Substituted (E)--(benzoyl)acrylic acids suppressed survival of neoplastic human HeLa cells

Z.JURANIC¹, LJ. STEVOVIC² B. DRAKULIC³, T.STANOJKOVIC¹, S.RADULOVIC¹ AND I JURANIC²

1. Institute for Oncology and Radiology, Belgrade;

2. Faculty of Chemistry, P.O.Box 158, Belgrade;

3. Center for Chemistry ICTM, Belgrade, Yugoslavia

KEYWORDS: Benzoylacrylic acids, HeLa cells, cytotoxicity, QSAR

Abstract

Bacteriostatic activity of some of alkyl substituted (E)--(benzoyl)acrylic acids was shown earlier. The aim of this study was to investigate the antiproliferative action of 19 alkyl-, or halogeno-, or methoxy-, or acetamido substituted (E)--(benzoyl)acrylic acids, against human cervix carcinoma, HeLa, cells.

Target HeLa cells, were continuously treated with increasing concentrations of substituted (E)--(benzoyl)acrylic acids during two days. MTT test was used for assessment of antiproliferative action of this group of compounds.

Treatment of HeLa cells with 4-methyl-, 4-fluoro-, 4-chloro-, 4-bromo- and 4-methoxy derivatives of (E)--(benzoyl) acrylic acid lead to the expression of cytostatic activity against HeLa cells (IC50 were in the range from 31-40 M). Their antiproliferative action was less than that of the basic compound of (E)--(benzoyl)acrylic acid whose IC50 was 28.5 M. 3,4-Dimethyl-, 2,4-dimethyl- and 2,5-dimethyl- substituents, as well as 4-ethyl- and 3,4-dicloro- and 2,4-dichloro- derivatives, have stronger cytostatic activity than corresponding mono- substituted and parent compound. Their IC50 were 18.5 M; 17.5 M; 17.0 M; 17.5 M; 22.0 M and 18 M, in the already given order. 4-i-Propyl-, then 4-n-butyl- derivatives exerted higher cytostatic activity than compounds with lower number of methylene -CH₂- groups in the substituent. Their IC50 were 14.5 M and 6.5 M respectively. 2,5-Di-i-propyl- and 4-t-butyl- derivatives expressed the most strong antiproliferative action against investigated HeLa cells, IC50 being 4.5 M and 5.5 M in the already mentioned order.

Investigated compounds affected survival of HeLa cells, expressing strong structure-activity relationship of the Hansch type.

Introduction

In this study we investigated the cytostatic action of various (E)--(benzoyl)acrylic acids. Determination of the extent of bacteriostatic activity of a similar group of compounds was done earlier.¹ It was found a marked increase in activity against gram positive bacteria in para alkyl-substituted (benzoyl)acrylic acids with substituent group ranging from methyl to nonyl. para-Methoxy- and ethoxy- derivatives were of the same order of activity as the methyl- or ethyl- compounds. The p-chloro and p-bromo benzoyl acrylic acids were only moderately active. The antibacterial activity of this group of compounds determined on Staphilococcus aureus, Escherichia coli and Kllebsiella was ascribed to the presence of the highly conjugated benzoyl-acrylic system which may react with biologically essential -SH groups. Bowden et al.,² reported the data on the bacteriostatic activity of a series of (E)-3-(aroyl)acrylic acids and their methyl esters. They found that bacteriostatic activity measured as minimum inhibitory concentrations (C) are well correlated with the Hansch-type equation, where is the partition substituent constant. Results reported by Bowden et al.,³ performed on a series of substituted (E)-3-(4-phenylbenzoil)acrylic acids indicated that nucleophilic addition at the ketovinyl double bond is not the mode of the bacteriostatic action of these group of compounds. The receptor for bacteriostatic action appears to be related to the lipophylic region associated with the benzoylphenyl group.

On the other side, the cytostatic activity of similar compound, sodium salt of the (E)-[-bromo--(4-methoxy)benzoyl]acrylic acid, (Z)-Br,H (Cytembena) was extensively investigated in experimental models, as well as in patients with various malignancies.^4-7 Principal mode of antitumor action of this compound was ascribed to its inhibitory effect on tetrahydrofolate formylase. It was found that compound without halogen on C is inactive in inhibition of this enzyme.

In this study nineteen aroylacrylic acids were synthesized, and their cytostatic activity toward human cervix carcinoma, HeLa cells, was studied. Structures of the acids studied are given in Table 1.

Table 1. Structures of substituted (E)--(aroyl)acrylic acids studied in this work.

Compound	R1	R2	R3	R4
1	H	H	H	H
2	Me	Me	H	H
3	Me	H	Me	H
4	H	H	Me	Me
5	Me	H	H	H
6	Et	H	H	H
7	i-Pr	H	H	H
8	H	H	i-Pr	i-Pr
9	n-Bu	H	H	H
10	t-Bu	H	H	H
11	CH2CH2CH2CH2		H	H
12	Cl	H	Me	H
13	Cl	H	H	H
14	Br	H	H	H
15	Cl	Cl	H	H
16	Cl	H	Cl	H
17	OMe	H	H	H
18	AcNH	H	H	H
19	F	H	H	H

Material and Methods

Synthesis of aroylacrylic acids: (E)--(Aroyl)acrylic acids were prepared, according to the Papa et al.⁸ by a modification of the Friedel-Crafts reaction, by adding an aromatic substrate to a solution of maleic anhydride and anhydrous aluminum trichloride (molar ratio 1:2) in 1,1,2,2-tetrachloroethane. Instead of tetrachloroethane we used 1,2-dichloroethane and moderately higher yields were obtained.⁹

Typical experimental procedure

In a 100 ml two-necked flask equipped with magnetic stirrer, reflux condenser and dropping funnel, 6.125 g (62.5 mmol) of maleic anhydride was suspended in 25 ml of dry 1,2-dichloroethane. After 10 minutes 15.5 g (125 mmol) of powdered anhydrous aluminum trichloride was added and the reaction mixture was stirred for another 20 minutes, until a homogeneous yellow suspension was formed. An aromatic substrate (62.5 mmol) was added at such a rate to keep the temperature (below 50 C) and foaming under control. The reaction mixture was stirred for 9 h at 20 C; 0.5 h at 60 C; refluxed for another 0.5 h and then poured into 200 g of ice/water mixture (1:1) with 20 ml concentrated hydrochloric acid. The dichloroethane was removed by steam distillation. The crude acid was collected by filtration, dissolved at 20 C in water with sodium carbonate at pH 8.5-9.0 and traces of aluminum hydroxide were filtered off. The mother liquor was acidified with hydrochloric acid to pH 1.0 and the pure acid was collected by filtration, washed with water and dried in the open air. Yields, recrystallization solvents, and melting points are given in Table 2.

Table 2. Crystallization solvent, melting points and yields of compounds (1-19)

Compound	Solvent	M.p. C	Yield (%)
(1) H	C6 H6	98-99	74
(2) 3,4-dimethyl	EtOH	123	72
(3) 2,4-dimethyl	EtOH:H2O	113-114	70
(4) 2,5-dimethyl	EtOH:H2O	89-90	95
(5) 4-methyl	C6 H6	139-140	75
(6) 4-ethyl	C6 H6	105-106	94
(7) 4-iso-propyl	C6 H6	103-103.5	72
(8) 2,5-di-iso-propyl	cyclohexan : hexan	96-99	71
(9) 4-n-butyl	C6 H6	90-91	72
(10) 4-tert-butyl	C6 H6	125-127	75
(11) -tetralinoyl	EtOH	147-149	72
(12) 4-chloro-2-methyl	H2O	107-110	89
(13) 4-chloro	H2O	154-155	89
(14) 4-bromo	H2O	159-160	82
(15) 3,4-dichloro	cyclohexan : hexan	143	66
(16) 2,4-dichloro	H2O	190	22
(17) 4-methoxy	H2O	138-139	72
(18) 4-acetamido	EtOH	240	35
(19) 4-fluoro	C6 H6	142	91

Stock solution of examined compounds was made in dimethylsufoxide, at concentration range 42-62 M and then various dilutions were prepared in nutrient medium, (RPMI 1640 medium supplemented with l-glutamine (3 mmol/L), streptomycin (100 g/mL), and penicillin (100 IU /mL), 10% heat inactivated fetal bovine serum, FBS, and 25 mM Hepes, adjusted to pH 7.2 (by bicarbonate solution) to various final concentration (between 1.3 - 80 or 142 M). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Sigma Chemicals (St. Luis, MO, U.S.A.). MTT was dissolved, 5 mg/ml in phosphate buffer saline pH 7.2, and filtered through Millipore filter, 0.22 m, before use. RPMI 1640 cell culture medium and FBS were products of ICN Pharmaceuticals Co, USA.

Treatment of HeLa cells. HeLa cells, were seeded into 96-well microtiter plates, 2000 cells per well. After 20 hours, to one series of wells without or with HeLa cells, five different concentrations of investigated substituted (E)--(benzoyl)acrylic acid derivative were applied to the wells to various final concentrations, except to the control wells, the wells with cells grown in a nutrient medium only. All concentrations were set up in triplicate. Nutrient medium with corresponding concentrations of investigated agent, but without of cells was used as blank, in triplicate too.

Determination of cell survival. HeLa cell survival was determined by MTT test, according to Mosmann¹⁰ with modification by Ohno and Abe,¹¹ 44 hours upon addition of the drug, as it was described earlier.¹² Briefly, 20 L of MTT solution (5 mg/ml PBS) were added to each well. Samples were incubated for further four hours at 37 C in 5% CO₂ and humidified air atmosphere. Then, 100 L of 10% SDS in 0.01M HCl were added to the wells. Optical density (OD) at 570 nm was red the next day. Percent of control of OD was used as a measure of cell survival (%), therefore OD of a sample with cells grown in the presence of various concentration of substituted (E)--(benzoyl)acrylic acid derivative, OD, was divided with control optical density, ODc, (the OD of cells grown only in nutrient medium) x100. (It was implied that OD of blank was always subtracted from OD of a corresponding sample with target cells).

Results

Concentrations of examined agents that induced decrease in 50% in cell survival (IC50), determined under the exactly same experimental conditions, are given in Table3.

Table 3. IC50 values of the compounds studied

Compound	IC50 [M]	Compound	IC50 [M]	Compound	IC50 [M]	Compound	IC50 [M]
1	28.5	6	17.5	11	8.5	16	18
2	18.5	7	14.5	12	31.5	17	36
3	17.5	8	4.5	13	40	18	>117
4	17	9	6.5	14	31	19	35
5	40	10	5.5	15	22

It could be seen that 4-methyl-, 4-fluoro-, 4-chloro-, 4-bromo- and 4-methoxy-derivatives of (E)--(benzoyl)acrylic acid expressed similar cytostatic activity against HeLa cells (IC50 were 40; 35; 40; 31 and 36 M, respectively). This antiproliferative action was less than that of the basic compound of (E)--(benzoyl)acrylic acid whose IC50 was 28.5 M.

3,4-Dimethyl-, 2,4-dimethyl- and 2,5-dimethyl- substituted, as well as 4-ethyl- then 3,4-dicloro- and 2,4-dicloro- derivatives have stronger cytostatic activity than corresponding mono- substituted and parent compound: their IC50 were: 18.5; 17.5; 17.0; 17.5; 22.0 and 18 M in the already given order.

4-i-Propyl-, then 4-n-butyl- derivatives exerted higher cytostatic activity than compounds with lower number of methylene -CH₂- groups in the substituent. Their IC50 were 14.5 and 6.5 M respectively.

2,5-Di-i-propyl- and 4-tert-butyl- derivatives expressed the most strong antiproliferative action against investigated HeLa cells, IC50 being 4.5 and 5.5 M in the already mentioned order.

IC50 values have very good correlation with lipophylicity of studied compounds.

Estimation of logarithm of partition coefficient [n-Octanol/Water] Log(P)=log(K_OW) was done by Crippen's fragmentation method.¹³ For unsubstituted compound 1, the literature experimental value of log K_OW =1.530 was used.¹⁴ The plot of -log(IC50) versus log(P) is given in Figure 1.

Regression equation is;

log(1/IC50) = 0.418(+-0.042)*log(P) - 2.202(+-0.096)

having regression coefficient 0.924.

Discussion

Results obtained in this work showed that (E)--(benzoyl)acrylic acids expressed the cytostatic activity toward HeLa cells in vitro. A marked increase in cytostatic activity as 4-alkyl substituent in (E)--(benzoyl)acrylic acid changes from methyl- to ethyl-, i-propyl-, n-butyl-, and to t-butyl- was observed.

The similar increase in bacteriostatic activity against gram positive bacteria in para-alkyl- substituted (benzoyl)acrylic acids with substituent group ranging from methyl to nonyl was reported earlier.¹ para-Methoxy- derivative was of the same order of bacteriostatic activity as the methyl compounds.

Changes in the structure of the substituent could also lead to the suppression of the cytotoxic activity, as it was found for (E)--(4-acetamido)benzoyl acrylic acid.

Cytostatic activity of (E)--(aroyl)acrylic acids is not connected to the inhibition of the tetrahydrofolate formylase, as it was found⁶ that compound without halogen on C is inactive in the inhibition of this enzyme.

In conclusion, investigated substituted (E)--(aroyl)acrylic acids showed antiproliferative activity against human carcinoma cells in vitro. A simple Hansch-type correlation of IC50 with logP was found. Linear regression has high correlation coefficient (0.924), which means that biological activity is directly correlated with lipophylicity, and no additional effects need to be considered. Based on this result, the synthesis of more lipophylic (E)--(benzoyl)acrylic acids is in preparation.

List of abbreviations:

IC50 - concentration of agent that induces a 50 % decrease in cell survival.
FBS - fetal bovine serum
MTT - 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
HeLa cells - human cervix carcinoma cells
RPMI 1640 - standard nutrient medium

Acknowledgements: - The authors thank Mrs Tatjana Petrovi for her excellent technical assistance. The authors are grateful to The Serbian Ministry of Sciences and Technology and to the Boehringer Ingelheim Pharma for financial support

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Supstituisane (E)--(benzoil)akrilne kiseline umanjuju prezivljavanje neoplastickih humanih HeLa celija

Z.JURANIC¹, LJ. STEVOVIC² B. DRAKULIC³, T.STANOJKOVIC¹, S.RADULOVIC¹ AND I JURANIC²

1. Institut za onkologiju i radiologiju, Beograd;

2. Hemijski Fakultet, P. Fah 158, Beograd;

3. Centar za hemiju IHTM, Beograd, Jugoslavija

IZVOD

Jos ranije je pokazana bakteriostatska aktivnost nekih alkil supstitutisanih (E)--(benzoil)akrilnih kiselina. Cilj ovog rada je bilo proucavanje antiproliferativnog dejstva 19 razlicitih (E)--(benzoil)akrilnih kiselina; alkil-, ili halogeno-, ili metoksi-, ili acetamido-supstituisanih, na celije humanog karcinoma grlica materice, HeLa celije.

Ciljne HeLa celije, su kontinualno tretirane rastucim koncentracijama supstituisanih (E)--(benzoil)akrilnih kiselina tokom dva dana. MTT Test je koriscen za utvrdjivanje antiproliferativnog dejstva ove grupe jedinjenja.

Tretiranje HeLa celija sa 4-metil-, 4-fluoro-, 4-hloro-, 4-bromo- i 4-metoksi derivatima (E)--(benzoil)akrilne kiseline dovelo je do ispoljavanja citostatske aktivnosti prema HeLa celijama (IC50 su izmedju 31-40 M). Njihovo antiproliferativno dejstvo je bilo manje nego kod osnovnog jedinjenja, (E)--(benzoil)akrilne kiseline, cije IC50 je bilo 28,5 M. 3,4-Dimetil-, 2,4 dimetil- i 2,5-dimetil- supstituisani, kao i 4-etil- te 3,4-dihloro- i 2,4- dihloro- derivati, imaju jacu citostatsku aktivnost od odgovarajuceg mono- supstituisanog i osnovnog jedinjenja. Njihove IC50 vrednosti su 18,5 M; 17,5 M; 17,0 M; 17,5 M; 22,0 M i 18 M, u navedenom redosledu. 4-i-Propil-, i 4-n-butil- derivati pokazuju visu citostatsku aktivnost od jedinjenja sa manjim brojem metilenskih -CH₂- grupa u supstituentu. Njihove IC50 vrednosti su 14,5 M odnosno 6,5 M. 2,5-Di-i-propyl- i 4-t-butyl- derivati ispoljavaju najjace antiproliferativno dejstvo prema ispitivanim HeLa celijama, IC50 su 4,5 M i 5,5 M u navedenom redosledu.

Proucavana jedinjenja uticu na prezivljavanje HeLa celija, ispoljavajuci izrazitu relaciju Hansch-ovog tipa izmedju strukture i bioloske aktivnosti.