. HPLC purity: 98.73%. White solid. ¹H NMR

(

2

400 MHz, DMSO-d

H), 7.59 (d, J ¼ 8.8 Hz, 1H), 7.17 (d, J ¼ 8.5 Hz, 2H), 6.66 (d,

6

):

d

8.10 (d, J ¼ 4.0 Hz, 1H), 7.70 (d, J ¼ 8.8 Hz,

with compounds (10 mM), RSG (10 mM), Met (1.0 mM), and AICAR

J ¼ 8.6 Hz, 1H), 6.61e6.55 (m, 1H), 6.16 (s, 2H), 4.63 (s, 2H), 4.25 (t,

J ¼ 5.8 Hz, 2H), 4.14 (q, J ¼ 7.1 Hz, 2H), 3.95 (t, J ¼ 5.8 Hz, 2H), 3.50 (s,

(0.5 mM). After 24 h, wash the 3T3-L1 cell with precooling PBS

twice and then drying, join 50 Mm KOH liquor in cracking reaction

13

ꢀ

3

(

H), 3.09 (s, 3H), 1.20 (t, J ¼ 7.1 Hz, 3H), 1.08 (s, 9H). C NMR

under 60 C, 10 min later collecting the supernatant after centri-

100 MHz, DMSO-d

6

):

d

177.92, 167.48, 160.23, 157.99, 153.74,

fugation. The determination of TG was performed by commercial

Triglyceride Assay Kit (Biovision, San Francisco Bay, USA.). The re-

sults were recorded for three independent experiments. The TG

152.55, 150.47, 148.02, 147.53, 137.30, 130.46, 119.81, 114.99, 111.55,

1

2

07.02, 105.72, 65.76, 64.53, 61.57, 60.96, 48.34, 47.56, 41.71, 37.05,

9.52, 13.91. MS(ESI), m/z: 593.3 [MþH] .

þ

inhibition ratio was calculated using (1

TGcompound) ꢁ 100%.

ꢃ

TGmodel/

4.2.32. Ethyl-4-(3-methyl-8-(4-(2-(methyl(pyridin-2-yl)amino)

ethoxy)phenyl)-2,6-dioxo-7-((pivaloyloxy)methyl)-2,3,6,7-

tetrahydro-1H-purin-1-yl)butanoate (15c)

4.3.2. Animal model and treatment of DIO mice

C57BL/6J mice were obtained from Beijing HFK Bioscience CO.,

LTD and housed individually in a room maintained at 25 C on a

Yield 56.9%. HPLC purity: 98.92%. White solid. ¹H NMR

ꢀ

(

2

1

400 MHz, DMSO-d

6

):

H), 7.51 (t, J ¼ 7.7 Hz,1H), 7.15 (d, J ¼ 8.5 Hz, 2H), 6.66 (d, J ¼ 8.6 Hz,

H), 6.61e6.55 (m, 1H), 6.16 (s, 2H), 4.25 (t, J ¼ 5.6 Hz, 2H), 4.01 (t,

d

8.09 (d, J ¼ 4.0 Hz, 1H), 7.67 (d, J ¼ 8.4 Hz,

light/dark schedule. For DIO mice, 3-week-old male C57BL/6J mice

were fed a high fat diet (HFD, Research Diets, D12492) for 12 weeks,

thus their weight is higher 20% in the normal group. Then these

animals were assigned to ﬁve groups randomly consisting of 6 mice

each. The mice received a normal diet with 18.94% of energy

derived from fat, 31.67% from protein, and 49.39% from carbohy-

drates and received a high fat diet with 60.0% of calories from fat,

20.0% from protein, and 20.0% from carbohydrates. HFD þ 11c

(50 mg/kg/day), HFD þ 11d (50 mg/kg/day), HFD þ 11c$HCl (20 mg/

kg/day), HFD þ 11c$HCl (50 mg/kg/day), and HFD þ Met (150 mg/

kg/day) in 1% tween 80 were orally administered per day for 4

weeks. After the experimental period was completed, all mice were

anesthetized and samples were collected. Meanwhile, body, liver,

and epididymal white adipose tissues weight of mice were recor-

ded. Serum levels of glucose, insulin, AST, ALT, TG, LDL, HDL and FFA

were measured using a multifunctional biochemistry analyzer

Olympus AU2700 (Olympus, Tokyo, Japan). The liver content of TG

was analyzed using a diagnostic kit (Jiancheng, Nanjing, China)

according to the manufacturer's instruction. The percentages were

calculated using [(VHFD ꢃ Vtreatment)/VHFD] ꢁ 100%.

J ¼ 5.6 Hz, 2H), 3.97 (t, J ¼ 6.6 Hz, 2H), 3.90 (q, J ¼ 7.2 Hz, 2H), 3.46 (s,

H), 3.09 (s, 3H), 2.34 (t, J ¼ 6.8 Hz, 2H), 1.89 (m, 2H), 1.16 (t,

3

13

J ¼ 7.1 Hz, 3H), 1.11 (s, 9H). C NMR (100 MHz, DMSO-d

73.62, 159.51, 153.94, 152.06, 150.96, 149.71, 148.34, 143.25, 136.76,

28.06, 121.58, 114.76, 112.30, 112.03, 107.29, 65.27, 64.53, 61.47,

6

): d 177.92,

1

5

6

9.86, 49.39, 43.21, 38.47, 37.03, 31.17, 29.69, 13.92. MS(ESI), m/z:

þ

43.31 [MþNa] .

4

.2.33. 2-(3-Mmethyl-8-(4-(2-(methyl(pyridin-2-yl)amino)ethoxy)

phenyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)acetic acid

(

16a)

Yield 68.4%. HPLC purity: 97.63%. White solid. ¹H NMR

400 MHz, DMSO-d ): 13.73 (s, 1H), 12.92 (s, 1H), 8.08 (d,

(

6

d

J ¼ 4.2 Hz,1H), 7.70 (d, J ¼ 8.4 Hz, 2H), 7.58 (t, J ¼ 7.7 Hz, 1H), 7.08 (d,

J ¼ 8.5 Hz, 2H), 6.77 (d, J ¼ 8.6 Hz, 1H), 6.61e6.65 (m, 1H), 4.56 (s,

2

(

1

H), 4.24 (t, J ¼ 5.4 Hz, 2H), 3.96 (t, J ¼ 5.4 Hz, 2H), 3.51 (s, 3H), 3.11

s, 3H). ¹³C NMR (100 MHz, DMSO-d

52.06, 150.96, 149.71, 148.54, 143.25, 136.76, 128.06, 121.58, 114.76,

12.30, 112.03, 107.29, 65.27, 50.39, 37.07, 29.69, 23.08. MS(ESI), m/

6

): d 173.92, 159.51, 153.93,

4.3.3. Glucose and insulin tolerance tests

þ

z: 451.29 [MþH] .

Oral glucose tolerance test (OGTT) was performed on mice fas-

ted for 16 h. Glucose levels of blood collected from the tail vein were

determined at 0, 5,15, 30, 60, 90, and 120 min after an oral glucose

load of 2.0 g/kg. For the insulin tolerance test (ITT), mice fasted for

9 h were injected intraperitoneally with 0.5 U/kg of body weight

and glucose levels were measured at 0, 5, 15, 30, 60, 90, and

120 min postinjection. The insulin sensitivity was also evaluated by

the formula of HOMA-IR ¼ fasting glucose (mmol/L) ꢁ fasting in-

4

.2.34. 4-(3-Methyl-8-(4-(2-(methyl(pyridin-2-yl)amino)ethoxy)

phenyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)butanoic acid

(

16b)

Yield 50.9%. HPLC purity: 98.51%. White solid. ¹H NMR

400 MHz, DMSO-d ): 13.73 (s, 1H), 11.02 (s, 1H), 8.08 (d,

(

6

d

J ¼ 4.0 Hz,1H), 7.73 (d, J ¼ 8.4 Hz, 2H), 7.58 (t, J ¼ 7.7 Hz,1H), 7.08 (d,

J ¼ 8.8 Hz, 2H), 6.77 (d, J ¼ 8.6 Hz, 1H), 6.61e6.65 (m, 1H), 4.24 (t,

J ¼ 5.5 Hz, 2H), 3.96 (t, J ¼ 5.4 Hz, 2H), 3.86 (t, J ¼ 5.5 Hz,2H), 3.51 (s,

sulin (mU/mL)/22.5.

13

3

H), 3.11 (s, 3H). 2.27 (t, J ¼ 5.5 Hz, 2H), 1.86 (m, 2H). C NMR

4.3.4. Histology evaluation

For the histological examination, liver and epididymal fat tissues

were ﬁxed in 10% buffered formalin overnight and then embedded

(

1

100 MHz, DMSO-d

49.71, 148.34, 143.25, 136.76, 128.06, 121.58, 114.76, 112.30, 112.03,

07.29, 65.27, 59.86, 49.39, 43.21, 37.07, 29.69, 23.58. MS(ESI), m/

6

):

d

173.62, 159.51, 153.94, 152.06, 150.96,

in parafﬁn, cut at a thickness of 4

mm, and stained with

þ

z:479.27 [MþH] .

hematoxylin-eosin (H&E). Frozen livers were sectioned into 10 mm

thick sections using a microtome, and lipid deposition was detected

by two ways of staining. Oil red O staining was performed using

standard protocols, and together with hematoxylin staining. All cell

morphology and size were analyzed.

4

.3. Biological evaluation

.3.1. Determination the content of TG in 3T3-L1 adipocytes

Murine 3T3-L1 preadipocytes (ATCC, Rockville, MD) were plated

and grown to 2 days after conﬂuence in 96-well culture plates in

DMEM containing 10% fetal bovine serum. Preadipocytes were

induced to differentiate by replacing the medium with serum-

containing DMEM containing 0.5 Mm ethyl-3-isobutylxantine

4.3.5. Pharmacokinetics in SpragueeDawley rats

Two groups (n ¼ 5) of male and female SpragueeDawley rats

(200e250 g) were fasted overnight and received 11c and 11c$HCl as

an intravenous dose (5 mg/kg) or by oral gavage (20 mg/kg). Blood

samples (0.4 mL)were obtained from retro-orbital bleeding at

(

IBMX), 0.25 mM dexamethasone (DEX), and 10 mg/mL insulin.

6

10

L. He et al. / European Journal of Medicinal Chemistry 87 (2014) 595e610

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5

min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, and 24 h

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identi ﬁ cation of new anti-diabetic compounds, Biochem. Pharmacol. 84

post dose for the po group. At each time point, three mice were bled

resulting in a composite pharmacokinetic proﬁle. The tubes were

inverted several times to ensure mixing and placed on ice. The

blood samples were centrifuged to obtain the plasma fraction. The

plasma samples were deproteinized with acetonitrile containing an

internal standard. After centrifugation, the supernatant was diluted

and centrifuged again. The compound concentrations in the su-

pernatant were measured by a high performance liquid chroma-

tographyetandem mass spectrometry (LC/MS/MS).

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[

Acknowledgments

We greatly appreciate the ﬁnancial support from National Key

R&D Programs of China (No. 2012ZX09103101-033), and China

Postdoctoral Science Foundation (2014M552373).

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Appendix A. Supplementary data

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Supplementary data related to this article can be found at http://

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Synthesis and lipid-lowering evaluation of 3-methyl-1 H-purine-2,6-dione derivatives as potent and orally available anti-obesityagents

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