TITLg

NITROSOUREA DERIVATIVES HAVING ANTITUMOR ACTIVITY

RELATED APPLICATIONS This application is -a continuatio -i - art: of U.S.

Serial No. 117,940 filed 14- August 1980.

• TECHNICAL FIELD

The present invention relates to novel nitrosourea derivatives and pharmaceutically acceptable salts thereof useful for their antitumor and anticoagulant activity. The invention also includes pharmaceutical compositions containing these compounds and methods of using them.

BACKGROUND ART

In the past decade, nitrαsoureas have gained acceptance as potent antitumor agents (T.P. Johnston, et al, J. Med.

Che . _> 14, 600 (1971)).. The accepted mode of action appears

.to involve the release of isocyanate in vivo. The compounds

.most frequently used clinically are 1-cyclohexyl-5-(Z-chlαrα-

^• ethyl)-3-nitrosourea (CCNU) , 1,3-bis- (2-chloroethyl) -3- nitrosourea (3CNU) and MeCCNU (1- (4--trans-me hylcyclohexyl) - 3-(2-chloroethyl) -3-nitrosourea which release in vivo an isocyanate derived from the unnitrosated side of the molecule, and an al ylating agent from the other side thereof. MeCCNU

C PI

has been found to possess the highest degree of activity against most tumor systems, especially solid tumor systems. Numerous studies have been directed toward the meta¬ bolic products produced in vivo and in vitro in aqueous media, S which consist mainly of 2 ^' -chloroethanol, vinyl chloride, acetaldehyde, and dichloroethane from the nitrσsated side of the molecule. (_T. ? . Johnston et al, J. Med. Che . , 13: 634 (1975)). It is also known that the N-nitrαso-N-alkyl ureido portion of the molecule alkylates DNA (deσxyribonucleic acid) 0 i vivo and in vitro (Frei et al, 3iσchem. J. , 174: 1031

(197S)). In fact, is has bee shown that the carcinogenic ' effectiveness of agents such as N-m hy1-N- itrosourea cor¬ relates with the extent of alkylation αf the guanine moiety in DNA of target tissues at the oxygen- atom. 3 Alkylation of DNA occurs within an hour of.administra¬ tion of the nitrosourea, and the half-life of the alle lated products is about 24 to 43 ^" hours (D.J. Reed et al, Cancer Res. 55: 568 (1975)). The study indicated that low doses of nitrσ- soureas pose only a small threat as utagens, and hence, are 0 not significantly carcinogenic.

It has recently been shown that a number of isocyanates are potent inhibitors of transglut minase- (Gross et al, J. 3iol. Chem. , 250: 7693 (1975)), a calcium-de endent enryme which may catalyse lysine-gluta ine erasslinking of certain 5 proteins present onneoplastic cell surfaces. This enryme (Yancey et al, Ann. N.Y. Acad. Sci. , 202: 544 (1972)), and other similar enzymes such as gamma-glutamyl tran≤peptidase (Novogrσdsky et al, Proc. Natl, Acad. Sci. U.S.A., 75: 2414 (1976); Fiala et al, J. Natl Cancer nst. , 57: 591 (1976); 0 Cameron et al, Cancer Res . , 53: 323 (1978)), have been impli¬ cated in the uncontrolled proliferation αf cancer cells and fibrin crosslinking. It is generally accepted that these crosslinked proteins form an extracellular coating causing the cell to be unrecognised by the cellular immune system,

^■ 35 thus preventing normal destruction of the foreign neαplastic

tissue. These enzymes are fairly specific toward glutamine and gluta ic acid residues as substrates, and isocyanates resembling these residues have been found to be the most effective inhibitors (Gross, et al, J. Siol. Chem., 250: 7693 (1975)). -Other enzymes with similar functions and specifi¬ cities may also be involved,, such as other glutamyl cycle enzymes. From this it may be postulated that the more the isocyanate resulting' from the decomposition of the nitro¬ sourea shows specificity toward such enzymes, the lower the required dose, resulting in -a reduced risk of carcinogenesis from the antitumor agent.

The structure of the active site of trans lutaminase has been found to contain the pentapeptide sequenc --Ty -Gly- Glπ-Cys-Trp-- and has* the shape of a pocket S x S Angstroms in dimension (Folk et al J. Biol. Chem. , 241: 5253 (I960)).

Gaπma _^ gluta yl tran≤peptxdase may be expected to have a somewhat similar active site structure..

Toxic side effects- such as myelosuppression are αf major concern in chemotherapy when πitrosαureas are employed. Apparently, this effect is not directly related to the alkyl- ating agent released in vivo from the nitrosated side αf the nitrosourea molecule.. This is. supported by the fact that large doses of the nitrσ oureas Stre.ptσzαtocin and Chlorozotαcin relative to other nitrosou asr produce a comparatively minute level of myelosuppression (Schein et al, Cancer, 54: 935

(1974)). Compounds such as CCNH and 3C U exhibit myelosup¬ pression as their most toxic side effects. All four agents release similar alkylating- agents in vivo. It has further been demonstrated that Streptozotocin. and Ghlαrozotocin release the same isocyanate which then undergoes intramolecular carba- moylation, forming a cyclic carba ate (Montgomery et al, Cancer Treat. Rep., 60: 651 (1976)). This intramolecular react may therefore be eliminated by use of a cyclic carbamate, or other carbamate directly.

DISCLOSURE OF THE INVENTION

According to the hypothesis upon which the invention is

based, a superior inhibitor αf transglutaminase and related enzymes would have, in view- of the shape and size indicated above , hydrophobic moieties directed away from, but in proximit to, the pocket at the active site. The substrate may have S groups which aid hydrogen bonding to the active site tyrosine hydroxyl group. Other compounds, designed previously by the inventor of the present application (U.S. Patent Application S.M ^" . 68,470) using this approach have been shown to be suc¬ cessful antitumor agents and anticoagulants. 0 In accordance with the present invention, some 1- cyclαalkyl methyl-3-(2-chlαroethyl)-3-nitrσsoureas bearing hydroxyl,. ^* rhalogen*. or other hydrogen bonding groups at the tertiary position on the cycloalkyi ring were synthesized for evaluation as antitumor agents and anticoagulants. The hypα- 5 thesis αf the invention is supported by the fac that hydro- phobic groups attached to the beta- or gamma-carbon atαm αf glutamine produce superior substrates for transglutaminase CGross _. et al _* J. Bioi. Chem. , 243; 130 (1375)} and- similar enzymes, and nitrosoureas which release isσcyanates analogous 0 to these substrates are potent antitumor agents (U.S. Patent Application S.N'. 68,470). The hydroxyl group attached to tyrosine in the active site provides a location where hydrogen bonding may occur, and the hydroxy- and halogenated isσcyanates releas-ed from the nitrosoureas of the present invention should 5 therefore have a much higher affinity far the active site of transglutaminase and possibly other similar enzymes than alkyl isocyanates which are not substituted. This may tend to ex¬ plain the efficacy of compounds such as BCTϋ and CC U, since 3CNU contains a chloro group which may hydrogen bond to -the 0 active-site tyrosine hydroxyl group, and CQΪU and MeCCNU are known to be hydrαxylated in the liver to derivatives which may contribute to its ^* binding to the active site in a similar manner. This hydroxylation, however, lowers iipid solubility and hinders crossing αf the blσod»brain barrier. S The mechanism of inhibition of the isσcynate is via the alkyl thiαcarbamate ester formation at the single sulfhydryl

group at the active site of the enzyme (Gross et al, J . Biol Chem., 250: 7695 C T ) :

H 0

Enzyme-SH + ^• R-N*C-*0 -^ R-N-C-S-Enzyme wherein R represents an alkyl group.

In accordance with the present invention, nitrosourea derivatives release the following- cycloalkyl isocyanates:

wherein R is a group capable αf hydrogen bonding to the active site tyrosyl group of the enzyme, preferably a halogen, carboxyl, αr hydroxyl group, or a derivative thereof, and more preferably a chloro or hydroxyl grou .. The cycloalkyl ring may.be further substituted with 1 or more preferably 1 or 2 alkyl groups, preferably methyl groups, or 1 or more preferably-1 or 2 hydroxyl groups. Recent studies have shown that the activity of nitro¬ soureas is markedly enhanced by the 2-chloro thyl or the 2- fluorethyl groups present on the nitrσsated side of the com¬ pound ( ontgomery, Cancer ^' Treat, Rep., 60: 651 (1976); Johnston et al, JY Med. Chem. , 9: 892 (1966); Farmer et al, J. Med. C m.y 21: 314 (197S)). Since the 2-chiαrρethyl and 2-fluoroethyl groups also enchance solubility, they are the pref rred groups in the present invention.

In accordance with the present invention, novel compounds which possess the structural criteria necessary to selectively inhibit transgluta inase and similar glutamyl cycle enzymes and therefore be of potential significance as antitumor as well as anticoagulant agents are defined by the following formu

OMFI

wherein, n is 4 to 7 _t hai is ^' chlo-τάne'-or flourine and R is a group capable of hydrogen bonding to the active site tyrosyl group of the enzyme, preferably a halogen, carboxyl, or hydroxyl group, or a derivative thereof, and more preferably S a chloro or hydroxyl group. The resulting S to 8 carbon ring may be substituted with one or more, preferably 1 or 2 lower alkyl groups _r preferably methyl groups to enhance selectivity, or certain hydrophiiic groups, such as hydroxyl groups, to enhance solubility, or other groups, which are widely described 0 in the art.

While only one carbon atom is placed between the ring and the H group, the addition of one extra carbon is not to be considered beyond the scope of the present invention, since the isσcyanates released from these'compounds would S also be in accordance with the enzyme inhibition criteria, since "branching",, in effect, would occur at the gamma-carbon attached to the isocyanate group,

It is, therefore, an object of the present invention to provide novel compounds having antitumor activity in 0 humans and other mammals.

It is another object of the invention to provide novel compounds which inhibit the activity of transglutaminase and enzymes with similar functions having similar specifications. Still another object αf the invention is to provide S novel compounds which are effective antitumor agents at low doses, i.e., that have a lower toxicity than MeCCNU, so as to minimize adverse mutagenic and/or carcinogenic eff cts, and to provide compounds with minimal or nonexistent yelosuppres- sive eff cts. 0 An additional object αf the invention is to provide compositions containing the novel compounds and methods αf using them.

Yet another object of this invention is to provide novel nitrosourea derivatives which have anticoagulant activity.

BEST MODE QF THE- -INVEHTTON

Melting points were determined using a Thomas-Hoover capillary melting point apparatus and ^* are uncorrected.. Infra¬ red (IR) spectra were obtained using a Perkin Elmer 59? spectrophotometer, and NMR spectra were taken on a Nicolet 200 MHz instrument using CDC1- as the solvent and TMS as an internal standard.

EXAMPLE 1

₂ CH ₂ C1

eostatin l-teτt-Hydroxycyclohexylmethyl-j-(2-Chloroethyl)-S-Nitrosou rea

1-Aminomethyl-l-cyclohexanol hydrochloride (16.6 g,' 0.1 mol) was stirred with 13.9 ml (0.1 ol) of trie hylamine in 7S ml of anhydrous diethyl ether and cooled to less than 5" 2-Chloroethyl isocyanate (10.5 g, 0.1 mol) was dissolved in 2S ml of ether and added in small portions with stirring while maintaining a temperature αf less than 5 ^β . After 2 hr. ad¬ ditional stirring, the reaction mixture-was filtered and the residue was washed with two 20 ml portions αf ether and sus- pended in 20 ml of water, filtered, resuspended in 20 ml of water, refiltered, and dried in vacua over CaCl ₇ /K.QH. The yield of the urea was 19.3 g (83%) as a.white powder, MP 109- 110°.

An 11.7 g (0.0S mol) quantity of the above compound was dissolved in SO ml αf 98% formic acid and cooled to 0 ^β .

Sodium nitrite (6.9 g, 0.1 mol) was added in portions with vigorous stirring while maintaining a temperature less than S ^a . After stirring an additional hour, 100 ml of water was added. The mixture was extracted with 50 ml αf ether, may

be washed with 5% NaHCG- and was- vaporated to dryness in vacuo giving 13.2 g (82%) of the nitrosourea as an oily solid. I analysis showed bands at 1710 c (C ^a O) and 1530 cm (C-N-H) . NMR showed a triplet at 4.2 pp (2H, J*ό.5 Hz), a triplet at 3.5 ppm (2H, J*ό.S Hz), and a ultiplet at 1.6 ppm (12H) . The l-nitro"SO isomer was present also to the f- ^• - extent αf about 30%.

EXAMPLE 2

(l-Chloro-l-cyclohexyl)methyl-3-(2-C51oroethyl) -3-Nitrosourea

0

CH Cl ^■

The above compound was made using, the procedure in Ex¬ ample 1 replacing the hydroxy-starting material with the corresponding chloro-compound. Yields were similar with approximately 35% of the isomer present. Other compounds according to the present invention, such as methyl or hydroxyl substituted cycloalkyl derivatives may be synthesized according to the procedure described in Example 1 by using an appropriately substituted 1-aminomethyl- 1-cyclσalkanol hydrochloriάe which may be synthesized by known procedures.

Antitumor Screening

Antitumor-screening data were obtained through the National Cancer Institute, Drug Evaluation 3ranch, National Institutes of Health, 3ethesda, MD. Screening data for MeCCNU was generated simultaneously for comparison purposes. L-1210 lymphoid leukemia tumors, (10 ³ cells) were implanted in CDF. mice in accordance with NIH Protocols. The results reported in Table I are single dose responses with survival being evaluated S days after i.p. injection of

Neostatin (six days after implantation) . The compound was inj ected as a suspension in 10% EtOH, 10% emulphor, and 80% saline. Log kill data indicates the number of tumor cells killed, and 30-day survivors- are termed "cured" . The degree of antitumor- activity is expressed as a ratio of treated animals over control animals using NCI test evalua¬ tion numbers , as speci iced on individual protocols . Table II contains comparison data for MeCCNU, the most potent agent against this tumor system in use at the present time.

TAULϊi I

Neostatin v. 1.-12X0 Leukemia

Dose Sex Uose per . Sur- Log Cures No- Tu ored Weight Test

Injection vival Kill akes Survi ^¬ i»iff. T/C Eval Og/kg) vors (T/C)

1 100.00 3/6 Toxic 0 0 0 -3.9

2 SO.00 6/6 5.94 3 0 0 -3.1 253* 22.8 3 25.00 6/6 5.94 5 0 0 -2.0 318* 28.7

4 12.50 6/6 3.57 0 0 0 -1.5 142* 12.8

5 6.25 6/6 1.67 0 0 0 0.0 120 10.8 6 M 100.00 6/6 5.94 1 0 0 -3.9 187* 15.0 7 M 50.00 6/6 5.94 4 0 0 -1.4 316* 25.3 8 M 25.00 6/6 3.29 0 0 0 -1.2 143* 11.5 9 12.50 6/6 2.16 0 0 0 -0.8 128* 10.3 10 M 6.25 6/6 0.66 0 0 0 -0.2 108 8.7

txceeϊϊs ^" the mi imum T7C ^~ ° ^~ P V iii require ^' tO." fϊuhcate a potential carcmostatic agent

TABLE I (continued)

Dose DAY/DEATHS

1 5/3 7/2 8/1

2 6/1 10/1 28/1

3 17/1

4 ^β /i 12/1 13/2 15/1 16/1

5 10/2 11/3 12/1

6 6/2 10/1 17/1 20/1

7 14/2

10 8/4 9 1 U/l

Control (Female)

7/2 8/7 9/12 10/6 11/3

Control (Male)

7/1 8/21 9/2

TABLE π O

MeCCNU (NCS 95441) V, L-1210 Leuk :emia

Dose Sex Dose per Sur¬ Cures No- Tumored Weight Test ^" No. Injection vival Takes Survi¬ Diff. T/C Eval. (mg g) vors (T/C)

11 E 50.00 6/6 ^' 3 0 0 -4.0 214* 19.3

12 V 25.00 6/6 5 0 0 -0.8 314* 28..3

13 J- 12.50 6/6 0 0 0 -0.9 172* 15.5 S

14 P 6.25 6/6 0 0 0 -0.4 113 10.2

15 M 50.00 6/6 0 0 1 -4.2 227* 18.2

16 M 25.00 6/6 6 0 0 -2.4 375* 30.0

17 M 12.50 6/6 3 0 0 -0.9 310* 24.8

18 M 6.25 6/6 0 0 0 -1.7 135* 10.8

*Exceedi > the minimum 25 require*! to indicate a potential carcinostatic agent '

TABLE U (continued)

Dose DAY/DEATHS f

11 6/2 11/1

12 15/1

13 11/1 12/1 15/1 16/1 18/1 21/1

14 9/2 10/2 11/1 12/1

15 6/1 11/1 12/1 19/1 30/1

16

17 13/1 15/1 28/1

18 10/2 11/3 12/1

Control (Female)

7/2 8/7 9/12 10/6 11/3

Control (Male)

7/1 8/21 9/2.

"

Table III compares the results obtained using, a different tumor system (F-1S34- leukemia). I. . injections were made daily for S days. Survival was measured on day 5, and "cures" were determined 45 days after implanta¬ tions.

TABIB in Neostatin (NSC 32S624) and MeCCNU (NSC 9541) v. P-1S34 leukemia

Dose Sex t I Test

T/C pval.

Neostatin

19 V 16.00 6/6 P 0 0 3.5 153* 15.0 20 F 8.00 6/6 3 0 0 3.9 459* 45.0 21 P 4.00 6/6 0 0 0 2.6 191* 18.8

22 P 2.00 6/6 0 0 0 2.5 123 12.1 23 F 1.00 6/6 0 0 0 1.1 104 10,2

MeCCNU

24 F 16.00 5/6 0 0 0 4-3 115 11,3 25 P 8.00 6/6 1 0 0 3.4 225* 25.0 26 F 4.00 6/6 1 0 0 2.8 225* 25,0 27 1* 2.00 6/6 0 0 0 1.7 132* 13.0 28 P 1.00 6/6 0 0 0 1.2 111 10.9

^■ Exceeds the minimum 125 required to indicate a potential carcinostatic agent.

tτ.|

TABLE HI (continued)

Pose DAY/DEATHS

II

Neostatin

19 11/1 13/2 15/1 19/1 24/1

20 6/1 13/1 29/1

21 17/3 19/2 21/1

22 10/1 12/4 13/1

23 10/5 11/1

MeCCNU o*.

24 5/1 8/1 11/2 12/1 13/1

25 15/1 19/1 21/1 25/1 42/1

26 21/1 23/1 24/1 25/1 41/1

2 12/2 13/3 14/1

28 10/2 11/4

Control

8/1 9/13 10/16 11/6

Neostatin was found to have activity paralleling that α£ MeCCNU in the L-121Q system with lower toxicity at high doses as measured in terms of weight loss. Since L-1210 leukemia responds well to _. number of nitrosoureas, the additional, less responsive tumor system C-P-153**) was employed for further testing. Using thi system, fteostatin is seen to be far superior- to MeCCJTU at similar dose levels.

Similar clinical results can be obtained with the use of the other compounds of the invention. The nitrosoureas α£ th -invention can be formulated into a form suitable for administration by methods well known in the art. For example,, they can be admixed with pharmaceutical acceptable carriers or diluents such as ethanol, lactose, starch, magnesium stearate,. tragacanth, gelatin and sodium carboxymethylcellulose, and the resulting mixture or solution may be* processed, by conventional procedures to pharmaceutical dosage unit forms such as capsules, tablets, powders, pills, ampoules, suppositories and the like. ,

The compounds of the invention, such as Meostatin may be administered orally or parenterally. For example, the drug may be given intravenously by first dissolving the compound to be administered in 0.5-10 ml of ethanol and adding 50-90% water thereto. Further dilution may be made with physiological saline solution or S% dextrose- (USP) _} resulting in a solution slightly acidic in pK. Intravenous admini tration may be continued for a period of up to about two hours. The nitro¬ soureas may be administered parenterally oτ orally at dosages of about 0.5-5 g/kg.

Other parenteral'routes of adminis ration may be accom- pushed using any formulation known in the art that allows for the emulsification, dissolution and suspension of relatively water-insoluble drugs or other compounds prior to parenteral administr tion.

When given in unit dosage forms orally, compounds such as Neostatin are active when provided in a gelatin capsule or

13

tablet combined with pharmaceutically acceptable binders, fillers or other additives as known in the ^' art.

The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a. departure from the spirit and- scope αf the invention,, and all such modifications are intended to be included within, the.- scope of th following claims.