The Effect of Mixtures of Sulfonamides and Urea Derivatives upon Bacterial Growth in Vitro
Authors: Tenenberg D. J. , Tsuchiya H. M. , Clark W. G. , Larson Nora 
Published: January 2023

Summary and Discussion
Table 10 suggests a possible correlation between the structure and “synergistic” activity of the substances discussed in this paper. It appears that substituting =S for =O increases the activity of the compound; substituting =NH for =S increases the activity still more. Thus, thiourea is more active than urea, and guanidine and O-ethylisourea are more active than thiourea. Methylurea is inactive at 480 mg/100 ml concentration, but substitution of =S for =O, making the compound methylthiourea, results in an active “synergist” at approximately 128 mg/100 ml concentration. Urethane is inactive at 200 mg/100 ml concentration but substituting =NH for =O making the compound O-ethylisourea, results in a very active “synergist.” The order guanidine τ; thiourea τ; urea agrees with the findings of Lee et al (5).
Substitution in the amino group may or may not decrease activity. Thus, dicyandiamide is less active than guanidine. However, methylthiourea is slightly more active than thiourea. Substituting an entirely different group for the amino group can increase the activity. Thus O-ethylisourea is more active than guanidine. Another example of this might be the reported greater activity of urethane (1–2 per cent) as compared to urea (14, 15).
All of the inactive compounds, with the exception of succinamide or cyanamide, may be considered as derivatives of urea,—either cyclic derivatives through the amino groups, or urea with substitutions in/for one of the amino groups. None of these compounds is “synergistic” in the highest concentrations employed, concentrations within the range of the more active compounds tested. Therefore substitution in or for the amino group appears to be less effective in increasing the activity of urea than does substitution for the =O in the carbonyl group.
These points suggest, therefore, that there is a relationship between the structure of urea and its derivatives and “synergistic” activity when added to sulfonamides.
The nature of these experiments cannot reveal the mode of action of these “synergisms.” Perhaps the polar nature of solutions of these compounds, as suggested by Schmelkes (8), is intimately involved in their “synergistic” activity.
The nature of these experiments also does not permit one to say whether or not the “synergism” is due to an antisulfonamide inhibitor (PAB) activity, a direct enhancement of sulfonamide activity, or both. If sulfonamide resistance by sulfonamide-fast staphylococci is not due to the production of inhibitors of sulfonamides, then the “synergism” noted against sulfonamide-fast strains of Staph. aureus would indicate that the latter effect (direct enhancement of sulfonamide activity) does occur. Also, the experiments of Weinstein and McDonald (15), in which no sulfonamide-inhibitor was added to the medium, showed that urea and urethane are “synergistic”, and thus may directly enhance sulfonamide activity by mechanisms not directly involving neutralization of sulfonamide-inhibitor. It is recognized, however, that sulfonamide inhibitors may play a role, even though not added to the medium, since the bacteria may produce them. So it can merely be stated that in the present experiments these “synergists” tend to neutralize the observable inhibition of sulfonamides by para aminobenzoic acid, and that urea (as previously reported (13)), thiourea, N-methylthiourea, and O-ethylisourea-HCl “synergized” sulfonamide against sulfonamide resistant Staph. aureus.
In regard to the failure of Kirby (3) to find a urea synergism, it must be emphasized that the synergisms described in the present and previous papers may fail to be demonstrable under other, experimental conditions. In addition to the importance of the size of the inoculum, as emphasized by Lee et al (4), the medium employed is of extreme importance. For example, employing a basal medium other than those described in the present paper, guanidine-HCl failed to “synergize” NaSAT against E. coli while S-ethylisothiourea and guanidine CO3 showed striking “synergistic” activity. At present the reasons for this “medium difference” are unknown. Thus, failure to demonstrate the “synergism” described in the present paper may be accurate, and due to different experimental conditions.