354 Journal of Medicinal Chemistry, 1971, r o l . 14, S o . /t
FL-JII, T.~saic.\,TK:CAIY.I, . t w COOK
to give a partial solii of finely divided Xa3PSO3. DhIF (10 nil) was added, and the mixt was allowed to warm to 25". Powdered 12a (7.68 g, 20.0 mmoles) was then added in portions. Complete sohi occurred after 10-15 min. Stirring wab contd, and after 30-40 min cryst 13b began separating. EtOIl (500 ml) was added ; and, after overnight refrign, the solid was collected, washed with EtOH, and recrystd twice from H20(30 m1)-EtOH (300 ml). The collected product, washed successively with EtOH and Et&), was air-dried and then allowed t o equilibrate at corist 5 0 5 , relative humidit'y . 8-2- [3 (cis 1,2 Cyclohexanedicarboximido)propylamino] ethyl sodium hydrogen phosphorothioate (13d) was prepd from 12b (15.0-mmole scale) as described above for the conversioii
- - -
-
of 12a into 13b. The product that pptd from the EtOH-dild reaction mixt wab recrystd from refiigd HZO (50 m1)-EtOH (50 ml) $oln, air-dried, and equilibrated a t SOc; relative humidity. 5-2- [4-( czs-l,2-Cyclohexanedicarboximido)butylamino] ethyl Sodium Hydrogen Phosphorothioate (13f).--Adapiatioii of the procedure descrihed for the prepn of 13d ieadily affoided 13f
Acknowledgments.-The author- 'ire indebted t o Dr. D. 1'. ,Tacobus arid Dr. T. R Yv,eent.\ for nntiradiation data arid to Dr. IT. C. Coburri, J r ~ i menid bers of thi. 1Zolecular Spectroscopy Section of Southern Research Institute for spectral data. ~
Antistaphylococcal and Antifibrinolytic A4ctivitiesof a-Amino Acids and Their L-Histidine Dipeptides's' AKIRAFUJII,ICINJI
TAXAKA, 'ITOSHIKI
TSUCHITA, .4SD ELTON s. C O O K *
Divisim of Chemistry and Biochemistry, St. Thomas Institute, Cmcznnati, Ohio 452206 Received Xay 7 , 1970 The relationship was investigated between the molecular structure and the antistaphylococcal and antifibrinolytic actions of w-amino acids and their bhistidine dipeptides, of which 6-aminovaleryl-L-histidine (9) and e-aminocaproyl-L-histidine (10) are newly synthesized. The antistaphylococcal properties were demoristrated through their protective effects against staphylococcal infections in mice. The antifibrinolytic activities were determined in vitro by measuring prolongation of lysis time of a fibrin clot. The order of antistaphylococcal potencies of these compounds was: (a) glycine (1) < B-alanine (2) < v-aminobutyric acid (3) < e-aminocaproic acid (5j < 6-aminovaleric acid (4) and (b) glycyl-L-histidine (6) < p-alanyl-L-histidine (7) < r-aminobutyryl-L-histidine ( 8 ) < 6-aminovaleryl-L-hi&idine (9j < e-aminocaproyl-L-histidine (10). Comparing a and b, the protective power of w-aminoacyl-L-histidines was much higher than that of the corresponding w-amino acids. The order of antifibrinolytic potencies of a-amino acids was identical with that of antistaphylococcal action except that 4 < 5 in the former. Practically no antifibrinolytic activity of w-aminoacyl-bhistidiiies was observed i n vitro under the conditions we employed.
It was previously reported that, by a prophylactic procedure, homocarnosine (8) and carnosine (7) protected C3H/HeJ m i ~ e but ~ ~only , ~ 8~ protected Swisalbino nice^^'^ from death b j Staphylococcus aureus infections. Iri this work, a series of component w-amino acid:, WL:, also examined arid compared with the peptides by a combined prophylactic-therapeutic procedure with S w i s albino mice.4 The compounds discussed in this paper are. w-amino acid., H,K(CH,),COOH, where n = 1, glycine ( 1 ) ; n = 2 , (?-alanine (2); n = 3, y-aminobutyric acid (3); rb = 4, 6-aminovaleric acid (4) ; n = 5 , t-aminocaproic ncid ( 5 ) ; and w-aminoacyl-~-histidines,H2S(CH2),COHis, where n = 1,glycyl-L-histidine (6); 1% = 2 , p-alanylL-histidine (7) (carnosine) ; n = 3, y-amiriobutyryl-Lhistidine (8) (homocarnosine) ; 71 = 4, 6-aminovaleryl-Lhistidine (9) ; 12 = 5 , E-aminocaproyl-L-histidine (10). The results indicate that 4 arid 5 , higher homologs of 3, were more effective against staphylococcal infections than 3. Comparing 8 with 3, and also 7 with 2, both histidine dipeptides were more potent than their com(1) hided by grants from Stanley Drug Products, I n c . , Division of Bperti Drug Corp., a n d t h e Lt. David Van Alstyne I11 Foundation. (2) Presented in p a r t a t t h e 1st Central Regional Meeting of t h e American Chemical Society, Akron. Ohio, M a y 1968 (Abstracts, N o . 2 1 ) , and t h e 158th National hleeting of t h e American Chemical Society, New York, K . Y., Sept 1969, Abstract No. MEDI 50. ( 3 ) (a) K. T a n a k a , Y. Tsuchiya, N . J. Berberich, Jr., L. G . Nutini, a n d E. S. Cook, Abstracts of t h e 37th General hleeting of t h e Japanese Biochemical Society, Kagoya, J a p a n , Oct 1964: Seikayaku, 36, 660 (1964): (11) R . T a n a k s , Y. Tsuchiya. 5 . J. Berberich, Jr., A . J. M u k k a d a , L. G . Kutini, and E. S. Cook, A p p l . Microbbol., 16, 1457 (1968): (e) A. J. M u k kada, L. G. Nutini, a n d E. S . Cook, ibid., 18, 641 (1969). (4) T.Tsuchiya, K . T a n a k a , E . S . Cook, a n d L. G. Nutini, i b i d . , 19, 813 (19iO).
porierit w-amino acids. These t v o fact3 suggested to us that both 9 arid 10 might have cveri higher activities than 8, as verified by the data in l'igure 1 The mechanism of the anti~taphylococcalaction ulao has been considered because none of the w-amino acids and w-amirioac!.l-L-histidiries tested showed bactericidal or bacteriostatic effects zn u i f l o . Ji-e have been especially interested in the possible relationship between antistaphylococcal arid antifibrinolytic activities, sirice 4 and 5 were reported to have tiritifibririolytic activity.' Chemistry.-Ti\ o general .;\ rithetic procedures M c w used to preparr the w-aniiiioacyl-L-histidilles in this study. Thc first 11 us the phthalyl method, a modification of the one described by Sheehan and I'raiih6 and similar to that reported b? T u r ~ i e r ,except ~ for the filial purification proce-s, in 1% hicli me uied ion-exchange chromatograph;\ arid the phenol-calcium h j pochlorite color reaction for the isolation arid detectlori of w-amino acids arid their histidine dipeptides. The carbobenzoxy method, ubed the iecond procedure, TKLS modified from Bergmarin arid Zervas,8 Sifferd arid duVigneaud,Y and Pisano, et a / . l n The yields, melting points, specific ( 5 ) (a) R I . Mangyo, Y e i k a g a k u , 36, 735 (1964); (b) S.Okamoto a n d U . Okamoto, Keio J , Med., 8 , 211 (1959): (e) S. Sherry, A. P . Fletcher, N . Alkjaersig, a n d TV. D . Sawyer, Trans. Ass. Amer. Physicians, 7 2 , 62 (1959): (d) .'I.Alkjaersip, A . P. Fletcher, a n d S. Sherry. J . B i d . Chem., 134, 832 (1959): (e) A. Sjoerdsma and I. hI. Milsson, Proc. SOC.E x p . B i d . M e d . , 103, 533 (1960). (6) J. C . Sheehan a n d \'. F r a n k , J . .4mer. Chem. Soc.. 71, 1856 (194'J). (7) R . -4. T u r n e r , ibid., 7 6 , 2388 (1953). (8) hI. Bergmann a n d L. Zervas, Chem. Rer., 65, 1192 (1932). (9) R . H. Sifferd a n d V. duvigneaud, J . B i d . Chem., 108, 753 (1935). (10) J. J. Pisano, J. D. Wilson, L. Cohen, D. Abraham, and 5. Udonfriend, i b i d . , 136, 499 (1961).
Journal of Xedicinal Chemistry, 1971, Vol. 14, hTo.4
ACTIVITIESOF w-AMINOACIDS
355
TABLE I W-AMINOACYL-L-HISTIDINES AND RELATED COMPOUNDS Yield,
Method
Compd
%"
degc ( e 2, Hz0)
[.]SD,
Mp,
O C b
Formulad
30 173-174e3f +17.6f CaH12Na03 6 +21. 4h CsHiaNaOa 25 251-252 decg 7 +23.2 CioHlsNaOa 16 242-243 dec 8 Z 84 242-244 deet f4.01 CioHlsNaOa ' &So4 9k Pht 21 238-239 dec +23.2 CiiHi8NaOs * HzO' Z 72 239-240 dec +23.5 CiiHmN403.H20 +25.0 CizHnoNaOs 10m Pht 40 236-237 dec Z 76 238-239 dec +25.4 CizH20Na03 HisOhfe .2HC1 96 198- 199 +3 5 * Melting points were determined by the capillary tube method and were uncorrected. Specific Based on histidine in 6-10. optical rotations were taken with a Laurent polarimeter. Analytical results for C, H, N were within *0.4y0 of the theoretical values, f AI. Hunt except where indicated. e G. Losse and G. Muller [Chem. Be?., 94, 2768 (196l)l give mp 170-175". Lit.7 mp 175-176'. and V. duVigneaud [ J . Biol. Chem., 127, 43 (1939)l give mp 175" and [ c Y ] ~ ~+25' D (c 1, HpO) for C8H12N403.HC1.Hz0. Lit.7 mp D (c 1.1, Hp0); lit.g [ c Y ] ~ ~+20.5" D (c 2, HtO); lit.e [ a l Z 0 D 253-256' dec; lit.g mp 260" dec; lit.e mp 260-262" dec. * Lit.' [ c Y ] ~ ~+21.7" I r peaks (cm-1) were 672, 726, 742, +21.9" (c 1, Hp0). * Lit.l0 mp 240' dec; 1it.f mp 235' dec. 1Lit.f [ c Y ] ~ ~+So D (c 1, H20). 752, 800. C : calcd, 48.52; found, 49.05. N : calcd, 20.58; found, 20.10. I r peaks (em-') were 666, 693, 747, 796, 815. * E. Fischer and L. H. Cone [Justus Liebigs Ann. Chem.. 363, 107 (1908)l give mp 197-198; N. C. Davis [ J . Biol. Chem., 223, 935 (1956)l gives mp 200-201; lit.e mp 199-200'. Pht Pht Pht
Q
TABLE I1 Rf VALUESOF a-AMINOACYL-L-HISTIDINES AND RELATED COMPOUNDS ( R f X 100) 7-
Compd
Solv 1
Solr 2
Tic'-----------. --Rb-Solv 3 Solr 4 Solv 5 Solv 6 S o l r 7
24 34 44 36 46 26 75 40 34 52 20 62 67 45 48 36 56 16 60 79 56 42 54 60 14 83 65 62 J 50 64 63 16 87 71 70 24 10 16 50 85 6 63 15 29 09 14 15 78 7 76 19 11 8 36 15 08 62 83 22 38 12 9 18 06 87 25 61 10 46 14 18 07 90 30 58 L-Histidine 42 18 16 49 66 22 84 33 40 46 L-Alanine 39 59 41 80 Silica gel G (E. RIerck) 250-p plates, purchased from Analtech, Inc., were used for tlc. Solvent 1, PhOH-H20 (75325, w/v), pH 2.0; 2, n-BuOH-AcOH-HpO (60:20:20), pH 2.4; 3, i-PrOH-formic acid-HzO (77:4: 19), pH 2.7; 4, sec-BuOHMeCOEt-dicyclohexylamine-H20 (55: 15: 10:20), pH 10.3; 5, CHC13-hIeOH-17% "aOH (40:40: 20), pH 11.6. b Whatman No. 1 papers were used for pc. Solvent 6, PhOH-HpO (80:20, w/v), pH 1.6; 7, n-BuOH-AcOH-HpO (60: 15:25), pH 2.7. 1
* O .c E
2 3 4
70
-,*
0
1
60
0
zrK
m
50 iZ I I
0
40 w
r+
30 v,
> 20
-1
IO U
optical rotations, and analytical data of the compounds are given in Table I. Table I1 gives the Rf values on tlc and paper chromatography (pc). Testing Procedures.-In vivo antistaphylococcal activity was examined using female Swiss albino mice, 8-10 weeks old and weighing 20-25 g each. The challenge organism was a penicillin-resistant strain of Staphylococcus aureus, originally isolated from an infected tonsil, which has been maintained in our laboratories for years. It was preserved under refrigeration in the lyophilized state, and stock cultures were grown on Bacto Staphylococcus Medium 110 (Difco) slants once in every 6 months. The inoculum was prepared from 24-hr cultures on slants a t 37". The cells were centrifuged and washed twice with physiological saline (TC Tyrode Solution, Difco), then suspended in the same soln. Usually 0.5 ml of the suspension adjusted to give a 70y0 transmission on a nephelometer (2.6 X lo8 organisms) caused SO-SO% mortality in 4 days after subcutaneous challenge in the groin of mice. A total of 5 mg of each sample was given in equally divided
a LENGTH BETWEEN CkCOOH) AND NCNHZ), A
Figure 1.-Antistaphylococcal and antifibrinolytic activities of w-amino acids and their bhistidine dipeptides. Numbers refer to compounds. The antistaphylococcal activity of each compound was established with 10 mice in each of 3 or more experiments: w-amino acids (e-.), w-aminoacyl-L-histidines (0-0). The antifibrinolytic activity was determined in 0.05 M phosphate M samples: w-amino acids buffer-saline solution with (H-M), w-aminoacyl-L-histidines (0-0); and with M samples : u-amino acids (A-A), w-aminoacyl->histidines (A-A).
doses sc 2 hr before and 4 hr after the injection of S . aweus. Antifibrinolytic activity in vitro was determined by adopting the profibrinolysin-streptokinase system with expectation that these compounds would most likely affect the activation of profibrinolysin." I n a series of test tubes [8 (id.) X 74 mm] at 0" were mixed 0.1 nil of standard human serum (Microbiological Associates, (11) S. Okamoto a n d U. Okamoto, Keio J . M e d . , 11, 105 (1962)
FT-JII, TAN.\K.~, TSUCHIYA, AND Coo~i
356 Journal of Nedicinal Chemistry, 1971, Vol. 14, KO.4
Inc.), 0.4 nil of saniple dissolved in 0.05 M phosphate buffer saline, and 0.1 ml of saline containing 100 units of streptokiiiase (Yaridase, 1,ederle 1,aboratories). Then 0.03 in1 of saline containing 5 units of bovine thrombin (hlochida Pharmaceutical Co., Ltd.) and 0.3 in1 of a 0.33% s o h of bovine fibrinogen (Fraction I, Culbiochem) were added to the test tubes. The mixtures were incubated a t 2.5' for 3 min. Then the test tubes were inclined :It a n angle of -20" to the 1iorizont:il and rapidly vibrated (70 vibrations/min, amplitude 15 mm)l* to minimize the influence of the test tubes arid other factors. The lysis time was measured by the number of minutes required for the clot to flow out from the test tubes. Biological Results and Discussion.-The antistaph!lococcal and antifibrinolytic activities of w-amino acids and their histidine dipeptides are hummarized in Figure 1. In a series of straight-chain w-amino acids, 4 aiid 5 were demonstrated to provide better protection for mice against X. aureus than the smaller homologs, 3, 2, and 1. .211 of the w-arninoacyl-L-histidine.. were found to be more effective than their respective component w-umirio acids. Two newly ..nithesized histidine dipeptideb, 9 aiid 10, were shown to have greater untistaphylococcal activity than homocarnosirie which previously w:i< reported to he the most potelit ailtistaph! lococcul compound of this type. 31, T h e gre:ite-t antifibrinolytic activity for the w-amino fouiid when the length of the CH2 chniii hclneeii tbc C02H mid the ISH? group was approxiinatcly 7 This is almost the .:imc pattern obtained i n t h c :iiitistal)liylococcal activity. 1:urther verificntioii of thib relationship w a i obtained rvith the very ~)otc~ritniitihbrinol c agent, ti.ans--l-amiiioiiieth! 1c j clolicxaiie-l-c:irbo ic acid. which a t 5 m g produced 725; protectioii ng st yt:iphylococcal infections (a\ effective :ih 8). I t \ \ a h thc most effective :mtifiljriiiolytic agent studied, 2% nui! he Yeeii by comparing ly4. time- of 48 xiid > 180 miii for 10-o and mol(>.;. reqpectivelj , n ith the value, in Vigure 1. From :Lcoild e r a t i o n of the iiiterre1:ttionilii~) betweeii the :intistnphylococcnl and antifibrinolytic wtivitie- of w-amino wid., it i, poinible that thwe w-:miiiio :icidi might inhibit the action of fibriiiol!+.iii u-hich i. :ictivated by Thu-, t h r hcteriohiiiase, in this c:ise -t:iphyloBinaic piwtcctioii of mice from ,t:iph!.lococc:il infection I)! thew w-:tmiiio acidh m:q. be pnrtiallj. due to the :intifibriiiolytic action nhich ma! locnlizc the bacterin1 infection, :ilthough direct observation uiidcr the micro~ c o ~lis* ) c riot been attempted as >.et. I l o w v e r , all of t lie w-aniiiioacyl-L-histidiiieiwere showii to hnve greater aiiti.;ta~)hylococcalactivity than thcir respective compon w-amino acids :ind !ct 110 :mtifibriimlytic activity 1 obtained. The mechaniirn of the :ictioii of t h e dipeptide.; has not been established :iiicl niust :in-ait further pharmacological 5tudie.. l'(~-ibl> tlie w-nmiiio:ic? 1-L-liistidineb may itay longer iii the mice, being incorporated iiito the ti a1 ~ v a r n ito room lenip with vigoruu. ,-1irriiig : i i d colicti t( in ~ I U C I ( O . n-PrOH (40 nil) w a y ;rddrtl $11 in m c u o tvas repeated. The rehidue wviis nnd rect,y-id from aq l I e O l l : yir.lti, [a]% +l.5,0" (c 1, ). A / L U / . ~(~,~~I320X4Oi) IT, s : ( % : ralcd, 59.37; furuid, 5s 6-Aminovaleryl-L-his e (9).---Alrolii c i i 3.0 g of Pht-8aniiii(,valel.yl-r.-hiht idiiie i i i 1L' 1111 of I 1 2 0 \vas of .i 111 sohi of hgdrazitic hydrate i i i EtOI1. niixt of wy-t Phi-hi-drazirie d i i i f 8-nniiiio aiid its sohi >vas diluted x i t h 25 nil of IliO. 1.O nil of glaciul h c O l I c n u r e d pptu of Phi-hydrazirie, which \vas then filtered OH' ;iiid Tvashetl well Rith cold II?O, The filtrate \vas wricd aiitl pitriiied by ineaii. of ioii-exclinnge c1irotn:rtogrnphy. For ihe separnriuii O I I :ii ~ ( ~ 1 r i i i i(1.8 ti x 30 cm) of Amberlite CG-120 resiii, 0.1 .lI L'>&littiditlo hi1ffei.1'~was ri-ed. T h i r PhOH-CatOCl2~ culur reacIioii14,'j was erriployetl for the tl?tectioii of the prire fractioii> of 4, 9, t t i i d hist,idiiie. Coinpoulid. 4 and 9 were identified by the dear blue color and histidiiie b y :t brow1i color. The color reactioti was perforiiied in the fullowiiig niaiiiier: 0.5 nil of B sample froni each fractioli W I S mixed with 1 .O 1111 Of 1 ' ; PliOIT. The tvell-mixed soln, after addit'ioli of 0.1 g of C:a(OC'1)2 poa.der, was heated 5 miii iti :I boiling water h i I i :tiid theii cooled ill riiiiiiirig H2O. The peak q[iatit,iiy of 4 :tilpeared iii the efiiueiit at approx 2660 nil, histidine a t 600 1111, n ~ i c l 9 at, 1300 nil. The piire 9 fraction$, 1100-1700 nil, were pooled and coiicd in t v c u o . T h e dry residue was treated with 111 EtOH to produce oryhtals: yield, S9.0:;; nip 238-239" d [ a ] % $23.2" ( C 2, 1120). A n ~ l . ~ C I I H I ~ X & ~ . H Z11; O) calcd, 48.52; foiind, 19.03. N : c.alcd, 20.58; fo~uld,20.10.
(13) (a) 13. .\l,ral,am, ,i..r. I'isanu, a n d s. Iltlenfricnd, .trcii. R ~ o c h t ~ r ~ . B z u p i , ? , ~ , 99, , 210 (1962); (1,) 13, I . hIcYIanus, 1. H i d . Ciiern.. 235, 18!18
< 1'360), (12) T h e necessity of vibration \\as demonstrated b y t h e following experimental results. W i t h ribration fibrin clots in 83.37, of tlie control test tubes n.ere dissolved in i-9 min; xitilout vibration, 85.0C;, n e r e dissolved in 8-13 min.
~
'14) J. \V. Scliick a n d 1:. F. Ilegering, 2 n d . 1 3 n ~ Ciir,7ri.. . 39, '306 (1917). 1 1 5 ) ( a ) 6 . Iiitaoka. l i . Siiziiki, anti 11. Itoli, .\IJstracts of tlle 10111 General N e e t i n g of t h e Japanese TIiociielnical Society, Osaka, .Jakian, ;io\. 1967, Selkrigokzc, 3 9 , ,580 (196;): (11) 8 . Iiitaoka a n d N . S a k a n o , .llistract*
of t h e 4 l s t General Meeting of t h e Japanese 13ioctiemical Society, T(?k\.o, .lapan, Ocr 1968, S r i k n g a k w , 40, 551 (1'368).
Journal of Medicinal Chemistry, 1971, Vol. 14, No. 4 357
ANTIULCERAGENTS. I The acid hydrolysis (6 K HCl, 110", 24 hr) of 9 produced 4 and histidine. Phthalyl-e-aminocaproic Acid.-The mixture of 14.8 g of phthalic anhydride and 13.1 g of 5 was allowed to react by the same procedure described for the synthesis of Pht-&aminovaleric acid: yield, 96.9%; mp 105-106'. Anal. (ClaH16N04) C, H , N. Phthalyl-e-aminocaproyl chloride was prepd as described before: yield, 93.0%; mp 66-67". Phthalyl-e-aminocaproyl-L-histidinewas prepd as described before: yield, 46.0%; mp 225-226"; [a]"D +16.6" (c 1, HzO). Anal. ( C Z ~ H Z Z NH, ~ ON~; ) C: calcd, 60.29; found, 57.37. e-Aminocaproyl-L-histidine ( 10) was prepd and purified as described before: yield, 86.0%l,;mp 236-237' dec; [ a I z 5 D +25.0' ( c 2, HZO). Anal. (ClzHz0N40a) C, H, N. The acid hydrolysis as described gave 5 and histidine. B. Carbobenzoxy Method. Carbobenzoxy-6-aminovaleric Acid,-To a soln of 20.6 g of 4 in 100 ml of 2 S NaOH in a flask cooled in an ice bath, 34 g of benzyl chloroformate and 50 ml of 4 NaOH were added simultaneously to the vigorously stirred soln over a period of 20-25 min. The mixt was stirred for an additional 10 min. The s o h was cooled in an ice bath and acidified to congo red with concd HC1. The ppt was filtered, washed with a small portion of cold H20, and dried in the desiccator in vacuo; yield, 60.47,; mp 102-103". Anal. (C13H~N04) C, H, N. L-Histidine Methyl Ester.%HCI.-A sohi of 22.4 g of dry powdered L-histidine.2HCl and 320 ml of anhyd MeOH was heated on a water bath and dry HC1 gas was introduced. The heating was continued for 3 hr. The resulting soln was cooled and allowed to stand in the ice bath and treated with Et20 to furnish fine, colorless crystals which were recrystd from MeOH~ (c 2, HzO). EtzO: yield, 96.07,; mp 198-199'; [ a I z 5+3.5" 6-Aminovaleryl-L-histidine (9).-To a soln of 12.5 g of Z-6aminovaleric acid in 250 ml of CH2ClZwas added 7.0 ml of Et3N. After the resulting soln had been chilled to -5", 4.8 ml of ethylene chloroformate was added and the mixt was kept a t the same temp for 10 min. To this soln was added rapidly a soln of Lhistidine XIe ester prepared by the addition of 21 ml of EtsN to a s o h of 12.2 g of L-histidine Me ester'2HCl in 250 ml of
CHzClZ which had been chilled to 0'. The resulting mixt was stored a t 25" for 2 days. It was then washed with 200 ml of HzO and 200 ml of 1 N aq NaHCOa, dried (Na2S04),and concd to a syrup. I t was dissolved in 100 ml of MeOH and 50 ml of 1 h' aq NaOH was added. After storage for 3 hr a t room temp, the soln was adjusted to pH 5 with 2 N HzSOr and concd to dryness in vacuo. The syrupy residue was extd with two 50-ml portions of hot EtOH, and 50 ml of HzO was added to the ext. After addition of 1.0 g of 107, Pd/C, the mixt was hydrogenated. The formation of COZgas was checked occasionally until it ceased after 6 hr. The soln was filtered and concd in vacuo. The residual syrup was dissolved in 20 ml of Hz0 and 2 N HC1 added to give a pH below 5.0. The purification process by means of ionexchange chromatography was the same as described before: yield, 71.8% (based on histidine Me ester.2HCl; mp 239-240' dec; [aIz6o $23.5" (c 2, HzO). Anal. (Cl1Hl8N4Oa~HzO) C, H, N. e-Aminocaproyl-L-histidine ( lo).-Z-e-Aminocaproic acid ( 13 g ) was treated in the same way: yield, 76.0%; mp 238-239" dec; [ a I 2 5+25.4" ~ (c 2, Hs0). Anal. (ClzHzoNaOa)C, H, N. y-Aminobutyryl-L-histidine (S).-Z--pAminobutyric acid (12 g) was treated in the same way: yield, 83.6y0; mp 242-244' dec; [ a I z 5+4.0" ~ ( c 2, Hz0). Anal. (CloHlaNlOj.HzSO4)C , H, N.
Acknowledgments.-We thank Dr. Leo G. Nutini, Professor of Biology and Experimental Medicine at the St. Thomas Institute, who established the antistaphylococcal assay procedure, Dr. Floyd Green, of Matheson Coleman and Bell, for ir spectra, and Dr. Utako Okamoto, of the School of Medicine, Keio University, Tokyo, Japan, for valuable suggestions regarding the antifibrinolytic assay procedure. We are also indebted to the Ono Pharmaceutical Company, Ltd., Osaka, Japan, for a gift of y-aminobutyryl-Lhistidine monohydrate and the Daiichi Seiyaku Company, Ltd., Tokyo, Japan, for a gift of trans-4-aminomethylcyclohexane-1-carboxylicacid.
New Antiulcer Agents. 1. Syntheses and Biological Activities of 1-Acyl-%, -3-, and -4-substituted Benzamidopiperidines TSUTOMU IRIKURA* AND KAZUNORI KASUGA Kyorin Chemical Laboratory, Kyorin Pharmaceutical Co., Ltd., Tokyo, Japan Received August 17, 1970 A variety of 2-, 3-, and 4-substituted benzamidopiperidines was prepared from the corresponding benzamidopyridines by a Pd-catalyzed hydrogenation of the ring; syntheses of 1-acyl-2-, -3-, and -4-substituted benzamidopiperidines are reported. These compounds were tested for curative activity on the chronic gastric ulcer in rats using the clamping-cortisone method. Some of these compounds, particularly l-(p-aminobenzoyl)-4-(3,4,5-trimethoxybenzamido)piperidine, showed some antiulcer activity. Structure-activity relationships are discussed.
Pharmacologically active piperidines having one substituent at the 2, 3, or 4 position of the ring are mainly of the following 3 types.
0"' I
Rz 1, Ri 11, OR, 111, "R,
Compounds I and 11, where RI and Rz signify various alkyl-, arylalkyl-, or CO-containing groups, have antihistaminic and antispasmodic activities. Aminopi-
peridine derivatives 111, where R1 and Rz are various alkyl or arylalkyl groups, have been described as having antihistaminic and spasmolytic activities. Hoxever, diacyl compounds of type I11 do not appear to have been studied, although 1-benzoyl-3-benzamidopiperidine, l-acetyl-4-acetamidopiperidineJ and l-benzoyl-4benzamidopiperidine were obtained by Xienburg' and Tomita2 in the course of the confirmation of the structure of aminopiperidine derivatives. We synthesized a series of 1-substituted benzoyl-%, -3-, and -4-substituted benzamidopiperidines (IVa-IVc) and tested them for antiulcer activity by the clamping-cortisone method. (1) H. Nienburg, Chem. Be?., 10, 635 (1937). (2)
K.Tomita, Yakugaku Zasshi, 11, 1053 (1951).
