Abstract:
New nitrogen mustard analogs with arms of unequal reactivity on different nitrogen atoms have been made. The new analogs are less toxic, potent, cancer chemotherapentic agents.

Description:
BACKGROUND OF THE INVENTION 
     1. Technical Field 
     The present invention is related to nitrogen mustard analogs with arms of unequal reactivity on different nitrogen atoms. More particularly, the present invention is related to 1-(2-chloroethyl)-4-(3-chloropropyl)-piperazine, dihydrochloride or similar halo-derivatives and method of preparing the same. The new analogs are less toxic, potent, cancer chemotherapeutic agents. 
     2. State of the Art 
     Tertiary amine alkylating agents with one or two beta-haloethyl groups attached to nitrogen have been used in a variety of pharmacologic regimens. The former are employed principally as alpha adrenergic blockers and the latter, frequently described as nitrogen mustards, are cytotoxic agents. Analogs of nitrogen mustards containing a beta-haloethyl group and a gamm-halopropyl group attached to the same nitrogen have been known and reported to be effected cancer chemotherapeutic agents. Piperazine mustard, 1,4-bis(2-chloroethyl)piperazine, has also been prepared and found to be active against murine leukemia (Burchenal et al., Cancer, 4: 353, 1951), but its analog containing a beta-chloroethyl group and a gamma-chloropropyl group on different nitrogens has not been produced. 
     SUMMARY OF INVENTION 
     It is, therefore, an object of the present invention to provide new analogs of nitrogen mustard with arms of unequal reactivity on different nitrogen atoms. 
     It is a further object of the present invention to provide new analogs of nitrogen mustard with decreased host toxicity (LD50) but with equal or greater chemotherapeutic activity compared to mechlorethamine. 
     Other objects and advantages will become evident as the detailed description of the present invention proceeds. 
     DETAILED DESCRIPTION OF INVENTION 
     The above and various other objects and advantages of the present invention are achieved by new analogs of nitrogen mustard with arms of unequal reactivity on different nitrogen atoms, a preferred analog being 1-(2-chloroethyl)-4-(3-chloropropyl)-piperazine, dihydrochloride. 
     Unless defined otherwise, all technical or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. 
    
    
     EXAMPLE 1 
     Preparation of 1-(2-chloroethyl)-4-(3-chloropropyl)piperazine, dihydrochloride 
     Preparation of 1-(2-hydroxyethyl)-4-(3-hydroxypropy)piperazine [I]: 
     1-(2-hydroxyethyl)-piperazine, 26 grams, freshly distilled allyl alcohol, 36 g and sodium hydroxide, 8 g are heated to 118°-120° C. for 36 hours. The viscous reaction mixture is taken up into 250 ml water, and solid potassium carbonate is added to saturate the warmed solution. A yellow oil, the product, rises to the top. It is extracted with tetrahydrofuran, the solvent evaporated, the residue dissolved in ethanol, the solution filtered, and the ethanol evaporated under vacuum to a thick oil, which solidifies on standing to give the isolated product, [I], above. 
     EXAMPLE 2 
     Preparation of 1-(2-chloroethyl)-4-(3-chloropropyl)piperazine, dihydrochloride [II]: 
     The dihydroxy compound, [I], 10 g, described above, is dissolved in 100 ml dimethylformamide and the solution is cooled in an ice-bath. To the solution is added carbon tetrachloride (25 ml) and triphenylphosphine (28 g) and the mixture stirred for four hours at room temperature (22°-25° C.). The solution is evaporated under vacuum and the residue taken up in 2N hydrochloric acid. The precipitate, triphenylphosphine oxide, is removed by filtration and the clear solution brought to pH 8 with 2N sodium hydroxide. It is extracted 3 times with ethyl acetate, dried with anhydrous sodium sulfate and the solvent removed by evaporation in vacuo. The oil obtained is dissolved in a minimal volume of ethyl acetate and the solution mixed with hydrochloric acid in dioxane to precipitate a white solid, the product, (II). 
     Of course, the amounts shown in the above examples are only illustrative. Upgrading and adjustment of various parameters described in the above methods for large scale production can be easily achieved by one of ordinary skill in the art to which the present invention belongs. 
     Animal studies 
     Male CDF 1  mice (Balb/c×DBA/2), 8-12 weeks old and weighing 22-31 grams, were placed in groups of equivalent weight, 5 or 6 animals per plastic cage with wood chip bedding and were given laboratory chow ad libitum. L1210 cells of the NCI strain were maintained in female DBA/2 mice and transplanted intraperitoneally (i.p.) into male CDF mice for experiments. They were harvested on day seven following the inoculation of 1×10 5  cells. 
     Chemotherapy of L1210 leukemia bearing mice 
     The most effective treatment schedule for mice with 1-(2-chloroethyl)-4-(3-chloropropyl)piperazine was obtained by daily intraperitoneal injections, which resulted in an increase in survival of 67% over controls (Table 1). Increased survival with the standard nitrogen mustard, mechlorethamine, was only 61%. 
     
                       TABLE 1______________________________________Treatment of Mice Bearing L1210 Leukemia with1-(2-Chloroethyl)-4-(3-Chloropropyl)piperazine 2HClL1210 cells, 1 × 10.sup.5, were injected i.p. on day 0and treatment was begun i.p. on day 1.There were 6 mice per group.T/C is survival in days of treated animalsdivided by survival of controls.          Mean Survival Time                         T/CTreatment (mg/kg)          (days)         (%)______________________________________0                  8.2            100100    (day 1)     7.0             85*50     (day 1)     11.4           13925     (day 1)     10.0           12210     (days 1-5)  11.5           1405      (days 1-5)  9.8            1202.5    (days 1-5)  9.2            1121.25   (days 1-5)  8.8            1070                  8.2            10030     (days 1-5)  8.0             98*20     (days 1-6)  12.0           14610     (days 1-7, 10)              13.7           1670                  8.0            10010     (days 1-5)  12.6           158______________________________________ *T/C less than 100% is due to toxicity. 
    
     Chemotherapy against a spectrum of tumors: 
     Tables 2 and 3 show a drug screening summary of NSC 344007 (the compound of the present invention). It can be seen that the compound is surprisingly effective in the human tumor colony forming assay, against a number of murine tumors and in a human tumor xenograft test. 
     
                                           TABLE 2__________________________________________________________________________HUMAN TUMOR COLONY FORMING ASSAY         **CONFIRMATION TESTING**         (DOSE RESPONSE)         EVALUABLE/TOTAL         ASSAYS      RESPONSES__________________________________________________________________________TUMOR                             TUMOR   MGROUP                             GROUP   O                                     DMELANOMA      5/5         4 (80%) MEL     ROVARIAN CARCINOMA         4/4         1 (25%) OVATOTAL         9/9         5 (55%) ALL GROUPSOVERALL RESPONSE         5/9           (55%)RATE AT 10OR LESS MCG/MLAN OVERALL RESPONSE RATE OF 20% OR GREATER IS THE CURRENTACCEPTABLE GUIDELINE.__________________________________________________________________________ 
    
     
                                           TABLE 3__________________________________________________________________________                          RATING                OD   CURES/SYSTEM  NAME OF TUMOR          ++/+/-                                RT  SCHEDULE                                           T/C  MG/KG                                                     TOT__________________________________________________________________________3B131   B16 MELANOMA           ++    IP  QD1-9  260                                                10   3/10                                                3LE31 L1210                                                     LEUKEMIA ++ IP Q                                                     D1-9 209                                                     10 0/10                                                       HUMAN MAMMARY                                                     TUMOR IN                                                3MBG5  ++ SC Q4DX3 -6                                                     1                                                     15 2/6                                                      SUBRENAL                                                     CAPSULE                                                3M531 M5076                                                     SARCOMA ++ IP Q4                                                     DX4 240                                                     20 5/10                                                3PS31 P388                                                     LEUKEMIA ++ IP Q                                                     D1-5 238                                                     10 0/5                                                3PO31 CYTOXAN                                                     RESISTANT P388                                                     LEUKEMIA ++ IP Q                                                     D1-5 197                                                      9 0/10                                                3C872 COLON 38                                                     CARCINOMA                                                     + IP Q7DX2 16                                                     20 0/10                                                3CDJ2 CD8F.sub.1                                                     MAMMARY                                                     TUMOR ++ IP Q1DX                                                     1 -43                                                     40 0/10                                                3LE32 L1210                                                     LEUKEMIA                                                     + IP QD1-9 138                                                     10 0/10                                                3CP31 CIS PLATIN                                                     RESISTANT P388                                                     LEUKEMIA                                                     + IP QD1-5 170                                                     13 0/10__________________________________________________________________________ 
    
     In summary, the compound of the present invention when tested according to the protocols of the Development Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, is found to be highly active (++) against the following mouse tumors: the B16 melanoma, the CD8F1 mammary adenocarcinoma, the P388 and L1210 leukemias, the M5076 sarcoma, the cytoxan resistant P388 leukemia and active (+) in the colon 38 carcinoma, the cis-platin and L-PAM resistant P388 leukemias. It gave reproducible cures for the B16 melanoma and the M5076 sarcoma and was highly active (++) in the human mammary subrenal capsule xenograft (MX-1) and scored positive in 4 of 5 tests in the human melanoma colony forming assay (Tables 2 and 3). The results indicate that the compound of the present invention has both carcinocidal as well as carcinostatic properties. 
     Of course, the compounds of the present invention can be used in a pharmaceutical composition comprising a chemotherapeutically effective amount of the compound in a pharmaceutically acceptable carrier such as sterile water, physiological saline, non-toxic physiological fillers and/or buffers and the like well known in the art. The composition can be in any suitable form such as a liquid, a solid, a capsule, a tablet, a paste or creamy mixture and the like. 
     It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.