**3. Carcinogenicity causes of ER ligands**

176 Steroids – Basic Science

The introduction of relatively small linear substituents (4-5 carbon atoms) at position 11 of steroid skeleton may lead to the appearance of antagonistic activity to ER at agonistic properties to ER (steroids **14** and **15**), whereas analogue **16** is agonist to both receptors

Steroids with small substituents at position 11 without aromatic ring possess significantly higher affinity and transcriptional activity of ER (**Loosen еt al., 2000**), for example,

**OH**

Finally, there are compounds, which fully block the binding of estradiol **1** with known ERs, for example steroids **20-23** (**Jordan, 2003**). A number of compounds with large substituents at С-7 () and С-11 () have been synthesized, such analogues are of great interest for the treatment of estrogen-dependent oncological diseases and hormones imbalance, caused by

**CF2CF3**

**OH**

**CH3 H**

**OH**

**F**

**OH**

**H**

**OH**

**O**

**N**

**H**

**<sup>H</sup> <sup>H</sup> <sup>N</sup>**

**O**

**OH**

**H H H** **CH3**

**OH**

(**Loosen, 1999**).

**OH**

**OH**

 **17 18 19** 

**O**

**OH**

**H H H**

the increased formation of estrogens in the body.

**S O O**

ICI 182,780 (fulvestrant) ICI 164,384  **20 21** 

**CF2CF3**

RU 58,668 RU 39,411  **22 23** 

**OH OH**

analogues **17**-**19** have been found**.**

**OH**

**<sup>H</sup> <sup>H</sup> <sup>S</sup>**

**H**

**OH**

**O**

The establishment of at least major reasons of carcinogenicity of estrogens, their agonists/antagonists has significant importance, since this understandings lies in the fundament of the creation of medications with the improved properties.

At the present time two main types of carcinogenesis are known - promotive and genotoxic (mutagenic), action of them may add up. Existence of the fist one is confirmed by wellknown facts of tumor formation under the estrogen action (**Russo et al., 2002**) and promotion of tumor induction under the action of various agents, for example, nitrosobutylurea (**Sumi et al., 1984**). The same results have been obtained in other models. For example, 17α-ethynylestradiol **24** enhances the tumor formation in rat males under the action of diethylnitrosamine (**Yoshida & Fukunishi, 1981).** Estradiol increased dysplasia of breast on androgenized rats, induced by 7,12-dimethylbenz[a]anthracene.

The explanation of this negative influence of estrogens is quite clear – when in active state the cells are vulnerable to attacks by reactive compounds.

Lately using 2-fluoroestradiol **25** as an example it was shown that there is no correlation between carcinogenicity and hormonal action of estrogens (**Liehr, 1983**). It is important that 17-ethynylestradiol **24** possesses high hormonal activity and lowered carcinogenicity (**Li, J.J. et al., 1995**). These investigations (and earlier ones) became the basis for searching of other mechanisms of estrogens' carcinogenicity.

Approaches for Searching

(**Lewis et al., 2001**).

of Modified Steroid Estrogen Analogues with Improved Biological Properties 179

**OH**

It is also known that metabolites with hydroxyl group at C-6 of types **33** and **34** may have carcinogenic properties (**Itoh et al., 1998**). Thus, compounds which can not be hydroxylated at position 6 are perspective potential candidates for synthesis and further investigations. Another metabolite of estrogens - 16-hydroxyestrone **35** is also considered as inductor of tumors' formation (**Lewis et al., 2001; Seeger et al., 2006).** Hydroxylation of estrone at position 16 is one of main directions of estrogen metabolism, which is elevated in women with high risk of breast cancer, as well with other oncological diseases. Estriol **3** content is informative as well: there is the correlation between breast tumors' development in mice

Products of hydroxylation of equilenin and equilin also possess carcinogenic properties (**Zhang, F. et al., 1999; Shen et al., 1997).** Corresponding quinones may damage DNA, and the investigation of this class of compounds with substituent at position 4 is of a great importance. Authors concluded that 4-fluoroequilenin derivatives **36** are promising alternatives to traditional estrogen replacement therapy due to their similar estrogenic

**N**

The hydroxylation of selective modulators of ERs and the following reaction products transformations may cause DNA depurination that leads to carcinogenicity of drugs. It was shown on the example of the formation of -hydroxytamoxifen **37** and its possible

It is not surprising that during last years the efforts are directed to the methods of steroid estrogen level determination, because the exact value of their content in various tissues helps to diagnose differentially various hormone-sensitive diseases (**Arai et al., 2010; Blair et al., 2010**).

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

**OH**

**F**

**NH**

**NH**

**N**

**O**

**N N**

**O**

**OH**

**OH**

**O**

**H H H**

 **3 36** 

properties with less overall toxicity (**Liu, X. et al., 2003**).

**N**

**OH**

 **37 38** 

transformation into compounds of type **38** (**Jordan, 2007**).

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

**O**

**OH**

**OH**

Specifically, it was established that estrogen metabolites may covalently bind with hamster microsomic proteins, and this process is inhibited by ascorbinic and cathehol-Omethytransferase action (**Haaf et al., 1987**). Therefore authors proposed that «active» metabolites are о-quinones, which possess heightened reactivity. In next 20 years it was clarified that the hydroxylation of estrogens in the body leads to the formation of 2- or 4 hydroxyestrogens of types **26** and **27**, which then converted to о-quinones of type **28** and **29** (**Liehr et al., 1995; 1996; Bolton et al., 1998; Bolton, 2002; Bolton & Thatcher, 2008; Zhang, F. et al., 1999; Liu, X. et al., 2002; Zhang, Q. et al., 2008).** Irreversible depurination of DNA that could not be regenerated by reparases may take place under the action of oquinones. Compounds of types **31** and **32** are possible depurination products**.** Quinones of type **28** do not cause the damage of DNA. It is assumed that the reason of this is their heightened reactivity; therefore they may be deactivated before their migration into cell nucleus.

From our point of view one of possible variants for the decreasing of estrogens' carcinogenicity is the introduction of a substituent in the position 1, which blocks the interaction with DNA and thus prevents its depurination. In the case when compounds with substituent at position 1 will lead to decreasing of estrogenic activity, such modification (in dependence from the task) will be very perspective.

**O**

**30**

**H 2NH**

Specifically, it was established that estrogen metabolites may covalently bind with hamster microsomic proteins, and this process is inhibited by ascorbinic and cathehol-Omethytransferase action (**Haaf et al., 1987**). Therefore authors proposed that «active» metabolites are о-quinones, which possess heightened reactivity. In next 20 years it was clarified that the hydroxylation of estrogens in the body leads to the formation of 2- or 4 hydroxyestrogens of types **26** and **27**, which then converted to о-quinones of type **28** and **29** (**Liehr et al., 1995; 1996; Bolton et al., 1998; Bolton, 2002; Bolton & Thatcher, 2008; Zhang, F. et al., 1999; Liu, X. et al., 2002; Zhang, Q. et al., 2008).** Irreversible depurination of DNA that could not be regenerated by reparases may take place under the action of oquinones. Compounds of types **31** and **32** are possible depurination products**.** Quinones of type **28** do not cause the damage of DNA. It is assumed that the reason of this is their heightened reactivity; therefore they may be deactivated before their migration into cell

From our point of view one of possible variants for the decreasing of estrogens' carcinogenicity is the introduction of a substituent in the position 1, which blocks the interaction with DNA and thus prevents its depurination. In the case when compounds with substituent at position 1 will lead to decreasing of estrogenic activity, such modification (in

> **H H H**

**OH**

**OH**

**OH**

**O**

**C-8-adenine adduct**

**31**

**H H H**

**OH**

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

**32**

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

**OH**

**N NH**

**N N**

**OH**

**OH**

**N N**

**N N O**

**OH**

**2NH**

**H H H +1e adenine**

**O**

**O**

**N**

**H**

**2NH**

nucleus.

**OH**

**O**

**N N**

**N**

**O**

**O**

**H H H**

**\_**

**O**

**O**

**.**

**H H H**

**O**

**dR**

**O**

dependence from the task) will be very perspective.

**OH**

**OH**

 **33 34 35** 

**H H H**

**OH**

It is also known that metabolites with hydroxyl group at C-6 of types **33** and **34** may have carcinogenic properties (**Itoh et al., 1998**). Thus, compounds which can not be hydroxylated at position 6 are perspective potential candidates for synthesis and further investigations.

Another metabolite of estrogens - 16-hydroxyestrone **35** is also considered as inductor of tumors' formation (**Lewis et al., 2001; Seeger et al., 2006).** Hydroxylation of estrone at position 16 is one of main directions of estrogen metabolism, which is elevated in women with high risk of breast cancer, as well with other oncological diseases. Estriol **3** content is informative as well: there is the correlation between breast tumors' development in mice (**Lewis et al., 2001**).

Products of hydroxylation of equilenin and equilin also possess carcinogenic properties (**Zhang, F. et al., 1999; Shen et al., 1997).** Corresponding quinones may damage DNA, and the investigation of this class of compounds with substituent at position 4 is of a great importance. Authors concluded that 4-fluoroequilenin derivatives **36** are promising alternatives to traditional estrogen replacement therapy due to their similar estrogenic properties with less overall toxicity (**Liu, X. et al., 2003**).

The hydroxylation of selective modulators of ERs and the following reaction products transformations may cause DNA depurination that leads to carcinogenicity of drugs. It was shown on the example of the formation of -hydroxytamoxifen **37** and its possible transformation into compounds of type **38** (**Jordan, 2007**).

It is not surprising that during last years the efforts are directed to the methods of steroid estrogen level determination, because the exact value of their content in various tissues helps to diagnose differentially various hormone-sensitive diseases (**Arai et al., 2010; Blair et al., 2010**).
