Steroid sulfatase (STS) catalyzes the desulfation of biologically inactive sulfated steroids to yield biologically active desulfated steroids and is currently being examined as a target for therapeutic intervention for the treatment of breast and other steroid-dependent cancers. A series of 17-arylsulfonamides of 17-aminoestra-1,3,5(10)-trien-3-ol were prepared and evaluated as inhibitors of STS. Introducing n-alkyl groups into the 4´-position of the 17-benzenesulfonamide derivative resulted in an increase in potency with the n-butyl derivative exhibiting the best potency with an IC50 of 26 nM. A further increase in carbon units (to n-pentyl) resulted in a decrease in potency. Branching of the 4´-n-propyl group resulted in a decrease in potency while branching of the 4´-n-butyl group (to a tert-butyl group) resulted in a slight increase in potency (IC50 = 18 nM). Studies with 17-benzenesulfonamides substituted at the 3´- and 4´-positions with small electron donating and electron withdrawing groups revealed the 3´-bromo and 3´-trifluoromethyl derivatives to be excellent inhibitors with IC50’s of 30 and 23 nM respectively. The 17-2´-naphthalenesulfonamide was also an excellent inhibitor (IC50 = 20 nM) while the 17-4´-phenylbenzenesulfonamide derivative was the most potent inhibitor with an IC50 of 9 nM. Kinetic studies with 3´-bromo derivative revealed it to be a non-competitive inhibitor and so these types of inhibitors might be capable of binding at the active site and also at a secondary site outside the active site. The amide analogs of some of these compounds were found not to be as potent inhibitors as the sulfonamides. Introducing a nitro group or fluorine atom into the 4-position of the 17-arylsulfonamide inhibitors resulted in an increase in potency. Some of these compounds are the most potent reversible STS inhibitors ever reported with apparent Ki’s as low as 1 nM. 3-O-Sulfation of these compounds did not significantly alter their potency. It is not known if 3-O-sulfated derivatives were acting as inhibitors or reversible suicide inhibitors. Docking studies were performed on selected inhibitors to gain insight into how they might interact with STS. Selected 17-arylsulfonamide inhibitors were sent to the NCI (USA) for in vitro screening with a panel of 60 human tumor cell lines (NCI-60 panel). Almost all of the compounds exhibited GI50’s in the 1 to 10 M range with all 60 cell lines and so were only moderately potent in terms of their ability to inhibit the growth. None of the compounds stood out in terms of their ability to inhibit the growth of any breast cancer, prostate cancer or any other cancer cell line studied. The thiadiazolidinedione group was proposed as a sulfate mimic for obtaining STS inhibitors. A new approach to the synthesis of 3-aminoestrone was achieved as part of an attempt to prepare the thiadiazolidinedione target. 3-O-Sulfamoylation of one of the 17-arylsulfonamide inhibitors was attempted using a variety of reaction conditions but was unsucce

Steroid sulfatase (STS) catalyzes the desulfation of sulfated steroids such as estrone sulfate to the corresponding steroid such as estrone. Inhibitors of STS are believed to have potential for treating estrogen-dependent breast cancer. A new class of potential irreversible suicide inhibitors of STS, based on aryl sulfates bearing a monofluoromethyl or difluoromethyl group ortho to the sulfate group, was synthesized. Key to the success of these syntheses was the use of new sulfation methodology recently developed in the Taylor group. A new and efficient route to 4-formyl estrone, a time-dependent, irreversible STS inhibitor, is also reported. Several new classes of potential, reversible STS inhibitors were synthesized. These compounds are analogs of known STS substrates in which the sulfate group is replaced with an ???,??????-difluoromethylenesulfonamide group, a boronic acid group or a sulfinic acid group. We also report the synthesis of estrone sulfate analogs that bear a carboxylate moiety at the 17-position and a sulfate surrogate at the 3-position. It is anticipated that these compounds will inhibit STS by interacting with Arg98 which lies at the periphery of the active site. Key to the success of this synthesis was the use of the t-butyl group as a protecting group for the 2-position of estrone. Finally, our preliminary investigations into the synthesis of a new class of chiral electrophilic fluorinating agents are presented. These reagents are based on a chiral binaphthyl sulfonimide scaffold and are expected to be capable of performing enantioselective electrophilic fluorinations. Such reagents may be useful in synthesizing organofluorines of biological significance including STS inhibitors.

Steroid sulfatase (STS) catalyzes the desulfation of sulfated steroids such as estrone sulfate to the corresponding steroid such as estrone. Inhibitors of STS are believed to have potential for treating estrogen-dependent breast cancer. A new class of potential irreversible suicide inhibitors of STS, based on aryl sulfates bearing a monofluoromethyl or difluoromethyl group ortho to the sulfate group, was synthesized. Key to the success of these syntheses was the use of new sulfation methodology recently developed in the Taylor group. A new and efficient route to 4-formyl estrone, a time-dependent, irreversible STS inhibitor, is also reported. Several new classes of potential, reversible STS inhibitors were synthesized. These compounds are analogs of known STS substrates in which the sulfate group is replaced with an ,-difluoromethylenesulfonamide group, a boronic acid group or a sulfinic acid group. We also report the synthesis of estrone sulfate analogs that bear a carboxylate moiety at the 17-position and a sulfate surrogate at the 3-position. It is anticipated that these compounds will inhibit STS by interacting with Arg98 which lies at the periphery of the active site. Key to the success of this synthesis was the use of the t-butyl group as a protecting group for the 2-position of estrone. Finally, our preliminary investigations into the synthesis of a new class of chiral electrophilic fluorinating agents are presented. These reagents are based on a chiral binaphthyl sulfonimide scaffold and are expected to be capable of performing enantioselective electrophilic fluorinations. Such reagents may be useful in synthesizing organofluorines of biological significance including STS inhibitors.

Steroid sulfatase (STS) is considered to be one of the key enzymes contributing to the development of breast cancer. It catalyzes the hydrolysis of inactive sulfated steroids such as estrone sulfate (ES) to inorganic sulfate active steroids such as estrone (E1), a precursor to estradiol (E2), a key stimulator for breast cancer development. Inhibitors of STS are currently being pursued in both academia and industry as potential drugs for treating breast cancer. A series of 4-substituted estrone and estradiol derivatives were examined as inhibitors of STS. Inhibition of STS with 4-FE1, an irreversible inhibitor of STS previously studied in the Taylor group, can be enhanced by introducing a hydrophobic benzyl group at the 17-positon of 4-FE1. As with 4-FE1, the inhibition was concentration and time-dependent. Only 14% of the activity could be recovered after extensive dialysis. Introducing substituents at the 2-position of 4-formyl estrogen derivatives resulted in loss of concentration and time-dependent inhibition and a considerable decrease in inhibitor affinity. Studies with estrogen derivatives substituted at the 4-position with groups other than a formyl revealed that a relatively good reversible inhibitor can be obtained simply by introducing an electron withdrawing group at this position. These types of inhibitors are non-competitive inhibitors suggesting an alternative steroid binding site. A series of estrone derivatives were examined as photoaffinity labels of STS. 4-azidoestrone suflate and 4-azidoestrone phosphate exhibited properties that are suitable for photoaffinity labeling studies with STS. These labels may be useful for ascertaining pathways of substrate entry into the STS active site. 16-diazoestrone phosphate was not a photoaffinity label of STS. 2- and 4-azido estrone and 16-diazoestrone all acted as photoaffinity labels of STS. These compounds may be useful for ascertaining pathways of product release from the STS active site.

Steroid sulfatase (STS) is considered to be one of the key enzymes contributing to the development of breast cancer. It catalyzes the hydrolysis of inactive sulfated steroids such as estrone sulfate (ES) to inorganic sulfate active steroids such as estrone (E1), a precursor to estradiol (E2), a key stimulator for breast cancer development. Inhibitors of STS are currently being pursued in both academia and industry as potential drugs for treating breast cancer. A series of 4-substituted estrone and estradiol derivatives were examined as inhibitors of STS. Inhibition of STS with 4-FE1, an irreversible inhibitor of STS previously studied in the Taylor group, can be enhanced by introducing a hydrophobic benzyl group at the 17-positon of 4-FE1. As with 4-FE1, the inhibition was concentration and time-dependent. Only 14% of the activity could be recovered after extensive dialysis. Introducing substituents at the 2-position of 4-formyl estrogen derivatives resulted in loss of concentration and time-dependent inhibition and a considerable decrease in inhibitor affinity. Studies with estrogen derivatives substituted at the 4-position with groups other than a formyl revealed that a relatively good reversible inhibitor can be obtained simply by introducing an electron withdrawing group at this position. These types of inhibitors are non-competitive inhibitors suggesting an alternative steroid binding site. A series of estrone derivatives were examined as photoaffinity labels of STS. 4-azidoestrone suflate and 4-azidoestrone phosphate exhibited properties that are suitable for photoaffinity labeling studies with STS. These labels may be useful for ascertaining pathways of substrate entry into the STS active site. 16-diazoestrone phosphate was not a photoaffinity label of STS. 2- and 4-azido estrone and 16-diazoestrone all acted as photoaffinity labels of STS. These compounds may be useful for ascertaining pathways of product release from the STS active site.

The development of reversible and irreversible inhibitors of steroid sulfatase (STS) and protein tyrosine phosphatase 1B (PTP1B) is reported herein. STS belongs to to the aryl sulfatase family of enzymes that have roles in diverse processes such as hormone regulation, cellular degradation, bone and cartilage development, intracellular communication, and signalling pathways. STS catalyzes the desulfation of sulfated steroids which are the storage forms of many steroids such as the female hormone estrone. Its crucial role in the regulation of estrogen levels has made it a therapeutic target for the treatment of estrogen-dependent cancers. Estrone sulfate derivatives bearing 2- and 4-mono- and difluoromethyl substitutions were examined as quinone methide-generating suicide inhibitors of STS with the goal of developing these small molecules as activity-based probes for proteomic profiling of sulfatases. Kinetic studies suggest that inhibition by the monofluoro derivatives is a result of a quinone methide intermediate that reacts with active-site nucleophiles. However, the main inhibition pathway of the 4-difluoromethyl derivative involved an unexpected process in which initially formed quinone methide diffuses from the active site and decomposes to an aldehyde in solution which then acts as a potent, almost irreversible STS inhibitor. This is the first example where this class of inactivator functions by in situ generation of an aldehyde. 6- and 8-mono- and difluoromethyl coumarin derivatives were also examined as quinone methide-generating suicide inhibitors of STS. The 6-monofluoromethyl derivative acted as a classic suicide inhibitor. The partition ratio of this compound was found to be very large indicating that this class of compounds is not likely suitable as an activity-based probe for proteomic profiling of sulfatases. Boronic acids derived from steroid and coumarin platforms were also examined as STS inhibitors with the goal of improving our understanding of substrate binding specificity of STS. Inhibition constants in the high nanomolar to low micromolar range were observed for the steroidal derivatives. The coumarin derivatives were poor inhibitors. These results suggest that the boronic acid moiety must be attached to a platform very closely resembling a natural substrate in order for it to impart a beneficial effect on binding affinity compared to its phenolic analog. The mode of inhibition observed was reversible and kinetic properties corresponding to the mechanism for slow-binding inhibitors were not observed. PTP1B catalyzes the dephosphorylation of phosphotyrosine residues in the insulin receptor kinase and is a key enzyme in the down regulation of insulin signaling. Inhibitors of PTP1B are considered to have potential as therapeutics for treating type II diabetes mellitus. The difluoromethylenesulfonic (DFMS) acid group, one of the best monoanionic phosphotyrosine mimics reported in the literature, was examined as a phosphotyrosine (pTyr) mimic in a non-peptidyl platform for PTP1B inhibition. The DFMS-bearing inhibitor was found to be an approximately 1000-fold poorer inhibitor than its phosphorus analogue. It was also found that the fluorines in the DFMS inhibitor contributed little to inhibitory potency. In addition, [sulfonamido(difluoromethyl)]-phenylalanine (F2Smp) was examined as a neutral pTyr mimic in commonly used hexapeptide and tripeptide platforms. F2Smp was found to be a poor pTyr mimic. These inhibition studies also revealed that the tripeptide platform is not suitable for assessing pTyr mimics for PTP1B inhibition. Taken together, the kinetic data on the inhibition of STS and PTP1B provide valuable information relevant for future design of inhibitors of these two therapeutic targets.

The development of reversible and irreversible inhibitors of steroid sulfatase (STS) and protein tyrosine phosphatase 1B (PTP1B) is reported herein. STS belongs to to the aryl sulfatase family of enzymes that have roles in diverse processes such as hormone regulation, cellular degradation, bone and cartilage development, intracellular communication, and signalling pathways. STS catalyzes the desulfation of sulfated steroids which are the storage forms of many steroids such as the female hormone estrone. Its crucial role in the regulation of estrogen levels has made it a therapeutic target for the treatment of estrogen-dependent cancers. Estrone sulfate derivatives bearing 2- and 4-mono- and difluoromethyl substitutions were examined as quinone methide-generating suicide inhibitors of STS with the goal of developing these small molecules as activity-based probes for proteomic profiling of sulfatases. Kinetic studies suggest that inhibition by the monofluoro derivatives is a result of a quinone methide intermediate that reacts with active-site nucleophiles. However, the main inhibition pathway of the 4-difluoromethyl derivative involved an unexpected process in which initially formed quinone methide diffuses from the active site and decomposes to an aldehyde in solution which then acts as a potent, almost irreversible STS inhibitor. This is the first example where this class of inactivator functions by in situ generation of an aldehyde. 6- and 8-mono- and difluoromethyl coumarin derivatives were also examined as quinone methide-generating suicide inhibitors of STS. The 6-monofluoromethyl derivative acted as a classic suicide inhibitor. The partition ratio of this compound was found to be very large indicating that this class of compounds is not likely suitable as an activity-based probe for proteomic profiling of sulfatases. Boronic acids derived from steroid and coumarin platforms were also examined as STS inhibitors with the goal of improving our understanding of substrate binding specificity of STS. Inhibition constants in the high nanomolar to low micromolar range were observed for the steroidal derivatives. The coumarin derivatives were poor inhibitors. These results suggest that the boronic acid moiety must be attached to a platform very closely resembling a natural substrate in order for it to impart a beneficial effect on binding affinity compared to its phenolic analog. The mode of inhibition observed was reversible and kinetic properties corresponding to the mechanism for slow-binding inhibitors were not observed. PTP1B catalyzes the dephosphorylation of phosphotyrosine residues in the insulin receptor kinase and is a key enzyme in the down regulation of insulin signaling. Inhibitors of PTP1B are considered to have potential as therapeutics for treating type II diabetes mellitus. The difluoromethylenesulfonic (DFMS) acid group, one of the best monoanionic phosphotyrosine mimics reported in the literature, was examined as a phosphotyrosine (pTyr) mimic in a non-peptidyl platform for PTP1B inhibition. The DFMS-bearing inhibitor was found to be an approximately 1000-fold poorer inhibitor than its phosphorus analogue. It was also found that the fluorines in the DFMS inhibitor contributed little to inhibitory potency. In addition, [sulfonamido(difluoromethyl)]-phenylalanine (F2Smp) was examined as a neutral pTyr mimic in commonly used hexapeptide and tripeptide platforms. F2Smp was found to be a poor pTyr mimic. These inhibition studies also revealed that the tripeptide platform is not suitable for assessing pTyr mimics for PTP1B inhibition. Taken together, the kinetic data on the inhibition of STS and PTP1B provide valuable information relevant for future design of inhibitors of these two therapeutic targets.

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