recently (Rake et al., 1949). p-acetamidobenzaldehyde with thiosemicarbazide and related compounds; table

Transcription

1 THE CHEMOTHERAPY OF EXPERIMENTAL TUBERCULOSIS II. THIOSEMICARBAZONES AND ANALoGUES IN EXPERIMENTAL TUBERCULOSIS IN THE MOUSE DOROTHY HAMRE, JACK BERNSTEIN, AND RICHARD DONOVICK The Squibb Institute for Medical Research, New Brunswick, New Jersey Received for publication February 1, 1950 The claims for antituberculous activity among the thiosemicarbazones of several substituted benzaldehydes (Domagk et al., 1946) have stimulated experimental studies both in this country and abroad (Donovick and Bernstein, 1949; Hoggarth et al., 1949). We wish to report at this time the data acquired to date in our standardized test using the mouse (Rake et al., 1949) on a series of related compounds. In this test 17- to 19-g mice of the CFI strain in groups of 10 are inoculated intravenously with the Ravenel strain of Mycobacterium tuberculosis. The mice are given the test drug in the powdered diet at two or three levels for 21 days beginning 48 hours before inoculation. In each experiment groups of infected mice given powdered diet containing no drug as well as those treated with PAS (p-aminosalicylic acid) at per cent and 0.75 per cent in the diet are used as controls. In the current tests the T60, time of survival of 50 per cent, of the control mice given no drug is still very close to 20 days. However, the mean increase over the controls in T5o for mice treated with PAS is +1.8 days at per cent in the diet and +5.5 days at 0.75 per cent in the diet. These increases in T5o are significantly less than those observed during the first year of the tests and reported recently (Rake et al., 1949). The data on the thiosemicarbazones are collected in five tables. Table 1 lists all the derivatives of benzaldehyde thiosemicarbazones carrying one or more substituents on the benzene ring; table 2 lists all the condensation products of p-acetamidobenzaldehyde with thiosemicarbazide and related compounds; table 3 lists all the thiosemicarbazones of aldehydes and ketones not covered in table 1; table 4 lists the remaining structurally related but otherwise miscellaneous compounds tested to date. Those compounds showing definite, reproducible antituberculous activity in vivo in our hands are collected in table 5. In each table we record the structure of the compound, the approximate maximal acceptable level of drug in the diet on which the mice will gain weight, the actual drug intakes averaged over the first 7 days in the therapeutic tests, the corresponding increases in T5o, and the minimal inhibiting concentration in mg per ml in our standardized in vitro test (Donovick et al., 1950) for the BCG strain of M. tuberculosis. Examination of the tables reveals that, of the compounds studied by us, in vivo activity in tuberculosis in the mouse is found only in para-substituted benzaldehyde thiosemicarbazones. Of the various substituting groups, the order of 675

2 676 HAMRE, BERNSTEIN, AND DONOVICK [VOL. 59 TABLE 1 Ar-substituted benzaldehyde thiosemicarbazones CH=N-NH-C-NH2 3 2 ~~~s MAXnIMAL S<UBSTITUNT DRUG ACCEPTABLE INTAKE DRUG INTAKE DAILY INCREASE EC FOR B G INT1* C DAILY mg/kg mg/kg days Ag/mi None CH CH(CH,) ' Cl, 4 Cl Cl, 4 Cl OH I, 4-OH, I5-2, CH,--O CH CH, OH, 3-OCH, 2, OCH,, 4-OH 1, OCH,, 3-OCH, OCHs, 4-OCHs SO2-C2H, > SO2NH b NH ' N(CHs) NH-C-CH, I ', ' b 3-NO NO2, * The reciprocal of the survival time has a distribution approximately Gaussian and it is permissible to use the Student's t test for comparing mean reciprocal survival times. This has been done above and those tests giving significant results are marked with b or 6 corresponding to significance of 1.0 per cent and 0.1 per cent. We are indebted to Mr. K. A. Brownlee for this analysis of the data. activity as expressed in terms of the inverse molar intake required to effect a minimal response is: ethyl sulfonyl = isopropyl > amino = acetamido = di-

3 1950] CHEMOTHERAPY OF EXPERIMENTAL TUBERCULOSIS 677 TABLE 2 Derivatives of p-acetamidobenzaldehyde CH,-C-NH- CHR MAXIMAL DUNAE ICES SUBSTITUENT ACCEPTABLE DAUGILTAY EIN Re mc FOR BCG DRUG INTAKE DIY I a mg/kg mg/kg days mg/ml -o 52,000 1, ==N-NH-C-NH =N-NH-C-S-CH3 52, N-NH-C-NH Il S =-N-NH-C=NH I SCH3 =N-NH-C=NH I +NH2- CL- =N-NH-C-NH-CH =N-NH-C-NH-CH2-CH=CH2 52, N-NH-C-N(C2H6) N-NH-C-NH-CoH 5>2,000 1, CHsS I =-SN-N-C-NH-CH I I S -N-NH]2 C-S =C[-C NH]2 C=IS 52,000, II 0 Significant at 0.1 per cent. methylamino > nitro = sulfamyl = methoxy. We do not wish to imply that all other compounds listed are thereby less active than those just cited. In many

4 678 HAMRE, BERNSTEIN, AND DONOVICK [VOL. 59 TABLE 3 Thiosemicarbazones of miscellaneous aldehydes and ketones ALDERYDE OR KETONE ACCEPTABLE DRUG INTAKE INCREASE KIC FOR BCG DRUG INTAKE DAILY IN T6se ICFR C mg/gk mg/kg days Ag/mi a-amylcinnamaldehyde Glucose Furfural Nitrofurfural Thiophenecarboxaldehyde Cyclopentanone Cyclohexanone Acetophenone p-aminoacetophenone Phenyl-2-propanone * None of these increases is significant. TABLE 4 Miscellaneous compounds MAXIaAL STRUCTURE ACCEPTABLE DRUG INTAKE INCREASE DRUG INTAKE DAILY IN mic FOR BCG Ti.* CFOUC DAILY mg/kg mg/kg days g1/mi H2N-NH-C-NH <7w-NH-C-NH-NH-C-NH S H2N-C-NH-NH-C-NH GJ NH-C-NH-N H-C-NH H2N-NH-C-NH * None of these increases is significant. cases the drugs were so poorly accepted by the mice that only very small drug intakes could be attained. Still, in all such cases no response could be elicited at the maximum administered drug level. Of the 8 drugs giving measurable increase in T50, the maximal increases were

5 1950] CHEMOTHERAPY OF EXPERIMENTAL TUBERCULOSIS 679 achieved with the p-acetamido-, p-dimethylamino-, p-ethyl sulfonyl-, and p-methoxybenzaldehyde thiosemicarbazones. For the remaining 4 the spread between the minimal effective and mal acceptable doses was considerably less. Until we have data relating the maximal acceptable drug level in the diet to the chronic toxicity of the drug when administered orally by stomach tube, we cannot evaluate the significance of these differences in the active drugs. Starting with the highly active p-acetamidobenzaldehyde thiosemicarbazone, it is of interest to report that the following chemical operations lead to complete loss of activity in vivo: (1) replacement of the sulfur by oxygen or (2) by nitrogen, (3) replacement of the terminal - NH2 group by -SCHy, (4) substitution of an alkyl group for a hydrogen atom on the sulfur or (5) terminal amino group. The TABLE 5 Benzaldehyde thiosemicarbazones active in vivo GJCH CH==N-NH-C-NH2 S OIF MAXIMLU MOLARDRUGKAXrAL INILEAE INFRACTION SUIBSTITUENT MOLA DRUG MAIA IAS ACCEPTABLE LEVEL EQUIVALENTSTio, DAY ~TESTED 4-NH-C-CH3 1.00* 30 1/1 0 4-S02-C2H, 4.0 >40 1/32 4-CH(CH3) /2 4-NH /1 4-N(CH3) /4 4-NO /1 4-SO0-NH /4 4-OCH /1 * Drug equivalent equals the ratio of molar intake of standard drug (4-acetamidobenzaldehyde, 3-thiosemicarbazone) to the molar intake of the test drug producing the same proportionate increase in T,o. equivalence in activity of the p-acetamido and -amino analogues suggests that the one will be rapidly converted to the other in vivo, and indeed thelesser activity of the p-nitro analogues could also be attributed to reduction to the amino derivative. The rather remarkable specificity of structures showing antituberculous activity is, indeed, an outstanding property in the thiosemicarbazone series. Comparison of the active compounds reported by Hoggarth et al. (1949) with ours shows excellent agreement in the order of increasing activity except for the p-amino analogue, which although highly active in our test gave only a minimal response in that of the English investigators even at a much higher drug intake. However, it should be pointed out that differences between the test used by Hoggarth et al. (1949) and our test prevent direct comparison of data on any one active thiosemicarbazone. For example, they calculated the survival time of mice living beyond 30 days as 31 days (Martin, 1946), whereas we recorded the actual

6 680 HAMRE, BERNSTEIN, AND DONOVICK [VOL. 59 survival time. Also, the mice in the English test were treated (in most experiments by mouth twice daily and in one experiment in the diet) for 14 days, whereas ours were kept on the drug diet for 21 days. It should be noted that the limitation in maximal drug intake when the drug is incorporated in the diet does not necessarily reflect the inherent toxicity of the substance; indeed, we have evidence for certain drugs that far larger quantities can be given by cannula orally than the mice will accept in the diet. Comparison of the in vitro activities with in vivo data likewise shows no obvious quantitative relationship. Although all compounds showing activity in vivo had minimal inhibiting concentrations, in vitro, equal to or less than 1.5 micrograms per ml, one of the most active in vitro, the p-methoxy analogue, was one of the least active in vivo. It seems quite clear that in vitro data can at best serve as a guide to the selection of chemical types for in vivo screening. ACKNOWLEDGMENT We are greatly indebted to W. A. Lott, G. W. Rake, and F. Y. Wiselogle for helpful suggestions throughout the work. SUMMARY Of nearly 100 thiosemicarbazones and related compounds tested for antituberculous activity in the mouse only eight, all of these being p-substituted benzaldehyde thiosemicarbazones, showed significant reproducible activity. In order of activity expressed in terms of inverse molar intake required to effect a minimal response, the substituents rank as follows: ethyl sulfonyl = isopropyl > amino = acetamido = dimethylamino > nitro = sulfamyl =methoxy benzaldehyde, 3-thiosemicarbazone. There is no quantitative relationship between in vivo and in vitro antituberculous activity, even among active compounds within the series of the thiosemicarbazones; however, thus far only compounds active in vitro have been found active in vivo. REFERENCES DOuAGK, G., BEHNISCH, R., MIETZSCH, F., AND SCHMIDT, H tjber eine neue, gegen Tuberkelbazillen in vitro wirksame Verbindungsklasse. Naturwissenschaften, 33, 315. DONOVICK, R., AND BERNSTEIN, J On the action of thiosemicarbazones in experimental tuberculosis in the mouse. Am. Rev. Tuberc., 60, 539. DONOVICK, R., PANSY, F., STRYKER, G., AND BERNSTEIN, J The chemotherapy of experimental tuberculosis. I. In vitro activity of thiosemicarbazides and thiosemicarbazones and related compounds. J. Bact., 59, HOGGARTH, E., MARTIN, A. R., STOREY, N. E., AND YOUNG, E. H. P Studies in the chemotherapy of tuberculosis. V. Thiosemicarbazones and related compounds. Brit. J. Pharm. Chemother., 4, MARTIN, A. R The use of mice in the examination of drugs for chemotherapeutic activity against Mycobacterium tuberculosis. J. Path. Bact., 58, RAKE, G., JAMBOR, W., MCKEE, C. M., PANSY, F., WISELOGLE, F. Y., AND DONOVICK, R The use of the mouse in a standardized test for antituberculous activity of compounds of natural or synthetic origin. III. The standardized test. Am. Rev. Tuberc., 60,