Investigation of Machilus macrantha Nees. II*

Investigation of Macrbihs mucrantrbu Nees. I1* Pharmacological Action and Chemical Constitution of Machiline By K. N. GAIND and S. K. BAVEJA Machiline, isolated as the hydrochloride from the root of Machilus macrantba Nees., has been studied for its pharmacological action and chemical characteristics. It has been found t o lower blood pressure of experimental animals b y a direct depressant effect o n the myocardium. It has n o antibacterial activity. A number of derivatives have been prepared. Based o n the results of (a) microanalysis for elements of the base, its hydrochloride, icrate, acetyl, and benzoyl derivatives, ( b ) chemical tests for functional grou s, microestimation of -0CHa in the base and =NCH5 in the methiodide, and &) infrared and ultraviolet absorption spectra, two structures are proposed for machiline

(fp

report (l), t h e isolation of a base “machiline” as a hydrochloride from the root of Machilus m c r a n t h Nees. was reported. The present investigation was undertaken t o study the pharmacological action and chemical constitution of this base.

I

N THE PREVIOUS

METHODS AND RESULTS Warmacological Actions.-Before undertaking chemical characterization, the isolated hydrochloride was studied for its effect on the cardiovascular system and on smooth muscle and for its antibacterial activity. Cardiovascular System.-Arterial blood pressure was recorded from the common carotid artery in dogs weighing 7-9 Kg., anesthetized with pentobarbital sodium (32 mg./Kg.) intravenously. The dose of machiline hydrochloride solution was injected through the cannulated femoral vein. With a dose of 0.1 to 1.0 mg., there was a fall in blood pressure (2 to 11 mm.) proportionate to the quantity administered. With larger doses the fall was only slightly greater, the maximum for a dose of 50 mg. being 18 mm. Machiline hydrochloride (10 mg.) caused a decrease in the amplitude of contraction of the intact heart in an anesthetized dog with open chest. On the isolated heart (Langendorffs method) (2) of rabbits weighing 1-1.5 Kg., a dose of 0.1 mg. caused slowing and irregular rhythm and a dose of 1.0 mg almost stopped the heart by slowing and gradual decrease in the amplitude of contraction. Subsequently the heart recovered gradually in about ten minutes. In the intact heart of a pithed frog, a dose of 0.1 mg. caused varying grades of heart block, and a dose of 1.0 mg. caused arrest in diastole. Subsequently the heart recovered completely in about five minutes through gradually diminishing grades of heart block. In perfusion experiments, with 0.1 t o 1.0 mg. of machiline hydrochloride the flow of frog Ringer’s fluid through the peripheral vessels of a frog in-

*

Received September 10. 1959, from the Department of Pharmacy, Panjab University, Chandigarb, India. The authors are sincerely grateful to Dr. K . C. Verma of the Pharmacology Department, Medial College, Amrttsar, for his assistance in the pharmacologlcal study and to the Director. National Chemical Laboratory, Pmna, for providing microanalytical data.

creased for a few minutes, while the flow of Locke’s solution through the coronary vessels of an isolated rabbit’s heart decreased for several minutes, but the magnitude of the effects was slight and insignificant. The kidney volume and blood pressure of an anesthetized dog showed simultaneous fall with doses of 1 to 15 mg. and this effect was not blocked by previous injection of atropine. These observations suggest that the hypotensive effect of machiline is due to its direct depressant effect on the myocardium. Action on Smooth Muscle.-A dose of 1.0 mg. of machiline hydrochloride had a depressant effect on the strips of intestine (Magnus’ method) (3) of rabbits weighing 1.0-1.5 Kg.. in an isolated organ bath with inner bath of 25 ml. capacity. Antibacterial Activity.-In concentrations up to 1 in 500, machiline hydrochloride failed to inhibit the growth of Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Salmonella typhi in nutrient broth and of Mycobacterium tuberculosis var. hominis in the medium suggested by Isao Yamane (4). Chemical Characterization of Machiline.Anal.-Calcd. for ClaH17N03.HCI: C, 62.41 ; H, 5.90; N, 4.55; C1, 11.58. Found: C, 61.46; H, 6.32; N, 4.35; C1, 11.13. The picrate from 50 mg. of the hydrochloride was precipitated with picric acid in aqueous solution and crystallized from water as yellow crystals (51 mg.) in the form of elongated rectangular plates, melting with decomposition between 205-207”. Anal.-Calcd. for C~~H~TN~~.C&IH~(OH)(NO*)~: C, 52.78; H, 4.03; N, 11.20; Mol. wt., 500.2. Found: C, 53.61; H, 4.40; N, 11.47; Mol. wt., 465 (Rast method). The base from 100 mg. of the hydrochloride was precipitated with ammonia in aqueous solution and crystallized from 50% alcohol as a microcrystalline white powder (60 mg.) melting with decomposition a t 210-211O. Anal.-Calcd. for CleHnN03: C, 70.81; H, 6.32; N, 5.16; Mol. wt., 271.2. Found: C, 70.29; H, 6.60; h’,4.74; Mol. wt., 301 (Rast method). Machiline hydrochloride did not respond to tests with alkaloidal reagents as has already been reported (1). The murexide test for purines was also negative. Heating with dilute as well as concentrated hydrochloric acid did not split the molecule of machiline. It decolorized bromine in aqueous, alcoholic, and glacial acetic acid solutions, and de-

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colorized potassium permanganate in aqueous solution. It did not reduce Fehling’s solution, but a silver mirror was formed with ammoniacal silver nitrate. I t gave a negative test with Schiff’s reagent and also with 2.4-dinitrophenylhydrazine reagent (5). The base neither gave effervescence with, nor dissolved in sodium bicarbonate solution. I t dissolved in caustic soda solution and also in ammonia solution, but gave no color with ferric chloride in aqueous or alcoholic solutions. I t did not give a green color with gallic acid reagent (6) or a red color with the phloroglucinol-sulfuric acid reagent of Gaebel (7). And-Calcd. for one methoxy in Cl&;S03: - 0 C H 3 , 11.3; Found: -0CH;. i.5. On heating the base with soda lime, vapors having an ammoniacal odor were evolved and when these were passed into a solution of hydrochloric acid gas in ether a precipitate was formed, which was identified as ammonium chloride. On treating the base with ice-cold nitrous acid, a gas was evolved and a yellow solid precipitated. The latter gave a negative Liebermann test. \\’hen the yellow supernatant liquid was made alkaline, no solid separated out. Rimni’s (5) and the carbon disulfide reagent (5) tests were also negative. Acetylmachiline.-Machiline hydrochloride ( 100 mg.), acetic anhydride (1.5 ml.), and pyridine (5 drops) were boiled together under reflux on a sand bath for one hour, cooled, poured into water (80ml.), set aside overnight, and the precipitate formed was crystallized from 50’;-, alcohol. A crop of white crystals in the form of elongated rectangular plates, m. p. 197-199’, was obtained. Anal.-Calcd. for C d i ~ s S O a(CHaC0)2: . C. 67.57; H , 5.96; S, 3.91. Found: C, 66.iO; H, 6.00; h’, 3.73. Bemyhachiline.-Benzoylation was effected by the Schotten-Baumann process and the product crystallized from absolute alcohol as elongated rectangular plates, m. p. 214-216”. Anal.-Calcd. for C ~ ~ H I ~ S ~ : ( C & C O )c?,:i5.12 H, 5.26; N, 2.92. Found: C, i6.20; H, 5.54; N, 2.88. Machiline Methiodide.-Machiline (100 mg.) in methanol (5 ml.) and methyl iodide (0.5 ml.) were warmed together under reflux for one hour. The volatile components of the reaction mixture were volatilized off and the residue dried to constant weight a t 120’ (154 mg.) was crystallized from dry acetone in a desiccator containing ether. The crystallized compound. after drying a t 120’. melted between 1&1-18i0. Anal.-Calcd. for ClbH17S0~. CHaI : -CHJ (attached to h’),3.63. Found: -CHI (attached to S ) . 3.51. Infrared Spectrum.-The infrared spectrum of machiline (Fig. 1) was taken in a Kujol mull on a single beam Perkin-Elmer spectrophotometer making necessary corrections for interferences due t o atmospheric water and carbon dinside.’ The brief analysis (Table I ) of the infrared record indicates the groups responsible for some of the absorption bands; the assignments were done on an empirical basis. Ultraviolet Spectra.-The ultraviolet spectra (Fig. 2) were taken on a Hilger Uvispeck photo-

electric spectrophotometer equipped with a fused silica prism.2 Both machiline and its hydrochloride showed approximately the same absorption maxima in the ultraviolet region and no absorption in the visible region of light. The base showed absorption masima a t 226 and 285 mp in absolute alcohol; its hydrochloride a t 200, 221, and 282 mp; and there was no inflexion near 315 mp even in a more concentrated solution. The maxima were comparable to those of naphthalene and quinoline except for the lack of inflexion. The inflexion could be absent if ( a ) a quinoline structure were present as its tetrahydro form or ( b ) a naphthalene structure were present as 1-phenylnaphthalene (or 2’-hydroxy-l-phenylnaphthalene or 2’-methoxy-l-phenylnaphthalene, etc.). The latter structure would make it rather clifficult to fit in nitrogen present as an amine group which in such cases would shift the maxima too much in the acidic condition, whereas not much shifting was observed in the curve of base hydro-

1The spectrum was obtained through the courtesy of Dr. M. L. Dhar of the Central Drug Research Institute, Lucknow.

* The spectra were prepared in the laboratory of the Royal Institute of Technology, Glasgow. through the courtesy of Dr. H. C. Mital.

-1000 3000 2000 1600 1200 900 3.500 2500 1800 1400 1000 800

700

0

100

2.5

3.0

4.0

6.0

8.0 10.0 12.0 14.0 7.0 9.0 11.0 13.0 WAVELENGTH I N p 5.0

Fig. 1.-Infrared

spectrum of machiline.

TABLEI.-Ehmrucu ASSIGNMENT OF GROUPS FOR ABSORPTION BANDSIN THE INRESPONSIBLE FRARED SPECTRUM OF MACHILINE Absorption Band Peak. cm.3

Assigned Grouping

3175 2381 2 105 2000 1835J

=N (these should be weak bands but

1600

CGHG, C=C, C= N in N heterocyclic

1504 1399 1333 1250

CeH6 substituted, C6HsN ( ? ) CsHsOH, -C-CHz (?) CsH5-?d=, CeHsOH, -OH CsHjOR

1058 1020 9761 813

1:2, 1:4, 1:2:4 substituted C6Hs

7 19

CII2 were found as strong bands due to faulty compensation)

(?I

S (heterocyclic), (CH3)zC (?),

C=CHf?) N (heterocyclic)

SCIENTIFIC EDITION

Octobcr 1960

2.1

-

2.0

-

quaternary ammonium compound with methyl iodide accompanied with increase in weight of 100 mg. of the base to 154 mg. of the methiodide, and microanalytical data for -CHs (attached to IY) in the methiodide, all point t o the tertiary nature o f the nitrogen function. Further the infrared spectrum indicates the nitrogen function to be in a heterocyclic system. The ultraviolet spectra corroborate this finding; specifically, there is a tetrahydroquinoline (or tetrahydroisoquinoline with a phenyl group attached to N) structure. This, together with OCH, and OH groups, accounts for CloH1zNO~leaving CsH6 which evidently constitutes a phenyl group and must be attached to a nitrogen function to account for the tertiary nature of the latter in the tetrahydroquinoline (or tetrahydroisoquinoline) nucleus. This conclusion is further strengthened by the maxima a t 1,333 cm.-l of the infrared spectrum. On this basis the likely structures for machiline arc those represented by I and 11.

I I

1.9 I

1.8 -

2 1.0 - I 0.9 0.8

665

-

0.7 0.6 -

r

0.5 0.4

1

H,

-

190

200

210 230 250 270 290 310 330 220 240 260 280 300 320 h IN Mrr

Fig. 2.-Ultraviolet spectra of machiline.machiline hydrochloride in water 0.000SSl8~~ ; _ - - _ machiline hydrochloride in decinormal sodium hydroxide 0.0009818u/o;-.-.-.- machiline base in ethanol 0.0014374~o.

I

-9H chloride. Taking the quinoline structure, its tetrahydro form would not show marked shift in maxima and is in agreement with the observed maxima of base hydrochloride. In alkaline solution the spectrum of machiline hydrochloride was markedly influenced (by the alkali) above 230 mfi. indicating that nitrogen function is near a benzene ring, i.e., there is a quinoline rather than an isoquinoline system or else a phenyl group is attached to nitrogen in the isoquinoline form.

-OCH:,

I1

The investigated alkaloids of laurels are known (8)to possess an aporphine ring structure, which has tetra1ij.droisoquinolinenucleus and on this analogy, structure I1 may be considered more likely for machilinc

DISCUSSION The microanalytical data of the base and its derivatives show that the molecular formula of machiline is CI6H1TNO3and it has two OH groups and one OCHa group. The solubility of the base in alkali hydroxides and the infrared spectrum indicate that a t least one of the OH groups is phenolic though it may be in a somewhat hindered position. It does not give the characteristic test for phenols with ferric chloride solution but many other phenolic and enolic compounds behave similarly (7) and, therefore, phenolic OH cannot be considered absent. Chemical tests, infared spectrum, formation of a

REFERENCES (1) Gaind. K. N li59 ( 1960)

, and Baveja,

S K , THISJOURNAL, 49,

” Blackwell S c ~ ~ ~ t i l ~ P ; b i i c a t i o ’Oxford,~l952, ns, 25. (3) Gaddum, J. H.. Pharmacology,” 4th e d . . Oxford University Press, London, 1953, p. 213. (4) Isao Y. J . Bacferiol.. 73, 172(11957). 1.5) Vo&I. A. I . . “Textbook of Practical Organic Chemis ~~, try,” 1st-ed’,, Longmans, Green and C o . . London, 1948. p 917. (6) Nadkarny-Kothare, “Organic Chemistry for Advanced Students, Part I , The Popular Book Depot, Bombay. 1931. p. 383. (7) Gaebel, G. O . , Arch. Pharm. 248 225(1910). (8) Henry, T. A. “The Plant Alkalbids,” 4th ed.. J . A . Churchill Ltd., London, 1949, p. 319.

(7) R n m

1

H

“Practical Pharmacolunv

u.