Chemical and biological studies on some natural and synthetic antimicrobial substances

Abstract

The eventful developments of chemotherapy and the growth in our concepts of the treatment of infective diseases, from Ehrlich to the present day, have been briefly traced, stressing the outstanding practical achievements. Attention has been focused on the inadequacy of chemotherapeutic measures for combating viral infections and neoplastic conditions and, in this connection, it has been suggested that screening of higher plants for possible chemotherapeutic agents deserves more intensive efforts than had been hitherto expended. The object of the present investigation, which forms part of a programme of studies on Indian medicinal plants, has thus been defined as to cover the constituents of (a) Garcinia morella and (b) substances allied to pterygospermin, an antiviral principle isolated by the author’s colleagues from the roots of Moringa pterygosperma.

In Part I of the thesis, the chemistry and biological properties of a few constituents of G. morella forming chelates with copper are described, while Part II includes attempts to disclose any structural relationship between antiviral activity and metal-chelating capacity of substances. In addition, this screening of antiviral compounds includes those containing the N–C=S grouping found in the pterygospermin molecule.

Part I - and -Guttiferins Chapter I -Guttiferin

Isolation, characterization, antibacterial spectrum and pharmacological properties of - and -guttiferins are described. These are two of the constituents of the seed coat and gamboge, the resinous exudation of G. morella, that form characteristic red complexes with copper. In pure state these principles possess no significant antiviral activity against vaccinia and influenza PR8 viruses in eggs or in mice (vide Part II). Nevertheless, their high antibacterial activity, relatively low toxicity and striking relationship to morellin warranted the chemical and biological investigations described in the following chapters.

Guttiferin (CHO; m.p. 113–115°) has been isolated from the raffinate left after the removal of morellin. Previously known orange-yellow pigments from the seed coat of G. morella have been described. Constituents of the raffinate form sparingly soluble red copper complexes but do not crystallize readily. Conditions for the preparation of (I) in a pure state have been stated. (I) dissolves in aqueous sodium carbonate but not in sodium hydrogen carbonate, and forms a characteristic sparingly soluble pyridine complex, CHO·CHN (m.p. 115–117°), similar to-guttiferin.

The close kinship of (I) with-guttiferin and morellin in physicochemical properties has been clearly demonstrated. Total loss of antistaphylococcal activity of (I) follows methylation with dimethyl sulphate in aqueous sodium carbonate solution at 24°, yielding a monomethyl ether (II) (m.p. 160°), insoluble in aqueous alkalis but forming readily a copper complex. On drastic methylation with methyl iodide and potassium carbonate in boiling acetone, a dimethyl ether (m.p. 117°), differing from (I) and (II) in spectral and other physicochemical properties, is produced and designated C-dimethyl-pseudoguttiferin.

Acetylation of (I) under mild conditions gives monoacetyl-guttiferin (m.p. 173°), while any diacetyl derivative formed has been found to be too unstable for isolation in a pure state. While the monoacetyl derivative does not form a copper complex, it possesses, unlike the monomethyl ether (II), one-fifth of the antistaphylococcal activity of (I), thus indicating no possible correlation between copper-binding capacity and antistaphylococcal effects.

The general physicochemical properties of (I) and (II) favour exclusion of a carboxyl group, the solubility of (I) in sodium carbonate being explained by the presence of an acidic phenolic group situated para to a carbonyl moiety. The presence of at least three double bonds has been demonstrated by catalytic hydrogenation (Adams’ catalyst), and two additional ethylenic linkages by the colour reactions and physicochemical properties of hexahydro-guttiferin (m.p. 153°).

Evidence in favour of a carbonyl group reacting with the usual carbonyl reagents is not clear-cut due to complex secondary reactions. The presence of an -unsaturated lactone group in (I) has been deduced by a study of the acids, viz., guttiferinic acid and pseudoguttiferic acid (m.p. 142–144°), formed on treatment of (I) with potassium hydroxide solutions. Methyl C-monomethyl-guttiferate and methyl C-dimethyl-pseudoguttiferate, the two esters formed from guttiferinic acid, have been recognized as identical with two substances previously described by the author’s colleagues during their study of amorphous fractions of the raffinate.

Like morellin, (I) does not react with diazoaminobenzene nor couple with diazonium salts, thus supporting its formulation as a fully substituted phloroglucinol. Acetic, isovaleric and homophthalic acids, besides acetone and methylheptenol, have been found among the products of fusion of (I) with alkali. Formation of hydroxy-isobutyric acid during oxidation of (I) with alkaline permanganate and a bromo-acid (m.p. 199–200°) on treatment of (I) with alkaline hypobromite has been described. Since guttiferin and guttiferin give the same bromo-acid, structural relationship between them is thereby indicated. Oxidation of hexahydro-guttiferin by potassium permanganate leads to acetic and isocaproic acids along with traces of a volatile lactone, possibly identical with that formed from perhydromorellin under similar conditions.

These reactions, along with spectral data, have been discussed and a partial structure for -guttiferin has been proposed.

Chapter II - Guttiferin (Garcinia morella)

The extensive chemical investigations on the component of gamboge since the time of Buchner have been critically reviewed. Guttiferin (m.p. 94–95°), that forms a red copper complex like guttiferin, has been isolated as a crystalline pyridine complex (m.p. 145–149°). This is tentatively designated -guttiferin in view of its close resemblance to guttiferin. The identity of guttiferin with gambogic acid characterized by J. S. Sorensen and -guttiferin could not be feasibly excluded even though the presence of a free carboxylic group in the latter has been postulated.

Guttiferin is distinguished from-guttiferin by the extraordinary difficulty with which its derivatives crystallize. Despite this complication, the striking similarity of the two substances is borne out by the formation of analogous derivatives and products. Among the crystalline derivatives of guttiferin newly well characterized may be mentioned (a) monoacetyl-guttiferin (m.p. 204–205°) and (b) diacetyl-guttiferin (m.p. 173–175°). These two resemble closely the corresponding derivatives of guttiferin in physicochemical properties.

While the chemical reactions of guttiferin have not been studied as extensively as those of guttiferin, they have, however, been interpreted on the basis of a partial structure which differs from that of guttiferin only in the moiety yet to be elucidated.

Chapter III – Antimicrobial Properties of and-Guttiferins

The range of antimicrobial activity of and guttiferins has been determined. Both these substances resemble closely in exerting high specific inhibitory effect on the growth of Gram-positive bacteria with lower activity against a few Gram-negative species and a strain of Helminthosporium. They display little or no activity toward a variety of other pathogenic and non-pathogenic organisms, viz., Gram-negative bacteria, fungi, yeasts and actinomycetes.

Their antistaphylococcal activity is similarly influenced by factors such as the presence of blood serum, while the presence of growth-promoting factors and metallic ions in general exerts little or no influence on the activity of guttiferin. Pyridoxine, thiamine and cyanocobalamin form an exception in that they seem to act synergistically. On the contrary, addition of methionine reverses the antistaphylococcal activity of guttiferin. In this respect, the antibiotic resembles morellin.

Although the precise mechanisms of their antibacterial action have yet to be elucidated, the reversal of activity by methionine seems to suggest interference with biological methylations as one of the possible steps involved in the mode of action.

Chapter IV – Toxicity of Guttiferin

Data indicating the relatively low toxicity of guttiferin and possibly of guttiferin following administration by oral, subcutaneous, intraperitoneal or intravenous routes in aqueous and oil solutions to rats and mice are summarized.

Acute toxicity of guttiferin (LD mg/kg):

Rats – Oral (oil and buffer) 800; Subcutaneous (oil) 400; Subcutaneous (buffer) 279 ± 22.5; Intraperitoneal (oil) 95 ± 4.1; Intraperitoneal (buffer) 91 ± 3.6; Intravenous (buffer) 105 ± 3.1. Mice – Intraperitoneal (buffer) 33 ± 4.5; Intravenous (buffer) 37 ± 5.7.

Acute toxicity of guttiferin (LD mg/kg):

Rats – Intraperitoneal (buffer) 85 ± 3.4; Intravenous (buffer) 107 ± 3.4.

Histopathological examination of liver, spleen and kidney removed from treated animals supports these results. Chronic oral toxicity of guttiferin could not be precisely determined. At 40 mg/kg/day for 40 days, no mortality nor striking histopathological changes were observed in rats; at 120 mg/kg/day for 40 days, significant decrease in growth rate, erythrocyte and leucocyte counts and haemoglobin concentration was observed.

A transient drop in blood pressure (in anaesthetized dogs), accompanied by increase in respiratory rate with decrease in amplitude, is produced by guttiferin at levels of 12 mg/kg and above. Contractions of isolated ileum (rat and guinea pig) induced by known spasmogens are inhibited by guttiferin at concentrations of 40 µg/ml and higher. Similarly, contractions of isolated nulliparous rat uterine horn are abolished at concentrations of 80 µg/ml.

Chapter V – In Vivo Activity Against Experimental Staphylococcal Infection

The therapeutic potentialities of and guttiferins are suggested by the in vivo experiments described. The two substances protect mice against lethal infections of S. pyogenes var. aureus following intraperitoneal and intravenous administration at a dosage level of 35 mg/kg/day for two days. Successful control of experimental septic wounds in mice by application of guttiferin ointment has been demonstrated.

Part II – Chemotherapy of Influenza and Vaccinia Virus Infections

The present status and approaches to chemotherapy of viral infections have been critically reviewed. The extreme stage of parasitism of viruses, as defined by their size and habitat, mode of replication and complete dependence on host-cell metabolism for multiplication, limits chemotherapeutic avenues for their control. Their restricted enzymic make-up offers fewer chances of selective interference.

After mentioning the present concepts of classification, composition, infectivity and probable mode of interference of potential antiviral agents at different phases of viral multiplication, the varied chemical types screened hitherto for antiviral activity against different groups of viruses are shown in Tables XXVII–XXXIX.

Naturally occurring and synthetic compounds forming metal chelates or containing the N–C=S group (e.g., and guttiferins, resacetophenone-oxime, substituted aminothioureas and sulphonates related to pterygospermin) have been screened for antiviral activity against vaccinia and influenza PR8 viruses in eggs and animals.

The results disclose no relationship between antiviral activity and capacity to form metal chelates. On the other hand, a few members containing the benzyl isothiocyanate skeleton, also found in pterygospermin, possess anti-influenza activity and protect chick embryos against lethal vaccinia infections. While five compounds exhibit pronounced anti-vaccinia activity, only two show significant inhibitory effect on influenza PR8 growth in chorioallantoic membrane or mouse lung tissue.

The antiviral activity of these compounds, particularly of BAD, is profoundly influenced by the presence of growth factors such as thiamine, pyridoxal and riboflavin. The limited data presented suggest possible interference with altered metabolic regulation of the host cell consequent upon viral invasion by means of analogues of growth factors and indicate the desirability of pursuing chemotherapeutic investigations on viral diseases and possibly neoplastic manifestations along these lines.