5'-substituted, 4',5'-dihydropsoralen compounds (5) bearing tertiary amines (and salts thereof), quaternary ammonium moieties or organomercurial moieties are described. ##STR1## Also described are 2-substituted mercurimethyl-2-3-dihydro-benzofurans of forumla (7): ##STR2## Also reported are versatile direct syntheses through a hitherto unknown compounds such as 3-R-4,8-dimethyl-4',5'-dihydro-5'-bromomethylpsoralen or a 3-R-4,8-dimethyl-4', 5'-dihydro-5'-iodomethylpsoralen to prepare a structurally diverse array of partially reduced psoralens and benzofurans. The presence of a permanent ammonium charge in these psoralens precludes membrane passage and the mono-unsaturation precludes the cross-linking of nuclear DNA, thereby minimizing the mutagenic/carcinogenic side effects long associated with psoralen-derived therapies. The presence of a mercury functionality provides a reactive cell-binding group on these psoralens with unique cytotoxicity without light activation and an enhancement of cytotoxicity activity upon light activation. The invention also relates to These partially reduced and quaternized psoralens, amino-substituted psoralens, and mercurio psoralens display impressive pharmacology against PAM 212 keratinocytes, a model cell line employed as a test system to indicate epidermal cytotoxicity and cancer. The compounds of the invention also have antimicrobicidal activity useful in pharmacologic agents for mammals (e.g. the treatment of tuberculosis) as well as in controlling the growth of microorganisms on substrates and in aqueous systems.

FIELD OF THE INVENTION
 This invention relates to amino- and mercurio-substituted
 4',5'-dihydropsoralens and 2-substituted
 mercurimethyl-2-3-dihydro-benzofurans and their use as phototherapeutics.
 Methods for preparing the amino- and mercurio-substituted
 4',5'-dihydropsoralens and the 2-substituted
 mercurimethyl-2-3-dihydro-benzofurans via ring closure reactions and
 synthetic intermediates are also described.
 BACKGROUND OF THE INVENTION
 Linear fluorocoumarins, also known as psoralens, have been used in
 combination with ultraviolet light for centuries in cosmetics and for the
 treatment of proliferative skin diseases such as, for example, vitiligo,
 eczema, mycosis fungoides, and psoriasis. Terms such as
 photosensitization, photochemotherapy, photopheresis and PUVA (psoralens
 ultra violet A radiation) are commonly used to refer to such methods.
 Recently it was discovered that by modifying the administration of
 psoralen and ultraviolet light to an offending condition, psoralens can be
 used to treat cancer (e.g., T cell lymphoma), autoimmune diseases, and
 microbial infection.
 The basic structure of psoralen, with the ring numbering structure used
 throughout the specification, is shown below:
 ##STR3##
 All psoralens contain two photo-activatable functions (absorbing in the UVA
 range)--an aryl-conjugated unsaturated pyrone (the coumarin portion) and
 an aryl-conjugated vinyl ether (the furan portion). All of the
 commercially available psoralens are highly lipophilic, non-nitrogenous,
 uncharged small molecules with minimal water solubility. Commercial
 psoralens are used in over-the-counter cosmetic creams, prescription
 pharmaceuticals, and as investigational candidates for many of the uses
 described above. The commercial psoralens in cosmetic/medical use include
 methoxsalen (also known as xanthotoxin, 8-methoxypsoralen or 8-MOP),
 trisoralen (also called 4,5',8-trimethylpsoralen, TMP, or trioxsalen), and
 bergaptan (alternatively named 5-methoxypsoralen or 5-MOP).
 The phototherapeutic action of psoralens has been discussed for example, by
 J. E. Hearst, "Photochemistry of the Psoralens," Chemical Research in
 Toxicology, 2, 69, 1989 and T. F. Anderson and J. J. Vorhees, Annual
 Reviews of Pharmacol. and Toxicol., vol. 10, p. 177, 1982. According to
 these articles, the highly lipophilic psoralens penetrate the target
 cell's membrane, intercalate into nuclear DNA, and photo crosslink the
 double helix through bis-cyclobutanes generated from the 3,4-double bond
 and the 4',5'-double bond [see numbering shown above] to double bonds in
 DNA's pyrimidine bases. Thus, because the crosslinked DNA is unable to
 uncoil and function as a template for new gene expression, the target cell
 is rendered non-viable.
 A severe limitation to the acceptance of psoralen-based photochemotherapy
 or cosmetic skin pigment enhancement, however, is the risk of genetic
 mutations induced by DNA damage since the natural cellular level repair
 processes of bi-functional DNA-crosslinks are highly error-prone. Errors
 in cellular repair processes of true crosslinks translate to
 mutagenic/carcinogenic events and, in the clinical use of psoralens,
 represent a significant post-treatment risk of cancer. See, for example,
 R. S. Stern et al, "Cutaneous Squamous-cell Carcinoma in patients treated
 with PUVA," New England J. of Med., 1984, pp. 1156-116; R. S. Stern et al,
 "Malignant Melanoma in Patients Treated for Psoriasis with Methoxsalen and
 Ultraviolet A Radiation (PUVA)," New England Journal of Medicine, vol.
 336, 1997, pp 1041-1045; and W. L. Morrison et al. "Consensus Workshop of
 the Toxic Effects of Long-Term PUVA Therapy," Arch. Dermatol., vol. 134,
 1998, pp. 595-598.
 The use of nonlinear furocoumarins (known as angelicins) for the treatment
 of psoriasis and other skin diseases is taught, for example by U.S. Pat.
 No. 4,312,883. According to the patent, nonlinear furocoumarins are an
 effective photochemotherapeutic compounds that does not have the risks
 associated with psoralens. Nonlinear furocoumarins, however, are limited
 by their structural geometry, forming only non-crosslinked mono-adducts
 which have diminished mutagenic behavior. See, for example, R. S. Cole,
 "Repair of DNA Containing Interstrand Crosslinks in E. Coli," Proc. Nat.
 Acad. Sci., volume 70, 1973, p. 1064. Further, lipophilic linear
 psoralens, capable of forming only monoadducts, can be phototoxic to
 malignant cells. See J. VanDongen, N. D. Heindel et al., "Synthesis of
 Psoralen Analogs and Evaluation of their Inhibition of Epidermal Growth
 Factor Binding," J. Pharm. Sci., volume 80, No. 7, July 1991, pp. 686-689.
 Despite such risks, an alternative mechanism exists, not involving DNA, by
 which psoralens can act as phototoxins to a cell. A 22 kDa receptor
 protein present on psoralen-sensitive cells has been identified as a
 binding site for photo-activated psoralens. Binding a psoralen to this
 non-nuclear receptor follows UVA light activation of the psoralen and
 blocks subsequent binding of epidermal growth factor (EGF) to that
 receptor. The existence of this non-nuclear target has been described in
 J. D. Laskin et al., "A Possible Mechanism of Psoralen Phototoxicity Not
 Involving Direct Interaction with DNA," Proc. Nat. Acad. Sci., vol. 82,
 pp. 6158-6161, September 1985.
 U.S. Pat. Nos. 5,473,083 and 5,216,176 report that reduced and quaternized
 psoralens are valuable photo-activated therapeutics. Although promising as
 therapeutics, these dihydro quaternary compounds have often been extremely
 difficult to synthesize. Furthermore, the reported method of synthesis
 does not permit access to 5'-(N-pyridiniummethyl) psoralens.
 5'-N-pyridiniummethyl)psoralens had been found (in the related fully
 unsaturated psoralens) to be potent members (active at the lowest
 concentration levels) of the fully unsaturated psoralens. See Table 1,
 U.S. Pat. No. 5,216,176. Thus, a need exists for a new synthetic route
 suitable for 4',5'-dihydro psoralens bearing pyridinium, alkyl amino,
 alkyl ammonium, or other nitrogen-heterocyclic groups at the 5'-methyl
 locus.
 U.S. Pat. No. 5,473,083 reports the synthesis of 5'-bromomethyl and
 5'-quaternary ammonium psoralens (2, with T.dbd.Br or R.sub.3 N+). But, as
 Scheme 1 below shows, hydrogenolysis of the pendant leaving
 group-to-carbon bond, not hydrogenation of the 4',5'-double bond, is the
 dominant outcome when catalytic hydrogenation or exchange hydrogenation
 are attempted. This unfortunate consequence leads to the recovery of the
 parent psoralen.
 ##STR4##
 U.S. Pat. No. 5,356,929 discloses the preparation of less labile (to
 hydrogenolysis) tertiary aminomethylpsoralen. Reduction and subsequent
 methylation can produce methylated quaternary compounds in low overall
 yields (35-40%). This is shown in Scheme 2.
 ##STR5##
 Obviously, however, niether 5'-N-pyridiniummethyl
 4',5'-dihydropsoralens--nor any quaternary psoralens derived from aromatic
 heterocyclic amines--are available by this route. Furthermore, as
 mentioned above, hetero-aromatic quaternary amine groups have been shown
 to possess beneficial properties in fully unsaturated psoralens. Thus, a
 need exists for a facile synthetic route to manufacture such quaternary
 amine-containing 4',5'-dihydropsoralens.
 Manufacturing the commercial psoralen, trioxsalen (1)
 (4,5',8-trimethylpsoralen or TMP), involves alkene bromination in
 chloroform of the unsaturated allylic bond in
 4,8-dimethyl-6-allyl-7-acetoxycoumarin (3, R.dbd.H, R'=Ac). However, if
 the 7-hydroxyl is not acetylated prior to bromination of the allyl moiety,
 bromination on C#3 occurs simultaneously with addition of the bromine to
 the double bond yielding
 3-bromo-6-(2,3-dibromopropyl)-4,8-dimethyl-7-hydroxycoumarin (4, R.dbd.Br,
 R'=H).
 ##STR6##
 In the Kaufman process, the
 6-(2,3-dibromopropyl)-4,8-dimethyl-7-acetoxycoumarin (4, R.dbd.H, R'=Ac)
 is ring-closed by uncapping the 7-acetoxy with sodium ethoxide with
 concomitant cyclization and double dehydrobromination yielding TMP in 48%
 yield. See K. D. Kaufman, J. Org. Chem., 1961, 26, 117-121. This reaction
 involves the loss of two molar equivalents of HBr. Theoretically, this
 might occur in two separable distinct steps in which one HBr loss is
 followed by a second through a possible intermediate such as
 4,8-dimethyl-4',5'-dihydro-5'-bromomethyl-psoralen (5, R.dbd.H, T.dbd.Br).
 ##STR7##
 As noted above, this theoretically possible intermediate is the very target
 compound which could not be obtained despite numerous attempts by
 catalytic hydrogenation or exchange hydrogenation to reduce
 4,8-dimethyl-5'-bromomethylpsoralen to
 4,8-dimethyl-4',5'-dihydro-5'-bromomethylpsoralen; hydrogenolysis to (1)
 being the dominant outcome. Alas, careful duplication of Kaufman's
 synthesis revealed no trace of
 4,8-dimethyl-4',5'-dihydro-5'-bromomethyl-psoralen (5, T.dbd.Br), even as
 a potential transient. NMR analysis of commercially available TMP prepared
 by Kaufman's route similarly showed none of this
 4',5'-dihydro-5'-bromomethyl material as a contaminant. A ready synthesis
 of this and other valuable intermediates to prepare quaternary ammonium
 4',5'-dihydropsoralens remains as a currently unmet need in the art.
 SUMMARY OF THE INVENTION
 The invention provides a 5'-substituted, 4,8-dimethyl-4',5'-dihydropsoralen
 of formula (5):
 ##STR8##
 wherein
 R is hydrogen, a halogen, NO.sub.2, or CN;
 T is a halogen, NR.sup.1 R.sup.2, (N.sup.+ R.sup.1 R.sup.2 R.sup.3)X.sup.-,
 or HgR.sup.4 ;
 R.sup.1 and R.sup.2 are independently a C.sub.1 -C.sub.6 alkyl, or R.sup.1
 and R.sup.2 together with the nitrogen form a 5-8 member heterocyclic
 ring, or when T is (N.sup.+ R.sup.1 R.sup.2 R.sup.3)X.sup.-, R.sup.1 and
 R.sup.2 together with the nitrogen form a 5-8 member heterocyclic ring or
 heterocyclic aromatic ring;
 R.sup.3 is hydrogen, a C.sub.1 -C.sub.12 alkyl, or, when R.sup.1 and
 R.sup.2 together with the nitrogen form a heterocyclic aromatic ring,
 R.sup.3 is a double bond within the heterocyclic aromatic ring;
 X.sup.- is a halide;
 R.sup.4 is OC(O)(C.sub.1 -C.sub.6 alkyl), OC(O)(C.sub.1 -C.sub.6
 fluoroalkyl), or a halogen;
 with the proviso that the 5'-substituted, 4,8-dimethyl-4'5'-dihydropsoralen
 is not 4,8-dimethyl-5'-(N-pyridiniummethyl)-4',5'-dihydropsoralen iodide
 salt.
 The invention also relates to a process for preparing a 5'-substituted,
 4,8-dimethyl-4',5'-dihydropsoralen of formula (5):
 ##STR9##
 wherein R and T are as defined above. The process comprises the step of
 contacting a 4,8-dimethyl-6-allyl-7-hydroxycoumarin of the formula:
 ##STR10##
 with a cyclization reagent under conditions to form said 5'-substituted,
 4,8-dimethyl-4',5'-dihydropsoralen. When T is Br, the cyclization reagent
 is selected from N-bromosuccinimide. When T is I, the cyclization reagent
 is selected from N-iodosuccinimide, I.sub.2, ICl, and IBr. When T is
 HgR.sup.4, where R.sup.4 is R.sup.4 is OC(O)(C.sub.1 -C.sub.6 alkyl),
 OC(O)(C.sub.1 -C.sub.6 fluoroalkyl), or a halogen, the cyclization reagent
 is Hg(R.sup.4).sub.2.
 The invention also provides a process for preparing a
 3-R-4,8-dimethyl-4',5'-dihydro-5'-bromomethylpsoralen (5) having the
 formula:
 ##STR11##
 where T is Br. This method comprises the steps of brominating a compound of
 formula
 ##STR12##
 where R' is acetyl to form
 4,8-dimethyl-6-(2,3-dibromopropyl)-7-acetoxycoumarin (4)
 ##STR13##
 and cyclizing the resulting
 3-R-4,8-dimethyl-6-(2,3-dibromopropyl)-7-hydroxycoumarin to yield the
 compound (5).
 To prepare 5-N-aminomethyl substituted 4,8-dimethyl-4',5'-dihydropsoralen
 of the formula (5):
 ##STR14##
 where R is hydrogen, a halogen, NO.sub.2, or CN and T is NR.sup.1 R.sup.2
 or (N.sup.+ R.sup.1 R.sup.2 R.sup.3)X.sup.-, one method of the invention
 contacts a compound of formula (5) where T is Br or I with a secondary
 amine of the formula of HNR.sup.1 R.sup.2 or N.sup.+ R.sup.1 R.sup.2
 R.sup.3. In these amines R.sup.1 and R.sup.2 are independently a C.sub.1
 -C.sub.6 alkyl, or R.sup.1 and R.sup.2 together with the nitrogen form a
 5-8 member heterocyclic ring, or when T is (N.sup.+ R.sup.1 R.sup.2
 R.sup.3)X.sup.-, R.sup.1 and R.sup.2 together with the nitrogen form a 5-8
 member heterocyclic ring or a 6-member heterocyclic aromatic ring; R.sup.3
 is hydrogen, a C.sub.1 -C.sub.12 alkyl, or, when R.sup.1 and R.sup.2
 together with the nitrogen form a heterocyclic aromatic ring, R.sup.3 is a
 double bond within the heterocyclic aromatic ring; and X.sup.- is bromo or
 iodo.
 In another embodiment, the invention relates to a 2-substituted
 mercurimethyl-2-3-dihydrobenzofuran of the forumla (7):
 ##STR15##
 wherein T is a mercurial group HgR.sup.4 and R.sup.4 is OC(O)(C.sub.1
 -C.sub.6 alkyl), OC(O)(C.sub.1 -C.sub.6 fluoroalkyl), or a halogen.
 Preferably, R.sup.4 is OC(O)CH.sub.3, OC(O)CF.sub.3, Cl, Br, or I.
 The 2-substituted mercurimethyl-2-3-dihydrobenzofuran of forumla (7)
 according to the invention may be prepared by contacting 2-allylphenol
 with a cyclization reagent selected from Hg(R.sup.4).sub.2 where R.sup.4
 is OC(O)(C.sub.1 -C.sub.6 alkyl), OC(O)(C.sub.1 -C.sub.6 fluoroalkyl), or
 a halogen under conditions to form a 2-substituted
 mercurimethyl-2-3-dihydrobenzofuran. Accordingly, the invention also
 relates to a process for preparing a 2-substituted
 mercurimethyl-2-3-dihydrobenzofuran of forumla (7) as described above.
 The compounds of the invention have beneficial pharmaceutical properties
 and can be used alone or in pharmaceutical compositions used to treat a
 proliferative skin disorder and to treat microbial infections in a mammal
 by administering to the mammal an effective amount of a compound of the
 invention and then irradiating the mammal with ultraviolet light.
 Similarly, compounds of the invention and pharmaceutical compositions
 containing them may be used to treat a disease of the blood or bone marrow
 or to treat microbial infections in a mammal. Such a method comprising the
 steps of: obtaining cells from the blood or marrow of the mammal,
 introducing in vitro into the cells an effective amount of a compound
 according to the invention, exposing the cells containing the compound to
 ultraviolet radiation, and returning the cells to the blood or bone marrow
 of the mammal. For the mercurio-substituted compounds of the invention,
 the irradiation step is optional.
 The compounds of the invention also have anti-microbial properties and can
 be used to control the growth of microorganisms on substrates and in
 aqueous systems.
 DETAILED DESCRIPTION
 1. Compounds of the Invention
 This invention relates to 5'-substituted,
 4,8-dimethyl-4',5'-dihydropsoralens of the formula:
 ##STR16##
 In formula (5), the substituent R at the 3-position is hydrogen, a halogen,
 NO.sub.2, or CN. In a preferred embodiment, R is hydrogen, F, Br, I,
 NO.sub.2, or CN.
 The 5' substituent, T, may be a halogen, an amine of the formula NR.sup.1
 R.sup.2 or (N.sup.+ R.sup.1 R.sup.2 R.sup.3)X.sup.-, or a mercurial group
 HgR.sup.4. When T is a halogen, Br and I are preferred.
 When T is an amine of the formula NR.sup.1 R.sup.2, R.sup.1 and R.sup.2 are
 independently a C.sub.1 -C.sub.6 alkyl, or R.sup.1 and R.sup.2 together
 with the nitrogen form a 5-8 member heterocyclic ring. An alkyl, according
 to the invention, may be straight chain or branched, substituted or
 unsubstituted with common substituents such as hydroxyl, halo, nitro,
 sulfonyl, nitro, amino, etc. The heterocyclic ring may likewise be
 substituted or unsubstituted. Preferably, R.sup.1 and R.sup.2 are
 independently methyl, ethyl, hydroxyethyl, or propyl, or R.sup.1 and
 R.sup.2 together with the nitrogen form a heterocyclic ring selected from
 N-pyrrolidinyl, N-2-pyrrolidinonyl, N-pyrrolinyl, N-pyrazolidinyl,
 N-pyrazolinyl, N-pyrazolyl, N-imidazolidinyl, N-imidazolinyl,
 N-imidazolyl, N-oxazolidinonyl, N-piperidinyl, N-piperazinyl,
 N-morpholinyl, N-pyridinyl, N-4-(C.sub.1 -C.sub.4 alkyl)pyridinyl,
 N-hexamethyleneiminyl, N-heptamethyleneiminyl, N-quinolinyl or
 N-isoquinolyl. Most preferably, R.sup.1 and R.sup.2 are independently
 methyl, 2-hydroxyethyl, or propyl, or R.sup.1 and R.sup.2 together with
 the nitrogen form a heterocyclic ring selected from N-morpholinyl,
 N-pyridinyl, N-4-ethylpyridinyl, N-methylpyridinyl, or N-quinolinyl.
 In dihydropsoralens of formula (5) when T is (N.sup.+ R.sup.1 R.sup.2
 R.sup.3)X.sup.-, R.sup.1 and R.sup.2 together with the nitrogen form a 5-8
 member heterocyclic ring or heterocyclic aromatic ring, as just discussed.
 When T is (N.sup.+ R.sup.1 R.sup.2 R.sup.3)X.sup.-, R.sup.1 and R.sup.2
 have the same preferred embodiments as when T is NR.sup.1 R.sup.2. R.sup.3
 is hydrogen, a C.sub.1 -C.sub.12 alkyl, or, when R.sup.1 and R.sup.2
 together with the nitrogen form a heterocyclic aromatic ring, R.sup.3 is a
 double bond within the aromatic ring and X.sup.- is a halide. Preferably,
 R.sup.3 is hydrogen, methyl, ethyl, or dodecyl and X.sup.- is a bromide or
 iodide. Most preferably, R.sup.3 is hydrogen, methyl, or dodecyl.
 As mentioned, the 5'-substituent T may also be a mercurial group HgR.sup.4.
 In that mercurial group, R.sup.4 is OC(O)(C.sub.1 -C.sub.6 alkyl),
 OC(O)(C.sub.1 -C.sub.6 fluoroalkyl), or a halogen. Preferably, R.sup.4 is
 OC(O)CH.sub.3, OC(O)CF.sub.3, Cl, Br, or I.
 Examples of dihydropsoralen compounds of formula 5 are:
 4,8-Dimethyl-5'-(bromomethyl)-4',5'-dihydropsoralen;
 4,8-Dimethyl-5'-(iodomethyl)-4',5'-dihydropsoralen;
 4,8-Dimethyl-5'-(N-pyridiniummethyl)-4',5'-dihydropsoralen bromide salt;
 4,8-Dimethyl-5'-(N-4-ethylpyridiniummethyl)-4',5'-dihydropsoralen bromide
 salt;
 4,8-Dimethyl-5'-(N-quinoliniummethyl)-4',5'-dihydropsoralen bromide salt;
 4,8-Dimethyl-5'-(N,N,N-trimethylammonium methyl)-4',5'-dihydropsoralen
 bromide salt;
 3-Bromo-4,8-Dimethyl-5'-(bromomethyl)-4',5'-dihydropsoralen;
 3-Bromo-4,8-Dimethyl-5'-(iodomethyl)-4',5'-dihydropsoralen;
 3-Bromo-4,8-dimethyl-5'-(N-pyridiniummethyl)-4',5'-dihydropsoralen, bromide
 salt;
 3-Bromo-4,8-dimethyl-5'-(N-4-ethylpyridiniummethyl)-4',5-dihydropsoralen,
 bromide salt;
 3-Bromo-4,8-dimethyl-5'-(N-pyridiniummethyl)-4',5'-dihydropsoralen, iodide
 salt;
 3-Bromo-4,8-dimethyl-5'-(N-quinoliniummethyl)-4',5'-dihydropsoralen,
 bromide salt;
 3-Bromo-4,8-dimethyl-5'-(N,N,N-trimethylammoniummethyl)-4',5'-dihydropsoral
 en, bromide salt;
 3-Bromo-4,8-dimethyl-5'-(N,N,N-trimethylammoniummethyl)-4',5'-dihydropsoral
 en, iodide salt;
 3-Nitro-4,8-dimethyl-5'-(bromomethyl)-4',5'-dihydropsoralen;
 3-Nitro-4,8-dimethyl-5'-(N-pyridiniummethyl)-4',5'-dihydropsoralen, bromide
 salt;
 3-Nitro-4,8-dimethyl-5'-(N-4-ethylpyridiniummethyl)-4',5'-dihydropsoralen
 bromide salt;
 3-Nitro-4,8-dimethyl-5'-(N-quinoliniummethyl)-4',5'-dihydropsoralen,
 bromide salt;
 3-Nitro-4,8-dimethyl-5'-(N,N,N-trimethylammonium
 methyl)-4',5'-dihydropsoralen bromide salt;
 3-Cyano-4,8-dimethyl-5'-(bromomethyl)-4',5'-dihydropsoralen;
 3-Cyano-4,8-dimethyl-5'-(iodomethyl)-4',5'-dihydropsoralen;
 3-Cyano-4,8-dimethyl-5'-(N-pyridiniummethyl)-4',5'-dihydropsoralen, bromide
 salt;
 3-Cyano-4,8-dimethyl-5'-(N-pyridiniummethyl)-4',5'-dihydropsoralen, iodide
 salt;
 3-Cyano-4,8-dimethyl-5'-(N-4-ethylpyridiniummethyl)-4',5'-dihydropsoralen
 bromide salt;
 3-Cyano-4,8-dimethyl-5'-(N,N,N-trimethylammoniummethyl)-4',5'-dihydropsoral
 en bromide salt;
 3-Cyano-4,8-dimethyl-5'-(N,N,N-trimethylammoniummethyl)-4',5'-dihydropsoral
 en iodide salt;
 3-Fluoro-4,8-dimethyl-5'-(bromomethyl)-4',5'-dihydropsoralen;
 3-Fluoro-4,8-dimethyl-5'-(iodomethyl)-4',5'-dihydropsoralen;
 3-Fluoro-4,8-dimethyl-5'-(N-pyridiniummethyl)-4',5'-dihydropsoralen,
 bromide salt;
 3-Fluoro-4,8-dimethyl-5'-(N-4-ethylpyridiniummethyl)-4',5'-dihydropsoralen
 bromide salt;
 3-Fluoro-4,8-dimethyl-5'-(N-quinoliniummethyl)-4',5'-dihydropsoralen,
 bromide salt;
 3-Fluoro-4,8-dimethyl-5'-(N,N,N-trimethylammonium
 methyl)-4',5'-dihydropsoralen bromide salt;
 3-Fluoro-4,8-dimethyl-5'-iodomethyl-4',5'-dihydropsoralen;
 3-Iodo-4,8-dimethyl-5'-iodomethyl-4',5'-dihydropsoralen;
 3-Fluoro-4,8-dimethyl-5'-acetomercurimethyl-4',5'-dihydropsoralen;
 3-Iodo-4,8-dimethyl-5'-acetomercurimethyl-4',5'-dihydropsoralen;
 4,8-Dimethyl-5'-acetomercurimethyl-4',5'-dihydropsoralen;
 4,8-Dimethyl-5'-trifluoroacetomercurimethyl-4',5'-dihydropsoralen;
 4,8-Dimethyl-5'-chloromercurimethyl-4',5'-dihydropsoralen;
 4,8-Dimethyl-5'-iodomercurimethyl-4',5'-dihydropsoralen;
 3-Cyano-4,8-dimethyl-5'-acetomercurimethyl-4',5'-dihydropsoralen;
 3-Fluoro-4,8-dimethyl-5'-acetomercurimetbyl-4',5'-dihydropsoralen;
 4,8-Dimethyl-5'-(N,N-diethanolaminomethyl)-4',5'-dihydropsoralen;
 4,8-Dimethyl-5'-(N,N-dimethylaminomethyl)-4',5'-dihydropsoralen hydroiodide
 salt;
 4,8-Dimethyl-5'-(N-morpholinomethyl)-4',5'-dihydropsoralen; and
 4,8-Dimethyl-5'-(N-2,6-dimethylmorpholinomethyl)-4',5'-dihydropsoralen.
 The photochemotherapeutic and/or chemotherapeutic compounds of the
 invention also include physiologically acceptable salts of the compounds
 of formula 5. Preferred physiologically acceptable salts are acid-addition
 salts. Common acceptable acid-addition salts include but are not limited
 to hydroiodic and hydrochloric acid salts, oxalate salts and tartrate
 salts.
 The invention also relates to a 2-substituted
 mercurimethyl-2-3-dihydrobenzofuran of the forumla (7):
 ##STR17##
 wherein T is a mercurial group HgR.sup.4 wherein R.sup.4 is OC(O)(C.sub.1
 -C.sub.6 alkyl), OC(O)(C.sub.1 -C.sub.6 fluoroalkyl), or a halogen.
 Preferably, R.sup.4 is OC(O)CH.sub.3, OC(O)CF.sub.3, Cl, Br, or I. A
 2-substituted mercurimethyl-2-3-dihydro-benzofuran according to the
 invention also includes physiologically acceptable salts of the compounds
 of formula 7. Preferred physiologically acceptable salts are acid-addition
 salts as described above.
 2. Preparation of Compounds of the Invention
 The compounds of the invention may be prepared according to general
 synthetic procedures. The examples below demonstrate the general synthetic
 procedures, as well as the specific preparation, of 5'-substituted,
 4',5'-dihydropsoralen and 2-substituted
 mercurimethyl-2-3-dihydro-benzofuran compounds according to this
 invention. The examples are illustrative, and are not intended to limit in
 any manner, the claimed invention.
 Preparation of T.dbd.Bromo Derivatives via Allyl Bromination
 One embodiment of the invention relates to a
 3-R-4,8-dimethyl-4',5'-dihydro-5'-bromomethylpsoralen (5, T.dbd.Br) having
 the formula:
 ##STR18##
 This process comprises the steps of brominating a compound of formula (3,
 R'=Ac)
 ##STR19##
 to form 4,8-dimethyl-6-(2,3-dibromopropyl)-7-acetoxycoumarin (4, R.dbd.H,
 R'=Ac) and
 ##STR20##
 cyclizing to form a mono-dehydrohalogenated dihydro psoralen intermediate
 (5, R.dbd.H, T.dbd.halo). Ring closure may be effected by the use of
 anhydrous sodium carbonate in dry acetone. The 7-acetoxycoumarin (4,
 R.dbd.H, R'=Ac) should first be deacetylated with sodium borohydride
 before cyclization to (5, R.dbd.H, T.dbd.Br). Freeing the 7-hydroxyl from
 the 7-acetoxy is not a trivial achievement. As shown in Example 8 below,
 treatment with sodium borohydride successfully freed the 7-hydroxyl from
 the 7-acetoxy compound. Attempted deblocking by promoting an ester
 interchange with 0.2% p-toluenesulfonic acid in methanol at reflux for 4
 hours returned starting material. Aqueous base (1% NaOH in water) at
 reflux for 7 days gave a complex inseparable yellow-brown oily mixture.
 When the 7-acetoxy was slurried with anhydrous silica gel in refluxing
 methanol for 4 hours it was recovered unchanged.
 The unacetylated compound (4, R.dbd.Br, R'=H), of course, needs no
 unblocking and cyclizes directly with anhydrous sodium carbonate in
 acetone in high yield to (5, T.dbd.Br). Careful examination of the .sup.1
 H-NMR spectra revealed that cyclizations of (4) yielded 4:1 mixtures of
 the desired psoralen (5, R.dbd.H or Br, T.dbd.Br) and the six-membered
 bromobenzodipyranone isomer (6, R.dbd.H or Br). Chromatographic separation
 was possible, as noted in Example 8, but, as discussed below, was
 unnecessary for subsequent amination.
 ##STR21##
 Attempting direct bromination-addition of the double bond without
 concomitant bromination-substitution at C#3 failed. Lower reaction
 temperatures, more dilute bromine concentrations, and solvent variations
 were unsuccessfully explored. Unless the phenolic hydroxy was first
 acetylated, concomitant bromination at C#3 could not be avoided. However,
 access to such ring-brominated dihydro quaternary psoralens is a marked
 synthetic advantage since bromo-substituted psoralens have recently shown
 promise as photo-viricides (see S. Rai)
 Preparation of T.dbd.Br and I Derivatives via Allyl Cyclization
 In another embodiment, 5'-substituted, 4,8-dimethyl-4',5'-dihydropsoralens
 of formula 5 may be prepared by contacting a
 4,8-dimethyl-6-allyl-7-hydroxycoumarin of the formula:
 ##STR22##
 with a cyclization reagent under conditions to form a 5'-substituted,
 4,8-dimethyl-4',5'-dihydropsoralen, 5 as represented by the following
 reaction Scheme 3:
 ##STR23##
 The cyclization reagent used in a process of the invention differs for
 different substituents T. For example, when T is Br, the cyclization
 reagent is selected from N-bromosuccinimide and when T is I, the
 cyclization reagent is selected from N-iodosuccinimide, I.sub.2, ICl, and
 IBr. Preferred processes are shown in Scheme 4 and described in more
 detail below and in the examples.
 ##STR24##
 Because as noted above, the bromine facilitated cyclization route on the
 4,8-dimethyl-6-allyl-7-hydroxycoumarin 3, (R'=H) lead to benzopyran
 formation in addition to the desired dihydrofuran formation, alternative
 electrophiles were explored in the halocyclization reaction to form the
 dihydrofuran portion of the psoralen ring with higher regioselectivity.
 Regiospecific bromination-cyclization was achieved by use of three molar
 equivalents of NBS. This synthesis of
 3-bromo-4,8-dimethyl-5'-bromomethyl-4',5'-dihydropsoralen (5, R.dbd.Br,
 T.dbd.Br) from 4,8-dimethyl-6-allyl-7-hydroxycoumarin (3, R.dbd.H, R'=H),
 used 3 moles of N-bromosuccinimide (NBS) to selectively brominate the
 3-position and ring close to the dihydrofurocoumarin in high yields with
 the absence of the benzopyran formation, a significant advantage.
 Variations in temperature (from -80.degree. C. to 25.degree. C.) and the
 presence or absence of light did not affect this reaction. One mole of NBS
 in chloroform or one mole of pyridinium hydrobromide perbromide in acetic
 acid selectively substituted on 4,8-dimethyl-6-allyl-7-hydroxycoumarin (3,
 R.dbd.H , R'=H) to give 3-bromo-4,8-dimethyl-6-allyl-7-hydroxycoumarin (3,
 R.dbd.Br, R'=H), previously generated by the addition of bromine from one
 mole of Br.sub.2. No ring closures occurred under these conditions.
 Alternate routes to the
 4,8-dimethyl-4',5'-dihydro-5'-halomethylfurocoumarin ring system (some
 with 3-position substitutions) were also developed. One preferred route
 involved the use of N-iodosuccinimide (NIS), to selectively ring close
 4,8-dimethyl-6-allyl-7-hydroxycoumarin (3, R.dbd.H, R'=H), to the
 4,8-dimethyl-5'-iodomethyl-4',5'-dihydropsoralen (5, R.dbd.H, T.dbd.I) in
 a single step with the absence of benzopyran formation and absence of
 halogenation at the 3-position. The
 3-iodo-4,8-dimethyl-5'-iodomethyl-4',5'-dihydropsoralen (5, R.dbd.I,
 T.dbd.I) may be generated by reacting
 4,8-dimethyl-5'-iodomethyl-4',5'-dihydropsoralen (5, R.dbd.H, T.dbd.I)
 with ICl in acetic acid at 50.degree. C. overnight.
 A second preferred route to the
 4,8-dimethyl-5'-iodomethyl-4',5'-dihydropsoralen (5, R.dbd.H, T.dbd.I) by
 tin (IV) chloride assisted iodocyclization was based on the synthesis of
 the 5-iodobenzofurans from 2-allyl phenol. The reaction occurs
 regioselectively in a 5-exo-Trig type cyclization. Applied to psoralen
 synthesis, this route gave the similar advantage of the NBS and NIS
 cyclizations in that reactions proceed in high yield with no competing
 benzopyran formation. Additional iodinating reagents used to successfully
 ring-close include iodine/NaHCO.sub.3 /CH.sub.3 CN, ICl/CH.sub.2 Cl.sub.2
 (prone to halogen exchange and to multiple reaction products) and
 IBr/CH.sub.2 Cl.sub.2 (also prone to multiple products). Attempts to ring
 close using N-chlorosuccinimide failed.
 The bromine- and iodine-based allylcyclization reactions of the invention
 offer new methods for the synthesis of readily substituted
 dihydropsoralens to form tertiary amines or quats.
 Nitration at the at C#3 of psoralen (5, R.dbd.H, T.dbd.Br) [in mixture with
 its six-membered bromobenzodipyranone (6, R.dbd.H)] was achieved with
 concentrated nitric acid in glacial acetic acid giving
 3-nitro-4,8-dimethyl-5'-bromomethyl-4',5'-dihydropsoralen (5,
 R.dbd.NO.sub.2, T.dbd.Br) and nitration product(s) of (6, R.dbd.H).
 Through recrystallization of the mixture, pure (5, R.dbd.NO.sub.2,
 T.dbd.Br) was obtained in 66% yield. One can note that nitration of (5,
 R.dbd.H, T.dbd.Br) at the C#5 seems to not proceed. Finally, the bromide
 salt (5, R.dbd.NO.sub.2, T.dbd.pyridinium Br salt) was prepared in 69%
 yield by refluxing (5, R.dbd.NO.sub.2, T.dbd.Br) in anhydrous pyridine.
 While the nitro and the bromo substituents at C#3 of psoralen (5,
 R.dbd.NO.sub.2 or Br, T.dbd.Br) were introduced directly from coumarin or
 psoralen precursors, the cyano and fluoro group were incorporated at an
 earlier stage in the synthetic design. See Scheme 5.
 ##STR25##
 As shown in Example 12, 3-Cyano-4,8-dimethyl-6-allyl-7-hydroxycoumarin (3,
 R.dbd.CN, R'=H) was prepared in 3 steps from
 2,4-dihydroxy-3-methylacetophenone through a Knoevenagel condensation with
 ethyl cyanoacetate as the nitrile carrier (42% overall yield). Then, in a
 similar fashion to the preparation of the 3-bromopsoralen derivatives,
 their 3-cyano counterparts were obtained by initial bromination of the
 allyl double bond [92% of (4, R.dbd.CN, R'=H)] and subsequent cyclization
 with sodium carbonate in acetone to a 78% yield of a 90:10 mixture of
 3-cyano-5'-bromomethyldihydropsoralen (5, R.dbd.CN, T.dbd.Br) and its
 pyrano isomer (6, R.dbd.CN). Pure target compound (5, R.dbd.CN,
 T.dbd.pyridinium, Br salt) was obtained in 88% yield from the above 90:10
 mixture. The 3-cyano-4,8-dimethyl-5'-iodomethyl -4',5'-dihydropsoralen (5,
 R.dbd.CN, T.dbd.I) could be obtained in 71% yield from the NIS ring
 closure of 3-cyano-4,8-dimethyl-6-allyl-7-hydroxycoumarin (3, R.dbd.CN,
 R'=H). Ring closure is also possible by the I.sub.2 /SnCl.sub.2 route
 described above.
 Similarly the fluorine atom was introduced during the formation of the
 pyrone moiety through a Pechmann reaction with ethyl 2-fluoro-acetoacetate
 as the fluorine carrier. Thus,
 3-fluoro-4,8-dimethyl-6-allyl-7-hydroxycoumarin (3, R.dbd.F, R'=H) was
 prepared in 3 steps from 2-methylresorcinol (36% overall yield). Then,
 ring closure was achieved in two steps in a 66% overall yield following
 the same route employed with the 3-cyano derivatives giving a mixture
 89:11 of (5, R.dbd.F, T.dbd.Br) and its pyrano isomer (6, R.dbd.F) in
 favor of the expected psoralen. Finally, this 89:11 mixture was reacted
 with refluxing anhydrous pyridine affording pure target compound (5,
 R.dbd.F, T.dbd.pyridinium, Br salt) in 73% yield. Alternatively, the
 3-fluoro-6-allyl-7-hydroxycoumarin (3, R.dbd.F, R'=H) could be ring closed
 with NIS in 91% yield to give
 3-fluoro-4,8-dimethyl-4',5'-dihydro-5'-iodomethylpsoralen (5, R.dbd.F,
 T.dbd.I). The resulting
 3-fluoro-4,8-dimethyl-4',5'-dihydro-5'-iodomethylpsoralen was reacted with
 pyridine to form the pyridinium iodine salt (5, R.dbd.F, T.dbd.pyridinium
 iodide). Ring closure of 3-fluoro-6-allyl-7-hydroxycoumarin (3, R.dbd.F,
 R'=H) with mercury acetate in ethanol gave the
 5'-acetomercurimethyl-4,8-dimethyl-4',5'-dihydropsoralen (5, R.dbd.F,
 T.dbd.HgOAc) in 92% yield.
 All of the above allyl cyclization pathways leading to various
 3-substituted dihydro psoralens--proceed via highly reactive
 5'-bromomethyl-4',5'-dihydro psoralens (5, T.dbd.Br) or the
 5'-iodomethyl-4',5'-dihydro psoralens (5, T.dbd.I). A very useful
 expansion of that cyclization to other affinity-labeling psoralens is now
 at hand.
 Preparation of T.dbd.HgR.sup.4 X derivatives via Allyl Cyclization
 The invention also relates to a process for obtaining
 3-R-4,8-dimethyl-4',5'-dihydro-5'-R.sup.4 mercurimethylpsoralens (5,
 T.dbd.HgR.sup.4). According to the invention,
 3-R-4,8-dimethyl-4',5'-dihydro-5'-R.sup.4 mercurimethylpsoralens may be
 prepared by contacting a 4,8-dimethyl-6-allyl-7-hydroxycoumarin of the
 formula:
 ##STR26##
 with a cyclization reagent Hg(R.sup.4).sub.2 under conditions to form said
 5'-substituted, 4,8-dimethyl-4',5'-dihydropsoralen, where R is hydrogen, a
 halogen, NO.sub.2, or CN; and T is HgR.sup.4. R.sup.4 is OC(O)(C.sub.1
 -C.sub.6 alkyl), OC(O)(C.sub.1 -C.sub.6 fluoroalkyl), or a halogen, the
 cyclization reagent is Hg(R.sup.4).sub.2. Preferred substituents R and
 R.sup.4 are the same as those described above. As shown in Example 29,
 mercury acetate effectively ring closed the
 3-cyano-6-allyl-7-hydroxycoumarin (3, R.dbd.CN, R'=H) in 80% yield to
 3-cyano-4,8-dimethyl-5'-acetomercurimethyl-4',5'-dihydropsoralen (5,
 R.dbd.CN, T.dbd.HgOAc). Mercury chloride, however, even with heating did
 not yield a cyclized product.
 Iodomercurimethyl compounds may be prepared by anionic exchange with other
 3-R-4,8-dimethyl-4',5'-dihydro-5'-Xmercurimethylpsoralens (e.g. 5,
 T.dbd.HgR.sup.4 wherein R.sup.4 =chloride, trifluoroacetate, acetate) with
 iodide. Preparing iodomercurimethyl compounds in this manner represents a
 further embodiment of the invention. Suitable iodide sources include, for
 example, potassium iodide, sodium iodide, and the like. Suitable
 mercury-anion derivatives include, for example, HgX with X being aceto,
 trifluoroaceto, and chloro.
 While all the processes and synthetic procedures have been described using
 4,8-dimethyl-substituted coumarins, considerable structural variation is,
 in fact, possible. The classic Pechmann synthesis (R. Elderfield,
 "Heterocyclic Compounds", II, Wiley & Sons, N.Y., 1951, p. 181 and 251)
 and the versatile Kostanecki reaction (C. Hauser et al., "Organic
 Reactions", Vol. 8, 1954, p. 91) are capable of generating a wider variety
 of different 7-hydroxycoumarins to be employed as starting materials.
 These 7-hydroxycoumarins may be O-allylated, subjected to the Claisen
 rearrangement, and may be employed as other examples of .sup.6
 -allyl-7-hydroxycoumarins (viz. 3, R'=H) in the cyclization methods of
 leading to the 4',5'-dihydropsoralens.
 Furthermore, the 7-hydroxycoumarins prepared by Pechmann's or Kostanecki's
 methods may be nitrated, halogenated, sulphonated, or chloromethylated
 prior to be subjected to ring closure (i.e. the 3 to 5 pathways). The
 readily available structural variants of 5 may contain at the
 dihydropsoralen ring carbon loci numbered 3, 4, 5, 4', and 8, alkyls
 (e.g., methyl, ethyl, etc.); aryls (e.g., phenyl); alkoxys (e.g., methoxy,
 ethoxy, etc); aryloxys (e.g., phenoxy); halo groups (F, Cl, Br, I); nitro
 groups; sulfonyl groups; haloalkyls (e.g., chloromethyl, trifluoromethyl,
 etc.); sulfonamido groups; amino groups; and alkylamino groups.
 Accordingly, in another embodiment the invention relates to a process for
 preparing a 5'-substituted,-4',5'-dihydropsoralen substituted at the 3, 4,
 5, 8, or 4' position of formula (8):
 ##STR27##
 wherein T is a halogen, or HgR.sup.4 wherein R.sup.4 is OC(O)(C.sub.1
 -C.sub.6 alkyl), OC(O)(C.sub.1 -C.sub.6 fluoroalkyl), or a halogen. The
 process comprises contacting a 6-allyl-7-hydroxycoumarin substituted at
 the 3, 4, 5, or 8 position of the formula:
 ##STR28##
 with a cyclization reagent under conditions to form said 5'-substituted,
 4',5'-dihydropsoralen substituted at the 3, 4, 5, 8, or 4' position. When
 T is Br, the cyclization reagent is selected from N-bromosuccinimide; when
 T is I, the cyclization reagent is selected from N-iodosuccinimide,
 I.sub.2, ICl, and IBr; and when T is HgR.sup.4, the cyclization reagent is
 Hg(R.sup.4).sub.2 wherein R.sup.4 is as defined above.
 Preparation of T.dbd.Tertiary Amino and Quaternary Ammonium Derivatives
 As shown in the Examples, tertiary amino and quaternary ammonium
 derivatives of 5'-substituted, 3-R-4,8-dimethyl-4',5'-dihydro-psoralens
 (5) may be prepared by displacing the bromine or iodine (5, T.dbd.Br or I)
 with an appropriate secondary or tertiary amine. As shown in Example 30,
 derivatization with pyridine (5, R.dbd.CN, T.dbd.pyridinium, I salt)
 occurred in 78% yield from the iodomethyl dihydropsoralen (5, R.dbd.CN,
 T.dbd.I) derivative.
 As discussed above the unacetylated compound (4, R.dbd.Br, R'=H) cyclizes
 directly with anhydrous sodium carbonate in acetone in high yield to (5,
 T.dbd.Br) but also contained the six-membered bromobenzodipyranone isomer
 (6, R.dbd.H or Br). In general, these cyclizations of (4) yielded 4:1
 mixtures of the desired psoralen (5, R.dbd.H or Br, T.dbd.Br) and the
 six-membered bromobenzodipyranone isomer (6, R.dbd.H or Br). Though
 possible, chromatographic separation before amination was unnecessary.
 Because (6) contains a bromine on a secondary carbon and (5) is a primary
 --CH.sub.2 --Br system, amination proceeds more rapidly on (5, T.dbd.Br)
 and the only product isolated, under the amination conditions employed, is
 the quaternary psoralen (5, T.dbd.N+, bromide salt).
 Some limitations were seen in that the quaternary ammonium derivative from
 triethylamine did not form and attempts to react the
 5'-iodomethyl-4,8-dimethyl-4',5'-dihydrofurocoumarin (5, R.dbd.H, T.dbd.I)
 with dodecyl amine did not prove successful. Trimethylpsoralen (1) was
 recovered when reactions were attempted with
 5'-iodomethyl-4,8-dimethyl-4',5'-dihydrofurocoumarin (5, R.dbd.H, T.dbd.I)
 and imidazole, diisopropanolamine, piperidine or an amine in the presence
 of a strong base in an attempt to force the addition of the amine. Thus,
 elimination dominated over substitution with more basic nucleophiles.
 In a preferred embodiment, 5-N-aminomethyl substituted
 4,8-dimethyl-4',5'-dihydropsoralen of the formula (5):
 ##STR29##
 where R is hydrogen, a halogen, NO.sub.2, or CN; and T is NR.sup.1 R.sup.2
 or (N.sup.+ R.sup.1 R.sup.2 R.sup.3)X.sup.- (as defined above) are
 prepared by contacting a 4,8-dimethyl-6-allyl-7-hydroxycoumarin of the
 formula:
 ##STR30##
 with a cyclization reagent under conditions to form a compound of formula
 (5) where T is Br or I, and contacting the resulting bromo or iodo
 compound of formula (5) with a secondary amine of the formula of a
 secondary amine, HNR.sup.1 R.sup.2, or a tertiary amine, NR.sup.1 R.sup.2
 R.sup.3.
 The bromide and iodide anions in the quaternary ammonium salts may be
 exchanged for other anions (e.g., chloride or another pharmaceutically
 acceptable anion) by ion exchange on a resin charged with the desired
 anion.
 Preparation of 2-substituted Mercurimethyl-2-3-Dihydrobenzofuran
 Derivatives
 The invention also relates to a process for obtaining 2-substituted
 mercurimethyl-2-3-dihydrobenzofuran of the forumla (7, T.dbd.HgR.sup.4). A
 2-substituted mercurimethyl-2-3-dihydrobenzofuran according to the
 invention may be prepared by contacting 2-allylphenol with a cyclization
 reagent Hg(R.sup.4).sub.2 under conditions to form a 2-substituted
 mercurimethyl-2-3-dihydrobenzofuran as represented by the following
 reaction scheme:
 ##STR31##
 wherein T is a mercurial group HgR.sup.4 wherein R.sup.4 is OC(O)(C.sub.1
 -C.sub.6 alkyl), OC(O)(C.sub.1 -C.sub.6 fluoroalkyl), or a halogen.
 Preferably, R.sup.4 is OC(O)CH.sub.3, OC(O)CF.sub.3, Cl, Br, or I. The
 cyclization reagent is Hg(R.sup.4).sub.2. Preferred substituents R and
 R.sup.4 are the same as those described above. As shown in Example 38,
 mercury chloride effectively ring closed 2-allylphenol in 89% yield to
 2-chloromercurimethyl-2,3-dihydrobenzofuran (7, T.dbd.HgCl). Further, as
 shown in Example 39, mercuric acetate effectively ring closed
 2-allylphenol in 60% yield to 2-chloromercurimethyl-2,3-dihydrobenzofuran
 (7, T.dbd.HgOAc).
 Iodomercurimethyl-2,3-dihydrobenzofuran compounds may be prepared by
 anionic exchange with the mercury-anion derivatives of other 2-substituted
 mercurimethyl-2-3-dihydrobenzofurans (e.g 7, T.dbd.HgR.sup.4 wherein
 R.sup.4 =chloride, trifluoroacetate, acetate) with iodide. Preparing
 iodomercurimethyl-2,3-dihydrobenzofuran compounds in this manner
 represents a further embodiment of the invention. Suitable iodide sources
 include, for example, potassium iodide, sodium iodide, and the like.
 Suitable mercury-anion derivatives include, for example, HgX with X being
 aceto, trifluoroaceto, or chloro. As shown in Example 40, anionic exchange
 of 2-acetomercurimethyl-2,3-dihydrobenzofuran to form
 2-iodomercurimethyl-2,3-dihydrobenzofuran occurred in 50% yield.
 3. Pharmacological Activity
 The 5'-substituted, 4',5'-dihydropsoralens and 2-substituted
 mercurimethyl-2-3-dihydrobenzofurans according to the invention are
 photochemotherapeutic compounds, and in the case of the mercurial
 compounds chemotherapeutic compounds, useful to prevent or treat skin,
 blood, marrow diseases, and microbial infections in a mammal. Treatment of
 a disease according to the invention encompasses not only treating an
 existing condition but treatment to prevent the disease condition from
 occurring. The partially reduced and quaternized psoralens,
 amino-substituted psoralens, and mercurio psoralens display impressive
 photopharmacology against PAM 212 keratinocytes, a model cell line
 employed as a test system to indicate epidermal cytotoxicity in these
 candidate phototherapeutics. Examples of diseases treatable by compounds
 of the invention include cancer, infections, Acquired Immuno Deficiency
 Syndrome, HIV, cutaneous T-cell lymphoma, scleroderma, vitiligo,
 myasthenia gravis, multiple sclerosis, rheumatoid arthritis and other
 arthridides, psoriasis, inflammation, lupus erythematosus, tuberculosis,
 and the like.
 Compounds of the invention have demonstrated photo-induced activity in an
 in vitro growth inhibition assay against PAM 212 keratinocytes. Psoriasis,
 mycosis fungoides, eczema, cancer, and similar proliferative diseases are
 often characterized by abnormal cell growth regulation. Application of
 PUVA therapy to correct proliferative disorders on the skin or internally,
 especially psoriasis, is one clinical expression of photochemotherapy. The
 use of the assay described in Example 42 is based on the observation that
 phototherapeutics are extremely potent inhibitors of binding of cell
 growth in mammalian cells including humans and this inhibition arrests the
 proliferative disorder. This assay was performed in the cell culture
 laboratory. For a discussion of cell growth assays see, e.g., J. Laskin et
 al., Cancer Research 1979, vol. 39, pp. 383-390 and E. Yurkow and J.
 Laskin, Cancer Chemotherapy and Pharmacology, vol. 27, pp. 315-319, 1991.
 Inhibition of cell growth is dependent on dose of the phototherapeutic and
 on the quanta of light in the 320-400 nm wavelength (ultraviolet light A).
 It is also structure-dependent, that is, there is a direct correlation
 between those specific phototherapeutics currently used that are
 clinically active and their ability to inhibit the growth of the cells.
 Advantageously, the mercurio-substituted compounds of the invention exhibit
 chemotherapeutic activity without ultraviolet light activation. The
 presence of a mercury functionality provides a reactive cell-binding group
 on these psoralens with unique cytotoxicity without light activation and
 an up to 2.5-fold enhancement of cytotoxicity activity upon light
 activation. All non-mercury psoralens required photoactivation to
 demonstrate any beneficial toxicity.
 Accordingly, one embodiment of the invention relates to a method of
 treatment of a skin disease in a mammal in recognized need thereof. The
 method comprises administering to the mammal an effective amount of a
 5'-substituted, 4',5'-dihydropsoralen or a 2-substituted
 mercurimethyl-2-3-dihydrobenzofuran and irradiating the mammal with
 sufficient UVA light to effect photochemical sensitation on the skin. For
 the mercurio-substituted compounds the irradiation step is optional. The
 5'-substituted, 4',5'-dihydropsoralen or 2-substituted
 mercurimethyl-2-3-dihydrobenzofuran may be administered topically or
 systemically. Generally, the dosage of UV applied is that conventionally
 used in the photochemical treatment of skin and preferably ranges from
 about 0.2 to about 15 joules/cm.sup.2. The amount and duration of
 irradiation will depend upon a number of factors including the type and
 the extent of the disease being treated, the age of the patient, and will
 be apparent to one skilled in the art. The frequency of treatment will
 also depend upon such factors and will also be apparent to one skilled in
 the art.
 The 5'-substituted, 4',5'-dihydropsoralens and 2-substituted
 mercurimethyl-2-3-dihydrobenzofurans of the invention may also be used to
 treat diseases of the blood or bone marrow in a patient. Accordingly, the
 invention also relates to a process for the treatment of diseases of the
 blood or bone marrow in a patient in need of such treatment. The process
 comprises obtaining cells from the blood or bone marrow of the patient,
 contacting the cells in vitro with a 5'-substituted, 4',5'-dihydropsoralen
 according to the invention, exposing the cell in contact with the
 5'-substituted, 4',5'-dihydropsoralen with sufficient UVA to activate the
 therapeutic effect of the 5'-substituted, 4',5'-dihydropsoralen and
 returning the cell to the patient's blood stream or bone marrow. In
 another embodiment, the patient is dosed in vivo with the psoralen and the
 cells of the blood or bone marrow subsequently removed, irradiated
 ex-vivo, and subsequently returned to the patient.
 The compounds of the invention which are capable of intercalating into
 double-stranded nucleic acid, i.e., 4',5'-dihydropsoralens, may also be
 used to eliminate or reduce the levels of infectious agents in blood.
 Blood may be treated with such a compound under the conditions described
 above and be subsequently irradiated with UVA. This treatment has
 advantages over known treatments which use psoralen compounds that also
 form crosslinks in double stranded nucleic acid. In the later case,
 residual psoralen remaining in the blood sample is potentially quite
 mutagenic to a patient receiving such blood, e.g., during a transfusion.
 In the former case, residual 4',5'-dihydropsoralen is potentially far less
 mutagenic to a recipient of the blood because of the inability of these
 compounds to form crosslinks in the DNA.
 Methods for treating blood cells and marrow are known in the art and
 taught, for example, by U.S. Pat. No. 5,356,929, the disclosure of which
 is herein incorporated by reference. Blood cells may be obtained from a
 patient using any ordinary conventional technique. Bone marrow may be
 obtained using established protocols available to those in the art and
 described, for example, in Kitano et al. (1991, Blood 77:1699-1705), or
 Folks et al (1988, Science 242:919-922). White blood cells may be
 separated from pigmented cells (red blood cells) and other factors using
 the common technique of leukopheresis. If necessary, subpopulations of
 cells of interest from either the blood or bone marrow may be separated
 from the remainder of cells in the sample using a combination of
 techniques including centrifugation and flow cytometry. Cells so isolated
 are then either irradiated (in the case of the patient to whom the drug
 has already been administered), or they are treated with the compound of
 choice in a manner similar to that described above for the treatment of
 cells in culture followed by irradiation. Essentially, the photherapeutic
 compound is dissolved in isotonic buffered solution and is added to the
 cells in a therapeutically effective amount to be determined by the extent
 and type of disease being treated, and the number of cells in the sample.
 After a period of incubation, treated cells are exposed to ultraviolet
 light (UVA, 320-400 nm) as described above. In some cases, depending on
 the compound involved, different wavelengths of light may also be used.
 After exposure to light, the cells are washed in an isotonic, buffered
 solution and are returned to either the patient's blood or bone marrow
 using conventional technology.
 The 5'-substituted, 4',5'-dihydropsoralen derivatives and 2-substituted
 mercurimethyl 2,3-dihydrobenzofuran derivatives according to the invention
 also have antimicrobial effects. Accordingly, the invention provides a
 method of treating microbiological infections in a mammal in recognized
 need thereof. The method comprises administering to the mammal an
 effective amount of a 5'-substituted, 4',5'-dihydropsoralen derivative or
 a 2-substituted mercurimethyl 2,3-dihydrobenzofuran derivative according
 to the invention. Examples of organisms that can be treated by a process
 according to the invention include A. Niger, Chlorella, Mycobacterium
 tuberculosis and fungal organisms, such as dermatophytes, Trichophyton,
 Microsporum and Epidermophyton, different Candida species, Trichoderma,
 Cryptococcus, Aspergillus Zygomyetes, Fusarim which can cause infections
 in humans and animals. Histoplasmosis, Blastomyces, and Coccidioides, for
 example, cause lower respiratory infections. Trichophyton rubrum causes
 difficult to eradicate nail infections. Hendersonula toruloidea and
 Scopulariopsis brevicaulis are known to cause tinea pedis, tinea captitis,
 tinea cruris and different ring worm infections. As shown in Example 44,
 compounds according to the invention exhibit anti-tuberculosis activity.
 Due to their valuable pharmacological properties, the compounds of the
 invention or their physiologically acceptable salts, are particularly
 suitable for use as active compounds in pharmaceutical compositions. The
 5'-substituted, 4',5'-dihydropsoralens and 2-substituted
 mercurimethyl-2-3-dihydrobenzofurans of the invention can be either
 administered alone or in mixtures with one another or with other
 therapeutic agents. As mentioned above, the 5'substituted,
 4',5'-dihydropsoralens and 2-substituted
 mercurimethyl-2-3-dihydrobenzofurans of the invention may be applied
 topically in the form of an ointment or lotion, administered orally,
 intravenously, or parenterally. Methods for preparing clinically-ready
 compositions are conventional in this art and include gelatin capsules or
 tablets for oral administration, solutions or ointments for external use,
 as described, for example, in U.S. Pat. No. 5,356,929. The compounds
 according to the invention can be administered orally, topically,
 rectally, anterally, internally, by boluses or, if desired, parenterally.
 Topical or oral administration may be preferred.
 The invention also relates to photochemotherapeutic and chemotherapeutic
 pharmaceutical compositions for use in treating diseases such as those
 discussed above. A pharmaceutical composition according to the invention
 comprises a therapeutically effective amount of a 5'-substituted,
 4',5'-dihydropsoralen or a 2-substituted
 mercurimethyl-2-3-dihydrobenzofuran with or without a pharmaceutically
 acceptable carrier or agent. Preferably, a pharmaceutical composition
 according to the invention contains a 5'-substituted,
 4',5'-dihydropsoralen or a 2-substituted
 mercurimethyl-2-3-dihydrobenzofuran in a therapeutically effective amount
 to treat a disease of the skin, blood or marrow of a mammal, in
 particular, a human. For treatment of microbiocidal infections, such as
 tuberculosis, the pharmaceutical composition contains 5'-substituted,
 4',5'-dihydropsoralen or a 2-substituted
 mercurimethyl-2-3-dihydrobenzofuran in a therapeutically effective amount
 to treat the microbiocidal infection. Pharmaceutically acceptable carriers
 are known in the art and are described, for example, in U.S. Pat. Nos.
 4,124,598 and 4,130,568, the disclosures of which are herein incorporated
 by reference.
 Pharmaceutical compositions of the invention may further include excipient,
 stabilizers, emulsifiers, therapeutic adjuvants, diluents and the like and
 may be provided in sustained release or timed release formulations.
 Suitable solid or liquid formulations are, for example, granules, powders,
 coated tablets, microcapsules, suppositories, syrups, elixirs,
 suspensions, emulsions, drops or injectable solutions. Commonly used
 additives in protracted release preparations are excipients,
 disintegrates, binders, coating agents, swelling agents, glidants, or
 lubricants, flavors, sweeteners or solubilizers. More specifically,
 frequently used additives are, for example, magnesium stearate, magnesium
 carbonate, titanium dioxide, lactose, mannitol and other sugars, talc,
 lactalbumin, gelatin, starch, cellulose and its derivatives, animal and
 vegetable oils, polyethylene glycols and solvents. Common solvents include
 sterile water and monohydric or polyhydric alcohols such as glycerol.
 Acceptable carriers, agents, excipient, stabilizers, diluents and the like
 for therapeutic use are well known in the pharmaceutical field, and are
 described, for example, in Remington's Pharmaceutical Sciences, Mack
 Publishing Co., ed. A. R. Gennaro (1985). If appropriate, the compound may
 be administered in the form of a physiologically acceptable salt, for
 example, an acid-addition salt.
 The pharmaceutical compositions are preferably produced and administered in
 dosage units, each unit containing as an active component an effective
 dose of at least one compound of the present invention and/or at least one
 of its physiologically acceptable salts. In the case of mammals, the
 effective dose to treat diseases such as those discussed above can range
 from about 1 to about 100 mg/kg of body weight per day.
 The pharmaceutical compositions according to the 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 5'-substituted 4',5;-dihydropsoralen or 2-substituted
 mercurimethyl-2-3-dihydrobenzofurans compound according to the invention
 and a pharmaceutically acceptable carrier or diluent. Such compositions
 are well known in the art and taught, for example, by U.S. Pat. Nos.
 4,124,598 and 4,130,568, the disclosures of which are herein incorporated
 by reference.
 For oral treatment, the active ingredient is generally formulated in
 tablets or in gelatin capsules. In such a case, the diluent may, if
 desired, may be used. For topical applications, solutions or ointments may
 be prepared and employed. These may be formulated with any one of a number
 of pharmaceutically acceptable carriers, as is well known in the art.
 Administration may be, for example, in the form of tablets, capsules,
 powders, syrups, or solutions, or as already stated in the form of
 ointments, creams, or solutions for topical use.
 For tablet preparation, the usual tablet adjuvants such as cornstarch,
 potato starch, talcum, magnesium stearate, gelatin, lactose, gums, or the
 like may be employed, but any other pharmaceutical tableting adjuvants 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, preferably from about 5 to about 10 mg, per kg of body
 weight. Such administration and selection of dosage and unit dosage will
 of course have to be determined according to established medical
 principles and under the supervision of the physician in charge of the
 therapy involved.
 Topical formulations comprise an effective amount of the active ingredient
 per unit area. Preferably, the topical formulation is in the form of a one
 percent solution, suspension or ointment and is applied on the skin at
 about 0.1 mL per square centimeter. The formulations contain a suitable
 carrier, such as, ethanol or any of the pharmaceutically acceptable
 carriers described above. A typical formulation for a 1% phototherapeutic
 lotion comprises:
 (A) 25 ml of propylene glycol;
 (B) 1 ml of triethanolamine;
 (C) 12 ml of water;
 (D) 1.5 grams of oleic acid;
 (E) 10.5 grams of polyethylene glycol 400 monostearate;
 (F) 10 ml of silicon fluid DC-200;
 (G) 10 ml of CARBOPOL 934, 2% mucilage; and
 (H) 1 gram of at least one 5'-substituted, 4',5'-dihydropsoralens according
 to the invention.
 4. Microbicidal Activity and Methods of Use
 The 5'-substituted 4',5'-dihydropsoralens according to the invention are
 useful in preventing the microbiological attack, degradation, or
 deterioration of various types of raw materials and products such as
 leather, textiles, plastics, plastic containers, pulp, paper and
 paperboard, coatings, lumber, as well as agricultural products such as
 seeds and crops. Antifungal activity of the compounds of the invention
 were demonstrated by measuring the inhibitory effects of compounds of the
 invention against Aspergillis niger. Antimicrobial activity effects were
 determined by measuring the inhibitory effects of compounds of the
 invention against Chlorella with minimum inhibitory concentrations of less
 than 100 ppm. Advantageously, the 5'-substituted 4',5'-dihydropsoralens
 and 2-substituted mercurimethyl-2-3-dihydrobenzofurans may be used in
 various industrial processes used to prepare or manufacture these
 products. Accordingly, additional embodiments of the invention employ the
 combination to control the growth of microorganisms on or in such
 industrial products, raw materials or processes.
 Accordingly, another embodiment of the invention provides a microbicidal
 composition. The composition contains a 5'-substituted,
 4',5'-dihydropsoralen or a 2-substituted
 mercurimethyl-2-3-dihydrobenzofurans in an amount effective to control the
 growth of at least one microorganism. The invention also provides a method
 for controlling the growth of a microorganism on a substrate. This method
 contacts a substrate susceptible to the growth of microorganisms with an
 effective amount of a 5'-substituted 4',5'-dihydropsoralen or a
 2-substituted mercurimethyl-2-3-dihydrobenzofuran to control the growth of
 at least one microorganism on the substrate. The invention further
 provides a method for controlling the growth of microorganisms in an
 aqueous system capable of supporting growth of a microorganism. This
 method treats the aqueous system with an amount of a 5'-substituted
 4',5'-dihydropsoralen or a 2-substituted
 mercurimethyl-2-3-dihydrobenzofuran effective to control the growth of at
 least one microorganism in the aqueous system.
 Depending on the application, microbicidal compositions according to the
 invention may be prepared in various forms known in the art. For example,
 the composition may be prepared in liquid form as an aqueous solution,
 dispersion, emulsion, or suspension, a dispersion or suspension in a
 non-solvent, or as a solution by dissolving a 5'-substituted
 4',5'-dihydropsoralen in a solvent or combination of solvents. Suitable
 solvents include, but are not limited to, methyl ethers of glycols,
 M-pyrol or 1-methyl pyrrolidinone, or petroleum distillates. The
 microbicidal composition may be prepared as a concentrate for dilution
 prior to its intended use. Common additives such as surfactants,
 emulsifiers, dispersants, and the like may be used as known in the art to
 increase the solubility of the 5'-substituted 4',5'-dihydropsoralen or
 2-substituted mercurimethyl-2-3-dihydrobenzofuran in a liquid composition
 or system, such as an aqueous composition or system.
 Microbicidal compositions of the invention may also be prepared in solid
 form, for example as a powder or tablet, using means known in the art. For
 example, a liquid product containing a 5'-substituted
 4',5'-dihydropsoralen or a 2-substituted
 mercurimethyl-2-3-dihydrobenzofuran is deposited on carriers such as
 diatomaceous earth or kaolin. The resulting solid or solids may be mixed
 together or one solid may be mixed with the other component, or a solution
 or liquid formulation containing the component, to form a powder or
 tablet.
 According to the invention, control of the growth of a microorganism on a
 substrate or in an aqueous system means control to, at, or below a desired
 level and for a desired period of time for the particular substrate or
 system. This can vary from the complete prevention or inhibition of
 microbiological growth to control at a certain desired level and for a
 desired time. The 5'-substituted 4',5'-dihydropsoralens and 2-substituted
 mercurimethyl-2-3-dihydrobenzofurans described here can, in many cases,
 reduce the total microbiological count to undetectable limits and maintain
 the count at that level for a significant period of time. Accordingly, the
 combination may be used to preserve a substrate or system.
 The effective amount or percentage of a 5'-substituted
 4',5'-dihydropsoralen or 2-substituted
 mercurimethyl-2-3-dihydrobenzofurans necessary to achieve the desired
 result will vary somewhat depending on the substrate or aqueous system to
 be protected, the conditions for microbial growth, the particular
 microbicide, and the degree of protection desired. For a particular
 application, the amount of choice may be determined by routine testing of
 various amounts prior to treatment of the entire affected substrate or
 system. In general, an effective amount used on a substrate ranges from
 about 0.0001% to about 4% (w/w); preferably about 0.0001% to about 1.0%.
 With aqueous systems, an effective amount may range from about 0.5 to
 about 10,000 parts per million, more preferably from about 5 to about 5000
 parts per million of the aqueous system, and most preferably from, about
 10 to about 1000 parts per million. Similar amounts effectively control
 slime formation. For slime control, effective amounts preferably range
 from about 1 to about 1000 parts per million, and more preferably, from
 about 1 to about 200 parts per million of the aqueous system.
 A microbicidal composition containing a 5'-substituted
 4',5'-dihydropsoralen, 2-substituted mercurimethyl-2-3-dihydrobenzofuran
 or a mixture thereof may be applied in a variety of industrial uses and
 processes for microorganism control. The 5'-substituted
 4',5'-dihydropsoralens and 2-substituted
 mercurimethyl-2-3-dihydrobenzofurans may be used in place of and in the
 same manner as other microbiocides traditionally used in the particular
 industry. As discussed above, such industries include, but are not limited
 to the leather industry, the lumber industry, the papermaking industry,
 the textile industry, the agricultural industry, and the coating industry.
 The 5'-substituted 4',5'-dihydropsoralens and 2-substituted
 mercurimethyl-2-3-dihydrobenzofurans may also be used with aqueous systems
 such as those previously discussed which are subject to microbiological
 attack and degradation. The problems caused by microbiological attack and
 deterioration in these various applications has been described above.