Crystal structure and Biological evolution of 5-bromothiophene based 3,4-dihydropyrimidin-2-(1H)-thi(ones)

Herein, Solvent-free synthesis of 5-bromothiophene based 3,4-dihydropyrimidin-2-(1H)-thi(ones) was explored. The main advantages of this study are that pure product comes in hand with easy workup and without using any separation technique, a good amount of yield, and formation of product in crude form. We developed a single crystal of 4c, and we analysed crystal structure by Hirshfeld surfaces analysis. All the synthesised compounds were screened for antifungal and antibacterial assay. Among all synthesised compounds, 4a, 4b and 4j have potent antibacterial activity. While 4f, 4g, 4h and 4i show good antifungal activity among all synthesised compounds. We also carried out ADMET prediction for the 4(a-j), and calculated data show that all the compounds have prominent drug-like nature.

For this purpose, we decided to utilise the Biginelli reaction to develop 5-bromothiophene based 3,4-dihydropyrimidin-2-(1H)thi(ones). Like many other reactions, [19][20][21][22][23][24][25] Biginelli reaction is a three-component reaction involving the condensation of an aldehyde, urea and a β-ketoester under acidic conditions. 26 In our previous work, we synthesised 5-bromothiophene based 1,4-dihydropyridine via solvent-free protocol for CAN (Ceric ammonium nitrate) catalysed Hantzsch reaction. 17 In the current study, we utilised the same protocol for the Biginelli reaction and successfully produced 5-bromothiophene based DHPMs with potent biological activity. We further screened the novel 5-bromothiophene based DHPMs for antibacterial and antifungal activity and established the in silico ADME prediction which shows promising drug-likeness for the synthesised molecules.
In optimisation of reaction, our rst target is to carry out the reaction at ambient temperature, but with this reaction we got lower yields at ambient temperature (Entry 2 and 3, Table 1). So, we carried out the reactions at 60°C with different amounts of catalyst at different reaction times, in which we got the best result with 20 mol % of CAN at 70 minutes (Entry 6, Table 1).
The reaction was also optimised with different solvents, but we got the sticky product on work up except DMF, which is tedious to handle, while we got the crude product in solvent-free condition.
We successfully carried out the solvent-free synthesis of compounds 4(a-j) using CAN as a catalyst within 60-90 minutes of heating at 60°C. Structures of the synthesised compound were con rmed by spectral analysis, for which spectral data are given in supplementary material.
In 1 H-NMR spectra, signals at δ 0.8-1.5 ppm con rm the presence of methyl group at C-1, whereas, for methyl, ethyl, isobutyl, tert-butyl of ester give signals in a range of δ 1.5-3.0 ppm. A signal that appears as a singlet at a range of δ 5.0-6.0 ppm con rms the presence of proton at γ-position. For aromatic protons, we observe signals in the range of δ 6.8-7.6 ppm. For 2 -NH groups in some of the compounds, we observe singlet for both after an aromatic region in between δ 8.0-9.5 ppm and in some cases, we got singlet for 1 -NH around δ 6.0 ppm and for another around δ 8.5 ppm.
In 13 C-APT spectra, signals in a range of δ 16-18 ppm con rm the presence of a methyl group, whereas signal for other aliphatic carbons of the ester group appears in the range of δ 30-55 ppm. For the quaternary carbon of the ring, we observe signals in a range of δ 100-110 ppm and signals in a range of δ 160-170 ppm that con rms the presence of the carbonyl group. Single-crystal XRD analysis of 4c also con rmed its structure.

Description of crystal structure
The single-crystal structural data indicate that 4c was crystallised in P1 space group. The molecule of 4c is not symmetrical (Fig. 1a). The asymmetric unit revealed two molecules of 4c, and one of them contains a distorted thiophene ring. Pyrimidinone ring in 4c is observed in half chair con guration. Triclinic unit cell of crystal with cell dimensions a = 7.3244(3) Å, b = 13.5742(6) Å, c = 14.8486(7) Å, α = 94.044(2)°, β = 103.959(2)°, γ = 99.511(2)° show in Fig. 1b. In the crystal lattice, molecules of 4c have connected via strong N-H ····O = C hydrogen bonding interactions in a zigzag fashion and overall, a layered structure is formed (Fig. 1b). Each carbonyl oxygen able to interact with two neighbour atom's hydrogens simultaneously with H ····O ····H bond angle 116.78° and 118.51°. Thus, each molecule interacts with two neighbour molecules by four hydrogen-bonding interactions having bond lengths 2.070 Å, 2.153 Å, 2.158 Å and 2.159 Å. XRD data were deposited online to Cambridge Crystallographic Data Centre (CCDC). CCDC deposition number 1970503 contains the Supporting Information crystallographic data for this paper.

Hirshfeld surfaces analysis
Hirshfeld surfaces for 4c were calculated as per established procedures. 27 These calculation help to understand effect of weak intermolecular interactions into the molecular packing. In Fig. 1c Hirshfeld surfaces mapped over d norm in the − 0.5530 to + 1.3583 arbitrary unit range. Broad and bright-red spots on pyrimidone ring surface and near hydrogen atom of amino groups and oxygen atom of carbonyl group respectively recognise donor and acceptor of potential N-H...O hydrogen bond. Whereas the presence of diminutive and faint-red spots on the surface characterises weak intermolecular interactions in 4c. On thiophene ring surface, short interatomic Br···S/S···Br contacts in crystal packing of 4c are viewed as the faint-red spots. Diminutive-red spots are also observed which indicated short interatomic H···H contacts. The antibacterial activity of compounds 4(a-j) was performed on gram-positive and gram-negative bacteria by broth dilution method. The screening results were summarised in Table 3. The MIC values of compounds 4(a-j) are between 50-500 µg/mL against tested microorganisms. In the case of gram-negative bacteria, 4h shows good antibacterial activity against E.coli than the standard drug ampicillin, while 4b show good antibacterial activity against P.aeruginosa than standard drug ampicillin. In the case of gram-positive bacteria, 4a, 4b, 4d, 4g and 4i show good antibacterial activity against S.aureus than standard drug ampicillin, while 4a show good antibacterial activity against S.pyogenus. Overall, 4a, 4b and 4h exhibited maximal antibacterial activity.

Antifungal assay
Fungicidal activities of 4(a-j) were evaluated against three fungi C.albicans, A.niger, and A.clavatus and the minimum inhibitory concentrations were derived. The screening results were summarised in Table 4. Tabulated data show that compounds 4f, 4g, 4h and 4i are more effective on C.albicans than the standard drug griseofulvin. Entire antifungal data indicate that dihydropyrimidinthione 4(f-j) are more potent than dihydropyrimidinones 4(a-e).

ADMET prediction
ADMET properties of the compound are important for its drug-like pro le. It might be important for novel drug discovery. 28 Physicochemical properties of the compounds correlate to their drug-likeness via various rules like Lipinski's, Ghose's, Veber's Egan's and Muegee's rule. Physicochemical properties must stay within limits de ned in each rule. We evaluate our synthesised compounds for their ADMET properties with the help of the online web server SwissADME. (http://www.swissadme.ch) Calculated physicochemical properties of 4(a-j) are summarised in Table 5. The value of physicochemical properties remains in the limits de ned for Lipinski's, Ghose's, Veber's, Egan's, and Muegee's rule. Thus, all 5bromothiophene based DHPMs incorporate all rule of drug-likeness, so they have good drug-like potential. Bioavailability radar of the compounds were derived based on six physicochemical properties namely lipophilicity, size, polarity, solubility, exibility and saturation. The bioavailability radar showed that all 4(a-j) molecules exhibited drug-like radar plots (Bioavailability radar graph of 4(a-j) were included in supporting information le).
To predict the gastrointestinal absorption and blood-brain barrier permeability of the compounds, BOILED-Egg delineation was used. BOILED-Egg delineation of all the synthesised compounds is illustrated in Fig. 3. The white, elliptical region of the egg depicts a high probability of gastrointestinal absorption, while the yellow (Yolk) region of the egg depicts blood-brain permeation of molecule. 29 All 5-bromothiophene based DHPMs show high gastrointestinal absorbance, and they have no blood-brain permeability. Red dots for all the 4(a-j) denoted that they are not P-gp substrate.

Conclusion
In summary, we successfully carried out the synthesis of compounds 4(a-j) in solvent-free condition using CAN as an e cient catalyst with easy workup procedure, eco-friendly synthetic route and without the use of any hazardous solvent or any separation technique. Antibacterial data show that our synthesised 5-bromothiophene based DHPMs have good antimicrobial activity. Furthermore, it also showed that dihydropyrimidinthione 4(f-j) have more potent antifungal activity compared to dihydropyrimidinones 4(a-e). In silico ADME prediction con rmed that 5-bromothiophene based DHPMs have a good drug-like pro le.

General
All chemicals were purchased from commercially available sources and were used without further puri cation. The progress of the reaction was monitored by silica gel 60 F254 (Merck) coated TLC plates. Reported R f values correspond to elution with 1:4 (n-hexane: ethyl acetate) mobile phase. Melting points were determined by the open capillary tube method and are uncorrected. IR spectra were recorded on an ABB MB3000 spectrophotometer. 1 H NMR and 13 C-APT spectral analyses were recorded using a BRUKER AVANCE II 400 NMR spectrometer. Abbreviations used for the 1 = 16.7, 34.6, 50.1, 100.2, 110.61, 122.8, 124.0, 129.0, 129.4, 148.7, 149.8, 153.6, 166.0 ppm; IR: 3489, 3442, 3061,  2996, 2950, 1735, 1642, 1586, 1029, 1122, 1254, 1484, 1379, 1336, 568 cm -1 ; Anal. Calcd for C 11  Author contribution MGS was involved in the synthesis, conception and design of the study. RMV was involved in acquisition of data, analysis and interpretation of data and in silico study. DPR was involved in in vitro antibacterial and antifungal activity. VR and RLG were involved in single-crystal XRD study. SB and HMP were involved in drafting and revising of the manuscript.

Con icts of interests
The authors declare that there are no con icts of interests.       Two-dimensional ngerprint plot for 4c which deline into various contacts.