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First published on August 11, 2005; DOI: 10.1124/mol.105.015669

0026-895X/05/6805-1254-1270$20.00
Mol Pharmacol 68:1254-1270, 2005

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Design of PAP-1, a Selective Small Molecule Kv1.3 Blocker, for the Suppression of Effector Memory T Cells in Autoimmune Diseases

Alexander Schmitz, Ananthakrishnan Sankaranarayanan, Philippe Azam, Kristina Schmidt-Lassen, Daniel Homerick, Wolfram Hänsel, and Heike Wulff

Pharmaceutical Institute, University of Kiel, Kiel, Germany (A.Sc., K.S.-L., W.H.); and Department of Medical Pharmacology and Toxicology, University of California, Davis, Davis, California (A.Sa., P.A., D.H., H.W.)

Received June 10, 2005; accepted August 10, 2005


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
The lymphocyte K+ channel Kv1.3 constitutes an attractive pharmacological target for the selective suppression of terminally differentiated effector memory T (TEM) cells in T cell-mediated autoimmune diseases, such as multiple sclerosis and type 1 diabetes. Unfortunately, none of the existing small-molecule Kv1.3 blockers is selective, and many of them, such as correolide, 4-phenyl-4-[3-(methoxyphenyl)-3-oxo-2-azapropyl]cyclohexanone, and our own compound Psora-4 inhibit the cardiac K+ channel Kv1.5. By further exploring the structure-activity relationship around Psora-4 through a combination of traditional medicinal chemistry and whole-cell patch-clamp, we identified a series of new phenoxyalkoxypsoralens that exhibit 2- to 50-fold selectivity for Kv1.3 over Kv1.5, depending on their exact substitution pattern. The most potent and "drug-like" compound of this series, 5-(4-phenoxybutoxy)psoralen (PAP-1), blocks Kv1.3 in a use-dependent manner, with a Hill coefficient of 2 and an EC50 of 2 nM, by preferentially binding to the C-type inactivated state of the channel. PAP-1 is 23-fold selective over Kv1.5, 33- to 125-fold selective over other Kv1-family channels, and 500- to 7500-fold selective over Kv2.1, Kv3.1, Kv3.2, Kv4.2, HERG, calcium-activated K+ channels, Na+,Ca2+, and Cl- channels. PAP-1 does not exhibit cytotoxic or phototoxic effects, is negative in the Ames test, and affects cytochrome P450-dependent enzymes only at micromolar concentrations. PAP-1 potently inhibits the proliferation of human TEM cells and suppresses delayed type hypersensitivity, a TEM cell-mediated reaction, in rats. PAP-1 and several of its derivatives therefore constitute excellent new tools to further explore Kv1.3 as a target for immunosuppression and could potentially be developed into orally available immunomodulators.

Potassium (K+) channels in humans are encoded by an extended superfamily of 76 genes (Gutman et al., 2003Go). Each K+ channel has a unique expression pattern that allows cells to "fine tune" their membrane potential and their excitability according to their respective functions. Specific K+ channel modulation therefore offers an enormous potential for the development of new drugs. One channel that constitutes an especially promising target is the voltage-gated Kv1.3 channel. Homomeric Kv1.3 channels are found in human T and B lymphocytes and their expression is up-regulated in terminally differentiated effector memory T cells (Wulff et al., 2003bGo) and class-switched memory B cells (Wulff et al., 2004Go). Kv1.3 blockers have therefore been proposed as potential new therapeutics for the treatment of autoimmune diseases (such as multiple sclerosis, type-1 diabetes, psoriasis, rheumatoid arthritis, transplant rejection, graft-versus-host disease, Sjögren's syndrome, and systemic lupus erythematosus) in which effector memory T cells and/or class-switched memory B cells are involved in the pathogenesis (Markovic-Plese et al., 2001Go; Hansen et al., 2002Go; Viglietta et al., 2002Go; Wulff et al., 2003bGo; Dorner and Lipsky, 2004Go; Fasth et al., 2004Go; Vissers et al., 2004Go; Yamashita et al., 2004Go; Pearl et al., 2005Go). In proof of this hypothesis, the Kv1.3 blocking peptides kaliotoxin (Beeton et al., 2001aGo), ShK (Beeton et al., 2001bGo), and its recently identified more specific derivative ShK(L5) (Beeton et al., 2005Go) have been shown to treat experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. Kv1.3 has been further validated as a target for the treatment of multiple sclerosis by the demonstration that myelin-reactive T cells in the blood from patients with multiple sclerosis are predominantly Kv1.3high effector memory T cells (Wulff et al., 2003bGo). Contrary to the aforementioned peptides, a small-molecule Kv1.3 blocker would have the advantage that it could be taken orally.

The first small molecules that were found to inhibit the Kv channel in human T cells were the classic Kv channel inhibitors 4-aminopyridine and tetraethylammonium, the Ca2+-activated K+ channel blocker quinine, and the Ca2+ channel inhibitors verapamil, diltiazem, and nifedipine (for recent reviews, see Wulff et al., 2003aGo; Chandy et al., 2004Go). After the cloning of the Kv1.3 gene in the early 1990s (Grissmer et al., 1990Go), several pharmaceutical companies started screening their libraries for more potent and more selective small molecule Kv1.3 blockers and identified a number of compound classes that roughly fall into two groups: 1) typical combinatorial library compounds, such as the iminodihydroquinoline CP-339818 (Nguyen et al., 1996Go), the benzylpiperidine UK-78282 (Hanson et al., 1999Go), the cyclohexyl-substituted benzamides (Schmalhofer et al., 2002Go), and the sulfamidebenzamidoindanes (Wulff et al., 2003aGo), which are of a relatively simple structure and low molecular weight and rich in nitrogen and halogen atoms, and 2) natural products or natural product derivatives, such as the triterpenoid correolide (Hanner et al., 1999Go) and the candelalides (Singh et al., 2001Go), which are rich in oxygen atoms and have a more complex stereochemistry. Although some of these compounds are quite potent Kv1.3 blockers, with EC50 values of 30 to 200 nM, they all lack the required selectivity over other ion channels, especially the closely related Kv1 family channels.

In contrast to the high-throughput screening approaches applied by pharmaceutical companies, we took an approach that is termed "retroactive exploitation of observations made in man" (Wermuth, 2004Go). Following up on anecdotal reports that tea prepared from the leaves of Ruta graveolens, the Common Rue (Herb of Grace), alleviated the symptoms of multiple sclerosis (Bohuslavizki et al., 1993Go) we extracted R. graveolens and identified a number of Kv1-family channel blocking psoralens and furoquinolines in this plant (Bohuslavizki et al., 1994Go). In subsequent single-case "off label" trials, the psoralen 5-methoxypsoralen (5-MOP; Fig. 1A), one of the major K+ channel blocking compounds of R. graveolens (Bohuslavizki et al., 1994Go), was found to improve the functional deficits in multiple sclerosis patients (Bohuslavizki et al., 1993Go), but its phototoxic activity and its low affinity to Kv1.3 precluded its use as a therapeutic for multiple sclerosis. We therefore started a traditional medicinal chemistry program to improve the potency and selectivity of the psoralens for Kv1.3 and identified 5,8-diethoxypsoralen (EC50,10 µM) (Wulff et al., 1998Go), 6-(2,5-dimethoxyphenyl)-psoralen (EC50, 700 nM) (Wernekenschnieder et al., 2004Go), and Psora-4 (EC50, 3 nM) (Vennekamp et al., 2004Go). Psora-4 (Fig. 1A), our most potent compound, was 17- to 70-fold selective for Kv1.3 over other Kv1 family channels, except for the cardiac channel Kv1.5 (EC50 ~8 nM), raising concerns about potential side effects in vivo.



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  		Fig. 1. A, R. graveolens was the source of 5-MOP, which served as a template for the Kv1.3 blocker Psora-4. B, design strategy for new Psora-4 analogs.

 
In the present study, we describe how we have used Psora-4 as a template and have successfully separated the affinities for Kv1.3 and Kv1.5 through further derivatization. Our new psoralen Kv1.3 blocker phenoxyalkoxypsoralen-1 (PAP-1) exhibits 23-fold selectivity over Kv1.5, potently suppresses the proliferation of human TEM cells in vitro, and prevents delayed type hypersensitivity (DTH) in rats when administered intraperitoneally or orally.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Chemistry. 5-MOP was purchased from Aldrich Chemical Co. (Milwaukee, WI) and dealkylated with magnesium and iodine at 165°C (Schoenberg and Aziz, 1953Go) to obtain 5-hydroxypsoralen (5-HOP).

General method A. 5-HOP was reacted with the respective alkyl halides in the presence of an excess of anhydrous potassium carbonate and catalytic amounts of potassium iodide under reflux in a dry organic solvent. The progress of the reaction was monitored by thin-layer chromatography. After completion of the reaction, the crude product mixture was diluted with water and acidified to pH 1 with concentrated hydrochloric acid. The aqueous slurry was extracted with dichloromethane. The organic phase was washed first with 1% sodium hydroxide to separate the unreacted 5-HOP and then with acidic water before it was dried over anhydrous sodium sulfate and concentrated to dryness. The resulting residue was decolorized with charcoal and recrystallized.

General method B. 5-HOP was alkylated with 4-chloroiodobutane in dry acetone in the presence of an excess of potassium carbonate for 24 to 28 h at 25°C. After completion of the reaction, the product, 5-(4-chlorobutoxy)psoralen, was isolated according to general method A and then reacted with mercapto-, amino-, and hydroxy-substituted heteroaromatics or with substituted phenols to obtain the heteroaromatic and phenoxy-substituted compounds shown in Figs. 1B and 2.



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  		Fig. 2. Top, EC50 values of compounds synthesized under strategy a (see Fig. 1B for chemical structures). Middle, chemical structures and EC50 values of compounds from strategy b. Bottom, EC50 values and structures of compounds that were synthesized through a combination of strategies b + c. All compounds were tested three to five times at four to six concentrations and EC50 values were determined by fitting the Hill equation to the reduction of area under the current curve at 40 mV. The standard deviations were smaller than 10% in each case.

 
General method C. To obtain phenoxy-substituted alkyl halides as reactants for general method A, elemental sodium was stirred in 120 ml of dry ethyl alcohol, and an equivalent amount of the phenol component was added drop-wise. After addition of an excess of 1,4-dibromobutane, the reaction mixture was refluxed for 4 h. The solvent was evaporated, and the residue was dissolved in 6% sodium hydroxide solution and extracted with ether. The organic layer was dried over sodium sulfate and evaporated to obtain the crude product.

All newly synthesized compounds were characterized by melting point, 1H and 13C NMR, mass spectrometry, and combustion analysis. 13C data are given only for PAP-1, PAP-17, and PAP-17a, where it was necessary to determine the structure of the two reaction products. It is available upon request for other compounds.

5-(4-Chlorobutoxy)psoralen. 5-HOP (817 mg, 4.0 mmol), 4-chlorobutyl iodide (1.4 g, 6.4 mmol) and potassium carbonate (3.0 g) were stirred at 25°C in 80 ml of acetone for 30 h. After completion of the reaction, the acetone was evaporated and the resulting residue suspended in petroleum ether, filtered to separate the excess 4-chlorobutyl iodide, and dried under suction (1.10 g, 92.9%): m.p. = 115.6°C; 1H-NMR (500 MHz, CDCl3): {delta} [ppm] = 8.15 (d, 1H, 3J = 9.8 Hz, 3-H), 7.60 (d, 1H, 3J = 2.6 Hz, 5'-H), 7.17 (s, 1H, 8-H), 6.95 (d, 1H, 3J = 2.2 Hz, 4'-H), 6.29 (d, 1H, 3J = 9.8 Hz, 4-H), 4.52 (t, 2H, 3J = 5.5 Hz, 5-OCH2CH2CH 2CH2Cl), 3.68 (t, 2H, 3J = 5.8 Hz, 5-OCH2CH2CH2CH2Cl), 2.08 (p, 4H, 3J = 3.0 Hz, 5-OCH2CH2CH2CH2Cl).

5-(3-Cyanopropoxy)psoralen (ACP-3). 5-HOP (800 mg, 3.9 mmol), 4-chlorobutyronitrile (655.5 mg, 6.3 mmol) and potassium carbonate (2.6 g) were refluxed in 50 ml of 2-butanone for 48 h. The crude product was recrystallized from methanol (710 mg, 66.7%): m.p. = 155.2°C; 1H NMR (500 MHz, CDCl3): {delta} [ppm] = 8.13 (d, 1H, 3J = 9.8 Hz, 3-H), 7.63 (d, 1H, 3J = 2.0 Hz, 5'-H), 7.21 (s, 1H, 8-H), 6.98 (d, 1H, 3J = 2.0 Hz, 4'-H), 6.32 (d, 1H, 3J = 9.5 Hz, 4-H), 4.58 (t, 2H, 3J = 5.8 Hz, 5-OCH2CH2CH2CN), 2.72 (t, 2H, 3J = 5.7 Hz, 5-OCH2CH2CH2CN), 2.26 (p, 2H, 3J = 6.3 Hz, 5-OCH2CH2CH2CN); MS (70 eV) m/z: 269 (100%, M+), 202 (74%, [C11H6O4]+), 174 (63%, [202-CO]+), 145 (26%), 118 (7%), 89 (14%); calculated for C15H11NO4 (269.26): C, 66.91%; H, 4.12%; N, 5.01%; O, 23.77%. Found: C, 66.52%; H, 4.03%; N, 5.01%.

5-(4-Pentynyloxy)psoralen (AP-1). 5-HOP (500 mg, 2.4 mmol), 5-chloro-1-pentyne (405.8 mg, 3.9 mmol) and potassium carbonate (2.0 g) were refluxed in 30 ml of acetonitrile for 24 h. The crude product was recrystallized from methanol (55 mg, 8.3%): m.p. = 145.1°C: 1H NMR (500 MHz, CDCl3): {delta} [ppm] = 8.16 (d, 1H, 3J = 9.8 Hz, 3-H), 7.60 (d, 1H, 3J = 2.3 Hz, 5'-H), 7.17 (s, 1H, 8-H), 7.01 (d, 1H, 3J = 1.5 Hz, 4'-H), 6.29 (d, 1H, 3J = 9.8 Hz, 4-H), 4.56 (t, 2H, 3J = 6.1 Hz, 5-OCH2CH2CH2C{equiv}CH), 2.51 (q, 2H, 3J = 2.6 Hz, 5-OCH2CH2CH2C{equiv}CH), 2.09 (p, 2H, 3J = 6.5 Hz, 5-OCH2CH2CH2C{equiv}CH), 2.03 (t, 1H, 4J = 2.6 Hz, 5-OCH2CH2CH2C{equiv}CH); MS (70 eV) m/z: 268 (88%, M+), 203 (15%), 202 (100%, [C11H6O4]+), 175 (8%), 174 (11%, [202-CO]+), 173 (14%), 146 (7%), 145 (21%), 118 (8%), 89 (10%), 67 (5%, C5H7); calculated for C16H12O4 (268.27): C, 71.64%; H, 4.51%; O, 23.86%. Found: C, 71.25%; H, 4.38%; LogP, 2.88.

5-[4-(1-N-Pyrazolyl)butoxy]psoralen (PH-1). 5-HOP (500 mg, 2.4 mmol), 4-iodo-1-chlorobutane (893 mg, 4.1 mmol) and potassium carbonate (2.0 g) were stirred at 25°C in 30 ml of acetone for 28 h. After completion of the reaction, the solvent was evaporated and the resulting residue refluxed for 50 h with pyrazole (400 mg, 5.9 mmol) and potassium carbonate (2.0 g) in 30 ml of 2-butanone. The crude product was recrystallized from an ethyl acetate/petroleum ether mixture (20:80) to yield white crystals (108 mg, 13.5%): m.p. = 145.6°C; 1H NMR (500 MHz, DMSO-d6): {delta} [ppm] = 8.17 (d, 1H, 3J = 9.1 Hz, 3-H), 8.02 (s, 1H, 5'-H), 7.74 (s, 1H, 5-OCH2CH2CH2CH2C3-H3N2), 7.43 (s, 1H, 5-OCH2CH2CH2CH2C3H3N2), 7.34 (s, 1H, 8-H), 7.29 (s, 1H, 4'-H), 6.32 (d, 1H, 3J = 9.1 Hz, 4-H), 4.47 (s, 2H, 5-OCH2CH2CH2CH2C3H3N2), 4.20 (s, 2H, 5-OCH2CH2CH2CH2C3H3N2), 2.0 (s, 4H, 5-OCH2CH2CH2CH2C3H3N2), 1.75 (s, 2H, 5-OCH2CH2CH2CH2C3H3N2); MS (70 eV) m/z: 324 (29%, M+), 202 (6%, [C11H6O4]+), 174 (6%, [202-CO]+), 123 (99%), 81 (26%), 69 (13%); calculated for C18H16N2O4(324.34): C, 66.66%; H, 4.97%; N, 8.64%; O, 19.73%. Found: C, 66.53%; H, 4.96%; N, 8.56%; LogP, 2.42.

5-[4-N-(4-Pyridinyl)aminobutoxy]psoralen (PH-3). 5-(4-Chlorobutoxy)psoralen (390 mg, 1.3 mmol) and 4-aminopyridine (628 mg, 6.7 mmol) were refluxed in 20 ml of acetonitrile for 45 h. The crude product was recrystallized from 2% acidic acetone (172 mg, 30.4%): m.p. = 133.9°C; 1H NMR (500 MHz, DMSO-d6): {delta} [ppm] = 8.27 (s, 1H, 5-OCH2CH2CH2CH2NHC5H4N), 8.25 (d, 2H, 3J = 7.41 Hz, 5-OCH2CH2CH2CH2NHC5H4N), 8.18 (d, 1H, 3J = 9.8 Hz, 3-H), 8.05 (d, 1H, 3J = 2.6 Hz, 5'-H), 7.36 (s, 1H, 8-H), 7.33 (d, 1H, 3J = 2.3 Hz, 4'-H), 6.85 (d, 2H, 3J = 7.3 Hz, 5-OCH2CH2CH2CH2NHC5H4N), 6.32 (d, 1H, 3J = 9.8 Hz, 4-H), 4.52 (t, 2H, 3J = 6.1 Hz, 5-OCH2CH2CH2NHC5H4N), 4.22 (t, 2H, 3J = 7.0 Hz, 5-OCH2CH2CH2CH2NHC5H4N), 1.99 (p, 2H, 5-OCH2CH2CH2CH2NHC5H4N), 1.77 (p, 2H, 5-OCH2CH2CH2CH2NHC5H4N); MS (70 eV) m/z: 350 (12%, M+), 202 (99%, [C11H6O4]+), 174 (60%, [202-CO]+), 184 (20%), 145 (11%), 123 (15%), 107 (46%), 94 (7%, C5H6N2); calculated for C20H18N2O4 as dihydrochloride salt (423.38): C, 56.68%; H, 4.72%; N, 6.61%; O, 18.27%. Found: C, 56.69%; H, 4.84%; N, 6.58%; LogP, 1.79.

5-[4-(5-Methyl-1,3,4-thiadiazol-2-thiolyl)butoxy]psoralen (PH-4). 5-(4-Chlorobutoxy)psoralen (500 mg, 1.7 mmol), 2-mercapto-1,3,4-thiadiazole (361 mg, 2.7 mmol) and potassium carbonate (2.0 g) were refluxed in 30 ml of 2-butanone for 66 h. The oily residue was recrystallized from a petroleum ether/ethyl acetate (80:20) mixture (107 mg, 16.13%): m.p. = 92.1°C; 1H NMR (500 MHz, CDCl3): {delta} [ppm] = 8.15 (d, 1H, 3J = 9.8 Hz, 3-H), 7.59 (d, 1H, 3J = 2.5 Hz, 5'-H), 7.16 (s, 1H, 8-H), 6.95 (d, 1H, 3J = 2.5 Hz, 4'-H), 6.29 (d, 1H, 3J = 9.8 Hz, 4-H), 4.51 (t, 2H, 3J = 5.8 Hz, 5-OCH2CH2CH2CH2S-), 3.43 (t, 2H, 3J = 6.9 Hz, 5-OCH2CH2CH2CH2S-), 2.74 (s, 3H, 5''-CH3), 2.09 (m, 4H, 3J = 3.0 Hz, 5-OCH2CH2CH2CH2S-); MS (70 eV) m/z: 388 (62%, M+), 202 (14%, [C11H6O4]+), 187 (96%, C7H11N2S2), 174 (12%, [202-CO]+), 145 (10%), 133 (32%), 99 (34%, C3H3N2S), 87 (8%, C4H7S), 55 (28%, C4H7); calculated for C18H16N2O4S2 (388.47): C, 55.65%, H, 4.15%; N, 7.21%; O, 16.47%; S, 16.51%. Found: C, 55.44%; H, 4.15%; N, 7.39%; S, 16.77%; LogP, 2.85.

5-[4-(7-Coumarinyloxy)butoxy]psoralen (PH-5). 5-(4-Chlorobutoxy)psoralen (500 mg, 1.7 mmol), 7-hydroxycoumarin (443 mg, 2.7 mmol) and potassium carbonate (2.0 g) were refluxed in 30 ml of 2-butanone for 68 h. The oily residue obtained was recrystallized from a methanol/acetone (70:30) mixture (134 mg, 18.8%): m.p. = 147°C; 1H NMR (500 MHz, CDCl3): {delta} [ppm] = 8.15 (d, 1H, 3J = 9.8 Hz, 3-H), 7.64 (d, 1H, 3J = 9.5 Hz, 5-OCH2CH2CH2CH2OC9H5O2), 7.61 (d, 1H, 3J = 2.1 Hz, 5'-H), 7.36 (dd, 1H, 3J = 8.6 Hz, 53J = 2.5 Hz, 5-OCH2CH2CH2CH2OC9H5O2), 7.16 (s, 1H, 8-H), 6.99 (d, 1H, 3J = 2.1 Hz, 4'-H), 6.83 (m, 2H, 5-OCH2CH2CH2CH2OC9H5O2), 6.26 (d, 1H, 3J = 9.5 Hz, 4-H), 6.20 (d, 1H, 3J = 9.8 Hz, 5-OCH2CH2CH2CH2OC9H5O2), 4.57 (t, 2H, 3J = 5.4 Hz, 5-OCH2CH2CH2CH2OC9H5O2), 4.15 (t, 2H, 3J = 5.0 Hz, 5-OCH2CH2CH2CH2OC9H5O2), 2.11 (m, 4H, 3J = 2.6 Hz, 5-OCH2CH2CH2CH2OC9H5O2); MS (70 eV) m/z: 418 (34%, M+), 378 (68%), 217 (89%), 202 (20%, [C11H6O4]+), 175 (100%), 174 (14%, [202-CO]+), 187 (16%), 145 (32%), 134 (26%), 89 (30%), 55 (48%, C4H7); calculated for C24H18O7 (418.41) C, 68.90%; H, 4.34%; O, 26.77%. Found: C, 69.08%, H, 4.46%; LogP, 3.34.

5-[4-(5-Methoxy-1,3-benzothiazol-2-thiolyl)butoxy]psoralen (PH-8). 2-Mercapto-5-methoxy-1,3-benzothiazole (539 mg, 2.7 mmol) and potassium hydroxide (161 mg, 2.8 mmol) were refluxed in 25 ml of methanol until the solution was clear. The solution was then concentrated to dryness under vacuum to obtain the dry potassium salt. To this potassium salt was added 20 ml of anhydrous acetonitrile, 5-(4-chlorobutoxy)psoralen (500 mg, 1.7 mmol), and sodium iodide (333 mg, 2.2 mmol), and the resulting mixture was refluxed for 69 h. The oily residue was recrystallized from a petroleum ether/acetone (80:20) mixture (600 mg, 48.8%): m.p. = 134.8°C; 1H NMR (500 MHz, CDCl3): {delta} [ppm] = 8.09 (d, 1H, 3J = 9.8 Hz, 3-H), 7.61 (d, 1H, 3J = 8.9 Hz, benzothiazole), 7.58 (d, 1H, 3J = 2.3 Hz, 5'-H), 7.35 (d, 1H, 4J = 2.2 Hz, benzothiazole), 7.14 (s, 1H, 8-H), 6.97 (dd, 1H, 3J = 8.9 Hz, 4J = 2.1 Hz, benzothiazole), 6.95 (d, 1H, 3J = 2.8 Hz, 4'-H), 6.18 (d, 1H, 3J = 9.8 Hz, 4-H), 4.53 (t, 2H, 3J = 5.8 Hz, 5-OCH2CH2CH2CH2S-), 3.87 (s, 3H, O-CH3), 3.48 (t, 2H, 3J = 6.6 Hz, 5-OCH2CH2CH2CH2S-), 2.11 (m, 4H, 5-OCH2CH2CH2CH2S-); MS (70 eV) m/z: 455 (6%, M+), 453 (44%), 328 (28%), 252 (100%, C12H14NOS2), 201 (6%), 196 (12%, C8H6NOS2), 174 (14%, [202-CO]+), 145 (8%), 89 (6%), 55 (28%, C4H7); calculated for C23H19NO5S2 (455.56): C, 60.70%; H, 4.65%; N, 3.07%; O, 17.64%; S, 14.08%. Found: C, 60.70%; H, 4.59%; N, 3.09%; S, 13.94%; LogP, 4.36.

5-[4-(Pyrimidin-2-thiolyl)butoxy]psoralen (PH-9). 2-Mercaptopyrimidine (306 mg, 2.7 mmol) and potassium hydroxide (163 mg, 2.8 mmol) were refluxed in 50 ml of methanol until the solution was clear. The solution was then concentrated to dryness under reduced pressure to obtain the potassium salt. To the potassium salt was added acetonitrile (30 ml), 5-(4-chlorobutoxy)psoralen (500 mg, 1.7 mmol), and sodium iodide (333 mg, 2.2 mmol), and the mixture was refluxed for 67 h. The oily residue was recrystallized from methanol (244 mg, 38.78%): m.p. = 107.1°C; 1H NMR (500 MHz, CDCl3): {delta} [ppm] = 8.51 (d, 2H, pyrimidine), 8.14 (d, 1H, 3J = 10.1 Hz, 3-H), 7.59 (d, 1H, 3J = 2.4 Hz, 5'-H), 7.16 (s, 1H, 8-H), 7.0 (t, 1H, 3J = 4.9 Hz, pyrimidine), 6.96 (d, 1H, 3J = 1.5 Hz, 4'-H), 6.25 (d, 1H, 3J = 9.8 Hz, 4-H), 4.52 (t, 2H, 3J = 5.8 Hz, 5-OCH2CH2CH2CH2S-), 3.29 (t, 2H, 3J = 6.8 Hz, 5-OCH2CH2CH2CH2S-), 2.01 (m, 4H, 5-OCH2CH2CH2CH2S-); MS (70 eV) m/z: 368 (27%, M+), 202 (8%, [C11H6O4]+), 167 (100%, C8H11N2S), 125 (34%), 113 (37%), 55 (26%, C4H7); calculated for C19H16N2O4S (368.41): C, 61.94%; H, 4.38%; N, 7.60%; O, 17.37%; S, 8.70%. Found: C, 61.85%; H, 4.34%; N, 7.41%; S, 8.66%; LogP, 3.00.

5-[4-(N-Phthalimido)butoxy]psoralen (PP-1). 5-HOP (500 mg, 2.5 mmol), N-(4-bromobutyl)phthalimide (1.12 g, 3.9 mmol), and potassium carbonate (2.2 g) were refluxed in 50 ml of acetonitrile for 72 h. The crude solid was recrystallized from methanol (260 mg, 26.1%): m.p. = 177.9°C; 1H NMR (500 MHz, CDCl3): {delta} [ppm] = 8.14 (d, 1H, 3J = 9.8 Hz, 3-H), 7.8 (dd, 4H, 3J = 5.4 Hz, 4J = 3.0 Hz, 5-OCH2CH2CH2CH2NC8H4O2), 7.6 (d, 1H, 3J = 2.4 Hz, 5'-H), 7.13 (s, 1H, 8-H), 6.95 (d, 1H, 3J = 1.6 Hz, 4'-H), 6.30 (d, 1H, 3J = 9.8 Hz, 4-H), 4.5 (t, 2H, 3J = 5.7 Hz, 5-OCH2CH2CH2CH2NC8H4O2), 3.82 (t, 2H, 3J = 6.6 Hz, 5-OCH2CH2CH2CH2NC8H4O2), 1.98 (p, 4H, 3J = 3.2 Hz, 5-OCH2CH2CH2CH2NC8H4O2); MS (70 eV) m/z: 403 (13%, M+), 202 (72%, [C11H6O4]+), 174 (8%, [202-CO]+), 160 (99%), 148 (6%), 130 (14%), 55 (5%, C4H7); calculated for C23H17NO6 (403.40): C, 68.48%; H, 4.25%; N, 3.47%; O, 23.80%. Found: C 68.17%; H, 4.32%; N, 3.36%; LogP, 3.21.

5-(4-Pyrimidynyloxybutoxy)psoralen (PAP-17). 5-(4-Chlorobutoxy)psoralen (500 mg, 1.7 mmol), sodium iodide (512 mg, 3.4 mmol), 4-hydroxypyrimidine (263 mg, 3.4 mmol), and potassium carbonate (3.0 g) were refluxed in 30 ml of acetonitrile for 90 h. The crude product mixture was separated by column chromatography on silica gel (60-200 mesh, 50 g) by gradient elution using a petroleum ether/ethyl acetate mixture and yielded PAP-17 and PAP-17a. PAP-17 (99.6 mg, 19.92%, m.p. = 126.4°C) was formed by O-alkylation of 4-hydroxypyrimidine: 1H NMR (500 MHz, CDCl3): {delta} [ppm] = 8.80 (s, 1H, 2''-H), 8.47 (d, 1H, 3J = 9.0 Hz, 6''-H), 8.17 (d, 1H, 3J = 9.7 Hz, 3-H), 7.60 (d, 1H, 3J = 2.2 Hz, 5'-H), 7.17 (s, 1H, 8-H), 6.96 (d, 1H, 3J = 1.8 Hz, 4'-H), 6.74 (d, 1H, 3J = 5.4 Hz, 5''-H) 6.30 (d, 1H, 3J = 9.7 Hz, 4-H), 4.54 (t, 2H, 3J = 5.3 Hz, 5-OCH2CH2CH2CH2OC4H3N2), 4.50 (t, 2H, 3J = 5.1 Hz, 5-OCH2CH2CH2CH2OC4H3N2), 2.2 (m, 4H, 5-OCH2CH2CH2CH2OC4H3N2); 13C-NMR (DMSO-d6, 75 MHz): {delta} [ppm] = 25.38 and 26.68 (5-OCH2(CH2)2CH2OC4H3N2); 65.90 and 72.29 (5-OCH2(CH2)2CH2OC4H3N2); 93.97 (C-8); 105.01 (C-4'); 106.66 (C-4a); 112.61 (C-3 and C-5''); 113.18 (C-6); 139.16 (C-4); 144.84 (C-5'); 148.74 (C-5); 152.63 (C-8a); 156.98 (C-6''); 158.20 (C-2'') 158.34 (C-7); 161.15 (C-2); 168.83 (C-4''). MS (70 eV) m/z: 352 (24%, M+), 202 (58%, [C11H6O4]+), 174 (29%, [202-CO]+), 151 (100%, C8H11N2O), 145 (12%, [174-CO]), 109 (8%, [C6H5O2]+), 97 (60%); molecular weight calculated for C19H16N2O5, 352.10592; found by high-resolution mass spectrometry, 352.10596.

5-[4-(4-Oxopyrimidin-3-yl)butoxy]psoralen (PAP-17a). PAP-17a was formed as a side product during the synthesis of PAP-17. During the alkylation reaction, 4-hydroxypyrimidine tautomerises to 4-oxopyrimidine which through N-alkylation formed PAP-17a (248 mg, 49.5%, m.p = 178.1°C): 1H NMR (500 MHz, CDCl3): {delta} [ppm] = 8.21 (s, 1H, 6''-H), 8.13 (d, 1H, 3J = 10.1 Hz, 3-H), 7.91 (d, 1H, 3J = 6.3 Hz, 3''-H), 7.60 (d, 1H, 3J = 1.7 Hz, 5'-H), 7.17 (s, 1H, 8-H), 6.94 (d, 1H, 3J = 2.6 Hz, 4'-H), 6.48 (d, 3J = 6.1 Hz, 4''-H), 6.29 (d, 1H, 3J = 9.9 Hz, 4-H), 4.49 (t, 2H, 3J = 6.0 Hz, 5-OCH2CH2CH2CH2OC4H3N2), 4.06 (t, 2H, 3J = 7.35 Hz, 5-OCH2CH2CH2CH2OC4H3N2), 2.2 (m, 4H, 5-OCH2CH2CHCH2OC4H3N2); 13C-NMR (DMSO-d6, 75 MHz): {delta} [ppm] = 26.01 and 27.06 (5-O-CH2(CH2)2CH2OC4H3N2); 46.92 and 72.01 (5-OCH2(CH2)2CH2OC4H3N2); 94.28 (C-8); 104.88 (C-4'); 106.81 (C-4a); 112.87 (C-3); 113.44 (C-6); 116.16 (C-3''); 139.03 (C-4); 145.03 (C-5'); 148.50 (C-5); 151.02 (C-4''); 152.63 (C-8a); 153.11 (C-6'') 158.17 (C-7); 160.82 (C-2''); 161.11 (C-2). MS (70 eV) m/z: 352 (13%, M+), 202 (12%, [C11H6O4]+), 174 (10%, [202-CO]+), 151 (100%, C8H11N2O), 145 (5%, [174-CO]+), 109 (6%, [C6H5O2]+), 97 (27%); molecular weight calculated for C19H16N2O5, 352.1059; found by high-resolution mass spectrometry, 352.1066.

5-(4-Phenoxybutoxy)psoralen (PAP-1). 5-HOP (700 mg, 3.5 mmol), 4-phenoxybutyl bromide (600 mg, 3.5 mmol), and potassium carbonate (2.0 g) were refluxed in 30 ml of 2-butanone for 24 h. The crude product was recrystallized from methanol-acetone (80:20) as a white solid (734 mg, 60.5%): m.p. = 104°C; 1H NMR (500 MHz, CDCl3): {delta} [ppm] = 8.13 (d, 1H, 3J = 9.7 Hz, 3-H), 7.59 (d, 1H, 3J = 2.0 Hz, 5'-H), 7.30 (m, 5H, 5-OCH2CH2CH2CH2OC6H5), 7.15 (s, 1H, 8-H), 6.91 (d, 1H, 3J = 2.0 Hz, 4'-H), 6.25 (d, 1H, 3J = 9.8 Hz, 4-H), 4.56 (t, 2H, 3J = 6.14 Hz, 5-OCH2CH2CH2CH2OC6H5), 4.09 (t, 2H, 3J = 5.80 Hz, 5-OCH2CH2CH2CH2OC6H5), 2.09 (m, 4H, 3J = 4.21 Hz, 5-OCH2CH2CH2CH2OC6H5); 13C-NMR (DMSO-d6, 75 MHz): {delta} [ppm] = 25.26 and 26.18 (5-O-CH2(CH2)2CH2OC6H5); 66.91 and 72.29 (5-OCH2(CH2)2CH2OC6H5); 93.18 (C-8); 105.62 (C-4'); 105.98 (C-4a); 112.29 (C-3); 112.92 (C-6); 114.39 (C-3'' and C-5''); 120.39 (C-4''); 129.41 (C-2'' and C-6''); 139.44 (C-4); 145.89 (C-5'); 148.72 (C-5); 152.11 (C-8a); 157.63 (C-1''); 158.48 (C-7); 160.07 (C-2); MS (70 eV) m/z: 350 (20%, M+), 202 (9%, [C11H6O4]+), 201 (5%), 174 (13%, [202-CO]+), 173 (4%), 150 (11%), 149 (100%), 145 (8%), 107 (100%, [149-C3H6]+), 94 (9%, C6H6O), 89 (4%), 77 (37%, C6H5), 65 (6%, C5H5); calculated for C21H18O5 (350.37): C, 71.99%; H, 5.18%; O, 22.83%. Found: C,71.92%; H, 5.08%; LogP, 4.03.

5-(3-Phenoxypropoxy)psoralen (PAP-3). 5-HOP (700 mg, 3.5 mmol), 3-phenoxypropyl bromide (750 mg, 3.5 mmol), and potassium carbonate (3.0 g) were refluxed in 30 ml of 2-butanone for 36 h. The oily residue was recrystallized from methanol/ethyl acetate (10:90) as a white solid (390 mg, 33.5%): m.p. = 108.4°C; 1H NMR (500 MHz, CDCl3): {delta} [ppm] = 8.13 (d, 1H, 3J = 9.8 Hz, 3-H), 7.59 (d, 1H, 3J = 2.3 Hz, 5'-H), 7.31 (t, 3H, 3''-H, 4''-H, 5''-H), 7.16 (s, 1H, 8-H), 6.99 (d, 1H, 3J = 2.4 Hz, 4'-H), 6.93 (d, 2H, 2''-H, 6''-H), 6.24 (d, 1H, 3J = 9.5 Hz, 4-H), 4.66 (t, 2H, 3J = 5.9 Hz, 5-OCH2CH2CH2OC6H5), 4.26 (t, 2H, 3J = 6.0 Hz, 5-OCH2CH2CH2OC6H5), 2.38 (p, 2H, 3J = 6.0 Hz, 5-OCH2CH2CH2OC6H5); MS (70 eV) m/z: 336 (91%, M+), 203 (7%), 202 (57%, [C11H6O4]+), 201 (11%), 174 (16%, [202-CO]), 173 (11%), 145 (14%), 135 (90%), 134 (9%), 108 (8%), 107 (100%), 95 (8%), 89 (9%), 77 (62%, C6H5), 65 (9%, C5H5); calculated for C21H18O5 (336.35): C, 71.42%; H, 4.79%; O, 23.78%. Found: C, 71.09%, H, 4.74%. LogP: 3.76.

5-(2-Benzyloxyethoxy)psoralen (PAP-5). 5-HOP (600 mg, 2.9 mmol), benzyl-2-bromoethyl ether (1.0 g, 4.6 mmol), and potassium carbonate (2.0 g) were refluxed in 30 ml of 2-butanone for 16 h. The oily residue was recrystallized from 70% methanol to obtain a white solid (123 mg, 12.3%): m.p. = 90.9°C; 1H NMR (500 MHz, CDCl3): {delta} [ppm] = 8.19 (d, 1H, 3J = 9.7 Hz, 3-H), 7.59 (d, 1H, 3J = 2.2 Hz, 5'-H), 7.37 (m, 5H, 5-OCH2CH2OCH 2C6H5), 7.19 (s, 1H, 8-H), 6.95 (d, 1H, 3J = 2.0 Hz, 4'-H), 6.25 (d, 1H, 3J = 9.7 Hz, 4-H), 4.64 (s, 2H, 5-OCH2CH2OCH2C6H5), 4.58 (t, 2H, 3J = 4.62 Hz, 5-OCH2CH2OCH2C6H5), 3.88 (t, 2H, 3J = 4.56 Hz, 5-OCH2CH2OCH2C6H5); MS (70 eV) m/z: 336 (35%, M+), 105 (5%), 91 (100%, [C7H7]+); calculated for C20H16O5 (336.35): C, 71.42%; H, 4.79%; O, 23.78%. Found: C, 70.65%; H, 4.73%; LogP, 3.34.

5-(5-Phenoxypentoxy)psoralen (AS-121). 1-Bromo-5-phenoxypentane was synthesized according to general method C using 1,5-dibromopentane instead of 1,4-dibromobutane. 5-HOP (300 mg, 1.5 mmol), 1-bromo-5-phenoxypentane (600 mg, 2.6 mmol), and potassium carbonate (2.0 g) were then refluxed in 30 ml of acetone for 24 h. The crude product was recrystallized from methanol/water (80:20) as a white solid (232 mg, 42.4%): m.p. = 91°C; 1H NMR (300 MHz, DMSO-d6): {delta} [ppm] = 8.19 (d, 1H, 3J = 9.7 Hz, 4-H), 8.02 (s, 1H, 5'-H), 7.25 to 7.33 (m, 4H, 8-H, 4'-H and 5-OCH2CH2CH2CH2CH2OC6H5), 6.90 to 6.93 (m, 3H, 5-OCH2CH2CH2CH2CH2OC6H5), 6.30 (d, 1H, 3J = 9.7 Hz, 3-H), 4.52 (t, 2H, 3J = 6.0 Hz, 5-OCH2CH2CH2CH2OC6H5), 4.00 (t, 2H, 3J = 6.2 Hz, 5-OCH2CH2CH2CH2CH2OC6H5), 1.77 to 1.93 (m, 4H, 5-OCH2CH2CH2CH2CH2OC6H5), 1.63 to 1.71 (m, 2H, 5-OCH2CH2CH2CH2CH2OC6H5); MS (70 eV) m/z: 364 M+ (9%, M+), 202 (22%, [C11H6O4]+), 163 (44%, [C11H15O]+), 107 (40%, [C6H5O-CH2]+), 69 (100%, [C5H9]+), 41 (52%, [C3H5]+); calculated for C22H20O5 (364.40): C, 72.51%; H, 5.53%; O, 21.96%. Found: C, 72.74%; H, 5.68%; LogP, 4.40.

5-(4-Benzyloxybutoxy)psoralen (PAP-6). 5-HOP (700 mg, 3.5 mmol), benzyl-4-bromobutyl ether (850 mg, 3.5 mmol), and potassium carbonate (2.0 g) were refluxed in 30 ml of 2-butanone for 24 h. The oily residue was recrystallized from 80% methanol (171 mg, 13.4%): m.p. = 78.4°C; 1H NMR (500 MHz, CDCl3): {delta} [ppm] = 8.14 (d, 1H, 3J = 9.8 Hz, 3-H), 7.55 (d, 1H, 3J = 2.5 Hz, 5'-H), 7.34 (m, 5H, 5-OCH2CH2CH2CH2OCH2C6H5), 7.13 (s, 1H, 8-H), 6.91 (d, 1H, 3J = 2.4 Hz, 4'-H), 6.25 (d, 1H, 3J = 9.8 Hz, 4-H), 4.54 (s, 2H, 5-OCH2CH2CH2CH2OCH2C6H5), 4.49 (t, 2H, 3J = 6.5 Hz, 5-OCH2CH2CH2CH2OCH2C6H5), 3.59 (t, 2H, 3J = 6.1 Hz, 5-OCH2CH2CH2CH2OCH2C6H5), 2.00 (p, 2H, 3J = 6.9 Hz, 5-OCH2CH2CH2CH2OCH2C6H5), 1.87 (p, 2H, 3J = 6.8 Hz, 5-OCH2CH2CH2CH2OCH2C6H5); MS (70 eV) m/z: 364 (37%, M+), 292 (10%), 202 (7%, [C11H6O4]+), 174 (6%, [202-CO]+), 163 (12%), 91 (100%, C7H7), 71 (8%); calculated for C22H20O5 (364.40): C, 72.51%; H, 5.53%; O, 21.95%. Found: C, 72.36%; H, 5.46%; LogP, 4.02.

5-(3-Benzyloxypropoxy)psoralen (PAP-7). 5-HOP (1.0 g, 4.9 mmol), benzyl-3-bromopropyl ether (1.36 g, 5.9 mmol), and potassium carbonate (3.4 g) were refluxed in 30 ml of 2-butanone for 24 h. The oily residue was recrystallized from 70% methanol (700 mg, 40.4%): m.p. = 75.2°C; 1H NMR (500 MHz, CDCl3): {delta} [ppm] = 8.06 (d, 1H, 3J = 9.7 Hz, 3-H), 7.57 (d, 1H, 3J = 2.2 Hz, 5'-H), 7.29 (m, 5H, 3J = 6.3 Hz, 5-OCH2CH2CH2OCH2C6H5), 7.15 (s, 1H, 8-H), 6.98 (d, 1H, 3J = 2.2 Hz, 4'-H), 6.21 (d, 1H, 3J = 9.8 Hz, 4-H), 4.58 (t, 2H, 3J = 6.1 Hz, 5-OCH2CH2CH2OCH2C6H5), 4.55 (s, 2H, 5-OCH2CH2CH2OCH2C6H5), 3.73 (t, 2H, 3J = 5.7 Hz, 5-OCH2CH2CH2OCH2C6H5), 2.18 (p, 2H, 3J = 6.1 Hz, 5-OCH2CH2CH2OCH2C6H5); MS (70 eV) m/z: 350 (25%, M+), 202 (9%, [C11H6O4]+ 174 (5%, [202-CO]+), 91 (100%, [C7H7]+); calculated for C21H18O5(350.37): C, 71.99%; H, 5.18%; O, 22.83%. Found: C, 71.64%; H, 5.34%; LogP, 3.66.

5-(4-Phenyl-3-oxobutoxy)psoralen (KP-1). 4-Chlorobutyrophenone (497 mg, 2.7 mmol) and sodium iodide (445 mg, 2.9 mmol) were refluxed in 30 ml of acetone for 1.5 h to obtain the iodo derivative. To this slurry were added 5-HOP (500 mg, 2.5 mmol), potassium carbonate (2.0 g), and the resulting mixture was then refluxed for 140 h. The solid residue was recrystallized from a petroleum ether/acetone (90:10) mixture (295 mg, 34.3%): m.p. = 129.1°C; 1H NMR (500 MHz, CDCl3): {delta} [ppm] = 8.13 (d, 1H, 3J = 9.9 Hz, 3-H), 8.01 (d, 2H, 3J = 7.9 Hz, 4J = 0.95 Hz, 2''-H, 6''-H), 7.62 (t, 1H, 3J = 7.4 Hz, 3''-H, 5''-H), 7.60 (d, 1H, 3J = 2.4 Hz, 5'-H), 7.50 (t, 2H, 3J = 7.7 Hz, 4''-H), 7.15 (s, 1H, 8-H), 6.99 (d, 1H, 3J = 2.5 Hz, 4'-H), 6.26 (d, 1H, 3J = 9.8 Hz, 4-H) 4.59 (t, 2H, 3J = 6.2 Hz, 5-OCH2CH2CH2COC6H5), 3.28 (t, 2H, 3J = 6.8 Hz, 5-OCH2CH2CH2COC6H5), 2.38 (p, 2H, 3J = 6.5 Hz, 5-OCH2CH2CH2COC6H5); MS (70 eV) m/z: 348 (34%, M+), 202 (5%, [C11H6O4]+), 147(99%, C10H10O), 174, (5%, [202-CO]+), 105 (71%, C3H6), 77 (33%, C6H5); calculated for C21H16O5 (348.36): C, 72.41%; H, 4.63%; O, 22.96%. Found: C, 72.18%; H, 4.75%; LogP, 3.31.

5-[4-(4-Methoxyphenoxy)butoxy]psoralen (AS-67). 5-HOP (300 mg, 1.5 mmol), 1-bromo-4-(4-methoxyphenoxy)butane (600 mg, 2.5 mmol, obtained by general method C), and potassium carbonate (2.0 g) were refluxed in 30 ml of acetone for 24 h. The crude product was recrystallized from methanol-water (80:20) as a white solid (201 mg, 35.2%): m.p. = 111.5°C; 1H NMR (300 MHz, DMSO-d6): {delta} [ppm] = 8.18 (d, 1H, 3J = 9.8 Hz, 4-H), 8.03 (d, 1H, 3J = 2.3 Hz, 5'-H), 7.32 to 7.34 (m, 2H, 8-H and 4'-H), 6.81 to 6.87 (m, 4H, 5-OCH2(CH2)2CH2OC6H4OCH3), 6.81 to 6.87 (m, 4H, 5-OCH2(CH2)2CH2OC6H4OCH3), 6.30 (d, 1H, 3J = 9.8 Hz, 3-H), 4.57 (t, 2H, 3J = 5.7 Hz, 5-OCH2(CH2)2CH2OC6H4OCH3), 4.00 (t, 2H, 3J = 5.8 Hz, 5-OCH2(CH2)2CH2OC6H4OCH3), 3.69 (s, 3H, -OCH3), 1.91 to 1.99 (m, 4H, 5-OCH2(CH2)2CH2OC6H4OCH3); MS (70 eV) m/z: 380 (14%, M+), 257 (8%), 215 (7%), 202 (5%, [C11H6O4]+), 179 (69%, [C11H15O2]+), 145 (6%), 137 (100%, [CH3OC6H4OCH2]+), 109 (29%), 107 (18%, [C6H5OCH2]+), 77 (23%, [C6H5]+_, 55 (61%, [C4H7]+), 41 (15%, [C3H5]+); calculated for C22H20O6 (380.4): C, 69.46%; H, 5.30%; O, 25.24%. Found: C, 69.52%; H, 5.39%; LogP, 3.89.

5-[4-(3-Methoxyphenoxy)butoxy]psoralen (AS-68). 5-HOP (300 mg, 1.5 mmol), 1-bromo-4-(3-methoxyphenoxy)butane (600 mg, 2.5 mmol, obtained by general method C), and potassium carbonate (2.0 g) were refluxed in 30 ml of acetone for 24 h. The crude product was recrystallized from methanol-water (80:20) as a white solid (178 mg, 31.2%): m.p. = 102.5°C; 1H NMR (300 MHz, DMSO-d6): {delta} [ppm] = 8.18 (d, 1H, 3J = 9.8 Hz, 4-H), 8.02 (d, 1H, 3J = 1.9 Hz, 5''-H), 7.33 (s, 2H, 8-H and 4'-H), 7.15 (t, 1H, 3J = 8.14 Hz, 8-H), 6.45 to 6.51 (m, 3H, 2''-H, 4''-H and 6''-H), 6.29 (d, 1H, 3J = 9.8 Hz, 3-H), 4.58 (t, 2H, 3J = 5.4 Hz, 5-OCH2(CH2)2CH2OC6H4OCH3), 4.05 (t, 2H, 3J = 5.4 Hz, 5-O-CH2(CH2)2CH2OC6H4OCH3), 3.71 (s, 3H, -OCH3), 1.91 to 1.99 (m, 4H, 5-OCH2(CH2)2CH2OC6H4OCH3); MS (70 eV) m/z: 380 (14%, M+), 257 (8%), 202 (5%, [C11H6O4]+), 179 (84%, [C11H15O2]+), 145 (6%), 137 (100%, [CH3OC6H4OCH2]+), 109 (14%), 107 (32%, [C6H5OCH2]+), 77 (27%, [C6H5]+), 55 (63%, [C4H7]+), 41 (12%, [C3H5]+); calculated for C22H20O6 (380.4): C, 69.46%; H, 5.30; O, 25.24%. Found: C, 69.89%; H, 5.38%; LogP, 3.99.

5-[4-(3,5-Dimethoxyphenoxy)butoxy]psoralen (AS-69). 5-HOP (300 mg, 1.5 mmol), 1-bromo-4-(3,5-dimethoxyphenoxy)butane (700 mg, 2.6 mmol, obtained by general method C) and potassium carbonate (2.0 g) were refluxed in 30 ml of acetone for 24 h. The crude product was recrystallized from methanol/acetone (80:20) as a white solid (182 mg, 29.6%): m.p. = 139°C; 1H NMR (300 MHz, DMSO-d6): {delta} [ppm] = 8.19 (d, 1H, 3J = 9.8 Hz, 4-H), 8.03 (d, 1H, 3J = 2.1 Hz, 5'-H), 7.34 (s, 2H, 8-H and 4'-H), 6.31 (d, 1H, 3J = 9.8 Hz, 3-H), 6.07 (s, 3H, 2''-H, 4''-H and 6''-H), 4.58 (t, 2H, 3J = 5.4 Hz, 5-OCH2(CH2)2CH2OC6H3(OCH3)2), 4.05 (t, 2H, 3J = 5.4 Hz, 5-OCH2(CH2)2CH2OC6H3(OCH3)2), 3.69 (s, 6H, -(OCH3)2), 1.93 to 1.97 (m, 4H, 5-OCH2(CH2)2CH2OC6H3(OCH3)2); MS (70 eV) m/z: 410 (12%, M+), 209 (100%, [C12H17O3]+), 202 (5%, [C11H6O4]+), 167 (75%, [(CH3O)2C6H3OCH2]+), 137 (34%, [CH3OC6H4OCH2]+), 122 (15%), 107 (10%, [C6H5OCH2]+), 77 (11%, [C6H5]+), 55 (46%, [C4H7]+), 41 (6%, [C3H5]+); calculated for C23H22O7 (410.43): C, 67.31%; H, 5.40%; O, 27.29%. Found: C, 66.92%; H, 5.60%; LogP, 3.94.

5-[4-(4-Nitrophenoxy)butoxy]psoralen (AS-78). 5-HOP (300 mg, 1.5 mmol), 1-bromo-4-(4-nitrophenoxy)butane (700 mg, 2.7 mmol, obtained by general method C), and potassium carbonate (2.0 g) were refluxed in 30 ml of acetone for 24 h. The crude product was recrystallized from methanol/acetone (80:20) as a yellow solid (291 mg, 49.1%): m.p. = 132°C; 1H NMR (300 MHz, DMSO-d6): {delta} [ppm] = 8.16 (d, 2H, 3J = 9.1 Hz, 3''-H and 5''-H), 8.15 (d, 1H, 3J = 9.6 Hz, 4-H), 8.00 (d, 1H, 3J = 2.1 Hz, 5'-H), 7.30 (d, 1H, 3J = 1.9 Hz, 4'-H), 7.28 (s, 1H, 8-H), 7.10 (d, 2H, 3J = 9.2 Hz, 2''-H and 6''-H), 6.27 (d, 1H, 3J = 9.8 Hz, 3-H), 4.56 (s, 2H, 5-OCH2(CH2)2CH2OC6H4NO2), 4.05 (s, 2H, 5-OCH2(CH2)2CH2OC6H4NO2), 1.99 (s, 4H, 5-OCH2(CH2)2CH2OC6H4NO2); MS (70 eV) m/z: 395 (12%, M+), 202 (30%, [C11H6O4]+), 194 (100%, [C10H12O3N]+), 174 (26%, [202-CO]+), 152 (82%, [O2NC6H4OCH2]+), 133 (17%), 106 (17%), 89 (13%), 75 (12%), 55 (84%, [C4H7]+), 41 (11%); calculated for C21H17O7 (395.34): C, 63.80%; H, 4.33%; N, 3.54%; O, 28.33%. Found: C, 63.79%; H, 4.46%; N, 3.60%; LogP, 3.91.

5-[4-(4-Chlorphenoxy)butoxy]psoralen (AS-84). 5-HOP (300 mg, 1.5 mmol), 1-bromo-4-(4-chlorophenoxy)butane (700 mg, 2.8 mmol, obtained by General Method C), and potassium carbonate (2.0 g) were refluxed in 30 ml of acetone for 24 h. The crude product was recrystallized from methanol/water (80:20) as a white solid (247 mg, 42.8%): m.p. = 142.5°C; 1H NMR (300 MHz, DMSO-d6): {delta} [ppm] = 8.15 (d, 1H, 3J = 9.8 Hz, 4-H), 8.01 (d, 1H, 3J = 2.0 Hz, 5'-H), 7.31 (s, 2H, 8-H and 4'-H), 7.29 (d, 2H, 3J = 8.9 Hz, 3''-H and 5''-H), 6.93 (d, 2H, 3J = 8.9 Hz, 2''-H and 6''-H), 6.28 (d, 1H, 3J = 9.8 Hz, 3-H), 4.55 (m, 2H, 5-OCH2(CH2)2CH2OC6H4Cl), 4.05 (m, 2H, 5-OCH2(CH2)2CH2OC6H4Cl), 1.95 to 1.96 (m, 4H, 5-OCH2(CH2)2CH2OC6H4Cl); MS (70 eV) m/z: 384 (10%, M+), 202 (14%, [C11H6O4]+), 183 (80%, [C10H12OCl]+), 174 (11%, [202-CO]+), 141 (100%, [ClC6H4OCH2]+) 113 (18%), 111 (23%), 89 (7%), 77 (9%, [C6H5]), 55 (72%, [C4H7]+), 41 (5%); calculated for C21H17ClO5 (384.82): C, 65.55%; H, 4.45%; Cl, 9.21%; O, 20.79%. Found: C, 65.23%; H, 4.57%; LogP, 4.50.

5-[4-(4-Phenoxyphenoxy)butoxy]psoralen (AS-85). 5-HOP (300 mg, 1.5 mmol), 1-bromo-4-(4-phenoxyphenoxy)butane (800 mg, 2.5 mmol, obtained by general method C), and potassium carbonate (2.0 g) were refluxed in 30 ml of acetone for 24 h. The crude product was recrystallized from ethanol/acetone (70:30) as a white solid (165 mg, 24.9%): m.p. = 137°C; 1H NMR (300 MHz, DMSO-d6): {delta} [ppm] = 8.17 (d, 1H, 3J = 9.8 Hz, 4-H), 8.02 (s, 1H, 5'-H), 7.31 to 7.35 (m, 4H, 8-H, 4'-H and -O-C6H4OC6H5), 7.06 (t, 1H, 4'''-H), 6.89 to 6.96 (m, 6H, -OC6H4OC6H5), 6.29 (d, 1H, 3J = 9.7 Hz, 3-H), 4.57 (s, 2H, 5-OCH2(CH2)2CH2OC6H4OC6H5), 4.05 (s, 2H, 5-OCH2(CH2)2CH2OC6H4OC6H5), 1.97 (s, 4H, 5-OCH2(CH2)2CH2OC6H4OC6H5); MS (70 eV) m/z: 442 (25%, M+), 257 (16%), 241 (100%, [C6H5OC10H12O]+), 215 (12%), 199 (100%, [C6H5OC8H8O]+), 186 (100%, [C6H5OC7H5O]+), 171 (12%), 148 (38%), 129 (13%), 115 (17%), 93 (10%), 77 (49%, [C6H5]+), 55 (70%, [C4H7]+), 41 (7%); calculated for C27H22O6 (442.47): C, 73.29%; H, 5.01%; O, 21.7%. Found: C, 73.24%; H, 5.09%; LogP, 5.03.

5-[4-(4-Methylphenoxy)butoxy]psoralen (AS-96). 5-HOP (300 mg, 1.5 mmol), 1-bromo-4-(4-methylphenoxy)butane (600 mg, 2.5 mmol, obtained by general method C), and potassium carbonate (2.0 g) were refluxed in 30 ml of acetone for 24 h. The crude product was recrystallized from methanol/acetone (80:20) as a white solid (253 mg, 46.3%): m.p. = 128°C; 1H NMR (300 MHz, DMSO-d6): {delta} [ppm] = 8.15 (d, 1H, 3J = 9.8 Hz, 4-H), 8.02 (d, 1H, 3J = 2.1 Hz, 5'-H), 7.31 (s, 2H, 8-H and 4'-H), 7.06 (d, 2H, 3J = 8.4 Hz, 2''-H and 6''-H), 6.80 (d, 2H, 3J = 8.5 Hz, 3''-H and 5''-H), 6.28 (d, 1H, 3J = 9.8 Hz, 3-H), 4.56 (t, 2H, 3J = 5.6 Hz, 5-OCH2(CH2)2CH2OC6H4CH3), 4.02 (t, 2H, 3J = 5.7 Hz, 5-OCH2(CH2)2CH2OC6H4CH3), 2.22 (s, 3H, -CH3), 1.92 to 2.01 (m, 4H, 5-OCH2(CH2)2CH2OC6H4CH3); MS (70 eV) m/z: 364 (10%, M+), 202 (5%, [C11H6O4]+), 163 (87%, [C11H15O]+), 121 (100%, [CH3C6H4OCH2]3), 91 (33%, [C7H7]+), 65 (9%), 55 (35%, [C4H7]+), 41 (4%); calculated for C22H20O5 (364.40): C, 72.51%; H, 5.53%; O, 21.96%. Found: C, 72.59%; H, 5.65%; LogP, 4.42.

5-[4-(4-Fluorphenoxy)butoxy]psoralen (AS-111). 5-HOP (300 mg, 1.5 mmol), 1-bromo-4-(4-fluorophenoxy)butane (600 mg, 2.5 mmol, obtained by general method C), and potassium carbonate (2.0 g) were refluxed in 30 ml of acetone for 24 h. The crude product was recrystallized from methanol/water (80:20) as a white solid (192 mg, 34.7%): m.p. = 121°C; 1H NMR (300 MHz, DMSO-d6): {delta} [ppm] = 8.14 (d, 1H, 3J = 9.8 Hz, 4-H), 8.01 (d, 1H, 3J = 2.0 Hz, 5'-H), 7.30 (s, 2H, 4'-H and 8-H), 6.89 to 6.94 (m, 2H, 5-OCH2(CH2)2CH2OC6H4F), 6.27 (d, 1H, 3J = 9.8 Hz, 3-H), 4.53 to 4.55 (m, 2H, 5-OCH2(CH2)2CH2OC6H4F), 4.01 to 4.05 (m, 2H, 5-OCH2(CH2)2CH2OC6H4F), 1.94 to 1.96 (m, 4H, 5-OCH2(CH2)2CH2OC6H4F); MS (70 eV) m/z: 368 (11%, M+), 202 (11%, [C11H6O4]+), 174 (11%, [202-CO]+), 167 (83%, [C10H12OF]+), 125 (100%, [FC6H4OCH2]+), 95 (23%), 89 (5%), 55 (46%, [C4H7]+), 41 (3%); calculated for C21H17FO5 (368.37): C, 68.47%; H, 4.65%; F, 5.16%; O, 21.72%. Found: C, 68.15%; H, 4.65; LogP, 4.11.

5-[4-(3-Trifluormethylphenoxy)butoxy]psoralen (AS-118). 5-HOP (300 mg, 1.5 mmol), 1-bromo-4-(3-trifluoromethylphenoxy)-butane (700 mg, 2.4 mmol, obtained by general method C), and potassium carbonate (2.0 g) were refluxed in 30 ml of acetone for 24 h. The crude product was recrystallized from methanol/acetone (60:40) as a white solid (142 mg, 22.6%): m.p. = 118°C; 1H NMR (300 MHz, DMSO-d6): {delta} [ppm] = 8.15 (d, 1H, 3J = 9.8 Hz, 4-H), 8.01 (d, 1H, 3J = 2.3 Hz, 5'-H), 7.49 (t, 1H, 3J = 7.93 Hz, 5''-H), 7.18 to 7.32 (m, 5H, 2''-H, 4''-H, 6''-H, 8-H and 4'-H), 6.26 (d, 1H, 3J = 9.8 Hz, 3-H), 4.57 (s, 2H, 5-OCH2(CH2)2CH2OC6H4CF3), 4.15 (s, 2H, 5-O-CH2(CH2)2CH2OC6H4CF3), 1.98 (s, 4H, 5-OCH2(CH2)2CH2OC6H4CF3); MS (70 eV) m/z: 418 (14%, M+), 217 (79%), 202 (19%, [C11H6O4]+), 175 (100%, [C12H15O]+), 145 (37%), 127 (7%), 109 (14%), 89 (6%), 55 (75%, [C4H7]+), 41 (4%); calculated for C22H17F3O5 (418.37): C, 63.16%; H, 4.10%; F, 13.62%; O, 19.12%. Found: C, 63.19%; H, 4.09%; LogP, 4.65.

5-(4-[2-Methoxy-4-nitrophenoxy]butoxy)psoralen (PAP-10). 5-(4-Chlorobutoxy)psoralen (500 mg, 1.7 mmol) and sodium iodide (741 mg, 4.9 mmol) were refluxed in 15 ml of anhydrous acetonitrile for 60 min to obtain the iodo derivative. To this solution were added 4-nitroguaicol (837 mg, 4.9 mmol), potassium carbonate (3.0 g), and 10 ml of acetonitrile, and the mixture was refluxed for 72 h. The solid residue was recrystallized from a methanol/acetone (80:20) mixture (382 mg, 52.6%): m.p. = 170.5°C; 1H NMR (500 MHz, CDCl3): {delta} [ppm] = 8.15 (d, 1H, 3J = 9.8 Hz, 3-H), 7.91 (d, 1H, 4J = 2.6 Hz, 3''-H), 7.75 (dd, 1H, 3J = 2.7 Hz, 5''-H), 7.60 (d, 1H, 3J = 2.2 Hz, 5'-H), 7.16 (s, 1H, 8-H), 6.98 (d, 1H, 3J = 2.4 Hz, 4'-H), 6.93 (d, 1H, 3J = 2.2 Hz, 6''-H), 6.26 (d, 1H, 3J = 9.7 Hz, 4-H), 4.59 (t, 2H, 3J = 6.0 Hz, 5-OCH2CH2CH2CH2OC6H3), 4.23 (t, 2H, 3J = 5.7 Hz, 5-OCH2CH2CH2CH2OC6H3), 3.915 (s, 3H, 2''-OCH3), 2.14 (m, 4H, 5-OCH2CH2CH2CH2OC6H3); MS (70 eV) m/z: 425 (24%, M+), 395 (11%, [C22H19O7]+), 257 (12%), 224 (100%), 202 (44%, [C11H6O4]+), 182 (68%,), 174, (26%, [202-CO]+), 145 (18%, [174-CO]+); molecular weight calculated for C22H19NO8, 425.1110; found by high-resolution mass spectrometry, 425.1112

5-(4-[4-Methyl-2-nitrophenoxy]butoxy)psoralen (PAP-11). 5-(4-Chlorobutoxy)psoralen (500 mg, 1.7 mmol) and sodium iodide (741 mg, 4.9 mmol) were refluxed in 15 ml of acetonitrile for 60 min to obtain the iodo derivative. To this solution were added 2-nitro-p-cresol (523 mg, 3.4 mmol), potassium carbonate (4.0 g), and 10 ml of acetonitrile. The mixture was refluxed for 69 h. The solid residue was recrystallized from a methanol/acetone (80:20) mixture (447 mg, 64.0%): m.p. = 124.5°C; 1H NMR (500 MHz, CDCl3): {delta} [ppm] = 8.14 (d, 1H, 3J = 9.7 Hz, 3-H), 7.63 (d, 1H, 4J = 1.8 Hz, 3''-H), 7.59 (d, 1H, 3J = 2.4 Hz, 5'-H), 7.31 (d, 1H, 3J = 8.2 Hz, 5''-H), 7.14 (s, 1H, 8-H), 6.98 (d, 1H, 3J = 2.5 Hz, 4'-H), 6.96 (d, 1H, 3J = 8.76 Hz, 6''-H), 6.26 (d, 1H, 3J = 9.8 Hz, 4-H), 4.56 (t, 2H, 3J = 5.7 Hz, 5-OCH2CH2CH2CH2OC6H3), 4.17 (t, 2H, 3J = 6.0 Hz, 5-OCH2CH2CH2CH2OC6H3), 2.34 (s, 3H, 4''-CH3), 2.05 (m, 4H, 3J = 4.216 Hz, 5-OCH2CH2CH2C-H2OC6H3); MS (70 eV) m/z: 409 (22%, M+), 379 (20%, [C22H19O6]+), 257 (90%), 224 (100%), 202 (86%, [C11H6O4]+), 174, (52%, [202-CO]+), 145 (30%, [174-CO]+); molecular weight calculated for C22H19NO7, 409.1161; found by high-resolution mass spectrometry, 409.1170

5-(4-[2-Nitrophenoxy]butoxy)psoralen (PAP-12). 5-(4-Chlorobutoxy)psoralen (500 mg, 1.7 mmol) and sodium iodide (512 mg, 3.4 mmol) were refluxed in 15 ml of acetonitrile for 1 h to obtain the iodo derivative. To this solution were added 2-nitrophenol (475 mg, 3.4 mmol), potassium carbonate (4.0 g), and 15 ml of acetonitrile. The mixture was refluxed for 29 h. The solid residue was recrystallized from a methanol/acetone (80:20) mixture (380 mg, 56.3%): m.p. = 121.8°C; 1H NMR (500 MHz, CDCl3): {delta} [ppm] = 8.14 (d, 1H, 3J = 9.7 Hz, 3-H), 7.82 to 7.86 (overlapping dd, 2H, 4J = 1.6 Hz, 3J = 8.3 Hz, 3J = 7.9 Hz, 3''-H, 6''-H), 7.60 (d, 1H, 3J = 2.5 Hz, 5'-H), 7.52 to 7.55 (t, 2H, 3J = 7.6 Hz, 4J = 1.0 Hz, 3''-H, 4''-H), 7.15 (s, 1H, 8-H), 6.99 (d, 1H, 3J = 2.4 Hz, 4'-H), 6.26 (d, 1H, 3J = 9.7 Hz, 4-H), 4.57 (t, 2H, 3J = 5.8 Hz, 5-OCH2CH2CH2CH2OC6H4, 4.23 (t, 2H, 3J = 2.8 Hz, 5-OCH2CH2CH2CH2OC6H4), 2.09 to 2.16 (m, 4H, 5-OCH2CH2CH2CH2OC6H4); MS (70 eV) m/z: 395 (36%, M+), 365 (14%, [M-30]+), 332 (78%), 283 (8%), 202 (9%, [C11H6O4]+ 194 (60%, [C10H12NO3]+), 122 (48%), 109 (8%), 92 (10%); molecular weight calculated for C21H20O5, 395.10050; found by high-resolution mass spectrometry, 395.10053.

5-(4-[3-Nitrophenoxy]butoxy)psoralen (PAP-13). 5-(4-Chlorobutoxy)psoralen (500 mg, 1.7 mmol) and sodium iodide (512 mg, 3.4 mmol) were refluxed in 15 ml of acetonitrile for 1 h to obtain the iodo derivative. To this solution were added 3-nitrophenol (475 mg, 3.4 mmol), potassium carbonate (4.0 g), and 15 ml of acetonitrile, and the mixture was refluxed for 29 h. The solid residue was recrystallized from a methanol/acetone (80:20) mixture (286 mg, 42.4%): m.p. = 140.3°C; 1H NMR (500 MHz, CDCl3): {delta} [ppm] = 8.15 (d, 1H, 3J = 9.7 Hz, 3-H), 7.84 (dd, 1H, 3J = 8.1 Hz, 4J = 1.6 Hz, 4''-H), 7.61 (d, 1H, 3J = 2.3 Hz, 5'-H), 7.42 (t, 1H, 3J = 8.3 Hz, 5''-H), 7.41 (t, 1H, 4J = 2.2 Hz, 2''-H), 7.22 (dd, 1H, 3J = 8.1 Hz, 4J = 2.3 Hz, 6''-H) 7.16 (s, 1H, 8-H), 6.97 (d, 1H, 3J = 2.2 Hz, 4'-H), 6.27 (d, 1H, 3J = 9.8 Hz, 4-H), 4.56 (t, 2H, 3J = 5.8 Hz, 5-OCH2CH2CH2CH2OC6H4), 4.16 (t, 2H, 3J = 5.6 Hz, 5-OCH2CH2CH2CH2OC6H4), 2.12 (m, 4H, 5-OCH2CH2CH2CH2OC6H4); MS (70 eV) m/z: 395 (32%, M+), 365 (14%, [M-30]+), 292 (56%), 202 (100%, [C11H6O4]+), 194 (28%, [C10H12NO3]+), 174 (48%, [202-CO]+), 145 (16%, [174-CO]+), 109 (8%, [C6H5O2]+), 93 (14%, C6H5O); molecular weight calculated for C21H17NO7, 395.10050; found by high-resolution mass spectrometry, 395.10103.

5-(4-[2,4-Dinitrophenoxy]butoxy)psoralen (PAP-14). 5-(4-Chlorobutoxy)psoralen (500 mg, 1.7 mmol), sodium iodide (512 mg, 3.4 mmol), 2,4-dinitrophenol (629 mg, 3.4 mmol), and potassium carbonate (3.0 g) were refluxed in 30 ml of acetonitrile for 50 h. The solid residue was recrystallized from a methanol/acetone (80:20) mixture (82 mg, 11.0%): m.p. = 134.2°C; 1H NMR (500 MHz, CDCl3): {delta} [ppm] = 8.78 (d, 1H, 4J = 2.8 Hz, 3''-H), 8.45 (dd, 1H, 3J = 9.0 Hz, 4J = 2.8 Hz, 5''-H), 8.15 (d, 1H, 3J = 9.7 Hz, 3-H), 7.61 (d, 1H, 3J = 2.0 Hz, 5'-H), 7.22 (d, 1H, 3J = 9.5 Hz, 6''-H), 7.17 (s, 1H, 8-H), 6.98 (d, 1H, 3J = 2.1 Hz, 4'-H), 6.29 (d, 1H, 3J = 9.8 Hz, 4-H), 4.57 (t, 2H, 3J = 5.3 Hz, 5-OCH2CH2CH2CH2OC6H3), 4.36 (t, 2H, 3J = 5.1 Hz, 5-OCH2CH2CH2CH2OC6H3), 2.2 (m, 4H, 5-OCH2CH2CH2CH2OC6H3); MS (70 eV) m/z: 440 (44%, M+), 384 (18%, M+-[C2H2+NO]), 283 (16%), 202 (100%, [C11H6O4]+), 174 (34%, [202-CO]+), 145 (12%, [174-CO]+); molecular weight calculated for C21H16N2O9, 440.08558; found by high-resolution mass spectrometry, 440.08598.

5-(4-Phenoxybutoxy)-4',5'-dihydropsoralen (AS-77). PAP-1 (175 mg, 0.5 mmol) was dissolved in 40 ml of ethanol. After addition of 80 mg of 10% palladium on carbon, it was agitated in a hydration bomb for 5 h under 2.1 bar of pressure. The Pd/C was then filtered off, the solution evaporated, and the remaining residue was purified by medium-pressure lipid chromatography with cyclohexane/ethyl acetate (80:20). Yield: (110 mg, 62.9%); m.p. = 93°C; 1H NMR (300 MHz, DMSO-d6): {delta} [ppm] = 7.99 (d, 1H, 3J = 9.7 Hz, H-4), 7.25 to 7.30 (m, 2H, 5-OCH2(CH2)2CH2OC6H5), 6.90 to 6.94 (m, 3H, 5-OCH2(CH2)2CH2-O-C6H5), 6.13 (d, 1H, 3J = 9.7 Hz, H-3), 6.53 (s, 1H, H-8), 4.63 (t, 2H, 3J = 8.5 Hz, H-5'), 4.31 (s, 2H, 5-OCH2(CH2)2CH2OC6H5), 4.05 (S, 2H, 5-OCH2(CH2)2CH2OC6H5), 3.42 (t, 2H, 3J = 8.5 Hz, H-4'), 1.91 (s, 4H, 5-OCH2(CH2)2CH2OC6H5); MS (70 eV) m/z: 352 (18%, M+), 204 (4%, [C11H8O4]+), 176 (6%, [204-CO]+), 149 (65%, [C10H13O]+), 107 (100%, [C6H5OCH2]+), 95 (5%), 77 (28%, [C6H5]+), 55 (25%, [C4H7]+); molecular weight calculated for C21H20O5, 352.13107; found by high-resolution mass spectrometry, 352.13091.

LogP Values. LogP (log of octanol-water partition coefficient, a measure of the hydrophobicity) values of compounds were determined by high-performance liquid chromatography with a binary high-performance liquid chromatography pump (Waters 1525; Waters, Milford, MA), a Kromasil 100 C18 column (5 µM, 60 x 4.6 mm; EKA Chemical Separation Products, Bohus, Sweden), and a Waters 2475 multiwavelength fluorescence detector. Compounds were eluted with a gradient changing from 30% acetonitrile and 70% Sørensen's phosphate buffer, 11 mM, pH 7.4, to 78% acetonitrile and 22% buffer over 70 min. 4-Methylbenzaldehyde, toluene, ethyl-, n-propyl-, n-butyl-, n-pentyl-, n-hexyl-, n-heptyl, and n-octylbenzene were used as standards.

Cells, Cell Lines, and Clones. Throughout the article, we are using the new IUPHAR nomenclature for ion channels (Gutman et al., 2003Go). Cells stably expressing mKv1.1, rKv1.2, mKv1.3, hKv1.5, and mKv3.1 have been described previously (Grissmer et al., 1994Go). Cell lines stably expressing other mammalian ion channels were gifts from several sources: mKv1.7 in CHL cells and hKCa2.3 in COS-7 cells (Aurora Biosciences Corp., San Diego, CA); hKv1.4 and rKv4.2 in LTK cells (Michael Tamkun, University of Colorado, Boulder, CO); hKv2.1 in HEK293 cells (James Trimmer, University of California Davis, Davis, CA); Kv11.1 (HERG) in HEK293 cells (Craig January, University of Wisconsin, Madison, WI); hKCa1.1, rKCa2.1 and hKCa3.1 in HEK-293 cells (Khaled Houamed, University of Chicago, Chicago, IL); hNav1.4 in HEK-293 cells (Frank Lehmann-Horn, University of Ulm, Germany); and Cav1.2 in HEK-293 cells (Franz Hofmann, Munich, Germany). RBL-2H3 (expressing Kir2.1) and N1E-115 neuroblastoma cells (expressing Nav1.2) were obtained from the American Type Culture Collection (Manassas, VA). hKv1.6 and rKv3.2 (both in pcDNA3) were obtained from Protinac GmbH (Hamburg, Germany) and transiently-transfected into COS-7 cells with Fugene-6 (Roche) according to the manufacturer's protocol. The hKv1.3-H399T and A413C mutants were a generous gift from Tobias Dreker and Stephan Grissmer (University of Ulm, Germany), whereas the H399Y and A413V (Panyi et al., 1995Go) mutants were kindly supplied by Carol Deutsch (University of Pennsylvania, Philadelphia, PA).

Electrophysiology. All compounds used for electrophysiological testing were >98% pure as determined by combustion analysis. All experiments were conducted in the whole-cell configuration of the patch-clamp technique with a holding potential of -80 mV unless otherwise stated. Pipette resistances averaged 2.0 M{Omega}, and series resistance compensation of 80% was employed when currents exceeded 2 nA. Kv1.3 currents were elicited by repeated 200-ms or 2-s pulses from -80 to 40 mV, applied at intervals of 30 or 60 s. Kv1.3 currents were recorded in normal Ringer solution with a Ca2+-free pipette solution containing 145 mM KF, 10 mM HEPES, 10 mM EGTA, 2 mM MgCl2, pH 7.2, 300 mOsM. EC50 values and Hill coefficients were determined by fitting the Hill equation to the reduction of area under the current curve measured at 40 mV. Kv1.1, Kv1.5, Kv1.4, Kv1.6, Kv1.7, Kv2.1, Kv3.1, Kv3.2, and Kv4.2 currents were recorded with 200-ms depolarizing pulses to 40 mV applied every 10 s (Grissmer et al., 1994Go; Wulff et al., 2000Go; Bardien-Kruger et al., 2002Go). For Kv1.2, we applied 200-ms pulses to 40 mV every 5 s to allow for the channel's use-dependent activation (Grissmer et al., 1994Go). HERG (Kv11.1) currents were recorded with a 2-step pulse from -80 mV first to 20 mV for 2 s and then to -50 mV for 2 s (Zhou et al., 1998Go) and the reduction of both peak and tail current by the drug was determined.

The inward rectifier (rKir2.1) in RBL cells was studied with ramp pulses from -150 to 0 mV applied every 10 s and a Na+ aspartate Ringer external and a K+ aspartate-based pipette solution containing 50 nM free Ca2+. For measurements of IKCa (KCa3.1) SKCa (KCa2.3), and BKCa (KCa1.1) currents, we used an internal pipette solution containing 145 mM K+ aspartate, 2 mM MgCl2, 10 mM HEPES, 10 mM K EGTA, and 8.5 mM CaCl2 (1 µM free Ca +), pH 7.2, 290 to 310 mOsM, and an external solution containing 160 mM Na+ aspartate, 4.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH 7.4, 290 to 310 mOsM. BKCa currents were elicited by 200-ms voltage ramps from -80 to 80 mV applied every 10 s, and channel block was measured as a reduction of slope conductance at 35 mV. IKCa and SKCa currents were elicited by 200-ms voltage ramps from -120 mV to 40 mV applied every 10 s, and the reduction of slope conductance at -80 mV by drug was taken as a measure of channel block (Wulff et al., 2000Go). Nav1.2 currents from N1E-115 cells and Nav1.4 currents from stably transfected HEK cells were recorded with 100-ms pulses from -80 to 0 mV every 10 s with a KCl-based pipette solution and normal Ringer's solution as an external solution. Cav1.2 currents were elicited by 600-ms depolarizing pulses from -80 to 20 mV every 10 s with a CsCl-based pipette solution and an external solution containing 30 mM BaCl2 (Kolski-Andreaco et al., 2004Go). Blockade of both Na+ and Ca2+ currents was determined as reduction of the current minimum. Swelling-activated chloride currents were elicited from activated human T cells with a hyperosmolar cesium glutamate-based pipette solution (420 mOsM) and ramp pulses from -120 to 60 mV every 10 s (Ross et al., 1994Go).

Cytotoxicity Assays. Jurkat E61 and MEL cells were seeded at 5 x 105 cells/ml in 12-well plates. Compounds were added at concentrations of 10 nM, 100 nM, and 10 µM in a final DMSO concentration of 0.1%, which was found not to affect cell viability. After 48 h, the cells in each well were well mixed and resuspended, and the number of trypan blue-positive cells in three aliquots from each well was determined under a light microscope. The test was repeated twice.

Ames Test. The mutagenic activity of PAP-1 was determined on the Salmonella typhimurium tester strains TA97A, TA98, TA100, TA102, and TA1535 by Nelson Laboratories (Salt Lake City, UT).

Receptor Screening. PAP-1 was screened at 1 µM for potential affinity to a panel of 56 receptors by displacement of standard radioligands at MDS Pharma (http://www.mdsps.com). The following receptors were included: adenosine (A1, A2A, A3), adrenergic ({alpha}1A, {alpha}1B, {alpha}1D, {alpha}2A, {beta}1, {beta}2), bradykinin (B1, B2), dopamine (D1, D25, D3, D4.2), endothelin (ETA, ETB), epidermal growth factor, estrogen ({alpha}), GABAA (agonist site), GABAB (benzodiazepine and nonselective), glucocorticoid, glutamate (kainate, NMDA agonism, NMDA glycine, NMDA phencyclidine), histamine (H1, H2, H3), imidazoline (I2), interleukin-1, leukotriene (CysLT1), muscarinic (M1, M2, M3), neuropeptide Y (Y1, Y2), nicotinic acetylcholine, opiate ({delta}, {kappa}, µ), phorbol ester, platelet activating factor, purinergic (P2X, P2Y), serotonin (5-HT1A, 5-HT3), {sigma} ({sigma}1, {sigma}2), tachykinin (NK1), testosterone, and transporters (dopamine, GABA, norepinephrine, serotonin). Because significant activity was witnessed only on the dopamine transporter, PAP-1 was retested at 0.01, 0.1, 0.25, and 1 µM to determine an IC50 and a Hill coefficient. All assays were performed in duplicate.

Inhibition of Cytochrome P450-Dependent Enzymes. Inhibition of the catalytic activity of purified recombinant human cytochrome P450 2A6 and 3A4 in microsomes (BD Gentest, Woburn, MA) was assayed on the turnover of coumarin (CYP2A6) or 7-benzyloxy-4-trifluoromethyl-coumarin (CYP3A4) by the detection of their fluorescent metabolites (Henderson et al., 1999Go), and IC50 values were determined by testing multiple compound concentrations ranging from 10 nM to 200 µM. All experiments were conducted in duplicate and results are reported as percentage inhibition. Tranylcypromine (CYP2A6) and ketoconazole (CYP3A4) were run as positive controls on the same plates and rendered IC50 values of 170 and 37 nM, respectively.

DNA Cross-Linking. The DNA cross-linking potency of selected compounds was determined by measuring melting profiles of poly(dA-dT)-poly(dA-dT) DNA in the presence and absence of compound with and without UVA irradiation according to Dall'Acqua et al. (1971Go). Incubation mixtures containing 10 µM concentrations of the compound, 0.1 units of poly(dA-dT)-poly(dA-dT) (Sigma-Aldrich, Deisenhofen, Germany), and 1% DMSO and MOPS buffer (0.1 M; pH 7.2) were prepared in 2-ml quartz cuvettes. Samples were irradiated with a Heraeus Fluotest lamp for 30 min at a distance of 20 cm with an intensity of 3.5 mW/cm2 or stored in the dark for 30 min. Afterward, samples were placed into HP 8845A diode-array spectrophotometer fitted with a Julabo F20-C heating controller. The temperature was directly measured in the cuvettes with a digital thermometer. Heating was applied at a rate of 1°C min-1, with absorbance (260 nm) and temperature data sampling at 1-min intervals until the temperature reached 80°C.

Photoproduction of Singlet Oxygen. The production of singlet oxygen (1O2) by selected psoralen compounds was determined according to Kraljic and El Moshni (1978Go). Compound (10 µM) was mixed with 4 µM N,N-dimethyl-4-nitroso-aniline, 10 mM histidine, and 1% methanol in oxygen-saturated phosphate buffer (0.01 M; pH 7.0) and irradiated with a Heraeus Fluotest lamp for 5 h at a distance of 20 cm with an intensity of 3.5 mW/cm2. Singlet oxygen mediated photobleaching of the yellow N,N-dimethyl-4-nitrosoaniline was followed by photometric measurements (440 nm) every 30 min. Results are given as percentage reduction of initial absorption at 440 nM before irradiation and are corrected for "self-bleaching" in the absence of compounds (11.4% ± 0.3%, n = 9).

Proliferation Assays. Untouched CD3+ T cells were isolated from peripheral blood of healthy volunteers with RosetteSep (StemCell Technologies, Vancouver, BC, Canada) and then incubated with a biotinylated anti-CCR7 Ab (Clone 3D12; BD PharMingen, San Diego, CA). After two washes with PBS, the labeled cells were first incubated with an anti-biotin tetramer complex (StemCell Technologies) and then with magnetic microbeads. The cells were placed onto a negative selection column in a magnet (all StemCell Technologies) and pure CCR7- cells were eluted (97% CCR7- as determined by flow cytometry). CCR7- T cells were then seeded at 5 x 104 per well together with 5 x 104 autologous irradiated peripheral blood mononuclear cells (PBMCs; 2500 rad) in RPMI culture medium into round-bottomed, 96-well plates. PAP-1 was added at different concentrations, and the cells were stimulated with 25 ng/ml soluble anti-CD3 Ab (Biomeda) 30 min later. To determine the effect of PAP-1 on naive and central memory T cells, human PBMCs were seeded at 2 x 105 cells per well in flat-bottomed, 96-well plates, preincubated with increasing concentrations of PAP-1 for 30 min, and then stimulated with 25 ng/ml anti-CD3 monoclonal Ab (Biomeda, Foster City, CA). [3H]Thymidine (1 µCi/well) was added for the last 12 h of the 60-h assay. Cells were harvested onto glass-fiber filters, and radioactivity was measured in a {beta}-scintillation counter.

DTH. Nine- to 11-week-old female Lewis rats were purchased from Charles River Laboratories (Wilmington, MA) and housed in microisolator cages with irradiated rodent chow and autoclaved water ad libitum. All experiments were in accordance with National Institutes of Health guidelines and approved by the University of California, Davis, Institutional Animal Care and Use Committee. Rats were immunized at the base of the tail with 200 µl of an emulsion of egg albumin grade II (Sigma) in complete Freund's adjuvant (Difco, Detroit, MI). The emulsion was prepared with 50% complete Freund's adjuvant and 50% saline containing a final concentration of 1 mg/ml albumin. Seven days later, the thickness of both ears was measured using a spring-loaded micrometer (Mitutoyo, Spokane, WA). The rats were then challenged by an injection of 10 µl of albumin (2 mg/ml) dissolved in saline in the pinna of one ear and saline in the other ear. In two experiments, rats received i.p. injections of 0.3, 1.0, or 3.0 mg/kg of PAP-1 or of vehicle [saline with 25% Cremophor EL (Sigma)] three times daily for 48 h starting 24 h before the challenge. In another experiment, rats were gavaged three times daily with peanut oil or 5, 20, or 80 mg/kg PAP-1 dissolved in peanut oil (volume 0.5 ml). Ear swelling was measured 24 h later. Values are given as differences in the thickness (µm) of the albumin-injected ear measured before the challenge and 24 h later. The other ear was used as a control for the injection, and the rats were excluded from the experiment if the thickness of the saline-injected ear increased by more than 10%.


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Probing of the Alkoxypsoralen Pharmacophore to Increase Selectivity <