Bioorganic & Medicinal Chemistry Letters 24 (2014) 2324–2328

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Synthesis and biological evaluation of compounds which contain pyrazole, thiazole and naphthalene ring as antitumor agents Ji-Wen Yuan  , She-Feng Wang  , Zhong-Liang Luo, Han-Yue Qiu, Peng-Fei Wang, Xin Zhang, Yong-An Yang, Yong Yin, Fei Zhang, Hai-Liang Zhu ⇑ State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China

a r t i c l e

i n f o

Article history: Received 9 January 2014 Revised 5 March 2014 Accepted 24 March 2014 Available online 3 April 2014 Keywords: EGFR inhibitors Anti-tumor activity Cell migration Structure–activity relationship

a b s t r a c t A series of compounds which contain pyrazole, thiazole and naphthalene ring (1a–7a, 1b–7b, 1c–7c, 1d–7d) were firstly synthesized and their anti-proliferative activity, EGFR inhibitory activity, cytotoxicity and inhibition to Hela cell migration were evaluated. Compound 2-(3-(3,4-dimethylphenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4(5H)-one (7d) displayed the most potent inhibitory activity (IC50 = 0.86 lM for Hela and IC50 = 0.12 lM for EGFR). Structure–activity relationship (SAR) analysis showed that the anti-proliferative activity was affected by A-ring-substituent (–OCH3 > –CH3 > –H > –Br > –Cl > –F). Docking simulation of compound 7d into EGFR active site showed that naphthalene ring of 7d with LYS721 formed two p–p bonds, which enhanced antitumor activity. Therefore, compound 7d may be developed as a potential antitumor agent. Ó 2014 Elsevier Ltd. All rights reserved.

Pyrazole ring was a prominent skeleton motif in a lot of pharmaceutically active compounds, and pyrazole derivatives possess a wide range of biological activities, such as anti-inflammatory,1 antifungal,2,3 antimicrobial,4,5 anticoagulants,6 analgesic,7 antithrombolic,8 anti-tumor activity,9,10 and so on. A series of pyrazoleoxime ether derivatives were prepared and examined as cytotoxic agents, among which 5-phenoxypyrazole exhibited very potent cytotoxicity against XF 498 and HCT15.9 While 1-arylmethyl-3aryl-1H-pyrazole-5-carbohydrazide derivatives were showed inhibitory effects on the growth of A549 cells.10 Thiazole and its derivatives were widely used in pesticides and medicine. For example, 4-(4-chlorophenyl)-2-(3-(3,4-dimethylphenyl)-5-p-tolyl-4,5-dihydro-1H-pyrazol-1-yl) thiazole displayed the potent EGFR TK inhibitory activity and anti-proliferative activity against MCF-7,11 4-substituted methoxybenzoyl-aryl-thiazoles exhibited anti-proliferative activity of against melanoma and prostate cancer cells from lM to nM range.12 Besides, thiazole and its derivatives also showed other biological activities in antimicrobial,5 antifungal,13 antifilarial,14 anti-inflammatory,15 antiviral,16 and so on. Compounds containing naphthalene ring displayed potent bioactivity in anti-arrhythmia,17 anti-tumor18 and antioxidant.19 Due to potent biological activities and low toxicities,20,21,4,22–25 we designed and synthesized a series of compounds containing ⇑ Corresponding author. Tel.: +86 25 83592572; fax: +86 25 83592672.  

E-mail address: [email protected] (H.-L. Zhu). These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.bmcl.2014.03.072 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

pyrazole, thiazole and naphthalene ring and evaluated their antiproliferative activity. As everyone knows, EGFR (epidermal growth factor receptor) plays a very important role in cell proliferation, survival, migration, differentiation and metastasis of many tumors. EGFR was often used as targets for the development of novel anticancer agents, such as Gefitinib and Elotinib.26,27 To further understand the antitumor effect of designed compounds, docking simulation was performed to position compound 7d into the EGFR active site to determine the probable binding model. The synthetic procedures of compounds 1a–7a, 1b–7b, 1c–7c and 1d–7d were outlined in Scheme 1, Tables 1 and 2 and their structures were confirmed by one 1H NMR and MS. The structures of compounds 1d and 5d were demonstrated by X-ray diffraction analysis. The crystal data were presented in Table 3, Figures 1 and 2 and have been deposited at the Cambridge Crystallographic Data Centre as supplementary publication Nos. 987465 and 978466. The title compounds were evaluated for their ability to inhibit cell proliferation against Hela, BGC823 and HepG2 cell lines using CCK8 assay. The results in Table 4 showed good anti-proliferative activities on Hela cancer cell line (IC50 values between 0.86 and 12.35 lM). Among them, compound 7d showed the most potent inhibiting activity with IC50 0.86 lM. SAR studies were carried out to determine how compounds affected the anti-proliferative activity. Firstly, compound 7d (IC50 = 0.86 lM) with two -CH3 in A-ring displayed stronger anti-proliferative activity than those with one -OCH3 (5d,

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O S

S

N N N A

A

R1

1c-7c

R1

1d-7d

R2

Br

R2

O

A R1

O

i

NH2 A

ii

2

R

R1 R2

CHO

A

NH2

N

N

S N H

O

iii S

O H2N

Br

O

iv

CHO

N

N N

S

R1

O

R2

S i

H2N

A

N H

N

NH2

N

NH2

R1 ii

R2

A

R1

R2 O O

Br

O

O iii

iv

S

S

N N

N

N N

A R

N

A

R1 2

Br

R1

1b-7b

1a-7a

R2

Scheme 1. General synthesis compounds 1a–7a, 1b–7b, 1c–7c, 1d–7d. Reagents and conditions: (i) 40% aqueous KOH solution, ethanol, rt; (ii) thiosemicarbazide, KOH, ethanol, reflux; (iii or iv) bromoacetic acid (or 2-bromo-1-phenylethanone), acetic anhydride, sodium acetate, acetic acid, 80 °C, 7–9 h.

Table 1 Chemical structure of compounds 1a–7a, 1c–7c

Table 2 Chemical structure of compounds 1b–7b, 1d–7d

S S

O N

N N

N

N N

A R1 R2

A R1 R2 Compounds

R1

R2

Compounds

R1

R2

1a 2a 3a 4a 5a 6a 7a

H F Cl Br OCH3 CH3 CH3

H H H H H H CH3

1c 2c 3c 4c 5c 6c 7c

H F Cl Br OCH3 CH3 CH3

H H H H H H CH3

IC50 = 1.48 lM), –CH3 (6d, IC50 = 1.76 lM), –H (1d, IC50 = 3.48 lM), –Br (4d, IC50 = 4.22 lM), –Cl (3d, IC50 = 6.32 lM) and F (2d, IC50 = 9.17 lM), the order was 5d > 6d > 1d > 4d > 3d > 2d. The

Compounds

R1

R2

Compounds

R1

R2

1b 2b 3b 4b 5b 6b 7b

H F Cl Br OCH3 CH3 CH3

H H H H H H CH3

1d 2d 3d 4d 5d 6d 7d

H F Cl Br OCH3 CH3 CH3

H H H H H H CH3

same results were also found in compounds 1a–7a, 1b–7b and 1c–7c. This meant that compounds with stronger activating substituents in A-ring showed better anti-proliferative activity. The results indicated that the anti-proliferative activity was affected by A-ring-substituent (–OCH3 > –CH3 > –H > –Br > –Cl > –F). Secondly,

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Table 3 Crystal data for compounds 1d and 5d

Empirical formula Molecular weight Crystalsize (mm3) Temperature (K) Radiation Crystalsystem Space group a (Å) b (Å) c (Å) a (°) b (°) c (°) V (Å3) Z Dc (g cm 3) l (mm 1) absort. coeff F(0 0 0) h rang (deg) Reflections collected Indep. reflns Refns obs. [I > 2r(I)] Data/restr./paras Goodness-of-fit on F2 R1, wR2 (all data) R1, wR2[I > 2r(I)] Larg. peak/hole (e Å)

1d

5d

C22H17N3OS 371.1 0.19 ⁄ 0.23 ⁄ 0.16 273(2) Mo-Ka (0.7103 Å) Monoclinc P21/n 11.96241(8) 8.9920(6) 18.2136(13) 90.00 109.171(2) 90.00 1850.5(2) 24 1.617 0.773 912 2.46–25.72 17,883 (Rint = 0.0502) 3515 2396 3515/0/244 1.058 0.1138/0.2535 0.0790/0.2255 0.640/ 0.200

C23H19N3O2S 401.2 0.3 ⁄ 0.35 ⁄ 0.26 273(2) Mo-Ka (0.7103 Å) Monoclinic P21/c 11.7565(6) 9.0469(5) 19.2557(10) 90.00 104.632(2) 90.00 1981.61(2) 26 1.636 0.782 988 2.45–25.73 18,236 (Rint = 0.0318) 3761 2869 3761/0/262 1.069 0.0955/0.2628 0.0776/0.2441 0.675/ 0.450

Table 4 Inhibition (IC50) of BGC823, Hela and HepG2 cells proliferation by compounds 1a–7a, 1b–7b, 1c–7c, 1d–7d IC50(lM)

Compounds

1a 2a 3a 4a 5a 6a 7a 1b 2b 3b 4b 5b 6b 7b 1c 2c 3c 4c 5c 6c 7c 1d 2d 3d 4d 5d 6d 7d Gefitinib

BGC823

Hela

HepG2

14.25 22.73 19.55 17.37 9.51 13.34 8.46 11.16 19.26 16.38 14.62 8.21 10.01 7.11 9.62 17.34 14.63 12.96 7.25 8.94 6.57 8.86 14.93 13.20 11.72 6.94 7.79 6.02 —

5.32 12.35 9.94 6.65 3.31 4.29 1.72 4.62 11.25 8.84 5.81 2.54 3.21 1.33 3.86 9.73 7.56 4.96 1.86 2.46 0.95 3.18 9.17 6.32 4.22 1.48 1.76 0.86 2.67

13.21 23.15 18.71 15.56 8.74 10.43 7.25 10.07 19.52 16.45 13.42 7.85 8.66 5.94 17.19 26.53 22.14 20.31 12.56 14.73 7.37 15.73 24.12 20.43 18.22 10.36 12.69 8.49 6.42

Table 5 EGFR inhibitory activity of synthetic compounds Compounds

EGFR (IC50 lM)

1d 5b 5d 6c 6d 7a 7b 7c 7d Gefitinib

6.31 5.53 2.80 5.26 3.25 3.96 1.32 0.65 0.12 0.02

Figure 1. Crystal structure diagram of compound 1d.

7

EGFR inhibitory activity/IC50

6

2

R =0.91125

5 4 3 2 1 0 1.0

1.5

2.0

2.5

3.0

3.5

Antiproliferation activity IC 50

Figure 2. Crystal structure diagram of compound 5d.

Figure 3. Correlation between the anti-proliferation against Hela cell line and EGFR inhibitory activity, R2 = 0.911.

J.-W. Yuan et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2324–2328

the same trends were observed in compounds with naphthalene ring. The anti-proliferative activities of compounds bearing 1-substituted naphthalene ring were better than those with 2-substituted, such as 1d (IC50 = 3.18 lM) > 1b (IC50 = 4.62 lM), 1c (IC50 = 3.86 lM) > 1a (IC50 = 5.32 lM). The results suggested that naphthalene ring played an important role in the anti-proliferative activity. Thirdly, when 4-thiazolinone ring was substituted with 4-phenylthiazoline, anti-proliferative activities were higher (xd > xc, xb > xa, x = 1–7). Ten compounds (1d, 5a–5d, 6d, 7a–7d) with better anti-proliferative activities were used to test inhibitory EGFR activities. As result in Table 5, all tested compounds exhibited good inhibitory EGFR activities with IC50 between 0.12 and 6.31 lM. Among, compound 7d showed highest inhibitory EGFR activity (IC50 = 0.12 lM). SAR analysis of ten compounds showed a moderate correlation between anti-proliferation and EGFR inhibitory, as showed in Figure 3, with R2 value was 0.911.

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To further understand the potency of compound 7d and guide SAR studies, the molecular docking of 7d and EGFR (PDB code 1M17) was performed by using the Discovery Studio 3.5. The 2D and 3D binding model of 7d with EGFR were shown in Figures 4 and 5. In the binding model, naphthalene ring of 7d with LYS721 formed two p–p bonds, which enhanced the binding affinity, resulting in the increased antitumor activity. In order to evaluate cytotoxicity of the potent anti-tumor agents, we determined cytotoxicity data (CC50) of the ten compounds against human macrophage by the CCK-8 assay. As shown in Table 6, no cytotoxicity was observed. Metastases are the dissemination of cancer cells from the primary tumor to a distant organ. Studies showed that more than 90% of cancer patients die from tumor metastasis, so inhibiting cancer cell migration was an effective way for curing cancer.28 We analyzed the relationship between 7d’s concentration with Hela cell survival ratios by CCK8 assay to test inhibition of cell migration. The result showed that 7d

Figure 4. 2D molecular docking of compound 7d with 1M17.

Figure 5. 3D model of the interaction between compound 7d and 1M17 bonding site.

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Table 6 The median cytotoxic concentration (CC50) data of tested compounds Compounds

CC50a (lMa)

Compounds

CC50a (lMa)

1d 5c 6c 7a 7c Gefitinib

345.3 284.8 289.6 342.2 382.4 280.6

5b 5d 6d 7b 7d

273.4 363.2 396.1 382.7 397.3

a Minimum cytotoxic concentration required to cause a microscopically detectable alteration of normal cell morphology.

Acknowledgments This work was supported by Major Projects on Control and Rectification of Water Body Pollution (2011ZX07204-001-004), and by Henan Educational Committee (14B150036). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.03. 072. References and notes

Cell migration (%)

150

*

100

*

*

50

12 0.

10 0.

08 0.

co

nt

ro

l

0

Concentration (µM) Figure 6. Inhibition to Hela cell migration of compound 7d. Values are expressed as a percentage of the control, which was defined as 100%. Date are potted as mean SD (⁄p < 0.05 vs control).

had no effect on Hela cell survival ratios at 0.14 lM, so we further evaluated inhibition of 7d against Hela cell migration at 0.08, 0.10 and 0.12 lM. As shown in Figure 6, the amount of migrating cells in 7d group was obviously less than that in control group, indicating 7d may be a potential anti-metastasis agent. In conclusion, a series of compounds containing pyrazole, thiazole and naphthalene ring (1a–7a, 1b–7b, 1c–7c, 1d–7d) were synthesized and their anti-proliferative activity, EGFR inhibition, cytotoxicity were evaluated. Among them, compounds 1d and 5d were determined by X-ray. SAR analysis showed that the antiproliferative activity was affected by A-ring-substituent (–OCH3 > –CH3 > –H > –Br > –Cl > –F) and the activity with 4-thiazolinone were higher than that with 4-phenylthiazoline (d > c, b > a). Compound 7d displayed the most potent inhibitory activity (IC50 = 0.86 lM for Hela and 0.12 lM for EGFR). Docking simulation of compound 7d into EGFR active site showed that naphthalene ring of 7d with LYS721 formed two p–p bonds, which enhanced antitumor activity. Therefore, compound 7d may be developed as a potential antitumor agent.

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