Synthesis of pyrazolo[4,3-c ][1,2,6]benzothiadiazocine, a new ring system as potential COX inhibitor

Derivatives of the new ring system 1,4-dihydropyrazolo[4,3-c ][1,2,6] benzothiadiazocin-11(10 H )one 5,5-dioxide were synthesized in five or six steps in 57-66% overall yields and tested as COX inhibitors.


Introduction
Cyclooxygenases (COXs) are key enzymes in the synthesis of prostaglandin H2 which is a precursor for the biosynthesis of prostaglandins, thromboxanes, and prostacyclins. 1 COX enzymes exist in two isoforms, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2).COX-1, the constitutive form of the enzyme, is present in the stomach, intestines, kidneys, and platelets.This form is mainly responsible for the physiological production of prostanoids.COX-2, although it is also constitutively expressed in brain, spinal cord, and kidneys, is an inducible form and its expression is triggered under pathological conditions such as inflammation.
Several studies have reported the development of new pyrazole analogues for the development of novel drugs with better safety profiles that could be used long-term to relieve chronic inflammatory conditions. 7

Figure 1
Recently some of us described the synthesis of pyrazolo [3,4-c] [1,5]benzodiazocine derivatives that showed inhibitory profile against both COX1 and COX2. 8ur starting point was synthesizing new condensed heterocycles containing a pyrazoles moiety in order to investigated how structural changes affect the anti-inflammatory properties.The analgesic and anti-inflammatory activity of substituted dibenzo[c,g][1,2,6]thiadiazocines has also been reported. 9n view of the above facts, and in continuation of our search for anti-inflammatory compounds, we here report the synthesis of new ring system 1,4-dihydropyrazolo [4,3-c] [1,2,6]  benzothiadiazocin-11(10H)one 5,5-dioxide of type 5 a potential COXs inhibitor.
Reduction of the nitro group of compounds 3a-c with stannous chloride in hydrochloric acid led to the corresponding amines 4a-c in good yields (79-89%).
The structure of the tricyclic derivatives 5a-c was confirmed by spectral data as well as elemental analysis.Thus, in the IR spectra, the NH stretchings were found in the range 3188-3298 cm -1 and the carbonyl absorptions were observed in the range 1645-1687 cm -1 .The 1 H NMR spectra, beside the benzene protons, showed the methyl group in position 3 as a singlet in the range 2.02-2.12δ; the pyrazole N-ethyl protons were found at 1.08-1.18δ and 4.00-4.04δ, while the substituent protons next to N-4 nitrogen were observed in the range 3.18-3.65δ; the amide hydrogen protons, instead, resonated at 10.09-10.64δ.In the 13 C NMR spectra the carbonyl singlets were observed at 161.6-162.2ppm.
Compounds 5a, 5b and 5c were tested for their ability to inhibit COX-1 and COX-2 unfortunately none of them showed significant anti-inflammatory activity showing COX-1 inhibition of 5.2, 5.6, and 6.4% respectively and COX-2 inhibition of 5.3, 5.5, and 6.6% respectively.

Experimental Section
General.All melting points were taken on a Büchi-Tottoli capillary apparatus and are uncorrected; IR spectra were determined in bromoform with a SHIMADZU IR Affinity-1 spectrophotometer; 1 H and 13 C NMR spectra were measured at 200 and 50.3 MHz, respectively in DMSO-d 6 solution, using a Bruker Avance II series 200 MHz spectrometer (TMS as internal reference).Mass spectra were recorded on a JEOL JMS-OI-SG-2 spectrometer at 75 eV (100 μA).Elemental analyses (C, H, N), performed with a VARIO EL III elemental analyzer, were within ±0.4% of the theoretical values.

Biology
In vitro cyclooxygenase inhibition assay.The reference compounds (indomethacin and NS398) were purchased from Cayman Chemical, Ann Arbor, MI (cat.N. 70270 and 70590 respectively).
They are used at the final concentration of 0.2 μM, according to the manufacturer's instructions.Compounds 5a-c were tested for their ability to inhibit COX-1 and COX-2 using a COX-(ovine) inhibitor screening kit (Catalogue No 560101, Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions.Cyclooxygenase catalyzes the first step in the biosynthesis of arachidonic acid (AA) to PGH2 PGF2α produced from PGH2 by reduction with stannous chloride is measured by enzyme immunoassay (ACE competitive EIA).Stock solutions of test compounds were dissolved in a minimum volume of DMSO.Briefly, to a series of supplied reaction buffer solutions (950 μL, 0.1 M Tris-HCI pH 8.0 containing 5 mM EDTA and 2 mM phenol) with either COX 1 or COX-2 (10 μL) enzyme in the presence of heme (10 μL) was added 20 μL of 10 μM concentration of test drug solutions.These solution were incubated for a period of 2 min at 37 °C after which 10 μL of AA (100 μM) was added, and the COX reaction was stopped by the addition of 50 μL of 1 M HCI after 2 min.PGF2 produced from PGH2 by reduction with stannous chloride, was measured by enzyme immunoassay.This assay is based on the competition between PGs and a PG-acetylcholinesterase conjugate (PG tracer) for a limited amount of PG antiserum.The amount of PG tracer that is able to bind to the PG antiserum is inversely proportional to the concentration of PGs in the wells, since the concentration of the PG tracer is held constant while the concentration of PGs varies.This antibody-PG complex binds to a mouse anti-rabbit monoclonal antibody that had been attached to the well previously.The plate is washed to remove any unbound reagents and then Ellman's Reagent, which contains the substrate to acetylcholinesterase is added to the well.The product of this enzymatic reaction produces a distinct yellow color that absorbs at 405 nm.The intensity of this color, determined spectrophotometrically, is proportional to the amount of PG tracer bound to the well, which is inversely proportional to the amount of PGs present in the well during the incubation absorbance ∝ [bound PG tracer] ∝ 1/PGs.Percent inhibition was calculated by comparison of compoundtreated to various control incubations (duplicate determinations).