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PARP-2 is a member of the poly(ADP-ribose) polymerase family, with some activities similar to those of PARP-1 but with other distinct roles. Two series of isoquinolin-1-ones were designed, synthesized, and evaluated as selective inhibitors of PARP-2, using the structures of the catalytic sites of the isoforms. A new efficient synthesis of 5-aminoisoquinolin-1-one was developed, and acylation with acyl chlorides gave 5-acylaminoisoquinolin-1-ones. By examination of isoquinolin-1-ones with carboxylates tethered to the 5-position, Heck coupling of 5-iodoisoquinolin-1-one furnished the 5-CH═CHCO(2)H compound for reduction to the 5-propanoic acid. Alkylation of 5-aminoisoquinolin-1-one under mildly basic conditions, followed by hydrolysis, gave 5-(carboxymethylamino)isoquinolin-1-one, whereas it was alkylated at 2-N with methyl propenoate and strong base. Compounds were assayed in vitro for inhibition of PARP-1 and PARP-2, using FlashPlate and solution-phase assays, respectively. The 5-benzamidoisoquinolin-1-ones were more selective for inhibition of PARP-2, whereas the 5-(ω-carboxyalkyl)isoquinolin-1-ones were less so. 5-Benzamidoisoquinolin-1-one is the most PARP-2-selective compound (IC(50(PARP-1))/IC(50(PARP-2)) = 9.3) to date, in a comparative study.

Poly(ADP-ribose)polymerase-1 is an important target enzyme in drug design; inhibitors have a wide variety of therapeutic activities. A series of quinoline-8-carboxamides was designed to maintain the required pharmacophore conformation through an intramolecular hydrogen bond. 3-Substituted quinoline-8-carboxamides were synthesized by Pd-catalyzed couplings (Suzuki, Sonogashira, Stille) to 3-iodoquinoline-8-carboxamide, an efficient process that introduces diversity in the final step. 2-Substituted quinoline-8-carboxamides were prepared by selective Pd-catalyzed couplings at the 2-position of 2,8-dibromoquinoline, followed by lithium-bromine exchange of the intermediate 2-(alkyl/aryl)-8-bromoquinolines and reaction with trimethylsilyl isocyanate. The intramolecular hydrogen bond was confirmed by X-ray and by NMR. The SAR of the 3-substituted compounds for inhibition of human recombinant PARP-1 activity showed a requirement for a small narrow group. Substituents in the 2-position increased potency, with the most active 2-methylquinoline-8-carboxamide having IC(50) = 500 nM (IC(50) = 1.8 microM for 5-aminoisoquinolin-1-one (5-AIQ, a standard water-soluble inhibitor)).

The synthesis, SAR, and preclinical evaluation of 17-cyanated 2-substituted estra-1,3,5(10)-trienes as anticancer agents are discussed. 2-Methoxy-17beta-cyanomethylestra-1,3,5(10)-trien-3-ol ( 14), but not the related 2-ethyl derivative 7, and the related 3- O-sulfamates 8 and 15 display potent antiproliferative effects (MCF-7 GI 50 300, 60 and 70 nM, respectively) against human cancer cells in vitro. Investigation of the SAR reveals that a sterically unhindered hydrogen bond acceptor attached to C-17 is most likely key to the enhanced activity. Compound 8 displayed significant in vitro antiangiogenic activity, and its ability to act as a microtubule disruptor was confirmed. Inhibitory activity of the sulfamate derivatives against steroid sulfatase and carbonic anhydrase II (hCAII) was also observed, and the interaction between 15 and hCAII was investigated by protein crystallography. The potential of these multimechanism anticancer agents was confirmed in vivo, with promising activity observed for both 14 and 15 in an athymic nude mouse MDA-MB-231 human breast cancer xenograft model.

Estradiol-3,17-O,O-bis-sulfamates inhibit steroid sulfatase (STS), carbonic anhydrase (CA), and, when substituted at C-2, cancer cell proliferation and angiogenesis. C-2 Substitution and 17-sulfamate replacement of the estradiol-3,17-O,O-bis-sulfamates were explored with efficient and practical syntheses developed. Evaluation against human cancer cell lines revealed the 2-methyl derivative 27 (DU145 GI(50) = 0.38 microM) as the most active novel bis-sulfamate, while 2-ethyl-17-carbamate derivative 52 (GI(50) = 0.22 microM) proved most active of its series (cf. 2-ethylestradiol-3,17-O,O-bis-sulfamate 4 GI(50) = 0.21 microM). Larger C-2 substituents were deleterious to activity. 2-Methoxy-17-carbamate 50 was studied by X-ray crystallography and was surprisingly 13-fold weaker as an STS inhibitor compared to parent bis-sulfamate 3. The potential of 4 as an orally dosed anti-tumor agent is confirmed using breast and prostate cancer xenografts. In the MDA-MB-231 model, dramatic reduction in tumor growth or regression was observed, with effects sustained after cessation of treatment. 3-O-Sulfamoylated 2-alkylestradiol-17-O-carbamates and sulfamates have considerable potential as anticancer agents.

By introducting the steroid sulfatase inhibitory pharmacophore into aromatase inhibitor 1 (YM511), two series of single agent dual aromatase-sulfatase inhibitors (DASIs) were generated. The best DASIs in vitro (JEG-3 cells) are 5, (IC50(aromatase) = 0.82 nM; IC50(sulfatase) = 39 nM), and 14, (IC50(aromatase) = 0.77 nM; IC50(sulfatase) = 590 nM). X-ray crystallography of 5, and docking studies of selected compounds into an aromatase homology model and the steroid sulfatase crystal structure are presented. Both 5 and 14 inhibit aromatase and sulfatase in PMSG pretreated adult female Wistar rats potently 3 h after a single oral 10 mg/kg dose. Almost complete dual inhibition is observed for 5 but the levels were reduced to 85% (aromatase) and 72% (sulfatase) after 24 h. DASI 5 did not inhibit aldosterone synthesis. The development of a potent and selective DASI should allow the therapeutic potential of dual aromatase-sulfatase inhibition in hormone-dependent breast cancer to be assessed.

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyze the interconversion between the oxidized and reduced forms of androgens and estrogens at the 17 position. The 17beta-HSD type 1 enzyme (17beta-HSD1) catalyzes the reduction of estrone to estradiol and is expressed in malignant breast cells. Inhibitors of this enzyme thus have potential as treatments for hormone dependent breast cancer. Here we report the syntheses and biological evaluation of novel inhibitors based on the estrone or estradiol template. These have been investigated by modification at the 6, 16 or 17 positions or combinations of these in order to gain information about structure-activity relationships by probing different areas in the enzyme active site. Activity data have been incorporated into a QSAR with predictive power, and the X-ray crystal structures of compounds 15 and 16c have been determined. Compound 15 has an IC50 of 320 nM for 17beta-HSD1 and is selective for 17beta-HSD1 over 17beta-HSD2. Three libraries of amides are also reported that led to the identification of inhibitors 19e and 20a, which have IC50 values of 510 and 380 nM respectively, and 20 h which, having an IC50 value of 37 nM, is the most potent inhibitor of 17beta-HSD1 reported to date. These amides are also selective for 17beta-HSD1 over 17beta-HSD2.

17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) are an important class of steroidogenic enzymes that regulate the bioavailability of active estrogens and androgens and are as yet a relatively unexploited therapeutic target. Based on our investigations and those of others, E-ring modified steroids were identified as a useful template for the design of inhibitors of 17beta-HSD type 1, an enzyme involved in the conversion of estrone into estradiol. The synthesis and biological evaluation of a new series of N- and C-substituted 1,3,5(10)-estratrien-[17,16-c]-pyrazoles and the corresponding SAR are discussed. Among the N-alkylated analogues, the most potent inhibitor was the 1'-methoxyethyl derivative, 41, with an IC(50) of 530 nM in T47-D human breast cancer cells. The X-ray crystal structure of the 1'-isobutyl derivative, was determined. Further optimization of the template using parallel synthesis resulted in a library of C5'-linked amides from which 73 emerged. This pyridylethyl amide had an IC(50) of 300 nM and its activity, with that of 41, suggests the importance of hydrogen bond acceptor groups in the pyrazole side chain. Both 41 and 73 displayed selectivity over 17beta-HSD type 2, and preliminary investigations showed 41 to be nonestrogenic in vitro in a luciferase reporter gene assay in contrast to the parent pyrazole 25. Molecular modeling studies, which support these findings, and a QSAR, the predictive power of which was demonstrated, are also presented.

Efficient and flexible syntheses of 2-substituted estrone, estradiol and their 3-O-sulfamate (EMATE) derivatives have been developed using directed ortho-lithiation methodology. 2-Substituted EMATEs display a similar antiproliferative activity profile to the corresponding estradiols against a range of human cancer cell lines. 2-Methoxy (3, 4), 2-methylsulfanyl (20, 21) and 2-ethyl EMATEs (32, 33) proved the most active compounds with 2-ethylestradiol-3-O-sulfamate (33), displaying a mean activity over the NCI 55 cell line panel 80-fold greater than the established anticancer agent 2-methoxyestradiol (2). 2-Ethylestradiol-3-O-sulfamate (33) was also an effective inhibitor of angiogenesis using three in vitro markers, and various 2-substituted EMATEs also proved to be inhibitors of steroid sulfatase (STS), a therapeutic target for the treatment of hormone-dependent breast cancer. The potential of this novel class of multimechanism anticancer agents was confirmed in vivo with good activity observed in the NCI hollow fiber assay and in a MDA-MB-435 xenograft mouse model.

Structure-based drug design using the crystal structure of human 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) led to the discovery of novel, selective, and the most potent inhibitors of 17beta-HSD1 reported to date. Compounds 1 and 2 contain a side chain with an m-pyridylmethyl-amide functionality extended from the 16beta position of a steroid scaffold. A mode of binding is proposed for these inhibitors, and 2 is a steroid-based 17beta-HSD1 inhibitor with the potential for further development.

The high affinity of adenophostin A for 1D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] receptors may be related to an alteration in the position of its 2'-phosphate group relative to the corresponding 1-phosphate group in Ins(1,4,5)P(3). To investigate this possibility, two bicyclic trisphosphates 9 and 10, designed to explore the effect of relocating the 1-phosphate group of Ins(1,4,5)P(3) using a novel fused-ring system, were synthesized from myo-inositol. Biological evaluation of 9 and 10 at the Ins(1,4,5)P(3) receptors of hepatocytes showed that both were recognized by hepatic Ins(1,4,5)P(3) receptors and both stimulated release of Ca(2+) from intracellular stores, but they had lower affinity than Ins(1,4,5)P(3). This finding may be explained by considering the three-dimensional structures of 9 and 10 in light of recent studies on the conformation of adenophostin A.

Steroid sulfatases regulate the formation of estrogenic steroids which can support the growth of endocrine-dependent breast tumors. Therefore, the development of potent steroid sulfatase inhibitors could have considerable therapeutic potential. Several such inhibitors have now been developed including estrone 3-O-sulfamate (EMATE, 1), which shows potent active site-directed inhibition. However, EMATE was subsequently shown to be also a potent estrogen. In an attempt to reduce the estrogenicity while retaining the potent sulfatase inhibitory properties associated with this type of molecule, (E)-17-oximino-3-O-sulfamoyl-1,3,5(10)-estratriene (5) (estrone oxime 3-O-sulfamate, OMATE) was synthesized. The X-ray crystal structure of (E)-17-oximino-3-hydroxy-1,3,5(10)-estratriene (4) (estrone oxime) demonstrated the presence of only one geometrical isomer [anti-isomer, (E)]. OMATE potently inhibited estrone sulfatase (E1-STS) activity and was similar to EMATE (>99% inhibition at 0.1 microM in MCF-7 breast cancer cells). It was also evaluated in vivo for its estrogenicity and ability to inhibit sulfatase activity. While it was equipotent with EMATE in vivo as a sulfatase inhibitor, it surprisingly had a stimulatory effect on uterine growth in ovariectomized rats about 1.5-fold greater than that of EMATE. Thus, OMATE possesses potential as a superestrogen and modification at C-17 is identified as a useful route for enhancement of estrogenicity in sulfamate-based estrogens.