Synthesis and antibacterial activity of men 10700, a new penem antibiotic

Bkwrgmic

Pergamon

& Medicinal

Chmimy

Leaen, Vol. 5, No. 6. pp. 555-558, 1995 Copyright 0 1995 Blscvicr science La Printed in Great Britain. All rigb mcrwd 0960-894X195 $9.5OtO.W

0960-894X(95)00072-0

SYNTHESIS

AND ANTIBACTERIAL A NEW PENEM

ACTIVITY ANTIBIOTIC

OF MEN 10700,

Maria Altamura *, Enzo Perrotta, Piero Sbraci, Vittorio Pestellini and Federico Arcamone IndustrieFarmaceutiche Riunite S. r. I., Via dei Sette Santi 3, I-SOI Firenze (Italy)

“A. Menarini”

GiuseppeCascio Llrsochimica

S. p. a., Via Carnia 26, I-10232 Milan0

(Italy)

GiuseppeSatta,Graxia Morandotti,RobertaSpeming Universitd Cattolica de1 Sacro Cuore, Large F. Vito I, I-00168 Roma (Italy)

The new penemantibiotic Men 10700(3, bearingan aminoacidderivedamideasC-2 sidechain, wassynthesized.Men 10700exhibited high potencyand a broadspectrumof activity againstGrampositive andGramnegativemicroorganisms.

Abstract.

The penemsarea syntheticclassof highly potent,broadspectruml3-lactamantibioticsstructurallyrelated to the naturallyoccurringpenicillins,cephalosporins andcarbapenems 1.The penemskeleton,born asa hybrid betweenpenicillinsandcephalosporins, hasbeentheobjectof extensivesyntheticwork sinceits first synthesis in 19782 anda numberof penemderivatives,suchasritipenemb, furopenem3bandsulopenem k, arenow in clinical trials. While the presenceof the l(R)-hydroxyethyl moiety at C-6. asin 1, is now consideredto be a fundamentalrequisiteto confer chemicaland Dlactamasestability, a largedifferentiation is possiblein the natumof C-2 substituent.In the courseof our researchprogramon newpenems,we focusedour attentionon 2-CHS substitutedpenems1 bearinga heteroatomlinkedto thebicyclic skeletonthrougha methylenespacer. As a part of thesestudies,we reported4 on the synthesisandantibacterialactivity of penemdithiocarbamates 2: thesecompoundsexhibited potent in vitro antibacterialactivity againstGram positive bacteria,including heterogeneous methicillin resistantStaphylococcusaureus strains.However, their activity againstGram negativestrainswasconsiderablylower.

In the searchfor 2-substitutedpenemsendowedwith a betterbalancedantibacterialspectrum,we turned thenour attentionto nitrogensubstitutedmethyl penemsbearingaminoacidderivedsidechains.We reasoned thatinsertionof aminoacidderivedmoietiesassmall,polargroups,shouldimprovethepenetrationthroughthe outermembrane of Gram negativebacteria:in facts,in the previousseries,our bestresultswereobtainedwhen 555

556

M.

&TAMURA

et

al.

a N-methyl glycinamide was inserted (2, R’ = CH3, R” = CH2CONH2) as the terminal group on the dithiocarbamate. In addition,naturaland unnaturalaminoacidsshouldprovide us a largepool of sidechains with tunablebiologicalandchemicalfeatures:incorporationof aminoacidderivedside-chains in otherseriesof O-lactamcompounds hasbeenusedto improvespectrum5 or pharmacokinetic properties,asoral absorption.6 We have synthesized7 a number of new amino acid derived, amido substituted penems (1, X = -NR1-(CIiR2),-CONR3R4):amongthem,the N-methylglycinamidoderivative 3 (Men 10700)emergedas apotent,broadspectrum,antibacterialagent. CJl-BDMS I

‘*I,

CR S

0F+

CH2Y

N

/J

‘R,,

(b)

COOCH&H=CH,

0*

y3

N

4 Y=OH

6 R=TBDMS

5

7 R=H

Y=OSO$H,

Scheme 1. TEtDMS = text-butyldimethylsilyl. Reagents: (a): CH3SO2Cl. TEA, DMSO, 16 h, I. t. (c): TBAF, AcOH, THF, 24 h. r. t. (d): Pd(pph3)4.

CH,-N-CHaCONH,

03

COOCH,CH=CH,

TEA, THF, O-SQC. lh. (b): CH3NH-CHflONH2, triphenylphcsphine. THF, 30 min. 35%.

Synthesisof’3 (Scheme1) startedfrom well-known Zhydroxymethyl penemintermediate4 8: the primary hydroxy group in 4 was activated as the mesylate5 and allowed to react with sarcosinamide hydrochlorideto give 6 9 (65%overall yield from 4). Deprotectionat C-8 with tetrabutylammonium fluoride and removal of the ally1 ester moiety by palladium catalysis,lo followed by reverse phasecolumn chromatography,gave (JR.6S)-2-[N-methyl-N-(2-acetamido)]-aminomethyl-6-[(1R)-hydroxyethyl]-penem-3carboxylic acid 3 (56%overall yield from 6) asa white solid; mp 956°C. IH NMR (200 MHz, D20) 6 1.25 (3H, d, J = 6.2 Hz, CH3-C),2.90 (3H, s, N-CHs), 3.98 (2H, s, N-CH2), 3.98 (lH, dd, J = 1.4,6.4 Hz, H-6), 4.13-4.28(lH, m, H-8), 4.25 (2H, s,CH2-2’),5.69(lH, d, J = 1.4 Hz, H-5). I3C NMR (50 MHz, D20) 6 24.9 (CH3-C),46.1 (N-CH3), 57.4(CH2-2’).60.5 (N-CH2), 68.1 (C-5), 69.5 (C-8), 75.1 (C-6), 135.7(C-2). 141.1 (C-3), 169.7(COOH), 173.7(CONHz), 180.2(8lactam C=O). IR (KBr) cm-l 1761(O-lactamC=O), 1682, 1605.FAB-MS (m/z) 316 (M + H)+. [a]*‘,: +96’ (c 0.1, H20). UV: kmax(H20) 254, 315 nm. Chemical half-lives(HPLCdeterminationat 37°C):4 h @H1.7),50 h (pH 3.7), 120h (pH 7.0). 30h @H9.0). Antibacterialactivity of 3 wasfirst testedagainsta numberof standatdandmodifiedGrampositiveand Gram negativestrains(Table 1). Compound3 showedoutstandingactivity againststandardS. aureu.rstrains, heterogeneous methicillin resistantS.aureus, and againstE. coli standardstrain. Against all thesestrains activity of 3 wasvery similarto ritipenemandimipenem,by far exceedingthat of ampicillin-sulbactam. It is interestingto note how the differencein minimuminhibitory concentrationsbetweenstandardand hyperpermeabile E. coli strains,previouslyshowedfor 2, andindicatingin that casea problemin permeability throughtheouterGram negativebacterialmembrane, disappeared for 3, supportingthe hypothesisof enhanced permeabilityof the compound,whenbearingthe small,polar, sarcosinamide derived. sidechain.Activity of 3 againstE. coli did not showany significantchangewhenE. cofi strainsproducingknown beta-lactamases were usedand constantly remainedat the imipenemlevel. Comparisonwith two close analoguesof 3, the N,N-dimethylamide 8 and the n-butylester9 (X = -N(CH3)CH$ON(CH3)2 and -N(CI-I~)CH$!OOCqH~, respectively,in generalformula 1) broughtinto evidencethe importanceof a primaryamidomoiety:in fact, the

Men 10700, a new penem antibiotic

557

ester compound did not show any significant activity against Gram negative strains, while dimetbylamide 8 led to a neat decrease of potency all over the spectrum, confirming the subtle influence on biological properties of a change in the amino acid derivative. As most of penem antibiotics 11, 3 did not show activity against Pseudomonas aeruginosa.

Table 1. In vitro antibacterialactivity * of compounds2 and3 (Men 10700)in comparisonwith ritipenem. imipenemandampicillin-sulbactam, asdeterminedby the agardilutiontechnique. COtll pou”(

MIc hw-1 S.a. S.a. S.a. S.a. E.f. E. coli E. coli E. coli ATCC Al-CC MR13 MR09 A-% A-KC DC2 TEMl 2921325923 29212 25922

2

0.12 0.12 0.03 2

a

3

0.06

0.06

I

8

0.25

0.12

0.25

4

9

0.5

0.25

0.5

RITl

0.12

0.06

co.03 0.25

IMI A-S

* Minimum Incubation:

E. coli TEM2

E. coii SHV

E. coli L

Ps.aer. ATCC 27853

Ps. ser. Ps.aer. VR5 G242

16

~32

4

a

8

0.5

8

8

16

4

0.25

0.25

0.25

0.5

0.5

0.25

z-32

0.5

4

8

0.5

I

1

1

1

0.5

~32

4

0.5

16

8

32

1

>32

>32

>32

>32

z-32

>32

>32

0.12

2

2

0.5

1

1

1

1

1

~32

0.5

1

CO.03

0.12

0.25

0.5

0.12

0.5

0.25

0.25

0.25

0.12

1

0.25

0.25

0.03

0.12

4

1

8

1

>32

8

>32

8

32

0.12

>32

4.03

Inhibitory Concenuations (ME) 24 hours at 37°C. Abbreviations:

determined in Mueller Hinton 2 Medium, S. a. : Staphylococcus aureus; S. a. MR 13: aureus; S.a. MR09:homogeneous methicillin resistantS. aurew E.f.: Enrerococcus faecalis; seain; E. coli TEMl. TEM2. SHV: strains producing known beta-lactamases; E. coii L: Pseudomonas aeruginosa; Ps. ser. VR5: LX-lactamase Iacking hypex-permeable strain: Ps. au.

ritipenem, IMI = imipenem, A - S= ampicillin - sulbactam.

bioMerieox. Inoculum: 104 cells/ml. heterogeneous me.thicillin resistant S. E. co/i DC2z hyper-permeable E. cofi B-lactamase lacking strain; Ps. au.: G242: hyper-permeable strain. RITI =

The high potency and broadspectrumof activity exhibitedby Men 10700asa new antibiotic become evident in light of the data of Table 2, which collectsminimuminhibitory concentrationsvalues(asMICw) againstclinical isolates,comparedwith imipenem(asoneof the mostpotent andbroadestspectrumD-lactam antibiotic now available), ampicillin-sulbactamand amoxicillin. Tested microorganismsincluded Gram positive, Gram negative, aerobicand anaerobicstrains.Men 10700 showedhigher activity than standard compounds, comprisingimipenem,againstbothmethicillin-sensitive andmethicillin-resistant S. aureus strains. Activity of Men 10700againstS. epidermidis wasten-fold greaterthan imipenemand morethan 100.fold greaterthan ampicillin-sulbactam and amoxicillin. Our compoundexhibited equalpotency whencompared with standardsagainst a range of different Srreprococcus species,although it was lesspotent against Cfosfridium petfringens. As for Gram negative spectrum,Men 10700 was constantly morepotent than ampicillin-sulbactamand amoxicillin, reaching imipenemMICgo valuesagainstHaemophilus it&enzae. Klebsiella pneumoniae, Citrobacter freundii, Escherichia coli andProteus spp. Especiallynoteworthyis the activity againstEnrerobacter, generallyconsidered asa highly resistantnosocomialpathogen. Furtherevaluationsarenowin progressin orderto establishthein vivo efficacy of the newcompound. Acknowledgment.

We wish to thank Dr. Laura Lorenzi

and Mr. Daniele Caglio for synthetic

work and Dr. Antonio

Triolo for the

regisuation of mass speclra. Manythanks aredueto Prof.Roberta Fontana (University of Verona) forhelpfuldiscussions. ThisWC& was supported in part by MURST

(IMI Grant No. 53529).

M. ALTAMURA et al.

Table 2. In vitro antibacterial activity * (MB&J) of Men 10700(3) against clinical isolates Micro-mganism (no. of strains) Staphylococcus aureus US (6) Staphylococcus aureus MR (20) Swphylococcus epidermiiis (13) Strepwcoccus pyagenes A (3 1) a strepwcoccw pneumoniae (23) a Strepwcoccus a@actiae B (21) c105tridiu?n perfrinRcns (27) Enterococctts faecalis (47) Enterococcus faecium (47) Listeria mo#wcytogeneS(20) Branhamella catarrhalis (27) Haemophilus iyluenzae (21) b Eacteroides fragilis (15) Aeromonas spp. (21) Kkbsiella pneumoniae (20) Acinewbacter anitratus (20) Pseudomonas aer&noso (11) Xanthomonas malwphilia (9) Enterobacter aerogenes (19) Escherichia coli (21) Citrobacter fieundii (20) Yersinla spp. (20) Protercs mkbilis (16) Proteus vulgaris (10) Providencia rettgerii (10) Providencia stuartii (10) Morganella mor*anii (10)

Men 10788 0.0075 4 0.015 0.06 0.06

0.06 0.5 8 %4

MIc90 (ClghnL) lmipenem Amp. - Sulb. 0.015 1 >32 32 0.25 4 0.06 0.03 0.06 0.03 0.015 0.06 0.03 0.12 2 2 %4

0.5 0.25

2 2 1 0.5 16 244

%4

0.03 0.03 1 0.25 0.06 0.25 0.5

8

0.5 0.5 1 4 ~32 16 32 x54 %4 a64

Amoxicillin

1

>32 4 0.015 0.015 0.06 0.06

0.5 32 0.5 2 1 32 ~32 a64

n. t. n. t.

n. t. 4 n. 1. 0.12 8 n. t. 1 n. t. 0.5 %I 0.25 0.5 32 x54 1 2 32 n. t. 2 2 8 n. t. 2 1 32 n. t. 2 2 64 n. t. 2 2 16 n. t * MJCs determined in Mueller Hinton 2 Medium, bioMerieux (aerobes), inoculum: 104 cells/ml, incubation: 24 hours at 37°C: in Wilkins-Chalgren Agar, Oxoid (anaerobes). inoculum: Id cetls/ml, incubation: 24-48 hours at 37°C. a) MJCs determined in Tryptone Soya Agar. Oxoid + 1% Supplement B, Bacto; inoculum: 104 cells/ml, incubation: 24-48 hours at 37°C. b, MJCs determined in Haemophilus Test Medium Bsse. Oxoid + Haemophilus Test Medium Supplement, Oxoid; inoculum: 104 cells/ml, ineubatioa: 24-48 hours at 37°C. z-64

>64

2 0.25

Referencesand Notes 1.

2. 3. 4. 5. 6.

;: 9.

10. Il.

(a) Dthckheimer. W.: Blumbsch. J.: Lattrell. R.: Scheunemsnn.K.H.: Atmew. Chen. Iti. Ed. EnsI. 1985.24.180. Ib) Penone, E:; Franceschi, 6. Synthesisof Pe&ms. In kecent Progress k the khekcal Synthesis of Antikotics &k&s, G.; Ghno, M.; Ms.; Springer Verlaa: Berlin-Heidelberg. 1990; -_ pp 613-704. (c) McCombie, S.W.: Ganguly. - - A.K. Medicinal Research Review; 1988,8.393.EmeSt. I.: Gosteli, J.; Greengrass. C.W.; Holick. W.; Jackman, D.E.; Pfaendler, H.R.; Woodward, R.B. J. Am. Chem. Sot. 1978,100,8214. (a) Franceschi,G.; Foglio. M.; Alpegiani. M.: Battistini, C.; Be&&i, A.; Parone, E.; Zarini. F.; Arcamone, F. J. Antibiotics 1983.36.938. (b) Nishino. T.: Maeda. Y.: Ohtsu. E.: Koixuka. S.: Nishihsm. T.: Adzhi. H.: Okamoto. K.: Hhiauro. M. J. .kibiorics ISib, 42,977.~(c)‘Volkmann, k. A.; Kelbhgh, P. R:; N&on. D. M:; Jt&ys,V. J:J. &g. Chem.~19&,57~4352. Laura, M.; Giannotti, D.; Perrotta.E.: Sbraci, P.; Pestellini, V.; Arcsmone, F.: Sat&G. BieUed. C/rem.L+rff.1993.3.2159. Altamtrm. M.; Perrotta, E.; Sbraci, P.; Pestellini, V.; Arcamone, F.: Cascio, G.: F.; Satta, G. 4th International Symposium on Ihe Chemical synlhesis of Antibiotics and Related Microbiai Products, Nashvilte. lndians (USA), 1994, PosterNo. 1. (a) Alpegiani, M.; Bedeschi, A.; Perrone, E.: Zarini, F.; Frsncesehi. G. Heterocycles 1988 27. 1329. (b) Corrax. AJ.; Dsx, S.L.; Dunlap. N.K.; Georgopapadakou, N.H.; Keith, D.D.: Pruess. D.L.: Rossman, P.L.; Then, R.; Unowsky, J.: Wei. C. J. Med. Chem. 1992,35,1828. Watanabe, N.; K~LSU, K. J. Antibiotics 1993.46, 1707. Hughes, R. A.; Toth, I.; Ward, P.; McCohn. A. M.: Cox, D. ht.; Andemo& G. J.; Gibbons, W. A. J. Pharm. Sci. 1992.81.845. ‘H NMR (288 MHZ. CDCl3) 6 0.04 (6H. s, Si(CH3h. 0.84 (9H. s,Si(CH3)3), 1.21 (3H, d, J 5 6.2 Hz, CH3-C). 2.33 (3H, s, N-CM 3.06 (2H. s, N-CH2). 3.65 (lH, dd, J = 1.6.4.5 Hz, H-6). 3.73 and 3.85 (W, ABq. J = 16 Hx, CH2-2’). 4.14-4.28 UH, m. H-8),4.54475(m, m, COOCH2). 5.15-5.45 (2H. m. CH=!X2). 5.52 (lH, d, J = 1.6 HZ. H-5). 5.756.00 (1H. m, CH=CH2). 6.5 (1H. brs,N-H), 6.8 (1H. br s,N-H). Jeffrey.P.D.; McCombie, S.W. J. Org. Citem. 1982,47,587. Nishi, T.; Higashi, K.; Takemura, M.; Saw, M. J. Antibiotics 1993,46,1740.

(Received in Belgium 28 November 1994; accepted 16 January 1995)