J . Med. Chem. 1989,32, 1062-1069
1062
NH4CH3C02)a t 2 mL/min and peak areas measured. Yields (Figure 1) are given as area (product)/area (all peaks). Determination of Specific Activities and Radiochemical Purities. An aliquot of the solution was transferred to a thin glass vial and its radioactivity measured with a dose calibrator. Another aliquot (of the same volume) was removed by the same syringe and injected onto the analytical HPLC column and the UV peak area of the product measured. The mass of the product was calculated by comparison to a standard curve. The sensitivity of the analyses was maximized by setting the wavelength of the UV detector to 239 nm-the A, of 21. The specific activity in mCi/pmol was then calculated by dividing the number of millicuries in the aliquot by the number of micromoles in the same aliquot. The radiochemical purity of the final product was always >99%; some hydrophilic radioactive impurities (13%) were detected in '9-labeled 21 following storage for 3 months at -10 "C. Binding Assays. Male CD-1 mice were killed, and the whole brain minus cerebellum was rapidly homogenized in 50 mM phosphate-buffered saline (pH = 7.4, 37 "C). The tissue homogenate (8 mg/mL buffer) was then incubated with 1 nM [3H]N-methylscopolamine (New England Nuclear, 70 Ci/mmol) alone or in the presence of 1.0 pM atropine and in the presence of increasing concentrations of the compounds listed in Table I. Samples were incubated for 30 min a t 37 "C in a shaking water bath to achieve equilibrium. All samples were incubated in triplicate. Samples containing atropine defined nonspecific binding. Following the incubation period samples were rapidly filtered over Whatman glass fiber filters under vacuum and rapidly washed with 15 mL of ice-cold phosphate-buffered saline. Filters were then counted in the presence of 10 mL of Formula 963 scintillation fluid (New England Nuclear) by using standard techniques a t efficiencies of ca. 40%. Radioactivity in the presence of atropine (nonspecific binding) was subtracted from that in other samples to compute net specific binding. Net specific binding at each drug concentration was then expressed as percent of control specific binding. Log-logit analysis was then used to compute the 50% inhibitory concentrations (IC,) for each test compound. Dexe-
timide was always studied in parallel with other compounds to assure experimental reproducibility. The values shown in Table I represent average results from two to three experiments; values varied less than 20%. Saturation experiments using ['%I]- and [1231]iododexetimidewere carried out in a similar fashion using lo4 M atropine to define nonspecific binding. Biodistribution studies were carried out on male CD-1 mice according to literature procedures."
Acknowledgment. We t h a n k Dr. Pierre L a d u r o n for samples of a u t h e n t i c dexetimide and levetimide and Annslie S m i t h for carrying o u t the binding a n d biodistribution studies. T h i s work was supported b y USPHS G r a n t No. CA32845 and NS-15080. Registry No. (S)-1, 21888-98-2; (R)-2, 21888-99-3; (R,S)-3, 119391-55-8; (R,S)-3*HCl,6767-69-7; (S)-4, 115216-87-0; (R)-5, 115216-88-1; (R,S)-6, 119477-43-9; (R,S)-7,119391-56-9; (R,S)7.HC1, 119391-73-0;(R,S)-8,119391-57-0;(R,S)-8*HCl,119391-74-1; (R,S)-9, 119391-58-1; (R,S)-9*HCl, 119391-75-2; (R,S)-10, 119391-59-2; (R,S)-10.HC1, 119391-76-3; (R,S)-11, 119391-60-5; (R,S)-ll.HCl, 119391-77-4;(R,S)-12, 119391-61-6;(R,S)-12+HCl, 119391-78-5; (R,S)-13, 119391-62-7; (R,S)-13*HCl,119391-79-6; (R,S)-14, 119391-63-8; (R,S)-14*HC1, 119391-80-9; (R,S)-15, 119391-64-9; (R,S)-15*HCl,119391-81-0; (R,S)-16, 119391-65-0; (R,S)-16*HCl,119391-82-1;(R,S)-17,119391-66-1;(R,S)-17*HCl, 119391-83-2; (R,S)-18, 119413-94-4; (R,S)-18.HCl, 119391-84-3; 19, 17903-42-3; 20, 16004-15-2; 20 (1251),105644-30-2; 20 ('231), 105644-31-3; 21, 119478-57-8; '251-21, 119391-69-4; '231-21, 119391-70-7; 22, 119477-44-0; '251-22, 119391-71-8; '231-22, 119391-72-9; (S)-23, 119391-67-2; (R)-24, 119391-68-3; 4-iOdOtoluene, 624-31-7; 3-fluorobenzyl bromide, 456-41-7; 3-iodobenzyl bromide, 49617-83-6; 3-bromobenzyl bromide, 823-78-9; 3chlorobenzylbromide, 766-80-3;4-chlorobenzylbromide, 622-95-7; 2-fluorobenzyl bromide, 446-48-0; 4-bromobenzyl bromide, 58915-1; 4-fluorobenzyl bromide, 459-46-1; 2-chlorobenzyl bromide, 611-17-6; 2-bromobenzyl bromide, 3433-80-5; 2-iodobenzyl bromide. 40400-13-3.
Aromatic Dienoyl Tetramic Acids. Novel Antibacterial Agents with Activity against Anaerobes and Staphylococci' T e r r y Rosen,**t P r a b h a v a t h i
B. Fernandes, M a r y A. Marovich, Linus Shen, James Mao, and AndrB G. Pernet
Abbott Laboratories, Abbott Park, Illinois 60064. Received January 2 2, 2988 Streptolydigin (1) and tirandamycin A (2) are typical members of the naturally occurring class of 3-dienoyl tetramic acids. These compounds, which possess potent antibacterial activity particularly against anaerobes, have been shown to inhibit bacterial RNA polymerase. In contrast, tenuazonic acid (5), which lacks a complex dioxabicyclononane moiety and diene chromophore present in 1 and 2, exhibits essentially no antimicrobial activity and has no effect on bacterial RNA polymerase, suggesting that one or both of these structural features may be critical for antibacterial activity. In this paper, we report on a novel series of synthetic dienoyl tetramic acids that lack a complex dioxabicyclononane unit. Several of these compounds, particularly 8T-W, exhibit potent antimicrobial activity against Gram-positive and Gram-negative anaerobes as well as staphylococci. We will discuss the structureactivity relationship for this series of compounds which, in contrast to their natural counterparts, do not inhibit significantly RNA polymerase. We will also discuss preliminary results on the biochemical and microbiological properties of this series of compounds, several of which moderately inhibit supercoiling by DNA gyrase isolated from E . coli H560, although this enzyme has not been established as their target in whole cells. Compound 8W, which is not cross-resistant with DNA gyrase subunit A or B inhibitors or tirandamycin, has also been demonstrated to be rapidly bactericidal. Streptolydigin (1),2tirandamycin A (2): BU2313A (3), and BU2313B (4)4 are members of the naturally occurring
a n d tirandamycin A have been shown t o be inhibitors of bacterial RNA p ~ l y m e r a s e . ~In , ~ contrast, tenuazonic acid
class of 3-dienoyl tetramic acids. These compounds possess potent antibacterial activity, particularly against anaerobes and some Gram-positive aerobes. Streptolydigin
(1) This work was presented, in part, at the 1987 ICAAC. Rosen,
Current address: Pfizer, Medicinal Chemistry Department, Central Research Division, Groton, CT 06340. 0022-2623/89/1832-1062$01.50/0
T.; Fernandes, P. B.; Shen, L.; Marovich, M. A.; Mao, J.; Pernet, A. Abstracts of the 27th Interscience Conference on Antimicrobial Agents and Chemotherapy 1987, Abstract No. 1003. 0 1989 American Chemical Society
Aromatic Dienoyl Tetramic Acids
(5) exhibits essentially no antimicrobial activity and has
Journal of Medicinal Chemistry, 1989, Vol. 32, No. 5 1063
Table I. Structures of Compounds
J-NH
0
0
5
no effect on bacterial RNA p o l y m e r a ~ e . ~ !It~ has been postulated that the dioxabicyclononane skeleton or the diene unit present in 1-4 may be critical to the antibiotic properties of these compounds.' Although tirandamycin was reported initially to be ineffective for the treatment of bacterial infections in mice, more recently tirandamycin and the BU2313 compounds have been shown to be effective in mouse protection tests.8 We have also demonstrated that tirandamycin is effective in a mouse abscess model for Bacteroides f r ~ g i l i s . ~ There has been a structure-activity study on a series of tetramic acids lacking the diene chromophore, and these compounds are generally devoid of substantial antibacterial activity.l0 In this paper, we report our preliminary results on the synthesis and biological activity of a series of dienoyl tetramic acids. Several of these compounds possess potent antibacterial activity. However, unlike their natural counterparts, these compounds do not inhibit appreciably RNA polymerase. These compounds show moderate inhibition of supercoiling by DNA gyrase, although this enzyme has not been established as the target of these agents in whole cells. Results and Discussion Chemistry. The tetramic acids in this study were synthesized by employing general methodology analogous to that reported by Schlessinger et al. in their synthesis of tirandamycin A.l1 The key step in this convergent methodology involves Wadsworth-Emmons condensation of a tetramic acid keto phosphonate 7 and an aldehyde 6 (Scheme I, eq 1). In Schlessinger's synthesis of 2, it was necessary to employ a protecting group on the nitrogen of the tetramic acid 7 (R3 = 2,4-dimethoxybenzyl, R4 = H) in order to better facilitate the coupling reaction. Deblocking is accomplished with neat trifluoroacetic acid to afford 8 (R3 = R4 = H). We used this sequence to prepare compounds in which R3 in 8 is hydrogen. However, we also utilized the keto phosphonate 7 (R3 = CH,, R4 = H), which affords compounds with the N-CH, moiety present in BU2313A (31, obviating the need for a subsequent deprotection step. The general route to the keto phosphonate reagents (7) Eble, T. E.; Large, C. M.; DeVries, W. H.; Crum, G. F.; Shell, J. W. Antibiotics Annual; Medical Encyclopedia, Inc.: New York, 1956; p 893. Reusser, F. In Antibiotics Vol. I: Mechanism of Action of Antibacterial Agents; Hahn, F. E., Ed.; Springer-Verlag: New York, 1979; p 361. Tsukiura, H.; Tomita, K.; Hanada, M.; Kobaru, S.; Tsunakawa, M.; Fujisawa, K.-I.; Kawaguchi, H. J. Antibio. 1980, 33, 157. Reusser, F. Antimicrob. Agents Chemother. 1976, 10, 618. (a) Gitterman, C. 0. J. Med. Chem. 1965,8,483. (b) Suzuki, S.; Sano, F.; Yuke, H. Chem. Pharm. Bull. 1967, 15, 1120. Lee, V. J.; Branfman, A. R.; Herrin, T. R.; Rinehart, K. L. J. Am. Chem. SOC. 1978, 100, 4225. Kawaguchi, H.; Tsukiura, H.; Tomita, K. U.S. Patent 4,169,096, 1979. Fernandes, P. B. Unpublished results. Matsuo, K.; Kimura, M.; Kinuta, T.; Takai, N.; Tanaka, K. Chem. Pharm. Bull. 1984,32,4197. (a) Schlessinger, R. H.; Bebernitz, G. R. J. Org. Chem. 1985, 50, 1344. (b) Schlessinger, R. H.; Bebernitz, G. R.; Lin, P. J. Am. Chem. SOC. 1985,107, 1777.
8
80 A
Bb
E
R, i-C3H7 n-C11H23
R, CH3 CH3 C6Hll CH3 CH3 CBHllCHZ C ~ H ~ I C H ( C H ~ )CH3
R, CH3 CH3 CH3 CH3 CH3 CH3
R, H H H H H
CH3 CH3 H CH3 H CH3 H CH3 H CH3 CH3 CH3 CH3 H CH3 H CH3 CH3 CH3 CH3 CH3
H H H H H H H H H H H H H H H H H H H H H
v
-
formula CidH19N03 C22H35N03 C17H23N03*1/2H20 Cl8H25N03 ClgH27N03 C16H21N04
(CH2)3 CH3 CH3
C23H21N03 C22H21N03
CH3
C21H19N04
CH3 H
C25H27N0,y1/4Hz0
a Unless noted otherwise, all new compounds had high field 'H NMR spectra, mass spectra, and combustion analysis (C, H, N f 0.4% of theoretical value) and/or high-resolution mass spectra that were consistent with the indicated structures. Compound characterized by high field 'H NMR spectrum and low-resolution mass spectrum. MEM = methoxyethoxymethyl.
is shown in Scheme I (eq 2). Treatment of the bromo acid bromide 9 with an amino ester 10 gives the adduct 11. Exposure of 11 to the potassium salt of diethyl phosphite results in displacement of bromide and intramolecular cyclization to furnish 7. The reagent 7 (R3 = CH3, R4 = H) is obtained from N-methylglycine ethyl ester (10, R3 = CH,,R4 = H, R5 = C2H5) (see the Experimental Section). The aldehydes employed in the condensation reaction were obtained either from commercial sources or by a homologation sequence employing an aldehyde and an appropriate stabilized Wittig reagent ((carbethoxyethy1idene)triphenylphosphorane or (carbethoxymethylene)triphenylphosphorane,Aldrich) followed by manipulation to the desired oxidation state (Scheme I, eq 3).l59I6 The structures of the 3-acyltetramic acids (8) thus
1064 Journal of Medicinal Chemistry, 1989, Vol. 32, No. 5
Rosen et al.
2
Table 11. Minimum Inhibitory Concentrations of Tetramic Acids 8 organism B. fragilis ATCC 25285 B. thetaomicron ATCC 29741 C. perfringens ATCC 13124 C. difficile ATCC 9689 organism B. fragilis ATCC 25285 B. thetaomicron ATCC 29741 C. perfringens ATCC 13124 C. difficile ATCC 9689 organism B. fragilis ATCC 25285 B. thetaomicron ATCC 29741 C. perfringens AtCC 13124 C. difficile ATCC 9689
A 31 62 31 31 N 16 16 8 8
B 125 125 31 4 O 4 4 8 4 AA 16 31 8 8
C 8 8 4
4 P 4 8 8 4 BB 2 4 2 2
Q 62 125 16 31
MIC. m / m L D E F G H 62 8 8 125 125 31 62 125 62 62 2 2 125 31 62 2 2 125 31 62 MIC, Mg/mL R S T U 8 62 1.0 10.5 16 125 1.0 2.0 125 31 1.0 10.5 125 8 1.0 10.5 CC 125 62 125 125
obtained are shown in Table I, and further physical and spectral properties are provided in the Experimental Section.
MIC, pg/mL EE FF 62 31 62 31 31 16 62 16
DD 31 62 31 16
I 50 50 25 25
J 62 62 125 16
V 10.5 50.5 10.5 50.5
W 10.5 31 2 10.5
GG 125 250 125 8
K 8 8 31 8
L 16 16 8 16
X 62 125
Y
2
62 31
2 2
4
TIR 0.1-0.8 0.2-0.8 10.05-0.1 1.6-3.1
M 8 8 4 4 Z 8 125 31 10.5
CLIN 2
8 10.5 1-2
Scheme I. Synthesis of 3-Dienoyl Tetramic Acids" R2
RlC H
