**6. Osteoptotective action of estrogen receptors ligands**

Several animal modes are used to test new compounds with osteoprotective properties. Ovariectomized mice are the ones most commonly used. Although aging in rats does not trigger osteoporosis development, ovariectomy-driven low estrogen concentration leads to

We carried out the search for modified steroid estrogen analogues in the series of steroids with unnatural rings junction having antioxidant properties and lowered/depressed uterotropic activity. Such properties belong to compounds **102** (**Pison et al., 2009**) and **103**. Some results of investigations of antioxidant properties of steroid **102** are presented in the

**OAc**

**OH**

**O**

Triene conjugates, conventional units / mg of phospholipids

> 0.1520.022 Р 0.05

**H**

**H**

Klein coefficient

0.710.05 Р 0.05

Malonic dialdehyde, nmol/mg of phospholipids

> 1.800.18 Р 0.05

**O**

 **102 103** 

**CH3O**

Schiff bases, conventional units / mg of phospholipids

Р0.05

animals were treated by olive oil in equal volume.

with antioxidant properties will be decreased.

**H**

**H**

**H**

Conjugated diene, nmol/mg of phospholipids

> 9.51.0 Р0.05

Control 12910 12.20.7 0.2090.022 0.700.01 1.650.14

Table 2. Results of investigation of action of steroid **102** on lipid peroxidation in brain of rats Steroid was given *per os* in olive oil in dose 5 mg per 100 g of weight of rats, for the day before the slaughter. Solutions of the steroids contain 5 mg in 0.3 ml. Control group of

Solely further investigations of such compounds may show the perspectives of this class of steroids. Depressed hormonal activity of investigated steroids may be a negative factor, inasmuch as during their using the capacity for the induction of the formation of enzymes

The presence of antioxidant activity leads to expect neuroprotective action of such modified estrogens, in spite of the fact that exact mechanisms of these properties have not yet been established. A huge amount of patents in this area is a proof of it. We shall cite some of those works (**Simpkins et al, 1994; Covey, 2002; Wuelfert et al, 2002; Peri et al., 2005; Pei, 2005).** Unfortunately clinical trials did not confirm neuroprotective action. Most probably it is connected with multifunctional activity of estrogens, and as a result their positive properties are "compensated" by negative action, but how and which ones are unknown. Further progress in this area must be connected with success in detailed investigation of mechanism

Several animal modes are used to test new compounds with osteoprotective properties. Ovariectomized mice are the ones most commonly used. Although aging in rats does not trigger osteoporosis development, ovariectomy-driven low estrogen concentration leads to

of neuroprotective properties and synthesis of analogues with restricted action.

**6. Osteoptotective action of estrogen receptors ligands** 

Table 2.

Experimental conditions

Steroid **102** 966

P – Student' coefficient.

changes in bone tissue, which mimic those occurring in aged women. At first phase of fast bone tissue loss triggered by accelerated bone remodeling occurs. Later on this process slows down. Trabecular tissue becomes depleted more so then cortical tissue. It should be noted that bone breaking is rarely observed in ovariectomized rats, thus is probably better to call this process osteopeny.

There are known several models for testing steroids on animals. When investigating the action of compounds in mature rats at the age of 3 months intensive growth of bones in longitudinal direction should be taken into account. 75 Days old (**Turner et al., 1994**) rats are more suitable for the investigation; rats model (6-12 months), which has very few skeletal changes, are much harder to work with. To test bone state several parameters are used, such as «bone mineral density» (g/cm3) and «bone mineral content» (g/sm. length, g/sm2). Last parameter has linear correlation with bone mass (r=0.999) (**Sato et al., 1995**) and calcium contain in ash after bone burning (r=0.90) **(Gaumet M., 1996)**. By measuring dry weight of the bone (after drying at high temperature until the constant mass is obtained), ash weight after burning **(Bauss et al., 1996)**, and also by investigating the ratio of this two parameters (**Broulik & Schreiber, 1994**) changes in mineral bone content can be measured. Results of dual-energy X-rays absorptiometry BMD correlate with fracture incidence and useful in evaluation progression of osteoporosis (**Sharp et al., 2000**). Yet, the final conclusion about steroid activity can only be given after the investigation of their influence on mechanical durability of the bone (**Turner et al., 1994; Sato et al., 1999**). All the works in this part were investigating naturally occurring steroids.

As we have already noted the significant structural similarity between natural steroids and their 8-analogues, the osteoprotective and uterotropic action of these compounds were investigated. In experiments with ovariectomized Wistar rats parallel change in those effects was observed. It was suggested that osteoprotective activity is dependent on nuclear receptor of estrogens (**Morozkina et al., 2007**). Hence analogues were chosen as model compounds, despite the fact that presence of methoxyl group at С-3 was expected to trigger the lowering of activity. However in this case carcinogenicity was also expected to be lowered. In the experiments with Sprague-Dowley rats compounds **104** and **105** were shown to possess the same osteoprotective activity as ethynylestradiol, although they were administered in higher doses **(Morozkina et al., 2008**). The presence of substituents in positions 2 and 4 cancels the osteoprotective effect.

**106** 

Approaches for Searching

(**Silverman et al., 2010**).

Group of experimental rats (number of animals in the

Ovariectomized, treated

Ovariectomized, treated

Ovariectomized, treated

Ovariectomized, reated with raloxifen and steroid

group)

**69a** (15)

Table 3.

influence on prostate (**Ke et al., 2000**).

investigation was stopped (**Vogelvang et al., 2006**).

3. 17-ethynylestradiol was used as standard.

of Modified Steroid Estrogen Analogues with Improved Biological Properties 197

lowering properties, decreased body weight in the experiments on ovariectomized rats. This compound had osteoprotective action in adult male orchideectomized rats, and has not

Droloxifene showed interesting biological properties in the experiments with animals, such as osteoprotective action in aged female rats (**Chen, H.K. et al., 1995**). However it was shown to be less effective then tamoxifen in breast cancer treatment clinical studies, so its

Lasoxifen showed osteoprotective and cholesterol-lowering action while lowering body weight of ovariectomized rats. This compound also displayed osteoprotective properties in adult male orchideectomized rats while having no effect on prostate (**Ke et al., 2000**). As far

Idoxifene was used in clinical trials on patients, whose tumors were resistant to tamoxifen, however third-generation aromatase inhibitors took its place, clinical data about its

Preliminary studies of breast cancer treatment by HMR3339 and its osteoprotective

Bazedoxifen possesses promising properties. As a result of 5 years long clinical study on 4216 patients in postmenstrual period good osteoprotective properties were found

We have already mentioned the possibility of agents' synthesis in 8-analogues of steroid estrogens series with osteoprotective properties. Preliminary results are presented in Table

Sham-operated (10) 29±3\* 154 ± 4\* 0.432±0.007\* 0.258±0.005\* Ovariectomized (15) 60±5 22.7 ± 0.5 0.398 ±0.005 0.231±0.003

with EE (10) 12±4\* 140± 6\*\* 0.433±0.005\* 0.255±0.006\*

with steroid **69a** (10) 7 ± 4\* 148± 6\*\* 0.425±0.006\* 0.258±0.007\*

with raloxifen (10) 33±5\* 46.5 ± 2.6\* 0.430±0.007 0.245±0.006\*

According to the data, this compound possesses osteoprotective properties. Also, in contrast to action of 17-ethynylestradiol and steroid, combination of compound and raloxifene did not trigger hypertriglyceridonemy, yet cholesterol-lowering effect remained. There is no doubt that investigations regarding properties of 8-series steroids are of great importance.

Signs \* and \*\* mean statistically significant difference between studied group and group of

ovariectomized animals, р<0.05 and р<0.01 (Students t-criterion).

Uterine weight, mg/100 g of body weight

Ash femur weight/wet femur weight

38±5\* 31.2 ± 1.2\* 0.425±0.005\* 0.248±0.006\*

Bone mineral density, g/cm2

properties provided results that may be considered positive (**Vogelvang et al., 2006**).

as we are aware, effectiveness of lasofoxifen have not undergone clinical studies.

osteoprotective properties are conflicting (**Vogelvang et al., 2006).** 

Change of body weight,

We assumed that steroid **106** may have osteoprotective action on the analogy with tibolone. It was confirmed experimentally; however compound **106** has embriotoxic action in experiments of contraceptive properties investigations which possibly decreases significantly the perspectives of such analogues.

As ER modulators are capable of displaying antagonistic properties in uterus and mammary gland while retaining agonistic features in bone, perspectives of using such compounds as osteoprotectors was investigated. Raloxifene has very effective osteoprotective properties. Clinical studies have shown that, aside from osteoprotective effects, the risk of breast cancer was also lowered (**Fontana & Delmas, 2001**). Interestingly that osteoprotective effect of raloxifene is not always mediated by ERs (**Miki et al., 2009**), and as the result the properties of its analogues may be quite interesting. However it was established that raloxifene in this case has some negative properties, peculiar to typical estrogens (high risk of hot flashes, leg crumps and venous thrombosis (**Deal & Draper, 2006**). In view of the aforesaid it is worth to remember about earlier synthesized compound LY357489 **(Grese et al., 1998),** whose osteoprotective action is significantly higher in comparison with raloxifene. This compound is the conformationally restricted raloxifene' analogue; its selective action was explained by specificity of interaction with ERs. Probably, it is necessary to investigate the mechanism of this compound' action.

Lately a huge number of SERMs with osteoprotective action have been synthesized, for example droloxifene **107 (Ke et al., 1995; Chen, H.K. et al., 1995**), LY 353381 (arzoxifene **110**) (**Sato et al., 1998; Ma et al., 2002**), lasofoxifen (CP-336,156) **111** (**Ke et al., 1998; 2000, 2004)**, GW5638 **109** (**Wilsson et al, 1997**) and many other compounds with interesting biological properties. For example, lasoxifen (CP-336,156) showed osteoprotective and cholesterollowering properties, decreased body weight in the experiments on ovariectomized rats. This compound had osteoprotective action in adult male orchideectomized rats, and has not influence on prostate (**Ke et al., 2000**).

Droloxifene showed interesting biological properties in the experiments with animals, such as osteoprotective action in aged female rats (**Chen, H.K. et al., 1995**). However it was shown to be less effective then tamoxifen in breast cancer treatment clinical studies, so its investigation was stopped (**Vogelvang et al., 2006**).

Lasoxifen showed osteoprotective and cholesterol-lowering action while lowering body weight of ovariectomized rats. This compound also displayed osteoprotective properties in adult male orchideectomized rats while having no effect on prostate (**Ke et al., 2000**). As far as we are aware, effectiveness of lasofoxifen have not undergone clinical studies.

Idoxifene was used in clinical trials on patients, whose tumors were resistant to tamoxifen, however third-generation aromatase inhibitors took its place, clinical data about its osteoprotective properties are conflicting (**Vogelvang et al., 2006).** 

Preliminary studies of breast cancer treatment by HMR3339 and its osteoprotective properties provided results that may be considered positive (**Vogelvang et al., 2006**).

Bazedoxifen possesses promising properties. As a result of 5 years long clinical study on 4216 patients in postmenstrual period good osteoprotective properties were found (**Silverman et al., 2010**).

We have already mentioned the possibility of agents' synthesis in 8-analogues of steroid estrogens series with osteoprotective properties. Preliminary results are presented in Table 3. 17-ethynylestradiol was used as standard.


Signs \* and \*\* mean statistically significant difference between studied group and group of ovariectomized animals, р<0.05 and р<0.01 (Students t-criterion).

Table 3.

196 Steroids – Basic Science

We assumed that steroid **106** may have osteoprotective action on the analogy with tibolone. It was confirmed experimentally; however compound **106** has embriotoxic action in experiments of contraceptive properties investigations which possibly decreases

Droloxifene Toremifene GW5638 LY353381 (arzoxifene)

 **107 108 109 110** 

**OH**

 CP335156 (Lasoxifen) HMR3339 Bazedoxifene  **111 112 113** 

**<sup>O</sup> <sup>N</sup>**

**Cl**

**CO2H**

**OH**

**H H H**

As ER modulators are capable of displaying antagonistic properties in uterus and mammary gland while retaining agonistic features in bone, perspectives of using such compounds as osteoprotectors was investigated. Raloxifene has very effective osteoprotective properties. Clinical studies have shown that, aside from osteoprotective effects, the risk of breast cancer was also lowered (**Fontana & Delmas, 2001**). Interestingly that osteoprotective effect of raloxifene is not always mediated by ERs (**Miki et al., 2009**), and as the result the properties of its analogues may be quite interesting. However it was established that raloxifene in this case has some negative properties, peculiar to typical estrogens (high risk of hot flashes, leg crumps and venous thrombosis (**Deal & Draper, 2006**). In view of the aforesaid it is worth to remember about earlier synthesized compound LY357489 **(Grese et al., 1998),** whose osteoprotective action is significantly higher in comparison with raloxifene. This compound is the conformationally restricted raloxifene' analogue; its selective action was explained by specificity of interaction with ERs. Probably, it is necessary to investigate the mechanism of

Lately a huge number of SERMs with osteoprotective action have been synthesized, for example droloxifene **107 (Ke et al., 1995; Chen, H.K. et al., 1995**), LY 353381 (arzoxifene **110**) (**Sato et al., 1998; Ma et al., 2002**), lasofoxifen (CP-336,156) **111** (**Ke et al., 1998; 2000, 2004)**, GW5638 **109** (**Wilsson et al, 1997**) and many other compounds with interesting biological properties. For example, lasoxifen (CP-336,156) showed osteoprotective and cholesterol-

**OH S**

**OH**

**O**

**N**

**O**

**N**

**O**

**OMe**

**N**

**OH**

significantly the perspectives of such analogues.

**NMe2**

**Cl**

**NMe2 <sup>O</sup>**

**<sup>O</sup> <sup>N</sup>**

**OH**

**OH**

this compound' action.

**O**

According to the data, this compound possesses osteoprotective properties. Also, in contrast to action of 17-ethynylestradiol and steroid, combination of compound and raloxifene did not trigger hypertriglyceridonemy, yet cholesterol-lowering effect remained. There is no doubt that investigations regarding properties of 8-series steroids are of great importance.

Approaches for Searching

**Karas (1999**).

**2011**).

function.

(**Koren et al., 1996**).

of Modified Steroid Estrogen Analogues with Improved Biological Properties 199

experimental data, showing the cardioprotective activity of estrogens, which is not mediated

Estrogens increase vasodilatation and inhibit the response of blood vessels to injury and the development of atherosclerosis, this action is referred to non genomic, this develops maximum after 20 min after estrogen introduction (**Mendelsohn & Karas, 1999**). Fast vasodilatation is possible due to the influence on both calcium-activated potassium ionchannel function (**Ghanam et al., 2000**), and on the synthesis of nitric oxide (the last relaxes vascular smooth muscle and inhibits platelet activation (**Holm et al., 1997; Node et al., 1997**). More detailed ways of non-genomic effects have been considered by **Mendelsohn &** 

Of crucial importance is the influence of agents on hemodynamic functions (**Borissoff et al.,** 

One more factor of risk of arising of atherosclerosis and thrombosis is higher content of oxidized lipoproteins, possible mechanism of these diseases have been considered

Antioxidant action of estrogens has been widely studied *in vivo* and *in vitro*. Besides its effects on LDL-oxidation (**Maziere et al., 1991; Markides et al., 1998; Ruiz-Larrea et al., 2000; Badeau et al., 2005**), it has reported that estrogens decreased lipid peroxidation in brain homogenates and neuronal cultures (**Vedder et al., 1999; Thibodeau P. et al., 2002**), reduced the superoxide anion production of different cells (**Bekesi et al., 2000**; **Florian et al., 2004).** All these effects may contribute to the beneficial consequences of estrogen replacement on the cardiac and vascular function, bone and mineral metabolism, brain

In the experiments on rabbits with hypercholesterinemic diet it was shown that 17 dihydroequilin sulphate and 17-ethynylestradiol significantly reduce atherosclerosis by 35% in the aortic arch and 75-80% in the thoracic and abdominal aorta, in spite of high level of LDL cholesterol (**Sulistiany at al., 1995**). High ration between HDL and LDL level is important but is not absolute parameter in prognosis of cardio-heart diseases risk. Clinical trials of estrone sulphate (at the same time 17-ethynylestradiol has been investigated) in postmenopausal women have shown the significant improvement of this parameter. It is important that the action of estrone sulphate did not result in triglycerides level increasing

Thus, numerous investigations on animal models (we described only few of them) give the evidence about the advisability of application of estrogens for HRT. First clinical trials for using of estrogens with this aim gave optimism, however wide-ranging investigations did not confirm the expectations. In older postmenopausal women with established coronaryartery atherosclerosis, 17β-estradiol had no significant effect on the progression of atherosclerosis (**Hodis et al., 2003**). Moreover, the combination: estrogen plus progestin may increase the risk of CHD (**Manson et al., 2003**). This is very important effect, because the content of triglycerides in blood is the independent factor of risk of cardio-vascular diseases

This difference is very important because the increased level of triglycerides in blood is the

From other side, main and side effects of any medication depend on/from methods of the introduction into the body. Therefore the positive results obtained during transdermal introduction of estradiol takes attention **(Sumino et. al., 2006)**. Oral estrogens raised triglycerides whilst transdermal estradiol lowered those (**Nerbrand et al., 2004**). The

independent factor of risk of cardiovascular diseases (**Koren et al., 1996**).

by nuclear receptors (**Shavva et al., 1987; Karas et al., 2001**).

**(Steinberg et al., 1989; Holvoet & Collen, 1994).**

in contrast to 17-ethynylestradiol (**Colvin et al., 1990**).

The new area in synthesis of compounds with osteoprotective properties should be noted – the creation of hybrid molecules having steroid and peptide fragments, for example compounds (**Wang et al., 2003**). Such compounds have stronger action in the experiments on rats in comparison with sum of action of these compounds.

Peptidylsteroid **114** has very interesting properties (**Yokogawa et al., 2006**). This hybrid compound during intranasal administration has a potent antiosteoporotic effect without side effects in experiments on female ddY mice.

In the treatment of estrogen-sensitive conditions aromatase inhibitors are to be used sometimes, which triggers estrogen concentration to drop, increasing osteoporosis and cardiovascular disease risk (**Ponzone et al., 2008; Reid, 2009; Gennari et al., 2011**). In each case the decision in using a drug depends on individual features of the patient. Usage combined drugs, which have calcitriol as a part of them, might prove useful to lower osteoporosis (**Krishnan et al., 2010).** 
