Synthesis of mono, bis [1, 3] Oxazines and Schiff base of naphthalene-2-thiol possessing anti-cancer activity

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1 Chapter 3 Synthesis of mono, bis [1, 3] Oxazines and Schiff base of naphthalene-2-thiol possessing anti-cancer activity 3.1 INTRODUCTION In this chapter, we report the extension of classical Betti reaction for the synthesis of mono, bis [1, 3] oxazines and Schiff base of naphthalene-2-thiol. In recent years, ammonium acetate has been utilized as a suitable source of ammonia during the formation of several important heterocyclic skeletons. Hence, we have used ammonium acetate for the synthesis of oxazines starting from electron-rich aromatic compound and aryl aldehydes. Interestingly, the use of ammonium acetate in this reaction played a major role because it led to synthesis of unsymmetrical mono and bis [1, 3] oxazines in a single step i.e. mono and bis [1, 3] oxazines bearing two identical or two different aryl substituent on either side of the oxazine ring were obtained. Extensive studies were carried out to evaluate the behavioral change of ammonium acetate in this reaction and it was found that it acted as dual role (reactant as well as catalyst). The mechanism for the formation of [1, 3] oxazines were got from theoretical and experimental studies. One more fascinating chemistry of [1, 3] oxazines was observed that the synthesized compounds exhibited ring-chain tautomerism which depended on the electronic character of the substitution on the benzaldehyde as well as the solvent used. The existence of ring-chain tautomerism for the synthesized compounds has been extensively studied with various solvents using UV-Visible spectra, HPLC and NMR. Structurally similar oxazines were found to posses enormous medicinal properties. So, initially we screened the synthesized compounds in various cancer cell lines to study their cytotoxicity, and finally IC 50 values were obtained. Docking studies were also carried out to find the mechanism involved between the cancer receptor and the synthesized ligand moieties. Figure 3.1 Synthesised mono, bis [1, 3] oxazines and Schiff base of naphthalene- 2-thiol 61

2 3.2 LITERATURE DISCUSSION Betti synthesis emerged in 19 th Century when the reaction was carried out between ammonia or amines, formaldehyde and enolisable carbonyl compounds. The reaction between formaldehyde and ammonia or amines was found to yield an imine which then reacted with the carbonyl compound to yield a Mannich product. In 1912, Betti developed a synthetic method for the synthesis of hydroxyalkylimines starting from benzaldehyde, aniline and 2-napthol (Betti et al., 1912). These methods are commonly classified as Mannich aminoalkylations. In 1900, initially, Betti assumed that 2- naphthol could be used as a carbon nucleophile towards the imine for the formation of Mannich product, and later he proved that 2-naphthol could be a used as a good carbon nucleophile towards the formation of imine as represented in Scheme 3.1. Scheme 3.1 Synthesis of Betti base from 2-naphthol and an imine Betti proposed a possible mechanism for the formation of Betti base. Initially ammonia reacts with benzaldehyde to yield corresponding imine, that subsequently reacts with 2-naphthol. The proposed mechanism for the synthesis of Betti base is represented in Scheme3.2. Scheme 3.2 Proposed mechanism for the formation of Betti base In recent years, many reactions have been explored to synthesise [1, 3] oxazine via Betti base. For the synthesis of [1, 3] oxazine three mechanisms are possible. One possible mechanism is the formation of Betti base in the first step, and the second step is the reaction between this Betti base and the benzaldehyde to yield the desired product. Synthesis of [1, 3] oxazine from Betti base is presented in Scheme 3.3. Scheme 3.3 Synthesis of oxazine from Betti base 62

3 In 1930, Littman and Brode (Littman and Brode et al., 1930) proposed the second possible mechanism involving the formation of benzylidinediamine intermediate from benzaldehyde and secondary amines in the first step. In the later step, this benzylidinediamine intermediate attacks 2-naphthol to yield aminobenzylnaphthol, after the elimination of an amine molecule. Finally, this intermediate can react with benzaldehyde to yield [1, 3] oxazine as shown in Scheme 3.4. Scheme 3.4 Betti reaction with secondary amines The third and most favorable mechanism would be the formation of aldimine ion in the first step and ortho-quinone methides (O-QMs) intermediate in the second step. These two intermediates react to form the desired product. In 1964, Burke and Nasutavicus (Burke et al., 1964) reported the synthesis of [1, 3] oxazine using this method and proposed the mechanism as shown in Scheme 3.5. Scheme 3.5 A different approach for the synthesis of oxazine Betti later reported a modified Mannich reaction for the synthesis of [1, 3] oxazines from a three component involving 2-naphthol, an ethanolic solution of ammonia and benzaldehyde (2 equiv). Smith and Cooper, in the later stage, reported that the product [1, 3] oxazine exists in equilibrium form (Smith and Cooper et al., 1970) (i.e.) it exists in ring-chain tautomerism. A general schematic representation for the preparation of [1, 3] oxazine is presented in Scheme 3.6. Scheme 3.6 General schematic representation for the synthesis of [1, 3] oxazine 63

4 In the earlier report, [1, 3] oxazine were illustrated as naphthoxazines, but in later studies - Smith & Cooper in 1970, and Mohrle et al in showed that [1, 3] oxazine existed in three tautomeric mixture, possessing two epimeric naphthoxazines and one Schiff base in CDCl 3 solution (Mohrle et al., 1974; Smith & Cooper 1970). Thus, they reported that [1, 3] oxazine exists only in tautomeric mixture when the compound is present in protic solvent as shown in Fig 3.2. Smith and Cooper were the first researchers to identify the ring-chain tautomeric interconversion between N- unsubstituted 1, 3-O, N-heterocycles and the corresponding hydroxyalkylimines in [1, 3] oxazines when the compound was dissolved in protic solvent. Figure 3.2 Ring-chain tautomerism of [1, 3] oxazine in protic solvent In earlier studies, the tautomeric equilibrium was explained with the aid of IR and UV spectroscopy. But these methods cannot give the exact tautomeric ratio of [1, 3] oxazine. The ring-chain tautomerism in [1, 3] oxazine strongly depends on the solvent used and the temperature, and this equilibrium occurs very rapidly in a few seconds. Hence, Pihlaja (Pihlaja et al., 1987) developed a method to find out the exact tautomeric ratio using NMR data obtained for [1, 3] oxazine in CDCl 3 solvent. In 1987, Pihlaja et al, carried out a comparative study of various [1, 3] oxazine on the ring-chain tautomerism, where they described a simple equation for the formation of equilibrium in all cases as follows: log K = ρ σ + + log K x = H where K x = [ring] / [chain] (X H) and σ+ is the Hammett-Brown parameter of substituent X on the 2-phenyl group. In 1999, Naso et al., synthesized several Betti bases from 2-napthol, aldehydes and n- butylamine which was resolved as enantiomerically pure Betti base using tartaric acid (Naso et al., 1999). Enantiomerically pure Betti base was used as a chiral catalyst for enantioselective addition of diethylzinc to aryl aldehyde as represented in Scheme 3.7 and

5 Scheme 3.7 Synthesis of Betti base and separated enantiomers Scheme 3.8 Enantioselective addition of diethylzinc to aryl aldehydes using enantiomerically pure Betti base as a chiral catalyst In 2006, Alfonsov et al., reported the separation of [1, 3] oxazine tautomeric mixture into enantiomers by reacting oxazine with the enantiomerically pure tartaric acid (Alfonsov et al., 2006). Enantiomerically pure oxazine can be used as a chiral reagent or catalyst in stereoselective organic synthesis. Scheme 3.9 Resolution of Betti base using enantiomerically pure tartaric acid Many research groups have established several ways to synthesize unsymmetrical oxazine in several steps. Initially, symmetrical oxazines were synthesized from 2- napthol, benzaldehyde in the presence of ammonia at room temperature and the product was hydrolyzed under acidic conditions to yield aminobenzylnaphthols. Finally, these Betti bases were be allowed to react with heteroaldehyde, giving unsymmetrical oxazine. Szatmari et al., in 2006, and Turgut et al., in 2007, (Szatmari 65

6 et al., 2006; Turgut et al., 2007) reported the synthesis of unsymmetrical oxazine in three steps via Betti base (Scheme 3.10). Scheme 3.10 Synthesis of unsymmetrical [1, 3] oxazine in several steps Sapkal et al., in 2009 developed a green approach for the synthesis of [1, 3] oxazine, using dual role of ammonium acetate under solvent free condition (Sapkal et al., 2009). In this reaction they used ammonium acetate as a source of nitrogen instead of ammonia which performed a dual role - as a reactant as well as a catalyst. They established the synthesis of unsymmetrical oxazine in several steps as presented in Scheme Scheme 3.11 Synthesis of unsymmetrical [1, 3] oxazine using dual role of ammonium acetate In 2012, Ghandi et al. reported the synthesis of Betti base from three component reactions of 2-napthol, aryl aldehydes and heteroaryl amine in water (Ghandi et al., 2012). They studied the enantiomeric resolution of 1-(p-methyl phenyl (2- pyrazinylamino) methyl) naphthalene-2-ol using chiral europium shift reagent in 1 H- NMR spectroscopy. They developed a convenient method using water as solvent medium; a green approach was carried out to synthesize Betti base as shown in Scheme Scheme 3.12 A green approach for the synthesis of Betti base In the current work, we report a simple, straight forward and efficient approach for the synthesis of mono and bis [1, 3] oxazine derivatives which exist in ring-chain tautomerism via O-QMs successfully under mild reaction condition in a short time. 66

7 3.3 RESULTS AND DISCUSSION For the first time, we report the synthesis of mono, bis [1, 3] oxazine and Schiff s bases of naphthalene-2-thiol derivatives (9, 10, 11 and 12) possessing symmetrical / unsymmetrical units via a domino reaction of 2-napthol / naphthalene-2, 3-diol / naphthalene-2, 7-diol / naphthalene-2-thiol, aromatic aldehyde and ammonium acetate. When the reaction was carried out in the presence of ammonium acetate instead of liquid ammonia, the efficiency of the reaction between benzaldehydes and naphthalene-2, 3-diol increased and the free energy of intermediate-3 (int3) (Fig.3.5) reduced by kcal/mol because of the liberation of ammonia from ammonium acetate. It is more effective than the direct use of ammonia from the liquid ammonia. Since no report for the synthesis of unsymmetrical unit of [1, 3] oxazine in a single step is available, we explored the synthetic method for the synthesis of [1, 3] oxazine derivatives by the formation of carbon-carbon, carbon-nitrogen, carbon-sulphur and carbon-oxygen bond as shown in Fig 3.3. This reaction possesses several advantages such as low cost, less reaction time, easy work up and use of simple precursors to synthesize bio-active molecule in one-pot fashion. Figure 3.3 Synthesis of compound 9a 12e OPTIMISATION OF REACTION CONDITION Initially, the reaction condition was optimised to evaluate the efficiency of catalyst for the reaction between naphthalene-2, 3-diol and aromatic aldehyde under various conditions. It was found that 1 mmol of ammonium acetate, as a source of ammonia, 67

8 provided high yield in short time. In the initial screening, we used liquid ammonia as reported. However, the yield obtained was too low after long time. Finally, 1 mmol of ammonium acetate was used in the absence of liquid ammonia, and it was found to provide excellent yield in short reaction time (25 min). Figure 3.4 Screening of catalyst and solvent for the reaction of naphthalene-2, 3- diol (8a) with benzaldehyde (2a) The solvent effect is another factor that affects the yield. Thus, experiments were carried out in various solvents like MeOH, EtOH, isopropanol, n-butanol, THF and hexane. Only polar protic solvents helped this reaction, whereas the reaction did not proceed in aprotic solvents. This is because the proton from the protic solvent plays a major role in the reaction mechanism. Thus, the best yield in short time was achieved by employing 1 mmol of ammonium acetate in ethanol. Table 3.1 Synthesis of compound 9a 12e. Entry ArCHO (2) Product Time (min) Yield (%) b 1 R 1 =R 2 =H 9a R 1 =R 2 =2-Cl 9b R 1 =R 2 =4-Cl 9c R 1 =R 2 =4-NO 2 9d R 1 =R 2 =2-F 9e R 1 =R 2 =4-F 9f R 1 =R 2 =1-napthaldehyde 9g R 1 =R 2 =2-Me 9h R 1 =R 2 =4-Me 9i R 1 =R 2 =2-OMe 9j R 1 =R 2 =4-OMe 9k

9 12 R 1 =R 2 =4-OEt 9l R 1 =R 2 =3,4-OMe 9m R 1 =R 2 =3,4,5-OMe 9n R 1 =2Cl, R 2 =4-OMe 9o R 1 =4Cl, R 2 =2-OMe 9p R 1 =4M, R 2 =2-OMe 9q R 1 =2Cl, R 2 =4-Me 9r R 1 =2F, R 2 =4-Me 9s R 1 =4Cl, R 2 =2-Me 9t R 1 =2Me, R 2 =4-Me c 10a R 1 =2Me, R 2 =4-OEt c 10b R 1 =2Cl, R 2 =4-Cl c 10c R 1 =2F, R 2 =4-Cl c 10d R 1 =2F, R 2 =4-F c 10e R 1 =R 2 = H d 11a R 1 =R 2 = 2-F d 11b R 1 =R 2 = 4-F d 11c R 1 =R 2 = 2-CF 3 d 11d R 1 =R 2 = 4-Cl d 11e R 1 =R 2 = 2-Me d 11f R 1 =R 2 = 4-OEt d 11g R 1 =R 2 = H e 12a R 1 =R 2 = 4-Me e 12b R 1 =R 2 = 4-OMt e 12c R 1 =R 2 = 4-Cl e 12d R 1 =R 2 = 4-F e 12e a 4 mmol when R 1 R 2 or 2 mmol when R 1 =R 2 ; ammonium acetate (1 mol %) in ethanol (10 ml). b Isolated yield. c substrate 8a as 2-naphthol (1.0 mmol) d as naphthalene-2-thiol (1.0 mmol). e as naphthalene-2,7-diol (1.0 mmol). 69

10 To investigate the generality of the reaction in other system, these reactions were carried out using a diverse range of aromatic aldehyde with 2-napthol / naphthalene-2, 3-diol / naphthalene-2, 7-diol or naphthalene-2-thiol in the presence of ammonium acetate. The results obtained are summarized in Table MECHANISTIC INSIGHT AND DFT CALCULATION EXPERIMENTAL STUDIES The mechanism involved in this reaction was investigated both experimentally and theoretically. The reaction between 2-napthol and aromatic aldehyde in the presence of catalyst gives ortho-quinone methides (o-qms) as reported in the literature (Hamid et. al., 2008; Singh et al., 2012). In the same way, 2-napthol / naphthalene-2, 7-diol / naphthalene-2, 3-diol / naphthalene-2-thiol condensed with highly reactive aryl aldehyde to form, corresponding benzylidene intermediate via nucleophilic addition in the presence of ammonium acetate catalyst. The remaining unreactive aryl aldehyde reacts with ammonium acetate to form aldimines intermediate via imine synthesis. Figure 3.5 A detailed reaction mechanism for the synthesis of bis [1, 3] oxazines (where X=Y=OH, Z=H) (int1, int2 etc. are intermediates during reaction) Ammonium acetate acted as a reagent in the first step and as a catalyst in the second step (i.e. intermediate int3 was formed only in the presence of a catalyst). These two intermediates get bonded to form the desired product via cycloaddition reaction. 70

11 Hence, from the Table 3.1, it is clear that the reaction proceeded at a faster rate in the case of electron withdrawing groups or small groups present in the aromatic aldehyde and gave excellent yield, whereas it was slower in the case of electron donating groups or bulkier groups present in the aromatic aldehyde, longer reaction time and lower yields were observed. A practical explanation can be given by considering the fact that 2, 4-disubstituted bis [1, 3] oxazines with two different aryl substituents are formed using 2 equivalents of two different aryl aldehydes instead of four equivalents of the same aryl aldehyde in the case of naphthalene-2, 3-diol as substrate. Initially, 4-dibenzylidene naphthalene- 2, 3-dione intermediate was formed from the reaction between one of the aldehydes and naphthalene-2, 3-diol; aldimine ion was formed from the reaction between second aldehyde and ammonium acetate. These two intermediates react to form the desired product. When two different aldehydes were used in the reaction, the products formed were indeed 2, 4-disubstituted bis [1, 3]-oxazines with two different aryl substituents. This was confirmed initially from the melting point which differed from the product obtained with either of the benzaldehydes. The reaction mechanism was elucidated by recording LC-MS for the crude product formed during the reaction between parent benzaldehyde with naphthalene-2, 3-diol in the presence of ammonium acetate, which strongly confirms the presence of intermediates 2, 4 and 6. Hence, undoubtedly this reaction follows the proposed reaction mechanism. LC-MS spectra can be obtained from the Spectra 3.6. A detailed reaction mechanism for the formation of bis [1, 3] oxazines 9a to 9t is shown in Fig 3.5, and the same mechanism was followed for all the substrates THEORETICAL STUDIES Unusual behaviour of ammonium acetate led to the synthesis of unsymmetrical bis [1, 3]-oxazines in a single step, which motivated us to investigate further on the mechanism involved. DFT calculations were carried out to investigate the exact role of ammonium acetate in this reaction. Calculated reaction and transition-state enthalpies (ΔH) and free energies (ΔG) for reactions (Fig 3.5) are described in Fig The first step was the formation of aldimine from benzaldehyde, ammonium acetate, and its potential energy profile is presented in Fig 3.6. Initially, the energy profile was investigated in gas phase at B3LYP/6-31G** and in order to check the solvent effect for the formation of aldimine, we calculated the energy profile using 71

12 ethanol as solvent at B3LYP/6-31G** level of theory. The free energy obtained for int1 in gas phase (ΔG = kcal/mol) differed 0.98 kcal / mol compared to solvent effect (ΔG = 7.25 kcal / mol). The free energy of int1, TS1 and int2 calculated in ethanol simulated PCM was lower than in the gas phase (ΔG: 18.01>7.25, 29.06>21.71, 3.12>-2.60 respectively). Activation energy obtained for TS1 at gas phase is thermodynamically not feasible for a reaction carried out at room temperature. Thus, the calculation for ethanol as solvent at B3LYP/6-31G** favoured the first step with lower energy values which indicated that the protic solvent played a role in this step. Thus all the calculations were carried out using EtOH solvation. Figure 3.6 Potential energy surface (in kcal / mol) generated at the B3LYP/6-31G** level of theory including solvent effect (ethanol) - (step 1) Figure 3.7 Potential energy surface (in kcal / mol) generated at the B3LYP/6-31G** level of theory using EtOH solvation (step 2). The second step was the formation of 4-dibenzylidene naphthalene-2, 3-dione (int4) from naphthalene-2, 3-diol and benzaldehyde. This transformation proceeded via an int3 for which energy values were calculated without any catalyst and with ammonia and ammonium acetate as catalysts. The free energy was lower for ammonium acetate catalysed than from non-catalysed reaction and liquid ammonia catalyzed (ΔG: 72

13 4.47<11.68<12.39 kcal/mol respectively). This step is favoured when the reaction is carried out with ammonium acetate as catalyst. The potential energy profile of step 2 is presented in Fig 3.7 calculated at B3LYP/6-31G** level of theory. TS1 TS2 TS3 Figure 3.8 The transition state structures (TS1, TS2 and TS3) for the reactions as described in Fig2 (distances are in Angstroms). The final step was the cycloaddition between int2 (acting as dienophile) and int6 (acting as diene) to yield the corresponding product. Conversion of two weak π bonds into two strong σ-bonds proceeded via transition state TS3 and TS4 with the activation barriers of 1.99 and 1.39 kcal / mol respectively. This reaction was exothermic in nature which possessed and kcal / mol free energy for the formation of int7 and product 4. The observed distance between aldimine CH carbon and int6 carbonyl oxygen in TS2 was Å, and the distance between aldimine NH nitrogen and vinylic carbon was Å. In TS3, the observed distance between aldimine NH nitrogen and vinylic carbon was Å and the distance between 73

14 aldimine CH carbon and int7 carbonyl oxygen was Å, as shown in Fig 3.8. The potential energy profile of the final step calculated at B3LYP/6-31G** level of theory is presented in Fig 3.9. Figure 3.9 Potential energy surface (in kcal / mol) generated at the B3LYP/6-31G** level of theory using EtOH solvation (step3). Palmieri et. al., were the first to explore the mechanism of this type of Mannich reaction and to propose the most favoured possible mechanism for the synthesis of aminoalkylnapthols using theoretical calculations and experimental methods (Palmieri et. al., 2001). The first step was the formation of aldimine and the second step was the formation of o-qms intermediate. Finally, both intermediates reacted by means of cycloaddition to yield the product. Similarly, the reaction described above also followed the same mechanism via o-qms intermediate followed by the cycloaddition to yield the final product 9. All these experimental and theoretical evidence allow us to conclude that the ammonium acetate played a dual role in this reaction - as a reactant and as a catalyst. It was also inferred that it favoured the proposed mechanism via o-qms RING-CHAIN TAUTOMERISM Interestingly, CDCl 3 has an effect on the ring-chain equilibrium in 1 H NMR spectroscopy. In a solution of CDCl 3, all the compounds exhibit ring open-chain tautomerism. The 1 H NMR spectra of all of these compounds show the presence of cis-1,3-oxazine A, trans-1,3-oxazine B, and the Schiff base tautomer C (Fig 3.10). The 1 H NMR spectra of all the compounds have signals at δ = ppm (N-H, cyclic), δ = ppm (OH), and δ = ppm (N=C, Schiff base), which indicates that there was a continuous shift of protons to give ring open chain equilibrium in CDCl 3 solution (HCl impurity) at K. The other signals in the 1 H NMR and IR spectra are in complete agreement with the assigned structures. The 74

15 mass spectra of these compounds have molecular ion peaks at appropriate mass values, which were in accordance with the respective molecular formulas. Ring-openchain tautomer ratios were calculated using the ratio of integral values of the [1, 3] oxazine and Schiff base protons. The NMR data clearly indicates that the synthesised compounds show ring open-chain tautomerism in solution, as shown in Table 3.2. Fig.3.10 Ring-Chain tautomeric possibilities of mono [1, 3] oxazines in CDCl 3 Table H-NMR data and Ring-Chain tautomeric ratios using chemical shifts. [1,3]oxazines (cyclic) Schiff base (acyclic) Ring-chain Code C-1 and C-3 proton NH tautomeric ratio Cis Trans CH-N N=CH OH b 10a 5.75, , a --- a 10b 5.72, a --- a 10c 5.54, , a 88:12 10d 5.58, , a 86:14 10e 5.54, , :11 NMR measured at 400 MHz. a Not detected. b assigned using the ratio of integral values of cyclic [1,3]oxazines and Schiff base protons. Figure 3.11 Solvent effects on 9n using UV Visible and HPLC spectroscopy. Synthesised products 9, 10 and 12 thus obtained show ring-chain tautomerism. Form the synthesised products, mono [1, 3] oxazine s ring-chain tautomeric ratio was calculated using 1 H-NMR whereas the synthesized bis [1, 3] oxazine was a complex macrocyclic compound making it cumbersome to calculate the tautomeric ratio using 75

16 1 H-NMR. So, we studied various spectroscopic techniques (UV and HPLC) by choosing a compound 9 to investigate the ring-chain tautomeric ratio using different solvents. In UV spectroscopy, the sample prepared using DMSO solvent showed only one λ max, which clearly indicated the absence of tautomers whereas with other solvents like CHCl 3, HCl, TFA, IPA, acetic acid and EtOH showed two to three λ max as shown in Fig This variation in the ring-chain tautomerism for different solvents was due to the presence of H + in the solvents. Even though CHCl 3 is aprotic, it always contains acidic impurities. In HPLC, when CHCl 3, EtOH and MeOH were used for sample preparation, 90% of one form and 10% of the other two forms were observed due to the presence of tautomerism. However, when the sample was prepared in TFA and CH 3 CN 51%: 36%: 13% and 68%: 32% respectively of the ring chain tautomerism was observed. Thus, the formed product showed continuous tautomeric shift in a solution phase. This was very clear from FT-IR and NMR spectra; FT-IR spectra of the compounds (9a to 9t) have a sharp absorption at cm -1 which indicates all the compounds possess NH. Compound 9b clearly showed a sharp -NH absorption at 3323 cm -1 and vinylic C-C stretching peak at 1697 cm -1 in FT-IR spectra and in 1H- NMR spectra vinylic proton showed a peak at 9.34 ppm and NH proton showed at 1.30 ppm which indicated the presence of both NH and vinylic group (acyclic), but the crystal structure of 9b clearly showed that both sides of naphthalene moiety were closed (cyclic). 1 H-NMR showed that there was a continuous proton shift in CDCl 3 solution (HCl impurity) at K to yield ring open chain equilibrium. Figure Possible ring-chain tautomerism for compound 9a-9t in solvents 76

17 In earlier studies, we calculated the ring-chain tautomeric ratio of all the compounds, but in the extension towards macrocycles it was more complicated to calculate the ring-chain tautomeric ratio for compound (9a to 9t). Possible ring-chain tautomerism for the compounds (9a to 9t) in solvent is shown in Fig To find out the ring chain tautomerism in other system, we used naphthalene-2-thiol containing active alpha hydrogen in the same reaction conditions but we got Schiff s base of naphthalene-2-thiols (acyclic). This was very clear from the obtained FT-IR and NMR spectra. In the presence of sulphur atom, ring-chain tautomerism was hindered due to the orbital interaction between 3p of sulphur and 2p of carbon, whereas in the case of bis [1, 3] oxazine orbital interaction was between 2p of oxygen and 2p of carbon. Thus, in this case, it clearly showed that ring-chain tautomerism was only observed in [1, 3] oxazines and was absent in naphthalene-2-thiol derivatives as shown in Fig The proportion of the ring-closed forms in bis [1, 3] oxazines strongly depends on the electronic character of the substitution on benzaldehyde as well as the solvent used. Figure 3.13 Ring chain tautomeric possibilities in the synthesised compound IN-VITRO ANTI-CANCER SCREENING The [1, 3]-oxazine ring is incorporated in numerous biological compounds as a substructure. Hence, designing a more appropriate synthetic route is a great deal of importance for the synthetic community to achieve a novel therapeutic drug against human cancers. It is well-known that 6-membered nitrogen and oxygen containing [1, 3]-oxazines are of great biological and pharmacological interest such as antiinflammatory, anti-thrombotic, and antibacterial activities. Figure 3.14 Biologically active compound containing oxazine unit. 77

18 Efavirenz (Sustiva) - a non-nucleoside reverse transcriptase inhibitor is one of the great examples, which is presently used for the treatment of AIDS (Vrouenraets et al., 2007). Human 5-HT6 receptor inhibitor contains the oxazine moiety which can be developed to use as an anti-depressant (Zhao et al., 2007). The [1, 3]-oxazine ring structure is incorporated in numerous biological compounds as a sub-structure CYTOTOXICITY STUDIES FOR THE SYNTHESISED COMPOUNDS. In order to explore the anti-cancer activity for the synthesised compounds in vitro, anti-cancer activity towards a panel of five human cancer cell lines for the synthesized molecules (9a - 9t) at two different concentrations (1 μm and 10 μm) was evaluated. The human tumour cell lines of panc1 (Pancreatic cancer), ACHN (Renal cancer), HCT116 (Colon cancer), H460 (Non small cell lung cancer) and Calu1 (Lung cancer) and MCF10A (normal breast epithelium) were used for evaluating anticancer activity on the high throughput screening platform using Cell Counting Kit (CCK8), cell proliferation and cytotoxicity assay. All the cancer studies were carried out using DMSO solvent because of the absence of ring-chain tautomerism of product (9). Initially cytotoxicity of bis [1, 3] oxazines was screened against five different cancer cell lines at 1 µm concentration to evaluate the toxicity on various cancer receptors. Out of twenty derivatives, eight compounds showed moderate to less toxicity on various cancer receptors. A pictorial representation of an active compound against these cancer cell lines at 1 μm concentration is shown in Fig Figure 3.15 Cytotoxic activity of active compound at 1 µm concentration. 78

19 ANTI-CANCER STUDIES FOR THE PRELIMINARILY ACTIVE COMPOUNDS A preliminary screening of cytotoxicity studies showed that some of the synthesised derivatives exhibited a moderate to strong anti-cancer activity on various cancer cells. Hence, IC 50 values of primarily active compounds at a single lower concentration were evaluated to study the activity against cancer cell lines at 1 μm concentration. The results revealed that the compound 9a and 9d showed potent IC 50 in the range of 0.56 to 0.83 μm and 0.63 to 0.86 μm in cancer cell lines possessing IC 50 of normal breast epithelium cells at 4.83 and 4.16 μm respectively. Hence, compound 9a and 9d showed potent activity against these cancer cell lines, with less cytotoxicity. Compound 9l showed moderate activity with less cytotoxicity, whereas all other compounds like 9e, 9j, 9k, 9m and 9n showed moderate activity against these cancer cell lines with moderate cytotoxicity, as shown in Table 3.3. The results obtained for the active analogues are reported in the following table. The activity of the compounds on cancer cell lines was moderate when aromatic aldehydes bore EDG, and potent activity was observed when aromatic aldehydes bore EWG. Table 3.3 In vitro anti-cancer studies for the synthesised analogue against five cancer cell lines. Concentration (IC 50 in μm) [a] Entry code ACHN Renal Cancer PANC1 Pancreatic cancer CALU-1 Lung cancer H460 Non-small cell lung cancer HCT116 Colon cancer MCF10A Normal breast epithelium HTS Remarks 1 9a 0.59 ± ± ± ± ± ± 0.60 Active 2 9d 0.72 ± ± ± ± ± ± 0.48 Active 3 9e 0.82 ± ± ± ± ± ± 0.79 Active 4 9j 1.36 ± ± ± ± ± ± 0.79 MA 5 9k 1.31 ± ± ± ± ± ± 0.40 MA 6 9l 2.21 ± ± ± ± ± ± 0.87 MA 7 9m 0.92 ± ± ± ± ± ± 0.70 MA 8 9n 2.23 ± ± ± ± ± ± 0.71 MA [a] Experiment was performed in triplicate for three repeats, and IC 50 values were expressed as Mean ± SEM. 79

20 DOCKING STUDIES Computational prediction of structural interaction between ligand and protein complexes can be studied using docking process which is an important method for drug design called structure-based drug design. A few of the synthesised compounds showed good anti-cancer activity. Thus, for further understanding of the mechanism involved between the ligand and the receptor, we carried out docking studies. A molecular Docking study shows good binding interaction between the synthesised bis [1, 3] oxazines on various cancer receptors. Compound 9n showed the highest intermolecular interactions between the receptor and the ligand with least binding energy indicating the strong inhibition of the receptor as shown in Table 3.4. Figure 3.16: Homology model of 3TWJ ROCK1 (breast cancer) and its docking model (3TWJ ROCK1 9n complex) 80

21 Table 3.4 Van der Waals interactions between ligands and the receptor residues S.No Ligand 3TWJ ROCK1 - Breast 4FLH pi3k - Colon cancer 1MOX - lung cancer cancer Total Total Energy Z score VDWa VDW a Total VDW a Energy Z score Energy Z score 1 9a b c d e f g h i j k l m n o p q r s t a Van der Waals (VDW) interactions between ligands and the receptor residues 81

22 Figure 3.17: Homology model of 3B8Q vgfr2 (renal cancer) and its docking model (3B8Q vgfr2 9n complex) In the 3B8Q vgfr2 9n complex, compound 9n stabilized by N-H O and hydrophobic interactions in the centre of the active site. Amide group of LYS residues (N-H O) interacted with the 9n methoxy oxygen and showed an intermolecular distance of 99Å. One more interaction was observed between ASP residues of oxygen and secondary amine of [1, 3] oxazine as shown in Fig

23 3.4 SPECTRAL DISCUSSION Characterization of compound 9b is discussed as a representative compound of this chapter. Figure 3.18 Structure of 6-((2-chlorobenzylideneamino) (2-chlorophenyl) methyl)-1, 3-bis (2-chloro phenyl) -2, 3-dihydro-1H-naphtho [1, 2-e] [1, 3] oxazin- 5-ol (9b) 6-((2-chlorobenzylideneamino) (2-chlorophenyl) methyl)-1, 3-bis (2-chloro phenyl) - 2, 3-dihydro-1H-naphtho [1, 2-e] [1, 3] oxazin-5-ol (9b) is a colourless crystal obtained by slow evaporation from ethanol and THF (1:1) mixture. Yield and melting point are 89 and ⁰ C respectively. FT-IR spectra of compound 9b, showed a sharp single absorption band at 3324 cm -1 representing the N-H stretching. A band at 3066 cm -1 indicated aromatic C-H stretching. A band at 2920 cm -1 indicated the aliphatic C-H stretching (Spectra 3.1). 1 H and 13 C-NMR spectra were recorded in 400 MHz Bruker using CDCl 3 as solvent. 1 H-NMR spectra of compound 9b showed singlet peak at 1.30 ppm corresponding to NH proton. Peak at ppm showed three singlet peaks corresponding to three CH protons. The hydroxyl OH proton appeared as singlet at 7.01 ppm. The aromatic protons exhibited in the range of ppm. Vinyl CH proton showed a singlet peak at 9.34 ppm (Spectra 3.2). 13 C- NMR spectra of compound 9b showed peaks at 29.73, 52.44, and ppm corresponding to aliphatic CH carbon. Aromatic carbon showed a signal at a range of ppm (Spectra 3.3). Additional information of 9b structure was obtained from single crystal X-ray diffraction analysis. The structures of all the synthesised compounds (9, 10, 11 and 12) were confirmed using FT-IR, 1 H, 13 C-NMR and HRMS analysis (Table 3.5). 83

24 84 Spectra 3.1: FT-IR of 6-((2-chlorobenzylideneamino) (2-chlorophenyl) methyl)-1, 3-bis (2-chlorophenyl)-2, 3-dihydro-1Hnaphtho [1, 2-e][1,3]oxazin-5-ol (9b) 4000 DI2cL /cm %T

25 Spectra 3.2: 1 H NMR of 6-((2-chlorobenzylideneamino) (2-chlorophenyl) methyl)-1, 3-bis (2-chlorophenyl)-2, 3-dihydro-1Hnaphtho [1, 2-e][1,3]oxazin-5-ol (9b) 85

26 Spectra 3.3: 13 C NMR of 6-((2-chlorobenzylideneamino) (2-chlorophenyl) methyl)-1, 3-bis (2-chlorophenyl)-2, 3-dihydro-1Hnaphtho [1, 2-e][1,3]oxazin-5-ol (9b) 86

27 Spectra 3.4: Mass spectra of compound 6-((2-chlorobenzylideneamino) (2-chlorophenyl) methyl)-1, 3-bis (2-chlorophenyl)-2, 3- dihydro-1h-naphtho [1, 2-e][1,3]oxazin-5-ol (9b) 87

28 Spectra 3.5: HPLC data for compound (9b) in TFA 88

29 Spectra 3.6: LC-Ms spectra for the reaction mixture of compound 9a 89

30 Spectra 3.7: ORTEP diagram of 6-((2-chlorobenzylideneamino) (2-chlorophenyl) methyl)-1, 3-bis (2-chlorophenyl)-2, 3-dihydro- 1H-naphtho [1, 2-e] [1, 3] oxazin-5-ol (9b) 90

31 Product code 9a 9b 9c 9d 9e 9f 9g FT-IR (cm -1 ) 3315, 3061, 2922, , 3066, 2920, , 3061, 2900, , 3307, 2885, , 2891, 1691, 3311, 3066, , 3309, Rf - TLC (EA : hexane) (60%) Table 3.5 Spectral Data of compound 9a-12e 1 H-NMR (δ) in ppm 13 C-NMR (δ) in ppm 2.17 (s, NH, 1H), (s, CH, 3H), 29.69, 30.90, 54.14, 82.47, , 1294, (s, OH, 1H), (ArH, 24H), (Vinylic H, 1H) , , , , , , , , , , , (s, NH, 1H), (s, CH, 3H), 29.73, 52.44, 71.30, 81.60, , , (s, OH, 1H), (ArH, , 1296, , , , , 20H), 9.34 (Vinylic H, 1H) , , , , , (NH, 1H), (CH, 3H), (ArH, 20H), 7.50 (Vinylic H, 1H), 8.72 (s, OH, 1H) 21.16, 78.58, 78.92, 79.25, , , , , , (CH, 2H), (ArH, 20H), 56.45, , , , , , 7.33 (vinylic H, 1H), 7.09 (vinylic H, 1H), 12111, 12127, , , , , (OH, 2H) , , , (NH, 2H), (CH, 4H), (ArH, 20H) 21.12, 47.56, 77.67, 79.14, , , , , , , , , , , , , (NH, 1H), (CH, 2H), 6.81 (CH, 1H), (ArH, 20H), 9.87 (OH, 1H) 21.74, 79.13, , , , , , (OH, 2H), (CH, 2H), , 52.11, 76.69, 77.33, 81.35, , 8.17 (ArH, 32H), (vinylic H, 2H) , , 1293, , , , 91 HRMS Calculated Found C38H30N2O C38H26Cl4N2O C38H26Cl4N2O C38H26N6O C38H26F4N2O C38H26F4N2O C54H38N2O2

32 9h 9i 9j 9k 9l 9m 3045, , , , 13112, , , 3313, (CH3, 6H), (CH3, 6H), (NH, 1H), (CH, 3H), 17.48, 18.78, 50.85, 78.93, 80.46, , , , , , , , C42H38N2O2 3064, (30%) (ArH, 20H), 7.18 (Vinylic H, 1H), 7.74 (OH, 1H) , , , , , , , , , 3313, 3014, (60%) (CH3, 12H), (NH, 1H), 6.6-6,75 (CH, 3H), (ArH, 20H), 7.81 (OH, 1H), 9.87 (Vinylic H, 1H) 17.90, 19.27, 52.11, 80.76, 81.68, , , , , , , , , , , , , C42H38N2O , 3068, (OCH3, 12H), 5.32 (NH, 1H), 5.54 (CH, 1H), 5.65 (CH, 2H), 6.28 (OH, 1H), (ArH, 20H), 9.78 (OH, 1H) 55.45, 58.23, , 11127, , , , , , , , , , C42H38N2O , 2999, (NH, 1H), (OCH3, 12H), (CH, 1H), (CH, 1H), 55.46, 55.61, 68.12, , 111,08, , , , , , , , C42H38N2O6 2858, 6.78 (CH, 1H), (ArH, 20H), , , , , , , (OH, 1H), (vinylic H, 1H) , , 3068, 2895, (CH3, 12H), (OCH2, 8H), (CH, 3H), 6.38 (NH, 1H), (ArH, 20H), 7.00 (OH, 1H), , 17.78, 19.01, 51.99, 63.90, 80.26, , , , , , , , , , , , , C46H46N2O (vinylic H, 1H) , , 3315, 2937, (OCH3, 24H), (CH, 3H), (NH, 1H), (ArH, 16H), 7.89 (OH, 1H), 9.82 (Vinylic H, 1H) 21.15, 55.45, 55.84, 77.26, , , , , , , , , , , C46H46N2O

33 9n 9o 9p 9q 9r 3159, 2935, , 3308, 3034, 2872, , 3298, 3021, 2867, , 3327, 3051, 2905, , 3322, 3034, 2823, (60%) (OCH3, 36H), (CH, 3H), 4.54 (NH, 1H), 5.62 (OH, 1H), (ArH, 12H), 9.92 (Vinylic H, 1H) 1.25 (NH, 1H), 125 (CH, 1H), 3.95 (OCH3, 3H), 4.05 (OCH3, 3H), (CH, 2H), (ArH, 20H), 9.29 (vinylic H, 1H), (OH, 1H) 2.08 (NH,1H), 3.88 (OCH3,3H), 4.00 (OCH3,3H), (CH,1H), (CH, 1H), 6.13 (CH, 1H), (ArH, 20H), 8.68(vinylic H, 1H), (OH, 1H) 2.25 (NH, 1H), (CH3, 6H), (OCH3, 6H), (OCH3, 6H), (CH, 2H), 6.41 (CH, 1H), (ArH, 20H), (vinylic H, 1H), 9.89 (OH, 1H) (CH3, 3H), (OCH3, 3H), (OCH3, 6H), (CH, 1H), (CH, 1H), 6.45 (CH, 1H), (ArH, 20H), 8.55 (vinylic H, 1H), 9.96 (OH, 1H) , 55.90, 59.89, , , , , , , , , , , , 52.5, 80.87, , , , , , , , , , , , , , , , , 5103, 55.76, , 11.46, , , , , , , , , , , , , 21.77, 55.35, 55.73, , , 1299, , , , , , , , , 31.06, , , , , , C50H54N2O C40H32Cl2N2O C40H32Cl2N2O C42H38N2O C40H32Cl2N2O

34 9s 9t 10a 10b 10c 10d 3461, 3343, 3022, 2854, , 3317, 3064, , 3067, 2918, , 3027, 2950, , 3069, 2975, , 3061, 2967, (25%) 0.79 (25%) 0.69 (25%) 0.41 (30%) (CH3, 6H), 2.40 (CH, 1H), 3.85 (NH, 1H), (CH, 1H), (CH, 1H), (ArH, 20H), 8.65 (vinylic H, 1H), 9.96 (OH, 1H) (Me, 6H), 1.96(NH, 1H), (CH, 1H), 2.77 (CH3, 3H), (CH, 1H), (CH, 1H), 5.94 (OH, 1H), (ArH, 20H), 7.81 (vinylic H, 1H) (14H, Ar), 7.75 (1H, s, N=CH), (2H, CH), (6H, CH3) (14H, Ar), (H, CH), (2H, q, CH2), 2.68(H, CH), 1.84 (3H, s, CH3), (3H, t, CH3) (14H, Ar), (H, CH), (H, CH), 2.72 (1H, s, NH) (14H, Ar), (1H, CH), (1H, CH), 1.62 (1H, s, NH) , 77.35, 81.72, , , , , , 12106, , , , , , 19.73, 52.53, 80.92, 81.93, , , , , , , , , , , , , , , , , , , , , , , , , 80.30, 77.01, 53.99, 21.10, , , , , , , , , , , , , , , 80.26, 63.90, 51.99, 19.01, 17.78, , , , , , , , , , , , , , , , , 80.12, ( 1 JC-F = 176 Hz), , , , , , , , , , , , , , , 81.64, 67.50, 53.34, C17H15Br2NOS C17H15Br2NOS C26H23NO C27H25NO C24H17Cl2NO C24H17ClFNO

35 10e 11a 11b 11c 11d 11e 11f 11g 3319, 3062, 2972, , 3050, , 2952, , 2935, , 2924, , 2925, , 2927, , 3051, 1602, 0.32 (30%) (14H, Ar), (1H, CH), 4.78 (1H, CH), (1H, s, NH). 13 C NMR (CDCl3, 100 MHz) 4.95(s, CH, 1 H), 5.45(s, SH, 1 H), (m, ArH, 16 H), 8.50 (s, Vinylic - H, 1 H) (SH, 1H), 7.24 (CH, 1H), (ArH, 14H), 7.96 (Vinylic H, 1H) (SH, 1H), (CH, 1H), (ArH, 14H), 7.97 (Vinylic H, 1H) (SH, 1H), (CH, 1H), (ArH, 14H), 7.98 (Vinylic H, 1H) (SH, 1H), (CH, 1H), (ArH, 14H), 7.97 (Vinylic H, 1H) 1.03 (s, CH3, 3H), 1.18 (s, CH3, 3H), 4.01 (SH, 1H), 7.19 (CH, 1H), (ArH, 14H), 7.91 (Vinylic H, 1H) (m, CH2, 2 H), (m, CH, 1 H), (m, CH, 1 H), (m, CH, 1 H), (m, CH, 1 H), 3.77(s, ( 1 JC-F = Hz), ( 1 JC-F = Hz), , , , , , , , , , , 81.72, 77.35, 55.50, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , 63.89, , , , , , , , , , 63.91, , , , , , , , , , , 63.87, , , , , , , , , , , , , 19.34, , , , , , , , , , , , 30.94, 41.98, 55.28, 62.08, , , , , , , , , C17H15Br2NOS C24H17F2NO C24H17F2NS C24H17F2NS C26H17F6NS C24H17Cl2NS C26H23NS C28H27NO2S

36 12a 12b 12c 12d 12e 1579 OCH3, 3 H), 6.78 (s, CH, 1 H), (d, J = 8.8 Hz, 2 H), (m, ArH, 7 H) 3322, 3.28 (s, NH, 1H), 5.06 (s, CH, 1 H), 5.61 (s, 3019, 2872, 0.42 CH, 1H), 6.79 (s, CH, 1H), 7.22 (s, OH, 1H), (ArH, 20H), 8.86 (vinylic H, H) 3286, 3.24 (s, NH, 1H), 3.79 (s, OCH3, 12H), , 2782, 0.44 (s, CH, 1H), 5.69 (s, CH, 1H), 5.78 (s, CH, 1H), 6.45 (s, OH, 1H), (ArH, H), 8.37 (s, vinylic H, 1H) 3279, 2.19 (s, CH3, 12H), 2.64 (s, NH, 1H), , 0.48 (s, CH, 1H), 5.43 (s, CH, 1H), 5.59 (s, CH, 2932, H), 6.39 (s, OH, 1H), (ArH, 20H), 8.29 (s, vinylic H, 1H) 3301, 2.10 (s, NH, 1H), 5.19 (s, CH, 1H), 5.43 (s, 3124, 0.51 CH, 1H), 5.70 (s, CH, 1H), 6.11 (s, OH, 2972, H), (ArH, 20H), 8.74 (s, vinylic H, 1H) 3307, 3018, (s, NH, 1H), 4.93 (s, CH, 1H), 5.22 (s, CH, 1H), 5.43 (s, CH, 1H), 6.95 (s, OH, 2987, 1H), (ArH, 20H), 8.58 (s, vinylic 1656 H, 1H) , 68.79, 84.16, , , , , , , , , , , , , , , , , , , , , 57.92, 65.75, 84.11, , , , , , , , , , , , , , , , , , , , 57.99, 68.70, 84.15, , , , , , , , , , , , , , , , , , , , 68.78, 84.16, , , , , , , , , , , , , , , , , , , , , 68.70, 84.13, , , , , , , , , , , , , , , , , , , , , C38H30N2O C42H38N2O C42H38N2O C38H26F4N2O C38H26Cl4N2O

37 3.5 CRYSTALLOGRAPHY DISCUSSION Conformations and interaction patterns in bis [1, 3] oxazine compounds were investigated using X-ray crystallography. Crystal structure of a representative compound 9b is shown in spectra 3.5. Attempts to grow diffraction quality crystals for other compounds were not successful. In the reported centrosymmetric structure, the stereogenic centers C2 C3, C12 and C13 adopted S, S, R and R configurations, respectively. Molecule, 9b, possessed approximate mirror symmetry with the mirror plane bisecting the C7-C8/C5-C10/C1-C14 bonds of the naphthalene ring. Table 3.6 Inter-molecular interactions observed in (9b) D-H A D-H (Å) H A (Å) D A (Å) D-H A (º) N2 H1...N2 (i) 0.72(5) 2.48(6) 3.180(4) 165(5) C1P2-H1P2...O (10) 169 Symmetry codes: (i) 2-x, 2-y,-z, The conformation of fused oxazine rings (O1/N1/C1-C4; O2/N2/C11-C14) on either side of the naphthalene (C1/C4-C11/C14) was a distorted semi-chair having C2 carbon (C2 & C13) and nitrogen (N1 & N2) residing above and below the plane formed by naphthalene. The strain in the ring allows ring opening polymerization to take place. Table 3.7 Intra-molecular interactions observed in (9b) Interactions D-H A D-H (Å) H A (Å) D A (Å) D-H A (º) Intra-molecular C2 H2A...Cl (3) 103 C3 H3...Cl (3) 105 C12-H12...Cl (3) 109 C13-H13...Cl (3) 101 C20-H20...O (4) 100 Symmetry codes: (i) 2-x, 2-y,-z, C38-H38...O (5)