Novel 4H-pyrimido[2,1-b]benzothiazoles derivatives: Camphorsulphonic acid catalyzed enantioselective synthesis, optimization, and biological study

Abstract An efficient, highly convergent route for the synthesis of novel fourteen single isomer derivatives of ethyl-2-methyl-4-(pyridin-2-yl)-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carboxylate have been developed by one-pot, three-component reaction between pyridine 2-aldehyde, β-ketoester and various derivatives of 2-amino benzothiazole in the presence of D-(+)-10-camphorsulphonic acid (D-(+)-10-CSA) as an effective chiral acid catalyst. Under the optimized conditions with 20 mol% catalyst loading, a wide range of optically active 4H-pyrimido[2,1-b]benzothiazole derivatives were obtained in good yields (up to 82%) with good to excellent enantioselectivities (84-99%ee). Their structures were elucidated by various spectroscopic methods such as 1H-NMR, 13C-NMR, Chiral HPLC, mass and elemental analysis. The attractive feature of this approach is one-pot synthesis, good yield, environment benign, mild reaction condition, simple, efficient, excellent stereoselectivity, water soluble catalyst and easy workup. All the final scaffolds have been screened for antibacterial activity. Among them, 4a’, 4c’, 4d’, 4e’, 4f’, 4g’, 4j’,4m’ and 4n’ were proven to possess enhanced antibacterial properties as compared to the standard drug. Graphic Abstract


Introduction
The chiral scaffold plays a vital role not only in our daily life but also in nature, as the different stereoisomers of a chiral molecule may show entirely diverse biological activities. [1]herefore, it is an aim for synthetic chemists to design selective and efficient methodologies that allow them to synthesize the desired enantiomer. [2]The design, synthesis, and construction of molecules having value as human therapeutic agents are necessary objectives of medicinal chemistry.The number of chiral, non-racemic pharmaceuticals on the market was tremendously increasing for the past 10 years.Many innovative single enantiomer drugs with an opportune chiral switch were formed to offer enhanced therapy, more anticipatable pharmacokinetics, and reduced toxicity. [3]any drugs contain a heterocyclic scaffold in their structures. [4]Among them, benzothiazole derivatives are the most widely existing ones and have shown various biological activities [8] and many applications. [5]In recent years, polyheterocyclic compounds with fused hetero-systems have gained attention because of their diverse range of potential therapeutic activities. [6,7]he multi-component reaction (MCRs) is one of the liveliest fields for the synthesis of complicated heterocyclic scaffolds from readily available starting materials. [8]Biginelli reaction is one of the well-known MCRs for the synthesis of Dihydropyrimidine (DHPM) by the reaction of various aldehydes derivatives, 1,3dicarbonyl compounds, and urea or thiourea by Italian chemist Pietro Biginelli since 1893. [9]DHPM is a nitrogencontained heterocycle that possesses various biological properties [10] such as anticancer, anti-inflammatory, calcium channel antagonists, antibacterial, anticonvulsant, antitubercular agents, antimalarial agents, antihypertensive, antioxidant, anti-HIV, antitumor, antimicrobial, and antiepileptic activities. [10]Only minor structural variations in all the three building blocks have been reported [11] apart from a very recently reported major structural variation where the urea component was replaced by a guanidine system. [12]Many researchers used 2-amino benzothioazoles [5b] and 2-aminobenzimidazoles [13] derivatives in the place of urea. [14]More efficient conditions have been found for the Biginelli reaction using various catalysts, [15] microwave irradiation, [16] solid-phase, and fluoro-phase techniques [17] facilitating this synthesis have also become increasingly widespread.Chiral DHPMs have displayed extensive importance in pharmaceutical activities and developed the key structural units in many drugs, [18] such as (S)-Monastrol, [19] (S)-L-771688, [20] SNAP-794130 [21] and SQ 3292630 [22] (Figure 1), in addition to some natural alkaloids.Therefore, the various methods to access optically active DHPMs by the use of asymmetric catalytic Biginelli reactions have received much more attention nowadays. [4,17,23]Many groups of researchers have used chiral reagents such as chiral Lewis acid catalyst containing ytterbium, [17a] BINOL-phosphoric acids, [17b] chiral secondary amines, [4,23b-e] primary amines, [23f] bifunctional primary aminethioureas, [23g] and NbCl 5 /primary amines [ 23h] and SPINOL-phosphoric acid [24a] for the enantioselective Biginelli reactions for the synthesis of DHPMs.The synthesis of 4H-pyrimido [2,1-b]benzothiazoles is one of the examples of the Biginelli reaction.Many researchers have synthesized 4H-pyrimido [2,1-b]benzothiazoles by using the various catalysts such as FeF 3 , Kaolin, N,N-dichlorobis (2,4,6trichlorophenyl)urea, hydrotalcite, TBAHS, AlCl 3 and Zn(ClO 4 ) 2 6H 2 O have shown to be effective for the synthesis of 4H-pyrimido [2,1-b]benzothiazole. [5b] Camphorsulfonic acid [24a,b] (CSA), also known as Rachel's acid, is an aprotic acid.24b] It also used as water soluble catalysts for several reactions such as synthesis of a-hydroxy, a-amino phosphonates, b-amino carbonyl compounds, various 1,3,4-oxadiazoles derivatives, pseudoglycosides.coumarins, spirocyclic compounds, rearrangement of 1,2dialkynylallyl alcohols and used in the optical resolution.24b-d] The development of efficient and environmentally friendly chemical processes is of considerable interest in organic chemistry. [24]24b,c] Consequently, an efficient protocol for the synthesis of single isomers of 4H-pyrimido [2,1-b]benzothiazoles under mild conditions using efficient catalysts is still needed.
Given the importance of all the above factors, our main objective is to prepare a single isomer of 4H-pyrimido [2,1b]benzothiazoles using a one-pot, environmentally benign, and mild reaction condition by a conventional method.Therefore, herein we reported the chiral synthesis of ethyl-2-methyl-4-(pyridin-2-yl)-4H-benzo [4,5]thiazolo [3,2-a]pyrimidine-3-carboxylate derivative by the reaction of b-ketoester with various derivative of 2-amino benzothiazole and heterocyclic aldehydes using 20 mol% of D-(þ)-10-CSA as a chiral catalyst in moderate to excellent chemical yields.To the best of our knowledge, such a work has not been reported in the literature so far.

Chemistry
As per our previous study, we have initially reported an efficient, one-pot three-component synthesis of ethyl 2-methyl- 4-(pyridin-2-yl)-4H-benzo [4,5]thiazolo [3,2-a]pyrimidine-3carboxylate derivatives [8b] by the reaction of various derivatives of 2-amino benzothiazole (3a-3n) with pyridine 2-aldehyde 1 and ethyl acetoacetate 2 by using PdCl 2 as a catalyst under microwave-assisted solvent-free conditions, with better yield and short reaction time.It was introduced to develop new structural motifs with promising biological activity.The compounds 4a-4n have been analyzed through chiral HPLC analysis to know the optical purity of the compounds.Compound 4a seems like a racemic compound (50.37% major isomer and 49.62% minor isomer) which was confirmed by chiral HPLC using a Daicel Chiralpak AD-H, 250 Ã 4.60 mm, k ¼ 254 nm.The measured optical rotations (a) and specific rotation ([a] k [25] value of compound 4a was À0.004 and À2.898 (c ¼ 0.0069 g/10 mL THF) respectively (see Supplementary Content -chiral HPLC of racemic compound (4a-4n)).We have reported racemic mixture of 4a-4n in our previous study.
In the present study, it has been understood and practically noticed that the catalyst plays a significant role in organic synthesis, especially in the chiral synthesis of various molecules, not only by increasing the rate of the reaction but also any other aspects relating to the concern reaction.Initially, we screened the effect of various chiral catalysts (such as D-(þ)-10-camphorsulphonic acid, quinidine, cinchonidine, quinine, (þ)-cinchonine, and (þ)-mandelic acid) (Figure 2) on Knoevenagel and Michael type addition reaction (K&M reaction) on this model reaction between pyridine 2-aldehyde 1, ethyl acetoacetate 2 and 2-aminobenzothiazole 3a in CH 2 Cl 2 as a solvent at room temperature, as shown in Table 1 (entries 1-6).All aforementioned catalysts could promote the reaction through the diverse activation modes, for example, hydrogen bonding, [25] conjugated nucleophilic addition. [26]Unusually, the used catalysts affected the chemical yield significantly at a specific temperature.
Based on encouraging results as shown (entries 1-9) in Table 1, we have subsequently studied the effect of six catalysts in the reaction for the synthesis of 4a'.By this assessment, the product yield of compound 4a' highly depended on the structural nature of the catalysts examined.When (þ)-10-CSA and (þ)-cinchonine were used as catalysts for this reaction, correspondingly a maximum of 45% (70% ee) and 40% (61% ee) yields were obtained in 40 h (entries 1 and 5).By using cinchonidine and quinine as catalyst, the comparative yield of 4a' were 36% (<40% ee), and 38% (49% ee) respectively after 40h (entries 3 & 4).The reaction was carried out using quinidine, and (þ)-mandelic acid catalyst, the yield of 4a' were 33% (42% ee)  and 32% (<40% ee) individually (Table 1, entries 2 and 6) (See Figure S53 Supplemental Materials).We observed that the reaction did not proceed efficiently without a catalyst with low yield (19%).Furthermore, it was noted that increasing the reaction temperature could increase the chemical yield of 4a' (51% and 57%) in the different degrees (Table 1, entries 7 and 8).
We further optimized the reaction conditions to increase the reaction yields by changing the various solvent.In the presence of 10 mol% of (þ)-10-CSA at 60 C for 24 h, we explored the effect of various seven organic solvents (such as toluene, THF, MeOH, CHCl 3 , 1,4-dioxane, CH 3 CN, and DMF) on the reaction between pyridine 2-aldehyde 1, ethyl acetoacetate 2 and 2amino-benzothiazole 3a as presented in entries 10-16 of Table 1 (See Figure S54 in supplementary Content).1,4-dioxane used as a solvent at 60 C temperature to get 64% yield of compound 4a' (entry 14).The product yield of 4a' were 63% and 62% respectively by using DMF and toluene as solvent at 60 C. (Table 1, entries 16 and 10).But when the reaction was carried out in MeOH at 60 C temperature, the yield of product 4a' could be improved to 71% (Table 1, entry 12).When we used THF as solvent we obtained 60% yield of 4a' (entry 11).We observed that the reaction did not progress efficiently in other remaining solvents (Table 1, entries 13, and 15).In all the solvents checked, the reaction went efficiently in MeOH furnishing 4a' in excellent product yields (86%) (Table 1, entry 12).
Encouraged by the above result from Table 1, we have further improved the reaction conditions to increase the reaction temperature in range 15-90 C at 10 mol% fixed catalyst concentration of (þ)-10-CSA (Table 2, entries 1-9).Firstly we carried out the reaction with an temperature from 15 C to 55 C with increasing % yield (<10 to 65%) of the product 4a' (Table 2, entries 1-5) (See Figure S55).However, the chemical yield of the product 4a' obtained was 82% at 60 C (entry 10).Moreover, an increase in the reaction temperature dramatically decreased the chemical yield of 4a' due to charring of reaction (Table 2, entries 6 vs 7-8).
We have further optimized the reaction conditions by the screening of % concentration of (þ)-10-CSA catalyst with fixed reaction time and temperature.Our main aim is to maximize product yield (along with higher optical purity) with minimum reaction time as well as a minimum amount of catalyst loading in the reaction mixture.The selected catalyst ((þ)-10-CSA), was then further optimized.The catalytic loading of (þ)-10-CSA was screened in the range from 10 to 60 mol% (See Figure S56).When we loaded (þ)-10-CSA (20 mol%) in in the reaction mixture to get highest yield (82%) of compound 4a' (Table 2, entry 10) in 20 h.When we loaded the catalyst in a concentration of 25-30 mol% of (þ)-10-CSA catalyst a yield in the range of 70-73% was achieved.The % yield of product decreased with increasing concentration of the catalyst in a steady manner.Therefore, at the current stage, we determined the optimal reaction conditions for the synthesis of compound 4a': 1/2/3a, 20 mol% (þ)-10-CSA, methanol, and 60 C temperature stirred for 24 h to obtain an optimum yield.
With the optimized reaction conditions in hand, we further investigated the substrate scope for the synthesis of compounds 4a'-4n'.As shown in Table 3, a variety of 2-amino-benzothiazole derivatives were investigated as potential substrates (Table 3 entries 1 -14), and the results demonstrated that different substituents, including electron-donating, electron-withdrawing, and halogen groups on substituted benzothiazoles, affording the desired cyclized products in good yields (71-82%) with diverse reaction time (Scheme 1).The desired product structure of 4a'-4n', is illustrated in Table 3.
The structures of all the synthesized compounds (4a'-4n') were confirmed by their 1 H-NMR, 13 C-NMR, and mass spectra analysis, and the obtained spectra for each compound are found to be in agreement proposed molecular structures.As an example in the 1 H NMR spectrum of 4a', pyridine ring proton (Py-H) displayed peaks at d 8.59 (1H, dd, J ¼ 7.5, 1. 3 Hz)  4 which indicates the existence of 4a'.Molecular ion peak i.e., M þ 1 or Mþ 2 peak confirms the formation of the product as it was found appropriate to the molecular weight of the compound.The structure of 4a' was also confirmed through 13 C NMR analysis.The pyridine ring carbon atoms attached to the chiral center resonated at d 146.8, 121.6, 139.4,and 127.9 ppm.The carbon attached with the chiral carbon atom showed a peak at d 55.9 ppm.In compound 4a', the characteristic peaks for protons and carbons in 1 H-NMR and 13 C-NMR appeared at specific chemical shift values were in good agreement with the molecular formula of the synthesized compounds.The structure of compound 4a' was also confirmed by single-crystal X-ray diffraction studies.Fine crystals of compound 4a' were obtained by slow evaporation method using 80% ethyl acetate: hexane mixture.It revealed the formation of pure 4a', which crystallizes as a monoclinic crystal system with a space group P21/n (CCDC No.: 1048059).
Furthermore, the structure of compound 4a' was confirmed via single-crystal X-ray crystallography (Figure S57).The enantiomeric purity of compound 4a' was determined by chiral HPLC studies.As per the chiral HPLC result of 4a', the major isomer was eluted at retention time (t r ) 96.83 minutes, while the minor isomer eluted at retention time 3.1722 minutes.We can calculate the percentage enantiomeric excess (% ee) by using below mentioned equation.The percentage enantiomeric excess of compound 4a' was 94% which was presented in Table 3.
The absolute configuration of compound 4a' (major enantiomer) formed for this reaction was determined by Xray crystallographic analysis of the product 4a' (CCDC No.: 1048059) (See Figure 7 in supplementary Content).As per the CIF file of single-crystal X-ray crystallography analysis, the absolute configuration of the chiral carbon atom of 4a' formed during the reaction is determined to be R. Based on the relative stereochemistry of 4a', the configurations of other title compounds (4b'-4n') were assigned as (R) by analogy.The other parameter of crystal is Crystal Data for C 20 H 18 N 2 O 3 S 0.25 (M ¼ 351.43 g/mol): monoclinic, space group P21/n (no.14), a ¼ 10.3341 (19)  (6.54  2H 58.3 ), 3813 unique (R int ¼ 0.1089, R sigma ¼ 0.1060) which were used in all calculations.The final R 1 was 0.1018 (I> ¼2u(I)) and wR 2 was 0.2965 (See Supplemental Materials Tables S2-S7 for other X-Ray data).
Taking into consideration the entire outcome, a plausible mechanistic pathway for the synthesis of 4a' is represented in Scheme 2. In transition state (TS), one face of the ethyl 2-( (2-iminobenzo[d]thiazol-3(2H)-yl)(pyridin-2-yl)methyl)-3-oxobutanoate was efficiently shielded by the steric hindrance of the bulky D-(þ)-10-camphorsulphonic acid moiety ((þ)-10-CSA), whereas the other face was available to attack the imine.During mechanism, promising hydrogen bonding connections between the oxygen of acetyl group and (þ)-10-CSA, and the -NH atom of the benzo[d]thiazol-2(3H)-imine, the two oxygen atom of the (þ)-10-CSA, and the acetyl moiety positioned the (þ)-10-CSA activated imine moiety, which allowed the enamine double bond to approach the one face of the imine via a stable six-membered-ring transition state.This resulted pyridine ring set in front side during cyclization process including TS.5b]

Antibacterial activity
We have performed antibacterial activity of 4a'-4n' using Nutrient-agar plates by well-diffusion assay against bacterial cell lines.All the test cultures were stimulated in Nutrient broth.The zone of inhibition was determined in terms of zone diameter with respected to that zone index was calculated.During calculation, streptomycin (1000 mg/mL concentration) was used as standard drug.

Determination of activity index
The activity index of the probiotic culture was calculated as:

Mean of zone of inhibition of derivative Zone of inhibition obtained for standard antibiotic drug
The result of antimicrobial activity of all synthesized derivatives 4a'-4n' is shown in Table S1 (Supplemental Materials).Gram negative bacterial strains were more inhibited as compared to Gram Positive bacteria.Also, 4a', 4c', 4d', 4e', 4f', 4g', 4j', 4m'and 4n' were proved to have more  antibacterial property as compared to the streptomycin while contrastingly 4b' had a lower activity.

Chemistry
The nomenclature of the compounds was performed using CambridgeSoft.ChemOffice.2010.v12.All Starting materials and other reagents were purchased from commercial suppliers and were used without any further purification unless otherwise indicated.The reactions were examined by thin-layer chromatography (TLC) and terminated as arbitrated by the consumption of starting material.TLC was performed on silica gel G 60 F254 (Merck) plate.It was visualized by UV radiation or exposure to iodine vapors.Gravitational column chromatography was conducted over silica gel 60 (60-120 mm).The melting points were recorded on an Optimelt automated melting point system that was uncorrected.IR spectra were recorded at room temperature in the region from 500-3950 (cm À1 ) on a Perkin-Elmer 377 spectrophotometer in KBr with absorption in cm À1 .Elemental analysis was performed as per standard protocol on vario MICRO cube, Elementar CHNS analyzer serial no.: 15084053.1 H-NMR and 13 C-NMR spectra were recorded on Bruker AV 400 and 100 MHz using DMSO-d 6 as solvent and TMS as an internal standard.Chemical shifts are reported in parts per million (d in ppm) relative to the designated referenced peaks: DMSO-d 6 at 2.5, and 3.33 (residual water) ppm.Coupling constants (J) were reported in (Hertz) Hz.Percentage enantiomeric excess (% ee) was determined by chiral HPLC using Daicel   13 C NMR and chromatographic spectra for 4a' -4n' (Figures S1-S52).

X-ray crystallography
The crystallographic data, details of data collection and some important features of the refinements for compounds 4a' is given in Table S2 and selected bond lengths and angles and hydrogen atom coordinates and isotropic displacement parameters are given in Table S3-S7.Yellow crystals of a suitable size, obtained by slow evaporation method using 80% ethyl acetate: hexane mixture, were selected and mounted.Data were collected with MoKa radiation (k ¼ 0.71073 Å) at 293 K for compound 4a' on an Xcalibur, Eos, Gemini diffractometer equipped with a CCD area detector.Using Olex2, [27] the structure was solved with the SIR2004 [28] structure solution program using Direct Methods and refined with the XH [29] refinement package using CGLS minimization.All nonhydrogen atoms were refined anisotropically.The positions of the hydrogen atoms were calculated from the difference Fourier map.

Conclusion
In summary, we have reported a highly efficient, one-pot, novel, regio and stereoselective asymmetric synthesis for the synthesis of single isomer of ethyl 2-methyl-4-(pyridin-2-yl)-4H-benzo [4,5]thiazolo [3,2-a]pyrimidine-3-carboxylate derivatives catalyzed by 20 mol% of D-(þ)-10-Camphorsulphonic acid ((þ)-10-CSA) with moderate to good chemical yields with excellent enantioselectivities. Significant features of the reaction include the mild reaction conditions, simple, efficient, environmentally benign, easy workup, and excellent regio-, stereo-and enantioselectivities.The synthesized compounds were evaluated in vitro antibacterial activity and revealed that they were found to be active against reference strains compared to standard drugs.

Figure 2 .
Figure 2. Chiral cinchona alkaloid and other catalysts screened in this work.

Scheme 2 .
Scheme 2. Proposed Mechanism for the Formation of 4a' with transition state.

Table 1 .
Screening of catalyst and solvent for the synthesis a of 4a'.

Table 3 .
Extension of the Reaction Scope for the synthesis of 4a'-4n' a .