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Title:
PROCESS FOR PREPARATION OF 5-SUBSTITUTED TETRAZOLES
Document Type and Number:
WIPO Patent Application WO/2010/133909
Kind Code:
A2
Abstract:
The present invention is a process for preparing sterically hindered 5-substituted tetrazole, which comprises of reacting a nitrile with an organotin halide and an azide in presence of a phase transfer catalyst, in an organic solvent and a co- solvent at reflux temperature for 4 to 20 h.

Inventors:
POTLURI, Ramesh, Babu (417 Nilgiri, Aditya EnclaveAmeerpet, Hyderabad 8, 500 03, IN)
DEHURY, Sanjay, Kumar (417 Nilgiri, Aditya EnclaveAmeerpet, Hyderabad 8, 500 03, IN)
VENKATA SUBHRAMANIAN, Harihara Krishnan (417 Nilgiri, Aditya EnclaveAmeerpet, Hyderabad 8, 500 03, IN)
Application Number:
IB2009/005660
Publication Date:
November 25, 2010
Filing Date:
May 20, 2009
Export Citation:
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Assignee:
SMS PHARMACEUTICALS LIMITED (417 Nilgiri, Aditya EnclaveAmeerpet, Hyderabad 8, 500 03, IN)
POTLURI, Ramesh, Babu (417 Nilgiri, Aditya EnclaveAmeerpet, Hyderabad 8, 500 03, IN)
DEHURY, Sanjay, Kumar (417 Nilgiri, Aditya EnclaveAmeerpet, Hyderabad 8, 500 03, IN)
VENKATA SUBHRAMANIAN, Harihara Krishnan (417 Nilgiri, Aditya EnclaveAmeerpet, Hyderabad 8, 500 03, IN)
International Classes:
C07D257/04
Domestic Patent References:
2005-06-09
2005-06-02
2005-06-09
Attorney, Agent or Firm:
VISHWANATH, Ramesh, R. (Vishwanath & Global Attorneys, # 42-839, 2nd Floor, Meghanaakash,Arcade, Moulal, 0 Hyderabad, 50004, IN)
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Claims:
We claim;

1. A process for making 5-3ubstituted tetrazoles of formula I: Formula wherein R is selected from moieties of the formulae:

where R1 is Bz or H.

2. T he process according to claim 1, wherein the synthesis 5-substituted tetrazoles of formula I comprises a. reacting a compound of formula II,

Formula Il wherein R is as defined hereiπabove, with a sodium azide and tributyl tin azide in presence of a phase transfer catalyst, in an organic solvent and a oo solvent at reflux temperature for from Λ to 20hourε, b. hydrolyziπg with a diluted aqueous solution of metal hydroxide, c. washing the aqueous layer with an organic solvent, d. acidifying aqueous layer and recovering the 5-substituted tetrazole of formula I so produced.

Formula

3. The process according to claim 2a, wherein the phase transfer catalyst is a quaternary ammonium halide selected from the group of tetrabutyl ammonium bromide, tetrabutyl ammonium chloride, tetraethyl ammonium bromide, cetyltrimsthyl amnhonium bromide, benzyltriethyl ammonium bromide, benzyltriethyl ammonium chloride, more preferably tetrabutyl ammonium bromide or cetyltrimethyl ammonium' bromide.

4, The process according to claim 2a, wherein the phase transfer catalyst is 0.0001 to 0.01 , preferably 0.001, mole equivalent to the compound of formula I.

5. The process according to claim 2a, wherein the organic solvent is selected from the group toluene, xylene, ethyl acetate, chloroform and ketonic solvent.

G. The process according to claim 2a, wherein the co-solvent is selected from the group dimethyl formamide, dimethyl acetamide, N-methyl pyrrolidinone, dimethyl sulfoxide and sulfolane either singly or in a combination of two or more of the solvent.

7, The process according to claim 2a, wherein the reaction mixture was refluxed for 4 to 20 h, preferably 6-16h.

8. The process according to claim 2d, wherein the reaction mixture is acidified by addition of an acid selected from the group of hydrochloric acid, acetic acid or sulfuric acid.

9. The process according to claim 2d, wherein the product is isolated by filtration.

10. The process according to claim 1, wherein acute oral toxicity of a sample of ((S1 S)-N-valeryl-N-({2'-(1H-tetrazole- 5-yl)biphenyl-4- yi}methyl)isoleucine (ISB V) was evaluated.

11. The process according to claim 9, wherein the maximum Tolerance Dose (MTD) of ISB-V by oral route, was found to be > 2000mg/KG in Wistar rats and 1250 mg/KG in Swiss albino mice.

Description:
Process for preparation of 5-substituted tetrazoles

Field of the invention

The present invention relates to a process for preparing sterically hindered 5- substituted tetrazoles from nitrites with ease and safely at low costs. The process finds its scope in the synthesis of very useful compounds having pharmaceutical importance viz. synthesis of valsartan and its impurities.

Background of the invention

The. synthesis of valsartan and some of its impurities are reported in US Patent No. 5,339,578 and US Patent Publication No. US2005/0010053 respectively. One of the most important steps of the synthesis of these compounds involves the reaction of a .nitrile intermediate with an azrde species to render the tetrazole moiety.

There has been research interest on processes for preparing tβtrazolθs from nitriles and azides, and many processes are known. For example, in the synthesis of 5-phenyl-IH-tetrazole, dimethylformamide (DMF) is used as a solvent and NH 4 CI as a catalyst (J. Am. Chβm. Soc. t 1958, 80, 3908). If NH 4 CI is used as a catalyst in this process, it reacts with NaN 3 to give explosive NH 4 N 3 as a sublimate attached to a condenser tube. Thus, the process involves a great risk in industrial manufac ;ure of tetrazoles. However, if NH 4 CI is not used, a tetrazole is produced In a significantly low yield. Stated more specifically, if a cataiysl other than NH 4 Ci, e.g. LiCl, is used, the yield would be reduced. Further the process requires a high reaction temperature and a prolonged reaction time as a whole.

A process is known wherein an acid such as acetic acid is used as a catalyst and wherein HN 3 is generated in the presence of a solvent and is reacted with a πitrile {J. Org. Chem., 1957, 22, 1142). However, the process entails a relatively low reaction rate, a high reaction temperature, a prolonged reaction time and α low yield. Furthermore, toxir. HN 3 is likely to escape from the reaction system. That is, this process is problematic from the viewpoint of industrial manufacture.

If DMF is used as a solvent in the preparation of a tetrazole from a nitrile having a complicated structure, the yield of the tetrazole may be pronouncedly reduced because of the reaction of the nitrile with DMF and the decomposition of DMF. In this case, a process is employable which uses an aromatic solvent or polar solvent and an organotin compound, organic silicon compound or the like as a catalyst to increase the organic properties of an azide for the facilitated production of the tetrazole (J. Org. Chem., 1991, 56, 2395). The process uses trimethyltin or tributyltin azide to produce tetrazoles in good yield. Trimethyltin azide must be prepared in advance and tributyltin azide is prepared in situ.

Japanese Patent Publication No. 53,489/1995 discloses a preparation for tributyltin azide from tributyltin chloride and sodium azide in presence of toluene. Further, on completion of the reaction of nitrile with tributyltin azide, the reaction mixture is hydrolyzed with a diluted aqueous solution of sodium hydroxide, followed by neutralization of the aqueous layer with an acid, to afford the corresponding tetrazole.

Reference is made to United States Patent Number 5, 502, 191/1996 wherein an invention reveals a process of tetrazole synthesis using a azide e.g. sodium azide or trimethylsilyl azide and equivalents thereof or a preformed metal azide complex, zinc azide bispyridine complex, in the presence of a lewis acid such as zinc chloride, magnesium chloride, aluminum chloride, aluminum isopropoxide or tin tetrachloride.

Reference is made to United States Patent 5.744,612/1998, which discloses a process of tetrazole sysnthesis by reacting a nitrile with an inorganic azide salt in an aromatic hydrocarbon solvent in the presence of an amine salt.

Recently Sharpless et a/. (J. Org. Chem. 2001 , 66, 7945) have developed a process for the synthesis of tetrazole using sodium azide in presence of a stoichiometric amount of zinc salt in water. Further Pizzo et a/. (J, Org. Chem.

2004, 69, 2896) have reported the efficient use of trimethylsilyl azide in presence of TBAF as catalyst. Very recently Lakshmi Kantam et al. (Adv. Synth, Catal.

2005, 347, 1212) have disclosed a process for using nanocrystlline ZnO as efficient heterogeneous catalyst for the synthesis of 5-substituted tetrazole. However some of the main drawbacks of the abovemeπtioned processes are low yield and longer reaction time in the case of sterically hindered substrates.

US Pharmacopoeia has reported three impurities for valsartan as follows 1. Impurity Λ;(R) N valoryl N ({2' (1H tctrαzolo B yl)biphcnyl 4 yljmcthyljvoilino, 2.lmpurity B: (S)-N-butyryl-N-({2'-(1 H4eLrazoIe-5-yl)biphenyl-4-yl}melhyl)valine 3 Impurity C 1 (S)-M-valeryl-N-({2'-(1H-tetrazole- 5-yl)biphenyl-4~yl}rnethyl)valme

» benzyl ester

Hence, one of very useful issues of contemporary Importance Is to develop a method for easy access to the aforementioned compounds.

In addition, two other major impurities, i.e, the L-Ieucine and L-isoleucine analogues of L-valine, are detected In many Instances owing to the fact that the samples of L-valine contain L-isoleucine and L-leucine. Hence one of the ways out could be synthesis, characterization and toxicity study of these analogues. ■

Objectives of the present; invention

An objective of the present invention is to provide a facile and easy to operate process for preparing sterically hindered 5-substituted tetrazoles of formula I in high yields.

I he inventors of this invention carried out extensive researches on industrially advantageous processes to achieve the foregoing objects and found that an inorganic azide salt is reacted with a organic tin compound in the presence of an aromatic hydrocarbon as a solvent, and a quaternary ammonium halide compound as phase transfer catalyst. Further findings were that the reactions are made facile by using an aprotic dipolar solvent as a co-solvent.

According to the present invention, there is provided a process for preparing a sterically hindered 5-substituted tetrazole represented by the formula I 1

the process comprising the step of reacting a nitrile represented of formula Il

Formula Ii with an inorganic azide salt in an aromatic hydrocarbon solvent in the presence of a co-solvent and phase transfer catalyst, wherein R is selected from moieties of the formulae:

where R' is Bz or H.

Another objective of the present invention is to develop a process for the synthesis of Valsartan.

Yet another objective of the present invention is development of a process for the synthesis of one of the impurities of Valsartan Wz -1 (R)-N-vaIeryl-N-({2'-(1 H- tetrazole-5-yl)biphenyl-4-yl}methyl)valine, which is reported in US

Pharmacopoeia. Yet another objective of the present invention is development of a process for the synthesis of one of the Impurities of Valsartan viz., (S)-N-butyry!-N-({2'-( 1 H- tetrazote-5-yl)biρhenyl-4-yl}rnethyl)valiπθ r which is reported in US

Pharmacopoeia.

Yet another objective of the present invention is development of a process for the synthesis of one of the impurities of Valεartan viz., (S)-N-valeryl-N-({2'-(1H- tetrazole- 5-yl)biphenyl-4-yl}methyl)valine benzyl ester, which is reported in US Pharmacopoeia.

Still another objective of the present invention iβ development of a process for the synthesis of one of the impurities of Valsartan viz., (S)~N-valeryl-N-({2'-(1H- tetrazole- 5-yl)biphenyl-4-yl}methyl)isoIeucine , which is not reported in US Pharmacopoeia.

Still further objective of the present invention is development of a process for the synthesis of one of the impurities of Valsartan viz., (S)~N-valeryl-N-({2'-(1 H- tetrazole- 5-yl)biphenyl-4-yl}methyl)leucine , which is not reported in US Pharmacopoeia.

Still further objective of the present invention i$ evaluation of acute oral toxicity of a (S, S)-N-valeryl-N-({2'-(1H-tetrazoie- -5-yl)biphenyl-4-yl}methyl)isoleucine (ISB- V). Summary of the invention

A process for preparing sterically hindered 5-substituted tetrazole, which comprises of reacting a nitrile with an organotin haiide and an azide in the presence of a phase transfer catalyst, in an organic solvent and a co-solvent at reflux temperature for 4 Io 20 h is disclosed. The process finds its scope in the synthesis of very useful compounds having pharmaceutical importance viz. synthesis of valsartan and its impurities. In addition, it discloses the evaluation of acute oral toxicity of one of the impurities of vasartan viz, (S, S)-N-valery|-N-({2'- (1 H-tetrazole- 5-yl)biphenyl-4-yl}methyl)isoleucine.

Description of the invention

There has been research interest not only on the development easy to operate process for valsartan but also on the development of process for the synthesis of its related substances and acute toxicity evaluation thereof. US Pharmacopoeia has reported three impurities for Valsartan viz. (R)-N-valeryl-N-({2'-(1H-tetrazole- 5-y[)biphenyl-4-yl}methyl)valine, (S)-N-butyryl-N-({2"'(1H-tetrazole-5-yl)biphenyl- 4-y | }methyl)valine, (S)-N-valeryl-N-({2'-(1 H-tetrazole- 5-yl)biphenyl-4- yl}methyl)valine benzyl esler. In addition, two other major impurities, I.e. the L- teuciπe and L-isoleucins analogues of L-valinc, are detected in many instances owing to the fact that the samples of L-valine contain L-isoleucine and L-leucine. Hence one of the ways out could be synthesis, characterization and toxicity study of these analogues. One the important steps for the syntheses involves reaction of a nitrite intermediate with an inorganic azide salts in presence of an organotin halide to afford the corresponding tetrazole derivative. Examples of useful inorganic azide salts include azides of alkali, metals or alkaline earth metals such as sodium, potassium, lithium, calcium, magnesium, etc. Azides of alkali metals are suitable and sodium azide are industrially more suitable. The amount of the inorganic azide salt used is 1 to 6 moles, preferably 3 to 5 moles, as calculated as hydrogen azide, per mole of a nitrile of the formula II. Desirably the inorganic azide salt is used In higher molar amount relative to an organo tin compound.

The phase transfer catalyst used for the invention are quaternary ammonium halides selected from the group of tetrabutyl ammonium bromide, tetrabutyl ammonium chloride, tetraethyl ammonium bromide, cetyltrimethyl ammonium bromide, benzyltricthyl ammonium bromide, benzyltriethyl ammonium chloride, more preferably tetrabutyl ammonium bromide or cetyltrimethyl ammonijum bromide. The amount of the phase transfer catalyst used is sufficient if it is a minimum amount required for the progress of the reaction.The amount of phase transfer catalyst used is 0.0001 to 0.01, preferably 0.001, mole equivalent to the compound of formula II.

Solvents preferred in the reaction include aromatic hydrocarbons inert to the reaction. Aromatic hydrocarbons which are Industrially suitable and proper for the reaction are, for example, benzene, toluene, xylene, mesitylene, ethylbenzene, chlorobenzene, nitrobenzene, cumene, chlorotoluene, etc. among which toluene and xylene are preferred. A mixture of at least two of these aromatic hydrocarbons can be used in the reaction. The amount of the aromatic hydrocarbon used may be a minimum amount which allows the reaction to proceed. In many instances, it is assisted by a co-solvent of aprotic dipolar nature. In the present process the co-solvent is selected from the group dimethyl formamide, dimethyl acetamide, N-methyl pyrrolidinone, dimethyl sulfoxide and sulfolane either singly or in a combination of two or more of the solvent. More preferably dimethyl formamide is used. The amount of co-solvent used is is 0,0001 to 0.01 , preferably 0.001 , mole equivalent to the compound of formula IL The reaction was carried out by refluxing a mixture of nitrile of formula I!, with an organo tin halide and inorganic azide in presence of a phase transfer catalyst, in an organic solvent and a co-solvent at reflux temperature for 4 to 2Oh, preferably 6-16h.

The acids to be used for precipitaion of the product include, for example, inorganic acids such as hydrochloric acid, hydrogen bromide, sulfuric acid, nitric acid, phosphoric acid, boric acid, hydrogen sulfide and the like; and organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid and the like to which useful acids are not limited, Preferred acids are, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, acetic acid, trifluoroacetic acid, more prepfarably hydrochloric acid. Further the ester moiety may be hydro lyzed with a hydroxide of alkali metal or alkaline earth melal and water after the reaction. Examples of useful hydroxides of alkali metals are lithium hydroxide, sodium hydroxide, potassium hydroxide, etc. Among them, sodium hydroxide is preferred. Useful hydroxides of alkaline earth metals are, for example, barium hydroxide and calcium hydroxide among which calcium hydroxide is favorable. The reaction temperature in the hydrolysis is not critical, but preferably 0 to 40 0 C. The reaction time in the hydrolysis is not critical, but preferably 1 to 5 hours. Moreover the benzyl esters are debenzoylated by catalytic hydrσgeπolysis using palladium on charcoal catalyst. After completion of the hydrolysis, the aqueous layer is washed with an organic solvent such as an aromatic hydrocarbon or ether. Further the pH of the aqueous layer is adjusted to an acidity range. The organic solvent layer contains the unreacted ester derivative which can be recovered by evaporation of the solvent. In addition, the aromatic hydrocarbon solvent can be easily recovered by distillation and can be re-used.

Advantages of the present invention

1. The reagent does not have to be prepared in advance;

2. The yields are higher overall;

3. The work-up procedure is simple;

4. The products are purer and do not need to be chromatographed before they are used;

5. The reaction times are shorter;

6. The reagents are cheaper;

7. Sterically hindered 5-substituted-1 H-tetrazoles can be synthesized in high yield.

8. It provides an improved process for the synthesis of valsartan.

9. It discloses the synthesis of three impurities of valsartan, which are reported in US Pharmacopoeia.

10. The present invention reports the synthesis of two impurities of valsartan, which are not reported in US Pharmacopoeia.

11. In addition it provides evaluation of acute oral toxicity of an impurity of valsartan viz. ■ (S)-N-valeryI-N-({2'-(1 H-tetrazole- 5-yl)bipheπyl-4- yl}methyl)iso!eucine (ISB-V).

The following examples illustrate the invention, which should not be construed in limiting the scope of the present invention.

Example 1

(S)-N-valeryl-N-({2'-(1H-tetrazole- 5-yl)bipheny!-4-yl}methyl)valine

a. 46 g of N-[(2'-cyanobiphenyl-4-yl)methyl]-N-valeryl-(L)-vaIine benzyl ester, 108 g of tributyltin chloride, 34g of sodium azide, 0.5g of TBAB and 1 mL of DMF in 200 ml of o-xylene were heated to boiling with stirring for 6 hours, After completion of the reaction, the reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated to remove o-xylene and DMF. 450 mL ϊsl,heτ was added lυ the residue follpwed by treatment with 1 N aqueous sodium hydroxide solution. The reaction mixture was intensively stirred for 2 h. The aqueous phase was separated off and rendered acidic with 1N aqueous hydrochloric acid. The precipitated product was isolated by filtration. The crude was dissolved in ethyl acetate and dried with anhydrous Na 2 SO 4 . It was concentrated to render 38g of N-[(2'-(1H4etrazol-5-yl)biphenyl~4-yl)methyl]~N- valeryl-(L)-valine benzyl ester with [ctjo 20 39,5 and melting interval of 50-54 0 C. A solution of 42.2 g of N-[(2'-(1 H-tetrazol~5^yl)biphenyl-4-yl)methyl]-N-valeryl-(L)- valine benzyl ester in 300 ml of methanol was hydrogenated at room temperature with the addition of 8 g Of 1 PdZC (10% ) until the calculated amount of hydrogen had been absorbed (24 h). The crude acid was obtained by filtration and evaporation of the solution. It was partitioned between 2N sodium bicarbonate solution and ethyl acetate. The aqueous phase was separated off and rendered acidic, and the title compound is isolated by extraction with ethyl acetate. It is obtained from ethyi acetate in crystalline form and has a melting interval of 103- 105 0 C and an optical rotation [αfo 20 -64.95° (c=1% in methanol), b. Recrystalfisation of (S)-N-(I -Ca rboxy-2~methyl-prop-1-yl)-N-pentanoyl-N-[2'- (1 H-teirazol-5-yl)bi phenyl-4-ylmethylJ-amine

55 g of crude valsartan was charged into a round bottomed flask containing ethyl acetate (330 ml) and the contents were heated to reflux under stirring to form a clear solution. To the clear solution, 5.5 g of activated charcoal was added and stirred at reflux for about 35 minutes. The reaction mass was filtered hot through a flux calcined diatomaceouβ earth (Hyflow) bed and the bed was washed with ethyl acetate (27.5 ml). The resultant filtrate was cooled to about 35 0 C. and seeded with 2.75 g of pure valsartan. The seeded solution was stirred for 1 hour, 15 minutes at 35 0 C. and subsequently cooled stepwise: to 30 0 C. for 1 hour, 15 minutes; further to 15 0 C. for about 40 minutes; and further to about 5 0 C. for about 2 hours, 20 minutes; all accompanied by stirring. The separated solid was filtered and washed with ethyl acetate (27.5 ml) to get solid compound. It was followed by washing with 3OmL of hexane. The dry solid was suspended in 100 ml_ of hexane and was refϊuxed for 2h. It was then cooled to room temperature and separated by filtration followed by washing with 20 ml_ of hexane to render 41g of the products Purity by HPLC: 99.83%) The starting material could.be prepared, for example, as follows: a) N-[(2'-Cyanobiphenyt-4-yI)methyl]-(L)-valine benzyl ester A solution of 85 g of 4-bromomethyI-2'-cyanobiphenyl, 65 g of (L)-Valine benzyl ester (free base) and 66.4g of Na2CO 3 in 460 ml of acetone was stirred at 55 0 Q for 4 hour. The reaction mixture was then cooled, poured into 300 ml of ice wator and extracted with 150 ml of ethyl acetate. By washing the extract with aqueous potassium bicarbonate solution, drying and evaporating, the crude title compound was obtained a$ an oil which formed 55g of N-[(2'-cyanobiphenyl-4- yl)methyl]"(L)-valine benzyl, ester hydrochloride of m.p : . 169-173 0 C,

b) N-[(2'-Cyanobiphenyl-4-yl)methyI]"N-valeryl-(L)-valine benzyl ester

50 g of N-f(2'-cyanobiphenyl-4-y!)methyl]-(L)-valine benzyl ester hydrochloride and 50g of Na 2 CO 3 were charged to 500 mi of methylene chloride. The reaction mixture was further treated with 25g of valeryl chloride with stirring. Further work up with water and concentration of the dichloromethane layer rendered 5Og of N- [(2 l -Cyanobipheny!-4-yl)methyl3-N-valeryl"(L)-valine benzyl ester as pale yellow liquid.

Example 2

The process of Example 1 was repeated, using D-valine benzyl ester instead of

L-valine benzyl ester to (R)-N-valeryl-N-({2'-(1H4etrazole- 5-yl)biρheπyl-4- yl}methyl)valiπe.

Example 3 The process of Example 1 was repeated, using L-leucine benzyl ester instead of L-valine benzyl ester to {S)-N-valeryl-N-({λ'-(iH-tetrazole- 5-yi)biphenyl-4- yl}methyl)leucine.

Example 4

The process of Example 1 was repeated, using L-isoleucine benzyl ester instead of L-valine benzyl ester to (S, S)-N-valeryl-N-({2'-(1H-tθtrazole- 5-yl)biphenyl-4- yl}methyl)isoleucine.

Example 5

The process of Example 1 was repeated, using butyryl chloride instead of valeroyl chloride to render (S)-N-butyryl-N-({2'~(1H-tetrazole- 5-yl)bipheπyl-4- y|}methyl)valiπe.

Acute oral toxicity of a sample of (S, S)-N-valeryI-N-({2'-(1H-tetrazole-- 5- yl)biphenyl-4-yl}methyl)isoleucine (ISB V) was evaluated and the results are shown below;

Rats:

1 , In the sighting study eyen at the dose of 2000rng/KG, none of the animals

(rats) showed appreciable symptoms of toxicity and there were no death,

2, The maximum dose 2000mg/KG was seleted based on the results of the sighting study. None of the animals showed signs of toxicity. No mortality was observed. The 'hematology and biochemical ' parameters obtained on the termination day were well with in the normal range and comparable to lhe values obtained on "O" day. 3 The maximum Tolerance Dose (MTD) of the ISB-V by oral route, was found to be > 2000mg/KG in Wistar rats. Mice:

1. The compound was showing toxicity at the dose of 2000 mg/KG. The sighting study was conducted at an intermediate dose of 1250 mg/KG in mice. None of the mice showed appreciable symptoms of toxicity and there were no doubt at this dose.

2. In main study the maximum dose of 1250 mg/KG was used in the mice. None of the animals showed any symptoms and signd of toxicity. No mortality was observed. The hematology and biochemical parameters obtained on the termination day were with in the range and comparable to the values obtained on "0"day.

Conclusion:

1. The maximum Tolerance Dose (MTD) of the ISB-V by oral route, was found to be 2000 mg/KG in Wistar rats.

2, The maximum Tolerance Dose (MTD) of the ISB-V by oral route, was found to be 1250 mg/KG in Swiss albinυ mice.




 
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