Synthesis and COX inhibition of 7-R 1 -8-R 2 -1-ethyl-3,4-dimethyl-4,10-dihydro-1 H -pyrazolo[3,4-c ][1,5]benzodiazocine-5,11-diones

The title compounds were easily synthesized by reacting the 4-aminopyrazole hydrochloride 2 and the substituted 2-nitrobenzoyl chlorides 3a-d . The obtained 2-nitrobenzamides 4a-d were methylated and then reduced to give the corresponding amines 6a-d . These were hydrolyzed then directly converted into 4,10-dihydro-1 H -pyrazolo[3,4-c ][1,5]benzodiazocine-5,11-diones 1a-d by the action of SOCl 2 in benzene. These were tested for their COX inhibitory activity, showing an inhibitory profile against both COX-1 and COX-2, being slightly more selective against COX-2 with a percentage of inhibition, at the concentration of 10 µM, in the range 42.0 – 55.0.


Introduction
The main part of our research has been devoted to synthetic methods leading to new ring systems, containing the pyrazole nucleus, as a pharmacophore moiety for potential drugs.In particular, the pyrazole nucleus represents a very attractive scaffold to obtain new molecules endowed with anti-inflammatory activity.3][4][5] Finally, selective COX-2-inhibiting activity for 1H-pyrazolylbenzo[d]thiazoles, 1Hpyrazolylbenzo [d]oxazoles, and 1H-pyrazolylbenzo [d]imidazoles, as well as bicyclic pyrazoles have been described. 6,7ecently, in the course of our studies on pyrazolodiazocines, we have reported the successful procedure affording novel pyrazolo [3,4-c] [1,6]benzodiazocin-11-ones 8 and pyrazolo [3,4c] [1,5]benzodiazocine-5-ones. 9s an extension of our research, in order to gain more insight into the pyrazolobenzodiazocine series, and particularly with regard to their analgesic and/or antiinflammatory activity, we sought to synthesize novel pyrazolo [3,4-c] [1,5]benzodiazocine -5,11dione derivatives of type 1 and screen them for their ability to inhibit selectively the cyclooxygenase of type 2 (COX-2).Thus, in the present investigation pyrazolobenzodiazocines of type 1 were synthesized and evaluated for their in vitro COX-1/COX-2 isoenzyme inhibition.We believe that the efficiency of the described synthesis of compounds 1 (see Scheme 1) could give access to a series of 4,10-dihydro-1H-pyrazolo[3,4-c][1,5]benzodiazocine-5,11diones, for further study in order to investigate their SAR and to verify the promising results with particular regard to the "in vivo" anti-inflammatory activity.

Results and Discussion
Compounds 1a-d were obtained as shown in the Scheme 1.We started our synthesis by reacting 4-aminopyrazole hydrochloride, 2, with the substituted 2-nitrobenzoyl chlorides 3a-d to give the corresponding 2-nitrobenzamides 4a-d in good yields (60-69%).Reaction of these latter with methyl iodide in alkaline medium afforded the methyl derivatives 5a-d in 53-60% yields.Reduction of these compounds with iron and hydrochloric acid in aqueous ethanol led to the corresponding amines 6a-d in reasonable yields (53-59% ).

ISSN 1551-7012
Page 3 © ARKAT USA, Inc.These products, in turn, were transformed by hydrolysis to the corresponding acids which were not isolated but converted directly into 4,10-dihydro-1H-pyrazolo [3,4-c]- [1,5]benzodiazocine-5,11-diones, 1a-d, by the action of SOCl 2 in benzene solution, in 55-70 % yields.The structures of compounds 1, and 4-6 were confirmed by spectroscopic data as well as elemental analysis.Both the 1 H-NMR spectra of compounds 5a-d (in d 6 -dimethyl sulfoxide, and the 13 C-NMR spectrum of compound 5a, taken as an example, provided evidence for the presence of a rotational isomerism because of the hindered rotation due both to the partial double bond character of the amide C-N bond, and to the steric hindrance of the substituents in the positions 5 (ester group) and 2' (nitro group) (Tables 1 and 2).In fact, the double signals for each of the carbon atoms in the 13 C-NMR spectrum of compound 5a gave an account of the relative abundance of the two conformers (59-70 % for the more abundant isomer).Thus, two signals appeared for the NCH 3 carbon atom, the upfield ones being more abundant.According to the literature, 10 the upfield resonance was assigned to the conformer bearing the methyl group syn-to the carbonyl.Also, the amide carbonyl carbon resonance of the more abundant conformer resulted in an upfield shift with respect to the corresponding isomer.
Finally, in the case of derivatives 1, the 1 H-NMR spectra indicated hindered internal rotation of the N-ethyl group.In fact, the methylene protons of the ethyl group bonded at the 1-position of the tricyclic system are diastereotopic, and showed signals as multiplets in the 3.92-4.12ppm region, due to geminal and vicinal coupling.
The ability of compounds 1a-d to inhibit the conversion of arachidonic acid to prostaglandin H 2 (PGH 2 ) was determined using a COX-1/COX-2 inhibitor screening assay kit (Kit No. 560101;Cayman Chemical, Ann Arbor, MI, USA).The results reported in Table 3 show that compounds 1 are endowed with a good inhibitory activity against both COX-1 and COX-2.All compounds 1 were slightly more selective against COX-2, showing at a 10 µM concentration, a percentage of inhibition in the range 42-55.The 8-chloro derivative 1c was the most active, with 55% of COX-2 inhibition.The most active against COX-1 was the unsubstituted derivative 1a, with a percentage 43.1.
These preliminary results are quite encouraging and further investigation of the structureactivity and toxicity of these kind of compounds are currently underway in our group, with the aim of obtaining more COX-2-selective compounds.

General procedure for the synthesis of methyl 4-[(2-aminobenzoyl(methyl) amino]-1-ethyl-3-methyl-1H-pyrazole-5-carboxylate (6a-d)
Compounds 5a-d (8 mmol) were dissolved in 95% ethanol (10 mL) diluted with water (3.5 mL) and cooled to 25 °C.Iron powder (2.0g, 36 mmol) was added, followed by HCl (36%, 1 mL).An exothermic reaction resulted for the next 15 minutes; after this reaction subsided, the mixture was stirred under reflux for another 2 h.The cooled reaction mixture was filtered using Celite.The filter cake was washed well with ethanol, and the filtrate was evaporated under reduced pressure.The residue was diluted with ether (100 mL), and the solution extracted with water and dilute NaOH (4%).The ether solution was dried over Na 2 SO 4 and after evaporation a residue was obtained which was purified from a small amount of ethanol.6a: yield 50%; mp 114-115 °C (beige crystals); IR (KBr) (cm -1 ) 3471 and 3363 (NH 2 ), 1717 and 1645 (CO); 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 emymatic 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 compound-treated to various control incubations (duplicate determinations).

1 .
Scheme 1. Synthetic pathway for the formation of the compounds 1a-d.

Table 1 .
1 H-NMR chemical shifts of compounds 5: δ H [ppm] (the signals related to the minor conformers are in round brackets)

Table 3 .
Percent All melting points were taken on a Büchi-Tottoli capillary apparatus and are uncorrected.IR spectra were recorded on a Perkin-Elmer Infracord 137 spectrophotometer as Nujol mulls; 1 H-and 13 C-NMR spectra were obtained in DMSO-d 6 at 300.13 and 75.47 MHz respectively, using a Bruker AC series 300 MHz spectrometer (TMS as internal reference).Mass spectra were recorded on a JEOL jms-0I-SG-2 spectrometer at 75 eV (100µA).