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J. Med. Chem. 2002, 45, 3032-3040
Highly Antibacterial Active Aminoacyl Penicillin Conjugates with Acylated Bis-Catecholate Siderophores Based on Secondary Diamino Acids and Related Compounds Lothar Heinisch,*,† Steffen Wittmann,†,‡ Thomas Stoiber,†,§ Albrecht Berg,† Dorothe Ankel-Fuchs,| and Ute Mo¨llmann† Hans Kno¨ ll-Institute for Natural Products Research, Jena, Germany, and Gru¨ nenthal GmbH, Division of Anti-Infectives/New IndicationsAachen, Germany Received December 3, 2001
New acylated bis-catecholates and 1,3-benzoxazine-2,4-dione derivatives based on secondary diamino acids (N-(aminoalkyl)glycines, N-aminopropyl-alanine, and N-aminopropyl-4-aminovaleric acid), on N-(aminoalkyl)aminomethyl benzoic acids, on N-(aminoalkyl)aminomethyl phenoxyacetic acids, or on 3,5-diaminobenzoic acid were synthesized as artificial siderophores. The corresponding diamino acids were obtained from the diamines and oxocarboxylic acids by catalytic hydrogenation. The acylated bis-catecholates and 1,3-benzoxazine-2,4-diones were coupled with ampicillin or amoxicillin to new siderophore aminoacylpenicillin conjugates. These conjugates exhibited very strong antibacterial activity in vitro against Gram-negative bacterial pathogens including Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Escherichia coli, Klebsiella pneumoniae, and Serratia marcescens. The ampicillin derivative 7b (HKI 9924154) and the corresponding amoxicillin derivative 8 (HKI 9924155) represent the most active compounds. The conjugates can use bacterial iron siderophore uptake routes to penetrate the Gram-negative outer membrane permeability barrier. This was demonstrated by assays with mutants deficient in components of the iron transport systems. New siderophore penicillin V conjugates with the siderophore component attached to the phenyl ring of penicillin V are inactive against these Gram-negative bacteria. Introduction The outer membrane permeability barrier of bacteria is one important reason for β-lactam antibiotic resistance of Gram-negative bacterial pathogens. This applies especially to Pseudomonas aeruginosa and Stenotrophomonas maltophilia strains. To overcome this membranemediated resistance, siderophore structures covalently bound to the antibiotic can function as a shuttle for active transport into the bacterial cell. Siderophores are microbial iron chelators excreted to sequester extracellular ferric ions under iron starvation conditions such as found in infected tissues. Specific outer membrane receptors recognize the siderophore iron complex and initiate its active transport into the cell. Many natural siderophores contain two or three catecholate groups as chelating ligands based on di- or triamines.1 Examples are bis-catecholate derivatives of spermidine2 or of diamino acids such as N,N′-bis-(2,3-dihydroxybenzoyl)L-lysine.3 Artificial siderophores of these structures based on spermidine or norspermidine were published.4-6 Recently, we synthesized artificial siderophores based on amino acids or dipeptides7 and mono-1,3-benzoxazine-2,4-dione derivatives as masked catecholates. These compounds can act as siderophore components in β-lactam conjugates.8 Examples of siderophore anti* To whom correspondence should be addressed. Tel.: (+49)3641 656714. Fax: (+49)3641 656705. E-mail:
[email protected]. † Hans Kno ¨ ll-Institute for Natural Products Research. ‡ Present address: Jenapharm GmbH & Co. KG, Jena, Germany. § Present address: Institute of Molecular Biotechnology, Jena, Germany. | Division of Anti-Infectives/New IndicationsAachen.
biotic conjugates with one catecholate moiety and enhanced in vitro antibacterial activities have been published.9,10 Ampicillin conjugates with natural pyoverdins were specifically active only against that Pseudomonas strain producing the corresponding pyoverdin.11 Pyoverdin-quinolone adducts were synthesized, too.12 β-Lactam conjugates with a series of different noncatecholate siderophore structures seem to act independently of iron transport systems and can reach activities near that of the cephalosporin moiety.13 In this paper, we report on the synthesis of N-(aminoalkyl)amino acids from diamines and oxocarboxylic acids by catalytic hydrogenation, on the acylation of these compounds or of 3,5-diaminobenzoic acid to acylated biscatecholate and 1,3-benzoxazine-2,4-dione derivatives as new artificial siderophores and on their condensation with aminoacyl penicillins and on the investigation of the biological activities of the siderophores and their conjugates with ampicillin and amoxicillin. We used acylated catecholates and the corresponding benzoxazine derivatives as siderophore components to facilitate the synthesis and to decrease pharmacological side effects in comparison to free catecholates. Results and Discussion Synthesis of the Siderophores. As the backbone for new artificial siderophores, we synthesized N(aminoalkyl)amino acids 3a-d,o,p, N-(aminoalkyl)aminomethyl benzoic acids 3k,l, and N-(aminoalkyl)aminomethyl phenoxyacetic acids 3m,n from the diamines 1a-e and oxocarboxylic acids 2a-f by hydro-
10.1021/jm010546b CCC: $22.00 © 2002 American Chemical Society Published on Web 06/06/2002
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Journal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3033
Figure 1. Syntheses of the siderophore components 5 and 6 and of the antibiotic conjugates 7-9; Ac ) COCH3; Moc ) COOCH3; 1a: n ) 0, R1 ) H; 1b: n ) 1, R1 ) H; 1c: n ) 1, R1 ) CH3; 1d: n ) 2, R1 ) H; 1e: n ) 3, R1 ) H; 2a: X ) CH; 2b: X ) C-CH3; 2c: X ) C-CH3-(CH2)2-; 2d: X ) CH-oC6H4; 2e: X ) CH-pC6H4-OCH2; 2f: X ) CH-oC6H4-OCH2; 4a: R2 ) 2,3-OAc, R3 ) H; 4b: R2 ) 2,3-OMoc, R3 ) H; 4c: R2 ) 2,3-OMoc, R3 ) 5-Cl; 4d: R2 ) 2,3-OMoc, R3 ) 5-Br; 4e: R2 ) 2,3-OMoc, R3 ) 5,6-di-Cl; 4f: R2 ) 3,4-OAc, R3 ) H; 6a, 9a: n ) 2, X ) CH2; 6b, 9b: n ) 1, X ) CHCH3; 6c, 9c: n ) 1, X ) CH(CH3)CH2CH2; other substituents, see Table 1.
Figure 2. Structure of the aminoacyl penicillin components; R ) siderophore components of Figures 1 and 4; R1 ) H: ampicillin derivatives, Ap; R1 ) OH: amoxicillin derivative, Ax.
genation catalyzed by palladium on active carbon. The synthesis of N-(aminoethyl)glycine 3a by this procedure was known.14 The intermediate in this reaction is possibly an N-heterocyclic acid like hexahydro-pyrimidine-2-carboxylic acid as a derivative of 1,3-diamino-npropane15 and not the corresponding azomethine, because no CHdN signal was found in 1H nuclear magnetic resonance (NMR) spectra. Compounds 3 were purified by codistillation with toluene to separate unreacted diamines or via the synthesis of CBZ derivatives with benzyl chloroformate, separation from impurities by preparative high-performance liquid chromatography (HPLC), and hydrogenolysis with Pd/C. The diamino acids 3a-n were acylated with diacyloxybenzoyl chlorides 4a-f in aqueous sodium hydrogen carbonate solution to the bis-catecholate compounds 5a-n (Figure 1). The 4,5-dichloro-2,3-di-methoxycarbonyloxy-benzoyl chloride 4e was prepared analogously to 4b 13 from 4,5dichloro-2,3-dihydroxybenzoic acid16 and methyl chloroformate by reaction of the obtained 4,5-dichloro-2,3di-methoxycarbonyloxy-benzoic acid with PCl5. Compounds 5a-c,l-n and the corresponding β-lactam conjugates 7a-c,l,m, 8, and 10a,b obviously exist in two rotameric forms because two triplets for CONH were found in the 1H NMR spectra, which change to singlets at 360 K studied on compounds 5b,m and 5b and 10a, respectively. The 1,3-benzoxazine-2,4-dione derivatives 6a-c were synthesized from the diamino acids 3b,o, or p and 2,3dimethoxycarbonyloxybenzoyl chloride 4b (Figure 1). Bis-2,3-di-(methoxycarbonyloxy)-benzoyl intermediates
Figure 3. Penicillin Vsderivatives 10a,b, Ac ) COCH3.
according to structure 5 were cyclized to structure 6 by reaction in acetonitrile. These compounds represent a heterocyclic acylated form of catecholates, which can be transformed enzymatically into the free catecholates. β-Lactam conjugates with one benzoxazindione moiety showed high antibacterial activity.8 As a further diamino acid, we used 3,5-diaminobenzoic acid as the backbone for siderophores, which were acylated with 4a to compound 11 and with 4b, with [8-(methoxycarbonyloxy)-1,3-benzoxazine-2,4-dione-3-yl]acetyl chloride 4g and the corresponding propionyl chloride 4h,8 respectively, to the compounds 13a-c (Figure 4). Synthesis of the β-Lactam Conjugates. The compounds 5a-m, 6a-c, 11, and 13a-c were coupled with ampicillin or amoxicillin via their mixed anhydrides with isobutyl chloroformate to the ampicillin conjugates 7a-m, 9a-c, 12, and 14a-c and to the amoxicillin conjugates 8 and 15c (Figure 2). The bis-catecholates 5m,n were also coupled with 6-aminopenicillanic acid to give derivatives of penicillin V 10a (p-derivative) and 10b (o-derivative) (Figure 3, the structure of penicillin V is marked). In these compounds, the siderophore component is placed on the phenyl of the phenoxy-
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Journal of Medicinal Chemistry, 2002, Vol. 45, No. 14
Heinisch et al.
Figure 4. Synthesis of 3,5-diaminobenzoic acid derivatives 11-15, Ac ) COCH3, Moc ) COOCH3; 13a and 14a: X ) direct bond; 13b and 14b: X ) CH2CONH; 13c, 14c, and 15c: X ) CH2CH2CONH-. Table 1. Substituent Pattern of Compounds 3, 5, 7, 8, Ac ) COCH3, and Moc ) COOCH3 compd
n
R1
3a 5a 7a 3b 5b 7b 8 3c 5c 7c 3d 5d 7d 5e 7e 5f 7f 5g 7g 5h 7h 5i 7i 3k 5k 7k 3l 5l 7l 3m 5m 7m 3n 5n 3o 3p
0 0 0 2 2 2 2 3 3 3 1 1 1 1 1 1 1 1 1 1 1 2 2 1 1 1 2 2 2 2 2 2 2 2 1 1
H H H H H H H H H H CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 H H CH3 CH3 CH3 H H H H H H H H H H
R2
R3
2,3-OAc 2,3-OAc
H H
2,3-OAc 2,3-OAc 2,3-OAc
H H H
2,3-OAc 2,3-OAc
H H
2,3-OAc 2,3-OAc 2,3-OMoc 2,3-OMoc 2,3-OMoc 2,3-OMoc 2,3-OMoc 2,3-OMoc 2,3-OMoc 2,3-OMoc 3,4-OAc 3,4-OAc
H H H H 5-Cl 5-Cl 5-Br 5-Br 5,6-di-Cl 5,6-di-Cl H H
2,3-OAc 2,3-OAc
H H
2,3-OAc 2,3-OAc
H H
2,3-OAc 2,3-OAc
H H
2,3-OAc
H
X CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2-(o-C6H4) CH2-(o-C6H4) CH2-(o-C6H4) CH2-(o-C6H4) CH2-(o-C6H4) CH2-(o-C6H4) CH2-(p-C6H4)-OCH2 CH2-(p-C6H4)-OCH2 CH2-(p-C6H4)-OCH2 CH2-(o-C6H4)-OCH2 CH2-(o-C6H4)-OCH2 CH3CH CH3CH-(CH2)2
acetylpenicillanic acid representing a new type of siderophore β-lactam conjugate. Siderophore Activity. Siderophore activity of the synthesized compounds was determined by the assay of their growth-promoting activity in Gram-negative bacteria under iron-limited conditions. According to Table 2, the bis-catecholates of N(aminoalkyl)glycines 5b-d,f-h and 6a,b efficiently act as siderophores and promote the growth of the bacterial test strains. The growth promotion activities of compounds 5e,i, 6c, 11, and 13b exhibited decreased
Table 2. Growth Promotion of the Bis-catecholate Derivatives 5a-n, 6a-c, 11, and 13a-c of Gram-Negative Bacteria under Iron Limitation (5 µL of a 2 mM Solution Was Applicated on a 6 mm Paper Disk) and the Results of CAS Assaya P. aeruginosa compd 5b 5c 5d 5e 5f 5g 5h 5i 5k 5l 5m 5n 6a 6b 6c 11 13a 13b 13c desferal a
E. coli
ATCC NCTC ATCC K799/ ATCC CAS 27853 SG 137 10662 9027 WT 25922 assay 20 30 19 15 25 22 25 25 20 22 22 18 18 15 13 0 15 15 18 35
26 28 15 9 26 25 30 10 19 21 19 nt 27 19 15 20 25 20 23 30
25 30 23 18 25 27 30 14 18 23 23 27 24 25 20 17 18 20 20 30
20 20 18 13 24 20 25 19 nt nt 25 26 nt nt nt 10 20 20 20 35
25 25 22 25 25 27 25 20 17 15 12 25 23 22 14 nt nt nt nt 36
28 27 20 20 30 32 30 21 27 34 27 26 30 22 15 27 20 15 20 38
++ +++ +++ + + + ++ ++ + + + + + ++ + ++ + (+) (+)
nt ) not tested.
activity for some of the strains. The acylated catecholates active as siderophores obviously were transformed to free catecholates in bacterial cultures. Iron-Complexing Capacity. All bis-catecholates showed a positive chromazurol-S (CAS) reaction (Table 2), which is associated with iron chelation, a basic requirement for siderophore activity. Antibacterial Activity. Compounds 7-10, 12, 14, and 15c were tested for their antibacterial activity in vitro against the Gram-negative bacteria P. aeruginosa SG 137 and ATCC 27853, S. maltophilia GN 12873, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 10031, Serratia marcescens SG 621, and against the Gram-positive strain Staphylococcus aureus SG 511 (Table 3). The compounds 7a-h, 8, and 9a exhibited extremely high antibacterial activity against the Gramnegative test strains, by far exceeding the activity of the ampicillin moiety, azlocillin, and partly also the activity of meropenem, especially against S. maltophilia.
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Journal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3035
Table 3. Antibacterial Activities In Vitro of the Siderophore-β-Lactam Conjugates 7-15 (MIC in mg/L) P. aeruginosa
E. coli
K. pneumoniae
S. maltophilia
S. marcescens
S. aureus
compd
SG 137
ATCC 27853
ATCC 25922
ATCC 10031
GN 12873
SG 621
SG 511
7a 7b 7c 7d 7e 7f 7g 7h 7i 7k 7l 7m 8 9a 9b 9c 10a 10b 12 14a 14b 14c 15c azlocillin ampicillin meropenem
0.4
