Discovery of N-(3-((7H-purin-6-yl)thio)-4-hydroxynaphthalen-1-yl)- sulfonamide derivatives as novel protein kinase and angiogenesis inhibitors for the treatment of cancer: Synthesis and biological evaluation. Part III

Article abstract of DOI:10.1016/j.bmc.2013.11.052

A novel series of N-(3-((7H-purin-6-yl)thio)-4-hydroxynaphthalen-1-yl)- sulfonamides were designed and synthesized. Biological characterization revealed that several compounds exerted enhanced anti-proliferative activity against human umbilical vein endothelial cells (HUVECs) and several cancer cell lines and high specific protein kinase and angiogenesis inhibitory activities. Compared with our previously synthesized compounds, the substitution of sulfonamide structure for amide fragment played an essential role for the advance of inhibitory activities. In addition, the replacement of 1H-1,2,4-triazole ring by 7H-purine did not result in obvious decrease of inhibition efficacy, indicating that the sulfonamide structure contributes even more to the inhibition efficacy than the 1H-1,2,4-triazole ring. Among these compounds, compound 9n demonstrated comparable in vitro antiangiogenic activities to pazopanib in both HUVEC tube formation assay and the rat thoracic aorta rings (TARs) test. Meanwhile, compound 9n was identified to inhibit Akt1 (IC₅₀ = 1.73 μM) and Abl tyrosine kinase (IC₅₀ = 1.53 μM) effectively.

Full text of DOI:10.1016/j.bmc.2013.11.052

Bioorganic & Medicinal Chemistry 22 (2014) 1487–1495
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of N-(3-((7H-purin-6-yl)thio)-4-hydroxynaphthalen-1-yl)
-sulfonamide derivatives as novel protein kinase and angiogenesis
inhibitors for the treatment of cancer: Synthesis and biological
evaluation. Part III
a,
Fuming Xu a, Hao Xu b, Xuejian Wang c, Lei Zhang d, Qingli Wen e, Yingjie Zhang a, , Wenfang Xu
⇑
⇑
a Department of Medicinal Chemistry, School of Pharmacy, Shandong University, Ji’nan 250012, PR China
b Department of Breast and Thyroid Surgery, Shandong Provincial Hospital Affiliated to Shandong University, 324 Jingwu-Weiqi Road, Ji’nan 250021, PR China
c Department of Pharmacology, Pharmacy and Biological Science College, WeiFang Medical University, Weifang 261053, PR China
d Department of Pharmacy, School of Medicine, Qingdao University, Qingdao 266071, PR China
e Affiliated Hospital of Weifang Medical University, Weifang, Shandong 261031, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of N-(3-((7H-purin-6-yl)thio)-4-hydroxynaphthalen-1-yl)-sulfonamides were designed
and synthesized. Biological characterization revealed that several compounds exerted enhanced anti-pro-
liferative activity against human umbilical vein endothelial cells (HUVECs) and several cancer cell lines
and high specific protein kinase and angiogenesis inhibitory activities. Compared with our previously
synthesized compounds, the substitution of sulfonamide structure for amide fragment played an essen-
tial role for the advance of inhibitory activities. In addition, the replacement of 1H-1,2,4-triazole ring by
7H-purine did not result in obvious decrease of inhibition efficacy, indicating that the sulfonamide struc-
ture contributes even more to the inhibition efficacy than the 1H-1,2,4-triazole ring. Among these com-
pounds, compound 9n demonstrated comparable in vitro antiangiogenic activities to pazopanib in both
HUVEC tube formation assay and the rat thoracic aorta rings (TARs) test. Meanwhile, compound 9n was
Received 12 September 2013
Revised 26 November 2013
Accepted 29 November 2013
Available online 8 December 2013
Keywords:
Angiogenesis
HUVEC tube formation
Rat thoracic aorta rings
Protein kinase B/Akt
Abl
identified to inhibit Akt1 (IC50 = 1.73
lM) and Abl tyrosine kinase (IC50 = 1.53
lM) effectively.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
agents used in combination are much more successful in cancer
chemotherapy.1,16 We hypothesized that one agent possessing
both cytostatic protein kinase and angiogenesis inhibition and
the cytotoxic activities to tumors would enhance the clinical treat-
ment outcome with reduced drug resistance, like sorafenib (BAY
43-9006, receptor tyrosine kinase inhibitor),17 ZD6474 (vandeta-
nib, receptor tyrosine kinase inhibito),18,19 combretastatin A-4
(CA-4, vascular targeting agent),20 etc.
Angiogenesis is a normal process in growth and development of
new capillary blood vessels involved in physiological events such
as pregnancy, organ development, menstrual cycle and wound
healing.1–3 Aberrant angiogenesis, the process that leads to the for-
mation of newly abnormal vessels, is a prerequisite for tumor cell
proliferation, invasion and metastasis.4–6 Tumor-induced blood
capillaries can help to acquire enough oxygen and nutrients, to re-
move metabolic waste and to facilitate metastasis formation for
several solid tumors and haematological malignancies.5,7
Thus, inhibition of tumor angiogenesis affords attractive targets
for the development of novel antitumor agents.8–10 Because tu-
mors have abundant signaling pathways for angiogenesis and often
develop resistance to agents that target one specific pathway, anti-
angiogenic therapy targeting multi-protein kinases has been of
great interest.11–15 Moreover, it has been demonstrated in recent
years that angiogenesis inhibitors and conventional cytotoxic
In our previous work, we have reported a series of N-(4-hydro-
xy-3-mercaptonaphthalen-1-yl)amide derivatives, of which the
biological characterization revealed that the most active compound
6d (see Fig. 1) displayed potent inhibitory effect and can be used as
a leading antiangiogenic and anticancer agent for further structure
optimization.21 In an effort to search for small molecular entities
with both anti-protein kinase and anti-angiogenesis activities and
conventional anticancer property for cancer treatment, we modi-
fied the structure of lead compound 6d as follows, Figure 1: (i) sul-
fonamide moiety plays prominent roles in biology, biochemistry,
and pharmaceutical areas. So the amide structure can be replaced
with sulfonamide structure fragment on considering its high
cytotoxicity, antitumor activity, and broad spectrum of biological
⇑
Corresponding authors. Tel./fax: +86 531 88382264.
E-mail addresses: zhangyingjie@sdu.edu.cn (Y. Zhang), xuwenf@sdu.edu.cn
activities in medicinal chemistry.22–26 (ii) The
R
group
(W. Xu).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.11.052

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F. Xu et al. / Bioorg. Med. Chem. 22 (2014) 1487–1495
nine structure of ATP and to play a role in the strong interaction
with the active site of protein kinases. The possible mechanism
for the antiangiogenic and antitumor activity was examined under
in vitro and ex vivo conditions.
2. Results and discussion
2.1. Chemistry
Target compounds 9a–9p were synthesized using the proce-
dures described in Scheme 1. First, hydroxyl group was introduced
into nitronaphthalene 1 in the presence of t-BuOOH and KOH.27,28
The nitro-group on compound 2 was then deoxidized to the amino
group by Pd-C/H2 in methanol solution to give compound 3,29
which was then treated with various substituted sulfonyl chlorides
to generate compounds 7a–7p.30 Then, compounds 7a–7p were
oxidated using NaIO4/SiO2 to obtain quinone type structures 8a–
8p.31 At last compounds 8a–8p took Michael addition reaction to
gain the target compounds 9a–9p.32 Both analytical and spectral
data for all compounds are in full agreement with the proposed
structures.
Figure 1. Strategy for the design of N-(3-((7H-purin-6-yl)thio)-4-hydroxynaphtha-
len-1-yl)-sulfonamides.
corresponding to the 2-nitrobenzene group of compound 6d can be
methyl or substituted aromatic fragments. (iii) The 1H-1,2,4-tria-
zole ring was substituted by 7H-purine structure to mimic the ade-
O
O
R
R
S
S
NO2
HN
NO2
NH2
OH
N
O
O
OH
S
O
O
a
b
c
d
e
N
S
N
R
N
H
OH
OH
7a 7p
O
N
HN
8a 8p
1
2
3
-
9a 9p
-
-
7a 9a
7g-9g
7h-9h
7l -9l
-
, R = phenyl
,
,
R = 1-naphthyl
,
R = 4-(trifluoromethyl)phenyl
, R = methyl
7b 9b
7m-9m
-
, R = 4-methoxyphenyl
R = quinoline-8-yl
7c-9c, R = 4-methylphenyl
7i -9i, R = 5-chlorothiophene-2-yl
7n-9n, R = 3,4-dimethoxyphenyl
7d-9d
7e-9e
7j 9j
7o-9o
7p-9p
, R = 4-chlorophenyl
, R = 3-chloro-4-fluorophenyl
-
,
R = 4-fluorophenyl
,
,
R = 5-(dimethylamino)-1-naphthyl
R = 4-(1,1'-biphenyl)
7k-9k
,
R = 2,6-dichlorophenyl
7f -9f, R = 4-(tert-butyl)phenyl
Scheme 1. Reagents and conditions: (a) t-BuOOH, KOH, DMSO/H2O(3/1); (b) Pd/C, H2, MeOH; (c) Pyridine, 0 °C; (d) NaIO4/SiO2, DCM, rt; (e) DMF, rt.
Table 1
Antiproliferative activities of compounds 9a–9p and sorafenib against HUVECs and three tumor cell lines
OH
N
N
O
S
O
S
N
N
H
R
HN
a
Compd.
R
IC50 (lM)
HUVECs
MDA-MB-231
HCT116
PC-3
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
Phenyl
4-Methoxyphenyl
4-Methylphenyl
7.34 0.55
10.24 0.48
8.72 0.65
6.29 0.41
5.43 0.26
15.42 0.65
6.73 0.35
7.33 0.46
9.04 0.27
10.44 0.82
5.56 0.37
6.39 0.32
8.41 0.67
4.05 0.43
9.53 0.35
10.76 0.64
6.42 1.13
8.06 0.11
3.92 0.31
7.66 0.32
5.8 0.34
3.77 0.77
86.35 2.88
4.25 0.14
3.68 0.47
14.95 0.60
5.97 0.56
4.2 0.65
15.06 0.31
32.79 0.14
2.69 0.09
24.79 0.86
32.57 1.21
14.62 0.96
6.51 0.72
7.59 0.44
6.69 0.21
7.54 0.42
4.68 0.18
27.04 0.86
9.52 0.36
7.26 0.21
8.63 0.96
10.06 0.41
4.75 0.36
10.77 0.78
7.46 0.89
3.32 0.14
8.25 1.05
10.97 0.54
8.08 0.86
44.27 0.53
41.45 0.40
88.72 3.93
31.55 2.34
57.38 0.75
>100
55.69 4.15
39.26 0.24
56.75 0.16
59.52 0.62
27.61 0.36
51.86 0.63
32.75 0.87
54.66 0.71
55.64 1.24
43.31 1.37
24.20 1.03
4-Chlorophenyl
3-Chloro-4-fluorophenyl
4-(tert-Butyl)phenyl
1-Naphthyl
Quinoline-8-yl
5-Chlorothiophene-2-yl
4-Fluorophenyl
2,6-Dichlorophenyl
4-(Trifluoromethyl)phenyl
Methyl
3,4-Dimethoxyphenyl
5-(Dimethylamino)-1-naphthyl
4-(1,10-Biphenyl)
9p
Sorafenib
a
Data represent the mean SD of two experiments.

F. Xu et al. / Bioorg. Med. Chem. 22 (2014) 1487–1495
1489
2.2. In vitro antiproliferative activity
activities (IC50 of HUVECs, MDA-MB-231 and HCT116 are
4.05 0.43/5.43 0.26, 2.69 0.09/3.77 0.77 and 3.32 0.14/
In vitro antiproliferative activity of the N-(4-hydroxy-3-merca-
ptonaphthalen-1-yl)amide derivatives against HUVECs, MDA-MB-
231 (breast cancer), HCT116 (colon cancer) and PC-3 (prostate can-
cer) cell lines were evaluated by MTT assay using sorafenib as a po-
sitive control. HUVECs were used to evaluate their in vitro
inhibitory effects on the proliferation of endothelial cells that are
closely related to angiogenesis, while MDA-MB-231, HCT116 and
PC-3 cell lines were used as target cancer cells. As is shown in Ta-
ble 1, most compounds exhibited enhanced antiproliferative activ-
4.68 0.18
synthesized compounds against PC-3 cells were morderate
(IC50 > 24.20 M). Comparing the data in Tables 1 and 2, we ob-
lM, respectively). The antiproliferative activity of all
l
tained some following basic structure–activity relationships
(SAR): (1) the sulfuryl structure are critical to the antiproliferative
activities. For instance, when the sulfuryl structure was replaced
by carbonyl, compound 9d almost lost its proliferative inhibition
activity (compared with 6r). Comparing compounds 9d, 6c and
6r, the replacement of 1H-1,2,4-triazole ring by 7H-purine did
not result in obviously decrease of inhibition efficacy, indicating
that the sulfonamide structure contributes even more to the inhi-
ities with low
l
M of IC50 values compared with previous work.21
Among them, compounds 9n and 9e showed the most potent
Table 2
Antiproliferative activities of previously reported compounds 6c, 6d and 6r against HUVECs and three tumor cell lines21
a
Compd.
Structure
IC50 (lM)
HUVECs
>100
MDA-MB-231
25.71 0.84
HCT116
PC-3
OH
S
O
N
N
6c
43.27 1.35
5.52 0.36
N
H
N
H
Cl
OH
N
NO2
O
6d
6r
5.34 0.76
21.37 0.72
10.19 0.65
52.52 1.15
N
N
N
H
S
N
H
OH
S
O
N
NDb
22.97 0.74
>100
66.43 3.14
N
H
N
HN
Cl
a
Data represent the mean SD of two experiments.
ND means not determined.
b
Figure 2. Representative images of the tube formation assay depicting the formation of a HUVEC capillary-like tubular network by treatment with synthesized compounds,
Pazopanib (positive control) and 0.5% DMSO (vehicle control). The scale bar indicates 200 m of length.
l

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F. Xu et al. / Bioorg. Med. Chem. 22 (2014) 1487–1495
bition efficacy than the 1H-1,2,4-triazole ring. (2) The size and
shape of R group may influence the proliferation inhibitory activi-
ties: (a). Compounds with plane geometrical conformation of R
group (such as phenyl and 1-naphthyl fragments) exhibited much
better inhibitory activities compared to the compounds with spa-
tial structures of R group (such as 4-(tert-butyl)phenyl and 4-
(1,10-biphenyl) groups). For example, compounds 9f and 9p
showed less inhibition activities than compounds 9a and 9g. It
was noteworthy that smaller substituent methyl (9m) exhibited
equivalent efficacy in proliferation inhibition as phenyl (9a). (b).
Disubstituted groups attached to the phenyl ring of R displayed
much more inhibitory performances than compounds with mono-
substituted groups (9e, 9k, 9d vs 9j, 9n, 9b).
2.3. In vitro HUVECs tuber formation assay
In order to find one agent possessing both cytotoxic activities
and cytostatic angiogenesis inhibition to tumors, we employed
in vitro HUVEC tube formation first as a model to investigate the
antiangiogenic activity of the most cytotoxic compounds 9n and
9e, as vascular endothelial tube network formed in vitro has many
similarities with capillary vessels formed in vivo.33,34 While the
HUVECs were plated, elongated and robust tube-like structures
were well established after incubation for 6 h in the negative con-
trol group; but treatment of HUVECs with compounds at 20
most inhibited the tuber formation completely (data not shown),
while at 10, 5 and 1 M suppressed the formation of tubular
lM al-
l
Figure 3. Representative images of rat aortic rings treated with synthesized compounds, Pazopanib (positive control) and 0.5% DMSO (vehicle control). The scale bar indicates
200 m of length.
l
Figure 4. Kinase inhibition (%) assay results at 10 lM for 9n. Values are the mean of two independent experiments performed in triplicate.

F. Xu et al. / Bioorg. Med. Chem. 22 (2014) 1487–1495
1491
mation model in view of multi-steps involved in angiogenesis:
sprouting, proliferation, migration and differentiation. As pre-
dicted, compound 9e having weak HUVECs tuber formation inhib-
itory efficacy still could not reduce microvessel outgrowth at
10 lM in the TARs assay, while compound 9n appeared to reduce
microvessel outgrowth in a concentration-dependent manner. As
we can see in Figure 3, compound 9n demonstrated almost com-
plete inhibition of angiogenesis at 5 lM, and comparable angio-
genesis inhibitory activity to pazopanib at 1 lM.
2.5. In vitro protein kinase assay
As many protein kinases are involved in oncogenic signaling,
the in vitro kinase assay was carried out to test the kinase inhibi-
tion activity of compound 9n at the concentration of 10 lM against
a panel of 22 angiogenesis associated protein kinases by measuring
the levels of phosphorylation of the kinase-specific ligand peptides
(Fig. 4). The pan-kinase inhibitor Staurosporine was used as a po-
sitive reference compound. As shown in Figure 4, compound 9n
showed weak or moderate kinase inhibitory activity to most of
the selected kinases, while potent efficacy to Akt1 and Abl kinases
Figure 5. IC50 curves for 9n and Staurosporine against Abl and Akt1 kinase. Dose–
response inhibition curves are shown and plotted as normalized percent activity.
(% inhibition was 92.1% to Akt1 and 76.2% to Abl at 10 lM, respec-
tively), indicating good kinase selectivity to Akt1 and Abl. To fur-
ther verify this conclusion, we evaluated the IC50 values of 9n
against Akt1 and Abl kinases. As shown in Figure 5, The IC50 values
of staurosporine were 0.13 lM against Akt1 and 0.21 lM against
Abl, meanwhile 9n exhibited favorable dose-dependent manner
structures in different ranks. Surprisingly, compound 9n exhibited
dose-dependent inhibition of tube formation which was compara-
ble to Pazopanib, the reference standard (see Fig. 2). But compound
9e showed no obvious tuber formation inhibitory activity even at
the concentration of 10 lM.
in inhibiting the phosphorylation of Akt1 and Abl kinases with
the IC50 values being 1.73 and 1.53 lM, respectively.
2.4. Ex vivo rat thoracic aorta rings (TARs) assay
2.6. Molecular modeling analysis
Ex vivo TARs assay was then employed to evaluate the antian-
giogenic activity of compounds 9n and 9e, for TARs model being
more close to in vivo condition compared with HUVEC tuber for-
In order to better understand the protein kinase and angio-
genesis inhibitory mechanism of synthesized compounds,
a
Figure 6. Molecular docking of synthesized compounds with target proteins. (a) Compound 9n with Akt1; (b) Compound 9n with Abl; (c) Compound 9d with Abl; (d)
Compound 6r with Abl. Hydrogen bonds were indicated by red dash lines.

1492
F. Xu et al. / Bioorg. Med. Chem. 22 (2014) 1487–1495
molecular docking study was performed by means of FlexX mod-
ule of SYBYL 8.0. Derivative 9n was identified as the most potent
compound among all prepared derivatives, and was taken as a
representative compound to study the N-(3-((7H-purin-6-
yl)thio)-4-hydroxynaphthalen-1-yl)-sulfonamide binding models
by docking into the ATP-binding pocket of the Akt1 (PDB ID:
3CQU) and Abl (PDB ID: 2HYY) kinase domains. According to
our docking simulation as shown in Figure 6a and b, hydrophobic
interaction and hydrogen bonds were characterized clearly be-
tween 9n and the ATP binding pocket. 9n was nicely bound to
the Akt1 binding domain (Fig. 6a), of which one methoxyl of R
group formd a hydrogen bond with the crucial residue Thr291
(2.0 Å). It was consistent with the result that compound 9n
showed more potency than any other methoxyl free compound
as an Akt1 kinase inhibitor. The –NH– and two –N@ moieties
of 7H-purine structure formed the other three hydrogen bonds
with Asp292 (2.1 Å), Ser7 (2.6 Å) and Lys276 (2.2 Å) residues,
respectively, contributing much to the kinase inhibition. In addi-
DMSO-d6 solutions. ESI-MS were determined on an API 4000
spectrometer.
4-Nitronaphthalen-1-ol (2) and 4-aminonaphthalen-1-ol (3)
were synthesized as described previously.21
4.1.1. General procedure for the preparation of 7a–7p
Under the condition of ice bath, 4-aminonaphthalen-1-ol 3
(1.59 g, 10 mmol) was dissolved in pyridine (50 mL). After substi-
tuted sulfonyl chloride (12 mmol) was added drop by drop, the
mixture was stirred at room temperature for 5 h (detected by
TLC). Solvents were evaporated with the residues being taken up
in EtOAc (50 mL). Then the organic layer was washed with satu-
rated 1 N HCl (20 mL ꢀ 2), distilled water (20 mL ꢀ 2), brine
(20 mL ꢀ 2), dried over anhydrous MgSO4 and evaporated under
vacuum. The residues were purified by EtOH/H2O to give desired
intermediate 7a–7p as white to yellow powder.
4.1.1.1.
(7a).
N-(4-Hydroxynaphthalen-1-yl)benzenesulfonamide
tion, a type of face to face aromatic–aromatic (
p–p
stacking)
Recrystallized from EtOH/H2O as a white crystal, yield:
interaction between the naphthalene structure of 9n and benzene
ring of Phe161 was observed, which may enhance the Akt1 bind-
ing affinity and stability of compound 9n. Figure 6b showed that
compound 9n penetrated deeply into the Abl ATP binding do-
main and formed significant hydrogen bonds with Asp381
(2.2 Å to –SO2–, 1.9 Å to –OH) and Thr315 (2.7 Å to –OMe of R
group), respectively. In order to compare the superposition of
sulfonamide moiety and previously reported amide structure
with the target proteins, compounds 9d and 6r were selected
to undergo further docking study. As shown in Figure 6, the sul-
fonamide moiety of 9d can form an hydrogen bond with MET-
318 of Abl kinase (Fig. 6c), while the amide structure of 6r
showed no obvious interactions with this kinase (Fig. 6d). The
computational data is consistent with the in vitro experimental
protein kinase inhibition results and supports the further investi-
gation of this class of compounds as potential Akt1 and Abl ki-
nase inhibitors.
88%; 1H NMR (DMSO-d6) d 6.71 (d, J = 8.08 Hz, 1H), 6.86 (d,
J = 8.08 Hz, 1H), 7.37–7.39 (m, 2H), 7.46–7.50 (m, 2H), 7.56 (d,
J = 7.36 Hz, 1H), 7.63 (d, J = 7.20 Hz, 2H), 7.85 (d, J = 7.64 Hz, 1H),
8.07 (d, J = 8.60 Hz, 1H), 9.82 (s, 1H), 10.28 (s, 1H).
4.1.1.2. N-(4-Hydroxynaphthalen-1-yl)-4-methoxybenzenesulf-
onamide (7b).
Recrystallized from EtOH/H2O as a white crys-
tal, yield: 91%; 1H NMR (DMSO-d6) d 3.78 (s, 3H), 6.70 (d,
J = 8.04 Hz, 1H), 6.84 (d, J = 8.08 Hz, 1H), 7.00 (d, J = 7.08 Hz, 2H),
7.38–7.40 (m, 2H), 7.55 (d, J = 7.08 Hz, 2H), 7.89–7.91 (m, 1H),
8.05–8.08 (m, 1H), 9.63 (s, 1H), 10.24 (s, 1H).
4.1.1.3. N-(4-Hydroxynaphthalen-1-yl)-4-methylbenzenesulf-
onamide (7c).
Recrystallized from EtOH/H2O as a white crys-
tal, yield: 74%; 1H NMR (DMSO-d6) d 2.33 (s, 3H), 6.70 (d,
J = 7.80 Hz, 1H), 6.83 (d, J = 7.80 Hz, 1H), 7.29 (d, J = 8.40 Hz, 1H),
7.38–7.42 (m, 2H), 7.51 (d, J = 7.80 Hz, 1H), 7.90 (dd, J = 7.80 Hz,
2.40 Hz, 1H), 8.07 (dd, J = 7.80 Hz, 2.40 Hz, 1H), 9.72 (s, 1H),
10.28 (s, 1H).
3. Conclusions
4.1.1.4. 4-Chloro-N-(4-hydroxynaphthalen-1-yl)benzenesulfon-
In conclusion, our studies have shown that N-(3-((7H-purin-6-
yl)thio)-4-hydroxynaphthalen-1-yl)-sulfonamide is a novel scaf-
fold possessing both anti-protein kinase and anti-angiogenesis
activities and conventional anticancer property for cancer treat-
ment. The SAR indicated that the sulfuryl structure and plane geo-
amide (7d).
Recrystallized from EtOH/H2O as a white crystal,
yield: 83%; 1H NMR (DMSO-d6) d 6.75 (d, J = 8.08 Hz, 1H), 6.89 (d,
J = 8.08 Hz, 1H), 7.39–7.42 (m, 2H), 7.57 (d, J = 8.68 Hz, 2H), 7.63
(d, J = 8.68 Hz, 2H), 7.85–7.87 (m, 1H), 8.08–8.11 (m, 1H), 9.93 (s,
1H), 10.33 (s, 1H).
metrical conformation of
R
group are critical to the
antiproliferative activities. The results of kinase assay and molecu-
lar docking study exhibited that 7H-purine structure of synthe-
sized compounds can form special interaction with Akt1 and Abl
kinases and contributed much to the inhibition of the two kinases.
Among all the compounds, 9n comprehensively showed strong
inhibitory effect, which deserved further in vitro and in vivo stud-
ies on its mechanism of anti-cancer therapy.
4.1.1.5. 3-Chloro-4-fluoro-N-(4-hydroxynaphthalen-1-yl)ben-
zenesulfonamide (7e).
Recrystallized from EtOH/H2O as a
6.76 (d,
white crystal, yield: 78%; 1H NMR (DMSO-d6)
d
J = 8.08 Hz, 1H), 6.88 (d, J = 8.08 Hz, 1H), 7.41–7.43 (m, 2H), 7.55–
7.60 (m, 2H), 7.75 (dd, J = 6.80 Hz, 2.04 Hz, 1H), 7.83–7.85 (m,
1H), 8.09–8.11 (m, 1H), 9.99 (s, 1H), 10.36 (s, 1H).
4.1.1.6. 4-(tert-Butyl)-N-(4-hydroxynaphthalen-1-yl)benzene-
4. Experimental section
4.1. Chemistry
sulfonamide (7f).
Recrystallized from EtOH/H2O as a white
crystal, yield: 85%; 1H NMR (DMSO-d6) d 1.23 (s, 3H), 1.24 (s,
3H), 1.27 (s, 3H), 6.72 (d, J = 8.08 Hz, 1H), 6.92 (d, J = 8.08 Hz,
1H), 7.30–7.37 (m, 2H), 7.47 (d, J = 8.60 Hz, 2H), 7.53 (d,
J = 8.60 Hz, 2H), 7.75 (d, J = 8.32 Hz, 1H), 8.06 (d, J = 8.08 Hz, 1H),
9.73 (s, 1H), 10.25 (s, 1H).
Unless otherwise noted, all solvents and reagents were com-
mercially available and used without further purification. All reac-
tions were monitored by thin-layer chromatography on 0.25 mm
silica gel plates (60 GF-254) and visualized with UV light, or iodine
vapor. Proton NMR spectrums were determined on a Brucker DRX
spectrometer (600/300 MHz), d in parts per million and J in Hertz,
using TMS as an internal standard. Measurements were made in
4.1.1.7.
N-(4-Hydroxynaphthalen-1-yl)naphthalene-1-sulfon-
amide (7g).
Recrystallized from EtOH/H2O as a white crystal,
yield: 81%; 1H NMR (DMSO-d6) d 6.61 (d, J = 8.40 Hz, 1H), 6.73 (d,
J = 7.80 Hz, 1H), 7.19 (t, J = 6.60 Hz, 1H), 7.32 (t, J = 6.60 Hz, 1H),
7.47 (t, J = 7.80 Hz, 1H), 7.67 (t, J = 7.80 Hz, 1H), 7.71 (d,

F. Xu et al. / Bioorg. Med. Chem. 22 (2014) 1487–1495
1493
J = 7.80 Hz, 1H), 7.77 (d, J = 8.40 Hz, 1H), 7.89 (dd, J = 7.20 Hz,
1.2 Hz, 1H), 8.01 (d, J = 8.40 Hz, 1H), 8.07 (d, J = 7.80 Hz, 1H), 8.16
(d, J = 8.40 Hz, 1H), 8.81 (d, J = 8.40 Hz, 1H), 10.10 (s, 1H), 10.22
(s, 1H).
4.1.1.16. N-(4-Hydroxynaphthalen-1-yl)-[1,10-biphenyl]-4-sul-
fonamide (7p).
Recrystallized from EtOH/H2O as a white
crystal, yield: 66%; 1H NMR (DMSO-d6) d 6.72 (d, J = 7.80 Hz, 1H),
6.91 (d, J = 7.80 Hz, 1H), 7.37–7.40 (m, 2H), 7.41–7.44 (m, 1H),
7.49 (t, J = 7.20 Hz, 2H), 7.70 (d, J = 8.40 Hz, 4H), 7.80 (d,
J = 8.40 Hz, 2H), 7.89 (dd, J = 7.20 Hz, 1.80 Hz, 1H), 8.07 (dd,
J = 7.20 Hz, 1.80 Hz, 1H), 9.85 (s, 1H), 10.27 (s, 1H).
4.1.1.8. N-(4-Hydroxynaphthalen-1-yl)quinoline-8-sulfonamide
(7h).
Recrystallized from EtOH/H2O as a white crystal, Yield:
68%; 1H NMR (DMSO-d6) d 6.58 (d, J = 8.08 Hz, 1H), 6.64 (d,
J = 8.08 Hz, 1H), 7.30 (d, J = 8.08 Hz, 1H), 7.28–7.31 (m, 1H), 7.34–
7.38 (m, 1H), 7.60 (t, J = 7.76 Hz, 1H), 7.76–7.79 (m, 1H), 8.05 (d,
J = 8.60 Hz, 2H), 8.11 (d, J = 6.76 Hz, 1H), 8.24 (d, J = 8.20 Hz, 1H),
8.56 (dd, J = 8.32 Hz, 1.36 Hz, 1H), 9.24 (s, 1H), 9.62 (s, 1H), 10.21
(s, 1H).
4.1.2. General Procedure for the Preparation of 8a–8p
To a solution of compounds 7a–7p (10.0 mmol) in 200 mL of
CH2Cl2, was added 22 g (15 mmol) of NaIO4/SiO2(0.68 mmol of
NaIO4/1 g of SiO2).31 After stirring at room temperature for 2 h,
the solid particles were filtered from the solution and washed with
20 mL of CH2Cl2, and the filtrates were evaporated in vacuum to
obtain reddish-brown crude compounds 8a–8p which was directly
used for the subsequent reaction without further purification.
4.1.1.9.
5-Chloro-N-(4-hydroxynaphthalen-1-yl)thiophene-2-
sulfonamide (7i).
Recrystallized from EtOH/H2O as a white
crystal, yield: 77%; 1H NMR (DMSO-d6) d 6.79 (d, J = 8.08 Hz, 1H),
7.01 (d, J = 8.08 Hz, 1H), 7.14 (d, J = 4.00 Hz, 1H), 7.23 (d,
J = 4.00 Hz, 1H), 7.42–7.45 (m, 2H), 7.85–7.88 (m, 1H), 8.09–8.12
(m, 1H), 10.17 (s, 1H), 10.38 (s, 1H).
4.1.3. General procedure for the preparation of 9a–9p
To a solution of 7H-purine-6-thiol (3.80 g, 25 mmol) in 10 mL of
DMF, was added 10 mmol of compounds 8a–8p, slowly. After stir-
ring at room temperature for 4 h, the solution was poured into
100 mL of distilled water with slight yellow solid precipitation.
After standing for 20 min the slight yellow solid was filtered with
suction, pressed to remove excess water, and allowed to dry in air.
The dry residues were recrystallized with MeOH to give title com-
pounds 9a–9p as white or slight yellow powder.
4.1.1.10.
4-Fluoro-N-(4-hydroxynaphthalen-1-yl)benzenesul-
fonamide (7j).
Recrystallized from EtOH/H2O as a white crys-
tal, yield: 94%; 1H NMR (DMSO-d6) d 6.74 (d, J = 8.08 Hz, 1H), 6.87
(d, J = 8.08 Hz, 1H), 7.30–7.35 (m, 2H), 7.38–7.41 (m, 2H), 7.65–
7.68 (m, 2H), 7.82–7.85 (m, 1H), 8.07–8.10 (m, 1H), 9.89 (s, 1H),
10.31 (s, 1H).
4.1.3.1.
yl)benzenesulfonamide (9a).
white crystal, yield: 74%; 1H NMR (DMSO-d6)
d
J = 7.80 Hz, 1H), 7.20 (d, J = 8.40 Hz, 1H), 7.27–7.31 (m, 2H), 7.50–
7.53 (m, 2H), 7.63–7.67 (m, 3H), 7.90 (d, J = 7.80 Hz, 1H), 7.95 (s,
1H), 8.16 (d, J = 7.20 Hz, 1H), 8.30 (s, 1H), 8.61 (s, 1H), 10.50 (s,
1H), 13.76 (s, 1H). LC–MS (ESI) 450.4 [M+H]+. HRMS (AP-ESI) m/z
calcd for C21H15N5O3S2 [M+H]+ 450.0695, found 450.0651.
N-(3-((7H-Purin-6-yl)thio)-4-hydroxynaphthalen-1-
Recrystallized from MeOH as
7.14 (d,
4.1.1.11. 2,6-Dichloro-N-(4-hydroxynaphthalen-1-yl)benzene-
sulfonamide (7k).
Recrystallized from EtOH/H2O as a white
a
crystal, yield: 80%; 1H NMR (DMSO-d6) d 6.73 (d, J = 8.08 Hz, 1H),
6.92 (d, J = 8.08 Hz, 1H), 7.43–7.47 (m, 2H), 7.48–7.52 (m, 1H),
7.57–7.59 (m, 2H), 8.01 (d, J = 7.44 Hz, 1H), 8.11 (d, J = 7.44 Hz,
1H), 10.30 (s, 1H), 10.37 (s, 1H).
4.1.1.12.
methyl)benzenesulfonamide
N-(4-Hydroxynaphthalen-1-yl)-4-(trifluoro-
(7l). Recrystallized from
4.1.3.2. N-(3-((7H-Purin-6-yl)thio)-4-hydroxynaphthalen-1-yl)-
EtOH/H2O as a white crystal, yield: 90%; 1H NMR (DMSO-d6) d
6.76 (d, J = 8.08 Hz, 1H), 6.91 (d, J = 8.08 Hz, 1H), 7.36–7.41 (m,
2H), 7.79 (d, J = 8.00 Hz, 1H), 7.82–7.84 (m, 2H), 7.88–7.90 (m,
2H), 8.10 (d, J = 8.00 Hz, 1H), 10.10 (s, 1H), 10.35 (s, 1H).
4-methoxybenzenesulfonamide (9b).
Recrystallized from
MeOH as a white crystal, yield: 81%; 1H NMR (DMSO-d6) d 3.59
(s, 3H), 6.75 (d, J = 9.00 Hz, 1H), 6.84 (d, J = 7.20 Hz, 2H), 7.44 (d,
J = 9.00 Hz, 1H), 7.50 (d, J = 8.40 Hz, 1H), 7.62 (d, J = 9.00 Hz, 2H),
7.72 (s, 1H), 7.99 (d, J = 8.40 Hz, 1H), 8.19 (d, J = 7.80 Hz, 1H),
8.30 (s, 1H), 8.61 (s, 1H), 10.39 (s, 1H), 13.72 (s, 1H). LC–MS (ESI)
480.4 [M+H]+. HRMS (AP-ESI) m/z calcd for C22H17N5O4S2 [M+H]+
480.0800, found 480.0816.
4.1.1.13.
(7m).
N-(4-Hydroxynaphthalen-1-yl)methanesulfonamide
Recrystallized from EtOH/H2O as a white crystal, Yield:
77%; 1H NMR (DMSO-d6) d 3.59 (s, 3H), 7.55–7.60 (m, 2H), 7.69–
7.71 (m, 2H), 8.14–8.16 (m, 1H), 8.30–8.31 (m, 1H), 9.86 (s, 1H),
10.30 (s, 1H).
4.1.3.3. N-(3-((7H-Purin-6-yl)thio)-4-hydroxynaphthalen-1-yl)-
4-methylbenzenesulfonamide (9c).
Recrystallized from
4.1.1.14.
zenesulfonamide (7n).
N-(4-Hydroxynaphthalen-1-yl)-3,4-dimethoxyben-
Recrystallized from EtOH/H2O as a
MeOH as a white crystal, yield: 83%; 1H NMR (DMSO-d6) d 2.73
(s, 3H), 7.37 (d, J = 7.80 Hz, 1H), 7.41 (d, J = 8.40 Hz, 1H), 7.52 (d,
J = 7.80 Hz, 1H), 7.5–7.59 (m, 2H), 7.96 (s, 1H), 8.07 (d,
J = 7.80 Hz, 1H), 8.13 (d, J = 9.00 Hz, 1H), 8.19–8.22 (m, 2H), 8.23
(d, J = 8.40 Hz, 1H), 8.47 (s, 1H), 13.64 (s, 1H), 13.76 (s, 1H). LC–
MS (ESI) 464.3 [M+H]+. HRMS (AP-ESI) m/z calcd for C22H17N5O3S2
[M+H]+ 464.0851, found 464.0863.
white crystal, yield: 86%; 1H NMR (DMSO-d6) d 3.64 (s, 3H), 3.77
(s, 3H), 6.73 (d, J = 8.08 Hz, 1H), 6.87 (d, J = 8.08 Hz, 1H), 7.00 (d,
J = 8.48 Hz, 1H), 7.11 (s, 1H), 7.18 (dd, J = 8.40 Hz, 2.00 Hz, 1H),
7.39–7.41 (m, 2H), 7.92–7.94 (m, 1H), 8.07–8.09 (m, 1H), 9.62 (s,
1H), 10.25 (s, 1H).
4.1.1.15.
5-(Dimethylamino)-N-(4-hydroxynaphthalen-1-
Recrystallized from
4.1.3.4. N-(3-((7H-Purin-6-yl)thio)-4-hydroxynaphthalen-1-yl)-
yl)naphthalene-1-sulfonamide (7o).
4-chlorobenzenesulfonamide (9d).
Recrystallized from
EtOH/H2O as a white crystal, yield: 89%; 1H NMR (DMSO-d6) d
2.81 (s, 6H), 6.61 (d, J = 7.80 Hz, 1H), 6.75 (d, J = 8.40 Hz, 1H),
7.18 (t, J = 8.40 Hz, 1H), 7.26 (d, J = 7.20 Hz, 1H), 7.32 (t,
J = 7.80 Hz, 1H), 7.43 (t, J = 8.40 Hz, 1H), 7.60 (t, J = 7.80 Hz, 1H),
7.74 (d, J = 8.40 Hz, 1H), 7.87 (d, J = 7.20 Hz, 1H), 8.00 (d,
J = 8.40 Hz, 1H), 8.39 (d, J = 8.40 Hz, 1H), 8.48 (d, J = 8.40 Hz, 1H),
10.04 (s, 1H), 10.20 (s, 1H).
MeOH as a white crystal, yield: 72%; 1H NMR (DMSO-d6) d 6.89
(s, 1H), 7.44 (d, J = 7.80 Hz, 2H), 7.54–7.57 (m, 1H), 7.62 (d,
J = 9.00 Hz, 1H), 7.67 (d, J = 7.80 Hz, 2H), 8.01 (d, J = 9.00 Hz, 1H),
8.24 (d, J = 7.80 Hz, 1H), 8.41 (s, 1H), 8.49 (d, J = 8.40 Hz, 1H),
10.05 (s, 1H), 10.23 (s, 1H), 13.63 (s, 1H), 13.76 (s, 1H). LC–MS
(ESI) 484.3 [M+H]+. HRMS (AP-ESI) m/z calcd for C21H14ClN5O3S2
[M+H]+ 484.0305, found 484.0318.

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F. Xu et al. / Bioorg. Med. Chem. 22 (2014) 1487–1495
4.1.3.5. N-(3-((7H-Purin-6-yl)thio)-4-hydroxynaphthalen-1-yl)-
3-chloro-4-fluorobenzenesulfonamide (9e). Recrystallized
J = 7.80 Hz, 1H), 7.61 (d, J = 8.40 Hz, 1H), 7.95 (s, 1H), 8.12 (d,
J = 8.40 Hz, 1H), 8.41 (s, 1H), 10.26 (s, 1H), 10.48 (s, 1H), 13.55 (s,
1H). LC–MS (ESI) 517.4 [M+H]+. HRMS (AP-ESI) m/z calcd for C21-
H13Cl2N5O3S2 [M+H]+ 517.9915, found 517.9936.
from MeOH as a white crystal, yield: 63%; 1H NMR (DMSO-d6) d
7.35 (s, 1H), 7.43 (s, 1H), 7.46 (d, J = 8.40 Hz, 1H), 7.54 (d,
J = 7.80 Hz, 1H), 7.56 (d, J = 7.80 Hz, 2H), 7.97 (d, J = 7.80 Hz, 1H),
8.08 (d, J = 7.20 Hz, 1H), 8.23–8.26 (m, 1H),8.42 (d, J = 8.40 Hz,
1H), 10.12 (s, 1H), 13.58 (s, 1H), 13.76 (s, 1H). LC–MS (ESI) 502.3
[M+H]+. HRMS (AP-ESI) m/z calcd for C21H13ClFN5O3S2 [M+H]+
502.0211, found 502.0223.
4.1.3.12.
N-(3-((7H-purin-6-yl)thio)-4-hydroxynaphthalen-1-
Recrystal-
yl)-4-(trifluoromethyl)benzenesulfonamide (9l).
lized from MeOH as a white crystal, yield: 87%; 1H NMR (DMSO-
d6) d 7.51–7.53 (m, 1H), 7.55–7.58 (m, 1H), 7.74 (d, J = 8.40 Hz,
2H), 7.84 (d, J = 7.80 Hz, 1H), 7.89 (d, J = 7.20 Hz, 1H), 7.96 (s,
1H), 8.00 (dd, J = 6.00 Hz, 2.40 Hz, 1H), 8.24–8.26 (m, 1H), 8.51 (s,
1H), 10.18 (s, 1H), 10.28 (s, 1H), 13.59 (s, 1H). LC–MS (ESI) 518.3
[M+H]+. HRMS (AP-ESI) m/z calcd for C22H14F3N5O3S2 [M+H]+
518.0568, found 518.0374.
4.1.3.6. N-(3-((7H-Purin-6-yl)thio)-4-hydroxynaphthalen-1-yl)-
4-(tert-butyl)benzenesulfonamide (9f).
Recrystallized from
MeOH as a white crystal, yield: 84%; 1H-NMR (DMSO-d6) d 1.06
(s, 9H), 7.31 (d, J = 8.40 Hz, 1H), 7.41 (s, 1H), 7.48–7.50 (m, 2H),
7.57 (d, J = 8.40 Hz, 2H), 7.64 (d, J = 7.80 Hz, 1H), 8.12 (d,
J = 7.80 Hz, 1H), 8.26 (s, 1H), 10.41 (s, 1H), 13.78 (s, 1H), 13.96 (s,
1H). LC–MS (ESI) 506.4 [M+H]+. HRMS (AP-ESI) m/z calcd for C25-
H23N5O3S2 [M+H]+ 506.1321, found 506.1348.
4.1.3.13.
yl)methanesulfonamide (9m).
N-(3-((7H-Purin-6-yl)thio)-4-hydroxynaphthalen-1-
Recrystallized from MeOH as
a white crystal, yield: 80%; 1H NMR (DMSO-d6) d 2.74 (s, 3H),
7.40–7.43 (m, 2H), 7.55–7.59 (m, 1H), 7.85 (d, J = 7.80 Hz, 1H),
7.96 (s, 1H), 8.06 (d, J = 7.80 Hz, 1H), 8.67 (m, 1H), 9.95 (s, 1H),
10.34 (s, 1H), 13.64 (s, 1H). LC–MS (ESI) 388.4 [M+H]+. HRMS
(AP-ESI) m/z calcd for C16H13N5O3S2 [M+H]+ 388.0538, found
388.0593.
4.1.3.7.
N-(3-((7H-Purin-6-yl)thio)-4-hydroxynaphthalen-1-
Recrystallized from
yl)naphthalene-1-sulfonamide (9g).
MeOH as a white crystal, yield: 73%; 1H NMR (DMSO-d6) d 6.86
(s, 1H), 7.37–7.41 (m, 2H), 7.45–7.49 (m, 1H), 7.62 (t, J = 7.60 Hz,
1H), 7.92–7.95 (m, 2H), 8.00 (d, J = 8.00 Hz, 1H), 8.07 (d,
J = 8.24 Hz, 1H), 8.19 (d, J = 8.240 Hz, 1H), 8.47 (s, 1H), 8.53 (s,
1H), 8.82 (d, J = 8.60 Hz, 1H), 10.29 (s, 1H). LC–MS (ESI) 500.3
[M+H]+. HRMS (AP-ESI) m/z calcd for C25H17N5O3S2 [M+H]+
500.0851, found 500.0837.
4.1.3.14.
N-(3-((7H-Purin-6-yl)thio)-4-hydroxynaphthalen-1-
Recrystallized
yl)-3,4-dimethoxybenzenesulfonamide (9n).
from MeOH as a white crystal, yield: 73%; 1H NMR (DMSO-d6) d
2.73(s, 3H), 2.89(s, 3H), 6.86 (d, J = 7.20 Hz, 1H), 6.92 (s, 1H), 7.27
(d, J = 8.40 Hz, 1H), 7.64 (d, J = 8.40 Hz, 1H), 8.04–8.07 (m, 1H),
8.25 (t, J = 7.80 Hz, 2H), 8.44–8.49 (m, 2H), 9.72 (s, 1H), 10.16 (s,
1H), 13.58 (s, 1H), 13.76 (s, 1H). LC–MS (ESI) 510.3 [M+H]+. HRMS
(AP-ESI) m/z calcd for C23H19N5O5S2 [M+H]+ 510.0906, found
510.0915.
4.1.3.8.
N-(3-((7H-Purin-6-yl)thio)-4-hydroxynaphthalen-1-
Recrystallized from MeOH
yl)quinoline-8-sulfonamide (9h).
as a white crystal, yield: 78%; 1H NMR (DMSO-d6) d 6.78 (s, 1H),
7.42–7.50 (m, 2H), 7.56 (t, J = 7.74 Hz, 1H), 7.68–7.71 (m, 1H),
8.11–8.19 (m, 4H), 8.38 (s, 1H), 8.44 (dd, J = 8.40 Hz, 1.64 Hz, 1H),
8.49 (s, 1H), 9.16-9.18 (m, 1H), 9.78 (s, 1H), 10.09 (s, 1H), 13.55
(s, 1H). LC–MS (ESI) 501.3 [M+H]+. HRMS (AP-ESI) m/z calcd for C24-
H16N6O3S2 [M+H]+ 501.0804, found 501.0787.
4.1.3.15.
yl)-5-(dimethylamino)naphthalene-1-sulfonamide
(9o). Recrystallized from MeOH as a white crystal, yield: 79%;
N-(3-((7H-Purin-6-yl)thio)-4-hydroxynaphthalen-1-
1H NMR (DMSO-d6) d 2.69 (s, 6H), 7.20 (d, J = 7.20 Hz, 1H), 7.42–
7.45 (m, 2H), 7.59 (d, J = 7.20 Hz, 1H), 7.74 (t, J = 8.40 Hz, 1H),
7.96 (d, J = 7.20 Hz, 1H), 8.02 (d, J = 8.40 Hz, 1H), 8.17 (d,
J = 7.80 Hz, 1H), 8.24 (d, J = 8.40 Hz, 1H), 8.39 (d, J = 7.80 Hz, 1H),
8.46 (d, J = 8.40 Hz, 1H), 10.16 (s, 1H), 10.76 (s, 1H), 13.56 (s, 1H),
13.75 (s, 1H). LC–MS (ESI) 543.4 [M+H]+. HRMS (AP-ESI) m/z calcd
for C27H22N6O3S2 [M+H]+ 543.1273, found 543.1264.
4.1.3.9. N-(3-((7H-Purin-6-yl)thio)-4-hydroxynaphthalen-1-yl)-
5-chlorothiophene-2-sulfonamide (9i).
Recrystallized from
MeOH as a white crystal, yield: 69%; 1H NMR (DMSO-d6) d 7.04
(d, J = 4.20 Hz, 1H), 7.07 (s, 1H), 7.27 (d, J = 4.20 Hz, 1H), 7.56–
7.59 (m, 2H), 8.03 (d, J = 7.80 Hz, 1H), 8.27 (d, J = 8.40 Hz, 1H),
8.50 (s, 1H), 8.52 (s, 1H), 10.30 (s, 1H), 10.33 (s, 1H), 13.60 (s,
1H). 13C NMR (150 MHz, DMSO-d6) d: 105.01, 123.16, 123.30,
123.43, 125.67, 127.84, 127.90, 130.08, 132.37, 132.77, 134.90,
138.33, 143.44, 149.79, 151.61, 155.97, 158.07. LC–MS (ESI)
490.3 [M+H]+. HRMS (AP-ESI) m/z calcd for C19H12ClN5O3S3
[M+H]+ 489.9869, found 489.9868.
4.1.3.16.
N-(3-((7H-Purin-6-yl)thio)-4-hydroxynaphthalen-1-
Recrystallized from
yl)-[1,10-biphenyl]-4-sulfonamide (9p).
MeOH as a white crystal, yield: 80%; 1H-NMR (DMSO-d6) d 7.35
(s, 1H), 7.43–7.47 (m, 2H), 7.49 (d, J = 8.40 Hz, 1H), 7.58–7.63 (m,
4H), 7.72 (d, J = 8.40 Hz, 1H), 7.99 (d, J = 8.40 Hz, 1H), 8.15 (d,
J = 8.40 Hz, 1H), 8.20 (s, 1H), 8.25 (d, J = 8.40 Hz, 1H), 8.48 (s, 1H),
10.11 (s, 1H), 13.61 (s, 1H), 13.77 (s, 1H). LC–MS (ESI) 526.4
[M+H]+. HRMS (AP-ESI) m/z calcd for C27H19N5O3S2 [M+H]+
526.1008, found 526.1027.
4.1.3.10.
N-(3-((7H-Purin-6-yl)thio)-4-hydroxynaphthalen-1-
Recrystallized from
yl)-4-fluorobenzenesulfonamide (9j).
MeOH as a white crystal, yield: 76%; 1H NMR (DMSO-d6) d 7.01
(s, 1H), 7.26 (t, J = 8.72 Hz, 2H), 7.55–7.61 (m, 2H), 7.75 (t,
J = 6.84 Hz, 2H), 8.06 (d, J = 8.48 Hz, 1H), 8.29 (d, J = 7.36 Hz, 1H),
8.55 (s, 2H), 10.02 (s, 1H), 10.29 (s, 1H), 13.63 (s, 1H). 13C NMR
(100 MHz, DMSO-d6) d: 105.59, 116.39, 116.61, 123.53, 123.93,
124.37, 126.15, 126.36, 128.12, 130.38, 130.47, 132.66, 133.12,
136.41, 136.44, 152.05, 156.02, 163.37, 165.87. LC–MS (ESI)
468.3 [M+H]+. HRMS (AP-ESI) m/z calcd for C21H14FN5O3S2
[M+H]+ 468.0600, found 468.0603.
4.2. MTT assay
HUVECs, PC-3, HCT116 and MDA-MB-231 cells were, respec-
tively, grown in RPMI 1640 medium containing 10% FBS at 37 °C
in 5% CO2 humidified incubator. Cell proliferation was determined
by the MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetra-
zolium bromide) assay. Briefly, cells were plated in a 96-well plate
at 5000 cells per well, cultured for 4 h in complete growth med-
ium, then treated with serial dilutions of the compounds for
4.1.3.11.
N-(3-((7H-Purin-6-yl)thio)-4-hydroxynaphthalen-1-
yl)-2,6-dichlorobenzenesulfonamide
(9k).
Recrystallized
from MeOH as a white crystal, yield: 65%; 1H NMR (DMSO-d6) d
7.05 (s, 1H), 7.38–7.42 (m, 1H), 7.50 (d, J = 8.40 Hz, 1H), 7.57 (d,
48 h. During the last 4 h, 10 lL of 0.5% MTT solution was added

F. Xu et al. / Bioorg. Med. Chem. 22 (2014) 1487–1495
1495
to each well. After further incubation for 4 h, formazan formed
from MTT was extracted by adding 200 L of DMSO and mixed
Acknowledgments
l
for 15 min. Optical density was read with an ELISA reader at
570 nm.
This work was supported by National Nature Science Founda-
tion of China (Grant No. 21172134), National Science and Technol-
ogy Major Project (Grant No. 2011ZX09401-015), Academic Award
for Young Doctoral Postgraduate of Ministry of Education of China
(Grant No. 20110131110037) and Independent Innovation Foun-
dation of Shandong University, IIFSDU (Grant No. 2013GN013).
4.3. In vitro HUVEC tuber formation assay
Forty eight-well slide chambers were coated with 100 lL of
Matrigel (BD Biosciences, NJ) and allowed to gel at 37 °C and 5%
CO2 for 30 min. HUVECs were then seeded at 40,000 cells/well in
M199 (5% FBS) containing either the vehicle (0.5% DMSO), Pazopa-
nib or synthesized compounds and incubated at 37 °C and 5% CO2
for 6 h. The morphological changes of the cells and HUVEC tubes
formation were observed under a phase-contrast microscope and
photographed at 200ꢀ magnification. The corresponding area
was measured as the number of pixels using Adobe PhotoShop.35,36
Experiments were repeated three times.
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ˇ
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