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We decided to optimize both the N-methyl groups simultaneously using a factorial design strategy, representing pi (lipophilicity), sigma (electronics), and L (length). This required a total of 9 X 9 or 81 compounds to test every possible combination. Since the synthesis of this class had been worked out, it seemed to us to be an ideal candidate for optimizing activity using an "indexed" combinatorial library [3,4].
To prepare the 81 compound library, a total of 18 compounds were required as starting materials; a set of nine pyrazoles (pyrazole prelibrary) and a set of nine piperidones (piperidone prelibrary).
X = CH(CH3)2, CH2C(CH3)3, CH2CF3, CH2CH2CN,
CH2CH2OCH3, CH2C6H5, CH3, C6H5, CH2CH2CH2CH3
Y = COCH2CH3, COC6H5, CH(CH3)2, CH2CH2F, CH2CH2OCH3,
CH2CN, CH3, CO2CH(CH3)2, CH2CH2CH2CH2CH3
Each prelibrary was pooled and divided into nine separate flasks and each reacted with the appropriate reagents as indicated in Figure 1 (piperidone pool) and Figure 2 (pyrazole pool) to generate the following mixtures:
Due to synthesis problems, Mixture number 3 was not tested in the screen. The remaining 17 mixtures were tested in our mosquito assay and the results are shown in Table 1 or in 3D graphical representation. The more active mixtures were mixture's number 7, 12-14.
Red = data for mixtures (relative potency, 4=most active)
Blue = pure compounds (pLC50)
To confirm the results obtained from the relative potency data, the three compounds predicted to be the more active were synthesized independently along with the three theoretical "best" from mixture number 3. In addition, we prepared the compound predicted to be the least active, having R = butyl (mixture number 8) and R' = isopropylcarboxy (mixture number 17).
The major advantage of using an "indexed" combinatorial library is to improve synthesis efficiency; not having to synthesize every compound of interest independently. A limitation, however, is the requirement of relying on ranking activity data of mixtures. As we found in this study, if the absolute range in activity for the mixtures is small, it becomes increasingly difficult to differentiate between mixtures that are close inactivity, such as a rank order of 3 versus 4 due to the variance in the assay data (± 0.3 pLC50 ).