An expedient synthesis of novel 2-substituted thiazolo[4,5-f ]isoquinolines/quinolines and benzo[1,2-d :4,3-d  ]bisthiazoles and their potential as inhibitors of COX-1 and COX-2

An efficient, general synthesis of 2-substituted thiazolo[4,5-f ]isoquinolines, thiazolo[4,5-f ]- quinolines and benzo[1,2-d :4,3-d′ ]bisthiazoles has been accomplished from 5-nitroisoquinoline/quinoline and 6-nitrobenzothiazole, respectively, and all the products have been thoroughly identified spectroscopically (IR, 1 H and 13 C NMR, LR/ HR EI/ FAB/ ESI-MS). The synthesis of thiazolo[4,5-f ]isoquinolines constitutes the first synthesis of this class of heteroarenes. Eighteen compounds, covering all three types, were screened for inhibition of COX-1 and COX-2, and some of them showed moderate activities.


Introduction
The thiazole ring is an important pharmacophore, 1 and many thiazolyl compounds 2 and annulated thiazoles 3 are used in human therapeutics, veterinary medicine and as lead molecules for drugs. 4Additionally, thiazoles find application in other fields like polymers, liquid crystals, photonucleases, fluorescent dyes, insecticides, antioxidants, etc. 1 The reported diverse biological activities and industrial usefulness of annulated thiazoles of both natural and synthetic origins drew our attention to condensed thiazoles.Although a variety of such compounds have been synthesised, 3 we became particularly interested in thiazoloisoquinolines since, of the twelve possible isomeric structures of this class, only three isomers, viz.thiazolo[4,5-f]-, -[4,5-g]-and - [5,4-g]isoquinolines have not yet been synthesised.Amongst these three, the thiazolo[4,5-f]isoquinolines became our target molecules simply because Taurins et al. had earlier failed to synthesise this isomer by a unified approach, [5][6][7] which they successfully applied in synthesising five other isomeric thiazoloisoquinolines, viz. the [4,5c]-, [5,4-c]-, [5,4-f]-, [4,5-h]-and [5,4-h]-isomers.This route comprised the acid (EtOH-HCl)catalysed cyclisation of ortho-amino-thiocyanato-isoquinolines.Our motivation was to overcome this failure, and we achieved our goal by developing a brief, three/four-step synthesis of 2substituted thiazolo [4,5-f]isoquinolines starting from commercially available 5-nitroisoquinoline. Indeed, Taurins' group had used the same starting material, viz. the derived aminoisoquinoline and attempted to thiocyanate it at C-6 by treatment with potassium thiocyanate and bromine in acetic acid in cold, which resulted in the formation of, contrary to expectation, the 8-thiocyanato isomer, which was clearly not cyclisable. 7Taurins' work is shown in Scheme 1.

Scheme 1
We have developed a general synthesis of a number of 2-alkylamino/anilino-, 2-alkylthio-and 2alkyl/phenylthiazolo [4,5-f]isoquinolines and later extended this methodology successfully to the synthesis of similarly substituted thiazolo [4,5-f]quinolines and benzo [1,2-d:4,3-d′]bisthiazoles.A few products of each type were screened for their anti-inflammatory potential by measuring their ability to inhibit cyclooxygenase (both COX-1 and COX-2).The details of the syntheses and the results of screening for bioactivity are presented in this communication.

Scheme 2
To the best of our knowledge, this piece of work constitutes the first ever synthesis of the thiazolo[4,5-f]isoquinoline ring.Pertinently, in this and all subsequent classes of novel compounds, the individual 1 H and 13 C NMR assignments of one member of each type were ascertained by analysing their HMQC and HMBC correlations. 13These assignments have been shown in the data of the relevant compounds in the Experimental.
This protocol was next extended to the synthesis of similarly fused thiazolo[4,5-f]quinolines and benzo [1,2-d:4,3-d′] / [1,2-d:4,5-d′]bisthiazoles starting from 5-aminoquinoline and 6-aminobenzothiazole, respectively.The reason for choosing these thiazoloheteroaryl nuclei was threefold.Firstly, some members of particularly the thiazolo [4,5-f]quinolines and quinolones were reported to display significant bioactivities, e.g.mutagenic, cardiotonic and dopaminergic properties.14a-f Secondly, although a few members of these two classes of condensed thiazoles had earlier been synthesised using different routes, 15 a general method was still lacking.Thirdly, the starting amines could easily be prepared by reduction of the corresponding nitro compounds which are cheap and commercially available.

Scheme 3
been prepared from 6-nitrobenzothiazole by reduction with tetrabutylammonium borohydride in dimethylsulfoxide. 18The methylene protons flanked between NH and S appeared unusually downfield at δ 6.83 (s).Each of 4 and 5 was treated, as in the isoquinoline series, with methyl, ethyl, n-propyl and benzyl isothiocyanates separately in methanol under reflux to form the respective 5-(N′-alkylthioureido)quinolines 7a-d and 6-(N′-alkylthioureido)benzothiazoles 8a-d as the sole products.In these thioureides too, the Ar-NH-C=S protons appeared downfield (δ 9.6-9.8,br s) than the R-NH-C=S protons (δ 7.5-7.8,br s for 7,8a-c; δ 8.2, br s for 7d, 8d) whereas the C=S carbons appeared at the same range of ca.δ 181-183 as in the case of 2a-d.
The thioureides 7a-d were then cyclised by bromine in chloroform to the 2-alkylamino derivatives of thiazolo[4,5-f]quinolines 9a-d which displayed similar spectroscopic behaviour as in the case of cyclisation of 2 to 3. The angular modes of cyclisation were similarly ascertained from the appearance of two ortho-coupled (J=8.5 / 9 Hz), one-proton doublets at ca. δ 7.65 (H-8) and 8.0 (H-9) in 9a-d.

Synthesis of 2-alkylthiothiazolo[4,5-f]isoquinolines, -thiazolo[4,5-f]quinolines and -benzo-[1,2-d:4,3-d′]bisthiazoles
We next adopted a related approach for achieving a general synthesis of 2-alkylthio derivatives of the three classes of heteroarenes.Each of 1, 4 and 5 was first converted to its methyl and ethyl dithiocarbamates 12-14a,b by successive treatments with carbon disulfide-pyridine and methyl/ethyl iodide. 19Some of their significant 1 H and 13 C NMR spectroscopic data are discussed later.These were then cyclised by bromine in acetonitrile or bromine in chloroform at room temperature to the respective 2-alkylthio derivatives of thiazolo [4,5-f]  2, see next page).

Scheme 5
The mode of cyclisation in each case was discernible from a comparison of the NMR data of the dithiocarbamates and the products.Thus, in all the three series, (i) the ArNHCS signals observed at δ 11.8-11.9for 12-14a,b disappeared, (ii) two one-proton doublets with ortho-couplings appeared at around δ 8.0 corresponding to H-8 and H-9 of the isoquinoline/quinoline series and H-4 and H-5 of the benzothiazole series, and (iii) the thiocarbonyl carbon signal at ca. δ 199-202, observed for 12-14a,b disappeared and a non-protonated carbon (C-2) appeared at δ 167-169.

Synthesis of 2-anilinothiazolo[4,5-f]isoquinolines, -thiazolo[4,5-f]quinolines and -benzo-[1,2-d:4,3-d′]bisthiazoles
We further developed a general synthesis of the three types of 2-anilinothiazoloheteroarenes by cyclisation of the respective N′-phenylthioureides 2e, 7e and 8e which were attempted to be prepared from 1, 4 and 5 by separate condensations with phenyl isothiocyanate in methanol under reflux.But, different results were obtained for 1 and 5 on one hand and for 4 on the other hand.For 1 and 5, a number of products were formed and the reactions never went to completion even after a prolonged period.We, therefore, resorted to a different protocol. 20Thus, 2e (expected from 1) and 8e (expected from 5) were prepared in excellent yields by a reasonably fast (1/2 h) reaction of aniline with ethyl N-(5-isoquinolinyl/6-benzothiazolyl)dithiocarbamates 12b / 14b (Scheme 6).

Scheme 6
From 4 were obtained two different, somewhat unexpected products, viz. the known N,N′diphenylthiourea 18 21 (38%) and the novel methyl N-(5-quinolinyl)thiocarbamate 19 (46%) were formed.The EI-MS of 19 expectedly recorded the base peak at m/z 186.i.e. at [M-MeOH] + .We believe, 18 and 19 were formed from 5-(N′-phenylthioureido)quinoline 7e, the expected initial condensation product, by thermal cleavage to aniline and 5-isothiocyanatoquinoline 20.While the condensation of aniline with phenyl isothiocyanate led to the formation of 18, the condensation of 20 with methanol resulted in the formation of 19 (Scheme 7).

Scheme 7
The desired intermediate 7e was, therefore, prepared in the same way as 8e was prepared from 2e.Thus, aniline was condensed efficiently with ethyl N-(5-quinolinyl)dithiocarbamate 13b in methanol under reflux to furnish 7e as the only product.Each of the thioureides 2e, 7e and 8e was then cyclised by bromine in acetic acid (for 2e) or in chloroform (for 7e and 8e), which furnished the 2-anilino derivatives of thiazolo[4,5-f]isoquinoline 3e, thiazolo[4,5-f]quinoline 9e and benzo[1,2-d:4,3-d′]bisthiazole 10e, respectively.Angular cyclisation was observed in all the three compounds.Thus, H-8 and H-9 of the isoquinoline 3e and the quinoline 9e appeared downfield (δ 7.8 and 8.1) whereas C-2 appeared upfield (δ 164) than those in the 2-alkylamino derivatives 3a-d and 9a-d.However, in the case of the benzobisthiazole 10e, no such distinctive feature was observed.The preparation of 3e, 9e and 10e from 12-14b via 2e, 7e and 8e are shown in Scheme 6 (see previous page) and the results in Table 3 below.
In this series too, the angular mode of cyclisation in each case was evident from a similar appearance of two ortho-coupled, one-proton doublets at around δ 8.0, corresponding to H-8 and H-9 for 27 and 28 and H-4 and H-5 for 29.

Synthesis of 2-aminobenzo[1,2-d:4,3-d′]bisthiazole
The 2-amino derivatives of all the three series were the next target molecules since the amino group can very well be converted to a number of other functionalities.These compounds had earlier been either prepared or attempted to be prepared from the corresponding heteroaryl amines.Thus, 2-aminothiazolo[4,5-f]quinoline 30 had previously been synthesised from 5aminoquinoline 4 by treatment with potassium thiocyanate and bromine in glacial acetic acid.15b,d But, as shown earlier (Scheme 1), when 5-aminoisoquinoline 1 was treated in a similar manner, thiocyanation took place at C-8 of 1, and 5-amino-8-thiocyanatoisoquinoline was the sole product. 7-Aminobenzo[1,2-d:4,3-d′]bisthiazole 31 had previously been prepared from 5 in two steps -treatment with ammonium thiocyanate, acetic acid and N,N′-dichlorourea, followed by hydrochloric acid-catalysed cyclisation of the resulting 6-amino-7-thiocyanatobenzothiazole.15i Clearly, there was room for improvement in the synthesis of 31 from 5, and we did it efficiently in one step by treatment with potassium thiocyanate and bromine in glacial acetic acid.
We have recorded for the first time the spectroscopic data of 31.Here too, angular cyclisation was indicated by two signals at δ 7.52 and 7.91 (d, J=8.5 Hz, 1H each), corresponding to H-4 and H-5, respectively of the 31.Syntheses of 30 and 31 are depicted in Scheme 9.

Scheme 9 Biological results. In vitro experiments. COX-1 inhibition
In view of the reported anti-inflammatory activities of substituted isoquinolines, 24a quinolines, 24b condensed quinolines 24c,d and benzothiazoles, 24e we checked the anti-inflammatory potential of the synthesised products by measuring their ability to inhibit cyclooxygenase (COX).Accordingly, eighteen assorted products, viz. the thiazoloisoquinolines 3a,d,e, 15a, 27a,d, the thiazoloquinolines 9a,d, 16a, 28a,d and the benzobisthiazoles 10a,d,e, 17a, 29a,d, 30 were screened for inhibiton of both COX-1 and COX-2 using naproxen 25 as the standard following a recent protocol. 26These compounds were added to the assay mixture at 200 µM using arachidonic acid at a concentration of 0.1 µM.The results are presented in Table 5.From the data presented in Table 5, the following observations could be made.With the exception of compound 27d (entry 6), all compounds inhibit COX-1 in the range of 8% 3d to 75% 3a.COX-1 inhibitory activity was found to be strongly dependent on the nature of the rings and on the substituents at C-2.As regards the first point, thiazoloisoquinolines are more active than the thiazoloquinolines and the benzobisthiazoles.As regards the second point, a high level of activity (62-75%) was observed for the 2-methylamino compounds in all the three series.
In the case of thiazoloisoquinolines in particular, the presence of methylamino, anilino and methylthio groups were found to be favourable for COX-1 inhibitory activity, whereas methyl and phenyl groups led to decrease in activity.The lowest inhibitory activity was observed for the benzylamino derivative 3d.In the case of the thiazoloquinolines, the most active compounds are the 2-methylamino 9a and the 2-phenyl 28d derivatives while the benzylamino derivative 9d was completely void of COX-1 inhibition.In the benzobisthiazole series, the methylamino derivative 10a was most active, the anilino 10e and the methylthio 17a derivatives were less active and the rest displayed poor activity.

COX-2 Inhibition
All the compounds showed no or low (0-36%) inhibition of COX-2 when they were added to the reaction mixture at 200 µM using an arachidonic acid (substrate) concentration of 0.1 µM.
2-Methylthiobenzobisthiazole 17a exhibited the best COX-2 inhibition (36%), followed by the 2-amino derivative 30.Although the 2-substituted benzobisthiazoles possessed the lowest COX-1 inhibitory activity, they displayed better COX-2 inhibition than shown by the two other groups.All our synthesised compounds are less potent COX-1 and COX-2 inhibitors than naproxen.
No correlation could be found between the activity of the compounds and their lipophilicity, which is reflected in their ClogP values. 27The computer predictions of biological activity spectra of the compounds were ascertained using PASS software.

Conclusions
We have accomplished the first general synthesis of 2-alkylamino/anilino-, 2-alkylthio-and 2alkyl/phenylthiazolo[4,5-f]isoquinolines in three/four steps from a commercially available starting material and extended the protocols to the synthesis of novel, similarly substituted thiazolo[4,5-f]quinolines and benzo[1,2-d:4,3-d′]bisthiazoles.All compounds, mostly new, were unambiguously identified by thorough spectroscopic analyses.In all the cases, angular cyclisation did take place, which was concluded from 1 H NMR data.Moreover, this study also contains the definitive 1 H and 13 C NMR assignments of at least one member of each class.Some of the final products, viz.3a,e, 9a, 10a and 15a showed cognizable inhibition of COX-1 while only 17a and 30 displayed noticeable inhibition of COX-2.
Since the steps throughout are brief and simple, the starting materials and the reagents are cheap and the yields of the products are very good to excellent, the present piece of work is significant and holds the promise of being useful in designing the synthesis of similarly substituted other classes of thiazoloheteroarenes.

Experimental Section
General.The nitro compounds and all reagents were procured commercially.All solvents were dried and purified as per literature. 28Melting points were determined in open capillaries on a Toshniwal apparatus and are uncorrected.FT-IR spectra were recorded on KBr pellets (unless stated otherwise) in a Perkin-Elmer RX 1 FT-IR spectrophotometer or in nujol mull in a Nicolet Impact 410 spectrophotometer.NMR ( 1 H, 500 MHz; 13 C, 125 MHz; DEPT 135, HMQC and HMBC) spectra were recorded in a Bruker DRX-500 spectrometer.The LR EI/FAB-MS spectra were recorded on a JEOL JMS-AX505HA mass spectrometer and the HR EI/FAB-MS and ESI-MS spectra on a JEOL JMS-700 Mstation and a Q-TOF MICRO YA263 mass spectrometer, respectively.GC EI-MS were recorded in a Thermo Scientific Trace GC Ultra -POLARIS Q 230LT mass spectrometer.Elemental analyses were carried out in a Perkin Elmer 2400 Series II C, H, N Analyser.TLCs were carried out on silica gel G (Merck, India) plates and column chromatographies (CCs) on silica gel (60-120 mesh, Qualigens, India).Organic layers were dried using anhydrous Na2SO4.PE, DCM and EA stand for petroleum ether, bp.60-80 °C, dichloromethane and ethyl acetate, respectively.
In the cases of 13 and 14, since the products did not precipitate, the reaction mixtures were separately extracted with EA (3×20 mL) and CHCl3 (3×20 mL), respectively.The residues resulting from the pooled extracts were crystallised to furnish pure 16, 17a,b.

General procedure for thionation of the amides (21a-d, 22a-d, 23a-c)
A solution of the amide (1 mmol) in dry C6H6 (25-30 mL) containing Lawesson's reagent (0.484 g, 1.2 mmol) was refluxed until (see Table 4) the starting material was consumed.The solvent was distilled off from the reaction mixture, the residue was worked up in the usual way 22 and the resulting crude product was purified either by CC, followed by crystallisation (for 24b-d, 26a-c), or directly by crystallisation (for 24a, 25a-d).4) the reaction was complete (TLC).The reaction mixture was then extracted with EA (3×20 mL).The residue from the duly treated pooled extract was purified by CC (for 27c) or by crystallisation (for the rest).

Biological assay. In vitro experiments
In the in vitro assays, each experiment was performed in triplicate and the standard deviation of absorbance was less than 10% of the average values.

Screening of assorted products for inhibition of COX-1 and COX-2
The inhibitory activities of the compounds were measured using bovine COX-1 and human recombinant COX-2 enzymes included in the "COX Inhibitor Screening Assay" kit provided by Cayman (Cayman Chemical Co., Ann Arbor, MI).The assay directly measures PGF2α produced by SnCl2 reduction of COX-derived PGH2.The prostanoid productions were quantified via enzyme immunoassay using a broadly specific antibody that binds to all the major prostaglandins.The final estimation of % inhibition (Table 5) was performed at a substrate concentration of 0.1 µM.