Investigational New Drugs 2, 35-47 (1984) �9 1984, Martinus Nijhoff Publishers. Printed in the Netherlands

Antitumor cell and antimetabolic effects of 5-ethyl-2'-deoxyuridine and 5'-substituted 5-ethyl-2'-deoxyuridine derivatives

Jan Balzarini, 1 Erik De Clercq, 1 Gebhard Kiefer, 2 Klaus Keppeler, 2 and Alfred Buchele 2

Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium

2 Robugen Pharmaceuticals, P.O. Box 266, D-7300 Esslingen, West Germany

Key words: 5-ethyl-2'-deoxyuridine, routine leukemia L1210, DNA synthesis

Summary

A series of forty 5'-ester derivatives of 5-ethyl-2'-deoxyuridine (EDU) have been evaluated for their in- hibitory effects on the growth and metabolism of routine leukemia L1210 cells. Several EDU esters proved as potent as EDU in their inhibitory effects on L1210 cell growth (inhibitory dose-50: 5-10/ ,g /ml) , suggesting that these esters were readily hydrolyzed to release the parent compound EDU. That the EDU esters had to be hydrolyzed first to EDU was further suggested by the dependence of their antiproliferative action on the thymidine kinase activity of the cells. It was further ascertained that EDU and its esters acquired their an- tiproliferative effects by an interaction with dCTP biosynthesis, possibly at the CDP ribonucleotide reductase step. Under conditions where thymidine was readily incorporated, we were unable to demonstrate any incor- poration of EDU into L1210 cell DNA.

Introduction

Several 5-substituted 2'-deoxyuridine (dUrd) nu- cleoside analogs have proven efficacious in the treatment of cancer in either humans or experimen- tal tumor models. For example, 5-fluoro-dUrd has shown high activity against murine leukemia L1210 cells, Walker carcinoma 256, adenocarcinoma 75, sarcoma 180 and Lewis lung carcinoma in animals (1). In its free base form, 5-fluoro-dUrd has since several years been used in the treatment of patients with disseminated breast and colon cancers (2). Another dUrd derivative, 5-trifluoromethyl-dUrd, has also been the subject of clinical trials. It was found effective against breast carcinoma and cer- tain pediatric malignancies (3). Other 5-substituted 2'-deoxyuridines have not been submitted to exten-

sive antitumor studies. Some dUrd derivatives, i.e. 5-ethynyl-dUrd (4), 5-vinyl-dUrd (4), 5-mercapto- dUrd (5); 5-formyl-dUrd (6), 5-nitro-dUrd (7, 8), 5-hydroxymethyl-dUrd (9, 10) and 5-ethyl-dUrd (11, 12), were reported to inhibit the growth of

either L1210 or other tumor (Ehrlich ascites car- cinoma, B5 59 melanoma, namalva, raji) cells in culture. However, the mechanism of antitumor ac- tion of these dUrd derivatives has not been clearly established.

We now report on the antitumor cell and anti- metabolic activities of a series of 5'-substituted derivatives of EDU, including 5'-0-pivaloyl-, 5'- 0-butyryl-, 5'-0-valeryl-, 5'-0-benzoyl-, 5'-0-(1- adamantanecarbonyl)-, 5 '-0-carbamoyl-EDU and various other EDU ester derivatives. These EDU esters were evaluated for their inhibitory effects

Address for reprints: Prof. Dr. Erik De Clercq, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroeders- straat 10, B-3000 Leuven, Belgium

36

on the growth of several murine leukemia L1210 cell lines. Our studies were conducted with murine leukemia L1210 cells since (i) these cells are widely used in the evaluation of new anticancer agents as compared to known active drugs, (ii) this test system has proven predictive for the growth-in- hibiting effects of 5-substituted 2'-deoxyuridines on human lymphoblast cell lines (12), and (iii) several mutant L1210 cell lines have been developed which are deficient in dThd kinase or dCyd kinase activity. This enabled us to investigate the mechanism of antitumor action of the test com- pounds. Several EDU esters showed an antipro- liferative effect that was comparable to that of the parent compound. Additional experiments were designed to establish the mechanism of action of EDU and its esters.

Materials and methods

Ce//s

Murine leukemia L1210 cells were grown in 75 cm a tissue culture flasks (Falcon 3024F; Becton Dickin- son France S.A., Grenoble, France) in Eagle's minimal essential medium, supplemented with 10~ (v/v) inactivated fetal calf serum (Gibco Bio-Cult, Glasgow, Scotland, U.K.) and 2 mM L-glutamine (Flow Laboratories, Irvine, Scotland, U.K.).

Two sublines (referred to as L1210/BdUrd and L1210/araC) have been selected from the parental L1210/0 cells for their ability to grow in the presence of 5-bromo-dUrd (260 txg/ml) and araC (1 ~g/ml), respectively (12, 13). The L1210/BdUrd cell line is deficient for deoxythymidine kinase (12); the Ll210/araC cell line is deficient for deoxycyti- dine kinase (13).

Compounds

2'-Deoxyuridine (dUrd), 2'-deoxythymidine (dThd) and 2'-deoxycytidine (dCyd) were obtained from Sigma Chemical Company, St. Louis, Mo. The 5'-derivatives of 5-ethyl-2'-deoxyuridine (EDU) that were tested for their cytotoxic and anti- metabolic effects are depicted in Fig. 1. The source

of the test compounds was as follows: the prepara- tion of the compounds listed in Fig. 1 has been de- scribed earlier. Compound 1 is the active compo- nent of Aedurid | -Gel. The 5'-phosphorus deriva- tives of EDU 2 - 6 were prepared according to literature methods (14-16), the 5'-sulphur derivative 7 was prepared by the appropriate reac- tion of the 5 '-iodo derivative of EDU with sodium sulphite (17). The 5 '-ester derivatives 8, 12-26 and

3 1 - 3 5 were obtained by reaction of EDU with the corresponding acid chloride in a mixture of pyri- dine/DMF as described elsewhere (18). 5'-Carba- moyl-EDU (36) was synthesized following the pro- cedure used for the preparation of nucleoside 5'- carbamates (19). Of the derivatives modified at the 5' and 3'-position, compound 29 was obtained by reaction of compound 26 with diethyl chlorophos- phate in pyridine, compounds 26 and 28 by the method of Sowa et al. (14), and compound 30 by the method of Verheyden et al. (15). The 5 '-carbamoyl derivatives 37 and 38 were prepared by the reaction of compound 1 with the appropriate isocyanates (20). Compound 39 was obtained from the 5'-azido derivative of 1 by hydrogenolysis on Pd/C catalyst, as described for analogous com- pounds (21). Likewise, compounds 40 and 41 were prepared from compound 39 (22).

Radiochemicals

The radiolabeled nucleosides (1', 2'-3H) dUrd (specific radioactivity, 31 Ci/mmole), (methyl) H) dThd (specific radioactivity, 47 Ci/mmole) and (5 -3H)dCyd (specific radioactivity, 22 Ci/mmole) were obtained from the Radiochemical Centre (Amersham, U.K.).

(2-14C)EDU (specific radioactivity, 0.2 mCi/ mmole) was provided by Robugen GmbH, Ess- lingen, West Germany.

Test procedures

The methods for evaluating antitumor cell and anti- metabolic activity have previously been described (23).

Incorporation of (2)4C)EDU or (methyl )H) dThd into L1210/0 cell DNA was evaluated by

O

o H2cH3 RICH2 0

Number Compound

I EDU

2 EDU-5'-monophosphate disodium salt

3 EDU-5'-diethylphosphate

4 EDU-5'-methylphosphonate sodium salt

5 EDU-5'-carboxylatomethylphosphonate diammonium salt

6 5'-cyclophosphamide-EDU

7 5-ethyl-2',5'-dideoxyuridine-5'-sulfonic acid sodium salt

8 5'-C-pivaloyl-EDU

9 5'-O-pivaloyl - EDU - 3'-monophosphate disodium salt

I0 5'-O-pivaloyl - ZDU - 3'-monophosphate diammonium salt

]I 5'-O-pivaloyl - EDU - 3'-methylphosphonate sodium salt

]2 5'-O-n-butyryl-EDU

13 5'-O-isobutyryl-EDU

]4 5'-O-,(2-phenylbutyryl)-EDU

15 5'-O-(2-(ethoxycarbonyl)hutyryl)-EDU

16 5 ' -O- (3,3-dimethylbutyryl) -EDU

/rig. f. 5'-Derivatives of 5-ethyl-2'-dcoxyuridine (EDU).

_R 1

HO-

O-P-C-

O

C2HsO-P-0-

0

C2H 5

o G ~ Na O-P-O-

I CH 3

O 0

%c~ J-o- o(-~ 2 ; |

~4 | NH~ @

NH

N~ @ @O-!-

CH3-C-C-O-

CH 3

N3~ o CN3-C-C-O-

CH 3

CH3-C-C-O-

CH 3

CH3-~-C-O-

CH 3

0

%-~c~24-c-o-

CH3-CH-C-O-

CH3~Ctt~C-O-

fi Clt3~CH2-CH-C-0-

C=C I 0 C2H 5

CH3-C-CH2-C-O-

CH 3

_R 2

HO =

HO-

HO-

NO-

NO-

HO-

HO-

q ~ | | o-~'-o-

Na +~

~:4 | @o-P-o-

o ~a| |

CH 3

HO-

HO-

HO-

37

38

17 5'-O-(4-(me~hoxycarbonyl)butyryl)-EDU

18 5'-O-valeryl-EDU

19 5'-O-isovaleryl-EDU

20 5,-O-~methoxycarbonyl)pentanoyl)-EDU

2) 5'-O-lauryl-EDU

22 5'-O-(2,4-dichlorobenzoyl)-EDU

5'-O-(2,6-dichlorobenzoyl)-EDU

5'-O-(3,4-dichlorobenzoyl)-EDU

25 5'-O-(2-furanecarbonyl)-EDU

26 5'-O-(l-adamantanecarbonyl)-EDU

27 5'-O-(l-adarmantanecarbonyl) - EDU - 3'-monophosphate disodium salt

28 5'-O-(l-adamantanecarbonyl) - EDU - 3'-monophosphate dian~nonium salt

29 5'-O-(l-adamantanecarbonyl)- EDU - 3'-diethylphosphate

5'-O-(l-adamantanecarbonyl)- EDU- 3'-methylphosphonate sodium salt

31 5'-O-(phenoxycarbonyl)-EDU

32 5'-O-(4-chlorphenoxyacetyl)-EDU

33 5'-O-(ethoxycarbonyl)-EDU

Fig. 1. (cont.)

o

~ H2-(CH2) 2-C-O- C=O

'o CH 3

o

CH3-(CH2) 3-C-O-

CH3-CH-CH2-C-O-

0

~H2-(CH 2) 3-C-

C=O,

%3 CH3-(CH 2 ) ]0-C-O-

CI

Cl @i_o_ e l

Cl

O

C-O-

o

C-O-

0

C-O-

o

C-O-

o

C-O-

Cyo-i-0- O-CH2-C-O-

?, C2H~-C-O-

HO-

HO-

HO-

HO-

HO-

HO-

HO-

HO-

HO-

N~ | �9 O-P-O-

| | o-P-o-

NH 4

0

C2H5-O-P-O-

OC2H 5

0 Na Q ~) O-P-O-

I CH 3

HO-

HO-

HO-

39

34 5'-O-methyloxalyl-EDU

35 5'-H-echylmalonyl-EDU

36 5'-O-carbamoyl-EDU

37 3',5'-di-O-(2-chloroethylcarbamoyl)-EDU

38 5'-H-(tert-butylcarbamoyl)-EDU

5'-amino-5-ethyl-2',5'-dideoxyuridine

5'-N-sulfamino~5-ethyl-2',5'-dideoxyuridine sodium salt

51-O-(l-adamantylthiocarbamoylamino)-5-ethyl-2',5 '- dideoxyuridine

F&. 1. (cont.)

CsC1 density gradient analysis. Therefore, L1210 cells were seeded in tissue cluster 3524 Costar cups at 5 • 105 cells/ml/cup and incubated with either (methyl-3H)dThd at 1 /xCi/5 nmole/ml, or (me- thyl -3 H)dThd at 1/xCi/5 nmole/ml + EDU at 2.25 ~mole/ml, or (2-14C)EDU at 0.45/xCi/2.25/xmole/

ml for 24 h at 37~ in a humidified, CO2- control- led incubator. The specific activities of (methyl -3 H) dThd and (2-14C)EDU in these experiments were

chosen in such a way that the cpm inputs of both radiolabeled nucleoside were comparable. After the incubation period, cells were pelleted by centrifuga- tion, washed and finally lysed by addition of 100/xl of 2~ sodium dodecylsulfate in 0.15 M NaCI and 0.1% EDTA (pH 8.4). The cell lysate was then brought on top of a CsC1 solution (~ = 1.7241 g/ml, pH 8.0) and centrifuged for 24 h at 19~ and 200,000 x g. Twenty fractions of ten drops each were collected from the bottom of the tubes and assayed for radioactivity.

Results and discussion

Inhibitory activities o f 5 '-substituted E D U derivatives on L1210/0 cells

The structures of the 5 '-substituted EDU derivatives

o o II II

CH30-C-C-~)-

o

C2H50-C-CH2-C-0-

o

H2N-C-O-

HN-C-O- k

(CH2) 2

Cl

H37 H ~, CH 3- IC-N-C-O-

CH 3

H2N-

Na @ (2) ~H v H-S-N- g

XH HN-C-N-

HO-

HO-

HO-

HN-C-O- I

(i CH2) 2 C1

HO-

HO-

H0-

are depicted in Fig. 1, and the results of the anti- tumor tests are presented in Table 1. The indicated values represent data, obtained for at least three separate determinations. The reference compound 5-ethyl-dUrd (EDU) was included in all assays. The IDs0 (inhibitory dose-50) of EDU for L1210 cell

proliferation was 4.65/xg/ml. Among the 5'-substituted derivatives of EDU,

whose substituent consisted of a phosphate group, linked to the C-5' atom via a phosphomonoester bound (compounds 1-6) , only the 5 ' -monophos- phate was as active as the parent compound in in- hibiting the growth of L1210 cells. Compounds 4 and 5, both 5 '-methylphosphonate derivatives, and compound 6, a 5 '-cyclophosphamide-EDU deriva- tive, were 24 to 70 times less active, while the 5 '-die- thylphosphate (3) was totally devoid of any inhibit- ory activity towards L1210 cell proliferation.

Among the 5'-alkanecarbonyl derivatives (com- pounds 8-21) , 5'-0-pivaloyl-EDU (8) and its 3 ' -monophosphate derivatives (9, 10) were 6 t o 7 times less inhibitory than EDU. The 3'-methyl- phosphonate derivative (11) lacked any inhibitory activity. 5'-0-n-Butyryl-EDU and 5'-0-isobutyryl- EDU were slightly less inhibitory than 5'-0-piva- loyI-EDU. However, introduction of a phenyl or ethoxycarbonyl group on C-2, or a methoxycarbo- nyl group on C-4 of the butyryl chain brought

40

Table 1. Effects of 5'-derivatives of 5-ethyl-2'-deoxyuridine on the proliferation of L1210 cells.

Compound IDs0 a (#g/ml) for cell growth

As such On addition of On addition of On addition of dUrd (125/xg/ml) b dThd (5/xg/ml) b dCyd (500 #g/ml) u

1 4.65 (• 0.30) 2 4.69 (• 0.59) 3 > 1000 4 365 (• 78) 5 146 (• 30) 6 43O ( • 6O) 7 773 (_+ 152) 8 34.5 (• 15) 9 42.8 (• 14)

10 33.8 (• 5.3) I1 > 1000 12 110 (• 36) 13 63.6 (• 20) 14 21.9 (• 14) 15 5.05 (2 0.53) 16 220 (• 35) 17 5.86 (+_ 0.98) 18 21.8 (• 14) 19 55.8 (2 13) 20 6.90 (2 1.7) 21 42.9 (• 2.5) 22 619 (2 256) 23 > 1000

24 14.3 (2 3.7) 25 71.5 (• 16) 26 16.2 (2 4.4) 27 30.2 (+ 2.0) 28 13.2 (• 5.1) 29 38.4 (2 5.1) 3O 265 ( • 92) 31 8.56 (• 5.6) 32 5.66 (• 1.8) 33 17.8 (• 5.6) 34 5.25 (+_ 0.9) 35 19.3 (• 11) 36 423 ( • 82) 37 > 1000 38 173 (• 22) 39 > 1000 40 > 1000 41 156 (• 97)

150 (• 30) 87 (• 17) >1000 262 (• 32) 134 (+ 40) >1000

> 1000 > 1000 > 1000 > 1000 > 1000 > 1000 > 1000 > 1000 > 1000 > 1000 > 1000 > 1000 > 1000 > 1000 > 1000

105 (• 20) 101 (• 8.0) 181 (• 3.8) 379 (• 16) 419 (+ 51) 533 (• 47) 213 (• 84) 260 (• 75) 349 (• 46)

> 1000 > 1000 > 1000 207 (• 44) 166 (• 62) 302 (• 50) 251 (• 22) 213 (• 21) 288 (• 13) 185 (_+ 29) 230 (• 57) 372 (+_ 88) 154 (+ 46) 133 (• 12) 297 (• 55) 59.6 (• 9.7) 291 (• 51) 284 (• 14)

287 (• 44) 175 (• 30) > 1000 272 (• 12) 221 (• 99) 342 (• 18) 280 (• 120) 324 (_+ 93) 362 (• 72.5) 261 (• 1.0) 103 (_+ 11) >1000

56.2(_+ 1.3) 51.7 (+_ 6.7) 56 .2(• 7.9) > 1000 > 1000 > 1000 > 1000 > 1000 > 1000

52.5 (• 7.7) 100 (+_ 22) 374 (• 284) 291 (• 26) 250 (• 22) 317 (_+ 42)

16.0 (+_ 0.66) 13.5 (• 3.5) 19.5 (• 2.2) 31.7 (• 3.3) 30.9 (_+ 3.1) 38.3 (+_ 2.7)

5.8 (• 0.99) 13.4 (• 3.3) 12.4 (• 3.8) 39 (• 1.5) 46.05 (_+ 0.45) 38.3 (+ 3.0)

238 (• 60) 195 (• 64) 224 (_+ 66) 46.8 (• 4.9) 44.3 (_+ 6.4) 121 (+ 23)

100 (+_ 15) 69.25 (_+ 2.7) 557 (• 167) 429 (• 97) 431 (• 189) 648 (_+ 102) 154 (_+ 17) 130 (_+ 3.0) >1000 529 (+_ 50) 466 (+ 84) >1000

> 1000 > 1000 > 1000 > 1000 > 1000 > 1000

255 (+ 127) 115 (• 15) 207 (+ 65) - - > 1 0 0 0

> 1000 > 1000 > 1000 39.9 ( • 6.4) 175 (• 69) 180 (• 43)

a Inhibitory dose-50 or dose required to inhibit cell proliferation by 50o70. b Maximum concentration of dUrd, dThd and dCyd that were themselves not inhibitory to L1210 ceil proliferation.

41

Table 2. Effects of 5'-derivatives of 5-ethyl-2'-deoxyuridine on the incorporation of (1 ' , 2 ' -3H)dUrd, (methyl-3H)dThd and (5-3H)dCyd into DNA of L1210 cells.

Compound IDso a ~ g / m l ) for DNA synthesis

As monitored by As monitored by As monitored by incorporation of incorporation of incorporation of (1 ' , 2'-3 H)dUrd (methyl -3 H)dThd (5 -3 H)dCyd

1 6.4 (_+ 0.4) 73 (_+ 11) >1000 2 28.5 (_+ 6.8) 307 (_+ 92) >1000 3 > 1000 752 (_+ 247) > 1000 4 513 (_+ 76) >1000 >1000 5 366 (_+ 17) > 1000 > 1000 6 > 1000 > 1000 > 1000 7 > 1000 > 1000 > 1000 8 186 (_+ 29) 183 (+ 4.2) 465 (_+ 80) 9 210 (_+ 97) 276 (_+ 15) 732 (_+ 62)

10 290 (_+ 15) 276 (+_ 51) _>1000 11 > 1000 > 1000 > 1000 12 17.2 (+ 7.2) 395 (_+ 308) _+ 1000 13 57.1 (+ 24) 270 (_+ 16) _+ 1000 14 161 (_+.48) 194 (_+ 47) 637 (_+ 125) 15 20.3 (_+ 4.7) 162 (_+ 36) 855 (_+ 57) 16 244 (_+ 11) 53.4 "(_+ 6.4) 16.3 (_+ 2.5) 17 11.3 (_+ 4.3) 48.9 (_+ 25) >1000 18 33.5 (_+ 6.5) 118 (_+ 43) 835 (_+ 67) 19 260 (_+ 6.5) 200 (_+ 20) 650 (_+ 45) 20 8.1 (_+ 0.3) 103 (_+ 7) > 1000 21 172.5 (_+ 95) 470 (_+ 95) >1000 22 579 (_+ 11) >1000 >1000 23 > 1000 > 1000 _+ 1000 24 37 (_+ 23) 67.8 (_+ 14) >1000 25 49.0 (_+ 13) 203 (_+ 9.0) 665 (_+ 63) 26 8.18 (_+ 5.5) 3.09 (_+ 0.69) 1.88 (_+ 0.66) 27 37 (_+ 9.0) 15.5 (_+ 3.0) 9.33 (_+ 4.8) 28 27.2 (_+ 3.7) 10.1 (_+ 1.5) 5.04 (_+ 1.5) 29 31.2 (_+ 8.0) 66 (_+ 21) 28.3 (_+ 12) 30 _+ 1000 295 (_+ 50) 205 (_+ 37) 31 28.3 (_+ 8.1) 59.8 (_+ 15) 703 (_+ 56) 32 6.81 (_+ 0.49) 66.5 (_+ 9.5) >1000 33 6.98 (_+ 0.48) 100 (_+ 46) >1000 34 6.18 (_+ 0.72) 65 (_+ 10) >1000 35 45.4 (_+ 8.7) 398 (_+ 96) >1000 36 551 (_+ 9.0) >1000 >1000 37 > 1000 > 1000 > 1000 38 309 (_+ 86) 222 (_+ 16) 156 (_+ 12) 39 > 1000 > 1000 > 1000 40 > 1000 > 1000 > 1000 41 89.1 ( + 11) 28 (_+ 4.2) 14.05 (_+ 3.7)

a Inhibitory dose-50 or dose required to inhibit the incorporation of labelled nucleoside into LI210 cell DNA by 50~

42

about a marked increase in the inhibitory activity of these derivatives (IDs0 for L1210 cell growth: 21.9 t,g/ml, 5.05 #g/ml and 5.86/xg/ml, respectively, as compared to 110/xg/ml for the unsubstituted 5 '-0-n- butyryl-EDU). A similar observation was made for

5'-0-valeryl- and 5'-0-(5-(methoxycarbonyl)penta- noyl)-EDU. There were no dramatic differences in the antitumor cell activity between the 5 '-0-pivaloyl- EDU (8), 5'-0-valeryl-EDU (18), 5'- 0-isovaleryl- EDU (19) and 5'-0-1auryl-EDU (21). The IDs0 values of these compounds ranged from 21.8/xg/ml to 55.8 ~g/ml. For 5'-0-n-butyryl- (12), 5'-0-iso- butyryl- (13), and 5'-0-(3,3-dimethylbutyryl)-EDU (16), the IDs0 values were comprised between 63.6 and 220 txg/ml.

The effect of several cyclic substituents at C-5' of EDU were also evaluated (compounds 22-32). Among the 5'-0-benzoyl substituted EDU deriva- tives (compounds 22-24), only 5'-0-(3,4-dichlo- robenzoyl)-EDU (24) showed a considerable anti- tumor activity (IDso: 14.3 txg/ml). While 5'-0-(2- furanecarbonyl)-EDU (25) was 10-fold less in-

hibitory than EDU, 5'-0-(1-adamantanecarbonyl)- EDU (26) and its 3 '-monophosphate- (27, 28) and 3'-diethylphosphate (29) derivatives were about 2 to 6 times less active than EDU. However, the 3 '-methylphosphonate analog was significantly less active (IDs0 = 265 ~g/ml). Both 5'-0-phenoxycar- bonyl-EDU (31) and 5'-0-(4-chlorphenoxyacetyl)- EDU (32) were as active as EDU.

The antitumor cell activity of the EDU deriva- tives with a short aliphatic chain (compounds 33- 35) was comparable or only slightly inferior to that of EDU itself. However, upon introduction of a carbamoyl group (36) or a substituted carbamoyl group (37, 38) the antitumor cell activity was re- duced 30- to 160-fold.

Among the four 2 ' , 5 '-dideoxy-EDU derivatives (compounds 7, 39, 40, 41) that were tested, none demonstrated an appreciable inhibition of L1210 cell proliferation.

Role of dThd kinase in the cytotoxic activities of 5 '-substituted EDU derivatives

The cytotoxicity of the 5 '-substituted EDU deriva- tives could be partially or completely reversed by

the addition of dUrd or dThd. The degree of reduc- tion of the antitumor cell activity upon addition of dUrd and dThd was quite similar for compounds 2, 15, 17, 20, 32 and 34 as it proved to be for EDU and became less important when the compounds were less cytotoxic. The 5'-substituted derivatives of EDU were more inhibitory to the incorporation of (1 ' , 2 '-3H)dUrd into DNA than to the incorpora- tion of (methyl-3H) dThd (Table 2). The (l-ada- mantane)carbonylesters of EDU (compounds 26- 30) exert their cytotoxic and antimetabolic effects clearly in a different way. These compounds proved considerably inhibitory towards L1210 cell prolifer- ation, their cytotoxicity could not be reversed by the addition of dUrd or dThd and (methyl-3H) dThd incorporation into DNA was even more in- hibited than (1' , 2'-3 H)dUrd incorporation.

The observation that (i) addition of dUrd or dThd reversed the cytotoxic effects of EDU at a similar degree, and (ii) EDU inhibited (1' , 2' 3 H) dUrd and (methyl-3H)dThd incorporation into DNA, could be explained either by an interaction of EDU with dThd kinase and/or by the interference of EDU with one of the successive metabolic steps that lead to the incorporation of dUrd or dThd into DNA. Since EDU is phosphorylated to EDUMP by thymidine kinase from L1210 cells with a Ki/Km of 35.6 (24) and since EDU is inactive against the dThd kinase deficient L1210/BdUrd cell line (12), one may infer that the conversion of EDU to EDUMP by dThd kinase is essential for its antitumor cell ac- tivity. However, since the Ki value (617 IxM) of EDU for dThd kinase (24) is about 19 times higher than its IDs0 value for L1210 cell proliferation (33.17/,M) (23), dThd kinase must be considered as an activating enzyme rather than as a target enzyme for the cytotoxic activity of EDU.

The 5 '-substituted derivatives of EDU proved as a rule considerably less inhibitory towards the pro- liferation of Ll210/BdUrd cells than of L1210/0 cells (Table 3). As illustrated in Fig. 2, there was a close parallelism between the inhibitory effects of the compounds on L1210/0 cell proliferation and their dependence on phosphorylation by dThd kinase. Indeed, we found a close correlation be- tween the log IDs0 values for L1210/0 cell pro- liferation and the ratio of the log IDs0 for LI210/

43

Table 3. Inhibitory effects of 5 '-derivatives of 5-ethyl-2'-deoxyuridine on the proliferation of several murine leukemia L1210 cell lines.

Compound IDso a (/,g/ml) for cell growth

L 1210/0 b L1210/BdUrd ~ L1210/araC d

1 4.65 (• 0.30) >1000 >1000 2 4.69 (• 0.59) > lO00 > 1000 3 >1000 713 (• 178) >1000 4 365 (+_ 78) 780 (_+ 147) >1000 5 146 (+ 30) 378 ( • 52) >1000 6 430 (+ 60) 423 (+ 56) >1000 7 773 (• 152) > 1000 > 1000 8 34.5 (_+ 15) 70 (• 11) 167 (• 11) 9 42.8 (• 14) 250 (• 20) 270 (_+ 27)

10 33.8 (• 5.3) 252 (_+ 5.7) 278 (+_ 14) 11 > 1000 947 (+ 38) > 1000 12 110 (• 36) 287 (• 24) 98 (_+ 15) 13 63.6 (• 20) >1000 173 (+ 145) 14 21.9 (_+ 14) 273 (• 28) 165 (• 83) 15 5.05 (• 0.53) 310 (+ 13) 195 (• 24) 16 220 ( • 35) 130 ( • 6.6) 205 (-- 23) 17 5.86 (_+ 0.98) 853 ( • 36) 317 (• 51) 18 21.8 (_+ 14) 313 (_+ 22) 203 (_+ 24) 19 55.8 (_+ 13) 407 (• 58) 200 (+_ 33) 20 6.90 (+_ 1.7) 477 (• 89) 221 (• 39) 21 42.9 (• 2.5) 983 (+_ 22) 503 (_+ 37) 22 619 (• 256) > 1000 > 1000 23 > 1000 793 (• 207) > 1000 24 14.3 (• 3.7) 52 (• 16) >1000 25 71.5 (• 16) 337 (• 49) 154 (• 84) 26 16.2 (_+ 4.4) 12.4 (• 0.35) 25.1 (• 2.4) 27 30.2 (_+ 2.0) 21 (• 4.0) 24 ( • 3.0) 28 13.2 (+ 5.1) 19.7 (_+ 0.6) 21.8 (+ 2.8) 29 38.4 (• 5.1) 149 (• 61) 71 (_+ 24) 30 265 (_+ 92) 345 (_+ 40) 573 (• 69) 31 8.56 (• 5.6) 400 (+ 20) 175 ( • 23) 32 5.66 ( • 1.8) 480 (• 33) 190 (• 43) 33 17.8 (_+ 5.6) >1000 >1000 34 5.25 (_+ 0.9) 737 (• 44) 698 (• 266) 35 19.3 (• 11) >1000 >1000 36 423 (_+ 82) > 1000 > 1000 37 > 1000 > 1000 > 1000 38 173 (_+ 22) 357 (• 24) 420 (• 93) 39 > 1000 > 1000 763 (• 158) 40 > 1000 > 1000 > 1000 41 156 (_+ 97) 47 (_+ 3.3) 116 (_+ 11)

a Inhibitory dose-50 or dose required to inhibit cell proliferation by 50%. b Data taken from Table 1. c L1210/BdUrd is a murine leukemia L1210 cell line selected from the parental L1210/0 cell line for its ability to grow in the presence

of 5-bromo-dUrd (260/~g/ml). This cell line is shown to be deficient for dThd kinase (13). d L1210/araC is a murine leukemia LI210 cell line selected from the parental L1210/0 cell line for its ability to grow in the presence

of araC (1 /,g/ml). This cell line is shown to be deficient for dCyd kinase (19).

44

N ~ N N ' Y = - 1. 097X' + 2. 634 '

3 ~ 17

2 Q32

% 10 ! �9

o ~ 75

~ o {~1%1 I'~ 8 "13~ 27 ~~

0 1 2 3

Log IOso L1210 I 0 ( ,Ug/rot)

Fig. 2. Linear regression line for the log ID50 values of various

5 '-substituted EDU derivatives for L 1210/0 cell proliferation as

a function of the ratio of the log IDs0 values of these compounds for L1210/BdUrd cell proliferation to the log ID~o values for L1210/0 cell proliferation. The IDso values were taken from Table 3. For the measurement of the regression line, compounds 26-30 (indicated on the graph between parentheses) and all compounds that showed IDs0 values > 1000 ~g/ml for L1210/0 or L1210/BdUrd cell proliferation were not taken into account.

BdUrd cell proliferation to the log IDs0 for L1210/0 cell proliferation (r = -0.744). The latter

ratio can be considered as a parameter for the dependence of the compounds on phosphorylat ion

by dThd kinase. Since this enzyme acts on the free

5 ' -OH moiety of EDU, the data indicate that the ester bound at C-5 ' of those compounds that are

highly dependent on dThd kinase, must be easily hydrolysed to release the parent compound EDU. This is particularly true for the following com-

pounds: 5 '-0-(2-ethoxycarbonylbutyryl)-EDU (15), 5'-0-(4-methoxycarbonylbutyryl)-EDU (17), 5'- 0-(5-(methoxycarbonyl)-pentanoyl)-EDU (20), 5'- 0-methyloxalyl-EDU (34) and 5'-0-ethylmalonyl- EDU (35), 5'-0-(phenoxycarbonyl)-EDU (31), 5'-0-(4-chlorphenoxyacetyl)-EDU (32) and 5'-0- ethoxycarbonyl-EDU (33). The role of dThd kinase

in the anti tumor cell activities of the 5 '-substituted EDU derivatives becomes less important when the compounds are less cytotoxic; thus, when hydrolysis of the C-5' ester bound proceeds more

slowly or only partially. Among all compounds tested, 5 ' -0-(1-adamantanecarbonyl)-EDU (26) and its 3 ' -monophosphates (27, 28, 29) and 3'-

phosphonate (30) may be considered as an excep- tion to this rule; these compounds were fairly active

towards L1210/0 cell proliferation, and demon-

strated similar IDs0 values towards L1210/BdUrd cells. It is possible that these compounds were not

hydrolysed to the parent compound EDU, and that

phosphorylation by dThd kinase may not be re- quired for these compounds to exert their cytotoxic action. When compounds 26-30 were not taken into account for the measurements of the correla-

tion coefficient between the log IDs0 values for

L1210 cell proliferation and the ratio of the log

IDs0 for L1210/BdUrd cell proliferation to the log

IDs0 for L1210/0 cell proliferation, an even higher

correlation coefficient could be obtained (r = -0.888).

Compound 2, which represents the 5 ' -mono-

phosphate of EDU, was completely inactive against L1210/BdUrd cell growth. This means that the 5 '-

phosphate group must be hydrolysed before EDUMP is taken up by the cell. The same observa- tions have been made for 5-f luoro-dUMP and for 5-nitro-dUMP, two nucleotide analogs which are

also highly dependent on dThd kinase activity to exert their antiproliferative effects (our unpublish-

ed data).

Interaction o f E D U and its 5 '-substituted derivatives with deoxycytidine metabolism

Silagi et al. (11) suggested that incorporation of EDU into DNA of B5 59 melanoma cells is essential

for its biological properties. In contrast, we could not observe any incorporation of (2 -14 C)EDU into

L1210 cell DNA, under conditions where (methyl- 3 H)dThd was perfectly well taken up in the DNA (Fig. 3).

The observations that addition of dCyd com- pletely reversed the anti tumor cell activity of EDU (Table 1), and that EDU stimulated (5 -3 H)dCyd in- corporation into DNA (Fig. 4), suggest that EDU exerted its antiproliferative effect through interfer- ence with dCyd metabolism. Indeed, if the de novo biosynthesis of dCTP is inhibited, the intracellular

I I I I J

E 7 170 .-'2- r

c ) >" 5

-> 4

~o 1 / \o ~,

1 5 10 15 20

F r a c l i o n N u m b e r

Fig. 3 CsC1 equilibrium density gradient profile of L1210 cell

DNA labeled with either (methyl -3 H)dThd at 1/~Ci/5 nmole /mI

(O 0), or (methy# ~ H)-dThd at 1 /zCi/5 nmole/ml + 2.25 #mole EDU/ml �9 � 9 or (2-14C)EDU at 0.45 /zCi/2.25 ~mole/mI (1 II).

lO01

60-

o =

i.,7-,,~ / / . 0 . -~

r /,0-

o

: 20' g 2 r

J

S ~ ~",~ %,.

ho

I I I I

0 I 2 3

Log r of EDU (,ug/ml)

c a

u o o

J i

o i "E ,

300 "=

t ~

c o ~ 100

o

o u c

Fig. 4. Effect of EDU on the inhibition of L1210 cell prolifera-

tion and incorporation of (5 -3 H)dCyd into L1210 cell DNA.

dCTP pool will decrease. Since dCTP is an allos- teric feedback inhibitor of dCyd kinase (25, 26), shortage of dCTP will lead to an increased dCyd kinase activity so as to provide dCTP via the sal- vage pathway.

Hypothetically, EDU may interfere with the de novo biosynthesis of dCTP at the cytidine 5'-diphosphate (CDP) reductase level. This would occur after the intracellular conversion of EDU to its 5 '-triphosphate. In this sense, EDU may act like

45

dThd, whose 5'-triphosphate dTTP is known to be an allosteric inhibitor of CDP reductase (27-29).

This would explain the decrease in the inhibitory ef- fects of dThd (13) and EDU (Table 3) towards the dCyd kinase-deficient L1210/araC cell line. We have shown that the mutant L1210/araC cells possess a 3-fold higher level of CDP reductase ac- tivity than the parent L1210/0 cells (our unpublish- ed data), and these increased CDP reductase levels may be a plausible reason for the decreased in- hibitory effects of dThd and EDU towards pro- liferation of the L1210 /a raC cells.

The stimulatory effects on (5-3H)dCyd incor-

poration that have been observed for EDU (Fig. 4) could also be demonstrated for most of the 5 '-sub- stituted EDU derivatives. All compounds that

showed an IDs0 value close to the IDs0 of EDU for L1210 cell proliferation (i.e. compounds 2, 15, 17, 20, 31, 32, 34) stimulated the incorporation of (5 -3 H)dCyd into cellular DNA in a similar way as

the parent compound EDU (data not shown). The inhibitory effects of these compounds on L1210 cell

proliferation were reversed by the addition of dCyd (Table 1), and they were 20 to 200 times less cyto- toxic for L1210/araC cells than for L1210/0 cells. Also compounds 33 and 35 behaved in a similar way (Table 3):

However, compounds 2 6 - 3 0 did not show any stimulation of (5 -3 H)dCyd incorporation, and their inhibitory effects towards L1210 cell proliferation could not be reversed by the addition of dCyd (Table 1). These compounds were equally in- hibitory towards L1210/0 and L1210/araC cells (Table 3). These observations are consistent with the hypothesis that the cytotoxic effects of the 5'-0-(1-adamantanecarbonyl)-EDU derivatives are achieved by a mechanism that is different from that of EDU and the other EDU derivatives.

C o n c l u s i o n

Structure function analysis revealed that the anti- tumor cell activity of the 5'-substituted EDU derivatives depended clearly on the nature of the substituent. Introduction of a monophospha te group at the 5' position of EDU (compare corn-

46

pound 2 with 1) or 3' position of several 5'-sub- stituted EDU derivatives (compare compounds 9 and 10 with 8, and 27 and 28 with 26) did not affect the inhibitory effects of the parent compound. In contrast, introduction of a phosphonate group on C-5' or C-3' decreased considerably the antitumor effect (compare compound 4 with 1, 11 with 8 and 30 with 26). Most 5 '-substituents, i.e. pivaloyl (8), butyryl (12), valeryl (18), lauryl (21), dichloroben- zoyl (22-24), (1-adamantane)carbonyl (26) or car- bamoyl (36), decreased the inhibitory effects of EDU on L1210 cell proliferation. However, several 5'-substituted EDU derivatives, i.e. those with a 5'-substituent like 2-(ethoxycarbonyl)butyryl, 4- (methoxycarbonyl)butyryl, 5-(methoxycarbonyl) pentanoyl, phenoxycarbonyl, 4-chlorphenoxyace- tyl or methyloxalyl, demonstrated antitumor cell and antimetabolic activities that were equivalent to those of EDU. These compounds will be compared with EDU for their in vivo antitumor properties.

From the studies aimed at deciphering the mode of antitumor action of EDU and its 5 '-substituted derivatives in the murine L1210 cell system it can be inferred that: (i) the inhibitory effects of all EDU esters, except for the (1-adamantane)carbonyl esters (26-30), is mediated by the release of free EDU; (ii) to achieve its cytotoxic action EDU must first be phosphorylated by the cellular dThd kinase; (iii) under conditions where dThd is effectively in- corporated into DNA, EDU is not; (iv) the cyto- toxic activity of EDU is completely reversed by dCyd, and, furthermore, EDU stimulates the incor- poration of dCyd into DNA, suggesting that EDU interferes with dCyd metabolism. As a likely target for the antitumor action of EDU we propose CDP reductase. EDU may act as an allosteric inhibitor of this enzyme after it has been converted intracellu- larly to its 5'-triphosphate.

Acknowledgements

The authors thank Lizette Van Berckelaer and Miette Stuyck for excellent technical assistance, and Christiane Callebaut for fine editorial help.

This study was supported by grants from the Bel- gian F.G.W.O. (Fonds voor Geneeskundig Weten-

schappelijk Onderzoek) (Krediet No 30048.75), and the Geconcerteerde Onderzoeksacties (Conventie No 81/86-27).

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