A QUINAZOLINE DERIVATIVES AND PREPARATION THEREOF

Abstract

A QUINAZOLINE DERIVATIVES AND PREPARATION THEREOF The present disclosure relates to an anti-tuberculosis quinazoline derivative, its solvate, its stereoisomer, or a pharmaceutically acceptable salt thereof. Also, the present disclosure relates to the process for preparing quinazoline derivatives. The present process is eco-friendly and provides high yields and pure compounds. Further, the present disclosure provides a compound with good binding affinity to the target protein and is endowed with potent In-vitro anti-tuberculosis activity against Mycobacterium tuberculosis by the MGIT-Mycobacteria Growth Indicator Tube method to combat the drug resistance and toxicity. Compound of formula I

Specification

Description:TECHNICAL FIELD:
The present disclosure relates to the field of pharmaceuticals. More specifically, the present disclosure relates to an anti-tuberculosis quinazoline derivative, its solvate, its stereoisomer, or a pharmaceutically acceptable salt thereof. Also, the present disclosure relates to the process for preparing quinazoline derivatives. The present process is eco-friendly and provides high yields and pure compounds. Further, the present disclosure provides a compound with good binding affinity to the target protein and is endowed with potent In-vitro anti-tuberculosis activity against Mycobacterium tuberculosis by the MGIT-Mycobacteria Growth Indicator Tube method to combat the drug resistance and toxicity.

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.

Tuberculosis (TB) is a major communicable disease with extremely serious problem on global health threat. According to WHO statistics report, nearly 10.6 million people were affected and 1.6 million people died from this infectious disease in 2021. It is one of the second leading infectious killer after COVID-19 in Worldwide. Multidrug-resistant TB (MDR-TB) and Extensively-resistant TB (XDR-TB) remains as a major public health crisis. For three decades, the continuing spread of drug resistance-TB are the most urgent and difficult challenges facing global TB control.

A major clinical problem in the Treatment of TB is multidrug-resistant (MDR)-TB and extensively drug-resistant (XDR)-TB relied on medications that are less potent and more toxic than first-line TB therapy and number of drugs available is limited. So, the diagnostic delay leads to a more severe disease, with increased risks of spreading and mortality. WHO suggested Fluoroquinolones (levofloxacin or moxifloxacin), Bedaquiline and Delamanid strongly recommended for longer MDR-TB regimen but there is an evidence of drug resistance and a risk of toxicity. In the initial trial, bedaquiline and delamanid were associated with a higher number of unexplained deaths along with the risk of hepatotoxic and QT-interval prolongation leads to cardio-toxicity resulted in a black box warning by the FDA. Additional problems for uptake of bedaquiline and delamonid include the expensive cost. Due to this side effects and low clinical effects, safer and more effective treatments need to be Develop novel multitarget compounds tackle the problem in emergence of resistance against target enzymes for effective therapeutic strategies for the recurrence of MDR TB.

In continuation search in development of newer drug molecules potentially bioactive scaffold of novel quinazoline derivatives were designed based on Structure activity relationship (SAR) involved quinazoline ring substituted with halogen group (electron withdrawing groups) & various aromatic amino group containing compounds, Fragment replacement, and Lipophilic aromatic insertion at 2nd position showing the significant antitubercular activity as per reported literature Review.

Thus, there is an ongoing need for novel potential anti-tuberculosis drugs against the targeted enzyme to combat the drug resistance and toxicity of existing antitubercular drugs. The present disclosure relates to an anti-tuberculosis quinazoline derivative, its solvate, its stereoisomer, or a pharmaceutically acceptable salt thereof. Also, the present disclosure relates to the process for preparing quinazoline derivatives. The present process is eco-friendly and provides high yields and pure compounds. Further, the present disclosure provides a compound with good binding affinity to the target protein and is endowed with potent In-vitro anti-tuberculosis activity against Mycobacterium tuberculosis by the MGIT-Mycobacteria Growth Indicator Tube method.

OBJECTIVES:
An object of the present disclosure is to provide novel compounds that exhibit potent anti-tuberculosis activity.

Another object of present disclosure is to provide novel potential anti-tuberculosis drugs against the targeted enzyme to combat the drug resistance and toxicity of existing antitubercular drugs.

In another object of present disclosure is to provide a novel an anti-tuberculosis quinazoline derivatives.

Yet another object of present disclosure is to provide an anti-tuberculosis quinazoline derivatives, its solvate, its stereoisomer or a pharmaceutically acceptable salt thereof for potential In-vitro anti-tuberculosis activity against Mycobacterium tuberculosis by the MGIT-Mycobacteria Growth Indicator Tube method.

Further, object of the present disclosure is to provide a process of preparing the anti-tuberculosis quinazoline derivatives.

Furthermore, object of present disclosure is to provide a process is eco-friendly and provides compounds with higher yield and purity.

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, the present disclosure provides a quinazoline derivative of compound of formula I, its solvate, its stereoisomer or a pharmaceutically acceptable salt thereof, comprising:


Compound of formula I

wherein:
R1 is selected from hydrogen, amino, halogen;
R2 is selected from hydrogen, alkyl, cycloalkyl, cycloalkyl alkyl, alkoxy alkyl, arylalkyl, aryl alkoxy, aryl, heteroaryl alkyl, substituted aryl, substituted heteroaryl alkyl, wherein substituted aryl, substituted heteroaryl alkyl are substituted with one to three substituents independently selected from alkyl, cycloalkyl, cyano, halogen, haloalkyl or alkoxy group;
R3-R5 is selected from hydrogen;
R6 is selected from hydrogen, and halogen.
R7 is hydrogen
R8 is selected from
a) alkoxy, alkyl, alkoxy carbonyl, alkylcarbonylamino, alkyl-S= imine group and organosulfur; or
b) hydrogen, alkyl, cycloalkyl, cycloalkyl alkyl, alkoxy alkyl, arylalkyl, aryl alkoxy, arylalkoxyalkyl, aryl, heteroaryl alkyl, substituted aryl, substituted heteroaryl alkyl, wherein substituted aryl, substituted heteroaryl alkyl are substituted with one to more substituents independently selected from alkyl, alkyl sulfonyl group, cycloalkyl, cyano, halogen, haloalkyl or alkoxy.

Another aspect of the present disclosure provides a process of preparing the anti-tuberculosis quinazoline derivatives.

Various features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.

BRIEF DESCRIPTION OF THE 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.

Figure 1 shows Mass spectra of RNSUMBR
Figure 2 shows Mass spectra of RSUL35
Figure 3 shows NMR spectra of RSUL35
Figure 4 shows NMR spectra of RNSUMBR
Figure 5 shows sample tubes containing test compound for anti-tubercular evaluation
Figure 6 shows determination of Mycobacterial growth in the test compounds against the standard & control compounds.
Figure 7 shows toxicity studies of synthesized compounds.

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.

The phrase "pharmaceutically acceptable salt(s)', as used herein, unless indicated, includes salts of acidic or basic groups which may be present in the compounds of the present invention. The compounds of the present invention that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of Such basic compounds are those that form nontoxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate. Succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, Saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate i.e., N-[(4-nitro-1H- benzimidazol-2 yl)methyl]pyridine-3-carboxamide salts.

The chemical bases that may be used as reagents to prepare pharmaceutically acceptable base salts of those compounds of the compounds of the invention that are acidic in nature are those that form non-toxic base salts with such compounds.

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."

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 5 reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified.

An aspect of the present disclosure provides a quinazoline derivative of compound of formula I, its solvate, its stereoisomer or a pharmaceutically acceptable salt thereof, comprising:


Compound of formula I

wherein:
R1 is selected from hydrogen, amino, halogen;
R2 is selected from hydrogen, alkyl, cycloalkyl, cycloalkyl alkyl, alkoxy alkyl, arylalkyl, aryl alkoxy, aryl, heteroaryl alkyl, substituted aryl, substituted heteroaryl alkyl, wherein substituted aryl, substituted heteroaryl alkyl are substituted with one to three substituents independently selected from alkyl, cycloalkyl, cyano, halogen, haloalkyl or alkoxy group;
R3-R5 is selected from hydrogen;
R6 is selected from hydrogen, and halogen.
R7 is hydrogen
R8 is selected from
a) alkoxy, alkyl, alkoxy carbonyl, alkylcarbonylamino, alkyl-S= imine group and organosulfur; or
b) hydrogen, alkyl, cycloalkyl, cycloalkyl alkyl, alkoxy alkyl, arylalkyl, aryl alkoxy, arylalkoxyalkyl, aryl, heteroaryl alkyl, substituted aryl, substituted heteroaryl alkyl, wherein substituted aryl, substituted heteroaryl alkyl are substituted with one to more substituents independently selected from alkyl, alkyl sulfonyl group, cycloalkyl, cyano, halogen, haloalkyl or alkoxy.

An embodiment of the present disclosure provides the R1 cycloalkyl group is selected from phenyl.

Another embodiment of the present disclosure provides the R6 halogen is selected from bromine, iodine, chlorine and fluorine.

Yet another embodiment of the present disclosure provides compound of formula I is selected from;
4-[(6-iodo-2-phenylquinazolin-4-yl) amino] benzene-1-sulfonic acid [RSUL35]

4- [(3-amino-6-bromo-2-phenyl-3,4 dihydroquinazolin-4-yl) amino]-N-(5- methyl-1,2-oxazol-3-yl) benzene-1-sulfonamide. [ RNSUM8989]

Further embodiment of the present disclosure provides a process for preparing compound of formula I, the process comprising the steps of:
a. iodination of anthranilic acid with iodine monochloride solution to obtain an intermediate I;
b. obtained intermediate I react with benzoyl chloride in the presence of anhydrous pyridine to form an intermediate II;
c. the intermediate II reacts with formamide or hydrazine hydrate to form an intermediate III;
d. the intermediate III reacts with phosphorous pentachloride and phosphorous oxy chloride to obtain an intermediate IV; and
e. the intermediate IV reacts with primary amino compounds to form a compound of formula I.
Furthermore, embodiment of the present disclosure provides the halogenation is iodination, bromination, chlorination and fluorination.

In an embodiment of the present disclosure provides the primary amino compounds are sulfamethoxazole and sulphanilic acid.

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.

The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

EXAMPLES:
The following Working Examples represent preferred embodiments of the present invention. All temperatures are expressed in degrees Centigrade unless otherwise indicated.
Example 1: Synthetic Scheme:
SCHEME -I:
I. Synthesis of Intermediate I:

i. 0.08 mole of anthranilic acid dissolved in distilled water (10 ml) and conc. HCl (0.8 ml) allowed to cool at 20 0C. To this, add conc. HCl (1.4ml) with 5ml of cold water (diluted solution) were mixed with crushed ice and allow to cool at 5 0C for 2 minutes and stirring with 1.29g of Iodine monochloride solution.

ii. Combined mixing of 50C containing Iodine monochloride solution mixture and 200C of anthranilic acid mixture to form step-I mixture as tan to violet colour precipitate.
iii. Intermediate-I mixture was stirred for an hour which reaches the room temperature and filtered off. The reaction mixture was washed with three parts of 100 ml portions of cold water and allow to dried at 90-1000C. The yield was obtained at 88-90 % with brown to purple acid which melts at 185-1900C.

II. Synthesis of Intermediate-II

i. Intermediate-I product (0.1 mole) was dissolved in dry pyridine (30 ml) by shaking. To this solution, Benzoyl Chloride (0.2 mole, 28 ml) taken in dry pyridine was added slowly with constant stirring. When the addition was completed, the resultant solution was subjected to vigorous stirring for one hour mechanically, subsequently it was left as such for one hour at room temperature and treated with a solution of 10% sodium bicarbonate.

ii. The addition of sodium bicarbonate solution was continued till the neutral to litmus. The precipitate obtained was collected and recrystallized using absolute alcohol. Its yield is 80% with white crystalline powder and melting point 182-190ºC.

III. Synthesis of Intermediate-III:

Intermediate-II product (0.1 mole) was heated under reflux in Formamide for three hours. The solid obtained was filtered and dried. It was recrystallized using hot ethanol. White Crystalline mass, yield was 70% and Melting point 186-190 ºC.

IV. Synthesis of Intermediate IV:

i. Intermediate-III product (0.04 mole), Phosphorous pentachloride (0.2 mole) and Phosphorous oxy chloride (25ml) was heated under reflux for 4 hours at 115-118 ºC.

ii. Excess of phosphorous oxy chloride was distilled off and the reaction mixture was quenched in ice with efficient stirring, solid precipitate was obtained which was filtered and washed with ice water. The precipitate obtained was recrystallized using ethyl acetate to obtain white crystalline powder.

V. Synthesis of Compound of formula I:
Equimolar concentration of intermediate-IV product and various primary amino compounds were refluxed for three hours. Then, the resultant reaction mixture was poured into crushed ice to obtain a compound of formula I.

Intermediate - IV Compound of formula - I

SCHEME -II
I. Synthesis of Intermediate I:


i. Appropriate quantity of anthranilic acid dissolved in sufficient quantity of glacial acetic acid. It was kept under cold condition with mixing of conc. HCl. (25ml).
ii. Bromine in acetic acid was run into the cold solution of anthranilic acid and conc. HCl with distilled water with continuous shaking. Abundant separation of colorless crystals took place.
iii. After cooling, product was filtered off. The precipitate obtained boiled with 5 times with water and filtered.
iv. Insoluble residue containing dibromo precipitate whereas filtrate containing cooling to get mono bromo anthranilic acid precipitate.

II. Synthesis of Intermediate II:

i. Intermediate-I product (0.1 mole) was dissolved in dry pyridine (30 ml) by shaking. To this solution, Benzoyl Chloride (0.2 mole, 28 ml) taken in dry pyridine was added slowly with constant stirring. When the addition was completed, the resultant solution was subjected to vigorous stirring for one hour mechanically, subsequently it was left as such for one hour at room temperature and treated with a solution of 10% sodium bicarbonate.

ii. The addition of sodium bicarbonate solution was continued till the neutral to litmus. The precipitate obtained was collected and recrystallized using absolute alcohol. Its Yield is 80% with white crystalline powder and melting point 182-190ºC.

III. Synthesis of Intermediate III:

Intermediate-II product (0.1 mole) was heated under reflux in hydrazine hydrate for three or six hours. The solid obtained was filtered and dried. It was recrystallized using hot ethanol. White Crystalline mass, yield was 70% and Melting point 186-190ºC.

IV. Synthesis of Intermediate IV:


i. Intermediate-III product (0.04 mole), Phosphorous pentachloride (0.2 mole) and Phosphorous oxy chloride (25ml) was heated under reflux for 4 hours at 115-118 ºC.
ii. Excess of phosphorous oxy chloride was distilled off and the reaction mixture was quenches in ice with efficient stirring, solid precipitate was obtained which was filtered and washed with ice water. The precipitate obtained was recrystallized using ethyl acetate. White crystalline powder.

V. Synthesis of Compound of formula I:



Intermediate-IV Compound of formula-I
Equimolar concentration of intermediate IV and various primary amino compounds were refluxed for three hours. Then, the resultant reaction mixture was poured into crushed ice to obtain a compound of formula I.



Table:1 PRECISE HERG ACTIVITY PREDICTIONS
Sl. No Compound Code P IC50 Prediction Type of Blocker Predicted Toxicity
1. RSUL35 5.357 Non-blocker Non-cardiotoxicity
2. M RNSUMBR 5.402 Non-Blocker Non-cardiotoxicity
3. Isoniacid 5.572 Blocker Cardiotoxicity

4. Bedaquiline 5.470 Blocker Cardiotoxicity

From the Protox-II & pred Herg cardiotoxicity results, it was observed that most of the studied compounds showed (pIC50 = less than 5.47) non-cardiotoxicity which acts as a non-blocker of hERG encoded gene whereas the standard drugs showed cardiotoxicity effect depicted in Fig.7. (pIC50 = more than 5.47) leads to potassium channel blocker causes hERG blockage.

Table: 2 PROTOX-II Hepatotoxicity
Sl. No Compound Code Probability Predicted Hepatotoxicity
1. RSUL35 0.58 Inactive
2. RNSUM89 0.50 Inactive
3. Bedaquiline 0.68 Mild active
4. Isoniacid 0.94 Hepatotoxicity-Highly ACTIVE
From the Hepatotoxicity results, it was observed that most of the studied compounds showed non-hepatotoxicity (Inactive) depicted in fig.10.0. (probability = less than 0.70) whereas the standard drugs showed highly active Hepatotoxic effect (probability = more than 0.70)

Table.3. Physico -chemical properties of the synthesized compounds:
Sl.
No Compound Code Molecular Formula Molecular Weight
(gms) %
of
yield Melting point
(◦C) Rf Value Appearance
1 RSUL35 C20H14IN3O3S 503.31 73 230-235 0.62 Pale yellow solid
3 RNSUMBR C24H21BrN6O3S 553.43 75 102-110 0.70 Pale brown solid

Example 2. Evaluation of In-Vitro Anti-Tubercular Activity
BACTEC MGIT METHOD:
Using the BD BBLTM MGITTM 960 and BD BBLTM MGIT TM 320 systems, the BD BBLTM MGIT TM Mycobacteria Growth Indicator Tube supplemented with BD BactecTM MGITTM Growth supplement and BD BBLTM MGITTM PANTATM antibiotic mixture is designed to detect and recover mycobacteria.

7 ml of Modified Middlebrook 7H9 Broth base are contained in the MGIT-Mycobacteria Growth Indicator Tube. One of the most often used liquid media for mycobacteria cultivation is the complete medium, which contains an OADC enrichment and a PANTA antibiotic mixture.

Principle of the procedure:
MGIT Medium:
The liquid broth medium used in the MGIT (Mycobacteria Growth Indicator Tube) is proven to promote recovery and increase mycobacteria growth. 7.0 millilitres of modified Middlebrook 7H9 broth base are included in the MGIT. Autoclaving is used to terminally sterilize this medium. To complete the medium, an enrichment, such as MGIT 960 Growth supplement or MGIT OADC (Oleic acid, Albumin, Dextrose, and Catalase), is added. Numerous mycobacteria, particularly those that are part of the M. TB complex, require this growth supplement in order to grow. Both tubercle bacteria and mycobacteria use oleic acid, which is crucial to their metabolism. By binding free fatty acids that may be harmful to Mycobacterium species, albumin serves as a protective factor and speeds up their recovery. One source of energy is dextrose. Catalase degrades harmful peroxides that may be present in the media. The addition of the MGIT PANTA is required to prevent contamination.

Reagents:
Approximate formula per L of Purified water:
Modified Middlebrook 7H9 Broth base : 5.9 g.
Casein peptone : 1.25 g.

BACTEC MGIT Growth supplement contains 15 mL Middlebrook OADC enrichment
Approximate formula per L of Purified water:
Bovine albumin : 50.0 g
Dextrose : 20.0g
Polyoxyethylene Stearate (POES): 1.1 g
Catalase : 0.03 g
Oleic acid : 0.1 g

The BBL MGIT PANTA vial contains a lyophilized mixture of anti-microbial agents.
Appropriate formula per vial Lyophilized PANTA
Polymyxin B : 6,000 units
Amphotericin B : 600 µg
Nalidixic acid : 2,400 µg
Trimethoprim : 600 µg
Azlocillin : 600 µg

Principle of detection and drug susceptibility testing:
In addition to Middlebrook 7H9 liquid media, the MGIT tube contains an oxygen-quenched fluorochrome, tris-4,7-diphenyl-1,10-phenonthroline ruthenium chloride pentahydrate embedded in silicone at the bottom of the tube. During bacterial growth within the tube, the free oxygen is utilized and is replaced with carbon dioxide. With depletion of free oxygen, the fluorochrome is no longer inhibited, resulting in fluorescence within the MGIT tube when visualized under UV light. The intensity of fluorescence is directly proportional to the extent of oxygen depletion.

MGIT tubes may be incubated at 37ºC and read manually under a UV light or entered into a MGIT 960 instrument where they are incubated and monitored for increasing fluorescence every 60 minutes. Growth of bacteria as well as mycobacteria increases the fluorescence. In case of M. tuberculosis, at the time of positivity, there are approximately 105 to 106 colony forming units per millimetre (CFU/mL). The instrument declares a tube negative if it remains negative for six weeks (42 days). The detection of growth can also be visually observed by the presence of a non-homogeneous light turbidity or small granular/flaky appearance in the medium. Growth of some NTM (most commonly rapid growers) results in light turbidity, while contaminating bacteria generally produce heavy turbidity.

Drug susceptibility testing can be performed based on the same principle. Two MGIT tubes are inoculated with the test culture. A known concentration of a test drug is added to one of the MGIT tubes and growth is compared with the MGIT tube without the drug (growth control). If the test drug is active against the isolated mycobacteria, it will inhibit the growth and thus there will be suppression of fluorescence, while the growth control will grow uninhibited and will have increasing fluorescence. Growth is monitored by the BACTEC instrument which automatically interprets results as susceptible or resistant.

Specimen collection:
All specimens should be collected as recommended by the CDC (Centre for Disease control and prevention).

Digestion, Decontamination and Concentration:
Sputum specimen should be processed using the NALC-NaOH method as recommended by the CDC's Public Health Mycobacteriology. A Phosphate buffer solution (pH 6.8) should be used to QS the sample decontaminant mixture to 50 mL prior to centrifugation. Resuspension of pellet must also be done using a fresh preparation of phosphate buffer solution (pH 6.8).

Materials required:
BD BBL MGIT Mycobacteria Growth Indicator Tubes and BD BACTEC MGIT 960 Supplement kit containing BD BACTEC MGIT Growth supplement and BBL MGIT PANTA Antibiotic Mixture. Falcon TM brand 50 mL centrifuge tubes, 4% sodium hydroxide, 2.9% sodium citrate solution, Phosphate buffer pH 6.8, vortex mixer, 37◦C incubator, 1 mL sterile pipettes, sterile transfer pipettes.
Preparation of specimen suspension:
1. If the specimen is not collected in a 50 ml centrifuge tube, transfer it to a 50 ml centrifuge tube with a screw cap.
2. Add NaOH-NALC-Sodium citrate solution in a volume equal to the quantity of specimen. Tighten the cap.
3. Vortex lightly or hand mix for about 15-30 seconds. Invert the tube so the whole tube is exposed at the NaOH-NALC solution.
4. Wait 15-20 minutes (up to 25 minutes maximum) after adding the NaOH-NALC solution. Vortex lightly or hand mix/invert every 5-10 minutes or put the tubes on a shaker and shake lightly during the whole time.
5. Make sure the specimen is completely liquefied. If still mucoid, add a small quantity of NALC powder (30-35 grams) directly to the specimen tube. Mix well.
6. At the end of 15-20 minutes, add phosphate buffer (pH 6.8) up to the top ring on the centrifuge tube (plastic tube has a ring for50 ml mark). Mix well (lightly vortex or invert several times). Addition of sterile water is not a suitable alternative for the phosphate buffer.
7. Centrifuge the specimen at a speed of 3000 or more for 15-20 minutes. Use of refrigerated centrifugation at a higher speed is known to increase recovery of mycobacteria.
8. After centrifugation, allow tubes to sit for 5 minutes to allow aerosols to settle. Then carefully decant the supernatant into a suitable container containing a mycobactericidal disinfectant. Make sure the sediment is not lost during decanting of the supernatant fluid. Add a small quantity (1-2 ml) phosphate buffer (pH 6.8) and resuspend the sediment with the help of a pipette or vortex mixer.
9. Use the resuspended pellet for making smears and for inoculation of MGIT tube and other media.
Inoculation of MGIT Tubes:
BD BBL MGIT 7mL Tubes must be used with BD BACTEC MGIT instrument
1. Reconstitute a lyophilized vial of BD BBL MGIT PANTA Antibiotic mixture with 15 mL of BD BACTEC MGIT Growth supplement.
2. Label the MGIT tube with the specimen code.
3. Unscrew the cap and aseptically add 0.8 mL of Growth supplement/ PANTA Antibiotic Mixture.
4. Add 0.5 mL of concentrated specimen suspension prepared above.
5. Tightly recap the tube and mix well.
6. Tubes entered into the instrument will be automatically tested for the duration of the recommended 42 days testing protocol.
7. Positive tubes, identified by the BD BACTED MGIT instrument should be sub-cultured and an acid-fast smear prepared.


Table 4. In Vitro Anti-Tubercular Activity for the synthesized RNSUMBR compound by MGIT method:
Sample code Strains Concentration (μg/ml)


RNSUMBR Conc. 12.5 25 50 75 100 Isoniazid
300 Rifampin
450
H37Rv strain
(wild- sensitive Strain) Status R R R S S S S
Growth unit 400 400 400 78 0 0 0
Status R R R S S S S
Growth unit 400 400 180 86 87 0 0
MDR Strain
(Multi drug resistant Strain) Status R R R R S R R
Growth unit 400 400 400 353 0 400 400
Status R R R R S R R
Growth unit 400 400 400 197 0 400 400

Table 5. In Vitro Anti-Tubercular Activity for the synthesized RSUL35 compound by MGIT method:
Sample code Strains Concentration (μg/ml)


RSUL35 Conc. 12.5 25 50 75 100 Isoniazid
300 Rifampin
450
H37Rv strain
(wild- sensitive Strain) Status R R R R S S S
Growth unit 400 400 400 259 0 0 0
Status R R R R S S S
Growth unit 400 400 400 367 0 0 0
MDR Strain
(Multi drug resistant Strain) Status R R R R S R R
Growth unit 400 400 400 367 0 400 400
Status R R R R S R R
Growth unit 400 400 400 400 0 400 400
S: Sensitive R: Resistant
From the results, anti-tubercular activity revealed that the compounds RNSUMBR and RSUL35 showed sensitivity in both H37Rv strain (wild- sensitive Strain) and MDR Strain (Multi drug resistant Strain) compared to standard drugs (Isoniazid & Rifampicin) which showed resistant effect against MDR strain.

From the Fig.6, they were observed that, RNSUMBR showed sensitivity against H37Rv strain (wild- sensitive Strain) at the concentration of 75 and 100 μg/ml and MDR Strain (Multi drug resistant Strain) showed sensitivity at the concentration of 100 μg/ml. RSUL35 showed sensitivity against H37Rv strain (wild- sensitive Strain) and MDR Strain (Multi drug resistant Strain) at the concentration of 100 μg/ml.

ADVANTAGES:
1. The present disclosure provides eco-friendly.
2. The present disclosure provides compounds with high yield and purity.
3. The present disclosure provides novel compounds that exhibit potent anti-tuberculosis activity.
4. The present disclosure provides potential anti-tuberculosis drugs against the targeted enzyme to combat the drug resistance and toxicity of existing antitubercular drugs.
, Claims:We Claim:

1. A quinazoline derivative of compound of formula I, its solvate, its stereoisomer or a pharmaceutically acceptable salt thereof, comprising:


Compound of formula I

wherein:
R1 is selected from hydrogen, amino, halogen;
R2 is selected from hydrogen, alkyl, cycloalkyl, cycloalkyl alkyl, alkoxy alkyl, arylalkyl, aryl alkoxy, aryl, heteroaryl alkyl, substituted aryl, substituted heteroaryl alkyl, wherein substituted aryl, substituted heteroaryl alkyl are substituted with one to three substituents independently selected from alkyl, cycloalkyl, cyano, halogen, haloalkyl or alkoxy group;
R3-R5 is selected from hydrogen;
R6 is selected from hydrogen, and halogen.
R7 is hydrogen
R8 is selected from
a) alkoxy, alkyl, alkoxy carbonyl, alkylcarbonylamino, alkyl-S= imine group and organosulfur; or
b) hydrogen, alkyl, cycloalkyl, cycloalkyl alkyl, alkoxy alkyl, arylalkyl, aryl alkoxy, arylalkoxyalkyl, aryl, heteroaryl alkyl, substituted aryl, substituted heteroaryl alkyl, wherein substituted aryl, substituted heteroaryl alkyl are substituted with one to more substituents independently selected from alkyl, alkyl sulfonyl group, cycloalkyl, cyano, halogen, haloalkyl or alkoxy.

2. The compound as claimed in claim 1, wherein the R1 cycloalkyl group is selected from phenyl.
3. The compound as claimed in claim 1, wherein the R6 halogen is selected from bromine, iodine, chlorine and fluorine.

4. The compound of formula I is selected from;
4-[(6-iodo-2-phenylquinazolin-4-yl) amino] benzene-1-sulfonic acid
4- [(3-amino-6-bromo-2-phenyl-3,4 dihydroquinazolin-4-yl) amino]-N-(5- methyl-1,2-oxazol-3-yl) benzene-1-sulfonamide.

5. A process for preparing compound of formula I, the process comprising the steps of:
a. halogenation of anthranilic acid with iodine monochloride solution to obtain an intermediate I;
b. obtained intermediate I react with benzoyl chloride in the presence of anhydrous pyridine to form an intermediate II;
c. the intermediate II reacts with formamide or hydrazine hydrate to form an intermediate III;
d. the intermediate III reacts with phosphorous pentachloride and phosphorous oxy chloride to obtain an intermediate IV; and
e. the intermediate IV reacts with primary amino compounds to form a compound of formula I.

6. The process as claimed in claim 5, wherein the halogenation is iodination, bromination, chlorination and fluorination.

7. The process as claimed in claim 5, wherein the primary amino compounds are sulfamethoxazole and sulphanilic acid.

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