Five-membered heteroaromatic ring fused-pyrimidine derivatives: Design, synthesis, and hedgehog signaling pathway inhibition study

Article abstract of DOI:10.1016/j.bmcl.2014.05.066

A series of novel five-membered heteroaromatic ring fused-pyrimidine derivatives including purines, pyrrolo[2,3-d]pyrimidines, pyrrolo[3,2-d] pyrimidines, thieno[2,3-d]pyrimidines, thieno[3,2-d]pyrimidines and furo[3,2-d]pyrimidines have been identified to be potent inhibitors of hedgehog signaling pathway. The synthesis and SAR of these compounds are described. Among this new series of hedgehog signaling pathway inhibitors, most compounds exhibited significant inhibitory activity compared to vismodegib, indicating that the five-membered heteroaromatic ring fused-pyrimidines stand out as encouraging scaffolds among the currently reported structural skeletons for hedgehog signaling pathway inhibitors, deserving more exploration and further investigation.

Full text of DOI:10.1016/j.bmcl.2014.05.066

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Five-membered heteroaromatic ring fused-pyrimidine derivatives:
Design, synthesis, and hedgehog signaling pathway inhibition study
b
b
b
b
b
Liandi Zhang a,b, Minhang Xin c, , Han Shen , Jun Wen , Feng Tang , Chongxing Tu , Xinge Zhao ,
⇑
Ping Wei a,
⇑
a The College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, No. 30 Puzhu Road, Nanjing 211800, PR China
b Jiangsu Simcere Pharmaceutical Co. Ltd, Jiangsu Key Laboratory of Molecular Targeted Antitumor Drug Research, No. 699-18, Xuan Wu District, Nanjing 210042, PR China
c Department of Medicinal Chemistry, School of Pharmacy, Health Science Center, Xi’an Jiaotong University, No. 76, Yanta West Road, Xi’an 710061, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel five-membered heteroaromatic ring fused-pyrimidine derivatives including purines,
Received 2 April 2014
Revised 15 May 2014
Accepted 17 May 2014
Available online xxxx
pyrrolo[2,3-d]pyrimidines,
pyrrolo[3,2-d]pyrimidines,
thieno[2,3-d]pyrimidines,
thieno[3,2-d]
pyrimidines and furo[3,2-d]pyrimidines have been identified to be potent inhibitors of hedgehog signal-
ing pathway. The synthesis and SAR of these compounds are described. Among this new series of hedge-
hog signaling pathway inhibitors, most compounds exhibited significant inhibitory activity compared to
vismodegib, indicating that the five-membered heteroaromatic ring fused-pyrimidines stand out as
encouraging scaffolds among the currently reported structural skeletons for hedgehog signaling pathway
inhibitors, deserving more exploration and further investigation.
Keywords:
Hedgehog signaling pathway
Inhibitors
Ó 2014 Elsevier Ltd. All rights reserved.
Five-membered heteroaromatic ring fused-
pyrimidine
Synthesis
Hedgehog (Hh) signaling pathway has been found to be a key
signal transduction cascade which significantly affects embryonic
morphogenesis, tissue patterning, angiogenesis and tumorigene-
sis.1 The Hh signaling transduction is initiated by binding of the
Hh-protein, such as sonic hedgehog (Shh), Indian hedgehog (Ihh),
desert hedgehog (Dhh), to its cellular membrane receptor patched
(Ptch), thereafter leading to the relief of suppressed GPCR-like
receptor smoothened (Smo). The activated Smo can trigger a down-
stream signaling cascade, leading to activation of Gli-transcription
factors. And then the active Gli-1 and Gli-2 move into the nucleus
where they induce the target genes to regulate proliferation, differ-
entiation, and survival.2 Usually, the Hh signaling is inactive in
adulthood; however, when mutations or hyper-activations in this
signaling pathway occur, it becomes aberrantly activated and con-
sequently leads to tumor growth and aggravation. For examples,
loss-of-function mutations in Ptch1 occur in a majority of basal cell
carcinoma (BCC) and approximately 30% of sporadic medulloblas-
toma cases, while Hh overexpression by an autocrine or paracrine
manner in this pathway identified in the past few years is impli-
cated in many other cancer types, such as lung, pancreatic, gastric,
colorectal, prostate, breast and melanoma tumors.3 Hence, inhibi-
tion of Hh signaling pathway is considered to be an attractive
therapeutic strategy for anticancer intervention.4 In 2012, the small
molecule Hh signaling pathway inhibitor vismodegib (GDC-0449)
was approved by FDA for its significantly clinical efficacy in phase
II clinical evaluation of metastatic and locally advanced BCC.
Subsequently, the Hh signaling pathway inhibitors NVP-LDE225
and LY-2940680 were also quickly advanced into clinical phase III
and phase II, respectively, to validate their clinical profiles for
cancer therapy (Fig. 1).5
We have reported a novel series of potent 4-(2-pyrimidinylami-
no)benzamide-based inhibitors of Hh signaling pathway recently,
such as 1, which exhibited highly effective inhibition with an
IC50 = 1.3 nM in Gli-luciferase reporter assay.6 In the course of
extensively structural modification, we have described the SAR
details, and identified several compounds showing satisfactory
potency and significant pharmacokinetic properties. In addition,
on the basis of 4-(2-pyrimidinylamino)benzamide core, using con-
ceptual cyclization strategy, we have also developed two new
pyrimidine-based scaffolds, pyrrolo[2,1-f][1,2,4]triazines and dihy-
dro-5H-pyrano[2,3-d]pyrimidines.7 And in the comparison of these
two nucleuses derivatizing analogues, it showed that pyrrolo
[2,1-f][1,2,4]triazines exhibited more potent inhibition than
dihydro-5H-pyrano[2,3-d]pyrimidines. For examples, pyrrolo
[2,1-f][1,2,4]triazine 2 (IC50 = 1.6 nM) displayed over three-fold
more effective than dihydro-5H-pyrano[2,3-d]pyrimidine
(IC50 = 5.2 nM). From these discoveries, with substitution of the
3
⇑
Corresponding authors. Tel.: +86 2558139908 (P.W.).
E-mail addresses: xmhcpu@163.com (M. Xin), weiping@njtech.edu.cn (P. Wei).
http://dx.doi.org/10.1016/j.bmcl.2014.05.066
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhang, L.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.05.066

2
L. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Cl
H
O
Cl
N N
N
N
N
N
N
N
O
N
N
H
O
F
O
O
CF3
N
S
O
O
CF3
vismodegib
(GDC-0449)
NVP-LDE-225
(sonidegib; LDE-225)
LY-2940680
Lilly Ph II
Genentech/Roche
approved in 2012
Novartis Ph III
Figure 1. Chemical structure of several Hh signaling pathway inhibitors.
pyrimidine extensively explored, we became interested in
preparing other five-membered ring fused-pyrimidine heterocyclic
cores, such as purines, pyrrolo[2,3-d]pyrimidines, pyrrolo
[3,2-d]pyrimidines, thieno[2,3-d]pyrimidines, thieno[3,2-d]pyrimi-
dines and furo[3,2-d]pyrimidines. These novel alternative scaffolds
would allow us to maintain the optimized substitute profile, while
potentially attempting to improve the developability characteris-
tics, by altering lipophilicity of the core skeleton. Accordingly,
herein we describe the synthesis of several novel scaffolds of
five-membered heteroaromatic ring fused-pyrimidine, and their
inhibition toward the Hh signaling pathway (Fig. 2).
success in accomplishing the building blocks, we then focused on
coupling of the above blocks with 2-chloro-6-(4-trifluoromethoxy-
phenyl)purine (Scheme 1-B). However, straight Suzuki C–C
coupling of block 6 with 2,6-dichloro-9H-purine 14 gave complex
mixtures and was difficult to isolate the desired product 16a. Con-
sidering that such difficulty was likely due to the presence of free
NH group in purine backbone, we decided to introduce protecting
group at 9-position of purine. In this regard, 2,6-dichloro-9-(4-
methoxybenzyl)purine 15 was prepared by a general 4-methoxy-
benzyl protection procedure, which was subsequently reacted with
4-trifluoromethoxyphenylboronic acid under Pd-catalyzed condi-
tion to afford Suzuki coupling product 16. And then the coupling
reaction between 16 and the above aniline-blocks under Pd(OAc)2/
BINAP/Cs2CO3 catalytic system proceeded smoothly and afforded
4-methoxybenzyl protected products 19a–19d. Deprotection of
19a–19d with TFA provided the target compounds 20a–20d, and
methylation of 20a–20b with methyl iodide gave target compounds
21a–21b. Besides, the coupling of 16 with other aniline-like mate-
rials 1H-indazol-5-amine and 5-aminoindolin-2-one, followed
deprotection, afforded another target compounds 18a–18b
(Scheme 1).
The novel five-membered heteroaromatic ring fused-
pyrimidine derivatives (18a–18b, 20a–20d, 21a–21b, 26a–26c,
30a–30d, 44a–44d and 45a–45c) were prepared as depicted in
Schemes 1–4.
Scheme
1 detailed the synthesis of the desired purines
(18a–18b, 20a–20d and 21a–21b). We first prepared the four build-
ing blocks 6, 9, 13a and 13b, shown in Scheme 1-A. Condensation of
4-nitrobenzoic acid
4 with 2,6-dimethylaniline, 3-amino-4-
methylphenol and 3-amino-4-methylbenzyl alcohol provided
intermediates 5, 7 and 10, respectively. Reduction of 5 using cata-
lytic hydrogenation by hydrogen in the presence of 10% Pd/C led
to the block 6, while block 9 was obtained including nucleophilic
Scheme
2 depicted the synthesis of the desired pyrrolo
[2,3-d]pyrimidines (26a–26c). The similar synthetic procedures
substitution of
7
with commercially available 4-(2-chloro-
were employed. Briefly, from the commercially available
ethyl)morpholine hydrochloride, then following a similar catalytic
hydrogenation procedure. And blocks 13 were synthesized from
10 by three steps, through chlorination of 10 with thionyl chloride
to give 11, which was subsequently substituted by cyclic amines
and then catalytically hydrogenated in a similar procedure. With
2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine
22
and
4-trif-
luoromethoxyphenylboronic acid, the Suzuki coupling product 23
was constructed. Subsequently the free NH group of 23 was pro-
tected by another alternative protection group p-toluenesulfonyl,
to give product 24. Coupling of 24 with block 6 led to intermediate
OCF3
OCF3
A
OCF3
A
A
5
6
O
O
4
O
D
D
4
D
3
N
N
H
N
2
N
H
3
1
N
N
H
5
B
C
B
C
2
B
N
C
N
O
N
N
H
N
1
N
H
6
N
H
cyclization
3
IC50 = 5.2 nM
1
IC50=1.3nM
2
IC50 = 1.6 nM
N
B
N
our current design
B-ring
N
R
O
N
N
S
N
N
N
N
N
N
N
N
S
N
N
N
N
N
N
R
R
R = H, Me
novel five-membered heteroaromatic ring fused-pyrimidine derivatives
Figure 2. Novel five-membered heteroaromatic ring fused-pyrimidine derivatives design.
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L. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
(A)
O
OH
H2N
O
O
N
H
N
H
a
b
H2N
O
O2N
O
5
6
NO2
a
H2N
O
O
4
O
O
N
OH
b
N
Cl
N
N
H
O
N
H
O
NH2
N
OH
d
H
a
O2N
8
9
H2N
O2N
7
OH
O
O
O
O
HN
X
R
R
N
OH
c
Cl
N
H
b
H
N
H
N
H
H2N
d
13a-13b
O2N
O2N
12a-12b
O2N
11
10
a: R = 4-morpholinyl
a: R = 4-morpholinyl
b: R = 4-methylpiperazin-1-yl
b: R = 4-methylpiperazin-1-yl
OCF3
OCF3
OCF3
(B)
Cl
N
O
Cl
N
O
e
N
6, or 9, or 13,
N
h
N
R2
N
N
N
N
N
H
R2
N
N
N
N
N
H
f
g
R1
N
Cl
R1
N
H
N
H
N
N
H
Cl
N
N
Cl
N
N
H
20a-20d
14
O
O
16
19a-19d
a: R1 =CH3; R2 = H
b: R1 = H; R2
c: R1 = H; R2
d: R1 = H; R2
a: R1 =CH3; R2 = H
O
b: R1 = H; R2
c: R1 = H; R2
d: R1 = H; R2
=
O
N
N
f
O
N
N
=
N
g
15
=
ArNH2
O
N
=
N
=
O
OCF3
O
N
=
i
O
OCF3
OCF3
OCF3
O
N
N
N
N
N
N
R2
h
N
H
Cl
N
N
H
N
N
N
R1
N
N
N
H
Ar
Ar
N
N
16a
N
H
N
N
21a-21b
a: R1 =CH3; R2 = H
H
O
H
17a-17b
a: Ar = 1H-indazol-5-yl
b: Ar = 2-oxoindoline-5-yl
18a-18b
a: Ar = 1H-indazol-5-yl
b: Ar = 2-oxoindoline-5-yl
b: R1 = H; R2
=
O
N
Scheme 1. Reagents and conditions: (a) (1) SOCl2, reflux for 3 h; (2) THF, DIPEA, rt for 26 h, 2,6-dimethylaniline for 5, 46%; 3-amino-4-methylphenol for 7, 100%; 3-amino-4-
methylbenzyl alcohol for 10, 100%; (b) 10% Pd/C, H2, rt for 24 h, 84–96%; (c) SOCl2, CH2Cl2, rt for 2.5 h, 100%; (d) DMF, K2CO3, 85 °C for 2.5 h, 4-(2-chloroethyl)morpholine
hydrochloride for 8, 24%; morpholine for 12a, 100%; 1-methylpiperizine for 12b, 78%; (e) 4-methoxybenzyl chloride, K2CO3, DMF, rt, overnight, 50%; (f) 4-
trifluoromethoxyphenylboronic acid, Pd(PPh3)2Cl2, TEA, DMF, H2O, 80 °C for 4 h, 48%; (g) Pd(OAc)2, BINAP, Cs2CO3, dioxane, 150 °C, microwave for 4 h, 12–81% ; (h) TFA,
reflux for 4 h, 34–79%; (i) CH3I, K2CO3, DMF, rt for 6 h, 55–100%.
OCF3
OCF3
OCF3
OCF3
OCF3
Cl
N
O
d
a
c, 6
b
O
N
+
N
H
N
N
H
N
N
N
H
Cl
N
N
N
H
N
H
N
B(OH)2
N
Ts
N
Ts
Cl
N
N
H
N
N
Cl
22
H
25
24
26a
23
c, 13a
e
OCF3
OCF3
O
O
O
N
N
N
H
N
N
N
H
N
N
N
N
H
H
N
H
26b
26c
Scheme 2. Reagents and conditions: (a) Pd(PPh3)2Cl2, TEA, DMF, H2O, 80 °C for 4 h, 41%; (b) TsCl, NaOH, acetone/H2O, rt for 3 h, 87%; (c) Pd(OAc)2, BINAP, Cs2CO3, dioxane,
150 °C, microwave for 4 h, 39–46%; (d) NaOH, CH3OH, reflux for 1 h, 81%; (e) CH3I, K2CO3, DMF, rt for 6 h, 53%.
Please cite this article in press as: Zhang, L.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.05.066

4
L. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
OCF3
OCF3
OCF3
Cl
N
Cl
N
H
N
O
N
c
O
a
b
N
N
N
N
N
N
N
6
N
N
N
H
N
N
H
N
Cl
Cl
N
H
N
N
N
Cl
H
30b
27
28
29
30a
c, 13b
d
OCF3
OCF3
OCF3
OCF3
OCF3
g
e
f
b
h
O
O
Cl
N
O
Ts
N
H
N
H
N
H
N
Ts
N
OH
H
N
N
OH
N
N
N
H
N
N
N
O
N
N
N
N
N
H
N
Cl
N
N
Cl
H
Cl
H
36
35
31
32
33
34
c, 6
i
OCF3
OCF3
OCF3
O
O
O
j
H
H
N
H
Cl
N
N
N
N
N
N
N
N
H
N
N
N
H
H
N
N
H
N
H
N
H
N
30c
30d
37
Scheme 3. Reagents and conditions: (a) CH3I, K2CO3, DMF, rt for 6 h, 88%; (b) 4-trifluoromethoxyphenylboronic acid, Pd(PPh3)2Cl2, TEA, DMF, H2O, 80 °C for 4 h, 54–58%; (c)
Pd(OAc)2, BINAP, Cs2CO3, dioxane, 150 °C, microwave for 4 h, 36–58%; (d) TsCl, NaOH, acetone/H2O, rt for 3 h, 97%; (e) NaOH, CH3OH, reflux for 1 h, 95%; (f) methyl 4-
aminobenzoate, Pd(OAc)2, BINAP, Cs2CO3, dioxane, 150 °C, microwave for 4 h, 31%; (g) NaOH, MeOH/H2O, reflux overnight, 99%; (h) 3-amino-4-methylbenzyl alcohol, HATU,
DMF, DIPEA, 80 °C for 5 h, 38%; (i) SOCl2, CH2Cl2, rt for 2.5 h, 99%; (j) N-methylpiperazine, DMF, K2CO3, 85 °C for 2.5 h, 58%.
25, which was further deprotected to afford the target compound
26a. Methylation on the NH of 26a provided methylated compound
26b. Alternately, compound 26c was achieved straightly using non-
protected 23 as reactive reagent in 39% yield (Scheme 2).
Synthesis of pyrrolo[3,2-d]pyrimidine analogues 30a–30d was
illustrated in Scheme 3. Treatment of commercially available
2,4-dichloro-5H-pyrrolo[3,2-d]pyrimidine 27 with methyl iodide
or p-toluenesulfonyl chloride under basic condition gave N-methyl
product 28 and N-p-toluenesulfonyl product 31, respectively. At
this point, coupling of 28 using the above similar Suzuki condition
afforded compound 29, which was subsequently treated with 6 or
13b under Buchwald–Hartwig coupling condition to yield target
compounds 30a and 30b, respectively. Alternately, target
compound 30c was practically assembled from 31 by three
steps, which were Suzuki coupling of 31 with 4-trif-
luoromethoxyphenylboronic acid to give 32, followed by
OCF3
OCF3
OCF3
OCF3
Cl
c
a
b
d
Y
Z
O
O
O
N
OH
Y
Z
Y
Z
Y
Z
N
N
H
Y
Z
N
O
N
N
OH
N
Cl
N
N
N
N
N
H
N
H
N
Cl
39a-39c
H
42a-42c
38a-38c
a: Y = S, Z = CH
b: Y = CH, Z = S
c: Y = O, Z = CH
41a-41c
40a-40c
a: Y = S, Z = CH
b: Y = CH, Z = S
c: Y = O, Z = CH
a: Y = S, Z = CH
b: Y = CH, Z = S
c: Y = O, Z = CH
a: Y = S, Z = CH
b: Y = CH, Z = S
c: Y = O, Z = CH
a: Y = S, Z = CH
b: Y = CH, Z = S
c: Y = O, Z = CH
e
OCF3
OCF3
OCF3
6
b,
f
O
O
O
Cl
Y
Z
N
N
H
Y
N
N
H
Y
Z
N
H
N
N
N
N
H
Z
N
N
H
N
N
H
43a-43c
44a-44d
X
45a-45c
a: Y = S, Z = CH, X = NCH3
b: Y = CH, Z = S, X = NCH3
c: Y = CH, Z = S, X = O
a: Y = S, Z = CH
b: Y = CH, Z = S
c: Y = O, Z = CH
a: Y = S, Z = CH
b: Y = CH, Z = S
c: Y = O, Z = CH
d: Y = O, Z = CH, X = O
Scheme 4. Reagents and conditions: (a) 4-trifluoromethoxyphenylboronic acid, Pd(PPh3)2Cl2, TEA, DMF, H2O, 80 °C for 4 h, 2,4-dichlorothieno[3,2-d]pyrimidine for 39a
(76%); 2,4-dichlorothieno[2,3-d]pyrimidine for 39b (75%); 2,4-dichlorofuro[3,2-d]pyrimidine for 39c (70%); (b) methyl 4-aminobenzoate, Pd(OAc)2, BINAP, Cs2CO3, dioxane,
150 °C, microwave for 4 h, 43–77%; (c) NaOH, MeOH/H2O, reflux overnight, 90–100%; (d) 3-amino-4-methylbenzyl alcohol, HATU, DMF, DIPEA, 80 °C for 5 h, 64–89%; (e)
SOCl2, CH2Cl2, rt for 2.5 h, 42–94%; (f) N-methylpiperazine or morpholine, DMF, K2CO3, 85 °C for 2.5 h, 48–62%.
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L. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
5
Table 1
The IC50 of target compounds against Gli-luc reporter
OCF3
OCF3
OCF3
O
H
N
H
N
N
N
N
N
N
H
R2
B
O
R1
N
H
N
H
N
N
H
N
N
N
H
H
20a-20d, 21a-21b, 26a-26c,
30a-30d, 44a-44d and 45a-45c
18a
18b
Compds
B ring
R1
R2
Gli-luc reporter
IC50 (nM)
18a
18b
—
—
—
—
—
—
398.8
>1000
N
N
20a
20b
20c
20d
21a
21b
26a
26b
26c
Me
H
H
512.2
0.69
0.90
2.98
116.1
0.35
6.85
2.58
6.04
N
H
N
N
N
N
N
N
N
N
N
N
N
O
N
N
N
N
N
N
N
N
N
N
N
H
N
H
N
H
O
N
O
N
O
H
N
H
N
N
Me
H
H
N
N
N
Me
Me
H
H
H
N
H
N
N
N
O
N
N
H
N
N
N
N
N
N
30a
30b
30c
30d
44a
Me
H
H
H
1.17
5.10
12.84
1.39
9.39
N
N
N
N
N
N
H
N
Me
H
H
N
N
N
N
N
S
H
(continued on next page)
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L. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 1 (continued)
Compds
B ring
R1
H
R2
Gli-luc reporter
IC50 (nM)
N
N
N
N
O
O
N
N
N
N
N
N
44b
44c
44d
45a
45b
3.36
1.75
1.13
3.61
0.60
S
N
N
N
N
N
N
H
S
O
H
S
Me
Me
Me
H
H
H
S
O
45c
1.78
7.17
GDC-0449
deprotection to yield 33, and next by Buchwald–Hartwig coupling
to afford 30c. Otherwise, N-methylpiperazine derivative 30d was
prepared from intermediate 33 involving five steps. Coupling of
33 with methyl 4-aminobenzoate using the above Buchwald–
Hartwig condition gave 34, which was subsequently hydrolyzed
to free acid 35. Condensation 35 with 3-amino-4-methylbenzyl
alcohol provided 36, followed by chlorination with thionyl chloride
to afford 37, and next by nucleophilic substitution with N-methyl-
piperazine to provide 30c (Scheme 3).
The representative compounds of other scaffolds including thi-
eno[2,3-d]pyrimidine, thieno[3,2-d]pyrimidine and furo[3,2-
d]pyrimidine were prepared as described in Scheme 4. Employing
similar synthetic procedures, analogues 44a–44d and 45a–45c
were synthesized. Beginning with appropriately commercially
available materials (2,4-dichlorothieno[3,2-d]pyrimidine 38a, 2,
4-dichlorothieno[2,3-d]pyrimidine 38b and 2,4-dichlorofuro[3,
2-d]pyrimidine 38c), the desired compounds 44a–44d were
accomplished in six steps, including Suzuki coupling reaction (to
give 39a–39c), Buchwald–Hartwig coupling reaction (to give
40a–40c), hydrolysis (to give 41a–41c), condensation (to give
42a–42c), chlorination (to give 43a–43e), and subsequently nucle-
ophilic substitution with morpholine or N-methylpiperazine (to
give 44a–44d). While compounds 45a–45c were constructed by
Suzuki-coupling of intermediates 39a–39c with the above building
block 6, respectively (Scheme 4).
All the newly synthesized five-membered heteroaromatic ring
fused-pyrimidines, including purines (18a–18b, 20a–20d and
21a–21b), pyrrolo[2,3-d]pyrimidines (26a–26c), pyrrolo[3,
2-d]pyrimidines (30a–30d), thieno[3,2-d]pyrimidines (44a and
45a), thieno[2,3-d]pyrimidines (44b, 44c and 45b) and furo[3,2-
d]pyrimidines (44d and 45c) were evaluated for their ability to
inhibit the Hh signaling pathway by using a luciferase reporter in
NIH3T3 cell carrying a stably transfected Gli-reporter construct
(Gli-luciferase reporter cell lines).7 The in vitro IC50 values were
illustrated in Table 1 and GDC-0449 was used as a positive control.
In the initially designed purine derivatives, although several com-
pounds such as 18a (398.8 nM), 18b (>1000 nM) and 20a
(512.2 nM) showed very weak inhibitory activity against Hh, the
analogues 20b, 20c and 20d, bearing basic amine side chains,
exhibited significantly potency (20b: 0.69 nM, 20c: 0.90 nM, 20d:
2.98 nM). This result demonstrated the viability of purines as an
alternative to our previously reported pyrrolo[2,1-f][1,2,4]triazine
and pyrimidine scaffolds. Additionally, some purines of methyl
substituted at 9-position were explored, such as compounds 21a
and 21b. Interestingly, methyl substitution which lacked a hydro-
gen bond acceptor, appeared to be helpful for slightly improving
inhibitory activity (21a: 116.1 nM vs 20a: 512.2 nM, as well as
21b: 0.35 nM vs 20b: 0.69 nM). Considering purines to be good
surrogate scaffold, we tried to explore other scaffolds for enlarging
the scaffold alternatives. Some scaffolds such as pyrrolo[2,
3-d]pyrimidines, pyrrolo[3,2-d]pyrimidines, thieno[2,3-d]pyrimi-
dines, thieno[3,2-d]pyrimidines and furo[3,2-d]pyrimidines were
designed and synthesized and the selected examples were
depicted in Table 1. Two pyrrolo[2,3-d]pyrimidine derivatives such
as 26a and 26c showed satisfactorily equipotent potency compared
to GDC-0449 (26a: 6.85 nM, 26c: 6.04 nM vs GDC-0449: 7.17 nM),
while compound 26b attaching a methyl group displayed approx-
imate 3-fold more effective than GDC-0449. However, in pyrrol-
o[3,2-d]pyrimidines (30a–30d), it was found that the methyl 30a
showed better potency than no-methyl 30c, while methyl 30b
when bearing N-methyl piperazine in D-ring displayed decreased
activity compared to the no-methyl compound 30d. Furthermore,
when the N atom of five-ring adjacent to the ring junction was
replaced with isosteric S or O atom, thereby derivatizing scaffolds
such as thieno[2,3-d]pyrimidines, thieno[3,2-d]pyrimidines and
furo[3,2-d]pyrimidines. It showed very tolerant. These analogs
containing S or O on the scaffolds showed good Hh signaling inhib-
itory activity with IC50 varied from 0.60 nM to 9.39 nM. Interest-
ingly, the biological data between 44a and 44b (IC50: 9.39 nM vs
3.36 nM) showed that the thieno[2,3-d]pyrimidine one was more
potent than thieno[3,2-d]pyrimidine one. The same cases were
accounted by compounds 45a and 45b (IC50
: 3.61 nM vs
0.60 nM). In addition, Compared 45c with 45a (45c: 1.78 nM vs
45a: 3.61 nM), it appeared the furo[3,2-d]pyrimidines provided a
better potency than thieno[3,2-d]pyrimidines. Considering that
thieno[2,3-d]pyrimidine and furo[3,2-d]pyrimidine scaffolds
showed improving activity, the morpholine derivatives 44c and
44d were prepared, whereas both showed low nanomolar Hh
Please cite this article in press as: Zhang, L.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.05.066

L. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
7
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inhibitory activity with an IC50 of 1.75 nM and 1.13 nM, respec-
tively, more potent than GDC-0449 (7.17 nM). In a word, five-
membered heteroaromatic ring fused-pyrimidines appears to be
good alternatives for surrogate of pyrrolo[2,1-f][1,2,4]triazine and
pyrimidine scaffolds. The structural modification study showed
most of target compounds displayed better inhibitory activities
than positive control GDC-0449, of which 4 ones exhibited superior
activities with an subnanomolar IC50 values (<1 nM).
In summary, we have designed and synthesized a series of novel
five-membered heteroaromatic ring fused-pyrimidines targeting
the hedgehog signaling pathway. These five-membered
heteroaromatic ring fused-pyrimidine scaffolds including purine,
pyrrolo[2,3-d]pyrimidine, pyrrolo[3,2-d]pyrimidine, thieno[2,
3-d]pyrimidine, thieno[3,2-d]pyrimidine and furo[3,2-d]pyrimidine
turned out to be viable alternatives to the previously reported pyr-
rolo[2,1-f][1,2,4]triazine and pyrimidine cores, showing similar
inhibitive profile of Hh signaling pathway. Furthermore, structural
modification study showed most compounds exerted better inhibi-
tory activities than positive control GDC-0449. Compound 20b, 20c,
21b and 45b were found to be the most potent inhibitors with IC50
values below 1 nM, and deserved more exploration and further
investigation. Otherwise, what is important is that this study may
provide a potential route to design more effective hedgehog signal-
ing pathway inhibitors with novel structural skeletons.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.05.
066.
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