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9.1. Introduction

One reason for synthesizing and testing conjugates of a nitrofuran with a polyamine was the proposal that this might increase the antibacterial activity of the nitrofuran by increasing its uptake into the cell via active transport systems for polyamines (Introduction 2.4). Of the compounds synthesized, compounds I, II, III and IV (Figure 4.9.1) were shown to be more active than nitrofurantoin (Figure 4.9.1 ) against E coli (Tables 4.7.1 and 4.7.2). If the above hypothesis is correct, polyamine conjugates, such as compounds I, II and III, should be relatively less active against polyamine uptake-deficient mutants of E. co//than wild-type strains. Whereas compound IV, which is a benzoyl conjugate, and nitrofurantoin itself might be predicted to have similar activities against polyamine-deficient and -proficient strains.

O NO

O NO

III O.N. , 0

o NO

^ Jj—OH—N—^ ^ —CO—NH—CH^CH^NH —CO—CH3

r - i °

O2N. . 0 , ,NH

Nitrofurantoin

Figure 4.9.1. Structures of novel nitrofuran compounds I - IV and nitrofurantoin.

I = bis-5-nitrofurfurylidenylputrescine; II = bis-5-nitrofurfurylidenylspermine; III = bis-5-nitro- furfurylidenylspermidine; IV = 4-(N-5-nitrofurfurylidene)aminobenzoyl-2-acetylaminoethyl- amide

Kashiwagi et al. (1990) have isolated several polyamine transport deficient mutants of E. coli MA261 (Materials & Apparatus Table 2.1.1), a polyamine- requiring mutant. These are KK313 which is deficient in spermidine uptake, NH1596 which is deficient in spermidine and 90% deficient in putrescine uptake and KK313

potF/.Km which is deficient in spermidine and putrescine uptake.

9.2. Antibacterial activity of the novel nitrofuran compounds in liquid medium Volumes of nutrient broth containing 100 gg ml'"' of test compound or nitrofurantoin were inoculated with a 1 in 50 dilution of an overnight culture of the organisms. The concentration of 100 pg ml'^ was chosen because this concentration of nitrofurantoin is bactericidal to E. coli (Figure 4.1.11 ). Therefore, since the novel nitrofuran compounds have activities better than or similar to nitrofurantoin (Tables 4.7.1 and 4.7.2), the polyamine transport mutants should be more resistant to the respective novel nitrofuran compounds than the polyamine uptake-proficient parent strain. Compounds I and I V were dissolved in DMSO, compounds I I and I I I were dissolved in HOI and neutralized with NaOH, and nitrofurantoin was suspended in water and dissolved by the addition of NaOH ( Materials & Apparatus 4). Drug-free nutrient broth and nutrient broth containing 2% DMSO were used as controls. All mixtures were held in a water bath at 37°C for five hours and viable counts determined hourly on nutrient agar (Experimental Methods 2.3). The effect of the novel nitrofuran compounds upon the survival of these strains is shown in Figures 4.9.2 to 4.9.8.

<u

Time of exposure (hours)

-9

—

—

I —

^

Figure 4 . 9 . 2 . N u tr ie n t b ro th

Time of exposure (hours)

Figure 4.9.3. 2 % D M S O in n u tr ie n t b ro th

Figures 4.9.2 & 4.9.3. G r o w th o f E. coli s tr a in s M A 2 6 1 , K K 3 1 3 , N H 1 5 9 6 a n d K K 3 1 3 p o fF ;: K m in d r u g - f r e e n u tr ie n t b ro th o r n u trie n t b ro th c o n ta in in g 2 % D M S O .

Cells grew exponentially and at similar rates in both drug-free nutrient broth (Figure 4.9.2) and in nutrient broth containing 2% DMSO (Figure 4.9.3) showing that 2% DMSO did not affect growth. The initial rate of growth of strain KK313 pofF::Km was higher than the other three strains.

\ o

c /5

Time of exposure (hours)

Figures 4.9.4, 4.9.5 & 4.9.6.

Survival of E. coli strains MA261, KK313, NH1596 and KK313 potF:Km in 100 pg mT^ of nitrofurantoin or compounds I or II. □ =MA261; 0 = KK313; V = NH1596; O = KK313 pofF:;Km Figure 4.9.4. Nitrofurantoin G Q--- ©---Q--- o o- 10’ - c /5

Time of exposure (hours)

V \

c /5

10" ’ -

V

Time of exposure (hours) Figure 4.9.5. Compound Figure 4.9.6. Compound II

10^ - -

Time of exposure (hours)

— Q---O---o

- - - O --- . O

10’ -

co

Time of exposure [hours]

Figure 4.9.7. C o m p o u n d I I I Figure 4.9.8. C o m p o u n d I V

Figures 4.9.7 & 4.9.8. S u rv iv a l o f E. coli s tr a in s M A 2 6 1 , K K 3 1 3 , N H 1 5 9 6 a n d K K 3 1 3

p o tF /K m in 1 0 0 p g m M o f c o m p o u n d s I I I o r I V .

□ = M A 2 6 1 ; 0 = K K 3 1 3 ; V = N H 1 5 9 6 ; O = K K 3 1 3 p o fF ::K m

The kill kinetics of the polyamine transport proficient control strain MA261 in 100 pg ml'"' of nitrofurantoin were similar to those of E. coli AB1157, which had previously been used in bactericidal studies (compare Figures 4.9.4 and 4.1.11). Nitrofurantoin had similar activities against strains KK313 and KK313 potF::Km but strain NH1596 was significantly more sensitive.

The addition of 100 pg ml'"' of compound I, the putrescine conjugate (Figure 4.9.5) inhibited growth of strain KK313 potF::Km and was mildly bactericidal for strains MA261, KK313 and NH1596 (Figure 4.9.5). Since the compound is at least inhibitory to all four strains it can be assumed that it is able to enter cells of all of these strains regardless of their transport deficiency.

Compounds II and III, the spermine and spermidine conjugates respectively, were also bactericidal against all of the strains (Figures 4.9.6 & 4.9.7). As it has been suggested that spermine is transported by the same system as spermidine (Kashiwagi eta!., 1986), neither of these conjugates should have been bactericidal to any of the polyamine transport-deficient mutants as they all lack the spermidine

transporter. However, due to the relatively bulky nitrofuran groups at both ends of the spermine or spermidine spacer, it is possible that these compounds were not transported via the spermidine transport system but by some other method.

Compound IV, the benzoyl conjugate (Figure 4.9.8), gave results similar to compound I. It was bacteriostatic rather than significantly bactericidal against all strains over the five hour period. Since this compound is not a polyamine derivative it was expected that it might be as bactericidal as nitrofurantoin to all strains. However, it would appear that uptake of this benzoyl conjugate is otherwise affected.

9.3. Summary

The four novel nitrofuran compounds did not behave as predicted against the three polyamine transport deficient strains of E. coli. It was expected that if the compounds were taken up only through active polyamine transport systems then the strains that were deficient in the particular transport mechanisms would be resistant to the respective compounds. However, since compounds I and IV are bacteriostatic and II and III are bactericidal to all strains, it must be assumed that alternate routes of uptake are available for these compounds.