Novel Vancomycin Dimers with Activity against Vancomycin-Resistant Enterococci Uma N. Sundram and John H. Griffin* Department of Chemistry, Stanford UniVersity Stanford, California 94305-5080 Thalia I. Nicas Infectious Disease Research Lilly Research Laboratories, Lilly Corporate Center Indianapolis, Indiana 46285 ReceiVed June 24, 1996 Vancomycin typifies the glycopeptide family of antibacterial agents, which exhibit an unusual, receptor-like mode of action: by binding with high affinity and specificity to the C-terminal L-lysyl-D-alanyl-D-alanine portion of peptidoglycan precursors, vancomycin prevents their incorporation into the polymeric bacterial cell wall. 1 Williams et al. have detailed a second molecular recognition function of glycopeptides, that of self- association into homodimers. They have demonstrated that glycopeptide dimerization can be highly favorable and coopera- tive with the binding of peptide ligands. 2 They have also provided evidence that self-association can lead to enhancements in in Vitro antibacterial potency, 3 consistent with a model in which dimerization increases the intrinsic affinity of glycopep- tides for their peptidoglycan precursor targets, and increases the overall avidity of interaction between self-associated, ditopic glycopeptide receptors and polyvalent ligands presented by the bacterial cell. 3,4 Interestingly, vancomycin self-associates in solution only weakly (K dim ) 700 M -1 ), suggesting that dimerization may not play a significant role in the biological action of this clinically important agent. This prompted us to examine the effects that coValent dimerization would have on the molecular recognition and antibacterial properties of van- comycin. We now report the synthesis of a series of novel bis- (vancomycin)carboxamides and the discovery that some of these compounds exhibit promising in Vitro activity against vanco- mycin-resistant enterococci and selectively enhanced affinity for depsipeptide ligands mimicking peptidoglycan precursors from these organisms. Bis(vancomycin)carboxamides 1a-d (Figure 1) were pre- pared by HBTU-mediated coupling of vancomycin (2.2 equiv) with 1,6-diaminohexane, cystamine, homocystamine, and tri- ethylenetetramine, respectively. 5 The dimers were isolated in 44-68% yield by reversed-phase HPLC and characterized by high-field 1 H NMR spectroscopy and liquid secondary ion (LSI) or electrospray (ES) mass spectrometry. As controls, the monomeric adducts of vancomycin with cystamine (2b) and triethylenetetramine (2d) were prepared by use of excess amine. The in Vitro antibacterial properties of vancomycin, 1a-d, 2b, and 2d were determined by broth microdilution assays. It was found that both monomeric and dimeric vancomycin carboxamide derivatives retained activity against vancomycin- susceptible Gram-positive organisms, including staphylococci and enterococci (Table 1). Strikingly, dimers 1a-c display substantially enhanced in Vitro potency against strains of enterococci which exhibit high-leVel resistance to Vancomycin and other glycopeptides (Vancomycin-resistant enterococci (VRE, VanA phenotype). 6 The average minimum inhibitory concentration (MIC) displayed by bis(vancomycin)cystamide (1b) against VRE (19 μg/mL, 11 μM in vancomycin subunits) is more than a factor of 60 lower than that displayed by vancomycin against these strains and approximately 10-fold higher than the MICs displayed by vancomycin against sus- ceptible enterococci. Dimerization appears to be required for significant activity against VRE, since 2b and 2d did not inhibit (1) (a) Perkins, H. R.; Nieto, M. Pure Appl. Chem. 1973, 35, 371-381. (b) Reynolds, P. E. Eur. J. Clin. Microbiol. Infect. Dis. 1989, 8, 789-803. (2) (a) Mackay, J. P.; Gerhard, U.; Beauregard, D. A.; Maplestone, R. A.; Williams, D. H. J. Am. Chem. Soc. 1994, 116, 4573-4580. (b) Gerhard, U.; Mackay, J. P.; Maplestone, R. A.; Williams, D. H. J. Am. Chem. Soc. 1993, 115, 232-237. (c) Waltho, J. P.; Williams, D. H. J. Am. Chem. Soc. 1989, 111, 2475-2480. (3) Beauregard, D. A.; Williams, D. H.; Gwynn, M. N.; Knowles, D. J. C. Antimicrob. Agents Chemother. 1995, 39, 781-785. (4) Mackay, J. P.; Gerhard, U.; Beauregard, D. A.; Westwell, M. S.; Searle, M. S.; Williams, D. H. J. Am. Chem. Soc. 1994, 116, 4581-4590. (5) Sundram, U. N.; Griffin, J. H. J. Org. Chem. 1995, 60, 1102-1103. (6) (a) Leclerq, R. E.; Derlot, R. E.; Duval, J.; Courvalin, P. N. Engl. J. Med. 1988, 319, 157-161. (b) Uttley, A. H. C.; Collins, C. H.; Naidoo, J.; George, R. C. Lancet 1988, 1, 57-58. (c) Courvalin, P. Antimicrob. Agents Chemother. 1990, 34, 2291-2296. Figure 1. Bis(vancomycin)carboxamides 1a-d. Table 1. In Vitro Antibacterial Properties and Ligand-Binding Affinities of Monomeric and Dimeric Vancomycin Carboxamide Derivatives MIC (μM) a Kd b (μM) cmpd S. aureus (mean) c Enterococcus (mean) d VRE (mean) e Ac2KDADLac Ac2KDADA Van 0.30 1.0 690 1800 ( 65 1.1 ( 0.4 1a 2.0 0.51 32 180 ( 20 1.5 ( 0.1 1b 0.48 0.50 11 440 ( 70 1.9 ( 0.8 1c 3.2 0.29 15 680 ( 160 3.9 ( 0.6 1d 0.46 2.9 >69 1160 ( 380 2.9 ( 0.4 2b 0.08 1.4 >67 2600 ( 120 4.3 ( 0.1 2d 0.40 2.8 >60 3630 ( 230 8.4 ( 0.9 a Minimum concentration of derivative required to inhibit growth of bacterial cells in cation-adjusted Mueller-Hinton broth. For 1a-d, MIC values refer to the concentrations of individual vancomycin subunits. b Dissociation constants determined by competitive titration with AcK(F)DADA in 10 mM HEPES, 6 mM NaCl, pH 7.0, buffer. For 1a-d, Kd values refer to the noncooperative association of individual vancomycin subunits to the ligands. c Average MIC values for 10 vancomycin-susceptible strains of Staphylococcus aureus. d Average MIC values for 4 vancomycin-susceptible strains of Entero- coccus faecium and Enterococcus faecalis. e Average MIC values for 4 strains of E. faecium and E. faecalis exhibiting high-level resistance to vancomycin (VanA genotype confirmed by PCR). 13107 J. Am. Chem. Soc. 1996, 118, 13107-13108 S0002-7863(96)02129-4 CCC: $12.00 © 1996 American Chemical Society