[CANCER RESEARCH 35, 106-109,January19751 Activity of 6-Methyl-S-substituted against the 7, 12-Dimethylbenz[a]anthracene-induced Mammary Carcinoma'

Martin J. Sweeney, Gerald A. Poore. Edmund C. Komfeld, Nicholas J. Bach, Norris V. Owen, and James A. Clemens

The Lilly Research Laboratories, Eli Lilly and Company, indianapolis, indiana 46206 [M. J. S., G. A. P., E. C. K., N. J. B., J. A. C.), and Greenfleld Laboratories, Greenfield, Indiana 46140 [N. V. O.J

SUMMARY METHODS AND MATERIALS

The ability of a seriesof 8@-carboxamidoergolines, Measurementof AntitumorEffect. The DM BA-induced 8-formamido ergolines, and 8-methyl ergolines to cause mammary carcinoma in Sprague-Dawley rats was the regressions of established dimethylbenz[a]anthracene experimental tumor used because it is PL dependent (7). induced mammary carcinomas was compared to some The tumors were induced by a single p.o. dose of 20 mg of . Although most of the derivatives de DMBA (Calbiochem, La Jolla, Calif.) in 1.0 ml of corn oil pressed serum prolactin concentrations in rats, only a few by gavage in 50- to 54-day-old female Sprague-Dawley rats had pronounced effects against the dimethylbenz[ajan weighing 100 ±10g. The 1st tumors were seen in 7 weeks. thracene-induced mammary carcinoma in rats. Some deny Rats with tumor areas of 36 to 100 sq mm were put into atives from each of the three groups of substituted ergolines groups of S or 10 for testing. Many rats had more than 1 gave comparable activities against the dimethylbenz[a]an tumor. Each tumor was identified by diagram and its area thracene-induced mammary carcinoma. was measured by vernier calipers and recorded separately. Compounds were dissolved in corn oil and dosed s.c. at 3 mg/kg once daily for 13 days, and the tumors were INTRODUCTION measured on the 14th day. The following parameters were also included: the number of tumors that regressed and the Many postmenopausal women with metastatic breast percentage of change in the area, the number of tumors that carcinoma have a higher mean basal serum level of grew and the percentage of change in the area, the number prolactin than do postmenopausal women without breast of complete remissions, and the number of new tumors that cancer (6). Although the dependency of normal breast appeared during the treatment period. The volumes for development upon PL2 has been known for many years, the dosing were 0. 1 ml s.c., 0.5 ml p.o., 0.2 ml i.m., and 0.2 ml dependency of some breast cancer on PL has been estab i.v. (water-solublesaltsonly). lished recently (8, 9). Therefore, drugs that are capable of lowering serum PL concentrations may be useful in the treatment of PL-dependent breast cancer. RESULTS The 1stclassof compoundsthatwasfoundto inhibitPL was the ergot alkaloids (4, 5, 7). Studies by Nagasawa and Ergot Alkaloids Meites (7) and by Shaar and Clemens (1 1)showed the direct effect of on serum PL in rats. A synthetic Ergocornine and several other related ergot alkaloids derivative of , 2-bromo-a-ergocryptine, also were used as standard references for PL depression and inhibited lactation and experimental mammary tumors (3). activity against the DMBA-induced mammary carcinoma. A program of chemical modificationsof the ergoline Ergocornine (I) was the most active of the ergot alkaloids nucleus of the ergot alkaloids was begun to determine the tested. It depressed the PL serum levels by 76% and reduced effect of various chemical groups on the PL and tumor the area ofthe DMBA tumors by 31%. inhibition. Many of the derivatives were 9, lO-dihydro (II) causeda 56%loweringof serumPL but onlyprevented derivatives, because the reduction of this double bond the DMBA tumors from enlarging. Ergonovine (III) and reduced or eliminated the vasoconstnictive properties of the (IV) were less active in both respects (Table I). alkaloids (1). 8-Ergolines

I Presented in part at the 64th Annual Meeting of the American Several known ergolines and the new 6-methyl-8-sub Association of Cancer Research, Atlantic City, April 11 to 13, 1973. stituted derivatives were compared to ergocornine and the 2 The abbreviations used are: PL, prolactin; DMBA, 7,12-dimethylbenz [a)anthracene;LT, ; LTM, lergotrile mesylate. other . The PL inhibition data in Table 1 are those of ReceivedJuly 1, 1974;acceptedSeptember18, 1974. Clemens et a!. (2).

106 CANCER RESEARCH VOL. 35

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1975 American Association for Cancer Research. 6-Methyl-8-ergolines and DMBA Mammary Carcinoma

Table I Activity ofergoline derivatives (3 mg/kg s.c.; 13 days) against the DMBA-induced mammary carcinoma

3

6-Methylergoline nucleus In theseexperiments 101control rats were used.There were 20 partial and 5 completeremissionsamong the 121tumors. The averagearea of the control tumors increased by 98.2%, and 8 1 new tumors appeared during the 14-day test. The average gain in body weight was 10.4 g.

growth (+) or Double- prolactin remissions/ remissions/ tumors/ regression(—) bond inhibition total total total of tumorErgocornineCompoundR% position at I hrAWC' (g)Partial tumorsCompletetumorsNew rats%

76 Dihydroergo cornine II 47 +1.0 5/9 1/9 0/5 +3 Ergonovine Ill 9-10 35 +0.8 2/5 0/5 1/5 +34 ErgotamineI' IV9-10+45VCONHCH,CH1CH(CH1)OH8@-Carboxamides9-10 17+2.2 —0.89/12 2/72/12 0/75/l@Y 2/5—31

60—4.911/15I/IS8/9—32VICONH__1@@J9-109-10

58—5.65/61/62/5—35VIICONHCH2C(CH,),9-10

65—0.41/61/6I/S+30VIII

57

IXCONH__1@@+1108-FormamidesxCON39-10 9-10 37—0.4 +0.21/4 1/50/4 0/56/4 1/5+68

76 XI a-NHCHO-lO@ 70 +1.2 2/5 0/5 3/5 0 XII a-NHCHO-2,3-dihydro 9-10 41 +9.0 3/7 0/7 3/5 +36 XIII a-NHCHOCH 9-lO 77 —14.0 4/7 2/7 0/5 +27 +101Agroclavine XIVa-NHCHOfi-NHCON@@@9-10 16+6.8 +4.66/6 2/105/6 0/102/5 0/5—81

hylergolines 8-9 62 XVI CH,OH 8-9 71 —4.4 5/6 3/6 3/5 —41 LysergolXV XVII CH,OH 9-10 56 —0.5 11/18 0/18 5/10 —29 XVIII CH,OH 59 +4.8 1/12 0/12 6/10 +66 XIX CH,CI 45 —1.8 3/6 1/6 6/5 +32 xx CH,CN 85 —3.5 7/10 2/10 3/10 —38 XXI CH2CN,2-bromo 53 +9.9 7/10 1/10 3/10 —29 XXIICH,CH1CN,2-chloro8-Met 63+0.2 —4.01/6 6/100/6 1/101/S 1/5+49 —30

a AWC, average weight change in rats during treatment. b See Chart I for some of the structures of the ergot alkaloids.

(. Data from paper of Clemens ci a!. (2). d New tumors that appeared during treatment period.

Group 1, the 6-Methyl-8j8-carboxamideErgolines.The Group 2, the 6-Methyl-8-formamides. The most active 8@-carboxamides, V to IX, caused 37 to 65% depression of PL depressors in this group, X to XIV, were the unsub serum PL in rats after I hr. Only 2 of the carboxamides, V stituted formamides, X and XI, and the acetamide, XIII. and VI, had an effect against the DMBA tumor, with 32 and However, only X was active in causing regression of the 35% remission, respectively. DMBA tumor. XI maintained the tumors at a zero growth

JANUARY 1975 107

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1975 American Association for Cancer Research. M. J. Sweeneyet a!. rate, while XIII retarded the growth of tumors (27%) com Ca755 mammary carcinoma, X5563 plasma cell myeloma, pared to the control tumors, which grew by 96%. and the Mecca lymphosarcoma in mice. These tumors are Group3, the 6-Methyl- S-methylErgolines. not known to be PL dependent. (XV) the unsubstituted 8-methylergoline, depressed serum PL by 62% but it had little activity against the DM BA tumor (49% growth with I of 6 partial regressions). DISCUSSION Monosubstituted 8fi-methyl derivatives showed both PL depression and activity against the DMBA tumor. Previous studies have indicated that the ergot alkaloids (XVII), the 8fi-hydroxymethylergoline, caused a 56% PL can reduce the serum PL in animals (7, 11). The dependency depression and a 29% regression of the DM BA tumor. of some breast tumors on pituitary hormones, and espe Dihydrolysergol (XVIII), although as active as lysergol in cially PL, has been focused on as an area for chemotherapy depressing PL, was not effective against the DMBA tumor. of breast carcinoma. The complex side chain of the ergot The chloromethylergoline (XIX) was less active than agro alkaloidscomplicatesasyntheticapproachtothe availabil clavine in prolactin inhibition. ity of the ergots. The most active 8fi-methylergoline derivative was the Several of the 6-methylergoline 8-derivatives showed 8@-acetonitnile (XX) ( 10). It was also the most active of all ability to depress PL levels in serum and to cause a the compounds in Table 1 in lowering PL serum levels in regressionofthe PL-dependentDM BA-inducedmammary rats (85%) and was one of the best in causing an overall carcinoma. These compounds may have been able to regression of the DMBA tumor (38%). Seven of 10 tumors depress serum PL levels for long periods of time, whereas partially regressed while 2 tumors regressed completely. compounds that were inactive against the tumor may not The substitution of halogens at position 2 in other have been able to sustain the low PL levels. Therefore, those ergolines is known to reduce toxicity. The 2-bromo-8fi derivatives that would depress PL for 24 hr or longer would acetonitnile (XX I) and the 2-chloro-8@-acetonitrile (XXII) showed less inhibition of PL release (53 and 63%, respec tively). However, the 2-bromo-8@-acetonitrile and 2-chloro 8@-acetonitrile caused a number of regressions of the DMBA tumor, comparable to the regression seen with the parent compound. There was little difference in the weight changes in the rats treated with either the acetonitnile or 2-chloroacetoni tnile at 3 mg/kg. The 2-chloroacetonitnile derivative (XX II), LT, was favored for clinical trials. More extensive compani sons in rats with the acetonitnile showed that LT was less toxic and showed better activity than did 6-methylergoline R1= CH(CH3)2 R@=CH3 8@-acetonitnile at the lower doses against the DMBA tumor R2= CH(CH3)2 R2 CH2C6H5 (Table 2). The methane sulfonate salt of LT, LTM, was water I ERGOCORNINE iv: ERGOTAMINE soluble and it was active only when given p.o. or i.v. (Table [ii 9-10, DIHYDROERGOCORNINE] 3). Chart I. Structure of ergot nucleus.Differencesbetweensome LTM was inactive at 3 and 6 mg/kg p.o. against the ergots are indicated.

Table 2 A ctivity of acetonitrile and L T against the DMBA mammary carcinoma in rats

of (mg/kg x doses oftumors, oftumors, complete % change CompoundDoseareaAN s.c.)AWC' (g)New tumorsNo. increased areaNo. decreased areaNo. regressionsAv. in

x 13 AN 5 x 13 —17.8 1 3(192)― 2(58) 1—100 +65 AN 3 x 13 +0.4 2 6 (98) 1 (27) +80 Control7+106LT 0—24.8 +4.0lb 30 6(106)5(100)' 05

x 13 (114) LT 5x 13 —3.8 3 2(47) 7(39) 1 —20 LT 3x 13 —4.0 1 1(83) 9(54) 1—28 —40 Control7 0—1.4 +2.21 21 3 (219)4(63) 2 (48)1 +25

(2AWC, average weight change of the rats during treatment period, 5 rats/group: AN, acetonitrile.

h Number of new tumors that appeared during treatment.

(. % decrease. d % increase.

108 CANCER RESEARCH VOL. 35

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1975 American Association for Cancer Research. 6-Me:hyl-8-ergolines and DMBA Mammary Carcinoma

Table 3 JosephParton for their technical assistance;Dr. Richard Pioch and Dr. Activity of L TM (3 mg/kg: 13 days) against the DMBA-induced William Garbrecht for supplying severalof the derivativesstudiedin this mammary carcinoma report; and the Sandoz Company for samples of ergocornine and di five rats were used in each series; some had multiple tumors. hydroergocornine.

Av.%No.ofchange REFERENCES inNewNo. ofcompletetumorRoutetumorstumorstumorsregressionsareai.p.24(lO7)@4(69)'2+19s.c.I3ofNo. I. Bove,F. J. The Story of Ergot. Basel:S. Karger AG, 1970. 2. Clemens, J. A., Shaar, C. J., Smalstig, E. B., Bach, N. J., and Kornfeld, E. C. Inhibition of Prolactin Secretion by Ergolines. Endo crinology,94:1171-1176,1974. (100)1+38iv.007 (84)1 3. Heuson,J. C., Gayer, C. W., and Legros, N. Growth Inhibition of (48)1—48P.O.006 Rat Mammary Carcinoma and Endocrine Changes Produced by (73)3—73 2-Br-a-Ergocryptine, A Suppressor of Lactation and Nidation. EuropeanJ. Cancer,6: 353-356, 1970. a % increase in size. 4. Lu, K. H., Koch, Y., and Meites, J. Direct Inhibition by Ergo b % decrease in size. cornine of Pituitary Prolactin Release. Endocrinology, 89: 229- have kept the PL low until the next daily dose of the 233,1971. ergoline. 5. Malven, P. V., and Hoge, W. R. Effect of Ergocornine on Pro lactin Secretion by Hypophysial Homografts. Endocrinology, 88: The ergot alkaloids have an undesirable vasoconstnictive 445-449, 1971. effect that has limited their use in long-term therapy. This 6. Murray, R. M. L., Mozaffarian, 6., and Pearson,0. H. Prolactin effect can be eliminated by reduction of the double bond at Levels with L-DOPA Treatment in Metastatic Breast Carcinoma. positions 8 or 9. Dihydroergocornine lowers PL but is not a In: A. Boyns and K. Griffiths (eds.), Prolactin and Carcinogenesis, vasoconstnictor. Several of the ergolines studied in this p. 158-161.Cardiff, Wales, 1972. report were dihydro derivatives. They were effective PL 7. Nagasawa, H., and Meites, J. Suppression by Ergocornine and inhibitors and were active against the DMBA carcinoma. Iproniazid of Carcinogen Induced Mammary Tumors in Rats: One of the most effective ergoline derivatives, the 8-for Effects on Serum and Pituitary Prolactin Levels. Proc. Soc. Exptl. mamido(X), producedseverecentralnervoussystemtoxic Med., 135:469-472, 1970. 8. Salih, H., Flax, H., and Brander, W. Prolactin DependenceAmong ity in rats. The carboxamide series (V to IX) showed a Human Breast Cancers. Excerpta Med., 147-155, 1973. dependency on the nature of the side chain. 9. Salih, H., Flax, H., Brander, W., and Hobbs, J. R. Prolactin Dc LTM was selected as a clinical candidate and is now being pendencein Human Breast Cancers. Lancet, 2: 1103-1105, 1972. evaluated against several PL-dependent disorders and in 10. Semonsky, M., and Kucharczyk, N. Ergot Alkaloids. XXX. Syn PL-dependent mammary carcinoma. thesis of D-6-Methyl-8-Ergolin-l-ylacetic Acid and Some of Its Derivatives. Collection Czech. Chem. Commun.. 33: 577-582, 1968. ACKNOWLEDGMENTS I 1. Shaar, C. J., and Clemens,J. A. Inhibition of Lactation and Pro lactin Secretion in Rats by Ergot Alkaloids. Endocrinology, 90: We thank Virginia Bothwell, Steven Miller, Edward Priller, and 285-288,1972.

JANUARY 1975 109

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1975 American Association for Cancer Research. Activity of 6-Methyl-8-substituted Ergolines against the 7,12-Dimethylbenz[a]anthracene-induced Mammary Carcinoma

Martin J. Sweeney, Gerald A. Poore, Edmund C. Kornfeld, et al.

Cancer Res 1975;35:106-109.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/35/1/106

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/35/1/106. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1975 American Association for Cancer Research.