Design, Synthesis and Cytotoxic Activity of Spiro(oxindole-3-3

Send Orders for Reprints to [email protected] Letters in Drug Design & Discovery, 2018, 15, 000-000

1

RESEARCH ARTICLE

Design, Synthesis and pyrrolidine) Derivatives

Cytotoxic

Activity

of

Spiro(oxindole-3-3'-

Dilan Konyara,*, Cenk A. Andacb and Erdem Buyukbingola a b

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Ankara-06100, Turkey; Department of Pharmaceutical Chemistry, School of Pharmacy, Istinye University, Istanbul-34010, Turkey Abstract: Background: Spiro[pyrrolidine-3,3’-oxindole] compounds are reported to be highly bioactive natural and synthetic products. Initially, spirooxindole alkaloids were isolated from plants of the Apocynaceae and Rubiaceae families, which were found to have a common scaffold, spiro[pyrrolidine-3,3’-oxindole], exhibiting anticancer activities., we specifically aimed at the synthesis, characterization and anticancer activity of novel spiro[pyrrolidine-3,3' -oxindole] derivatives, compounds 6a-c and 7.

A R T I C L E H I S T O R Y Received: February 06, 2017 Revised: July 16, 2017 Accepted: July 24, 2017 DOI: 10.2174/1570180814666170810120634

Methods: The synthesis was initiated by Knovenegal condensation of indole-2-one with an appropriate benzaldehyde in presence of piperidine to afford compounds 3a-c. Compounds 6a-c were synthesized by an asymmetric 1,3-dipolar cycloaddition between compounds 3a-c and (2S, 3R)-2, 3, 5, 6-tetrahydro-2, 3-diphenyl-1, 4-oxazin-6-one, which is an intermediate compound formed by the Schiff base reaction between 3-methyl-butanal and (2S, 3R)-2, 3, 5, 6-tetrahydro-2, 3-diphenyl1,4-oxazin-6-one, in presence of molecular sieves (4Å) under argon atmosphere. Compound 6a was then reacted with ethylamine-HCl in THF at room temperature to yield compound 7. Results: Cytotoxic effects of the compounds synthesized were determined on Huh7, MV, HCT116 and MCF7 cancer cell lines by the NCI-60 Sulforhodamine B Assay, using (S)-(+)-Camptothecin as a positive control. In general, target compounds showed better cytotoxic activities against the MCF7 and HCT116 cancer cell lines. It was found that compound 7 exhibited the most potent inhibitory activity with IC50 values of 4.8 µM, 3.9 µM, 14.9 µM and 8.2 µM against the MCF7, HCT116, MV and Huh7 cell lines, respectively. Conclusion: It was determined that compounds 6a&6b possess C6'(S)|C8'(R)|C9'(R) stereochemistry and compound 7 adopts C2'(S)|C4'(R)|C5'(R) stereochemistry. Cytotoxicity studies suggest that compound 7 gave rise to the highest anticancer activity against MCF7, HCT116, and Huh7 cancer cell lines.

Keywords: Oxindole, spiro pyrrolidino oxindole, 1,3-dipolar cycloaddition reaction, cytotoxic activity, cancer, drug design. 1. INTRODUCTION Cancer has increasingly become a major cause of mortality throughout the world. 14.1 millions of cancer cases were reported in 2012 [1]. Despite all advances in cancer diagnosis and therapy, the mortality rate remains to be still high. Therefore, discovery of newer and safer anticancer agents with extended-spectrum cytotoxicity against tumor cells becomes an urgent necessity [2]. It is known that spirooxindole compounds have significant roles in drug design in terms of antimicrobial [3], anti-HIV [4], antimalarial [5] and anticancer *Address correspondence to this author at the Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100, Tandogan, Ankara, Turkey; Tel: +90 (312) 203 30 73; Fax: +90 (312) 213 10 81; E-mail: [email protected]

1570-1808/18 $58.00+.00

features [6] as well as being inhibitors of the MDM2 (Mürine Double Minute)-p53 interaction [3]. Particularly, spiro[pyrrolidine-3,3’-oxindole] compounds (Fig. 1) are reported to be highly bioactive natural and synthetic products [7]. Initially, spirooxindole alkaloids were isolated from plants of the Apocynaceae and Rubiaceae families, which were found to have a common scaffold, spiro[pyrrolidine3,3’-oxindole], exhibiting anticancer activities [8, 10]. For instance, many natural spirooxindole alkaloids, such as Spirotryprostatine A and B, horsifiline, elacomine etc. [9] exhibit anticancer activity [9-11] as well as inhibitory potentials for the p53-MDM2 interaction [10, 11] Synthesis of spirooxindole derivatives fused with a five-membered heterocyclic ring including pyrrolidines, pyrrolines, pyrroles, triazoles, and isoxazolidines were accomplished by the Huisgen cycloaddition reaction, [12] a 1,3-dipolar cycloaddi©2018 Bentham Science Publishers

2 Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1

Konyar et al.

Z

Y

' 9'/5 '/1' 5 N 8'/4' 6'/2' X O N H

Oxindole scaffold

O

O

O

O N

N O

O2N

O

NH

NH

6b

6a

O Cl

N

HO

O

HN

O O2N

N

O NH

6c

O N H 7

Fig. (1). X, Y, and Z substituents constituting novel spiro[pyrrolidine-3,3′-oxindole] derivatives, namely compounds 6a-c and 7.

tion reaction. Here, we specifically aimed at the synthesis, characterization and anticancer activity of novel spiro[pyrrolidine-3,3' -oxindole] derivatives, compounds 6ac and 7 in Fig. (1), in which positions 6', 8' and 5' -9' on compounds 6a-c and positions 2', 4' and 1' -5' on compound 7 are substituted with X, Y and Z. The novel spiro[pyrrolidine3,3’-oxindole] derivatives (6a-c) were synthesized by reaction of the oxazinone and aldehyde with 3-benzyldene-1,3dihydro-indol-2-one derivatives in toluene were by 1,3dipolar cycloaddition reaction is shown in Scheme 1. The stereochemistry of this class of compounds has a significant influence on the binding affinities. Wang et al. suggested that diastereomeric spirooxindole class of compounds had a significant effect on binding affinities to MDM2; There is a 100-fold difference between binding affinity of the strongest

and the lowest potent stereoisomers. They have shown that the binding affinities of the cis-cis isomers to MDM2 are better than the trans-cis isomers. And also cis-cis isomer is more effective than trans-cis isomer in p53 activation and apoptosis induction, in vivo [13]. Chirality of the carbons at positions 6', 8', 9' of compounds 6a-b and positions 2', 4', 5' of compound 7 was determined by 1H-NMR and ROESY NMR experiments. A previously reported spiro[pyrrolidine3,3'-oxindole] derivative, compound 6a [14], see Fig. (1), was also synthesized, characterized, recrystallized for X-ray crystallographic analysis and found to be the E-conformer and used as a reference compound in biological activity studies [14]. Anticancer activities of compounds 6a-c and 7 were determined on the Huh7 human liver, MCF7 human breast and HCT116 human colon cancer cell lines.

Synthesis of Spiro(oxindole-3-3'-pyrrolidine) Derivatives

Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1

Ph Ph

Ph Ph

O HN

O

4Ao

Ph

mol.sieves,tol.

4

Ph

O N

OHC

H

3

O N

O

O

H 5

OH

Scheme 1. Proposed reaction mechanism pathway of compound 6a-c synthesis. R1

O

O

Ph

N H

Ph

CHO O

+

N H

,

4

Piperidine O

R1

1

CHO

MeOH/80°C 2

N H

4 A°mol.sieves, toluene/70°C

3a-c 3a (R1= -H) 3b (R1= -p-NO2) 3c (R1= -o-Cl)

R1 4" 5"

Ph 2' 3" O 1' aO f 3' e 4' 2" b Ph 9' c d 8' 2 1N 5' 1'' 3 6" 5 6' 7' 4 3 O 2 1 N H

R1 H2N THF/r.t.

6a-c 6a (R1= -H) 6b (R1= -p-NO2) 6c (R1= -o-Cl) R1: -H, -p-NO2, -o-Cl

4"

HO 3" HN 2"

O 5'

5" 6"

1"

4'

N 1' 3 3' 2' O 2 1 N H 7

7 (R1= -p-NO2)

Scheme 2. Synthesis of 3-substituted-benzyldene-1,3-dihydro-indol-2-one (3a-c), 3',4'-diphenyl-8’-p-nitro-phenyl-6'-isobutyl-3',4',8,8'atetrahydrospiro[3H-indol-3,7'(6'H)-[1H]pyrrolo[2,1-c][1,4]oxazine]-1',2(1H)-dione (6a-c) derivatives and 2'-(2-methylpropyl)-1,2-dihydro1'-[(1R,2S)-2-hydroksi-1,2-diphenylethyl]-N-ethyl-2-oxo-4'-p-nitrophenyl-spiro[3H-indol-3,3'-pyrrolidin]-5'-carboxamide (7). Reagents and conditions: (a) Substituted benzaldehydes, piperidine, methanol, 80°C, 1.5 h, (b) (2S,3R)-2, 3, 5, 6-tetrahdro-2, 3-diphenyl-1, 4-oxazin-6one, 3-methyl-butyraldehyde, 4 A° mol.sieves, toluene, 70°C, (c) ethylamine-HCl, THF, r.t.

2. MATERIALS AND METHOD 2.1. Chemistry 2.1.1. Materials 2-Chloro benzaldehyde (Fluka), 4-nitro benzaldehyde (Aldrich), benzaldehyde (Aldrich), (2S,3R)-2,3,5,6-tetra hydro-2,3-phenyl-1,4-oxazin-6-one (Nantong Baihua BioPharmaceutical Co., Ltd.), oxindole (Aldrich), piperidine (BDH Chemicals Ltd.), 3-methyl butiraldehyde (Aldrich), 4Aº molecular sieves (Sigma-Aldrich), petroleum ether (Merck), acetone (Fluka), hexane (Sigma-Aldrich), ethyl acetate (Sigma-Aldrich), methanol (Riedel de Haen), toluene

(Merck), ether (Merck) were purchased in high purities and freshly used. Melting points were determined by a capillary melting point apparatus (Electrothermal 9100, Essex, UK). Elemental analysis was performed by a Leco932 CHNS analyzer (Leco, St. Joseph, MI, USA), which gave rise to elemental compositions within 0.4% error-range of the calculated values. 1H, 1H,1H-gCOSY, 1H,1H-ROESY, and gHSQC NMR experiments were performed on a Varian Mercury-400 FTNMR spectrometer operating at 400 MHz (Varian Inc., Palo Alto, CA, USA). All NMR spectra were acquired in CDCl3. 1 H NMR FID data were collected between -2 and 14 ppm


with 32k data points. 1H,1H-gCOSY and 1H,1H-ROESY NMR FID data were collected between -1 ppm and 9 ppm with 400 data points in F1 dimension and 586 data points in F2 dimension gHSQC NMR FID data were collected between -2 and 14 ppm with 960 data points in F2 (1H) dimension and between -10 and 180 ppm with 512 data points in F1 (13C) dimension. All NMR FID data were post-processed by Mestre-C(nD) v.1.1 (Mestre-C, Spain) and Varian NMR software. All 1H NMR FID data were zero-filled to 32K data points, linear-predicted and apodized with line-broadening function before Fourier transformation. All 1H,1H-gCOSY and 1H,1H-ROESY NMR FID data were zero-filled and linear-predicted to 2K data points in F2 and F1 dimensions before Fourier transformation. In addition, line-predicted ROESY FIDs were apodized with 90o Sine and 0.07 Hz Gaussian functions before Fourier transformation in both dimensions. Apodized ROESY FIDs were Fourier transformed in inverse and quadrature modes along t2 dimension and in inverse and quadrature modes using the hypercomplex protocol in t1 dimension. 1H NMR chemical shifts (δ) were reported in ppm, 3JHH and 2JHH peak coupling constants were reported in Hz, and peak splits were reported as s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). In NMR assignments, ortho/meta/para aromatic hydrogens are denoted o/m/p respectively. Mass spectral data were acquired on a Waters ZQ Micromass LC-MS spectrometer (Waters Corporation, Milford, MA, USA) using the Electrospray Ionization (ESI) method. The synthesis of compounds (6a-c, 7) is schematically shown in Scheme 2. 2.1.2. General Procedure for the Synthesis of 3-benzyldene1,3-dihydro-indol-2-one Derivatives (Compounds 3a-c)14 Piperidine (0.197 ml, 2 mmol) was added to a mixture of oxindole (1 mmol) and the benzaldehyde derivative of interest (1.1 mmol) in dry methanol. The reaction mixture was then stirred under Argon atmosphere at 80°C for 1.5 hours. The solvent of the reaction mixture was then evaporated to yield the crude product, which was purified by silica gel column chromatography (hexanes/EtOAc, 2:1). 3-Benzylidene-1,3-dihydro-indol-2-one (compound 3a) Obtained as a yellow solid; M.P.: 164-166 °C (lit. M.P.: 175176 °C)11 ; MS(ESI) m/z 222 [M+H]+. 3-(4'-Nitro)-benzylidene-1,3-dihydro-indol-2-one (compound 3b) Obtained as a red solid; M.P.: 239-242 °C (lit. M.P.: 245-250 °C)11; MS(ESI) m/z 267 [M+H]+. 3-(2'-Chloro)-benzylidene-1,3-dihydro-indol-2-one (Compound 3c) Obtained as a red solid; M.P.: 145-148 °C (lit. M.P.: 142 °C)11; MS (ESI) m/z 256 [M+H]+ (100%), 258 (M+H+2)+ (33%). 2.1.3. General Procedure for the Synthesis of the 3',4',8'triphenyl-6'-isobutyl-3',4',8,8'a-tetrahydrospiro[3H-indol3,7'(6'H)-[1H]pyrrolo[2,1c]-[1,4]oxazine]-1',2(1H)-dione Derivatives (Compounds 6a-c)14 3-Benzylidene-1,3-dihydro-indol-2-one compounds 3a/3b/3c, (0.475mmol), 3-methyl-butyraldehyde (0.0509 ml, 0.475mmol), and (2S,3R)-2,3,5,6-tetrahydro-2,3-diphenyl1,4-oxazine-6-one (0.1 g, 0.396 mmol) were dissolved in toluene in the presence of 2 g of freshly activated molecular

Konyar et al.

sieves (4Å) followed by stirring overnight at 70oC under argon atmosphere. After completion of the reaction, the solvent was evaporated in vacuo and the major crude product was purified by silica gel column chromatography (hexanes/EtOAc, 5:1). Compound 6a (yield 16%), compound 6b (yield 9%), and compound 6c (yield 5%). (3R,3'S,4'R,6'S,8'S,9'R)-3',4',8'-triphenyl-6'-(2-methylpropyl)3',4',8',9'-tetrahydro-1'H,6'H-spiro[2,3-dihydro-indole-3, 7'-pyrrolo[2,1-c][1,4]oxazine]-1',2(1H)-dione (Compound 6a) 1

H-NMR (400 MHz, CDCl3) δ(ppm): 7.64 (1H, s, NH), 7.34-6.94 (18H, m, HAr), 6.91 (1H, t, 3JHH= 8.0, 7.6 Hz, 6HAr), 6.56 (1H, d, 3JHH = 8.0 Hz, 7-HAr), 6.63 (1H, t, 3JHH = 8.0, 7.2 Hz, 5-HAr), 6.23 (1H, d, 3JHH = 7.2 Hz, 4-H Ar), 6.35 (1H, d, 3JHH = 4.0 Hz, 3'-H), 4.80 (1H, d, 3JHH = 4.4 Hz, 4'H), 4.39 (1H, d, 3JHH = 10.8 Hz, 9'-H), 4.25 (1H, d, 3JHH = 10.8 Hz, 8'-H), 3.20 (1H, dd, 3JHH = 10.0, 4.0 Hz, 6′-H), 2.07-2.0 (1H, m, 6'H-iBu(-CH2)), 1.75-1.68 (1H, m, 6′HiBu(-CH2)), 1.05-1.04 (1H, m, 6'H-iBu(-CH)), 0.80 (3H, d, 3 JHH = 6.8 Hz, 6'H-iBu(-CH3)), 0.47 (3H, d, 3JHH = 6.0 Hz, 6'H-iBu(-CH3)); COSY proton-proton correlations [δΗ/δΗ]: 6.91/6.56 [6-HAr/7-HAr], 6.91/6.63 [6-HAr/5-HAr], 6.63/6.23 [5-HAr/4-HAr], 6.35/4.80 [3'-H/4'-H], 4.39/4.25 [8a′-H/8'-H], 3.20/2.07-2.0 [6'-Heq/CH2], 3.20/1.75-1.68 [6'-H/CH2], 2.072.0/1.75-1.68 [CH2/CH2]; 13C-NMR (400 MHz, CDCl3 ) δ(ppm): 129.8, 128.1, 127.7, 126.9, 125.7, 125.0, 122.0, 109.2, 83.7, 66.1, 60.5, 60.3, 56.0, 36.5, 24.9, 23.8, 21.6; MS (ESI) m/z: 543 [M+H]+; Anal. (C36H34N2O3-0.5 H2O) requires C, 78.37; H, 6.40; N, 5.07; found C, 78.22, H, 6.62, N, 5.04. (3R,3'S,4'R,6'S,8'S,9'R)-3',4'-diphenyl,8'--p-nitrophenyl6'-(2-methylpropyl)-3',4',8',9'-tetrahydro-1'H,6'H-spiro [2,3-dihydro-indole-3,7'-pyrrolo[2,1-c][1,4]oxazine]-1', 2(1H)-dione (Compound 6b) 1

H-NMR (400 MHz, CDCl3) δ(ppm): 7.88 (2H, d, 3JHH = 8.4 Hz, 3''-HAr-5''-HAr), 7.35-7.16 (10H, m, H Ar), 7.59 (1H, s, NH), 7.10 (2H, d, 3JHH = 8.4 Hz, 2''-H Ar-6''-HAr), 6.93 (1H, t, 3 JHH = 8.0, 8.0 Hz, 6-HAr), 6.56 (1H, d, 3JHH = 8.0 Hz, 7HAr), 6.65 (1H, t, 3JHH = 7.6, 7.2 Hz, 5-H Ar), 6.17 (1H, d, 3 JHH = 7.6 Hz, 4-H Ar), 6.31 (1H, d, 3JHH = 4.0 Hz, 3'-H), 4.80 (1H, d, 3JHH = 4.4 Hz, 4'-H), 4.32 (1H, d, 3JHH = 10.8 Hz, 9'H), 4.28 (1H, d, 3JHH = 10.8 Hz, 8'-H), 3.16 (1H, dd, 3JHH = 10.4, 3.2, 2.8 Hz, 6'-H), 2.15 (1H, ddd, 3JHH = 13.6, 10.4, 3.6 Hz, 6'H-iBu(-CH2)), 1.72 (1H, ddd, 3JHH = 13.6, 10.8, 3.2 Hz, 6'H-iBu(-CH2)), 1.06-1.02 (1H, m, 6'H-iBu(-CH)), 0.83 (3H, d, 3JHH = 6.4 Hz, 6'H-iBu(-CH3)), 0.48 (3H, d, J= 6.0 Hz, 6'H-iBu(-CH3)); COSY proton-proton correlations [δΗ/δΗ]: 7.88/7.10 [3''-H Ar-5''-HAr /2''-HAr-6''-HAr], 6.93/6.56 [6-HAr/7HAr], 6.93/6.65 [6-HAr/5-HAr], 6.65/6.17 [5-H Ar/4-HAr], 6.31/4.80 [3'-H/4'-H], 3.16/2.18-2.11 [6'-H/CH2], 3.16/1.751.68 [6'-H/CH2], 2.18-2.11/1.75-1.68 [CH2/CH2]; MS(ESI) m/z 588[M+H]+; Anal. (C36H33N3O5-1 H2O) requires C: 71.38; H: 5.82; N: 6.93; found C: 71.38, H: 5.68, N: 6.80. (3R,3'S,4'R,6'S,8'R,9'R)-3',4'-diphenyl,8'-o-chlorophenyl6'-(2-methylpropyl)-3',4',8',9'-tetrahydro-1'H,6'H-spiro [2,3-dihydro-indole-3,7'-pyrrolo[2,1-c][1,4]oxazine]-1', 2(1H)-dione (Compound 6c) 1

H-NMR (400 MHz, CDCl3) δ(ppm): 8.42 (1H, s, NH), 7.53 (1H, d, 3JHH = 7.6 Hz, 3''-H Ar), 7.24-7.08 (13H, m, HAr), 7.05-6.98 (1H, m, 6-HAr), 6.76 (1H, d, 3JHH = 8.0 Hz, 7-HAr),


6.61 (1H, t, 3JHH = 8.0, 7.2 Hz, 5-HAr), 6.30 (1H, d, 3JHH = 7.6 Hz, 4-HAr), 6.73 (1H, d, 3JHH = 3.6 Hz, 3'-H), 4.90 (1H, d, 3JHH = 4.0 Hz, 4'-H), 4.78 (1H, d, 3JHH = 10.4 Hz, 9'-H), 4.74 (1H, d, 3JHH = 10.8 Hz, 8'-H), 3.32 (1H, t, 3JHH = 7.2, 6.8 Hz, 6'-H), 1.92-1.85 (1H, m, 6'H-iBu(-CH2)), 1.49-1.42 (1H, m, 6'H-iBu(-CH2)), 1.02-0.96 (1H, m, 6'H-iBu(-CH)), 0.64 (3H, d, 3JHH = 6.4 Hz, 6'H-iBu(-CH3)), 0.47 (3H, d, 3 JHH = 6.0 Hz, 6'H-iBu(-CH3)); COSY proton-proton correlations [δΗ/δΗ]: 7.04-6.98/6.76 [6-HAr/7-HAr], 7.04-6.98/6.61 [6-HAr/5-HAr], 6.61/6.30 [5-HAr/4-HAr], 6.73/4.90 [3'-H/4'H], 4.78/4.74 [9'-H/8'-H], 3.32/1.92-1.85 [6'-H/CH2], 3.32/ 1.49-1.44 [6'-H/CH2], 1.92-1.85/1.49-1.44 [CH2/CH2]. 13

C-NMR (400 MHz, CDCl3) δ(ppm): 129.5, 129.1, 127.9, 127.7, 126.2, 126.0, 124.9, 121.8, 109.3, 82.1, 69.5, 62.7, 61.5, 59.1, 53.4, 35.6, 29.6, 24.5, 23.8, 22.7, 22.2; MS(ESI) m/z 577 [M+H]+ (100%), 579 [M+H+2]+ (40%); Anal. (C36H33ClN2O3-0,25 C6H14-0,75 H2O) requires C: 73.57; H: 6.25; N: 4.57; found C: 73.41, H: 6.57, N: 4.47. 2.1.4. Synthesis of 2'-(2-methylpropyl)-1,2-dihydro-1'[(1R,2S)-2-hydroxy-1,2-diphenylethyl]-N-ethyl-2-oxo-4'-pnitrophenyl-spiro[3H-indol-3,3'-pyrrolidine]-5'-carboxamide (2'S,3'S,4'S,5'R)-N-ethyl-1’-[(1R,2S)-2-hydroxy-1,2diphenylethyl]-2'-(2-methylpropyl)-4'-p-nitrophenyl-2(1H)oxo-spiro[2,3-dihydro-indole-3,3'-pyrrolidine-5'carboxamide (Compound 7) Under nitrogen atmosphere, to a solution of compound 6 (0.587 g, 1 mmol) dissolved in 5 ml THF was added ethylamine-HCl (0.164 g, 1.5mmol), followed by stirring overnight. After brine was added into the reaction mixture, the crude products were extracted with ethyl acetate for several times. The combined organic phase was dried over anhydrous Na2SO4. The solvent was evaporated in vacuo and the desired product was purified by silica gel column chromatography (hexanes/acetone, 2:1, (yield: 45%) [14]. 1

H-NMR (400 MHz, CDCl3) δ(ppm): 7.53 (1H, s, indoleNH), 7.52-7.22 (10H, m, HAr), 7.75 (2H, d, 3JHH = 8.4 Hz, 3''-HAr-5''-H Ar), 6.82 (2H, d, 3JHH = 7.6 Hz, 2''-HAr-6''-HAr), 6.99 (1H, t, 3JHH = 8.4, 7.6 Hz, 6-HAr), 6.65 (1H, s , amide(NH)), 6.56 (1H, d, 3JHH = 8.0 Hz, 7-H Ar), 6.15 (1H, d, J= 7.6 Hz, 4-H Ar), 5.29 (1H, s, CH), 4.34 (1H, d, J= 8.8 Hz, 5'-H ), 4.41 (1H, d, J= 6.0 Hz, CH), 3.88 (1H, d, 3JHH = 8.8 Hz, 4'H), 3.59 (1H, d, 3JHH = 9.2 Hz, 2'-H), 3.36-3.28 (1H, m, 5'HN-Et(-CH2)), 3.06-3.00 (1H, m, 5'H-N-Et(-CH2)), 2.56 (1H, s, OH), 2.25 (1H, t, 2'H-iBu(-CH2)), 1.52 (1H, t, 2'H-iBu(CH2)), 1.10-1.08 (1H, m, 2'H-iBu(-CH), 0.99 (3H, t, 5'H-NEt(-CH3)), 0.82 (3H, d, 3JHH = 6.4 Hz, 2'H-iBu(-CH3)), 0.70 (3H, d, 3JHH = 6.0 Hz, 2'H-iBu(-CH3)); COSY proton-proton correlations [δΗ/δΗ]: 7.75/6.82 [3''-H Ar-5''-H Ar /2''-HAr-6''HAr], 6.99/6.56 [6-HAr/7-HAr], 6.99/6.65 [6-HAr/5-HAr], 6.65/ 6.15 [5-HAr/4-HAr], 6.65/3.36-3.28 [NH/CH2], 6.65/3.06-3.0 [NH/CH2], 5.29/4.41 [H2/H1], 5.28/2.56 [CH/OH], 4.34/ 3.88 [5'H/4'H], 3.59/2.25 [2'-H/CH2], 3.59/1.52 [2'-H/CH2], 3.36-3.28/0.99 [CH2/CH3], 3.06-3.0/0.99 [CH2/CH3], 2.25/1.52 [CH2/CH2]; 13C-NMR (400 MHz, CDCl3) δ(ppm): 131.4, 129.7, 128.7, 128.2, 128.1, 126.9, 125.5, 122.9, 122.3, 109.5, 84.2, 70.5, 67.1, 65.8, 60.7, 60.0, 59.8, 58.1, 55.5, 40.6, 36.6, 34.2, 25.5, 23.7, 21.7, 14.3; MS(ESI) m/z 633[M+H]+ ; Anal.(C38H40N4O5-0,25H2O) requires C, 71.62; H, 6.40; N, 8.79; found C, 71.67, H, 6.43, N, 8.45.


5

2.2. Determination of Anticancer Effects of Compounds 6a-c and 7 in Cell Culture Huh7 human hepatocellular carcinoma, MV (Mahlavu) human hepatocellular carcinoma, MCF7 human breast carcinoma and HCT116 human colon carcinoma cells were grown in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 1% penicillin and streptomycin (GIBCO, Invitrogen) at 37°C under 5% CO2 atmosphere. 2.3. NCI-60 Sulforhodamine B Assay Cells were plated in 96-well plates (2000-3000 cells/well in 150µl/well) and grown for 24 hours at 37 °C. Grown cells were then treated in duplicates with a series of dilutions (in DMSO), ranging from 40µM to 2.5 µM, of the compounds synthesized (6a-c and 7) as well as (S)-(+)-camptothecin (the positive control). After 72 hours of incubation, cells were fixed with 10% (v/v) trichloroacetic acid (MERCK) for an hour and stained with sulforhodamine B (SRB) solution (50 µl of a 0.4% (m/v) of SRB in 1% acetic acid solution). For the removal of unbound SRB, cells were washed out with 1% acetic acid and left for air-drying. SRB bound to protein was solubilized in 10 mM Tris-base prior to absorbance measurement (515 nm) using 96-well plate reader. Cells treated with DMSO alone were used as the control in percent inhibition and IC50 studies. Percent inhibition values were determined by the following equation [15]: %inhibition=[{(average(ODtreatment)/average(ODDMSO))X10}] (S)-(+)-Camptothecin (the positive control), compound 6a (the reference) and compounds 6b-c and 7 were tested for their antitumor activities at their IC50 concentrations, given in Table 1, against Huh7, MV, MCF7, HCT116 cancer cell lines. Percent inhibition of the cells was determined using 5 dilutions of the compounds synthesized, with concentrations ranging from 40 µM to 2.5 µM (in DMSO). 3. RESULTS AND DISCUSSION The reference compounds (6a and the target compounds 6b-c and 7) were synthesized as described in the methods section, following the steps shown in Scheme 2. The synthesis was initiated by Knovenegal condensation of indole-2one in Scheme 2 with an appropriate benzaldehyde in the presence of piperidine to afford compounds 3a-c [14]. Compounds 6a-c were synthesized by an asymmetric 1,3-dipolar cycloaddition between compounds 3a-c and (2S, 3R)-2, 3, 5, 6-tetrahydro-2, 3-diphenyl-1, 4-oxazin-6-one (compound 4 in Scheme 2), which is an intermediate compound formed by the Schiff base reaction between 3-methyl-butanal (in Scheme 1) and (2S, 3R)-2, 3, 5, 6-tetrahydro-2, 3-diphenyl1,4-oxazin-6-one (compound 4 in Scheme 1), in the presence of molecular sieves (4Å) under argon atmosphere. Compound 6a was then reacted with ethylamine-HCl in THF at room temperature to yield compound 7 [14]. After silica gel chromatography separation, the major products for compounds 6a (yield: 16%), 6b (yield: 9%), 6c (yield: 5%) and 7 (yield: 45%) were purified in the best possible highest yields with more than 95% purity. All the compounds purified were


Table 1.

Konyar et al.

Experimental 3JHH (Hz) values and the corresponding HLA (Haasnoot, De Leeuw and Altona)16 dihedral angles (HLAo) for the vicinal H8'-H9' proton pairs of compounds 6a-c and the vicinal H4'-H5' proton pair of compound 7. t stands for trans HLAo (~180o).

Table 2.

ROESY NOE cross-peak intensities for compounds 6a, 6c and 7. NOE cross-peaks were referenced to the prochiral geminal protons of –CH2- attached to C6' of compounds 6a-c or C2' of compound 7. NOE Intensities

3

JHH(Hz)/HLAo

6a

6b

6c

7

H8':H9'

10.7/t

10.8/t

n.d.

n.a.

H4':H5'

n.a.

n.a.

n.a.

9.1/t

n.d.: not determined due to spectral overlap. n.a.: not applicable.

6c

7

6'H-8'H

v.weak

n.d.

2'H-4'H

v.weak

6'H-9'H

weak

n.d.

2'H-5'H

weak

8'H-9'H

medium

n.d.

4'H-5'H

n.d.

n.d.: not determined due to spectral overlap; weak: weak NOE intenstiy; v.weak: very weak NOE intensity; medium: medium NOE intensity.

characterized by NMR, mass spectroscopy and elemental analysis techniques, see the corresponding experimental section. In ESI mass spectroscopy studies, all compounds yielded (M+H) + positive ions with 100% relative abundances. Elemental analysis results were found to be within the range of calculated values with less than 0.4% deviations, proving the chemical structure and purity of the compounds synthesized. Proton chemical shifts listed in the experimental section were determined by 1H and gCOSY NMR methods. 13C chemical shifts, listed in the corresponding experimental section, were determined by 13C and gHSQC NMR methods. Stereoisomerism for the C6, C8' and C9' atoms of the pyrrolo[2,1-c][1,4]oxazine heterocyclic group in compounds 6a and 6c, (Fig. (1)), was studied by the application of the Haasnoot, De Leeuw and Altona (HLA) Karplus equation [16, 17] on 3JH8'-H9' coupling constants for the H8'-H9' vicinal protons, see Table 1, as well as through-space NOE crosspeak intensities determined for the H6'-H8', H6'-H9' and H8'H9' proton pairs by ROESY NMR. In terms of compound 7, a hydrolyis product of compound 6b, stereoisomerism was determined for the C4' and C5' atoms of the pyrrole group, see Scheme 2, utilizing 3JH4'-H5', see Table 1, and NOE cross-peak intensities for the H2'-H4', H2'-H5' and H4'-H5' Table 3.

6a

proton pairs, (Table 2). Stereochemistry determined for compound 7 was directly correlated to compound 6b. As seen in Table 3, 3JH8'-H9' (compounds 6a & 6b) and 3JH4'-H5' (compound 7) are within the range of ~9-12 Hz, Table 1, giving rise to trans HLAo dihedral angles (~180o/t) [17], which means that the C8'/C4' atom will have a different stereoisomerism than the neighboring C9'/C5' atom. For instance; a C8' atom with an R stereoisomerism would be bound to a C9' atom with an R stereoisomerism or vice versa. Stereoisomerism for the C6', C8' and C9' atoms of compound 6c was not determined due to severe spectral overlap of the H8' and H9' proton peaks. In Table 2, NOE cross-peak intensities were correlated to proton-proton distances in three ranges: 1.8-2.8 Å for strong NOEs, 2.8-3.3 Åfor medium NOEs and 3.3-5.0 Å for weak NOEs [18]. Excluding homostereoisomeric possibilities with the C8'-C9' (compounds 6a-c) and C4'-C5' (compound 7) atom pairs, 4 sets of stereoisomerism involving the C6'/C8'/C9' atoms of compounds 6a & 6c and the C2'/C4'/C5' atoms of compound 7 were modeled and minimized by Discovery Studio v3.0 Visualizer (free trial version by Accelrys Software, San Diego, CA, USA), for which dihedral angles and protonproton distances are listed in Table 3.

Torsion (dihedral) angles and proton-proton through-space distances for modelled conformers of compounds 6a and 7 possessing possible different stereochemistries. Compound 6a torsion(o)

chirality

distance (Å)

C6'

C8'

C9'

8'H-9'H

6'H:8'H

6'H:9'H

8'H:9'H

R

S

S

178/t

1.91

3.50

3.00

+

R

R

R

166/g

3.74

1.86

3.09

S

S

S

-179/t

3.56

3.61

3.02

S

R

R

172/t

3.94

3.50

3.12

Compound 7 torsiono

chirality C2'

C4'

C5'

distance (Å)

4'H-5'H

2'H:4'H

2'H:5'H

4'H:5'H

R

S

S

176/t

1.93

3.52

3.04

R

R

R

173/g+

3.42

1.81

2.96

S

S

S

-176/t

3.47

3.75

3.05

S

R

R

172/t

3.84

3.57

3.14



7

Fig. (2). Huh7, HCT116, MCF7 and Mahlavu cells were incubated at 72 hours. All molecules and controls were implemented to the cells in dublicated with five different concentrations: 40, 20, 10, 5, and 2.5µM. After 72 h of incubation, SRB assays were generated and the IC50 values were calculated. mol. sieves, molecular sieves; tol, toluen; cycloadd., cycloaddition.

Looking at the computational dihedral angle (8'H-9'H) and through-space proton-proton distances (6'H:8'H, 6'H:9'H and 8'H:9'H) for compound 6a in Table 3, it is determined that C6'(S)|C8'(R)|C9'(R) stereochemistry matches best the experimental data for compound 6a with the corresponding H8′-H9′ dihedral angle in Table 1 and the NOE cross-peak

intensities in Table 2, which conforms to the X-ray crystallography data reported by Sebahar et al. [19], the spiro C7' atom possesses S stereochemistry in asymmetric 1,3-dipolar cycloaddition reaction products, therefore, so do compounds 6a-c &7.


Table 4.

Konyar et al.

IC50 values of camptothecin and compounds 6a-c & 7, determined on Huh7, MV (Mahlavu), MCF7 and HCT116 cancer cell lines. IC50 (µM) Compound

MCF7

HCT116

MV

Huh7

Camptothecin

˂1

˂1

˂1

˂1

10.1

8.4

48.6

32.1

6b

No inhibition

No inhibition

No inhibition

77.8

6c

11.2

10.5

26.3

36.6

7

4.8

3.9

14.9

8.2

6a

14

Computational dihedral angle (C8'H-C9'H) and throughspace proton-proton distances (6'H:8'H, 6'H:9'H and 8'H:9'H) determined for the C2'(S)|C4'(R)|C5'(R) stereochemistry of compound 7, listed in Table 3, points to the experimental data obtained for compound 7, listed in Tables 1 and 2. As compound 6b is the starting material for compound 7, the pyrrole ring of compound 6b possesses identical stereochemistry as the pyrrolidine ring of compound 7, which are now assigned as C6'(S)|C8'(R)|C9'(R) for compound 6b. Unfortunately, 8'H and 9'H proton peaks of compound 6c severely overlap at 4.76 ppm, disabling determination of 6'H:8'H and 6'H:9'H through-space distances. Nevertheless, a medium-to-weak NOE (~ 3.5 Å) and a strong NOE (< 3.5 Å) for the 4'H:6'H and 4'H:[C6'-CH2-] proton pairs, respectively, of the pyrrolo[2,1-c][1,4]oxazine heterocyclic ring were detected by ROESY, which is a condition met only when C6' adopts R stereochemistry that gives rise to through-space distances of 3.4 Å and 2.65 Å for the 4'H:6'H and 4'H:[C6'-CH2-] proton pairs, respectively. Otherwise, an S stereochemistry with C6'(S) would give rise to distances of 2.88 Å (with a very strong NOE cross-peak) and 3.22 Å (with a strong NOE cross-peak) for the 4'H:6'H and 4'H:[C6'-CH2-] proton pairs, respectively. This indeed makes sense because a large ortho-substituent on a phenyl group, such as chloride as in compound 6c (Fig. 1), is not sterically allowed to accommodate on the same side of the 2methylpropyl group bound to C6'(S). Therefore, C6' of compound 6c prefers to adopt R stereochemistry after the asymmetric 1,3-cycloaddition reaction due to steric reasons. Stereochemistry for C8' and C9' of compound 6c remains to be unknown, except that C8' and C9' are homesteroisomeric. Therefore we can explain with Cahn-Ingold-Prelog (CIP) priority rule the stereochemistry at position C8' of compound 6c. Note that at position C8' of compound 6c a molecule with the S assignment is produced because a Cl atom reverses the Cahn-Ingold-Prelog (CIP) priority order [20]. Cytotoxic effects of the compounds synthesized were determined on Huh7, MV, HCT116 and MCF7 cancer cell lines by the NCI-60 Sulforhodamine B Assay, using (S)-(+)Camptothecin as a positive control shown in Fig. (2). The cell inhibition (IC50) results are given in Table 4. In general, target compounds showed better cytotoxic activities against the MCF7 and HCT116 cancer cell lines. It was found that compound 7 exhibited the most potent inhibitory activity with IC50 values of 4.8 µM, 3.9 µM, 14.9 µM and 8.2 µM against the MCF7, HCT116, MV and Huh7 cell lines, re-

spectively. As compared to compound 6a [14], paranitrophenyl substitution in compound 6b knocked out cytotoxic activity against the MCF7, HCT116 and MV cell lines, while a 2.4-fold decrease in cytotoxic activity was observed against the Huh7 cell line. It is likely that p-nitrophenyl substitution increases repulsive electrostatic interactions at the site of action. Ortho-chlorophenyl substitution in compound c resulted in slight decrease in cytotoxic activity against the MCF7, HCT116 and Huh7 cell lines, due possibly to C6'(R) stereochemistry in compound 6c as compared to C6'(S) stereochemistry in compound 6a. CONCLUSION In this study, novel spiro(oxindole-3-3'-pyrrolidine) derivatives, compounds 6a-c & 7, were successfully synthesized in good yields, characterized and tested for their anticancer activities. It was determined that compounds 6a & 6b possess C6'(S)|C8'(R)|C9'(R) stereochemistry and compound 7 adopts C2'(S)|C4'(R)|C5'(R) stereochemistry. Compound 6c gains a C6'(R) stereochemistry while its C8'|C9' stereochemistry is unknown cytotoxicity studies suggest that compound 7, an amine hydrolysis product of compound 6b, gave rise to the highest anticancer activity against MCF7, HCT116, and Huh7 cancer cell lines, and therefore should be further studied as a potential drug candidate and as a lead compound for its novel derivatives. CONSENT FOR PUBLICATION Not applicable. CONFLICT OF INTEREST The authors declare that there is no conflict of interest amongst the authors involved in this study. ACKNOWLEDGEMENTS This study is funded by the Research Fund of Ankara University (Grant No. 10B3336001). The authors acknowledge Dr. Rengul Atalay at the Cancer System Biology Laboratory, Graduate School of Informatics, Middle East Technical University-Ankara, Turkey, and Deniz Cansen Yildirim at the Department of Molecular Biology and Genetics (DMBG), Faculty of Science, Bilkent University (BU) at Ankara, Turkey, for implementing the cytotoxic activity studies at the DMBG laboratories at BU-Ankara,


Turkey. We also would like to thank the instrumental Analysis Facility in the School of Farmacy at Ankara University-Ankara, Turkey for allowing us to use the NMR, LC-MS and Elemental Analysis instruments therein.


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