J. Med. Chem. 1992,35, 3731-3744
3731
Inhibition of Human Leukocyte Elastase. 4. Selection of a Substituted Cephalosporin (L-658,758) as a Topical Aerosol+ Paul E. Finke,'J Shrenik K. Shah: Bonnie M. Ashe,# Richard G. Ball," Thomas J. Blacklock,ll Robert J. Bonney,lR Karen A. Brause: Gilbert 0. Chandler? Meredith Cotton,' Philip Davies,l Pam S. Dellea,* Conrad P. Dorn, Jr.? Daniel S. Fletcher,l Laura A. O'Grady: William K. Hagmann,t Karen M. Hand,l Wilson B. Knight,# Alan L. Maycock,#+'Richard A. Mumford,#Donald G. Osinga,l Paul Sohar,ll Kevan R. Thompson? Hazel Weston,#and James B. Dohertyt Departments of Medicinal Chemical Research, Enzymology, Immunology and Inflammation, and Biophysical Chemistry and Process Chemistry Research and Product Research and Development, Merck Research Laboratories, P.O.Box 2000, Rahway, New Jersey 07065 Received April 22, 1992
Human leukocyte elastase (HLE) is a serine protease which has been implicated as a causative agent in several pulmonary diseases. The continued modification of our previously reported cephalosporin-based HLE inhibitors has led to the identification of a series of C-2 amides with potent, topical activity in an in vivo hamster lung hemorrhage model. While the most potent in vitro HLE inhibition had previously been obtained with lipophilic ester derivatives, it was found that the less active, but more polar and stable, amide derivatives were much more effective in vivo. The development of the structureactivity relations for optimization of these activities is discussed. These results led to the selection of 3-(acetoxymethyl)-2-[(2(S)-carboxypyrrolidino)carbonyl]7cu-methoxy-8-oxo-5-thia-l-azabicyclo[4.2.0loct-2-ene 5,5-dioxide (3, L-658,768) as a selective, potent, time-dependent HLE inhibitor suitable for formulation as atopical aerosol drug for possible clinical use. The inhibition of human leukocyteelastase (HLE) (EC 3.4.21.37) has been a major therapeuticgoal for some time. Since an excess of active HLE has been implicated in several disease states, such as emphysema,' chronic bronchitis,2 acute respiratorydistress syndrome(AFtDS),S and cystic fibrosis: an inhibitor of HLE has the potential of preventingor arrestingthese conditions.5 In most cases, the pathogenesis of these diseases has been correlatedwith the inactivation or an insufficient reserve of HLEs natural inhibitors, notably crl-protease inhibitor (crl-PI),in localized environments. The presence of this uninhibited elastase in the intercellular spaces then results in the To whom correspondence should be addressed. Thia paper is dedicated to Profensor Ralph Hirschmann on the occaeion of his 70th birthday in recognition of his many scientific accomplishments at Merck and throughout his career. t
3 Department of Medicinal Chemical Wearch. 4 Department of Enzymology.
I Proceee Chemietry Rasearch. 1 Department
uncontrolled proteolysis and damage of structural tissue. Given this hypothesis,considerableeffort has been directed toward finding treatments which would augment the activityof the natural inhibitors and restore the protease/ antiproteasebalance.'&* Two methods investigatedso far in this regard are the developmentof recombinant sources of the natural inhibitors for a replacement therapy' and the synthesisof mechanism-based peptide or low molecular weight inhibitors.* Since many of the proposed HLEmediated diseasesoccur in the lung, aerosolization of these agents has been employed. This route of administration circumvents the problems of absorption and metabolism associated with systemic administration and should also lessen any possible side effects.9 Modification of the cephalosporin nucleuslo to afford selective, patent, and time-dependent inhibitors of HLE has been previously reported by these laboratorie~.1~-15 From these studies 4-carboxybenzyl 3-(acetoxymethyl)7a-methoxy-8-oxo-5-thia-l-azabicyclo[4.2.0loct-2-ene-2-
of Immunology and Inflammation. Product Research and Development. Department of Biophysical Chemietry. X Preaent address: Briatol-MyersCo., 100Foreat Avenue, Buffalo, NY (6) Snider, G. L. ProteaeaAntiproteaee Imbalancein the Pathogene& 14213. of Emphysema and Chronic Bronchial I n j w A Potential Target for O Present address: Sterling ResearchGroup, 26GreatValley Parkway, Drug Development. Drug Dev. Res. 1987,10,236-253. (7) Schnebli,H. P. Recombinant ElastaeeInhibitorsforTherapy. Ann. Malvem, PA 19356. (1) (a) Janoff, A. Ebtaae and Emphysema Current Aaeessment of N . Y. Acad. Sci. 1991,624,212-218. the Proteaee-Antiprotease Hypothesis. Am. Rev. Respir. Die. 1986,132, (8) (a)Williame,J. C.; Stein,R L;Gilea,R. E.; Krell,R. D. Biochemistry 417-433. (b) Pulmonary Emphysema and Boteolysiq Taylor, J. C.; and Pharmacologyof IC1200,880,a SyntheticPeptideInhibitorof Human Mittman, C., Ekia.; Acndemic Press, Inc.: New York, 1987,pp 1-560. (c) Neutrophile Ebtaae. Ann. N. Y.Acad. Sci. 1991,624,230-243. (b) Pulmonary Emphysema,the Rationale for Intenention. Ann. N . Y.Acad. Skilas,J. W.; Fuche, V.; Mino, C.; Sorcek,R.; Grozinger, K.0.;Mauldin, Sci. 1991, 624, 1-370. 5.C.; Vitous, J.; Mui, P. W.;Jacober, S.; Chow, 0.;Maw,M.; Skoog, (2)Stockley,R. A.; Hill, S. L;Burnett, D. Proteinaaea in Chronic Lung M.; Wehn, S. M.; Poseanza, G.; Keirns, J.; Letts, G.; Roeenthnl, A. S. Inhibition of Human Leukocyte Ebtaae (HLE) by N-Subetituted Infection. Ann. N . Y.Acad. Sci. 1991,624,257-266. (3) Marritt,T.A.; Cochrane, C. G.; Holcomb, K.;Bohl, B.; Hallman, Peptidyl TriffluoromethylKetones. J. Med. Chem. 1992, 36,641-662 M.;Strayer,D.;Edwarde,D.;Gluck,L.ElasteseandolProteinaaoInhibitor and references therein. (c)Trainor, D. A. Synthetic Inhibitors of Human Activity in Tracheal Aspirates During Repiratory Distreee Syndrome. NeutrophilElaetaae. TrendsPharmacol. Sci. 1987,8,303-307. (d) Stein, J. Clin. Invest. l9S3,72,666-668. R. L.;Trainor, D. A,; Wddonger, R A. Neutrophil Elastase. Annu. Rep. (4)Jackeon, A. H.; Hill,9. L.;Afford, 5.C.; Stockley, R. A. Sputum Med. Chem. 1986,20,237-246.(e)Powers, J. C.; Gupton, B. F.; Harley, Soluble Phase Proteins and Elaetase Activity in Patients with Cystic A D.; Nighino,N.; Whitley,R. J. Specificityof PorcinePancreatic Elastase, Fibrosis. J. Respir. Dis. 1984,66, 114-124. Human Leukocyte Elaetaee and Cathepsin G. Inhibition with Peptide Chloromethylketmes. Biochem. Biophye. Acto 1977,484,166-166. (5) Davies, P.; Ashe, B. M.; Bonney,R. J.; Dorn, C.; Fmke, P.; Fletcher, (9)Travis, J.; Fritz, H. Potential Problem in Designine Ebtaee D.; Hanlon, W.A.; Humes, J. L.;Maycock, A.; Mumford, R. A,; Navia, O w , E. E.; Pacholok, 5.; Shah, 5 . ; Zimmerman, M. ; Doherty, J. Inhibitors for Therapy. Am. Rev. Respir. Die. 1991,143,1412-1415. M. A.; (10)The numbering sequence ueed in thia paper is shown in structure B. The Discovery and Biologic Properties of Cephaloeporin-Bd A. Inhibitors of PMN Ehtaee. Ann. N . Y.Acad. Sci. 1991,624,218-229.
0022-2623/92/1835-3731$03.00/0Q 1992 American Chemical Society
3732 Journal of Medicinal Chemistry, 1992, Vol. 35, No. 21
Finke et al.
carboxypyrrolidino)carbonyll-7a-methoxy-8-oxo-6-thial-azabicyclo[4.2.0loctd-ene5,5-dioxide (3, L-658,758) as a more stable, selective,and time-dependent HLE inhibitor having functional in vivo activity in this and several other efficacy models of HLETmediatedtissue damage. In this paper the development of this compound ie described in 1 l YOHO terms of the structure-activity relations (SAR)for enzyme 0 inhibition and lung hemorrhage activity as well as the chemical and physical properties required of an aerosol 5 candidate for possible clinical evaluation. ?+S'I4 It has been that large, polar compounds are retained in the lung environment better than small, 0 48 -1N* A 3' nonpolar entities which can be rapidly absorbed. However, experience has shown that HLE prefers a more hydroA phobic, nonpolar substratel8 or inhibitor.llJ3 As will be Cephalosprin Nunbing seen below, another crucial findingwas that the previously 0 optimized esters were in fact very sueceptibleto hydrolysis 3, L-658,758 of the @-lactam,while the correspondingless potent amides were found to have improved hydrolytic stability. These initial findings explained very well the above results with 1 and 2, and thus the focus of this work became the development of a more polar, stable derivative which retained the previous potent HLE inhibition. The criteria o , & S-R3, A H e C % used to evaluate these cephalosporin derivatives were (1) their activity in the hamster lung w a y , which was used OR>NR2>NHR>OH as the primary screen for efficacy as well as a pharmaB cokinetic tool, (2) their stability in pH 8 MOPS buffer to determine their relative chemical stability, and (3)their carboxylate 5,&dioxide (1) was idenWied13as avery potent, HLE inhibitory potency. As described below, 3was found time-dependent inhibitor of HLE (k,d[Il = 57 OOO M-I to have good overall activity both in vitro (kod[I1 = 3800 8-9 whereasthe sarcosineamide2 was comparatively much M-' 8-9 and in vivo (ED60 = 5 pg/animal with a 30411 less active (k,d[Il = 300 M-' 8') and the C-2carboxylic predose, TW = 3 h with a 200-pg dose), as well as better acid 19b was inactive. However, all of these inhibitors chemicalstability (tip = 21 h),suitablephyaicalproperties, were found to be rapidly degraded in blood and no oral ease of synthesis, and the required initial safety. In absorption could be detected in the rat. These problems addition, 3 has been founds to be potent in several were addressed with the subsequent establishent of an subsequent efficacy studies as well. intratracheally (IT) administered, HLE-induced lung hemorrhage assay in the hamster which afforded the Chemistry necessary means to evaluate the efficacy of our cephaAs reported in our initial investigations and depicted in losporin inhibitors directly in a lung environment.le In Structure B,a small, a-oriented chloro, methoxy, or ethyl contrast to the above in vitro inhibition values, 2 was found group was found to be optimal at the C-7position for HLE to be much more efficacious than 1 in the in vivo assay inhibition and the sulfone (oxidation state at S-5) was (Table I) when administered intratracheally prior to HLE considerably more potent than the sulfide derivative or instillation. "his pivotal finding then led to tRe synthesis either of the sulfoxide isomers.12 At the C-2 position, of several amide analogues and ultimately to the selection lipophilic esters were found to be the most potent inhibitors of the L-proline derivative 3-(acetoxymethyl)-2-[(2(S)while tertiary amides were considerably lese active and secondary amides were very poor inhibitors. The free (11)Doherty, J. B.; h h e , B. M.; Argenbright, L. W.; Barker, P. L.; Bonney, R. J.; Chandler, G. 0.;Dahlgren, M.E.; Dorn, C. P., Jr.; Finke, carboxylic acids (asis found in most @-lactamantibiotics) P. E.; Firestone, R. A.; Fletcher, D.; H a g " , W. K.; Mumford, R.; OGrady, L.; Maycock, A. L.; P m o , J. K.;Shah,S. K.;Thompson, K. R.; Zimmerman,M. ~phalosporinA n ~ ~ o t i ~ModiAedTo s B e Inhibit Human Leukocyte Elastase. Nature 1986,322,192-194. (12) Doherty,J.B.;hhe,B.M.;Barker,P.L.;Blacklock,T. J.;Butcher,
J. W.;Chan~er,G.O.;Dahlgren,M.E.;Davies,P.;Dorn,C.P.,Jr.;Finke, P. E.; Fireatone, R. A.; Hagmanu, W. K.; Halgren, T.;Knight, W. B.; Maycock, A. L.; Navia, M. A.; O'Grady, L; Pieano, J. M.; Shah,S. K.; Thompson, K. R.; Weston, H.; Zimmennan, M. Inhibition of Human Leukocyte Elastase. 1. Inhibition by C 7 Subetituted Cephalosporin tee-Butyl Esters. J. Med. Chem. 1990,33,2513-2621. (13) Finke, P. E.; h h e , B.M.; Knight, W. B.; Maycoek, A. L.; Navia,
(16) (a) Bonney, R. J.; h h e , B.; Maycock, A.; Dellea, P.; Hand, K.;
Oeinga, D.;Fletcher,D.; Mumford,R.;Daviea,P.;Frankenfield,D.;Nolnn,
T.; Schaeffer,L.; H a g " , W.;Finke, P.; Shah,S.; Dom, C.; Doherty,
J. PharmacologicalProme of the Substituted Beta-LactamL859,286: A Member of a New C h of Human PMN Elastase Inhibitors. J. Cell. Biochem. 1989,39,47-63. (b) Fletcher, D. S.; Oainga, D. G.; Hand, K.
M.;Dellea,P.S.;Ashe,B.M.;Mumford,R.A.;Daviea,P.;HEg",W.; Finke, P. E.; Doherty, J, 3.;Bonney, R. J. A Com&aon of alpha-lProteinase Inhibitor, Methoryeuccinyl-Ala-Ala-Pr+ValChlomMethylketone, and Specific Beta-Lactam Inhibitors in an Acute Model of M. A.; Shah,S. K.; Thompson, K. R.; Underwood, D. J.; Weeton, H.; Human PMN Elastase-Induced LungHemorrhagein the Hameter. Am. Zimmerman,M.; Doherty,J. B. Inhibition of Human LeukocyteElastase. Reo. Respir. Die. 1990,141,672477. (c) Haeesl, C. H.; Johneon, W. H.; 2. Inhibition by SubstitutedCephalosporinEeters and Amides. J.Med. Kennedy, A. J.; Roberta, N. A. A New Claw of inhibitors of Human Chem. 1990,33,2622-2528. Leukocyte Elastase. FEBS Lett. 1985,183,201-206. (14) Shah, S. K.; Brause, K. A.; Chandler, G. 0.;Finke, P. E.; Ashe, (17)Schauker, L. 5. Drug Absorption from the Lung. Biochem. B.M.;Weston,H.;Knight,W.B.;Maycock,AL.;Doherty,J.B.Inhibition Pharmacol. 1978,27,381-386. of HumanLeukocyte Elastase. 3. SyntheahandActivityof 3'-Subatituted (18)Harper, J. W.; Cook, R. R.;Roberts, C. J.; McLaughlin, B. J.; Cephalosporins. J. Med. Chem. 1990,33,2629-2636. Human Powers, J. C. Active Site Mapping of the Serine Pro(16) Hagmanu,W. K.; OGrady, L. A.; Ashe, B. M.;Dahlgren, M.E.; LeukocyteEhetase, Cathepsin G, PorcinePancreatic Elastase.Rat Maet Weston,H.; Maycock, A. L.; Knight, W. B.; Dobrty, J. E.Inhibition of Cell Proteasea I and II,Bovine ChymotrypsinAa, and Staphylococcus Human Leukocyte Elastase by C-2 Subatitutad CephalosporinSulfones. aureus Protease V-8 Using Tripeptide Thiobenzyl Eater Substrates. Eur. J. Med. Chem. 1989,24,699-604. Biochemistry 1984,23, 2996-3002.
Journal of Medicinal Chemistry, 1992, Vol. 35, No. 21 3733
Inhibition of Human Leukocyte Elastase
Table I. Chemical Stability and in Vitro and in Vivo HLE Activity of Selected Initial C-2 Ester and Amide Derivatives
lung hemorrhage activity time, min % inhibn (SD)f 18 OAc -5 98 (3) -30 25 (25) 28 NMeCHzC02H OAc 13 (1) 300 (100) -30 90 (10) -30 67 (27) -180 54 (30) 4ah O-t-Bu OAc ndi 16000 (1500) 50 premixj 98 (5) 4bk OCH2Ph-4-CO2H H >>24 Ki 0.5 pMI 100 -30 54 (12) 4d 'la-ethyl derivative of 1 40 (2) 5700 (1400) 100 -30 -32 (24) 4dm 4-methyl derivative of 1 e5 25000 (2300) 100 -30 29 (27) 4d O-t-Bu OCO(CHZ)&OZH nd 13800 (300) 400 -5 98 (2) 4fk O-t-Bu A nd 63900 (4000) 100 -30 87 (7) 100 -90 10 (31) 4& O-t-Bu B nd 8600 (500) 100 -30 90 (6) 100 -90 18 (16) 4h' O-t-Bu C nd 3700 (300) 100 -30 89 (13) 30 -30 77 (10) 4i8 NH-t-Bu OAc nd 2200 (100) 400 -30 -5 (41) 4js N(CHzCHz)20 OAc 13 (0.3) 35200" 100 -30 -21 (49) 4k8 NMeCH2Ph-4-COzH OAc nd 8600 (3200) 100 -30 65 (20) a See ref 16 for methodology. The compounds were administered IT at the given dose (per animal) and time prior toHLE. * See Experimental Section for methodology. See ref 12and 22 for methodology. d Standard deviation of experimentalpoints from the calculated first-order decay curve. Average of two or more determinations at different inhibitor concentrations. f Average of three animals at each dose and time. 8 See ref 13. See ref 12. i Not determined. j HLE was premixed with compound prior to IT administration. See ref 14. I No time-dependent inhibition was observed. See ref 15. Result of a single determination. compd
Ri OCHzPh-4-CO2H
Rz
tl/z,bh (SD)d 5 (0.1)
were inactive.13 Also, it was found that substitution at C-4 could enhance activity in some caseslS and the C-3' acetoxy group could be replaced by a variety of other oxygen- or sulfur-based leaving groups.14 The unsubstituted C-3methyl analogueswere muchless active, although they could still be time-dependent inhibitors even without a potential leaving group at this position.14 For this work the sulfonederivativesreceived the primary emphasis and the 7a-methoxy group at C-7 was chosen over the more potent 7a-ChlOrOso as to avoid possible problems inherent in an a-halocarbonylcompound. A few of the less potent, but more stable, 7a-ethyl analogues were also prepared for comparison. As implied from the results of 1 and 2 mentioned above, the substitution at C-2 was pivotal to the modification of both the in vitro and in vivo activities and was extensively explored. Then, once the SAR at C-2 was delineated, a variety of other (2-3' and C-4 analogues was evaluated. To help understand the above contradiction with 1and 2, a solution-stability assay was developed. Samples were dissolved at 1mg/mL in 0.5 M MOPS buffer at pH 8.0and incubated at 25 OC. Aliquota (50 pL) were removed at regular intervals, quenched with 2% aqueous trifluoroacetic acid, and analyzed by reverse-phase HPLC. The disappearance of compound was then monitored for one to two half-lives and the results fit to a first-order decay curve from which the reported t1p values were derived. Table I lists the structure and pH 8stability data for some selected, previously described esters as well as several of our initial amides. In general, the esters were readily hydrolyzed (1, t1p = 5 h) while the amides had significantly longer half-lives (2, t1/2 = 13h). The more potent, neutral
kobdVI )C
M-ls-l (SDP 57000 (6000)
dose, pg 100 100 100 10 200
amide 4j was equally stable (t1/2 = 13 h) but was totally inactive in the lung assay. As expected, the C-3 methyl (4b) and 7a-ethyl(4c)analogues were much more resistant to hydrolysis due to less activationof the 8-lactamcarbonyl. Also, the C-4 methyl-substituted compounds, such as 4d, were found to be more readily hydrolyzed compared to the unsubstituted parents, possibly due to steric repulsion of the acetoxy, making it a better leaving group. This also correlates with the improved HLE inhibition observed with some of these C-4 derivatives. For reference,the t1/2 of the C-2 carboxylic acid 19b increased to 85 h and that of cephalothin, a known antibiotic, was >lo0 h (data not shown). The amides prepared in this study are listed in Table I1 along with the hydrolysis results (tip) for selected compounds. The C-3' acetoxy amide analogues were routinely prepared from t-butyl 3-(acetoxymethyl)-7amethoxy-8-oxo-5-thia-l-azabicyclo[4.2.0] oct-2-ene-2-carboxylate (5)12as previously described13or as outlined in Scheme I. Since the C-2 carboxylic acid 6 was an oil and difficult to purify, the trifluoroacetic acid (TFA) deprotection of 5 (method A) was usually followed immediately by amide formation to give the sulfides 7 as a mixture of A2 and A3 isomers. This reaction was accomplished through dicyclohexylcarbodiimide (DCC) activation and active ester formation with N-hydroxysuccinimide followed by addition of the corresponding amine or amino acid tert-butyl ester. It was soon found that it was advantageous to convert the active ester intermediate completely to the A3 form by treatment with excess triethylamine (TEA) prior to addition of the secondary amine (method B). The conversion to the A3 isomer was
3734 Journal of Medicinal Chemistry, 1992, Vol. 35, No.21
Finke et al.
Table 11. Chemical Stability and in Vitro and in Vivo HLE Activity of C-2 Amide Derivatives
k o d [I3
compd
Rz
R1
CHiCOnH
h& t&
M-’
8-l
(SDIh
OAc 13 (1) 300(100) 93/86/77 21 (1) 3aoo(soo) OAc 99/97/78/32 24(2) 500(100) OAc 80/39/-1/33 OAc ndj 9ook 92/43/4/-12 OAc nd 500 (50) 96/89/78/50 94/81/47/21 OAc 97190129 nd 900 (400) 50 30(10) Ma Me CHzCOzH Me CHzCOzH E nd 100 (20) 04159157128 16b 4-Methyl Derivative OAc Me CHiCOiH 8 (0.3) 1400 (100) 18 - a See ref 16 for methodology. The compounds were administered IT at the given dose (per animal)and time prior to HLE. b See Experimental Section for methodology. See refs 12 and 22 for methodology. d The standard screen was administration of 100 pg of compound/animal30 min prior to instillation of 50 pg of HLE. Inhibition of the hemorrhage was determined after 3 h. e For active compounds from the screen, doses of 100,30,10, and 3 pgtanimal were given at 30 min prior to HLE and inhibition determined. On repeated assaying, an EDm could be determined. f For active compounds, the duration of the compound was determined by dosing at 200 pglanimal 1 , 2 , 3 , and 4 h prior to HLE instillation. The time at which 50% inhibition was still obtained was used as the Tm for efficacy. 8 Standard deviation of experimental points from the calculated first-order decay curve. h Average of two or more determinations at different inhibitor concentrations. i Average of three animals at each dose and time. Similar errors were obtained for the titration and duration experimenta. Not determined. Result of a single determination. I No time-dependent inhibition was observed. 2
Me
R3
lung hemorrhage activity: % inhibna titratione of durationf screend 100/30/10/3 of 200 a at (SD)’ pg at 30 min 1/2/3/4 h 90 (10) 90175165 97180154120
3 8a 8b 8c 8d 8e 8f
conveniently monitored by NMR analysis (A3 C-4 H, b = 6.54) and the product was determined to be almost exclusively the A3 isomer. Later, the use of l-hydroxybenzotriazolewas found to afford a better yield of A3 sulfide without the need for TEA (method C). No evidence was observed for an intermediate A3 ketene as proposedl9for the preparation of A3 esters from the A2acid chloride. The products were usually only partially purified by flash chromatography before oxidation to the sulfones 8 with m-chloroperbenzoicacid (m-CPBA) (method D).Treatment with a trace of pyridine before purification was required to completely convert the sulfones back to the more stable A2 isomers. The amino acid tert-butyl ester
derivativeswere then deesterified with TFA to afford the final amidocarboxylicacid products2,3,8a-e,g,i,j(method E). As hoped, these amides retained or had improved stability in the pH 8 hydrolysis w a y as observed with the @-alaninederivative8a (t112= 24 h). The important effect of the carboxylate position on the t1p values was clearly seen in the pyrrolidine series, with the neutral, unsubstituted compound 8f having a t l ~ 2of 16 h, and that of the L-proline derivative 3 improved to 21 h while that of the D isomer 88 was less than 5 h. The instability of the latter compound may actually imply internal catalysis by the carboxylate. Replacement of the C-3’acetoxy was accomplished by hydrolysis of the sulfone amides 8 with titanium isopro(19)Murphy, C. F.; Koehler, R. E. Chemistry of Cephalosporin Antibiotics. XVIII. Synthesisof 7-Acyl-Smethyl-2-cephem-4-carbox~c poxide (method F) before deblocking the carboxylic acid. Acid Esters. J. Org. Chem. 1970,35, 2429-2430. Treatment of the intermediate alcohols 9 with thionyl
Journal of Medicinal Chemistry, 1992, Vol. 35, No. 21
Inhibition of Human Leukocyte Elastase
3735
Scheme 14" w,,,
Meoh,,,
"\\&I
borc
a
Meo40 f&OAC
OAc
0
0
