Synthesis, X-ray Single Crystal, Conformational Analysis and Cholinesterase Inhibitory Activity of a New Spiropyrrolidine Sca ﬀ old Tethered Benzo[ b ]Thiophene Analogue

: Described herein is a one-pot protocol for the synthesis of a substituted spiropyrrolidine sca ﬀ old tethered benzo[ b ]thiophene analogue from ( E )-3-(benzo[ b ]thiophen-2-yl)-1-(4-ﬂuoro-phenyl)-prop-2-en-1-one. The described protocol has the advantage of the high purity of the cyclized adduct and high chemical yield. To assign the chemical structure, di ﬀ erent spectrophotometric tools have been applied, including 1 H-NMR, 13 C-NMR, FTIR, and the X-ray single crystal technique. The X-ray structure showed that the studied compound exist in two disordered parts with equal partial occupancies. The energies of the two conformers were found to be very similar and not exceed 1 kcal / mol, which justiﬁes their coexistence in the crystal with equal percentage. The molecular packing in the crystal was analyzed using Hirshfeld topology analysis. The packing described as two dimensional hydrogen bond network extended along the ac -plane in both conformers but the intermolecular interactions included in each conformer are not similar. The synthesized spiropyrrolidine sca ﬀ old tethered benzo[ b ]thiophene analogue was examined against cholinesterase inhibitory activity and show moderate activity compared to standard drug galantamine.


Introduction
Alzheimer's disease (AD) treatment is an exciting and interesting research area for chemists. Alzheimer's disease (AD) plays a crucial rule in dementia, affecting millions of people around the world, according to the Alzheimer's World Report [1]. The treatment of Alzheimer's disease symptoms is based on inhibiting the cholinesterase enzymes (ChEs) which exist in the central nervous system [2,3]. To design a new, and safe pharmaceutical drug with long half-live time, high bioavailability, and high efficacy with less toxicity is a challenge in pharmaceutical research.

General Methods
"All melting points were determined using a Mel-Temp electrothermal apparatus (Electrothermal, Staffordshire, UK), and are uncorrected. Thin layer chromatography (TLC) was conducted on silica gel (Kieselgel G, Merck, Darmstadt, Germany) and spots were detected under UV light at 254 nm. IR spectra were recorded in a KBr matrix with a Spectrum 100 FT-IR spectrophotometer (Perkin Elmer, Waltham, MA, USA). 1 H-and 13 C-NMR spectra were recorded in DMSO-d 6 as solvent using a JEOL 400 MHz instrument (JEOL, Ltd, Tokyo, Japan), and the chemical shifts (δ) values are given in ppm. The X-ray crystallographic analysis was performed using the SMART APEX II D8 Venture diffractometer (Bruker D8 Venture, Karlsurehe, Germany) located at Karachi University."

Single-Crystal X-ray Diffraction Analysis
Single-crystal X-ray diffraction analysis of the studied compound was carried out by mounting an appropriate crystal with dimension 0.11 × 0.09 × 0.07 mm 3 on a Bruker D8 Venture instrument equipped with a CCD Photon II detector and graphite monochromator having Cu Kα radiation (λ = 1.54178 Å) at T = 100(2) K for data collection. For the integration, and reduction of data, Crystals 2020, 10, 120 3 of 14 SAINT (Bruker 1998) program was used [21]. The solved structure was done by direct method, and Fourier transformation techniques, and further refined by full-matrix least-squares techniques on F 2 using SHELXL-2018 program. PLATON [22], and SHELXL [23] programs were employed for the final refinement of solved structure (Table 1). "The final refinement was carried out by full-matrix least-squares techniques with anisotropic thermal data for non-hydrogen atoms on F. CCDC 1972061 contains the supplementary crystallographic data for this compound can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif."

Hirshfeld Surface Analysis
"The topology analyses were performed using the Crystal Explorer 17.5 program [24] in order to determine the percentages of the different intermolecular interactions in the crystal structure of the studied compound."

Computational Methods
All DFT calculations were performed with the Gaussian 09 software package [25,26] using the B3LYP/6-31G(d,p) method. The resulting optimized structures showed no imaginary frequencies.

Scheme 1:
The synthetic route of compound 5.
Subsequently, a one pot three component reaction of the synthesized chalcone 2 with isatin 3 and thioproline 4 afforded the cyclized compound 5 in excellent yield, regio-and diasetereoselectivity. Based on the 1 H-NMR spectrum, the protons have been assigned as follows: a singlet for the NH proton appears at δ 10.39 ppm and the 13 aromatic protons appeared as expected in the aromatic region, along with signals in the lower field chemical shift region between δ 4.75 until 3.19 ppm, that could be assigned for the protons belonging to fused pyrrolothiazole ring. The 13 C-NMR signals are also consistent with the assigned carbons. The FTIR spectrum shows characteristic peaks, including NH (3420), CH (3066, 2927), CO (1704) and C=C (1597) functionalities. Additionally, a suitable single crystal was grown and submitted for single crystal X-ray diffraction analysis and the data is reported here (an image of the single crystal and the ORTEP structure are shown in Figure 1). The relative configuration of the synthesized substituted spiropyrrolidine scaffold was assigned by the help of the X-ray single crystal technique, and a plausible reaction mechanism is shown in Scheme 2 [28][29][30][31][32][33][34]. The reaction proceeds following a one pot multicomponent protocol in which initially isatin reacted with thioproline to afford an intermediate that leads to an azomethine yilde after expulsion of carbon dioxide. Subsequently the azomethine yilde reacts with the chalcone Subsequently, a one pot three component reaction of the synthesized chalcone 2 with isatin 3 and thioproline 4 afforded the cyclized compound 5 in excellent yield, regio-and diasetereoselectivity. Based on the 1 H-NMR spectrum, the protons have been assigned as follows: a singlet for the NH proton appears at δ 10.39 ppm and the 13 aromatic protons appeared as expected in the aromatic region, along with signals in the lower field chemical shift region between δ 4.75 until 3.19 ppm, that could be assigned for the protons belonging to fused pyrrolothiazole ring. The 13 C-NMR signals are also consistent with the assigned carbons. The FTIR spectrum shows characteristic peaks, including NH (3420), CH (3066, 2927), CO (1704) and C=C (1597) functionalities. Additionally, a suitable single crystal was grown and submitted for single crystal X-ray diffraction analysis and the data is reported here (an image of the single crystal and the ORTEP structure are shown in Figure 1). All DFT calculations were performed with the Gaussian 09 software package [25,26] using the B3LYP/6-31G(d,p) method. The resulting optimized structures showed no imaginary frequencies. Natural population analysis was performed using NBO 3.1 program as implemented in the Gaussian 09W package [27].

Scheme 1:
The synthetic route of compound 5.
Subsequently, a one pot three component reaction of the synthesized chalcone 2 with isatin 3 and thioproline 4 afforded the cyclized compound 5 in excellent yield, regio-and diasetereoselectivity. Based on the 1 H-NMR spectrum, the protons have been assigned as follows: a singlet for the NH proton appears at δ 10.39 ppm and the 13 aromatic protons appeared as expected in the aromatic region, along with signals in the lower field chemical shift region between δ 4.75 until 3.19 ppm, that could be assigned for the protons belonging to fused pyrrolothiazole ring. The 13 C-NMR signals are also consistent with the assigned carbons. The FTIR spectrum shows characteristic peaks, including NH (3420), CH (3066, 2927), CO (1704) and C=C (1597) functionalities. Additionally, a suitable single crystal was grown and submitted for single crystal X-ray diffraction analysis and the data is reported here (an image of the single crystal and the ORTEP structure are shown in Figure 1). The relative configuration of the synthesized substituted spiropyrrolidine scaffold was assigned by the help of the X-ray single crystal technique, and a plausible reaction mechanism is shown in Scheme 2 [28][29][30][31][32][33][34]. The reaction proceeds following a one pot multicomponent protocol in which initially isatin reacted with thioproline to afford an intermediate that leads to an azomethine yilde after expulsion of carbon dioxide. Subsequently the azomethine yilde reacts with the chalcone The relative configuration of the synthesized substituted spiropyrrolidine scaffold was assigned by the help of the X-ray single crystal technique, and a plausible reaction mechanism is shown in Scheme 2 [28][29][30][31][32][33][34]. The reaction proceeds following a one pot multicomponent protocol in which initially isatin reacted with thioproline to afford an intermediate that leads to an azomethine yilde after expulsion of carbon dioxide. Subsequently the azomethine yilde reacts with the chalcone to provide the target compound in a regioselective and diastereoselective manner. The reaction proceeds regioselectively via path A to afford the regioisomeric product 5, while the second regioisomers 5 is Crystals 2020, 10, 120 5 of 14 not formed (path B). There are possible diastereoselective products that could be formed but in this case the only diastereoselective compound 5 was observed (path C). to provide the target compound in a regioselective and diastereoselective manner. The reaction proceeds regioselectively via path A to afford the regioisomeric product 5, while the second regioisomers 5 is not formed (path B). There are possible diastereoselective products that could be formed but in this case the only diastereoselective compound 5 was observed (path C).

Scheme 2.
Plausible reaction mechanism for the synthesized compound.

Crystal Structure Discription
Crystallographic data and refinement details for the studied compound, C28H21FN2O2S2.CH3OH are listed in Table. 1. The compound C28H21FN2O2S2 co-crystallized with one methanol molecule as crystallization solvent. It crystallizes as block colorless crystals in the orthorhombic crystal system and has the P212121 as a space group. The asymmetric unit comprised one molecular unit and Z = 4. A list of geometric parameters (bond distances and angles) is given in Table S1 (Supplementary Data). It is clear from the X-ray structure analysis that the compound has disorder over two positions as shown in Figure 2. The difference between the two conformers is a result of two types of disorder which are rotational, and positional disorders of the benzothiophene, and thiophene ring, respectively with partial occupancies of (50%:50%) for each, as shown in Figure 2. The interesting point is the disorder of the benzothiophene moiety in which the structure exhibited the two conformational structures indicated in the lower part of Figure 2. The main difference between the two conformations is that the benzothiophene moiety is rotated around the C12-C8 bond by 130°. Scheme 2. Plausible reaction mechanism for the synthesized compound.

Crystal Structure Discription
Crystallographic data and refinement details for the studied compound, C 28 H 21 FN 2 O 2 S 2 .CH 3 OH are listed in Table 1. The compound C 28 H 21 FN 2 O 2 S 2 co-crystallized with one methanol molecule as crystallization solvent. It crystallizes as block colorless crystals in the orthorhombic crystal system and has the P2 1 2 1 2 1 as a space group. The asymmetric unit comprised one molecular unit and Z = 4. A list of geometric parameters (bond distances and angles) is given in Table S1 (Supplementary Data). It is clear from the X-ray structure analysis that the compound has disorder over two positions as shown in Figure 2. The difference between the two conformers is a result of two types of disorder which are rotational, and positional disorders of the benzothiophene, and thiophene ring, respectively with partial occupancies of (50%:50%) for each, as shown in Figure 2. The interesting point is the disorder of the benzothiophene moiety in which the structure exhibited the two conformational structures indicated in the lower part of Figure 2. The main difference between the two conformations is that the benzothiophene moiety is rotated around the C12-C8 bond by 130 • .

Crystal Packing
Another interesting difference between the two conformers, which is the intermolecular hydrogen bonding interactions, occurred in each part which are not typically the same. The detailed list of the hydrogen bonding interactions is given in Table 2 while the packing diagram showing the most important hydrogen bonding interactions is shown in Figure 3. In this illustration, the weak C-H…O hydrogen bonding interactions are omitted for simplicity.
It is clear that, the packing of the molecular units of part A could be described as a twodimensional hydrogen bond network extended in the ac plane via N2-H2…O3 and O3-H3O…O1 hydrogen bonds in addition to the weak C7A-H7AA…S2A and C10A-H10A…S2A intermolecular interactions, with donor acceptor distances of 2.830(3), 2.775(3), 3.604(16) and 3.378(7) Å, respectively. On the other hand, the packing in the second conformer (part B) represent also two dimensional hydrogen bond polymer extended along the ac-direction. In addition to the N2-H2…O3 and O3-H3O…O1 hydrogen bonds, the molecular units are packed via weak C7B-H7BA…F1 and C4B-H4B…S1B intermolecular interactions with donor acceptor distances of 3.080(9) and 3.738(8) Å, respectively.

Crystal Packing
Another interesting difference between the two conformers, which is the intermolecular hydrogen bonding interactions, occurred in each part which are not typically the same. The detailed list of the hydrogen bonding interactions is given in Table 2 while the packing diagram showing the most important hydrogen bonding interactions is shown in Figure 3. In this illustration, the weak C-H. . . O hydrogen bonding interactions are omitted for simplicity.

Hirshfeld Analysis of the Molecular Packing
Based on the single crystal X-ray structure of the studied compound, there are two possible conformers for this molecule represented by parts A and B in Figure 4. The Hirshfeld surface mapped over dnorm, shape index (SI) and curvedness for this molecule is shown in Figure 5. Decomposition analysis of the different intermolecular contacts indicated that both parts have almost similar intermolecular contacts ( Figure 6). The H…H, O…H, S…H, and F…H contacts contributed by 44.2%, 11.8%, 9.4%, and 5.9%, respectively for conformer A and 42.5%, 12.4%, 11.4% and 6.8% for conformer B, respectively. Those are the most abundant contacts in the structure of the studied systems.

Hirshfeld Analysis of the Molecular Packing
Based on the single crystal X-ray structure of the studied compound, there are two possible conformers for this molecule represented by parts A and B in Figure 4. The Hirshfeld surface mapped over d norm , shape index (SI) and curvedness for this molecule is shown in Figure 5. Decomposition analysis of the different intermolecular contacts indicated that both parts have almost similar intermolecular contacts ( Figure 6)    As clearly indicated from the decomposed fingerprint of the most common contacts shown in Figure 7, the O…H hydrogen bonds are significantly strong in both conformers. The shortest O…H contacts are the O3-H3O…O1 and N2-H2…O3 ones with hydrogen-acceptor distances of 1.838 and 1.828 Å, respectively in both conformers. On other hand, the F…H contacts appeared stronger in conformer B than conformer A. The C7B-H7BA…F1 has hydrogen-acceptor distance of 2.262 Å while the corresponding value in conformer A for the C13-H13…F1 interaction is 2.648 Å. The latter is longer than the van der Waals radii sum of fluorine and hydrogen atoms (2.56 Å). In contrast, the S…H contacts are shorter in conformer A than conformer B. In the former there are three significant S…H interactions which are the C10A-H10A…S2A, C12-H12…S2A and C7A-H7AA…S2A with hydrogen acceptor distances of 2.426, 2.865 and 2.738 Å, respectively. In the conformer B, the weak C10B-H10D…S1B, C12-H12…S2B and C9A-H9A…S1B intermolecular interactions have longer S…H contact distances of 2.964, 2.963, and 2.944 Å, respectively. Interestingly, the Hirshfeld analysis detected very small amount (1.3%) of significantly short S…F interactions with F1…S1A distance of 3.172 Å. The shape index and curvedness maps revealed the absence of significant ring π-π stacking interactions.  As clearly indicated from the decomposed fingerprint of the most common contacts shown in Figure 7, the O…H hydrogen bonds are significantly strong in both conformers. The shortest O…H contacts are the O3-H3O…O1 and N2-H2…O3 ones with hydrogen-acceptor distances of 1.838 and 1.828 Å, respectively in both conformers. On other hand, the F…H contacts appeared stronger in conformer B than conformer A. The C7B-H7BA…F1 has hydrogen-acceptor distance of 2.262 Å while the corresponding value in conformer A for the C13-H13…F1 interaction is 2.648 Å. The latter is longer than the van der Waals radii sum of fluorine and hydrogen atoms (2.56 Å). In contrast, the S…H contacts are shorter in conformer A than conformer B. In the former there are three significant S…H interactions which are the C10A-H10A…S2A, C12-H12…S2A and C7A-H7AA…S2A with hydrogen acceptor distances of 2.426, 2.865 and 2.738 Å, respectively. In the conformer B, the weak C10B-H10D…S1B, C12-H12…S2B and C9A-H9A…S1B intermolecular interactions have longer S…H contact distances of 2.964, 2.963, and 2.944 Å, respectively. Interestingly, the Hirshfeld analysis detected very small amount (1.3%) of significantly short S…F interactions with F1…S1A distance of 3.172 Å. The shape index and curvedness maps revealed the absence of significant ring π-π stacking interactions.

DFT Studies
The crystal structures of both conformers A and B were optimized using B3LYP method and their total energies are listed in Table 3. It is clear from the results listed in this table that the energy difference is very small. It is found that conformer B is slightly more stable than conformer A by

DFT Studies
The crystal structures of both conformers A and B were optimized using B3LYP method and their total energies are listed in Table 3. It is clear from the results listed in this table that the energy difference is very small. It is found that conformer B is slightly more stable than conformer A by 0.4885 kcal/mol. Further, optimization of the structures of both conformers in absence of methanol as crystallized solvent gave almost the same results (0.6255 kcal/mol) indicating that both conformers have very close stability and could exist in very similar proportions to each other. Also, the crystallization solvent has a little effect on the solid state conformation. The X-ray structure analysis supported this point where the two parts have almost equal partial occupancies. Not only the structures of A and B are energetically very similar but also their polarities are close to each other. The calculated dipole moment values of the two conformers were predicted to be 2.6886 and 2.5838 Debye for conformers A and B, respectively. The solvated species have also close polarities of 3.6435 and 4.3095 Debye, respectively. Similar observation could be noted for the frontier molecular orbital energies ( Table 3). The reactivity indices [35][36][37][38][39][40][41][42][43] of both conformers such as ionization potential (I), electron affinity (A), chemical potential (µ), hardness (η), softness (S), and electrophilicity (ω) indices were calculated based on the HOMO and LUMO energies and were found very close to each other (Table 3). These descriptors could be used to understand the pharmacological and biological reactivity of the compounds. The optimized structures of the organic molecules were matched with the experimental structure in Figure 8 while the calculated and experimental bond distances and angles are listed in Table S2 (Supplementary Data). The correlation coefficients were found in the range of 0.969-0.983 and 0.940-0.959 for bond distances and angles, respectively, which indicated good agreement between the calculated and experimental results. The natural charge distribution at the different atomic sites were calculated and the results were listed in Table S3 (Supplementary Data). The S-atoms have a positive natural charge where the benzothiophene S-atom is more electropositive than that in the thioproline moiety. In contrast, the oxygen atoms are electronegative with natural charges ranging from −0.5487 to −0.6354 e. The most important conclusion that could be concluded from this table is that the methanol molecule has a net charge density of −0.0167 and −0.0178 e for A and B, respectively. The rest organic fragments have the opposite of these values. As a result of the hydrogen bonding interactions, methanol molecule is acting as an electron acceptor while the organic fragments acts as electron donor.

Biological Activity
To show the biological activity of the synthesized compound based on the literature survey an assay has been conducted. The cholinesterase inhibitory activity of the synthesized compound was assessed in vitro [44]. The result is summarized in Table 4. The spiropyrrolidine scaffold tethered benzo[b]thiophene analogue analogue has an IC50 value of 62.25 µg/mL compared to galantamine (IC50 = 0.98 µg/mL). This result shown some activity so further studies must be investigated by synthesis of another analogues and report the SAR.

Biological Activity
To show the biological activity of the synthesized compound based on the literature survey an assay has been conducted. The cholinesterase inhibitory activity of the synthesized compound was assessed in vitro [44]. The result is summarized in Table 4. The spiropyrrolidine scaffold tethered benzo[b]thiophene analogue analogue has an IC 50 value of 62.25 µg/mL compared to galantamine (IC 50 = 0.98 µg/mL). This result shown some activity so further studies must be investigated by synthesis of another analogues and report the SAR.

Conclusions
The proceeding study reported the design and synthesis of spiropyrrolidine scaffold tethered benzo[b]thiophene analogue 5 as a potential AChe inhibition agent. The structure features of 5 were elucidated using different techniques. The energies of the two conformers observed experimentally based on the disorder model of the X-ray structure of the studied compound are very close, indicating that the two conformers could exist in equal proportions. Different intermolecular interactions were analyzed using Hirshfeld analysis. Also, the dipole moment, frontier molecular orbitals energies, reactivity descriptors and natural atomic charge populations were discussed based on the B3LYP/6-31G(d,p) method. The synthesized spiropyrrolidine-based benzo[b]thiophene exhibited moderate activity compared to galantamine as standard. Further investigations are underway in our laboratory.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4352/10/2/120/s1, protocol for the Acetylcholinesterase (AChE) inhibitory assay; Figure S1: 1 H-NMR of 5 in DMSO-d 6 ; Figure S2: 13 C-NMR of 5 in DMSO-d 6 ; Figure S3: FTIR of 5 in KBr; Figure S4: Atom numbering of the optimized structures of conformers A and B (see Table S1).  Table S2: The calculated geometric parameters of the studied conformers with and without the crystallized methanol molecule a; Table S3: Natural charge populations at the different atomic sites of the studied conformers.