In order to form metal complexes, for example, (III), isoniazid must first form the anion (II), which is the chelating species. No anion can be formed by 1-tsonicotinyl-l-methylhydrazine (I, R = CH3). This substance has recently been synthesized and found to be virtually inactive3, thus giving strong support to the hypothesis of chelation. In the present communication, the determination of the avidity of other hydrazides for heavy metals is recorded. It is seen from Table 1 that of the two isomers of isoniazid, nicotinic hydrazide (which is biologically inert) has approximately the same affinity for metals as isoniazid, whereas picolinic hydrazide, which is about eight times less effective biologically, has 103-105 times the affinity for metals. Table 1 also includes an aromatic, and an aliphatic, hydrazide which have much the same avidity for metals as isoniazid but have far less biological activity. Thus, ' although the ability to form chelate compounds seems to be essential for the functioning of isoniazid, it now becomes clear that some other factor is of over-riding importance. The incorporation, by the living cell, of isoniazid into diphosphopyridine nucleotide4 (in the place of nicotinamide) does not explain its antitubercular action, because cyanacetic hydrazide is not a structural analogue of nicotinamide. Yet cyanacetic hydrazide, although weak, has been used successfully in human tuberculosis5, and is apparently acting in the same way as isoniazid, because organisms resistant to one are resistant to the other6.Table 1. PHYSICAL AND ANTI-TUBERCULAR PROPERTIES OF HYDRAZIDESJsonico- Nico- Pico- Benzoic Cyan- Ioniz ti tinic tinic linic aceticstants (as pKa) NHa l-851-861 3 032-340-04 0 03 0-01 0-02 Ring-N 3-543-292-86none none 0 04 0-04 0 01Acidic 10-77ll-4712-2712-4511-17 j. 0 05 0 04 0 04 0 03 0 04Stability con- stantsCu++ log K' 8-7 12-4 9-0 8-5 log K" 7-5 9-17 1 log Ks .-. 16-2 21-5 - 15-6Ni++ log K' 5 5 6 0 10-7 6-3 6 0 log K" c. 4-3 4-7 9-5o log Kc. 9-8 10-7 20-2 __ .__. Co++ log K' 4-8 5-4 9-6 5-3log X"7-8 -log JEt .__. .__. 17-4 - - Zn++ log K' 5-48-4 + log K"__ 7-2 .-. log K" - - 15-6 - -Fe++ log K'8-4log JK" .- -.-. ,-- logK, - - - - _Biological action In vitro ^gm. per ml.greatest dilution permitting sur-vival (a) 0-025 25 0 1-5 12-5 3 0 In vivomgm./kgm.~) smallest (6) daily dose }- 3 no 25 no ?? action actionpermitting J (c) survival J 210Precipitate.Not attempted. - Calculation impossible because of precipitate.(a) Prof. R. Knox, unpublished results on H 37 Rv. strain in Dubos medium (semi-solid). All substances were tested simultaneously. (6) Berenstein, J., Loft, L., Steinberg, B., and Yale, H., Amer. Rev. Tuberc.% 65, 357 (1952). Survival of mice, H 37 Rv. strain (drug given orally).(c) Barnett, M., Bushby, S., Goulding, R., Knox, R., and Robson, M., Brit. Med. J.t ii, 647 (1955). Survival of mice, CN 3679 strain (human). Drug given subcutaneously, the isoniazid-treated mice survived longer than those treated with cyanacetic hydrazide. The specificity of these hydrazides for M. tuberculosis has yet to be explained : none of the substances in Table 1 was bacteriostatic to Staph. aureus or Strept. haemolyticus in broth at pil 73 (37 for 24 hr.) even at 10~2 M, whereas 8-hydroxy-quinoline (control) inhibited both organisms at 10-5M (Marshall, J. H., unpublished work). The ionization constants were determined by potentiometric titration at 20, the two basic functions being separated by the method of Noyes7. The stability constants (Kf for the 1 : 1 complex, for example, III, K" for the complex formed by adding one more ion of ligand, and K>.hich is the product of K' and K") were obtained by potentiometric titration in the presence of one equivalent of the metal ion8.Prof. R. Knox and Dr. J. H. Marshall are thanked for permission to quote from their unpublished biological results ; and Mr. E. P. Serjeant for skilled technical assistance ; also Mr. D. E. Seymour, of Herts Pharmaceuticals, Ltd., for gifts of nicotinic and picolinic hydrazides, and Dr. Maxwell Savage, of S. Maw, Son and Sons, Ltd., for the cyanacetic hydrazide.