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Chapter 12. Synthetic Antibacterial Agents
Chapter 12. Synthetic Antibacterial Agents Robert G. Shepherd and Arthur Lewis Lederle Laboratories, American C y a n d d Co., P e a r l River, New York Perspective.-Work on these agents i n 1966 w a s directed toward improvement i n the l e v e l of a c t i v i t y or i n the pharmacology of known active struct u r e s . No new a n t i b a c t e r i a l type a c t i v e i n animals or i n m a n was reported. Several sulfanilamides and other synthetic a n t i b a c t e r i a l s as well as the a n t i b i o t i c s a r e involved with "infectious drug resistance. 2,3 Infectious drug resistance denotes t h e transmission of d r u g resistance from one bacterium ( s o far only Gram-negatives) t o another of the same or d i f f e r e n t s t r a i n , species, or genus. This i s accomplished by conjugation: formation of a temporary i n t e r c e l l u l a r connection between two sex types and unidirect i o n a l t r a n s f e r of the resistance genes and a t r a n s f e r a b i l i t y factor, usua l l y associated with an extra-chromosomal sex-determining f a c t o r . Conjugation leads d j r e c t l y t o a new r e c i p i e n t s t r a i n with a comparable l e v e l of resistance. The surprising f a c e t of t h i s phenomenon i s the simultaneous t r a n s f e r of r e s i s t a n c e t o a whole s e r i e s of chemically and biochemically unrelated antimicrobials (most c m o n l y sulf anilami de s t e t r a c y c l i n e s streptomycin, and chloramphenicol but a l s o more recently p e n i c i l l i n s , kanamycin, neomycin, n a l i d i x i c a c i d and nitrof'urans). There appears t o be a close genetic r e l a t i o n s h i p between the resistance genes which leads t o t h e i r beccaning arranged imme a t e l y adjacent t o one another i n the genetic element ( D W ) t r a n ~ f e r r e d . 5 , ~ I snp i t e of requiring several minutes and the proper physiological conditions, conjugation does occur i n vivo, even though l e s s r e a d i l y l ~ ~ t h ainn v i t r o . Resistant b a c t c r i a can be ffcured'fof i n f e c t i o u s drug r e s i s t a n c e with various acridines i n v i t r o , a l b e i t with low frequency;l,2,3,5usually t h e cure applies t o a l l the resistance f a c t o r s but some segregation has been achieved.5~6 The t r u e chemotherapeutic significance of this phenomenon i s not defined a t t h e moment8 but it requires c a r e f u l scrutiny since it involves so many drugs and so many organisms (Coli, Salmonella, Klebsiella, Shigella, Vibrio). Perhaps, by means of t h i s new look a t a b a c t e r i a l l y o l d process, we will now be able t o devise a cure f o r t h i s " i n f e c t i o u s disease'' of b a c t e r i a which will be e f f e c t i v e i n vivo and w i l l f i n a l l y enable us t o deal with the important problem of b a c t e r i a l resistance ( c f . a c l i n i c a l study9 of a t e b r i n i n combination therapy of multi-drug-resistant urinary infections). I n Vivo Actives.-The Gram-negative a c t i v i t y of n a l i d i x i c acid I i s seen from f u r t h e r work t o be very s t r u c t u r a l l y specific. Several 1,5-naphthyridine analogs apparently had l i t t l e , i f any, a c t i v i t y i n v i t r o or i n vivo while one 1,6-ana1og (3-carboxy-boxo-1,5,7-trimethyl-l, 6-naphthyridine) was p a r t i a l l y e f f e c t i v e against Kleb. pneumoniae i n mice (7@0 survival with subcut. 2OOmg/kg/day) .loThe intermediate d i e t h y l N-( 2,6-dimethyl-bpyridy1)amino-methylenemalonate had some i n vivo Staph. a c t i v i t y (6@ survival
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with subcut. lOOmg/kg/day) Curiously, t h r e e 1,7-naphthyridinesl1 had only Gram-positive a c t i v i t y : 1,6,8-trimethy1-3- carboxy-boxo-lJ7-naphthyridine was a c t i v e against Staph., Strep. and Pneumococcus ( p a r t l y e f f e c t i v e ) a t 25, 100 and bOmg/kg/day o r a l l y i n mice; the lY8-dimethyl-6-ethyl and t h e 1e t h y l analogs were much l e s s active.
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I1 I11 IV I Several types of nitrof'urans (NF = 5-nitrc-2-fbyl) with i n t e r e s t i n g l e v e l s of i n vivo a c t i v i t y were reported but margins of s a f e t y were not given. The 4-aminoquinazolines I1 [Z = N(CH3)C2H40HY N(C2H)+OH)2and NHC)+H8-1-pyrrolidinyl] had a broad i n v i t r o spectrum, including seudmonas, Proteus and Aerobacter, and high a c t i v i t y (0.01-12mcg/ml) .15 However, only a c t i v i t y i n vivo against Staph. aureus was reported ( E i n t r a p e r i t o n e a l 1-15mg/kg) ; a c t i v i t y c o r r e l a t e d poorly with data. Homologs of CH3 were l e s s active. Pyridazinone I11 and i t s dihydro intermediate13 were about equal i n vim and i n v i t r o i n Salm.-Staph. activi t y ; 2-alkylation lowered t h e i n v i t r o a c t i v i t y of both. I n contrast, it increased t h e i n vivo a c t i v i t y of t h e former and decreased t h a t of t h e l a t ter. Their i n v i t r o a c t i v i t y (min. inhib. concn. 2-6mcg/d) was l e s s than t h a t of I1 (0.05-0.8mcg/d) while t h e i n vivo a c t i v i t y was the same. Also poorer i n a c t i v i t y i n v i t r o and i n spectrum was t h e t r i a z o l e I V which had comparable Salm.-Staph. a c t i v i t y i n vivo (ED50 20-40mg/kg) .I4 1-Acylation or 5-alkylation decreased i t s a c t i v i t y which w a s g r e a t e r than t h a t of t h e upen-chain N-formylamino nitrof'uramidine, i n t u r n more a c t i v e i n vivo than t h e anddine i t s e l f . The l-methylcarbamoyl-5-imino derivative o f I V w a s a c t i d 5 against Staph., Salm. and Coli i n mice a t about 4Omg/kg with an oral LD 0 of 1 9 0 . A s e r i e s of 2-(5 '-nitro-2'-fluyl)CH=CHpyridines showed he following s i n g l e i n t r a p e r i t o n e a l anti-Salmonella a c t i v i t y ( EDF0 2-10mgjkg; oral ED5 5-75mg/kg) r e l a t i o n s h i p i n mice: 6-CH2OH-l-oxide = 5-CH20Ac-l-oxide > t-CH$Ac-l-oxide > 5-CFi2OAc > ~ - C H ~ O = H 5-CH OH > 6-CH20Ac with approximately t h e same r e l a t i o n s h i p of t o x i c i t y $ The 5methyl-1-oxide reported l a s t year had canparable a c t i v i t y . The a s - t r i a z i n e derivative V was p a r t l y effective17 against Staph. and Salm. i n f e c t i o n s i n mice a t lOOmg/kg. Activity of 5-methylmercapt methyl-+( 5 ' - n i t r o f b f u r y l ideneamino)-oxazolidin-2-one w a s unpromising18 i n various urinary infect i o n s i n a small c l i n i c a l trial. Further c l i n i c a l investigations have confirmed the e f f i c a c y of ethambut o 1 (EMB), (+)-N,N'-bis( l-hydroxy-2-butyl)ethylenedi~ne, i n treatment of pulmonary tuberculosis and a l s o demonstrated i t s use against r e n a l and e treatment of pulmonary i n f e c t i o n s due vesical tuberculosisY19 t o a t y p i c a l mycobacteria. I n controlled t r i a l s conducted i n the National Sanatoria of Japa.n2s t o determine t h e b e s t t h i r d component of a t r i p l e regimen with i s o n i a z i d (INH) and streptomycin (SM) i n o r i g i n a l treatment, cycloserine and p-amino-salicylic a c i d (PAS) were l e s s e f f e c t ive and more t o x i c than EX3 while ethionamide w a s as e f f e c t i v e s EMB but l e s s well t o l e r a t e d ( c f . a dose-response c a p a r i s o n i n monkeys2'). In a
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companion trial, 25 ethambutol ( l g d a i l y ) cambined with e i t h e r cycloserine (0.5g d a i l y ) o r ethionamide (O.5g d a i l y ) was well t o l e r a t e d and h i g h l y e f f e c t i v e i n t u b e r c u l o s i s re-treatment ( r e s i s t a n t t o SM, INH and PAS). I n t h e s e t r i a l s a diminution i n v i s u a l a c u i t y occurred i n about 8 2 of t h e pat i e n t s receiving l g d a i l y ; t h e e f f e c t was r e v e r s i b l e . h i s i cidence and r e v e r s i b i l i t y w a s observed i n o t h e r c l i n i c a l s t u d i e s . 2 ?27,2j29 A reduced dosage regimen30 avoided t h e v i s u a l disturbance While EMB by i t s e l f i s e f f e c t i v e a g a i n s t a c u t e and chronic t u b e r c u l o s i s , 28,30J 31 combined t h e r apy i s r e c m e n d e d t o avoid emer ence of r e s i s t a n t mycobacteria. I n man, about 10,;of EMF3 i s metabolized 27 t o t h e i n a c t i v e d i c a r b o x y l i c a c i d by t h e sequence -CH;zOH + -CHO j -C02H. A s t r u c t u r e - a c t i v i t y has defined t h e very s p e c i f i c requirements f o r a c t i v i t y i n an extensive s e r i e s of EMB analogs
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V VIII I n combination with SM, ethionamide (ETH, l g d a i l y ) was as e f f e c t i v e as IhW (0.3g d a i l y ) i n m a n by t h r e e t h e r a p e u t i c c r i t e r i a . However, t h e ETH
regimen w a s considered t o be t o o t o x i c t o be c l i n i c a l l y acceptable.33 C l i n i c a l incidence of s i d e - e f f e c t s from ETH ( p l u s INH). decreas d sharply with dosage: 31% f o r lg, 18;; f o r 0.75g and 87: f o r O.5g d a i l y . 3E Separation and c h a r a c t e r i z a t i o n o f ETH m e t a b o l i t e s has been achieved and t h e i r quantit a t i v e i n t e r r e l a t i o n i s under s t u The m a j o r i t y of ETH i s metabolized and r a p i d l y e x c r e t e d i n t h e u r i n e 3 5 A s u b s t a n t i a l m o u n t i s converted t o i t s sulfoxide VI which has now been shown t o be e u a l y a c t i v e and i n t e r ,3~ metaboc o n v e r t i b l e w i t h ETH i n man and o t h e r s p e ~ i e s . 3 ~ ~ 3 7Extensive l i s m t o i n a c t i v e compounds a l s o occurs by d e t h i a t i o n ( d i r e c t l y or, more probably, through t h e s u l f o x i d e ) t o t h e carboxamide, deamidation leading t o 2-ethyl i s o n i c o t i n i c acid, and by 1-methylation t o give VII, which i s p a r t l y converted t o 11. Dethiated VII and VIII and t h e sulfoxide of VIII were a l s o observed. Continued work on sulfanilamides includes s t u d i e s on t e s t s t o guide t h e i r use, new uses, new compounds, metabolism, p r o t e i n binding, and a t o x i c i t y warning. An improved method f o r determining b a c t e r i a l s e n s i t i v i t y t o s u l f a d r u g s permits r e l i a b l y t & i n g advantage o f t h e i r a c t i v i t y a g a i n s t 80-9@ of E. c o l i and Proteus m i r a b i l i s ur2nary i n f e c t i o n s i n gene r a l p r a c t i c e , i n b a c t e r i u r i a of pregnancy and i n h o s p i t a l patients.39 Synergism between t h e i n e f f e c t i v e polymyxins and t h e b a c t e r i o s t a t i c s u l f a d r u g s i n v i t r o , using a number of s t r a i n s of Proteus m i r a b i l i s and vulgari s , l e d t o an 8- t o 6 b f o 1 d decrease i n a c t i v e concentration and t o bactericidal actionb, 41 ( c f . i n vivo work i n 1963). N o such i n t e r a c t i n occurred Plaswith e i t h e r of t h e s e agents when combined with f i v e modium falciparum s t r a i n s r e s i s t a n t t o chloroquine and quinine r e c l i n i c a l l y s u s c e p t i b l e t o sulfanilamides and diaminodiphenylsulfone. &2j43 Sulfasymazine "appea:c4to be a usef'ul agent i n t h e treatment of b a c t e r i u r i a during pregnancy. U s i n g t h e computer method r e c e n t l y reported, The
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dosing i n t e r v a l and the maintenance dose f o r sulfasymazine has been calculated.45 A s i n other s u l f a n i l m i d o heterocycles, introduction of bromine or chlorine i n t o the heterocycle increased t h e persistence& of 4-sulfa-2,6dimethoxypyrimidine; acute t o x i c i t y and p r o t e i n binding were a l s o increased. The isomeric 5-sul a- 3 - e t h y l - l , 2 , b t h i a d i a z o l e and 2 - e t h y l - l , 3 , b t h i a d i a zole were compared 7 as t o d e t a i l e d differences i n r e n a l elimination chara c t e r i s t i c s independent of p r o t e i n binding. C l i n i c a l and pharmacological studi@ were done on extremely long-acting 4- sulfa-5,6- dimethoxypyrimidine, and on t h i s and four other long-acting sulfa drugs (3-sulfa-6-methoxypyridazine, 4-sulfa-2,6-dime hoxypyrimidine, 2- sulfa-5-methoxypyrimidine and 2-sulfa-3-methoxypyrazine). 89 A study50 of 2-sulfanilamidopyrimidine i t s e l f rounds out the p i c t u r e of the metabolism of ulfapyrimidines : 5 l l a r g e variations i n N4-acetylation, i n N4-sulfonate, N -glucuronide and N1-glucuronide formations occur i n closely r e l a t e d compounds. The 5,6-dimethoxy- and 2,6-dimethoxy- &sulfapyrimidines a r e $ and 8G6 converted t o glucuronides. I n contrast t o these 2-sulf&4,6-dimethoxypyrimidine i s not N4and other sulfapyrimidine acetylated i n vivo; i t s - a c e t y l derivative is, i n f a c t , r a p i d l y deacetylated. Further study of s u l f a drug p r o t e i n binding has indicated t h a t t h e r e i s more t o i t s e f f e c t on a n t i b a c t e r i a l a c t i v i t y than ,just the Dercent bound a t equilibrium under s p e c i a l conditions. Human serum w a s found t o increase the minimal i n h i b i t o r y concentration f o r both sulfadiazine and sulfadimethoxine but the increase for each was eight times as great; f o r Shig. dysent e r i a e as f o r E, c 0 l i . 5 ~In addition, the r a t i o of t h e increases i n m.i.c.'s of the two s u l f a s i n the presence of 20-5C$ human serwn was too s m a l l ( 2 ) t o correspond t o the r a t i o (42) of percentage of unbound sulfa. The binding capacity of human serum p r o t e i n determined by equilibrium dialysis53 w a s nearly the same (ca. 2 moles/mole protein) f o r 8 sulfanilanides (pKa's 5.77.2), t h i s s m a l l capacity supporting chemical. r a t h e r than absorptive binding; p r o t e i n a f f i n i t i e s varied a hundred-fold. A quite d i f f e r e n t number ( ) of binding s i t e s f o r one of these, sulfamethoxypyridazine, was calculated from thermodynamic data based on e q u i l i b r a t e d Sephadex columns; the energ e t i c s indicated binding by van der Waal's forces. R warning has been issued t o physicians t h a t the long-acting sulfcnild d e s , sulfamethoxypyridazine and sulfadimethoxine, have been implicated i n the development of Stevens-Johnson syndrome.55 Over t h e years, many other d m g s and c e r t a i n conditions ccination, foods, i n f e c t i o n s themselves) have a l s o been i m p l i ~ a t e d , 5 ~ 'a,nd t h i s very low incidence syndrome may be an a l l e r g i c r e a c t i n with multiple etiology o r may have a single, s t i l l undetermined cause. 5 Quinoxaline di-N-oxides, which were extensively investigated 15-20 years ago as antiviral, amebicidal and a n t i b a c t e r i a l agents, have received some f u r t h e r attention. Compounds such as 2,3-dimethylquinoxaline-l, &dioxide were shown57 e a r l i e r t o be a c t i v e the r e s u l t of rapid metabolism t o t h e hydroxymethyl analog. Recent work5 has disclosed a l s o the b i s ( hydroxymethyl) metabolite, which w a s s t i l l more active. However, t h e t o x i c i t y increased as d i d t h e a c t i v i t y and precluded chemotherapeutic use.@ The mono and b i s acetoxymethyl analogs were found59 t o have reasonable Gram-negative a c t i v i t y (5C$ survival a t 20-12Omg/kg i n t r a p e r i t o n e a l l y ) i n mice but near t o x i c levels. A quinoxaline-l,h-dioxide-2-aldehyde hydrazone (R-CH=N-r-mx-
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olidin-2-on-3-yl), o r a l l y a c t i v e against Pasteurella i n mice ( ~13mg/kg) 4 ~ and i n a Proteus urinary i n f e c t i o n model i n r a t s (min. effective'aose 3m@;/kg)," i s possibly r e l a t e d more t o i t s nitrofbrfurylidene analog, i'urazolidone, than t o the dioxides above, since the active s e r i e s here comprises only hydrazones. No t o ' c i t y e l a t i o n s h i p t o e i t h e r w a s indicated. reported the a c t i v i t y of three ammonium bisThree recent papers where Z i s CllH23, COOC12H25 and quaternaries, ZCH2(Me2)N-C6H&M(Me2)CH2Z COOCloH21, against a l e t h a l Klebsiella rhinoscleramatis i n f e c t i o n i n mice, i n t r a p e r i t o n e a l dosage of 2-5mg/kg twice d a i l y f o r 1 2 days giving 100% surand no data or vival. The LD50 5-iwl-e dose values given were comment on a c t i v i t y against o t h e r orgmisms was given. Antiseptics ( I n V i t r o Actives) .-Compounds a r e included here i f reasonably complete experimental i n d t r o data have been reported-inactivity i n vivo i s assumed unless t h e a r t i c l e s t a t e s o t h e n d s e . For assessing p r a c t i cal a p p l i c a b i l i t y , t h e e f f e c t of p r o t e i n and l i p i d on i n v i t r o a c t i v i t y and t o x i c i t y data a r e necessary but a r e frequently lacking. I n v i t r o structurea c t i v i t y s t u d i e s on i n vim a c t i v e s a r e covered i n t h a t section. N o i n v i t r o antituberculous r e p o r t s have been included.
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The b' y c l i c IX (f'urazolium chloride as i t s salt) had broad-spectrum a c t i v i t & t (O.b12mcg/ml) s u b s t a n t i a l l y superior t o t h a t of nitrof'urazone and comparable t o t h a t of nitrofhrylacrylamide. C-Methyl s u b s t i t u t i o n or r i n g expansion decreased a c t i v i t y while i n s e r t i o n of a vinyl group increased a c t i v i t y against Staph., Strep. and Aerobacter. The b i s ( n i t r o v 1 ) X had Coli-Staph. a c t i v i t y comparable t o I X but a narrower spectrum. 5 Same aryl and heteroaryl pyrazoles and isoxazoles X I were about as active66 (0.2t'vireported e a r l i e r . The a s - t r i a z i n e X I 1 had broad but apparently not as broad as t h e vinyl a n a 1 0 g , ~ ~ , or ~g the ~ - N ( C H ~ O A Cderivative )~ of t h e vinyl analog. 70
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XI11 XIV xv X V I SR Further work on nitrof'uryl viny168,70 and butadienyl compounds has been reported. 3-hino-6- [ 3 ' -methyl- 4 ' 1( 5-nitro- 2- f'uryl ) but a d i e i y l ] -as- t riazine, t h e butadien 1 analog of X I I , w a s very active i n v i t r o ( C o l i - S t a s . a t 0.03I n 2- d i subst i t u t e d amino- 1,3,h- oxadia zole s the n i t rofurylO.O6mcg/a) 3'-methylbutadienyl compound w a s l e s s a c t i v e than t h e v i n y l analog, and the thiadiazole analog of t h e best (0.8-2mcg/ml) compound w a s l e s s a ~ t i v e . 7 ~ Branching of the butadiene, e s p e c i a l l y with a l k y l s l a r g e r than methyl, decreased a c t i v i t y 7 3 of the 2-R2N-oxadiazoles, a s dld a 2-alkyl or phenyl group on t h e 0xadiazole.7~ 4-Methyl-3-methylthio-5-(5 '-nitro-2'-f'urylvinyl)1,2, h-triazole75 had Coli-Staph. a c t i v i t y a t l-l&cg/ml. A ten-fold increase i n Coli-Staph. a c t i v i t y ( t o l - a c g / k ~ )r ~ e s~u l t e d on c y c l i z a t i o n of t h e acyl semicarbazide t o the 2-amSno-oxadiazole X I I I . The 2-0x0 analog had Coli-Staph. a c t i v i t y a t lmcg/ml while i t s 2-ethoxy and
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2-chloro analogs were much l e s s active. Various 3-acyl-2-0x0 analogs of X I 1 1 were equally active but l e s s t o x i ;77 t h e 3-CH3-2-0x0 analog w a s more act i v e (Coli-Staph. a t O.lmcg/ml) .78 The 3 I-methylbutadlenyl 2-0x0 analog79 shifted i n a n t i b a c t e r i a l spectrum (Staph. 0.4 and Coli 5Omcg/ml). The pyrh i d i n o n e X I V w a s broadly a c t i v e but only a t lO-3Omcg/ml except f r Shigella (0.5mcg/ml) ; cross-resistance with fwazolidone was not observed. 80
I n the 3-(5'-nitro-2'-f'uryl)acryloyl g ou XV, a c t i v i t y w a s poor when Z was pyrazoline, a l k y l or carbethoxyalkyl.fjl,82 When Z was 2-imidazoline, Staph. a c t i v i t y w a s 10s without change i n Coli a c t i v i t y r e l a t i v e t o the When bromine i s present ( Z = CH$r o r NF-CH== simple amide8&8; RH~).'~ C ( Br )- CHO ) the compounds a c t by a d i f f e r e n t mechanism and a r e un table i n vivo t o SH groups. Then Z was " H - d i s u b s t i t u t e d ~ y r i m i d i n y 1 , 7 ~ , ~ ~ Coli-Staph. a c t i v i t y 1" good (lmcg/ml) but not when Z was NH-5-CH2pyrimidor " H - c e r t a i n a ~ y l s . ~The 7 spectrum of 60 n i t r o inyl-2,4-R2 (lOmcg/ f'urans w a s studied:@ classes of compound i n decreasing order of a c t i v i t y
,
)%
NF-CH=N-NH-hetero, NF-CH=N-hetero, IF-hetero, NFa r e NF--CH=CH-Z, C(=NH)--"KR. I n the aldoxime, the aldehyde guanylhydrazone and vinyl- quinoline comparisons89 of Coli-Staph. a c t i v i t y i n vitro, changing 5-nitro- t o 5-methylsulfonyl- f'uryl produced 4- t o 100-fold decreases i n a c t i v i t y , i n contrast t o the same v a r i a t i o n on chlormphenicol. The s u s c e p t i b i l i t y of almost all s t r a i n s of various species of Neisseria (gonorrhoeae, meningitidis, flava) t o i n v i t r o i n h i b i t i o n by acetazo1azni.de (2-acetanino-1 3, bthiadiazole-5-sulfonamide) i s unique r e l a t i v e t o other organisms, go, g i but apparently does not carry over t o i n vivo infections. I n h i b i t i o n of only N. eon. occurred a t high C02 concentrations; the other N. and r e s i s t a n t b a c t e r i a appear t o have low concentrations of carbonic anhyb a s e . 90 A miscellaneous group of a n t i s e p t i c s reported i s mostly reminiscent of known a n t i b a c t e r i a l types : 2-guanidino-6-substituted quinazolines92 vs. Staph. and Salmonella a t lO-lOOmcg/ml; N, N'-di( cyclohexylpropyl)-l, 4-bis (aminmethy1)cyclohexane~~vs. Staph., S t r p., Salm. and Coli a t 3-lOmcg/ml; 3-(p-bromophenyl)-6-brcano-l, 3-benzoxazineg' vs. Salm. and Klebsiella a t 204Omcg/ml; and sodium dodecyldi- (and mono) chlorodiphe loxidesulfonateg5 vs. Staph. a t 5mcg/ml. TI e a c t i v i t y of t h e pyridazinonesg XVI ( R = H and CH3) i s curiously specific: 0.5-3mcg/ml against Shigella species but much l e s s e f f e c t i v e against Coli and Salmonella. Mechanism of Action.-Nalidixic acid I s e l e c t i v e l y i n h i b i t s DNA synthesis i n growing E. c o l i , acting d i r e c t l y against i t s r e ~ l i c a t i o n . 9The ~ bacteric i d a l phase of i t s action can be prevented by i n h i b i t i o n of the synthesis of p r o t e i n (e.g. by chloranrphenicol) o r of RNA. Similarly, the addition of seve r a l , but not a l l , nitrof'urans antagonized i t s i n v i t r o a n t i b a c t e r i a l a c t i v i t y against various Proteus-Providence species, g8 but n a l i d i x i c acid did not oppose the action of the nitroflrrans. The l a t t e r a l s o antagonized i t s a c t i v i t y against Coli and Salmonella s t r a i n s , 99 as did t e t r a c y c l i n e and chloramphenicol against a number of coliform organisms.100 Against B. subt i l i s , one of the few Gram-positive b a c t e r i a susceptible t o it, the mode of action i s a l s o s e l e c t i v e i n h i b i t i o n of DNA synthesis.lol The various hy-potheses on the mechanism of a c t i o n of isoniazid have been reviewed. 102 Research Techniques And Screening Methods.-A v a r i e t y of chromatographic techniques continue t o be applied t o separation and i d e n t i f i c a t i o n of syn-
--
118
Sect. I11 - Chemotherapeutics
Cheney, Ed.
t h e t i c a n t i b a c t e r i a l agents and t h e i r metabolites. G a s chromatogra hy of 60 n i t r o f u r y 1 and n i t r o f l u y l v i n y l heterocycles has been a~complished'~3on a s i l i c o n e column generally without decomposition. Nitrof'urantoin, nitrof'urazone, and f b a z o l i d o n e as well as a l a r g e group of othe nitrofurans were studied i n a v a r i e t y of solvent systems by t h i n - l a y e r l o t and by paperlo? chromatography. About 20 sulfanilamide derivatives have been characterized by t h e i r thinl a y e r chromatographic behavior i n a dozen solvent systems (CHCl ROH mixtures with or without a t h i r d solvent) usin detection with iodine,l' p-dimethyla m i n o b e n ~ a l d e h y d e , ~or ~ 7 C U S O ~ - N H ~ O and H ~ ~quantitative ~ u l t r a v i o l e t spectrophotometry. lo6, 109 A two-dimensional t h i n - l a e r q u a l i t a t i v e method using a basic and an a c i d i c system was a l s o reported.f1o Work c o n t i n u e s l l l on automated analysis of sulfa drug samples (20 per hour) based on the usual diazotization and coupling method; reproducibility f o r therapeutic concentrat i o n s was s a t i s f a c t o r y (l-$) Paper chromatography of t u b e r c u l o s t a t i c agents containing a C = S moiety employed rapid development with methanolwater and detection by means of iodine-azide or mercury-fluorescein reagents.112 D i f f e r e n t i a l thermal analysis has been applied t o the examination of the i d e n t i t y and p u r i t y of ethambutol, s u l f a d r u g s and other synthetic pharmaceuticals. 113 Analog computers were used f o r model calculations on the influence of p r o t e i n b'nding on the clearance of a long-acting b-sulfa-5,6-dimethoxypyri m i d i el1$ and f o r devising optimal therapeutic regimens f o r sulfasymazine N o new a n t i b a c t e r i a l screening methods were reported. Recent success i n experimental animals and i n v i t r o with growing the lepros b a c i l l u s has l e d t o s i g n i f i c a n t and promising additions t o our knowlecige.lf5 Over 100 Myco. leprae s t r a i n s from a l l over the world and from the several c l i n i c a l types of leprosy have now produced mouse foot-pad infections. The r e l a t i o n s h i p of b a c t e r i a l morphology, administration of a n t i l e p r o t i c agents, BCG vaccination, and immune-response suppression t o production of increased infectivi t y i n mice has been studied. The neural involvement produced by t h i s organism i n t h e mouse and hamster i n f e c t i o n s i s unique among the mycobacteria and i s c h a r a c t e r i s t i c of t h e c l i n i c a l disease. Reviews.-The followi have been reviewed: species differences amon drofolate reductases, ethambutol laboratory and c l i n i c a l studies, $9, 'vstructure-activity r e l a t i o n s h i p s i n ethambutol analogs, 32 mode of act i o n of isoniazid,lo2 progress i n l e rosy research,115 lorg-acting s u l f a n i l amides i n urinary t r a c t infections, 48 and i n f e c t i o u s drug resistance. 1 23 39 4
.
.k
8%
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d,
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"
Chap. 1 2
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A. Forkman and A. B. Laurell, Rcta Pathol. Microbiol. Scand. 65, 450
(1965). 91. T. C. Eickhoff, 6 t h I n t e r s c i . Conf. Antimicrobial Agents Chemother.
p. 1. P. Brown, B r i t i s h patent 1,024,907 (1966). G. Humber, U. S. patent 3,244,750 (1966). Urbanski and J. Venulet, French patent 1,429,328 (1966). F. Steinhauer and J. C. Valenta, U. S. patent 3,252,856 (1966). Takahashi and T. Maki, Japanese patent 103( '66). 97. T\i. H. Dietz, T. M. Cook and W. 2.. Goss, J. Bacteriol. 91, 768 (1966). 98. Y. Kanazawa and T. Kuramata, Jap. J. Bacteriol. 21, 21F(1966). 99. W, S t i l l e and K. H. Ostner, Klin. Wochschr. 44, 3 5 (1966). 100. R. P. Mouton and A. Koelman, Chemotherapia l c 10 (1966). 101. T. M. Cook K. G. Brown, J. V. Boyle and W. A. Goss, J. Bacteriol. 92, 1510 (lg66). 102. J. Nestler, Arzneimittel-Forsch. 16, 1442 (1966). 103. K. Nakamura, Y. Utsui and Y. N i n a y a , Y a k u g a k u Zasshi 86, 404 (1966). 104. R. Neidlein, E. Hohndorf and J. Rosenblath, Pharm. Z t g . Ll l , 874 (1966). 105. V. Cohen, J. Chrcanatog. 23, 446 (1966). 106. M. Sarsunova, V. SchwarzTE. Feketeova and J. Protiva, P h m a z i e 21, 219 (1966). 107. C. H. Pao, Chem. Abstr. 65, 6996g (1966). 108. K. C. Guven and 0. PekinTChem. Abstr. 65, 1992813 (1966). 109. G. Wagner and J. Wandel, Pharmazie 21, 105 (1966). 110. W. Kamp, F'harm. Weekblad 101, 181 (1966). 1ll. H. P. Probst, W. F. Rehm H .Spoerri and J. P. Virilleumier, Chem. Abstr. 64 11,703f (1966). 112. D. G o e c d i t z , Pharmazie 21, 668 (1966). U3. L. M. Brancone and H. J. Errari, Microchem. J. 10, 370 (1966). 114. E. Krueger-Thiemer, Arzneimittel-Forsch. 16, 1 4 3 r (1966). 115. Editors, Chronicle WHO 20, 98 (1966). 116. G. H. Hitchings and J. Burchall, Federation Proc. 25, 881 (1966). 117. N. Y. Acad. Sci. Conference, L m . N. Y. Mad. Sci. 680-939, 107% 1084, 1098- 1u8 ( 1966)
1966, Abstr.
92. J. 93. L. 94. T. 95. A. 96. T.
-
c
.
ax
,
,
Chap. 12
Antibacterials
.
Shepherd and Lewis
113 -
with subcut. lOOmg/kg/day) Curiously, t h r e e 1,7-naphthyridinesl1 had only Gram-positive a c t i v i t y : 1,6,8-trimethy1-3- carboxy-boxo-lJ7-naphthyridine was a c t i v e against Staph., Strep. and Pneumococcus ( p a r t l y e f f e c t i v e ) a t 25, 100 and bOmg/kg/day o r a l l y i n mice; the lY8-dimethyl-6-ethyl and t h e 1e t h y l analogs were much l e s s active.
H
0
I1 I11 IV I Several types of nitrof'urans (NF = 5-nitrc-2-fbyl) with i n t e r e s t i n g l e v e l s of i n vivo a c t i v i t y were reported but margins of s a f e t y were not given. The 4-aminoquinazolines I1 [Z = N(CH3)C2H40HY N(C2H)+OH)2and NHC)+H8-1-pyrrolidinyl] had a broad i n v i t r o spectrum, including seudmonas, Proteus and Aerobacter, and high a c t i v i t y (0.01-12mcg/ml) .15 However, only a c t i v i t y i n vivo against Staph. aureus was reported ( E i n t r a p e r i t o n e a l 1-15mg/kg) ; a c t i v i t y c o r r e l a t e d poorly with data. Homologs of CH3 were l e s s active. Pyridazinone I11 and i t s dihydro intermediate13 were about equal i n vim and i n v i t r o i n Salm.-Staph. activi t y ; 2-alkylation lowered t h e i n v i t r o a c t i v i t y of both. I n contrast, it increased t h e i n vivo a c t i v i t y of t h e former and decreased t h a t of t h e l a t ter. Their i n v i t r o a c t i v i t y (min. inhib. concn. 2-6mcg/d) was l e s s than t h a t of I1 (0.05-0.8mcg/d) while t h e i n vivo a c t i v i t y was the same. Also poorer i n a c t i v i t y i n v i t r o and i n spectrum was t h e t r i a z o l e I V which had comparable Salm.-Staph. a c t i v i t y i n vivo (ED50 20-40mg/kg) .I4 1-Acylation or 5-alkylation decreased i t s a c t i v i t y which w a s g r e a t e r than t h a t of t h e upen-chain N-formylamino nitrof'uramidine, i n t u r n more a c t i v e i n vivo than t h e anddine i t s e l f . The l-methylcarbamoyl-5-imino derivative o f I V w a s a c t i d 5 against Staph., Salm. and Coli i n mice a t about 4Omg/kg with an oral LD 0 of 1 9 0 . A s e r i e s of 2-(5 '-nitro-2'-fluyl)CH=CHpyridines showed he following s i n g l e i n t r a p e r i t o n e a l anti-Salmonella a c t i v i t y ( EDF0 2-10mgjkg; oral ED5 5-75mg/kg) r e l a t i o n s h i p i n mice: 6-CH2OH-l-oxide = 5-CH20Ac-l-oxide > t-CH$Ac-l-oxide > 5-CFi2OAc > ~ - C H ~ O = H 5-CH OH > 6-CH20Ac with approximately t h e same r e l a t i o n s h i p of t o x i c i t y $ The 5methyl-1-oxide reported l a s t year had canparable a c t i v i t y . The a s - t r i a z i n e derivative V was p a r t l y effective17 against Staph. and Salm. i n f e c t i o n s i n mice a t lOOmg/kg. Activity of 5-methylmercapt methyl-+( 5 ' - n i t r o f b f u r y l ideneamino)-oxazolidin-2-one w a s unpromising18 i n various urinary infect i o n s i n a small c l i n i c a l trial. Further c l i n i c a l investigations have confirmed the e f f i c a c y of ethambut o 1 (EMB), (+)-N,N'-bis( l-hydroxy-2-butyl)ethylenedi~ne, i n treatment of pulmonary tuberculosis and a l s o demonstrated i t s use against r e n a l and e treatment of pulmonary i n f e c t i o n s due vesical tuberculosisY19 t o a t y p i c a l mycobacteria. I n controlled t r i a l s conducted i n the National Sanatoria of Japa.n2s t o determine t h e b e s t t h i r d component of a t r i p l e regimen with i s o n i a z i d (INH) and streptomycin (SM) i n o r i g i n a l treatment, cycloserine and p-amino-salicylic a c i d (PAS) were l e s s e f f e c t ive and more t o x i c than EX3 while ethionamide w a s as e f f e c t i v e s EMB but l e s s well t o l e r a t e d ( c f . a dose-response c a p a r i s o n i n monkeys2'). In a
€
28,&, 29
Sect. 111 - C h e m o t h e r a p e u t i c s
114 -
Cheney, Ed.
companion trial, 25 ethambutol ( l g d a i l y ) cambined with e i t h e r cycloserine (0.5g d a i l y ) o r ethionamide (O.5g d a i l y ) was well t o l e r a t e d and h i g h l y e f f e c t i v e i n t u b e r c u l o s i s re-treatment ( r e s i s t a n t t o SM, INH and PAS). I n t h e s e t r i a l s a diminution i n v i s u a l a c u i t y occurred i n about 8 2 of t h e pat i e n t s receiving l g d a i l y ; t h e e f f e c t was r e v e r s i b l e . h i s i cidence and r e v e r s i b i l i t y w a s observed i n o t h e r c l i n i c a l s t u d i e s . 2 ?27,2j29 A reduced dosage regimen30 avoided t h e v i s u a l disturbance While EMB by i t s e l f i s e f f e c t i v e a g a i n s t a c u t e and chronic t u b e r c u l o s i s , 28,30J 31 combined t h e r apy i s r e c m e n d e d t o avoid emer ence of r e s i s t a n t mycobacteria. I n man, about 10,;of EMF3 i s metabolized 27 t o t h e i n a c t i v e d i c a r b o x y l i c a c i d by t h e sequence -CH;zOH + -CHO j -C02H. A s t r u c t u r e - a c t i v i t y has defined t h e very s p e c i f i c requirements f o r a c t i v i t y i n an extensive s e r i e s of EMB analogs
=
.
TzH5
8
8
?!2H5 s"
"H2
\
s" t\ d/
m2 VI
V VIII I n combination with SM, ethionamide (ETH, l g d a i l y ) was as e f f e c t i v e as IhW (0.3g d a i l y ) i n m a n by t h r e e t h e r a p e u t i c c r i t e r i a . However, t h e ETH
regimen w a s considered t o be t o o t o x i c t o be c l i n i c a l l y acceptable.33 C l i n i c a l incidence of s i d e - e f f e c t s from ETH ( p l u s INH). decreas d sharply with dosage: 31% f o r lg, 18;; f o r 0.75g and 87: f o r O.5g d a i l y . 3E Separation and c h a r a c t e r i z a t i o n o f ETH m e t a b o l i t e s has been achieved and t h e i r quantit a t i v e i n t e r r e l a t i o n i s under s t u The m a j o r i t y of ETH i s metabolized and r a p i d l y e x c r e t e d i n t h e u r i n e 3 5 A s u b s t a n t i a l m o u n t i s converted t o i t s sulfoxide VI which has now been shown t o be e u a l y a c t i v e and i n t e r ,3~ metaboc o n v e r t i b l e w i t h ETH i n man and o t h e r s p e ~ i e s . 3 ~ ~ 3 7Extensive l i s m t o i n a c t i v e compounds a l s o occurs by d e t h i a t i o n ( d i r e c t l y or, more probably, through t h e s u l f o x i d e ) t o t h e carboxamide, deamidation leading t o 2-ethyl i s o n i c o t i n i c acid, and by 1-methylation t o give VII, which i s p a r t l y converted t o 11. Dethiated VII and VIII and t h e sulfoxide of VIII were a l s o observed. Continued work on sulfanilamides includes s t u d i e s on t e s t s t o guide t h e i r use, new uses, new compounds, metabolism, p r o t e i n binding, and a t o x i c i t y warning. An improved method f o r determining b a c t e r i a l s e n s i t i v i t y t o s u l f a d r u g s permits r e l i a b l y t & i n g advantage o f t h e i r a c t i v i t y a g a i n s t 80-9@ of E. c o l i and Proteus m i r a b i l i s ur2nary i n f e c t i o n s i n gene r a l p r a c t i c e , i n b a c t e r i u r i a of pregnancy and i n h o s p i t a l patients.39 Synergism between t h e i n e f f e c t i v e polymyxins and t h e b a c t e r i o s t a t i c s u l f a d r u g s i n v i t r o , using a number of s t r a i n s of Proteus m i r a b i l i s and vulgari s , l e d t o an 8- t o 6 b f o 1 d decrease i n a c t i v e concentration and t o bactericidal actionb, 41 ( c f . i n vivo work i n 1963). N o such i n t e r a c t i n occurred Plaswith e i t h e r of t h e s e agents when combined with f i v e modium falciparum s t r a i n s r e s i s t a n t t o chloroquine and quinine r e c l i n i c a l l y s u s c e p t i b l e t o sulfanilamides and diaminodiphenylsulfone. &2j43 Sulfasymazine "appea:c4to be a usef'ul agent i n t h e treatment of b a c t e r i u r i a during pregnancy. U s i n g t h e computer method r e c e n t l y reported, The
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dosing i n t e r v a l and the maintenance dose f o r sulfasymazine has been calculated.45 A s i n other s u l f a n i l m i d o heterocycles, introduction of bromine or chlorine i n t o the heterocycle increased t h e persistence& of 4-sulfa-2,6dimethoxypyrimidine; acute t o x i c i t y and p r o t e i n binding were a l s o increased. The isomeric 5-sul a- 3 - e t h y l - l , 2 , b t h i a d i a z o l e and 2 - e t h y l - l , 3 , b t h i a d i a zole were compared 7 as t o d e t a i l e d differences i n r e n a l elimination chara c t e r i s t i c s independent of p r o t e i n binding. C l i n i c a l and pharmacological studi@ were done on extremely long-acting 4- sulfa-5,6- dimethoxypyrimidine, and on t h i s and four other long-acting sulfa drugs (3-sulfa-6-methoxypyridazine, 4-sulfa-2,6-dime hoxypyrimidine, 2- sulfa-5-methoxypyrimidine and 2-sulfa-3-methoxypyrazine). 89 A study50 of 2-sulfanilamidopyrimidine i t s e l f rounds out the p i c t u r e of the metabolism of ulfapyrimidines : 5 l l a r g e variations i n N4-acetylation, i n N4-sulfonate, N -glucuronide and N1-glucuronide formations occur i n closely r e l a t e d compounds. The 5,6-dimethoxy- and 2,6-dimethoxy- &sulfapyrimidines a r e $ and 8G6 converted t o glucuronides. I n contrast t o these 2-sulf&4,6-dimethoxypyrimidine i s not N4and other sulfapyrimidine acetylated i n vivo; i t s - a c e t y l derivative is, i n f a c t , r a p i d l y deacetylated. Further study of s u l f a drug p r o t e i n binding has indicated t h a t t h e r e i s more t o i t s e f f e c t on a n t i b a c t e r i a l a c t i v i t y than ,just the Dercent bound a t equilibrium under s p e c i a l conditions. Human serum w a s found t o increase the minimal i n h i b i t o r y concentration f o r both sulfadiazine and sulfadimethoxine but the increase for each was eight times as great; f o r Shig. dysent e r i a e as f o r E, c 0 l i . 5 ~In addition, the r a t i o of t h e increases i n m.i.c.'s of the two s u l f a s i n the presence of 20-5C$ human serwn was too s m a l l ( 2 ) t o correspond t o the r a t i o (42) of percentage of unbound sulfa. The binding capacity of human serum p r o t e i n determined by equilibrium dialysis53 w a s nearly the same (ca. 2 moles/mole protein) f o r 8 sulfanilanides (pKa's 5.77.2), t h i s s m a l l capacity supporting chemical. r a t h e r than absorptive binding; p r o t e i n a f f i n i t i e s varied a hundred-fold. A quite d i f f e r e n t number ( ) of binding s i t e s f o r one of these, sulfamethoxypyridazine, was calculated from thermodynamic data based on e q u i l i b r a t e d Sephadex columns; the energ e t i c s indicated binding by van der Waal's forces. R warning has been issued t o physicians t h a t the long-acting sulfcnild d e s , sulfamethoxypyridazine and sulfadimethoxine, have been implicated i n the development of Stevens-Johnson syndrome.55 Over t h e years, many other d m g s and c e r t a i n conditions ccination, foods, i n f e c t i o n s themselves) have a l s o been i m p l i ~ a t e d , 5 ~ 'a,nd t h i s very low incidence syndrome may be an a l l e r g i c r e a c t i n with multiple etiology o r may have a single, s t i l l undetermined cause. 5 Quinoxaline di-N-oxides, which were extensively investigated 15-20 years ago as antiviral, amebicidal and a n t i b a c t e r i a l agents, have received some f u r t h e r attention. Compounds such as 2,3-dimethylquinoxaline-l, &dioxide were shown57 e a r l i e r t o be a c t i v e the r e s u l t of rapid metabolism t o t h e hydroxymethyl analog. Recent work5 has disclosed a l s o the b i s ( hydroxymethyl) metabolite, which w a s s t i l l more active. However, t h e t o x i c i t y increased as d i d t h e a c t i v i t y and precluded chemotherapeutic use.@ The mono and b i s acetoxymethyl analogs were found59 t o have reasonable Gram-negative a c t i v i t y (5C$ survival a t 20-12Omg/kg i n t r a p e r i t o n e a l l y ) i n mice but near t o x i c levels. A quinoxaline-l,h-dioxide-2-aldehyde hydrazone (R-CH=N-r-mx-
f
6
8
?(i
6
2
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Cheney, Ed.
olidin-2-on-3-yl), o r a l l y a c t i v e against Pasteurella i n mice ( ~13mg/kg) 4 ~ and i n a Proteus urinary i n f e c t i o n model i n r a t s (min. effective'aose 3m@;/kg)," i s possibly r e l a t e d more t o i t s nitrofbrfurylidene analog, i'urazolidone, than t o the dioxides above, since the active s e r i e s here comprises only hydrazones. No t o ' c i t y e l a t i o n s h i p t o e i t h e r w a s indicated. reported the a c t i v i t y of three ammonium bisThree recent papers where Z i s CllH23, COOC12H25 and quaternaries, ZCH2(Me2)N-C6H&M(Me2)CH2Z COOCloH21, against a l e t h a l Klebsiella rhinoscleramatis i n f e c t i o n i n mice, i n t r a p e r i t o n e a l dosage of 2-5mg/kg twice d a i l y f o r 1 2 days giving 100% surand no data or vival. The LD50 5-iwl-e dose values given were comment on a c t i v i t y against o t h e r orgmisms was given. Antiseptics ( I n V i t r o Actives) .-Compounds a r e included here i f reasonably complete experimental i n d t r o data have been reported-inactivity i n vivo i s assumed unless t h e a r t i c l e s t a t e s o t h e n d s e . For assessing p r a c t i cal a p p l i c a b i l i t y , t h e e f f e c t of p r o t e i n and l i p i d on i n v i t r o a c t i v i t y and t o x i c i t y data a r e necessary but a r e frequently lacking. I n v i t r o structurea c t i v i t y s t u d i e s on i n vim a c t i v e s a r e covered i n t h a t section. N o i n v i t r o antituberculous r e p o r t s have been included.
1%1,62,G +
The b' y c l i c IX (f'urazolium chloride as i t s salt) had broad-spectrum a c t i v i t & t (O.b12mcg/ml) s u b s t a n t i a l l y superior t o t h a t of nitrof'urazone and comparable t o t h a t of nitrofhrylacrylamide. C-Methyl s u b s t i t u t i o n or r i n g expansion decreased a c t i v i t y while i n s e r t i o n of a vinyl group increased a c t i v i t y against Staph., Strep. and Aerobacter. The b i s ( n i t r o v 1 ) X had Coli-Staph. a c t i v i t y comparable t o I X but a narrower spectrum. 5 Same aryl and heteroaryl pyrazoles and isoxazoles X I were about as active66 (0.2t'vireported e a r l i e r . The a s - t r i a z i n e X I 1 had broad but apparently not as broad as t h e vinyl a n a 1 0 g , ~ ~ , or ~g the ~ - N ( C H ~ O A Cderivative )~ of t h e vinyl analog. 70
yo
"
NF- CH=CH- C Z'
XI11 XIV xv X V I SR Further work on nitrof'uryl viny168,70 and butadienyl compounds has been reported. 3-hino-6- [ 3 ' -methyl- 4 ' 1( 5-nitro- 2- f'uryl ) but a d i e i y l ] -as- t riazine, t h e butadien 1 analog of X I I , w a s very active i n v i t r o ( C o l i - S t a s . a t 0.03I n 2- d i subst i t u t e d amino- 1,3,h- oxadia zole s the n i t rofurylO.O6mcg/a) 3'-methylbutadienyl compound w a s l e s s a c t i v e than t h e v i n y l analog, and the thiadiazole analog of t h e best (0.8-2mcg/ml) compound w a s l e s s a ~ t i v e . 7 ~ Branching of the butadiene, e s p e c i a l l y with a l k y l s l a r g e r than methyl, decreased a c t i v i t y 7 3 of the 2-R2N-oxadiazoles, a s dld a 2-alkyl or phenyl group on t h e 0xadiazole.7~ 4-Methyl-3-methylthio-5-(5 '-nitro-2'-f'urylvinyl)1,2, h-triazole75 had Coli-Staph. a c t i v i t y a t l-l&cg/ml. A ten-fold increase i n Coli-Staph. a c t i v i t y ( t o l - a c g / k ~ )r ~ e s~u l t e d on c y c l i z a t i o n of t h e acyl semicarbazide t o the 2-amSno-oxadiazole X I I I . The 2-0x0 analog had Coli-Staph. a c t i v i t y a t lmcg/ml while i t s 2-ethoxy and
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2-chloro analogs were much l e s s active. Various 3-acyl-2-0x0 analogs of X I 1 1 were equally active but l e s s t o x i ;77 t h e 3-CH3-2-0x0 analog w a s more act i v e (Coli-Staph. a t O.lmcg/ml) .78 The 3 I-methylbutadlenyl 2-0x0 analog79 shifted i n a n t i b a c t e r i a l spectrum (Staph. 0.4 and Coli 5Omcg/ml). The pyrh i d i n o n e X I V w a s broadly a c t i v e but only a t lO-3Omcg/ml except f r Shigella (0.5mcg/ml) ; cross-resistance with fwazolidone was not observed. 80
I n the 3-(5'-nitro-2'-f'uryl)acryloyl g ou XV, a c t i v i t y w a s poor when Z was pyrazoline, a l k y l or carbethoxyalkyl.fjl,82 When Z was 2-imidazoline, Staph. a c t i v i t y w a s 10s without change i n Coli a c t i v i t y r e l a t i v e t o the When bromine i s present ( Z = CH$r o r NF-CH== simple amide8&8; RH~).'~ C ( Br )- CHO ) the compounds a c t by a d i f f e r e n t mechanism and a r e un table i n vivo t o SH groups. Then Z was " H - d i s u b s t i t u t e d ~ y r i m i d i n y 1 , 7 ~ , ~ ~ Coli-Staph. a c t i v i t y 1" good (lmcg/ml) but not when Z was NH-5-CH2pyrimidor " H - c e r t a i n a ~ y l s . ~The 7 spectrum of 60 n i t r o inyl-2,4-R2 (lOmcg/ f'urans w a s studied:@ classes of compound i n decreasing order of a c t i v i t y
,
)%
NF-CH=N-NH-hetero, NF-CH=N-hetero, IF-hetero, NFa r e NF--CH=CH-Z, C(=NH)--"KR. I n the aldoxime, the aldehyde guanylhydrazone and vinyl- quinoline comparisons89 of Coli-Staph. a c t i v i t y i n vitro, changing 5-nitro- t o 5-methylsulfonyl- f'uryl produced 4- t o 100-fold decreases i n a c t i v i t y , i n contrast t o the same v a r i a t i o n on chlormphenicol. The s u s c e p t i b i l i t y of almost all s t r a i n s of various species of Neisseria (gonorrhoeae, meningitidis, flava) t o i n v i t r o i n h i b i t i o n by acetazo1azni.de (2-acetanino-1 3, bthiadiazole-5-sulfonamide) i s unique r e l a t i v e t o other organisms, go, g i but apparently does not carry over t o i n vivo infections. I n h i b i t i o n of only N. eon. occurred a t high C02 concentrations; the other N. and r e s i s t a n t b a c t e r i a appear t o have low concentrations of carbonic anhyb a s e . 90 A miscellaneous group of a n t i s e p t i c s reported i s mostly reminiscent of known a n t i b a c t e r i a l types : 2-guanidino-6-substituted quinazolines92 vs. Staph. and Salmonella a t lO-lOOmcg/ml; N, N'-di( cyclohexylpropyl)-l, 4-bis (aminmethy1)cyclohexane~~vs. Staph., S t r p., Salm. and Coli a t 3-lOmcg/ml; 3-(p-bromophenyl)-6-brcano-l, 3-benzoxazineg' vs. Salm. and Klebsiella a t 204Omcg/ml; and sodium dodecyldi- (and mono) chlorodiphe loxidesulfonateg5 vs. Staph. a t 5mcg/ml. TI e a c t i v i t y of t h e pyridazinonesg XVI ( R = H and CH3) i s curiously specific: 0.5-3mcg/ml against Shigella species but much l e s s e f f e c t i v e against Coli and Salmonella. Mechanism of Action.-Nalidixic acid I s e l e c t i v e l y i n h i b i t s DNA synthesis i n growing E. c o l i , acting d i r e c t l y against i t s r e ~ l i c a t i o n . 9The ~ bacteric i d a l phase of i t s action can be prevented by i n h i b i t i o n of the synthesis of p r o t e i n (e.g. by chloranrphenicol) o r of RNA. Similarly, the addition of seve r a l , but not a l l , nitrof'urans antagonized i t s i n v i t r o a n t i b a c t e r i a l a c t i v i t y against various Proteus-Providence species, g8 but n a l i d i x i c acid did not oppose the action of the nitroflrrans. The l a t t e r a l s o antagonized i t s a c t i v i t y against Coli and Salmonella s t r a i n s , 99 as did t e t r a c y c l i n e and chloramphenicol against a number of coliform organisms.100 Against B. subt i l i s , one of the few Gram-positive b a c t e r i a susceptible t o it, the mode of action i s a l s o s e l e c t i v e i n h i b i t i o n of DNA synthesis.lol The various hy-potheses on the mechanism of a c t i o n of isoniazid have been reviewed. 102 Research Techniques And Screening Methods.-A v a r i e t y of chromatographic techniques continue t o be applied t o separation and i d e n t i f i c a t i o n of syn-
--
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t h e t i c a n t i b a c t e r i a l agents and t h e i r metabolites. G a s chromatogra hy of 60 n i t r o f u r y 1 and n i t r o f l u y l v i n y l heterocycles has been a~complished'~3on a s i l i c o n e column generally without decomposition. Nitrof'urantoin, nitrof'urazone, and f b a z o l i d o n e as well as a l a r g e group of othe nitrofurans were studied i n a v a r i e t y of solvent systems by t h i n - l a y e r l o t and by paperlo? chromatography. About 20 sulfanilamide derivatives have been characterized by t h e i r thinl a y e r chromatographic behavior i n a dozen solvent systems (CHCl ROH mixtures with or without a t h i r d solvent) usin detection with iodine,l' p-dimethyla m i n o b e n ~ a l d e h y d e , ~or ~ 7 C U S O ~ - N H ~ O and H ~ ~quantitative ~ u l t r a v i o l e t spectrophotometry. lo6, 109 A two-dimensional t h i n - l a e r q u a l i t a t i v e method using a basic and an a c i d i c system was a l s o reported.f1o Work c o n t i n u e s l l l on automated analysis of sulfa drug samples (20 per hour) based on the usual diazotization and coupling method; reproducibility f o r therapeutic concentrat i o n s was s a t i s f a c t o r y (l-$) Paper chromatography of t u b e r c u l o s t a t i c agents containing a C = S moiety employed rapid development with methanolwater and detection by means of iodine-azide or mercury-fluorescein reagents.112 D i f f e r e n t i a l thermal analysis has been applied t o the examination of the i d e n t i t y and p u r i t y of ethambutol, s u l f a d r u g s and other synthetic pharmaceuticals. 113 Analog computers were used f o r model calculations on the influence of p r o t e i n b'nding on the clearance of a long-acting b-sulfa-5,6-dimethoxypyri m i d i el1$ and f o r devising optimal therapeutic regimens f o r sulfasymazine N o new a n t i b a c t e r i a l screening methods were reported. Recent success i n experimental animals and i n v i t r o with growing the lepros b a c i l l u s has l e d t o s i g n i f i c a n t and promising additions t o our knowlecige.lf5 Over 100 Myco. leprae s t r a i n s from a l l over the world and from the several c l i n i c a l types of leprosy have now produced mouse foot-pad infections. The r e l a t i o n s h i p of b a c t e r i a l morphology, administration of a n t i l e p r o t i c agents, BCG vaccination, and immune-response suppression t o production of increased infectivi t y i n mice has been studied. The neural involvement produced by t h i s organism i n t h e mouse and hamster i n f e c t i o n s i s unique among the mycobacteria and i s c h a r a c t e r i s t i c of t h e c l i n i c a l disease. Reviews.-The followi have been reviewed: species differences amon drofolate reductases, ethambutol laboratory and c l i n i c a l studies, $9, 'vstructure-activity r e l a t i o n s h i p s i n ethambutol analogs, 32 mode of act i o n of isoniazid,lo2 progress i n l e rosy research,115 lorg-acting s u l f a n i l amides i n urinary t r a c t infections, 48 and i n f e c t i o u s drug resistance. 1 23 39 4
.
.k
8%
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