Journal ofMedicina1 Chemistry, 1973, Vol. 16, No. 12 1411
Notes frared spectrophotometer, Model IR-20, a t Matheson Coleman and Bell, Norwood, Ohio. Elementary analyses were made by Galbraith Laboratories, Inc., Knoxville, Tenn. 6-Guanidinovaleric Acid ( 4 ) . To the solution of S-ethylisothiourea dihydrogen sulfate (10.0 g, 0.05 mol) dissolved in 25 ml ' NaOH was added 6.0 g (0.05 mol) of 6-aminovaleric acid of 2 LV dissolved in 10 ml of H z 0 under ice cooling and vigorous stirring. The mixture stood for 5 hr in the ice bath and was kept overnight at room temperature. The turbid solution was filtered and acidified ( p H 4) with concentrated HC1. Absolute EtOH was added to the solution and the precipitate of NaC1 was filtered off. The filtrate was concentrated and subjected to ion-exchange chromatography, using a 2.0 x 40 cm column of Amberlite CG-120, 200-400 mesh. pyridine form. For elution, first 500 ml of H20 and then 0.1 M pyridine were used. The peak quantity of 4 appeared in the effluent at approximately 1800 ml. The .1 fractions, 1600-2100 ml, were pooled and concentrated in cucuo. The dry residue was treated with HZO-EtOH-Et20 to produce crystals: yield, 28.070; mp 2677271". e-Guanidinohexanoic Acid ( 5 ) . S-Ethylisothiourea dihydrogen sulfate (10.0 g, 0.05 mol) and 6.5 g (0.05 mol) of e-aminohexanoic acid were treated in the same way as 4 except for the following modifications. After the reaction was completed the precipitate was filtered and washed with cold HzO. The crystals were then dissolved in 20 ml of 2 HCI. The p H was adjusted to 8.0 by the addition of 2 iV NH4OH. After standing at 5" overnight, the white crystals were filtered and washed with cold HzO and EtOH and then dried in a vacuum desiccator: yield, 68.070;mp 274-275". Guanidinoacetyl-L-histidine Hydrate ( 6 ) . The solutions of 1.50 g (0.007 mol) of S-ethylisothiourea dihydrogen sulfate in 5 ml of HzO and 1.74 g (0.007 mol) of Gly-His.HC1 in 3 ml of HzO were adjusted to pH 8-9 with 4 N NaOH. They were then mixed and kept at room temperature for a week. The cloudy suspension was filtered and the filtrate concentrated to dryness in cacuo. The residue, after addition of a small amount of EtOH, was dried in cacuo. A small amount of HzO was added to dissolve the dry residue and resulting solution was poured onto the column (2.5 cm i.d. X 30 cm Amherlite CG-120, 200-400 mesh, pyridine form). HzO. 1.0 M pyridine, 2.0 M pyridine, and 1.0 M pyridine-0.5 M NH40H were used as the effluent solutions. Each fraction was tested for ninhydrin, Pauli, and Sakaguchi reactions. The 6 fractions were pooled and concentrated to dryness in cacuo: crystals ' from H20-EtOH; yield, 69.270; mp 108-111" dec. Hydrolysis (6 LV HCI, 110", 24 hr) gave 1 and His, confirmed by tlc. 6-Guanidinopropionyl-L-histidineHydrate ( 7 ) . S-Ethylisothiourea dihydrogen sulfate (5.0 g, 0.025 mol) and d-Ala-His (5.3 g, 0.025 mol) were treated in the same way as 6 : yield, 38.8%; m p 121-124" dec. Hydrolysis as described above gave 2 and His (tlc). y-Guanidinobutyryl-L-histidine Dihydrogen Sulfate (8). SEthylisothiourea dihydrogen sulfate (10.0 g, 0.05 mol) and y-AbuHis (11.0 g, 0.05 mol) were treated in the same way as 6 except for the following changes. For the final crystallization, the aqueHzS04 and absolute ous solution was adjusted to p H 5 with 2 EtOH was added to form fine white crystals; yield, 51.770; m p 119-124" dec. Hydrolysis as described gave 3 and His (tlc). 6-Guanidinovaleryl-L-histidineDihydrogen Sulfate Hydrate (9). S-Ethylisothiourea dihydrogen sulfate (0.8 g, 0.0037 mol) and 6-Avl-His (1.0 g, 0.0037 mol) were treated in the same way as 8: yield, 50.6%; m p 80-82" dec. Hydrolysis as above gave 4 and His (tlc). c-Guanidinohexanoyl-L-histidine i10). S-Ethylisothiourea dihydrogen sulfate (1.4 g, 0.0063 mol) and c-Ahx-His (1.7 g, 0.0063 mol) were treated in the same way as 6: yield, 56.1%; mp 102103" dec. Hydrolysis as above gave 5 and His (tlc). A\J
A\T
Acknowledgments. We t h a n k D r s . Leo G. N u t i n i and K i n j i T a n a k a for t h e i r interest, Drs. Floyd Green and T h o m a s H i e b e r of M a t h e s o n C o l e m a n and Bell for i r s p e c t r a , a n d J o s e p h J. Mukkada for t e c h n i c a l assistance. References (1) (a) E . S. Cook, Breu, Chem. N e u s L e t t . , 13, 4 (1953); Breu. Guardian, 82, 17 (1953); ( h ) G. S. Sperti, Ed., "Probiotics,"
Avi, Westport, Conn., 1971; (c) E . S. Cook and K. Tanaka, 164th Kational Meeting of the American Chemical Society, New York, N. Y., Aug 1972, Abstract No. HIST 9. ( 2 ) Abstracts, 164th National Meeting of the American Chemical Society, New York, N. Y., Aug 1972, Abstracts No. MEDI 25. (3) ( a ) Y. Tsuchiya, K. Tanaka, E . S. Cook, and L. G. Nutini,
Appl. Microbiol., 19, 813 (1970). (b) A . Fujii, K. Tanaka, Y. Tsuchiya, a n d E . S. Cook, J . Med. Chem., 14,354 (1971). A . Fujii, K. Tanaka, and E. S. Cook, ibid., 15,378 (1972). A. Fujii and E. S. Cook, 3rd Central Regional Meeting of the American Chemical Society, Cincinnati, Ohio, May 1971, Abstracts N o . 186. H. Takahashi, M . Shimizu, and K . Koshi, Japanese Patent 20564 (1963); Chem. A bstr., 60, 2786h (1964). E . Schutte, Hoppe-Seyler's 2. Physiol. Chem., 279, 52 (1943). R. Pant, ibid., 335,272 (1964). Horlicks Ltd., French Patent M 5924 (1968); Chern. Abstr., 71,80752a (1969). U. Okamoto, Rinsho Kensa, 8,966 (1964).
Synthetic Trypanocides. 3. S t r u c t u r e - A c t i v i t y Relationships? Nora Poliakoff, Sem M. Albonico,* Manuel Alvarez, Jorge Gallo Pecca, and Marcel0 J . Vernengo
Facultad de Farmaciay Bioqkimica, JuAin 956, Buenos Aires, Argentina, and Instituto National de Farmacologiay Bromatologia, Caseros 2161, Buenos Aires, Argentina. Received November 6, 1972 W e h a v e previously reported the t r y p a n o c i d a l activity of several s u b s t i t u t e d 1,2,3,4-tetrahydrocarbazoles~(THC) and dihydro[c]benzocarbazoles.2 C o n t i n u i n g w i t h t h e s e a r c h for more active c o m p o u n d s and the e s t a b l i s h m e n t of a relation between c h e m i c a l s t r u c t u r e and activity, we h a v e p r e p a r e d several n e w c o m p o u n d s active a g a i n s t Trypanosoma cruti, the p a r a s i t e responsible for Chagas's disease. In the s t u d y o n structure-activity relationships, the biological effect produced b y c h e m i c a l modification at t h r e e regions of the c o m p o u n d s c o n t a i n i n g an N - s u b s t i t u t e d indole nucleus w a s considered, Le., the benzene ring, the alicyclic ring, and the heterocyclic ring. Previously,z we h a v e f o u n d that in c o m p o u n d s containing an N - s u b s t i t u t ed indole nucleus, a m e t h o x y or chlorine s u b s t i t u e n t i n the benzenoid portion of the a r o m a t i c ring provides c o m p o u n d s w i t h higher activity. F r o m the several s u b s t i t u e n t s checked o n the a r o m a t i c nitrogen, the 2-piperidinoethyl has proven t o e n h a n c e t h e activity b o t h on the e p i m a s t i gote and trypomastigote s t a g e of T. cruti. M a i n t a i n i n g the m o s t active s t r u c t u r e , w h e n the alicyclic ring w a s d e h y d r o g e n a t e d , t h e fully a r o m a t i c c o m p o u n d s 6-8, a l t h o u g h w i t h good activity o n the e p i m a s t i gote s t a g e , lose completely t h e i r activity on the h e m a t i c form. The a m o u n t required of the m o r e active c o m p o u n d 4 t o prevent the transmission of the disease b y an average blood transfusion' is slightly below that of Crystal V i ~ l e t , ~ w i t h the a d v a n t a g e of b e i n g '20-fold less toxic and colorless. C o m p o u n d s 9 and 10 and t h e 8 - s u b s t i t u t e d 5,6-dihydrobenzo[a]carbazoles (DHBC) a r e w a t e r insoluble and cannot be checked in vitro. T h e i r in c k o activity is low e v e n at a high dose. Biological Results. T h e activities r e p o r t e d i n T a b l e I were measured o n the e p i m a s t i g o t e s t a g e of the T u l a h u e n s t r a i n of T. cruzi i n a liquid c u l t u r e m e d i u m . 5 When t h e s a m e test w a s performed in blood, on t h e t r y p o m a s t i g o t e s t a g e , the activity of 4 is 200 p g / m l and therefore only 100 m g will be required for an average transfusion. In the s a m e conditions t h e activity for Crystal Violet is 250 pg/ ml. The toxicity of 4 w a s t e s t e d o n Rockland mice weighing 18-20 g and t h e LD50 w a s 200-220 m g / k g ( i p ) , whereas s4
t This investigation was supported by grants from the National Research Council (Argentina).
141.2 Journal ofMedicinal Chemist?, 1973, Vol 26, No 12
Table I
.A
€3
I)
(
Min' useful concn, wug m l
7c Compd
RI
1 2 3 4 5
R?
RJ
M p , "C
yield
Formular
S u b s t i t u t e d 1,2,3,4-Tetrahydrocarbazole Fumarates N(Et). 280-282 dec 50 N C Hi, 256-259 dec 50 N(Me)? 196-198 42 OCHI NC 31, 194-196 65 OCHj N C Hi, 232-233 60 H
400 250
H H
123
70 500
S u b s t i t u t e d Carbazole F u m a r a t e s (B) N(CH,), 180-182 73 N (CzH,), 170-172 62 NC,Hio 194-195 77
6 7 8
100 60 40
S u b s t i t u t e d 1,2,3,4-Tetrahydrocarbazoles(C)
9" 10
H CF,
85 87
68 90
S u b s t i t u t e d 5,6-Dihydrobenzo [a]carbazoles (D)
llt 12 13 14 15d 16 17 28
19
158-160 138-140 156-158 157-159 187-189 dec 166-168 186-188 284-285 278-280
95 95 90 80 76 87 84
86 82
'IConcentrations were considered t o b e useful w h e n all epimastigotes were killed a f t e r n o longer t h a n 16 hr in t h e liciuid m e d i a containing the t e s t e d compound. "Lit.' 83-84'. S u b l i m e d (130°,30 p ) . "it. 163-164":C. V. R o g e r s a n d B. B. Corson, J . Amer. Chem. Soc., 69, 2910 (1947). d I t decomposed rapidly and could n o t b e analyzed. < A l lc o m p o u n d s were analyzed for C , H , and N, a n d t h e analytical results o b t a i n e d f o r t h e s e elements were within 0.4% of t h e theoretical values. T h e UV, ir, a n d n m r s p e c t r a also were in a g r e e m e n t w i t h t h e proposed structures. M e l t i n g points were t a k e n in capillaries a n d a r e uncorrected. All c o m p o u n d s except 9 a n d 1 0 were recrystallized f r o m ethanol. t h e dose a d m i n i s t e r e d t o a p a t i e n t with an average blood transfusion will h e a r o u n d 1 m g / k g . T h e a c u t e toxic s y m p t o m s of 1 were those of c e n t r a l e x c i t a t i o n ; d e a t h occurred with respiratory paralysis. Il'ith 0.5 LD5o ( i p ) t a c h y p n e a and t r a n s i t o r y depression was observed. Infected blood a f t e r t r e a t m e n t with 200 F g / m l o f 1 w a s inoculated i n t o w h i t e m i c e and n o infection was d e t e c t e d . Controls d i e d (100%) 15-20 d a y s after inoculation. T h e in cicv s t u d y of 13 w a s m a d e o n m i c e (18-20 g ) infected w i t h t h e MI strain of ?'. cruzi (4 x 105). a dose t h a t kills 90% of t h e control mice. In m i c e receiving. 2 d a y s after infection. five daily injections ( i p ) of 100 m g / k g of 13, t h e survival increased t o 60%.
Experimental Section The ii-substituted THC derivatives were prepared by the procedure descrihed by Borsche'j for 6-nitro-THC. The 6-trifluoromethyl-THC was purified by sublimation (130". 30 M ) and the 8methoxy-THC was purified by distillation (190", 15 m m J . Trifluoromethylphenylhydrazine Hydrochlorides. N a K 0 2 (2.1 g. 3.1 x 10 - 2 mol) in water (10ml) was added dropwise with stirring to 2-aminohenzotrifluoride (4.27 g. 3.0 X mol) in concentrated HCI (12ml) at -5". After 30 min of stirring, stannous chloride ( 2 0 g. 8.9 x lo-* mol) in concentrated HC1 ( 2 2 mi) was added dropwise. The reaction mixture was kept 16 hr at 0". \vater i100 ml) was added. and the solution was made alkaline ( p H 8) (concentrated "3). The suspension was extracted (Et20. 200 ml); the combined extracts were dried and saturated with dry gaseous HCl, affording 5.8 g (90%) of crystals of mp 216-217"Ilit.: '18").
The 4-trifluorome~hylphenylhydrazinehydrochloride was oh-
tained in 80% yield b y the same procedure: nil, 2103 20.ib ! l i t : 206-?0O;"), I-Methoxycarbazole. A w l u t ion of 8-niethoxS 2.5 mmol) in xylene ( 5 mli and 0.2 g of :3OR Pd on charcoal i v a i refluxed for 12 hr. The filtered bolution was evaporated and the residue recrystallized (aqueous Et OHI: yield 51:1 mg !UO% I : mp 66-68" (lit.869-O;0CJ. Preparation of %Substituted 5,6-Dihydrobenzoja]carbazoles. .4solution of ct-tetralone (0.02 mol) in acetic acid ( I ? mli \ + a i refluxed and the 4-substituted ~ihenylhgdrazone I 0.02 mol ) was added during 1 hr. Heating was maintained for 1 hr longer. Alter cooling the precipitated DHBC \vas filtered anti Lvorkcd i i i 1 :is ubual. The 6-nitro-DHBC and r j - t r i f l u o r o m e t h y l - ~ H ~werc ( ~ prv1itired hh- this modification. The corresponding 4-substituted phenylh> drazine 10.0,') mol) u a s added to a boiling solution rjt ~t-tetraloiic 10.02 mol) in 107~sulfuric acid in acetic acid 170 nilr The > i i l i i t i o n was refluxed tor 90 min anti cooled and the 1)recipiiatt\ worked up as usual. N-Alkylations. The alkylation 01 cumpounds I >I 'i\t i s i n r n e t i out as previously described.'
References .I. Gallo f'ecca and S. XI. Alhonico. ./ . L f d
('hem
,
13, ,{27
1970). .I. Gallo Pecca and S.11. Alhonico. [ b i d . , 1-1, 448 I1971 1 . (
.J. Kloetzei. K p i . Inst. M e d . Trop Sao Pauio, 3, 254 1961 i . G . C . Vilaseca. .I. A . Cerisola. .I. A . Olarte. and A . Z o t h r k t r . Iyo,sSan#., 11, 711 (19661. E. P. Camargci. KFL..I m t . ,l.led. Trop. Sao I'nuio. 6 , 0:i 191i.l I \Y, Borsche.Justu,s Liehigs Ann. Chpm., 359, 19 (l9OX). E. .J. Forhes. LI. Stacey. .J. C . Tatlow. and R. T. \Yragg. Y'P! mhcdron. h , 67 119601.
Notes (8) C. S. Barnes, K . H. Pausacker, and C. I. Schubert, J. Chem. Soc., 1381 (1949).
Synthesis of Cephapirint a n d Related Cephalosporins from 7 - (a-Bromoacetamido)cephalosporanicAcid Leonard B. Crast, Jr.,* Robert G. Graham, and Lee C. Cheney
Reaearch Diuision, Bristol Laboratories, Diucsion of Bristol-Myers Company, S y a c u s e , N e u York 13201. Receiued July 5, 1973
Since the structural elucidation of cephalosporin C the discovery of ingenious and remarkably efficient chemical methods3-6 for its N-deacylation has rendered the novel fused dihydrothiazine-P-lactam nucleus, 7 aminocephalosporanic acid (7-ACA, 2), readily available for the synthesis of semisynthetic cephalosporins which now complement the penicillins in chemotherapeutic interest . 7 The purpose of this report is to describe a convenient and versatile synthesis of cephapirin ( 4 ) 8 and related a heterosubstituted acetamidocephalosporanic acids by nucleophilic displacement of the bromine atom in 7 - ( a bromoacetamido)cephalosporanic acid (3)9 with mercapto- and aminopyridines8.10 and various 1,3-disubstituted and trisubstituted thioureas.11 Treatment of an aqueous-acetone suspension of 7-ACA (2) with bromoacetyl bromide in the presence of sodium bicarbonate afforded crystalline 7 4 a-bromoacetamido)cephalosporanic acid (3) in 77% yield. The interaction of 3, 4-mercaptopyridine, and triethylamine in methylene chloride at room temperature produced 7-[a-(4-pyridylthio)acetamido]cephalosporanic acid (cephapirin, 4 ) in 82% yield (Scheme I ) . A stirred suspension of 4 and N,N-diisopropylethylamine in aqueous acetone reacted with methyl iodide at ambient temperature to provide a fair yield of i - [ a - ( l methyl-4-pyridylthio)acetamido]cephalosporanic acid betaine (5).:-12 3-Mercaptopyridinel3 and commercially available 2mercaptopyridine were converted by procedures analogous to the one used for the preparation of 4 into the corresponding cephapirin isomers 6 and 7 , respectively (Table I ). Chemical properties and spectral data observed for cephapirin ( A ) , 6, and 7 were in accord with the assigned structures. However, the remote possibility remained that the tertiary nitrogen atom instead of the thiol function in 4-mercaptoppridine had preferentially undergone reaction with 2 to form 7-[a-(4-thiopyridon-l-yl)acetamido]cephalosporanic acid rather than the thioether 4. This question was definitely settled by the independent synthesis of cephapirin by Silvestri and Johnson.14 Compound 4 and the product of unequivocal structure which they obtained by coupling 7-ACA (2) with ~-(4-pyridylthio)acetylchloride hydrochloride were identical. In contrast to the behavior of 4-mercaptopyridine, 4aminopyridine reacted with 3 to form the betaine 8 as the only product isolated. Since thiols are generally stronger nucleophiles than the corresponding amines, this result was not unexpected. Compound 8 was reported by other investigatorsl5 after the completion of our work. Their method of synthesis was analogous to our own. In order to obtain the desired amino analog 9 of cephapirin (4), N-(4-pyridyl)glycine16 was converted into (1).1.2
tCephapirin is the nonproprietary name for 7-[a-(4-pvridylthioiacetamido]cephalosporanic acid (4). code number BL-P1322; CEFADYL is the trade name of Bristol Laboratories for this compound, IThe code number for 5 is BL-S217.
Journal ofMedicinal Chemistry, 1973, Vol. 16, No. 12 1413
its acid chloride hydrochloride and coupled in an anhydrous medium with silylated 7-ACA to obtain a 59% yield of 9. 7 - [ ( ~1,3-Diethylformamidino-2-thio)acetamido]cephalo-( sporanic acid (10) is a representative of a series of this class of cephalosporins prepared by displacement of the bromine atom in 3 by various acyclic and cyclic 1,3-disubstituted and trisubstituted thioureas in methylene chloride in the presence of a tertiary amine. Other investigators have reported17 that $ - [ ( a formamidinothio)acetamido]cephalosporanicacid was not obtained from the reaction of 7-(a-chloroacetamido)cephalosporanic acid with thiourea in aqueous solution. The products isolated were 7-ACA and pseudothiohydantoin formed by intramolecular displacement of the N chloroacetyl group by thiourea. Structure-Activity Relationships. Microbiological reports relating t o cephapirin ( 4 ) , t J 8 its 1-methyl betaine 5,1919 and the class of substituted formamidinothioacetamidocephalosporanic acids20 exemplified by 10 have disclosed the potent antimicrobial properties of these semisynthetic cephalosporins, some of which compare favorably with commercially important cephalothin (11). 2 1 , 2 2 Some comparative minimal inhibitory concentrations (MIC's) data, expressed in pglml, are given in Table I. The MIC's were determined by the standard twofold broth dilution method after overnight incubation at 37". Although all of the compounds shown have broad-spectrum activity, 10 and 5 stand out in overall activity. Crystalline 10 is unstable in aqueous solutions and, to a lesser degree, in the dry state. In general, these compounds derived from the substituted thioureas had good resistance toward P-lactamases produced by cephalothin-sensitive strains of Klebsiella p n e u m o n i a e and Escherichia coli but were found to be somewhat more toxic to animals than the other cephalosporins reported in this paper.20 Experimental Section Melting points were determined on a Mel-Temp apparatus and are uncorrected. The elemental analyses are within 10.470 of theoretical values unless stated. The ir spectra were recorded on a Beckman IR-9 spectrometer. The nmr spectra were run on a Varian A-60 spectrometer at a sweep width of 500 Hz with SilanorD20-TSP as the internal standard. 7-(e-Bromoacetamido)cephalosporanic Acid ( 4 ) . To a viporously stirred suspension of 200 g (0.731 mol) of 7-aminocephalosporanic acid (7-ACA, 2) in 2 1. of water and 750 ml of acetone was added 200 g (2.38 mol) of KaHC03. in portions. to prevent excessive foaming. The resulting solution was cooled to 3" and 200 g (0.99 mol) of bromoacetyl bromide was added in portions over a 10-min period. After 1 hr of stirring with the cooling bath removed, the pH was adjusted to 4 with 40% H3P04. Decolorizing carbon (40 g, Darko-KB) was added and after 15 min the slurry was filtered through a Celite pad, with suction. and the pad washed with 600 ml of water. The combined filtrates were then brought to pH 2 with 40% and after stirring for 1 hr in an ice bath the crystalline product was filtered. washed well with cold water, and air-dried. The yield was 200 g ( 7 i Y o i . An analytical sample was obtained by recrystallization from acetone-water. m p 173". Anal. (Cl2HI3BrN2O6S)C. H. N,Br. 7-[a-(~-Pyridylthio)acetamido]cephalosporanic Acid (4).X TO a stirred suspension of 39.3 g (0.1 mol) of 3 in 500 mi of CH2C12 was added 14 ml (0.1 mol) of triethylamine. To the resulting solution was added 11.1 g (0.1 mol) of 4-mercaptopyridine. A nearly clear solution resulted with precipitation of the product beginning after a few minutes. After stirring 4 hr the crystalline product was filtered off, washed well with CHzC12. and air-dried. The white product weighed 35 g (82%). Recrystallization from boiling acetone-water (1:l) gave an analytical sample, m p 155". Nmr indicated 0.5 mol of acetone of solvation: nmr (D20 + KHC03) 6 7.77-[a-( 1-Methyl-4-pyridylthio)acetamido]cephalosporanic
Acid Betaine (5).12To a stirred suspension of 42.3 g (0.1 mol) of 4 in 50 ml of water and 600 ml of acetone at 22" was added 12.9 g
