D-2-(
l,'&cYCLOHEXADIENYL)GLYCYL
Journal of Medicinal Chemistry, 1971, Vol. 14, No. d 117
PENICILLINS
A New Class of Semisynthetic Penicillins and Cephalosporins Derived from D-%( 1,4-Cyclohexadienyl)glycine JOSEPH E. DOLFINI,"HAROLD E. APPLEGATE, GEORGES BACH,HAROLD BASCH,JACK BERNSTEIN, JOSEPH SCHWARTZ, AND FRANK L. WEISENBORN Squibb Institute for Medical Research, New Brunswick, New Jersey 08903
Received June 16, 1970 Derivatives of 1,4-cyclohexadienyl-substituteda-amino acids have been made from 6-aminopenicillanic acid, 7-aminocephalosporanic acid, and 7-aminodesacetoxycephslosporanicacid. The compounds exhibit considerable activity against a variety of Gram-positive and Gram-negative organisms in vitro.
As part of our study of new semisynthetic penicillins and cephalosporins, we became interested in structures derived from 1,4-cyclohexadienyl-a-aminoacids. We were curious to see whether 1,4-cyclohexadienyl structures might exhibit activities different from those of I their aromatic analogs. It mas expected that the 1,4cyclohexadienyl ring would be planar, similar, in that respect, to the benzene ring, but yet, chemically much different. Therefore, differences between the aromatic and 1,4-cyclohexadienyl analogs might be expected in their affinities for active sites on enzymes, dependent on the electrophilic and lipophilic properties involved. I Contrasting enzyme site affinities may result not only CO,H rv in modified potency but also in differences in patterns II,X = OAc of metabolism, absorption, excretion, and tissue pene111,X'= H tration. During the studies, several publications appeared describing a similar interest in the comparative properties of systems containing aromatic us. 1,4-cyclohexadienyl structures. For instance, one1 describes the antimicrobial action of 3- (1,4-~yclohexadienyl)alanine against Leuconostoc dextranicum 8056, an effect that 7- [~-2-amino-2(1,4-cyclohexadienyl)acetamido]can be competitively reversed by phenylalanine. The cephalosporanic acid (11) was prepared in several steps authors also suggest that the inhibitory action of the from 7-aminocephalosporanic acid (VI), and its descyclohexadienyl compound is due to its structural acetoxy (111) was prepared from 7-aminodesacetoxysimilarity to the aromatic relative, and propose that cephalosporanic acid (VII). Variations of published the C atoms of the 1,4-cyclohexadiene ring are coplanar, procedures5 which employ the N-tert-butoxycarbonyl a suggestion substantiated by nmr studies2 on 1,4group to protect an amino acid during the coupling cyclohexadiene and X-ray crystallographic studies3 on process were used for preparing amphoteric cephal2-(1,4-cyclohexadienyl)glycine. We synthesized the osporins. ( cephalosporins and penicillins derived from ~ - 2 -1,4cyclohexadieny1)glycine (I, 11, 111)for comparison with their aromatic analogs. D-a-Phenylglycine is commercially available. It was 0 reduced by the well-known Birch reduction technique, C02H without extensive racemization, to provide its D-1,4cyclohexadienyl analog IV. VI, X = OAc The synthesis of 6- [~-2-amino-2-(1,4-cyclohexadiVII, X = H eny1)acetamidolpenicillanic acid (I) was achieved by While the physical properties of the cyclohexadienyl the method of Dane and D ~ c k n e ras , ~described in the compounds cited here are generally similar to those of Experimental Section. The method utilizes an acetthe aromatic analogs, a distinct trend toward greater oacetic ester enamine derivative of an amino acid to basicity should be noted. The pK2 values for I and protect it during the coupling process with 6-aminoI11 are 7.62 and 7.57, respectively; for both ampicillin penicillanic acid (V). and cephalexin the comparable value is 7.24. The results of a number of in vitro comparisons with * T o whom correspondence should be addressed. (1) M. L . Snow, C. Lauinger, a n d C. Ressler, J. Org. Chem., 33, 1774 other penicillins and cephalosporins are listed in Tables (1968). I and 11. These data were determined in a twofold,
H2Ns&
(2) E. TV. Garbisch, Jr. a n d M. J. Griffith, J. Amer. Chem. Soc., 90, 3590 (1968). (3) B. A . Shoulder, R . M . Gipson, R. J. Jandacek, S. M. Simonsen, a n d W.Shive, zhid., 90, 2992 (1968). (4) E. D a n e a n d T. Dockner, Angew. Chem., 1 6 , 342 (1964).
(5) J. I. Spencer, E. H . Flynn, R. W.Roeske. R. Y. Siu, a n d R. R . Chauvette, J. M e d . Chem., 9, 746 (1966); C. W. R y a n , R . L. Simon, a n d E. M. Van Heyningen, ihid., 12, 310 (1969).
118 Journal of Medicinal Chemistry, 1971, Vol. 14, S o . 2
I~OLFINI, et nl.
fication was conveniently performed by in vacuo conen of a n aq soln of the amino acid in 28%, NHa, resulting in optically pure, OF white, cryst material: [ C X ] ~ ~-D 117.0" 12 -1: YaOH). A n d . 6 - [ D - 2 - ~ ~ ~ l S O - 2 - ( ~ , ~ - C k . C L O H F ~ ~ . ~ D I ~ ~ ~PI~SICILLANIC ~L)~~CI~T.~~~lD~] (CjHiiKO?)C, H, N. ACID ( I ) WITH OTHERPP:SICII,LIS~ Methyl Acetoacetic Ester Enamine of ~-2-(1,4-CyclohexaSquibL dienyl)glycine, Na Salt (VI).-Compd I V (306 mg, 2.00 mmoles), culture Penicillin was dissolved by warming in a sohi of 108 mg of XaOMe (2.00 Organism S3. I G .\mnicillin Oxacillin mmoles) in 4.3 nil of reagent grade ;\IeOH. Methyl acetoacet,ate Staphylococcus (235 mg, 0.24 ml, 2.20 mmoles) was added, and the niixt was reaureus 1276 0.02 0.01 0.03 0.05 fiiised for 4.5 niiii. The 3ieOII was almost totally stripped off in S t a p h . aureus 2399 0.16 0.03 0.08 0.16 w"o. PhH !. ml) i wah added and distd off to a small residual vol. S t a p h . aureus' 2100 >50.0 >50.0 >50.0 0.0:) The tidditioii and dibtii of PhH wa, repeated to eiisiire complete Streptococcus pyo. removal ( J f the AIeOH and €LO. The product crystd overnight genes 3862 0.004 0.001 0.00G 0.012 Salmonella from a small residual vol of PhH. It was filtered off, Tvashed sciiottmuelleri 3850 0.14 0.6 0.10 >50.0 with PhH, arid dried in z'acuo, yielding 463 mg, mp 66-68". This I'seudomonab material vas used without fiirther purification. aeruginoaa 3840 50.0 >50.0 >50.0 >50.0 6- [o-Z-Amino-2-( 1,4-~yclohexadienyl)acetamido] penicillanic PR.aeruginosa 8329 12.3 130.0 3i.3 >50.0 Acid (I).--& ~ l i ofi 3 % mg (1.66 mmoles) of Y arid 0.23 ml of 2975 Escherichia cola 0.51 18.7 1.b >X.0 Et13?; iii 2.5 nil of IIpO was prepared, with the final pH being 7.4: Candida altican 5314 >50 0 >50.0 >50.0 >so 0 0.83 nil of l\Ie,CO rvas added, arid the s o h was kept at - 10". il Staph. aureus SC 2400, penicillinase producer. solti of 469 mg of methyl acetoacet,ate enamine of u-2-amino-2(1,4-cyclohesadienyljaceticacid, X a salt (1.72 mmoles) in 4.23 TABLEI1 A microdrop of .\--methylnil of MenCO r a s chilled to -20". morpholiiie v a s added, followed by the &JK addition of 1\18 mg of In J'ii?'O ;\IICl3OI3lOLOGlC.~L COX'.~RISOX O F 7-[D-2-LkMIXO-2ice-cold ethyl rhloroformale, IlnO (0.43 nil) ah added, and a L)ACl:T.IXIIDO] CEPH.ILOSPORIlYIC A C I D (11) turbid iolti re5ulted. The mist was stirred for 10 miii at -20". .IXD ITSDES.ICETOXT ASALOG(111)WITH OTHER The tiirbid b o l i i of mixed anhydride xva< then added t o the 6CEPHALOSFORISS APA , G o h i , The clear soli1 observed was ,tined for 30 niin at S < iuibli - l o " , then raised to room tenip, :tiid acidified t o pI12.0 with dil culture Cephal- CeglialIICl? and, withgood stirring, the pII wa> kept at that level for 10 Organism no. I1 I11 oglycin exin niiii. The s o h was theii estd with 5 ml of sylene. The sy phase Staph. a u r e m 12iD 0.27 0.27 0.27 0.3T \vas layered xvith 5 nil of lIeCO-i-Bu, aiid the pH was adjiirted t o S t a p h . aureus 2399 3.1 3.1 2 1 :3.1 .i.O with 1 S S a O H aiid (shilled overnight, The resiiltirig crystals S t a p h . aureu,P 15.6 2400 31.2 lL3 3; 3 were filtered off. washed rvith H 2 0 , aiid air-dried to vield 272 mg Strept. pyogenes 3862 0.0.1 0.01 0 01 0.01 (44: C!, dec 20"", iodonictric penicillin litrat ion,g 97.4c1. :lrial. S . schottrnuelleri 3850 1.6 4.i 0.6 -.> .I ,ClsHii?iaOiS.O..iH:OI C , 1-1, S , 8. P,?. aeruginoea 3840 >50 0 >50 0 >50 0 >50.0 * . E. coli 2975 $1.4 18.i B.1 .), 50 0 >50.0 >50.0 >j0 0 acetic Acid (VIII).---.4 solii of 30 g (0,201 mole) of I V in 201.5 ml Staph. aureus SC 2400, penicillinase producer. of 1 ATXaOI-I w a b di1iit.edn-ith 100 nil of R,O and 300 ml of 95 EtOH. To t,hir iv.a.j ndded 2S.K g (20.21 nil, 0.201 mole) of iP~t-hutoxycarhori.1azide, witth n o significalit rise in t>enip. T tube-dilution assay in antibiotic assay brothGor brain> o h wai mired for 16-18 h r . .I total of 13.9 g of uiireacted heart infusion broth6 as previously described.' I t may ainiiio acid pptd diiring thi.. period. After filtration, the white be seen that these are highly potent, mediuni-spectrum ninterinl i v r i ~vashed v i t h H,O, mid the wash wvns combined the firit fil~rate. The nq - i j l i i via> diluted with 100 in1 of antibiotics with activities comparable to those of the H 2 0 ,aiid the pH was lowered from 8.5 to 2 , using about 46 nil of aromatic analogs. Additional microbiological proper(i -\. HCl. The ppt, mx wahliecl thoroiighly with €Id) yielding ties, as n-ell as the chemotherapeutic values of these 24.6 g !40'; ! [if white ( 1-, 1111) 6 4 4 7 " . . I n n [ . ~Ci~HloNJO,) compounds,811-illbe reported separately. c, €I>3 . 7- 1~4% [L\~-terl-Butoxycarbonyl)amino] -24 1,4-~yclohexadienyl)acetamido]desacetoxycephalosporanic Acid (IX).-A s o h of 4.77 Experimental Section g (19.0; mmoles) of VI11 in 100 ml of THF cont,aiiiirig Et& (1.96 g, 2.7 nil) ivas chilled to - 10". K i t h good agit,atioii, isobiityl Melting poiiits (corrected) were takeii oii a Kofler hot stage. chloroformate (2.5i g, 2.6 nil) wx-i added over a period of 2 niiri; Proton nmr spectra were obtained oil a Variaii A-60 iiistrument, L t , S ~ € I Cpptd l f r o m the solii. The ~ l u r r ywas niised a i -%j0 for T M S standard. Combiibtioii analyses vere in accord with the w i a ndded a c~ild.solii of 4 g (18.ti8 iiirnolesj of calcd percentages withiri i 0.4$fcunless otherwise indicated. THF-H20 c.oiitg 1.9 g (2.G.i mli of Et!?;. The 0-2-( 1,4-Cyclohexadienyl)glycine (IV).--A s o h of 11.0 g tenip wa,. kept betweeii 0 a d - 103 for 1 hr. A c1e:ti. 5oln re(72.7 mmoles) of u-pheiiylglyciiie in 900 ml of distd XH3 (xhich hulted s h e i i the temp was allowed t o rise t o 25'. Agitation was had been t,reat,ed with 43 mg of I,i after distil to remove traces of coiitiiliied for aii ndditioiial 2 hr. then 100 nil of 1110 was added, moisture) was sloivly diluted u-ith 370 ml of dry t-BuOH. Over :I aiid the ..oh W:I\ estd :itimes with 75 in1 of E.tO.Lc. The r t ~ l period of 2 hr, 1.65 g of Li was added in .mall portions until a fiaci ioii v:~.layered with 1(;'~-71 of EtOAc. and the pIl was lowered permanent blue color appeared. The blue reacticn mixture was t o :2,u*iiig ti nil of :j -1-IIC1. l>ikL::,g thi? estn, some >olids formed, then treated with 38 g of Et& .HC1. The XH3 was alloTved to a i i d ueparatioii of the lnyers became tiifficiilt. !t was necesary evap a t room temp overnight, mid the residual solvent was evapd t o filter the Inistiire to coiitiiiiie the estn with two .?O-ml aliquots at reduced pressure. The white re4due wa> takeii up in a small of EtOXc. The conibiiied EtOXc fractions were dried (N amt of AIeOH-H& aiid added to 4 1. of cold 1: 1 CHC13-31e2C0 before beiiig roiicd o n a rotary evaporator. The result wa-; to precipitate the crude product. After 20 min of stirring, the yellow foam weighing 5.37 g (64"; ), which w a s used w subpension was filtered, and the white filter cake was dried in fiirther purificatioii. vacuo; it was pulverized, dissolved in ;LIeOH-H20, and submitted 7- [~-2-Amino-2-(1,4-~yclohexadienyl)acetamido] desacetoxyonce more to the pptii process from 1: 1 CHCI3-Me~CO. A nearly cephalosporanic Acid, Hydrate (III).-At 0 " ) 10 ml of cold quant yield of white, cryst product, 11.8 g, was obtained: dec FaCC0211was added I . 3 i g of I S . Swirling the flask for several 297"; [ O ] ' ~ D - 89.7" (2 NaOH); iimr (NaOD, D20) 6 :i.83 miii wsultecl i l l :I clear s o h , n-hich r v a ~kept, at, 25" for It? min (b, 3, vinyl), 3.79 (s, 1, HC), 7.30 (11, 4, allylic). Further puribefore strippiiig olf the esress acid. The clear reRidue was triturated with EtsO and mixed very well t,o form small colorless tal-. This product ,lightly hygroscopic and was filtered 16) Supplied b y t h e Ijaltimore Liiological Laboratories. iuider dry S ? . The r q - , 7 t d s were washed a-ith EtnO. The yield ( T ) F. Pansy, 11, 13ascli, I T , Jambor, G . X a e s t o n e , H . Semar, a n d I?. \vas 4.1.i g, i 9 . 1 ( ;, nip 140-142O dec. A satisf:tctory anal. was Donovick, Antzmicrob. A Q . Chemother., 399 (1966).
TABLE I I n Vitro ~IICROBIOLOGICIL Com.misox
9
(1
(1
(8) F. L. Tveisenborn, 0 . G . Uach, J . E. Dolfini, a n d J. Bernstein, C . PatenL 3,485,819, Dec 23, 1969.
s.
.. . d j
~
~
.
~
J, 1.. .ilicino, C'hcm. E n g . S e l ~ s 648 , (1961).
IRREVERSIBLE ENZYME INHIBITORS. 180
Joumal of Medicinal Chemistry, 19’71, Vol. 14, No. B 119
not obtained. A soln of 7.88 g of the CFaCOOH salt in 350 ml of HzO required filtration to remove a small amt of insol material, The pH of this clear soln was raised from 2 to 5 by gradually adding about 50 g of Amberlite IR4B resin that had been washed several times with HzO. Good agitation was necessary, and the time required for conversion was about 10 min. Darco (I g) was added to the soln after filtration and mixed in for 5 min. The light yellow soln resulting from this treatment was concd on a rotary evaporator using a vacuum pump. The concn was stopped when crystn of the product began, a t avo1 of approx 25 ml. Storage in the cold for several hours, followed by the addition of 150 ml of EtOH with strong agitation, led to the formation of small particles that were easily filtered. The product was washed with EtOH and dried in vacuo a t 45-50’. The anhyd product absorbed 1 mole of H10 from the atm. The yield was 3.56 g
(58%) of colorless crystals; iodometric assay, 97.9%, &s monohydrate. A second crop (0.77 g) was obtained by concn of the C, H, N. mother liquor. Anal. (ClsH1SNa04S.H~0) 7- [~-2-Amino-2-( 1,4-~yclohexadienyl)acetamido] cephalosporanic acid (11) was prepd in the same manner as I11 using VI11 and isolated as light tan crystals: mp 265-270’ dec. Anal. (CisHziN30eS.HzO)C, H, N.
Acknowledgment.-We are indebted to Joseph Alicino and Joseph Hydro and to Mrs. Mary Young for analytical data, to Dr. Allen Cohen and Miss Barbara Keeler for spectroscopic studies, and to Dr. Harold Jacobson for pK determinations.
Irreversible Enzyme Inhibitors. 180.lv2 Irreversible Inhibitors of the C’la Component of Complement3Derived from m-(Phenoxypropoxy)benzamidine and Phenoxyacetamide B. R. BAKERAND MICHAEL CORY Department of Chemistry, University of California at Santa Barbara, Santa Barbara, California 98106 Received August 6 , 1970
A new assay for irreversible inhibition of the C‘la component of complement has been established. A series of substituted pyridines quaternized with fluorosulfonylbenzyl bromide related to 1 in structure were previously shown to be good inhibitors of whole guinea pig complement; many of these compounds are excellent irreversible inhibitors of the C’la component of complement. The good correlation in irreversible inhibition of C’la and inhibition of whole complement by analogs of 1 strongly suggests that the main site of action by compounds of type 1 is inhibition of C’l. In contrast, the lack of correlation of irreversible inhibition of C’la and inhibition of whole complement by benzamidines of type 5 strongly suggests that the main site of action of the benzamidines benzis one of the other 8 components of complement. m-[m-(p-Fluorosulfonylphenylureido)phenoxypropoxy] amidine ( 5 ) is the most potent inhibitor of guinea pig complement yet observed; 5 is about 1000 times as potent as benzamidine and 3000 times as potent as N-tosyl-L-arginine Me ester (TAME).
Inhibitors of the serum complement system could have medicinal utility for organ transplantation and in treatment of some arthritic state^.^,^ The serum complement system is a mixture of 11 distinct protein^.^-^ One of the functions of complement is to kill foreign cells such as bacteria and protozoa; however, it can also lyse foreign mammalian cells and causes rejection of organ transplant^.^-' Some of the proteins of the complement system are proteases with “tryptic” or “chymotrypic” proper tie^;^-^ therefore it is not surprising that complement is inhibited by certain inhibitors of trypsin4 or c h y m ~ t r y p s i nwhen ~ ~ ~measured by the lysis of sheep red blood cells (RBC) by guinea pig complement and antibody.l , l o Two types of chymotryptic inhibitors of complement have emerged from this laboratory as exemplified by Is (1) This work was generously supported by Grant CA-08695 from the National Cancer Institute, U. 9. Public Health Service. (2) For the previous paper in this series see B. R. Baker and W. T.Ashton, J . M e d . Chem., 13, 1165 (1970). (3) For the previous paper on complement see B . R. Baker and M.Cory, rbid., 18, 1053 (19691, paper 165 of this series. (4) B . R. Baker and E. H . Erickson, ibid., 18, 408 (1969), paper 152 of this series. (5) H . J. Muller-Eberhard, Aduan. Immunol., 8, 1 (1968). (6) P. H.Schur and K. F . Austen, Annu. Rea. Med., 19, 1 (1968). (7) Ciba Foundation Symposium, “Complement,” G. E. W. Wolstenholme and J. Knight, Ed., Little, Brown and Co.,Boston, Mass., 1965. (8) (a) B . R. Baker and J. A. Hurlbut, J . Med. Chem., 18, 677 (1969), paper 156 of this series. (b) B . R. Baker and J. A. Hurlbut, ibid., 18, 902 (1969), paper 161 of this series. (9) B . R. Baker and J. A . Hurlbut, %bid.,18, 415 (1969), paper 153 of this series. (10) E. A . Kabat and M . M . Mayer, “Experimental Immunochemistry,” 2nd ed, C. C. Thomas Co., Springfield, Ill., 1967, pp 149-153.
c1 I
2
and 2;$ in both cases, removal of the SOzF moiety resulted in loss of their activity, indicating that the SOzF group was necessary for activity, presumably by irreversible inhibition” of one of the complement enzymes. That 1 was an irreversible inhibitor of the C’la component of complement was shown by BeckerS8 Benzamidine, a strong trypsin i n h i b i t ~ r , ’ is ~ ~a ’weak ~ inhibitor of ~ o m p l e m e n tinhibition ;~ is enhanced 6-fold by introduction of a m-phenoxypropoxy substituent (3)l which is further enhanced to 400-fold by substitution of m-(p-nitrophenylurea) on the phenoxy moiety (4)3(Table 111). Notable is the fact that 3 and 4, which are most probably reversible inhibitors in con(11) B . R . Baker, “Design of Active-Site-Directed Irreversible Enzyme Inhibitors,” Wiley, New York, N . Y . , 1967. (12) M . Mares-Guia and E . Shaw, J . B i d . Chem., 840, 1579 (1964). (13) B . R. Baker and E. H . Erickson, J . Med. Chem., 10, 1123 (1967), paper 106 of this series.
