**4. Inhibition of estrone formation in the body**

It is known that about two thirds of all the breast cancers occur in postmenopausal women, when estrogen is no longer synthesized in ovaries. However the estradiol content in

Approaches for Searching

**O**

properties.

of Modified Steroid Estrogen Analogues with Improved Biological Properties 181

Steroid **41** and **42** properties investigation results could be used as an example (**diSalle & Robinson, 1990**). The IC50 values in the experiments on human placenta cells are approximately equal - 42.5±4.3 and 20.3±2.2 nM correspondingly. However, in the experiments on rats the values of ED50 (dose, at which the inhibition is 50% from highest possible one) are 3.7 and more 100 mg/kg under *per os* injection. Necessity of compounds investigation can be illustrated by huge number of examples in various animal models and by methods of steroids introduction into the body. In particular, compound **42** under subcutaneous introduction in doses 30 and 150 mg/kg blocks the growth of bearing DMBAinduced mammary tumors in female rats (**Nishino et al., 1989**) and after numerous

The main position for attack during demethylation of androst-4-en-3,17-dione is methyl group at С-19. Numerous derivatives at С-10 have been synthesized. Among them the most effective one is compound **43** with Ki 3 nМ to human placenta enzyme (**Cole & Robinson, 1990).** It comes as no surprise, that this compound is clinically used (**Numazava et al., 2005**). Compounds **44** (**Greway et al., 1990**) and **45** (**Peet et al., 1992**) demonstrated good

Detailed search of aromatase inhibitors led to the creation of medications of steroid nature for the treatment of oncological diseases in clinic, from them, exemestane **41** and formestane **46** are also widely known. However clinical application of aromatase inhibitors revealed the presence of a number of non-specific toxic side effects: asthenia, nausea, headache etc. Certain endocrinological side effects in postmenopausal women are notable, namely hot flashes and vaginal dryness (**Goss, 1999**). Therefore the search of aromatase inhibitors in

**O**

**OH**

**H H H**

**H H H**

**Ph**

**OH**

**F**

**OH**

**H H H** **O**

**O**

**OH**

**O**

**O**

**H H H**

**CH3**

**OH**

**H H H**

**O**

**O**

 **44 45 46** 

investigations is clinically used with the trade name atamestane.

steroid series must be done in series that is not belonging to androgens.

**OH**

**H H H**

 **47** a) R=Cl, b) R=Br, c) R=CH3 **48 49** 

**O**

**H H H**

**F**

**OH**

 **25 50** 

**R**

**OH**

**O**

**H H H**

estrogen-dependent tumors is significantly higher than in blood plasma and in normal tissues on bourdes with tumors. Partly it can be explained by the ability of tumor to accept estradiol from the blood **(Pasqualini et al., 1996**), because estrogen synthesis may be enhanced in extragonadal organs. Furthermore, local synthesis of estrogens in tumor may be on high level (**Pasqualini et al., 199**7). In view of the aforesaid it is necessary to consider the ways of synthesis of these hormones and understand the influence on the formation of reactive estrogens in tumor (scheme).

It is well-known, that estrogens are formed in the body from testosterone or androst-4-en-3,17-dione **39**. Aromatization is multistep process, proceeding under the action of enzymes of cytochrome Р-450 system. The main steps of aromatization, key stage in estrogen formation are considered in many publications (**Jordan & Brodie, 2007** and citations herein). It is reasonable that the great attention is directed to the search of irreversible inhibitors of aromatase.

Most often the initial investigations are carrying out on human placenta cells, the criterion for the evaluation of effectiveness of investigated compounds is value of IC50 (concentration, at which the inhibition of enzyme activity is 50% from highest possible one under experimental conditions). Compounds having IC50 in the range of nanomolar concentrations have perspectives to be further investigated. Next step for the evaluation of inhibitory activity of perspective compounds is the investigation on animal models, and very often the preliminary evaluation of effectiveness is in contrast with data obtained in second series.

estrogen-dependent tumors is significantly higher than in blood plasma and in normal tissues on bourdes with tumors. Partly it can be explained by the ability of tumor to accept estradiol from the blood **(Pasqualini et al., 1996**), because estrogen synthesis may be enhanced in extragonadal organs. Furthermore, local synthesis of estrogens in tumor may be on high level (**Pasqualini et al., 199**7). In view of the aforesaid it is necessary to consider the ways of synthesis of these hormones and understand the influence on the formation of

**H**

**H H**

**H**

**H H**

**Estradiol (E2)**

It is well-known, that estrogens are formed in the body from testosterone or androst-4-en-3,17-dione **39**. Aromatization is multistep process, proceeding under the action of enzymes of cytochrome Р-450 system. The main steps of aromatization, key stage in estrogen formation are considered in many publications (**Jordan & Brodie, 2007** and citations herein). It is reasonable that the great attention is directed to the search of irreversible inhibitors of

Most often the initial investigations are carrying out on human placenta cells, the criterion for the evaluation of effectiveness of investigated compounds is value of IC50 (concentration, at which the inhibition of enzyme activity is 50% from highest possible one under experimental conditions). Compounds having IC50 in the range of nanomolar concentrations have perspectives to be further investigated. Next step for the evaluation of inhibitory activity of perspective compounds is the investigation on animal models, and very often the preliminary evaluation of effectiveness is in contrast with data

**O**

**O**

**H H H**

**Estrone**

**OH**

**OH**

**Aromatase**

**O**

**OH**

**17-Hydroxysteroid dehydrogenase**

**Estrone sulphatase Sulfotransferase estrone**

**HO3SO**

**H**

**H H**

**Sulphate estrone (E1C)**

**40**

**O**

**H H H** **O**

reactive estrogens in tumor (scheme).

**O**

**39**

**H**

**H H**

**Androst-4-en-3,17-dione**

**O**

**(Adione)**

aromatase.

obtained in second series.

**O**

**CH2**

**H H H** **O**

**O**

**Exemestane 41 42 43** 

Steroid **41** and **42** properties investigation results could be used as an example (**diSalle & Robinson, 1990**). The IC50 values in the experiments on human placenta cells are approximately equal - 42.5±4.3 and 20.3±2.2 nM correspondingly. However, in the experiments on rats the values of ED50 (dose, at which the inhibition is 50% from highest possible one) are 3.7 and more 100 mg/kg under *per os* injection. Necessity of compounds investigation can be illustrated by huge number of examples in various animal models and by methods of steroids introduction into the body. In particular, compound **42** under subcutaneous introduction in doses 30 and 150 mg/kg blocks the growth of bearing DMBAinduced mammary tumors in female rats (**Nishino et al., 1989**) and after numerous investigations is clinically used with the trade name atamestane.

The main position for attack during demethylation of androst-4-en-3,17-dione is methyl group at С-19. Numerous derivatives at С-10 have been synthesized. Among them the most effective one is compound **43** with Ki 3 nМ to human placenta enzyme (**Cole & Robinson, 1990).** It comes as no surprise, that this compound is clinically used (**Numazava et al., 2005**). Compounds **44** (**Greway et al., 1990**) and **45** (**Peet et al., 1992**) demonstrated good properties.

Detailed search of aromatase inhibitors led to the creation of medications of steroid nature for the treatment of oncological diseases in clinic, from them, exemestane **41** and formestane **46** are also widely known. However clinical application of aromatase inhibitors revealed the presence of a number of non-specific toxic side effects: asthenia, nausea, headache etc. Certain endocrinological side effects in postmenopausal women are notable, namely hot flashes and vaginal dryness (**Goss, 1999**). Therefore the search of aromatase inhibitors in steroid series must be done in series that is not belonging to androgens.

Approaches for Searching

residues (mainly it is hydrophobic amino acids).

enzyme with inhibitors of steroid series (**Ahmed S., 2001**).

an unusual amino acid - formylglycine in 75 position.

specificity of estrone sulfatase of various natures.

comparison with estrone **(Elger et al., 1995**).

**H**

**R**

**1**

**R**

**2**

**NH2SO2O**

**H H**

**O**

**H O**

 **51 52 53** 

**O**

of Modified Steroid Estrogen Analogues with Improved Biological Properties 183

Estrone sulfatase from human placenta is suitable model object for searching for corresponding inhibitors. It is the transmembrane protein that consists of 583 amino acid

On the basis of data about primary structure of this enzyme and taking into account its homology with human arylsulfatase A and human arylsulfatase B (X-Ray data of arylsulfatase were known), the structure of catalytic centre of estrone sulfatase was proposed (**Howard et al., 2002).** X-Ray data of estrone sulfatase has been obtained a little bit later and it was shown that its tertiary structure is formed by two domains – globular polar domain, containing the active center and transmembrane domain, formed by two antiparallel α-helixes. Polar domain consists of two sub-domains, one of them has the active centre of enzyme. Transmembrane domain opens the entrance into the active center of enzyme, which is situated deep under the "mashrooms hat", close to the membrane surface. Near the active center entrance a big amount of hydrophobic amino acids is concentrated, which are a part of TD (Phe178, 182, 187, 230, 233 and 237), and the polar domain (Phe104 and 557, Leu554). Side chains of these amino acids form a tunnel, which leads to the active center (**Hernandes-Guzman et al., 2003**). Catalytic area of active center of estrone sulfatase is highly homologous to active centers of arylsulfatase A and В. Nine from ten catalytically important amino acids Asp35, Asp36, FG75, Arg79, Lys134, His136, His290, Asp342 and Lys368 are in analogous positions to all three sulfatases. It is in quite correlation with the experimental data about the importance of hydrophobic interactions during the binding of

Conceptions concerning estrogen sulfates hydrolysis mechanisms and ferment deactivation are presented in series of articles (**Reed et al., 2005**), where an important role is assigned to

Investigation of kinetic parameters of hydrolysis of estrone sulphate and dehydroepiandrosterone sulphate have shown that values of Vmax for these compounds are correspondingly 9.95 and 1.89 μM/min, values of Km are 72.75 and 9.59 μM (**Hernandez-Guzman et al., 2001**). These values could be used as standard for the evaluation of substrate

In several investigations it was established that for the effective inhibition of estrone sulfatase molecules must have sulphamate group at position 3. Firstly the necessity of this group was demonstrated on the example of steroid **51a**, widely known as EMATE (**Purohit et al., 1995).** However this compound did not reach the clinical application as anti-tumor agent because its estrogenic activity under *per os* introduction is in 5 times higher in

**H**

**H H**

The investigations of Professor M. Reed' group are examples of the successful creation of estrone sulfatase inhibitors with lowered hormonal activity (**Purohit et al., 1998**), authors have synthesized compounds **51a-j**. The selection of modifications in EMATE structure was done on the basis of experimental facts about significant fall of uterotropic activity of

**O**

**B OH**

**OH**

**OH**

**H**

**H H**

Because estrone **2** or estradiol **1** are formed as result of aromatization process it was possible to assume that these compounds will be reversible inhibitors of aromatase. Indeed, later it was found that even estradiol is anti-aromatase agent in human breast cancer cells (**Pasqualini & Chetrite, 2006);** the same was found for estrone sulfatase inhibition **(Pasqualini & Chetrite, 2001).** Simultaneously, reasoning from the same assumption, the synthesis of modified estrogens with substituents at С-2, С-4 or С-6 was carried out (**Numazava et al., 2005**).

These analogues are competitive inhibitors of aromatase. 2-Substituted steroids **47 a,b,c,** have the best properties, they are significantly active in comparison with estrone **2**, Ki values are 0.130±0.017, 0.224±0.024, 0.360±0.019 and 2.50±0.22 μM correspondingly. Steroids **48** and **49** have similar activity - Ki values are 0.10±0.006 and 0.21±0.009 μM, however their synthesis is more complicated. Inhibitory activity of estradiol derivatives is significantly lower. From our point of view, its worth to pay attention to 2-fluoroestradiol **25,** in spite of the value of Ki 9.04±0.5 μМ being higher in comparison to other compounds and the fact, that its synthesis is more complicated. It is more important that this analogue does not possess carcinogenic properties in the experiments on rats (**Liehr, 1983**). In the series of 4 substituted estrone derivatives – 4-fluoroanalogue **50** has the best properties (Кi = 1.28±0.07). All presented investigations had extensive character and it was impossible to take into account many details of biological properties. X-Rays analysis of complex of aromatase from human placenta with androst-4-en-3,17-dione **39** has been obtained (**Ghosh et al., 2009**), that opened the perspectives for the targeted search of aromatase inhibitors. Using the data obtained resulted in the docking of new reversible inhibitors in aromatase structure that allowed to explain their activity (**Yadav et al., 2011**).

Aromatase inhibitors are widely used for the treatment of estrogen-dependent oncological diseases; however they have a number of side-effects. Thus, the decreasing of estrogen content in the body automatically results in the increasing of cholesterol level in blood, and hence to the increased risk of cardiovascular diseases. This is the main reason for the creation of inhibitors on the basis of steroids in comparison with non-steroid ones. For example, exemestane has minimal negative effects on bone and lipid metabolism in animal and clinical studies (**Carpenter & Miller, 2005**). There is an opinion that exemestane may improve lipid profile (**Bundred, 2005**).

Molecular bases of interactions of various groups of ligands with aromatase have been considered relatively not long ago, that may lead to the creation of inhibitors with improved properties (**Hong et. al., 2008**).

Aromatase inhibitors are important, but not sufficient for blocking of growth of hormonesensitive tumors. Point is that there is at least one more way for estrogens' formation in tumors.

It is known that estrone sulphate concentration in blood plasma of postmenopausal women is in 5-10 times higher in comparison with free hormone concentration. Assumption was made that estrogen sulphates may play a role of "source" of free estrogens in various organs, because its «half-life time» is longer than life-time of free estrogens (**Reed & Potter, 199**9). Estrogen sulphates may migrate into hormone-depended tumor and then converted to free hormones under estrone sulfatase action. It was found that estrone sulfatase activity in tumors is in 130-200 times higher in comparison with aromatase activity (**Chetrite et al., 2000**). This was the basis for initiation of investigations for the search for irreversible estrone sulfatase inhibitors.

Because estrone **2** or estradiol **1** are formed as result of aromatization process it was possible to assume that these compounds will be reversible inhibitors of aromatase. Indeed, later it was found that even estradiol is anti-aromatase agent in human breast cancer cells (**Pasqualini & Chetrite, 2006);** the same was found for estrone sulfatase inhibition **(Pasqualini & Chetrite, 2001).** Simultaneously, reasoning from the same assumption, the synthesis of modified estrogens with substituents at С-2, С-4 or С-6 was carried out

These analogues are competitive inhibitors of aromatase. 2-Substituted steroids **47 a,b,c,** have the best properties, they are significantly active in comparison with estrone **2**, Ki values are 0.130±0.017, 0.224±0.024, 0.360±0.019 and 2.50±0.22 μM correspondingly. Steroids **48** and **49** have similar activity - Ki values are 0.10±0.006 and 0.21±0.009 μM, however their synthesis is more complicated. Inhibitory activity of estradiol derivatives is significantly lower. From our point of view, its worth to pay attention to 2-fluoroestradiol **25,** in spite of the value of Ki 9.04±0.5 μМ being higher in comparison to other compounds and the fact, that its synthesis is more complicated. It is more important that this analogue does not possess carcinogenic properties in the experiments on rats (**Liehr, 1983**). In the series of 4 substituted estrone derivatives – 4-fluoroanalogue **50** has the best properties (Кi = 1.28±0.07). All presented investigations had extensive character and it was impossible to take into account many details of biological properties. X-Rays analysis of complex of aromatase from human placenta with androst-4-en-3,17-dione **39** has been obtained (**Ghosh et al., 2009**), that opened the perspectives for the targeted search of aromatase inhibitors. Using the data obtained resulted in the docking of new reversible inhibitors in aromatase structure that

Aromatase inhibitors are widely used for the treatment of estrogen-dependent oncological diseases; however they have a number of side-effects. Thus, the decreasing of estrogen content in the body automatically results in the increasing of cholesterol level in blood, and hence to the increased risk of cardiovascular diseases. This is the main reason for the creation of inhibitors on the basis of steroids in comparison with non-steroid ones. For example, exemestane has minimal negative effects on bone and lipid metabolism in animal and clinical studies (**Carpenter & Miller, 2005**). There is an opinion that exemestane may

Molecular bases of interactions of various groups of ligands with aromatase have been considered relatively not long ago, that may lead to the creation of inhibitors with improved

Aromatase inhibitors are important, but not sufficient for blocking of growth of hormonesensitive tumors. Point is that there is at least one more way for estrogens' formation in

It is known that estrone sulphate concentration in blood plasma of postmenopausal women is in 5-10 times higher in comparison with free hormone concentration. Assumption was made that estrogen sulphates may play a role of "source" of free estrogens in various organs, because its «half-life time» is longer than life-time of free estrogens (**Reed & Potter, 199**9). Estrogen sulphates may migrate into hormone-depended tumor and then converted to free hormones under estrone sulfatase action. It was found that estrone sulfatase activity in tumors is in 130-200 times higher in comparison with aromatase activity (**Chetrite et al., 2000**). This was the basis for initiation of investigations for the search for irreversible estrone

(**Numazava et al., 2005**).

allowed to explain their activity (**Yadav et al., 2011**).

improve lipid profile (**Bundred, 2005**).

properties (**Hong et. al., 2008**).

tumors.

sulfatase inhibitors.

Estrone sulfatase from human placenta is suitable model object for searching for corresponding inhibitors. It is the transmembrane protein that consists of 583 amino acid residues (mainly it is hydrophobic amino acids).

On the basis of data about primary structure of this enzyme and taking into account its homology with human arylsulfatase A and human arylsulfatase B (X-Ray data of arylsulfatase were known), the structure of catalytic centre of estrone sulfatase was proposed (**Howard et al., 2002).** X-Ray data of estrone sulfatase has been obtained a little bit later and it was shown that its tertiary structure is formed by two domains – globular polar domain, containing the active center and transmembrane domain, formed by two antiparallel α-helixes. Polar domain consists of two sub-domains, one of them has the active centre of enzyme. Transmembrane domain opens the entrance into the active center of enzyme, which is situated deep under the "mashrooms hat", close to the membrane surface. Near the active center entrance a big amount of hydrophobic amino acids is concentrated, which are a part of TD (Phe178, 182, 187, 230, 233 and 237), and the polar domain (Phe104 and 557, Leu554). Side chains of these amino acids form a tunnel, which leads to the active center (**Hernandes-Guzman et al., 2003**). Catalytic area of active center of estrone sulfatase is highly homologous to active centers of arylsulfatase A and В. Nine from ten catalytically important amino acids Asp35, Asp36, FG75, Arg79, Lys134, His136, His290, Asp342 and Lys368 are in analogous positions to all three sulfatases. It is in quite correlation with the experimental data about the importance of hydrophobic interactions during the binding of enzyme with inhibitors of steroid series (**Ahmed S., 2001**).

Conceptions concerning estrogen sulfates hydrolysis mechanisms and ferment deactivation are presented in series of articles (**Reed et al., 2005**), where an important role is assigned to an unusual amino acid - formylglycine in 75 position.

Investigation of kinetic parameters of hydrolysis of estrone sulphate and dehydroepiandrosterone sulphate have shown that values of Vmax for these compounds are correspondingly 9.95 and 1.89 μM/min, values of Km are 72.75 and 9.59 μM (**Hernandez-Guzman et al., 2001**). These values could be used as standard for the evaluation of substrate specificity of estrone sulfatase of various natures.

In several investigations it was established that for the effective inhibition of estrone sulfatase molecules must have sulphamate group at position 3. Firstly the necessity of this group was demonstrated on the example of steroid **51a**, widely known as EMATE (**Purohit et al., 1995).** However this compound did not reach the clinical application as anti-tumor agent because its estrogenic activity under *per os* introduction is in 5 times higher in comparison with estrone **(Elger et al., 1995**).

The investigations of Professor M. Reed' group are examples of the successful creation of estrone sulfatase inhibitors with lowered hormonal activity (**Purohit et al., 1998**), authors have synthesized compounds **51a-j**. The selection of modifications in EMATE structure was done on the basis of experimental facts about significant fall of uterotropic activity of

Approaches for Searching

**OH**

**OH**

**O**

**O**

Oxathiazine analogues showed weak inhibitory activity.

values of IC50 - 1 nM (**Fischer et al., 2003**).

**H**

**H H**

**H H H**

crucial importance **(Suzuki et al., 2003).**

biological properties of both series.

**OH**

**NH2SO2O**

**NH2SO2O**

**O**

 **62 63 64** 

of Modified Steroid Estrogen Analogues with Improved Biological Properties 185

Compounds of type **54** were chosen as an example and authors used another analogue (oxatiazine ring, condensed with A ring) instead of sulphamate group at C-3 **(Peters et al., 2003)**. In the experiments *in vitro* in an intact MCF-7 human breast cancer cells model steroids have shown high inhibitory activity. *In vivo*, agent **54** showed moderate antitumor activity against MCF-7 breast cancer xenografts in BALB/c athimic nude mice. 3,4-

Compounds **55-57** effectively inhibit estrone sulfatase (**Tanabe et al, 1999**). It indicated specifically on the possibility for the creation of inhibitors in the series of D-homo analogues

The development of this direction led to the creation of strong inhibitors **60** and **61** with

One more group of perspective inhibitors of estrone sulfatase was found – sulphamates of estrogens with methoxy- group at С-2 (**Raobaikady et al., 2003**). At the time of these investigations it was known that 2-methoxyestradiol **62** induces apoptosis in many various lines of tumor' cells, including prostate cancer cells, exhibits antiproliferative activity and blocks angiogenesis processes, which became a basis for the selection of model compounds.

**H**

**H H**

**H**

**H H**

 **65 66 67** a) R = SO2NH2, b) R = H The fact that bis-sulphamates of 2-methoxyestradiol **63** and 2-ethylestradiol **66** inhibit the growth of breast cancer cells, which are resistant to the action of many medications, has a

We have considered the ways for the search for modified estrogens for the inhibition of steroids metabolism enzymes which have been done in series of derivatives with natural rings junction. Obvious perspective direction is the investigation of analogues with unnatural rings junction since peculiarities of their structure have the number of important characteristics, comparable with ones in series of natural estrogens and comparable

It is known that high effectiveness of binding of estradiol **1** with ER is a result of high hydrophobicity of steroid molecule and possibility to form hydrogen bonds of phenolic hydroxyl group at С-3 with Glu353, Arg394 and water molecule. Hydroxyl group at С-17 forms hydrogen bond with His524 (**Brzozowski et al, 1997**). Modified 8-analogues of steroid estrogens have relative high affinity to ER, and model explaining the binding with

**OSO2NH2**

**OSO2NH2**

**NH2SO2O**

**R**

**O**

**O O**

**H H H**

> **H H H**

**O**

**O**

of estrogens that was confirmed using sulphamates **58** and **59** (**Reed & Potter, 1999**).


estrone derivatives when substituents like alkyl-, allyl- or nitro- groups were introduced at positions 2 and (or) 4 (**Purohit et al., 1999**). Some results on human placenta microsomes enzyme are presented in the Table 1.

Table 1.

Irreversible E-ST inactivation has been observed in all cases. Most active inhibitor from this series is – 4-nitro derivative **51d** – in 5 times stronger than ЕМАТЕ *in vitro* and had comparable activity with ЕМАТЕ activity *in vivo*. The investigation of uterotropic activity has shown that this steroid increases uterus weight of ovariectomized rats, although to lesser degree than ЕМАТЕ (158% in comparison with control, and 414% - ЕМАТЕ). Monoallylic analogues are weaker inhibitors estrone sulfatase, but they are more active in comparison with propyl- ones. The presence of large substituents at С-2 and/or С-4 leads to decreasing of inhibitory activity, which may be caused by shielding of oxygen atom at С-3. Estrone formiate **52** (**Schreiner & Billich, 2004**) and boronic acids **53** (**Ahmed, V., 2006**) have shown irreversible inhibitory activity, however these investigations had no further progress.

estrone derivatives when substituents like alkyl-, allyl- or nitro- groups were introduced at positions 2 and (or) 4 (**Purohit et al., 1999**). Some results on human placenta microsomes

Steroid R1 R2 IC50, μM Steroid R1 R2 IC50, μM a Н Н 0.004 f H CH3(CH2)2 > 100 b OMe H 0.03 g CH3(CH2)2 CH3(CH2)2 > 100 c NO2 H 0.07 h Allyl H 2.5 d H NO2 0.0008 i H Allyl 9 e CH3(CH2)2 H 29 j Allyl Allyl > 100

Irreversible E-ST inactivation has been observed in all cases. Most active inhibitor from this series is – 4-nitro derivative **51d** – in 5 times stronger than ЕМАТЕ *in vitro* and had comparable activity with ЕМАТЕ activity *in vivo*. The investigation of uterotropic activity has shown that this steroid increases uterus weight of ovariectomized rats, although to lesser degree than ЕМАТЕ (158% in comparison with control, and 414% - ЕМАТЕ). Monoallylic analogues are weaker inhibitors estrone sulfatase, but they are more active in comparison with propyl- ones. The presence of large substituents at С-2 and/or С-4 leads to decreasing of inhibitory activity, which may be caused by shielding of oxygen atom at С-3. Estrone formiate **52** (**Schreiner & Billich, 2004**) and boronic acids **53** (**Ahmed, V., 2006**) have shown irreversible inhibitory activity, however these investigations had no further progress.

> **H H H**

> > **NH2O2SO**

**N H**

**O**

**NH2O2SO**

**H H H** **N**

**O N**

**O**

**H H H**

**R'O**

**H H H**

> **H H H**

**COOCH2CH3**

**O O**

enzyme are presented in the Table 1.

**R**

**NH2O2SO**

**O**

**H**

**H H H**

**O**

**H**

 **54 55 56** 

**NH2O2SO**

**N**

**O**

 **60 61** 

**R' = Bn, H, SO2NH2**

**O**

 **57 58 59** 

**H H H**

**N S O**

**NH2O2SO**

**R'O**

**O O**

Table 1.

Compounds of type **54** were chosen as an example and authors used another analogue (oxatiazine ring, condensed with A ring) instead of sulphamate group at C-3 **(Peters et al., 2003)**. In the experiments *in vitro* in an intact MCF-7 human breast cancer cells model steroids have shown high inhibitory activity. *In vivo*, agent **54** showed moderate antitumor activity against MCF-7 breast cancer xenografts in BALB/c athimic nude mice. 3,4- Oxathiazine analogues showed weak inhibitory activity.

Compounds **55-57** effectively inhibit estrone sulfatase (**Tanabe et al, 1999**). It indicated specifically on the possibility for the creation of inhibitors in the series of D-homo analogues of estrogens that was confirmed using sulphamates **58** and **59** (**Reed & Potter, 1999**).

The development of this direction led to the creation of strong inhibitors **60** and **61** with values of IC50 - 1 nM (**Fischer et al., 2003**).

One more group of perspective inhibitors of estrone sulfatase was found – sulphamates of estrogens with methoxy- group at С-2 (**Raobaikady et al., 2003**). At the time of these investigations it was known that 2-methoxyestradiol **62** induces apoptosis in many various lines of tumor' cells, including prostate cancer cells, exhibits antiproliferative activity and blocks angiogenesis processes, which became a basis for the selection of model compounds.

The fact that bis-sulphamates of 2-methoxyestradiol **63** and 2-ethylestradiol **66** inhibit the growth of breast cancer cells, which are resistant to the action of many medications, has a crucial importance **(Suzuki et al., 2003).**

We have considered the ways for the search for modified estrogens for the inhibition of steroids metabolism enzymes which have been done in series of derivatives with natural rings junction. Obvious perspective direction is the investigation of analogues with unnatural rings junction since peculiarities of their structure have the number of important characteristics, comparable with ones in series of natural estrogens and comparable biological properties of both series.

It is known that high effectiveness of binding of estradiol **1** with ER is a result of high hydrophobicity of steroid molecule and possibility to form hydrogen bonds of phenolic hydroxyl group at С-3 with Glu353, Arg394 and water molecule. Hydroxyl group at С-17 forms hydrogen bond with His524 (**Brzozowski et al, 1997**). Modified 8-analogues of steroid estrogens have relative high affinity to ER, and model explaining the binding with

Approaches for Searching

properties.

site-directed substitution.

we know such investigations have not yet been done.

**O**

**73 74** 

of Modified Steroid Estrogen Analogues with Improved Biological Properties 187

635 and133% in comparison with estradiol (**Langer et al., 1959**). Values of Km and Vmax of 2 hydroxyestradiol **72** are comparable with these values for estradiol **1** (**Chernyaeva et al., 1972**)**,** and such modification may be important during the search of inhibitors of 17 hydroxysteroid dehydrogenase. Hence, the presence of free hydroxyl group at С-3 is not necessary for significant substrate specificity of such steroids. Modified androgens are also substrates for this enzyme, although they are notably less specific (**Chernyaeva et al., 1972**). The abovementioned fact gives a persuasion that the search of inhibitors of investigated enzyme is most perspective in the series of steroids with unnatural rings junction. As far as

It is quite interesting, that 2F- and 4F-estradiols (correspondingly **25** and **50**) are not substrates for 17-hydroxysteroid dehydrogenase (**Langer et al., 1959)**, which may be important during the search of compounds with different spectrum of biological

Primary structure of this enzyme was determinated after the analysis of corresponding сDNA **(Peltoketo et al., 1988**), lately X-rays analysis was obtained **(Ghosh et al., 1995**). The comparison of spatial structures of 17-hydroxysteroid dehydrogenase and investigated earlier 3,20-hydroxysteroid dehydrogenase (**Ghosh et al., 1994**) led to the conclusion about participation of His in the binding of hydroxyl group at C-3 of steroid and structure of transition state with the participation of triad Tyr-Ser-Lys. Authors justly mention the importance of the data obtained for modeling of interactions of 17-hydroxysteroid dehydrogenase with various ligands for the creation of effective inhibitors of the enzyme. Interesting developing of these investigations was the modeling of spatial structures of the enzymes of humans and rats, which are significantly different in primary structure and substrate specificity. Rat enzyme structure was modeled by the replacement of corresponding amino acids in the structure of human enzyme crystal (**Ghosh et al., 1995**) and following minimization of energy was done (**Putanen et al., 1997**). Results of calculations of rat enzyme are in good correlation with obtained experimental data. Clarification of mechanism of action of both enzymes was done using chimeric enzyme and

**OH**

**OH**

**H O**

X-rays data of the complexes of 17-hydroxysteroid dehydrogenase with estradiol **1** (**Azzi et al., 1996**), 5-dihydrotestosterone **73** and 20-hydroxy-analogue of progesterone **74** (**Lin et al., 1999**) have been obtained lately. Nevertheless the preliminary evaluation of characteristics for the searching the specific potential inhibitors is quite a difficult task because in human body types 1, 7 and 12 catalyze the transformation of estrone into estradiol (**Blanchard & Luu-The, 2007**). Substrate specificities of 17-hydroxysteroid dehydrogenases type 1 and 2 are similar, but in the case of second enzyme the transformation reaction estradiol – estrone is mainly directed to estrone formation (**Miettinen et al., 1996**), therefore the inhibition of this enzyme during the treatment of hormone-sensitive breast cancer is quite undesirable. The search of specific inhibitors of

ER has been proposed **(Shavva et al., 2002** and citation herein). Metabolism of steroids with unnatural rings junction may be quite different from metabolism of natural steroids, which will allow to find the alternative solutions of many medicinal-biological tasks.

The perspectives for the creation of new inhibitors of steroid metabolism have been demonstrated using 6-oxa-D-homo-8-analogues of estrogens **67** as an example (**Gluzdikov et al., 2007**). Authors accomplished the docking of different compounds of this series into ligand-binding domain of ER. And after the analysis the assumption was made that this compound must have weak binding affinity to ER or must have none in total. Sulphamate **67а** has been synthesized and the analogue showed a quite good inhibitory activity to estrone sulfatase. Steroid **67b** - potential product of hydrolysis has no uterotropic activity. It became the basis for the improvement of synthesis scheme of 7-methyl-D-homo-8 analogues of steroid estrogens (**Morozkina et al., 2009**) and investigation of peculiarities of their spatial structure (**Shavva et al., 2008**). Steroids of such structure may be used for the solution of other tasks (for example, as vectors for the transport of other classes of compounds into estrogen target-tissues).

Finally, 17-hydroxysteroid dehydrogenase type 1 plays the important role in the induction and development breast cancer (**Vihko et al, 2002**). In ER+ breast cancer cells under the action of this enzyme the dominate direction of the reaction is the transformation of estrone into more dangerous estradiol. In ER-positive breast cancer cells the reaction tends to proceed in the reverse direction. Another reason of tumor growth is inactivation of dehydroepiandrosterone **68**, which blocks tumor growth (**Aka et al., 2010**).

Substrate specificity of available enzyme from human placenta was investigated and it was found that steroids of unnatural series are also substrates for this enzyme (**Egorova et al., 1973**). High substrate specificity was observed only in the case of *trans*-junction of C and D rings. Values of Vmax of 8α-series steroids may be more than ones for compounds of natural series. For example, Vmax value of 8-estradiol **69а** is 219% of Vmax value of estradiol. Dhomo-8-estradiol **70а** has value of Km in 5 times less than estradiol value, whereas values of Vmax are approximately equal. Methylation of phenolic group (analogue **69b**) leads to the decreasing of Km value in one order, steroid **70b** has value of Km in 2.5 times less than compound **70а.** It takes attention big values of Vmax of steroids **71** and **72**, correspondingly

ER has been proposed **(Shavva et al., 2002** and citation herein). Metabolism of steroids with unnatural rings junction may be quite different from metabolism of natural steroids,

The perspectives for the creation of new inhibitors of steroid metabolism have been demonstrated using 6-oxa-D-homo-8-analogues of estrogens **67** as an example (**Gluzdikov et al., 2007**). Authors accomplished the docking of different compounds of this series into ligand-binding domain of ER. And after the analysis the assumption was made that this compound must have weak binding affinity to ER or must have none in total. Sulphamate **67а** has been synthesized and the analogue showed a quite good inhibitory activity to estrone sulfatase. Steroid **67b** - potential product of hydrolysis has no uterotropic activity. It became the basis for the improvement of synthesis scheme of 7-methyl-D-homo-8 analogues of steroid estrogens (**Morozkina et al., 2009**) and investigation of peculiarities of their spatial structure (**Shavva et al., 2008**). Steroids of such structure may be used for the solution of other tasks (for example, as vectors for the transport of other classes of

Finally, 17-hydroxysteroid dehydrogenase type 1 plays the important role in the induction and development breast cancer (**Vihko et al, 2002**). In ER+ breast cancer cells under the action of this enzyme the dominate direction of the reaction is the transformation of estrone into more dangerous estradiol. In ER-positive breast cancer cells the reaction tends to proceed in the reverse direction. Another reason of tumor growth is inactivation of

> **H H H**

**H H H**

Substrate specificity of available enzyme from human placenta was investigated and it was found that steroids of unnatural series are also substrates for this enzyme (**Egorova et al., 1973**). High substrate specificity was observed only in the case of *trans*-junction of C and D rings. Values of Vmax of 8α-series steroids may be more than ones for compounds of natural series. For example, Vmax value of 8-estradiol **69а** is 219% of Vmax value of estradiol. Dhomo-8-estradiol **70а** has value of Km in 5 times less than estradiol value, whereas values of Vmax are approximately equal. Methylation of phenolic group (analogue **69b**) leads to the decreasing of Km value in one order, steroid **70b** has value of Km in 2.5 times less than compound **70а.** It takes attention big values of Vmax of steroids **71** and **72**, correspondingly

**OH**

**OH**

**RO**

**OH**

**F**

**H H H**

**OH**

**H H H**

**OH**

dehydroepiandrosterone **68**, which blocks tumor growth (**Aka et al., 2010**).

**RO**

**OH**

**71 72 50** 

**68 69** a) R=H, b) R= CH3 **70** a) R=H, b) R= CH3

which will allow to find the alternative solutions of many medicinal-biological tasks.

compounds into estrogen target-tissues).

**H H H** **O**

**OH**

**OH**

**OH**

635 and133% in comparison with estradiol (**Langer et al., 1959**). Values of Km and Vmax of 2 hydroxyestradiol **72** are comparable with these values for estradiol **1** (**Chernyaeva et al., 1972**)**,** and such modification may be important during the search of inhibitors of 17 hydroxysteroid dehydrogenase. Hence, the presence of free hydroxyl group at С-3 is not necessary for significant substrate specificity of such steroids. Modified androgens are also substrates for this enzyme, although they are notably less specific (**Chernyaeva et al., 1972**).

The abovementioned fact gives a persuasion that the search of inhibitors of investigated enzyme is most perspective in the series of steroids with unnatural rings junction. As far as we know such investigations have not yet been done.

It is quite interesting, that 2F- and 4F-estradiols (correspondingly **25** and **50**) are not substrates for 17-hydroxysteroid dehydrogenase (**Langer et al., 1959)**, which may be important during the search of compounds with different spectrum of biological properties.

Primary structure of this enzyme was determinated after the analysis of corresponding сDNA **(Peltoketo et al., 1988**), lately X-rays analysis was obtained **(Ghosh et al., 1995**). The comparison of spatial structures of 17-hydroxysteroid dehydrogenase and investigated earlier 3,20-hydroxysteroid dehydrogenase (**Ghosh et al., 1994**) led to the conclusion about participation of His in the binding of hydroxyl group at C-3 of steroid and structure of transition state with the participation of triad Tyr-Ser-Lys. Authors justly mention the importance of the data obtained for modeling of interactions of 17-hydroxysteroid dehydrogenase with various ligands for the creation of effective inhibitors of the enzyme. Interesting developing of these investigations was the modeling of spatial structures of the enzymes of humans and rats, which are significantly different in primary structure and substrate specificity. Rat enzyme structure was modeled by the replacement of corresponding amino acids in the structure of human enzyme crystal (**Ghosh et al., 1995**) and following minimization of energy was done (**Putanen et al., 1997**). Results of calculations of rat enzyme are in good correlation with obtained experimental data. Clarification of mechanism of action of both enzymes was done using chimeric enzyme and site-directed substitution.

X-rays data of the complexes of 17-hydroxysteroid dehydrogenase with estradiol **1** (**Azzi et al., 1996**), 5-dihydrotestosterone **73** and 20-hydroxy-analogue of progesterone **74** (**Lin et al., 1999**) have been obtained lately. Nevertheless the preliminary evaluation of characteristics for the searching the specific potential inhibitors is quite a difficult task because in human body types 1, 7 and 12 catalyze the transformation of estrone into estradiol (**Blanchard & Luu-The, 2007**). Substrate specificities of 17-hydroxysteroid dehydrogenases type 1 and 2 are similar, but in the case of second enzyme the transformation reaction estradiol – estrone is mainly directed to estrone formation (**Miettinen et al., 1996**), therefore the inhibition of this enzyme during the treatment of hormone-sensitive breast cancer is quite undesirable. The search of specific inhibitors of

Approaches for Searching

**R**

**OH**

**NH2SO2O**

6-hydroxyestrogens (**Itoh et al., 1998**).

perspectives for its using in clinic is not clear at the moment.

**F**

**NH2SO2O**

of Modified Steroid Estrogen Analogues with Improved Biological Properties 189

**O**

**H H H**

**Ph**

**N**

**O**

**Alk**

**O**

**H H H**

**(CH2) 5 Br**

**N Me Bu**

**NH2SO2O**

**OH**

**H H H**

**NH2SO2O**

Both substrate specificity of enzymes and potential ways of inhibition metabolism must be taken into account during the search for steroid hormone synthesis inhibitors. The fine example of inhibitors which are able to inhibit the activity of aromatase and estrone sulfatase at the same time was demonstrated by Japanese authors (**Numazava et al., 2005; 2006).** In earlier investigation it was demonstrated that steroids **25**, **47a,b,c** and **48-50** are good inhibitors of aromatase. Sulphamates **80a,b,c** and **81-83** having higher activity in comparison with EMATE have been synthesized. Authors justly think that these results may be useful for the development a new class of drugs having a dual function for the treatment of breast cancer. Additional benefits of the modifications at positions 2-, 4-, and 6 of steroid skeleton are perspective in respect of the fact that they may decrease the potential carcinogenicity of hydrolysis products (if they have) since the decreased possibility of formation of danger о-quinones of type **29** (**Liehr et al., 1995, 1996; Bolton et al., 1998; Bolton & Thatcher, 2008; Zhang, F. et al., 1999**; **Liu, X. et al, 2002; Zhang, Q. et al., 2008)** or

Search for non-steroidal inhibitors of aromatase and sulfatase is of interest as well. Such properties belong to heterocyclic sulphamates (**Reed & Potter, 2006**), however the

The attempts of synthesis of steroids with multifunctional action, in particular, inhibitors of ERs and 17-hydroxysteroid dehydrogenase type 1 were done (**Tremblay & Poirier, 1996**). Steroid **84** was selected as model compound by authors on the basis of previous data. Earlier it was established that in the series of 16-(bromoalkyl)estradiols for the realization of

**MeO**

**O**

**H H H**

**CH3**

**O**

**NH2SO2O**

**80 a)** R=Cl, b) R=Br, c) R=CH3 **81 82** 

**O**

**OH**

**N**

**O**

**Alk**

**O**

**H H H**

**83 84** 

**H H H**

**85 86** 

**H H H**

17-hydroxysteroid dehydrogenases type 1 is complicated task, the attempts to solve this task by replacement oxygen-containing groups at position 17 and introduction of fluorine atoms into the same position of steroid skeleton were unsuccessful (**Deluca et al., 2006**).

Steroid **75** with big value of IC50 (530±7 nM) to this enzyme was synthesized, and this analogue has no hormonal activity (**Fisher et al., 2005**). Authors also obtained X-Ray data for the complex of compound **76** with 17-hydroxysteroid dehydrogenase type 1 that has importance for the solution of this task.

Investigation of inhibitory activity of estrogen analogues with large substituents at С-16 in -position led to find steroids **77** and **78** with selective action (**Lawrence et al., 2005**). Analogue **77** has value of IC50 0.29 μM to 17-hydroxysteroid dehydrogenase type 1 (**Purohit et al., 2006**).

Parallel investigations of properties of modified estrogens with substituents in -position at С-16 led to obtain the compounds of type **79** (**Laplante et al., 2008**), this steroid has value of IC50 44±7 nM for the transformation estrone **2** into estradiol **1** in T-47D intact cells. Unfortunately, analogue **79** possesses estrogenic activity, although this activity is decreased.

Obviously, clinical application of specific inhibitors of all three discussed enzymes is an intermediate step in the search for medications for the treatment of estrogen-sensitive oncological diseases. The most perspective direction is the search for compounds with simultaneous/synchronous inhibition activity to aromatase, estrone sulfatase and 17βhydroxysteroid dehydrogenase. The basis for this approach is the possibility for overexpression of aromatase in tumors, which was shown in experiments on MCF-7 cell lines **(Santen et al., 1999**). Aromatase may stimulate the growth of tumors through both autocrine and paracrine pathways (**Chen et al., 1999**). Moreover, long-term estrogen deprivation increases sensitivity to estradiol and enhances aromatase activity in MCF-7 cells (**Yue et al., 1999**).

17-hydroxysteroid dehydrogenases type 1 is complicated task, the attempts to solve this task by replacement oxygen-containing groups at position 17 and introduction of fluorine atoms into the same position of steroid skeleton were unsuccessful (**Deluca et** 

Steroid **75** with big value of IC50 (530±7 nM) to this enzyme was synthesized, and this analogue has no hormonal activity (**Fisher et al., 2005**). Authors also obtained X-Ray data for the complex of compound **76** with 17-hydroxysteroid dehydrogenase type 1 that has

Investigation of inhibitory activity of estrogen analogues with large substituents at С-16 in -position led to find steroids **77** and **78** with selective action (**Lawrence et al., 2005**). Analogue **77** has value of IC50 0.29 μM to 17-hydroxysteroid dehydrogenase type 1

Parallel investigations of properties of modified estrogens with substituents in -position at С-16 led to obtain the compounds of type **79** (**Laplante et al., 2008**), this steroid has value of IC50 44±7 nM for the transformation estrone **2** into estradiol **1** in T-47D intact cells. Unfortunately, analogue **79** possesses estrogenic activity, although this activity is

**N N**

**OH**

**H H H** **O**

**MeO**

**H H H** **O**

**O**

**N H** **O N H**

**O NH2**

**N**

**H H H**

**N**

**OH**

Obviously, clinical application of specific inhibitors of all three discussed enzymes is an intermediate step in the search for medications for the treatment of estrogen-sensitive oncological diseases. The most perspective direction is the search for compounds with simultaneous/synchronous inhibition activity to aromatase, estrone sulfatase and 17βhydroxysteroid dehydrogenase. The basis for this approach is the possibility for overexpression of aromatase in tumors, which was shown in experiments on MCF-7 cell lines **(Santen et al., 1999**). Aromatase may stimulate the growth of tumors through both autocrine and paracrine pathways (**Chen et al., 1999**). Moreover, long-term estrogen deprivation increases sensitivity to estradiol and enhances aromatase activity in MCF-7 cells

**75 76 77** 

**O**

**78 79** 

**N H**

**al., 2006**).

(**Purohit et al., 2006**).

decreased.

**OH**

**OH**

(**Yue et al., 1999**).

**Et**

importance for the solution of this task.

**N N**

**H H H** **(CH2)2OCH3**

**OH**

**O**

**H H H**

Both substrate specificity of enzymes and potential ways of inhibition metabolism must be taken into account during the search for steroid hormone synthesis inhibitors. The fine example of inhibitors which are able to inhibit the activity of aromatase and estrone sulfatase at the same time was demonstrated by Japanese authors (**Numazava et al., 2005; 2006).** In earlier investigation it was demonstrated that steroids **25**, **47a,b,c** and **48-50** are good inhibitors of aromatase. Sulphamates **80a,b,c** and **81-83** having higher activity in comparison with EMATE have been synthesized. Authors justly think that these results may be useful for the development a new class of drugs having a dual function for the treatment of breast cancer. Additional benefits of the modifications at positions 2-, 4-, and 6 of steroid skeleton are perspective in respect of the fact that they may decrease the potential carcinogenicity of hydrolysis products (if they have) since the decreased possibility of formation of danger о-quinones of type **29** (**Liehr et al., 1995, 1996; Bolton et al., 1998; Bolton & Thatcher, 2008; Zhang, F. et al., 1999**; **Liu, X. et al, 2002; Zhang, Q. et al., 2008)** or 6-hydroxyestrogens (**Itoh et al., 1998**).

Search for non-steroidal inhibitors of aromatase and sulfatase is of interest as well. Such properties belong to heterocyclic sulphamates (**Reed & Potter, 2006**), however the perspectives for its using in clinic is not clear at the moment.

The attempts of synthesis of steroids with multifunctional action, in particular, inhibitors of ERs and 17-hydroxysteroid dehydrogenase type 1 were done (**Tremblay & Poirier, 1996**).

Steroid **84** was selected as model compound by authors on the basis of previous data. Earlier it was established that in the series of 16-(bromoalkyl)estradiols for the realization of

Approaches for Searching

hormone treatment.

**Manthey Behl, 2006**).

(NO•), peroxynitrite (ONOO-

in each other.

hydrogen atom (**Prokai et al., 2001**; **Perez et al., 2005**).

of Modified Steroid Estrogen Analogues with Improved Biological Properties 191

catecholestrogens, that have another antioxidant property and can produce prooxidants (**Picazo et al., 2003**). Most animal studied have utilized rodents (especially rats), that have a very high rate of estrogen degradation. Further, in case of brain the synthetic estrogens that are candidates for neuroprotective antioxidants in vivo should be able to cross the bloodbrain barrier after their systemic administration. In vivo estrogens probably do not exert their own antioxidant action but interact synergistically with other antioxidants or reductants (**Hwang et al., 2000**). They can also affect the redox state of the cell through alteration of glutathione concentration and enhance the production of high energy compounds, stimulate antioxidant enzymes, such as SOD, catalase or glutathione transferase (**Perez et al., 2005**; **Akcay et al., 2006; Siow et al., 2007; Kumtepe et al., 2009**). Using in vivo methodology we can not understand molecular mechanisms of certain hormone antioxidant action, but it allows studying the manifestation of complex action of

More than 12 different types of neuronal cells against the 14 different toxicities were used to investigate neuroprotective effect of estrogens, their analogues and derivatives (**Green Simpkins, 2000; Wise et al., 2001**). The concentrations of estrogens that have produced protective action in these models vary from physiological (0.1 nM) to pharmacological (50 M) and it is suggested that different neuronal types may have different sensitivities to estrogen-mediated protection. For example physiological concentrations of 17-estradiol were neuroprotective in cultures that contain multiple cell types and maintain intact cellular architecture (**Dhandapani Brann, 2007)**. Several studies provide a positive correlation between in vitro neuroprotective potency and antioxidant activity of estrogens: they inhibited lipid peroxidation induced by glutamate, iodoacetic acid, amyloid peptide (**Perez et al., 2005; Prokai-Tatrai et al., 2008**), reduced iron-induced lipid peroxidation (**Vedder, et al., 1999**), prevented intracellular peroxides accumulation induced by different toxicities (**Behl et al., 1997**). The neuroprotective antioxidant activity dependents on the presence of OH-group in the C-3 position on the A ring of the molecule. The formation of ether derivatives at C-2 position reduces effect because it abolishes the ability to donate a

However, adjoin electron-donating methoxy groups to the phenolic ring may enhance antioxidant potency by weakening the phenolic O-H bond and provide stability of the formed phenoxyl radical (**Prokai Simpkins, 2007**). Nevertheless generally higher concentrations of the hormones were required for antioxidant action than were needed for neuroprotection. This observation indicates that antioxidant effect is not a significant mechanism involved in the neuroprotective activity of estrogens in vivo (**Wise et al., 2001;** 

The simplest level of in vitro study of radical scavenging activity is investigation using cellfree models. Oxidation is induced by different systems of free radical generation and different types of prooxidants in order to gain a more precise view of the mechanism of inhibition. This methodology allows to investigate basic chemical properties (antioxidant or prooxidant) of natural estrogen molecules or synthetic analogues and compare compounds

There are multiple reactive oxygen and nitrogen species (ROS and NOS) and free radicals: superoxide radical (O2•-), hydroxyl radical (HO•), hydrogen peroxide (H2O2), nitric oxide

lipoperoxy radical (LOO•). The reactivity toward various ROS or NOS can be measured by

), hypochlorous acid (HOCl), peroxyl radical (ROO•),

inhibitory action the presence of side chain (3-4 carbon atoms) at position 16 is optimal. Such substituents contribute to the appearance of antagonist activity to ER. It is desirable to have the tertiary amide group with substituents on the amide nitrogen. Authors assumed that primary bromide will inactivate 17-hydroxysteroid dehydrogenase. Thus, steroid was expected to be inhibitor of 17-hydroxysteroid dehydrogenase and agonist-antagonist to nuclear estrogen receptor. These suppositions have been confirmed: model steroid caused 25% stimulation of cellular growth at concentration 0.1 μM, at the same concentration steroid inhibited by 45% the 0.1 nM estradiol-stimulated growth of ZR-75-1 cells. It means, that compound **84** is partial agonist of ER and inhibitor of 17-hydroxysteroid dehydrogenase with moderate activity.

Sulphamates **85** and **86** are inhibitors of estrone sulfatase and 17-hydroxysteroid dehydrogenase (**Potter & Reed, 2002; Messinger et al., 2006).**

The selection of ways for the treatment of oncological diseases mainly depends from individual peculiarities of patients, as result the methods of definition of aromatase, estrone sulphatase, 17-hydroxysteroid dehydrogenase or mRNA (which realizes their synthesis), content have a crucial importance (**Irahara et al., 2006**).
