Synthesis, Characterization, Crystal Structure, Hirshfeld Surface, Electronic Excitation, Molecular Docking, and DFT Studies on 2-Amino Thiophene Derivative

Abstract Ethyl-2-amino-5,6,7,8-tetrahydro-4H-cyclo-hepta[b]thiophene-3-carboxylate (EACHT) has been synthesized, characterized via single-crystal X-ray diffraction at 293 K and investigated quantum chemically by DFT approach, surface analysis by Hirshfeld and spectrochemically by NMR (1H-NMR and 13C-NMR), FTIR, and UV–Visible. The compound crystallizes in monoclinic crystal system with P21/c space group with Z = 4, with following unit cell dimensions: a = 9.5956 (3) Å, b = 9.5607 (4) Å, c = 13.7226 (7) Å. To get the optimized structure which is base for all the other calculations (vibrational frequency, NBO, natural hybrid orbital, nonlinear optical, frontier molecular orbital, etc.), B3LYP method with 6-311++G(d,p) basis set was used. Complete potential energy distribution assignments were done successfully by VEDA. 1H-NMR and 13C-NMR shifts were estimated by GIAO method and results were compared with experimental spectra. TDDFT method and PCM solvent model was utilized for electronic property analysis such as UV–Vis (in gas phase, ethanol, and DMSO) and compared with the experimental UV–Vis spectra. The HOMO and LUMO energy results emphasize adequate charge transfer was happened within the molecule. NBO analysis MEP surface analysis electron localization function Diagram and Fukui function analysis were done. Hole and Electron density distribution maps were drawn in two different excited states of higher oscillatory strength with DMSO, MeOH as solvents. Docking with seven different receptors, and drug likeness was also carried out.


Introduction
Ethyl-2-amino-5,6,7,8-tetrahydro-4H-cyclo-hepta[b] thiophene-3-carboxylate (EACHT) is a medically important heterocyclic molecule, one of the 2-aminothiophene derivatives and significant building block in pharmaceuticalfield. 1 In medicinal chemistry, heterocyclic derivatives have been recognized as significant structural moieties, as well as in bio-molecules such as natural products, enzymes, vitamins, and biological active compounds such as antifungal, anti-inflammatory, DFT approach has been used in this work because this technique can accurately predict the chemical shifts in NMR, structural parameters, chemical reactivity, and Hirshfeld surface. Also, this technique has been widely used by organic chemists in their research work. Along with this, molecular electrostatic potential (MEP), Frontier molecular orbital (FMO) analysis, and NBO analysis were also done. We believe that the interesting results of the NBO analysis will provide more insight into the possible intermolecular interaction of the titled molecule. Along with molecular docking and druglikeness, the above investigation helps in the rationalization of intermolecular drug receptor interactions showing a path for the purpose of designing new drug with the biologically active 2-amino thiophene bearing molecules.

Synthesis of EACHT
A reaction flask was charged with (10 mmol) of cycloheptanone, (10 mmol) of ethylcyanoacetate, (10 mmol) of sulfur powder, and 20 mol% of catalyst in 5 mL of EtOH. The reaction mixture was heated to 50 C with stirring for the required time, and progress of reaction was monitored by TLC. The reaction was completed within 6 h. After completion of reaction the corresponding product was worked up by 10 mL of ethanol and catalyst was separated by simple filtration. The filtrate product was concentrated on rotovap. The Yellow color solid residue was purified by silica gel column chromatography (solvent hexane: ethyl acetate 7:3).

Theoretical analysis details
To investigate the arrangements of intra and intermolecular interactions in the crystal and Hbond interaction Hirshfeld was done. Analysis is based upon 3D graph representation in the atoms was illustrated in Figure 1. The calculated numeric value of the bond length (Å) and the bond angles ( Å) is compared with the structural parameters values which were obtained from experimental findings of single crystal X-ray analysis 22 detailed in Table 1. The point group of the studied compound comes out to be C1 point group. Dihedral angle values of some planes C2-S1-C3-N6, S1-C3-N6-H12, C7-C3-N6-H13, S1-C3-C7-C14, N6-C3-C7-C5, C7-C14-O22-C27, O22-C27-C28-H33, H29-C27-C28-H31 are around 179.9 , which shows that thiophene ring is in planarity with side chains amino and acetate group, while the cycloheptane ring is nonplanar as shown in Figure 2. The RMSD and R 2 values of the calculated and experimental bond length were 0.9552 and 0.972, respectively. Similarly, RMSD and R 2 values of calculated and experimental bond angles were 0.987 and 0.998, respectively. After evaluating the values of RMSD and R 2 (correlation) we can infer that there was an excellent correlation between experimental and calculated values. The computational calculations were performed in gas phase whereas experimental study was done in solid phase which creates a slight variance between the experimental and computational values. In general, the bond length and bond angles were very well predicted. The  Table 2. The prominent calculated value of bond length between S-C bond was 1.767 Å (exp. 1.737 Å), C-N bond was 1.354 Å (exp. 1357 Å). The bond length of C-C bond lies in the range of 1.34 Å-1.50 Å (exp. 1.35 Å-1.50 Å), N-H bond 1.00 Å-1.101 Å (exp. 0.835 Å-0.862 Å), N6 atom and H13 atom in this structure shows shortest bond length of 1.0059 Å. As the N6-H13 and N6-H12 bond of the amino possess same value of 1.016 Å, we can say that N6-H13 and N6-H12 bonds are strongest among all. One can conclude from the above parameters that the bond length of N-H, C-C bond remains consistent due to the similarity in their surroundings. Dihedral angles of different planes such as C2-S1-C3-N6, C5-C2-C4-H10, S1-C2-C5-C11, C4-C2-C5-C7, C7-C3-N6-H13, N6-C3-C7-C5, O22-C27-C28-H33 were calculate to be around 179.9 that shows that thiophene part of the compound is planar with other side chains. The interaction between the thiophenes delocalized electrons and the nitrogen, Sulfur, oxygen atoms lone pair leads to the overlap of the lone pair with the delocalized ring system. The donation of the nitrogen, oxygen lone pair into the ring system leads to an increase in the density of electrons around the ring. Due to the presence of oxygen, acetoacetate, and amino group (NH 2 ) as a side chain and Sulfur atom in the ring, the experimental bond angles were slightly distorted out of a perfect structure.

Hirshfeld surface analysis
Three dimensional Hirshfeld surface and 2D fingerprint plots were drawn by Crystal Explorer 17 27 software. Hirshfeld surfaces can be analyzed in crystals only. To clearly examine interactions between atoms in crystal of EACHT Hirshfeld surface analysis was done. Hishfeld surface of EACHT mapped with d norm , d i , d e , shape index, curvedness, and fragment patch is shown in Figure 3-F, respectively. d i , d e pair sum explains the distance separating the atoms contributing to this point. The hydrogen bonding and close contact of the atoms in crystal with neighboring atoms is shown by dashed lines by different colors. It was found that d norm value comes from À0.4862 to 1.4298 Å, shape index vary from À0.998 to 0.9958 Å, curvedness from À3.5326 to 0.4975 Å were attained as minimal to maximal value, respectively.
The d norm surface shows brick red circular region at some areas in the Hirshfeld surface that indicated the close contact with the neighboring molecules, hydrogen bond was shown between O-H as shown in Figure 3A, (3.34) in contrast with the electronegativity of N9 (3.04), it could be suggested that electron density on the H was affected mainly by the oxygen atom. d norm varies from À0.4862 to 1.4298 Å. Shape index is one of the main features of Hirshfeld analysis that gives identification of complementarily in between the molecules in crystal packing, it is a property to measure p-p stacking interactions in the compound indicated by the presence of red and blue patches on adjacent positions, it they are not present at adjacent places then p-p stacking interactions are not present. 39 Patches on shape index surface with different colors shows intermolecular complementarily areas as seen in Figure 3D. The red marked patches show concave regions of atoms of pstacked molecule above molecule. Blue marked patches show convex region which indicates ring structure of molecule inside the crystal surface. Shape index varies from À0.455 to 0.554 Å. Curvedness is a measure of surface area of shape of molecule. 40 Curvedness shows the electrostatic interaction present in between the molecules. The curvedness of molecule EACHT is depicted in Figure 3E, curvedness shows range from -3.5326 to 0.4975 Å, flat disk like areas on the surface corresponds to low value of curvedness, while sharp edge like curvature corresponds to high value of curvedness and tend to distribute the surface into patches, shows interaction between neighboring molecules. Blue outline that separates flat patches indicates p-p stacking interactions. It could be recognized that the outcome from the curvedness analysis correlate well to those presented by means of d norm and shape index.
Two dimensional fingerprint images reveals about type of intermolecular contact between atoms and also analyze the difference in these patterns and represent a major intermolecular connection in entire crystal structure. 41 Fingerprint plot particularly shows close contact between all the elements in the molecule. The 2D fingerprint plots of various intermolecular contacts with percentage of intermolecular contacts contributed by each atom are shown in Figure 4. Highest contribution percentage 62.3% of contact is attributed by H-H from the total contribution. These 2D plots indicated narrow spikes like pseudo symmetrical area with high concentrated blue color  which approximately surround the total area contributed. 42 Almost all the interactions aforementioned give information regarding 3D network of EACHT. 43 To confirm the clear picture about nature and contribution of bonds formed in the molecule the E enrichment ratio was determined. E enrichment ratio of a pair of elements may be described as ratio in between the actual contact percentage in the crystal and the theoretically present percentage of the random contacts. 44 Table 3 shows the list of enrichment ratio shown by intermolecular contacts in the crystal, H-N/N-H (ratio ¼ 2.6) clearly provides evidence of the formation of hydrogen bonds of type N-H. The H-H contacts are second most persistent interactions due to the large number of hydrogens on the molecular surface (62.3%). Calculations revealed that most of the molecular surface about 62.3% was generated by H atoms, then C-H with 11.6%, for N-H value of 2.6% was attributed while that of C-O was around 1.5% as shown in Table 3.

NMR analysis
C 13 and H 1 NMR experimental value for 3-PPIA were obtained in chloroform solvent on Bruker Advance 400/Avll HD-300 (FT-NMR) instrument from SAIF, CDRI, Lucknow, India, shown in Supporting Information Figure S11, respectively, whereas, Gaussian with Gauge Independent Atomic Orbital (GIAO) method 45 were used for calculating theoretical C 13 and H 1 chemical shifts as shown in Figure 5. Experimental and theoretical data of 13 C and 1 H NMR for target molecule were presented in Table 4. Dissimilarities were seen between theoretical and experimental values due changes in solvent used. Titled molecule (EACHT) shows12 carbon NMR peaks. Organic compounds usually show 13 C NMR chemical shift >100 ppm whereas, 1 H NMR shows <100 ppm. Isotropic shielding values were used to evaluate isotropic chemical shifts with respect to tetramethylsilane (TMS). Experimental and computed chemical shifts values were summarized in Table 4.  EACHT showed 13 C chemical shifts at 167.37 and 161.25 ppm for the carbon atoms C14 and C3, respectively. In comparison simulated value comes at 154.91 and 153.926 ppm which were C-O and C-N group peaks. Higher value of chemical shift at C14 and C3 was due to shielding effect of carbonyl group and NH 2 near C3. The Experimental proton NMR shows that all the cyclohepta protons come at around 1.7-1.3 ppm and NH 2 protons shows different values due to presence of N atom. The difference in the NMR values of experimental and theoretical peaks was theoretical calculations were carried out in gas phase but experimentally is recorded in solid state. 24

Molecular electrostatic potential analysis
The 3D map of Molecular electric potential surface shows the interaction in between the electrons and nuclei of the compound and also charge distributions of molecule which is used to predict the molecular interaction and the nature of the chemical bond. It plays a very important role as it portraits the negative, positive and the neutral electrostatic potential along with molecular shape and size through color grading. Therefore, by using MEP surface of a molecule we can easily evaluates the physiological properties of a molecule 46,47 such as biological recognition processes with drug-receptor, enzyme-substrate interaction processes; reactive properties of nucleic acids, including their component basis; chemical carcinogenesis, indicating polycyclic aromatic compound, and halogenated olefins and their epoxides and many more. 48 Since different color is used to show various electrostatic potential values; red color denotes highest positive electrostatic potential (strongest repulsion), i.e. red region is prone to electrophilic, 49,50 blue color denotes the highest negative electrostatic potential (strongest attraction), i.e. blue region is prone to neutrophilic attack while green color denotes the zero electrostatic potential. Figure 6 shows the mapped molecular electrostatic potential surface of the titled molecule with different colors. Yellow color at S1 indicates formation of C-H-S bond with neighboring molecules and C7 shows low value of electrostatic potential. The range of the dispersion of potential with color code of the studied molecule lies between À4.327e-2 (deepest red) to 4.327e-2 (deepest blue). The trend of electrostatic potential color code bar is in the increasing order, i.e. red (highest positive electrostatic potential) < orange < yellow < green < blue (highest negative electrostatic potential). From Figure  6, we can infer that the point at which oxygen atom is attached to the ring is prone to electrophilic attack because of the electronegative behavior of oxygen, increasing the electron density in this region. The point where the amino group is attached is electron deficit due to subsidiary side chain molecules (amino and carbonyl) which are highly negative, reducing the electron density cloud around on the amino group by forcing the electron cloud toward them hence this area is prone to neutrophilic attack. Therefore, by analyzing the MEP surface one can infer the intermolecular interaction, chemical complex building capability, hydrophilic and hydrophobic nature which is very useful information for pharmaceutical applications. Therefore, the reactive regions of EACHT can be readily obtained qualitatively by MEP surface analysis. Table 3. Finger print percentage of the total surface area for closed contact between atoms inside and outside the surface for 3-Picoline.

Electron localization function diagram
To harmonize the chemical nature of the molecule with quantum chemical postulates, many procedures were introduced, of which quantum chemical analysis of ELF by Becky and Edge combe 51 gives the direct relation between the chemical structure and the electron density distribution. By characterizing the corresponding electron density, ELF constitutes an useful relative assessment of the electron pair localization. Using the positive definite local Pauli and Thomas Fermi kinetic energy densities, ELF (density-based property), can be interpreted within the given framework. In the validity of such framework, these quantities provide key information to assess the relative local excess of kinetic energy associated with Pauli's principle. The ELF values lies within the range of 0-1. The highest ELF values have been associated with the spatial positions with higher relative electron localization. 51 The measurement of ELF was justified by Pauli repulsion theory which results in the excess of kinetic energy density. Important information of the molecules like chemical structure, reactivity, and molecular bonding is obtained by the analysis of ELF which was greatly used for quantitative analysis of aromaticity. 52 The probability of finding opposite pair behavior or single electron was highest in the region of space. Here, the value of ELF was close to 1, which indicates the region of maximum Pauling repulsion where ELF close to 0 indicates minimum Pauling repulsion. Atomic shells, chemical bonds, and the lone pair electrons can be used to identify the strongest Pauli repulsion region corresponding to well-localized electrons. N6, C3, S1 atoms of compound were chosen in one plane and to plot ELF color filled map and shaded surface map with projection effect The ELF values (coded in a color scale in analogy to a geographical map and as a shaded contours) has been characterized by three dimensional graphical representations illustrated in the Supporting Information Figure S12 High ELF values ($1.4-1.0) are colored red followed by yellow to green for middle ELF values (ca. 0.7) and the lower end of the scale is represented by blue which indicates the lowest ELF. The red region was used to depict the regions around the hydrogen with single electron that has the maximum Pauli repulsion. While the blue region is used to depict the region around nitrogen and carbon having similar spin electrons close together. So, in between C-H bond, the density was down due to the repulsion of carbon and hydrogen atoms. The area around N and C having similar electrons close to each other were depicted by Blue color. The C-C and C-N bond area because of the presence of double bonded character in C-C bond had highest degree of localization. Blue ring like area about around nucleus showed less localization of electrons in between valence electron shell and inner electron shells of heavier atoms.

NLO analysis
Nonlinear properties (NLO) of an isolated EACHT molecule in the gas phase were studied. DFT technique at B3LYP/6-311þþG(d,p) level was used to calculate polarizabilities (a 0 ), 1st mean hyperpolarizability (bo), and dipole moment (m) from which we get information for NLO property of the studied compound accordingly. Molecules having high polarizability possessed strong NLO potential and could be used for optoelectronics and in various optical instruments and device. [53][54][55][56][57] The assumption of the high sensitivity of hyperpolarizibility on the basis used and the theoretical values gives information about the changes in the value of hyperpolarizability due to electron correlation. 50,52 Due to hydrogen bonding (hydrogen bond interaction) present in the organic compound, hyperpolarizibility, and the mechanical stability increases. 58 As the studied compound have sulfur in the ring, acetoacetate and amino group as a side chain, compound possessed hydrogen bonding, therefore, the hyperpolarizibility and the mechanical stability of the compound increases. The values of calculated dipole moment, polarizability, and first-order hyperpolarizibility were mentioned in Table 5. The values of dipole moment of the studied molecule in x, y, and z direction were À0.5032 D, À0.6270 D, and À0.0100 D, respectively, and the total static dipole moment was 0.8041 D. The high value of total static dipole moment reflects strong intermolecular interaction in the molecule. The reference threshold value of NLO for comparative studies was taken from the studied value of the NLO properties of molecular system of the prototypical molecule named urea. 59 The calculated value of polarizability and hyperpolarizibility obtained from Gaussian output file was in atomic unit (AU) and were converted to electrostatic unit (e.s.u). The conversion factor for polarizability and hyperpolarilizibility was 1 au ¼ 0.148 Â 10 À24 e.s.u and 1au ¼ 08.6393 Â 10 À33 e.s.u, respectively. We can conclude from the values given in Table 3, the first-order hyperpolarizibility of the studied compound was much higher than that of the reference molecule urea (0.3728 Â 10 À30 esu). Thus, due to the high polarizability value of the studied compound reflects that the molecule had considerable NLO properties, and therefore, future research on the titled molecule's NLO properties can be possible.

Natural bond orbital and natural hybrid orbital analysis
Natural bond order (NBO) explains the interaction between occupied and unoccupied orbitals as well as intermolecular and intramolecular interaction of molecule. The localized small-centered orbitals which explain the Lewis-like molecular bonding pattern of electron pairs (or of individual electrons in the open-shell case) in optimally compact form are called natural bond orbitals (NBOs). The covalency effect and the hybridization in polyatomic wave function were interpreted by using the technique named NBO analysis. 59 The electronic wave function in the NBO analysis was studied in terms of sets of occupied Lewis type (bond or lone pair) and a set of unoccupied non-Lewis type (antibonding or Rydberg) localized NBO orbitals. Between the NBO, the delocalization of electron density (ED) was correlated with the stabilizing donor-acceptor interaction. To evaluate the stabilizing energies of all the possible interaction between donor and acceptor orbital, second-order perturbation theory was used. The loss in occupancy is resulted due to change in the interactions of the localized NBO of the idealized Lewis structure into empty non-Lewis orbitals. By using off diagonal elements of Fockmatrix in the NBO basis, the delocalization effect (or donor acceptor charge transfer) was estimated. For each donor (i) and acceptor (j), the stabilization energy (E 2 ) associated with the delocalization of i ⟶ j is determined by Eq. (3): where (F ij ) 2 is off diagonal Fock matrix element between the i and j NBO orbitals, er & er Ã represents the energies of bonding and antibonding NBO, respectively, and n r represents the population of the donor orbital. 60 To investigate intramolecular charge transfer interactions, re-hybridization and delocalization of electron density within the molecules, the natural bonding orbital analysis had been performed by DFT technique B3LYP method and 6-311þþG(d,p) basis set. In NBO analysis all the orbital details were mathematically evaluated and include the highest percentage of electron density (ED), therefore, it provide the most accurate "natural Lewis structure" picture of i. According to Godnman and Sauers, on using balanced-basis set NBO results were more accurate. 61,62 A useful aspect of the NBO method was that it provides efficient information about the interactions of the both filled and virtual orbital space which could enhance the analysis of intra and inter molecular interaction. The large E2 value in NBO analysis represents the intensive interaction between electron-donor and electron-acceptor and had greater extent of conjugation in the whole system.
The second-order perturbation theory analysis of Fock matrix in NBO basis shows strong intramolecular hyperconjugative interaction of p electrons and was given in Supporting Information Table ST1. The hyper-conjugative r Ã interaction in NBO analysis indicates the weak departure from strictly localized natural Lewis structure that constitutes the primary "noncovalent effect." From Supporting Information Table ST1, we can conclude that E values was high for r C3-C7 ⟶ p Ã C2-C5, r C4-C8 ⟶ p Ã C14-O21, LP(2) S1 ⟶ p Ã C2-C5, LP(2) S1 ⟶ p Ã C3-C7, LP(2) S1 ⟶ p Ã C3-C7, LP(2) O21 ⟶ p Ã C7-C14, LP(2) O21 ⟶ p Ã C14-O22, LP(2) O22 ⟶ p Ã C14-O21, 18.50, 29.35, 16.58, 24.74, 50.96, 14.47, 30.94, 46.52 kJ/mol, respectively, which shows that there was a strong intramolecular hyper-conjugative interaction hence provide stronger stabilization to the structure. Due to the presence of intermolecular hyper-conjugation of s and p electrons of C-C, C-N, C-S, and C-O bonds contributes in stabilization in some parts of the ring which can be evident from Supporting Information Table ST2. It can be further noted from Supporting Information Table ST1 that occupancies of most of the NBO's are more than the threshold occupancy between 1.89 and 1.98, therefore, the Lewis structure of the titled molecule was accepted. Maximum stabilization energy shown given above is the basis for the calculation of interaction energy of resonance in the molecule. The information for the formation of complex is associated with the changes in NBO bond polarization and hybridization and the percentage changes of the studied compound, given in Supporting Information Table ST2 one can infer that the most important interaction between filled (donor) Lewis type NBO's and empty (acceptor) non-Lewis NBO's. For example from Supporting Information Table ST2 r S1-C2 bond is formed from sp 4.48 hybrid of S which is the mixture of s (18.16%), p (81.28%), d (0.56%) atomic orbital and sp 3.31 hybrid of carbon which is the mixture of s (23.16%), p (76.72%), d (0.12%) atomic orbital. Thus, the bond between rS1-C2 is the result of overlapping of sp 4.48 hybrid of S1 and sp 3.31 hybrid of C2. The larger polarization coefficient of C2 indicates that the fluorine is more electronegative than S1. This can be represented as: rsc ¼ 0:6978ðsp 4:48 Þ S1 þ 0:7163ðsp 3:31 Þ C2 The direction of a hybrid is indentified by evaluating the polar (h) and azimuthal (A) angles of the vector describing its p-component. The deviation angles of hybrid A and hybrid B orbital describes the bending nature between the bonds. The data present in Supporting Information Table ST3 describes the geometrical changes and the bending nature of natural hybrid orbital (NHO). The NHO rS1-C2, rS1-C3 are bent by 4.0 and 6.2, 3.4 and 6.8, respectively. The direction of geometry varies because of the stable optimized geometry.

Population analysis
The calculation of effective atomic charges, which portrays the charges of each atom, distribution of positive and negative charges in molecules is very crucial to increase or decrease the bond length between the atoms. It is also important as atomic charges creates dipole moment effect, molecular polarizability, electronic structure, acidic and basic behavior, molecular reactivity, electrostatic potential surface, and a lot of molecular system properties. [63][64][65] Other than all the aforementioned importance it also provides useful information for NMR chemical shifts of the atom. The values of charges of the atom of the studied molecule was calculated by Mulliken population analysis (MPA) function, B3LYP method, and 6-311þþG(d,p) and were mentioned in Table 6 along with the graph shown in Supporting Information Figure S13. The charges were calculated with three charge components as neutral charge ¼ 0, multiplicity ¼ singlet (N), anionic charge ¼ -1 (N þ 1), multiplicity ¼ doublet, cationic charge ¼ þ1 (N -1), multiplicity ¼ doublet, in the Mulliken population analysis Mulliken population analysis is one of the best approaches for logical justification for differences in electronegativity of atoms present in a molecule and is often used to support the MEP contour mapping. Both MEP and Mulliken population was used to predict the behavior of a wide range of chemical system in both electrophilic and nucleophilic reaction. 66 From Table 6, we can conclude that N6, C4, C6, C7, C8, C11, O21, O22 have negative charge as they are electronegative elements than other he electronegative atoms, C2, C3, S1 were positively charged. There were nine carbon atom, one sulfur, and one nitrogen atom in the ring, where at C3, C14, amino group and oxygen were attached, respectively. The analysis of the studied compound done by MPA was accurate and precise; the presence of the respective charges on the studied molecule was explained. Electron donated by the amino group (electron donating group) present at C3 carbon, along with the electron donated by the S present in the ring having lone pair gets accumulated round C creating more electron density around C carbon hence C4 have negative charge. The charge present on the hydrogen atom of the studied molecule shows slight variance due to the surrounding atoms to which they were attached. The hydrogen atom present in the studied molecule plays an important role in the formation of hydrogen bonding network in crystalline state. The Fukui function analysis was also done on the titled compound as Fukui function was an important parameter to determine the nucleophilic and electrophilic behavior of the compound.
By utilizing Mulliken population analysis (MPA) the separate charge value of the Fukui functions can be achieved. Fukui functions can be calculated by the following formulas, The value of Fukui function analysis was obtained from the NBO charges and was mentioned in Table 6. The negative values of the Fukui function indicate that when an electron is added to a molecule, the electron density gets reduced at some regions where as when an electron is removed from the molecule, electron density gets increased at some regions. The calculated value mentioned in Table 6 reveals the reactivity order for electrophilic and nucleophilic site in the compound. The order is as follows: a. Electrophilic reactivity order: > C5. b. Nucleophilic reactivity order: S1 > C2 > C3 > C27.
The position of the electrophilic reactive sites and nucleophilic reactive sites was consistent with the total electron density surface and the chemical behavior. If we compare all the three types of attack, we can observe that the electrophilic attack was more reactive than the nucleophilic and radical attack within the molecule. The local softness was also studied and the values were given in Table 6 which are derived from the Fukui function, helps in studying and provides information about biological studies, ligand-protein interaction, and protein folding 68,69 which are of great importance in medicinal field.

FMO and UV-Vis spectra
The UV-Vis spectra of the molecule using methanol was recorded experimentally on UV-Vis instrument. The UV-Vis spectrum of the EACHT depicted in Figure 7, was calculated in Gas phase and DMSO, methanol as solvent phases.TD-DFT by B3LYP method and 6-311þþG(d,p) basis set was employed by including IEPCM with two solvents DMSO and methanol upto six excited states, and the electronic spectra was imitated. 70 The three main excited states with k max (maximum wavelength absorbed), excitation energy, band gap, oscillatory strength were summarized in Table 7. The Experimental k max was found to be at 229 nm with methanol as solvent. The calculated theoretical absorption k were calculated k at 347, 299.47, 270.24 nm in gas phase while 311.40, 291.33, and 267.17 nm in DMSO and were practically equal in gas phase as well as in DMSO, which showed no impact of solvent upon optical activity of compound. The stability of compound, its chemical activity and other variables can be analyzed by HOMO to LUMO energy band gap. 71 HOMO ⟶ LUMO energy gap decides the chemical stability of molecule. The HOMO energy value was -À7.918 eV and the LUMO energy value was located at À4.027 eV. The HOMO to LUMO energy gap found to be 3.891 eV, (HOMO -1) to (LUMO þ 1) was 4.947 eV, (HOMO -2) to (LUMO þ 2) was 7.616 eV, and (HOMO -3) to (LUMO þ 3) was 8.226 eV as depicted in Figure 8.
Many other significant variables such as ionization potential (IE), electron affinity (EA), electronegativity (E), chemical hardness, chemical softness, chemical potential, and electrophilicity index were calculated by HOMO ⟶ LUMO energy gap values and given in Table 8. This band gap verified that EACHT was a stable molecule having bioactive nature and transfer of charge can also takes place inside the molecule. Besides band gap, chemical hardness was also providing the chemical stability values. The chemical hardness of EACHT was 2.4218 as shown in Table 8, large value of chemical hardness indicates the chemical stability of substance. Likewise, electronegativity value estimates the attraction of electrons in a covalent bond, was estimated to be 5.96.
The maximum flow of electron between HOMO ⟶ LUMO leads to high electrophilicity index of the EACHT was 8.97.
Various parameters like electrophilicity, chemical hardness, and chemical potential were used to explain conceptual density functional theory (CDFT), used to analyze biological characteristics and to recognize active sites. 44 Electrophilicity index is the key indicator based on CDFT used in investigating bio-activity. 72 The titled compound found with an extremely less chemical softness value 0.51 was, therefore, considered to be nontoxic.
DOS reveals the number of states in unit energy interval and their participation to the chemical bonding through the COOP diagrams. The OPDOS reveals the bonding, antibonding and nonbonding interacting nature of two different orbitals, atoms or group of atoms. Positive value indicates a strong bonding cooperation (because of the positive population overlapping), negative values indicates strong anti-bonding participation (because of negative overlap population) and zero value means nonbonding interactions. The OPDOS diagrams contrast the donor-acceptor characteristics of ligands and determine the bonding, anti-bonding, nonbonding. The configuration of fragment orbitals aiding to the molecular orbital was portrayed by the PDOS.

Thermodynamical properties
The thermodynamic properties of EACHT was also computed at different temperature using ORCA software, on the basis of vibrational analysis at B3LYP/6-311þþG(d.p) level and the results had been detailed in Supporting Information Table ST4. The thermodynamic parameters like enthalpy (H o ), entropy (S o ), and Gibbs free energy (G o ) were calculated using ORCA programe for a range of temperature from 100 K to 700 K. when there is an increase in temperature, the molecular vibrational intensities increases along with that translational and rotational energy  also increases which intern increases the thermodynamic function such as entropy (S 0 ) and enthalpy (H o ). This relation between thermodynamic function with increase in temperature was very well explained by equipartition 73,74 But Gibbs free energy decreases because it shows reverse behavior with increase in temperature in comparision with entropy and enthalpy. The dependency of the thermodynamic function with the temperature was represented graphically shown in Figure 9. Quadratic and linear equations were used to determine the correlation between the thermodynamic function and the temperature. The correlation R 2 among the thermodynamic properties/function such as entropy, enthalpy, and Gibbs free energy was calculated using quadratic and linear formulae were 1.00, 0.9652, and 0.9856, respectively.
S ¼ 0:0288T 2 À 32:731T þ 9809:5608 (5) The quadratic equation used to find correlation was given below: Thermodynamic properties also used to determine the binding interaction mode between noncovalent interactions which includes electrostatic interactions, multiple hydrogen bonds, van der Waals interactions, and hydrophobic effects. 75 This thermodynamic data can be used to determine other thermodynamic energies and direction of chemical reaction in according with second law of thermodynamics. 76 Plots of quadratic equations were given in Supporting Information S7-S9.

Vibrational spectral analysis
The titled molecule of which vibrational analysis had been done was a hetrocyclic compound and has planar structure of C1 point group, therefore, the vibrational frequency assignment shows some level of similarity with the vibrational frequencies of benzene, pyridine and pyrimidine compounds. The titled molecule (EACHT) shows 93 modes of vibration as it has 33 atoms (3n -6). Both Gaussian 3.0 and VEDA4.0 program package 34 was used simultaneously to study all modes of vibration of the studied molecule. Calculated frequency was scaled by 0.961 because approximation causes calculated frequency to be in higher region than experimental results. 77 The calculated frequency along with normalized IR and Raman intensities and PED assignment at DFT/B3LYP/6-311þþG(d,p) level and the experimental FTIR and FT-Raman 78 have been summarized in Supporting Information Table ST5. The calculated modes of vibration were numbered in descending order (from largest to the smallest) within each fundamental wave number. The Raman and IR graph was also plotted using multiwfn program using same level theory illustrated in Figures 10 and 11. The experimental, calculated IR spectra shown in Figure 10A,B, experimental FT-Raman is shown in S11 and theoretical Raman in Figure 11. The calculated intensity was plotted against harmonic vibrational wave numbers. There is no negative (imaginary) frequency, hence, all the 93 modes of vibration were both IR and Raman active. From Supporting Information Table ST5 we can infer that, great mixing of the ring vibrational modes and also between the ring vibrational modes and also between the ring and substituent modes was seen. The low symmetry of the studied molecule makes the descriptions of the modes very complicated. Especially in the plane and out of plane modes were the most difficult modes and also with the substituent modes. There are same strong frequencies of the studied molecule, which were helpful to characterize in the IR and Raman spectra and were worth mentioning. The R 2 value within experimental FTIR and theoretical FTIR was 0.997, likewise for FT-Raman R 2 was 0.996. This reveals that the theoretical as well as experimental analysis was showing good correlation. Harmonic theoretical frequencies are calculated in gas phase of an isolated compound, while the experimental frequencies are obtained in its solid phase. So, in some modes there is a disagreement in between the observed and calculated frequencies. Some important modes are explained below:

Thiophene vibrations
The C-S mode cannot be identified in thiophenes. That is due to the short bond length and high polarity of the C-S bond in thiophene. 79 Klots et al. 80 ascribe mode at 882 cm À1 , 763 cm À1 , and 880 cm À1 ; 755 cm À1 to vapor and liquid phases, respectively. The Predicted C-S stretching mode found at 691 cm À1 , 707 cm À1 and recorded at 757 cm À1 and 411 cm À1 . 81 In this work, the C-S stretching modes observed in FTIR at 757 cm À1 (IR), and 411 cm À1 , bending mode is observed at 566 cm À1 . In plane bending modes NCS, SCC with 18%, 20% PED was calculated at 566, 416 cm À1 by scaled B3LYP, but this mode was not observed experimentally in FTIR. Out-of-plane bands NCSC were observed at 557 cm À1 (FTIR), 472 cm À1 . These modes were calculated to be at 643 cm À1 , 477 cm À1 . Torsion out-of-plane modes are sSCCC. These modes were observed at 552 cm À1 in FT-Raman spectrum. This modes was not observed experimentally in FTIR, and was calculated to be at 628 cm À1 by scaled B3LYP method.

Amino group vibrations
Usually, the N-H stretching vibrational modes in primary amines, occur in the region of 3550-3320 cm À1 . 82,83 The NH 2 group has two modes; one is symmetric and asymmetric. The vibrational frequency of asymmetric vibration is on higher side than that of symmetric vibration. In this study, the asymmetric and symmetric NH stretching vibrations were observed at 3437, 3330 cm À1 , respectively. The corresponding bands were calculated to be at 3692 and 3500 cm À1 by B3LYP/6-311þþG(d,p) method. These two modes are pure stretching modes, as it is clear from PED assignment, they are contributing almost 90%. As expected and also observed that, the asymmetric mode is more intense than symmetric. These assignments are in line with the literature. 84,85 The in-plane N-H bending vibrations (scissoring) are observed in the region of 1610-1630 cm À1 , vibrations (rocking) are assigned in between 1100-1200 cm À1 and the N-H out of plane bending (wagging and twisting) vibrations are identified in range of 900 cm À1 . [86][87][88][89] In this case, in plane bending mode HNH was observed at 1588 cm À1 (FTIR) and was calculated to be at 1614 cm À1 by B3LYP. likewise, in-plane rocking modes were observed at 1494 cm, 1256, 1129 cm À1 . In concurrence to the literature, these assignments are well agreed.

Electron excitation analysis (electron and hole density distribution)
Process to excite single-electron involve delocalization of electron from a to b where a and b demonstrates the real space functions. One commonly used prototype of excited state resembles excitation of an electron out of an occupied MO to virtual MO. Although excited states calculated represent the electronic structures described in terms of multiorbital electronic states with a combination of various occupied-to-virtual MO excitations. In view of the nonapplicability of a single orbital pair excitation prototype, the issue was evaded by conceptualizing multimolecular orbital excitations in terms of EDD and HDD (electron density distribution and hole density distribution) maps, giving very clear impression of photoexcited states. Electronic states calculation for EACHT done at TD-DFT-B3LYP/6-311Gþþ(d,p) level with IEPCM in MeOH, DMSO forecast two major electronic transitions (1st and 4th) depicted in Supporting Information Figure S14 Figure S13 shows more density shift toward phenyl ring, while EDD maps show more density shift toward anhydride group. Hence, the discovered changes in the surface densities are attributed to electron density relocation from phenyl toward the more polarizing anhydride group. Else, the matching of EDD and HDD map with the ground state LUMO for first ES and HOMO⟶ LUMO for fourth ES, surfaces of the EACHT can be ascribed to a single MO pair excitation process.
The calculated centriod coordinates of HDD and EDD for EACHT in DMSO and Methanol with two excited states transitions was summarized in Table 9.

Molecular docking
One of the most widely used technique to study the structure-activity relationship and in the drug discovery is molecular docking 92 as it gives the result with a substantial degree of accuracy. It gives information about the strength of interaction of small molecule ligand with the Figure 9. Graphs representing dependence of entropy, Gibbs free enthalpy and enthalpy on temperature of EACHT. macromolecular targets (receptor), i.e. the binding energy and the binding site of the ligand and the macromolecule. 93 It is very important in pharmaceutical research and the development as it investigate the crucial molecular events including ligand binding modes, stability of the ligand-receptor complex by its corresponding intermolecular interaction and also predicts the binding affinity of the ligand-receptor complexes. 70,94 The software named Autodoc-Vina 74 is an open source programme for molecular docking. The suitable target protein ID was selected from Swiss ADME-Target prediction site and downloaded from protein data bank (PDB). 21,95,96 The  receptors 3KIA, 3WZE, 4GJ2, 4MVH, 4ZGX, 7KKQ of transferase domain was docked with EACHT, illustrated in Figure 12 with the bond distance of Hydrogen bond interaction between EACHT and the target proteins. The least binding energy and Ki value comes out to be À6.9 kcal/mol and 9.00, respectively, given in table Table 10 with the hydrogen bond distance (2.355, 2.224, 2.505 Å) between the bonded residue and the ligand which determines the stability of the ligand and the receptor protein. The resulted high value of binding energy shows that the studied molecule was biologically active. There were three residue present in all docked proteins. The EACHT compound interacts with various different receptors as depicted in Figure 12A-F and Supporting Information Figure S12. Least value of binding energy shows the antibacterial protein 7KKQ can properly interacted with EACHT.

Drug-likeness
Drug-likeness was carried out to acquire efficacious and well-organized results in drug development, structural characteristics of the ligands. Drug likeness was based on rules like Lipinski's rule, MDDR-like rule, Veber rule, Ghose filter, BBB rule, CMC-50 rule, and QED. 97 The EACHTand its derivatives possesses antibacterial properties so, they were applied to the druglikeness rule and the efficacy was analyzed.
The significant ADME variables such as hydrogen bond acceptors (HBA), Hydrogen bond donors (HBD), molar refractivity (MR), Topological polar surface area (TPSA), Blood-brain barrier penetration (BBB), logkp and bioavailability score of the common derivatives of compound EACHT were calculated and data recovered was summarized in Table 11. From the literature it was analyzed that values of HBD and HBA must be less than 10. Here in this study it ranges from 1 to 3 for all the derivatives. The maximum value of TPSA must be 140 Å 2 . Here, the value ranges in between 38 and 42.23 for all the derivatives of EACHT. Likewise, the value of the molar refractivity must be between 40 and 130. 98 The MR value of EACHT was <40, all the derivatives were showing different values. The table reflects that GI absorption was on high side, BBB permeant was attainable for all the derivatives, skin permeability (log Kp) falls within À5.58 to À6.78 and bioavailability value scored by EACHT and its derivative were same as 0.55. The aforementioned analogy reflects that compound EACHT has adequate biological properties. The figure showing Drug likness properties of EACHT and one of its derivatives was given in Supporting Information S11 and S12.

Conclusion
Herein, the medicinally important molecule generally named: Ethyl-2-amino-5,6,7,8-tetrahydro-4H-cyclo-hepta[b]thiophene-3-carboxylate (EACHT) has been synthesized, characterized investigated Quantum chemically via DFT/B3YYP/6-311þþG(d,p) level. Arrangement of intramolecular and intermolecular interactions in the crystal and H-bond interaction Hirshfeld surface analysis was carried out. Absolute geometry optimization was carried out, the compound posses C1 point symmetry. Three dimensional Hirshfeld surfaces and 2D Fingerprint plots with d norm view of the contacts shows the close contact in crystal structure, p-p stacking interactions in the crystal indicated by the presence of red and blue patches. EDD and HDD maps were plotted for first excited state and fourth excited state with DMSO, MeOH to evaluste shift of electron density in case of hole and electron pair excitation from occupied to unoccupied MO as in this case from HOMO ⟶ LUMO for first and HOMO⟶ LUMO þ 3 for fourth excited state. The reactivity, the chemical nature as well as the attacking sites via MEP and ELF which uses different color codes, showing that the studied molecule is electron rich hence prone to electrophilic attacking species in the order N6 > O21 > C11 > C7 > C15 > C4 > C18 > C28 > C5, hence it can interact with other proteins. The UV-Visible spectra in the gas phase and in DMSO, Methanol shows similar absorption wavelength and were also consistent with the experimental data. The maximum stabilization was shown by electron donated from the LP(2) S1 ⟶ p Ã C3-C7, LP(2) O21 ⟶ p Ã C7-C14, LP(2) O21 ⟶ p Ã C14-O22, LP(2) O22 ⟶ p Ã C14-O21, i.e. 50.96, 14.47, 30.94, 46.52 kJ/mol which is the basis for calculation of interaction energy. The HOMO energy value was À7.918 eV and the LUMO energy value was located at À4.027 eV. The HOMO to LUMO energy gap found to be 3.891 eV, (HOMO -1) to (LUMO þ 1) was 4.947 eV and (HOMO -2) to (LUMO þ 2) was 7.616 eV which indicate that there is high charge transfer within the molecule. The confirmation on the nontoxic and biological active behavior of molecule is provided by the FMO analysis. The studied compound is considered as a new NLO compound as the value of hyperpolarizability is high than that of the reference NLO compound urea. The information of hyperconjugation is given by NBO analysis. Molecular docking studies carried out on the 3KIA, 3WZE, 4GJ2, 4MVH, 4ZGX, 7KKQ proteins. The resultant binding energy comes out to be À6.9 kcal/mol after docking for three protein, indicating that the titled compound can be studied further for its medicinal application. Drug-likeness study confirms that the titled molecule shows promising result in the treatment of many diseases.

Disclosure statement
No potential conflict of interest was reported by the authors.