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Synthesis and Antimicrobial Activity of Novel Piperidinyl Tetrahydrothieno[2,3-c]isoquinolines and Related Heterocycles

A novel series of 1-amino-2-substituted-5-piperidinyl-6,7,8,9-tertahydrothieno[2,3-c]isoquinolines (4a–e) was synthesized upon treatment of 4-cyano-1-piperidinyl-5,6,7,8-tetrahydroisoquinline-3(2H)-thione (2) with α-halo carbonyl compounds such as chloroacetone, ethyl chloroacetate, 2-bromoacetophenone, chloroacetamide, and chloroacetanilide. Construction the pyrrolyl ring associated with the thienotetrahydroisoquinoline moiety was achieved by treatment of compounds 4a, b with 2,5-dimethoxytertahydrofuran in acetic acid. 1-Pyrrolyl-2-substituted-thieno[2,3-c]isoquinolines 5a and 5b which in turn were used as multipurpose precursors for synthesis of other new heterocycles. Assignments of the chemical structures of the respectively synthesized thienotetrahydroisoquinolines and their derivatives were established on the bases of elemental and spectral techniques (Fourier transform infrared, 1H NMR, 13C NMR, and mass spectroscopy). Furthermore, certain compounds were screened for their antimicrobial activity which revealed promising activities against various pathogenic strains of bacteria and fungi.


■ INTRODUCTION
The tetrahydroisoqunioline ring system is a privileged fundamental scaffold of various alkaloids isolated from natural sources and abundantly found in various plants, soils, and marine microorganisms. 1 Molecules embedded with these scaffolds are important pharmaceutical intermediates in medicinal chemistry and have gained distinctive attention because of their broad spectrum of pharmacological properties. Many tetrahydroisoquinoline compounds are considered as antitumor, 2,3 anticonvulsant, antithrombotic, 4 analgesic, 5 antiinflammatory, 6 antifungal, and antibacterial agents. 7 Moreover, tetrahydroisoquinolines are useful compounds as antagonist to NMDA and D1 receptors, 8 Parkinson's disease, 9 enzyme inhibitory actions for glucosidases, 10 and monoamine oxidases. 11 Isolated alkaloids from natural sources containing the tetrahydroisoquinoline moiety are broadly abundant in many biologically active compounds ( Figure 1). Higenamine 1 extracted from the leaves of Aristolochia brasiliensis has been recognized as an agonist for the β2-adrenergic receptor 12−14 and antimycobacterial 12 and anti-inflammatory drugs. 14,15 The complex α-substituted tetrahydroisoquinoline, Noscapine 2, revealed an antiproliferative activity against human prostate cancer cells. 16,17 Oleracin E 3 has been established as an antioxidant and antidepressant agent. 18,19 The catechol-derived tetrahydroisoquinoline alkaloid Salsolinol 4 plays an important role in pathogenic treatment associated with Parkinson's diseases. 20−23 Despite thienoquinolines and thienoisoquinolines being poorly represented in nature, they are important compounds in medicinal chemistry. Related compounds exhibit antileukemic 24 and antibacterial activities; 25 also, they were proved as vasodilators. 26 In the light of the prior biological activities of tetrahydroisoquinolines and in resumption of our project for synthesis of new heterocycles containing the thienotetrahydroisoquinolines moiety, 27−34 we have announced here preparation of the new 1-amino-2-substituted and 1-pyrrolyl-2-substituted-5- piperidinyl -6,7,8,9-tetrahydrothieno [2,3-c]isoquinolines and their fused heterocycles which have proved promising antibacterial and antifungal activities parallel to the standard drugs.

■ RESULTS AND DISCUSSION
Our strategy is initiated with synthesis of 3-thioxo-1piperidinyl-5,6,7,8-tetrahydroisoquinoline-4-carbonitrile substrate (2) which is an essential intermediate for preparation Scheme 1. Synthesis of 1-Amino-2-substituted-5-piperidinyl- 6,7,8,9-tertahydrothieno [2,3-c]isoquinolines (4a−e) Scheme 2. Synthesis of the Pyrrolyl Derivatives 5a, b Starting with the Acetyl Derivative 5a To Synthesize Various Chalcones, Pyrazolyl, and Isoxazolyl Compounds 6−9 of the new tetrahydrothieno [2,3-c]isoquinolines by the reaction of isothiochromene 1 with piperidine via Dimroth 35 rearrangement in an excellent yield. The thione 2 was permitted to react with several α-halo carbonyl compounds (e.g. chloroacetone, ethyl chloroacetate, 2-bromoacetophenone, chloroacetamide, and chloroacetanilide) in refluxing ethanol and fused sodium acetate to afford the corresponding S-alkylated products 3a−e, which in turn underwent Thorpe− Ziegler cyclization 36 upon heating with sodium ethoxide solution in ethanol producing the target o-bifunctionally substituted thieno [2,3-c]isoquinolines 4a−e. The latter compounds were obtained via an alternative route, upon reaction of 2 with the mentioned α-halo carbonyl compounds using anhydrous potassium carbonate as a basic catalyst instead of sodium acetate. The products that were created by the two methods were identical in all features. Elucidation of the chemical structures of 4a−e was confirmed from their spectral analyses. Fourier transform infrared (FT-IR) of 4a revealed bands at 3449 and 3271 cm −1 specific for NH 2 and at 1589 cm −1 characteristic for CO. 1 H NMR in CDCl 3 exhibited two singlets at δ 2.39 and 7.20 ppm attributed to CH 3 and NH 2 groups. 13 C NMR of 4a displayed signal at δ 192.32 ppm for CO unique for the acetyl group (Scheme 1).
Furthermore, treatment of the amino-acetyl and amino-ester derivatives 4a and 4b with 2,5-dimethyl tetrahydrofuran afforded the corresponding pyrrolyl derivatives 5a and 5b. The IR spectrum of 5b emerged with the absence of bands at 3493 and 3337 distinctive to the NH 2 group with a band at 1657 specific for the CO ester. 1 H NMR of 5b affirmed the absence of the singlet signal attributed to NH 2 in 4b and the presence of multiplet signals at δ 6.20−6.33 and 7.15−7.46 particular for the pyrrolyl protons, in addition to triplet and quartet signals at δ 1.34−1.38 and 4.28−4.33 characteristic for CH 3 and CH 2 of the ethoxyl group, respectively. 13 C NMR in CDCl 3 exhibited signals at 14.55, 60.06, and 166.08 specific for CH 3 , CH 2 , and CO of the ethoxyl group. The pyrrolyl acetyl 5a was used as a basic intermediate for production of new heterocycles. When the pyrrolyl acetyl 5a was subjected to react with dimethylformamide dimethylacetal (DMFDMA) in DMF, the corresponding dimethyl amino-2-propenone 6 was obtained. Similarly, condensation of 5a with different aromatic aldehydes developed formation of the corresponding chalcone derivatives 7a−c. 1 H NMR of 7a emphasized disappearance of the signal at δ 1.95 ppm specific for CH 3 of the acetyl group and emergence of multiplet signals at δ 7.33−7.45 particular for aromatic protons. The 13 C NMR spectrum showed signals at 128.65−130.27 ppm characteristic for the carbon atoms of the phenyl ring. Also, the mass spectrum of 7a exhibited peaks at 467.34 and as a molecular ion and at 84.15 as a base peak. Therefore, reaction of chalcone 7a with phenyl hydrazine and hydroxyl amine hydrochloride afforded the pyrazolyl and isoxazolyl derivatives 8 and 9. The homogeneity of the chemical structures for the latter compounds was confirmed by elemental and spectral techniques. 1 H NMR of compound 8 represented doublet and triplet signals at δ 3.63−3.70 and 5.03−5.18 attributed to CH 2 and CHPh of pyrazole. Also, 13 C NMR exhibited signals at δ 51.05 and 64.81 for CH 2 and CH pyrazole (Scheme 2).
Moreover, hydrazinolysis of the ester 5b with hydrazine hydrate provided the corresponding carbohydrazide 10. 1 H NMR in CDCl 3 represented two singlets at δ 3.89 and 6.26 accredited to NH 2 and NH. 13 C NMR emerged signal at δ 162.70 specific for the CONH group. Cyclocondensation of the carbohydrazide 10 with acetyl acetone 37 in ethanol produced first the corresponding pentylidene Schiff's base 11 followed by cyclization upon reflux in acetic acid to produce the dimethyl pyrazolyl derivative 12 in a moderate yield. FT-IR of 12 showed absence of bands assigned to NH and COCH 3 groups. The 1 H NMR spectrum represented singlet signals at δ 1.27 and 1.65 ppm unique for the two pyrazolyl methyl groups. 13

■ CONCLUSIONS
In the current study, we have provided a facile approach for synthesis of unique piperidinyl tetrahydrothieno[2,3-c]isoquinolines based on various reactions of the pyrrolyl acetyl 5a and pyrrolylester derivatives 5b. The isolated products were easily purified by recrystallization from the proper solvents. Alternatively, most of the synthesized products revealed promising antibacterial and antifungal activities against various genera of bacteria and fungi. Therefore, this sophisticated strategy could be utilized for synthesis of compounds that are medicinally and pharmaceutically significant.

■ BIOLOGICAL ACTIVITY
Recently, developing new and more effective antimicrobial therapies have become one of the greatest concerns for human health, which has attracted considerable attention of the scientists worldwide as a result to the development of resistance to the present antimicrobial remedy. Therefore, one of the main targets of our study is synthesis of new compounds having superior importance in biological and medicinal chemistry. The selected compounds 5a, 6, 7a, 7b, and 7c were screened for their in vitro inhibitory effect versus a panel of pathogenic genera of Gram positive (Bacillus cereus and Staphylococcus aureus) and Gram negative bacteria ( Pseudomonas aeruginosa and Escherichia coli) in addition to four strains of pathogenic fungi Geotrichum candidum, Candida albicans, Trichophyton rubrum, and Aspergillus flavus. The inhibition zones (mm) and minimum inhibitory concentration (MIC) (μg/mL) of the tested compounds were compared with amoxicillin and clotrimazole as antibacterial and antifungal reference medicines.

■ ANTIBACTERIAL ACTIVITY
The antibacterial assessment results for the tested compounds indicated significant activity and were summarized in Table 1.

■ ANTIFUNGAL ACTIVITY
It is interesting to mention that the results of antifungal screening for the tested compounds revealed promising impacts against all kinds of fungi as indicated in Table 2. From the antifungal data, we found that compounds 5a, 6, 7b, and 7c exhibited intense activities against Geotrichum candidum (MIC 8.0−9.0 μg/mL) relative to the clotrimazole reference drug (MIC 5.0 μg/mL), while G. candidium was resistant to compound 7a. Subsequently, compounds 5a, 6, 7b, and 7c showed good activities against C. albicans, while compound 7a displayed moderate activity (MIC 10.0 μg/mL). In case of T. rubrum, compound 4a represented the best antifungal activity (MIC 7.0 μg/mL) which is almost the same as clotrimazole (MIC 6.0 μg/mL), while compounds 6, 7a, and 7b exhibited the highest inhibition zones (19−21 mm) which are very close to that of the reference drug (25 mm). However, compound 7c exhibited inferior activity (MIC 11 μg/mL) toward T. rubrum. Additionally, compounds 4a, 7a, and 7b revealed strong activities (MIC 9.0 μg/mL) againstA. flavus, while compound 7c displayed moderate activity. At the same time, A. flavus was resistant to the effect of compound 6 compared with clotrimazole (MIC 4.0 μg/mL).

■ STRUCTURE−ACTIVITY RELATIONSHIP
Tetrahydroisoquinolines hold a prominent place in organic and medicinal chemistry. Accordingly, we tried to investigate the influence of various chalcone compounds containing the piperidinyl thienotetrahydroisoquinoline moiety on the microbial inhibitory activity. From the data which are recorded in Table 3, we can deduce that the pyrrolyl-acetyl starting material 5a revealed strong antibacterial activity against all tested strains of bacteria. Formation of the chalcone compound 6 increases the antibacterial activity toward E. coli and slightly decreases the effect against B. cereus, S. aureus, and P. aeruginosa. Alternatively, reaction of the pyrrolyl-acetyl 5a with different aldehydes to form the corresponding chalcone compounds 7a, 7b, and 7c strongly affected on the antibacterial activity. Therefore, reaction with benzaldehyde to form the chalcone 7a strongly increases the effect against S. aureus and E. coli with moderate activity against B. cereus as well as no activity against P. aeruginosa. It is noticeable that substitution of the phenyl ring in benzaldehyde by the nitro group (electron withdrawing group EWG) 7c strongly enhances activity against P. aeruginosa, in addition to moderate or good activity against E. coli. However, substitution by the methoxyl group (electron-donating group) 7b increases the activity against S. aureus and E. coli with moderate to good activities against B. cereus and P. aeruginosa. Table 1. Antibacterial Activity (Inhibition Zone and MIC) of Compounds 5a, 6, 7a, 7b, and 7c Additionally, the recently synthesized compounds exhibited variable degrees of fungal inhibitory activity. Compound 5a revealed high activity against all genera of fungi, while reaction with DMFDMA to form the chalcone 6 slightly decreases the effect against C. albicans and T. rubrum. Antifungal activity of compound 6 against G. candidium is very close to the effect of 5a, showing inferior activity against A. flavus. Furthermore, reaction of 5a with benzaldehyde to form chalcone 7a slightly decreases the activity against C. albicans and T. rubrum. The effect of chalcone 7a is very similar to 5a against A. flavus with inferior activity versus G. candidium. Substitution of the phenyl ring of benzaldehyde with electron withdrawing group (NO 2 ) 7c or electron donating group (OCH 3 ) 7b strongly enhanced the activity against G. candidium by increasing the activity against C. albicans and T. rubrum. The methoxyl group in compound 7b slightly reduced the efficacy against A. flavus, while substitution by the nitro group did not influence the activity as compared to 7a.

■ DEVELOPMENT OF A 2D-QSAR MODEL
The descriptor reflects the molecular merits. The screening of various significant molecular descriptors from many other ones is an imperative procedure in the QSAR research. The best QSAR model should have appropriate number of descriptors.
N and D represent the number of compounds and descriptors, respectively. In our study, the number of compounds tested for antibacterial and antifungal activity is limited (only five compounds). Therefore, to meet the criteria the QSAR equation will contain only one descriptor.
A simple 2D-QSAR analyses for antibacterial activity and antifungal activities of the prepared derivatives (5a, 6, 7a, 7b, and 7c) was performed using MOE 2019 software.
A set of the synthesized compounds 5a, 6, 7a, 7b, and 7c was used as a training set with their measured MIC against B. cereus and C. albicans for QSAR modeling. MIC values against B. cereus and C. albicans were chosen for QSAR modeling; they were the training set compounds that showed best activity. A database for the training set compounds was established and different molecular 2D descriptors was calculated using MOE 2019 software. Next, the "best multilinear regression" was determined. In our study, PLS regression analysis was used to calculate the relationship between bioactivity and the descriptors. The QSAR model was validated employing leave one-out cross-validation.

■ 2D QSAR STUDY RESULTS
In the 2D-QSAR equation development for B. cereus, the regression equation indicated that the total hydrophobic vdw surface area (PEOE_VSA_HYD) of the training set compounds has a contribution to the inhibitory activity (eq 1). The correlation coefficient (r 2 ) is 0.81, and the root-mean-square error (RMSE) is 0.61. The predicted values are plotted versus experimental MIC results of the training set compounds against B. cereus, as given in Figure 2.
Method b. A solution of 2 (2.73 g, 0.01 mol) and the alkylating agent (0.01 mol) in ethanol (15 mL) and anhydrous potassium carbonate (3.00 g, 0.02 mol) in ethanol (15 mL) was refluxed for 3 h; then, the mixture was allowed to cool. The solid products were collected, washed with water, dried, and recrystallized from the ethanol/dioxane mixture (1:1). The physical constants, elemental analyses, and spectral data of compounds 4a−e are listed in Table 4.
3-(Dimethylamino)-1-(5-(piperidinyl)-1-(1H-pyrrolyl)-6,7,8,9-tetrahydrothieno[2,3-c]isoquinolin-2-yl)prop-2enone (6). The pyrrolyl acetyl 5a (2.00 g, 5.00 mmol) and DMFDMA (10 mL) was heated under reflux for 6 h. The solid precipitated, which separated out during reflux was filtered, dried, and recrystallized from ethanol as yellow crystals in 57% (1.30 g) yield, mp 204−206°C. Anal. Calcd for C 25  Procedure of In Vitro Antibacterial Assay. All microorganisms utilized were attained from the culture combination of Microbiology Department, Faculty of Medicine, Assiut University. A variety of Gram-negative (E. coli and Pseudomonas aeruginosa) and Gram-positive bacterial strains (S. aureus and B. cereus) was used to measure the efficacy of various synthesized compounds utilizing 5 mL solution of the synthesized compounds in dimethyl sulfoxide (DMSO) as a solvent. The examined compounds were primarily estimated by maximum concentration at 100 μg/mL in DMSO and amoxicillin as a reference. The sterile medium (Nutrient Agar Medium, 15 mL) in every Petri dish was uniformly smeared with cultures of Gram-positive and Gram-negative bacteria. The plates were incubated at 37 ± 2°C for 24 h.
Procedure of In Vitro Antifungal Assay. The fungal strains (C. albicans, A. flavus, G. candidium, and T. rubrum) were gained from selected conditions of human dermatophytosis (Assiut University Mycological Center, AUMC). The fungal kinds were developed in sterilized 9 cm Petri dishes containing Sabouraud's dextrose agar (SDA) supplemented with 0.05% of amoxicillin to inhibit contamination of bacteria. 40 The agar disks (10 mm diameter) containing spores from these cultures were aseptically transferred to screw-topped vials containing 20 mL sterile distilled water. After shaking, samples of the spore suspension (1 mL) were pipetted into sterile Petri dishes, followed by the addition of 15 mL liquefied SDA medium which was then left to solidify. The screened compounds 5a, 6, 7a, 7b, and 7c and the reference drug (clotrimazole) were dissolved in DMSO to provide 2.0% concentration. The inoculated plates were incubated at room temperature for 4 days.
Antibacterial and antifungal activities of the tested compounds were determined consistent with the strategy described by Kwon-Chung and Bennett 39 using 5 mm diameter wells loaded with 50 μL of the solution under study. Furthermore, stock solutions of the standard drugs (amoxicillin and clotrimazole) were prepared in DMSO and 100 μg/mL concentration utilized for antimicrobial and antifungal efficiency. The zones of inhibition were determined and listed in Tables 1 and 2, respectively.
Procedure of In Vitro Inhibition Zone and MIC. The examined compounds (5a, 6, 7a, 7b, and 7c) to be screened were dissolved in DMSO to afford a solution of 2% concentration. Filter paper discs (Whatman no. 3) with about 5 mm in diameter were saturated with 15 mL of the tested compound solutions and then placed on the surface of the previously prepared agar plates which seeded by the tested bacteria. To ensure complete contact with the agar surface, each disc was immersed down. Subsequently, the agar plates were incubated at 37°C for 16−18 h for bacteria and then at room temperature. The zones' diameters of the compound inhibition were measured and recorded in the previous table. A similar procedure 39,40 was implemented for commercial antibiotics amoxicillin which was utilized as the positive control for bacteria. The MIC of every compound was determined by the microdilution method. The biologically active compounds were successively diluted in DMSO and incubated with 10 mL broth tubes vaccinated with the examined culture for 24 h. MIC of each compound was measured as the lowest concentration (μg mL −1 ) that did not display any visible bacteria.