A PYRROLOPYRIMIDINEDIONE DERIVATIVE AND PREPARATION THEREOF

Abstract

The present disclosure relates to a novel pyrrolopyrimidinedione derivatives of compound of formula I, its solvate, its stereoisomer or a pharmaceutically acceptable salt thereof for highly selective anti-rheumatoid arthritis activity. Also, the present disclosure relates a process for preparing novel pyrrolopyrimidinedione derivatives. Further, the present disclosure is to provide compounds with simple, cost-effective and process provides high yield compounds.

Specification

Description:TECHNICAL FIELD:
The present disclosure relates generally to the field of anti-rheumatoid arthritis compounds. More specifically, the present disclosure relates to a novel pyrrolopyrimidinedione derivatives of compound of formula I, its solvate, its stereoisomer or a pharmaceutically acceptable salt thereof for highly selective anti-rheumatoid arthritis activity. Also, the present disclosure relates a process for preparing novel pyrrolopyrimidinedione derivatives. Further, the present disclosure is to provide compounds with simple, cost-effective and process provides high yield compounds.

BACKGROUND
Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic inflammation of the synovial joints, leading to pain, swelling, and progressive joint damage. In 2019, 18 million people worldwide were living with rheumatoid arthritis [1]. About 70% of people living with rheumatoid arthritis are women, and 55% are older than 55 years [1]. Despite advancements in treatment strategies, including disease-modifying antirheumatic drugs (DMARDs) and biologic agents targeting cytokines such as tumour necrosis factor-alpha (TNF-α), a significant proportion of RA patients fail to achieve adequate disease control or experience adverse effects with current therapies. Therefore, there is a unmet clinical need for the development of novel therapeutics with improved efficacy and safety profiles for the management of RA.

Janus kinases (JAKs) area family of intracellular tyrosine kinases that play crucial roles in signal transduction pathways mediated by various cytokines and growth factors involved in immune responses and inflammation [3]. Among the JAK family members, JAK3 has emerged as a promising therapeutic target for RA due to its selective expression in hematopoietic cells and its critical role in signaling pathways mediated by interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-15 (IL-15), and interleukin-21 (IL-21), which are implicated in the pathogenesis of RA. Inhibition of JAK3 has been shown to suppress immune responses and reduce inflammation in preclinical models of RA, highlighting its potential as a target for RA therapy.

In this study, we aimed to design and synthesize a series of novel heterocyclic analogues as potential dual inhibitors targeting both JAK3 enzyme and TAp63 transcription factor for the treatment of RA. We hypothesized that dual inhibition of JAK3 and TAp63 may exert synergistic Effects in suppressing immune responses and inflammation, leading to improved therapeutic outcomes in RA.

Need for new anti-rheumatoid arthritis drugs:
• To improve the current course of treatment by shortening its total duration.
• To facilitate drug compliance by providing less intensive supervision
• Drug with lesser frequency of administration which permits widely spaced intermittent treatment. To reduce the side effects which may include ringing in your ears, stomach irritation, heart problems and liver and kidney damage.
• To design a drug with higher efficacy and more specific in action by inhibiting the enzyme target which is responsible for inducing Rheumatoid Arthritis.

Therefore, there is an ongoing need to overcome the drawbacks of the prior art and to find a novel anti-rheumatoid arthritis drugs. Further the present disclosure provides compounds with simple, cost-effective and higher yield compounds. Thus, we have designed, synthesized and screened compounds for its potential anti-rheumatoid arthritis activity.

OBJECTIVES:
An object of present disclosure is to provide a novel anti-rheumatoid arthritis compounds.

An object of present disclosure is to provide a novel pyrrolopyrimidinedione derivatives.

Another object of present disclosure is novel pyrrolopyrimidinedione derivatives of compound of formula I, its solvate, its stereoisomer or a pharmaceutically acceptable salt thereof for highly selective anti-rheumatoid arthritis activity.

Yet object of the present disclosure is to provide a process for preparing novel pyrrolopyrimidinedione derivatives.

Further object of the present disclosure is to provide compounds with simple, cost-effective and process provides high yield compounds.

SUMMARY:
This summary is provided to introduce a selection of concepts in a simplified form that are further described below in Detailed Description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

An aspect of present disclosure provides a pyrrolopyrimidinedione derivative, its solvate, its stereoisomer or a pharmaceutically acceptable salt thereof


Compound of formula I

wherein the R is Aniline, 4-aminophenol, 4-chloroaniline, 3,4-dichloroaniline, 4-bromoaniline.

These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF DRAWINGS:
The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
1. IR Spectrum of compound JTI106
2. IR Spectrum of compound JTI107
3. IR Spectrum of compound JTI116
4. IR Spectrum of compound JTI120
5. IR Spectrum of compound JTI121

DETAILED DESCRIPTION
The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment.

An aspect of present disclosure provides a pyrrolopyrimidinedione derivative, its solvate, its stereoisomer or a pharmaceutically acceptable salt thereof


Compound of formula I

wherein the R is Aniline, 4-aminophenol, 4-chloroaniline, 3,4-dichloroaniline, 4-bromoaniline.

An embodiment of the present disclosure provides, the compound of formula I is selected from;
4-(furan-2-yl)-6-phenyl-3,4,6,7-tetrahydro-1H-pyrrolo[3,4-d]pyrimidine-2,5-dione (JTI 106);
4-(furan-2-yl)-6-(4- hydroxyphenyl)-3,4,6,7-tetrahydro-1H-pyrrolo[3,4- d]pyrimidine-2,5-dione (JTI 107);
6-(4-chlorophenyl)-4-(furan-2-yl)-3,4,6,7-tetrahydro-1H-pyrrolo[3,4-d]pyrimidine2,5-dione (JTI 116);
1-(3,4-dichlorophenyl)-3- (furan-2-yl-l3-methyl)-4-((oxo-l3-methyl)amino)-1,5- dihydro-2H-pyrrol- 2-one (JTI 120); and
6-(4-bromophenyl)- 4-(furan-2-yl)- 3,4,6,7-tetrahydro- 1H-pyrrolo[3,4- d]pyrimidine-2,5-dione (JTI 121).

In an embodiment of the present disclosure provides, the process comprising the steps of:
i) condensation of heteroaldehyde reacts with halo substituted urea in the presence of ethanol and hydrochloric acid to obtain a 6-chloromethyl derivative of dihydropyrimidine-2-one;

ii) the obtained 6-chloromethyl derivative of dihydropyrimidine-2-one reacts with substituted phenylamine in alcoholic solution to obtain a compound of formula I.

Another embodiment of the present disclosure provides, numbers have been used for quantifying weights, percentages, dosages, and so forth, to describe and claim certain embodiments of the invention and are to be understood as being modified in some instances by the term "about."

Accordingly, in an embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

As used in the description herein and throughout the claims that follow, the meaning of "a," "an," and "the" includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise.

Unless the context requires otherwise, throughout the specification which follow, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense that is as "including, but not limited to."

The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range.

Unless otherwise indicated herein, each individual value is incorporated into the specification
as if it were individually recited herein.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified.

The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.

It should also be appreciated that the present disclosure can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.
The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
In an embodiment, the pharmaceutically acceptable excipient includes but is not limited to adjuvants, preservatives, pharmaceutically acceptable carriers, wetting agents, dispersants, sweetening agents, flavouring agents, diluents and the like well-known to a person of skill in the art. The pharmaceutically acceptable excipient is safe, non-toxic and does not interfere with the therapeutic activities of the pharmaceutical active.

While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.

EXAMPLES:
General Scheme:
The synthesis of pyrrolo[3,4-d] pyrimidine33,34 involves both pyrimidine and pyrrole precursors almost evenly. The presence of a carboxylic ester at position 5 of a pyrimidine ring contributes to a series of fused pyrroles containing a carbonyl group. The pyrimidine containing a carboxylic ester at position 5 is heated at reflux conditions with primary amine providing pyrrolo[3,4-d] pyrimidine. The synthesis of pyrrolo[3,4-d] pyrimidine involves 2 steps.

Step 1 involves condensation of Heterosubstituted aldehyde, Halosubstituted urea and monohaloethylacetate leads to the formation of pyrimidine nucleus. Followed by the Step 2 involves reaction of step one product with substituted phenylamine to form a fused ring system of pyrrolo pyrimidine. Like 6-Chloromethyl functionalized dihydropyrimidine intermediates on treatment with an excess of primary amine in methanol provided the pyrrolo[3,4 d]pyrimidine. The corresponding amine was allowed to react with the appropriate chloromethyl dihydropyrimidine at ambient or at a slightly elevated temperature for 1-2 hours followed by heating under reflux for 5-6 hours. The solid pyrrolo[3,4 d]pyrimidine was directly precipitated from the solution upon cooling.

The reaction takes place in two steps. In the first step, the chloro group was displaced by excess amine nucleophile. In the second step, the cyclative nucleophilic cleavage (Lactam formation) by cyclization of ethyl ester with the resulting secondary amine. On the basis of different reactivity of the primary amines, the nucleophilic displacement was carried out at room temperature for alkylamines and phenylethylamines or at 50°C for benzylamines and sterically hindered amines.


Compound of formula I

SYNTHETIC PROCEDURE:

Step 1: Synthesis of 6-chloromethyl derivative of dihydropyrimidine-2-one
A mixture of Monohaloethylacetate like Ethyl-4-chloroacetoacetate (3.0mmol), appropriate heteroaldehyde like Furfuraldehyde(3.0mmol), Urea or Hydroxyurea or Halo substituted urea (3.5mmol) were refluxed in 30ml ethanol containing 0.5ml hydrochloric acid for 5-6hrs.The reaction mixture was cooled and poured into ice-cold water. The solid product which was obtained was filtered, dried and recrystallized from ethanol.

Step 2: Synthesis of compound of formula I:

To the product obtained in first step, solution of substituted phenylamine in methanol was added dropwise with stirring at 25-30°C. Stirring was continued for 1-2 hrs. Then ethanolic/ methanolic sodium hydroxide (5ml) was then added. The reaction mixture was refluxed for 6hrs and the excess of solvent was distilled. The reaction mixture was cooled to room temperature. The solid product which was obtained was washed with cold methanol, dried and recrystallized from suitable solvent.




Compound of formula I

Table 1. IUPAC name and other parameters of pyrrolopyrimidinedione derivatives
Compound code R IUPAC Mol formula
JTI 106 Aniline 4-(furan-2-yl)-6-phenyl-3,4,6,7-tetrahydro-1H-pyrrolo[3,4- d]pyrimidine-2,5- dione C16H13N3O3
JTI 107 4-aminophenol 4-(furan-2-yl)-6-(4- hydroxyphenyl)-3,4,6,7-tetrahydro-1H-pyrrolo[3,4- d]pyrimidine-2,5- dione C16H13N3O4
JTI 116 4-chloroaniline 6-(4-chlorophenyl)-4-(furan-2-yl)-3,4,6,7-tetrahydro-1H-pyrrolo[3,4-d]pyrimidine2,5-dione C16H14ClN3O3
JTI 120 3,4-dichloroaniline 1-(3,4-dichlorophenyl)-3- (furan-2-yl-l3-methyl)-4-((oxo-l3- methyl)amino)-1,5- dihydro-2H-pyrrol- 2-one C16H11Cl2N3O3
JTI 121 4-bromoaniline 6-(4-bromophenyl)- 4-(furan-2-yl)- 3,4,6,7-tetrahydro- 1H-pyrrolo[3,4- d]pyrimidine-2,5- dione C16H12BrN3O3

Table 2. Product profile of synthesized compounds
Product Code Mol. Wt. (g/mol) Docking score Inhibitory Constant Product Yield (%)
JAK3 TAp6 3 JAK 3 (nM) TAp63 (nM)
JTI106 295 -9.02 -10.35 246.27 26.04 90
JTI107 311 -9.69 -10.72 78.87 13.93 95
JTI116 329 -8.54 -10.79 554.14 12.28 91
JTI120 364 -8.68 -11.28 433.49 5.4 82
JTI121 374 -8.57 -10.91 519.25 10.11 86

All the newly designed 121 ligands have subjected to the above filtering method of good predictive activity with good docking scores and also drug likeness properties confirming that these leads are orally bioavailable. Based on synthetic feasibility and high docking score exhibiting effective binding efficacy the following ligands JTI106, JTI107, JTI116, JTI20, JTI121 were chosen for synthesis.

Example 2: CHARECTERIZATION
The purity of the synthesized compounds was checked by TLC method and the sharp melting point determination. Progress of the reaction was monitored by thin layer chromatographic technique. TLC was performed using Aluminum plated pre-coated with Silica gel 60F 254 (E-Merck); the mobile phase used was dichloroethane and methanol (9:1). Spots were detected using UV light chamber and Iodine chamber (solvent system-methanol). Melting points of all the synthesized compounds were determined by open-capillary tubes and values were uncorrected.

All the synthesized compounds were characterized by the following spectroscopic methods.

IR SPECTROSCOPY
IR spectroscopy (infrared spectroscopy) deals with the infrared region of the electromagnetic spectrum, i.e. light having a longer wavelength and a lower frequency than visible light. Infrared Spectroscopy generally refers to the analysis of the interaction of a molecule with infrared light. The major use of infrared spectroscopy is to determine the functional groups of molecules, relevant to both organic and inorganic chemistry. IR spectra was recorded using ABB MB3000-PH FT-IR Spectrometer. Most of the bands that indicate what functional group is present are found in the region from 4000 cm-1 to 1300 cm-1. Their bands can be identified and used to determine the functional group of an unknown compound.

Bands that are unique to each molecule, similar to a fingerprint, are found in the fingerprint region, from 1300 cm-1 to 400 cm-1. These bands are only used to compare the spectra of one compound with another.

MASS SPECTROMETRY
Mass spectrometry is an analytical technique used to establish the molecular structure and the molecular weight of the analyte under investigation. In this technique, the compound under investigation is bombarded with a beam of electrons producing ionic fragments of the original species. The relative abundance of the fragment ion formed depends on the stability of the ion and of the lost radical. The resulting charged particles are then separated according to their masses. Mass spectrum is a record of information regarding various masses produce their relative abundances. Waters-Xevo G2- XS QToF High Resolution Mass Spectrometer.

Table 3. IR interpretation of compound JTI106
S.No Functional group Frequency (cm-1)
1 C=O Stretching 1627.80
2 N-H Stretching 3417.61
3 C-H (Aromatic) Stretching 3093.59
4 C-H (sp3hybrid) Stretching 2923.87
5 C=C (Aromatic) Stretching 1596.94
6 C-O Stretching 1134.06
7 C-N Stretching 1242.07
Table 4. IR interpretation of compound JTI107
S. No Functional group Frequency (cm-1)
1 C=O Stretching 1689.52
2 N-H bending 1450.36
3 O-H Stretching 3309.61
4 C-H (Aromatic) bending 825.47
5 C-H (sp3hybrid) Stretching 2931.58
6 C=C (Aromatic) Stretching 1512.08
7 C-O Stretching 1095.49
8 C-N Stretching 1226.64
Table 5. IR interpretation of compound JTI116
S. No Functional group Frequency (cm-1)
1 C=O Stretching 1681.80
2 N-H Stretching 3417.61
3 C-H (Aromatic) Stretching 3093.59
4 C-H (sp3hybrid) Stretching 2923.87
5 C=C (Aromatic) Stretching 1596.94
6 C-O Stretching 1134.06
7 C-N Stretching 1234.35
8 C-Cl Stretching 856.33
Table 6. IR interpretation of compound JTI120
S. No Functional group Frequency (cm-1)
1 C=O Stretching 1627.80
2 N-H Stretching 3417.61
3 C-H (Aromatic) Stretching 3093.59
4 C-H (sp3hybrid) Stretching 2923.87
5 C=C (Aromatic) Stretching 1596.94
6 C-O Stretching 1134.06
7 C-N Stretching 1242.07
8 C-Cl Stretching 856.33
Table 7. IR interpretation of compound JTI121
S. No Functional group Frequency(cm-1)
1 C=O Stretching 1689.34
2 N-H Stretching 3365.18
3 C-H (Aromatic) bending 1459.85
4 C-H (sp3hybrid) Stretching 2923.56
5 C=C (Aromatic) Stretching 1592.91
6 C-O Stretching 1176.36
7 C-N Stretching 1222.65
8 C-Br Stretching 742.46

Example 3: BIOLOGICAL EVALUATION
a) MATERIALS
Animals Wistar rats (200-300g) either sex was acclimatized for 7 days under standard husbandry conditions, i.e. room temperature 25±10 C, relative humidity 45-55% and light/dark cycle 12/12 hrs.

The experimental protocols were approved by the Institutional Animal Ethical Committee (IAEC) of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) Husbandry Practices Caging Polypropylene rat cages covered with stainless steel grid top were Autoclaved clean rice husk was used as the bedding mate Water Bottle Each cage was supplied with a polypropylene water bottle with a stainless-steel nozzle.

Housing Single rat per cage Room Sanitation Each day, the floor of the experiment room was swept, and all work tops and the floor were mopped with a disinfectant solution. Animal Identification each rat was uniquely numbered on the tail using a tattoo machine. Appropriate labels were attached to the cages indicating the study number, sex, and dose, type of study, cage number and animal number.

b) METHODS
a. Acute oral toxicity (Acute toxic class method in Wistar rats)
b. In-vivo anti-rheumatoid arthritis activity.

Subsequently, all 121 ligands were subjected to docking simulations against the JAK3 enzyme and TAp63, utilizing the identified active sites and grid parameters. The docking process aims to predict the optimal binding poses of the ligands within the active sites of the target proteins, as well as estimate their binding affinities. Through this computational approach, potential lead compounds with favorable binding interactions and potential against JAK3 and TAp63 can be identified, laying the ground work for further experimental validation and drug development efforts. The docked results were listed below for some ligands and the remaining were filtered out based on the reasons like predicted toxicity, drugs likeness instability, abnormal docking score.

ACUTE ORAL TOXICITY (ACUTE TOXIC CLASS METHOD IN WISTAR RATS)
Acute oral toxicity defines to the adverse effects occurring following oral administration of single dose of substances or multiple doses given within 24 hrs.

The different methods used to evaluate the acute oral toxicity studies are as follows,
i. Fixed dose procedure (OECDGuidelines-420)
ii. Acute toxic class method (OECDGuideline-423)
iii. Ups and Down procedure (OECDGuideline-425)
Our study was done following acute toxic class method (OECD-423).

Acute toxic class method
In the present study the oral toxicity of the synthesized compounds was performed by acute toxic class method. In these methods the toxicity of the synthesized compounds was tested using a stepwise procedure, each step using three rats of a single sex. The various concentration of test drug as per OECD guidelines are as follows, the Wistar rats were fasted overnight prior to dosing (food but not water should be withheld). Following the period of fasting the animal should be weighed and the synthesized compounds administered orally at the dose of 2000 mg/kg body weight. Animals were observed individually after dosing at least during the first 4 hrs and daily thereafter, for a total of 14 days. As no mortality was observed with the above doses, a series of doses 200 and400 mg/kg body weight were selected for the further pharmacological evaluation. The test procedure with starting doses of 2000 mg/ kg body weight as per OECD- 423 guide.

Oral Acute Toxicity Study OECD-423
Oral acute toxicity of newly synthesized compound JTI107 was tabulated.

Table no 8. Observation in Oral Acute Toxicity Study (OECD-423)
S. No Observation For 30 mins 4hrs 24hrs 48hrs 7days 14 days
1. Sedation Absent Absent Absent Absent Absent Absent
2. Excitation Absent Absent Absent Absent Absent Absent
3. Jumping Normal Absent Absent Absent Absent Absent
4. Writhing Absent Absent Absent Absent Absent Absent
5. Scratching Absent Absent Absent Absent Absent Absent
6. Grooming Absent Absent Absent Absent Absent Absent
7. Aggression Absent Absent Absent Absent Absent Absent
8. Ptosis Absent Absent Absent Absent Absent Absent
9. Loss of Writhing reflux Absent Absent Absent Absent Absent Absent
10. Loss of pineal Reflux Absent Absent Absent Absent Absent Absent
11. Loss of Corneal reflux Absent Absent Absent Absent Absent Absent
12. Excess Salivation Absent Absent Absent Absent Absent Absent
13. Lacrimation Absent Absent Absent Absent Absent Absent
14. Skin & fur Normal Normal Normal Normal Normal Normal
15. Color of eye Normal Normal Normal Normal Normal Normal
16. Tremors Absent Absent Absent Absent Absent Absent
17. Diarrhea Absent Absent Absent Absent Absent Absent
18. Coma Absent Absent Absent Absent Absent Absent
19. Inflammation Absent Absent Absent Absent Absent Absent
20. Urination Absent Absent Absent Absent Absent Absent
In acute toxicity study test, no mortality (sign of toxicity) was observed for all the selected doses during the study. The acute toxicity studies showed that the non-toxic nature of the newly synthesized heterocycle of pyrollo pyrimidinedione JTI 107 was up to the level of 2000 mg/kg body weight selected doses.
IN-VIVO ANTI-RHEUMATOID ARTHRITIC ACTIVITY
Animal
Protocol of the study was passed by Institutional Ethics Committee of Madras Medical College, Chennai-03. The study was carried out with adult Wistar rats (Male/Female) weighing 100-200 g. Animal were acclimatized to the experimental conditions in cages and kept under a standard environmental condition (22 +30C ;12/12hr light/dark cycle). Rats must allow to feed water and food.

Statistical analysis
Statistical analysis of difference between groups was evaluated by one-way ANOVA followed by student t test. The values P<0.05 were regarded as significant and the values P<0.01 were considered as highly significant.

Induction of CFA and Drug treatment
Adult Wistar rats with an initial body weight of 100-200 g were taken and divided into five groups each containing 6 animals. On zero day, all rats will to injected into the sub plantar region of left hind paw with 0.1 ml of Complete Freund's Adjuvant. This consist of Mycobacterium butyricum suspended in heavy paraffin oil by through grinding with mortar and pestle to give a concentration of 6 mg/ml.

Evaluation of development of Arthritis
It must be inspected daily for the onset of arthritis characterized by edema in the paws. The incidence and the severity of arthritis will be evaluated using a system of arthritic scoring. Measurement of bi-hind paw volumes every 3 days, when arthritic signs first visible. Animals will be observed for the presence or absence of nodules in different organs like ear, fore paw, hind paw, nose and tail. Animal were scored 0 for absence of nodules and 1 for its presence. Score 5 was given as the potential maximum of arthritic score per animal. Hind paw volume was measured using plethysmometer. Paw volumes of both hind limbs must record from day of Treatment started to 21st day at three-day interval using mercury column plethysmometer.
Table 9. Effect of Foot pad thickness
Treatment
Mean foot pad thickness(mm)
0 day 3rd day 7th day 14th day 21st day
Group I (NC) 4.51±0.004 4.49±0.002 4.50±0.003 4.52±0.02 4.55±0.001
Group II (DC) 4.23±0.003*** 14.20±0.004*** 14.13±0.005*** 13.63±0.003*** 13.01±0.001***
Group III (STD) 4.12±0.04*** 14.13± 0.04*** 13.51± 0.03*** 12.78± 0.02*** 11.03± 0.02***
Group IV
(JTI 107 low dose) 4.10±0.04*** 12.25±0.04*** 6.13±0.03*** 5.79±0.02*** 6.02±0.03***

Group V
(JTI 107 high dose) 4.15±0.03*** 12.01±0.02*** 8.16±0.02*** 7.05±0.02***
5.06±0.02***
The hind paw injected with Complete Freund's adjuvant became gradually swollen and reached its peak at 21st day. The result obtained for the two different doses of the test compound and the standard drug (Methotrexate 5mg/ kg) in the Complete Freund's adjuvant induced paw edema test at specific time intervals. When compared with Normal Control rats, the Disease Control rats showed significant increase (P < 0.001) in the paw volume after seven days of sub plantar CFA administration. In the primary phase of the arthritis i.e. form day 4 to 7, there was non-significant deceased in the paw volume was observed. Rats treated with test compound (200 and 400 mg/kg) showed significant and dose-dependent attenuation in paw volume from day 15 to 21 onward as compared to Disease Control rats. Rat treated with Methotrexate (1mg/kg) significantly decreased (P<0.001) paw volume from day 15 to 21toascomparedtocontrolrats. Rats treated with test compound JTI 107 (200 and 400 mg/kg) was found to have effective anti- arthritic activity.

Effect of Average body weight
Changes in body weight was also recorded to evaluate anti- arthritic potential of synthesized compound JTI 107. Rats treated with Methotrexate (1mg/kg) shows significantly increased in body weight(P<0.001) synthesized compound JTI107 (200mg/kg) and (400mg/kg) also showed significant changes in body weight with value of (P< 0.001) when compared with disease control.
Table 10. Effect of Average body weight
TREATMENT Average Body weight
0 day 3rd day 7th day 14th day 21st day
Group I (NC) 275.66±0.22 272±0.40 274.50±0.95 272.93±0.60 274.60±0.15
Group II (DC) 263.19±
0.34** 221.16±
0.56** 217.40±
0.34** 196.31±
0.72** 182.41±
0.45**
Group III
(STD) 296.18±
0.84** 281.16±
0.96** 276.40±
0.24** 287.30±
0.92** 272.41±
0.32**
Group IV
(Cpd low dose) 266.16±
0.764** 265.16±
0.56** 267.40±
0.84** 266.30±
0.42** 267.41±
0.62**
Group V (Cpd
High dose) 270.13±
0.24** 268.16±
0.87** 267.40±
0.97** 269.30±
0.32** 270.41±
0.92**
N=6, values were expressed as Mean±SEM,***p value<0.001significant, NC-Normal control. DC - Disease control; STD - Standard group; Cpd - Compound JTI 107.
Total leukocyte Count and Neutrophile count
Blood samples were collected by puncturing the retro-orbital plexus into heparinized vials and analyzed for total leucocyte counts (TLC) and differential leucocyte counts (DLC).

Effect of Hematological Parameter
The changes in hematological parameters in adjuvant induced arthritic rats are shown in Table.No.27. There was a significant (p < 0.001) decrease in RBC count and hemoglobin and increase in WBC count and ESR of arthritic rats as compared to control rats. Rats treated with Methotrexate (1mg/kg) showed changes in the hematological function when compared with disease-controlled rats. Rats treated with synthesized compound JTI 107 (200mg/kg and 400mg/kg) also shows significant changes in body weight with value of P<0.001.
Table 11. Effect of Hematological Parameter

Rheumatoid Factor
The latex turbidimetry method was used in the present study using RF kit of Infinite Company. Calibration was carried out for linear range up to 100 IU/ml. The reading of RF factor of all the groups obtained was compared with the control animals was expressed as IU/ml.
Table 12. Effect of Rheumatoid Factor
Group Rheumatoid Factor (IU/ml).
Group I (NC) 12± 0.82
Group II (DC) 48 ± 3.39***
Group III (STD) 37 ± 2.65***
Group IV (JTI200) 19± 0.32***
Group V (JTI400) 17± 0.18***

Arthritic Index
The arthritic index in rats is typically performed as part of experimental studies to assess the severity and progression of arthritis, often in the context of testing potential treatments.

The severity of arthritis is scored using an arthritic index, which is based on visual assessment and palpation of the joints. The scoring system may vary but typically includes the following:
• 0: No signs of arthritis.
• 1: Mild swelling and erythema (redness) in one joint.
• 2: Moderate swelling and erythema in one or more joints.
• 3: Severe swelling and erythema affecting the entire paw.
• 4: Maximum swelling with deformity and/or ankylosis (joint stiffness).

Table 13: Effect of Arthritic Index
Treatment Arthritic Index
Group I (NC) 0.00±0.000
Group II (DC) 3.76±0.24***
Group III (STD) 1.32±0.25***
Group IV (JTI107-200mg) 1.86±0.32***
Group V (JTI107-400mg) 2.63±0.18***

Sub plantar administration of CFA results in significant increased (P < 0.001) in arthritic score in all arthritic treated rats as compared to control rats. Albino rats treated with synthesized compound JTI107 showed significant and dose dependent decreased in arthritic score(P<0.001) as compared to arthritic diseased rats.

Radiography
Wistar rats have to be sacrificed on 21st day of CFA administration and legs will be removed and placed on formalin containing bags. This plastic bag was kept at 90cm from the X-ray source and Radiographic analysis of arthritic and treated animal hind paw was performed by X-ray machine with a 300-mAexposition for 0.01s. An investigator blinded for the treatment regimen performed radiograph score which will used as a quantal test for bone necrosis. The following radiograph criteria will be considered. Radiographs will carefully be examined using a stereo microscope and abnormalities will be graded as follows:
• Periosteaic reaction,0-3(None, Slight, Moderate, Marked);
• Erosions,0-3(None, Few, Many Small, Many Large);
• Jointspacenarrowing,0-3(None, Minimal, Moderate, Marked);
• Jointspacedestruction,0-3(None, Minimal, Extensive, Ankylosis).
• Bone destruction was scored on the patell a as described previously.

Bone destruction, which is a common feature of adjuvant arthritis was examined by radiological analysis. Treated rat shad developed definite joints pace narrowing of the intertarsal joints, diffuse soft tissues swelling that includes the digits, diffuse demineralization of bone, marked periosteal thickening and cystic enlargement of bone and extensive erosions produced narrowing or pseudo widening of all joints space. In contrast, rats treated with synthesized compound JTI 107attenuate abnormalities consisted of asymmetric soft tissue swelling and small erosions, periosteal thickening and minimal joint space narrowing areas of the paws and the result were shown below. Despite a similar chemical course of arthritis, disease control rats suffered more pronounced bone destruction than synthesized compound JTI107 treated animals as shown on radiograph taken on 21st day in CFA induced arthritis.

Histopathological study of joints:
Synthesized compound JTI 107 treated animals showed more pronounced decrease in bone density, destruction of bony structure, as compare to disease control. Abrogation of disease progression by synthesized compound JTI 107 was further supported by the Histopathologic analysis of the joints from these animals. Rats that had been treated with test compounds at the time CFA immunization showed no histological abnormalities with no evidence of cartilage erosion in their joints in contrast to the disease control rats that displayed completely destroyed joint architecture.

Computational Drug Discovery aims to produce novel chemical molecule which potentially prevents or treats a disease/infection. Therefore, the current study was aimed to design and synthesizes some newer anti-rheumatic agents. Based on the literature review, JAK3 and TAp63 are chosen as the effective targets for anti-rheumatic therapy. Reviewing the literature and with the knowledge of available inhibitors and its interaction with the enzyme, a scaffold library has been constructed with 121 newly designed ligands. The ligands were subjected to docking studies using the software Autodock Tools 4.2 (1.5.6) to find better JAK3 and TAp63 inhibitor. The ligands were further optimized by per forming drug likeness screening such as Lipinski's rule of 5 & prediction of good ADMET properties using online tools like Molinspiration and insilico toxicity study using Osiris property explorer. All the 121 designed ligands were found to be nontoxic and can be orally bioavailable. Further JTI106, JTI107, JTI116, JTI120 and JTI121 with the pyrrolo-pyrimidinedione moiety were selected for synthesis based upon high docking score and synthetic feasibility. All the five selected leads were synthesized. The purity of all the synthesized molecules were assessed by TLC and melting point determination. Characterization of the compounds was carried out using IR spectroscopy, 1HNMR spectroscopy and Mass spectroscopy analytical techniques. Acute oral toxicity study of the synthesized compounds was performed as per the OECD Guidelines 423. All the synthesized compounds were found to be safe up to the dose of 2000 mg/Kg body weight and no mortality was observed.

In hematological parameter, the result of P values obtained in one way ANOVA, synthesized compound JTI 107; 200 mg/kg, 400mg/kg was found to decreases the Hemoglobin, RBC counts and increases the WBC, ESR counts with the***P value<0.001and was found to be reduce the rheumatoid factor and compared with that of standard Methotrexate 1mg/kg. In spleen index, the result of P values obtained in one way ANOVA, synthesized compound JTI107 200mg/kg, 400mg/kg was found to inhibit the splenomegaly with the ***P value < 0.001 and was found to be reduce the spleen index and also compared with standard Methotrexate 1mg/kg. In radiography, the result of P values obtained in one way ANOVA, synthesized compound JTI107 200mg/kg, 400mg/kg was found to increases the joint space and decreases the erosion in joints with the ***P value < 0.001 and proved to be significant when compared with standard Methotrexate 1mg/kg. In histopathology, the result of P values obtained in one way ANOVA, synthesized compound JTI 107 200mg/kg, 400mg/kg was found to reduces the destruction of bony structure and increased the bone density with the ***P value < 0.001 significantly as that of standard Methotrexate 1mg/kg.

In RA, the immune system attacks the synovium, the lining of the membranes that surround the joints, leading to inflammation. This inflammation causes the synovium to thicken, resulting in pain, swelling, and stiffness in the affected joints. Overtime, the inflammation can damage the cartilage and bone within the joint, leading to joint deformity and loss of function.

In conclusion, we have successfully designed and synthesized a series of novel Pyrrolopyrimidinedione as potential dual inhibitors targeting both JAK3 enzyme and TAp63 protein for the treatment of RA. The synthesized compounds exhibited potent efficacy in ameliorating RA symptoms in preclinical models. These findings warrant further investigation of the lead compounds as potential therapeutics for RA.

Advantages:
1. Simple
2. Cost-effective process.
3. The process of present disclosure provides compound with high yield.
4. The present disclosure provides potential anti-rheumatoid arthritis compounds.
, Claims:1.A pyrrolopyrimidinedione derivative, its solvate, its stereoisomer or a pharmaceutically acceptable salt thereof:

Compound of formula I
wherein the R is Aniline, 4-aminophenol, 4-chloroaniline, 3,4-dichloroaniline, 4-bromoaniline.

2. The compound of formula I is selected from;
4-(furan-2-yl)-6-phenyl-3,4,6,7-tetrahydro-1H-pyrrolo[3,4-d]pyrimidine-2,5-dione (JTI 106);
4-(furan-2-yl)-6-(4- hydroxyphenyl)-3,4,6,7-tetrahydro-1H-pyrrolo[3,4- d]pyrimidine-2,5-dione (JTI 107);
6-(4-chlorophenyl)-4-(furan-2-yl)-3,4,6,7-tetrahydro-1H-pyrrolo[3,4-d]pyrimidine2,5-dione (JTI 116);
1-(3,4-dichlorophenyl)-3- (furan-2-yl-l3-methyl)-4-((oxo-l3-methyl)amino)-1,5- dihydro-2H-pyrrol- 2-one (JTI 120); and
6-(4-bromophenyl)- 4-(furan-2-yl)- 3,4,6,7-tetrahydro- 1H-pyrrolo[3,4- d]pyrimidine-2,5-dione (JTI 121).

3. The process for preparing compound of formula I, the process comprising the steps of:
i) condensation of heteroaldehyde reacts with halo substituted urea in the presence of ethanol and hydrochloric acid to obtain a 6-chloromethyl derivative of dihydropyrimidine-2-one;
ii) the obtained 6-chloromethyl derivative of dihydropyrimidine-2-one reacts with substituted phenylamine in alcoholic solution to obtain a compound of formula I.

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