Cytotechnology 6: 227-232, 1991. 9 1991 KluwerAcademic Publishers. Printed in the Netherlands.

Inhibitory effect of new imidazole derivatives on the proliferation and nucleic acid synthesis of leukemic cells J. Nafziger 1, J.J. Guillosson 1, Y. Adam 2, C. Hecquet 3, M. Payard2 and M. Loiseau2 1Laboratoire d'Hrmatologie, Facult# des Sciences Pharmaceutiques et Biologiques, 4 Avenue de l"Observatoire, 75006 Paris, France; 2Laboratoire de Chimie Th#rapeutique et de Chimie Pharmaceutique, Facult# des Sciences Pharmaceutiques et Biologiques, 35 Chemin des Marafchers, 31062 Toulouse, France; 3Laboratoire de Pharmacologie Cellulaire, E.P.H.E., 15 Rue de l'Ecole de M#decine, 75006 Paris, France Received 25 October 1990; accepted in revisedform 10 June 1991

Key words: Imidazobenzothiazole derivatives, leukemic cells, cell growth, cell cycle kinetics, DNA binding, D N A and RNA synthesis

Abstract New derivatives of imidazothiazole and imidazobenzothiazole were tested in vitro for their potential antiproliferative activity. Four imidazobenzothiazole derivatives exhibited a cytotoxic activity against two leukemic cell lines, compound I being the most effective. Cell cycle kinetics studies showed that this drug delays the progression of cells from G1 to S and G2 M phases. An inhibitory effect on DNA and R N A synthesis was also observed. The antiproliferative effect of this compound, analogue of immunosuppressive agents, suggested that it could be of interest for a therapeutic use and for the synthesis of new derivatives.

Introduction L

Derivatives close to levamisole containing imidazothiazole and imidazobenzothiazole rings have been synthesized by different laboratories in order to find immunomodulator compounds (Wyeth Labs, 1981; Bender et al., 1985; Mase et al., 1986, 1988; Andreani et al., 1988). In addition to their activity on the immune system, some compounds have proved to be anti-tumorous (Andreani et al., 1988). This effect has called to our attention, and has led us to screen the antiproliferative activity of new imidazo (2, l-b) thiazoles and imidazo (2, l-b) benzo-thiazoles which we

had already synthesized (Amarouch et al., 1987, 1988). In the present study, the antiproliferative properties of five derivatives were tested. Each compound was evaluated in vitro against two leukemic cell lines (HL60 and REH cells). Their cytotoxic activity was evaluated by measuring their effects on the cell growth, cell kinetics and DNA and RNA synthesis of the leukemic cells. DNA binding was also investigated. Four derivatives of the imidazobenzothiazole showed a cytotoxic effect and compound I was the most active of them.

228 Material and methods c~

Cells Two human leukemic cell lines were used to test the antiproliferative effects of the different drugs synthesized: promyelocytic cells (ECACC, 850 11431 HL60) and ceils originally derived from an acute lymphoblastic leukemia (ATCC CRL 886 REH). Cells were grown as suspension culture in RPMI (105 cells/ml) supplemented with 10% fetal calf serum in an atmosphere containing 5% CO2.

"~ N~" NS0

HCI

C6H5 II

OMe III

Drugs New compounds containing imidazobenzothiazole ring were substituted by a phenyl group (I, II, III) or by a benzodioxane (IV). Furthermore, dehydrogenation was performed on compound II, and compounds III and IV were substituted by a methoxy group (Fig. 1). One analogue of levamisole was also synthesized: compound V, a derivative of the imidazothiazole ring substituted by a phenyl group. The synthesis of these compounds was previously described (Amarouch et al., 1987, 1988). Different doses of each drug ranging from 10-4 to 10-7 M were added to cultured cells at seeding. Drugs were dissolved in DMSO. The DMSO concentration never exceeded 0.2% and was never observed to affect cell growth or cycle progression in control cultures.

Cell growth After a 72 h drug treatment, the growth of control and treated cells was measured by counting them in a hemocytometer. Cell viability was checked by trypan blue exclusion and cell counts were expressed as the number of viable cells per milliliter of culture medium.

Cell kinetics DNA analysis was performed by flow cytometry

c6n5 ~

C6H5

N..,~ S .

...: N~

s.,

~____~______J

HCI

Levmnisole

Fig. i. Differentcompounds tested:I to IV (imidazobenzothiazole derivatives)and V (imidazothiazole derivatives).

on HL60 control and treated cells with compound I at 10-5 M, according to the modified method of Crissmann and Steinkamp (1973). Briefly, after three days of drug treatment, culture medium was changed and a new dose of drug was added. Cultures were incubated at 37~ and cells were harvested every 3 h by centrifugation, washed with 0.9% NaC1 solution and fixed in 70% ethanol for at least 30 min. Cells were then monodispersed and rinsed with 0.9% NaC1. For DNA staining approximately 1.106 cells were incubated with 1 ml Tris-HC1 buffer (0.1%, pH 7.4) containing 10 btg propidium iodide and 50 btg RNase (SIGMA). D N A analysis was performed every 3 h over a 24 h period in order to determine exactly when compound I had the greatest effect on cell cycle kinetics.

229 DNA binding Two different methods were used to investigate a possible DNA binding of the imidazobenzothiazole, derivatives: drug competition with D N A bound-ethidium and circular dichroism measurements of drug-DNA complex, Drug competition with ethidium was performed according to a method previously described (Gaugain et al., 1978; Baguley et al., 1981). The decrease in fluorescence of DNA-ethidium complex by addition of drug is due to both drug displacement of bound ethidium and quenching of the fluorescence of bound ethidium by the drug. Ethidium displacement assays were carried out at pH 7.4 ('Iris buffer) with various concentrations of compound I (10 -5 to 10-7 M) employing an exciting wavelength of 520 nm and monitoring emission at 585 nm. Ethidium bromide (SIGMA) was used at 10-6 M concentration and mixed to calf thymus DNA (SIGMA, nucleotide concentration: 1.4.10 -6 M), Circular dichroism measurements were performed in buffer cacodylate 0.025 M (pH 7.0), NaC1 100 raM. Compound I was mixed to calf thymus DNA (nucleotide concentration: 50.10 -6 M; drug to nucleotide ratio (D/P) in the range of 0 to 0.2). Samples were placed in a quartz cell (3 ml, 1 cm path length) thermostated at 25~ and circular dichroism spectra were analyzed at wavelengths ranging from 220 to 350 nm. Data were stored in a Minc 11/23 computer and corrected for the dichroism of the buffer.

treated cells. For labelling of DNA, culture plates were incubated for 18 h at 37~ in an atmosphere containing 5% CO2 and then collected on 0.45 gm nitrocellulose filters (MILLIPORE) by vacuum filtration. Filters were transferred to scintillation vials and radioactivity was determined by liquid scintillation spectrometry. The incubation time for (3H) uridine incorporation was 1 h and cells were then washed twice with RPMI. R N A was precipitated by the addition of 1 ml of cold trichloracetic acid at 10% (V/V) to 1 ml of cell suspension (30 rain at 4~ Cells were then collected on filters as described for (3H) thymidine incorporation. Filters were washed with cold trichloracetic acid at 5%, rinsed with 95% ethanol and transferred to scintillation vials.

Results

Effects on cell growth A decrease in cell growth was observed on the two leukemic cell lines with the four imidazobenzothiazole derivatives I, II, III, IV at 10-4 and 10-5 M concentrations (Table 1). Compound I exhibited the highest antiproliferative activity since a significant inhibition of cell growth was still observed at 10-6 M: 25% and 27% decrease for HL60 and REH respectively (p < 0.01). The IC50 value (concentration of drug which inhibited the cell growth by 50%) was lower for I than for the other compounds (< 3 gg/ml). No inhibitory effect was observed with the imidazothiazole derivative (compound IV).

Incorporation studies HE60 cells treated with different doses of compound I (5.10 -5, 10-5, 5.10 -6, 10-6 M) and control cells were incubated as previously described, for a 48 h period. Cells were then washed in RPMI and cell viability was checked by trypan blue exclusion. Viable cells were resuspended at 3.105/ ml in RPMI supplemented with 10% FCS and then placed into microtiter plates. To initiate incorporation, (3H) thymidine (10 ~tci/ml) or (3H) uridine (2 ~tci/ml) were added to control and

Effects on cell cycle kinetics DNA analysis performed on HL60 ceils treated with compound I, the most effective of the different derivatives tested, showed a reduction in the number of cells in S and G 2 M (Table 2). The cell decrease was most noticeable from the 18th to the 24th h after seeding, This was associated with a higher proportion of cells in G0/1 at the same time.

230

Table 1. Cell growth of HL60 and REH cells treated with the different drugs Compound

Control

Control in DMSO

10-4 M

10-5 M

10-6 M

10-7 M

IC50 ~tg/ml

I

500 + 90 a

535 + 86 425 + 44

395 + 66 (25%) 385 + 80

2.9

510 + 60

III

452 + 47

473 + 32

525 + 80

517 __+.63

6.75

IV

508 + 75

540 + 69

510 + 47

470 + 51

8.15

V

504 + 76

506 + 85

270 + 78 (50%) 340 + 60 (20%) 330 + 68 (30%) 350 + 27 (35%) 544 + 136

460 _+ 84

!I

5 + 1.5 (99%) 175 + 25 (59%) 57 + 8 (88%) 162 + 20 (70%) 377 + 66

544 + 90

532 + 62

-

I

175 + 42

172 + 36 160 + 27

135 + 18

7.50

III

168 + 41

160 + 32

125 + 25 (27%) 125 + 28 (22%) 161 + 28

3.0

154 + 17

154 + 16

4.50

IV

210 + 22

215 + 48

189 --- 22

170 + 33

155 + 18 (28%) 175 + 13

187 + 21

V

88 + 15 (49%) 106 + 15 (33%) 97 + 22 (39%) 150 + 26 (30%) 174 + 16

140 + 32

II

28 + 9 (83%) 46 + 10 (71%) 9+ 3 (94%) 86 + 16 (60%) 127 + 11

HL60 cells

410 _+ 78

15

REH cells

173 + 38

17.0 -

The number of cells was determined after a 72 h treatment. A decrease in cell proliferation was observed at 10--4 and 10-5 M with the four imidazobenzothiazole derivatives tested. A reduction of cell growth was still observed with I at 10-6 M on the two leukemic cell lines and the lowest IC50 value was obtained with this compound. aMean + SEM of four determinations in duplicate (No. of viable cells/ml); ( ) Percentage of cell decrease.

Table 2. Cell cycle kinetics of control and compound I treated cells

G1 S G2M

T Compound I 10-5 M T Compound ! 10-5 M T Compound I 10-5 M

3H

6H

9H

12H

15H

18H

21H

24H

55 52 34 34.5 11 13.5

56.5 53.5 29 33 14.5 13.5

57.5 68.5 32 24 10.5 7.5

61.5 69 27 25.5 11.5 5.5

58 69 33.5 25 8.5 6

57 71.5 b 34 23 c 9.5 5c

57 73.5 b 34 23 c 9 3.5 c

60.5 71.5 b 32 26 e 7.5 2.5 e

The relative percentage of control and treated HL60 cells in G0/1, S and G 2 M phases were estimated according to a planimetric analysis of DNA per cell distribution histogram. A decrease in the number of cells in the S and G2 M phases was associated with an increase in the number of cells in G 1 probably as the result of the prolongation of this phase. aMean of three determinations in duplicate; bp < 0.01 statistically significant versus the control; cp < 0.02 statistically significant versus the control.

DNA binding

tions of compound

I. T h e s e r e s u l t s i n d i c a t e t h a t

this

displace

drug

did

not

No decrease in the fluorescence of DNA-ethidium

DNA-binding

complex

ism measurements

was observed with different concentra-

ethidium

from

its

sites. Furthermore, circular dichrodid not reveal any modifica-

231

I011]

50.............

lO-6M

~iA_J

i 5.10"-6M 10"5M'

5.10-5MDrugconcentratton )

Fig. 2. Nucleic acid synthesis in HL60cells treated with compound L Values(a) are plotted as the percentage of (3H) thymidine and (3H) uridine incorporation compared with control in the absence of drug. A dose-dependentreduction of DNA and RNA synthesis was observed after a 48 h drug treatment; aMean of three experiments in triplicate.

tion o f the spectra at wavelengths ranging from 220 to 350 nm.

E f f e c t s on D N A a n d R N A synthesis

The effects o f c o m p o u n d I on the rates o f D N A and R N A synthesis were assessed by measuring cellular incorporation o f tritiated thymidine and uridine in HL60 cells. A 48 h exposure to the drug induced a dose-dependent inhibition o f D N A and R N A labeling (Fig. 2) and the ICs0 values (1.7 ~tg/ml and 3.4 ~tg/ml respectively for 50% inhibition o f D N A and R N A synthesis) were in the range o f the 50% inhibitory concentration o f cell proliferation (3 ~tg/ml).

Discussion A n in vitro system was used in the present study to determine the potential cytotoxic effect of five imidazothiazole or imidazobenzothiazole derivatives. An antiproliferative effect was obtained on the two leukemic cells lines HL60 and REH, with

four of the five compounds tested, at 10 -4 and 10-5 M concentrations and at 10 -6 M with compound I. Examination of the structure-activity relationship showed that imidazobenzothiazole derivatives only exhibited an antiproliferative activity (I, II, III, IV compounds). Indeed, no inhibitory effect o f cell growth was obtained with the imidazothiazole derivatives V, as observed with its analogue, levamisole, in preliminary experiments (data not shown). The most effective o f the four imidazobenzothiazole derivatives tested was I. Dehydrogenation o f the molecule (II) or substitution by a m e t h o x y group (III, IV) and/or by a benzodioxane (IV) induced a reduction o f the activity. D N A flow cytometric analysis showed that the reduction in the growth rate observed with compound I was probably the result o f an accumulation o f cells in G0/1 at the expense of the S and G 2 M phases. Exposure to I led to a general slowingdown o f the cell cycle progression with a reduction of the number of dividing cells. The same alterations in cell cycle distribution were previously reported with other antineoplastic agents (Bachur et al., 1973; W e c k b e c k e r et al., 1987; Nafziger et al., 1989). Ethidium competition assay and circular dichroism measurements did not allow to disclose a drug-DNA binding. The decrease o f D N A and R N A synthesis observed in this study with compound I could result o f an interaction with enzymes which play a role in acid nucleic metabolism. W e can hypothesize that imidazobenzothiazole derivatives could act on ribonucleotide reductase, an e n z y m e which catalyzes the conversion o f ribonucleotides to deoxyribonucleotides, the rate-limiting step to D N A synthesis (Weber, 1983; Cory and Chiba, 1985). Indeed, Weckbecker showed with aminoguanidine derivatives the same alterations in cell cycle kinetics and a decrease o f D N A and R N A synthesis associated with an inhibition of the ribonucleotide reductase (Weckbecker et al., 1987). The lowest ICs0 value was obtained with I and this c o m p o u n d should be considered for the synthesis of new derivatives and for potential thera-

232 p e u t i c use. I n d e e d , w e c a n p o s t u l a t e that this d r u g c o u l d d i s c l o s e in vivo a d u a l action: firstly a n a n t i p r o l i f e r a t i v e activity, as s h o w n i n this study, secondly an immunomodulatory function. Indeed, this c o m p o u n d shares the i m i d a z o t h i a z o l e m o i e t y with levamisole, a welt-know immunomodulator. F u r t h e r m o r e d i f f e r e n t a u t h o r s h a v e also r e p o r t e d an i m m u n o l o g i c a l activity of a n u m b e r of imidaz o b e n z o t h i a z o l e d e r i v a t i v e s ( W y e t h L a b s , 1981; B e n d e r et al., 1985; M a s e et al., 1986, 1988). F o r these r e a s o n s this c o m p o u n d c o u l d b e o f i n t e r e s t for p o s s i b l e t h e r a p e u t i c u s e i n a s s o c i a t i o n w i t h other a n t i n e o p l a s t i c a g e n t s a c t i n g o n differe n t p h a s e s o f the cell cycle.

Acknowledgements W e are g r a t e f u l to G e n e v i e v e A v e r l a n t for h e r e x p e r t t e c h n i c a l assistance. W e t h a n k C. A u c l a i r for the c i r c u l a r d i c h r o i s m m e a s u r e m e n t s . ( I n s e r m U 140, C N R S U R A 147, I n s t i t u t G u s t a v e R o u s s y , Villejuif, France).

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Address for offprints: J. Nafziger, Laboratoire d'Hrmatologie, 4 Avenue de l'Observatoire, 75006 Paris, France