## 2 Results & Discussion
### Biological Studies
#### 2.2.2 Antifungal Activity  
The target hybrids were screened for antifungal activity against the fungal strains _A. niger_ and _C. tropicalis_ using Fluconazole as a positive control (Table 2). Among the monosubstituted derivatives with substituents at the 4\({}^{\prime\prime}\)-position of the phenyl ring, the MICs ranged from 97.34 to 214.94 \(\upmu\)M and among them, good activity was exhibited by nitro derivative (**25**) with an MIC of 97.34 \(\upmu\)M. In general, within the monosubstituted derivatives, compounds bearing electron-withdrawing substituents (F, Cl, & NO\({}_{2}\)) showed 2-4-fold better activity over compounds containing electron-releasing groups such as hydroxy (OH), methoxy (OCH\({}_{3}\)), methyl (CH\({}_{3}\)), and dimethyl amino (N(CH\({}_{3}\))\({}_{2}\)). The order of activity of the monosubstituted derivatives was 4\({}^{\prime\prime}\)-NO\({}_{2}\) > 4\({}^{\prime\prime}\)-Cl > 4\({}^{\prime\prime}\)-F > 4\({}^{\prime\prime}\)-N(CH\({}_{3}\))\({}_{2}\) >> 4\({}^{\prime\prime}\)-OH = 4\({}^{\prime\prime}\)-Me-4\({}^{\prime\prime}\)-OMe.  
Figure 3: Structure–activity relationship (SAR) features of chloropyrazine-tethered pyrimidine hybrids. EWD = Electron withdrawal; ERG: Electron-releasing group.  
Among the disubstituted compounds, when the phenyl ring was substituted with F (**30**, MIC = 50.04 \(\mu\)M) or Cl (**31**, MIC = 45.37 \(\mu\)M) at the ortho and para positions of the phenyl ring, the activity was improved by twofold over compounds **23** (MIC = 100.57 \(\mu\)M) & **24** (MIC = 106.06 \(\mu\)M). Interestingly, compound **32**, containing 3'',''''-dimethoxysubstitution, had fourfold (MIC = 46.52 \(\mu\)M) higher antifungal activity compared to its monosubstituted counter compound **28** (MIC = >200 \(\mu\)M) against _A. niger_. However, the activity of **32** was not favorable against _C. tropicalis_ (**32**, MIC = 186.17 \(\mu\)M). The best activity among all compounds tested was observed for the 2'',''-di-Cl-substituted compound **31** with an MIC of 45.37 \(\mu\)M, followed by compound **32** (MIC = 46.52 \(\mu\)M). Both compounds were found to be almost twice as potent as funconazole against _A. niger_ and around 1.4-times as potent against _C. tropicalis_. In this case, the order of activity was 2'',''-di-Cl > 3'',''-di-OMe > 2'',''-di-F > 3''-OMe > 4''-OH.  
Figure 4: Structures of the most potent chloropyrazine-tethered pyrimidine hybrids.  
Derivatives with bioisosteric heterocyclic substituents showed moderate activity, with MIC in the range of 110.44-200 uM. The order of activity of these compounds was 2''-thienyl > 2''-pyridinyl = 3''-pyridinyl > 2''-furfuryl! > 4''-pyridinyl = 5''-pyrrolyl. The six-membered heterocyclic-based compounds (**35**-37) fared better over the five-membered ring containing compounds (**38**-**40), and the best activity was seen for compound **35** (MIC = 56.19 uM) with MIC 1.2-fold higher than that of Fluconazole against C. _tropicalis_. None of them exceeded the activity of the reference drug. Thus, the structure-activity relationship study showed the same behavior for monosubstituted derivatives as in the case of antibacterial activity. In the case of disubstitution, the presence of 2'',4''-di Cl (**31**) was favorable for antifungal activity followed by 3'',4''-OMe (**32**). Introduction of 2'',4''-di-F (**30**) slightly decreased the activity compared to **32**, while the presence of 3''-OMe, 4''-OH substitution (**33**) was detrimental. In the case of heterocyclic substitution, 2''-thienyl was found to have a positive effect on antifungal activity, while 4''-pyridinyl and 5''-pyrrolyl had very negative influences on antifungal activity (Figure 4)