Iodine-mediated one-pot intramolecular decarboxylation domino reaction for accessing functionalised 2-(1,3,4-oxadiazol-2-yl)anilines with carbonic anhydrase inhibitory action

Abstract A practical and transition metal-free one-pot domino synthesis of diversified (1,3,4-oxadiazol-2-yl)anilines has been developed employing isatins and hydrazides as the starting materials, in the presence of molecular iodine. The prominent feature of this domino process involves consecutive condensation, hydrolytic ring cleavage, and an intramolecular decarboxylation, in a one-pot process that leads to the oxidative formation of a C–O bond. Fluorescence properties of some of the representative molecules obtained in this way were studied. The synthesised 2-(1,3,4-oxadiazolo-2-yl)aniline-benzene sulphonamides (8a–o) were screened for their carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity. Most of the compounds exhibited low micromolar to nanomolar activity against human (h) isoforms hCA I, hCA II, hCA IV, and XII, with some compounds displaying selective CA inhibitory activity towards hCA II with KIs of 6.4–17.6 nM.


Introduction
Construction of O-heterocyclic ring systems via intramolecular C-O bond formation has become an emerging tool in drug discovery. Accordingly, many efforts have been devoted to this activity, and remarkable results have been achieved to date. Among these, the traditional intramolecular Pd-catalysed Hartwig-Buchwald 1 and copper-catalysed 2 Ullmann-type C-O coupling of aryl halides with hydroxyl moieties, and in an alternative approach, the direct dehydrogenative coupling occurs between C-H and O-H bonds 3 , leading to various functionalised compounds. In most cases, these elaborative designs implied complex catalytic systems (based on Pd(II), Cu(II), Rh(III), and Ru(III) derivatives) and multi-step processes for the preparation of diversely functionalised derivatives, such as, furan, pyrrole, pyrazole, isoquinoline, indole, benzoxazole, and carbazole ring systems 4 . However, oxidative decarboxylation leading to construction of C-heteroatom bonds, particularly the C-O and the C-N bonds, has received significantly less attention. In recent years, in the perspective of green chemistry, most of the organic chemists have switched to metal-free reactions to reduce the burden of toxicity. In this context, iodine and hypervalent iodine reagents have emerged as inexpensive, versatile, and environmentally more friendly reagents 5 . Structural features and the reactivity pattern of these iodine compounds in many aspects are similar to those of the transition metal compounds applied for such purposes. Up until now, many efforts have been made to directly functionalise C-H bonds for the construction of C-C and C-heteroatoms bonds by employing iodine or hypervalent iodine reagents 6,7 . Wang et al. demonstrated a facile access to various heterocycles (quinazoline, oxazole, and pyridine) through the tandem oxidative coupling reactions using iodine as catalyst and tertbutyl-hydroperoxide (TBHP) as the oxidant 8 . Furthermore, Ma et al. proposed the synthesis of imidazo[1,2-a]pyridines via oxidative coupling of 2-aminopyridine with 1,3-diketones in the presence of tetra-butylammonium iodide (TBAI), TBHP, and BF 3 Áetherate 9 . Very recently Tang et al. reported iodine-catalysed radical oxidative annulation for the synthesis of dihydrofurans and indolisines 10 . Interestingly, I 2 (or hypervalent iodine derivatives) also promoted the oxidative decarboxylation of amino acids and b,c-unsaturated carboxylic acids 11 . Intrigued by these advances, herein we envisioned a metal-free, iodine-mediated domino strategy involving intramolecular decarboxylative coupling of isatins, and hydrazides for the synthesis of 2-(1,3,4-oxadiazol-2-yl)aniline derivatives (Scheme 1).

Carbonic anhydrase inhibition assay
An SX.18 MV-R Applied Photophysics (Oxford, UK) stopped-flow instrument has been used to assay the catalytic/inhibition of various CA isozymes 24 . Phenol Red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.4) as buffer, 0.1 M Na 2 SO 4 or NaClO 4 (for maintaining constant the ionic strength; these anions are not inhibitory in the used concentration), following the CA-catalysed CO 2 hydration reaction for a period of 5-10 s. Saturated CO 2 solutions in water at 25 C were used as substrate. Stock solutions of inhibitors were prepared at a concentration of 10 mM (in DMSO-water 1:1, v/v) and dilutions up to 0.01 nM done with the assay buffer mentioned above. At least seven different inhibitor concentrations have been used for measuring the inhibition constant. Inhibitor and enzyme solutions were preincubated together for 10 min at room temperature prior to assay, in order to allow for the formation of the E-I complex. Triplicate experiments were done for each inhibitor concentration, and the values reported throughout the paper are the mean of such results. The inhibition constants were obtained by non-linear least-squares methods using the Cheng-Prusoff equation, as reported earlier, and represent the mean from at least three different determinations. All CA isozymes used here were recombinant proteins obtained as reported earlier by our group 25,26 .

Chemistry
We commenced our investigation with a reaction using an equimolar ratio of isatin and 4-methyl benzohydrazide as model substrates using molecular iodine (100 mol%) and Cs 2 CO 3 (1.0 equiv.) in DMSO at 100 C ( Table 1). The desired product was obtained in 71% yield (Table 1, entry 1). No product was obtained in the absence of either catalyst or base which suggests that an iodine/ base combination is required for the reaction to occur (Table 1, entries 2-4). Exploring the possibility for improving the reaction efficiency, the effect of other alkali metal carbonates/other bases on the reaction efficiency was then examined. The transformation underwent smoothly in the presence of K 2 CO 3 to afford the desired product 3a in 80% yield after 12 h (Table 1, entry 5),whereas other bases, such as Na 2 CO 3 , K 3 PO 4 , and NaHCO 3 were found to be less effective (Table 1, entries 6-8). With an attempt to further optimise the yield of the product, we investigated the influence of various iodine reagents. TBAI, N-iodosuccinimide (NIS) and KI gave poor to moderate yields, i.e. of 18, 45, and 35%, respectively (Table 1, entries 9-11). However, phenyliodine(III) diacetate (PIDA), and hydroxy(tosyloxy)iodobenzene (HITB) did not at all lead to the formation of the desired product 3a (Table 1, entries 12-13). Furthermore, a series of experiments were also carried out in various other solvents, such as, DMF, MeCN, THF, 1,4dioxane, EtOH, MeOH, and H 2 O. From the obtained results, it can be seen that the use of DMSO and DMF at 120 C gave an almost identical result, albeit with a lower yield in the latter case (Table 1, entries 14-15), whereas, MeCN, THF, 1,4-dioxane, EtOH, MeOH, and H 2 O at reflux temperatures proved to be less effective (Table 1, entries 16-21). Furthermore, the iodine loading was also investigated in this reaction, and the yields were dropped to 59 and 52 at 0.75 and 0.5 equiv., respectively ( Scheme 2. One pot synthesis of the 2-(1,3,4-oxadiazo-2-yl)aniline derivatives. (a) Reaction conditions: 1 (1 equiv.), 2 (1.05 equiv.), I 2 (1.0 equiv.), K 2 CO 3 (1.5 equiv.) in DMSO (3 ml) under mW irradiation at 160 C for 30-40 min, (b) isolated yields, and (c) The reaction was performed on gram scale. and to a significantly lower value of 33% at 0.2 mol equiv. of I 2 (Table 1, entry 24). We also conducted a control experiment under nitrogen atmosphere, but the yield under these conditions was diminished to 25%. This indicated that atmospheric O 2 played an important role in the above transformation. Surprisingly, when the same reaction was performed under microwave irradiation gave better yield of 3a (91%) within a short span of time (40 min). Indeed, the use of microwave technology has never been mentioned in the literature for the synthesis of 2-(1,3,4-oxadiazol-2yl)aniline derivatives up until now. Thus the foregoing experiments led to the conclusion that the conditions used under entry 25 of Table 1 are the optimal ones for the reaction and, therefore, the microwave conditions were employed subsequently for all further reactions to generate compounds 3a-3u/6a-6g/8a-o.
With the optimised conditions in hand, we started our exploration towards finding the potential applicability of this intramolecular decarboxylating domino reaction by attempting to prepare a variety of 1,3,4-oxadiazoles, using a varied set number of substituted isatins and benzohydrazides. The results are summarised in Scheme 2. In this way, a diversified set of 1,3,4-oxadiazoles 3a-3u were obtained in moderate to excellent yields. It was found that the reactions were equally successful with both electron withdrawing (4-CF 3 , 4-F, 4-Br, and 3,5-dichloro) as well as electron donating substituents [4-Me, 2-Me-4-OMe, and 3,4,5-(OMe) 3 ] on the hydrazide component. However, it may be emphasised that in contrast to other electron-withdrawing substituents, the 4-nitro group-bearing substrates required longer time to complete the reaction satisfactorily (3h).
In order to obtain some mechanistic insights into the nature of the reaction, radical trapping experiments were performed by employing TEMPO (0.5 equiv.) under the set of optimised conditions mentioned above. Indeed, no desired product was obtained when the reaction was performed in the presence of TEMPO, suggesting clearly that the reaction took place through a radical pathway (Scheme 5).
In the light of the obtained results and the work reported in the literature 8,17 , a plausible reaction mechanism is proposed, which is shown in Figure 3, using 1a and 2a as the starting materials for the iodine-mediated domino reaction. Isatin (1a) condenses with the hydrazide (2a)  oxygen, oxidises B to form the radical intermediate C.
Subsequently, abstraction of a proton by the base, followed by a cyclisation gave the intermediate E, which by elimination of one molecule of CO 2 and HI, led to the formation of the final product 3a.
1,3,4-Oxadiazoles are well known for exhibiting a specific fluorescence 21 . In this regard, we investigated the excitation and emission spectra of some representative molecules described here in diluted DMSO as solvent. The excitation and emission spectra of compounds 3e and 3o showed prominent shifts to longer wavelengths, in contrast to the other cases in which the shifts were typically only marginal. Figure 4 shows the fluorescence emission spectra of compounds 3g, 3q, and 3s. Fluorescence properties of these compounds suggest that they may hold a potential for applications as chemical probes.

Carbonic anhydrase inhibition
Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of metalloenzymes, present in most living organisms, in which they catalyses a simple physiological reaction, i.e. the reversible hydration of CO 2 to bicarbonate and protons via a ping-pong mechanism. These enzymes are involved in many physiological and pathological processes, such as pH and CO 2 homeostasis, respiration and transport of carbon dioxide and bicarbonate between metabolising tissues and lungs, electrolyte secretion in various tissues and organs, biosynthetic reactions (gluconeogenesis, lipogenesis, and ureagenesis); calcification, bone resorption, and tumourigenicity (in mammals) 22,23 . Dysregulated activities of these carbonic anhydrases were proven to be connected with different human diseases, and inhibition of these enzymes by small molecules represents an efficient strategy in chemotherapeutic intervention.   Sulphonamides and their bioisosteres (sulfamates and sulfamides), represents the main class of pharmacologically relevant CA inhibitors. Hence, it was of interest to evaluate the CA inhibitory activity of 2-(1,3,4-oxadiazolo-2-yl)aniline-benzene sulfonamides (8a-o) reported here. Thus, compounds 8a-o were investigated as inhibitors of four catalytically active human (h) CA isoforms, i. e. widespread, cytosolic, hCA I, and hCA II, the membrane-anchored hCA IV, as well as the transmembrane hCA IX, using the clinically used compound acetazolamide as a standard inhibitor.
The inhibition data are shown in Table 2. The following structure-activity relationship (SAR) can be delineated from the data of Table 2: i. The slow cytosolic isoform hCA I was inhibited by all the examined sulphonamide derivatives 8a-o with inhibition constants (K I s) spanning between 81.4 and 5828.3 nM. Sulphonamides incorporating fluoro (8g) and dimethylaniline moieties (8h) showed medium nanomolar activity (K I of 81.4 and 89.1 nM, respectively). The other analogues were less potent and exhibited high nanomolar to low micromolar inhibitory potency against this isoform (K I s ranging between 222.2 and 5828.3, respectively) 25,26 . ii. hCA II, the dominant physiological isozyme, which is an antiglaucoma drug target, was inhibited by all the tested compounds, with efficacy spanning from the low to the high nanomolar range (K I s of 6.4-515.7 nM, Table 2). Among these, compound 8g displayed the highest inhibitory activity with a K I of 6.4 nM. Compounds 8a-d, 8f, 8h-k, and 8o also showed nanomolar inhibitory activity against this isoform, with K I s in the range of 16.4-86.3 nM, whereas remaining analogues exhibited high nanomolar inhibitory action. Among all these compounds, the 4-sulfamoyl derivatives showed a better CA II inhibitory activity compared to the 3-sulfamoyl derivatives. For example compounds, 8c and 8g were 31 and 13 times more potent than 8l and 8o, respectively. On the other hand, substitution on the aniline fragment also had a significant role on activity, i.e. o-fluoro substituted aniline bearing analogues 8g and 8o were more active in comparison to the psubstituted analogues 8d and 8m. From these observations, it can be clearly demonstrated that the substitution pattern on both phenyl rings had significant effect on the inhibitory activity against hCA II.
iii. hCA IV, which is a membrane-associated isoform majorly expressed in the eye, lungs, and kidneys, being involved among others in glaucoma and retinitis pigmentosa diseases, was not particularly prone to inhibition by the sulphonamides investigated here In fact, all screened molecules (8a-o) displayed micromolar inhibitory activity, except 8j-m and 8o, which showed high nanomolar CA inhibitory activity, with K I s of 437.3-548.3 nM (Table 2). iv. hCA IX, the tumour-associated isoform, was moderately inhibited by all tested compounds with K I s in the range of 140.3-2964.0 nM. Substitution on both phenyl rings did not significantly influence the inhibition profile of these compounds for this isoform. Of the screened compounds, 8c, 8n, and 8o exhibited better CA IX inhibitory profile against this isozyme, with K I s of 140.3-282.1 nM ( Table 2).

Conclusions
We have developed a more efficient and environmentally friendly protocol for the construction of 2-(1,3,4-oxadiazol-2-yl)aniline derivatives through one-pot domino decarboxylation by employing molecular iodine under microwave irradiation. This strategy works well with various substituted isatins and hydrazides belonging to both aryl and heteroaryl series, also showing good functional group tolerance. Furthermore, this protocol provided various oxadiazoles, which can be used for further functionalisation protocols. The reaction mechanism of this domino reaction was also delineated and presented in this article. Many of the synthesised molecules exhibited fluorescence properties that indicate their potential usefulness in the field of material chemistry. The synthesised 2-(1,3,4-oxadiazolo-2-yl)aniline-benzene sulfonamides (8a-o) were tested for their CA inhibitory activity and it was noticed that compounds 8c, 8g, and 8h displayed promising and selective activity against isoform hCA II with K I s of 16.4, 6.4, and 17.6 nM, respectively. Such compounds may be useful for various applications in which the CA activity must be inhibited, such as for the design of anti-glaucoma, antiobesity, or antitumor agents 27,28 .