Synthesis and analgesic potential of 4-[4–chloro-3- (trifluoromethyl)-phenyl]-4-piperidinol analogues against pain model in mice

In the study of designing pharmacophore models for analgesic, a series of 4-[4–chloro-3- (trifluoromethyl)-phenyl]-4-piperidinol (TFMP) derivatives were synthesized and characterized by physical and spectral method (HR-EIMS, HR-FABMS, 1H-NMR, 13C-NMR, UV, and FT-IR). The analgesic action of the synthesized derivatives was estimated by means of Hot Plate Method. Most of the compounds displayed potent analgesic efficacy and an ultrashort to long duration of action. The results indicate that these compounds are useful as analgesics. In the presence of naloxone they displayed pain reliving effect. In conclusion, among active compounds 3 (188%), 5 (137%), 6 (162%), and 8 (107%) respectively, emerged as most effective analgesic and they depressed peripheral and centrally mediated pain by opioid independent systems.


Introduction
Pain is a complex body response to noxious stimulus both chemical (acetic acid and formalin) or physical (heat and cold pressure) [1,2]. Primarily inflammation or tissue injuries have been connected with pain stimulation through the release of quite a lot of inflammatory mediators that sensitizes as well as amplify nociceptive responses [3]. Pain is together a major economic and clinical delinquent, distressing large population than diabetes, cancer and heart malady. Currently, variety of prescribed medicines are existing for managing of pain, opioid drugs, exclusively those substituted μ-opioid receptor and associated ways. They have established to be the utmost active, regardless of more or less severe side effects comprising addiction, dependence, respiratory depression, pruritus, nausea, hypotension, and constipation. As a result it is needed to develop novel μ opioid receptor analgesics that preserve their opioid analgesic properties but less or no adversative side effects [4][5][6].
Among the major classes of μ agonists piperidine substituted moiety e.g. Fentanyl, Pethidines (meperidine), Piminodine, 4-Anilidopiperidine etc. More advanced investigation has been accomplished for the substituted piperidine derivatives to develop superior molecules for the treatment of pain [7][8][9][10][11][12]. Pethidine is phenylpiperidine posseses morphine-like activity [13]. When compared with morphine, pethidine was thought to be safer, carry a lower risk of addiction, and to be superior in treating the pain associated with biliary spasm or renal colic [14]. Numbers of its derivatives having substitution at Nitrogen of piperidine ring have been reported possessing relatively better analgesic potential with minimal side effect e.g. phenoperidine, pheneridine, anileridine, piminodine, etoxeridine [13]. 4-[4-chloro-3-(trifluoromethyl)-phenyl]-4-piperidinol (TFMP) is also a phenylpiperidine with additional substitution at para and meta position as depicted in Fig. 1. These facts encouraged the preparation of several novel TFMP derivatives and the evaluation of the coIrrelation between the structure and the analgesic activity.
Opioid drugs, symbolized by morphine, yield their pharmacological activities, plus analgesia, by interim on neuronal cell membranes receptors. In CNS opioid receptors have three main classes namely μ, δ and κ. Later research directed the isolation and structure of the endogenous ligand of the ORL-1 receptor (opioid receptor-like receptor). Discovery of the new receptor provides chance in drug innovation for new compounds which can be administered as analgesic or additional syndromes moderated by this receptor [15][16][17]. As a consequence, searches for other substitutes appear essential and favorable to get novel analgesic agents. Hot plate and tail immersion procedure are well known to be selective for opioid like compounds in many animal species [5,18]. Screening of novel chemical compounds as analgesic agents by using animal models is a particularly complex assignment. In finding analgesic agents, researchers have developed a relationship between animal models and human clinical responses. These methods are simple, reproducible and sensitive to discover weak and potent analgesic agents.
Despite of all the researches and work which is already done on piperidine, still there is need to explore these types of molecules for analgesic potential with safe efficacy. In proposed study different derivatives of 4-[4-chloro-3-(trifluoromethyl)-phenyl]-4-piperidinol (TFMP) was synthesized and evaluated for in vivo analgesic potential. The compounds being synthesized will be added to the current information of piperidine substituted compounds in the development of drugs.

Synthesis
We report here the synthesis of 4-[4-chloro-3-(trifluoromethyl)-phenyl]-4-piperidinol (TFMP) derivatives 1-12 via the reactions of TFMP by nucleophilic substitution with a variety of alkylating agent by N-alkylation. We selected different halogens substituted compounds including acetophenone, propiophenone, butyrophenone acetonaphthone, oxane, oxalane uracil, and coumarin substituted moieties and with the purpose of study the consequence of substituents on the piperidine ring of parent molecule (Scheme 1). To the best of our information, all compounds are reported here for the first time. The structures of synthesized compounds 1-12 were established by  Table 1).

Analgesic activity
Hot plate methodology was utilized to investigate the analgesic potential of the synthesized derivatives 1-12. Hot plate method is generally used centrally umpired test grounded on thermal incentive with the contribution of spinal reflex action. In this model, centrally interim analgesic agents constrain pain transmission at dorsal horn and persist the latency time of animals to jumping response [19]. It is among derivatives 1-12, nine compounds were effective in antagonizing pain as depicted in Tables 1 & 2 and Figs. 2 & 3.
Compound 1 displayed delay in latency time from 30-180 min. and maximum effect (66%) was observed at 30 min. Compound 3 is most effective derivative of this series which antagonizes pain comparable to standard morphine. The thermally prompted jumping action of control mice revealed around 10-13 s from time interval of 0-180 mins. However, the compound showed highly significant analgesia from 30-150 min (113-120%) followed by gradual decline in latency time. Compound 4 showed analgesic response from 90-150 min. The maximum delay in latency time observed at 120 min (68%). Compound 5 showed analgesia from 30-150 min. Compounds 10, 12 and 2 showed weak inhibition of pain. Compound 11 showed significant analgesic activity at 30 min (52%) which was followed by decrease in latency time from 60-150 min and the effect was non-significant at 180 min. Compound 6 displayed gradual increase in antinociceptive activity from 30 min and the response is maximum at 120 min which was followed by gradual decrease from 150-180 min. The results were significant from 60-180 min.   13.9 ± 0.9 (2.9) 12.9 ± 1.0 (6.5) 12.1 ± 1.5 (−4.7) 12.9 ± 1.8 (7.5) 13 Naloxone (Nal) 2 mg/kg was administered 10 min prior to administration of test agents Compounds 3, 5, 6 and 8 which showed potent pain reliving effect were used to observed their effects in the presence of naloxone Asterisks indicate *(p < 0.05), **(p < 0.001) and ***(p < 0.005); Latency time (sec.) of control (n = 90) and treated groups (n = 15), which are mean ± SEM of animals in three independent experiment; Values represented in parenthesis are percent pain protection from centrally mediated pain Compound 7 showed 88% analgesia at 30 min which was gradually decreased up to 29%. Compound 8 showed onset of analgesic effect from 30 min and the result became highly significant (107%) at 60 min. Compound 9 exhibited significant inhibition of pain (76%) at 30 min followed by rapid fall in activity.
Morphine sulphate at a dose of 10 mg /kg, ip caused significant analgesia up to 30 min whereas gradual increase in latency time observed and the effect was highly significant from 60-180 min (61-176%).
Naloxone, a non-selective opioid antagonist for μ, δ and κ receptors [20] blocked the centrally mediated antinociceptive effect of morphine (Table 2) in hot plate animal pain model and is in agreement as described earlier [21,22] in mice. Thereby, implying that pain relieving effect is neurogenic probably via involvement of opioid receptors at spinal/ supraspinal level. However, participation of specific receptor (s) in this analgesic action needs to be determined. On the other hand, compounds 5, 6 and 8 significantly relieved pain by ≤50% during 60th to 150th minutes of administration ( Table 2, Figs. 4 and 5) suggesting non-opioidergic independent response. The non-opioidergic responses possibly governed by other centrally mechanisms such as analgesic effect through COX-II pathway or GABAergic system [23].
Regarding onset of action compounds 7, 8, 9, 1, 3, and 5 displayed quick onset of action at 30 minutes while compounds 6 and 4 exhibited delayed onset of action at 60 min and 90 min respectively.
In case of duration of action compounds 3, 6, 1 and 5 have long duration (2-2.5 h), compounds 4 and 11 exhibited moderate (1-1.5 h) while compounds 7 and 9 have short duration of action (0.5 h). The analgesic effect in terms of duration of action is given below.
Structure-activity relationship (SAR) study specified that the activity of these 4-[4-chloro-3-(trifluoromethyl)-phenyl]-4-piperidinol (TFMP) derivatives could be due to different acetophenone substituents. All the compounds possess three main regions as depicted in Scheme 2 where first region is same in all derivatives with variation is spacer and terminal ring system. All the synthesized derivatives are divided into two groups on the basis of SAR (Scheme 3). First group A (A 1 -A 3 ) share the same substituted piperidine ring and spacer with the only difference in the terminal region. In second group B piperidine and terminal ring are same with only difference in linking group. This only difference is responsible for varied results in analgesic activity.
In group A 1, compounds 1 and 2 have methyl linkage between terminal ring and piperidine of parent molecule.  Compound 1 showed significant analgesic activity due to the presence of uracil while substitution of coumarin moiety in compound 2, making the molecule completely inactive.
In group A 2, oxane (compound 3), 3, 5, 5 trimethyl oxane (compound 4), oxalane (compound 5) and phenyl group (compound 6) respectively are connected with piperidine ring via bridging of ethyl group. The presence of oxane in compound 3 and 3, 5, 5-trimethyl oxane in compound 4 displayed interesting results as compounds 3 produced most pronounced and comparable to standard among all derivatives. Addition of three methyl group which is electron donating in nature in the same ring of compound 4 dropped the activity from highly significant to significant level. Another similar molecule compound 5 exhibited good analgesia but the presence of (5-membered) oxalane ring in place of oxane (6-membered) ring plays a crucial role in decreasing antinociceptic effect. The presence of aromatic ring in compound 6, also produced comparable results as by compound 3 having oxane ring.
In group A 3, compounds7, 8, and 9 having substituted phenyl, naphthyl and adamantyl group respectively, showed almost same level of analgesic activity. Significant antinociceptive effect exhibited by all these compounds 7, 8, and 9 suggesting the comparable ability of these three different ring systems to produce analgesic response.
In group B all the compounds 6, 10, 11, and 12 have the same substituents but only difference is in connecting chain between the phenyl and piperidine ring. Among these only compound 6 produced highly significant effect, while the other provoked weaker analgesic response. SAR studies indicated the importance of ethyl linkage. Therefore, it can be suggested that by replacing the -(CH 2 ) 2 − with -CO-(CH 2 ) 2 −, -CO-(CH 2 ) 3 − and -O-(CH 2 ) 3 linkage analgesic activity was decreased.

General experimental conditions
All reagents used including TMFP were obtained from Sigma Aldrich Company and organic solvents utilized were of analytical grade. Watt man's filter paper was used for filtration. TLC plates, Kieselgel 60 (GF-254) were used for the development of reaction and purification of compounds. The spots of synthesized product were envisaged under Hitachi U-3200 spectrophotometer utilized to record ultraviolet (UV) spectra while methanol used as solvent. FT-IR spectra were investigated on a Jasco 302 Fourier transform infrared spectrophotometer by KBR disc method. Mass spectrometry were carried out on Varian Massen spectrometer MAT 311 A spectrometer, Varian Massen spectrometer MAT 312, MAT 113 DMASPEC system. 1 H-NMR and 13 C-NMR spectral analysis was carried out at Bruker AM 75, 125, 300, and 500 spectrophotometer. Chemical shifts (δ) were recorded in parts per million (ppm) and coupling constants J in Hertz.
General procedure for synthesis of TFMP derivatives      Research Institute of Chemistry, University of Karachi, Pakistan. The ethical strategies for the treatment of laboratory animals were followed as suggested by Association for Assessment and Accreditation of laboratory Animal Care International (AAALAC) through approval of animal use committee. The NMRI mice of both sexes (23-29 g) were retained at a controlled temperature (22 ± 2°C) and clamminess (50 ± 10%) with 12 h light and dark cycle and nourished with standard diet and water.

Hot plate method
Eddy's hot plate methodology was utilized to investigate the analgesic activity of the synthesized derivatives 1-12 [24]. Mice (n = 6/dose) were orally pretreated with each vehicle (0.5% gum tragacanth suspension), synthesized drugs (50 mg/Kg) and morphine sulphate (10 mg/kg). Each mice were placed on hot plate (Ugo Basile, model-DS 37, 25 × 25 cm, Italy) at 50 ± 1°C temperature, just after giving the dose. The responding time of the animal to the pain stimulus by jumping or flicking or licking of hind paw licking is the latency time which was recorded in seconds. The detach time for the reaction was 30 sec to evade tissue damages [25]. Analgesic activity was monitored at 0, 30, 60, 90, 120,150, and 180 min and outcomes were concise in the form of latency time. Naloxone (2 mg/kg) was injected i.p 10 min prior to most potent test agents (compound 3, 5, 6 and 8 among all twelve compounds) administration and latency time of response was noted. The average latency time displayed by the control animals (base line latency) and treated group (drug latency) were matched and stated as percent pain protection via following formula: Percentage pain protection ¼ Drug latency À base line latency Base line latency Â 100

Statistical analysis
One-way analysis of variance (ANOVA) analgesic activity was expressed as latency ± SEM in second. The alterations between the means were tested utilizing post hoc LSD and values of p < 0.05 were statistically determined as significant. Statistical analysis was done on SPSS for windows version 12.
In vivo biological potential of TFMP derivatives 1-12 was evaluated by using Eddy's hot plate methodology. All synthesized compounds except 2, 10, and 12 displayed considerable inhibition of pain in the mouse hot plate model following administration by the oral route. Compounds 3, 5, 6, and 8 induce potent analgesic effect. As the hot plate technique was employed thereby, imply that analgesic outcome is neurogenic possibly via participation of opioid receptors at spinal/supra spinal level. Hot plate method is use to screen either compound have centrally acting pain receiving effect or not. Overall, the preclinical data for this class of substituted piperidines signify them as novel class of effective analgesics, for potential therapeutic activity suggesting non-opioidergic independent response. Due to their encouraging results, they can be selected for relieving pain. On the other hand, contribution of particular receptor (s) in this analgesic potential needs to be determined.