Development of Anti-Cancer Agent

Design, ultrasound assisted synthesis and in-vitro anticancer activity of 3-(4-chlorophenyl) sydnone and 3-(4-chlorophenyl) sydnonecarboxaldehyde against 60 Human Tumor cell lines

Sachin K. Bhosale, Shreenivas R. Deshpande and Rajendra D. Wagh

Design, ultrasound asisted synthesis and in-vitro anticancer activity of 3-(4-chlorophenyl) sydnone and 3-(4-chlorophenyl) sydnonecarboxaldehyde against 60 Human Tumor cell lines

Abstract:

In an attempt to develop an effective and safer anticancer agent, 3-(4-chlorophenyl)-4-sydnonecarboxaldehyde 5 have been designed and synthesized under ultrasonication by formylation of 3-(4-chlorophenyl)-sydnone 4 and characterized by spectral studies. The ultrasonic method of synthesis was found to be simple, ecofriendly, reduces reaction time and gave good yield when compared with traditional methods of synthesis. Anticancer activity of the compounds were tested against 60 human tumor cell lines and compared with standard drug Vincristine sulphate. Cytotoxicity evaluation revealed that compound 5 is most potent against NCI-H23 (Non-Small cell lung cancer) with comparative activity of Vincristine sulphate. Compound 5 also had shown moderate cytotoxicity against T-47D (Breast cancer), CAKI-1, UO-31 (Renal cancer), HL-16TB (Leukemia), NCI-H23, NCI-H522 (Non-Small cell lung cancer), SNB-75 (CNS cancer), MALME-3 (Melanoma). In future, research and modification of compounds to different derivatives may lead to development of potent anticancer drug.

Keywords: Anticancer, 1, 2, 3-oxadiazol-5-olate, Formylation, Ultrasonication

INTRODUCTION

Mesoionic sydnone compounds have shown a variety of biological activities including antitumor ((Kier et al., 1964, Dunkley et al., 2003, Satyanarayana et al., 1995, Kavali et al., 2000, Dunkley et al., 2003, Fregly et al., 1964, Stewart et al., 1965, Roche et al., 1965). It is thought that the ionic resonance structures of the heterocyclic ring promote significant interactions with biological molecules. In 1992 a series of 4?-substituted-3?-nitrophenylsydnones were synthesized and evaluated by Grynberg et al for anticancer activity and it was found that the 4?-chloro and the 4?-pyrrolidino compounds significantly enhanced the survival of Sarcoma 180 (S180), Ehrlich carcinoma (Ehrlich) and Fibrous histiocytoma (B10MCII) tumor bearing mice(Dunkley et al., 2003). It was also found that the larger hetero rings; p-piperidino and p-morpholino, were less potent (Kier et al., 1964, Dunkley et al., 2003). Herein we report the design, synthesis, and biological evaluation of two para-chloro substituted analogues of sudnone molecules 4 and 5. These were tested for 60 human tumor cell lines at the National Cancer Institute for antitumor activity at a minimum of five concentrations at 10-fold dilutions.The synthesized chloro substituted sydnones (4 and 5) passed the primary assay by inhibiting the growth of at least one cell line to less than 32%, and both are proved to be moderate active and versatile against each cell line.

MATERIALS AND METHODS

All chemicals and reagents were purchased form from Sigma-Aldrich, Mumbai, India. Melting points of the intermediates and the final products were recorded using a Systolic melting point apparatus and are reported uncorrected. Thin layer chromatographies (TLC) were performed on E-Merck precoated 60 F254 plates and the spots were rendered visible by exposing to UV light. Infra red spectra were recorded in KBr discs using Jasco FTIR 1460 Plus spectrometer. NMR spectra were obtained on a BRUKER AVANCE II 400 NMR spectrometer at 500 MHz for ¹H and 125 MHz for ¹³C, the chemical shifts are expressed in ? (ppm) downfield from tetramethylsilane (TMS). Electron impact mass spectra were recorded on WATERS, Q-TOF MICROMASS (LC-MS) instrument. Elemental analyses (C, H, N) were in full agreement with the proposed structures within ±0.4% of the theoretical values. The ultrasonic irradiation was performed by using a Biotechnics India TM ultrasonic cleaner bath, model 1510, AC input 115 V, output 50 W, 1.9 liters with a mechanical timer (60 min with continuous hold) and heater switch, 47 KHz.

Synthesis of Ethyl N-(4-chlorophenyl) glycinate (1)

A mixture of Para-Chloroaniline (2.80 g, 0.02mol) and chloroethyl acetate (2.12 mL, 0.02mol) was added to the solution of ethanol (20 mL) and anhydrous sodium acetate (3.28 g, 0.04mol) under ultrasonication conditions and allowed to react for 120 min. The mixture was diluted with 20 mL of water and cooled in refrigerator for overnight and recrystallized in ethanol to yield 1 (83%), mp 114-116 ?C. IR (KBr): 3327, 2950, 2934, 2879, 1756, 1069; ¹H NMR (400 MHz, DMSO-d6): ? 1.21 (t, 3H, COOCH2CH3), 3.76 (s, 1H, NH), 4.29 (s, 2H, CH2), 4.54 (q, 2H, COOCH2CH3), 6.83-7.21 (m, 4H, Ar-H); ¹³C NMR (40 MHz, DMSO-d6): ? 14.65, 44.73, 62.07, 115.12, 123.22, 129.01, 146.26, 173.13.

Synthesis of N-(4-chlorophenyl) glycine (2)

Ethyl N-(4-chlorophenyl) glycinate (4.26 g, 0.02mol) and sodium hydroxide (1.2 g, 0.030mol) in ethanol was heated under ultrasonication conditions at 65 °C for 15 min and allowed to cool and acidified with dil HCl. Yield 87 %, mp 146-148 ?C. IR (KBr): 3319, 3277, 2951, 2937, 2879, 1703, 1063; ¹H NMR (400 MHz, DMSO-d6): ? 4.29 (s, 2H, CH2), 6.39 (s, 1H, COOH), 6.55 (s, 1H, NH), 6.79-7.27 (m, 4H, Ar-H); ¹³C NMR (40 MHz, DMSO-d6): ? 44.48, 115.02, 124.16, 130.02, 145.97, 171.98.

Synthesis of N-nitroso (4-chlorophenyl) glycine (3)

Ice cold solution of the N-(4-chlorophenyl) glycine (3.72 g, 0.02mol) was added in crushed ice water (40 mL). A solution of sodium nitrite (1.38 g, 0.02mol) in ice cold water (10 mL) was added dropwise under ultrasonication condition at 0 °C for 10 min. The reaction mixture was filtered and precipitated by adding concentrated hydrochloric acid to the filtrate. Precipitate was filtered and recrystallized in methanol to yield 3 (81%), mp 109-111 ?C. IR (KBr): 3255-2521, 2923, 2849, 1711, 1569, 1325, 1062; ¹H NMR (400 MHz, DMSO-d6): ? 5.02 (s, 2H, CH2), 6.93-7.45 (m, 4H, Ar-H), 11.55 (s, 1H, COOH); ¹³C NMR (40 MHz, DMSO-d6): ? 49.42, 120.75, 128.29, 130.43, 138.78, and 169.25.

Synthesis of 3-(4-chlorophenyl) sydnone (4)

Acetic anhydride (30 mL) was added to 3 (5.40 g, 0.0252 mol) under ultrasonication condition at room temperature for 60 min. The reaction mixture was left overnightat room temperature. The solution was poured slowly into cold water which was very well stirred. The crude sydnone was filtered, dried and recrystallized in ethanol to yield 4 (93%), mp 139-141 ?C. IR (KBr): 3181, 1748, 1053. ¹H NMR (400 MHz, DMSO-d6): ? 7.23 (s, 1H, sydnone), 7.49-8.13 (m, 4H, Ar-H). ¹³C NMR (40 MHz, DMSO-d6): ? 123.15, 126.49, 131.53, 136.13, 141.13, 170.07

Synthesis of 3-(4-chlorophenyl)-4-sydnonecarboxaldehyde (5)

N-Methylformanilide 2.84g (0.0210mol) and phosphoryl chloride (3.17g, 0.0205mol) were mixed under ultrasonication conditions for 10 min. After 0.5 h, 3.0 g (0.0186mol) of 4 was added portionwise with swirling and cooling under ultrasonication below 35⁰C. Hydrogen chloride was evolved vigorously. After standing overnight, the viscous, dark-brown mixture was dissolved in 15 ml. of acetone and poured (stirring) into 75 ml. of ice water. The yellow-orange precipitate was filtered, washed (cold water), and dried. Yield 56%, mp 75-77 ⁰C. MS (M⁺) (m/e) 223.99, 225.996, 225.002. IR (KBr): 1790 (^N?C=O sydnone ring), 1640 (^N?C=O aldehyde). ¹H NMR (400 MHz, DMSO-d6): ? 7.2, 7.2, 7.3, 7.3 (Cl-Ph-), 9.61 (Sydnone-4-CHO). ¹³C NMR (40 MHz, DMSO-d6): ? 127, 129, 129, 130, 130, 134 (Cl-Ph-) 190 (CHO). Element analysis: C; 48.13, H; 2.24, N; 12.47, O; 21.37

Scheme 1-Synthesis of 3-(substituted aryl)-4-sydnonecarboxaldehyde (5) under ultrasonication conditions

1.3. Anticancer screening

The synthesized compound was screened for preliminary anticancer assay by National Cancer Institute (NCI), Bethesda, USA in an in vitro 60 human tumor cell panel. The process utilized 60 different human tumor cell lines of the leukemia, Non-small cell lung, Colon, CNS, melanoma, ovarian, renal, Prostrate and breast cancers which was aimed in showing selective growth inhibition or cell killing of particular tumor cell lines by specific compound. The screening begins with the evaluation of selected compounds against these 60 cell lines at a single dose of 10^-5 M. The output from the single dose screen is reported as a mean graph of the percent growth of treated cells. It allows detection of both growth inhibition (values between 0 and 100) and lethality (values less than 0) (Roschke et al., 2003, Lorenzi et al., 2009, Mingyi et al., 2013, Al-Suwaidan et al., 2013, Senff-Ribeiro et al., 2004, Butkovic et al., 2011, Lorenzi et al., 2009, Mingyi et al., 2013, Al-Suwaidan et al., 2013, Senff-Ribeiro et al., 2004, Butkovic et al., 2011).

RESULT

Compund 5 showed highly cytotoxic activity against NCI-H23 cell line (Non-Small cell lung cancer) as compared to standard drug Vincristine sulphate. Compound 5 also had shown moderate cytotoxicity against T-47D (Breast cancer), CAKI-1, UO-31(Renal cancer), HL-16TB (Leukemia), NCI-H23, NCI-H522 (Non-Small cell lung cancer), SNB-75 (CNS cancer), MALME-3 (Melanoma). Details of % growth inhibition for comp. 4 (NSC 35759) 5 (NSC 774943/1) as shown in table 1.

Table1 One dose mean graph for compound 4 (NSC: 35759) and compound 5 (NSC: 774943 / 1) Conc: 1.00E-5 Molar

HUMAN TUMOR CELL LINE

% GROWTH INHIBITION

for Comp. 4 (NSC 35759)

% GROWTH INHIBITION

for Comp. 5 (NSC 774943/1)

Leukemia

CCRF-CEM

HL-60(TB)

K-562

MOLT-4

RPMI-8226

SR

-11.40

10.60

-06.10

-02.20

-00.80

02.10

-06.46

13.01

-09.03

23.02

-00.15

08.18

Non-Small Cell Lung Cancer

A549/ATCC

HOP-62

NCI-H226

NCI-H23

NCI-H322M

NCI-H460

NCI-H522

-08.20

08.70

-03.00

-06.00

05.30

-01.30

-22.30

-01.23

07.71

08.86

02.57

-00.32

-02.78

25.65

Colon Cancer

COLO 205

HCC-2998

HCT-116

HCT-15

HT29

KM12

SW-620

-02.70

-01.20

-02.50

00.60

-18.00

04.20

02.50

-02.11

-00.83

-02.63

-06.99

-02.79

00.37

-02.44

CNS Cancer

SF-268

SF-295

SF-539

SNB-19

U251

SNB-75

-00.10

-12.00

-19.40

04.10

09.60

-02.10

-03.19

00.48

-00.66

-10.34

-00.12

46.71

Melanoma

LOX IMVI

MALME-3

M M14

MDA-MB-435

SK-MEL-2

SK-MEL-28

SK-MEL-5

UACC-257

UACC-62

02.00

04.72

-04.50

00.00

-02.00

-09.40

04.40

-04.30

08.50

00.94

15.73

01.46

-06.52

-10.96

-22.01

-03.95

-05.67

-03.35

Ovarian Cancer

IGROV1

OVCAR-3

OVCAR-4

OVCAR-5

OVCAR-8

NCI/ADR-RES

SK-OV-3

19.30

-04.50

17.90

03.30

-05.50

-02.90

-04.90

05.88

-03.77

01.90

-11.93

08.87

-08.95

00.52

Renal Cancer

786-0

A498

ACHN

CAKI-1

RXF 393

SN12C

TK-10

UO-31

-08.40

01.30

-05.70

-07.69

-16.28

07.11

-16.10

15.20

-08.73

-02.95

-03.37

15.69

-03.28

-05.11

-27.65

31.52

Prostate Cancer

PC-3

DU-145

08.60

-06.60

-01.72

-08.20

Breast Cancer

MCF7

MDA-MB-231/ATCC

HS 578T

BT-549 97.23

T-47D

MDA-MB-468

15.10

19.90

-00.60

-06.90

16.50

01.30

13.38

-05.71

-06.69

02.77

02.22

-14.44

Mean

Delta

Range

–

–

–

99.68

46.39

74.36

Table 2 Percent Growth inhibition of synthesized compounds against most effective cancer cell lines

Cell lines

Compound 4

Compound 5

Standard

Vincristine sulphate

CAKI-1

-7.69

15.69

73.5

UO-31

15.2

31.52

72

HL-16(TB)

10.6

13.01

125.1

NCI-H23

-6

2.57

-190.4

NCI-H522

-22

25.65

113.1

SNB-75

-2.1

46.71

84.3

MALME-3

4.72

15.73

64.6

Figure1 Percentage growh inhibition for cytotoxic activity evaluation of synthesized ompounds

Figure 2 Percentage growh inhibitions for cytotoxic activity evaluation of compound 4

Figure 3 Percentage growh inhibitions for cytotoxic activity evaluation of compound 5

Figure 4 Percentage growh inhibitions for cytotoxic activity evaluation of standard anticancer drug Vincristine sulphate.

DISCUSSION

Molecules synthesized under ultrasonication conditions. The method provides several advantages over current reaction methodologies, including a simple work up procedure. The ultrasonic method of synthesis was also found to be simple, ecofriendly, reduces reaction time and gave good yield when compared with traditional methods of synthesis. Cytotoxicity evaluation revealed that out of 60 human tumor cell lines, compound 5 is highly effective against SNB-75 (CNS cancer) and UO-31 (Renal cancer) human tumor cell lines.Compund 5 showed highly cytotoxic activity against NCI-H23 cell line (Non-Small cell lung cancer) as compared to standard drug Vincristine sulphate. Compound 5 also had shown moderate cytotoxicity against T-47D (Breast cancer), CAKI-1, UO-31(Renal cancer), HL-16TB (Leukemia), NCI-H23, NCI-H522 (Non-Small cell lung cancer), SNB-75 (CNS cancer), MALME-3 (Melanoma).

CONCLUSION

In the process of anticancer drug discovery, to find new potential anti-lung cancer agent, we designed and synthesized molecule 5. As compund 5 showed highly cytotoxicity against NCI-H23 cell line (Non-Small cell lung cancer) in comparision to standard drug Vincristine sulphate, in future, research and development with designing desired modifications of molecule 4 and 5 may develop in safer and effective potential anticancer molecules.

ACKNOWLEDGEMENT

Authors are thankful to BCUD, University of Pune, INDIA (Project-13PHM000018) for financial assistance, NCI, USA for carrying out anticancer activity, SAIF Punjab University for spectral study, JNTU Hyderabad and Dr. Dhake A. S., SMBT College of Pharmacy, Dhamangaon, Nasik (MS) India for providing necessary facilities to carry out the research work.

COMPETING INTERESTS The authors declare that they have no competing interest.

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