Design, synthesis, and structure-activity relationship studies of novel 2,4,6-trisubstituted-5-pyrimidinecarboxylic acids as peroxisome proliferator-activated receptor γ (PPARγ) partial agonists with comparable antidiabetic efficacy to rosiglitazone

Article abstract of DOI:10.1021/jm100443s

A series of novel 2,4,6-trisubstitutedpyrimidine-5-carboxylic acid derivatives were designed and synthesized with the intent of producing a peroxisome proliferator-activated receptor γ (PPARγ) partial agonist for antidiabetic agents. A pharmacophore-driven approach of in-house screening identified compound 7, which led to the identification of compound 9 featuring a 2,4,6-trisubstituted pyrimidine-5-carboxylic acid core. Structure-activity relationship studies of 9 resulted in identifying 4,6-bisbenzylthio-2- methylthiopyrimidine-5-carboxylic acid (50) as the most attractive of all the screened compounds. The X-ray cocrystal structure of 50 bound on PPARγ revealed that the key hydrogen bond interactions, which are not related to the activation function 2 (AF-2) site, are different from those of the full agonist. Compound 50 showed typical PPARγ partial agonist properties in the PPARγ-GAL4 functional assay and weaker differentiation of adipocytes in 3T3-L1 cells than observed with rosiglitazone. Furthermore, 50 displayed comparable antidiabetic efficacy with rosiglitazone in db/db mice, although its potency is 10-fold weaker than that of rosiglitazone.

Full text of DOI:10.1021/jm100443s

5012 J. Med. Chem. 2010, 53, 5012–5024
DOI: 10.1021/jm100443s
Design, Synthesis, and Structure-Activity Relationship Studies of Novel 2,4,6-Trisubstituted-5-
pyrimidinecarboxylic Acids as Peroxisome Proliferator-Activated Receptor γ (PPARγ) Partial
Agonists with Comparable Antidiabetic Efficacy to Rosiglitazone
Shigeki Seto,* Kyoko Okada, Koichi Kiyota, Shigeki Isogai, Maki Iwago, Takehiro Shinozaki, Yoshiaki Kitamura,
Yasushi Kohno, and Koji Murakami
Discovery Research Laboratories, Kyorin Pharmaceutical Co., Ltd., 2399-1, Nogi, Nogi-machi, Shimotsuga-gun, Tochigi 329-0114, Japan
Received April 11, 2010
A series of novel 2,4,6-trisubstitutedpyrimidine-5-carboxylic acid derivatives were designed and
synthesized with the intent of producing a peroxisome proliferator-activated receptor γ (PPARγ)
partial agonist for antidiabetic agents. A pharmacophore-driven approach of in-house screening
identified compound 7, which led to the identification of compound 9 featuring a 2,4,6-trisubstituted
pyrimidine-5-carboxylic acid core. Structure-activity relationship studies of 9 resulted in identifying
4,6-bisbenzylthio-2-methylthiopyrimidine-5-carboxylic acid (50) as the most attractive of all the scree-
ned compounds. The X-ray cocrystal structure of 50 bound on PPARγ revealed that the key hydrogen
bond interactions, which are not related to the activation function 2 (AF-2) site, are different from those
of the full agonist. Compound 50 showed typical PPARγ partial agonist properties in the PPARγ-
GAL4 functional assay and weaker differentiation of adipocytes in 3T3-L1 cells than observed with
rosiglitazone. Furthermore, 50 displayed comparable antidiabetic efficacy with rosiglitazone in db/db
mice, although its potency is 10-fold weaker than that of rosiglitazone.
Introduction
Structurally, the majority of the known PPAR partial
agonists can be classified into three types, types A-C in
Figure 1. Type A, which includes 1 (balaglitazone),14 2 (FK-
614),15 and 3 (MK-0533),16 has similar structural features as
PPARγ full agonists such as rosiglitazone, which is made up
of an acidic head part and a lipophilictail part. Compound 1 is
currently included in an ongoing phase III clinical trial. Type
B, which includes 4 (metaglitazen)17 and 5 (nTZDpa),9 has
two lipophilic parts on either side of an acidic center. Com-
pound 4, the most advanced drug candidate of this type, is
currently included in a phase III clinical trial. The acidic parts
of the Type A and Type B partial agonists are known to be
important for key hydrogen bond interactions with PPARγ.20
In contrast, Type C, which includes 6 (L-764406),21 usually
has no acidic part to form hydrogen bonds with PPARγ. This
type of partial agonist has a reactive leaving group on an
electron-deficient ring; this leaving group is important to the
formation of covalent interactions withPPARγ. Compound 6
is known to bind directly to Cys313 in helix 3 of the LBD of
human PPARγ through the loss of a chlorine atom. To the
best of our knowledge, there has been no report of a clinical
trial involving Type C partial agonists.
The peroxisome proliferator-activated receptors (PPARs)a
are ligand-activated transcription factors belonging to the
nuclear receptor superfamily.1 So far, there are three PPAR
subtypes encoded by distinct genes: PPARR (NR1C1),
PPARδ (NR1C2), and PPARγ (NR1C3).2 These receptors
are important regulators in multiple physiological pathways
such as glucose homeostasis, fatty acid metabolism, inflam-
mation, and cellular differentiation.3,4
PPARγ is also the target protein for the currently mar-
keted thiazolidinedione (TZD) class of antidiabetic agents,
rosiglitazone and pioglitazone.5,6 Although this type of
PPARγ full agonist modestly ameliorates hyperglycemia,
undesirable side effects including peripheral edema and
weight gain are also associated with these drugs.7,8 There-
fore, a recent drug-discovery program has focused on
identifying PPARγ partial agonists that promise to surpass
the potential of currently available PPARγ full agonists
if their desirable efficacy profile can be maintained, while
their negative side effects are minimized.9-13 On the basis of
this concept, several PPARγ partial agonists have been
developed.14-19
Our research on PPARγ partial agonists has been initiated
from our screening identification of compound 7, which pos-
sesses a new pyrimidine-5-carboxamide skeleton, as shown in
Scheme 1. On the basis of the chemical structure, 7 can pro-
bably be categorized as a Type C partial agonist and can inter-
act with PPARγ in a covalent fashion through the loss of chlo-
rine. As expected, compound 8, a related analogue compound
lacking the chlorine, was inactive in this assay. Covalent
binding may cause toxicological risks over the long-term;22
*To whom correspondence should be addressed. Phone: þ81-280-56-
2201. Fax: þ81-280-57-1293. E-mail: shigeki.seto@mb.kyorin-pharm.
co.jp.
aAbbreviations: PPAR, peroxisome proliferator-activated receptor;
AF-2, activation function 2, TZD, thiazolidinedione; LDA, lithium
diisopropylamide; LBD, ligand-binding domain; aP2, adipose fatty acid
binding protein; PCR, polymerase chain reaction; IBMX, 3-isobutyl-1-
methylxanthine; DEX, dexamethazone, MIX, methylisobutyl xanthine;
SEM, standard error of the mean; DMF, dimethylformamide; THF,
tetrahydrofuran; mCPBA, meta-chloroperoxybenzoic acid
r
pubs.acs.org/jmc
Published on Web 06/09/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 5013
Figure 1. Structural classification of known PPARγ partial agonists.
Scheme 1
however, the novel structural features of compound 7 led us to
embark on further investigation. With the intent of finding a
noncovalent binder, we next focused our attention on the
similarity of the chemical structure between 7 and 5, which is
categorized as Type B. We synthesized 9 by transference of the
benzyl amide group of 7 to the 4-position on the pyrimidine
ring and the introduction of a sulfur atom from one of the
Type B compounds, 5, as in Scheme 1. Obviously, 9 belongs to
the Type B category, which has two lipophilic parts on either
side of an acidic center, with no reactive leaving group.
Moreover, 9 showed a 25-fold higher potency in the transac-
tivation assay than 7.
We wish to report herein the details of structure-activity
relationship (SAR) efforts toward further optimization of a
series of novel 2,4,6-trisubstituted-5-pyrimidinecarboxylic
acid derivatives. We report the X-ray cocrystal structure of
selected compounds bound to the PPARγ to reveal important
molecular insight into the ligand-binding site interactions and
encouraging in vivo efficacy results in db/db mice.
dichloride 11 with phenyl methanethiol under basic condi-
tions afforded the benzylthio ether 12. Similar conditions as
those used in the preparation of 12 were used for the synthesis
of benzylamine 13. Further transformation of 13 took place in
order to introduce various kinds of amino groups at C-2.
Oxidation of the sulfur atom of 13 using mCPBA afforded
methyl sulfone 14; a subsequent displacement reaction of a
sulfinyl group of 14 with various amine nucleophiles in
toluene provided 2-aminopyrimidines 15-18, which have a
chlorine atom at the 4-position for further transformation.23
Some of the 2-alkylthioanalogues were prepared using the
method described in Scheme 3. Alkylation24 of barbituric acid
19 with butyl bromide or octyl bromide using sodium hydro-
xide afforded 2-alkylthiopyrimidines 20 and 21, respec-
tively. The introduction of a carboxylic acid moiety into 20
and 21 using CO2 and LDA as well as subsequent methyl-
ation with iodomethane gave esters, which were coupled
with phenyl methanethiol to provide thioethers 26 and 27,
respectively.
The trisubstituted pyrimidine 31 was prepared as shown in
Scheme 4. Bell’s condition25 was used for the preparation of
carboxylic acid 29, which was transformed to thioether 31 by
the methylation of 29 and a subsequent coupling reaction of
30 with phenyl methanethiol.
Chemistry
Chloropyrimidine analogues, common key intermediates,
were synthesized as outlined in Scheme 2. The coupling of

5014 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Seto et al.
Scheme 2a
a Reagents: (a) MeI, K2CO3, DMF, rt, 1 h; (b) PhCH2SH, Et3N, THF, rt, 2 h; (c) PhCH2NH2, Et3N, THF, rt, 2 h; (d) mCPBA, THF, 0 °C, 5 h;
(e) R2H, toluene, 0 °C, 5 h.
Scheme 3a
a Reagents: (a) (i) butyl bromide or octyl bromide, 3 N NaOH, EtOH, reflux, 2 h, (ii) POCl3, N,N-dimethylaniline, reflux, 1 h; (b) (i) LDA, THF,
-78 °C, 5 h, (ii) CO2, 10 min; (c) MeI, K2CO3, DMF, rt, 2 h; (d) PhCH2SH, K2CO3, DMF, rt, 1 h.
Scheme 4a
Results and Discussion
The PPARγ transactivation activation activities of the
synthesized compounds were determined, and the results are
summarized in Tables 1 and 2, together with the results for
rosiglitazone. The PPARγ transactivation value was assessed
by potency and efficacy using PPAR-GAL4 chimeric recep-
tors in transiently transfected CHO-K1 cells. Efficacy values
were calculated as a percentage of the maximal reporter
activity value of rosiglitazone.
The results shown in Table 1 reveal the effects of the length
and bulkiness of the alkyl chain of the R1 group. The
des-(methylthio) analogue 43 was found to be inactive on
PPARγ, while the parent methylthio analogue 9 resulted in a
significant activation of the receptor (EC50 = 0.18 μM).
Further elongation of the alkyl chain of the R1 group, the
propyl group as in 41, and the octyl group as in 42, led to an
increase in efficacy: 54% (9, methyl), 76% (41, butyl), and
89% (42, octyl), respectively, without a significant change in
EC50, which ranged from 0.18-0.95 μM. A similar tendency
in efficacy was observed for the alkylamino derivatives 45
(65%, butyl) and 46 (85%, heptyl). These results suggest that
the length of the alkyl chain is important for adjusting the
efficacy of PPARγ activation. We anticipate that these com-
pounds, with a variety of efficacies, may bind to different sites of
PPARγ, on the basis of the previously reported binding mode of
indole analogues with different efficacies.20,28
a Reagents: (a) POCl3, DMF, 100 °C, 5 h; (b) NaClO2, 2-methyl-2-
butene, t-BuOH, 0 °C, 4 h; (c) MeI, K2CO3, DMF, rt, 2 h; (d) PhCH2SH,
K2CO3, DMF, rt, 1 h.
Having the chlorides 10-18, 26, 27, 31 in hand, the target
pyrimidine-5-carboxylic acids 9, 32-50 were synthesized as
shown in Schemes 5-7. The nucleophilic displacement reac-
tion of chlorides 12, 26, 27, 31 with various kinds of amines,
and then subsequent hydrolysis with potassium hydroxide
gave 9, 32-43, respectively (Scheme 5). Similarly, chlorides
13, 15 underwent coupling with benzyl alcohol, phenyl metha-
nethiol and subsequent hydrolysis with potassium hydroxide
afforded the corresponding carboxylic acids 44, 45 (Scheme 6).
A one-pot procedure was used for the synthesis of the
target compounds 46-48 by heating the reaction mixture of
phenyl methanethiol and chloride 16-18 in DMF at 80 °C for
1 h.26 Biaryl analogue 49 was prepared by using a Suzuki
coupling reaction27 of chloride 13 with 2-thienoboronic acid,
and then subsequent hydrolysis with potassium hydroxide
(Scheme 6). Scheme 7 shows the synthesis of symmetrical
compound 50. Bisbenzylthio ether 50 was prepared from the
direct coupling between carboxylic acid 10 and 2 equiv of
phenyl methanethiol.
The bulky dibutylamino analogue 48 showed drastically reduced
potency (EC50>10 μM) over monobutylamino analogue 45.
The effects of the substituents in positions 4 and 6 of the
pyrimidine nucleus were examined, as shown in Table 2.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 5015
Scheme 5a
a Reagents: (a) R2NH2, K2CO3, DMF, rt, 2 h; (b) KOH, THF-MeOH, reflux, 4 h.
Scheme 6a
a Reagents: (a) (i) R2H, K2CO3, DMF, rt, 1 h, (ii) KOH, THF-MeOH, reflux, 4 h; (b) PhCH2SH, K2CO3, DMF, 80 °C, 3 h. (c) (i) R2B(OH)2,
Pd(PPh3)4, Na2CO3, toluene, dioxane, H2O, reflux, 2 h, (ii) KOH, THF-MeOH, reflux, 4 h.
Scheme 7a
Table 2. Effects of C-4 and 6 Substituents
EC50
(μM)a
efficacy
(%)b
a Reagents: (a) PhCH2SH or PhCH2OH, K2CO3, DMF, rt, 1 h.
compd
R1
R2
32
9
NHPh
NHCH2Ph
SCH2Ph
SCH2Ph
SCH2Ph
SCH2Ph
SCH2Ph
SCH2Ph
SCH2Ph
SCH2Ph
SCH2Ph
SCH2Ph
1.6
0.18
0.67
0.25
0.74
2.7
58
54
50
35
55
45
37
35
25
65
Table 1. Effect of C-2 Substituents
33
34
35
36
37
38
39
40
NHCH2CH2Ph
NHCH2-2-pyridyl
NHCH2-3-pyridyl
NHCH2-4-pyridyl
NHMe
>10
>10
1.8
NH(CH2)2Me
NH-cyclopropyl
NHCH2-
compd
R1
EC50 (μM)a
efficacy (%)b
0.30
cyclohexyl
NHCH2Ph
43
9
H
SMe
>10
0.18
0.55
0.95
0.47
0.25
0.42
>10
33
54
76
89
65
85
57
71
44
OCH2Ph
2-thienyl
SCH2Ph
2.3
>10
0.14
0.10
50
iac
49
50
NHCH2Ph
SCH2Ph
41
42
45
46
47
48
S(CH2)3Me
S(CH2)7Me
66
100
Rosiglitazone
NH(CH2)3Me
NH(CH2)6Me
N(Me)(CH2)3Me
N((CH2)3Me)2
a EC50 values were the molar concentration of the test compounds
that cause 50% of the maximal reporter activity. b Efficacy values were
calculated as a percentage of rosiglitazone. c “ia” means inactive at a
concentration of 10 mM.
a EC50 values were the molar concentration of the test compounds
that cause 50% of the maximal reporter activity. b Efficacy values were
calculated as a percentage of rosiglitazone.
the potency. The anilino analogue 32 and the phenethylamino
analogue 33 showed 9- and 4-fold less potency than the parent
benzyl analogue 9, respectively. The 2-pyridyl analogue 34
retained the same level of potency compared to 9. Shifting
the nitrogen on the pyridine ring to the 3- or 4-position
resulted in a decrease in potency (4-fold, 35; 15-fold, 36).
We first examined the effects of the phenyl ring on the
benzylamino moiety. The length of the linker between the
terminal phenyl ring and the nitrogen atom attached to the
core pyrimidine ring appears to play an important role in

5016 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Seto et al.
The introduction of various kinds of substituents onto the
terminal phenyl ring resulted in comparable potency (see
S1-S10 in Supporting Information). For example, the
4-methoxy analogue, 4-methyl analogue, 4-chloro analogue,
4-phenyl analogue, 4-tert-butyl analogue, and 4-dimethyl-
amino analogue showed similar potencies compared to 9
at the range of EC50 = 0.27-0.50, indicating that the presence
of a substituent on the phenyl ring is not crucial to the
interaction with PPARγ. Removal of the terminal phenyl
ring negatively affected potency. Most of the alkyl analogues
without a terminal aromatic ring, for example, 37 and 38
as well as cyclopropyl analogue 39, did not respond to
PPARγ. However, the cyclohexylmethyl analogue 40 main-
tained its potency at almost the same level as the parent 9
(EC50 = 0.30 μM).
On the basis of the results of the SAR examination shownin
Tables 1 and 2, parent compound 9 appears to have the
greatest potency in this series of compounds. However, 9
showed extremely poor water solubility (less than 1 μg/mL),
which could obstruct the absorption process during oral
administration. We hypothesized that hydrogen bonding
could occur between a carboxylic acid group and benzylamine
NH of 9, which could lead to low water solubility in Scheme 8.
To test this hypothesis, we synthesized the bisbenzylthio
analogue 50. As a result, not only did 50 show good water
solubility (43.7 μg/mL), it showed PPARγ agonist activity
similar to that of 9. We therefore selected 50 as the most
attractive compound for further biological examinations.
To clarify the hydrogen bond network between 50 and
PPARγ, an X-ray cocrystal structure of 50 bound to an active
PPARγ site was obtained by X-ray crystallography to a
Next, the effects of the benzylthio moiety (R2) were exam-
ined. Conversion of the sulfur on the benzylthio moiety to
oxygen negatively affected the potency (9 vs 44). Thienyl
analogue 49, a conformationally restricted analogue, showed
a dramatic loss of activity.
˚
resolution of 3.0 A, as shown in Figure 2. Analysis of the
binding mode reveals that the oxygen atom of the carboxylic
acidpart of50 formsa hydrogenbondwiththe carboxylicacid
side chain of Glu343; the distance between the two oxygen
˚
atoms is 2.95 A. The oxygen atom of 50 also interacts with
Arg288 through a hydrogen bond; the distance between the
oxygen atom and the nitrogen atom of the guanidinium group
Scheme 8
˚
of Arg288 is 2.76 A. Thus, the acidic part of 50 does not
directly interact with three key amino acid residues, H323,
H449, and Y473, located on the C-terminal activation func-
tion 2 (AF-2) helix and interact with acidic head groups of
synthetic PPARγ full agonists.29
The species difference of 50 in PPARγ transactivation was
examined using dose response analysis in the human and mice
PPARγ-GAL4 functional assay. As shown in Table 3, 50
showed a partial agonist profile to both human and mice
PPARγ receptors with a relative efficacy of 50-60% of
Figure 2. Hydrogen bond network of compound 50 in complex with PPARγ.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 5017
rosiglitazone. Compound 50 also showed an antagonistic
profile with IC50 = 1.79 μM when treated in the presence of
1 μM rosiglitazone.30 Similar results were observed when mice
and rat PPARγ were used. These data indicate that 50
displayed typical PPARγ partial agonist properties without
a species difference.
exhibited by 3 and 10 mg/kg doses, respectively, of rosiglita-
zone. Partial activation of PPARγ probably brings about the
modest potency of 50 relative to the PPARγ full agonist,
rosiglitazone.37
Some PPARγ partial agonists have been reported to cause
no weight gain in db/db mice.38 In contrast, some reports
indicate that weight gain has been observed, and that even the
tested compounds showed a decreased induction of adipogen-
esis in cells.39 Unfortunately, there was no significant differ-
ence in the weight gain profiles between 50 and rosiglitazone
after 7-day treatment of db/db mice (Figure 5B).
Partial agonists exhibit reduced ability to recruit coactiva-
tors, diminished adipogenic capacity, and attenuated gene
signatures in cultured adipocytes.31,32 To characterize the
profile of 50, we examined the ability to promote adipogene-
sis in 3T3L1 cells.33 We first examined the levels of adipose
fatty acid binding protein (aP2) mRNA expression in differ-
entiating 3T3-L1 adipocytes (blast and mature), as shown
in Figure 3.34,35 Rosiglitazone, a full agonist, increased aP2
mRNA expression in a dose-dependent manner in both blast
and mature 3T3-L1 cell lines. In contrast, 50 did not affect the
expression of mRNA in blast cells. A partial increasing effect
was observed in mature cells over rosiglitazone. Next, we also
examined the effects of 50 on adipocyte differentiation in
murine fibroblast 3T3-L1 cells, as shown in Figure 4. Rosigli-
tazone promoted lipid accumulation according to the results
of oil red O staining, while the effects of 50 were found to be
very weak, suggesting that 50 does not induce weight and
adipose tissue gain.
Before conducting in vivo experiments in diabetic mice, the
pharmacokinetic profile of 50 in mice was evaluated and the
results are shown in Table 4. Compound 50 showed low total
clearance (CL, 1.88 mL/min/kg), moderate oral bioavailabil-
ity (35.8%), buta shorthalf-life (t1/2 = 0.76 h), suggestingthat
50 is an orally bioavailable compound.
The in vivo efficacy of 50 was examined using db/db mice
undergoing daily oral administration for 7 days.36 As shown
in Figure 5A, 50 dose-dependently lowered plasma glucose
levels and exhibited excellent efficacy in db/db mice. Com-
pound 50 exhibited 56% and 77% glucose normalization at
doses of 30 and 100 mg/kg, respectively, compared with lean
control animals. These responses are equivalent to the effects
Conclusion
In conclusion, we have succeeded in the design, synthesis,
and evaluation of 2,4,6-trisubstitutedpyrimidine-5-carboxylic
acid derivatives based on the in-house screening hit of com-
pound 7. Structure-activity relationship studies have revea-
led that (1) the maximum activation level for PPARγ agonism
can be adjusted by modulation of the length of the alkyl group
at the 2-position on pyrimidine; (2) sulfur atoms at the 4- and
6-positions contribute to the potency and chemical properties,
including water solubility. Among the synthesized pyrimi-
dine-5-carboxylic acid derivatives, 50 was selected for further
evaluation. The X-ray cocrystal structure of 50 on PPARγ
revealed that the key hydrogen bond interactions, which are
not related to the AF-2 site, are different from those of the full
agonist. Pharmacological study of 50 demonstrated its excel-
lent antidiabetic activity in db/db mice. These results indicate
that 50 is attractive candidate as a new lead compound for
design of more potent PPARγ partial agonist.
Experimental Section
Chemistry. Melting points were determined with a Yamato
MP-500 melting point apparatus and are uncorrected. 1H NMR
Table 3. Species Difference of 50 in the Transactivation of PPARγ
Isoform
transactivationa
species
EC50 (μM)
efficacy (%)
IC50 (μM)
human
mice
rat
0.108
0.158
0.158
59
59
45
1.79
1.77
0.539
Figure 4. Visualization of lipid accumulation in 3T3-L1 cells by oil
red O staining. After reaching confluence, cells were stimulated with
1 μM dexamethasone, 1 μM IBMX, and 2 μM insulin for 7 days. The
DEX/MIX medium was then removed, and cells were treated for 10
days with the following ligands: C, vehicle; D, rosiglitazone; E,
compound 50.
a Compounds were screened for agonist or antagonist activity on
PPARγ-GAL4 chimeric receptors in transiently transfected CHO-K1
cells in the absence or presence of 1 μM rosiglitazone.
Figure 3. Effects of rosiglitazone and 50 on the differentiation of 3T3-L1 cells. Total RNA was extracted after 7 days for A, and after 10 days
for B. Reverse transcription and real-time PCR were on cDNAs with an aP2-specific sample. One microgram of total RNA was used in each
case.

5018 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Table 4. Pharmacokinetic Profiles of 50 in Mice
Seto et al.
C
10 min (μg/mL)a
CL (mL/mim/kg)a
vd (L/kg)a
t1/2 (h)a
AUC iv (μg*h/mL)a
AUC po (μg*h/mL)b
F (%)
6.89
1.88
0.12
0.76
8.86
3.17
35.8
a These values were measured in mice after iv administration (1 mg/kg). b These values were measured in mice after po administration (1 mg/kg).
Figure 5. Effects of rosiglitazone and compound 50 with oral administration in db/db mice after 7 days. Data are shown as mean ( SEM n = 5.
(A) Plasma glucose. (B) Body weight gain.
spectra were measured in CDCl3 or DMSO-d6 with TMS and
the solvent peak as internal standards, on a JEOL ECA-400
(400 MHz) spectrometer. Mass spectra (MS) were obtained on a
Hitachi M-2000 mass spectrometer. Column chromatography
was carried out on Merck silica gel 60. Analytical thin-layer
chromatography (TLC) was performed on Merck precoated
silica gel 60F254 plates, and the compounds were visualized by
UV illumination (254 nm) or by heating after spraying with
phosphomolybdic acid in ethanol. The data for elemental analy-
sis are within (0.4% of theoretical values and were determined
by a Yanaco CHN corder MT-5. All tested compounds had a
purity of at least 95%.
Methyl 4-(Benzylthio)-6-chloro-2-(methylthio)pyrimidine-5-
carboxylate (12). To a solution of 1123 (17.6 g, 69.5 mmol)
in DMF (100 mL) were added potassium carbonate (9.70 g,
70.2 mmol) and phenymethanethiol (8.68 g, 69.9 mmol) at 0 °C,
and the mixture was stirred at room temperature for 3 h. The
reaction mixture was diluted with ethyl acetate, then washed
with water and brine, dried over Na2SO4, filtered, and concen-
trated in vacuo. Flash chromatography (hexane/AcOEt = 5:1)
of the residue gave 12 (22.9 g, 97%) as a pale yellow oil. 1H NMR
(400 MHz, CDCl3) δ 2.56 (3H, s), 3.92 (3H, s), 4.44 (2H, s),
7.24-7.40 (5H, m). HRMS (EI) calcd for C14H13ClN2O2S2
(Mþ) 340.0107, found 340.0117.
the cake with phosphoryloxy chloride (15.0 mL, 0.161 mol) and
N,N-dimethylaniline (5.2 mL, 41.0 mmol) was refluxed for 1 h.
The mixture was poured into ice-water and extracted with ethyl
acetate, then dried over anhydrous Na2SO4, filtered, and con-
centrated in vacuo. Flash chromatography (hexane/AcOEt =
30:1) of the residue gave 20 (1.61 g, 19%) as a pale yellow oil. 1H
NMR (400 MHz, CDCl3) δ 0.96 (3H, t, J = 7.3 Hz), 1.48 (2H,
qt, J = 7.3 and 7.3 Hz), 1.71 (2H, tt, J = 7.3 and 7.3 Hz), 3.15
(2H, t, J = 7.3 Hz), 7.01 (1H, s). HRMS (FABþ) calcd for
C8H11Cl2N2S (M þ Hþ) 237.0020, found 236.9995.
4,6-Dichloro-2-(octylthio)pyrimidine (21). The title compound
21 (696 mg, 7%) was prepared from thiobarbituric acid (5.22 g,
36.2 mmol) and iodooctane (6.30 mL, 36.2 mmol) in the same
manner as described for 20. Pale yellow oil. 1H NMR (400 MHz,
CDCl3) δ 0.88 (3H, t, J = 7.3 Hz), 1.21-1.38 (8H, m), 1.38-1.49
(2H, m), 1.72 (2H, tt, J = 7.3 and 7.3 Hz), 3.14 (2H, t, J = 7.3
Hz), 7.01 (1H, s). HRMS (FABþ) calcd for C12H19Cl2N2S (M þ
Hþ) 293.0646, found 293.0623.
2-(Butylthio)-4,6-dichloropyrimidine-5-carboxylic Acid (22).
A 2.6 M solution of n-butyllithium (4.0 mL, 10.4 mmol) in
hexanes was added to a solution of diisopropylamine (1.50 mL,
10.6 mmol) in THF (10 mL) at -10 °C under Ar. After the
solution was stirred for 30 min, a solution of 20 (1.59 g,
6.70 mmol) in THF (10 mL) was added to the LDA solution at
-78 °C. The mixture was stirred at the same temperature for
5 h, and CO2 gas was then introduced for 10 min. After the
reaction was quenched by the addition of water (10 mL) and 6 N
HCl (10 mL), the mixture was extracted with ethyl acetate. The
combined extracts were dried over anhydrous Na2SO4, filtered,
and then concentrated in vacuo. The residue was recrystallized
from MeOH to afford 22 (1.40 g, 74%) as a pale yellow solid.
Mp: 210-215 °C. 1H NMR (400 MHz, DMSO-d6) δ 0.91 (3H, t,
J = 7.3 Hz), 1.40 (2H, qt, J = 7.3 and 7.3 Hz), 1.63 (2H, tt, J =
7.3 and 7.3 Hz), 3.10 (2H, t, J = 7.3 Hz). HRMS (EI) calcd for
C9H10Cl2N2O2S (Mþ) 279.9840, found 279.9844. Anal.
(C9H10Cl2N2O2S 2.2H2O) C, H, N.
2-(Butylthio)-4,6-dichloropyrimidine-5-carboxylic Acid (23).
The title compound 23 (420 mg, 54%) was prepared from 21
(680 mg, 2.32 mmol) in the same manner as described for 22.
Pale yellow solid. Mp: 210-214 °C. 1H NMR (400 MHz,
DMSO-d6) δ 0.86 (3H, t, J = 7.3 Hz), 1.20-1.43 (10H, m),
1.65 (2H, tt, J = 7.3 and 7.3 Hz), 3.09 (2H, t, J = 7.3 Hz).
HRMS (EI) calcd for C13H18Cl2N2O2S (Mþ) 336.0466, found
336.0446. Anal. (C13H18Cl2N2O2S 2H2O) C, H, N.
Methyl 4-(Benzylamino)-6-chloro-2-(heptylamino)pyrimidine-
5-carboxylate (16). To a solution of 1423 (130 mg, 0.365 mmol) in
toluene (0.5 mL) was added a mixture of triethylamine (20 μL)
and heptylamine (42.1 mg, 0.365 mmol) in toluene (0.5 mL)
portion-wise under ice cooling. The mixture was stirred at 0 °C
for 5 h. The resulting mixture was diluted with ethyl acetate and
then washed with water and brine. The organic layer was dried
over anhydrous Na2SO4, filtered, and then concentrated in
vacuo. Flash chromatography (hexane/AcOEt = 5:1) of the
1
residue gave 16 (74.1 mg, 52%) as a colorless foam. H NMR
(400 MHz, CDCl3) δ 0.88 (3H, t, J = 6.7 Hz), 1.20-1.40 (8H,
m), 1.45-1.55 (2H, m), 3.30-3.45 (2H, m), 3.84 (3H, s),
4.60-4.72 (2H, m), 5.05-5.33 (1H, br), 7.22-7.37 (5H, m),
8.58-8.97 (1H, br). HRMS (FABþ) calcd for C20H27ClN4O2
(M þ Hþ) 390.1823, found 390.1816.
2-(Butylthio)-4,6-dichloro-pyrimidine (20). To a mixture of
thiobarbituric acid (5.22 g, 36.2 mmol) in EtOH (50 mL) was
added 3 N NaOH (12 mL) at room temperature, and the mixture
was stirred under reflux for 1 h. The mixture was added
iodobutane (4.20 mL, 36.9 mmol) at room temperature, and
the mixture was stirred under reflux for 2 h. The mixture was
cooled to room temperature. The precipitate was formed and
collected by filtration, and then dried in vacuo. The mixture of
Methyl 2-(Butylthio)-4,6-dichloropyrimidine-5-carboxylate (24).
Toa solutionof 22 (1.21 g, 4.30 mmol)inDMF(5 mL) wereadded
potassium carbonate (595 mg, 4.31 mmol) and iodomethane

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 5019
(0.30 mL, 4.82 mmol) at 0 °C, and the mixture was stirred at
room temperature for 2 h. The reaction mixture was diluted with
ethyl acetate, then washed with water and brine, dried over
Na2SO4, filtered, and concentrated in vacuo. Flash chromatog-
raphy (hexane/AcOEt = 5:1) of the residue gave 24 (1.00 g,
66%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) δ 0.96
(3H, t, J = 7.3 Hz), 1.47 (2H, qt, J = 7.3 and 7.3 Hz), 1.72 (2H,
tt, J = 7.3 and 7.3 Hz), 3.16 (2H, t, J = 7.3 Hz), 3.98 (3H, s).
HRMS (EI) calcd for C10H12Cl2N2O2S (Mþ) 293.9997, found
293.9977.
Methyl 4,6-Dichloro-2-(octylthio)pyrimidine-5-carboxylate (25).
The title compound 25 (400 mg, 86%) was prepared from 23
(445 mg, 1.32 mmol) and iodomethane (0.10 mL, 1.61 mmol) in
the same manner as described for 24. Pale yellow oil. 1H NMR
(400 MHz, CDCl3) δ 0.88 (3H, t, J = 7.3 Hz), 1.20-1.37 (8H,
m), 1.37-1.49 (2H, m), 1.72 (2H, tt, J = 7.3 and 7.3 Hz), 3.15
(2H, t, J = 7.3 Hz), 3.98 (3H, s). HRMS (EI) calcd for C14H20-
Cl2N2O2S (Mþ) 350.0623, found 350.0657.
C6H5Cl2N2O2 (M þ Hþ) 206.9728, found 206.9736. Anal.
(C6H4Cl2N2O2 0.3H2O) C, H, N.
Methyl 4-(Benzylthio)-6-chloropyrimidine-5-carboxylate (31).
To a solution of methyl 30 (115 mg, 0.556 mmol) in DMF (1 mL)
were added potassium carbonate (77.0 mg, 0.557 mmol) and
phenyl methanethiol (70.0 mg, 0.564 mmol) at 0 °C, and the
mixture was stirred at room temperature for 1 h. The reaction
mixture was diluted with ethyl acetate, then washed with water
and brine, dried over Na2SO4, filtered, and concentrated in
vacuo. Flash chromatography (hexane/AcOEt = 5:1) of the
1
residue gave 31 (147 mg, 90%) as a pale yellow oil. H NMR
(400 MHz, CDCl3) δ 3.96 (3H, s), 4.48 (2H, s), 7.24-7.35 (3H,
m), 7.38 (2H, d, J = 6.8 Hz), 8.75 (1H, s). HRMS (FABþ) calcd
for C13H12ClN2O2S (M þ Hþ) 295.0308, found 295.0297.
4-(Benzylamino)-6-(benzylthio)-2-(methylthio)pyrimidine-5-car-
boxylic Acid (9). Step 1. Methyl 4-(benzylamino)-6-(benzylthio)-
2-(methylthio)pyrimidine-5-carboxylate. To a solution of 12
(100 mg, 0.309 mmol) in DMF (1 mL) were added potass-
ium carbonate (85.5 mg, 0.619 mmol) and phenyl methanethiol
(42.5 mg, 0.342 mmol) at 0 °C, and the mixture was stirred at
room temperature for 1 h. The reaction mixture was diluted with
ethyl acetate, then washed with water and brine. The organic
layer was dried over Na2SO4, filtered, and then concentrated in
vacuo. Flash chromatography (hexane/AcOEt = 5:1) of the
residue gave 9 Step 1 as a pale yellow solid (126 mg, 99%). Mp:
Methyl 4-(Benzylthio)-2-(butylthio)-6-chloropyrimidine-5-
carboxylate (26). To a solution of 24 (96.0 mg, 0.325 mmol) in
DMF (1 mL) were added potassium carbonate (45.0 mg, 0.326
mmol) and phenyl methanethiol (41.0 mg, 0.330 mmol) at 0 °C,
and the mixture was stirred at room temperature for 5 h. The
reaction mixture was diluted with ethyl acetate, then washed
with water and brine, dried over Na2SO4, filtered, and concen-
trated in vacuo. Flash chromatography (hexane/AcOEt = 5:1)
1
91-93 °C. H NMR (400 MHz, CDCl3) δ 2.46 (3H, s), 3.84
1
(3H, s), 4.37 (2H, s), 4.74 (2H, d, J = 5.5 Hz), 7.20-7.37 (8H, m),
7.39 (2H, d, J = 7.3 Hz), 8.90 (1H, t, J = 5.5 Hz). HRMS
(FABþ) calcd for C21H22N3O2S2 (M þ Hþ) 412.1153, found
412.1112. Anal. (C21H21N3O2S2 0.2H2O) C, H, N.
of the residue gave 26 (107 mg, 86%) as a pale yellow oil. H
NMR (400 MHz, CDCl3) δ 0.93 (3H, t, J = 7.3 Hz), 1.45 (2H,
qt, J = 7.3 and 7.3 Hz), 1.71 (2H, tt, J = 7.3 and 7.3 Hz), 3.13
(2H, t, J = 7.3 Hz), 3.92 (3H, s), 4.43 (2H, s), 7.24-7.40 (5H, m).
HRMS (FABþ) calcd for C17H20ClN2O2S2 (M þ Hþ) 383.0655,
found 383.0696.
Methyl 4-(Benzylthio)-6-chloro-2-(octylthio)pyrimidine-5-car-
boxylate (27). The title compound 27 (110 mg, 70%) was
prepared from 25 (126 mg, 0.359 mmol) and phenyl metha-
nethiol (45.0 mg, 0.362 mmol) in the same manner as described
for 26. 1H NMR (400 MHz, CDCl3) δ 0.87 (3H, t, J = 7.3 Hz),
1.18-1.35 (8H, m), 1.35-1.45 (2H, m), 1.66-1.76 (2H, m), 3.12
(2H, t, J = 7.3 Hz), 3.92 (3H, s), 4.43 (2H, s), 7.21-7.40 (5H, m).
HRMS (EI) calcd for C21H27ClN2O2S2 (Mþ) 438.1202, found
438.1167.
4,6-Dichloropyrimidine-5-carboxylic Acid (29). Step 1. 4,6-
Dichloropyrimidine-5-carboxaldehyde. To a mixture of phos-
phoryl oxychloride (8.42 mL, 89.2 mmol) and DMF (2.76 mL,
35.7 mmol), 4,6-dihydroxypyrimidine (2.00 g, 17.8 mmol) was
added over 40 min and then stirred at 100 °C for 5 h. The mixture
was poured into ice-water and then extracted with Et2O. The
organic layer was washed with H2O and dried over Na2SO4,
then concentrated in vacuo. The compound 29 Step 1 (2.04 g,
65%) was given as a yellow solid. 1H NMR (400 MHz, CDCl3) δ
8.90 (1H, s), 10.46 (1H, s).
Step 2. 4,6-Dichloropyrimidine-5-carboxylic acid (29). To a
mixture of the compound of step 1 (2.00 g, 11.3 mmol),
2-methyl-2-butene (6.00 mL, 56.6 mmol), NaH2PO4 (6.14 g,
51.2 mmol), t-BtOH (80 mL), and H2O (20 mL) was added
NaClO2 (4.09 g, 45.2 mmol) and then stirred at 0 °C for 4 h.
After being warmed at room temperature, the mixture was
diluted with AcOEt, washed with H2O and brine, and dried
over Na2SO4. The title compound 29 (1.28 g, 59%) was given as
a pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.88 (1H, s).
Methyl 4,6-Dichloropyrimidine-5-carboxylate (30). To a solu-
tion of 29 (386 mg, 2.00 mmol) in DMF (2 mL) were added
potassium carbonate (277 mg, 2.00 mmol) and iodomethane
(0.15 mL, 2.41 mmol) at 0 °C, and the mixture was stirred at
room temperature for 2 h. The reaction mixture was diluted with
ethyl acetate, then washed with water and brine, dried over
Na2SO4, filtered, and concentrated in vacuo. Flash chromatog-
raphy (hexane/AcOEt = 5:1) of the residue gave 30 (217 mg,
52%) as a pale yellow solid. Mp: 47-49 °C. 1H NMR (400 MHz,
CDCl3) δ 4.03 (3H, s), 8.84 (1H, s). HRMS (FABþ) calcd for
Step 2. 4-(Benzylamino)-6-(benzylthio)-2-(methylthio)pyrimi-
dine-5-carboxylic acid (9). To a solution of the compound of
Step 1 (108 mg, 0.262 mmol) in THF-MeOH (1.5 mL; 1:2 v/v)
was added 3 N KOH (0.5 mL) at room temperature, and the
mixture was refluxed for 4 h. The resulting mixture was cooled to
room temperature, and water and 2 N HCl (1 mL) were then
added. The precipitate was formed and collected by filtration,
and dried in vacuo. 9 was given as a colorless solid (80.0 mg,
77%). Mp: 165-167 °C. 1H NMR (400 MHz, DMSO-d6) δ 2.39
(3H, s), 4.28 (2H, s), 4.68 (2H, d, J = 6.1 Hz), 7.20-7.42 (10H, m),
8.98-9.10 (1H, br), 13.65-14.05 (1H, br). HRMS (EI) calcd for
C20H19N3O2S2 (Mþ) 397.0919, found 397.0944. Anal. (C20H19-
N3O2S2 0.1H2O) C, H, N.
4-Anilino-6-(benzylthio)-2-(methylthio)pyrimidine-5-carboxylic
Acid (32). Step 1. Methyl 4-anilino-6-(benzylthio)-2-(methylthio)-
pyrimidine-5-carboxylate. The compound of Step 1 (35.6 mg,
28%) was prepared from 12 (100 mg, 0.323 mmol) and phenyl
methanethiol (45.0 mg, 0.362 mmol) in the same manner as
1
described for 9 Step 1. Pale yellow solid. Mp: 102-103 °C. H
NMR (400 MHz, CDCl3) δ 2.50 (3H, s), 3.92 (3H, s), 4.39 (2H, s),
7.12 (1H, d, J = 7.3 Hz), 7.22-7.37 (5H, m), 7.40 (2H, d, J =
7.3 Hz), 7.62 (2H, d, J = 7.3 Hz), 10.70 (1H, s). HRMS (FABþ)
calcd for C20H20N3O2S2 (M þ Hþ) 398.0997, found 398.1002.
Anal. (C20H19N3O2S2) C, H, N.
Step 2. 4-Anilino-6-(benzylthio)-2-(methylthio)pyrimidine-5-
carboxylic acid (32). The title compound 32 (26.5 mg, 92%) was
prepared from the compound of Step 1 (30.0 mg, 75.5 μmol) in
the same manner as described for 9. Colorless solid. Mp:
1
192-195 °C. H NMR (400 MHz, DMSO-d6) δ 2.48 (3H, s),
4.33 (2H, s), 7.09-7.16 (1H, m), 7.22-7.29 (1H, m), 7.29-7.45
(6H, m), 7.61 (2H, dd, J = 8.5 and 1.2 Hz), 10.74-10.85 (1H,
br), 13.95-14.75 (1H, br). HRMS (FABþ) calcd for
C19H18N3O2S2 (Mþ þ 1) 384.0840, found 384.0851. Anal.
(C19H17N3O2S2 0.2H2O) C, H, N.
4-(Benzylthio)-2-(methylthio)-6-(2-phenethylamino)pyrimidine-
5-carboxylic Acid (33). Step 1. Methyl 4-(benzylthio)-2-(methy-
lthio)-6-(phenethylamino)pyrimidine-5-carboxylate. The com-
pound of Step 1 (38.7 mg, 53%) was prepared from 12 (57.4 mg,
0.170 mmol) and phenyl methanethiol (23.2 mg, 0.359 mmol) in
the same manner as described for 9 Step 1. Pale yellow solid. Mp:

5020 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Seto et al.
152-153 °C. 1H NMR (400 MHz, CDCl3) δ 2.53 (3H, s), 2.92
(2H, t, J = 7.3 Hz), 3.77 (2H, dd, J = 7.3 and 5.5 Hz), 3.81 (3H, s),
4.37 (2H, s), 7.20-7.35 (8H, m), 7.39 (2H, d, J = 7.3 Hz), 8.51
(1H, t, J = 5.5 Hz). HRMS (FABþ) calcd for C22H24N3O2S2
(M þ Hþ) 426.1310, found 426.1327. Anal. (C22H23N3O2S2
0.1H2O) C, H, N.
Step 2. 4-(Benzylthio)-2-(methylthio)-6-(2-phenethylamino)-
pyrimidine-5-carboxylic acid (33). The title compound 33 (29.7 mg,
93%) was prepared from the compound of Step 1 (33.0 mg,
77.5 μmol) in the same manner as described for 9. Colorless
solid. Mp: 175-177 °C. 1H NMR (400 MHz, DMSO-d6) δ 2.49
(3H, s), 2.85 (2H, t, J = 6.7 Hz), 3.62-3.72 (2H, m), 4.28 (2H, s),
7.18-7.42 (10H, m), 8.65-8.77 (1H, br), 13.50-14.05 (1H, br).
HRMS (FABþ) calcd for C21H22N3O2S2 (Mþ þ 1) 412.1153,
found 412.1176. Anal. (C21H21N3O2S2) C, H, N.
4-(Benzylthio)-2-(methylthio)-6-(2-pyridylmethylamino)pyrimi-
dine-5-carboxylic Acid (34). Step 1. Methyl 4-(benzylthio)-2-
(methylthio)-6-(2-pyridylmethylamino)pyrimidine-5-carbox-
ylate. The compound of Step 1 (87.5 mg, 85%) was prepared
from 12 (85.3 mg, 0.250 mmol)and 2-pyridylmethylamine (30.0
mg, 0.277 mmol) in the same manner as described for 9 Step 1.
Pale yellow solid. mp: 107-109 °C. 1H NMR (400 MHz, CDCl3)
δ 2.44 (3H, s), 3.90 (3H, s), 4.38 (2H, s), 4.85 (2H, d, J = 5.5 Hz),
7.17-7.34 (5H, m), 7.39 (2H, d, J = 7.3 Hz), 7.65 (1H, td, J =
7.3 and 1.8 Hz), 8.60 (1H, dd, J = 3.1 and 1.8 Hz), 9.34 (1H, t,
J = 5.5 Hz). HRMS (FABþ) calcd for C20H21N4O2S2 (M þ Hþ)
413.1106, found 413.1129. Anal. (C20H20N4O2S2 0.2H2O) C, H, N.
Step 2. 4-(Benzylthio)-2-(methylthio)-6-(2-pyridylmethyla-
mino)pyrimidine-5-carboxylic Acid (34). The title compound
34 (60.0 mg, 82%) was prepared from the compound of Step 1
(76.0 mg, 0.184 mmol) in the same manner as described for 9.
Colorless solid. Mp: 192-194 °C. 1H NMR (400 MHz, DMSO-
d6) δ 2.36 (3H, s), 4.28 (2H, s), 4.76 (2H, d, J = 4.9 Hz),
7.20-7.35 (5H, m), 7.35-7.42 (2H, m), 7.75 (1H, td, J = 7.3 and
1.2 Hz), 8.52 (1H, dd, J = 4.9 and 1.8 Hz), 9.27-9.42 (1H, br),
13.50-14.10 (1H, br). HRMS (FABþ) calcd for C19H19N4O2S2
(Mþ þ 1) 399.0949, found 399.0952. Anal. (C19H18N4O2S2
0.1H2O) C, H, N.
4-(Benzylthio)-2-(methylthio)-6-(3-pyridylmethylamino)pyrimi-
dine-5-carboxylic Acid (35). Step 1. Methyl 4-(benzylthio)-
2-(methylthio)-6-(3-pyridylmethylamino)pyrimidine-5-carbox-
ylate. The compound of Step 1 (91.0 mg, 74%) was prepared
from 12 (102 mg, 0.299 mmol) and 3-pyridylmethylamine (34.0
mg, 0.314 mmol) in the same manner as described for 9 Step 1.
Pale yellow solid. Mp: 121-123 °C. 1H NMR (400 MHz,
CDCl3) δ 2.44 (3H, s), 3.86 (3H, s), 4.37 (2H, s), 4.75 (2H, d,
J = 6.1 Hz), 7.21-7.28 (2H, m), 7.31 (2H, t, J = 7.3 Hz), 7.39
(2H, d, J = 7.3 Hz), 7.64 (1H, d, J = 7.3 Hz), 8.52 (1H, d, J =
3.1 Hz), 8.59 (1H, d, J = 1.2 Hz), 8.99 (1H, t, J = 6.1 Hz).
HRMS (FABþ) calcd for C20H21N4O2S2 (M þ Hþ) 413.1106,
found 413.1149. Anal. (C20H20N4O2S2) C, H, N.
8.52-8.57 (2H, m), 9.05 (1H, t, J = 6.1 Hz). HRMS (FABþ)
calcd for C20H21N4O2S2 (M þ Hþ) 413.1106, found 413.1131.
Anal. (C20H20N4O2S2 0.2H2O) C, H, N.
Step 2. 4-(Benzylthio)-2-(methylthio)-6-(4-pyridylmethyla-
mino)pyrimidine-5-carboxylic acid (36). The title compound
36 (62.5 mg, 92%) was prepared from the compound of Step 1
(70.0 mg, 0.170 mmol) in the same manner as described for 9.
Colorless solid. Mp: 202-203 °C. 1H NMR (400 MHz, DMSO-
d6) δ 2.30 (3H, s), 4.26 (2H, s), 4.69 (2H, d, J = 5.5 Hz),
7.20-7.32 (5H, m), 7.35-7.40 (2H, m), 8.47 (2H, dd, J = 4.3
and 1.8 Hz), 9.15-9.32 (1H, br). HRMS (FABþ) calcd for
C19H19N4O2S2 (Mþ þ 1) 399.0949, found 399.0941. Anal.
(C19H18N4O2S2 0.6H2O) C, H, N.
4-(Benzylthio)-6-(methylamino)-2-(methylthio)pyrimidine-5-
carboxylic Acid (37). Step 1. Methyl 4-(benzylthio)-6-(methyl-
amino)-2-(methylthio)pyrimidine-5-carboxylate. The compound
of Step 1 (83.2 mg, 83%) was prepared from methyl 12 (102 mg,
0.300 mmol) and methylamine (0.2 mL, 2 M solution in THF,
0.4 mmol) in the same manner as described for 9 Step 1. Pale
yellow solid. Mp: 151-153 °C. 1H NMR (400 MHz, CDCl3) δ
2.53 (3H, s), 3.05 (3H, d, J = 4.9 Hz), 3.85 (3H, s), 4.38 (2H, s),
7.22-7.26 (1H, m), 7.31 (2H, t, J = 7.3 Hz), 7.39 (2H, d, J = 7.3
Hz), 8.42-8.50 (1H, br). HRMS (EI) calcd for C15H17N3O2S2
(Mþ) 335.0762, found 335.0739. Anal. (C15H17N3O2S2) C, H, N.
Step 2. 4-(Benzylthio)-6-(methylamino)-2-(methylthio)pyri-
midine-5-carboxylic acid (37). The title compound 37 (61.5 mg,
91%) was prepared from the compound of Step 1 (70.7 mg,
0.211 mmol) in the same manner as described for 9. Colorless
solid. Mp: 169-171 °C. 1H NMR (400 MHz, DMSO-d6) δ 2.48
(3H, s), 2.93 (3H, d, J = 3.7 Hz), 4.29 (2H, s), 7.23 (1H, t, J = 7.3
Hz), 7.30 (2H, t, J = 7.3 Hz), 7.37 (2H, d, J = 7.3 Hz), 8.50 (1H,
brs), 13.72 (1H, brs). HRMS (EI) calcd for C14H15N3O2S2 (Mþ)
321.0606, found 321.0579. Anal. (C14H15N3O2S2) C, H, N.
4-(Benzylthio)-2-(methylthio)-4-(propylamino)pyrimidine-5-
carboxylic Acid (38). Step 1. Methyl 4-(benzylthio)-2-(methyl-
thio)-4-(propylamino)pyrimidine-5-carboxylate. The compound of
Step 1 (82.8 mg, 76%) was prepared from 12 (102 mg, 0.300 mmol)
and propylamine (20.0 mg, 0.338 mmol) in the same manner as
1
described for 9 Step 1. Pale yellow solid. Mp: 94-96 °C. H
NMR (400 MHz, CDCl3) δ 0.97 (3H, t, J = 7.3 Hz), 1.58-170
(3H, m), 2.50 (3H, s), 3.45-3.53 (2H, m), 3.85 (3H, s), 4.37 (2H, s),
7.21-7.27 (1H, m), 7.30 (2H, t, J = 7.3 Hz), 7.39 (2H, d, J =
7.3 Hz), 8.55-8.65 (1H, br). HRMS (EI) calcd for C17H21N3O2S2
(Mþ) 363.1075, found 363.1060. Anal. (C17H21N3O2S2) C, H, N.
Step 2. 4-(Benzylthio)-2-(methylthio)-4-(propylamino)pyrimi-
dine-5-carboxylic acid (38). The title compound 38 (60.0 mg,
92%) was prepared from the compound of Step 1 (68.0 mg,
0.187 mmol) in the same manner as described for 9. Colorless
solid. Mp: 159-162 °C. 1H NMR (400 MHz, DMSO-d6) δ 0.89
(3H, t, J = 7.3 Hz), 1.50-1.61 (2H, m), 2.47 (3H, s), 3.37-3.45
(2H, m), 4.28 (2H, s), 7.23 (1H, t, J = 7.3 Hz), 7.30 (2H, t, J =
7.3 Hz), 7.37 (2H, d, J = 7.3 Hz), 8.60-8.68 (1H, br), 13.78 (1H,
brs). HRMS (EI) calcd for C16H19N3O2S2 (Mþ) 349.0919, found
349.0922. Anal. (C16H19N3O2S2) C, H, N.
Step 2. 4-(Benzylthio)-2-(methylthio)-6-(3-pyridylmethyla-
mino)pyrimidine-5-carboxylic Acid (35). The title compound 35
(25.0 mg, 35%) was prepared from the compound of Step 1 (73.0
mg, 0.177 mmol) in the same manner as described for 9. Color-
less solid. Mp: 197-200 °C. 1H NMR (400 MHz, DMSO-d6) δ
2.38 (3H, s), 4.27 (2H, s), 4.69 (2H, d, J = 5.5 Hz), 7.20-7.40
(6H, m), 7.70 (1H, dt, J = 7.9 and 1.8 Hz), 8.44 (1H, dd, J = 4.9
and 1.8 Hz), 8.54 (1H, d, J = 1.8 Hz), 9.09-9.20 (1H, br).
HRMS (EI) calcd for C19H18N4O2S2 (Mþ) 398.0871, found
398.0841. Anal. (C19H18N4O2S2 0.3H2O) C, H, N.
4-(Benzylthio)-6-(cyclopropylamino)-2-(methylthio)pyrimidine-
5-carboxylic Acid (39). Step 1. Methyl 4-(benzylthio)-6-(cyclo-
propyl)-2-(methylthio)pyrimidine-5-carboxylate. The com-
pound of Step 1 (64.8 mg, 60%) was prepared from 12 (102
mg, 0.299 mmol) and cyclopropylamine (19.0 mg, 0.333 mmol)
in the same manner as described for 9 Step 1. Pale yellow solid.
1
Mp: 107-110 °C. H NMR (400 MHz, CDCl3) δ 0.52-0.58
4-(Benzylthio)-2-(methylthio)-6-(4-pyridylmethylamino)pyrimi-
dine-5-carboxylic Acid (36). Step 1. Methyl 4-(benzylthio)-2-
(methylthio)-6-(4-pyridylmethylamino)pyrimidine-5-carboxylate.
The compound of Step 1 (81.3 mg, 79%) was prepared from 12
(85.3 mg, 0.250 mmol) and 4-pyridylmethylamine (30.0 mg,
0.277 mmol) in the same manner as described for 9 Step 1. Pale
yellow solid. Mp: 111-114 °C. 1H NMR (400 MHz, CDCl3) δ
2.37 (3H, s), 3.88 (3H, s), 4.37 (2H, s), 4.75 (2H, d, J = 6.1 Hz),
7.18-7.27 (4H, m), 7.31 (2H, t, J=6.7Hz),7.40(2H,d,J=6.7Hz),
(2H, m), 0.79-0.85 (2H, m), 2.54 (3H, s), 2.92-3.00 (1H, m),
3.84 (3H, s), 4.37 (2H, s), 7.20-7.27 (1H, m), 7.30 (2H, t, J = 7.3
Hz), 7.39 (2H, d, J = 7.3 Hz), 8.51 (1H, brs). HRMS (EI) calcd
for C17H19N3O2S2 (Mþ) 361.0919, found 361.0879. Anal.
(C17H19N3O2S2) C, H, N.
Step 2. 4-(Benzylthio)-6-(cyclopropylamino)-2-(methylthio)-
pyrimidine-5-carboxylic acid (39). The title compound 39 (52.0 mg,
95%) was prepared from the compound of Step 1 (57.0 mg, 0.158
mmol) in the same manner as described for 9. Colorless solid. Mp:

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13 5021
177-180 °C. 1H NMR (400 MHz, DMSO-d6) δ 0.50-0.56
(2H, m), 0.72-0.80 (2H, m), 2.87-2.96 (1H, m), 4.30 (2H, s),
7.23 (1H, t, J = 7.3 Hz), 7.30 (2H, t, J = 7.3 Hz), 7.37 (2H, d,
J = 7.3 Hz), 8.50-8.60 (1H, br), 13.65-14.00 (1H, br). HRMS
(FABþ) calcd for C16H18N3O2S2 (Mþ þ 1) 348.0840, found
348.0850. Anal. (C16H17N3O2S2 0.2H2O) C, H, N.
4-(Benzylthio)-6-(cyclohexylamino)-2-(methylthio)pyrimidine-
5-carboxylic Acid (40). Step 1. Methyl 4-(benzylthio)-6-(cyclo-
hexylamino)-2-(methylthio)pyrimidine-5-carboxylate. The com-
pound of Step 1 (110 mg, 43%) was prepared from 12 (208 mg,
0.610 mmol) and cyclohexylamine (80 μL, 0.615 mmol) in the
same manner as described for 9 Step 1. Pale yellow solid. Mp:
76-79 °C. 1H NMR (400 MHz, CDCl3) δ 0.92-1.04 (2H, m),
1.14-1.30 (4H, m), 1.62-1.82 (4H, m), 2.50 (3H, s), 3.37 (2H, t,
J = 6.1 Hz), 3.85 (2H, s), 4.36 (2H, s), 7.21-7.27 (1H, m), 7.31
(2H, d, J = 7.3 Hz), 7.37 (2H, d, J = 7.3 Hz), 8.66 (1H, t, J = 6.1
Hz). HRMS (EI) calcd for C21H27N3O2S2 (Mþ) 417.1545, found
417.1592. Anal. (C21H27N3O2S2) C, H, N.
8.95-9.10 (1H, br), 13.50-14.20 (1H, br). HRMS (FABþ) calcd
for C27H34N3O2S2 (Mþ þ 1) 496.2092, found 496.2107. Anal.
(C27H33N3O2S2) C, H, N.
4-(Benzylamino)-6-(benzylthio)pyrimidine-5-carboxylic Acid
(43). Step 1. Methyl 4-(benzylamino)-6-(benzylthio)pyrimidine-
5-carboxylate. The compound of Step 1 (90.5 mg, 67%) was
prepared from 31 (115 mg, 0.390 mmol) and benzylamine (50.0
mg, 0.467 mmol) in the same manner as described for 9 Step 1.
Pale yellow solid. Mp: 84-87 °C. 1H NMR (400 MHz, CDCl3) δ
3.88 (3H, s), 4.41 (2H, s), 4.76 (2H, d, J = 5.5 Hz), 7.21-7.36
(8H, m), 7.40 (2H, d, J = 6.8 Hz), 8.42 (1H, s), 8.83 (1H, t, J =
5.5 Hz). HRMS (EI) calcd for C20H19N3O2S (Mþ) 365.1198,
found 365.1191. Anal. (C20H19N3O2S) C, H, N.
Step 2. 4-(Benzylamino)-6-(benzylthio)pyrimidine-5-carboxy-
lic acid (43). The title compound 43 (63.0 mg, 91%) was
prepared from the compound of Step 1 (72.0 mg, 0.197 mmol)
in the same manner as described for 9. Colorless solid. Mp:
1
223-226 °C. H NMR (400 MHz, DMSO-d6) δ 4.32 (2H, s),
4.70 (2H, d, J = 6.1 Hz), 7.20-7.40 (10H, m), 8.39 (1H, s), 8.89
(1H, t, J = 6.1 Hz), 13.70-14.40 (1H, br). HRMS (FABþ) calcd
for C19H18N3O2S (Mþ þ 1) 352.1120, found 352.1156. Anal.
(C19H17N3O2S) C, H, N.
Step 2. 4-(Benzylthio)-6-(cyclohexylamino)-2-(methylthio)-
pyrimidine-5-carboxylic acid (40). The title compound 40
(80.1 mg, 93%) was prepared from the compound of Step 1
(89.3 mg, 0.214 mmol) in the same manner as described for 9.
Colorless solid. Mp: 168-170 °C. 1H NMR (400 MHz, DMSO-
d6) δ 0.87-1.00 (2H, m), 1.05-1.25 (3H, m), 1.48-1.72 (6H, m),
2.46 (3H, s), 2.40-2.55 (2H, m), 4.28 (2H, s), 7.20-7.26 (1H, m),
7.26-7.32 (2H, m), 7.32-7.42 (2H, m), 8.70 (1H, t, J = 5.5 Hz),
13.50-14.10 (1H, br). HRMS (FABþ) calcd for C20H26N3O2S2
(Mþ þ 1) 404.1466, found 404.1484. Anal. (C20H25N3O2S2)
C, H, N..
4-(Benzylamino)-6-(benzyloxy)-2-(methylthio)pyrimidine-5-
carboxylic Acid (44). Step 1. Methyl 4-(benzylamino)-6-(benzy-
loxy)-2-(methylthio)pyrimidine-5-carboxylate. The compound
of Step 1 (165 mg, 90%) was prepared from 13 (150 mg,
0.463 mmol) and benzylalcohol (100 mg, 0.925 mmol) in the
same manner as described for 9 Step 1. Pale yellow oil. 1H NMR
(400 MHz, CDCl3) δ 2.46 (3H, s), 4.72 (2H, d, J = 6.1 Hz), 5.34
(2H, s), 7.25-7.45 (10H, m), 8.72 (1H, t, J = 6.1 Hz). HRMS
(EI) calcd for C21H21N3O3S (Mþ) 395.1304, found 395.1306.
Step 2. 4-(Benzylamino)-6-(benzyloxy)-2-(methylthio)pyrimi-
dine-5-carboxylic acid (44). The title compound 44 (61.8 mg,
80%) was prepared from the compound of Step 1 (80.0 mg,
0.202 mmol) in the same manner as described for 9. Colorless
solid. Mp: 108-112 °C. 1H NMR (400 MHz, DMSO-d6) δ 2.32
(3H, s), 4.62 (2H, d, J = 5.5 Hz), 5.36 (2H, s), 7.13-7.33 (8H,
m), 7.39 (2H, d, J = 7.3 Hz), 9.20 (1H, t, J = 5.5 Hz),
11.50-13.50 (1H, br). HRMS (FABþ) calcd for C20H20N3O3S
(Mþ þ 1) 382.1225, found 382.1191. Anal. (C20H19N3O3S
0.5H2O) C, H, N.
4-(Benzylamino)-6-(benzylthio)-2-(butylthio)pyrimidine-5-carbo-
xylic Acid (41). Step 1. Methyl 4-(benzylamino)-6-(benzylthio)-
2-(butylthio)pyrimidine-5-carboxylate. The compound of Step
1 (95.0 mg, 98%) was prepared from 26 (82.0 mg, 0.214 mmol)
and benzylamine (27.5 mg, 0.257 mmol) in the same manner as
1
described for 9 Step 1. Pale yellow solid. Mp: 87-89 °C. H
NMR (400 MHz, CDCl3) δ 0.88 (3H, t, J = 7.3 Hz), 1.32-1.44
(2H, m), 1.60-1.70 (2H, m), 3.04 (2H, t, J = 7.3 Hz), 3.84 (3H,
s), 4.36 (2H, s), 4.74 (2H, d, J = 5.5 Hz), 7.20-7.35 (8H, m), 7.39
(2H, d, J = 6.7 Hz), 8.89 (1H, t, J = 5.5 Hz). HRMS (EI) calcd
for C24H27N3O2S2 (Mþ) 453.1545, found 453.1573. Anal.
(C24H27N3O2S2 0.2H2O) C, H, N.
4-(Benzylamino)-6-(benzylthio)-2-(butylamino)pyrimidine-5-
carboxylic Acid (45). Step 1. Methyl 4-(benzylamino)-6-(benzy-
lthio)-2-(butylamino)pyrimidine-5-carboxylate. The compound
of Step 1 (58.6 mg, 94%) was prepared from 15 (50.0 mg, 0.143
mmol) and phenyl methanethiol (21.5 mg, 0.173 mmol) in the
same manner as described for 9 Step 1. Pale yellow solid. Mp:
109-112 °C. 1H NMR (400Mz, CDCl3) δ 0.90 (3H, t, J = 7.3
Hz), 1.28-1.40 (2H, m), 1.46-1.58 (2H, m), 3.31-3.47 (2H, m),
3.80 (3H, s), 4.24-4.42 (2H, br), 4.61-4.75 (2H, br), 5.02-5.13
(1H, br), 7.20-7.42 (10H, m), 8.73-8.98 (1H, br).
Step 2. 4-(Benzylamino)-6-(benzylthio)-2-(butylthio)pyrimi-
dine-5-carboxylic acid (41). The title compound 41 (55.0 mg,
74%) was prepared from the compound of Step 1 (77.0 mg,
0.170 mmol) in the same manner as described for 9. Colorless
solid. Mp: 156-158 °C. 1H NMR (400 MHz, DMSO-d6) δ 0.80
(3H, t, J = 7.3 Hz), 1.28 (2H, qd, J = 7.3 and 7.3 Hz), 1.53 (2H,
tt, J = 7.3 and 7.3 Hz), 2.96 (2H, t, J = 7.3 Hz), 4.27 (2H, s), 4.69
(2H, d, J = 6.1 Hz), 7.20-7.40 (10H, m), 13.40-14.20 (1H, br).
HRMS (FABþ) calcd for C23H26N3O2S2 (Mþ þ 1) 440.1466,
found 440.1483. Anal. (C23H25N3O2S2) C, H, N.
Step 2. 4-(Benzylamino)-6-(benzylthio)-2-(butylamino)pyri-
midine-5-carboxylic acid (45). The title compound 45 (38.0 mg,
83%) was prepared from the compound of Step 1 (47.6 mg,
0.109 mmol) in the same manner as described for 9. Colorless
solid. Mp: 145-147 °C. 1H NMR (400 MHz, DMSO-d6) δ 0.83
(3H, t, J = 7.3 Hz), 1.18-1.30 (2H, m), 1.33-1.50 (2H, m),
3.15-3.30 (3H, m), 4.22 and 4.30 (2H, each s), 4.63 (2H, d, J =
6.1 Hz), 7.18-7.42 (11H, m), 8.81 and 8.94 (1H, each t, J = 6.1
Hz), 12.70-13.10 (1H, br). HRMS (FABþ) calcd for
C23H27N4O2S (Mþ þ 1) 423.1855, found 423.1880. Anal.
(C23H26N4O2S) C, H, N.
4-(Benzylamino)-6-(benzylthio)-2-(heptylamino)pyrimidine-5-
carboxylic Acid (46). To a solution of 16 (68.0 mg, 0.174 mmol)
in DMF (0.5 mL) were added potassium carbonate (72.1 mg,
0.522 mmol) and phenyl methanethiol (43.5 mg, 0.350 mmol) at
room temperature, and the mixture was stirred at 80 °C for 3 h.
To the resulting mixture was added 2 N HCl (1 mL) under ice
cooling. The precipitate was formed and collected by filtration,
and then dried in vacuo. The title compound 46 (48.7 mg, 60%)
4-(Benzylamino)-6-(benzylthio)-2-(octylthio)pyrimidine-5-car-
boxylic Acid (42). Step 1. Methyl 4-(benzylamino)-6-(benzy-
lthio)-2-(octylthio)pyrimidine-5-carboxylate. The compound
of Step 1 (112 mg, 88%) was prepared from 27 (110 mg, 0.251
mmol) and benzylamine (32.0 mg, 0.299 mmol) in the same
manner as described for 9 Step 1. Pale yellow solid. Mp: 65-
67 °C. 1H NMR (400 MHz, CDCl3) δ 0.87 (3H, t, J = 6.7 Hz),
1.18-1.40 (10H, m), 1.61-1.72 (2H, m), 3.03 (2H, t, J = 7.3
Hz), 3.84 (3H, s), 4.36 (2H, s), 4.74 (2H, d, J = 5.5 Hz),
7.21-7.41 (10H, m), 8.89 (1H, t, J = 5.5 Hz). HRMS (EI) calcd
for C28H35N3O2S2 (Mþ) 509.2171, found 509.2138. Anal.
(C28H35N3O2S2 0.25H2O) C, H, N.
Step 2. 4-(Benzylamino)-6-(benzylthio)-2-(octylthio)pyrimi-
dine-5-carboxylic acid (42). The title compound 42 (85.3 mg,
95%) was prepared from the compound of Step 1 (93.0 mg,
0.182 mmol) in the same manner as described for 9. Colorless
solid. Mp: 145-149 °C. 1H NMR (400 MHz, DMSO-d6) δ 0.83
(3H, t, J = 7.3 Hz), 1.10-1.30 (10H, m), 1.54 (2H, tt, J = 7.3
and 7.3 Hz), 2.96 (2H, t, J = 7.3 Hz), 7.20-7.40 (10H, m),

5022 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 13
Seto et al.
was given as a colorless solid. Mp: 141-144 °C. 1H NMR (400
MHz, DMSO-d6) δ 0.75-0.90 (3H, m), 1.12-1.32 (9H, m),
1.32-1.52 (2H, m), 3.12-3.23 (1H, m), 4.22 and 4.29 (2H, each s),
4.63 (2H, t, J = 5.5 Hz), 7.16-7.43 (11H, m), 8.80-9.00 (1H, br),
12.70-13.10 (1H, br). HRMS (FABþ) calcd for C26H33N4O2S
(Mþ þ 1) 465.2324, found 465.2351. Anal. (C26H32N4O2S 0.57-
H2O) C, H, N.
and cultured in Ham’s F12 medium containing 10% fetal
calf serum for 24 h at 37 °C. Cells were cotransfected for 2 h
with 40 ng of GAL4/human PPAR LBD, 400 ng of pFR-Luc,
and 5 ng of pRL-TK per well using lipofectamine (Gibco BRL,
Grand Island, NY). Transfected cells were treated with various
concentrations of test compounds at 37 °C for 20 h. Cells were
washed with phosphate-buffered saline and dissolved in passive
lysis buffer (Promega, Madison, WI). Luciferase activity was
determined with a microplate luminescence reader, LUCY2
(Authors, Salzburg, Austria). The EC50 values of tested com-
pounds were derived from the curve fitting using the Prism
program (GraphPad Software, San Diego, CA). The standard
reference full agonist is rosiglitazone (EC50 = 0.1 μM, 100%
maximum activation at 10 μM). All results were produced in
triplicate, and mean values are reported.
3T3-L1 Preadipocyte Differentiation Study. Confluent 3T3-
L1 preadipocytes were treated with complete medium contain-
ing 1 μM dexamethasone, 1 μM IBMX, and 2 μM insulin (DEX/
MIX medium). After 2 days, the DEX/MIX medium was
removed, and cells were washed three times with medium and
then treated with ligands at 37 °C for 48 h. The cells were stained
by Oil-red O using adipogenesis assay kit (Hokudo).
In Vivo Study. Male db/db mice (7-8 weeks old) and lean mice
were orally dosed once daily with test compounds and vehicle
(0.25% methylcellulose) by oral gavage for 7 days. Mice (seven
per group) received a once daily oral dosing of test compounds
with vehicle (0.25% methylcellulose). Plasma glucose levels were
measured before dosing on day 7. Each data point represents the
mean value ((SD) of five individual mice.
Structure Determination. Crystal was soaked for 2 weeks in
reservoir solution, which was prepared by the addition of 50 μL
of compound solution (5.6 mg/1 mL EtOH) to 0.95 M sodium
citrate and 100 mM HEPES buffer (pH 7.5). The crystals were
cryoprotected in glycerol and flash frozen in a cryo-stream.
X-ray data were collected using a Rikagaku RU-H2R X-ray
generator and a Rikagaku RAXIS-V detector. The data were
scaled and processed with the programs DPS-Mosflm and
SCALA.40,41
4-(Benzylamino)-6-(benzylthio)-2-(N-butyl-N-methylamino)-
pyrimidine-5-carboxylic Acid (47). The title compound 47 (19.0 mg,
39%) was prepared from 17 (40.4 mg, 0.111 mmol) and phenyl
methanethiol (28.0 mg, 0.225 mmol) in the same manner as
1
described for 46. Colorless solid. Mp: 140-142 °C. H NMR
(400 MHz, DMSO-d6) δ 0.74-0.86 (3H, m), 1.08-1.22 (2H, m),
1.30-1.52 (2H, m), 2.96-3.12 (3H, m), 3.40-3.60 (2H, m), 4.26
(2H, s), 4.63 (2H, t, J = 5.5 Hz), 7.17-7.41 (10H, m), 8.85-9.00
(1H, br), 12.85-13.10 (1H, br). HRMS (FABþ) calcd for
C24H29N4O2S (Mþ þ 1) 437.2011, found 437.1968. Anal. (C24H28-
N4O2S) C, H, N.
4-(Benzylamino)-6-(benzylthio)-2-(dibutylamino)pyrimidine-
5-carboxylic Acid (48). The title compound 48 (18.0 mg, 69%)
was prepared from 18 (22.0 mg, 54.3 μmol) and phenyl metha-
nethiol (13.5 mg, 0.109 mmol) in the same manner as described
for 46. Colorless solid. Mp: 138-139 °C. 1H NMR (400 MHz,
DMSO-d6) δ 0.70-0.85 (6H, m), 1.08-1.23 (4H, m), 1.30-1.55
(4H, m), 3.23-3.54 (14H, m), 4.27 (2H, s), 4.64 (2H, t, J = 4.9
Hz), 7.16-7.40 (10H, m), 8.85-9.00 (1H, br), 12.80-13.10 (1H,
br). HRMS (FABþ) calcd for C27H35N4O2S (Mþ þ 1) 479.2481,
found 479.2491. Anal. (C27H34N4O2S 0.1H2O) C, H, N.
4-(Benzylamino)-2-(methylthio)-6-(2-thienyl)pyrimidine-5-car-
boxylic Acid (49). Step 1. Methyl 4-(benzylamino)-2-(methy-
lthio)-6-(2-thienyl)pyrimidine-5-carboxylate. To a mixture of
13 (97.2 mg, 0.300 mmol) and 2-thienylboronic acid (57.6 mg,
0.450 mmol) in toluene (2 mL) and 1,4-dioxane (2 mL) were
added 2 M Na2CO3 (2 mL) and Pd(PPh3)4 (70.0 mg, 60.6 μmol),
and the mixture was stirred under reflux for 2 h. The reaction
mixture was cooled to room temperature and diluted with ethyl
acetate. It was then washed with 2 M Na2CO3 and brine, dried
over Na2SO4, filtered, and concentrated in vacuo. Flash chro-
matography (hexane/AcOEt = 5:1) of the residue gave 49 Step 1
The structure was determined by the molecular replacement
method using the model structure (PDBID: 1PRG).42 Refine-
ment was carried out using the program CNX.43 Model building
and validation took place using the programs TOM-FLODO
and PROCHECK.44,45 A summary of the crystallographic
statistics is shown in the Supporting Information.
1
(61.0 mg, 55%) as a pale yellow solid. Mp: 114-116 °C. H
NMR (400 MHz, CDCl3) δ 2.52 (3H, s), 3.66 (3H, s), 4.75 (2H,
d, J = 5.5 Hz), 7.06 (1H, dd, J = 3.7 and 1.2 Hz), 7.25-7.37
(6H, m), 7.46 (1H, dd, J = 4.8 and 1.2 Hz), 7.80 (1H, t, J =
5.5 Hz). HRMS (EI) calcd for C18H17N3O2S2 (Mþ) 371.0762,
found 371.0760. Anal. (C18H17N3O2S2) C, H, N.
Acknowledgment. The authors thank Dr. K. Hirai, Dr. T.
Ishizaki, and Dr. K. Iwase for their valuable suggestions and
encouragement. The authors also thank Mr. M. Suzuki and
Mr. N. Kobayashi for the biological experiments.
Step 2. 4-(Benzylamino)-2-(methylthio)-6-(2-thienyl)pyrimi-
dine-5-carboxylic acid (49). The title compound 49 (38.0 mg,
79%) was prepared from the compound of Step 1 (50.0 mg,
0.135 mmol) in the same manner as described for 9. Colorless
solid. Mp: 142-144 °C. 1H NMR (400 MHz, DMSO-d6) δ 2.40
(3H, s), 4.64 (2H, d, J = 5.5 Hz), 7.15 (1H, dd, J = 5.5 and
4.3 Hz), 7.20-7.27 (1H, m), 7.32 (4H, d, J = 4.3 Hz), 7.48 (1H,
dd, J = 3.7 and 1.2 Hz), 7.75 (1H, dd, J = 4.9 and 1.2 Hz), 8.08
(1H, t, J = 5.5 Hz), 13.55 (1H, s). HRMS (EI) calcd for
C17H15N3O2S2 (Mþ) 357.0606, found 357.0634. Anal.
(C17H15N3O2S2 0.3H2O) C, H, N.
Supporting Information Available: Experimental details of
compounds S1-S10; elemental analysis; summary of crystal-
lographic statistics. This material is available free of charge via
the Internet at http://pubs.acs.org.
4,6-Bis(benzylthio)-2-(methylthio)pyrimidine-5-carboxylic Acid
(50). To a solution of 10 (120 mg, 0.502 mmol) in DMF (1 mL)
were added potassium carbonate (138 mg, 0.999 mmol) and
phenyl methanethiol (124 mg, 1.00 mmol) at room temperature,
and the mixture was stirred at room temperature for 4 h. To the
resulting mixture was added 2 N HCl (1 mL) under ice cooling.
The precipitate was formed and collected by filtration, and dried
in vacuo. The title compound 50 (180 mg, 86%) was given as a
colorless solid. Mp: 187-189 °C. 1H NMR (400 MHz, DMSO-
d6) δ 2.55 (3H, s), 4.38 (4H, s), 7.22-7.42 (10H, m), 14.00-14.25
(1H, br). HRMS (FABþ) calcd for C20H19N2O2S3 (Mþ þ 1)
415.0609, found 415.0578. Anal. (C20H18N2O2S3) C, H, N.
PPARγ Transactivation Assay. Chinese hamster ovary K1
(CHO-K1) cells were seeded at 5 ꢀ 104 cells/well in 24-well plates
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