WO1990008529A2

WO1990008529A2 - 7-alkoxycoumarins, dihydropsoralens, and benzodipyranones as photo-activated therapeutic agents and inhibitors of epidermal growth factor

Info

Publication number: WO1990008529A2
Application number: PCT/US1990/000704
Authority: WO
Inventors: Ned Duane Heindel; Michele Carole Siller; Jeffrey Dean Laskin; Michael A. Gallo
Original assignee: Lehigh University; University Of Medicine And Dentistry Of New Jersey
Priority date: 1989-01-23
Filing date: 1990-01-23
Publication date: 1990-08-09
Also published as: WO1990008529A3

Abstract

A photochemotherapeutic compound of formula (I), wherein (i) n is zero, W is a (C1-16) alkyl, alkenyl, or alkynyl linear or branched chain hydrocarbon, having no more than four O, N, or S atoms in or attached to the chain; or (ii) n is 1, W is CR2, and R, R', and R'' are independently H or CH3; or (iii) n is 2, W is CR2, and R, R', and R'' are independently H or CH3; and A, B, C, and D are independently selected from hydrogen, alkyl, aryl, halogen, amino, aminoalkyl, nitro, alkoxy, aryloxy, hydroxy, carboxy, haloalkyl, or haloalkoxy.

Description

7-Alkoxycoumarins, Dihydropsoralensr and Benzodipyranones as Photo-activated Therapeutic Agents and Inhibitors of

Epidermal Growth Factor

Background of the Invention

A number of prolif erativ e skin disorders such as mycosis f ungoides, psoriasis, vitiligo, ecz ema, etc., and cancers, including cel l lymphomas, may be treated by the application of photosensitizing chemical s and ultraviol et light. These procedures are known as photocherao therapy or, in the specific case of psoriasis, as PUVA (psoral ens ultra viol et A radiation) . Chemical classes in which such phototherapeutic behavior hav e been observ ed are porphyrins, phthalocyanins, and psoralens. Each one of these classes possesses characteristics which makes it less than ideal in the phototherapeutic function: skin staining, suspected mutagenic/carcinogenic properties, poor

absorption rates, and systemic toxicity.

Whil e no singl e mechanism of photodermal action appears able to explain the behavior of all the known classes of photosensitizing chemicals, there is a widely-accepted mechanism for action of the threering heterocyclics known as psoral ens or furocoumarins [ S. T. Isaacs, C. J. Shen, J. E. Hearst, and H. Rapoport, Biochem., 16, 1058 (1977) ] . A psoral en with the essential structural requirements indicated by the ref erenced mechanism is

Psoral ens intercalate into DNA in the cell nucl eus and subsequently enter into photo-induced cross-linking with the DNA by forming 2+2 , eye lobutane-like fusions from double bonds 3-4 and 4 '-5' in the psoralens to doubl e bonds in the pyrimidine bases. This mol ecular action in the nucleus, which requires a pair of unsaturation loci in the psoral en therapeutic, has been cl aimed to be the origin of the established photopharmacology. It is al so the maj or limitati on to the more wide-spread clinical use of these agents since

mutagenic/carcinogenic activity is a potential side effect of DNA intercalation and subsequent al kylation or drug-linking. summary of the Invention

This invention describes the syntheses and pharmacological properties of new photochemotherapeutics . These agents displ ay benef icial phototherapy effects against sev eral kinds of malignancies and demonstrate the ability to hal t prol if eration of a variety of cells of epidermal origin. These compounds are generally described as psoral en anal ogs in which either the second site of unsaturation or the tricyclic ring system is not needed f or the beneficial photopharmacological effects. The general structure of the compounds of this invention is

wherein

(i) n is zero, W is a (C_1-16) alkyl,

alkenyl, or alkynyl linear or branched chain hydrocarbon, having no more than four O, N, or S atoms in or attached to the chain; or

(ii) n is 1, W is CR₂, and R, R', and R" are independently H or CH₃; or

(iii) n is 2, W is CR₂, and R, R', and R" are independently H or CH₃, and

A, B, C, and D are independently selected from hydrogen, alkyl, aryl, halogen, amino, aminoalkyl, nitro, alkoxy, aryloxy, hydroxy, carboxy, haloalkyl, or haloalkoxy.

Compounds which are representative of this general structure have central cores which include:

Detailed Description of the Invention

Suitable 7-alkoxycoumarin derivatives must bear an ether function at carbon #7. These side chains may hav e f rom one to 16 carbons and may be linear or branched. The side chains may specifically possess unsaturation (olefinic or acetylenic at any position) and may also possess polar functionalities such as hydroxyls, carboxyls, or amino moieties.

Variation of substituents at carbon # 7 alters the lipophilicity/hydrophilicity balance, apparently without modification, in the site-binding properties of the pharmaceutical. Potency is minimal f or the simple 7-hydroxy analogs and rises markedly for the alkyl ethers. All potent members of this group must also possess olefinic unsatur ation in the pyran-2- one ring (e.g., the double bond at carbon # 3) . Large

bulky groups at carbon # 3 and/or carbon # 4, which block the availability of the double bond, can

reduce the activity of the compounds (e.g. 4-t-butyl, 3-nitro, 3-carboethoxy, 3,4-diphenyl).

Suitable compounds include the hydrogen- substituted (bearing no ring-bound functionalities), the monoalkyl or monoaryl substituted analogs with groups at carbons 3,4,5,6, and 8; the multiply-substituted analogs with groups at more than one carbon selected from carbons 3,4,5,6, and 8; and/or ether-functionalized analogs bearing such groups as methoxy, ethoxy, i-propoxy, and n-propoxy at the 5- and/or 8-positions. Alkyl and aryl groups available for attachment at ring carbons 3,4,5,6, and 8 include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, phenyl, and substituted phenyl.

Nitro, amino, aminomethyl, halo, carboxyl-derivative, sulfhydryl or hydroxy groups may also be present. Solubility in plasma rises for those analogs bearing carboxyl, hydroxyl, or aminomethyl (compared to a completely unsubstituted parent molecule bearing no pendant functions) and falls for those bearing nitro, halo, carboethoxy, or similar hydrophobic functionalities. Variation of such groups allows micro alteration in lipophilic/hydrophilic balance and has ultimate clinical relevance in designing candidate

agents for oral vs transdermal dosage forms.

Examples include, but are not limited to:

7-ethoxycoumarin

7-n-propoxycoumarin 7-n-octyloxycoumarin

4,8-dimethyl-7-(omega-carboxyheptyloxy)coumarin

4,8-dimethyl-7-allyloxycoumarin

4-methyl-8-iodo-7-[(2-methyl-3-buten-2-yl)oxy]coumarin 7-[(2-octyn-1-yl)oxy]coumarin 4-methyl-7-n-propoxycoumarin

4,8-dimethy1-7-n-propoxycoumarin

Representative syntheses of these coumarin derivatives are described below. The candidate agents may be obtained by a Williamson-type ether synthesis, condensing an appropriate alkyl halide at the 7-hydroxy of the coumarin ring. EXAMPLE 1: Synthesis of 4,8-Dimethyl-7-allyloxycoumarin.

A solution of 4,8-dimethyl-7-hydroxy coumarin (4.85 g, 25.5 mmoles) in 200 ml of acetone was reacted with 3-bromopropene (4.62 g, 38.2 mmoles) in the presence of anhydrous potassium carbonate (10 g). The reaction mixture was heated under reflux for 4 hours, cooled and filtered. The collected solid was washed with fresh acetone. The filtrate and washings were combined and the solvent evaporated under reduced pressure. The crude product was recrystallized from methanol to yield the title compound as fluffy, white needles, m.p.107-108ºC, 3.5 g, 60% yield. ¹H NMR:

2.3 (s, 3H, C₈-CH₃), 2.4 (d, 3H, C₄-CH₃ J = 1.2 Hz), 4.6 (dt, 2H, O-CH₂ J = 1.5, 4.8 Hz), 5.4 (m, 2H, =CH₂

J = 4.8 Hz), 5-.9 (m, 1H, -CH=CH₂), 6.1 (d, 1H, C₃-H, J = 1.2 Hz), 6.8 (d, 1H, C₆-H, J = 9.0 Hz), 7.3 (d, 1H, C₅-H, J = 9.0 Hz)

Anal calcd for C₁₄H₂₄O₃: C, 73.03; H, 6.13.

Found: C, 73.02; H, 6.20.

EXAMPLE 2: Synthesis of 4-methyl-8-iodo-7-[2-methyl-3-buten-2-yl) oxylcoumarin.

A solution of 4-methyl-8-iodo-7-hydroxycoumarin (3.86 g, 12.8 mmoles) and 3-chloro-3-methyl-1-butene (1.96 g, 19.1 mmoles) in 200 ml of acetone was heated under reflux in the presence of 8 g of anhydrous potassium carbonate for 4 hours. After it was cooled to room temperature the reaction mixture w-as filtered and the collected solid was washed with fcesh acetone. The filtrate and washings were combined and the solvent was evaporated under reduced pressure. The. yellow residue was recrystallized from methanol to yield the title compound as a powdery white solid, m.p. 151.5- 153ºC, 2.56 g, 55% yield. ¹H-NMR: 1.78 (s, 6H,

CH₃'s), 2.4 (d, 3H, C₄-CH₃ J = 1.3 Hz), 4.65 (d, 2H,

=CH₂ J = 6.8 Hz), 5.5 (bt, 1H, =CH J = 6.8 Hz), 6.1 (d, 1H, C₃-H J = 1.2 Hz), 6.8 (d, 1H, C₆-H J = 8.8 Hz), 7.5 (d, 1H, C5-H J = 8.8 Hz). Anal calcd for C₁₅H₁₅O₃l: C, 48.67; H, 4.08; I, 34.28.

Found: C, 48.48; H, 3.86; I, 34.41.

EXAMPLE 3: Synthesis of 7-[(2-octyn-1-yl)oxylcoumarin.

A solution of 7-hydroxy coumarin (0.324 g, 1.99 mmol) and l-bromo-2-octyne (0.378 g, 1.99 mmol) in 200 ml of acetone was heated under reflux in the presence of anhydrous potassium carbonate (3 grams) for 24 hours. After the reflux period, the reaction mixture was cooled, filtered and the collected solid was washed with fresh acetone. The acetone washings and the filtrate were combined and the solvent evaporated under reduced pressure to yield a pale yellow viscous liquid. The crude product was dissolved in a minimum amount of ethyl acetate and petroleum ether added until the solution became cloudy. The cloudy solution was concentrated under reduced pressure and a white precipitate formed. This precipitate was collected by vacuum filtration and recrystallized from aqueous methanol to yield the title compound as shiny, colorless plates, m.p. 74-75°C, 0.245 g, 50%. ¹H-NMR: 0.8-2.2 (m, 11H, (CH₂)₄CH₃), 4.7 (t, 2H, OCH₂C≡ C) , 6.2 (d, 1H, C₃-H J=9.3 Hz), 6.8 (dd, 1H, C₆-H), 6.9 (d, 1H, C₈-H, J = 2.1 Hz), 7.4 (dd, 1H, C₅-H, J = 8.1 Hz), 7.6 (d, 1H, C₄-H, J = 9.3 Hz).

Anal calcd for C₁₇H₁₈O₃: C, 75.53; H, 6.66.

Found: C, 75.45; H, 6.76.

EXAMPLE 4: Synthesis of 4 ,8-dimethyl-7-(omega-carboxylheptyloxy) coumarin .

To pentane-washed sodium hydride (0.641 g) in 20 ml of N,N-dimethyl formamide was added a solution of 4,8-dimethyl-7-hydroxycoumarin, (1.52 g, 8.00 mol) and 8-bromooctanoic acid-, (1.78 g, 7.97 mol) in 20 ml of

DMF dropwise with stirring. The reaction mixture was dil uted with DMF to a final volume of 200 ml and heated at 80 ºC (oil bath) for 16 hours. Progress of the reaction was monitored by TLC (silica, 99% CHCl₃:1% isopropanol) . The reaction mixture was cooled, diluted with distilled H₂O, and acidified until pH 2. A tan precipitate formed and was collected by vacuum filtration. The crude product was treated with decolorizing carbon and recrystallized from methanol to yield the title compound as an off-white crystalline solid, m.p.

128-130ºC 1.7 g, 68%. ¹H-NMR: 1.2-1.8 (m, 12H,

(CH₂)6), 2-29 (s, 3H, C₈-CH₃), 2.38 (d, 3H, C₄-CH₃ J = 0.98 Hz), 4.0 (t, 2H, O-CH₂). 6.1 (d, 1H, C₃-H J = 0.98 Hz), 6.8 (d, 1H, C₆-H, J = 8.8 Hz), 7.4 (d, 1H, C₅-H, J = 8.8 Hz).

Anal calcd for C₁₉H₂₄O₅: C, 68.66; H, 7.28.

Found: C, 68.43; H, 7.34.

EXAMPLE 5: Synthesis of 4,8-dimethyl-7-methoxycoumarin.

A solution of 4, 8-dimethyl-7-hydroxy coumarin (0.190 g, 1.00 mmol) and methyl iodide (0.180 g, 1.30 mmol) in 50 ml of dry acetone was heated at reflux in the presence of 0.5 g of potassium carbonate for 14 hours. The solution was then cooled and filtered. The filtrate was evaporated under reduced pressure to yield a beige solid. The beige solid was taken up in 20% sodium hydroxide (aqueous) and collected by vacuum filtration. The crude product was recrystallized from methanol to yield shiny white crystals, m.p. 162-164ºC, 135 mg, 66% yield. ¹H-NMR: 2.27 (s, 3H, C₈-CH₃), 2.4 (d, 3H, C₄-CH₃, J = 1.2 Hz), 3.9 (s, 3H, OCH₃), 6.1 (q. 1H, C₃-H, J = 1.2 Hz), 6.8-6.9 (d, 1H, C₆-H, J = 8.8 Hz), 7.4-7.5 (d, 1H, C₅-H, J = 8.8 Hz).

Anal calcd for C₁₂H₁₂O₃: C, 70.59; H, 5.88.

Found: C, 70.34; H, 5.92.

In syntheses similar to that of EXAMPLE 2 above, the following analogs were prepared: 8-iodo-7-propargyloxycoumarin, 75% yield, mp 182- 183ºC

4-methyl-8-iodo-7-(2-methyl-3-butyn-2-yloxy)coumarin, 70% yield, mp 175-177ºC

Suitable dihydrobenzodiρyran-2-ones (consistent with the general structure shown) are those analogs bearing either hydrogen substitution or single alkyl or aryl group substitution at carbons 3, 4, 5, 6, 7, 8, or 10, polyalkyl or aryl substitution at these loci, and no olefinic unsaturation in the pyran ring involving carbons 6, 7, and 8. Nitro groups, halogens, aminomethyl functions and other structural moieties described above are included where their placement on the heterocyclic nucleus does not alter the fundamental structure shown. It should be noted, however, that substitution of certain polar functions at C#5 such as nitro, amino, sulfonic acid, or εulfonamide, can decrease the biologic response and such analogs are much poorer inhibitors of the binding of epidermal growth factor.

Unsaturation appears to be necessary in the pyran-2-one ring (e.g., a double bond at carbon # 3).

Also included are analogs with 5- and/or 10- alkoxy substituents such as methoxy, ethoxy, i-propoxy, and n- propoxy derivatives. Representative examples include, but are not limited to:

6,7-Dihydro-8-methyl-2H,8H-behzo[1,2-b;5,4-b']dipyran-2-one

6,7-Dihydro-4-methyl-2H,8H-benzo[1,2-b;5,4-b']dipyran-2-one

6,7-Dihydro-4-ethyl-10-n-propyl-2H,8H-benzo[1,2-b;5,4-b']-dipyran-2-one

6,7-Dihydro-8,8-diethyl-2H,8H-benzo[1,2-b;5,4-b']dipyran-2-one 6,7-Dihydro-4,6,8,8-tetramethyl-2H,8H-benzo[1,2-b;5,4-b']-dipyran-2-one

6,7-Dihydro-4-ethyl-8,8,10-trimethyl-2H,8H-benzo[1,2-b;5,4-b'3-dipyran-2-one Two convenient synthetic methods of choice exist f or the dihydrobenzodipyran-2-ones, hereafter designed Method A and Method B.

Method A prepares these substances by a unique catalytic selective internal hydrogen transfer reaction.

The transfer hydrogenation process itself is unexpected, facile, of high yield, and of great regio- specificity in that it exclusively reduces (under the conditions specified) the 6,7-unsaturation of 8-H- benzodipyran-2-one derivatives without reduction of the 3,4-unsaturation or the carbonyl unsaturation.

Description of th e Selective Reduction Process:

A solution is prepared of a suitable 8-H- benzodipyran-2-one in a low molecular weight alcohol (e.g., methanol, ethanol, i- or n-propanol) or ether (e.g., diethylether, 1, 4-dioxane, tetrahydrof uran) and brought quickly to reflux. A suspension of palladium catalyst in a solution of a labil e organic hydrogen donor (e.g., cycl ohexene, 1,3-cyclohexadiene, 1,4- cyclohexadiene, tetralin, decalin, indane, or limonene) and an al cohol or ether is added in one porti on and the resul ting suspension stirred at temperatures of 50 to 150º C f or 0.5 to 10.0 hours. The mixture is f iltered, evaporated, and the product isolated by crystallization, distillation, sublimation or other appropriate techniques consistent with the physical properti es of the substance being isolated and wel l-known to chemical practitioners. Typical reactant ratios employ the hydrogen donor in 1 to 5 mol ar equiv al ents to that of the mol ecul e to be reduced and the metal catalyst in 0.05 to 0.5 molar ratio to that of the hydrogen donor. The catalyst may be removed, washed with anhydrous methanol, dried without heating in v ac uo, and re-used 10-15 times with minimal loss in reaction yields.

Typical yields of the reduced products f all in the range of 40 to 75%. No hydrogen gas is required, no pressurized procedures are needed, and the reaction may be perf ormed in ordinary laboratory glassware. If temperatures are held under ca. 80ºC and if contact times ar e l ess than 12 hours for quantiti es up to 10 mmoles of reducible compound, reduction of the 3,4-doubl e bond is not observ ed. Hal ogens, alkoxy, amino, carboxyl-derivativ e, and hydroxy groups survive this selective catalytic exchange hydrogenation. Nitro groups, if present, however, are reduced.

Specific .examples of this method follow.

EXAMPLE 6: Preparation of 6,7-Dihydro-4,10-dimethyl-

2H,8H-benzo[1,2-b;5,4-b']dipγran-2-one. A mixture of 4,10-dimethyl-2H,8H-benzo[1,2-b;5,4-b']dipyran-2-one (100 mg, 0.44 mmoles), cyclohexene (0.5 ml, 4.94 mmoles) and palladium on activated carbon (10%, 50 mg) in ethanol (25 ml) was refluxed for 5 hr. The mixture was cooled, filtered and the solvent removed under reduced pressure. The residue was recrystallized from benzene/cyclohexene to afford colorless needles of 6,7-dihydro-4,10-dimethyl-2H,8H-benzo[1,2-b;5,4-b']dipyran-2-one (40 mg, 40%), mp 162-163º; ¹H NMR 1.93, mult, 2H, H₇; 2.11, s, 3H, C₁₀-Me; 2.24, d, J = 1.0 Hz, 3H, C₄-Me; 2.75, t, J = 6.5

Hz, 2H, H₆; 4.20, t, J = 5.2 Hz, 2H, H₈; 5.94, d, J = 1.0 Hz, 1H, H_3; 6.99, s, 1H, H₅. ¹³C NMR 7.88, q, C₁₀-Me; 18.52, q, C₄-Me; 21.85, 24.86, two t, C₆ and C_7; 66.90, t, C₈; 111.29, d, C₃; 112.73, 112.84, two d, C_5a and C₁₀; 118.31, s, C_4a; 122.078, d, C₅; 150.92, s,

C_9a; 152.55, s, C4; 155.74, s, C_10a; 161.54, s, C₂.

Anal. Calcd. for C₁₄H₁₄O₃: C, 73.02; H, 6.12

Found: C, 72.82; H, 6.24. Following the general synthetic method of EXAMPLE 6 representative analogs, which include but are not limited to, the substances indicated may be prepared:

6,7-dihydro-8,8-dimethyl-2H,8H-benzo[1,2-b;5,4- b']dipyran-2-one, 55% yield, mp 123-124ºC 6,7-dihydro-4,8,8-trimethyl-2H,8H-benzo[1,2-b;5,4- b']dipyran-2-one, 48% yield, mp 174-175ºC

6,7-dihydro-4,8,8,10-tetramethyl-2H,8H-benzo[1,2- b;5,4-b']-dipyran-2-one, 62% yield, mp.149-150ºC

Method B: An alternative method for preparation of the dihydrobenzodipyran-2-ones involves an acid-catalyzed cyclization of either a primary allylic alcohol or a primary allylic halide with a 7-hydroxycoumarin.

R and R' = variable functions. See NOTE under "mixtures of products" for situations in which C₈ bears a proton

The two reactants are heated at reflux with a trace of p-toluenesulfonic acid for 2-3 hours. The solvent employed may be toluene, xylene, ethyl benzene, cumene or any other similar appropriate miscible, unreactive solvent medium known to one skilled in the art. Other sulfonic acids, e.g., methane-, benzene-, or trifluoromethanesulfonic acid may also be employed. A specific example of this method follows.

EXAMPLE 7: Preparation of 6,7-Dihydro-4,8,8,10-tetra-methyl-2H,8H-bgnzo[1,2-b;5,4-b']dipyran-2-one.

The 4,8-dimethyl-7-hydroxy- coumarin (0.95 g, 5.0 mmol), 1-chloro-3-methyl-2-butene (1.4 g, 7.5 mmol) and p-toluenesulfonic acid (0.095 g, 0.50 mmol) in 25 ml of toluene were heated at reflux with stirring for 3 hours. Suitable alternatives to the 1-chloro-3-methyl-2-butene used herein are any other 1-substituted primary allylic system with a leaving group at carbon #1

(e.g., 1-iodo-3-methyl-2-butene, 3-methyl-2-buten-1-ol, or the p-toluenesulfonyl ester of 3-methyl-2-buten-1-ol). Other higher alkyl functions in lieu of the methyl groups provide similarly suitable substrates. The reaction mixture (dark brown in color) was cooled to room temperature and the solvent was removed under reduced pressure. The residue, a dark brown oil, was flash chromatographed on silica gel using methylene chloride as eluent. Fractions were analyzed by TLC (silica: CH₂CI₂), pooled and the solvent evaporated under reduced pressure. The crude product was recrystallized from aqueous methanol to yield white crystals (801 mg, 62%) mp 149-150ºC.1H-NMR: 1.4 (s, 6H, C₈-CH₃'s), 1.8 (t, J = 6.8 Hz, 2H, C₆-CH₂), 2.2 (s, 3H, C₁₀-CH₃), 2.4

(d, J = 1.2 Hz, 3H, C₄-CH₃), 2.8 (t, J = 6.8 Hz, 2H, C₇-CH₂), 6.1 (q, J = 1.2 Hz, 1H, C₃-H), 7.1 (s, 1H, C₅- H). Some representative examples of substances obtainable by this route follow.

6,7-Dihydro-8,8-dimethyl-2H,8H-benzo[1,2-b;5,4-b']dipyran-2-one, 53% yield, mp 123-124ºC

6,7-Dihydro-4,8,8-trimethyl-2H,8H-benzo[1,2-b;5,4-b']dipyran-2-one, 51% yield, mp 174-175ºC

Suitable 8-H-benzodipyran-2-ones are those typified by the generic structure and consist of all the structurally possible variations described previously herein for the dihydrobenzodipyran-2-ones consistent with the presence of a double bond at carbon #6. Some examples of this class include but are not limited to:

2H,8H-Benzo[1,2-b;5,4-b']dipyran-2-one 4-Methyl-2H,8H-benzo[1,2-b;5,4-b']dipyran-2-one 8,8,10-Trimethyl-2H,8H-benzo[1,2-b;5,4-b^,]dipyran-2-one

4,8,10-Trimethyl-2H,8H-benzo[1,2-b;5,4-b']dipyran-2-one

8,8-Dimethyl-2H,8H-benzo[1,2-b;5,4-b']dipyran-2-one

4,8,8-Trimethyl-2H,8H-benzo-l,2-b;5,4-b']dipyran- 2-one

4,8,8,10-Tetramethyl-2H,8H-benzo[1,2-b;5,4-b']dipyran-2-one

These materials can be obtained as previously described by Rodighiero et al., Journal of Heterocyclic Chemistry, 24, 485-488 (1987). This process requires the preparation of an intermediate alkynyl ether with subsequent thermal cyclization to the indicated product.

An improv ed, one-step synthesi s of sel ected geminal ly-substituted analogs is disclosed in this application. An acid-catalyzed cycl ization of a propargyl al cohol, or alternativ ely, a halide or simil arly functionaliz ed tertiary-carbon al kyne with a l eaving group on the tertiary carbon seat, with a 7-hydroxy- coumarin yields 8-H-benzodipyran-2-ones in good yield without i solation of any intermediate species and without the use of el ev ated temperatures which induce char and tar f ormation. The substances available by this route fall into the structural subtype indi cated:

R₁ and R₂ are alkyl or

aryl

R₃ - variable function

(wherein R₂ and R₃ are methyl or higher al kyl

functions)

The equation for the preparation of these materials is:

R₁ & R₂ = alkyl or aryl; R₃ = variable function; X = halide, OH, or other leaving group. See "mixtures of products" for cases where C₈ bears K. Compounds obtainable in this fashion include, but are not limited to, the following:

8,8-Dimethyl-2H,8H-benzo[1,2-b;5,4-b']dipyran-2-one 4,8,8-Trimethyl-2H,8H-benzo[1,2-b;5,4-b'3dipyran- 2-one

4,8,8,10-Tetramethyl-2H,8H-benzo[1,2-b;5,4-b']dipyran-2-one

8,8,10-Trimethyl-2H,8H-benzo[1,2-b;5,4-b']dipyran- 2-one

4,8,10-Trimethyl-2H,8H-benzo[1,2-b;5,4-b'3dipyran-2-one

Acids which prove suitable in this process are p-toluene-sulfonic acid, other sulfonic acids compatible with the solvent system, and 10% sulfuric acid. Toluene, xylene and cumene are suitable solvent systems. Reflux times of 2-4 hours at temperatures of 90-110ºC provide yields of 45-60% when hydroxycoumarin concentrations are in the 5-10 mmole range.

The method of synthesis is exemplified by the following.

EXAMPLE 8: Preparation of 8,8,10-trimethyl-2H,8H- benzo[1,2-b;5.4-b']dipyran-2-one. A solution prepared from 5.0 mmol of 8-methyl- 7-hydroxycoumarin and 6.0 mmol of 2-methyl-3-butyn-2-ol in 40 ml of xylene containing 0.095 g (0.50 mmol) of p- toluenesulf onic acid was refluxed with magnetic stirring for 4 hours. The solvent was distilled off in vacuo and the oily contents of the flask chilled in an ice-salt water bath to induce crystallization. The white microneedles, 33% yield, were recrystallized from methanol to analytical purity, m.p. 105-107ºC (lit.

m.p. 106-107ºC). The ¹H-NMR spectrum was also identical to that reported, see P. Rodighiero et al., reference cited above.

When the original 7-hydroxycoumarin being employed is unsubstituted in both carbon #6 and carbon #8, mixtures of the linear isomers (benzo[1,2-b;5,4-b']dipyran-2-ones) and the angular isomer (benzo[1,2- b;3,4-b']dipyran-2-ones) result.

Ring cyclization syntheses of both the 6,7- dihydro compounds and the unsaturated compounds may giv e rise to such mixtures if the substitution on the parent molecule permits it. (- - - ) implies presence of an optional double bond (i.e., a linear 8-H-benzodipyran- 2-one)

If linear isomers are desi red this may be achieved in three ways :

(1) selection of a coumarin bearing an alkyl or aryl f unction in position #8 to preclude closure to the angular isomer.

(2) introducing an iodide atom onto the C₈ position which is subsequently removed by the cyclization process. This method has been used by Ahluwalia and colleagues [Monatsh. Chem., 111, 877

(1980)] to force closure in one direction;

(3) chromatographic separation of the mixture of linear and angular isomers when they do form in the reaction.

As illustrative of the latter method (chromatographic purification), a synthesis performed according to Method B is given.

EXAMPLE 9: Synthesis and Purification of 6.7-Dihydro-4,8,8-trimethyl-2H,8H-benzo[1,2-b;5,4-b']dipyran-2-one

(linear isomer) and 9,10-Dihydro-4.8.8-trimethyl-2H,8H-benzon.2-b;3.4-b']dipyran-2-one (angular isomer).

A solution prepared from 5.0 mmol of 4-methyl-7-hydroxy coumarin, 7.5 mmol of 2-methyl-3-buten- 2-ol, and 0.50 mmol of trif luoromethanesulf onic acid in 25 ml of xylene was heated with stirring at reflux for 4 hours and the solvent removed by distillation in vacuo. The crude tan solids were dissolved in a minimum amount of methylene chloride and charged to a silica gel column. With methylene chloride elutant the angular isomer elutes first [29% yield, white needles, m.p. 160-162ºC after methanol recrystallization.3 followed by the linear one [36% yield, white needles, m.p.

174-175ºC after methanol recrystallization]. The angular products can be recognized by the coupled A-B double-doublet (J = ca. 6-9 Hz) for protons on C5 and C₆ on the benzenoid ring: ¹H-NMR for angular isomer in CDCI₃ with key features for identification underlined,

1.36 (s, 6H, C₈-CH₃'s), 1.80 (t, 2H, C₉-CH₂, J = 6.5

Hz), 2.40 (d, 3H, C₄-CH₃, J = 1.2 Hz), 2.79 (t, 2H,

C₁₀-CH₂, J = 6.5 Hz), 6.12 (q, 1H, C₃-H, J - 1.2 Hz),

6.80 (d, 1H, C₆-H, J = 8.8 Hz), and 7,29 (d, 1H, C₅-H, J = 8.8 Hz).

Suitable 4^,,5'-dihydropsoralens are those typified by the generic structure having either hydrogen substitution or single alkyl (or aryl) group substitution at carbons 3, 4, 5, 8, 4', or 5' and no olefinic unsaturation in the furan ring (involving carbons 4' and 5'). Nitro, amino, alkyl oxy, aryloxy, aminomethyl, halo functions and other structural variations described previously herein for the 7-oxycoumarins, but consistent with the presence of a saturated, five-membered furan ring, are included where their placement at any carbons on the three- ring system does not alter the fundamental structure shown for the 4^,,5'-dihydropsoralens. Also specifically included in this class are analogs with 5- and/or 8-alkoxy substituents such as methoxy, ethoxy, i-propoxy, and n-propoxy derivatives.

Certain highly polar groups attached at carbon #5 (e.g., nitro, amino, sulfonic acid, and sulfonamide) markedly reduce the biological activity of the class.

Methods for the syntheses of the 4',5'-dihydropsoralens have been published, [see N. D. Heindel, N. Foster, and M. Choudhuri, J. Org. Chem., 48,

3817-3819 (1983); N. D. Heindel, N. Foster, and T.

Varkey, J. Heterocyclic Chem., 23, 1579-1582 (1986); and N. D. Heindel, M. Choudhuri, J. Ressner, and N. Foster, 22, 73-76 (1985)3. These techniques constitute suitable synthetic approaches to these compounds.

Following these general synthetic methods, representative analogs, which include but are not limited to, the substances indicated below may be prepared:

5-N-phthalimidomethyl-4',5'-dihydro-8-hydroxypsoralen

5-N-phthalimidomethyl-4',5'-dihydro-8-methoxypsoralen

3-nitro-4',5'-dihydro-8-methoxypsoralen 3-amino-4',5'-dihydro-8-methoxypsoralen

4,5',8-trimethyl-4',5'-dihydroρsoralen

4,4',8-trimethyl-4',5'-dihydropsoralen

5-nitro-4',5'-dihydro-8-methoxypsoralen 5-amino-4',5'-dihydro-8-methoxypsoralen

4',5'-dihydro-8-methoxypsoralen

4',5'-dihydro-5-methoxypsoralen

4,5',8-trimethyl-4'-aminomethyl-4',5'-dihydropsoralen

4,4',8-trimethy1-5'-aminomethyl-4',5'-dihydropsoralen

3,5-dinitro-4',5'-dihydro-8-methoxypsoralen

5-iodo-4',5'-dihydro-8-methoxypsoralen

These substances mentioned above, and others which correspond to the general structure, have beneficial photo-cosmetic and photochemotherapeutic effects. The effects can be outlined as follows. In combination with light (UVA), they are able to induce a regional melanogenesis (tanning), to inhibit the binding of epidermal growth factor (EGF), and to act as anti-prolif erative agents for a wide variety of cells which possess EGF receptors. It is these photo-activated properties -- and their derivative clinical effects -- which constitute the utility of these compounds. The compounds are usually diluted prior to use and may be administered orally, intravenously, parenterally or topically, i.e. in the form of a lotion or ointment. The pharmaceutical compositions according to the present invention are suitable for use in effecting photochemical sensitivity on the skin of a mammal, particularly a human patient or subject, and comprise an effective amount of a compound of the invention in association with a pharmaceutically-acceptable carrier or diluent. Such compositions are well-known in the art, and reference is made to U.S.

Pat. Nos. 4,124,598 and 4,130,568 for representative examples, the disclosures of which are incorporated by reference herein. The procedure for preparation of such compositions is totally conventional in the art. For oral treatment, the active ingredient is generally formulated in tablets or in gelatin capsules. In such case the diluent may, if desired, be eliminated, although it is generally present. For topical application, solutions or ointments may be prepared and employed. These may be formulated with any one of a number of pharmaceutical ly-acceptabl e carriers, as is well known in the art. Administration may be, f or exampl e, in the form of tabl ets, capsul es, powders, syrups, or sol utions, or as already stated in the f orm of ointments, creams, or solutions for topical use. For tabl et preparation, the usual tablet adj uvants such as cornstarch, potato starch, talcum, magnesium stearate, gelatin, lactose, gums, or the like may be

employed, but any other pharmaceutical tabl eting adj uvants may also be used, provided only that they are compatible with the active ingredient. In general, an oral dosage regimen will include about 5 mg. to about 50 mg. per kg. of body wei ght, with a dose in the neighborhood of about 5-10 mg. per kg. general ly being pref erred. Such administration and sel ection of dosage and unit dosage will of course have to be determined according to established medical principl es and under the supervision of the physician in charge of the therapy involved. For topical use, only an effective amount of the active ingredient per unit area is involved, and this will illustrativ ely be in the form of a one percent sol ution, s-uspension, or ointment thereof , ill ustrativ ely applied on the order of one-tenth milliliter per square centimeter, in association with a suitable carri er, e.g., ethanol , or other carrier of types already mentioned. A typical f ormulation for a phototherapeutic lotion (1% lotion) is:

propylene glycol 25 ml

triethanolamine 1 ml

water 12 ml

oleic acid 1.5 grams polyethylene glycol 400

monostearate 10.5 grams

silicon fluid DC-200 10 ml

carbopol 934,

2% mucilage 50 grams psoralen or new

therapeutic 1 gram

Historically the uncov ering of clinically-promising phototherapeutics has arisen from the serendipitous discov eries of folk medicine utilization of natural products. For laboratory products, however, extensive animal trials (for which few good bioassay models exist for diseases of human skin) were required bef ore human trials could begin. The correlations between such animal studies and human photobiology hav e not been uniformly promising. We have discovered that certain biochemical assays at the cellular level hav e preclinical predictive merit f or beneficial photopharmacology. These assays are based on sound pharmacological principals of agonist-receptor interactions. [J.D. Laskin, E. Lee, E. Yurkow, D. Laskin, M. Gal lo, Proc. Nat. Acad . Sc i . (U. S.A. ) 82: 6158 (1985) ] .

Prior to the discovery of the methodologies described herein, structure-activity studies on thedrugs disclosed and claimed in this patent would have required both extensive animal and human testing. The reason was simply that no suitable in vitro models

exi sted f or the pharmacological prescreening of. l arge numbers of candidate phototherapeutics. Obviously, the use of live animal models is a serious limitation with regard to laboratory working-time, expense, and humane considerations. Rapid in vitro assays utilizing cells in culture are, if available, greatly advantageous, in the testing of l arge numbers of potential phototherapeutics. These methods , if successful , can be used as prescreens f or active compounds and thereby reduce the number of substances which must eventually undergo the more systemic, in vivo, chronic toxicity/ efficacy testing.

The primary in vitro screen for the phototherapeutics described in this patent is based on our discov ery that benef icial phototherapeuti cs compete for binding in cells of epidermal origin with epidermal growth factor (EGF) . Furthermore, the degree of effective competition in this EGF binding assay relates to the phototherapeutic eff ect of the test agent. [ J. Laskin, E. Lee, D. Laskin, M. Gall o, Proc. Nat. Acad . Sc i . (U.S.A.) 83 : 8211 (1986) 3

Epidermal growth factor is a low molecular weight polypeptide which binds to cell surface receptors and whi ch is known to be an important regul ator of growth in those cells which possess these particular cell surf ace receptors. Psoriasis, mycosis fungoides, eczema, cancer, and similar prolif erative diseases are often characterized by abnormal cell growth regulation whi ch may be related to the acti on of EGF on the cell s in question. Application of PUVA therapy to correct skin disorders, especially psoriasis, is one clinical expression of photochemotherapy. The use of the assay described herein is based on the observation that phototherapeutics are extremely potent inhibitors of binding of epidermal growth factor to cell surface receptors in mammalian cells including humans and that inhibition of this binding arrests the prolif erativ e disorder. This binding assay was perf ormed in the cel l culture laboratory.

Inhibition of EGF binding is dependent on dose of the phototherapeutic and on the quanta of light in the 320-400 nm wav el ength (ultrav iolet light A) . It is al so structure-dependent, that is, there is a direct correlation between those specific phototherapeutics currently used that are clinically active and their abil ity to inhibit the binding of epidermal growth factor to its receptor.

Representative examples of the compounds described and claimed in this patent, were tested in this assay for biological activity and found to be potent inhibitors of epidermal growth factor binding. Inhibition of EGF binding was rapid, dependent on concentration, and required light activation. These findings directly demonstrate that the newly synthesized compounds are potential phototherapeutics for human prolif erative diseases. A description of this assay follows.

The ability of the above compounds in the presence of ultraviolet light, to inhibit epidermal growth factor binding to its cell surface receptor is directly related to its phototherapeutic potential. To assay these compounds for this biological activity, human cells (HeLa) grown in vitro were used. Cells (1.8 × 10⁴/cm²) were inoculated into 5 cm culture dishes in growth medium consisting of Dulbecco' s modified Eagl e's medium suppl emented with 10% newborn calf serum. After 4-5 days at 37ºC in a humidified CO₂ incubator, the cell s were washed three times with 2 ml of phosphate-buff ered saline and then incubated with the different phototherapeutics in 2 ml of Earl e's salt sol ution suppl emented with 5.2 mM D-glucose/25 mM Hepes buff er , pH 7.2.

Control cultures were incubated in 2 ml of Earl e's salt solution in the absence of the test drugs.

After 30 minutes, the cells were then exposed to ultraviol et light (UVA, 320-400 nM) emitted from a bank of four BLB fluorescent light tubes (F40BL/Sylvania) placed approximately 10 cm above the cell culture plates. The incident light delivered onto the culture plates was 3.4 mW per cm² as measured with an

International Light UV-Radiometer and the cells

received 2.1 J/cm² of UVA light. After this light exposure, the cells were rinsed with phosphate-buffered saline and submitted for assay of epidermal growth factor binding. Phototherapeutic treated cells were then incubated for 2 hours at 4ºCwith 2 ml of binding buffer (Dulbecco's modified Eagle's medium/25 mM Hepes buffer, pH 7.2) containing 2 nM labeled epidermal growth factor (¹²⁵I-EGF, specific activity 200 Ci/g).

Nonspecific binding was determined by incubating separate plates of cells with buffer containing the radioligand and excess unlabeled epidermal growth factor (1 microgram/ml). The binding reaction of the radioligand to the cells was terminated by aspirating the binding buffer from the culture dishes and washing the cells four times with ice cold phosphate-buffered saline. The cells were then solubilized with 2 ml of

0.2 M NaOH and duplicate 0.5 ml aliquots were taken for gamma counting. Specific binding of epidermal growth factor to its receptor was calculated by subtracting non-specifically bound material from the total. Under the conditions of the assay, specific epidermal growth factor receptor binding represented 80% of the total bound to the cells. The assay may be performed on a variety of cells which possess EGF receptors.

As a specific example of this assay, HeLa cells were treated with 4 ,8-dimethyl-7-(propargyloxy) -coumarin, followed by ultraviolet light exposure and then by measurement for epidermal growth factor bind- ing. The data can be presented as a curv e of epidermal growth factor receptor binding to the cells as a percentage of untreated cells. Figure 1 is an example of ¹²⁵ I-BGF binding inhibition by 4, 8-dimethyl-7- (propargyloxy) coumarin) and ultraviolet light. The concentration inhibiting epidermal growth factor binding to the cells by 50% (IC₅₀) is determined from the curv e. This is shown in Table 1 for a variety of phototherapeutics. Note that each of the compounds tested were potent inhibitors of epidermal growth factor binding to the human cells. The IC₅₀ values are typically in the micromolar concentration range. In the absence of ultraviolet light, these compounds did not inhibit epidermal growth factor binding. Tabl e 1 also shows the. lack of biological activity of coumarin for comparison. This is a biologically inactive analog of the phototherapeutics described in this patent. Trioxsal en, methoxysalen and 5-methoxypsoralen and other phototherapeutics currently being used in the clinic show equivalent or higher (less potent) IC₅₀ values in this assay .

Table 1

Comparison of the Biological Activity of Novel Phototherapeutics Using Epidermal Growth Factor Binding

Inhibition Assay

Compounds** IC*₅₀

(Micromolar)

4,5',8-trimethylpsoralen 6.6

4',5'-dihydro-4,5',8-trimethylpsoralen 7.0 4',5'-dihydro-3-nitro-4,5',

8-trimethylpsoralen 24

4',5'-dihydropsoralen 11 4,8-dimethy1-7-hydroxycoumarin 60

4,8-dimethyl-7-(propargyloxy)coumarin 15

4,8-dimethyl-7-methoxycoumarin 20 4,8-dimethy1-7-butyloxycoumarin 25 6,7-dihydro-4,8,8,8,10-tetramethyl-2H,8H- benzo(l,2-b,5,4-b')dipyran-2-one 40 6,7-dihydro-4,10-dimethyl-2H,8H- benzo(1,2-b, 5,4-b')dipyran-2-one 7.4

6,7-dihydro-4,8,10-trimethyl-2H,8H- benzo(1,2-b, 5,4-b')dipyran-2-one 43

4,10-dimethyl-2H,8H- benzo(1,2-b, 5,4-b')dipyran-2-one 17 coumarin: >100***

*IC₅₀, concentration of each compound inhibiting ¹²⁵I-EGF binding to HeLa cells by 50%.

**Following treatment of the cells with phototherapeutic, they were pulsed with 2.1 J/cm² of ultraviolet light. ***Highest concentration tested.

Claims

We claim:

1. A photochemotherapeuti c compound of the f ormula

wherein

(i) n is zero, W is a (C_1-16) alkyl , alkenyl, or alkynyl linear or branched chain hydrocarbon, having no mor e than four O, N, or S atoms in or attached to the chain; or

(ii) n is 1, W is CR₂ , and R, R' , and R" ar e independently H or CH₃; or

(iii) n is 2 , W is CR₂ , and R, R' , and R" ar e independently H or CH₃; and

2. A compound of cl aim 1 in whi ch A, B, C, and D are independently selected from hydrogen, (C_1-4) alkyl, or halogen.

3. A compound of claim 2 in which at least two of A, B, C, and D are hydrogen.

4. A photochemotherapeutic compound of the formula

wherein W is a (C_1-16) alkyl , alkenyl, or

al kynyl linear or branched chain hydrocarbon, having no more than four O, N, or S atoms in or attached to the chain; and

R3, R4 , R5 , R6 and R8 are independently hydrogen, alkyl, aryl, nitro, amino, aminoalkyl, halogen, alkoxy, hydroxy, aryloxy, carboxy, haloalkyl, or haloal koxy.

5. A compound of claim 4 in which R3, R5 and R6 are hydrogen.

6. A compound of claim 5 in which W is a linear or branched (C_1-16) alkyl, alkenyl or alkynyl linear or branched chain hydrocarbon, having no more than four O, N, or S atoms in or attached to the chain.

7. A compound of claim 6 in which R4 and R8 are independently hydrogen, halogen, lower alkyl, amino, alkoxy, or aminoalkyl.

8. A compound of claim 7 in which W is a linear or branched chain (C_1-8) alkyl, alkenyl, or alkynyl, having no more than four O, N, or S atoms in or attached to the chain.

9. A compound of claim 8 in which R4 and R8 are independently hydrogen, (C_1-4) alkyl, or halogen.

10. A compound of claim 9 in which R4 is hydrogen or methyl.

11. A compound of claim 10 which is 4,8-dimethyl-7-(omega-carboxyheptyloxy) coumarin.

12. A compound of claim 10 which is 4,8-dimethy1-7-allyloxycoumarin.

13. A compound of claim 10 which is 4-methyl-8-iodo-7-[(2-methyl-3-buten-2-yl)oxy]coumarin.

14. A compound of claim 10 which is 7-[(2-octyn-1-yl)oxy3coumarin.

15. A compound of claim 10 which is 8-iodo-7-(propargyloxy)coumarin.

16. A compound of claim 10 which is 4-methyl-8-iodo-7-[(2-methyl-3-butyn-2-yl)oxy]coumarin.

17. A compound of claim 10 which is 4,8-dimethyl-7-ethoxycoumarin.

18. A compound of claim 10 which is 4,8-dimethy1-7-methoxycoumarin.

19. A compound of claim 10 which is 4,8- dimethyl-7-butyloxycoumarin.

20. A compound of claim 10 which is 4,8- dimethyl-7-propargyloxycoumarin.

21. A photochemotherapeutic compound of the formula

in which carbons 3, 4, 5, 6, 7, 8, and 10 are independently substituted with hydrogen, lower alkyl or alkoxy.

22. A compound of claim 21 which is 6,7-dihydro-8,8-dimethyl-2H,8H-benzo[1,2-b;5,4-b']dipyran-2-one.

23. A compound of claim 21 which is 6,7-dihydro-4,8,8-trimethyl-2H,8H-benzo[1,2-b;5,4-b']dipyran-2-one.

24. A compound of claim 21 which is 6,7-dihydro-4,8,8,10-tetramethyl-2H,8H-benzo[1,2-b;5,4-b']-dipyran-2-one.

25. A compound of claim 21 which is 6,7-dihydro-4,10-dimethyl-2H,8H-benzo[1,2-b;5,4-b']dipyran-2-one.

26. A method for producing a compound of claim 21 by palladium-catalyzed hydrogen transfer from a suitable hydrogen-donor molecule to the 6-7 double bond of an 8-H-benzodipyran-2-one.

27. A photochemotherapeutic compound of the formula

in which carbons 3, 4, 5 and 10 are substituted with hydrogen, alkyl, or alkoxy.

28. A method for producing 8,8-geminally-disubstituted compounds of claim 27 by direct, one-step condensation of an appropriately substituted 7-hydroxycoumarin with a propargyl alcohol or alcohol halide under acid-catalysis.

29. A compound of claim 27 which is 4,8,8,10-tetra-methyl-2H,8H-benzo[1,2-b;5,4-b'3dipyran-

2-one.

30. A compound of claim 27 which is 4,10- dimethyl-2H, 8H-benzo(1,2-b, 5,4-b-)dipyran-2-one.

31. A photochemotherapeutic compound of the formula

in which R1, R2, and R3 are selected from hydrogen. lower alkyl, and nitro.

32. A compound of claim 31 which is 4',5'-dihydro-4,5',8-trimethylpsoralen.

33. A compound of claim 31 which is 4',5'-dihydro-3-nitro-4,5',8-trimethyl-psoralen.

34. A compound of claim 31 which is 4',5'-dihydropsoralen.

35. A method for treating prolif erative skin disorders comprising the parenteral, oral, or topical administration of an effective amount of a compound of claims 1, 4, 21, 27 or 31.

36. A photochemotherapeutic formulation comprising an effective amount of a compound of claims 1,

4, 21, 27 or 31 in admixture with suitable carriers, stabilizers, or adjuvants.

37. A method for treating prolif erative disorders of the blood and bone marrow comprising

introducing a compound of claims 1, 4, 21, 27, or 31 into the blood or bone marrow and exposing the blood or bone marrow containing the compound to ultraviolet light.