Synthesis of chiral pyrazolo[4,3-e][1,2,4]triazine sulfonamides with tyrosinase and urease inhibitory activity

Abstract A new series of sulfonamide derivatives of pyrazolo[4,3-e][1,2,4]triazine with chiral amino group has been synthesized and characterized. The compounds were tested for their tyrosinase and urease inhibitory activity. Evaluation of prepared derivatives demonstrated that compounds (8b) and (8j) are most potent mushroom tyrosinase inhibitors whereas all of the obtained compounds showed higher urease inhibitory activity than the standard thiourea. The compounds (8a), (8f) and (8i) exhibited excellent enzyme inhibitory activity with IC50 0.037, 0.044 and 0.042 μM, respectively, while IC50 of thiourea is 20.9 μM.

Tyrosinase (EC 1.14.18.1) is a multifunctional copper containing enzyme from oxidase family, widely distributed in animals, plants and microorganisms 13 . It catalyzes two distinct reactions of melanin synthesis, the hydroxylation of L-tyrosine by monophenolase action and the oxidation of L-DOPA (3,4-dihydroxyphenylalanine) to o-dopaquinone 14 . Tyrosinase catalyzes melanin biosynthesis which plays a crucial role in protecting the skin from solar radiations. Melanin can serve as a photoprotector and is responsible for the pigmentation and color patterns of mammalian skin. It has been reported that effective tyrosinase inhibitors could ameliorate skin hyperpigmentation, neurodegenerative diseases, and could potentially improve cancer remedies 15,16 . However, when exposed to excessive ultraviolet light, melanin produced in excess causes various skin disorders 17 .
Urease (EC 3.5.1.5) is a nickel containing enzyme that catalyzes the hydrolysis of urea to ammonia and carbon dioxide or carbamate by at least 10 14 over the spontaneous reaction 18 . It is of our interest to focus on the discovery of novel urease inhibitors as it is already known that these compounds play a crucial part in the therapies both human and plants disorders. In agriculture, high urease activity during urea fertilization causes significant environmental and economic problems by releasing abnormally large amounts of ammonia into the atmosphere 19,20 . Moreover, it contributes to the development of peptic ulcers, pyelonephritis, kidney stones, hepatic encephalopathy, hepatic coma urolithiasis and urinary catheter encrustation 21 .
Our current work continuing our interest in the synthesis of new sulfonamide derivatives of pyrazolo [4,3-e] [1,2,4]triazine as new inhibitors of tyrosinase and urease enzymes. The synthesis pathway leading to the title compounds is depicted in Scheme 1.

Materials and methods
All chemicals used were of reagent grade quality and were used as received without any further purification and were obtained from Sigma-Aldrich. Melting points were determined on a Mel-Temp apparatus and are uncorrected. 1 H-and 13 C-NMR spectra were recorded on a Varian spectrometer (400 MHz for 1 H and 100 MHz for 13 C). The chemical shift values are expressed in ppm (part per million) with TMS as internal reference. The relative integrals of peak areas agreed with those expected for the assigned structures. Molecular weight of final compounds were assessed by electrospray ionization mass spectrometry (ESI/MS) on a Agilent Technologies 6538 UHD Accurate Mass Q-TOF LC/MS. Elemental compositions are within ±0.4% of the calculated values. For preparation and spectroscopic data of compounds 2-7 and 8a, 8b, 8 h, 8i see literature [22][23][24] .

Synthesis of sulfonamides 8a-j
A mixture of sulfonyl chloride 7 (100 mg, 0.29 mmol) and amine (100 mg, 1 mmol) in anhydrous acetonitrile (5 mL) was stirred overnight at room temperature, and then the reaction mixture was concentrated in vacuo to afford the crude sulfonamide, as a yellow solid 2,24 . The residue was purified on silica gel using a mixture of CH 2 Cl 2 :EtOH (25:1) as eluent to give the titled compounds as a yellow solid.

Mushroom tyrosinase inhibition assay
The mushroom tyrosinase (EC 1.14.18.1) (Sigma Chemical Co.) was used for in vitro bioassays as described previously with some modifications. 25,26 Briefly, 140 lL of phosphate buffer (20 mM, pH 6.8), 20 lL of mushroom tyrosinase (30 U/mL) and 20 lL of the inhibitor solution were placed in the wells of a 96well micro plate. After pre-incubation for 10 min at room temperature, 20 lL of L-DOPA (3,4-dihydroxyphenylalanine) (0.85 mM) was added and the plate was further incubated at 25 C for 20 min. Subsequently the absorbance of dopachrome was measured at 475 nm using a micro plate reader (OPTI Max , Tunable). Kojic acid was used as a reference inhibitor and for negative tyrosinase inhibitor phosphate buffer was used instead of the inhibitor solution. The extent of inhibition by the test compounds was expressed as the percentage of concentration necessary to achieve 50% inhibition (IC 50 ). Each concentration was analyzed in three independent experiments run in triplicate. The IC 50 values determined by the data analysis and graphing software Origin 8.6, 64-bit (OriginLab Corporation, Northampton, MA).

Urease inhibition assay
The urease activity was determined by measuring amount of ammonia produced with indophenols method described by Weatherburn 27 . The reaction mixtures, comprising 20 lL of enzyme (Jack bean urease, 5 U/mL) and 20 lL of test compounds in 50 lL buffer (100 mM urea, 0.01 M K 2 HPO 4 , 1 mM EDTA and 0.01 M LiCl 2 , pH 8.2), were incubated for 30 min at 37 C in 96-well plate. Briefly, 50 lL each of phenol reagents (1%, w/v phenol and 0.005%, w/v sodium nitroprusside) and 50 lL of alkali reagent (0.5%, w/v NaOH and 0.1% active chloride NaOCl) were added to each well. The absorbance at 625 nm was measured after 10 min, using a microplate reader (OPTI Max , Tunable). All reactions were performed in triplicate. The urease inhibition activities were calculated according to the following formula: Where OD control and OD sample represents the optical densities in the absence and presence of sample, respectively. Thiourea was used as the standard inhibitor for urease.

Kinetic study
Lineweaver-Burk plots of 1/absorbance versus 1/L-DOPA and 1/absorbance versus 1/Urea were used to determine the type of enzyme inhibition. A series of experiments were performed to determine the inhibition kinetics by following method 18,28,29 . Different inhibitor concentrations of compound 8b with 0, 15, 30, 61, 123 and 247 lM, 8j with 0, 14, 28 and 55.8 lM, respectively were used in case of mushroom tyrosinase inhibition. Substrate L-DOPA concentration was between 0.062 and 2 mM in all tyrosinase kinetic study. Pre-incubation and measurement time was the same as discussed in mushroom tyrosinase inhibition assay protocol. Maximal initial velocity was determined from initial linear portion of absorbance up to five minutes after addition of enzyme at a 30 s interval. The inhibition type on the enzyme was assayed by Lineweaver-Burk plots of inverse of velocities (1/V) versus inverse of substrate concentration 1/[S] mM À 1 , and the inhibition constant Ki was determined by two methods, by secondary replot of 1/V (y-intercept of Lineweaver-Burk plot) versus inhibitor concentrations and by Dixon plot of inverse of velocities (1/V) versus inhibitor concentrations. Urease inhibition was measured by varying the concentration of urea in the presence of different concentrations (0, 0.125, 0.25 and 0.5 lM) of compound 8a. Inhibitory constant (Ki) was determined as the intersection on the X-axis of the secondary replot of 1/V (y-intercept of Lineweaver-Burk plot) versus inhibitor concentrations and by Dixon plot of inverse of velocities (1/V) versus inhibitor concentrations. Briefly the urea concentration was changed from 3.12 to 100 mM for urease kinetics studies and remaining procedure was same as describes in urease inhibition assay protocol. Urease activity was determined by measuring ammonia production using the indophenol method as reported previously 30 . The results (change in absorbance per min) were processed by using SoftMaxPro software(Molecular Devices Corporation Sunnyvale, CA) and a Dixon plot was constructed by plotting (1/V) versus inhibitor concentration [I].

Chemistry
The synthesis of target sulfonamides 8a-i was achieved by a convenient multiple procedure starting from 3-methylsulfanyl-1,2,4-triazine (1) as shown in Scheme 1. Briefly, in the first step the reaction of 1 with nitroethane in KOH/DMSO mixture at room temperature gave appropriate oxime 2 as a main product 22 , which was readily transformed into ketone 3 in good yield 23 . Compound 3 was subjected to the reaction with methylhydrazine in the presence of acidic media according to standard procedure to give suitable hydrazone 4 as key intermediate for the preparation of 1H-pyrazolo[4,3-e][1,2,4]triazine derivative 5. The hydrazone 4 could be converted into derivative 5 under conventional heating (10% HCl, EtOH, reflux, 1 h) 31 or under solvent free reaction conditions according to our previous published procedure 32 . Using Guillaumet and co-worker's method 33 for the palladium-catalyzed cross-coupling reaction of 3-methylsulfanyl-1,2,4-triazine with boronic acid derivatives we have reacted 5-methylsulfanyl-1H-pyrazolo[4,3-e][1,2,4]triazines 5 with 2-ethoxyphenylboronic acid in the presence of copper (I) 3-methylsalicylate to obtain derivative 6 in excellent yield. Chlorosulfonylation reaction of compound 6 in neat chlorosulfonic acid at 0 C proceeded smoothly and selectively at the 5'-position of the phenyl ring to give the desired product 7 in excellent yield. The chlorosulfonyl derivative 7 was readily coupled with appropriate pure enantiomeric amines in acetonitrile at room temperature to produce the final chiral sulfonamides 8a-i in high yield.
Tyrosinase contains one binuclear copper complex in the catalytic center and the catechol hydroxyl groups of the substrate are suggested to be bound to the copper atoms during the enzymatic oxidation process. Phenolic hydroxyl groups are generally capable to coordinate copper atoms causing inhibition of the enzyme in competition to the catechol substrate. However, our previous study 10 showed that inhibitors with NH group in the sulfonamide part (as piperazinyl, homopiperazinyl) are more active as they reduce the enzyme activity of tyrosinase to 29.5%-33.5%. It was suggested, that NH or free NH 2 group are involved in the inhibition effect of tyrosinase activity. Our suggestion was confirmed by the activity of some inhibitor with two NH groups that reduced enzyme activity of tyrosinase to 20.2%. The inhibition of the compound 8j may arise with strong participation OH group due to its conformation. However, other tested derivatives with OH were only weak tyrosinase inhibitors. Therefore, we suggest that primary binding to one copper atom should be strengthened by additional interactions between the inhibitor e.g. p electrons of the ring and another copper atom or the amino acid residues of the enzyme's active site to accomplish the inhibition.
Urease assay The synthesized pirazolotriazine-sulfonamides (8a-j) have been evaluated for their inhibitory effects on urease. All of the synthesized compounds showed better inhibitory activity with IC 50 ranging from 0.037 to 0.084 lM than thiourea with IC 50 of 20.7 lM. The structures of amino alcohols in sulfonamide group were varied to study their role in enzyme inhibitory activity. The excellent activity was shown by derivatives (8a) and (8i) with IC 50 0.037 and 0.042 lM, respectively. In case of compound (8a) the presence of 2-hydroxy-1-methyl ethaneamine moiety with S configuration at sulfonamide group has central importance in urease inhibitory activity in the group of tested derivatives. This moiety in compound (8a) is responsible for its most potent urease inhibitory activity. On the other hand, isomeric derivative (8b) with R-configuration in sulfonamide part showed the lowest activity in the group of obtained compounds although it exhibited higher urease inhibitory activity than standard thiourea.
Mechanism underlying inhibitory effect of compounds (8a), (8b) and (8j) Since (8b) and (8j) against mushroom tyrosinase and (8a) against urease enzyme were the most potent inhibitors, we therefore further study mechanism underlying their inhibitory effect. The kinetic studies of the enzyme by the Lineweaver-Burk plot of (1/V) versus 1/[S] in the presence of different concentrations of inhibitors (8b),  (8j) and (8a) which gave a series of straight lines as shown in Figures 1-3(a). Furthermore, inhibition constant (Ki) were determined to gain insightful pathway by secondary replots and Dixon plots as shown in Figures 1-3(b,c). The results showed that compound (8b) behaved as a noncompetitive inhibitor (Figure 1), which means that it could combine with both the free enzyme and the enzyme-substrate complex. Thus, the interaction between (8b) and the enzyme was independent from the interaction between the substrate and the enzyme, and the inhibitor did not change substrate-enzyme affinity. With increasing concentrations, a series of lines was revealed with a common intercept on the 1/[S] axis, but with different slopes. On the other hand in Dixon plot (8b) was consistent with the characteristic patterns of noncompetitive inhibition with Ki value 40 lM and compound (8j) show mixed type inhibition with Ki value 20 lM as shown in Figure 2(a,b). In case of compound (8a) whose kinetic mechanism was studied against urease, by increasing the concentration of substrate (urea) gave family of straight lines, all of which intersected within the second quadrant. The analysis showed that V max decreased with increasing K m in the presence of increasing concentrations of (8a) This behavior of compound (8a), indicated that it is a mixed type inhibitor with respect to the substrate urea with Ki value 0.01 lM as shown in Figure  3(a-c). The results of inhibition type and inhibition constants are summarized in Table 2.

Conclusions
We have described facile and efficient method for the preparation of new chiral 1H-pyrazolo[4,3-e][1,2,4]triazine sulfonamides from simple available starting materials. The sulfonamides (8a-j) have been synthesized to validate their role in tyrosine and urease inhibitory activity. The most potent inhibitory activity against tyrosinase was displayed by compounds (8b) and (8j) with IC 50 30.76 and 27.90 lM, respectively. All of the obtained derivatives showed higher urease inhibitory activity than the standard thiourea. The kinetic analysis exhibited that compounds (8b) is noncompetitive inhibitor while (8j) is a mixed type inhibitor of tyrosinase and (8a) is a mixed type inhibitor of urease. According to the systematic investigation it could be deduced that pyrazolotriazine sulfonamides are a promising urease inhibitors for treatment of the urease related diseases.  Ki is enzyme inhibition constant.
is not determined.