## 3 Results and Discussion  
### Analysis of Assays  
Assay 1. The effect of the two fluorine atoms in 2,2-difluoro-3,4-methylenedioxy-\(\beta\)-methyl-\(\beta\)-nitrostyrene (**B**) reduced the antimicrobial activity compared to **A-1**. All of the test compounds showed very weak antimicrobial activity against _E. coli_. However, the activity of **B** against _S. aureus_ and _Bacillus subtilis_ was high and comparable to **A-1** (refer to Table 1). The activity of 2,2-difluoro-3,4-methylenedioxy-\(\beta\)-nitrostyrene was very weak compared with that of the same compound, including the \(\beta\)-methyl group (**B**), and therefore, the data were not included in Table 1.  
Assay 2. The highest activity was observed with 4-bromo-\(\beta\)-methyl-\(\beta\)-nitrostyrene **(D)**, which performed better than **A-1** against _E. coli_ and was comparable to **A-1** against the other species. Compounds **G** and **H** also showed good all-round activity. Improvement in activity is also observed by substitution of a \(\beta\)-methyl group (**G**) or a \(\beta\)-bromine group (**H**) to Compound **E**. Compound **C** (4-bromo-\(\beta\)-nitrostyrene) also showed fairly high activity, except against _E. coli_. It is, however, more active that **A-1**, **B** and **E** against this species. Between Compounds **D**, **F**, **G** and **H**, **D** is clearly superior against Gram-positive species, but there is little difference in activity against _C. albicans_. The fact that **D** is better than **F** suggests that 4-bromo substitution leads to higher activity than 4-chloro substitution. Only the 4-bromo derivative **(D)** was more active than the parent compound (**G**) against Gram-positive bacteria. Only **F** (4-chloro-), **G** (\(\beta\)-methyl) and **H** (\(\beta\)-bromo) derivatives gave satisfactory activity against the Gram-negative _E. coli_. Compounds **C** (4-bromo) and **D** (4-bromo-\(\beta\)-methyl), derivatives of nitrostyrene, are still significantly better than the methylenedioxy derivative (**A-1**) (refer to Table 1). The same comments apply to Compound **I**, which is evaluated in Assay 3. This compound has been evaluated in different assays, and the results in Table 3 are from an assay in which **I** was evaluated against 4-fluoro-\(\beta\)-nitrostyrene (without the \(\beta\)-methyl group), which \begin{table}
\begin{tabular}{c c c c c c c c c} \hline
**Strain** & **A-1** & **B** & **C** & **D** & **E** & **F** & **G** & **H** \\ \hline _Staphylococcus aureus_ ATCC 29213 & 16 & 16 & 32 & 16 & 128 & 32 & 32 & 32 \\ _Bacillus subtilis_ ATCC 6633 & 16 & 16 & 32 & 8 & 128 & 32 & 64 & 64 \\ _Enterococcus faecalis_ ATCC 29212 & 32 & 512 & 64 & 64 & 128 & 256 & 128 & 128 \\ _Escherichia coli_ ATCC 25922 & 362 & 512 & 197 & 181 & 362 & 128 & 128 & 128 \\ _Candida albicans_ ATCC 10231 & 23 & 44 & 23 & 23 & 362 & 23 & 44 & 23 \\ \hline \end{tabular}
\end{table} 
Table 1: Assays 1 and 2: minimum inhibitory concentrations (MICs) of \(\beta\)-nitrostyrene analogues. showed that **I** was much better than 4-fluoro-\(\beta\)-nitrostyrene and also **A-1**. In all of our work, we have found this to be the case (see [12, 13]). Table 1 shows the MICs in \(\mu\)g mL\({}^{-1}\) of \(\beta\)-nitrostyrene analogues tested against four strains of bacteria and a fungus. The values are the geometric means of two determinations. In our MIC data, the geometric mean represents a mean or average, indicative of the central tendency or typical value of duplicate-measured MIC values by using the product of their values. It is defined as the \(\
icefrac{{1}}{{2}}\) root of the product of two determinations [14]. Assay 3. In the third assay, 4-fluoro-\(\beta\)-methyl-\(\beta\)-nitrostyrene showed comparable activity to **A-1**, with the advantage against _E. coli_, but this was offset by lower activity against _E. faecalis_. No real advantage of fluorine over chlorine or bromine substitution could be claimed (refer to Table 2). All compounds showed equivalent activity to **A-1** against the bacteria and the fungus.