Development of (p-O-sulfamoyl)-N-alkanoyl-phenylalkyl amines as non-steroidal estrone sulfatase inhibitors.

Estrogen levels in breast tumors of postmenopausal women are as much as 10 times higher than estrogen levels in plasma, presumably due to in situ formation of estrogen. The major source of estrogen in breast cancer cells may be conversion of estrone sulfate to estrone by the enzyme estrone sulfatase. Thus, inhibitors of estrone sulfatase are potential agents for treatment of estrogen-dependent breast cancer. Several steroidal compounds have been developed that are potent estrone sulfatase inhibitors, most notably estrone-3-O-sulfamate. However, these compounds and their metabolites may have undesired effects, including estrogenicity. To avoid the problems associated with a potentially active steroid nucleus, we designed and synthesized a series of nonsteroidal estrone sulfatase inhibitors, the (p-O-sulfamoyl)-N-alkanoyl phenylalkyl amines. The compounds synthesized vary in the length of their alkanoyl chain and in the number of carbons separating the phenyl ring and the carbonyl carbon. The ability of these compounds to inhibit estrone sulfatase activity was tested using human placental microsomes and intact cultured human breast cancer cells. Estrogenicity was also evaluated, using growth of estrogen-dependent human breast cancer cells. All of the test compounds inhibited estrone sulfatase activity of human placental microsomes to some extent, with the most effective compound having an IC50 value of 72 nM. In general, compounds with longer alkanoyl chains (12-14 carbons) were more effective than those with shorter chains. The test compounds also inhibited estrone sulfatase activity in intact cultures of MDA-MB-231 human breast cancer cells. Again, the longer chain compounds were more effective. In both the placental and breast cancer cell sulfatase assays, the optimal distance between the phenyl ring and the carbonyl carbon was 1-2 carbons. The MCF-7 cell proliferation assay revealed that estrone and estrone-3-O-sulfamate were both estrogenic, but the (p-O-sulfamoyl)-N-alkanoyl phenylalkyl amines were not. Our data indicate the utility of (p-O-sulfamoyl)-N-alkanoyl phenyl alkylamines for inhibition of estrone sulfatase activity. Furthermore, our data support the concept that nonsteroidal estrone sulfatase inhibitors may be useful as therapeutic agents for estrogen-dependent breast cancers.

Synthesis and sulfatase inhibitory activities of (E)- and (Z)-4-hydroxytamoxifen sulfamates.

We report the development of (E)- and (Z)-4-hydroxytamoxifen sulfamates as estrone sulfatase inhibitors, potential therapeutic agents for the treatment of breast cancer. Both compounds competitively inhibit estrone sulfatase isolated from rat liver with apparent Ki of 35.9 microM for (E)-4-hydroxytamoxifen sulfamate and an apparent Ki of > 500 microM for the (Z) isomer.

Development of potent non-estrogenic estrone sulfatase inhibitors.

Estrogen levels in breast tumors of post-menopausal women are as much as 10 times higher than estrogen levels in plasma, presumably due to in situ formation of estrogen. The major source of estrogen in breast cancer cells may be conversion of estrone sulfate to estrone by the enzyme estrone sulfatase. Thus, inhibitors of estrone sulfatase have potential for the treatment of estrogen-dependent breast cancers. Several steroidal agents have been developed that are potent estrone sulfatase inhibitors, most notably estrone-3-O-sulfamate. These compounds may have undesired actions, especially estrogenicity. Recently, non-steroidal estrone sulfatase inhibitors have been designed that avoid the problems associated with an active steroid nucleus; however, these have not achieved the potency of estrone-3-O sulfamate. We have designed and synthesized a series of compounds, 17 beta-(N-alkylcarbamoyl)-estra-1,3,5(10)-trien-3-O-sulfamates (6a-d) and 17 beta-(N-alkanoyl)-estra-1,3,5(10)-trien-3-O-sulfamates (11a-d) that combine the structural features of the steroidal estrone sulfatase inhibitors with a membrane insertion region that should increase the affinity for the sulfatase enzyme and decrease the estrogenicity of the steroid. We tested the compounds for estrone sulfatase inhibition by measuring estrone sulfatase activity in intact cultures of human breast cancer cells (MDA-MB-231). We tested for estrogenicity by measuring growth of estrogen-dependent MCF-7 human breast cancer cells. All of the test compounds (10 nM) substantially inhibited estrogen sulfatase activity of intact MDA-MB-231 cells. Dose-response analysis indicated an IC50 of approximately 0.5 nM for two of the compounds (6a and 11a). In the test for estrogenicity, estrone and estrone-3-O-sulfamate significantly stimulated MCF-7 cell growth. In contrast, neither the 17 beta-(N-alkylcarbamoyl)-estra-1,3,5,(10)-trien-3-O-sulfamates++ + nor the 17 beta-(N)-alkanoyl)-estra-1,3,5,(10)-trien-3-O-sulfamates stimulated growth of MCF-7 cells at a concentration of 1 microM, indicating that they are not estrogenic at levels 2000 times greater than their IC50 for estrone sulfatase. Our data indicate the utility of the new compounds for inhibition of breast cancer cell estrone sulfatase activity. Further, our data support the concept that estrone sulfatase inhibitors may be useful as therapeutic agents for estrogen-dependent breast cancers.

Structure-activity relationship studies of the amide functionality in (p-O-sulfamoyl)-N-alkanoyl tyramines as estrone sulfatase inhibitors.

Recently, we reported the synthesis and biomedical studies of a series of (p-O-sulfamoyl)-N-alkanoyl tyramines as nonsteroidal estrone sulfatase inhibitors. One of the most potent inhibitors in this series is (p-O-sulfamoyl)-N-tridecanoyl tyramine 1 with an 1C50 value of 61.3 nM. In this study, we synthesized four analogs of 1 (compounds 2-5) to investigate the structure-activity relationships of the amide functionality in (p-O-sulfamoyl)-N-tridecanoyl tyramine. Replacement of the amide functionality in 1 with an ethylene moiety to form the alkyl analog 5 resulted in complete loss of sulfatase inhibitory activity (IC50 of 61.3 nM vs. > 20 microM). The keto, hydroxy, and ester analogs (inhibitors 2-4) are 8-15 times less in affinity to the sulfatase than inhibitor 1. However, their inhibitory activities are significantly higher than the alkyl analog 5. The results suggest that the amide functionality is favorable for sulfatase inhibitory activity and that there may be a hydrogen bonding component to the enzyme interaction in this region.

Synthesis of sodium androst-5-ene-17-one-3 beta-methylene sulfonate.

The synthesis of sodium androst-5-ene-17-one-3 beta-methylene sulfonate 2, a stable analog of memory-enhancing neurosteroid dehydroepiandrosterone sulfate, is described. The synthesis of compound 2 is carried out in six steps from dehydroepiandrosterone.

Exogenous transforming growth factor-beta 2 enhances connective tissue formation and wound strength in guinea pig dermal wounds healing by secondary intent.

The presence of transforming growth factor-beta (TGF-beta) at the site of acute injury, its ability to attract inflammatory and connective tissue cells, and its stimulatory effect on the deposition of connective tissue matrix combine to suggest that it may play a key role in the response to injury. The effect of exogenous TGF-beta form 2 on dermal wounds healing by secondary intent was investigated using a sponge composed of collagen and heparin as a delivery vehicle. Longitudinal lenticular-shaped wounds on the dorsum of adult guinea pigs were treated at the time of wounding with delivery vehicle containing 0.5, 1, or 5 micrograms of purified, bovine bone-derived TGF-beta 2, and were compared with wounds that received vehicle only or were untreated. At days 8 and 14 the amount of connective tissue in the wounds and the extent of epithelialization were determined by histomorphometric methods, and wound breaking strength was determined. At day 8, but not at day 14, wounds treated with 1 or 5 micrograms of TGF-beta 2 contained a significantly higher proportion of connective tissue than did wounds treated with vehicle only, and they also exhibited higher wound strength. No effect on wound size or re-epithelialization was detected. The observations provide evidence that a single treatment with exogenous TGF-beta 2 delivered in collagen/heparin sponge vehicle can accelerate repair in guinea pig dermal wounds allowed to heal by secondary intent.

Basement membrane collagen of renal glomerulus.

Collagen has been prepared from steer glomerular basement membrane by controlled pepsin solubilization. Four collagen polypeptides of potential alpha chain size have been isolated following denaturation and reduction with mercaptoethanol. Purification was obtained through sequential chromatography by gel filtration on agarose and by ion exchange on carboxymethyl- and diethylaminoethylcellulose and confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Fractions A and B resemble interstitial alpha chains in apparent molecular weight by gel filtration (93,000). Fractions C and D were released from a single high molecular weight fraction (II) by reduction with mercaptoethanol and had a larger apparent molecular weight by gel filtration (140,000). Amino acid composition of all fractions demonstrated that they are closely interrelated by generally distinctive from interstitial collagens. Cysteine was present in all fractions except Fraction A. Prior to reduction, all mercaptan groups were inaccessible to iodoacetate and p-chloromercuribenzoate but became completely titrable after treatment with mercaptoethanol. The starting material and all fractions contained large amounts of hexose. Glucose and galactose predominated; but mannose, glucosamine, and galactosamine were also present in substantial amounts. Small amounts of fucose and sialic acids were found in starting material only.