Isolation, Characterization and Pharmacological Evaluation of 8-chloro-3 (1-methyl-piperidene-4-yl) 5H-benzo [5, 6] Cyclohepta [1, 2b] Pyridene-11(6H)-one from Loratadine

INTRODUCTION

Researchers have paid attention towards the growing need for controlling and formation of impurities in the drug substances and have been gaining critical importance from various regulatory authorities world over (Roy, 2002). The Webster’s dictionary defines impurity as a “substance that is itself impure or makes something impure.” Impurities are the unwanted compounds present in the pharmaceuticals, that develop during their synthesis and remain with the Active Pharmaceutical Ingredients (APIs). It may even develop during their formulation or upon aging of both API and formulated APIs to other dosage forms (Ahuja and Alsante, 2003). Their presence has an influence on the pharmacological action and the safety of the formulated product. There are different spectroscopic techniques to identify and characterize impurities i.e., NMR, Mass, IR and FTIR (Eugene et al., 2006). Finally, the compound can be identified by LCMS, HPLC-DAD-MS (Ermer, 1998; Gomez-Caravaca et al., 2007; Abdul et al., 2008). Generally these methods are quite complicated and require expensive instruments (Bhaskara et al., 2011).

Impurity identification is easier when carried out at stages earlier to the formation of the final API, i.e., at an intermediates stage. It helps in dealing with the impurity at the point of its formation which provides ample time to address various aspects of its formation and control. However the thresholds and guidelines of the International Council of Harmonization do not apply to the impurities at the developing stages (Ahuja and Alsante, 2003).

A wide variety of pollens, house dust mites and fungi are responsible for inducing allergic reactions in humans (Sharma et al., 2009). These Allergic reactions are mainly mediated by histamine. It has been reviewed that histamine H₁ receptors are potential therapeutic targets of allergy and are most prominent in IgE mediated responses (Rahim, 2010). Antihistamines are the drugs which block the histamine messages from reaching their receptors. Loratadine is an antihistaminic drug belonging to the class of Second Generation Antihistamines. They are tricyclic antihistamines which are selective and inverse antagonists of histamine receptors. They are long lasting, non- sedative and peripherally acting. It has demonstrated effective control in allergic reactions over other antihistamines (Menardo et al., 1997).

Loratadine was initially developed by Schering and Plough due to the arising need of a nonsedative antihistaminic drug. Its chemical structure closely relates to that of tricyclic antidepressants such as Imipramine and has distant structural similarity with atypical antipsychotic Quetiapine (Kay and Harris, 1999).

Much work has been done on Loratadine of which most of them have been carried out for pharmacokinetic studies and applied them to assess Loratadine and its metabolite Desloratadine in plasma by HPLC (Zhong and Blume, 1994), GC-MS (Martens, 1995) and GC with NP detector (Johnson et al., 1994). It is highly essential to have a sensitive, reliable and at the same time a simple method for the determination of drugs in biological fluids for pharmacokinetic investigation (Jalalizadeh et al., 2006).

Simultaneous determination of Loratadine and Desloratadine in plasma has been done by various researchers using methods like liquid chromatography and Tandem Mass Spectroscopy and HPLC with fluorescence detection (Yin et al., 2003). The determinations of Loratadine impurities have been done by using various techniques like LCMS, preparative LCMS, preparative HPLC, UPLC etc. (Sutherland et al., 2001). The analytical works were carried out for the purpose of validation of the methods like preparative LC, HPLC and Capillary Electrophoresis (Ruperez et al., 2002). But the structures have not been characterized in this case. Impurities of loratadine have been isolated by using isocratic reverse phase HPLC from their mother liquor and then characterized using spectroscopic techniques (Reddy et al., 2003). The usual hydroxy contaminant of Loratadine has also been synthesized and characterized (Veronica et al., 2006). Some have been done to determine the amount of preservatives in Loratadine formulation by preparative TLC plates (Popovic et al., 2007).

At the 9th stage of the synthesis, the conversion of 8-chloro 10,11-dihydro-4-aza-5H-dibenzo (a, d) cycloheptane-5-one (L-07) to 11-(N-Methyl-4-piperidinyl)-11-hydroxy-8-chloro-6,11-dihydro-5H-benzo (5, 6) cyclohepta (1, 2-b) pyridene (L-08) is mediated by Grignard Reaction. Methyl Piperidyl Magnesium Bromide acts as the Grignard reagent. It adds to L-07 under highly basic conditions created by Tetrahydrofuran and low temperature conditions ranging from -80 to -90°C (Suri et al., 2004). At this stage quite a few impurities are formed out of which one is a critical impurity. This study is involved in isolation, characterization and evaluation of the critical impurity.

MATERIALS AND METHODS

Drugs and chemicals: various used drugs and chemicals include Acetonitrile, Isopropyl Alcohol, Ethyl acetate, Triethylamine, Methanol and Hexane obtained from S.D. Fine Chem. Ltd., Mumbai. Carageenan, Loratadine, Chlorpheniramine, Diclofenac Sodium were obtained from Spectrochem Pvt. Ltd., Mumbai. Crude samples of L-08 were obtained from Morepen laboratories Ltd., Parwanoo. All other chemicals were of analytical grade and were obtained from local sources.

Experimental animals: Wistar Alabino Rats of either sex weighing 110-150 g or Swiss Alabino mice weighing around 18-25 g were obtained from Guru Angad Dev Veterinary and Animal Sciences University. Animals were maintained at room temperature in standard cages under proper conditions and had free access to food and water. Twenty hours before the experiment the animals were starved however proper water supply was maintained. Animal experiments were carried out in accordance to the guidelines of the Committee for the purpose of Control and Supervision of Experiments on Animals with registration No. 874/ac/05/CPCSEA.

Instruments used: IR was recorded on PERKIN ELMER BX FTIR spectrometer 882, ¹H NMR on a 400 MHZ FT NMR Spectrometer (Model BRUKER ADVANCE).

¹³C NMR recording was done using 400 MHZ NMR Spectrometer (Model BRUKER ADVANCE) and LC-MS was on Finnegan Mat.

METHODS

Isolation of the impurity: To the Crude powdered sample of 11-(N-Methyl-4-piperidinyl)-11-hydroxy-8-chloro-6, 11-dihydro-5H-benzo (5, 6) cyclohepta (1, 2-b) pyridene (L-08) (70 g), 280 mL of Acetonitrile and 15 mL of water were charged. The mixture was then refluxed for an hour at temperatures ranging from 75 to 80°C. Then it was slowly cooled down for an hour upto 5°C for crystallization to take place. The crystals formed were separated by filtration and washed with a mixture of cold acetonitrile and water.

The filtrate and the residue were analysed for their impurity content. The filtrate was found to have excess of the impurity along with L-08 and most of the L-08 had been retained by the residue. Analysis was done by TLC (Isopropyl alcohol: Ethyl acetate: Triethylamine: Methanol /8:2:0.5:1). The filtrate was taken and the solvent was evaporated and dried. A small amount of the sample, about 20 g was taken and adsorbed on Silica Gel (100-200 mesh size) and loaded on to a column. The column was run using Hexane: Ethyl Acetate: Methanol (100: 2: 5) as the mobile phase and was run using gradient elution method. The elution was monitored by TLC. When pure spot of the impurity was observed in the TLC, the column fractions were collected and the excess solvent was evaporated at a temperature ranging from 50-60°C under a pressure of 200 mm Hg^-1.

PHARMACOLOGICAL EVALUATION

Anti-inflammatory activity: Carrageenan induced rat paw edema: The Carageenan induced rat paw edema assay was carried out according to the method of Winter et al. (1962).

The male alabino rats of either sex were randomly divided into four groups having four animals each:

A solution of Carageenan (1%, 0.05 mL) in physiological saline injected into the subplantar tissues of right hind paw of the rat was used to induce edema. The phylogistic agent was injected one hour after the administration of the vehicle, test and the standard drug. The paw volume was measured at intervals of 1, 2, 3 and 4 h by mercury displacement method using a plethysmometer and the percentage inhibition of inflammation by the drug was calculated using the formulae given below:

Where:

Spontaneous locomotor activity: Spontaneous Locomotor activity was carried out to evaluate the CNS depressant property of the impurity under study using an Actphotometer. Swiss Alabino mice of either sex were randomly divided into six groups of four animals each. Each animal was placed in the Actphotometer chamber individually for 25 min to note the basal readings:

After thirty minutes of oral administration of vehicle, standard, Loratadine and test, the animals were placed individually in the Actphotometer for 25 min and their locomotor activity was noted. Percentage inhibition of locomotor activity for each group was calculated by using the formulae as follows (Verma et al., 2010):

Where:

RESULTS AND DISCUSSION

Isolation: The residual impurity obtained was black in color and liquid in nature. The purity content of the sample was then analysed by HPLC which came out to be 70% pure. The sample was then characterized by spectroscopic techniques. The HPLC of the isolated impurity has been shown in Fig. 1.

Fig. 1:	HPLC showing the isolated impurity

Fig. 2:	IR Spectrum of the impurity

Characterization
IR (liquid): It shows characteristic peaks at 1651 cm^-1 (C = C Stretch, C = O stretch), 1416 cm^-1 (-CH₃ bend), 1350 cm^-1 (C-N), 737.2 cm^-1 (C-H aromatic oop.) (Fig. 2).

¹H NMR (400 MHZ CDCl₃): δ 8.5758-8.5707 (d, 1H, H₂) δ 8.04-8.02 (d, 1H, H₄) δ 7.48-7.47 (d, 1H, H₇) δ 7.337- 7.3110 (d, 1H, H₉) δ 7.27-7.25 (d, 1H, H₁₀) δ 2.66-2.96 (m, 4H, H_5a, H_5b, H_6a, H_6b), δ 2.42-2.43 (m, 3H, H_22a, H_22b, H_22c), δ 2.27-2.17 (m, 4H, H_18a H_18b H_20a H_20b) δ 2.03-1.87 (m, 5H, H_16a, H_16b, H_17a, H_17b, H₂₁).

¹³C NMR (400 MHZ, CDCl₃): δ 32.81, 32.02, 34.32, 38.82, 45.53, 55.30, 127.43, 129.42, 132.9, 135.51, 135.85, 137.14, 138.81, 143.10, 148.10, 152.15, 192.7 (C = O).

Table 1:	1H NMR data of (L-08)

Table 2:	1H NMR data of L-07

LC-MS: The major peaks were 341.1 (M⁺), 342.1 (M⁺+2), 343.1 (M⁺+3).

Structure elucidation of impurity of loratadine: The LCMS for the impurity shows the molecular ion peak at 341.1 which is exactly one mass unit less than that of 11-(N-Methyl-4-piperidinyl)-11-hydroxy-8-chloro-6,11-dihydro-5H-benzo (5, 6) cyclohepta (1, 2-b) pyridene which is 342.9 (Fig. 4). Interestingly the proton NMR spectrum of the impurity shows five signals in the aromatic region which is one less than that present in the proton NMR spectrum of the L-08 (Table 1). Moreover the ¹³C spectrum of the impurity shows similarity to the ¹³C spectrum of L-07 (Fig. 5) in the aromatic region i.e., there is a high field absorption peak at 192 ppm in L-07 which is present in the spectrum of the impurity also indicating the presence of a ketone (Table 4). The other half of it shows similarity to that of L-08 (Table 3). The IR shows a peak at 1651 cm^-1 which is similar to the absorption band present at 1664 cm^-1 of L-07 which corresponds to a ketone (Table 6). All these observations indicate that there is the presence of dihydro-4-aza-5H-dibenzo (a, d) cycloheptane-5-one and N-Methyl-4-piperidine in the impurity but the point of attachment is different from that of L-08. The piperidene ring is not attached to the 11th position of the cyclic ring but somewhere in the aromatic region.

The structure of the aromatic ring of L-07 (Fig. 5) has three electronegative elements i.e. Chlorine Nitrogen and Oxygen thus giving rise to inductive effect and its data is presented in Table 2. Oxygen being the most electronegative of the three, its effect dominates. Thus due to the delocation of the charges and resonance effect, position 3 was found to be the most probable position for the attachment of the Piperidene ring. The structure elucidated has been shown in Fig. 3.

It was analysed that the impurity of Loratadine is formed due to the highly basic and low temperature conditions involved in the Grignard reaction during the addition of N-methyl-4-Chloro piperidene magnesium bromide i.e. the Grignard reagent to the 8-chloro 10,11-dihydro-4-aza-5H-dibenzo(a,d)cycloheptane-5-one (L-07) (Fig. 5).

Fig. 3:	Structure of impurity

Fig. 4:	Structure of L-08

Fig. 5:	Structure of L-07

Table 3:	13C Data of L-08

L-07 is found to show Keto enol Tautomerism. IR spectroscopy data is shown in Table 5. In IR spectroscopy a normal ketone group shows stretching vibrations at 1751 cm^-1 but when in conjugation with a double bond (C = C), the double bond absorb at lower frequency near 1675 cm^-1 (Pavia et al., 2007). In L-07, the ketone group absorbs at 1661 cm^-1 and in the impurity it absorbs at 1651 cm^-1. It can be hypothesized that when the Grignard reagent comes in the vicinity of L-07 existing in the keto form the required product is formed as in Grignard reaction.

Table 4:	13C Data of L-07

Table 5:	IR data of L-08

Table 6:	IR data of L-07

But in the case when L-07 exist in the enol form, there are chances that the Grignard reagent would attack the next most reactive point of the molecule, found to be the 3rd position which appears to be electron deficient due to the electronegative nitrogen atom.

ANIMAL ACTIVITIES

Anti-inflammatory activity
Carageenan induced rat paw edema: The test sample showed a pronounced effect on Carageenan induced edema, the maximum being 58.6% inhibition at 5 mg kg^-1. It showed a constant inhibition of inflammation all through the experiment. Edema inhibition was found to be dose dependant, more inhibition at higher dose of the test drug (5 mg kg^-1) than at lower doses (2.5 mg kg^-1) (Table 7).

The pharmacological evaluation of the molecule establishes that the impurity has potent anti-inflammatory activity. Carageenan induced rat hind paw edema is a common model used to evaluate acute inflammation and is believed to be biphasic. Reviewers have suggested that this model has good correlation between the efficacy in the model and human activity (Mohamed et al., 2005; Garg and Paliwal, 2011). Anti-inflammatory activity carried out for pyridenedicarbonitrile and benzopyranopyridenes showed very high anti-inflammatory activity (Hosni and Abdulla, 2008).

Spontaneous locomotor activity: Locomotor activity exhibited significant decrease in case of standard drug Chlorpheniramine. Loratadine did not show any decrease in motility as it is a non sedative antihistaminic drug. The test sample at 50 mg kg^-1 did show major CNS depression while dosage at 10 mg kg^-1 showed less CNS depression (Table 8).

Table 7:	Anti-inflammatory activity of the Impurity
Values are Mean±SEM for 4 animals in each group. a = p<0.01, b = p<0.02, c = p<0.05 as compared to control, using one way ANOVA

Table 8:	Spontaneous locomotor activity of the impurity
Values are Mean±SEM for 4 animals in each group. p<0.01 using one way ANOVA

The impurity causes remarkable CNS depression at doses higher than 10 mg kg^-1. The locomotor activity using the Actophotometer measures the level of excitability of the central neurons and a decrease in activity may be due to sedation resulting from the depression of the CNS (Balamurugan et al., 2007; Pandy et al., 2009). Locomotor activity was studied on synthesized derivatives of benzocycloheptane pyridene derivatives. The derivatives showed considerable decrease in locomotor activity as compared to the standard drugs (Carceller et al., 1994). The Structure Activity Relationship of Loratadine states that the piperidene ring along with ethyl ester molecule attached to the cycloheptane ring is responsible for the prevention of the molecule from entering the blood brain barrier. In this case sedative properties attributes to the non attachment of the piperidene ring to the cycloheptane ring (Wilson et al., 2004).