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Antibacterial and Antifungal Properties of β-Naphthol Derivatives IV*
October 1957
SCIENTIFIC EDITION
both the present method and chromotography ; and C-1 was adopted as the reference standard. Assuming a purity of 100% for the standard, recoveries of reserpine from C-1 and C-2 after elution were 984970, when compared with unchromatographed standards. The values reported in Tables I and I1 are based upon procedural standards.e Of the twelve samples of crystalline reserpine analyzed, eight contained more than 96% reserpine. Samples C-9 and C-10 contained excessive quantities of deserpidine; samples C-11 and C-12 contained both deserpidine and significant quantities of more polar alkaloids; C-12 yielded the only sample blank with appreciable absorbance a t 390 mp. Fifteen of the commercial tablet samples were analyzed by both the proposed method of assay and the extended-nitrite procedure previously described (1). Twelve of the check analyses agreed within 2%. The greatest discrepancy between procedures 2 Solutions of reserpine in ethanol are chromogenically stable for several weeks when protected from light. Since the efficiency of the column is fairly uniform, i t is probably necessary to analyze procedural standards only occasionally. Unknowns may be compared with a standard ethanolic solution, diluted with the requisite volumes of chloroform and ethanol just prior to use, and the results corrected for differences between this standard and the procedural standard.
603
was 4%. Only Sample T-8contained quantities of 3-dehydroreserpates large enough t o give an appreciable blank a t 390 mp. Ultraviolet absorption data for 19 of the 24 prepared chloroform-ethanol solutions indicated that the recovered alkaloidal fractions consisted of reserpine almost exclusively. Paper chromatograms on extracts from the tablets in the most notable of the remaining five samples revealed the presence of other weakly basic alkaloids (rescinnamine and deserpidine). More polar alkaloids such as reserpic acid, methyl reserpate, ajmaline, and yohimbine are retained on the column quantitatively. Rescinnamine and reserpinine are extracted with reserpine, and yield similar colored derivatives when treated with nitrous acid. But these and other alkaloids can be detected by means of chromatography. The proposed method of assay is as selective as the previously published nitrite procedures (1, 2) and is simpler, quicker, and less vulnerable to manipulative errors. REFERENCES (1) Banes, D.. Wolff, J.,Fallscheer, H. O., and Carof, J., THIS JOURNAL, 45, 708(1956). ( 2 ) Szalkowski, C. R., and Mader, W. J., ibid., 45, 613
\'""",.
/lOKC\
(3) "United States Pharmacopeia," 15th ed., Mack Puhlishing Co., Easton, Pa., 1955, pp. 217-218.
Antibacterial and Antifungal Properties of ,+Naphthol Derivatives IV* By R. S . BAICHWALt, MALATI R. BAICHWALt, and M. L. KHORANAf. The bacteriostatic and fungistatic activities of a number of &naphthol and 2-hydroxy3-naphthoic acid derivatives are reported against Bacillus typhsus, Mimcoccus pyogenes, var. aureus, and Aspergillus niger. Selected com ounds from the initial sueening were further tested for their activity against Bacilks paratyphosus A, Bacillus coli, Bacillas dysenteriae Shiga, M icrococcuspyogenes, vat. albus, Streptococcus-8-haemolyticus, Clostridium tetani, Epidermophyton floccos~nt,Trichophyton rubrum, and Trichophyton gypseum. Few compounds exhibit activity against Gram-negative organisms at concentrations below 5 0 pg./cc., and none against C. tetani. The bacteriostatic concentration for Gram-positive organisms of 3,ddibromo-, 3,4 ',dtribromo-, 6-npentyl-, and 6-n-hexyl-8-na hthols and 1,4-dibromo-, 1,6-dibromo-, and 1,4,6tribromo-2-hydroxy-3-naphtianilides is between 0.2 and 10 pg./cc. Marked inhibition of all the fungous pathogens was shown b y 6,6'-dibromo-&dinaphthol and 1-bromo-+naphthol. 3-Chloro-, 1-bromo-, 6-bromo-, and 1,6-dinitro-naphthols inhibited the growth of E. ficcosum and T. rubrum at concentrations ranging between 1 and 10 pg./cc. HE BACTERICIDAL ACTIVITIES of a number of Tderivatives of p-naphthol have been reported previously (1-3) and the data might be useful in *Received March 4, 1957, from the Bombay University Department of Chemical Teehnology. Bombay 19, India. This paper is based in part on the theses submitted to the University of Bombay by R. S. Baichwal and Malati R. Baichwal for the degree of Doctor of Philosophy in the Faculty of Technology. t Present address: Faculty of Pharmacy, University of Toronto, Canada. $ Reader in Pharmaceutical Chemistry, Bombay University Department of Chemical Technology, Bombay 19, India. The authors wish to express their sincere appreciation to Dr. G. C. Walker of the Faculty of Pharmacy, University of Toronto, for his valuable suggestions in preparing this manuscript.
the evaluation of these derivatives as disinfectants and germicides. Much of the information reported on naphthalene and naphthol derivatives relate to their usefulness as insecticides and fungicides against plant pathogens, or as fumigants (4, 5). An extensive investigation of the bacteriostatic and fungistatic properties of pnaphthol derivatives has now been made to select active compounds that could be used as antiinfective agents and to examine the relationship between chemical constitution and anti-infective
Vol. XLVI, No. 10
JOURNAL OF THE AMERICANPHARMACEUTICAL ASSOCIATION
604
TABLEBACTERIOSTATIC AND FUNGISTATIC ACTIVITYOF &NAPHTHOL DERIVATIVES~
3'
/ /\K2 v
5
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
..
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
41 42 43 44
45
46 47 48 49 50 51 52 53 54 55 56 57 58
d
K~ = OH, fur cwqm.ulds 24-58 3
2
1 NO.
1 2 3 4
R
Ra = H, for compounds 1-23
Compound----
Ri
H H H H H H COCH, H H COCsH5 COCH3 H H H H H H H H H H H CH2CsHs Ri c1 Br I H H H c1 H Br Br H
c1
Br Brd c1 I c1 H NO NOz I' C1nHr OH(@) 1' - c,rJHsOY@Br6
-
H H H H H H H Br H Br Br
c1 Br
I _
R2
R6
Derivatives of @-naphthol OH H 0c.H~ H OCH~H=CH~ H OCHpCH2CI H OCOCH, H OCOCH; Br OCH, H OCH; COCHI CO-nPentyl OCH, OH H OH H OH COCH, OH coed% OH CO-isoPr CO-isoBu OH OH Et n-Pr OH n-Bu OH n-Pentyl OH n-Hexyl OH isoButyl OH OH isoPentyl OH H R8 Rs H H H H H H c1 H I H H Br H c1 c1 Cld H Br Br H Br Br c1 Cld Br Br Br Br H Br H Br c1 Br OH H H H H NO2 ~~
4
5
B.
M . gyoccnes var. aureusb
100 200, 50 200 200 50 50 50 50 50 100 50 200 200 200 100 200 200 100 100 200' 200J 200
100 200f 100
23 18
200f
19 19 14 15 14 10 13 21 17 18
t yghosusb
...
200 50 100 50 100 50 200f 50 50 50
50 10 10 10 ~. 5 2 50 10 10
A . nigerC
16
17 19 16 15 15 14 10 ~~
.. ..
19
50
50 200 200 20 50 100 100 50 200 50 200 100 20 50
50 Fi _ o_ 20
..
200 200 50 200
50 5 5 10 20 20 200 100 200
27 22 28 20 22 16 19 10 19 20 20 15 20 18 25
50
20 10
loo ~. . 5
-.
H
H
200
20
17
H
Br
50
5
26
Derivatives of 2-hydroxy-3-wdphthok acid COOCH, S _H_ -n _ COOChH5 H 50 COOnPr H 50 COOC~HF, H 50 coocsHs OCOCHi" 200 COO-&Cl-m-cresvl H 50 COOH Br 200f 2OOf 200f 200' 200f CONHCaH;C1(4') C1 20' CONHCsH4Br(4') Br 200f
200s 200f 200f 200 200f 200f
2% 5 20 0.5
0.5 10 0.2
13 14 13 10 17 11 18 10 10 10 10
..
..
See footnotes page 605 4
SCIENTIFICEDITION
October 1957
605
TABLE II.-INHIBITORY CONCENTRATIONSI N p g . / C C . properties. Preliminary ila vitro screening of 126 OF SELECTED COMPOUNDS~ AGAINSTGRAM-NEGAderivatives was carried out and the selected comTIVE ORGANISMS pounds tested against several other Gram-posiB . para- B . dysentive and Gramnegative bacteria as well as comtyphusus tcriz ComA. Shiga A. coli typhosus A pound5 mon dermatophytes. Detailed s t d i e s on the 24 50 50" 50 50" effective compounds will be published subse50" 50" 28 20 50 quently. 50" 50" 50 29 50 50" 50" 50 32 50 50 50" EXPERIMENTAL 37 20 50" 39 _.
40 46 47
50 .~ 50
50 50 50
49
50"
50
50"
50 50 50 50
50 50 50" 50"
50"
50" 50" 50"
Showing inhibition at 50 pg./cc. against Gram-negative organisms other than B. typhosus; Cultures obtained from Haffkine Institute, Bombay. 1, Numbers refer t o compounds included in Table I. c More than. (I
TABLEIII.-INHIBITORY
CONCENTRATIONS I N
5 5 10 20 10 10
5
53 55 56
50
5
58
1
19 20 34 36 37 4.5 ~.
SELECTED COMPOUNDS ORGANISMS
pG./CC. OF
M . pyogcnes var. aureusc (FDA 209)
Stvegt.-Phemolyticus5
Compounda
Synthesis.-Some of the compounds were available commercially, but for the majority of them synthetic methods reported in the literature were employed. Methods of synthesis are recorded in previous publications (13.6-8). Bacteriostatic Tests.-The broth dilution method using B. typhosus (Lister) and M. pyogenes var. aureus (FDA 209) was followed. Concentrations of 200,
M . pyogcnesc var. aureusc (Virulent)
5
M. Pyogenes var. albus
C.
telanid
50d
5 5 10 5 10 10 1 1 0.5
5 20 5 5 20
0.5 0.5 0.2
GRAM-POSITIVE
.5
2
2 2
AGAINST
2 2 0.5
50 50
... 50d ... ... 50d 50 50
Numbers refer to compounds included in Table I. 0 Isolated from patients at Nair Hospital Bombay. c Showing inhibition a t least a t 5 pg./cc.'against M. pyogencs var. a w e u s ( F D A Zag); Cultures obtained from Haffkine Institute, Bombay. d More than. a
TABLE IV.-FUNGISTATIC ACTIVITYOF SELECTED COMPOUNDSuAGAINST DERMATOPHYTES c
Compound/,
1 25 27 29
Agar Streak Method--E. T. T. floccosum rubrum gyPseum
50 10 10 10 50 50 ~. 10 50 10 50 50 10
30 31
32 37 38 39 42
43 45
50
50 10 10 10 10 50
10 50 10 1 50 10 10
50 50 50 50
50 50 50
-Agar
Cup-Plate Method&-
T.
T.
floccosum
rubrum
gypscum
16.5 48 18.5 19.5 16 17 11.6
22.5 47 31 23.5 21 18
20.5 28 19 26 15.5 16 13
36.5
28
E.
20
50
50 50 50 50 50
33.5
-
Showing inhibition of all the fungous pathogens a t least a t 50 ,ug./cc.; Cultures obtained from A.F.M.C. Poona, India. b Numbers refer t o compounds included in Table I. c Inhibitory concentration in pg./cc. d Mean diameter of zone ofcomplete inhibition in mm. at 0.25% concentration with a cup of IOmm. diameter. (I
a Compounds inhibiting growth of B. tyghosus and/or M. Pyogenes var. aurcus a t least at 50 rg./cc., and/or exhibiting 16 mm. or more clearance zone against A. niger. b Inhibitory concentration in pg./cc. c Mean diameter of zone of complete inhibition in mm. a t 0.26% concentration with a cup of 1Omm. diameter, d Substituent in 4 position. (I Substituent in 2 position. f More than: .not tested.
..
606
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION Vol. XLVI, No. 10
100, and 50 pg. fcc. were prepared by the addition of an alcoholic stock solution, and further dilutions such as 20, 10, 5, 2, 1, 0.5, 0.2, and 0.1 pg./cc. were made where necessary. The tubes were incubated for forty-eight t o seventy-two hours following inoculation with a loopful of a twenty-four-hour broth culture of the test organism. Controls indicated the absence of bacteriostasis by the concentrations of alcohol used. Compounds which inhibited the growth of the Organisms a t 50 pg./cc. or a t lower concentration are recorded in Table I. Further evaluation of the compounds inhibitory a t 50 pg./cc. was made using B. paratyphosus A, B.coli, B. dysenterza, Shiga, M . pyogenes var. aureus (virulent), M . pyogenes var. albus, Strept. 0 - h o l y t icus and C. tetuni. The results are recorded in Tables I1 and 111. Fungistatic Tests.-Ten cubic centimeters of Sabouraud‘s agar containing one cc. of a standardized spore suspension of A . nigev (NRRL 337) were poured over 20 cc. of agar and the plates incubated for six hours (9). Alcoholic solutions of concentration 1, 0.5 and 0.25ojO w/v were then added to the prepared cups and plates incubated for forty-eight t o seventy-two hours a t 25-27’, The mean diameters of the zones of complete inhibition for the conare reported in Table I. centration of Selected compounds showing activity against A . niger, were further tested against E . jfoccosum. T. rubrum and T . gypseum by the agar-streak (10) and the agar cup-plate (9)methods. In the streak method, streaks of spore suspension were drawn over agar plates containing the test substances in concentration of 200, 50, 10, and 1pg./cc.. Control plates containing alcohol exhibited no inhibition. Results obtained after incubation for five t o six days by both the methods are recorded in Table IV.
DISCUSSION The relationship between chemical structure and bactericidal activity has been discussed previously (1-3). Some of the observations on the data presented here appear similar. Blocking of the hydroxyl group depresses the bacteriostatic as well as fungistatic activity of the parent compound or its halogenated derivative. Introduction of a keto group slightly enhances the bacteriostatic activity, but increasing the chain length in the 6-substituted ketones does not further enhance the activity. Alkylation increases the bacteriostatic activity, while halogenation iduences the fungistatic activity as well. With an increase in the chain-length or the number of halogen atoms the activity is increased especially against M . pyogenes var. aureus. However, the position of substitution has a marked influence. Compared t o the straight-chain compounds, iso-alkyl derivatives were inferior; but halogenated tertiary-alkyl-@-naphthols have been recently reported (11)to be “staphylostatic” a t high dilutions. Methylation of alkyl-naphthols is detrimental as is the case with substitution of different halogen atoms in the parent naphthol. Groups such as hydroxy, nitro, amino, and sulphonic do not enhance the anti-infective power of pnaphthol. 1:6-Dinitro-p-naphtho1, however, exhibits marked inhibition of A . niger. Of the 8-
dinaphthols studied, B-dinaphthol and its dibromo derivative appear to be superior to the corresponding compounds in the &naphthol series. The alkyl and aryl esters of 2-hydroxy-3-naphthoic acid show preferential activity against B. typhosus which is not enhanced with increasing chain-length. The iso-alkyl esters or their halogenated derivatives are also inactive. Modification of the carboxyl group t o a n amide, anilide or hydrazide, or halogenation of the acid and its amide does not augment the activity. However, bromoanilides inhibit the growth of M . pyogenes var. aureus at very low concentration; but this depends markedly on the position occupied by the halogen atom. Most of the selected compounds (Table 11) are active against the Gram-negative organisms, excepting B. coli, at 50 pg./cc. but they are ineffective at 20 pg./cc. The inhibitory concentrations of all the compounds in Table I11 against the two strains of M . pyogenes var. aweus tested, do not differ significantly. Although M . pyogenes var. albus and Strept. hemolyticus require a higher concentration for inhibition, the specificity of activity shown by these compounds is noteworthy. Results of fungistatic activity (Table IV)indicate that halogenation of &naphthol enhances the activity but polyhalogenation or substitution of different halogen atoms fails to enhance it further. Although some derivatives exhibit fungistasis at 10 pg./cc. against E . jloccosum and T. rubrum by the streak method, the activity against each organism by the cup-plate method differs from compound t o compound and no generalizations can be made.
SUMMARY A bacteriostatic and fungistatic examination has been made of 126 compounds related t o 0-naphthol and 2-hydroxy-3-naphthoic acid using B. typlzosus, M. pyogenes var. aureus, and A . niger; and some conclusions on the relationship of chemical constitution and anti-infective properties are drawn. Whereas alkylation of 6-naphthol enhances the bacteriostatic activity, halogenation increases both bacteriostatic and fungistatic activity. However the position of substitution and a free phenolic group are influencing factors. Esters of 2-hydroxy-3-naphthoic acid show preferential activity against Gram-negative organisms. Bromoanilides, depending on the position occupied by the bromine atom, inhibit the growth of Grampositive organisms at low concentrations. Selected compounds were further evaluated using the organisms B. paratypkosus A, B. coli, B. dysenteric?, M . pyogenes var. albus, Strept. hemolyticus, C. tetani, E . jfoccosum, T. rubrum, and T . gypseum. For Gram-positive organisms, the inhibitory concentrations of a,fi-dibromo-, 3,4,6-tribromo-, fi-npentyl-, and 6-n-hexyl-@-naphtholsand 1,4’-dibromo1,6-dibromo-, and 1,4’,6-tribromo-2-hydroxy3-naphthanilides lie between 0.2 and 10 pg./cc. Results of the cup-plate method indicated l-bromonaphthol and 6,6’-dibromo-p-dinaphtho1as the most active compounds against the dermatophytes tested. Fungistasis at 10 pg./cc. against E.jloccosum and T. rubrum, by the streak method was exhibited by five halogenated derivatives and l&dinitro-&naphthol.
October 1957
SCIENTIFIC EDITION
GO7
REFERENCES 1. Baichwal, R. S., and Khorana, M. L., J. Sci. I n d . Reseavch ( I n d i a ) . , I I B , 189(1952). 2. Baichwal, R . S., Khorana, M. L., and Vangikar, M. B., ibid., lZB,43(1953). 3. Baichwal, R. S., Baichwal, M. R., and Khorana, M . L., I n d i a n J . Pharm., 16, 93(1954). 4. Baichwal, R. S., and Khorana, M. L., J. Sci. I n d . Reseavch ( I n d i a ) , I I A , 197(1952). 5. Block, S. S., “Industrial preservatives” in “Antiseptics, Disinfectants, Fungicides and Chemical and Physical Sterilization” edited by Reddish, G. F., Lea and Febiger, Philadelphia, 1954, pp. 603, 808, 617.
6. Baichwal, R. S., and Khorana, M L., J . Sci. Ind. Research ( I n d i a ) , IZB, 41(1953). 7. Baichwal, R. S., Baichwal, M. R., and Khorana, M. L., I n d i a n J. Pharm., 16, 90(1954). 8. Baichwal, R. S., Khorana, M. L., and Pishawikar, A. D., ibid., 18, 224(1958). 9. Burlingame, E. M., and Reddish, G. F , J . Lab. Clin. Med., 24,771(1939). 10. Schamberg, J. F . , and Kolmer, J. A , , Arch. Devmalol. and Syphilol., 6,746(1922). 11. Welsch, M . , Buu-Hoi, Ng. Ph., and Binon, F., E x p e r i entia, 11,350(1955).
Solution Rate of Theophylline Salts and Effects from Oral Administration* By EINO NELSON The solution rate of several theophylline salts was determined in buffer solutions covering a range of hydrogen ion concentrations somewhat greater than those to which the salts would be exposed after oral administration. It was found that marked differences existed in the rate with which the several salts dissolved and the rates were relatively independent of buffer pH. It was suggested that the fundamental reason for differences found by clinical workers in comparing blood levels from oral administration of the choline and isopropanolamine salts with the ethylenediamine salt was due to differences in solution rate. The in vitro solution rates determined strongly support this explanation. The effect of solution rate on maximum blood level and rate of build-up of blood level with time was discussed by means of a mathematical model relating blood level with solution rate and elimination rate from the blood stream. The solution rate of theophylline in acidic and basic media was determined and found to vary inversely with the hydrogen ion concentration of the diffusion layer. A relationship was developed between solution rate of theophylline and diffusion layer pH and it was shown to be capable of roughly predicting solution rate of salts from measurement of this pH.
the weak acid theophylline have been made available commercially and several are currently used in therapeutics. Part of the advantage claimed to be gained in the use of one particular salt in preference to another or the free acid itself is enhanced water solubility (1, 2). It is difficult to see how this factor could be of importance when the drug is administered orally. In the first place, the theophylline contained in the usual dose of most salts (0.2 Gm.) is soluble in 25-30 ml. of gastric fluid and in a slightly smaller volume of intestinal fluid. In the second place, the small amount of base introduced when any of the salts are taken could not possibly raise the pH of either gastric or intestinal fluid high enough to increase the solubility of theophylline in these fluids. In spite of the foregoing argument, there is
A
NUMBER OF SALTS of
-~
* Received February 22, 1957, from the School of Pharmacy, University of California Medical Center, San Francisco 22. Presented t o the Scientific Section, A. Pa. A., New York meeting, April-May, 1957.
convincing clinical evidence available to show that different blood levels of theophylline were obtained when several salts were studied comparatively. In one study, markedly higher levels were obtained from the choline salt when compared to the levels from the ethylenediamine salt (1). I n another, oral administration of the isopropanolamine salt resulted in higher average levels than those from the ethylenediamine salt ( 2 ) . The results obtained were not explainable on the basis of theophylline content of the preparations tested. This work was conducted to determine the rate with which several salts of theophylline dissolved in mediums possessing hydrogen ion concentrations in the physiological range since it was thought that this factor could logically explain the clinical results reported (1, 2 ) . A supporting purpose of this work was to consider the mathematics of the solution + absorption + elimination processes after oral administration of
SCIENTIFIC EDITION
both the present method and chromotography ; and C-1 was adopted as the reference standard. Assuming a purity of 100% for the standard, recoveries of reserpine from C-1 and C-2 after elution were 984970, when compared with unchromatographed standards. The values reported in Tables I and I1 are based upon procedural standards.e Of the twelve samples of crystalline reserpine analyzed, eight contained more than 96% reserpine. Samples C-9 and C-10 contained excessive quantities of deserpidine; samples C-11 and C-12 contained both deserpidine and significant quantities of more polar alkaloids; C-12 yielded the only sample blank with appreciable absorbance a t 390 mp. Fifteen of the commercial tablet samples were analyzed by both the proposed method of assay and the extended-nitrite procedure previously described (1). Twelve of the check analyses agreed within 2%. The greatest discrepancy between procedures 2 Solutions of reserpine in ethanol are chromogenically stable for several weeks when protected from light. Since the efficiency of the column is fairly uniform, i t is probably necessary to analyze procedural standards only occasionally. Unknowns may be compared with a standard ethanolic solution, diluted with the requisite volumes of chloroform and ethanol just prior to use, and the results corrected for differences between this standard and the procedural standard.
603
was 4%. Only Sample T-8contained quantities of 3-dehydroreserpates large enough t o give an appreciable blank a t 390 mp. Ultraviolet absorption data for 19 of the 24 prepared chloroform-ethanol solutions indicated that the recovered alkaloidal fractions consisted of reserpine almost exclusively. Paper chromatograms on extracts from the tablets in the most notable of the remaining five samples revealed the presence of other weakly basic alkaloids (rescinnamine and deserpidine). More polar alkaloids such as reserpic acid, methyl reserpate, ajmaline, and yohimbine are retained on the column quantitatively. Rescinnamine and reserpinine are extracted with reserpine, and yield similar colored derivatives when treated with nitrous acid. But these and other alkaloids can be detected by means of chromatography. The proposed method of assay is as selective as the previously published nitrite procedures (1, 2) and is simpler, quicker, and less vulnerable to manipulative errors. REFERENCES (1) Banes, D.. Wolff, J.,Fallscheer, H. O., and Carof, J., THIS JOURNAL, 45, 708(1956). ( 2 ) Szalkowski, C. R., and Mader, W. J., ibid., 45, 613
\'""",.
/lOKC\
(3) "United States Pharmacopeia," 15th ed., Mack Puhlishing Co., Easton, Pa., 1955, pp. 217-218.
Antibacterial and Antifungal Properties of ,+Naphthol Derivatives IV* By R. S . BAICHWALt, MALATI R. BAICHWALt, and M. L. KHORANAf. The bacteriostatic and fungistatic activities of a number of &naphthol and 2-hydroxy3-naphthoic acid derivatives are reported against Bacillus typhsus, Mimcoccus pyogenes, var. aureus, and Aspergillus niger. Selected com ounds from the initial sueening were further tested for their activity against Bacilks paratyphosus A, Bacillus coli, Bacillas dysenteriae Shiga, M icrococcuspyogenes, vat. albus, Streptococcus-8-haemolyticus, Clostridium tetani, Epidermophyton floccos~nt,Trichophyton rubrum, and Trichophyton gypseum. Few compounds exhibit activity against Gram-negative organisms at concentrations below 5 0 pg./cc., and none against C. tetani. The bacteriostatic concentration for Gram-positive organisms of 3,ddibromo-, 3,4 ',dtribromo-, 6-npentyl-, and 6-n-hexyl-8-na hthols and 1,4-dibromo-, 1,6-dibromo-, and 1,4,6tribromo-2-hydroxy-3-naphtianilides is between 0.2 and 10 pg./cc. Marked inhibition of all the fungous pathogens was shown b y 6,6'-dibromo-&dinaphthol and 1-bromo-+naphthol. 3-Chloro-, 1-bromo-, 6-bromo-, and 1,6-dinitro-naphthols inhibited the growth of E. ficcosum and T. rubrum at concentrations ranging between 1 and 10 pg./cc. HE BACTERICIDAL ACTIVITIES of a number of Tderivatives of p-naphthol have been reported previously (1-3) and the data might be useful in *Received March 4, 1957, from the Bombay University Department of Chemical Teehnology. Bombay 19, India. This paper is based in part on the theses submitted to the University of Bombay by R. S. Baichwal and Malati R. Baichwal for the degree of Doctor of Philosophy in the Faculty of Technology. t Present address: Faculty of Pharmacy, University of Toronto, Canada. $ Reader in Pharmaceutical Chemistry, Bombay University Department of Chemical Technology, Bombay 19, India. The authors wish to express their sincere appreciation to Dr. G. C. Walker of the Faculty of Pharmacy, University of Toronto, for his valuable suggestions in preparing this manuscript.
the evaluation of these derivatives as disinfectants and germicides. Much of the information reported on naphthalene and naphthol derivatives relate to their usefulness as insecticides and fungicides against plant pathogens, or as fumigants (4, 5). An extensive investigation of the bacteriostatic and fungistatic properties of pnaphthol derivatives has now been made to select active compounds that could be used as antiinfective agents and to examine the relationship between chemical constitution and anti-infective
Vol. XLVI, No. 10
JOURNAL OF THE AMERICANPHARMACEUTICAL ASSOCIATION
604
TABLEBACTERIOSTATIC AND FUNGISTATIC ACTIVITYOF &NAPHTHOL DERIVATIVES~
3'
/ /\K2 v
5
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
..
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
41 42 43 44
45
46 47 48 49 50 51 52 53 54 55 56 57 58
d
K~ = OH, fur cwqm.ulds 24-58 3
2
1 NO.
1 2 3 4
R
Ra = H, for compounds 1-23
Compound----
Ri
H H H H H H COCH, H H COCsH5 COCH3 H H H H H H H H H H H CH2CsHs Ri c1 Br I H H H c1 H Br Br H
c1
Br Brd c1 I c1 H NO NOz I' C1nHr OH(@) 1' - c,rJHsOY@Br6
-
H H H H H H H Br H Br Br
c1 Br
I _
R2
R6
Derivatives of @-naphthol OH H 0c.H~ H OCH~H=CH~ H OCHpCH2CI H OCOCH, H OCOCH; Br OCH, H OCH; COCHI CO-nPentyl OCH, OH H OH H OH COCH, OH coed% OH CO-isoPr CO-isoBu OH OH Et n-Pr OH n-Bu OH n-Pentyl OH n-Hexyl OH isoButyl OH OH isoPentyl OH H R8 Rs H H H H H H c1 H I H H Br H c1 c1 Cld H Br Br H Br Br c1 Cld Br Br Br Br H Br H Br c1 Br OH H H H H NO2 ~~
4
5
B.
M . gyoccnes var. aureusb
100 200, 50 200 200 50 50 50 50 50 100 50 200 200 200 100 200 200 100 100 200' 200J 200
100 200f 100
23 18
200f
19 19 14 15 14 10 13 21 17 18
t yghosusb
...
200 50 100 50 100 50 200f 50 50 50
50 10 10 10 ~. 5 2 50 10 10
A . nigerC
16
17 19 16 15 15 14 10 ~~
.. ..
19
50
50 200 200 20 50 100 100 50 200 50 200 100 20 50
50 Fi _ o_ 20
..
200 200 50 200
50 5 5 10 20 20 200 100 200
27 22 28 20 22 16 19 10 19 20 20 15 20 18 25
50
20 10
loo ~. . 5
-.
H
H
200
20
17
H
Br
50
5
26
Derivatives of 2-hydroxy-3-wdphthok acid COOCH, S _H_ -n _ COOChH5 H 50 COOnPr H 50 COOC~HF, H 50 coocsHs OCOCHi" 200 COO-&Cl-m-cresvl H 50 COOH Br 200f 2OOf 200f 200' 200f CONHCaH;C1(4') C1 20' CONHCsH4Br(4') Br 200f
200s 200f 200f 200 200f 200f
2% 5 20 0.5
0.5 10 0.2
13 14 13 10 17 11 18 10 10 10 10
..
..
See footnotes page 605 4
SCIENTIFICEDITION
October 1957
605
TABLE II.-INHIBITORY CONCENTRATIONSI N p g . / C C . properties. Preliminary ila vitro screening of 126 OF SELECTED COMPOUNDS~ AGAINSTGRAM-NEGAderivatives was carried out and the selected comTIVE ORGANISMS pounds tested against several other Gram-posiB . para- B . dysentive and Gramnegative bacteria as well as comtyphusus tcriz ComA. Shiga A. coli typhosus A pound5 mon dermatophytes. Detailed s t d i e s on the 24 50 50" 50 50" effective compounds will be published subse50" 50" 28 20 50 quently. 50" 50" 50 29 50 50" 50" 50 32 50 50 50" EXPERIMENTAL 37 20 50" 39 _.
40 46 47
50 .~ 50
50 50 50
49
50"
50
50"
50 50 50 50
50 50 50" 50"
50"
50" 50" 50"
Showing inhibition at 50 pg./cc. against Gram-negative organisms other than B. typhosus; Cultures obtained from Haffkine Institute, Bombay. 1, Numbers refer t o compounds included in Table I. c More than. (I
TABLEIII.-INHIBITORY
CONCENTRATIONS I N
5 5 10 20 10 10
5
53 55 56
50
5
58
1
19 20 34 36 37 4.5 ~.
SELECTED COMPOUNDS ORGANISMS
pG./CC. OF
M . pyogcnes var. aureusc (FDA 209)
Stvegt.-Phemolyticus5
Compounda
Synthesis.-Some of the compounds were available commercially, but for the majority of them synthetic methods reported in the literature were employed. Methods of synthesis are recorded in previous publications (13.6-8). Bacteriostatic Tests.-The broth dilution method using B. typhosus (Lister) and M. pyogenes var. aureus (FDA 209) was followed. Concentrations of 200,
M . pyogcnesc var. aureusc (Virulent)
5
M. Pyogenes var. albus
C.
telanid
50d
5 5 10 5 10 10 1 1 0.5
5 20 5 5 20
0.5 0.5 0.2
GRAM-POSITIVE
.5
2
2 2
AGAINST
2 2 0.5
50 50
... 50d ... ... 50d 50 50
Numbers refer to compounds included in Table I. 0 Isolated from patients at Nair Hospital Bombay. c Showing inhibition a t least a t 5 pg./cc.'against M. pyogencs var. a w e u s ( F D A Zag); Cultures obtained from Haffkine Institute, Bombay. d More than. a
TABLE IV.-FUNGISTATIC ACTIVITYOF SELECTED COMPOUNDSuAGAINST DERMATOPHYTES c
Compound/,
1 25 27 29
Agar Streak Method--E. T. T. floccosum rubrum gyPseum
50 10 10 10 50 50 ~. 10 50 10 50 50 10
30 31
32 37 38 39 42
43 45
50
50 10 10 10 10 50
10 50 10 1 50 10 10
50 50 50 50
50 50 50
-Agar
Cup-Plate Method&-
T.
T.
floccosum
rubrum
gypscum
16.5 48 18.5 19.5 16 17 11.6
22.5 47 31 23.5 21 18
20.5 28 19 26 15.5 16 13
36.5
28
E.
20
50
50 50 50 50 50
33.5
-
Showing inhibition of all the fungous pathogens a t least a t 50 ,ug./cc.; Cultures obtained from A.F.M.C. Poona, India. b Numbers refer t o compounds included in Table I. c Inhibitory concentration in pg./cc. d Mean diameter of zone ofcomplete inhibition in mm. at 0.25% concentration with a cup of IOmm. diameter. (I
a Compounds inhibiting growth of B. tyghosus and/or M. Pyogenes var. aurcus a t least at 50 rg./cc., and/or exhibiting 16 mm. or more clearance zone against A. niger. b Inhibitory concentration in pg./cc. c Mean diameter of zone of complete inhibition in mm. a t 0.26% concentration with a cup of 1Omm. diameter, d Substituent in 4 position. (I Substituent in 2 position. f More than: .not tested.
..
606
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION Vol. XLVI, No. 10
100, and 50 pg. fcc. were prepared by the addition of an alcoholic stock solution, and further dilutions such as 20, 10, 5, 2, 1, 0.5, 0.2, and 0.1 pg./cc. were made where necessary. The tubes were incubated for forty-eight t o seventy-two hours following inoculation with a loopful of a twenty-four-hour broth culture of the test organism. Controls indicated the absence of bacteriostasis by the concentrations of alcohol used. Compounds which inhibited the growth of the Organisms a t 50 pg./cc. or a t lower concentration are recorded in Table I. Further evaluation of the compounds inhibitory a t 50 pg./cc. was made using B. paratyphosus A, B.coli, B. dysenterza, Shiga, M . pyogenes var. aureus (virulent), M . pyogenes var. albus, Strept. 0 - h o l y t icus and C. tetuni. The results are recorded in Tables I1 and 111. Fungistatic Tests.-Ten cubic centimeters of Sabouraud‘s agar containing one cc. of a standardized spore suspension of A . nigev (NRRL 337) were poured over 20 cc. of agar and the plates incubated for six hours (9). Alcoholic solutions of concentration 1, 0.5 and 0.25ojO w/v were then added to the prepared cups and plates incubated for forty-eight t o seventy-two hours a t 25-27’, The mean diameters of the zones of complete inhibition for the conare reported in Table I. centration of Selected compounds showing activity against A . niger, were further tested against E . jfoccosum. T. rubrum and T . gypseum by the agar-streak (10) and the agar cup-plate (9)methods. In the streak method, streaks of spore suspension were drawn over agar plates containing the test substances in concentration of 200, 50, 10, and 1pg./cc.. Control plates containing alcohol exhibited no inhibition. Results obtained after incubation for five t o six days by both the methods are recorded in Table IV.
DISCUSSION The relationship between chemical structure and bactericidal activity has been discussed previously (1-3). Some of the observations on the data presented here appear similar. Blocking of the hydroxyl group depresses the bacteriostatic as well as fungistatic activity of the parent compound or its halogenated derivative. Introduction of a keto group slightly enhances the bacteriostatic activity, but increasing the chain length in the 6-substituted ketones does not further enhance the activity. Alkylation increases the bacteriostatic activity, while halogenation iduences the fungistatic activity as well. With an increase in the chain-length or the number of halogen atoms the activity is increased especially against M . pyogenes var. aureus. However, the position of substitution has a marked influence. Compared t o the straight-chain compounds, iso-alkyl derivatives were inferior; but halogenated tertiary-alkyl-@-naphthols have been recently reported (11)to be “staphylostatic” a t high dilutions. Methylation of alkyl-naphthols is detrimental as is the case with substitution of different halogen atoms in the parent naphthol. Groups such as hydroxy, nitro, amino, and sulphonic do not enhance the anti-infective power of pnaphthol. 1:6-Dinitro-p-naphtho1, however, exhibits marked inhibition of A . niger. Of the 8-
dinaphthols studied, B-dinaphthol and its dibromo derivative appear to be superior to the corresponding compounds in the &naphthol series. The alkyl and aryl esters of 2-hydroxy-3-naphthoic acid show preferential activity against B. typhosus which is not enhanced with increasing chain-length. The iso-alkyl esters or their halogenated derivatives are also inactive. Modification of the carboxyl group t o a n amide, anilide or hydrazide, or halogenation of the acid and its amide does not augment the activity. However, bromoanilides inhibit the growth of M . pyogenes var. aureus at very low concentration; but this depends markedly on the position occupied by the halogen atom. Most of the selected compounds (Table 11) are active against the Gram-negative organisms, excepting B. coli, at 50 pg./cc. but they are ineffective at 20 pg./cc. The inhibitory concentrations of all the compounds in Table I11 against the two strains of M . pyogenes var. aweus tested, do not differ significantly. Although M . pyogenes var. albus and Strept. hemolyticus require a higher concentration for inhibition, the specificity of activity shown by these compounds is noteworthy. Results of fungistatic activity (Table IV)indicate that halogenation of &naphthol enhances the activity but polyhalogenation or substitution of different halogen atoms fails to enhance it further. Although some derivatives exhibit fungistasis at 10 pg./cc. against E . jloccosum and T. rubrum by the streak method, the activity against each organism by the cup-plate method differs from compound t o compound and no generalizations can be made.
SUMMARY A bacteriostatic and fungistatic examination has been made of 126 compounds related t o 0-naphthol and 2-hydroxy-3-naphthoic acid using B. typlzosus, M. pyogenes var. aureus, and A . niger; and some conclusions on the relationship of chemical constitution and anti-infective properties are drawn. Whereas alkylation of 6-naphthol enhances the bacteriostatic activity, halogenation increases both bacteriostatic and fungistatic activity. However the position of substitution and a free phenolic group are influencing factors. Esters of 2-hydroxy-3-naphthoic acid show preferential activity against Gram-negative organisms. Bromoanilides, depending on the position occupied by the bromine atom, inhibit the growth of Grampositive organisms at low concentrations. Selected compounds were further evaluated using the organisms B. paratypkosus A, B. coli, B. dysenteric?, M . pyogenes var. albus, Strept. hemolyticus, C. tetani, E . jfoccosum, T. rubrum, and T . gypseum. For Gram-positive organisms, the inhibitory concentrations of a,fi-dibromo-, 3,4,6-tribromo-, fi-npentyl-, and 6-n-hexyl-@-naphtholsand 1,4’-dibromo1,6-dibromo-, and 1,4’,6-tribromo-2-hydroxy3-naphthanilides lie between 0.2 and 10 pg./cc. Results of the cup-plate method indicated l-bromonaphthol and 6,6’-dibromo-p-dinaphtho1as the most active compounds against the dermatophytes tested. Fungistasis at 10 pg./cc. against E.jloccosum and T. rubrum, by the streak method was exhibited by five halogenated derivatives and l&dinitro-&naphthol.
October 1957
SCIENTIFIC EDITION
GO7
REFERENCES 1. Baichwal, R. S., and Khorana, M. L., J. Sci. I n d . Reseavch ( I n d i a ) . , I I B , 189(1952). 2. Baichwal, R . S., Khorana, M. L., and Vangikar, M. B., ibid., lZB,43(1953). 3. Baichwal, R. S., Baichwal, M. R., and Khorana, M . L., I n d i a n J . Pharm., 16, 93(1954). 4. Baichwal, R. S., and Khorana, M. L., J. Sci. I n d . Reseavch ( I n d i a ) , I I A , 197(1952). 5. Block, S. S., “Industrial preservatives” in “Antiseptics, Disinfectants, Fungicides and Chemical and Physical Sterilization” edited by Reddish, G. F., Lea and Febiger, Philadelphia, 1954, pp. 603, 808, 617.
6. Baichwal, R. S., and Khorana, M L., J . Sci. Ind. Research ( I n d i a ) , IZB, 41(1953). 7. Baichwal, R. S., Baichwal, M. R., and Khorana, M. L., I n d i a n J. Pharm., 16, 90(1954). 8. Baichwal, R. S., Khorana, M. L., and Pishawikar, A. D., ibid., 18, 224(1958). 9. Burlingame, E. M., and Reddish, G. F , J . Lab. Clin. Med., 24,771(1939). 10. Schamberg, J. F . , and Kolmer, J. A , , Arch. Devmalol. and Syphilol., 6,746(1922). 11. Welsch, M . , Buu-Hoi, Ng. Ph., and Binon, F., E x p e r i entia, 11,350(1955).
Solution Rate of Theophylline Salts and Effects from Oral Administration* By EINO NELSON The solution rate of several theophylline salts was determined in buffer solutions covering a range of hydrogen ion concentrations somewhat greater than those to which the salts would be exposed after oral administration. It was found that marked differences existed in the rate with which the several salts dissolved and the rates were relatively independent of buffer pH. It was suggested that the fundamental reason for differences found by clinical workers in comparing blood levels from oral administration of the choline and isopropanolamine salts with the ethylenediamine salt was due to differences in solution rate. The in vitro solution rates determined strongly support this explanation. The effect of solution rate on maximum blood level and rate of build-up of blood level with time was discussed by means of a mathematical model relating blood level with solution rate and elimination rate from the blood stream. The solution rate of theophylline in acidic and basic media was determined and found to vary inversely with the hydrogen ion concentration of the diffusion layer. A relationship was developed between solution rate of theophylline and diffusion layer pH and it was shown to be capable of roughly predicting solution rate of salts from measurement of this pH.
the weak acid theophylline have been made available commercially and several are currently used in therapeutics. Part of the advantage claimed to be gained in the use of one particular salt in preference to another or the free acid itself is enhanced water solubility (1, 2). It is difficult to see how this factor could be of importance when the drug is administered orally. In the first place, the theophylline contained in the usual dose of most salts (0.2 Gm.) is soluble in 25-30 ml. of gastric fluid and in a slightly smaller volume of intestinal fluid. In the second place, the small amount of base introduced when any of the salts are taken could not possibly raise the pH of either gastric or intestinal fluid high enough to increase the solubility of theophylline in these fluids. In spite of the foregoing argument, there is
A
NUMBER OF SALTS of
-~
* Received February 22, 1957, from the School of Pharmacy, University of California Medical Center, San Francisco 22. Presented t o the Scientific Section, A. Pa. A., New York meeting, April-May, 1957.
convincing clinical evidence available to show that different blood levels of theophylline were obtained when several salts were studied comparatively. In one study, markedly higher levels were obtained from the choline salt when compared to the levels from the ethylenediamine salt (1). I n another, oral administration of the isopropanolamine salt resulted in higher average levels than those from the ethylenediamine salt ( 2 ) . The results obtained were not explainable on the basis of theophylline content of the preparations tested. This work was conducted to determine the rate with which several salts of theophylline dissolved in mediums possessing hydrogen ion concentrations in the physiological range since it was thought that this factor could logically explain the clinical results reported (1, 2 ) . A supporting purpose of this work was to consider the mathematics of the solution + absorption + elimination processes after oral administration of