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The initially developed compounds, 3 phenylureido 1, 5 benzodiazepines which were the first reported non-peptidyl CCK-A agonists and 3 (phenyl amide)1,5 benzodiazepine lacked oral activity in rodent feeding model. Activity of these compounds was found to be dependent upon the C-3 pharmacophore. Hence a series of isosteric replacements for C-3 pharmacophore were carried out in order to develop a full selective CCK-A agonist with oral activity. The 3 indolylmethyl compounds developed as a result maintained the agonist activity of the previous one. Further modifications in this class lead to the development of 3 indazolyl series. The activity of this series of compounds was evaluated using functional and binding assay, where compound 7 demonstrated full agonist activity and moderate binding selectivity. It was further evaluated for in vivo activity where it decreased food intake in rat feeding model and thus was found to mediate satiety.
Cholecystokinin (CCK) is a gastrointestinal peptide harmone synthesized in the intestine by endocrine cell . Different molecular forms of CCK have been isolated and identified. Out of these different forms , the minimum sequence required for bioactivity is that of C terminal octapeptide CCK-8 (H-Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2) which shows full bioactivity (Sugg et al, 1995). Cholecystokinin receptors are pharmacologicaly classified into 2 types based on their affinity for endogenous peptide agonists , as CCK-A (ailmentary) and CCK- B (brain) (Noble et al, 1999). CCK-A receptors are mainly located in the peripheral regions such as gallbladder, pancreas, pyloric sphincter and vagal afferent fibres and in small numbers in brain regions. Whereas CCK-B receptors are predominantly present in the brain and gastric glands (Sugg et al, 1995).
Figure 1: Schematic representation of the rat CCK1Â receptor showing the postulated transmembrane topology, sites for putative NH2-linked glycosylation (tridents), serine and threonine phosphorylation by PKC and protein kinase A (PO3), and conserved cysteines in the first and second ECLs, possibly forming a disulfide bridge, and a possible palmitoylated conserved cysteine in the cytoplasmic tail. NH2-, N terminus; COOH-, C terminus. (Noble et al 1999, adapted)
CCK-A receptors in pancreas and gallbladder are mainly responsible for satiety. CCK-A receptors in the pancreas are stimulated by CCK which inturn stimulates the digestive pancreatic amylase whereas, in gallbladder CCK-A receptors mediate contraction which causes release of bile which aids in fat and protein digestion (Noble et al, 1999).
Figure 2: Schematic representation of the mechanism of action of CCK in the regulation of feeding behaviour. NTS, nucleus tractus solitarius; PVN, paraventricular nucleus; PBN, parabrachial nucleus; VMH, ventromedial nucleus of the hypothalamus (Noble et al, 1999, adapted)
The basic mechanism involved in hunger suppression is based on the delivery of CCK from intestine to stomach via circulation after meal. It acts on afferent nerve fibres which signal brain and result in sensation of stomach fullness (Noble et al, 1999). Thus it causes decrease in meal size and shortens meal duration (Aquino et al, 1996). Hence efforts were made to develop compounds which could mimic CCK or have CCK agonist activity and thus can be used satiety agent in obesity treatment.
3-(phenylureido)-1,5 benzodiazepines were the first reported CCK-A agonists which were non-peptidyl in nature .
This series of compounds had no binding selectivity for CCK-A Vs. CCK-B receptors as well as compounds did not show any activity as oral satiety agent when tested in rat feeding model (Henke et al, 1996).
3(phenylureido) 1,5 benzodiazepine
Modification of the C-3 phenyl ureido group was carried out and ureido group was converted to amide (compound-1) (Wilson et al, 1996).
3(phenylamide) 1,5 benzodiazepine
Compound-1 exhibited selective partial agonist activity for CCK-A receptors. This agonist activity was tested using mouse gallbladder emptying assay after oral administration.But this compound failed to show oral activity in rat feeding model.The reason for this lack of activity of compound 1 was thought to be the partial agonist activity of this compound and hence efforts were made to develop a compound with full agonist activity and CCK binding selectivity (Henke et al, 1996).
For this purpose a series of isosteric replacements for C-3 phenyl amide moiety with groups such as 3 indolyl ,3 indazolyl were carried out .
Scheme 1 is employed for synthesis of this series of compounds (2- 10) .Scheme 1 is a general synthetic approach which allows easy variation of N1 and C-3 substituents (Henke et al, 1996 ).
Scheme 1: Reagents:Â (i) 2-bromo-N-isopropyl-N-phenylacetamide (R1Â = H) or 2-bromo-N-isopropyl-N-(4-methoxy)phenylacetamide (R1Â = OCH3), K2CO3, DMF, 18 h; (ii) malonyl dichloride, THF, 0 Â°C to room temperature, 18 h; (iii) NaN(TMS)2Â or KN(TMS)2, room temperature, DMF, 15 min; Ar-CH2-Br, DMF, room temperature, 2âˆ’5 h; (iv) KN(TMS)2, DMF, 0 Â°C, 15 min; MeI, DMF, room temperature, 3 h; (v) deprotection (if necessary) (Henke et al, 1996 adapted).
The starting compound N- phenyl 1,2 phenylenediamine was alkylated using 2-bromo-N-isopropyl-N-phenylacetamide or 2-bromo-N-(4-methoxy) phenylacetamide. This took place due to the complete chemoselectivity towards primary aniline. Potassium carbonate and di methyl formamide were employed for this step. 18 h were required for this reaction.Further cyclisation was achieved by condensation using malonyl chloride. This lead to the formation of benzodiazepine intermediate in rapid manner. This step was affected simply by stirring the two in THF at 0Ëš C to room temperature for 18 h. C-3 substituent was introduced in this intermediate (11) by deprotonation at the C-3 position by treatment with a base like KN(TMS)2 or NaN(TMS)2 at room temperature for 15 minutes.This was followed by addition of proper heteroaromatic alkyl halide (Ar-CH2-Br) using DMF at room temperature for 2-5 h. This resulted into development of the desired compound (2-10). Deprotonation was carriedout if necessary (Henke et al 1997). C-3 quaternary analogues were formed by deprotonation of second hydrogen followed by quenching with methyl iodide. Deprotonation was carried out if necessary.
The sequence employed here for the alkylation can be reversed without any change in yield. But the above sequence of alkylation was only preferred because C-3 H and C-3 CH3 analogues containing a common C3 indazole derivative can be obtained (Henke et al 1997).
2.2 Out of the series of 10 compounds which were synthesized compound number 7 was found to be the best compound because of the following reasons (Henke et al 1996) :
It was the only compound which demonstrated full agonist activity.
It also had moderate binding selectivity.
It was found to be as efficacious as CCK-8.
Activity of this compound is maintained when administered by ip and po routes.
2-[3-(1H-indazol-3-ylmethyl)-2,4-dioxo-5-phenyl-2,3,4,5- terahydrobenzo[b][1,4]diazepin-1-yl]-N-isopropyl-N-(4-methoxyphenyl) acetamide
Several groups such as Merck, Lilly, Glaxo worked on the development of CCK related compounds (Bock et al). Asperlicin , a selective CCK-A antagonist was used as a template for modification and development of various compounds such as selective CCK-A antagonists (Chang et al).
In research carried out at Merck and Lilly it was found that the 3-indolinylmethyl group of asperlicin was very essential for activity.Development of 1,4 benzodiazepines at Merck suggested that the benzodiazepine substructure is essential feature for interaction with CCK-A receptor (Henke et al 1996). Asperlicin moiety strucrurally resembles an important amino acid L- tryptophan. L-tryptophan is essential for agonist activity in peptide sequence of CCK and various other peptidomimetics (Shiosaki et al 1990).
Futher research carried out by Glaxo (Glaxo Wellcome Research and Development) lead to the development of 3-(1H- Indazol-3-ylmethyl)-1,5-benzodiazepines( CCK-A agonists).
2.4 STRUCTURE ACTIVITY RELATIONSHIP
Compound 1 Common structure
Table: 1 Structures (Henke et al, 1996)
When the Phenyl amide moiety in compound 1 is replaced by 3- indoylmethyl group , no change in the activity and efficacy occurs and it remains equal to amide 1. In the earlier work carried out it was found that replacement of para hydrogen in a the anilide (trigger portion of the molecule) with methoxy group causes increase in activity. But this replacement in indole series results in small decrease in the efficacy with slight increase in CCK-A affinity (compound 2 vs. 3). The removal of C-3 methyl group in this series leads to increase in efficacy (compound3 vs. 4) .
If the 3- indole moiety is replaced by 3 indazole modest increase in efficacy occurs. Indazole moiety can act as a bioisostere of indole and hence it was chosen for replacement (3 vs.6 , 4 vs.7) (Fludzinski et al 1987). Compound 7 which is the best compound is of indazole series and was the only compound to have full agonist activity and efficacy equal to CCK-8. Replacement of p-methoxy group of trigger portion in indazole series causes a small decrease in efficacy (7 vs.8). Methylation of N-1 nitrogen in indazole compounds cause decrease in efficacy (7 vs.9). N-benzylation causes decrease in agonist activity(7vs.10). In both indole and indazole series removal of C-3 methyl group causes decrease in CCK- A Vs. CCK-B selectivity (6 vs. 7). Optimal CCK-A Vs. CCK-B selectivity in this series can be obtained by addition of p- methoxy group in anilide portion and a C-3 methyl group in 1,5 benzodiazepine ring (2 vs.3 in indole series) , (3 vs.4, 6 vs. 7 in indazole series). Another example is compound 6 which shows greater then 5000 fold increase in selectivity for CCK- Areceptor. Direct replacement of phenylamide by indazole causes 5 fold decrease in selectivity (1 vs. 5) (Henke et al 1996)
This series of compounds synthesized were evaluated for their activity by performing in-vitro and in-vivo assays.
3.1 In vitro assays:
Two types of in vitro assays were performed for evaluation of activity.
3.1a Functional assay
Functional assay was performed on guinea pig gallbladder to evaluate the agonist activity of the compounds. Guinea pig gallbladder was isolated and cut into 2 rings of 2-4 mm each. These rings were then suspended in physiological salt solution using an organ bath. The conditions essential for tissue(37ËšC, 95% aeration, 7.4 pH) were maintained. Gold chains and stainless steel mountings were used to connect tissue to the isometric force displacement transducers. Polygraph was used to record response. Out of the 2 tissues from each animal one did not receive test compound and served as control. A basal ring tension of 1g was applied throughout the experiment. To verify the tissue contractility rings were exposed to Ach and the baline was re-established using sulphated CCK (Sugg et al, 1995).
The test ligand was then incubated with the tissue for 30 minutes period at 37ËšC. ED50 and % max values were calculated for the compounds from the response obtained. ED50 is the "concentration at which 50% of maximal contraction is observed". Where as % max is the "relative efficacy as determined by the maximal contraction observed at 30Âµm standardised to CCK-8(1Âµm=100%)". To verify that the response obtained is CCK-A mediated, reversal contraction was carried out using 1Âµm antagonist devazipide(MK329) (Sugg et al,1996)
3.1b Binding assay
CHO-K1 cells are employed for this assay. They are transfected with human CCK-A and CCK-B receptors (Brad et al, 1996). Radiolabelled CCK-8(125I Bolton Hunter CCK-8) was dissolved in appropriate solvent(DMSO). The stock concentration was adjusted such that its concentration is 100 times the final assay concentration and dilutions were prepared using binding buffer. Test ligand(25ÂµL) , labelled CCK-8 (25 ÂµL), buffer(150 ÂµL)and recptor preparation(50 ÂµL) was added to 96 well plate and assay was performed in triplicate manner. MK-329 was used to determine non-specific binding. Plates were incubated at 30ËšC for 3h and termination of incubation was carried out using glass filter. Unbound ligand was removed by washing .Radioactivity was measured by Î³ counting (Sugg et al, 1995).
3.2 In vivo assay
3.2a Mouse gall bladder emptying assay
Male CD-1 mice (10 animals per dose) were employed for this essay. They were fasted for a night and were treated ip or po with test drug dissolved in vehicle (ethanol/propylene glycol/water , 2:3:5, 1 mL/kg). After 30 minutes of drug treatment mice were sacrificed using CO2 and their gallbladders were isolated and weighed. Weights of the gallbladders of treated animals were normalised to that control group. ED50 and % empty values were determined uing this (Henke et al 1997).
3.2b Rat feeding model assay
2 weeks prior to the treatment male Long Evans rats (225-300g) were habituated to a palatable liquid diet after 2 h deprivation of food. On one day prior to treatment , rats were deprived of food for 100 minutes and then administered po with drug vehicle (propylene glycol, 1mL/kg). This was followed by oral preload of saline (8 mL/kg). After 20 minutes , consumption of liquid diet was measured at interval of 30,90 and 180 minutes. The rats which consumed atleast 8 mL of diet in first 30 minutes were qualified for drug treatment study. On the treatment day the same procedure was followed using various concentration of drug sample dissolved in the vehicle . Dunnett's multiple comparision test was used to normalise the data for each rat with respect to values obtained on pretreatment day (Henke et al 1997).
RESULTS AND DISSCUSSION
A series of compounds (2-10) were synthesized by employing scheme 1. They were thoroughly studied for their structure activity relationship and it was found that replacement of the C-3 pharmacophore can alter activity of these compounds. Hence a number of isosteric replacements for C-3 pharmacophore and variation in substituents at C-3 position and para position of trigger molecule were carried out.
These compounds were evaluated for their activity by performing in vitro and in vivo assays. The results obtained can be summarised as below (Henke et al, 1996).
4.1 In vitro assay
4.1a Functional assay
Table 2: Functional assay (Henke et al, 1996)
2 Â± 1(5)
190 Â± 20(4)
70 Â± 10(2)
Functional assay contraction of guinea pig gallbladder was considered as a measure of its efficacy. ED50 and % max values for compounds 1-10 and CCK-8 were calculated. Comparing the ED50 values of these compounds with that of standard CCK-8 it was observed that compound 7 of indazole series had high efficacy. % max value for compound 7 was equal to that of CCK-8. These values indicated that compound 7 was a full agonist of CCK-A receptors (Henke et al, 1996).
4.1b Binding assay
Table 3: Binding assay (Henke et al, 1996)
8.88 Â± 0.22(8)
9.46 Â± 0.04(8)
7.12 Â± 0.02(3)
5.08 Â± 0.04(3)
6.97 Â± 0.09(3)
8.08 Â± 0.24(4)
4.76 Â± 0.26 (5)
7.96 Â± 0.14(3)
5.28 Â± 0.08 (3)
6.92 Â± 0.12(3)
5.58 Â± 0.05 (3)
7.88 Â± 0.02(3)
4.16 Â± 0.23 (4)
7.64 Â± 0.12(5)
6.00 Â± 0.23 (4)
7.06 Â± 0.05(3)
6.75 Â± 0.10 (3)
7.37 Â± 0.43(3)
5.62 Â± 0.20 (3)
6.51 Â± 0.16(5)
4.00 Â± 0.0 (2)
Binding assay was used to evaluate the selectivity of these compounds for the CCK-A and CCK-B receptors. The pIC50 value of compound 7 was found to be comparable to that of CCK-8 which indicates it potency. pIC50 values were determined. pIC50 is "log of the concentration that displaced of 50% of Bolton- Hunter CCK-8 from membrane preparation" IC50(A)/IC50B gives selectivity for CCK-A. Thus from this in vitro assay data compound 7 was found to be the only compound to have full agonist activity and moderate binding selectivity (Henke et al, 1996).
4.2 In vivo assay
As compound 7 was the only compound to demonstrate full agonist activity , in vivo assay was performed to examine whether the in vitro activity of the compound 7 transformed to in vivo activity.
4.2a Mouse gallbladder emptying assay
Table 4: Mouse gallbladder emptying assay (Henke et al,1996)
Emptying of the mouse gallbladder was observed after ip and po administration of these compounds. ED50 and % empty values for each route were calculated. It was observed that the compound 7 was more potent and efficacious than compound 1and had efficacy equal to that of CCK-8 (Henke et al, 1996).
4.2b Rat feeding model assay
Figure:3 Graphical representation of food suppression in rat
(Â·) vehicle; (â-¡)Â 7, 0.1 Î¼mol/kg; ()Â 7, 1.0 Î¼mol/kg; ()Â 7, 10 Î¼mol/kg; ()Â d-Amphetamine, 2 mg/kg (Brad et al, 1996)
This assay is an important measure of in vivo activity of compound 7. Amphetamine was used as a positive control in this experiment. This assay demonstrates a dose dependent decrease in food intake in rats after oral administration of compound 7. The highest suppression in food intake(%) was observed for dose of 10Âµmol/kg, which can be clearly observed in graph (Henke et al, 1996).
A series of 10 compounds, comprising of compound 1 (amide), compounds 2 to 4 of indole series and compounds 5to10 indazole series was synthesized by carrying out replacements at C-3 position as C-3 pharmacophore was found to be most important for activity. Through functional and binding assays (in vitro) , indazole series was found to be most potent as compound 7 (G5823) of this series was the only compound which exhibited full agonist activity with moderate binding selectivity and was efficacious as CCK-8. It also distinctively demonstrated its in vivo oral activity in mouse gallbladder emptying assay and rodent feeding model, where it successfully suppressed food intake in a dose dependent manner.
Thus this compound (G5823) was found to be a potential orally active satiety agent which can be employed in obesity treatment.