The ion transport activity of the sarco/endoplasmic reticulum calcium ATPase (SERCA) is specifically and potently inhibited by the small molecule 2,5-di-tert-butylhydroquinone (BHQ). In this study, we investigated the relative importance of the nature and position of BHQ's four substituents for enzyme inhibition by employing a combination of experimental and computational techniques. The inhibitory potencies of 21 commercially available or synthesized BHQ derivatives were determined in ATPase activity assays, and 11 compounds were found to be active. Maximum inhibitory potency was observed in compounds with two para hydroxyl groups, whereas BHQ analogues with only one hydroxyl group were still active, albeit with a reduced potency. The results also demonstrated that two alkyl groups were an absolute requirement for activity, with the most potent compounds having 2,5-substituents with four or five carbon atoms at each position. Using the program GOLD in conjunction with the ChemScore scoring function, the structures of the BHQ analogues were docked into the crystal structure of SERCA mimicking the enzyme's E(2) conformation. Analysis of the docking results indicated that inhibitor binding to SERCA was primarily mediated by a hydrogen bond between a hydroxyl group and Asp-59 and by hydrophobic interactions involving the bulky inhibitor alkyl groups. Attempts to dock BHQ into crystal structures corresponding to the E(1) conformation of the enzyme failed, because the conformational changes accompanying the E(2)/E(1) transition severely restricted the size of the binding site, suggesting that BHQ stabilizes the enzyme in its E(2) form. The potential role of Glu309 in enzyme inhibition is discussed in the context of the computational results. The docking scores correlated reasonably well with the measured inhibitory potencies and allowed the distinction between active and inactive compounds, which is a key requirement for future virtual screening of large compound databases for novel SERCA inhibitors.

2,3-DIHYDR0-7H-DIBENZO [de, h]QUINOLINESrespectively. No unreacted aziridine could be detected after these prolonged reactions at high temperature.Reversibility of the Thermal Valence Isomerization of 1-Cyclohexyl-6-(cyclohexylimino)-la-phenylindano [ 1,241 aziridine. A. Reaction in the Absence of Dipo1arophile.-A deoxygenated solution of 1.15 g (3 mmol) of 4 in 30 ml of toluene was heated to 135-145" for 24 hr giving a deep red color. Evaporation of the solvent and trituration of the residue with ethanol gave recovered indano[l,2-6] aziridine 4 as a slightly yellow solid, mp 158-159", 0.702 g (6lY0 recovery). Evaporation of the ethanol mother liquor afforded 0.068 g (19'%) of 28, 123-124".B. Parallel Reaction in the Presence of a Dipolarophi1e.-A similar experiment was performed with 1.5 g (3 mmol) of 4 and 0.519 g (3 mmol) of N-phenylmaleimide under exactly comparable conditions and resulted in the isolation of adduct 6 (see above), 1.34 g (8O' %), mp 235-236", but no aziridine 4 could be recovered.Reversibility of Photochemical Valence Isomerization of 1-Cyclohexyl-6-(6-cyclohexylimino)-la-phenylindano [ 1,241 aziridine -A solution of 1.063 g (2.8 mmol) of the indano[l,2-b]aziridine 4 in 100 ml of tetrahydrofuran was irradiated under nitrogen a t 0 to -10" in a quartz cell using a 450-W Hanovia high-pressure lamp for 8 hr. A deep red solution was obtainedz4 and when exposed to visible light the color rapidly faded to a pale yellow resulting in a virtually complete restoration of the infrared absorption spectrum of the original compound 4.Photochemical Reaction between l-Cyclohexyl-6-(cyclohexylimino)-la-phenylindano [ 1,2-b] aziridine and Cyclohexylamine Hydrobromide.-A mixture of 1.15 g (3 mmol) of the indano[l,2-b]-aziridine 4 and 1.08 g (6 mmol) of cyclohexylamine hydrobromide in 150 ml of ether and 20 ml of methanol was irradiated for 8 hr under nitrogen with a 450-W high-pressure Hanovia lamp in a quartz reaction vessel. The resulting yellow solid 29 was colected and recrystallized from ether-methanol, 0.527 g (38%): mp 298-299"; ir (CHC13) 1617 (C=N), 3230 cm-l (NH); nmr (24) The very close similarity of the absorption spectrum of this species [Amax (dioxane) 500 mN, 520 (sh), 565 (ah)] to that obtained by heating 4 [Amax (xylene) 505 mN, 534 (ah), and 570 (ah) ] may be noted. ~T M B (CDCla) 0. 20, CeHl,), 3.0-3.6 (m, 1, CHNHC=), 3.9-4.5 (m, 1, =N+CH), 6. 9, aromatic protons), 8.51 (d, 1, D2O exchangeable XH), 9.65 (d, 1, J = 7 Hz, aromatic protons); uvmax (95'3$ EtOH-HBr) 393 mp (log e 4-12), 335 (3.77), 324 (sh, 3.72), 288 (3.86), 279 (3.89); mass spectrum (70 eV) 384.Anal. Calcd for C2,Ha3N2Br: N, 6.02. Found: N, 5.56. The identical compound was obtained by heating the indano-[ 1,241 aziridine with cyclohexylamine hydrobromide or ammonium bromide in toluene.Photochemical Reaction between l-Cyclohexyl-6-(cyclohexylimino)-la-phenylindano [ 1,241 aziridine and cis-1 ,Z-Dichloromethylene.-A mixture of 1.15 g (3 mmol) of the indano[l,2-b]-aziridine 4 and 20 ml of cis-1,2-dichloroethylene in 100 ml of ...

Analogues of the compound 2,5-di-tert-butylhydroquinone (BHQ) are capable of inhibiting the enzyme sarco/endoplasmic reticulum ATPase (SERCA) in the low micromolar and submicromolar concentration ranges. Not only are SERCA inhibitors valuable research tools, but they also have potential medicinal value as agents against prostate cancer. This study describes the synthesis of thirteen compounds representing several classes of BHQ analogues, such as hydroquinones with a single aromatic substituent, symmetrically and unsymmetrically disubstituted hydroquinones, and hydroquinones with ω-amino acid tethers attached to their hydroxyl groups. Structure-activity relationships were established by measuring the inhibitory potencies of all synthesized compounds in bioassays. The assays were complemented by computational ligand docking for an analysis of the relevant ligand/receptor interactions.

A series of chemically reactive derivatives of the nonsteroidal estrogen hexestrol have been synthesized as potential affinity labels for the estrogen receptor or as cytotoxic agents with selective activity against receptor-containing cells. These compounds are hexestrol ethers with halo ketone, halohydrin, or epoxide functions or 4-substituted deoxyhexestrols with halo ketone, benzyl halide, nitro, azide, sulfonyl fluoride, or sulfonyl azide groups. The alkylating activity of the electrophilic derivatives was measured using the colorimetric reagent nitrobenzylpyridine, the bromo derivatives being considerably more reactive than the chloro ones. Their reversible binding to the lamb uterine estrogen receptor was measured by competitive binding assays, and their irreversible reaction with receptor was measured by exchange assays that determine the rate and extent of receptor inactivation. In general, monoetherification of hexestrol or substitution of deoxyhexestrol produces compounds with relatively low affinity for the estrogen receptor (0.3-10% that of estradiol). Most of the electrophilic derivatives are rapid and effective inactivators of receptor (24-70% inactivation within 0.5-5 h at 25 degrees C). Of the photosensitive derivatives, 4-azidodeoxyhexestrol appears to be the most efficient receptor inactivator (49%). The high reactivity of these compounds toward the estrogen receptor and the lack of interference by their reaction with other cellular nucleophiles suggest that these compounds may be useful as affinity-labeling agents or as selective cytotoxic agents in intact systems.

The lysine analogue (4) of methotrexate (1) was treated with 5-(dimethylamino)naphthalene-l-sulfonyl chloride to give the corresponding dansyl analogue 5. The product isolated by preparative TLC was further purified on a preparative HPLC column. Analogue 5 exhibited fluorescence properties characteristic of the dansyl moiety and was a potent inhibitor of dihydrofolate reductase purified from Lactobacillus casei, chicken liver, and a human lymphoblastoid cell line.