344 Journal of Medicinal Chemistry, 1975, Vol. 18, N o 4 and d. 4.72, about 1 3 aromatic protons. d n n i . (C2:rH210;N:a. H2Oi H , N; C: calcd, 58.84; found, 59.28. Hydrolysis Procedure. Exactly 0.4 mmol of the ester was dissolved in 200 ml of acetone contained in a 250-ml volumetric flask. T h e solution was diluted t o the mark with H.0 and thoroughly mixed to give a 0.0016 M solution of, the ester. Aliquots (25 ml) were transferred cia a volumetric pipet to 50-ml erlenmeyer flasks, the flasks were stoppered, and the contents were equilibrated in a bath maintained a t 40 0 . 2 O . Into each aliquot was rapidly pipetted exactly ,i ml of 0.100 N NaOH. This gave a n initial concentration of the ester of 0.00133 M. T h e mixture was stirred magnetically. and at time intervals the hydrolysis was stopped by rapidly pipetting exactly 5 ml of 0.100 n; HCI into the flask. T h e contents were cooled to room temperature, a few drops of phenolphthalein solution were added, and the mixture was titrated to the end point with 0.100 N NaOH. T h e amount required to reach the end point. minus the blank value, was equivalent to that consumed during the hydrolysis. Blank values were determined by pipetting 5 ml of the standard 0.1 N NaOH into 2 5 m I aliquots of the acetone-H-0 solvent, pipetting into this mixture 5 ml of the standard 0.1 :Y HCI. and titrating this mixture to the phenolphthalein end point with the standard 0.1 N NaOH. The same pipets were used for the hlanks that were used for the hydrolysis. hlolar concentrations of the ester a t time intervals during t h e hydrolysis were calculated from the hydrolysis data, and the -10:: C values were plotted a s a function of time a s shown in Scheme I. T h e hydrolysis constant fi was calculated from the relationship h = 2.3 log C i t . Determination of Partition for the Palmitate Ester 2. ' ' C Palm-0-ora-C ( 2 ) (19.2 HCi/mg. labeled in the 'L position of the pyrmidine ring) was added to a stoppered erlenmeyer flask containing 15 ml each of 1-octanol and sodium phosphate aqueous huffer ( p H 7.0. 0.0:15 M.ionic strength 0.1). After vigorous shaking ( 3 7 O . 1 hr) and subsequent phase separation, radioactivity in 1.0ml aliquots of each phase was determined using a liquid scintillation spectrometer. T h e aqueous phase was removed and replaced \vith fresh buffer. T h e shaking procedure was repeated with fresh huffer until a constant partition coefficient was obtained. T h e value obtained was 127.3 (octanol-water) and the octanol concentration of Palm-0-ara-C ( 2 ) in octanol at equilibrium was 1.5 pg,' ml. (1
*
Acknowledgment. We gratefully acknowledge the technical assistance of Mr. R. J. Reischer and Mr. A. J. Taylor. We thank Dr. E. D. Purich for determining the p value for the palmitate ester and cyclocytidine.
Schaad. Werner Ddlon. Field, Tatc
References and Notes
'r. Gish. B. A. Court, E. E. Eidson. and W.J . Wechter, J . Med. Chem , 16, 754 (197:3), ( 2 ) T h e results and conclusions contained in this paper were presented as part of a presentation before the APhA Academy o i Pharmaceutical Sciences at the 120th Annual Meeting in Boston. .July 21-27. 1973. An abstract appears in .-tbstr Anier. Pharm. A w . , 3, 70, (1973). ( 3 ) Thesis presented by A. E. Berger as partial fulfillment of the requirements for the B.A. degree, Kalamazoo College, 1970. (1)B.A. Thesis presented by A. Harnach (A. H. M'enzel) as partial fulfillment of the requirements for the B.S. degree. K a lamzoo College. 1970. (5)\V.d. LVechter. J. Mr,d C'heni., 10,762 (1967). 16) ('. C. Smith, H. H. Buskirk. and LV. 1,. Lumis, J . M e d . ( ~ / ) o m 10,774 (1967). (7)A. R. Hanze. J A m e r C h c m . Soc., 89,6720 (1967). 18) C. I,. Neil, P. F. LViley. R. C. Manak. and T . E. Moxley. ( ' o n . ccr fi'es.. 30, 1047 (1970); G. L. Neil, H. H. Buskirk. T, E. Moxley. K. C. Manak. S. I,. Kuentzel. and H. I(.Bhuyan. & r J ciicni. Phnrniac,ol.. 20, 3295 (1971). ( 9 ) D. T. Girh. R. C. Kelly. G. M'.Camiener. and LV. .J. Wechter. .J. 22icd. ( ' h ~ r t . 14, , 1159 (1971). 110) G. D. Gray. F. K. h'icol, M. M. Mickelson, G. LV. Camiener. I>. T. (;ish. R. C . Kelly, \V. J . iyechter. T. E. Moxley. and G 1.. Keil. Hif~c,hr,n7. Phnrniacoi., 21, 465 (1972)). I1 1 D. 'r. IVarner. G.I,. Neil, A. J . Taylor. and IV. .I. Wechter. M c d . ('horn.. 1.7, 790 (1972j. 1 2 ) ( a ) C. Hansch and S. M.Anderson. J O r g ('hcm.. 32, 'L (1967): ( h ) -1. I\vasa, T. Fjita. and C. Hansch, J . i2.lcd. ( ' h ( , n i 8, l;iO (19651. 13) R. C'. Letsinger. 1'. S.hliller. and (i. \V. \V. Graus. Trsfrni7c.dron I,ett., 22, 2621 (1968). 141 Ll. S. Newman. Ed.. "Steric Effects in Organic Chemistry." \Vile?. Kew Yurk. N.Y.. 1956. p p 587-599. 13) T. C. Bruice and 'r. H. Fife. .J Amar ('h~ni.S O C , Xd, 197:3 (19ti2). (ifi) L. .J. Hanka, S. L. Kuentzel, and C;. I,. Neil. ('anccTr ( ~ h ~ m o f h c r . R P ~ 35, . . :39,'1 (1970). ( 1 7 1 KO attempt was made to control particle size; the compounds were simply milled in a tissue grinder. In the t w o cases in which this parameter was examined. compounds 2 and 4, the effect W H ~lehs dramatic than the animal to animal variat ion. (1) Nucleic Acids. 14. H. E. Renis, D.
Linear Regression Analysis of Inhibitory Potency of Organic Disulfides against Histoplasma capsula t u m f L. J. Schaad,* R. H. Werner,' Lynn Dillon. Lamar Field,* and C. Emory Tate Department of ('hcmistr?', Vanderbilt L'nipersit?. A'ashcilic, Tcnnc.ssct, 3 i 2 3 Keceiiied Jul? 2.5. 1974 The Free-LVilson equations are deri\ed for the case of symmetrical substitution and are applied, in four modifications. to in i,itro inhibitory activity of 77 organic disulfides against I f i s t o p i n s m a capsulaturn. Substituent constants are listed t o aid in the design of new inhibitory agents against this human pathogen (and perhaps other fungal organismsl.
As part of a search for improved inhibitory agents against Histoplasma capsulatum, the causative organism of histoplasmosis, a regression analysis of the in vitro activity against H. capsulatum was carried out for 71 organic disulfides. There are two main approaches to the problem of correlating biological activity with chemical structure. The one, due to Hansch,' correlates biological activity with other physical parameters, especially the partition ratio between octanol and water. The other, by Free and Wilson,2 estimates biological activity from empirically fitted substit'This ~nvestiyatiiin \ \ a s +upported in part l)y S I H Rehearch ( ; r a n i S ~ I AM 1188> from the N a t i o n a l I n s ~ i t i i t e,copathol. Mycol. A p p l . , 37,349 (1969). (8)L. Field, W. S. Hanley, I. McVeigh, and Z. Evans. J . Med. Chern., 14,202 (1971). (9) L. Field, W . S. Hanley, and I. McVeigh, J . M r d . C h c m . . 14, 995 (1971). (10) I. McVeigh, S. Evans, L. Field, LV. S. Hanley, and C. E. Tate. J . M e d . Chem., 15,431 (1972). (11) L. Field, W.S. Hanley, and I. McVeigh, J . Org. C h e m , , 36, 2735 (1971). (12) L. Field, unpublished data kindly supplied by I. McVeigh and her associates.
Substituted Thiadiazolines as Inhibitors of Central Nervous System Carbonic An hydrase James J. Lukes* Metabolic Laboratories, Rose Research and Diagnostic Center, S t . Vincent Hospital, Worcester, Massachusetts 016'10
and Karl A. Nieforth Section ofMedicinal Chemistry and Pharmacognosy, School of Pharrnacj,, Unii,ersitj,of Connecticut. Storrs. Conncrtic~ut06268 Rcceii,ed August 28?1971 A series (24-30) of substituted thiadiazolines was synthesized and tested for in c,itro carbonic anhydrase inhibition and for protective ability against pentylenetetrazole-induced convulsions. ED;,) (pentylenetetrazole protection). TD,o, and LD:,, values are reported for each compound. With the exception of 30, all compounds approximated the model compound methazolamide as i n uitro carbonic anhydrase inhibitors. Several of the compounds produced extended protection against pentylenetetrazole-induced convulsions. Ring methoxy substitution in the ortho position appeared to produce maximum activity.
Mann and Kellin' demonstrated the inhibitory effects of unsubstituted sulfonamides on carbonic anhydrase in 1940. Subsequent work led to the development of clinically important diuretics, a few of which revealed potential clinical usefulness as anticonvulsants. In a series of thiadiazole derivatives methazolamide (1) (2-acetylimino-3-methyl-A41,3,4-thiadiazoIine-5-sulfonamide, Neptazane, Lederle Laboratories) showed the highest concentration in the brain.? This compound served as a model for the design of the compounds in this paper. Previous work.' had revealed increased carbonic anhydrase inhibition in those compounds bearing an aromatic ring in the 2-substituents. Aromatic ring methoxy substitutions were modeled after known hallucinogenic compounds. Halogen substitutions were prepared to provide opposite electronic effects for SAR comparisons and the unsubstituted compound was prepared as a standard.
Experimental Section Methazolamide was obtained through the courtesy of Lederle Laboratories. Melting points were observed on a Thomas-Hoover capillary melting point apparatus and are uncorrected. Infrared spectra were taken on a Beckman Microspec Model 1485 in Nujol mull. Nmr spectra were determined on a Hitachi Perkin-Elmer spectrometer Model R-24. Elemental analyses were performed by Baron Consulting Company, Orange, Conn. Synthesized compounds are summarized in Tables I and 11. 2-Amino-5-benzylmercapto-1,3,4-thiadiazole (W4 KOH pellets (24 g, 0.43 mol) were added to a slurry of 2-amino-1,3,4-thiadiazole-5-thiol (40 g, 0.3 mol) in 100 ml of water. After cooling, 50 ml EtOH was added and benzyl chloride (48 ml, 0.38 mol) was added dropwise. T h e mixture became viscous and a "curdled" white product separated. After stirring for a n additional 30 min at loo, the mixture was diluted with 200 ml of water. Filtration yielded 64 g (95%) of white crystals (EtOH) melting a t 157-158O. Anal. (CgHgN3Sz)C, H,N.
Substituted 2-Benzamido-5-benzylthio-l,J,4-thiadiazoles (3-9)" ( G e n e r a l Procedure). To 25 ml of pyridine was added the appropriate acid (0.02 mol) and 2 (4.5 g, 0.02 mol). U'ith constant stirring S K I 4 ('2 g, 0.023 mol) was added dropwisc, resulting in temperature elevation to ca. 85". Stirring was continued at room temperature for 10 hr and the mixture was poured into ice water. Silica was removed by filtration and the filtrate was concentrated. The residual solid was recrystallized from EtOH-HIO. Yields ranged from 46 to 5SYo. The same amides were synthesized by reacting 2 with acid anhydrides in aqueous acid (5-16% yield), with acid chlorides in aqueous hydroxide (14-32'30 yield), and with acid chlorides in pyridine lJ,.l-thiadiazoIines (10-16)- ( G e n e r a l Procedure). The appropriate thiadiazole 3-9 (0.01 mol) was dissolved in 60 ml of water containing KOH (0.7 g, 0.0125 mol) and the solution was diluted with 35 mi of EtOH. Dimethyl sulfate (1.3 g, 0.01 mol) was added. the mixture was refluxed for 20 min, and it was then cooled to 10'. Cold NaOH solution (100 ml. 1.5 M ) was added until a slurry formed. Water (150 ml) was added and the mixture refrigerated for 1 hr. The precipitate was collected by filtration and recrystallized from MeOH-HZO. Yields ranged hetween 46 and 62"sb. Similar methylations with CH,J and K.&O{ in ;icetone resulted in yields between 29 and 43%. Substituted 2-Benzoylimino-3-methyl-A1-l,:3,4-thiadiazoline-5-sulfonyl C h l o r i d e (17-23) ( G e n e r a l Procedure). The appropriate thiadiazoline 10-16 (0.05 mol) was added to 40 ml of HOAc in a three-necked flask fitted with a thermometer. a n inlet tube, and an outlet tube leading to a water trap. T h e reaction wah maintained at 5' as chlorine was introduced by the inlet tuhe terminating about 1 in. above the reaction mixture. Chlorine wa5 added in excess over a period of 15 min until the compound went into solution and the solution attained a yellow color. The solutioi) was poured in water and the precipitate filtered and hlotted d r y . Yields ranged from 47 to 58%. T h e compound5 were not purified for subsequent reactions. Substituted 2-Benzoylimino-3-methyl-A1-l,:~,4-thiadiazoline-5-sulfonamide (24-30) (General Procedure). The :ippro-
