J. Med. Chem. 1993,36, 1971-1976
1971
Discovery and Structure-Activity Relationship of a Series of 1-Carba-1-dethiacephems Exhibiting Activity against Methicillin-Resistant Staphylococcus a ureus Robert J. Ternansky,. Susan E. Draheim, Andrew J. Pike, F. William Bell, Sarah J. West, Christopher L. Jordan, C. Y. Ernie Wu, David A. Preston, William Alborn, Jr., Jeffrey S. Kasher, and Brenda L. Hawkins Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 Receiued February 18, 1993
The synthesis and antimicrobial activity of several new 1-carba-1-dethiacephalosporins is described. The discovery of unique activity of some of the analogues against methicillin-resistant Staphylococcus aureus led to the development of a structureactivity relationship designed to optimize this activity. The results of this investigation along with the pharmacokinetic characteristics of select compounds are described. With the introduction of Lorabid (loracarbef; 1, Figure 1) as a commercial product, the carbacephems have established themselves as a new class of fl-lactam antibiotics available for clinical u8e.l Our efforts in the carbacephem arena have led us to the discovery of a group of compounds which not only display broad-spectrum antibacterial activity but have unique activity against methicillin-resistant Staphylococcus uureus. In this report, we detail our efforts to optimize this activity while maintaining pharmacokinetic characteristics suitable for clinical utility. The carbacephem nucleus is unique in ita ability to provide derivativeswhich are hydrolytically stable relative to comparable cephalosporin analogues,2 which have a sulfur in the 1-position. Thus, 3-substituted-1-carbacephems such as the 3-sulfonyl-1-carbacephems3 or 3-quaternary ammonium4 1-carbacephems (2 and 3, Figure 1) have been prepared and evaluated even though the corresponding cephem derivatives would not be anticipated to be particularly stable. With the consideration of enhanced stability in mind, we set out to prepare a series of 1-carbacephems bearing an aromatic sulfide directly attached to C-3 of the n u c l e u ~ . ~
N
coiCI
C02’
1 (loracarbef)
2
3
Figure 1.
Scheme I HS-AI
NOH
5
Chemistry In order to explore the structure-activity relationships of 3-substituted-1-carbacephems bearing a sulfur atom at the 3-position, we utilized the chemistry depicted in Scheme I. Thus, the previously described4 enol triflate 4 was reacted with the desired thiol 5 in the presence of base to provide the 3-substituted derivative 6. This conversion was sometimescomplicated by formation of the A2adduct (7) which could be separated via chromatography. Removal of the blocking groups present in 6 gave rise to the desired product 8, suitable for antibacterial evaluation. Purification of the final compounds was typically accomplished by reverse-phase chromatography of the sodium salts. Utilizing this synthetic sequence, we were able to prepare a large number of aromatic and heteroaromatic sulfides due to the availability of the corresponding mercaptans. For thiols which were not commercially available, literature procedures were followed for their preparation.
* Present address of corresponding author: La Jolla Pharmaceutical, 6455 Nancy Ridge Dr., San Diego, CA 92121.
7
6
In Vitro Evaluation The initial group of aryl and heteroaryl sulfides prepared is shown in Table I along with their MICs (in pg/mL) against a variety of Gram-positive and Gram-negative organisms. Although these compounds represent very potent antimicrobial agents, there was little advantage seen for any of the compounds when compared with currently marketed antibacterials such as cefotaxime. Importantly, none of the new carbacephalosporin analogues demonstrated any significant activity againststrains of Pseudomonas or methicillin-resistant S.uurew (MRSA; strain X400)(Table I). For these reasons we chose not to pursue further development of these derivatives.6
0022-262319311836-1971$04.00/0 0 1993 American Chemical Society
Ternansky et al.
1972 Journal of Medicinal Chemistry, 1993, Vol. 36, No.14 Table I*
S.aureus compd methicillin cefotaxime
9 lo
X
LY#
X1.l 2 2
- - s a - S C N
LY275837 0.5
E.cl. Pseudomaa K. EB5 X239 PS18 X26 32 >128 >128 >128 128 32
8
0.06
0.03
0.06
0.06
0.5
LY269092 0.5
>128
4
0.03
0.03
0.5
0.06
2
LY242189 0.5
128
64
8
2
0.015 0.03
0.06
0.06
0.5
32
32
64
2
2
1
128 32
8
128
128
4
128
32
LY20984l 0.5
>128
32
LY233742 0.5
-S$I{
S-epi. ~ . p n . ~ . i . E. c. EC14 270 222 PARK C.L. >128 4 4 0 . 5 4 0.03 8 2 0.015 0.015 0.5 2 2 0.015 0.03 0.06
LY233687 0.5 LY242436 2
12
X400 S13E 64 8 64 8 128 64
128
128
16 >128
I
LY269879 2
LY206513 0.125
19
-s'c"n
LY206514 2
128 16
0.03
128 >128 128
16
>128 >128
128 >128
N
LY242551 4
22
-s-q
S
>128 >128
LY231301 0.125
64
32
2
0.5
99.2 36.0 94.0
Test compound concentration = 20 pg/mL.
The exciting results presented herein are a prelude to definitivein uiuo data in a suitable animal model. Should the results of such a s t u d y prove positive, consideration willbe given for possible clinical evaluation of 47 in h u m a n subjects. Experimental Section All reactions described herein were performed under an inert atmosphere of dry nitrogen in flame-dried glaseware unless otherwise noted. All reagents were used as suppliedunless stated otherwise. Melting points were recorded on a Thomas-Hoover apparatus and are uncorrected. 1H NMR spectra were recorded at 300 MHz with a General Electric QE-300 instrument, at 270
1
4
6
(hours)
Figure 2. Plasma levels of antibacterial activity in rata (20 mg/ kg) and monkeys (30mg/kg) following iv administration. MHz with a Brucker W-M instrument, and at 90 MHz with a JOEL FX-90instrument. Chemical shifts are recorded in parta per million ( 6 ) relative to tetramethylailane. IR spectra were recorded on a Nicolet MX-1 FT-IR, optical rotations were measured on a Perkin-Elmer 241 spectrometer, and UV spectra were obtained on a Cary 219. The mass spectral data were obtained on either a CEC-21-140 or a Varian MAT-731 spectrometer. All W L C separations were conducted on Merck Lobar columns (LiChroprep RP-18) with the help of a Fluid Metering Inc. pump. Analytical HPLC separations were performed on a Varian chromatographic system utilizing a MicroPak MCH-5Ncap 15cm X 4 mm column and avariable-wavelength UV detector set to record at 254 nm. General Procedure for t h e Preparation of l-Carba-1dethiacepheme. The compounds described in this report were prepared according to the method outlined below. Preparation of Compound 4. Compound 4 was prepared in a manner described in ref 4 utilizing [2-(tritylamino)-4thiazolyl][ (2-fluoroethyl)oximino]acetic acid.18
1976 Journal of Medicinal Chembtry, 1993, Vol. 36,No.14
Ternaneky et 01.
References m763 .-"&,.".
1
I
0
2
4
TIME
'
CpTRlAYoM
I
6
.
I
8
.
1
10
(hours)
Figure 3. Plasma levels of antibacterial activity in male rhesus monkeys followingiv administration of compounds at 30 mg/kg. Preparation of Compound 6. Sodium hydride (1.6 equiv) and the appropriate heterocyclic thiol (1.5equiv) were stirred in THF followed by the triflate 4. This solution was allowed to stir overnight at room temperature. The solvent was removed in UUCLU), and the crude was taken up in CH2C12,fiitered, and purified by flash chromatography on silica using&75% EtOAc/hexanes. Preparation of Compound 8. Triethylsilane (5 equiv) was dissolved in formic acid (1:lO) and added to 6. After ca. 1 h, diethyl ether was added, and the volatile8were removed in vacuo. The resulting solid was triturated with diethyl ether and purified as the sodium salt by MPLC (MeOH/HIO solvent system). Pharmacology. Male SpragueDawley rats (n= 4) were dosed intravenouslywith LY206763at 20 mg/kgin 0.9 % saline. Dosing and blood samplingwere carried out through an indwellingjugular vein cannula, thus permitting serial sampling from individual rate. Plasma levels and cumulative urinary recoveries were determined from samples collected over a 6-h time course. Male rhesus monkeys (n = 4) were dosed intravenously with test compounds at 30 mg/kg in 0.9% saline. Plasma samples were collected from the cephalic vein over an 8-h time course, along with the 0-24-h cumulative urinary recovery. Urine was collected in 0.1 M sodium citrate buffer, pH 6.6, from animals placed in metabolism cages. Plasma and urine samples were stored at -70 O C prior to analysis. Plasma half-life was calculated as 0.693/8, where 8 is the slope of the terminal portion of the plasma vs time curve. The area under the curve was calculated using Simpson's rule. Urinary recovery was calculated as the percent of the administered dose recovered in the urine. In vitro plasma protein binding was determined in pooled samples of ICR mouse, Sprague-Dawley rat, rhesus monkey, or human plasma at a drug concentration of 20 &I&, using an ultrafiltration technique (Centrifree micropartition system). Antibiotic concentrations were determined with an agar well diffusion assay (bioassay) employing Escherichia coli (ATCC4157) or Micrococcus luteus (ATCC9241) as the bacterial test strain. Standard curves from rat, monkey, or human plasma spiked with the compound under study were employed for analysis of plasma samples. Urine samples were analyzed by comparison to a standard curve prepared in 0.1 M sodium citrate buffer, pH 6.5. Urine samples were diluted with citrate buffer so that the drug concentration would fall into the range of the standard curve.
Acknowledgment. The authors would like to express their appreciation to Dr. F. T. Counter and his associates for the antibacterial screening and the Physical Chemistry department for spectral data. SupplementaryMaterial Available: 1H NMR,IR,and MS for final compounds reported herein (8 pages). Ordering information is given on any current masthead page.
(1) Cooper, R. D. G.In The Chemistry of Beta-Lactam; Page, M. I., Ed.; Chapman and Hall: New York, 1992; pp 272-306. (2) Blnacz&L.C.;Brown,R.F.;Cook,G.K.;Hornback, W. J.;Hoying, R. C.; Indelicato, J. M.; Jordan, C. L.; Katner, A. 5.;Kinnick, M. D.; McDonald, J. H., Du,Morin, J. M., Jr.; Munroe, J. E.; Pasini, C. E. ComparativeReactivity of 1-Carba-1-dethiacephalosporins with Cephalosporins. J. Med. Chem. 1990,33,1666-1662. (3) Crowell, T. A.; Hnlliday, B. D.; McDonald, J. H., Du,Indelicato, J. M.; Pasini, C. E.; Wu, C. Y. E. 3-Sulfonyl-1-dethiacephem. J. Med. Chem. 1989,32,2436-2442. (4) Cook, G.K.; McDonald, J. H., III; Alborn,W., Jr.; Boyd, D. B.; Eudaly, J. A.; Indelicato, J. M.; Johnson, R.; Kaaher, J. 5.;Pasini, C. E.; Preston, D. A.; Wu, C. Y. E. 3-Quarternary Ammonium 1-Carba-1-dethiacephem. J. Med. Chem. 1989, 32, 2442-2460. (5) (a) Ternaneky, R. J. US. Patent # 6,077,287, 1991. (b) Cephem analogues of some of the compounda reported herein have been recentlydeacribed: Tsushima,M.; Tamura,A.; H.sra,T.; Iwamatau, K.; Shibahara, S. 32nd Interscience Conferenceon Antimicrobial Agents and Chemotherapy,1992, Abstract no. 394. (c) Farina,V.; Baker, S. R.; Hauck, S. I. A General Route to 8-bctionalized 3-Norcephalosporins. J. Org. Chem. 1989,64,4962-4966. (6) Although the hydrolytic stability of these cephalosporinswas not measur-ed, they appeared to be quite etable d aqueous manipulation as was anticipated. (a) Crossley,K.; Loeach, D.;Landeaman,B.; Mead, K.; Chern, M.; Strate, R. An outbreak of infections c a d by strains of Staphylococcus aureus resistant to methicillin and aminoglymidee. 1. Clinical studies. J. Infect. D i . 1979,139,273-279. (b) Klimek, J. J.; Mamik, F. J.; Bartlett, R. C., Weir, B.; Shea, P.; Quintilkmi, R Clinical, epidemiologic and bacteriologic observations of an outbreak of methicillin-resistant Staphylococcusaurew at a large communityhospital. Am. J. Med. 1976,61,340-34& (c) Sapico, F. L.; Montgomery, J. Z., Canawati, H. N.; Aeilta, G.Methicillinresistant Staphylococcus aureus bacteriuria. Am. J. Med. Sci. 1981,281,101-109. (d)Barrett, F. F.; McGehee,R. F., Jr.;Finland, M. Methicillin-resistant Staphylococcus aureus at Boston City Hoepital: Bacteriologicand epidemiologicobservations. N.Engl. J. Med. 1968,279,441-448. (e) Myers,J. P.; Linnemaun,C. C. Jr. Bacteremia due to methicillin-resistant Staphylococcus aureus. J. Infect. Die. 1982,146,632-636. Klooe, W. E.; Jorgensen,J. H. In Manwl of ClinicalMicrobiology, 4th ed.;Lennette, E. H., Balom, A., Haueler, W. J., Shadomy, H. J., Ede.; American Society for Microbiology: Washington D.C., 1987; p 143. Fontana, R. Activity of penems against methicillin-resistant Staphylococcusaureus. J. Antimicrob. Chemother. 1988,22,88. (a)Tompset,R;Schultz,S.; McDermott,W.The Relationof Protein Binding to the Pharmacology and Antibacterial Activity of Penicillins X,G,Dihydro F, and K1. J. hcteriol. 1947,63,681-696. (b) Merrikin, D.J.; Briant, J.; Rolinaon, G.N. Effect of protein binding on antibiotic activity in vivo. J. Antimicrob. Chemother. 1983,11,233-238. (c) Tawara, S.; Mataumoto, S., Kamimura, T.; Gob, 5.Effect of Protein Bindingin Serum on TherapeuticEfficacy of CephemAntibiotics. Antimicrob. Agents Chemother. 1992,36, 17-24. PBP2a is unique to MRSA and by virtue of ita low binding affinity for lacta tams is thought to be a mediator of the observed mistance: Hartman, B. J.; Tomasz, A. Low-Affinity PenicillinBinding Protein Associated with &Lactam Resistance in Staphylococcus aureus. J. Bacteriol. 1984,168, 613-616. (a) Chambers,H. F.; Sachdeva, M.; Kennedy, S. Binding Affiity for Penicillin-BindingProtein 2a Correlate with In Vivo Activity of @-LactamAntibiotics against Methicillin-Reeitant Staphylococcus aureus. J. Infect. Dis. 1990,162,706-710. (b) Pierre, J.; Williameon, R.; Bornet, M.; Gutmann,L. Presenceof an Additional Penicillin-BindingProtein in Methicillin-ReaidantStaphylococcus epidermis,Staphylococcwhoemolytkua,Staphylococcushominia, and Staphylococcuss h u k r n s with a Low Affiity for Methicillin, Cephalothin and Cefamandole. Antimicrob. Agents Chemother. 1990,34,1691-1694. (c)T o w , A,; Drugeon, H. B.; delencastre, H. M.; J a b , D.;McDougal, L.; Bille, J. New Mechanism for Methicillin Resistancein Staphylococcusaureus: ClinicalIsolates ThatLackthePBP 2aCeneandContainNormalPenicillin-Bindine Proteins with ModifiedPenicillin-BindingCapacity. Antimicrob. Agents Chemother. 1989,33,1869-1874. Yoshida, C.; Tanaka, K.; Todo, Y.; Hattori, R; FuLuoka, Y.; Komateu, M.; Saiknwa, I. Studies on Monocyclic B-Lactam Antibiotics. J. Antibiot. 1986,39,90-100.
