Iron transport mediated drug delivery systems: synthesis and

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Bioconlugte a m . 1881,2, 281-291

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Iron Transport Mediated Drug Delivery Systems: Synthesis and Antibacterial Activity of Spermidine- and Lysine-Based Siderophore-&Lactam Conjugates Julia A. McKee, Sushi1 K. Sharma, and Marvin J. Miller' Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556. Received February 4,1991 Conceptually, the penetration of &lactam antibiotics through the bacterial cell wall can be enhanced by utilizing various active transport systems such as the active iron-transport system that exists in bacteria. Several 8-lactam-siderophore analogue (microbial iron chelators) conjugates were prepared to investigate this approach. Thus, monocyclic (oxamazin) and bicyclic (carbacephalosporin)&lactams were separately linked to dihydroxybenzoyl derivatives of spermidine and lysine. The antibacterial activity of these conjugates has been investigated and the potential use of the iron-transport system as a drug-delivery system is discussed.

INTRODUCTION The construction of naturally occurring and synthetic 8-lactams, with control of functional groups and stereochemistry, has been the goal of synthetic organic chemists for the past 50 years. The various ways of constructing the @-lactamring system have been reviewed (I). However, bacteria have evolved defense mechanisms to protect themselves from the intruding antibiotics. These include (2): (1)mutations that decrease the affinity of essential penicillin-bindingproteins (PBP)for &lactame,(2)&lactamase production, and (3) inhibition of 8-lactam penetration to the PBP targets in the cytoplasmic membrane. The last mode of defense is a serious problem associated with Gram-negative bacteria in which the outer cell membrane presents a significant permeability barrier to many 8-lactam antibiotics. This may be the primary cause of the high resistance of Pseudomonas aeruginosa to many 8-lactam antibiotics (3). The iron-binding proteins transferrin and lactoferrin, in combinationwith antibiotics, often show powerful bacteriostatic effects in vitro and are essential for protection against many infections (4). It has been established that iron-chelating compounds (siderophores)form complexes with Fe(II1) and are readily accepted into the bacterial cell via specific iron-transport systems (5). Neilands and Valenta have published an excellent review of ironcontaining antibiotics (6). The albomycins and ferrimycine A1 are natural examples of siderophore-containing antibiotics that are incorporated into bacterial cells by the use of the iron-transport system (7). Sulfonamides have been attached to the natural siderophores ferricrocin and ferrioxamine B,and these conjugates have been shown to inhibit the growth of Staphylococcus aureus (8). Recently, our laboratory reported the synthesis and antimicrobial activity of several b-lactam-hydroxamatebased siderophore conjugates and have established that these 8-lactam-siderophore conjugate systems may be incorporated into the microbial cell through the irontransport mechanism (9). The syntheses of semisynthetic &lactam derivatives having catechol moieties have been accomplished by several groups, and the biological activities of such derivatives against several Gram-positive and Gram-negativebacteria were reported (10). Several of these catechol-containingantibiotics have been shown to rely on the tonB iron-transport system for their activity (IOc,d,11). The succesefulincorporation of a new catechol-

containing cephalosporin, E-0702,into Escherichia coli using an iron-transportsystem has been recently reported (12). The albomycins are the best known examples of natural hydroxamate siderophore containing antibiotics; however, there are no examples of natural catechol siderophore antibiotics. This paper describes our first attempts to link 8-lactam antibiotics with catechol siderophore analogues and discusses the growth-promoting ability of the siderophore analogues and the preliminary studies of the antimicrobial activity of the siderophoreantibiotic conjugates. Agrobactin (la) and parabactin (lb) are catecholcontaining, spermidine-based siderophores (Figure 1). These microbial iron chelators are being studied as potential drugs to treat iron overload resulting from treatment of Cooley's anemia (131, and are very effective in deferration of mammalian cell lines (14). Recently, an analogue of these spermidine-basedsiderophores la and lb was synthesized in our laboratory (15). This synthetic siderophore, spermexatol [2] (2, Figure l), was shown to stimulate the growth of E. coli mutants and required the enterobactin receptor (16) for activity. Spermexatol [2] also stimulated growth of Vibrio cholerae but did not require the vibriobactin receptor. Due to its growthpromoting abilities, spermexatol [2]was chosen as the siderophore component for our initial synthetic efforts. In addition, a lysine-based catechol siderophore was (3)was chosen. Nu,M-Bis(2,3-dihydroxybenzoyl)-~-lysine isolated from iron-deficient cultures of Azotobacter uinelandii and its structure was established by synthesis (17). Another synthesis was recently reported by Chimiak and Neilands (18). A new class of monocyclic @-lactamantibiotics, [ [3(S)(acylamino)-2-oxo-l-azetidinyl]oxy]aceticacid (oxamazins), has been recently synthesized in our laboratory (19). These compounds have shownpotent activityagainst Gram-negative bacteria even though the ionizable carboxyl group is displaced one atom further from the @-lactam nitrogen than in the penicillins, cephalosporins, and norcardicins. Due to the bactericidal activity of the oxamazins, the oxamazin-siderophore conjugates 4 and 6 were chosen as synthetic target compounds (Figure 2). Since cephalosporin derivatives are generally broader spectrum antibiotics than monobactams, carbacephalosporin-siderophore conjugates 6 and 7 were also selected for study (Figure 3). In addition, neither the oxamazin @ 1991 American Chemlcal Soclety

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Bbcmmte Chem., Vol. 2, No. 4, 1991

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@H

1 a Agrobacth, R = OH l b Panbadin.R=H

6

H CONH T

2 Spermexatok[2]

Figure 3. Siderophore-carbacephalosporin conjugates.

3 Na,Nm-Bis(2,3dhydroxybenroyl)lysine

Figure 1. Catechol siderophores. CH3

HO

H 4

Figure 2. Siderophore-oxamazinconjugates. nor the carbacephalosporin contain sulfur, thereby allowing the use of hydrogenolysis as a convenient means of deprotection of the synthetic conjugates. EXPERIMENTAL PROCEDURES

General Methods. Melting points were taken on a Thomas-Hoover capillary melting point apparatus and are uncorrected. Infrared spectra (IR) were recorded on a Perkin-Elmer 727B or Perkin-Elmer 1420 spectrophotometer. Proton NMR spectra were obtained on a Magnachem A-200 or General Electric GN-300 spectrometer. Chemical shifts are reported in ppm relative to tetramethylsilane (6 units). Carbon-13 NMR were obtained on a General Electric GN-300 spectrometer. Carbon NMR referenceswere the center peak of deuterochloroform (77.0

ppm) or the center peak of deuteromethanol(49.0 ppm). Electron-impact mass spectra, chemical-ionization mass spectra, and fast atom bombardment were performed by Dr. Bruce Plashko on an AEI Scientific Apparatus MS 902 or Du Pont DP 102 spectrometer. Field-desorption mass spectra were obtained by Dr. John L. Occolowitz (Eli Lilly and Co.). Elemental analyses were performed by M-H-W Laboratories, Phoenix, AZ. Radial chromatographywas performed with a ChromatotronModel 7924 purchased from Harrison Research Inc., Palo Alto, CA. Flash silicagel chromatography was performed with Merck silica gel 60. Opti-up c18 glass-backed plates, purchased from Fluka Chemical Corp., were used for reversed-phase TLC. Whatman #1filter paper was used for paper chromatography. Solvents used were dried and purified by standard methods. The term "dried" refers to the drying of an organic solvent over anhydrous magnesium sulfate. Carbacephalosporin24 was kindly provided by Eli Lilly and Co., Indianapolis, IN. N-(tert-Butoxycarbonyl)-L-threonine(9). To a 60lution of L-threonine (40.50g,O.34 mol) and NaHCOs (29.40 g, 0.35 mol) in water was added a solution of BoczO (74.80 g, 0.34 mol) in THF at room temperature. The solution was stirred overnight. The solution was made acidic with 10% citric acid and extracted with ethyl acetate. The organic layer was dried and filtered and the solvent was removed under reduced pressure to give compound 9 in 81% yield (59.66 g) as a white solid: mp 76-77 OC (lit. (20) mp 76-80 "C); IR (TF) 3600-2200 (br), 1770 (br), 1720 cm-l, lH NMR (200 MHz, CDCh) 6 1.22 (d, J = 12 Hz, 3 H, CH3), 1.42 (s,9 H, C(CH&), 4.14-4.58 (m, 2 H, CHI, 5.74 (br 8, 1 H, NH), 7.58 (br 8, 1 H, COOH). O-BenzylN-(tert-Butoxycarbonyl)-L-threoninehydroxamate (11). N-Boc-(&threonine (9) (20.0 g, 91.24 mmol) and N-hydroxysuccinimide (11.51 g, 101 mmol) were dissolved in dry THF and cooled to 0 OC (ice bath). To this was added slowly a solution of DCC (20.63 g, 100 mmol) in dry THF. The reaction mixture was stirred for 14 h. Dicyclohexylurea was removed by filtration, and the solvent was removed under vacuum. The oily residue was taken up in dry THF and a solution of O-benzylhydroxylamine (10) (13.47 g, 110 mmol) in THF was added. The solution was stirred overnight at room temperature. The solvent was evaporated and the residue was taken up in ethyl acetate, washed with 10%citricacid, 5% NaHCOa, and water, dried, and filtered. The solvent was removed under reduced pressure and the product was recrystal-

~kconlugetechem.,VOI. 2, NO. 4, 1991 28)

Iron Transport Mediated Drug Dellvery Systems

lized from ethyl acetate/hexanes to provide the product in 77% yield (22.83 g) as a white solid: mp 86-88 "C; [ c X ] ~ D= -39.0" (c = 5.0, CHCb); IR (KBr) 3500-3180 (br), 2970,1670,1530 cm-1; 1H NMR (200 MHz, CDCl3) 6 1.12 (d, J = 10 Hz, 3 H, CH3), 1.44 ( ~ , H, 9 C(CH3)3),3.91-3.97 (m, 1 H, CHOH), 4.34-4.18 (m, 1 H, NHCHCO) 5.00 (8, 2 H, benzylic H), 6.02 (d, J = 10 Hz, 1 H, NH), 7.52 (m, 5 H, aromatic H), 10.3 (br 8, 1 H, NH); TLC (ethyl acetate/ hexanes, 1:l) Rf = 0.29, (ethyl acetate) Rf = 0.75. Anal. (C16HdzOd c , H, N. N-(Benzyloxy)-4(@-methyl-3(@-[ (tert-butoxycarbonyl)amino]-2-azetidinone(12). Hydroxamate 11 (12.0 g, 37 mmol), triphenylphosphine (9.703 g, 37 mmol), and CC& (5.7 mL, 37 mmol) were dissolved in 150 mL of dry acetonitrile under nitrogen and triethylamine (3.74 mL, 37 mmol) was added at room temperature. The reaction mixture was followed by normal-phase TLC on silica gel. The solution was stirred for 2 h, the solvent was removed under reduced pressure, and the oily residue was taken up in ethyl acetate. Some of the triphenylphosphine oxide was removed by filtration, and the solution was washed with water, brine, dried, and filtered. The solvent was evaporated and the product was purified by flash column chromatography from ethyl acetate/hexane (1:3) to provide the product in 75 % yield (8.43 g) as needles and plates after crystallization from ethyl acetate/hexanes: mp 7577 "C; [Ct]''D = -11.9' (c = 5.0, CHCl3); IR (KBr) 3300, 3040,2980,1780,1710 cm-l; lH NMR (200 MHz, CDC13) 6 1.20 (d, J 6 Hz, 3 H, CH3), 1.42 (9, 9 H, C(CH&), 3.50-3.82 (m, 1 H, CHCHs), 4.02-4.38 (m, 1 H, COCH), 5.08 ( 8 , 2 H, benzylic H), 5.90 (br 8, 1 H, NH), 7.58 ( 8 , 5 H, aromatic H); TLC (ethyl acetate/hexanes, 1:3) Rf = 0.44, (ethyl acetate),Rf = 0.82. Anal. (C16HzzN204)C, H, N. N-Hydroxy-4(@-methyl-3( @-[ (brt-butoxycarbonyl)amino]-2-azetidinone (13). O-Benzyl 8-lactam 12 (3.525 g, 11.52 mmol) was dissolved in 150mL of methanol and 10% Pd/C (20%w/w) was added. Hydrogen gas was bubbled through the solution. After 1 h, Pd/C was removed by filtration and the methanol was evaporated under reduced pressure to provide the product in 94% yield (2.318 g) as a white solid. There was no need for further purification: mp 153-154 "C; IR (KBr) 3360,2930, 1780,1690 cm-l; lH NMR (200 MHz, acetone-d6) 6 1.20 (d, J = 6 Hz, 3 H, CH3), 1.30 (8, 9 H, C(CH&), 3.00 (br 8, 1 H, OH), 3.58-3.88 (m, 1 H, NCH(CHa)), 3.90-4.18 (m, 1 H, NHCHCO),6.64 (br 8, 1 H, NH); TLC (ethyl acetate) Rf = 0.43. Anal. ( C ~ H I ~ N ZC, O~ H,) N. Benzyl [[4(s)-Methyl-3(@-[(tert-butoxycarbonyl)amino]-2-oxo-l-azetidinyl]oxy]acetate(14). NHydroxy 8-lactam 13 (2.07 g, 9.57 mmol) and K2CO3 (1.323 g, 9.57 mmol) were dissolved in water and a solution of benzyl bromoacetate (1.52 mL, 9.57 mmol) in THF was added. The mixture was stirred overnight. The solution was concentrated and extracted with ethyl acetate. The organic layer was separated and washed with 0.5 9% NaOH and water, dried, and filtered. The solvent was removed in uucuoto provide the crude product in quantitativeyield (3.596 g) as an oily residue. The product was purified by silica gel column chromatography using ethyl acetate/ hexanes (1:4) as the elutant to give 79% yield (2.746 g) of 14 as a colorless oil: [CtIBD = -41.3" (c = 5.2, CHCl3); IR (TF) 3350, 3040, 2980, 1780, 1710 cm-l; lH NMR (300 MHz, CDClS) 6 1.40-1.50 (m, 12 H, CHS and C(CH&), 3.84 (dq, J = 2 and 6 Hz, 1 H, NCH(CHd), 4.09 (m, 1 H, NHCHCO), 4.61 (AB q, J 17 Hz, 2 H, OCH&O), 5.21 (AB q superimposedover a br s , 3 H, NH and benzylic H), 7.36 (8, 5 H, aromatic H); 13C NMR (75 MHz, CDCl3) 6 15.82,27.93,60.12,63.71,66.70,72.38,79.83,128.19,128.28,

134.63, 154.73, 162.59, 167.89; TLC (ethyl acetate/hexanes, 1:2) Rf = 0.24. Anal. (CdIuNzO6) C, H, N. 2,3-Bis(benzyloxy)benzoic acid N-succinimide 8ster (15) was prepared by the method of Chimiak and Neilands (18)in 88% yield after crystallization from ethyl acetate/hexanes: mp 105.5-107.5 "C (lit (18)mp 112-114 "C);IR (KBr) 1770,1740 cm-l; 'H NMR (300 MHz, CDCls) 6 2.86 (a, 4 H, COCHz), 5.14 (s,4 H, benzylic H), 7.10-7.60 (m, 13 H, aromatic H); exact MS (EI) calcd for c~H21NOs mlz 431.1369, found m/z 431.1367. Na,N-Bis[2,3-bis(benzyloxy)benzoyl]-~-lysine ( 16). N-Succinimideactive ester 15 (3.000g, 6.95 mmol), L-lyaine monohydrochloride (0.631g, 3.45 mmol),and triethylamine (1.470 g, 14.54 mmol) were dissolved in a homogenous mixture of acetone/ water and stirred for 24 h. The solution was concentrated and the residue was dissolved in ethyl acetate, washed with 1 N HCl, water, and brine, dried, filtered, and concentrated to provide 2.69 g (100%) as a = +8.8" (c = 1.0, CHC13); IR (TF) colorless oil: 3380 (br),1710,1660 (br) cm-l; 'H NMR (300 MHz, CDCb) 6 1.00-1.95 (m, 6 H, CHz), 3.02-3.24 (m, 2 H, CH2), 4.65 (m, 1 H, CH2CHNH), 4.97-5.22 (merging singlets, 8 H, benzylic H), 7.03-7.80 (m, 26 H, aromatic H), 7.93 (br 8, 1 H, NH), 8.58 (br s, 1 H, NH); 13CNMR (75 MHz, CDCb) 623.01,28.70,39.43,71.01,71.10,75.82,76.12,77.20,116.84,

124.06,128.52(m), 136.45 (m),146.66,146.78,151.53; TLC (methanol/CHCls, 15) Rf = 0.56; MS (positive-ion FAB, glycerol) m/z 779 (M + l),817 (M K+).

+

N~,N-Bis[2,3-bis(benzyloxy)benzoyl]-~-lysine NSuccinimide Ester (17). To a mixture of compound 16 (0.508g, 0.651 mmol) and N-hydroxysuccinimide (0.075 g, 0.651 mmol) in anhydroustetrahydrofuran at 0 "C under nitrogen was added a solution of dicyclohexylcarbodiimide (0.148 g, 0.717 mmol) in anhydrous tetrahydrofuran dropwise. The solution was warmed to room temperature and stirred overnight. The solvent was evaporated and the residue was dissolved in benzene and fiitered to remove dicyclohexylurea. The fiitrate was concentrated to provide 0.569 g (100%)of an oil which was used without further purification: IR (TF)3480,2940,1740,1650~m-~; lH NMR (300 MHz, CDCls) 6 1.10-2.00 (m, 6 H, CH2), 2.75 (s,2 H, COCH2),3.05-3.15 (m,2 H, CH2),4.95 (m, 1 H, NHCHCO), 5.04-5.12 (merging s,8 H, benzylic H), 7.00-7.90 (m, 27 H, aromatic H and NH), 8.50 (d, J = 11.6 Hz, 1 H, NH); TLC (ethyl acetate/hexanes, 3:l) Rf = 0.51; MS (positiveion FAB, m-nitrobenzyl alcohol/glycerol) m/z 876 (M+ 1).

lW,N-Bis(2$-dihydroxybenzoyl)-~-lysine(3). Compound 16 (0.223 g, 0.286 mmol) and 10% Pd/C (0.045 g, 20% w/w) were dissolved in 2.0 mL of methanol and exposed to hydrogen at atmospheric pressure for 4 h. The catalyst was removed by filtration and the filtrate was concentrated to provide 0.106 g (100%) of a glassy oil: FeC13 positive (red-purple); IR (TF) 3650-2300 (br), 1730 cm-l; lH NMR (300 MHz, methanol-d4) 6 1.51-2.10 (m, 6 H, CHZ),3.30-3.42 (m, 2 H, CH2), 4.63 (m, 1 H, CHI, 6.666.75 (m, 2 H, aromatic H), 6.92-6.98 (m, 2 H, aromatic H), 7.20 (dd, J = 1.5 and 8.1 Hz, 1 H, aromatic H), 7.35 (dd, J = 1.5 and 8.1 Hz, 1 H, aromatic H); 13CNMR (75 MHz, methanol-&) 6 24.31, 29.88, 40.15, 53.94, 116.71, 116.92, 118.61,119.42,119.52,119.72,147.05,147.12,149.63,150.09,

170.86, 171.40; reverse-phase TLC (isopropyl alcohol/ water, 1:l) Rf = 0.45; MS (positive-ion FAB, m-nitrobenzyl alcohol/glycerol) m/z 419 (M + 11, 441 (M + Na+). With the procedure of Raymond (211, the formation constant for the 3:2 siderophore-iron (111) complex of compound 3 was measured. Spectrophotometriccompetition experiments were performed on the iron(1II) complex

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as 22 as an oil in 61% yield: IR (TF) 3380 (br), 1770,1710, 1680,1610 (br) cm-l; lH NMR (200 MHz, CDCl3) 6 1.101.78 (m, 9 H, NCH(CH3) and CHz), 3.05-3.22 (m, 2 H, CH2),3.88-4.01 (dq, J = 2 a n d 6 H z , lH,NCH(CH3)),4.24 (m, 1H, NHCHCO), 4.47-4.61 (m, 3 H, CHzCHNH and OCH&O), 5.00-5.24 (m, 10 H, benzylic H), 7.04-7.95 (m, 37 H, aromatic H and NH), 8.54 (d, 1H, NH); TLC (ethyl acetate) Rf = 0.54. Anal. (C~1HsoN4011)C, H, N. [[4(S)-Methyl-S(S)-[ [[N1JV8-bis(2,3-dihydroxybenzoy1)spermidin-N4-yl]succinyl]amino]-t-oxo-l-azetidinyl]oxy]acetic acid (4) was prepared from ita benzyl protected precursor 22 upon catalytic hydrogenation using 10%Pd/C (20%w/w) inmethanolfor 1h. After filtration to remove the catalyst and concentration, compound 4 was obtained in 97 7% yield as a colorless oil which required no purification: FeC13 positive (red-purple); IR (TF) 3380 (br), 1750 (br), 1660 (br) cm-I; 'H NMR (200 MHz, methanol-&) 6 1.10-1.77 (m, 11H, NCH(CH3) and CHd, 2.22-2.49 (m, 4 H, CHz), 3.02-3.25 (m, 8 H, CH2), 3.73 (m, 1 H, NCH(CHd), 3.98 (m, 1 H, NCHCO), 4.30 ( 8 , 2 H, OCH&O), 6.47 (m, 2 H, aromatic H), 6.70 (m, 2 H, aromatic H), 6.98 (m, 2 H, aromatic H); reverse-phase TLC (2-propanol/HzO, 1:l)Rf = 0.71; paper chromatography(2-propanol/HzO, 3:l) Rf = 0.61; MS (FD) m/z 674 (M + 1). [[4( S)-Methyl-3(S)-[[Na,Nc-bis(2,3-dihydroxybenzoyl)-~-lysyl]amino]-2-oxo-1-azetidinyl]oxy]acetic acid (5) was prepared from 23 in a manner similar to that for 4 and was obtained in quantitative yield as a colorless oil 28.87,29.51,30.21,37.59,37.99,39.82,40.00,44.35,46.68, which required no further purification: FeCl3positive (red47.83,48.54,116.55,116.68,118.53,118.62,119.62,147.17, purp1e);IR (TF) 3700-2300 (br),1780-1720 (br),1650(br) 150.15 (m), 171.43,171.56,173.64,174.22,176.66; reversecm-l; lH NMR (200 MHz, methanol-d4) 6 1.33-2.05 (m, 11 phase TLC (isopropyl alcohol/water, 1:l)Rf = 0.64); MS H, NCH(CH3) and CH2), 4.01-4.13 (4, J = 5 Hz, 1 H, (positive-ion FAB, m-nitrobenzyl alcohol/glycerol) m/z NCH(CHs)),4.31 (m, 1H, NHCHCO), 4.52-4.60 (m, 3 H, 535 (M+ HzO), 517 (M+), 499 (M - H2O); SpectrophoOCHzCO and CH2CHC0);6.70-7.31 (m, 7 H, aromatic H tometric competition experiments were performed on the and NH), 8.15 (br s, 1H, COOH); reverse-phase TLC (2ferric iron complex of 20 at pH 7 with a slight excess of propanol/HzO, 1:l)Rf = 0.82; paper chromatography (2EDTA (21). The calculated formation constant for the propanol/HzO, 3:l) Rf = 0.76; MS (FD) m/z 575 (M + 11, ferric complex of 20 was 1027. 597 (M + Na+). Benzyl [[4( S)-Methyl-3(S)-[[[Nl,N s-bis[2,3-bis4-Nitrobenzyl7&[ [[N1,N8-Bis[2,3-bis( benzyloxy)(benzyloxy)benzoyl]spermidin-N4-yl]succinyl]amibenzoyl]spermidin-N4-yl]succinyl]amino]-l-carbano]-2-oxo-l-azetidinyl]oxy]acetate(22). In an oven3-chloro-3-cephem-4-carboxylate (25). Compound 18 dried flask, N-Boc-oxamazin benzyl ester 14 (0.705 g, 1.93 (0.173 g, 0.197 mmol) and carbacephalosporin 24 (0.077 g, mmol) was dissolved in dry methylene chloride under 0.198 mmol) were suspendedin l.OmL of anhydrousmethnitrogen. The solution was cooled to 0 "C (ice bath) and ylene chloride under nitrogen. Triethylamine (0.012 g, trifluoroacetic acid (TFA) (4-6 equiv) was added slowly. 0.197 mmol) and EEDQ (0.063g, 0.255 mmol) were added, The reaction mixture was allowed to warm up to room and the solution was stirred for 19 h. The solution was temperature and further stirred for 30 min. The solvent diluted with ethyl acetate and washed with 0.5 N HCl, and TFA were removed under a vacuum. Traces of TFA water, and brine, dried, filtered, and concentrated to were removed by evaporating the oily residue with toluene provide 0.273 g of a yellow oil. The oil was purified by and hexanes to provide the TFA salt 21 as a pale yellow radial chromatography eluting with 1:20 MeOH/ethyl solid. acetate to give 0.198 g (84%)of 25 as a yellow oil: [CulaaD 'The TFA salt 21 and NaHCOs (1.36 g, 1.93 mmol) were = +10.lo (c = 0.7, CHC13);IR (TF) 3420-2860 (br), 1770, dissolved in water. To this a solution of N-succinimide 1650,1520,1345cm-l; 'H NMR (300 MHz, CDCls) 6 1.20ester 19 (1.72 g, 1.76 mmol) in ethyl acetate was added at 2.00 (m, 10 H, CH2), 2.39-2.70 (m, 6 H, CH2 including room temperature. The reaction mixture was stirred allylic CHz), 3.00-3.34 (m, 8 H, CHd, 3.75 (m, 1 H, C8 overnight and the organic layer was separated. The ethyl CH&H), 5.00-5.40 (m, 11H, benzylic H and C7 NHCH), acetate layer was washed with 1 N HC1, water, 5% 7.11-7.70 (m, 27 H, aromatic H), 7.90-8.20 (m, 4 H, NaHCOs, water, and brine, dried, filtered, and evaporated. aromatic H and NH); l3C NMR (75MHz, CDCh, all signals The crude product was purified by silica gel chromatogat 25 "C reported) 6 21.80,24.84,25.75,26.47,26.60,27.47, raphy, eluting with ethyl acetate to provide 0.920 g (63% ) 28.34, 28.51, 30.98 and 31.60 (allylic), 36.90,38.79, 42.96, of 22 as an oil: IR (TF) 3382 (br), 1760, 1690 (br) cm-l; 45.18,45.25,47.04, 52.36,58.47,65.93,70.99,71.08,76.04 lH NMR (200 MHz, CDCls) 6 1.20-1.67 (m, 11H, NCH(m),77.20,116.54-116.94 (m) 122.72,122.94,123.51,124.29, (CH3) and CH2), 2.41-2.62 (m, 4 H, CHp), 3.01-3.34 (m, 126.78, 126.96, 127.31, 127.52, 128.07-128.61 (m), 130.91, 8 H, CHz), 3.81-3.97 (m, 1 H, NCH(CHa)), 4.11-4.25 (m, 131.18, 136.23,136.31,136.47, 142.13,146.41-146.66 (m), 1H, NHCHCO),4.52 (s,2H, OCHzCO),5.02-5.26 (merging singlets, 10 H, benzylic H), 7.06-8.01 (m,38 H, aromatic 147.53,151.59, 15938,159.91, 164.95-165.33 (m), 171.09, H and two NH); TLC (ethyl acetate) Rf = 0.12. Anal. 171.39,172.91;TLC (ethyl acetate) Rf = 0.13, (methanol/ ethyl acetate, 1:30) Rf = 0.20; MS (positive ion FAB, m-ni(CesHmNsOiz) C, H, N. trobenzyl alcohol/glycerol) m/z 1212 (M + 1). Benzyl [[4( S)-Methyl-3(8)-[[N*,Nc-bis[2,3-bis(benzyloxy)benzoyl]-~-lysyl]amino]-2-oxo-l-azetidi- 4-Nitrobenzyl 7&[[N*,Nc-bis[2,3-bis(benzyloxy)benzoyl]-~-lysyl]amino]l-carba-3-chloro-3-cephemnyl]oxy]acetate (23) was obtained in the same manner

of 3 with a slight excess of EDTA at pH 7. The formation constant for compound 3 was calculated to be lo2'. N4-( 3-Carboxypropanoyl)-Nl,N8-bis[2,3-bis(benzyloxy)benzoyl]spermidine (18) was prepared by the method of Sharma and Miller (15). N4-[3-[(Succinimidooxy)carbonyl]propanoyl]N 1,Ns-bis[2,3-bis(benzyloxy)benzoyl]spermidine (19) was prepared in the same manner as 17 in 88% yield as a colorless oil: IR (TF) 3380, 2920, 1810, 1770, 1730 cm-l; lH NMR (300 MHz, CDCls) 6 1.10-1.80 (m, 6 H, CH~),2.50-2.60(m, 2 H, CHz), 2.72 (s,4 H, COCHz), 2.95 (m, 2 H, CHd, 5.00-5.10 (m, 8 H, benzylic H), 7.00-8.00 (m, 28 H, aromatic H and NH); TLC (methanol/ethyl acetate, 1:30)Rf = 0.44; MS (positive-ionFAB, m-nitrobenzyl alcohol/glycerol) m/z 976 (M + 1). N4-(3-Carboxypropanoyl)-Nl,N8-bis(dihydroxybenzoy1)spermidine (20). Compound l8and 10%Pd/C (0.068 g, 20% w/w) were dissolved in methanol and exposed to hydrogen at atmospheric pressure for 4 h. The catalyst was removed by filtration and the filtrate was concentrated to provide 0.20 g (100%) of a colorless oil: FeCls positive (red-purple); IR (TF)3650-2300 (br), 1710 cm-l; lH NMR (300 MHz, methanol-d4) 6 1.15-2.00 (m, 6 H, CHz), 2.65 (br s, 4 H, CH3, 3.30-3.45 (m, 8 H, CHd, 6.65-6.75 (m, 2 H, aromatic H), 6.94 (d, J = 7.8 Hz, 2 H, aromatic H), 7.22 (d, J = 8.1 Hz, 2 H, aromatic H); 13C NMR (75 MHz, methanol-d4) 6 25.94,26.84,27.55,28.35,

Bbconjt@qte Chem., Vd. 2,

Iron Transport h4edlated Drug Dellvery SySt€"

4-carboxylate (26) was prepared by coupling 17 and 24 in the same manner as 22 to provide 26 in 84% yield as a colorless oil: [ a ] a=~-3.1O (c = 0.9, CHC13); IR (TF) 3380,1775, 1740, 1650, 1525 cm-l; lH NMR (300 MHz, CDCl3) 6 1.W1.40 (m, 6 H, CHd, 1.60-1.90 (m, 2 H, C1 CHCH2), 2.40-2.60 (m, 2 H, allylic CHd, 3.01-3.30 (m, 2 H, CHz), 3.82 (m, 1 H, C8 CH2CH), 4.49 (m, 1 H, NHCHCO), 5.05-5.41 (m, 13 H, C7 NHCH and benzylic H), 7.09-7.68 (m, 26 H, aromatic H), 7.86 (d, J = 8.1 Hz, 1 H, NH), 7.95 (t, J = 5.4 Hz, 1 H, NH), 8.17 (m, 2 H, aromatic H), 8.46 (d, J = 7.2 Hz, 1 H, NH); 13CNMR (75 MHz,CDC&)6 21.78,22.85,28.53,30.59,31.71,38.90,52.38, 53.21, 58.57, 66.05, 75.99, 76.25, 77.20, 116.87, 117.46, 122.96,123.03,123.10,123.61,124.31,124.53,126.15,127.21, 127.56,127.67,128.14,128.23,128.53,128.64,128.74,130.86, 136.30 (m), 142.20, 146.67, 167.88,147.67,151.62, 151.70, 159.96, 165.08, 165.54, 172.26; TLC (ethyl acetate/hexanes, 4 1 ) Rf = 0.53; MS (positive ion FAB, m-nitrobenzyl alcohol/glycerol) m/z 1112 (M+). 784[[N1,iV8-Bis[2,3-bis(benzyloxy)benzoyl]spermidin-N4-yl]succinyl]amino]-l-carba-3chloro-3-cephem-4-carboxylic Acid (6). To a solution of 25 (0.104 g, 0.086 mmol) in 1.0 mL of 5% aqueous dimethylformamide (made from deionized distilled water and HPLC grade DMF) was added 22.0 pL (0.294 mmol, 300 mol % ) of concentrated HC1 and 0.022 g of 10% Pd/C. This mixture-wasexposed to hydrogen at atmosphericpressure for 24 h. The catalyst was removed by filtration. The dimethylformamide/ water mixture was removed by evaporation under high vacuum. The residue was redissolved in methanol and was evaporated repeatedly in an attempt to remove the residual DMF. 'H NMR showed traces of DMF despite this attempt to remove it. Evidence that these types of compounds have a relatively high affinity for DMF has been reported (22). Product 6 was obtained in 100% yield as a dark amber oil: FeC13 positive (redpurple); IR (TF)3590-2600 (br),1760,1650 cm-'; lH NMR (300 MHz, methanol-d4) 6 1.40-2.00 (m, 10 H, CHz), 2.403.00 (m, 6 H, CH2), 3.25-3.50 (m, 8 H, CH2),4.80 (m, 1 H, C8 CHCHz), 5.25 (m, 1 H, C7 CHNH), 6.72 (m, 2 H, aromatic H), 7.20-7.40 (m, 3 H, aromatic H and NH); 13C NMR (75 MHz, methanol-d4,all signals at 25 "C reported) 6 23.22, 26.08, 26.98, 27.63, 28.50, 29.10, 29.61, 31.55 (m, allylic CHz), 32.04,37.64,38.00,39.82,40.07,44.44,46.72, 53.80,116.82,118.59,118.75,119.68,123.02,126.64,127.78, 130.28, 131.45, 147.22, 150.20 (m), 166.81, 171.45 (m), 173.61, 174.17, 175.26; Reverse-phase TLC (Zpropanol/ H20,15:1)Rf= 0.70;paper chromatography(bpropanol/ H20, 3:l) Rf = 0.67; MS (positive-ionFAB, glycerol) m/z 716 (M + 1); exact FAB (M + 1) calcd for CzsHmN4010Cl 617.1650, found 617.1641.

No. 4, 1991 2115

Scheme I

&kOH

0

13

14

glycerol) m/z 618 (M + l), 640 (M+ Na+);exact FAB (M + 1) for C33H&aOllCl calcd 716.2334, found 716.2328. Bioassay Testing Procedures. Plate Bioassay to Measure Siderophore Activity by Zones of Growth Stimulation. E. coli X580 was grown overnight in Luria broth at 300 rpm at 37 "C. The culture was diluted 1:200 OOO into Luria agar containing the deferrated iron chelator ethylenediaminebis(0-hydroxyphenylaceticacid) (EDDA) (100 pg/mL) and the agar poured into sterile Petri dishes and allowed to solidify. Siderophores 3 and 20werepreparedas10mMstocksolutionsin10%aqueous ammonium hydroxide and were diluted to the test concentrations with water and filtered through an AcroDisc 0.2-pmassembly. Sterile 6.5" filter-paper disks containing 5 pL of the compounds were placed on the surface of the agar and the Petri dishes were incubated for 24 h at 37 OC. Diameters of zones of stimulation were measured. Liquid Growth Bioassay for Antimicrobial Activity. The test compounds were prepared as 5 mM stock solutions in dimethylformamide and were added to sterile Luria broth or sterile Luria broth containing deferrated EDDA (100 pg/mL) to give 1.0 or 10 pM final concentration of the conjugates. Ten microliters of a 24-h-old culture of E. coli X580 in Luria broth was added per 50 mL of Luria broth. The bacteria were added immediately after addition of the conjugates or after preincubation of the test compound in the Luria broth for 12 h at 37 OC. The culture 78-[[Na,Ne-Bis(2,3-dihydroxybenzoyl)-~-lysyl]- flasks were then shaken at 37 OC at 300 rpm. Aliquota were removed every 2 h and the culture turbidity was amino]-l-carba-3-chloro-3-cephem-4-carboxylic acid measured at 600 nm. All the assays were performed in (7) was prepared in the same manner as 6 from 26 in 100% duplicate. yield as an amber oil: FeCl3 positive (red-purple); IR (TF) Minimum inhibitory concentration (MIC)values were 3 6 S 2 3 5 0 (br),1770,1650,1540 cm-l; lH NMR (300 MHz, determined by Eli Lilly and Co. using their standard cephmethanol-d4) 6 1.00-2.00 (m, 8 H, C1 CHCH2 and CH2), alosporin broad-screen assay. 2.02-3.30 (m,4H, CH2andallylicCH2obscuredby residual DMF), 3.85-3.95 (m, 1 H, C8 CHzCH), 4.60 (m, 1 H, RESULTS AND DISCUSSION NHCHCO), 5.30 (m, 1 H, C7 NHCH), 6.71 (m, 2 H, aromatic H), 6.98 (t,J = 6.9 Hz, 2 H, aromatic H), 7.28Synthetic Aspects. As in all multistep syntheses, an 7.42 (m, 4 H, aromatic and NH), 8.00 (e, 1 H, COOH); 13C important consideration in the syntheais of these conjuNMR (75 MHz, methanol-d4,all signals at 25 OC reported) gates was the careful choice of protecting groups. The most important factors in the choice of protecting groups 6 23.04, 24.26, 25.84, 26.53, 29.78, 32.27, 32.47 (allylic), were selective manipulation in high yields under mild 34.45, 40.02, 53.80, 53.09, 59.50, 116.69, 118.72, 119.54, conditions and, in the deprotedion sequence,the formation 119.76, 119.92, 122.90, 123.87,125.28, 129.92,131.45 (m), of volatile byproducta so that extensive purification of the 146.96,149.61, 150.08, 163.44,166.38, 170.95 (m), 171.37, final products could be avoided. Since the stability of the 174.87; Reverse-phaseTLC (Zpropanol/water, 1:l) Rf= target compounds was unknown, the use of protecting 0.66; MS (positive ion FAB, m-nitrobenzyl alcohol/

McKee et el.

288 Bbconlqpte Chem., Vol. 2, No. 4, lQQl

Scheme I1

LysineHCI, NE13 A&neHzO

0 15

3

20

groups that could be removed cleanly was important. Therefore, the tert-butoxycarbonyland benzyl groups were chosen. The ease of removal of a tert-butoxycarbonyl (t-Boc) group with trifluoroacetic acid (TFA) led to the use of t-Boc protection of the amino group of L-threonine enroute to a required modified synthesis of oxamazins (Scheme I). L-Threonine (8) was converted to its N-Bocprotected derivative 9 with di-tert-butyl dicarbonate (BoczO) in quantitative yield. N-Boc-threonine (9) was converted to hydroxamate11in 77 9% yield upon treatment with O-benzylhydroxylamine(10) (23),N-hydroxysuccinimide, and DCC. 8-Lactam 12 was obtained from hydroxamate 11 in 75% yield with PhaP/CC4/EtaN (24). The benzyl group of 8-lactam 12 was removed upon catalytic hydrogenation in methanolto provide N-hydroxy &lactam 13 in 96% yield. The N-hydroxy group of @-lactam 13 was then alkylated with benzyl bromoacetate to afford the desired N-Boc-oxamazin as ita benzyl ester 14 in 79% yield (35% overall yield from 8). N a J v ’ - B i s [ 2 , 3 - b i s ( ~ ~ l o ~ ) ~ ~ y l ) (16) l y s iwas n epre pared as described by Chimiak and Neilands (18). Thus, 2,3-dibenzoxybenzoic acid was converted to ita N-succinimide ester 15 with N-hydroxysuccinimide and DCC in 92% yield (Scheme 11). The active ester was then treated with L-lysine monohydrochloride and triethylamine to produce 16 in quantitative yield without further purification. Simple hydrogenolysis of 16 afforded the lysine siderophore 3 in quantitative yield. Synthesis of siderophore 18 from spermidine, 2,3-bis(benzyloxy)benzoicacid, and succinicanhydridewas previously reported (15).Again, hydrogenolysis of 18 gave deprotected 20 in 100% yield. The formation constants for the 3:2 siderophore-iron(II1) complexes of compounds 3 and 20 were measured by using

H

the procedure of Raymond (21). Spectrophotometric competition experiments were separately performed on the ferric iron complexes with a slight excess of EDTA at pH 7. The formation constants for both compounds 3 and 20 were calculated to be 1W. Siderophores 16 and 18 were converted to their N-succinimide active esters 17 and 19 by using the standard N-hydroxysuccinimide and DCC method in 100% and 88% yields, respectively. These active esters were not purified but directly used after filtration to remove dicyclohexylurea. With the two siderophore active esters in hand, efforts were directed at the synthesis of the siderophore-8-lactam conjugates. Coupling of N-succinimide esters 17 and 19 with oxamazin 14 was relatively straightforward (Scheme 111). The procedure involved the removal of the t-Boc protecting group from 14 using anhydrous trifluoroacetic acid in anhydrous methylene chloride to produce amine salt 21 as an oil. Treatment of 21 with esters 17 and 19 in a two phase ethyl acetate/water solvent system provided the protected oxamazin/siderophoreconjugates 22 and 23 in 63% and 61% yields, respectively. The desired oramazins 4 and 6 were obtained as highly viscous oils upon simple catalytic hydrogenation of rigorously purified 22 and 23 using 10% Pd/C in methanol in 97% and 100% yields, respectively. Compounds 4 and 6 were determined to be homogeneous by reverse-thin-layer and paper chromatographic analysis. The syntheses of the carbacephem conjugates 6 and 7 are shown in Scheme IV. The hydrochloride salt of carbacephalosporin 24 (26) was suspendedin anhydrousmethylene chloride and treated with triethylamine to liberate the free amine. The free amine was not isolated but was treated with siderophore 18 and 2-ethoxy-N-(ethoxycarr-

Bioconjugwte Chem., Vol. 2, No. 4, 1991 287

Iron Transport Mediated Drug Delivery Systems

Scheme 111

Hw\cH3

R-N

17, 19

I

t

wabr

V O B z I

&

NH. .O

Y

4

" 0'-N~ocH,CO,H

bonyl)-1,2-dihydroquinoline(26) (EEDQ) in anhydrous methylene chloride to provide protected conjugate 25 in 83 9% yield. Lysine siderophore 16 was also converted to its carbacephalosporin conjugate 26 in this manner, but although the desired product was obtained, the yield was unacceptable (20%). Compound 26 was obtained in satisfactory yield (84%) by coupling 24 and active ester 17 in the same manner as the oxamazin conjugates 22 and 23.

Deprotection of compound 26 proved to be a problem due to the presence of the double bond in the carbacephem. Initial deprotection using hydrogen and 10% palladium on carbon (20% w/w) in methanol reduced the double bond and reductively removed the chlorine atom. While it has been shown that carbacephalosporins without the vinylic chlorine demonstrate antibacterial activity, compounds without the double bond are devoid of activity (27). Successful deprotection was achieved with hydrogen, 10% palladium on carbon, concentratedhydrogen chloride, and 5 % aqueous dimethylformamide (28) to produce compound 6 in 73% overall yield from 18 and 24. Proof that the double bond was intact was facilitated by l3C NMR analysis, which showed the allylic carbon signal at 31.55 ppm. Compound 26 was also deprotected in the same manner, providing 7 as an amber oil in quantitative yield (84% overall yield from 16 and 24). The l3C NMR spectrumindicated the presence of the allylic carbon signal at 32.47 ppm, confirming the presence of the double bond. It is important to note that 8-lactam-siderophore conjugates 4-7 appear to have quadradentate ligands instead of the required hexadentate ligands for typical 1:1

metal-siderophore chelation to Fe(II1) and its subsequent transportation. However, the exact mode of coordination of these conjugates to ferric iron is not known. The quadradentate type siderophores have two binding sites for Fe(II1) ion. Therefore, quadradentate siderophore-iron complexes must have a minimum 3:2 stoichiometry. Nature has utilized quadradentate siderophores such as rhodotorulic acid and Na,N-bis(2,3-dihydroxybenzoy1)lysine (3) to transportFe(II1) efficiently. It has beenshown that rhodotorulic acid forms a 3:2 complex with ferric ions (29). Thus, it was anticipated that siderophore-8-lactam conjugates 4-7 would also chelate and transport ferric iron. Another important concept to consider is the effect (on siderophore activity) of the stereochemistry at the metal center. Studies have shown that one of the factors which may help regulate recognition of the iron complex by a receptor protein is the chirality about the metal center (30). The asymmetric character of the antibiotic component of the siderophore/&lactam conjugates may influence the chirality about the metal center, which in turn may influence the recognition of the conjugates at the receptor site. Biological Aspects. An objective of this work was to determineif effectiveantimicrobial agents could be formed from catechol siderophores and @lactam antibiotics. It was hoped that these conjugates would bypass the normal 8-lactam-transport system (3,31),and, instead, be transported through the iron-transport system. Several assays were used to study the ability of compounds 4-7 to act as siderophores and antimicrobialagents for several strainsof E. coli and other bacteria. The details

288

McKee et el.

Bioconlugete Chem., Vol. 2, No. 4, 1991

Scheme IV

&O2PNB 24

\

17, KHCh, EKIAeHpO

I / t OIB z l

Y O B r i

25

6

of these assays are given in the Experimental Procedures. The E. coli strains which were used includeE. coli RW193, E. coli RWB18, and E. coli X580. The RW193 strain is an E. coli K12 entA, fhuA positive organism which cannot synthesize enterobactin, but is able to use both hydroxm a t e and catecholate type siderophores. E. coli RWB18 is a mutant from E. coli RW193 which is unable to bind colicin B (32). The inability to bind colicin B has been attributed to the absence of an outer-membrane protein which also has been implicated in the transport of ferric enterobactin (32b). E. coli X580 (33) is a @-lactamhypersensitiveorganism which is commonly used to screen for antimicrobial activity. In addition, the organism and strain-selective antimicrobial activity was determined by the disk diffusion method (34). Before the antimicrobialactivity of conjugates 4-7 could be studied, it was necessary to establish that the siderophore portions of the conjugates were effective irontransport agents by promoting the growth of E. coli under EDDA-induced iron-deficient conditions. Table I shows the growth-promoting ability of siderophores 3 and 20 in E. coli X580 with the agar plate method. Both of the compounds were growth promoters as seen by a zone of growth stimulation. Neither one of these compoundswas tested as the preformed iron complex. This implies that these siderophores are able to scavenge the iron from the EDDA in the agar. A liquid growth assay showed that these siderophores 3 and 20 themselves are devoid of antimicrobial activity against E. coli X580. The 8-lactam-siderophore conjugates 4-7 were initially

Table I. Plate Bioarray for Siderophore Activity for the Siderophores 3 and 20 cOncn, mM

zone of stimulation," mm

concn,

zone of stimulation," mm

3 20 mM 3 20 13 NSb 10 31 26 28 0 E. coli X580 overnight culture, 1:200 OOO dilution into LuriaEDDA (100 pg/mL) agar. Filter-paper dish containing 5 pL of the siderophoresat the concentrations indicated wereplaced in the surface of the Luria agar. Plates were incubated at 37 O C for 24 h and examined for zones of stimulation of growth around the compounds. All plates were performed in duplicate. No zone of stimulation was observed.

1 5

tested by using the disk diffusion method. Of the four conjugates,only compound 6 exhibited any antimicrobial activity. Compound 6 showed moderate activity against Staphylococcus aureus X1.1(64) and V41(64)and Streptococcus A C283 (16) and Streptococcus pneumonae PARK (64) (MIC values in pg/mL in parentheses). Figure 4 shows the results of incubation of the siderophore-carbacephalosprin conjugates 6 and 7 with the 8-lactam-hypersensitive E. coli X680. Both of these conjugates show significant antimicrobial activity as shown by the delay in observedmicrobialgrowth compared to the control. The bacteria that eventuallydid grow were separately incubated again in the presence of each of test compounds 6 and 7. This time, there wae no observed delay in growth. These results may suggest that during

B/oconjugate Chepn., Vol. 2, No. 4, 1991 289

Iron Transport Medlated Drug Delivery Systems 1.0 0.8

,

-

0.6

-

1

e Control 10 pM 7 10 pM 6 0 1.0 M 6

Control

* 10pM7 10pM6

0.2 -

0.4

0.0 0

10

20

10

0

30

time (hrs)

Figure 4. Effects of conjugates 6 and 7 in Luria broth on the growth rate of E. coli X580.

the first incubation, the microbialgrowth that was observed after the delay w a due to selection of mutants from the parent E. coli strain. These mutants may be deficient in some form of iron transport since growth was observed in the presence of the conjugates. E. coli X580 survivors which were subsequently shown to be resistant to conjugate 6 have been isolated and a preliminary investigation to determine the mechanism of resistance has been performed (36). This type of overnight resistanceto iron-transported antibiotics has been reported for the natural antibiotic albomycin, as well as the catechol-containingcompound E-0702 (7c,12,37). The analogue of 6 that did not contain the double bond in the carbacephalosporin was also included in the assay. As expected (38), reduced 6 did display siderophore (growth promoting) activity, but exhibited no antimicrobial activity against E. coli X580. In order to determine if these conjugates were stable under these test conditions, compound 6 was preincubated in Luria both at 37 "C for 12 h prior to addition of the organisms. Results from the incubation produced a growth curve identical with that of the test flask which was not preincubated. Apparently, the result of the preincubation test reveals that the test compound is stable under the assay conditions, indicating that the observed results are not due to spontaneous decomposition of the compound in the culture broth. In addition, a sample of the carbacephaolsporin nucleus (deprotected 24) was incubated at 10 pM concentration against E. coli X580. The P-lactam, itself, did not inhibit the growth of the microorganisms, which provides further evidence of the conjugate's stability to the assay conditions. The liquid growth assay using an iron chelator, EDDA is shown in Figure 5. In these low-iron conditions, incubation with a 10pM final concentration of compound 6 resulted in no observed growth of the E. coli even after 30 h. Reinoculationof a loopful from this flask into fresh Luria broth resulted in normal growth of the organisms. It appears that 6 inhibited microbial proliferation but did not result in complete culture death (bacteriostatic activity). Incubation with 1.0 pM final concentration of 6 did result in observed growth after an initial delay. However, the organisms that did grow grew better than the control. These bacteriaapparentlywere not as affected by the low-iron conditions as were the parent strain. Further work with these selected mutants under the lowiron conditions is currently in progress in an attempt to elucidate this interesting result observed at the different concentrations. Figure 6 shows the results of incubation of conjugate 6 with the E. coli K12 strains RW193 and RWB18. After 12 h of incubation, the conjugation appears to promote

20

30

time (hrs)

Figure 5. Effect of conjugates 6 and 7 in Luria broth containing 100 pg/mL of EDDA on the growth rate of E. coli X580.

p

n

E

4

RWB18+6 RW193Control RWB18Control

0.0 0

20

10

30

time (hrs)

Figure 6. Effect of conjugate 6 (10 pM)in Luria broth with 100 pg/mL EDDA on the growth rate of E. coli RW193 and RWB18.

growth of the RW193 strain compared to the control. It may be argued that a P-lactamase might be responsible for the lack of antimicrobialactivity. However,this strain of E. coli was shown to be sensitive to both ampicillin and Loracarbef (a 7-(phenylglycyl)carbacephalosporin) (22). The observed growth-promoting activity is not easily explained,but may be attributed to the siderophoreportion of conjugate 6, which could be using a catechol irontransport system to deliver iron to the organism. This result is supported by the results from the liquid growth assay with 6 under similar conditions using the RWBl8 strain which lacks the catechol/colicin B receptor. After 12h of incubation,there was little observed growth, similar to the RWB18 control. However, if one continued to measure the turbidity for an additional 12 h, some observed growth was eventually seen. Compound 7 revealed a similar growth curve. The growth promoting ability of 6 and 7 with the RWBl8 strain is not without precedent. Other synthetic catechol-containingcompounds such as MECAM were able to promote growth in E. coli mutants lacking an enterobactin receptor protein (39). Recall also, that spermexatol[2] could promote the growth in Vibrio mutants which lacked the vibriobactin receptor (15).The observed growth with the RWB18 strain may have arisen for several reasons which are under active study. In addition to studying the potential antimicrobial activityof siderophore-carbacephalosporin conjugates4-7, the results described above also illustrate the importance of using a variety of assays and microbes to determine the biological activityof these types of compounds. Although, by the disk diffusion assay, compounds 6 and 7 showed only marginal or no activity, both appear to exhibit interesting activity as determined by a continuous liquid growth assay. The result of the liquid growth assay reveals

290 Bloconlugete Chem., Vol. 2, No. 4, 1991

that possibly many potential antimicrobial agents have been erroneously undetected in broad-screening techniques such as the "disk diffusion zone of inhibition method" due to mutant formation. Compounds 4 and 5 did not appear to have any biological activity. While mutant organisms or lack of peptidase activity may be responsible, this result was not surprising since other siderophore-oxamazin conjugates synthesized in our laboratory also showed no antimicrobial activity (9b), and oxamazin P-lactam antibiotics are known to be very sensitiveto the type of side chain contained in the molecule (19, 40). CONCLUSION

The synthesis and preliminary studies of the biological activity of several catechol siderophore-/3-lactam conjugates have been discussed. Two of the conjugates (6 and 7) exhibited significant antimicrobial activity against E. coli X580. In order to study the transport mechanism of the conjugates and their fate once inside the microbe in more detail, a photoaffinity probe approach (41) is being developed in our laboratory. Drug delivery using the siderophore iron-transport system to deliver a toxic substance to the microorganism shows promise and may be extended to other antibiotics, siderophores, and microorganisms. Work along these lines is in progress and will be reported in due course. ACKNOWLEDGMENT

We gratefully acknowledge support from the National Institutes of Health (Grant GM 25845). We thank Dr. Albert A. Minnick for help with the biological testing. Mass spectra were kindly obtained by Dr. Bruce Plashko. We thank Prof. Graham Lappin and Mr. Robert Warren for assistance with the measurement of the formation constants. The gift of carbacephalosporin from Eli Lilly and Co. and the performance of the broad-screenbacterial assays at Lilly were sincerely appreciated. LITERATURE CITED (1) (a) Miller, M. J. (1986) Hydroxamate approach to the

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