STUDIES ON THE MECHANISM OF NIDATION XIII. THE RELATIONSHIP BETWEEN CHEMICAL STRUCTURE AND BIODYNAMIC ACTIVITY OF CERTAIN ERGOT ALKALOIDS P. F. KRAICER and M. C. SHELESNYAK Biodynamics Institute, Weizmann Institute of Science, Rehovoth, Israel {Received 1st March 1965) Summary. Four ergot alkaloids were assayed for their potency to inter- rupt early pregnancy and concomittantly induce vaginal oestrus in the rat. A single s.c. injection was administered on Day L4 of pregnancy (day on which sperm are observed is Lo). The meds for termination of gestation were: ergokryptine, 175 \g=m\g; ergocornine, 335 \g=m\g;ergosine, 505 pg, and ergovaline, 945 pg per rat. From the alkaloids tested it was concluded that highest activity is obtained when the cyclolpeptide moiety has 2\g=b\-isopropyl-5\g=a\-isobutylsubstituents. INTRODUCTION The alkaloids of ergot are of biodynamic interest since they are capable of up¬ setting the hormone balance of progestation. Much of our recent work has centred on the use of ergocornine in the pregnant or pseudopregnant rat. We have previously reported the relationship between dose and response for ergocornine (Kraicer & Shelesnyak, 1964). It was shown that onset of oestrus and termination of gestation could both be used as end-points of response. The structure and stereochemistry of these alkaloids were elucidated and confirmed recently by the total synthesis of ergotamine (Hofmann, Frey & Ott, 1961; Hofmann, Ott, Griot, Stadler & Frey, 1963), ergosine and ergovaline (Stadler, Frey, Ott & Hofmann, 1964) in the Research Laboratories of Sandoz Ltd, Basle. In this report we have extended our analysis to three other ergot alkaloids: ergovaline, ergosine and ergokryptine (see Text-fig. 1 ). They are all D-lysergic acid derivatives of an amino-cyclolpeptide. Ergosine and the synthetic ergo¬ valine are congeners of ergotamine, i.e. the lysergic acid is linked to an oc- hydroxyalanine residue. Ergocornine and ergokryptine are ergotoxine alkaloids with a-hydroxyvaline residues. Both ergocornine and ergovaline have L-valine in their terminal positions; ergosine and ergokryptine have L-leucine. All contain L-proline. These alkaloids can be viewed as 2 ß, 5oc-dialkyl derivatives of 2a-d-lysergamido-12oc-hydroxyperhydrooxazolo [3, 2-a] pyrrolo [2a, 1-c] pyrazine-3, 6-dione. The substituent at the 2-position is methyl or isopropyl, at the 5-position isopropyl or isobutyl. In the present series all four possible 221 Downloaded from Bioscientifica.com at 09/30/2021 04:29:45AM via free access 222 P. F. Kraicer and M. C. Shelesnyak combinations of alkyl substituents were tested. One of these alkaloids, ergo¬ valine, does not occur naturally and it was prepared synthetically. Two other polypeptide alkaloids (ergotamine and ergocristine) have been known for a long time and a third one (ergostine) has been discovered recently (Schlientz, Brunner, Stadler, Frey, Ott & Hofmann, 1964). AU are 5oc-benzyl derivatives; ergotamine is characterized by 2ß-methyl, ergostine by 2ß-ethyl Lys. Class ofalkaloid Ä1 i?2 Name ofalkaloid Ergotamine Methyl Isopropyl Ergovaline Isobutyl Ergosine Ergotoxine Isopropyl Isopropyl Ergocornine Isobutyl Ergokryptine Text-fig. 1. General structural formula ofergot alkaloids. Lys. = lysergic acid. and ergocristine by 2 ß-isopropyl. These were not tested, since in previous studies both ergotamine and ergocristine were found to be relatively inactive (Shelesnyak, 1957, 1958). We have recently tested the activity of ergostine (unpublished) and found it also to be only weakly active. MATERIALS AND METHODS Virgin female rats from the Institute of Biodynamics colony, 3 to 4 months of age and weighing 167 ± 7-4 g (mean ± s.d.) were used. Their vaginal smears showed regular cycles during the 2 weeks preceding the experimental treatment. They were caged with males of known fertility and insemination was established by the finding of spermatozoa in the vaginal smear the morning after pro- oestrus. In our colony, 97% to 98% ofinseminated female rats become pregnant. The inseminated rats were divided into groups of twenty. Each group received one dose of one of the four alkaloids tested on Day L4 of pregnancy (the 4th day of the leucocyte vaginal smear, the day on which spermatozoa were found being Lo). The injected dose was dissolved in 0-25 ml of 70% (v/v) aqueous ethanol and administered by the subcutaneous route. Details are presented in Table 1. Downloaded from Bioscientifica.com at 09/30/2021 04:29:45AM via free access Mechanism of nidation 223 Following the injection of ergot alkaloid, the vaginal smears were studied at least once daily. Rats were killed at various times between Ls and L12 and their uteri were examined for implantation sites. Two end-points of ergot alkaloid action were sought: (1) induction of the oestrous vaginal smear, and (2) termination of pregnancy. The responses were considered independently since animals may give one response without the other (although usually Table 1 responses of pregnant rats to graded doses of ergot alkaloids No. females Drug Dose Body weight (g) (vg) (mean ± s.d.) Group Termination of Onset of vaginal size gestation oestrus Ergokryptine ms 125 157 ± 15-9 20 2 1 160 150 ± 15-5 20 11 11 200 151 ± 14-3 20 12 12 250 155 ± 17-7 20 14 16 315 147 ± 14-8 20 20 19 Ergocornine ms 200 162 ± 16-1 20 0 0 250 159 ± 13-0 20 7 8 315 159 ± 12-1 20 10 11 400 160 ± 15-1 20 12 14 500 155 ± 17-8 20 17 17 Ergosine ms 200 189 ± 13-0 20 1 1 250 151 ± 14-6 20 0 0 315 151 ± 13-5 20 2 2 400 152 ± 17-1 20 5 7 500 158 ± 16-2 20 11 9 Ergovaline ms 630 145 ± 19-2 20 2 2 800 154 ± 13-1 20 9 9 1000 163 ± 16-0 20 9 5 1260 160 ± 14-2 20 16 15 ms = Methanesulphonate. termination of gestation is associated with appearance of vaginal oestrus and vice-versa). The finding of blood in the uterine lumen or of resorbing implanta¬ tion sites was counted as termination of gestation. All specimens which could not be diagnosed with complete confidence macroscopically were examined histologically. Computations were made according to Emmens (1948). RESULTS The responses to the ergot alkaloids are presented in Table 1. Dose-response lines could be fitted after transformation to log of dose and probit of response in all but one case (Table 2). The resultant dose-response lines are shown in Text-fig. 2; the lines are approximately parallel, and hence it is possible to compare the activities of the compounds using the Median Effective Dose (med) . Since one dose-response curve is not available (induction of vaginal oestrus by ergovaline), we made these comparisons on the basis of termination of gestation only. In any case, this is the response of greatest interest to us. The alkaloids were effective in the following estimated ratio: ergokryptine/ergo- cornine/ergosine/ergovaline = 1 : 0-52 : 0-35 : 0-18. Downloaded from Bioscientifica.com at 09/30/2021 04:29:45AM via free access 224 P. F. Kraicer and M. C. Shelesnyak -\-1-tv]-1-1-1-1-1-r 20 19 18 ß 6· o te s s 12 i- M.E.D. 5 Ü el 1260 Text-fig. 2. Relationship between dose of ergot alkaloid, administered on Day L4 of gestation and the proportion of rats in which gestation was terminated. Dose ( pg/rat) of methanesulphonate salt of ergot alkaloid ; response as probit and as actual number responding. Twenty females tested per point. M.E.D. 5 315 «)0 1000 1260 Text-fig. 3. Relationship between dose of ergot alkaloid, administered on Day L4 of gestation and the proportion ofrats in which the vaginal smear became oestrous. Dose ( vgl rat) of methanesulphonate salt of ergot alkaloid; response as probit and as actual number responding. Twenty females tested per point. The broken line for ergovaline indicates statistical inhomogeneity of the results. Downloaded from Bioscientifica.com at 09/30/2021 04:29:45AM via free access Mechanism of nidation 225 The actual ratio of activity for ergokryptine and ergocornine was calculated and found to be antilog 0-754 ± 0-045 (s.e.) or 1-88 : 1 with a 95% fiducial interval from 1-54 : 1 to 2-3 : 1. Table 2 dose-response relationship of ergot alkaloids* Alkaloid Ergokryptine Ergocornine Ergosine Ergovaline Termination of gestation m.e.d. (vg¡ra.t) 175 335 505 945 95% Fiducial interval (ug/rat) 150-210 280-400 360-705 790-1140 Slopef 6-21 ± 1-09 6-01 ± 1-12 5-33 ± 1-42 5-96 ± 1-53 Onset of oestrus m.e.d. ^g/rat) 178 325 515 Not 95% Fiducial interval (Mg/rat) 150-213 273-390 355-740 homogeneous Slopef 6-60 ± 1-30 5-84 ± 1-09 5-06 ± 1-44 * Calculated from the log dose-probit of response equations, f Slopes of lines in Text-figs. 2 and 3 ± s.e. DISCUSSION The polypeptide-containing alkaloids of ergot are usually classified into two groups, the ergotoxine alkaloids and the ergotamine alkaloids. Both groups share certain common structural features : a D-lysergic acid moiety and a poly- peptide moiety. The essential difference between these two families resides in the substitution at the 2-position of the polypeptide moiety, where the ergo¬ cornine family has a methyl group and the ergotoxine family has an isopropyl group. A clear relationship between structure and biological activity exists among the four ergot alkaloids studied. The dose-response lines, following log dose: probit transformation, were parallel, allowing quantitative comparisons to be made. The rank order of effectiveness was ergokryptine, ergocornine, ergosine, ergovaline. In other words, the highest activity for inducing termination of gestation was obtained with the 2ß-isopropyl-5oc-isobutyl substituents on the cyclolpeptide moiety.