Water soluble derivatives and bifunctional analogs of the anticancer agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)

Europ. J. Cancer Vol. 13, pp. 937-945. Pergamon Press 1977. Printed in Great Britain

Water Soluble Derivatives and Bifunctional Analogs of the Anticancer Agent 1, 3-bis(2-chloroethyl)-l-nitrosourea (BCNU) H. H. FIEBIG,* G. EISENBRAND, W. J. ZELLER and T. DEUTSCH-WENZEL Institute for Toxicology and Chemotherapy of the German Cancer Research Center, Heidelberg, West Germany A b s t r a c t - - T h r e e water soluble and 6 bifunctional newly synthesized analogs of 1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) were screened /'or anticancer activity against subcutaneously and intracerebrally inoculated Walker carcinosarcoma 256 and 7,12-dimethylbenz(a)anthracene (DMBA) induced mammary cancer of the rat. The water soluble 1-(2-hydrox~ett~yl)-3-(2-chloroethyl)-3-nitrosourea showed a higher activity than B C N U in s.c. inoculated Walker carcinosarcoma 256 in several experiments. Treatment of i.c. inoculated Walker carcinosarcoma 256 with the bifunctional analogs and l-(2-hydroxyethyl)-3-(2-chloroethyl)-3-nitrosourea resulted in a marked increase in lifespan over untreated rats. However, statistically no differences were found in comparison to the treatment with B C N U . Against the D M B A induced mammary cancer of the rat, only 2 new analogs were examined; they produced a smaller tumor weight inhibition than B C N U . Further investigations are under work to examine the activily of the new compounds against chemically induced brain tumors.

INTRODUCTION

tively similar toxic effects. Clinically, the main toxic effects observed are acute nausea and vomiting and delayed, dose-limiting myelosuppression [7]. The bifunctional nitrosoureas described here were'synthesized to study the influence of bifunctional carbamoylating activity on chemotherapeutic effectiveness [11]. Water soluble compounds were prepared following a suggestion by Hansch et al. [12] that the synthesis of more hydrophilic nitrosoureas might uncover less toxic and chemotherapeutically more potent compounds [12].

THE NITROSOUREAS BCNU, 1-2(chloroethyl)-3cyclohexyl-l-nitrosourea (CCNU) and methylCCNU are active in a number of experimental and human tumours. They produce cures in L t210 leukemia and L 5222 leukemia after intraperitoneal or intracerebral inoculation [1-3] and also cause a marked inhibition of tumor weight or cures in the solid tumor systems B 16 melanoma and Lewis lung carcinoma [4]. In h u m a n tumors they have a broad spectrum of activity. BCNU, CCNU and M e C C N U are effective in advanced brain tumors, Hodgkin's disease, non-Hodgkin's lymphomas and melanomas [4-6]. Furthermore BCNU shows some activity against breast cancer [4], and CCNU against lung cancer [6]. In combination therapy they have not yet been investigated adequately [7]. The nitrosoureas are potent alkylators of nucleic acids [8-10]. They are cell cycle nonspecific in their mode of action and also active in slowly proliferating tumors. BCNU, CCNU and M e C C N U have qualita-

MATERIAL AND METHODS Animals. All investigations were carried out with Sprague Dawley rats kept under conventional conditions. They received Altromin ® pellets and water ad lib. Toxicological investigations. For determination of the LDso , at least 4 logarithmically spaced doses wele given to 5 male and 5 female rats (3-5 month old) respectively (total number of rats at least 40 for each compound tested). The observation period for the determination of the LD5o was 60 days; values were calculated

Accepted 6 December 1976. *Supported by the Deutsche Forschungsgemeinschaft. 937

938

H. H. Fiebig, G. Eisenbrand, W. J. Zeller and T. Deutsch-Wenzel

according to the m e t h o d described by S p e a r m e n and K•rber [13]. All rats were dissected, and the organs with pathological alterations were exa m i n e d histologically. Early toxicities within 14 d a y observation period were also given (Table 1).

induced by 7,12-dimethyl-benz(a) a n t h r a c e n e (DMBA). F e m a l e S p r a g u e - D a w l e y rats (50-55 days old) received 3 intravenous injections of D M B A in a total dose of 4 mg per rat within 1 week. T h e rats were checked weekly for palpable m a m m a r y tumors. Estimates of t u m o r

Table 1. Early toxicity* (observation period: 14 days) of 1, l'polymethylene-bis-[3-(2chloroethyl)]3-nitrosoureas, 1- (2-ehloroethyl) 3- (co-hydroxyalkyl) 1-nitrosoureas and 1,1 '- (4-methyl-m-phenylene) bis-nitrosourea in rats

Compound BCNU CNU (CH2)n-CNU ln=2 2n-=3 3n=4 4n=5 5n=6 CNU-(CH2)n-OH 6n=2 7n=3 8n=4

Solution in % ethanol/ cremophorq~ : 0.9 % saline

Application

(mg/kg)

5/5 : 90

i.p.

32

29-34

27

50 70 60 60 60

i.p. i.p. i.p. i.p. i.p.

58 45 46 45 50

51-64 43-47 41-52 42-48 48-52

44 40 36 38 46

100 100 100

i.p. i.p. i.p.

32 31 32

31-33 29-34 30-35

26 24 25

i.v.

20

18-23

16

25/25 15/15 20/20 20/20 20/20 0/0 0/0 0/0

50% lethality 95~ confidence interval

10~ lethality (mg/kg)

CH3 9 ~CNU

25/25 : 50

CNU OR

Ill

CNU = C1-CH 2 C H 2 - N - C - N - N = O . i.p. = intraperitoneal, i.v. = intravenous. rats. *LDso values including late toxicity see text.

Tumors. Male S p r a g u e - D a w l e y rats (5-7 weeks old) were inoculated subcutaneously with 4 x 10 6 W a l k e r ascites cells on day 0. T r e a t m e n t started on day 4, w h e n the t u m o r had reached a p p r o x 0.8 g. O n d a y 8, all rats were killed with ether and the tumors were excised mad weighed. I n t r a c e r e b r a l inoculat;~on of W a l k e r 256 ascites cells was carried out according to the m e t h o d described by Valzelli [14]. A needle (No. 20) with a glass sleeve limiting its penetration to 11 m m was inserted into the petrotympanic-fissure. T h e rats were anesthesized with ether. Ascites cells (2 x 10 4 in 25 /~1) were i m p l a n t e d into the thalamic area. Pathological examination of r a n d o m l y selected rats showed that the t u m o r was growing in a solid form practically exclusively in the target area of the brain (Fig. 1); in rare cases, some t u m o r cells could be detected additionally within the meninges. T u m o r take was 100%. T h e autochthonous m a m m a r y cancer was

Each dose was tested in 10

size were m a d e by p a l p a t i o n and comparison with plastilin models which had been copied from representative original tumors [15]. T h e tumors developed multicentrically along the milk-groin. T h e total t u m o r mass per rat which was o b t a i n e d by addition of estimated weights of individual tumors, was taken for evaluation. T h e first palpable tumors a p p e a r e d 3 weeks after the application of D M B A . T h e m e d i a n induction period until beginning o f t r e a t m e n t was 63 days ranging from 42 to 84 days. T u m o r s with a longer induction period were not used for c h e m o t h e r a p e u t i c experiments. T h e a n t i t u m o r activity was expressed as t u m o r weight inhibition, control-treated 100] control x and m e a n n u m b e r of tumors per rat. M o r t a l i t y and changes of b o d y weight were taken as a

Anticancer Agent 1,3-bis ( 2-chloroethyl)- 1-nitrosourea

Fig. 1. Intracerebral growth of Walker 256. Untreated rats died 6 days after intracerebral inoculation of 2 x 104 cells.

939

Anticancer Agent 1,3-bis (2-chloroethyl)- 1-nitrosourea measure for toxic effects. At the beginning of chemotherapy the mean total tumor mass was 3.6_+0.6 g and the mean number of tumors was 4.0 + 1.3 per rat. Each rat had at least one tumor exceeding a weight of 1 g. The rats were randomly assigned into different groups of treatment. All rats were killed 42 days after the beginning of chemotherapy and the tumors were excised and weighed. The tumors from the 18 control rats were examined histologically. Out of 131 tumors, 124 were classified as carcinomas, 5 as preneoplasms, and only 2 as adenomas.

Compounds Preparation of the compounds and chemical properties have been described elsewhere [11]. Nitrosoureas, not soluble in water, were first dissolved in a mixture of absolute ethanol and Cremophore'-EL * and then made to volume with 0.9°i, saline solution (Table I). All substances were given intraperitoneally, except for compound 9 which was given intravenously.

Statistics Tumor mass or survival time were statistically evaluated by the Kruskal-Wallis test, followed by multiple comparisons [16]. For the evaluation of BCNU against compound 6 the Wilcoxon rank sum test was used (Table 3). RESULTS

Toxicological investigation All 9 nitrosoureas showed qualitatively similar toxic effects. Bleedings of the stomach and intestines, lung oedema and a depression of the bone-marrow and lymphoid tissue were observed in animals which died 3-14 days after a single i.p. injection. A marked loss of body weight and moderate leucopenia was observed 40-60 days after the substances had been given. Rats which died' of late toxicity showed pronounced liver necrosis and bleedings of the stomach and intestines. Liver-necrosis was not observed after i.v. injection. The kDso values were determined after an observation period of 60 days to take care of late toxicity of the nitrosoureas. The hydroxylated substances 6 8 and BCNU all had LDs0 values of 25 mg/kg; LDs0 values for the bifunctional analogs 1-5 were around 35 mg/kg with a range of 32-40 mg/kg. The aromatic *A non-ionic solubilizing agent and surfactant based on polyethoxylated ricinus oil (BASF-Ludwigshafen, ~ . Germany).

941

compound 9 was more toxic with a LD50 of about 13 mg/kg after i.v. application; (after i.p. application compound 9 produced a peritonite and was therefore given i. v.). LD10 values were approximately 20°//o smaller than LDso values. The early toxicities of the 10 nitrosoureas (observation period of 14 days) are given in Table 1. For all compounds toxicities were similar in both sexes.

Anticancer activity, Tables 2 and 3 summarize the anticancer activity of BCNU analogs in s.c. inoculated Walker 256. BCNU and cyclophosphamide served as reference compounds. The bifunctional compounds 1-5 showed smaller chemotherapeutic effects than those of BCNU in the 2 schedules tested. One single dose (approx 800/0 of the LI~t0) given 4 days after transplantation was more effective than the fractionated schedule given on days 4-7. Single dose application however produced a reduction in body weight, which is a rough parameter of toxicity. The water soluble compounds 6-8 showed striking differences in their activities. Compound 6 was slightly more effective than BCNU in three dose-levels tested; in further experiments, the differences were statistically significant (Table 3). Compounds 7 and 8, however, were practically inactive, although their toxicities were equal to that of 6. The aromatic nitrosourea 9 showed a moderate tumor weight inhibition. Nitrosoureas used for clinical purposes are known to cross the blood-brain-barrier [17]. Therefore, the newly synthesized nitrosoureas were also tested against i.c. inoculated Walker 256. Control rats died 6-7 days after transplantations of 2 x 104 ascites cells. Treatment with BCNU and the newly synthesized nitrosoureas, listed in Table 4, caused a significant increase in lifespan. However, differences in survival time between BCNU and the new compounds 1- 6 were statistically not significant. In addition to transplantation tumors we used the DMBA-induced m a m m a r y cancer of the rat as a tumor model for screening experiments. Table 5 summarizes the results obtained by the treatment with BCNU and the new compounds 3 and 6. In this experiment the new analogs showed an activity which was somewhat smaller than that of BCNU. Schedules with repeated injections were more effective than those with one single injection; they were, however, also more toxic, causing a considerable loss of body weight and increase of mortality.

942

H. H. Fiebig, G. Eisenbrand, W. J. Zeller and T. Deutsch-Wenzel

7"able 2.

Anticanceractivity oJ'BCNU analogs against ,subcutaneously inoculated Walker 256 Day 8

Compound

Dose (mg/kg/day)

Scheduh" day

T u m o r weight* (g)

Tumor weight inhibitiont

Days 4-7 ~ body weight change

Control

14.9

-2.0

Control

18.6

-8.9

16 6.5 3.3

4 4--7 4-7

3.2§/18.6 3.2§/14.9 9.8 /14.9

83 79 34

-2.7 +2.3 +5.6

4

1.7§/18.6

91

-2.7

29 II.5

4 4 7

3.7§/14.9 3.9§/14.9

75 74

-3.4 -2.8

23 9 4.5

4 4-7 4 7

4.2 §/14.9 6.7:~/14.9 I2.7 /14.9

72 55 15

- 1.2 +5.5 +3.6

3n=4

23 9 4.5

4 4-7 4-7

3.5§/14.9 7.1 /14.9 12.7 /14.9

77 52 I5

-3.4 +6.8 +5.4

4n=5

23 9 4.5

4 4-7 4 7

4.8{}/14.9 7.1:~/14.9 14.4 /14.9

68 52 3

+ 0.9 +2.5 +5.8

5n=6

25 10

4 4-7

4.5:~/14.9 7.0 /14.9

70 53

0.0 +4.7

16 6.5 3.3

4 4- 7 4-7

2.7§/18.6 2.6§/14.9 7.9§/14.9

85 83 47

+2.2 - 2.5 -2.1

6.5

4 7

18.8 /18.6

0

-3.3

6.5

4-7

17.9 /18.6

4

-2.1

BCNU

Cyclophosphamide C N U (CH 2)n-CNU 1 n=2

2

1l ~

3

CNU (CH2)n-OH 6n=2

7 1l ~

3

8n=4

105

CNU

9 @ C N U

l0 4.0

4 4 7

9.7 /14.9 11.6 /18.6

35 38

-3.6 -2.5

CH3 Inoculation size 4 x 106 ascites cells. Each dose was tested against 10 S p r a g u e - D a w l e y rats. Compounds were given i.p. ; only c o m p o u n d 9 was injected intravenously. * M e a n of experimental over mean of controls for that experiment. control-test x 100. +P versus control < 0.05. §P < 0.01 (Kruskal-Wallis). ? control

Anticancer Agent 1,3-bis( 2-chloroethyl)- l-nitrosourea

943

Table 3. Comparison of anticancer activity of BCNU and 1-(2-hydroxyethyt)-3-(2chloroethyl)-3-nitrosourea (Compound 6) against subcutaneously inoculated Walker 256 Number of rats

Compound

Dose* (mg/kg)

T u m o r weight day 8 (g) T/C~"

P-value~.

BCNU Compound 6

22 22

16 16

2.7 + 1.3/15.4 + 3.2 2.0 + 1.3/15.4 + 3.2

P < 0.01

BCNU Compound 6

13 14

20 20

2.3 + 1.1/13.3 + 2.6 1.7 + 0.8/13.3 + 2.6

P < 0.1

BCNU Compound 6

14 14

12.5 12.5

4.6 + 1.9/13.3 + 2.6 2.9 + 1.5/13.3 _+ 2.6

P < 0.05

BCNU Compound 6

14 14

7.8 7.8

9.6 _+ 4.3/13.3 + 2.6 8.3 + 2.9/13.3 _+ 2.6

P < 0.1

*Single dose given i.p. on day 4. LDIo for b o t h c o m p o u n d s is 20 mg/kg. + M e a n + s t a n d a r d deviatiation of treated groups in c o m p a r i s o n to controls. ,+Wilcoxon rank sum analysis.

Table 4.

Anticancer activity of BCNU analogs against intracerebrally inoculated Walker 256 M S T * (days)

Compound

Dose (mg/kg/day)

Schedule (day)

Control

Median 6

BCNU

16 8

3 3-6

13§ 12.5§

Range

ILS'~ (%)

6-7 11-18 5-16

117 108

C N U ~.( C H 2)n-CNU 1 n = 2

6 15 12-17

117 125

11 12,+

4-14 7-18

83 100

3 3 6

11.5 13§

6-14 5-15

92 117

23 16

3 3-6

12~ 12.5§

5-14 12-16

100 108

25 20

3 3-6

13§ 13§

10-18 5-16

117 117

16 8

3 3-6

12§ 12.5~

10-18 5 15

100 108

29

3

13§

20

3-6

13.5§

2 n = 3

23 16

3 3-6

3 n = 4

23 16

4 n = 5 5 n = 6 6 CNU

(CH2)2-OH

I n o c u l a t i o n size 2 × 104 t u m o r ascites cells. E a c h dose was tested against 10 rats. Doses are e q u i v a l e n t to the o p t i m a l dose, d e t e r m i n e d against i n t r a m u s c u l a r W a l k e r c a r c i n o s a r e o m a 256. C o m p o u n d s were given i n t r a p e r i t o n a l l y . * M e d i a n survival time. ~'Increase in lifespan in % test over control. +*P vs control < 0.05. §P < 0.01 ( K r u s k a l ~,Vallis test).

944

H. H. Fiebig, G. Eisenbrand, W. ,1. Zeller and T. Deutsch-Wenze! DISCUSSION +

+11

+11

+It

w

t2~ m

~ ~.-~

i

0

a~

b MdM

m~m

~44

4m~

4~4

~ 4

d v

c5 ...<

v

~" > ++ >. © O

-g

>

O ;>-,

v:

.o

~o ca ×

×

X

'4 ×

~a

>-.

7~ e-

ll o

7Z ©

I

e~ e-,

T o

Z o

Z

Z

i

D

Screening for anticancer activity of BCNU and 9 newly synthesized analogs in the s.c. Walker 256 revealed one analog which produced a statistically higher tumor inhibition than BCNU. This was compound 6, 1-(2hydroxyethyl) - 3 - (2 - chloroethyl) - 3 - nitrosourea which is a water soluble analog of BCNU. The lipophilic compounds 1 5, however, were somewhat less active than BCNU. No relationship could be found between the length of the polymethylene bridges and antitumor or toxic effects of these bifunctional congeners of BCNU" The introduction of bifunctional carbamoyfating activity into the nitrosourea molecule therefore does not influence its biological properties significantly• Treatment of i.c. inoculated Walker 256 with BCNU, the bifunctional lipophilic analogs 1-5 and compound 6 showed no statistical differences in survival time: all compounds displayed equal activities. However, compounds 1-5 were somewhat less active against s.c. implanted Walker 256 than BCNU. Tile relatively better chemotherapeutic effectiveness of the latter compounds'against i.c. inoculated tumor cells might be attributed to an increased ability to cross the blood braiu barrier• Further investigations using slower growing models of brain tumors which better reflect the characteristics of human neoplasm are in progress• Acknowledgements--We thank Prof. Dr. K. Goerttler (Institute for exp. Pathologie, Deutsches Krebstbrschungszentrum Heidelberg) for carrying out the histological examinations. W e are greatly indebted to Mrs. B. Strobel and Mrs. A. Pohl for excellent technical assistance and to Mr. Paul E• Schurr for supplying 7 , 1 2 - D i m e t h y l b e n z (a) anthracen.

Artticancer Agent 1,3-bis ( 2-chloroethyl)- l-nitrosourea

REFERENCES 1.

2. 3.

4.

5. 6. 7. 8.

9. 10.

11.

12.

13. 14. 15.

16. 17.

F . M . SCHABEL,T. P. JOHNSTON, G. S. ]~CCALEB,J. A. MONTGOMERY, W. R. LASTER and H. SKIPPER, Experimental evaluation of potential anticancer agents. VIII. Effects of certain nitrosoureas on intracerebral L 1210 leukemia. Cancer Res. 23, 725 (1963). W . J . ZELLER, S. IVANKOVIC and D. SCHMAHL, Chemotherapy of the transplantable acute leukemia L 5222 in rats. Cancer Res. 35, 1168 (1975). H . H . FIEBIG, W. J. ZELLER, and D. SCHMAHL, An experimental model for meningeal leukemia in rats (L 5222). Effect of treatment with BCNU and cyclophosphamide. Int. J. Cancer 18, 710 (1976). S . K . CARTER, F. M. SCHABEL,JR., L. E. BRODER and T. P. JOHNSTON, 1, 3-bis(2-chloroethyl)-l-nitrosourea (BCNU) and other nitrosoureas in cancer treatment: a review. Advanc. Cancer Res. 16, 237 (1972). T . H . WASSERMAN, M. SLAVIK and S. K. CARTER,Methyl-CCNU in clinical cancer therapy. Cancer Treat. Rev. 1, 251 (1974). T. H. WASSERMAN, M. SLAVIK and S. K. CARTER, Review of CCNU in clinical cancer therapy. Cancer. Treat. Rev. 1, 131 (1974). T . H . WASSERMAN, M. SLAVIK and S. K. CARTER, Clinical comparison of the nitrosoureas. Cancer 36, 1258 (1975). G . P . WHEELER, B. J. BOWDON, J. A. GRIMSLEY and H. H. LLOYD, Interrelationships of some chemical, physicochemical, and biological activities of several 1- (2-haloethyl)- 1-nitrosoureas. Cancer Res. 34, 194 (1974). V . A . LEVIN, W. R. SHAPIRO, T. P. CLANCY and V. T. OLIVERIO, The uptake, distribution, and antitumor activity of 1-(2-chloroethyl)-3-cyclohexyl1-nitrosourea in the murine glioma. Cancer Res. 30, 2451 (1970). V. T. OLIVERIO, W. M. VIETZKE, M. K. WILLIAMS and R. H. ADAMSON, The absorption, distribution, excretion, and biotransformation of the carcinostatic 1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea in animals. Cancer Res. 30, 1330 (1970). G. EISENBRAND,H. H. FIEBIO and W. J. ZELLER, Some new congeners of the anticancer 1,3-bis (2-chloroethyl)- 1-nitrosourea (BCNU). Synthesis of bifunctional analogs and water soluble derivatives and preliminary evaluation of their chemotherapeutic potential. Z. Krebsforsch. 86, 279 (1976). C. HANSCH, N. SMITH, R. ENOLE and H. WOOD, Quantitative structure activity relationships of antineoplastic drugs: nitrosoureas and triazenoimidazoles. Cancer Chemother. Rep. 56, 443 (1972). G. K.~RBER, Beitrap zur kollektiven Behandlung pharmakologiseher Reihenversuche. Arch. exp. Path. Pharmacol. 162, 480 (1931). L . A . VALZELLI, A simple method to inject drugs intracerebrally. Med. exp. 11, 23 (1964). H. DRUCKREY, D. SCHM.g,HL, P. DANNEBERG, K. KAISER, H. A. NIEPER, H. W. Lo and R. MECKE, JR., Unter Mitarbeit yon J. yon Einem und W. Dischler.: Vergleichende Prtifung der chemotherapeutischen Wirkung von N-oxyd-Lost und anderen alkylierenden Substanzen auf Tumoren von Ratten. Arzneimittel-Forsch. 6, 539 (1956). O . J . DUNN, Multiple comparisons using rank sums. Technoraetrics. 6, 241 (1964). D . P . RALL and C. G. ZUBROD, Mechanism of drug absorption and excretion, passage of drugs in and out the central nervous system. Ann. Rev. Pharmacol. 2, 109 (1962).

945