AU730178B2 - Method for the preparation of (+-)-calanolide A and intermediates thereof - Google Patents

Description

S F Ref: 365926DI

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

*c a a. 4 a.

a.

Name and Address Name and Address of Applicant: Actual Inventor(s): Sarawak Medichem Pharmaceuticals, Inc.

12305 South New Avenue Suite 0 Lemont Illinois 60439 UNITED STATES OF AMERICA Michael T Flavin, Ze-Qi Xu, John D Rizzo, Alla Kucherenko, Albert Khilevich, Abram Kivovich Sheinkman, Vilayphone Wilaychack, Lin Lin, Wei Chen, William A Boulanger Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Method for the Preparation of (t)-Calanolide A and Intermediates Thereof Address for Service: Invention Title: The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845 METHOD FOR THE PREPARATION OF (±)-CALANOLIDE

A

AND INTERMEDIATES

THEREOF

FIELD OF THE INVENTION This invention relates to a method for the preparation of (±)-calanolide A, a potent inhibitor of HIV reverse transcriptase, and intermediates thereof. In 10 particular, this invention relates to a method for large scale production of (±)-calanolide A, chiral resolution of (±)-calanolide A into its optically active forms, and use of (±)-calanolide A and (-)-calanolide A for treating viral infections.

15 BACKGROUND OF THE INVENTION Human immunodeficiency virus (HIV), which is also called human T-lymphotropic virus type III (HTLV-III), lymphadenopathy-associated virus (LAV) or AIDS-associated retrovirus (ARV), was first isolated in 1982 and has been 20 identified as the etiologic agent of the acquired immunodeficiency syndrome (AIDS) and related diseases.

Since then, chemotherapy of AIDS has been one of the most challenging scientific endeavors. So far, AZT, ddC, ddl, and D4T have been approved by FDA and are being clinically used as drugs for the treatment of AIDS and AIDS-related complex. Although these FDA-approved drugs can extend the life of AIDS patients and improve their quality of life, none of these drugs are capable of curing the disease.

Bone-marrow toxicity and other side effects as well as the emergence of drug-resistant viral strains limit the longterm use of these agents.' On the other hand, the number of AIDS patients worldwide has increased dramatically within the past decade and estimates of the reported cases -2in the very near future also continue to rise dramatically. It is therefore apparent that there is a great need for other promising drugs having improved selectivity and activity to combat AIDS.' Several approaches including chemical synthesis, natural products screening, and biotechnology have been utilized to identify compounds targeting different stages of HIV replication for therapeutic intervention.

2 Very recently, the screening program at the National Cancer Institute has discovered a class of remarkably effective anti-HIV natural products, named calanolides, from the rain forest tree Calophyllum lanigerum, with calanolide A, 1, being the most potent compound in the i reported series.

3 For example, calanolide A demonstrated 100% protection against the cytopathic effects of HIV-1; one of two distinct types of HIV, down to a concentration of 0.1 AM. This agent also halted HIV-1 replication in human T-lymphoblastic cells (CEM-SS)(EC, 5 =0.1

AM).

3 More interestingly and importantly, calanolide A was 20 found to be active against both the AZT-resistant G-9106 strain of HIV as well as the pyridinone-resistant A17 virus.

3 Thus, the calanolides, known as HIV-1 specific reverse transcriptase inhibitors, represent novel anti-HIV chemotherapeutic agents for drug development.

The only known natural source of calanolide A, 1, was destroyed and other members of the same species did not contain the desired material.' Consequently, a practical synthesis of the natural product must be developed for further study and development to be carried out on this active and promising series of compounds. Herein, we describe a method for the synthesis and resolution of calanolide A and some related compounds.

OH 'O 1 Objects of the Invention Accordingly, one object of the present invention is to provide a simple and practical method for preparing (±)-calanolide A, 1, from readily available starting materials and resolving the same into its optically active forms via a chiral HPLC system or enzymatic acylation and hydrolysis.

Another object of the invention is to provide useful intermediates for preparing derivatives of (±)-calanolide A.

A further object of the invention is to provide a simple and practical method for large scale to preparation of (±)-calanolide A in high yields from key intermediate chromene 4.

An additional object of the invention is to provide a method for treating or preventing viral infections using (±)-calanolide A and (-)-calanolide A.

These and other objects of the invention will become apparent in view of the detailed description below.

1 5 Summary of the Invention In one aspect, the present invention relates to a process for preparing trans-10,11-dihydro- 6,6,10,11-tetramethyl-4-propyl-2H,6H,12H-benzo[1,2-b:3, 4-b':5,6-b"]tripyran-2,12-dione, or a Spharmaceutically acceptable salt thereof, the process comprising: reacting phloroglucinol with ethylbutyryl acetate in the presence of an acid catalyst to form 5,7-dihydroxy-4-propylcoumarin; acylating 5,7-dihydroxy-4-propylcoumarin with propionyl chloride in the presence of a Lewis acid to form 5,7-dihydroxy-8-propionyl-4-propyl coumarin; reacting 5,7-dihydroxy-8-propionyl-4-propyl coumarin under basic conditions with 4,4-dimethoxy-2-methylbutan-2-ol so as to produce 5-hydroxy-2,2-dimethyl-6-propionyl-10-propyl- 2H,8H-benzo[1,2-b:3,4-b']-dipyran-8-one; condensing 5-hydroxy-2,2-dimethyl-6-propionyl-10-propyl-2H,8H-benzol[1,2-b:3,4b']-dipyran-8-one with acetaldehyde in the presence of a base so as to form 5-hydroxy-2,2dimethyl-6-(2-methyl-3-hydroxybutyro)-10-propyl-2H,8H-benzo[1,2-b:3,4-b']-dipyran-8-one; (R:\LIBZZ]06193.doc:NJC ['Ar j. Q (R:\LIBZZ]06193.doc:NJC 3a cyclizing 5-hydroxy-2,2-dimethyl-6-(2-methyl-3-hydroxybutyro)-1 0-propyl-2H,8Hbenzo-[1 ,2-b:3,4-b']-dipyran-8-one under Mitsunobu conditions so as to produce trans-i 0,11 -dihydro- 6,6,10,11 -tetramethyl-4-propyl-2H,6H, 1 2H-benzo[1 ,2-b:3,4-b':5,6-b"]tripyran-2. 12-dione.

In another aspect, the present invention relates to a process for preparing (+)-calanolide A comprising the steps of: reacting phioroglucinol, in the presence of an acid catalyst, with ethyl butyrylacetate so as to form 5,7-dihydroxy-4-propylcoumarin; acylating 5,7-dihydroxy-4-propylcoumarin with a propionyl acylating agent so as to produce 5,7-dihydroxy-8-propionyl-4-propylcoumarin; reacting 5,7-dihydroxy-8-propionyl-4-propylcoumarin under basic conditions with 4,4-dimethoxy-2-methylbutan-2-ol so as to produce 5-hydroxy-2,2-dimethyl-6-propionyl- 2H ,8H-benzo[1 ,2-b:3,4-b']-dipyran-8-one; condensing 5-hydroxy-2,2-dimethyl-6-propionyl-1 0-propyl-2H,8H-benzo[1 ,2-b:3,4b']-dipyran-8-one with acetaldehyde in the presence of TiC1 4 so as to form an enantiomeric mixture of 5-hydroxy-2,2-dimethyl-6-(2-methyl-3-hydroxybutyro)-1 0-propyl-2H ,8H -ben zo-[1, ,24b3,4-b']-d ipyran-8one; enzymatically resolving the step enantiomeric mixture and isolating an optically active enantiomer of 5-hydroxy-2,,2-dimethyl-6-(2-methyl-3-hydroxybutyro)-10-propyl-2H,8H-benzo- 20 [1 ,2-b3,4-b]-dipyran-8-one; cyclizing step optically active enantiomer under Mitsunobu conditions so as to produce (+)-trans-1 0,1 1-dihydro-6,6, 10,1 1-tetramethyl-4-propyl-2H,6H, 12H-benzo[1 :5,6b"]tripyran-2,12-dione; and reducing (+)-trans-1 0,1 1-dihydro-6,6, 10,1 1-tetramethyl-4-propyl-2H,6H, 12Hben zo 2-b: 3,44: :5,6-b"ltri pyran-2, 1 2-d ione so as to produce (+)-calanolide A.

The present invention also relates to the syntheses of (±)-calanolide A and intermediates thereof, chiral resolution of (±)-calanolide and method of treating or preventing viral infections using (±-and (-)-calanolide A.

[R:\LIBZ.Z]O6193 .doc:NJC The method of the present invention for preparing (±)-calanolide A, 1, employs chromene 4 as the key intermediate. Chromene 4 is synthesized by the sequence depicted in Scheme I. Thus, 5,7-dihydroxy-4propylcoumarin, 2, 5 was prepared quantitatively from ethyl butyrylacetate and phloroglucinol under Pechmann conditions.

6 Product yield and purity were dependent on the amount of sulfuric acid used. The 8-position of 5,7dihydroxy-4-propylcoumarin, 2, was then selectively acylated at 8-10 0 C by propionyl chloride and AlCl 3 in a mixture of carbon disulfide and nitrobenzene to afford 5,7-dihydroxy-8-propionyl-4-propylcoumarin, 3.

In a alternative and preferred reaction, coumarin intermediate 3 can be produced in large scale quantities 15 and with minimal formation of undesirable 6-position acylated product and 6,8-bis-acylated product by selective acylation of 5, 7 -dihydroxy-4-propylcoumarin 2 with a mixture of propionic anhydride and AlC1 3 at about 70-75 0

C.

The chromene ring was introduced upon treatment of 20 compound 3 with 4,4-dimethoxy-2-methylbutan-2-ol,8 providing 4 in 78% yield (Scheme I).

As presented in Scheme II, Robinson-Kostanecki reaction 9 on 4 by using sodium acetate in refluxing acetic anhydride produced enone 5 in a 65% yield. This intermediate failed to afford calanolide A upon reduction with borohydride reagents such as NaBH 4 /CeCl 3 NaBH 4 /CuCl 2 L-selectride, 9-BBN, and DIP-chloride, and some transition metal reducing agents such as SmI 2 and [(Ph 3 P)CuH],, presumably because attack at the pyrone and ring opening occurred preferentially. Treatment of 5 with Baker's yeast also resulted in coumarin ring cleavage while tri-nbutyltin hydride 0 led to reduction of 5 to enol 6 in modest yield. However, treatment of 4 with acetaldehyde diethyl acetal in the presence of trifluoroacetic acid and pyridine with heating at 160 0 C produced chromanone 7 which can then be reduced into the final product.

OH

H

H

0 c Co IE t 2

C

2

H

6 cocI 4 le AIC1 Ho 0I" S. N4OA4 MGM8.

coS Jc3HouIl~ CFCc, 3

C

1 -6- Large scale production of L c an b eee productonrf chromanone 7 from chromene 4 can be effected under two different reactions conditions. In a one-step reaction, chromene 4 is treated With Paraldehyde, instead of acetaldehyde diethylacetal, and cyclized in the presence of an acid c i e t h y l t ta l, chromanone 7 in 2 l a c i catalyst to afford chromanone 7 in 27* yield along with 8% of the corresponding 10,11-cis d methl th of the step reaction -mune l der aivative 7a. In a two- Srene 4 nas reacted aldol condensation conditions, 1 chaon edol produected with acetaldehyde to form an openchain aldol product 7b. Aldol Product 7b was then cyclized under acidic conditions such asuct 7b ws th .:to form both chromanone 7 and 1 5 0 H -cisan-dime derivative 7a in a 1:1 ratio ith the former leading to 16 purified yield. How t h e f ormer leading to 16 urified yield. However, under neutral Mitsunobu"z conditions. 7b was reproducibly cyclized to give chromanone 7 as the predominant product and in 48% yield.

0 0

O

o H O O

:O

7a 7b -7- Finally, (±)-calanolide A was successfully formed with the desired stereochemical arrangement by subjecting chromanone 7 to Luche reduction 12 conditions (see Scheme II). (±)-Calanolide A was then resolved into optically active forms using a preparative HPLC chiral separating system 3 DESCRIPTION OF THE DRAWINGS Figures l(a) to l(e) illustrates in vitro MMT assay results, as described in Example 15, using G9106 HIV viral strain which is AZT-resistant.

Figures 2(a) to 2(e) illustrates in vitro MMT assay results, as described in Example 15, using H112-2 HIV viral strain which was not pre-treated with AZT.

Figures 3(a) to 3(e) illustrates in vitro MMT assay results, as described in Example 15, using A-17 HIV viral strain which is resistant to to non-nucleoside inhibitors such as TIBO and pyridinone but is sensitive to AZT.

Figures 4(a) to 4(e) illustrates in vitro MMT assay results, as described in Example 15, using IIIB cultivated 20 HIV viral strain.

Figures 5(a) to 5(e) illustrates in vitro MMT assay results, as described in Example 15, using RF cultivated HIV viral strain.

Figure 6 is an HPLC chromatogram of calanolide A on normal phase column; (+)-calanolide

A

on a chiral HPLC column; (+)-calanolide A on a chiral HPLC column and (-)-calanolide A on on a chiral HPLC column. The HPLC conditions are described in Example 13.

DETAILED DESCRIPTION OF THE INVENTION All patents, patent applications, and literature references cited herein are incorporated by reference in their entirety.

According to the method of the present invention, chromene 4 is a key intermediate in the preparation of (±)-calanolide A, 1. A preferred method for synthesizing chromene 4 from 5, 7 -dihydroxy-4-propylcoumarin, 2, is shown in Scheme I. According to this synthetic scheme, 7 -dihydroxy-4-propylcoumarin, 2, 5 was prepared quantitatively from ethyl butyrylacetate and phloroglucinol under Pechmann conditions.

6 In conducting this reaction, a volume of a concentrated acid is added in a dropwise manner to a stirring mixture of ethyl butyrylacetate and phloroglucinol with a mole ratio ranging between about 3:1 and about 1:3, with a preferable range being about 0.9:1.0. The dropwise addition of an acid was conducted at a rate such that the temperature of the reaction mixture is maintained at a temperature ranging between 15 about 0OC and about 120oC, preferably about 900C.

Suitable, but not limiting, examples of concentrated acid include sulfuric acid, trifluoroacetic acid, and methanesulfonic acid. In practicing this invention, concentrated sulfuric acid is particularly preferred. As 20 the product yield and purity appear to be dependent on the amount of concentrated sulfuric acid used, it is preferred that the amount of concentrated sulfuric acid range between about 0.5 and 10 mole, most preferably ranging between about 2 and about 3.5 mole, per mole of ethyl butyrylacetate.

The reaction mixture is then heated to a temperature ranging between about 40 0 C and about 150 0 C, preferably about 900C, until the reaction reaches completion as determined by TLC analysis. The reaction mixture is then poured onto ice and the precipitated product is collected by filtration and dissolved in an organic solvent.

Suitable, but non-limiting, examples of organic solvents include ethyl acetate, chloroform, and tetrahydrofuran.

A preferred solvent is ethyl acetate. The resulting solution is then washed with brine and dried over a -9suitable drying agent, sodium sulfate. The yields of this reaction are generally quantitative.

Thereafter, 5,7-dihydroxy-8-propionyl-4propylcoumarin, 3, was prepared by selectively acylating the 8 -position of 5, 7 -diydorxy-4-propyicoumarin, 2, with propionyl chloride in the presence of a Lewis acid catalyst. In conducting this reaction, a solution of propionyl chloride in a suitable solvent, carbon disulfide, was added in a dropwise manner to a vigorously stirred solution of 5, 7 -dihydroxy-4-propylcoumarin, 2, a Lewis acid and an organic solvent cooled in an ice bath.

Dropwise addition of propionyl chloride is conducted such that the temperature of the reaction mixture is maintained at a temperature ranging between o 0 C and about 30 0

C,

15 preferably between about 8 0 C and 10 0

C.

In practicing the invention, the amount of propionyl chloride used generally ranges between about 0.5 moles and about 6 moles, preferably ranging between about 1 mole and about 2 moles, per mole of 5, 7 -dihydroxy-4-propylcoumarin, 2.

Non-limiting examples of Lewis acid catalysts useful in the acylation reaction include AlCl 3 BF,, SnC1 4 ZnCl 2

POC

1 3 and TiCl 4 A preferred Lewis acid catalyst is AlC1 3 The amount of Lewis acid catalyst relative to 5,7- 25 dihydroxy-4-propylcoumarin, 2, ranges between about and about 12 moles, preferably ranging between about 2 and about 5 moles, per mole of 5, 7 -dihydroxy-4-propylcoumarin, 2.

Non-limiting examples of organic solvent for use in preparing the 5, 7 -dihydroxy-4-propylcoumarin, 2, solution include nitrobenzene, nitromethane, chlorobenzene, or toluene and mixtures thereof. A preferred organic solvent for use in this invention is nitrobenzene.

Upon completion of the addition of propionyl chloride, the vigorously stirred reaction mixture is maintained at a temperature ranging between about 0 C and about 1200C, preferably ranging between about 25 0 C and 0 C, until the reaction reaches completion as monitored by conventional means such as TLC analysis. The reaction mixture is then poured onto ice and extracted several times with a suitable solvent such as ethyl acetate, chloroform, methylene chloride, tetrahydrofuran, or a mixture of chloroform/methanol. A preferred solvent for this extraction is ethyl acetate. The extracts are then dried over a suitable drying agent, sodium sulfate, and the product may be purified by conventional means such as silica gel column chromatography.

On a small scale, the yield of 5,7-dihydroxy-8o° Propionyl-4-propylcoumarin 3, produced by the above described reaction is generally quantitative. However, on 15 a larger scale, the reaction was very difficult to controland did not exclusively afford the desired product.

A

route developed for the synthesis of Mammea coumarin was initially attempted for the preparation of compound 3, but it proved too awkward and low-yielding.

7 Since the desired 8 -position acylated product 3 was always accompanied by the formation of undesired 6position acylated product and 6 ,8-bis-acylated product, it was necessary to optimize the reaction conditions to minimize the formation of the undesired products and develop a more effective purification process to increase the purity and scale-up the quantities of the desired 5,7dihydroxy-8-propionyl-4-propylcoumarin 3. An alternative and preferred route for preparing 5,7-dihydroxy-8propionyl-4-propylcoumarin 3 in large scale quantities is then developed.

Preparation of 8-acylated coumarin 3 on a 5 gram scale as a single product (45% yield) has been achieved by adding a mixture of propionic anhydride, a Lewis acid, A1C1 3 and suitable solvent, 1,2dichloroethane, into a vigorously stirring pre-heated mixture of coumarin, a Lewis acid, A1C1 3 and -11suitable solvent, 1,2-dichloroethane, at a temperature ranging between about 40 and about 160 0

C,

preferably ranging between about 70 and about 75 0

C.

Dropwise addition of the propionic anhydride solution is conducted at a rate such that the temperature of the reaction mixture is maintained within the desired temperature range.

The amount of propionic anhydride used in the reaction generally ranges between about 0.5 moles and about 10 moles, preferably ranging between about 1 mole and about 2 moles, per mole of 5,7-dihydroxy-4propylcoumarin 2.

Non-limiting examples of Lewis acid catalysts useful in the acylation reaction include A1C13, BF 3

POC

3 SnCl 4 15 ZnCl 2 and TiC1 4 A preferred Lewis acid catalyst is A1Cl-.

The amount of Lewis acid catalyst relative to 5,7dihydroxy-4-propylcoumarin, 2, ranges between about and about 12 moles, preferably ranging between about 2 and about 4 moles, per mole of 5, 7 -dihydroxy-4-propylcoumarin, 20 2.

Suitable but nonlimiting examples of solvents for use in the invention include diglyme, nitromethane, 1,1,2, 2tetrachloroethane, and 1,2-dichloroethane (preferred).

Upon completion of the addition of propionyl anhydride, the vigorously stirred reaction mixture is maintained at a temperature ranging between about 40 0 C and about 160oC, preferably ranging between about 70 0 C and until the reaction reaches completion as monitored by conventional means such as TLC analysis. The workup procedure is the same as described above.

The product was purified without the use of column chromatography to afford the desired product 3. This procedure has been scaled-up to 1.7 kg of coumarin (for details see experimental section) and the yield for 8acylated coumarin 3 was 29% after recrystallization. The yield for 8-acylated coumarin 3 may be further improved by -12changing the purification processing. For example, the crude product may be recrystallized from solvent(s) other than dioxane, or a simple washing with an appropriate solvent may lead to product pure enough for the next reaction step.

Thereafter, chromene 4 was prepared by introducing the chromene ring into 5,7-dihydroxy-8-propionyl-4propylcoumarin, 3, using 4,4-dimethoxy-2-methylbutan-2-ol.

According to the method of the present invention, a solution of 5,7-dihydroxy-8-propionyl-4-propylcoumarin, 3, and 4 ,4-dimethoxy-2-methylbutan-2-ol in a suitable organic 0 solvent in the presence of a base was reacted at a temperature ranging between about 40 0 C and about 180 0

C,

preferably ranging between about 100 0 C and about 120 0

C,

S 15 until the reaction reached completion as determined by conventional means such as TLC analysis. Water and methanol formed during the reaction were removed azeotropically via a Dean-Stark trap.

SIn practicing this invention, the amount of 4,4dimethoxy-2-methylbutan-2-ol employed in the reaction generally ranges between about 0.5 and about 8 moles, preferably ranging between about 2 and about 4 moles, per mole of 5,7-dihydroxy-8-propionyl-4-propylcoumarin 3.

Suitable, but not limiting examples of organic solvents include pyridine, triethylamine,

N,N-

dimethylformamide (DMF), toluene, tetrahydrofuran (THF) or 1,2-dichloroethane. Suitable, but non-limiting examples of the bases include pyridine, 4-dimethylaminopyridine, triethylamine, N,N-diethylanailine, [4,3,0]-non-5-ene (DBN), 1,8-diazabicyclo-5,4,0]undec-7ene (DBU), sodium carbonate and sodium bicarbonate.

Pyridine was used as both base and solvent in this invention on a small scale; for scale-up, however, pyridine was used as a base and toluene was used as a solvent.

-13- Upon completion of the reaction, the solvent is removed under reduced pressure and the reaction products is dissolved in a suitable solvent, ethyl acetate.

The solution is then washed sequentially with water and brine and dried over a suitable drying agent, sodium sulfate. Thereafter, the crude chromene 4 product can be purified by conventional means such as silica gel column chromatography using 25% ethyl acetate/hexane as the elution solvent. The yields of chromene 4 generally fall with the range of about 60% and about 85%, usually resulting in about 78% yield.

Thereafter, chromanone 7 may be produced by reacting a solution of chromene 4, acetaldehyde diethylacetal, and an acid catalyst in organic solvent at a temperature 15 ranging between about 60 0 C and about 140 0 C, preferably *about 140 0 C, until the reaction is completed.

The amount of acetaldehyde diethylacetal used in the reaction generally ranges between about 0.5 and about moles, preferably ranging between about 3 and about moles, per mole of chromene 4.

Suitable, but non-limiting, examples of acid catalysts include trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, p-tosylic acid, acetic acid, hydrofluoric acid and their pyridinium salts 25 and mixtures thereof. A preferred acid catalyst for the use in this invention is trifluoroacetic acid. The amount of acid catalyst used generally ranges between about 2 and about 25 moles, preferably ranging between about 17 and about 22 moles, per mole of chromene 4.

Two alternative routes for preparing chromanone 7 from chromene 4 in large scale quantities were developed and which involve either a one-step reaction process (paraldehyde one-step reaction) or a two-step reaction processes (LDA/sulfuric acid process or LDA/Mitsunobu process).

Paraldehyde one-step reaction: -14- Instead of acetaldehyde diethylacetal, paraldehyde was used as the acetaldehyde equivalent. In the presence of one or more acid catalysts such as CF 3

SO

3 H, CF 3 COH, and pyridinium p-toluenesulfonate (PPTS),; chromene 4 was reacted with paraldehyde at elevated temperature in a suitable solvent to afford chromanone 7 as the major product and the corresponding derivative 7a as a minor product.

According to this reaction, paraldehyde was added to a stirring solution of chromene 4 and an acid catalyst, PPTS, at room temperature in a suitable solvent.

The resulting mixture was heated at the temperature ranging between about 40 and about 140 0 C, preferably i: ranging between about 60 and about 100 0 C, for a period of 15 time ranging between about 5 and about 36 hours, preferably about 20 hours. Thereafter,

CFCO

2 H, an additional equivalent of PPTS and paraldehyde was added and the resulting mixture was maintained at a temperature ranging between about 40 and about 140°C, preferably 20 ranging between about 60 and about 100 0 C overnight or until the reaction reached completion as determined by convention means,

TLC.

The amount of paraldehyde employed per mole of chromene 4 generally ranges between about 1 and about moles, preferably ranging between about 20 and about moles.

Non-limiting acid catalysts include trifluoromethane sulfonic acid, trifluoroacetic acid, methanesulfonic acid, p-tosylic acid and their pyridinium salts. In practicing this invention, pyridinium p-toluenesulfonate (PPTS) is the preferred acid catalyst. The amount of acid catalyst used in the reaction ranges between about 0.5 and about moles, preferably between about 1 and about 2 moles.

Representative solvents for use in the reaction include toluene, diglyme and 1,2-dichloroethane. In practicing the invention, 1,2-dichloroethane is the preferred solvent.

Upon completion of the reaction, the reaction was neutralized with saturated bicarbonate solution and extracted with a suitable solvent, ethyl acetate.

The crude product was then purified as described above.

The yields of chromanone 7 from this reaction generally range between about 20 and about 60%, usually about LDA/sulfuric acid two-step reaction: Under aldol condensation conditions, chromene 4 was reacted with acetaldehyde to form an open-chain aldol product 7b. According to the present invention, a solution of LDA in THF was added dropwise to a solution of 0chromene 4 in THF at a temperature ranging between about 15 78 0 C and about 0OC, preferably about -30 0 C and about 78 0 C. The amount of LDA added per mole of chromene 4 ranged between about 1 and about 4 moles preferably 0 ranging between about 2 and about 3 per mole of chromene 4. Dropwise addition LDA is conducted such that the 20 reaction temperature is maintained within the desired range.

Acetaldehyde was then added dropwise to the reaction mixture in amounts ranging between about 1 and about 12 moles, preferably ranging between about 4 and about 6 moles per mole of chromene 4. Dropwise addition of acetaldehyde is conducted such that the reaction temperature is maintained within the aforementioned range.

The reaction was monitored by conventional means, e.g., TLC analysis, until it reached completion.

One skilled in the art will appreciate that the aldol reaction of chromene 4 with acetaldehyde to form 7b can be carried out under conditions which employs bases other than LDA. For example, metal hydroxides such as NaOH, KOH and Ca(OH),, metal alkoxides such as MeONa, EtONa and t- BuOK, and amines such as pyrrolidine, piperidine, diisopropylethylamine, 1,5-diazabicyclo[4,3,0]non-5-ene 16 (DBN), 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), LDA, NaNH 2 and LiHMDS as well as hydrides such as NaH and KH can all be employed for the aldol reactions.

15 Also, aldol reactions can be mediated by metal complexes of Al, B, Mg, Sn, Ti, Zn and Zr compounds such as TiCl4, (i-pro)3TiCI, (i-pro)4Ti, PhBCI2, (n-bu)2BCI, BF3, (n-bu)3SnCI, SnCI4, ZnCl2, MgBr2, Et 2 AICI with or without chiral auxiliaries such as 1,1'-binaphthol, norephrenedrine sulfonate, camphanediol, diacetone glucose and dialkyl tartrate.

6 1 8 Thereafter, the reaction mixture was quenched at -30°C to -10°C with saturated aqueous ammonium chloride solution and extracted with a suitable solvent, eg., ethyl acetate. The pooled extracts were washed with brine and dried over a suitable drying agent, eg., sodium sulfate. The yields of aldol product 7b generally range between about 40% and about 80%, usually about It should be noted that there are two asymmetric centres in 7b. Therefore 7b, a secondary racemic alcohol, is a racemic mixture of two sets of enantiomers (four optically active forms) which may be resolved by conventional resolution methods such as chromatography or fractional crystallisation of suitable diastereomeric derivatives such as salts or esters with optically active acids camphor-10-sulfonic acid, camphoric acid, methoxyacetic acid or dibenzoyltartaric acid) or enzymatically catalysed acylation or hydrolysis of the racemic esters. Also chiral transition metal mediated aldol reaction 171 8 of chromene 4 with acetaldehyde may directly produce optically active one of the enantiomers of 7b. The resultant or synthetic enantiomer may then be transformed to enantioselective synthesis of (+)-calanolide A and its congeners.

20 The crude aldol product 7b was then cyclised under acidic conditions to form a mixture of both chromanone 7 and 10,11-cis-dimethyl derivative 7a in a 1:1 ratio. Suitable, but non-limiting, acids include one or more acids such as sulfuric acid, hydrochloric acid (aqueous or a.

*O°

o* oo** o~• IR:\UBZZ)06194.doc:NJC -17anhydrous), trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, p-tosylic acid, acetic acid or their mixture thereof. A preferred acid for use in the reaction is a 1:1 v/v mixture of acetic acid and 50% H 2

SO

4 The reaction mixture was cooled, ice water was added and the resulting mixture extracted with a suitable solvent, ethyl acetate. The pooled organic layers were washed with water, saturated bicarbonate solution and brine. The crude product was concentrated in vacuo and purified by conventional means, silica gel column using a 2:3 ethyl acetate/hexane solvent mixture.

The yields of chromanone 7 from this reaction generally range between about 10% and about 40%, usually about based on chromene 4.

So 15 One skilled in the are will also appreciate that 10,11-cis-chromanone 7A can be treated with with a base under thermodynamic (equilibrium) conditions so as to afford the corresponding trans-chromanone 7. The suitable, but non-limiting, bases include metal hydroxides 20 such as NaOH, KOH and Ca(OH),, metal alkoxides such as MeONa, EtONa and t-BuOK, amines such as triethylamine, diisopropylethylamine, pyridine, 4 -dimethylaminopyridine, N,N-diethylaniline, pyrrolidine, piperidine, diazabicyclo[4,3,0]non-5-ene (DBN), 1,8-diazabicyclo- [5,4,0]undec-7-ene (DBU), LDA and LiHMDS as well as metal hydrides such as NaH and KH.

LDA/Mitsunobu two-step reaction: In a preferred reaction, aldol product 7b may be converted to chromanone 7 as the predominant product under neutral Mitsunobu reaction conditions. In this reaction, diethyl azodicarboxylate (DEAD) was added dropwise to a solution containing crude aldol product 7b and triphenylphosphine at a temperature ranging between about 0 C and about 40 0 C, preferably about ambient temperature. The amount of DEAD used in the reaction generally ranges between about 1 mole and about 10 moles -18preferably about 1 mole and about 4 moles, per mole of aldol 7b. The amount of triphenylphosphine used in the reaction generally ranged between about 1 mole and about moles, preferably ranging between about 1 mole and about 4 moles, per mole of aldol 7b.

Instead of DEAD, other reagents reported in the literature can be employed such as diisopropyl azodicarboxylate (DIAD), dibutyl azodicarboxylate

(DBAD),

dipiperidinoazodicarboxamide, bis(N'-methylpiperazin-1- 10 yl)azodicarboxamide, dimorpholinoazodicarboxamide, N,N,N,N-tetramethylazodicarboxamide (TMAD) 19. Also, in addition to triphenylphosphine, tri-n-butylphosphine,9 triethylphosphine, trimethylphosphine and tris(dimethylamino)-phosphine have been used.

Thereafter, the reaction was quenched with saturated ammonium chloride upon completion and extracted with a suitable solvent, ethyl acetate. The pooled organic layers were washed with brine, concentrated in vacuo and the crude chromanone 7 was purified by conventional means 20 as discussed above. The yields of chromanone 7 from the LDA\Mitsunobu reaction generally range between about and about 60%, usually about 50% based on chromene 4.

Suitable, but non-limiting, examples of azo compounds for the Mitsunobu reaction include diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), dibutyl azodicarboxylate

(DBAD),

dipiperidinoazodicarboxamide, bis(N 4 -methylpiperazin-lyl)azodicarboxamide, dimorpholinoazodicarboxamide, and N,N,N',N'-tetramethylazodicarboxamide

(TMAD).

Suitable, but non-limiting, examples of phosphorous derivatives for the Mitsunobu reaction include triphenylphosphine, tri-n-butylphosphine triethylphosphine, trimethylphosphine and tris(dimethylamino)phosphine.

Finally, mild borohydride reduction of chromanone 7 in the presence of CeCl,(H 2 0) 7 produced (±)-calarolide

A

-19with the desired stereochemical arrangement. In conducting the reduction reaction, a solution of chromanone 7 was added dropwise into a solution of reducing agent, sodium borohydride and a metal additive, CeCl 3 (H20), in ethanol. The rate of addition is such that the reaction mixture temperature is maintained within a range of between about -40 0 C and about 0 C, preferably ranging between about 100C and about 0 C. Thereafter, the reaction mixture was stirred at a temperature ranging between about -40 0 C and about In general, the amount of metal additive, e.g., CeCl 3

(H

2 0)7 present in the reaction mixture ranged between about 0.1 and about 2 mole, preferably ranging between about 0.5 and about 1 mole, per mole of sodium S 15 borohydride. In addition, the amount of sodium reducing agent, borohydride employed in the reaction generally ranged between about 0.1 and about 12 mole, preferably ranging between about 2 and about 4 moles, per mole of chromanone 7. Suitable, but non-limiting, 20 examples of reducing agents include NaBH 4 LiAlH 4 Bu) 2 AlH, (n-Bu) 3 SnH,9-BBN, Zn(BH 4 2

BH

3 DIP-chloride, selectrides and enzymes such as baker yeast. Suitable, but non-limiting, examples of metal additives include CeCl 3 ZnCl 2 Al C1 3 TiC1 4 SnCl 3 and LnC13 and their mixture with triphenylphosphine oxide. In practicing this invention, sodium borohydride as reducing agent and CeCl 3

(H

2 0)7 as metal additive are preferred.

Thereafter, the reduction mixture was diluted with water and extracted with a suitable solvent, ethyl acetate. The extract was dried over a suitable drying agent, sodium sulfate, and concentrated. The resulting residue was then purified by conventional means such as silica gel chromatography, using ethyl acetate/hexane solvent mixtures.

Thus, (±)-calanolide A, 1, was successfully prepared with the desired stereochemical arrangement by treatment of the key intermediate chromene 4 with acetaldehyde diethyl acetal or paraldehyde in the presence of trifluoroacetic acid and pyridine or a two-step reaction including aldol reaction with acetaldehyde and cyclization either under acidic condition or neutral Mitsunobu condition to produce chromanone 7, followed by Luche reduction via chromanone 7 (see Scheme II).

An alternative route for preparing (±)-calanolide

A

from chromene 4 was attempted. A Robinson-Kostanecki reaction on 4 was conducted with sodium acetate in refluxing acetic anhydride and produced enone 5 in a 70% yield (see Scheme II). Enone 5, however, failed to .afford (±)-calanolide A when being reduced with borohydride reagents and some transition metal reducing agents, presumably because attack at the pyrone and ring opening occurred preferentially. Treatment of compound with Baker's yeast also resulted in coumarin ring cleavage while tri-n-butyltin hydride reduce enone 5 into enol 6 in .a modest yield.

20 In another embodiment of the invention, methods for resolving (±)-calanolide A into its optically active forms, (+)-calanolide A and (-)-calanolide A, are provided. In one method, (±)-Calanolide is resolved by high performance liquid chromatography (HPLC) with organic solvent system as a mobile phase. HPLC is performed on a column packed with chiral packing material. Suitable, but not limiting, examples of chiral packing material include amylose carbamate, D-phenylglycihe, L-phenylglycine, Dleucine, L-leucine, D-naphthylalanine, L-naphthylalanine, or L-naphthylleucine. These materials may be bounded, either ionically or covalently, to silica spherew which particle sizes ranging between about 5 um and about 20 um.

Suitable, but non-limiting, mobile phase includes hexane, heptane, cyclohexane, ethyl acetate, methanol, ethanol, or isopropanol and mixtures thereof. The mobile phase may be employed in isocratic, step gradient or Continuous gradient systems at flow rates generally rates generally ranging between about and about Another method for resolving (±)-calanolide A, a secondary racemic alcohol, into its optically cative forms involves enzyme catalysed acylation or hydrolysis. In practising this invention, enzyme catalysed acylation of (±)-calanolide A, a secondary racemic alcohol, is preferred. The enzymatic resolution method employs enzymes such as lipase CC (Candida cylindracea), lipase AK (Candida cylindracea), lipase AY (Candida cylindracea), lipase PS (Pseudomonas species), lipase AP (Aspergillus niger), lipase N (Rhizopus nieveus), lipase FAP (Rhizopus nieveus), lipase PP (porcine pancrease), pig (porcine) liver esterase (PLE), pig liver acetone powder (PLAP), or subtilisin. The preferred enzyme for use in the enzyme catalysed acylation reaction is lipase PS-13 (Sigma Corporation, St Louis, MO, USA). Immobilised forms of the enzyme on cellite molecular sieves or ion exchange resin are also contemplated for use in this method. The amount of enzyme used in the reaction depends on the rate of chemical conversion desired and the activity of the enzyme.

The enzymatic acylation reaction is carried out in the presence of an acylating agent. Suitable but not limiting examples of acylating agents include vinyl acetate, vinyl propionate, vinyl butyrate, acetic anhydride, propionic anhydride, phthalic anhydride, acetic acid, propionic acid, hexanoic acid or octanoic acid. The enzyme reaction employs at least one mole of acylating agent per mole of calanolide A. Acylating agent can be used as a solvent in the acylation reaction or as a co-solvent with another solvent such as hexanes, chloroform, benzene and THF.

S 20 One skilled in the art will appreciate that racemic esters of calanolide A can be made by conventional esterification means and selectively hydrolysed by the enzymes so as to produce, in high enantiomeric excess, *c *oo *o e~e *oo [R:LIBZZ]06194.doc:NJC -22optically active or (-)-calanolide A in free or esterified form. The esterified calanolide A may be hydrolyzed chemically or enzymatically into the free form.

Suitable, but not limiting examples of solvents for use in the enzymatic hydrolysis reaction include water, suitable aqueous buffers such as sodium phosphate buffers or alcohols such as methanol or ethanol.

In yet another embodiment of the invention, a method for treating or preventing viral infections using 0 and (-)-calanolide is provided. (±)-Calanolide A and (-)-calanolide A have not been reported before for their anti-HIV activity. It has been discovered that calanolide A inhibits human immunodeficiency virus type 1 (HIV-1) with EC 50 value being approximately half of that for (+)-calanolide A. Although calanolide A is inactive against HIV-1, it does not exhibit synergistic effect on toxicity of (+)-calanolide A. Therefore, one skilled in the art will appreciate to directly use the synthetic (±)-calanolide A as antiviral agent, without 20 further resolution into the optically pure (+)-calanolide A, to inhibit the growth or replication of viruses in a mammal. Examples of mammals include humans, primates, bovines, ovines, porcines, felines, canines, etc.

Examples of viruses may include, but not limited to, HIV- 1, HIV-2, herpes simplex virus (type 1 and 2) (HSV-1 and varicella zoster virus (VZV), cytomegalovirus

(CMV),

papilloma virus, HTLV-i, HTLV-2, feline leukemia virus (FLV), avian sarcoma viruses such as rous sarcoma virus (RSV), hepatitis types A-E, equine infections, influenza virus, arboviruses, measles, mumps and rubella viruses.

More preferably the compounds of the present invention will be used to treat a human infected with a retrovirus.

Preferably the compounds of the present invention will be used to treat a human exposed or infected in need of such treatment) with the human immunodeficiency virus, either prophylactically or therapeutically.

-23- An advantage of certain compounds of the present invention is that they retain the ability to inhibit certain HIV RT mutants which are resistant to other noncucleoside inhibitors such as TIBO and nevirapine or resistant to nucleoside inhibitors. This is advantageous over the current AIDS drug therapy, where biological resistance often develops to nucleoside analogs used in the inhibition of RT.

Hence the compounds of the present invention are 10 particularly useful in the prevention or treatment of infection by the human immunodeficiency virus and also in the treatment of consequent pathological conditions associated with AIDS. Treating AIDS is defined as including, but not limited to, treating a wide range of states of HIV infection: AIDS, ARC, both symptomatic and asymptomatic, and actual or potential exposure to HIV.

For example, the compounds of this invention are useful in treating infection of HIV after suspected exposure to HIV by blood transfusion, exposure to patient blood 20 during surgery or an accidential needle stick.

Antiviral (±)-calanolide A and (-)-calanolide A may be formulated as a solution of lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation is generally a buffered, isotonic, aqeuous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or in buffered sodium or ammonium acetate solution.

Such formulation is especially suitable for parenteral administration, but may also be used for oral administration. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.

-24- Alternatively, the compounds of the present invention may be encapsulated, tableted or prepared in an emulsion (oil-in-water or water-in-oil) syrup for oral administration. Pharmaceutically acceptable solids or liquid carriers, which are generally known in the pharmaceutical formulary arts, may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Solid carriers include starch (corn or potato), lactose, calcium sulfate dihydrate, terra alba, 1 0 croscarmellose sodium, magnesium stearate or stearic acid, talc, pectin, acacia, agar, gelatin, or collodial silicon dioxide. Liquid carriers include syrup, peanut oil, olive oil, saline and water. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about 10 mg to about 1 g per dosage unit.

The dosage ranges, for administration of antiviral (±)-calanolide A and (-)-calanolide A are those to produce 20 the desired affect whereby symptoms of infection are ameliorated. For example, as used herein, a pharmaceutically effective amount for HIV infection refers to the amount administered so as to maintain an amount which suppresses or inhibits secondary infection by syncytia formation or by circulating virus throughout the period during which HIV infection is evidenced such as by presence of anti-HIV antibodies, presence of culturable virus and presence of p24 antigen in patient sera. The presence of anti-HIV antibodies can be determined through use of standard ELISA or Western blot assays for, e.g., anti-gp41, anti-tat, anti-p55, anti-pl7, antibodies, etc. The dosage will generally vary with age, extent of the infection, the body weight and counterindications, if any, for example, immune tolerance.

The dosage will also be determined by the existence of any adverse side effects that may accompany the compounds. It is always desirable, whenever possible, to keep adverse side effects to a minimum.

One skilled in the art can easily determine the appropriate dosage, schedule, and method of administration for the exact formulation of the composition being used in order to achieve the desired effective concentration in the individual patient. However, the dosage can vary from between about 0.00i mg/kg/day to about 50 mg/kg/day, but preferably between about 0.01 to about 1.0 mg/kg/day.

The pharmaceutical composition may contain other pharmceuticals in conjunction with antiviral calanolide A and (-)-calanolide A, to treat (therapeutically or prophylactically) AIDS. For example, other pharmaceuticals may include, but are not limited to, other antiviral compounds AZT, ddC, ddl, D4T, 3TC-, acyclovir, gancyclovir, fluorinated nucleosides and nonnucleoside analog compounds' such as TIBO derivatives, nevirapine, a-interferon and recombinant CD4), immunostimulants pyridinones, BHAP, HEPTs, TSAOs, 20 a-APA various interleukins and cytokines), immunomodulators and antibiotics antibacterial, antifungal, anti-pneumocysitis agents). Administration of the inhibitory compounds with other anti-retroviral agents that act against other HIV proteins such as protease, intergrase and TAT will generally inhibit most or all replicative stages of the viral life cycle.

In addition, the compounds of the present invention are useful as tools and/or reagents to study inhibition of retroviral reverse transcriptases. For example, the instant compounds selectively inhibit HIV reverse transcriptase. Hence, the instant compounds are useful as a structure-activity relationship (SAR) tool to study, select and/or design other molecules to inhibit

HIV.

The following examples are illustrative and do not serve to limit the scope of the invention as claimed.

-26-

EXPERIMENTAL

All chemical reagents and solvents referred to herein are readily available from a number of commercial sources including Aldrich Chemical Co. or Fischer Scientific.

NMR

spectra were run on a Hitachi 60 MHz R-1200

NMR

spectrometer or a Varian VX-300 NMR spectrometer.

IR

spectra were obtained using a Midac M series

FT-IR

instrument. Mass spectral data obtained using a Finnegan MAT 90 mass spectrometer. All melting points are corrected.

9 EXAMPLE 1: 5,7-Dihydroxy-4-propylcoumarin 5 (2) Concentrated sulfuric acid (200 mL) was added into a mixture of phloroglucinol dihydrate (150 g, 0.926 mol) and ethyl butyrylacetate (161 g, 1.02 mol). The resulting 15 mixture was stirred at 90 0 C for two hours whereupon it was poured onto ice. The solid product was collected by filtration, and then dissolved in ethyl acetate. The :.solution was washed with brine and dried over Na 2

SO

4 After removal of the solvent in vacuo, the residue was triturated with hexane to provide essentially pure compound 2 (203 g) in quantitative yield, mp 233-235 0 c (Lit.

5 236-238 0

'H-NMR

5 (DMSO-d 6 0.95 (3H,t,J=6.9 Hz,

CH

3 1.63 (2H, apparent hextet, J=7.0 Hz, CH 2 2.89 (2H,t,J=7.5Hz,CH 2 5.85 (1H, s, 6.22 (1H, d, Hz, 6.31 (1H, d, J=2.0 Hz, Hs); 10.27 (1H, s, OH); 10.58 (1H, s, OH); MS 220(100, 205 (37.9, M- 192 (65.8, M-C 2

H

4 177 (24.8, M-C 3 164 (60.9, M- CHCO,+1); 163 (59.6 M-CHCO 2 IR (KBr): 3210 (vs and broad, OH); 1649 (vs, sh); 1617 (vs, sh); 1554 Anal.

calcd. for C, 2

H

24 0 4 C, 65.45; H, 5.49; Found: C, 65.61;

H,

5.44.

EXAMPLE 2: 5,7-Dihydroxy-8-propionyl-4-propylcoumarin (3) A three-neck flask (500 mL) equipped with an efficient methanical stirrer, thermometer and addition -27funnel was charged with 5, 7 -dihydroxy-4-propylcourmarin, 2, (25.0 g, 0.113 mol), aluminum chloride (62.1 g; 0.466 mol), and nitrobenzene (150 mL) and the mixture was stirred until a solution was obtained, which was cooled to 0°C in an ice bath. A solution of propionyl chloride (15.2 g; 0.165 mol) in carbon disulfide (50 mL) was added dropwise at such a rate that the reaction temperature was maintained at 8-10 0 C. Addition was completed over a period of 1 hour with vigorous stirring. The reaction was 10 monitored by TLC using a mobile phase of 50% ethyl acetate/hexane. After three hours, an additional portion of propionyl chloride (2..10 g; 0.0227 mol) in carbon o disulfide (10 mL) was added. Immediately after the TLC e* analysis indicated the total consumption of starting material, the reaction mixture was poured onto ice, and allowed to stand overnight. The nitrobenzene was removed by steam distillation, and the remaining solution was extracted several times with ethyl acetate. The extracts were combined and dried over Na 2

SO

4 The crude product obtained by evaporation in vacuo was purified by chromatography on a silica gel column eluting with ether/hexane to provide the desired propionylated coumarin 3, mp (corr) 244-246 0 C. H-NMR (DMSO-d) 6 0.96 (3H, t, J=7.3 Hz, 1.10 (3H, t, J=7.2 Hz, 1.60 (2H, m,

CH

2 2.88 (2H, t, J= 7.7 Hz, CH 2 3.04 (2H, q, J=7.2 Hz, CH) 5.95 (1H, s, H 3 6.31 (1H, s, 11.07 (1H, s, OH) 11.50 (1H, s, OH); MS 277 276 247 (100, M-C 2 IR (KBr): 3239 (s and broad, OH); 1693 1625 and 1593 cm'; Anal. calcd. for C 15

H,

6 0,: C, 65.21; H, 5.84; Found: c, 64.92; H, 5.83. The isomer assignment was made by analogy to precedent.

7 EXAMPLE 3: 2,2-Dimethyl-5-hydroxy-6-propionyl-10-propyl- 2H,8H-benzo[l,24-:3,4-b]dipyran-8-on (4) A mixture of 3 (2.60 g, 9.42 mmol) and 4,4-dimethoxy- 2-methylbutan-2-ol (5.54 g, 37.7 mmol) were dissolved in anhydrous pyridine (6.5 mL). The mixture was refluxed -28under nitrogen f or three days. Af ter removal of the solvent in vacuc, the residue was dissolved in ethyl acetate. The ethyl acetate was washed several times with 1. N HCl and brine. It was then dried over Na 2

SO

4 The crude product obtained by evaporation in vacuo was purified by silica gel column chromatography, eluting with ethyl acetate/hexane to afford 2.55 g of 4 in 78.6% yield, mp 96-98 0 C. 1 H-NI4R (CDCl 3 S 1. 05 (3H, t, J=7. 3 Hz, CHO); 1.22 (3H, t, J=7.5 Hz, CHO); 1.53 (6H, s, 2 CHO); 1.75 (2H, m, CH 2 2. 92 (2H, t, J-7. 1 Hz, CH 2 3. 35 (2H, q, J=7.l1 Hz, CH 2 5.56 (1H, d, J=10. 0 Hz, H0); 5.98 (1H, s,

H

9 6. 72 (1H, d, J=10.o0 Hz, 1-4) MS (El) 3 43 7, M+1); 342 (22.5, 327 (100, M-CH 3 IR (-KBr) 1728 (vs, C=O) cm-l; Anal. calcd. for C 20 H22O 5 C, 70. 16; H, 6.48;. Found: C, 70.45; H, 6.92.

EXAMPLE 4: 10 1 i e y r 2 o o a a o i e A A mixture of 4 76 g, 5. 11 mmol) and sodium acetate (0.419 g, 5.11 mmol) in acetic anhydride (12 mL) were ref luxed for 10 hours whereupon the solvent was removed in vacuo. The residue was purified by silica gel column chromatography, eluting first with 25% ethyl *.:acetate/hexane followed by 50% ethyl acetate/hexane to provide 1.16 g (62% yield) of enone 5 (6,6,10,11tripyran-2,22dione) as a white solid, mp 209-209.50C.

'H-

NKR- (CDCl 3 6 1.05 (3H, t, J=6.6 Hz, CHO); 1.56 (6H, s, 2 CHO 1.-7 3 (2H, m, CH 2 1.-9 8 (3H, s, CH 3 2. 38 (3H, s, CHO); 2. 91 (2H, t, J=7. 5 Hz, CHA); 5. 69 (1H, d, J=10. 0 Hz,

H

7 6. 11 (1H, s, HO); 6.71 (1H, d, J=10 Hz, H 8 MS (El) 3 66 (2 9. 6, 3 51 (10 0, M-CH 3 3 23 (16. 5, M-CAH)

IR

(KBr) 1734 (vs, C=O) 1657, 1640, 1610, and 1562 cm"; Anal. calcd. for CnH220 5 72.12; H, 6.05; Found: C, 72.14; H, 6.15.

-29- EXAMPLE 5: 1O,ll-Didehydrocalanolide A (6) A mixture of enone 5 (160 mg, 0.437 mmol) and tni-nbutyltin hydride (0.318 g, 1.09 mznol) in dry dioxane mL) was ref luxed under nitrogen for 12 hours. The solvent was then removed in vacuc and the residue was purified by preparative TLC using 25% ethyl acetate in hexane as the mobile phase. The product exhibited an Rf of about 0. 4.

Enol 6 (12-hydroxy-6,6, 10, ll-tetramethyl-4-propyl.

2

H,

6 H,l 2 H-benzol,2b:3,4-b:s,6..bsI]tripyran-2-.one) (13.3 mg, was isolated as an oil from the plate by ethyl .acetate elution. This elution may have been inefficient, and the actual yield higher, as indicated by analytical TLC of the crude product. 'H-NI4R (CDCl 3 0. 92 (3H, t, J=6. Hz, CHO;fU~ 1.26 1.39 (3H, s, 1.63 (2H, m, CH 2 1.96 (3H, s, CHO); 2.36 (3H, s, CII,); 2.45 t, J=6.0 Hz, CH 2 3.65 (1H, s, H, 2 5.51 (1H, d, *99*9.J=10.0 Hz, H 7 6.67 (1H, d, J 10.0 Hz, H 8 13.25 (1H, br s, OH) MS (El) 369 8, M+1) 368 367 99 9 M-1) 366 (28.4, 351 (100, M-OH); IR(KBr): 1651 20 1589 (m)cmir.

*9:EXAMPLE 6: 12-Oxocalanolide A (7) A solution containing chromene 4 (344 mg, 1.0 mmol), acetaldehyde diethylacetal (473 mg, 4.0 mmol), trifluoroacetic acid (1.5 mL, 19.4 mmol) and anhydrous 25 pryidine (0.7 mL) was heated at 140 0 C under N 2 The reaction was monitored by TLC analysis. After 4 hours, the reaction mixture was cooled to room temperature, diluted with ehtyl acetate and washed several times with aqueous NaHCO3 and brine. The organic layer was separated and dried over Na,S0 4 The solvent was removed in vacuo and the crude product was purified by silica gel column chromatography eluting with ethyl acetate/hexane Chromanone 7 (l0,1l-trans-dihydro66l0,lltetramethyl-4-propyl-2f, 6H1 2H-benzo(l,2-b:3,4-b' tripyran-2,12-dione) (110 mg, 30% yield) was obtained m.

-3o- 176-177- C. (Lit.

5 130-132- C) 'H-NItR 3 (CDCl 3 6 1.02 (3H, t, J=7. 5 Hz, CHO); 1. 21 (3H, d, J=6. 8 Hz, CHO); 1.-51 (3H, d, J=7.-0 Hz, CHO); 1.-55 (6H, 2 s, 2 CHO); 1. 63 (2H, sextet, J=7. 0 Hz, CHO); 2.55 (1H, dq, J=6. 9 J=11. 0 Hz, 2. 88 (2H, t, J=7. 6 Hz, CH 2 4. 28 (1H, dq, J=6. 3 Hz, J=1i1. 0 Hz, H1 0 5. 60 (1H, d, J=9. 9 Hz, Hs) 6. 04 (1H, s, H 3 6. (1H, d, J=11. 8 Hz, MS (CI) 3 69 (100, M+1) EXAMPLE 7: (±)-Calanolide A To a solution of chromanone 7 (11 mg, 0.03 mmol) in EtCH (0.4 mL) was added sodium borohydride (2.26 g, 0.06 :lImol) and CeCl 3

(H

2 0) 7 (11. 2 mg, 0. 03 .mmol) in EtCH (5 m.L) at room temperature. After stirring for 45 minutes, the mixture was diluted with H 2 0 and extracted with ethyl acetate. The organic layer was dried over Na 2 S0 4 and concentrated. The crude product was purified by preparative TLC eluting with ethyl acetate/hexane 1) to afford (±)-calanolide A (10.5 mg, m.p. 52-54 0

C,

which increased to 1021 0 C after it was dried thoroughly (Lit.' 56-58-C) 'H NMR (CDC1 3 6 1. 03 (3H, t, J=7. 3Hz, CHO) 1. 15 (3H, d, J=6.8SHz,

CH

3 1.46 (3H, d, j=6.8BHz,

CH

3 1~ .47~ (3H, s, CHOI, 1.51 (3H, s, CHO 1. 66(H ,C 1.93 (1H, m, H11) 2.89 (2H, m, CH 2 3.52 (1H, broad-s, OH), 3.93 (1H, m, H1 0 4.72 (1H, d, J=7. 8Hz H1 2 5.5 4 (1H, d, J=10.0OHz, H7) 5. 94 (1H, s, HA3), 6. 62 (1H, d, 5=9.9Hz, H 1 MS (CI) 371 (75.4, M+1) 370 (16.1, 353 (100, M-OH); Anal. calcd. f or C,2H25O: C, 71.33; H, 7.07:Found: C, 71.63; H, 7.21.

EXAMPLE 8: 5, 7 -Dihydroxy-4-propylcoumarin In this Example, kilogram scale preparation of intermediate 2 is described. Into a stirring suspension of phloroglucinol (3574.8 g, 28.4 mol, pre-dried to constant weight) and ethyl butyrylacetate (4600 M.L, 28.4 mol) was added concentrated sulfuric acid dropwise at such a rate that the internal temperature did not exceed 40 0 C. After -31- 100 mL of sulfuric acid was added, the temperature rose to 0 C and the suspension turned into a yellow solid.

Analysis of TLC indicated that the reaction had proceeded to completion.. The reaction mixture was diluted with water (10 L) and stirred at ambient temperature overnight.

The precipitated product was collected by filtration and then rinsed with water until the filtrate was neutral.

A

quantity of 4820 g (77% yield) of 5,7-dihydroxy-4propylcoumarin 2 was obtained after being dried, which was 1 0 identical with an authentic sample by comparsion of TLC, melting point and spectroscopic data.

EXAMPLE 9: 5,7-Dihydroxy-8-propionyl-4-propylcoumarin 3 In this Example, kilogram quantities of intermediate 3 was sythesized using propionic anhydride instead of propionyl chloride. 5, 7 -dihydroxy-4-propyl-coumarin, 2, (1710 g, 7.77 mol) and AlCl 3 (1000 g, 7.77 mol) were mixed in 1,2-dichloroethane (9 The resulting orange suspension was stirred and heated to 70oC until a solution was obtained. Then, a mixture of propionic anhydride 20 (1010 g. 7.77 mol) and AlCl 3 (2000 g, 15.54 mol) in 1,2dichloroethane (3.4 L) was added dropwise over 3 h. The reaction was allowed to stir at 70 0 C for an additonal hour. After being cooled down to room temperature, the reaction mixture was poured into a rapidly stirring mixture of ice water and IN HC1. The precipitated product was taken into ethyl acetate (30 L) and the aqueous solution was extracted with the same solvent (10 L x 2).

The combined extracts were successively washed with 1 N HC1 (10 saturated aq. NaHCO 3 (10 and water (10 L).

After being dried over MgS0 4 and concentrated in vacuo, a solid product (1765 g) was obtained which was washed with ethyl acetate (15 L) and recrystallized from dioxane L) to provide 514 g of pure compound 3. From the ethyl acetate washings, an additional 100 g of compound was obtained after recrystallization from dioxane. Thus, the -32combined yield for compound 3, which was identical with an authentic sample by comparison of TLC, melting point and spectroscopic data, was 29%.

EXAMPLE 10: 2,2-Dimethyl-5-hydroxy-6-propionyl-10-propyl- 2 H,SH-benzo[l,2-b:3,4-b']dipyran-8-one In this Example, intermediate 4 was prepared in half kilogram quantities from 3 via modification of the reaction conditions described in Example 3. A mixture of compound 3 (510.6 g, 1.85 mol) and 4,4-dimethoxy-2- 10 methylbutan-2-ol (305.6 g, 2.06 mol) were dissolved in a mixture of toluene (1.5 L) and dry pyridine (51 mL). This mixture was stirred and refluxed; water and MeOH formed .during the reaction were removed azeotropically via a Dean-Stark trap. The reaction was monitored by TLC.

After 6 days, the reaction had proceeded to completion.

S'The mixture was then cooled to ambient temperature and diluted with ethyl acetate and 1 N HC1 (1 The ethyl acetate solution was separated and washed with 1N HCI (500 mL) and brine After being dried over Na 2

SO

4 and evaporated in vacuo, a quantity of 590 g (93% yield) of compound 4 was obtained which was greater than 95% pure without further purification and was compared with an authentic sample by TLC and spectroscopic data. No trace of 6-acylated or 6,8-bisacylated product was observed, although a small amount of 7-monoester did form.

EXAMPLE 11: 12-Oxocalanolide A In this Example, chromanone 7 was prepared from two alternative pathways involving either a one-step reaction (procedure A) or a two-step reaction process (procedures B and C).

Procedure A. Paraldehyde One-Step Reaction: To a stirring solution of chromene 4 (350 mg, 1.0 mmol) and PPTS (250 mg, 1.0 mmol) in 1,2-dichloroethane (2 mL) at ambient temperature under N 2 was added 3 mL paraldehyde (22.5 mmol). The resulting mixture was refluxed for 7 h. Then, -33-

CF

3

CO

2 H (1 mL), an additional equivalent of PPTS and 1 mL of paraldehyde were added; the mixture was refluxed overnight. The reaction mixture was neutralized with saturated aqueous NaHCO 3 and extracted with ethyl acetate (50 mL x The crude product obtained by evaporation under reduced pressure was washed with hexane. The residue was purified by column chromatography eluting with ethyl acetate/hexane to afford 100 mg (27% yield) of chromanone 7 and 30 mg yield) of 7a. Chromanone 7 S 10 (10,11-trans-dihydro-6,6, 10,11-tetramethyl-4-propyl- 2

H,

6 H,12H-benzo[l,2-b:3,4-bI:5,6-b"]tripyran-2, 1 2-dione) obtained by this method was identical with an authentic sample by comparison of TLC, HPLC and spectroscopic data.

Procedure B LDA/Sulfuric Acid Two-Step Reaction: To a stirring solution of chromene 4 (5.0 g, 14.6 mmol) in THF mL) at -30 0 C under N 2 was added 18.3 mL (36.5 mmol) of 2 M LDA in THF. After 15 min at the same temperature, acetaldehyde (5.0 mL, 89.5 mmol) was added via syringe.

The reaction was monitored by TLC analysis. After 1 h, 20 the reaction mixture was quenched at -10oC.with saturated aqueous NH 4 Cl (75 mL) and extracted with ethyl acetate (125 mL x The combined extracts were washed with brine (125 mL) and dried over Na 2

SO

4 Removal of solvents in vacuo afforded a reddish oil of 7b (8.5 g).

The crude 7b was. dissolved in acetic acid (100 mL) and then 50% H 2

SO

4 (100 mL) was added with stirring. The resulting mixture was heated at 75 0 C for 2.5 h and then at 0 C for 4 h. TLC analysis indicated that the starting material had been consumed. The reaction mixture was determined to contain both chromanone 7 and 10,11-cisdimethyl derivative 7a in a 1:1 ratio. After cooling to ambient temperature, the reaction mixture was poured into a mixture of ice water (500 mL) and ethyl acetate (500 mL). The layers were separated and the aqueous layer was extracted with ethyl acetate (200 mL x The ethyl -34a a acetate solutions were combined and washed with saturated aqueous NaHC0 3 and brine. After being concentrated in vacuo, the product was purified by chromatography on a silica gel column eluting with ethyl acetate/hexane (2:3) to provide 850 mg (16% yield) of chromanone 7, which was further purified by recrystallization from ethyl acetate/hecane and was identical with an authentic sample by comparison of TLC, HPLC and spectroscopic data.

Procedure C. LDA/Mitsunobu Two-Step Reaction: Into a 10 stirring solution of THF (10 mL) containing triphenylphosphine (1.27 g, 4.80 mmol) and the crude 7b, obtained from chromene 4 (1.0 g, 2.34 mmol), equivalents of LDA and 6.0 equivalents of acetaldehyde by the procedure described above, was added dropwise diethyl 15 azodicarboxylate (DEAD, 0.77 mL, 4,89 mmol). The resulting reddish solution was stirred at ambient temperature under N 2 for 1 h, after which the reaction mixture was quenched with saturated aqueous NH 4 C1 and extracted with ethyl acetate (50 mL x The extracts 20 were washed with brine and dried over Na 2

SO

4 After removal of solvents, the crude product was purified by column chromatography on silica gel eluting with ethyl acetate/hexane to provide 412 mg (48% yield, based on chromene 4) of chromanone 7, the predominant product of the reaction, which was identical with an authentic sample by comparison of TLC, HPLC and spectroscopic data.

EXAMPLE 12: (t)-Calanolide A In this Example, (±)-calanolide A was prepared in multi-gram scale using the procedure described in Example 7. To a stirring solution of chromanone 7 (51.5 g, 0.14 mol) in EtOH (1.5 L) was added CeCl 3

(H

2 0) 7 (102 g, 274 mmol). The mixture was stirred for 1.5 h at room temperature under N, and then cooled to -300C with an ethylene glycol/H,0 (1:2 w/w) dry ice bath. After the temperature was equilibrated to -30oC, NaBH 4 (21.3 g, 563 mmol) was added and stirred at the same temperature for h, at which time the reaction was quenched with H 2 0 (2 L) and extracted with ethyl acetate L x The extracts were combined, washed with brine (2 L) and dried over Na 2

SO

4 The crude product obtained by removal of solvent under reduced pressure was passed through a short silica gel column to provide 53 g of mixture which contained 68% of (±)-calanolide A, 14% of calanolide B and 10 13% of chromanone 7 as shown by HPLC. This material was subjected to further purification by preparative HPLC to afford pure (±)-calanolide A EXAMPLE 13: Chromatographic Resolution of Synthetic (±)-Calanolide

A

15 The synthetic was resolved into enantiomers, (+)-calanolide A and calanolide A, by preparative HPLC. Thus, using a normal phase silica gel HPLC column (250 mm x 4.6 mm I.D. Zorbasil, 5 gm particle size, MAC- MOD Analytical, Inc., PA, USA), the synthetic appeared as one peak with a retention time of 10.15 minutes when hexane/ethyl acetate (70:30) was used as the mobile phase at a flow rate of 1.5 mL/min and a wavelength of 290 nm was used as the uv detector setting. However, on a chiral HPLC column packed with amylose carbamate (250 mm x 4.6 mm I.D. Chiralpak AD, 10 gm particle size, Chiral Technologies, Inc., PA, USA), two peaks with retention times of 6.39 and 7.15 minutes in a ratio of 1:1 were observed at a flow rate of 1.5 mL/min. The mobile phase was hexane/ethanol (95:5) and the uv detector was set at a wavelength of 254 nm. These two components were separated using a semi-preparative chiral HPLC column, providing the pure enantiomers of calanolide A. The chemical structures of the separated enantiomers, which were assigned based on their optical rotations and compared with the reported natural product, were characterized by spectroscopic data. HPLC chromatograms -36- (±)-calanolide A and its optical forms are shown in Figure 6.

(+)-Calanolide A mp 47-50 0 C 45-48 0 +68.80 (CHCl 3 C 0.7) (Lit. 1 4 rcr25D= +66.60 (CHC1 3 C 1H MMR (CDC1 3 6 1.03 (3H, t, J=7.3 Hz, CHOI, 1.15 (3H, d, J=6.8 Hz, CHO), 1.46 (3H, d, J=6.4 Hz, CHO), 1.47 (3H, s, CHO), 1. 51 (3H, s, CHO)1 1. 66 (2H, m, CHI) 1. 93 (1H, mn,

H,

11 2 89 (2 H, m, CHI) 3 .5 2 1H, d, J= 2. 9 H z, OH) 3 .9 3 (1H, m, H1 0 4.72 (1H1, dd, J=7.8 Hz, J=2.7 Hz, H1 2 5.54 10 (1H, d, J=9.9 Hz, H 7 5.94 (1H, s, HO), 6.62 (1H, d, J=9.9 Hz, Hg); 1 3 C NMR (CDCl 3 6 13 .99 (CHO), 15. 10 (CHO), 18.93 (CHO) 2 3. 26 (CHI) 2 7. 38 (CHO) 28. 02 (CHO) 38. 66 (CHI) 40.42 (CH) 67.19 (CM-OH), 77.15 77.67 5 104.04 106.36 and 110.14 116.51 (CS), 12 6.97 (C 7 151.14 15 3 .10 Ca) 15 4 .50 C 12), 158 .8 8

(C

4 160.42 CIMS: 371 (100, M+1) 370 (23. 6, .353 (66.2, M-OH) 1R: 3611 and 3426 (in, broad, OH) 1734 (vs. C=O) 1643 1606 (mn) and 1587 (vs) UJV X~(MeOM): 204 (32,100), 228 (23,200), 283 (22,200), 325 20 (12,700) nm; Anal. calcd. for %2H 2 ,0 5 -1/4H 2 0: C, 70.47; H, 7.12; Found: C, 70.64; H, 7.12.

(-)-Calanolide A mp 47-509C; Ca 2 1D= -75.6-(CHC1 c 0.7) Lit 1 4 ]15D= 660,(CHC 3 c 0.5) 'H NMR (CDCl 3 6 1.03 (3M, t, J=7.4 Hz, 1.15 (3H, d, J=6.8 Hz, CHO), 1.46 (3H, d, J-6.3 Hz, CM 3 1.47 (3H, s, CHO), 1.51 (3H, s, CHO), 1.66 (2H, mn, CHI) 1 1.93 (1H, mn, H 11 2.89 (2H, mn, CHI) 3 (1H, d, J=2. 9 Hz, OH) 3. 92 (1H, mn, H 10 4. 72 (1H, dd, J=7.8 Hz, J=2.7 Hz, H, 2 5.54 (1H, d, J=10.0 Hz, H 7 5.94 (1H, s, H 3 6. 62 (1H, d, J=10 -O Hz, "C NMR (CDC1 3 13. 99 (CHO), 15. 10 (CHO), 18.-93 (CH 3 23. 36 (CM 2 27.3 8 (CHO), 28. 02 (CHO), 38. 66 (CHI) 40. 42 (CH) 67. 19 (CH-OH) 7 7. 15 (CH-O) 77. 67 104. 04 106. 36 (Cq 8 and Cu) 110. 14 116.51 (Cs) 126.97

(C

7 151. 14 (C 4 b),1 153. 11 (Cab),1 154.50 (C2b 158.90 (C 4 160.44

CIMS:

-37- 371 (95.2, 370 353 (100, M-OH); IR: 3443 broad, OH), 1732 (vs, 1643 1606 and 1584 (vs) cm- 1 UV X. (MeOH): 200 (20,500), 230 (19,400), 283 (22,500), 326 (12,500) nm; Anal.: calcd. for (C,

H

26 0 5 /4H 2 0: C, 70.47; H, 7.12; Found: C, 70.27; H, 7.21.

EXAMPLE 14: Enzymatic Resolution of (±)-Calanolide

A

To a magnetically stirred suspension of Calanolide A, prepared by the method of the present invention, and vinyl butyrate (0.1 mL) in hexane (0.5 mL) 10 at ambient temperature was added 1 mg of lipase PS-13 S.(Pseudomonas Species) (Sigma Corporations, St. Louis, MO, USA). The reaction mixture was stirred and monitored by conventional means such as TLC analysis. At 10 days, an additional 1 mg of lipase PS-13 was added. After stirring 15 for a total of 20 days, the reaction was stopped because there was no obvious increase in ester formation. The enzyme was filtered out and the filtrate was concentrated to dryness. The residue was analyzed by HPLC (see Example 13), which showed that 21% of (-)-calanolide A had been 20 converted into its butyrate ester form (Scheme IV). The enriched (+)-calanolide A and the butyrate ester of calanolide A can be easily separated by conventional means such as column chromatography. The enriched calanolide A may be repeatedly treated with vinyl butyrate and lipase PS-13 as described above so as to obtain high e.e. of (+)-calanolide A.

EXAMPLE 15 In Vitro evaluation of and calanolide A This example illustrates the anti-HIV viral activity of the synthetic (±)-calanolide A and its pure enantiomers, (+)-calanolide A and (-)-calanolide A, were evaluated using the published MTT-tetrazolium method.

2 0 Retroviral agents, AZT and DDC, were used as controls for comparison purposes.

-38- The cells used for screening were the MT-2 and the human T4-lymphoblastoid cell line, CEM-SS, and were grown in RPMI 1640 medium supplemented with 10% fetal (v/v) heat-inactivated fetal calf serum and also containing 100 units/ml penicillin, 100 g/ml streptomycin, 25 mM HEPES and 20 Ag/ml gentamicin. The medium used for dilution of drugs and maintenance of cultures during the assay was the same as above. The HTLV-IIIB and HTLV-RF were propagated in CEM-SS. The appropriate amounts of the pure compounds 10 for anti-HIV evaluations were dissolved in DMSO, then "0 ~diluted in medium to the desired initial concentration.

"S The concentrations (ug drug/mL medium) employed were 0.0032 ug/mL; 0.001 ug/ml; 0.0032 ug/mL; 0.01 ug/mL; 0.032 S* ug/mL; 0.1 ug/mL; 0.32 ug/mL; 1 ug/mL; 3.2 ug/mL; ug/mL; 32 ug/mL; and 100 ug/mL. Each dilution was added to plates in the amount of 100 Il/well. Drugs were tested in Striplicate wells per dilution with infected cells while in duplicate wells per dilution with uninfected cells for evaluation of cytotoxicity. On day 6 (CEM-SS cells) and 20 day 7 (MT-2 cells) post-infection, the viable cells were measured with a tetrazolium salt, MTT (5 mg/ml), added to the test plates. A solution of 20% SDS in 0.001 N HC1 is Sused to dissolve the MTT formazan produced. The optical density value was a function of the amount of formazan produced which was proportional to the number of viable cells. The percent inhibition of CPE per drug concentration was measured as a test over control and expressed in percent The data is summarized in Figures 3 4 and Figures l(a) to l(e) illustrates in vitro MMT assay results using an isolate, G9106 HIV viral strain 21 which is AZT-resistant. The data shows that (-)-calanolide

A

was relatively non-toxic at concentrations of 1 ug/mL but exhibited very little antiviral effect. Moreover, calanolide A was effective as (+)-calanolide A in reducing -39viral CPE. As expected, AZT had little to no effect in reducing viral CPE and enhancing cell viability.

Figures 2(a) to 2(e) illustrates in vitro MMT assay results using H112-2 HIV viral strain which was not pretreated with AZT. As expected, the viral strain was sensitive to AZT. The data also showed that (-)-calanolide A was relatively non-toxic at concentrations of 1 ug/mL but exhibited very little antiviral effect. (±)-calanolide A was nearly as effective as (+)-calanolide A in reducing 10 viral CPE.

Figures 3(a) to 3(e) illustrates in vitro MMT assay results using A-17 HIV viral strain" which is resistant to to non-nucleoside inhibitors scuh as TIBO and pyridinone but is sensitive to AZT. The results here parallel those shown in Figures Figures and illustrate in vitro MMT assay results using lab cultivated HIV viral strains IIIB and RF, respectively. The results here also parallel those shown in Figures

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H.

McKee, T. Boyd, M. Resolution and Comparative Anti-HIV Evaluation of the Enantiomers of Calanolides A and B. Bioorg. Med. Chem.-ett. 1995, 1011-1014.

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S.F.;

Yan, Baker, D. Synthesis of Optically Active Calanolides A and B. J. Org. Chem. 1995, 60, 2964- 10 2965.

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Claims (2)

12-dione. 2. The process according to claim 1, wherein step base comprises LDA. The process according to claim 1, wherein step condensation is mediated by a metal complex. 4. The process according to claim 3, wherein said metal complex is TiCl 4 The process according to claim 1, wherein step cyclization is preformed with diethyl azodicarboxylate and triphenylphosphine. 6. The process according to claim 1, further comprising step enzymatically resolving and separating enantiomers of 5-hydroxy-2,2-dimethyl-6-(2-methyl-3-hydroxybutyro)-10-propyl-2H ,8H- benzo[1 ,2-b:3,4-b']-dipyran-8-one, prior to step 7. The process according to claim 1, further comprising step reducing trans-l0,ll- dihydro-6,6, 10,11 -tetramethyl-4-propyl-2H,6H, 12H-benzo[1 ,2-b:3,4-b':5,6-b"]tripyran-2, 12-dione to produce (±)-calanolide A. 8. A process for preparing (+)-calanolide A comprising the steps of: reacting phioroglucinol, in the presence of an acid catalyst, with ethyl butyrylacetate so as to form 5,7-dihydroxy-4-propylcoumarin; acylating 5,7-dihydroxy-4-propylcoumarin with a propionyl acylating agent so as to produce 5,7-dihydroxy-8-propionyl-4-propylcoumarin; 41 [R:\LIBZZ]061I94.doc:NJC reacting 5,7-dihydroxy-8-propionyl-4-propylcoumarin under basic conditions with 4,4- dimethoxy-2-methylbutan-2-ol so as to produce 5-hydroxy-2,2-dimethyl-6-propionyl-1 0-propyl-2H ,8H- benzo[1 ,2-b:3,4-b']-dipyran-8-one; condensing 5-hydroxy-2,2-dimethyl-6-propionyl-1 O-propyl-2H,8H-benzo[1 ,2-b:3,4-b']-dipyran-8- one with acetaldehyde in the presence of TiCI4 So as to form an enantiomeric mixture of 2,2-dimethyl-6-(2-methyl-3-hydroxybutyro)-1 0-propyl-2H ,8H-benzo-[1 ,2-b:3,4-b']-dipyran-8-one; enzymatically resolving the step enantiomeric mixture and isolating an optically active enantiomer of 5-hydroxy-2,2-dimethyl-6-(2-methyl-3-hydroxybutyro)-1 0-propyl-2H ,8H-benzo-[1 ,2- b:3,4-b]-dipyran-8-one; cyclising step optically active enantiomer under Mitsunobu conditions so as to produce trans-i 0,11 -dihydro-6,6, 10,11 -tetramethyl-4-propyl-2H,6H, 1 2H-benzo[l ,2-b:3,4-b':5,6-b"]tripyran- 2,12-dione; and reducing (+)-trans-1 0,1 1-dihydro-6,6, 10,11 -tetramethyl-4-propyl-2H,6H, 12H-benzo[1 ,2-b:3,4- b':5,6-b]tripyran-2,12-dione so as to produce (+)-calanolide A.

159. The process according to claim 8, wherein step is performed in the presence of diethyl azodicarboxylate and triphenylphosphine. The process according to claim 8, wherein step is performed with NaBH4 and CeC3. 11. (+)-trans-1 0,11 -dihydro-6,6, 10,11 -tetramethyl-4-propyl-2H,6H, 12H-benzo[1 ,2-b:3,4- b':5,6-b"]tripyran-2,12-dione. 12. A process for preparing trans-10,1 1-dihydro-6,6,10,1 1-tetramethyl-4-propyl-2H,6H,12H- ~benzo[1 :5,6-b"ltripyran-2, 12-dione, or a pharmaceutically acceptable salt thereof substantially as hereinbefore described with reference to the examples. *13. trans-i 10, 11 Adihydro-6,6, 10,11 -tetramethyl-4-propyl-2H,6H, 1 2H-benzo[, 3,4-b':5,6- b'ltripyran-2,12-dione, or a pharmaceutically acceptable salt thereof when produced by the process of any one of claims I to 7 or 12. 14. (+)-calanolide A when produced by any one of claims 8 to A method of preventing or treating a viral infection in a mammal which comprises administering to said mammal an antiviral effective, non-toxic amount of a compound as defined in any one of claims 11, 13 or 14. Dated 11 December 2000 SARAWAK M EDICHEM PHARMACEUTICALS, INC. Patent Attorneys for the Applicant/No *minated Person SPRUSON FERGUSON [R:1IBZZIO6I94.doc:NJC