Androstanes

The first reports on CYP17 steroidal inhibitors date back to 1971 when Arth et al. synthesized and evaluated testosterone derivatives against rat testicular CYP17, following the observation that testosterone acetate 6 (Fig. 3, Table 1, entry 1) was a potent inhibitor of the enzyme [75]. Almost total abrogation of the enzyme's activity was observed after treatment with 1.5 ||M of compounds 7, 8, and 10 (Table 1, entries 2-3, and 5), with the acetamide derivative 9 being less potent (Table 1, entry 4). Competitive inhibition of pig CYP17 was reported for the anabolic steroids mestanolone 11, stanozolol12, and furazobol 13 (Fig. 3) [76]. Week inhibition in the high |M range was found with compounds 11 and 13 against the C17,20-lyase activity whereas stanozolol 12 inhibited both enzyme activities with IC50 values of 2.9 |M and 0.74 |M, for the 17a-hydroxylase and C17,20-lyase activities, respectively.

The irreversible inhibition of CYP17 by compound 14 (Fig. 3, Table 1, entry 6) was reported to occur due to the presence of a cyclopropylamino moiety capable of being activated by the enzyme by one-electron oxidation of the nitrogen atom, which causes ring opening to afford a p-iminium radical that covalently binds to the enzyme, while the compound is still bound in the active site [77]. Other related irreversible inhibitors reported include compounds 15-18 (Fig. 3, Table 1, entries 7-10) [78-81]. Compounds 15-17 were potent inhibitors of the human CYP17 at 0.8 and 1 |M, after preincubation with the enzyme (Table 1, entries 7-9). The ki values of the 4-amino derivatives 16-17 and of the sulfoxide derivatives 19-20 were determined using cynomolgous monkey and porcine testicular CYP17, respectively (Table 1, entries 8-9 and 11-12) [82]. Compound 18 also potently inhibited the activity of the monkey cynomolgous CYP17 at 0.1 |M, after preincubation with the enzyme (Table 1, entry 10) [80].

The introduction of heterocyclic moieties into molecules is a commonly used strategy in drug discovery and the design of potent steroidal CYP17 inhibitors based on this feature is an example of success. Thus, several androstane derivatives have been synthesized bearing a heterocycle ring at C17 either connected to it by a carbon (Fig. 4, Compounds 21-50) or a nitrogen (Fig. 5, Compounds 53-60) atom. In 1995, Jarman et al. reported the synthesis of abiraterone 21 (Fig. 4), a 17-(3-pyridyl)androstane derivative and a potent irreversible inhibitor of human testicular CYP17 (Table 2, entry 1), about 16- and 9-fold more potent than ke-toconazole 5 for the inhibition of the hydroxylase and lyase activities, respectively, with IC50 values in the low nM range [86]. Its 3p-acetoxy derivative and prodrug, abiraterone acetate 3 (Table 2, entry 2) has helped to further evidence and establish the utility of specific CYP17 inhibition in metastatic PC (mPC) patients. In 2001, Hartmann et al. reported that the introduction of a pyrimidyl substituent at C17 originated compounds such as 22 and 23 (Fig. 4, Table 2, entries 3-4) which were more potent inhibitors of the human enzyme than both ke-toconazole 5 and abiraterone 21, under the same assay conditions, and that compound 23 effectively lowered T plasma concentrations to castrate levels after administration to mice [87, 88]. The thiazole and furan derivatives 24 and 25 were also synthesized and tested on the monkey cynomolgous enzyme (Fig. 4, Table 1, entries 13-14) [83, 85].

Entry

Compound

Inhibitor concentration (mM)

% Inhibition'

Ki (nM)

IC50 (MM)

Ref.

1

6

1.5

65

__

2

7

1.5

95

__

__

3

8

1.5

100

__

__

[75]

4

9

1.5

85

__

__

5

10

1.5

90

__

__

6

14

90b

4.6c

[77]

7

15

0.8

64

__

__

[78,79]

8

16

1

84

339b

__

[80,81]

9

17

1

86

286b

__

10

18

0.1

79b

__

__

[80]

11

19

380c d

1.9c

[82]

12

20

380c d

1.9c

13

24

0.1

58b

__

0.063b

[83-85]

14

25

0.1

53b

Table 1. Inhibition of CYP17 by androstane derivatives. 'Human CYP17; bDetermined on cynomolgous monkey testis enzyme; cPorcine testicular CYP17; dki for compound 14 under the same assay conditions was 3620 nM.

Table 1. Inhibition of CYP17 by androstane derivatives. 'Human CYP17; bDetermined on cynomolgous monkey testis enzyme; cPorcine testicular CYP17; dki for compound 14 under the same assay conditions was 3620 nM.

A series of interesting effects on PC cells other than just CYP17 inhibition was reported by Brodie et al. for the imidazolyl, pyrazolyl, and isoxazolylandrostane derivatives 26-32 (Fig. 4, Table 2, entries 5-11). The isoxazolyl compound 32 was not only a non-competitive inhibitor of human CYP17 but also a competitive inhibitor of 5a-reductase, with potency similar to finasteride, while in addition bearing antiandrogenic activity [89-93]. Its effects were confirmed using PC xenograftmodels, however, its short half-life and rela tively low bioavailability were reasoned to limit its efficacy in vivo [93-95]. Less successful attempts of CYP17 inhibitors design include the 5'-methyl-2'-thiazolyl androstane 33 (Fig. 4) which was a weak inhibitor of human CYP17 expressed in E. coli when compared to ketoconazole 5 [3]. In 2006, Wolfling et al. reported the synthesis of a series of dihydrooxazine derivatives 34-45 (Fig. 4) which low inhibitory activity of CYP17 is most likely due to the bulkiness of the C17 moieties and the absence of a double bond at C16 [96]. The same group later reported the synthesis of the oxazolidone derivative 46 (Fig. 4, Table 2, entry 12) which inhibited the activity of rat testicular C17,20-lyase with an IC50 value of 3 |M [97]. Similar inhibition of the enzyme was observed with the halogenated oxazoline derivatives 47 and 48 [98], and with the D-ring fused arylpyrazoline 51 (Fig. 4, Table 2, entries 13-14, and 17) [99]. The N-phenylpyrazolyl derivatives 49 and 50 were however much less active, with IC50 values in the high |M range [100], as was the steroidal D-ring fused oxazolidine 52 (Fig. 4, Table 2, entries 15-16, and 18) [99].

Figure 3. Androstane based CYP17 inhibitors.

In 1996, Njar et al. reported the first steroidal inhibitors of CYP17 bearing a heterocyclic moiety bound to C17 by a nitrogen atom [101], which included compounds 53-55 (Fig. 5, Table 2, entries 19-21), among which the imidazolyl derivative 53 was found to be the most promising [101-104]. Later, in 2005, the same group reported the synthesis of galeterone 4 and its A4-3-keto derivative 56 (Fig. 5, Table 2, entries 22-23) [104-106].

Entry

Compound

CYP17 inhibition (nM)

Ref.

1

21

Human (OHase): 4 Human (lyase): 2.9

2

3

Human (OHase): 18 Human (lyase): 17

Rat: 220

3

22

Human: 24 E.colia: 30

Rat: 1460

4

23

Human: 38 E.colia: 2500

5

26

Rat: 91 Human: 66

6

27

Rat: 49 Human: 24

7

28

Rat: 79 Human: 58

8

29

NDb Human: 21

[89, 90]

9

30

Rat: 28 Human: 42

10

31

Rat: 76 Human: 59

11

32

Rat: 32 Human: 39

12

46

Rat: 3000

[97]

13

47

Rat: 4800

[98] [98]

14

48

Rat: 5000

15

49

Rat: 22000

16

50

Rat: 59000

17

51

Rat: 5800

[99] [99]

18

52

Rat: 26000

Rat: 9

19

53

Human: 8 LNCaP-CYP17 cellsc: 1.25

20

54

Rat: 8 Human: 7 LNCaP-CYP17 cellsc: 2.96

[102, 103]

21

55

Rat: 10 Human: 13

Entry

Compound

CYP17 inhibition (nM)

Ref.

LNCaP-CYP17 cellsc: 7.97

22

4

E.colia: 300

23

56

E.colia: 915

24

61

LNCaP-CYP17 cellsc: 11500

25

62

LNCaP-CYP17 cellsc: 17100

Table 2. IC50 values for androstane CYP17 inhibitors. Recombinant human CYP17 expressed in E. coli; bND = Not Determined; Recombinant human CYP17 expressed in LNCaP cells.

Table 2. IC50 values for androstane CYP17 inhibitors. Recombinant human CYP17 expressed in E. coli; bND = Not Determined; Recombinant human CYP17 expressed in LNCaP cells.

Thus, in vitro results with compounds 53-55 revealed a high inhibitory potential of the human enzyme expressed in LNCaP cells. In addition, compounds 53 and 55 completely suppressed T and DHT stimulated growth of LNCaP cells below 5 |M, and displayed antiandrogenic activity [102, 108]. In vivo experiments confirmed these results and showed that the compounds were however less effective than castration [109]. The C17-benzimida-zole derivative 4 became the first example of a CYP17 inhibitor and antiandrogen that could effectively suppress androgen-dependent tumor growth better than castration [105]. In 2007, our group reported the synthesis of the 1H- and 2H-indazole androstanes 57-60 which despite being poor inhibitors of human CYP17 displayed selective inhibition of PC-3 cells suggesting that mechanisms other than interference with the AR could be involved in their cytotoxicity [5]. We also synthesized a series of steroidal carbamates out of which compounds 61 and 62 (Fig. 5, Table 2, entries 24-25) were inhibitors of human CYP17 with IC50 values of 11.5 and 17.1 |M, respectively [4].

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