Field of the invention:

The present invention relates to process for preparation of hispolon and its analogs.

Background of the invention:

In 1996 hispolon was isolated as yellow pigment from a parasitic fungus, Inonotus hispidus during bioactivity guided isolation of the extracts that showed immunomodulatory and antiviral activity (Ali N.A.A.; Pilgrim H.; Liberra K.; Lindequist U.; Jansen R. Hispolon, a yellow pigment from Inonotus Hispidus. Phytochemistry, 1996, 41, 927-929). Subsequently, hispolon was also isolated as an active ingredient from the fungus Phellinus igniarius, which exhibited anti-oxidant and anti-inflammatory activities (Shunyan Mo; Sujuan Wang; Guangxiong Zhou; Yongchun Yang; Yan Li; Xiaogiiang Chen; Jiangong Shi. Phelligridins C-F: Cytotoxic pyrano[4,3-c][2]benzopyran-l,6-dione and Furo[3,2-c]pyran-4-one derivatives from the fungus Phellinus igniarius. Journal of Natural Products, 2004, 67, 823-828). Recently, the ethanolic extracts of Phellinus merrillii containing hispolon have been found to exhibit analegesic and anti-inflammatory properties (Heng-Yuan Chang; Wen-Huang Peng; Ming-Jyh Sheu; Guan-Jhong Huang; Zong-Chung Leu; Mu-Chuan Tseng; Yu-Ling Ho; Yuan Shiun Chang. Analgesic and anti-inflammatory activities of Phellinus merrillii. Mid-Taiwan Journal of Medicine, 2007, 12, 76-82). Hispolon had been reported to exhibit apoptotic effect on human epidermis KB cells (Wei Chen; Fei-Yu He; Yong-Qvian Li. The apoptosis effect of hispolon from Phellinus lintels (Berkeley & Curtis) Teng on human epidermoid KB cells. Journal of Ethnopharmacology, 2006, 105, 280-285) and to exhibit antivirus activities (Awadha Ali N.A.; Mothana R.A.; Lesnau A.; Pilgrim H.; Lindequist U.; Antiviral activity of Inonotus hispidus. Fitoterapia, 2003, 74, 483-485). Hispolon was shown to inhibit the chemiluminescence response of human mononuclear cells and suppress mitogen-induced proliferation of spleen lymphocytes in mice (Ali N.A.A.; Ludtke J.; Pilgrim H.; Lindequist U.; Inhibition of chemiluminescence response of human mononuclear cells and suppression of mitogen-induced proliferation of spleen lymphocytes of mice by hispolon and hispidin. Pharmazie, 1996, 51, 667-670).Hispolon was found to be a very strong oxidant and weak antibacterial agent (Venkateswarlu, S.; Ramachandra, M.S.; Sethuramu, K.; Subbaraju, G.V.; Synthesis and antioxidant activity of hispolon, a yellow pigment Inonotus hispidius, 2002, 41B, 875-877).
Good bioactivity profile and limited availability from natural sources, we have reported the synthesis of hispolon for the first time in Indian Journal of Chemistry (Venkateswarlu, S.; Ramachandra, M.S.; Sethuramu, K.; Subbaraju, G.V.; Synthesis and antioxidant activity of hispolon, a yellow pigment Inonotus hispidius, 2002, 41B, 875-877), which synthesis comprises the following steps of:

i) Knoevenagel-Doebner condensation of veratraldehyde (1) with malonic acid affords 3,4-dimethoxycinnamic acid(2)

ii) Reaction of acid chloride of (2) with ethyl acetoacetate in the presence of sodium hydride (NaH) yields ethyl 4(E)-2-acetyl-5-(3,4-dimethoxy-phenyl)-3-oxo-4-pentenoate (3)

iii) Decarboxylation of (3) using DMSO/NaCl gives 4-hydroxy-6-(3,4-
dimethoxyphenyl) -hexa-3,5-diene-2-one (4)

iv) Demethylation of (4) using BBr3 gives hispolon.
The above described procedure is depicted in the following scheme 1.

But the earlier synthesis of hispolon is achieved through a four step sequence as shown above. The synthetic method has many limitations and suffers from the disadvantage of using hazardous reagents like sodium hydride and involves safety issues during scale up.
Summary of the invention:
The main object of the present invention is to provide an efficient synthetic process for the preparation of hispolon and its analogs having the general formula (I) from an aromatic aldehyde (II) and acetyl acetone boron complex.

Another object of the present invention is to provide novel hispolon analogs, which are prepared via the reaction of aromatic aldehyde (II) and acetyl acetone boron complex.
Yet another object of the present invention is to provide a simple procedure for the preparation of hispolon from a commercially viable vanillin or 3, 4-

Dihydroxybenzaldehyde using acetyl acetone boron complex.

Detailed description of the invention:

In the present invention, the main object is to provide an efficient synthetic process for the preparation of hispolon and its analogs having the general formula (I) from an aryl aldehyde (II) and acetyl acetone boron complex. The general formula (I), wherein R1 R2, R3 are indepnedly hydrogen, hydroxy and methoxy.

It is convenient to describe the invention herein in relation to particularly preferred embodiments relating to synthesis of hispolon and its analogs. However, it is to be appreciated that other constructions and arrangements are also considered as falling within the scope of the invention. Various modifications, alterations, variations and or additions to the construction and arrangements described herein are also considered as falling within the ambit and scope of the present invention.
The process of present invention is depicted in the following scheme 2

The synthesis involves activation of terminal methyl of acetyl acetone through a boron complex and condensation with aryl aldehyde in presence of an amine, if required the resulting compound is dealkylated by conventional methods to afford the desired compound.

The amine used for this purpose is selected from the group alkyl amine and aryl amine, wherein alkyl amine is selected from methyl amine, ethyl amine, propel amine, n-butyl amine and the like and aryl amine such as benzyl amine and the like, preferably n-butyl amine is used.

The object of the invention is to provide a simple and efficient synthetic process for the preparation of hispolon from a commercially available vanillin or 3, 4-dihydroxyben2aldehyde and acetyl acetone boron complex in presence of amine is required, and demthylatinon of resulting compound by conventional methods such as aluminum chloride/pyridne or BBra.

Amines used here are as described above. Thus the present invention becomes more simple efficient and economic as hispolon is accomplished here in one step and one pot.
The foregoing procedure is shown in the scheme 3

Similar synthetic sequence is utilized to prepare two other new hispolon analogs (4 & 5) as shown in scheme 4. The synthetic method is general and is applicable for preparing series of analogs.


Along with the hispolon a large number of hispolon analogs have also been reported in the literature. Hispolon and its analogs reported previously are believed to have low solubility properties. So it is our interest to make the novel hispolon analogs that have better solubility properties, are also part of the present invention, which novel compounds are prepared by the process of the present invention.
Here the hispolon analogs (4) and (5) are found to be novel and are said to have good solubility properties.

The present invention is best described with the following examples;
Example -1: Synthesis of 3,4 Dihydroxybenzaldehyde

Vanillin (70 g, 1 eq.) and ethylene dichloride (1400 ml) are taken at room temperature and slowly cooled to 0-5° C and added ammonium chloride (153.3g, 2.5 eq.) lot wise and then

pyridine (349 ml, 9.4 eq.) was added drop wise for 15 minutes and then the temperature was raised to reflux condition and refluxed for 2 hours. The reaction was monitored by TLC. After two hours the temperature was brought to 0-5°C and then HCl was added drop wise maintaining the inside temperature between 20-30°C. The organic layer was separated and the aqueous layer was isolated with ethyl acetate (four times) and then combined both the layers washed with water (thrice). Dried over anhydrous sodium sulphate. The solvent was filtered and evaporated the solvent. The crude product was purified by column chromatography using Hexane and ethyl acetate as eluents and followed by recrystallization with ethyl acetate and hexane to obtain 3, 4-Dihydroxybenzaldehyde (83% yield, HPLC purity: 98.9%).

Example -2: Synthesis of Hispolon methylether (2)

To a solution of acetyl acetone (335 ml, 5 eq) in ethyl acetate was added boric anhydride (32 g, 0.7 eq) and stirred for 30 minutes at 70° C. To the above solution was added vanillin (100 g, 1 eq) and tributyl borate (177 ml,l eq) and stirred for 30 minutes at 70° C. After 30 minutes the temperature was raised to 85° C and n-butyl amine (64.9 ml ,1 eq) in ethyl acetate was added drop wise and the stirring was continued for 1 h at 100° C. The mixture was cooled to 50° C and hydrolyzed by adding IN HCl and stirred for 30 minutes at 30° C. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined layers were washed with water until neutral and dried over sodium sulphate. After removal of the solvent in vacuum the crude product was purified by column chromatography using chloroform as eluent followed by recrystallization with ethyl acetate and hexane to obtain hispolon monomethyl ether (26% yield, HPLC purity: 99%).

Example -2: Synthesis of Hispolon (1) by demethylating of hispolon methylether (2)
Method 1: To an ice cold solution of hispolon methylether (11 g, 1 eq) in ethylacetate was added aluminium chloride (15 g, 2.3 eq) followed by the drop wise addition of pyridine (35 ml, 9.26 eq) for 15 minutes and the reaction mixture was heated under reflux for 7 h at 65°C. After 7 h the reaction mixture was cooled to 10°C, cold dil HCl (20%) was added to decompose Aluminum chloride complex and extracted with ethyl acetate. The combined ethyl acetate layer was washed with water, brine and dried over anhydrous sodium sulphate. The solvent was filtered and evaporated the solvent. The crude product was purified by column chromatography using chloroform as eluent followed by recrystallization with ethyl acetate and hexane to obtain hispolon (51% yield, HPLC purity: 99%).

Method 2: N, N-Dimethylaniline (43.94 ml, 5 eq) was taken in a flask at room temperature and slowly the temperature was raised to 40°C and aluminum chloride (48.42 g, 5 eq) was added lot wise to the N, N-Dimethylaniline and stirred for 30 min at 60°C. After 30 min ethyl acetate was added and stirred for 30 min later solution of hispolon methylether (17 g, 1 eq) in ethyl acetate was added to the above mixture and stirred for 12 h at 80°C. After 12 h the reaction mixture was

hydrolyzed with aqueous HCl and ethyl acetate layer was separated and the aqueous layer was extracted with ethyl acetate. The combined ethyl acetate layer was washed with water, brine and dried over anhydrous sodium sulphate. The solvent was filtered and evaporated the solvent. The crude product was purified by column chromatography using chloroform as eluent followed by recrystallization with ethyl acetate and hexane to obtain hispolon (41% yield, HPLG purity: 99%).

Example -3: Synthesis of Hispolon (1) from 3,4-dihydroxybenzaladehyde

To a solution of acetyl acetone (9.6 ml, 5 eq) in ethyl acetate was added tributyl borate (25 ml, 5 eq) and stirred for 30 minutes at 70°C. To the above solution was added 3,4-Dihydroxybenzaldehyde (2.6 g, 1 eq) and stirred for 30 minutes at 70°C. After 30 minutes the temperature was raised to 85° C and n-butyl amine ( 1.8 ml ,1 eq) in ethyl acetate was added drop wise and the stirring was continued for 1 h at 100°C and boric anhydride (0.9 g, 0.7 eq). The mixture was cooled to 50° C and hydrolyzed by adding 1N HCl and stirred for 30 minutes at 30°C. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined layers were washed with water until neutral and dried over sodium sulphate. After removal of the solvent in vacuum the crude product was purified by column chromatography using chloroform as eluent followed by recrystallization with ethyl acetate and hexane to obtain hispolon monomethyl ether (44 % yield, HPLC purity: 99%).

Example -4: Synthesis of Methoxyhispolon methyl ether (3)

To a solution of acetyl acetone (69.9 ml, 5 eq) in ethyl acetate was added boric anhydride (6.68 g, 0.7 eq) and stirred for 30 minutes at 70°C. To the above solution was added syringaldehyde (25g, 1 eq) and tributyl borate (37 ml, 1 eq) and stirred for 30 minutes at 70°C. After 30 minutes the temperature was raised to 85° C and n-butyl amine (13.5ml, 1 eq) in ethyl acetate was added drop wise and the stirring was continued for 1 h at 100° C. The mixture was cooled to 50° C and hydrolyzed by adding IN HCl and stirred for 30 minutes at 50° C. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined layers were washed with water until neutral and dried over sodium sulphate. After removal of the solvent in vacuum the crude product was purified by column chromatography using chloroform as eluent followed by recrystallization with ethyl acetate and hexane to obtain hispolon monomethyl ether (44% yield, HPLC purity: 99%).

Example -5: Synthesis of Methoxyhispolon (4) and hydroxyhispolon (5):

N,N-Dimethylaniline (34.5 ml, 12 eq) was taken in a flask at room temperature and slowly the temperature was raised to 40° C and aluminum chloride (36.3 g, 5 eq) was added lot wise to the N,N-Dimethylaniline and stirred for 30 min at 60° C, After 30 min ethyl acetate was added and stirred for 30 min later solution of Methoxy hispolon methylether (6 g, 1 eq) in ethylacetate was added to the above mixture and stirred for 12 h at 80°C. After 12 h the reaction mixture was

hydrolyzed with aqueous HCl and ethyl acetate layer was separated and the aqueous layer was extracted with ethyl acetate. The combined ethyl acetate layer was washed with water, brine and dried over anhydrous sodium sulphate. The solvent was filtered and evaporated the solvent. The crude product was purified by column chromatography using chloroform as eluent followed by recrystallization with ethyl acetate and hexane to obtain Methoxy hispolon (26%, HPLC purity 98%) and hydroxyhispolon (3.6%, HPLC purity: 98%).

What is claimed is:

1. A process for the preparation of hispolon and its analogs having the general formula (I)
Wherein R1 R2 R3 are independently hydrogen, hydroxyl and methoxy, which process comprises the steps of:
a) Reacting the aromatic aldehyde of formula (II)

with boron complex formed from acetyl acetone in presence of an amine to obtain the compound of formula (I)

b) optionally dealkylating the resulting compound to obtain the desired compound of
formula (I)

2. The process of claim 1, wherein borate complex of acetyl acetone is prepared from acetyl acetone and tribally borate optionally in presence of boric anhydride (B2O3) in presence of suitable organic solvent.

3. The process of claim 2, wherein trialkylborate is triethyl borate or tributyl borate

4. The process of claim 2, wherein the suitable organic solvent is ethyl acetate

5. The process of claim 1, wherein the amine is selected from the group alkyl amine such as methyl amine, ethyl amine, properly amine, n-butyl amine and the aryl amine such as benzyl amine and the like, preferably n-butyl amine is used.

6. The process for the preparation of hispolon, which process comprises the steps of:

a) reacting the vanillin with borate complex of acetyl acetone to obtain methoxy hisoplon

b) demethylating the step (a) compound to obtain hispolon.

7. The single step procedure for the preparation of hispolon, which process comprises the reaction of 3,4-Dihydroxybenzaldehyde with borate complex of acetyl acetone to obtain hisoplon