Design, Synthesis and Evaluation of Novel Phenothiazines as Antipsychotic Agents.
Ganesh Bhawal*, Meenakshi Deodhar, Ashok Bhosale and Deepak Lande
Department of Pharmaceutical Chemistry, S.G.R.S College of Pharmacy, Saswad, Pune, Maharashtra 413716, India
*Corresponding Author E-mail: ganesh_bhawal@rediffmail.com
ABSTRACT:
The present work relates to novel phenothiazines derivatives as antipsychotic agents. The introduction of the Chlorpromazine (a Phenothiazine class drug) in 1952 was positive advance in the treatment of psychosis. Phenothiazines constitute one of the most active classes of compounds possessing diversified biological applications and are the most commonly used class of antipsychotic agents. Phenothiazines mainly act by antagonism of the D2 receptors and are thus useful for the treatment of the positive symptoms and have very little effect on negative symptoms or cognitive deficits
In addition, blockade of dopamine D2 receptors leads to an increase in prolactin secretion and associated neuroendocrine disorders including gynecomastia, amenorrhea, and sexual dysfunction. The antipsychotic efficacy of typical neuroleptics is related to the D2 receptor blockade. D2 receptor is one of the G protein coupled receptors family that also includes the D3 and D4 receptor subtypes. And it was found that the D4 receptor density is elevated in schizophrenia.
About six fold increases in the D4 receptor subtype have been reported in schizophrenic brains, compared with normals. Autoradiographic analyses with the D4 selective ligand 3H-NGD 94-1 show D4 sites to be dense in rat and human hippocampus, hypothalamus, and neocortex, among other brain regions, and to be absent in the striatum. These findings suggested that a D4-specific compound might treat schizophrenia as effectively as Clozapine but without the D2 antagonist-mediated EPS or the Clozapine constellation of side effects.
We undertook a plan of building pyrazoline ring fused with the nucleus of phenothiazine. The preliminary pharmacological screening of the synthesized compounds revealed that out of the 12 compounds synthesized 6 compound (3a,3c,3d,3f,3j,3l) displayed significant activity.
KEYWORDS: Phenothiazine, Hydrazine hydrate, Pyrazoline, Antipsychotic activity.
Schizophrenia, one of the most devastating mental disabilities, affects around 1% of the total world population1. Schizophrenia affects a person’s day to day life and thus must be controlled through proper medication. Phenothiazine is one of the most widely used classes of typical antipsychotic agents. This class however suffers from various side effects, extrapyrimidal effects being one of the major one2. Even atypical antipsychotics like Clozapine suffer from side effects like agranulocytosis, weight gain and type II diabetes3, 4. There is thus a need for new antipsychotic agents with improved therapeutic properties
Literature survey revealed that Pyrazoline ring show variety of biological activity such as antipsychotic, antibiotic, antiviral activity thus reducing the extrapyrimidal side effects5,6,7. Thus it was thought interesting to synthesize phenothiazine based derivatives, incorporating pyrazoline ring in order to assess their antipsychotic activity.
EXPERIMENTAL:
Chemistry:
All chemicals used were of synthetic grade. The purity of compounds was ascertained by TLC on precoated silica F254 plates (Merck, Mumbai) using iodine vapors and UV light as detecting agents. The melting points of the synthesized compounds were determined by open capillary method and are uncorrected. The IR spectra of synthesized compounds were recorded on a Shimadzu FT-IR spectrophotometer in KBr. The 1H NMR were recorded in CDCl3 using NMR Varian-Mercury 300MHz spectrometer and chemical shifts(δ) are given in parts per million (ppm), downfield from tetramethylsilane (TMS) as an internal standard from University of Pune, Pune.
The starting material, N-Acetylphenothiazine (1) was prepared by acetylation of 10-H phenothiazine.9 Chalcones of N-acetylphenothiazine, (2a-2l) were synthesized using Claisen-Schmidt condensation reaction. Various substituted benzaldehydes were stirred with compound (1) in presence of 10% ehtanolic NaOH. On refluxing 2a-2l with hydrazine hydrate in ethanolic NaOH 10-(5- substituted aryl-3-pyrazolinyl) phenothiazine (3a-3l) were formed. All the compounds were characterized by analytical and spectroscopic methods. The physical data of the synthesized compounds is given in Table 1.
The IR spectrum of the compound 1 showed a sharp peak at 1670.41cm-1 assigned to the C=O stretch. The peak at 3064.99 cm-1 was due to aromatic stretch. The peak at 1477.52 cm-1 was attributed to C-N stretch.
Graph 1: Spontaneous motor activity for compounds 3a-3l
N=6, ** - P < 0.01 compared with vehicle treated group,
Data expressed as Mean ± SEM.
Data was analysed by one way ANOVA followed by Dunnett’s test.
Dose: Target compounds- 40mg/kg i.p.; Chlorpromazine – 4 mg/kg i.p
The IR spectrum of 2a-2l in KBr showed sharp carbonyl and aliphatic C=C stretching vibration for the chlacone compounds at around 1630-1690 cm-1. Further the peaks at 2900-3100 cm-1 are due to aromatic stretch. The stretching vibrations for C-N of phenothiazine are seen at around 1477.79 cm-1. The 1H NMR (δ ppm, CDCl3) spectrum of 2l bearing α, β-unsaturated carbonyl group, revealed characteristic peaks as two doublets at 5.656 and 6.234 ppm. The aromatic protons showed peaks at 6.584-7.210 ppm as multiplet.
The IR spectrum of 3a-l in KBr showed characteristic aromatic stretch between 2900-3100 cm-1. The C=N stretch of the pyrazoline ring revealed peaks at 1590-1599 cm-1, 3342.82 NH-stretch and C-N of phenothiazine ring showed stretching vibrations at around 1460-1478 cm-1. The 1H NMR (δ ppm, CDCl3) spectrum of 3l having pyrazoline 5-H revealed triplet at 3.983 ppm, pyrazoline 4-CH2 revealed quartet at 3.201 ppm, pyrazoline NH revealed singlet at 7.490 ppm. The aromatic protons showed peaks at 6.207-7.105 ppm as multiplet.
N=6, ** - P < 0.01, * - P < 0.05
Data expressed as Mean ± SEM.
Data was analysed by one way ANOVA followed by post-hoc Dunnett’s test.
Antipsychotic activity:
The antipsychotic potential of compounds was evaluated by behavioural effect, testing their ability to reduce locomotor activity after intraperitonial administration. The active compounds were also tested for induction of catalepsy in Swiss albino mice so as to assess their antipsychotic activity and ability to induce extra pyramidal side effects in terms of reduced catalepsy.
Table 1: Physical characterization data of synthesized compounds
|
Compound |
Rf* |
R1 |
Mol.Formula |
m.pa (oC) |
Yield (%) |
|
3a |
0.55 |
2-NO2 |
C21H16N4O2S |
188-190 |
67.48 |
|
3b |
0.65 |
2-Cl |
C21H16 ClN3S |
185-188 |
73.54 |
|
3c |
0.59 |
3,4,5(OCH3)3 |
C24H22N2O4S |
170-172 |
62.44 |
|
3d |
0.53 |
4-Br |
C21H16BrN3S |
168-170 |
64.45 |
|
3e |
0.58 |
4-CH3 |
C22H19N3S |
156-158 |
63.02 |
|
3f |
0.62 |
4-N(CH3)2 |
C23H22N4S |
184-186 |
58.58 |
|
3g |
0.54 |
3-NO2 |
C21H16N4O2S |
178-180 |
62.88 |
|
3h |
0.58 |
3-Cl |
C23H22ClN3S |
183-185 |
65.24 |
|
3i |
0.48 |
4 -OCH3 |
C22H19N3OS |
170-172 |
69.58 |
|
3j |
0.72 |
H |
C21H17N3S |
160-165 |
62.68 |
|
3k |
0.60 |
4-Cl |
C21H16ClN3S |
160-162 |
69.58 |
|
3l |
0.58 |
4-NO2 |
C21H16N4O2S |
186-188 |
74.74 |
a. The value is uncorrected, * Solvent system - toluene-methanol -9:1
Table 2: Behavioural effect
|
Compound |
Effect On |
||||
|
Body position behavior reflex |
Grooming |
Rearing |
Sleeping |
Righting |
|
|
Vehicle |
N |
N |
N |
- |
N |
|
3a |
N |
N |
N |
- |
N |
|
3b |
N |
↓ |
↓ |
- |
N |
|
3c |
N |
N |
N |
- |
N |
|
3d |
N |
N |
N |
- |
N |
|
3e |
N |
↓ |
↓ |
- |
N |
|
3f |
N |
↓ |
↓ |
- |
N |
|
3g |
N |
N |
↓ |
- |
N |
|
3h |
N |
N |
↓ |
- |
N |
|
3i |
N |
N |
↓ |
- |
N |
|
3j |
N |
↓ |
N |
- |
N |
|
3k |
N |
N |
↓ |
- |
N |
|
3l |
N |
N |
N |
- |
N |
|
CPZ |
N |
↓ |
↓ |
+ |
- |
N : Normal, ↓ : Decreased, + : Present, - : Not present, CPZ : Chlorpromazine
1. Behavioral effect10,11:
The method is applied in the beginning of pharmacological screening to detect psychotropic activities. It allows identifying and differentiating the profile pattern of various classes of pharmacological agents. It was observed that none of the synthesized compounds had effect on body position, righting reflex and none of the synthesized compounds induced sleep. This indicates the synthesized compounds would induce lesser side effects.
Some of synthesized compounds caused decrease in grooming behaviour and rearing which would indicate CNS depression. Observations of the behavioural assessment are given in the table 2.
2. Spontaneous motor activity10,12:
All compounds were screened for spontaneous motor activity using actophotometer. Some of the synthesized compounds were able to cause decrease in locomotor activity of mice indicating CNS depression. Most active of these were 3a, 3c, 3d, 3f, 3j and 3l. But these compounds were less active as compared to standard Chlorpromazine. Data for this study is given in table 3.
3. Catalepsy induction10:
It was important in the preliminary pharmacological screening to determine the propensity of the synthesized compounds to cause the extra pyramidal side effects (EPS)
in terms of catalepsy. Only most active compounds were tested for catalepsy and it was found that none of the tested compounds showed catalepsy as compared to Haloperidol. Data for the test is given in table 4.
Table 3: Spontaneous locomotor activity
|
Compound
|
Locomotor count at 30 min Mean ± S.E.M |
|
Control |
151.33 ± 4.163 |
|
3a |
144.67 ± 6.22 |
|
3b |
110 ± 4.619** |
|
3c |
120.67 ± 1.453** |
|
3d |
119.33 ± 1.202** |
|
3e |
146.33 ± 1.767 |
|
3f |
105.67 ± 3.528** |
|
3g |
135 ± 6.557 |
|
3h |
134.67 ± 3.18 |
|
3i |
154 ± 2.082 |
|
3j |
95.66 ± 3.528** |
|
3k |
153 ± 3.786 |
|
3l |
84.66 ± 8.373** |
N=6, ** - P < 0.01 compared with vehicle treated group,
Data expressed as Mean ± SEM.
Data was analysed by one way ANOVA followed by Dunnett’s test
Table 4: Catalepsy test
|
Compound |
Time (sec) spent with forepaws on rod after time interval of |
|||
|
30 min (Mean ± SEM) |
60 min (Mean ± SEM) |
90 min (Mean ± SEM) |
120 min (Mean ± SEM) |
|
|
Vehicle |
1.06 ±0.066 |
0.0 ±0.0 |
0.73 ±0.371 |
1.12 ±0.088 |
|
3b |
1.16 ±0.1528 |
2.60 ±0.2990 |
4.9 ±0.4041 |
3.86 ±0.4055 |
|
3c |
2.03 ±0.088 |
5.63 ±0.384 |
8.8 ±0.2082** |
5.16 ±0.2028 |
|
3d |
1.46 ±0.240 |
3.1 ±0.3606 |
6.96 ±0.1856** |
3.8±0.1732 |
|
3f |
2.13 ±0.1856* |
4.73 ±0.3883** |
8.16 ±0.5783** |
10.2 ±0.1856 |
|
3j |
2.81 ±0.3980* |
6.22 ±0.1764** |
8.86 ±1.794** |
10.33 ±0.2048 |
|
3l |
5.9 ±0.1732 |
6.56 ±0.3507* |
11.14 ±1.161** |
7.23 ±0.8212* |
|
Haloperidol |
222.66 ±3.930** |
242.33 ±3.055** |
272 ±3.712** |
282.66 ±2.906 |
N=6, ** - P < 0.01, * - P < 0.05, Data expressed as Mean ± SEM, Data was analysed by one way ANOVA followed by post-hoc Dunnett’s test.
Melting points are uncorrected and were recorded using open-end capillaries. Thin layer chromatography of synthesized compounds was performed on percolated silica gel G plates using toluene- methanol (9:1) solvent systems. Iodine and dilute H2SO4 spray was used as visualizing agent.
The IR spectra of synthesized compounds were recorded on Shimadzu 8400-S FT-IR Spectrophotometer. The 1H NMR was recorded in CDCl3 using NMR Varian-Mercury 300 MHz (internal standard TMS).
N-Acetylphenothiazine (1): To the solution of 10-H Phenothiazine (1.99 g, 0.01mole) in dry benzene (50ml) taken in a conical flask, acetyl chloride (0.8 ml, 0.01 mole) was added drop wise at 0-5°C. After the addition was completed, the reaction mixture was stirred for 3-4 hours at room temperature. The reaction mixture was then kept undisturbed overnight. The resulting mixture was distilled and the residue poured onto ice. The solid thus obtained was recrystallised from ethanol to get colorless crystals.
N-Acetylphenothiazine (1): Yield 80.46 %, mp 196-198°C, Rf -0.64 (toluene: methanol - 9:1). IR KBr, cm-1: 3064.99, 1670.41, 1477.52.
General procedure for substituted N-Benzylideneacetylphenothiazine derivatives (2a-i):
Compound (1) N-Acetylphenothiazine (2.39 g, 0.01 mole) in ethanol (25ml) was stirred with 2 ml (10%) alcoholic sodium hydroxide solution in a conical flask, kept in an ice bath for 10 min. Substituted benzaldehyde (0.01 mole) was added and the mixture was stirred for 3-4 hours. The resulting mixture was poured onto ice to get the solid product. The separated solid was filtered, washed with water and recrystalised from ethanol.
N-(4-Nitrocinnamoyl) phenothiazine (2l): Yield 67.25 %, mp 172-174°C, Rf- 0.63 (toluene: methanol - 9:1). IR KBr, cm-1 3078.49, 2930.32, 1670.11, 1647.26, 1460.16, 1534.16. 1H NMR d ppm, CDCl3 δ 6.584-7.210 m (Ar-H aromatic protons), δ 5.686-6.271 d (COCH=CH).
General procedure for synthesis of 3-(10H-phenothiazin-10-yl)-5-(substituted phenyl)-2-pyrazoline derivatives (3a-3l): A mixture of substituted N-Benzylideneacetylphenothiazine (2) (0.002 mol), hydrazine hydrate (0.2 ml, 0.002 mol) dissolved in ethanol (25 ml) was taken in RBF. Reaction mixture was heated under reflux for 10-12 hours. The resulting mixture was concentrated, cooled and poured onto the ice water. The solid separated was collected and washed several times with water and recrystallised from ethanol to get faint violet crystals.
5-(4-Nitrophenyl)-3-(10H-phenothiazin-10-yl)-2-pyrazoline (3l): Yield 74.74 %, mp 186-188°C, Rf - 0.58 (toluene-methanol -9:1). IR KBr , cm-1 3096.71, 2992.24, 1597.10, 1494.33. 1H NMR d ppm, CDCl3 δ 6.207-7.329 m (Ar-H aromatic protons), δ 3.983 t (pyrazoline 5-H), δ 3.201 dd (pyrazoline 4-CH2)
Pharmacology:
Behavioral effect:
Behavioral effect of synthesized compounds (40mg/kg) was assessed by the method described by Irwin et al, (1968)11. Mice of either sex weighing between 25-30 g were divided into groups of 6 mice each. One group of animals receiving the vehicle (saline solution) only, serves as control group. After the intaperitoneal (i.p.) administration animals were observed upto 2 hours for behavioral changes. The behavioral observation parameters consisted of body position, grooming behavior, rearing, sleeping and righting reflex.
Spontaneous motor activity:
The albino mice of either sex weighing 25-30 g were used for the test. They were divided into three groups each group containing 6 mice. Each group was injected with test drug (40mg/kg, i.p.) or vehicle (saline solution) (i.p.). Chlorpromazine (4mg/kg, i.p.) was used as reference drug. The spontaneous locomotor activity was recorded at every 30 minutes after drug administration using an actophotometer with automatic counting of animal movements on the cage floor. The locomotor count for each animal was recorded for 5 minutes at 30 minutes intervals for 2 hours. All the results were statistically evaluated using ANOVA followed by Dunnett’s test.
Catalepsy test:
Albino mice of either sex weighing 25-30g were divided into three groups containing 6 animals each. The control group received vehicle (saline solution) (10 ml/kg i.p.) whereas the test group received synthesized compounds (40 mg/kg i.p.) and standard group received haloperidol (1 mg/kg i.p). After the treatment, the forepaws of the mice were placed on rod of 0.9 cm diameter set at height of 2.5 cm. Duration for which the mice retain the forepaws on the elevated rod was noted down at the intervals of 30, 60, 90 and 120 minutes. The cut off time was 300 seconds. Between the measurements, the mice were returned to their home cages.
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Received on 28.04.2010 Modified on 18.05.2010
Accepted on 24.05.2010 © AJRC All right reserved
Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 906-910