A-ring modified steroidal azoles retaining similar potent and slowly reversible CYP17A1 inhibition as abiraterone

Article abstract of DOI:10.1016/j.jsbmb.2014.01.013

Abiraterone acetate is a potent inhibitor of human cytochrome P450c17 (CYP17A1, 17α-hydroxylase/17,20-lyase) and is clinically used in combination with prednisone for the treatment of castration-resistant prostate cancer. Although many studies have documented the potency of abiraterone (Abi) in a variety of in vitro and in vivo systems for several species, the exact potency of Abi for human CYP17A1 enzyme has not yet been determined, and the structural requirements for high-potency steroidal azole inhibitors are not established. We synthesized 4 Abi analogs differing in the A-B ring substitution patterns: 3α-hydroxy-Δ⁴-Abi (13), 3-keto- Δ⁴-Abi (11), 3-keto-5α-Abi (6), and 3α-hydroxy- 5α-Abi (5). We measured the spectral binding constants (K_s) using purified and modified human CYP17A1 along with the determination constants (K_i) applying a native human CYP17A1 enzyme in yeast microsomes for these compounds as well as for ketoconazole. For Abi, 3-keto- Δ⁴-Abi, 3-keto-5α-Abi, and 3α-hydroxy-5α-Abi, the type 2 spectral changes gave the best fit for a quadratic equation, since in these experiments K_s values were 0.1-2.6 nM, much lower than that for ketoconazole and 3α-hydroxy-Δ⁴-Abi (K_s values were 140 and 1660 nM, respectively). Inhibition experiments showed mixed inhibition patterns with K_i values of 7-80 nM. Abi dissociation from the CYP17A1-Abi complex was incomplete and slow; the t_1/2 for dissociation was 1.8 h, with 55% of complex remaining after 5 h. We conclude that Abi and the 3 related steroidal azoles (3-keto-Δ⁴-Abi, 3-keto-5α-Abi, and 3α-hydroxy-5α-Abi), which also mimic natural substrates, are extraordinarily potent inhibitors of human CYP17A1, whereas the 3α-hydroxy-Δ⁴-Abi is moderately potent and comparable to ketoconazole.

Full text of DOI:10.1016/j.jsbmb.2014.01.013

Accepted Manuscript
Title: A-Ring modified steroidal azoles retaining similar potent
and slowly reversible CYP17A1 inhibition as abiraterone
Author: Mariana Garrido Hwei-Ming Peng Francis K.
Yoshimoto Sunil K. Upadhyay Eugene Bratoeff Richard J.
Auchus
PII:
S0960-0760(14)00024-7
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jsbmb.2014.01.013
SBMB 4141
To appear in:
Journal of Steroid Biochemistry & Molecular Biology
Received date:
Revised date:
Accepted date:
31-10-2013
25-1-2014
28-1-2014
Please cite this article as: M. Garrido, H.-M. Peng, F.K. Yoshimoto, S.K. Upadhyay, E.
Bratoeff, R.J. Auchus, A-Ring modified steroidal azoles retaining similar potent and
slowly reversible CYP17A1 inhibition as abiraterone, Journal of Steroid Biochemistry
and Molecular Biology (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.01.013
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A-Ring modified steroidal azoles retaining similar potent and slowly reversible CYP17A1
inhibition as abiraterone
Mariana Garridoa*, Hwei-Ming Pengb*, Francis K. Yoshimotob, Sunil K. Upadhyayb, Eugene
Bratoeffa, and Richard J. Auchusb
aDepartment of Pharmacy, Faculty of Chemistry, National University of Mexico, Mexico, D.F.,
Mexico
bDivision of Metabolism, Diabetes, and Endocrinology, Department of Internal Medicine,
University of Michigan, Ann Arbor, MI 48019, Tel 7347647764, FAX 7349366684;
rauchus@med.umich.edu
Corresponding author present address:
Dr. Richard J. Auchus
Division of Metabolism, Diabetes, and Endocrinology, Department of Internal Medicine,
University of Michigan School of Medicine, Room 5560A, MSRBII, 1150 W. Medical Center
Drive, Ann Arbor, MI 48019
Tel 7347647764, FAX 7349366684
rauchus@med.umich.edu
*Both authors contributed equally to this manuscript
Page 1 of 30

Abstract:
Abiraterone acetate is a potent inhibitor of human cytochrome P450c17 (CYP17A1, 17α-
hydroxylase/17,20-lyase) and is clinically used in combination with prednisone for the treatment
of castration-resistant prostate cancer. Although many studies have documented the potency of
abiraterone (Abi) in a variety of in vitro and in vivo systems for several species, the exact
potency of Abi for human CYP17A1 enzyme has not yet been determined, and the structural
requirements for high-potency steroidal azole inhibitors are not established. We synthesized 4
Abi analogs differing in the A-B ring substitution patterns: 3α-hydroxy-Δ4-Abi (13), 3-keto-Δ4-
Abi (11), 3-keto-5α-Abi (6), and 3α-hydroxy-5α-Abi (5). We measured the spectral binding
constants (Ks) using purified and modified human CYP17A1 along with the determination
constants (Ki) applying a native human CYP17A1 enzyme in yeast microsomes for these
compounds as well as for ketoconazole. For Abi, 3-keto-Δ4-Abi, 3-keto-5α-Abi, and 3α-
hydroxy-5α-Abi, the type 2 spectral changes gave the best fit for a quadratic equation, since in
these experiments Ks values were 0.1-2.6 nM, much lower than that for ketoconazole and 3α-
hydroxy-Δ4-Abi (Ks values were 140 and 1660 nM, respectively). Inhibition experiments showed
mixed inhibition patterns with Ki values of 7-80 nM. Abi dissociation from the CYP17A1-Abi
complex was incomplete and slow; the t1/2 for dissociation was 1.8 hour, with 55% of complex
remaining after 5 hours. We conclude that Abi and the 3 related steroidal azoles (3-keto-Δ4-Abi,
3-keto-5α-Abi, and 3α-hydroxy-5α-Abi), which also mimic natural substrates, are
extraordinarily potent inhibitors of human CYP17A1, whereas the 3α-hydroxy-Δ4-Abi is
moderately potent and comparable to ketoconazole.
Page 2 of 30

1. Introduction
It has been 70 years since Huggins first introduced the concept of androgen-deprivation therapy
(ADT) for advanced prostate cancer [1], and at the present time, ADT is the primary treatment
for advanced prostate cancer. In parallel to this, there has been 30 years of off-label use of the
antimycotic drug ketoconazole for the treatment of androgen resistant prostate cancer, which is
also called castration-resistant prostate cancer (CRPC) [2], because of its off-target inhibition of
CYP17A1. This enzyme has both 17α- hydroxylase and 17,20-lyase activities, thus mediating the
biosynthesis of both cortisol and sex steroids [3]. Recently, the FDA approved abiraterone
acetate (Zytiga), a prodrug for the selective CYP17A1 inhibitor abiraterone (Abi), which is about
20 times more potent than ketoconazole, for the treatment of CRPC. Abi significantly suppresses
serum androgen concentrations derived from both adrenal and testicular sources within the
prostate cells, thereby improving the survival in men with metastatic CRPC both before and after
docetaxol failure [4]. Abi inhibition, however, also impairs cortisol synthesis, leading to a
compensatory increase in ACTH and mineralocorticoid excess, a clinical syndrome of 17-
hydroxylase deficiency, which includes edema, hypokalemia and hypertension[5]. As a result,
Abi must be co-administered with prednisone to prevent endocrine side effects. Nevertheless,
the significant reduction in androgens by Abi might also be useful in the management of
hormone-dependent breast cancer and in androgen excess disorders, including polycystic ovary
syndrome and congenital adrenal hyperplasia.
A general approach for the synthesis of potent steroidal CYP17A1 inhibitors is to modify the
steroid scaffold by attaching a heterocycle as a functional group onto the 17-position, which
forms a strong complex with the heme iron of the enzyme. Abi has a 17-(3-pyridyl) substituent
Page 3 of 30

together with a 16,17-double bond, and these structural features are a stringent requirement for
potent inhibition. Substituents having a 2- or 4-pyridyl function instead of the currently used 3-
pyridyl group showed a poor inhibitory activity; on the other hand, a reduction of the 16,17-
double bond also diminished potency [6]. The crystal structure of the CYP17A1 complex with
Abi at 2.6 Å resolution demonstrated that the C-17-(3-pyridyl) group of the inhibitor binds to the
heme iron, and forms a 60° angle with the steroid nucleus above the heme plane and packs
against the central I helix [7]. Furthermore, it was suggested that the hydrogen bonding
interaction between the 3β-OH of Abi and the conserved polar asparagine 202 in the F helix is
crucial for high-affinity binding. Nevertheless, the A ring stereochemistry and functionality of
the substituent at C-3 of the Abi skeleton on the inhibitory activity of CYP17A1 enzyme has not
yet been explored. In the present study, we report the design, the synthesis and characterization
of CYP17A1 inhibitors of a series of 16-dehydro-17-(3-pyridyl) steroidal derivatives
(compounds 5, 6, 11 and 13) based on known substrate tolerance (Figure 1).
2. Experimental procedures
2.1. Materials and methods
13
NMR spectra were obtained using Varian instruments at frequencies for 1H and C as specified
for each compound in the experimental section. High-resolution mass spectra were obtained on
a Micromass AutoSpec Ultima instrument. Chemical shifts were referenced to chloroform peak
in the 1H NMR and are assigned at 7.26 ppm; in the 13C NMR, this signal was assigned at 77.16
ppm. Reaction progress was determined either by TLC monitoring, or an aliquot analysis by
NMR. The purity of compounds 5, 6, 11, and 13 was assessed by HPLC analysis using the
method in section 2.3.2. NMR spectra, mass spectra, and HPLC tracings are provided in the
Page 4 of 30

supplementary data file. Progesterone and all other reagents and solvents were purchased from
Sigma Aldrich (St. Louis, MO), Steraloids (Newport, RI), ThermoFisher Scientific (Pittsburgh,
PA), or as specified. Protein determinations used the Coomassie Plus Reagent (Pierce,
Rockford, IL). Modified human CYP17A1 was expressed in E. coli JM109 cells and purified to
homogeneity as described [8]. Abi was synthesized as described [9].
2.2. Chemical synthesis
2.2.1. 3α-Acetoxy-5α-androsta-16-ene-17-yl-trifluoromethanesulfonate (3)
A solution of compound 2 (250 mg, 0.752 mmol) in tetrahydrofuran (3 mL) at -78 °C was treated
with potassium bis(trimethylsilyl)amide (KHMDS, 0.5 M, 1.5 mL, 0.75 mmol) and stirred for 1
h at -78 °C. N-phenylbis-(triflouromethanesulfonamide) (323 mg, 0.905 mmol), was added, and
the reaction was stirred at -78 °C for 2 h, then slowly warmed to room temperature and quenched
with saturated NH4Cl. The compound was extracted with ethyl acetate. The organic phase was
washed with water, dried over Na2SO4 and concentrated under reduced pressure. The compound
was purified by silica gel column chromatography (hexanes to 10% ethyl acetate in hexanes).
Yield: 58%. 13H NMR (400 MHz, CDCl3) δ: 5.5 (s, 1H), 4.9 (s, 1H), 1.9 (s, 3H), 0.9 (s, 3H), 0.7
(s, 3H).
2.2.2. 3α-Acetoxy-17-(3-pyridyl)-5α-androsta-16-ene (4)
A suspension of compound 3 (550 mg, 1.18 mmol), diethyl-3-pyridylborane (265 mg, 1.81
mmol), bis(triphenylphosphine)palladium (II) chloride (8.12 mg, 0.0116 mmol) in THF (8 mL)
was added to an aqueous solution of sodium carbonate (2 M, 5 mL). The mixture was refluxed
for 4 h under N2. The reaction was concentrated under reduced pressure, and the residue was
Page 5 of 30

extracted with diethylether; the organic phase was washed with brine, dried over Na2SO4 and
concentrated under reduced pressure. The compound was purified on florisil column
chromatography (hexanes to 50% ethyl acetate in hexanes). Yield: 42%. 13C NMR (400 MHz,
CDCl3) δ: 170.6, 151.7, 147.8, 143.7, 140.8, 133.6, 129.1, 122.9, 69.9, 21.5, 11.3, 9.1.
2.2.3. 17-(3-pyridyl)-5α-androsta-16-ene-3α-ol (5)
Compound 4 (470 mg, 1.2 mmol) was dissolved in methanol (5 mL) at room temperature. A
solution of KOH (10%) in methanol (3.5 mL) was added, and the mixture was stirred for 1.5 h,
then concentrated under reduced pressure. Dichloromethane (25 mL) and water (25 mL) were
added, and the mixture was stirred for 1 h. The organic phase was dried over Na2SO4. The
compound was purified on florisil column chromatography (hexanes to 50% ethyl acetate in
hexanes). Yield: 68%, purity 99.3% by HPLC. 1H NMR (400 MHz, CDCl3) δ: 8.6 (s, 1H), 8.4 (d,
J = 4.8, 1H), 7.6 (d, J = 7.6, 1H), 7.2 (dd, J1 = 4.8 Hz and J2 = 7.6 Hz, 1H), 5.9 (d, J = 1.6 Hz),
13
4.0 (m, 1H), 2.2 (d, 1H), 2.0 (t, 2H), 1.2-1.8 (m, 17H), 0.9 (s, 3H), 0.8 (s, 3H). C NMR (400
MHz, CDCl3) δ: 151.5, 147.4, 147.3, 133.9, 133.1, 129.3, 123.1, 66.3, 57.5, 54.5, 47.5, 39.3,
36.2, 35.3, 34.0, 31.8, 31.7, 29.0, 28.4, 20.7, 16.7, 11.2. Measured m/z for [M+H] = 352.2642
and predicted m/z = 352.2635.
2.2.4. 17-(3-pyridyl)- 5α-androsta-16-ene-3-one (6)
To a solution of compound 5 (50 mg, 0.14 mmol) in acetone (4 mL), chromic acid 10% (w/v)
(4.5 mL) was drop-wise added at 0 °C. The mixture was stirred at room temperature for 3 h, and
a solution of sodium bicarbonate was then added to a pH = 7. The reaction mixture was extracted
with ethyl acetate, and the organic phase was washed with water and dried over Na2SO4. The
Page 6 of 30

compound was purified on silica gel column chromatography (hexanes to 40% ethyl acetate in
hexanes). Yield: 30%, purity 99.4%. 1H NMR (400 MHz, CDCl3) δ: 8.6 (s, 1H), 8.4 (d, J = 4.8,
1H), 7.6 (d, J = 7.6, 1H), 7.2 (dd, J1 = 4.8 Hz and J2 = 7.6 Hz, 1H), 5.9 (d, J = 1.6 Hz), 2.4-1.2
(m, 20H), 1.1 (s, 3H), 1.0 (s, 3H). 13C NMR (400 MHz, CDCl3) δ: 211.8, 151.5, 147.4, 147.3,
134.2, 133.1, 129.3, 123.1, 66.3, 57.5, 54.5, 47.5, 39.3, 36.2, 35.3, 34.0, 31.8, 31.7, 29.0, 28.4,
20.7, 16.7, 11.2. Measured m/z for [M+H] = 350.2489 and predicted m/z = 350.2478.
2.2.5. 17-Iodoandrosta-5,16-dien-3β-ol (9)
Into a 100 mL round-bottomed flask, fitted with a magnetic stirring bar, was placed
dehydroepiandrosterone (7) (1 g, 3.47 mmol) and ethanol (18 mL). Hydrazine hydrate solution
(4.5 mL, 67.3 mmol) was added followed by a solution of hydrazine sulfate (0.05 g, 0.384 mmol)
in water (0.5 mL). After stirring at room temperature during 18 h, the mixture was poured into
cold water. The precipitate was collected by filtration and dried at room temperature to yield the
hydrazone 8 (96% yield). To a solution of compound 8 (2.3 g, 7.61 mmol) and triethylamine (9
mL, 64.82 mmol) in dioxane (42 mL) was added iodine (4.117 g, 16.22 mmol) in portions over
30 min. The mixture was stirred at room temperature for an additional 1 h, it was poured into
10% Na2SO3 (50 mL), and the precipitate was collected by filtration, washed with cold water,
and recrystallized from ethanol to yield the iodo derivative 9. Yield: 50%. 1H NMR (400 MHz,
CDCl3) δ: 6.1 (d, 1H), 5.3 (d, 1H), 3.5 (m, 1H), 1.(s, 3H), 0.7 (s, 3H).
2.2.6. 17-Iodoandrosta-4,16-dien-3-one (10)
A solution of 9 (300 mg, 0.7531 mmol) in toluene (60 mL) and N-methylpiperidone (12 mL) was
refluxed; 35 mL of toluene was distilled off using a Dean-Stark apparatus. Aluminium
Page 7 of 30

isopropoxide (800 mg, 3.9 mmol) was added, and the reaction was refluxed for 6 h. The reaction
mixture was extracted with ethyl acetate, it was washed with brine, dried over Na2SO4, and
concentrated under reduced pressure. The crude compound was purified by silica gel column
1
chromatography (hexanes to 15% ethyl acetate in hexanes). Yield: 75%. H NMR (400 MHz,
CDCl3) δ: 6.1 (d, 1H), 5.7 (s, 1H), 1.1 (s, 3H), 0.7 (s, 3H).
2.2.7. 17-(3-pyridyl)androsta-4,16-dien-3-one (11)
A stirred solution of 10 (0.1 g, 0.2729 mmol) in THF (1.2 mL) was purged with nitrogen, and
bis(triphenylphosphine)palladium (II) chloride catalyst (0.0017 g, 0.0024 mmol) was added,
followed by diethyl(3-pyridyl)borane (0.048 g, 0.3264 mmol). To the resultant orange solution
was added an aqueous solution of sodium carbonate (2M, 0.8 mL). After refluxing for 7 h,
diethyl ether was added, and the organic phase was washed with water and dried over sodium
sulfate. The compound was purified on florisil column chromatography (hexanes to 40% ethyl
acetate in hexanes). Yield: 39%, purity 99.2%. 1H NMR (400 MHz, CDCl3) δ: 8.6 (s, 1H), 8.4 (d,
J = 4.8, 1H), 7.6 (d, J = 7.6, 1H), 7.2 (dd, J1 = 4.8 Hz and J2 = 7.6 Hz, 1H), 5.9 (d, J = 1.6 Hz),
5.7 (s, 1H), 2.5-1.2 (complex, 16H), 1.2 (s, 3H), 1.0 (s, 3H). 13C NMR (400 MHz, CDCl3) δ:
199.3, 170.8, 151.3, 147.5, 147.4, 133.9, 132.8, 129.2, 123.9, 123.1, 56.7, 53.9, 38.6, 35.5, 35.0,
34.1, 33.9, 32.7, 31.7, 31.6, 20.8, 17.2, 16.5. Measured m/z for [M+H] = 348.2328 and predicted
m/z = 348.2322.
2.2.8. 17-Iodoandrosta-4,16-dien-3α-ol (12)
An ice-cold solution of sodium hydroxide (10 mg, 0.250 mmol) in water (0.1 mL) and methanol
(2.5 mL) was added to a vigorously stirred ice-cold solution of compound 10 (50 mg, 0.126
Page 8 of 30

mmol) in methanol (3 mL). An ice-cold solution of sodium borohydride (20 mg, 0.538 mmol) in
pyridine (1.1 mL) was added, and the mixture was stirred at room temperature for 1 h. The
reaction was quenched with 5% HCl and extracted with diethyl ether. The organic phase was
washed with a saturated solution of sodium bicarbonate and dried over anhydrous sodium
sulfate. The compound was purified on silica gel column chromatography (hexanes to 30% ethyl
acetate in hexanes). Yield: 46%. 1H NMR (400 MHz, CDCl3) δ: 6.1 (s, 1H), 5.3 (s, 1H), 4.1 (bs,
13
1H), 1.0 (s, 3H), 0.7 (s, 3H). C NMR (400 MHz, CDCl3) δ: 146.6, 137.4, 123.8, 112.9, 67.5,
18.8, 15.2.
2.2.9. 17-(3-pyridyl)androsta-4,16-dien-3α-ol (13)
To a stirred solution of 12 (500 mg), bis(triphenylphosphine)palladium(II) chloride catalyst (10
mg), and diethyl-(3-pyridyl)borane (250 mg) in 6 mL of THF was added 5 mL of 2 M aqueous
sodium carbonate. The mixture was refluxed for 4 h, diethyl ether was added, and the organic
phase was washed with water and dried over sodium sulfate. The crude compound was purified
1
on florisil column chromatography (hexanes to 40% ethyl acetate in hexanes). Yield: 66%. H
NMR (400 MHz, CDCl3) δ: 8.6 (s, 1H), 8.5 (d, J = 4.8, 1H), 8.1 (d, J = 7.6, 1H), 7.6 (dd, J1 = 4.8
Hz and J2 = 7.6 Hz, 1H), 6.2 (d, J = 1.6 Hz), 5.3 (s, 1H), 4.1 (bs, 1H), 2.4-0.9 (m, 16H), 1.1 (s,
3H), 1.0 (s, 3H). 13C NMR (400 MHz, CDCl3) δ: 13C NMR (400 MHz, CDCl3) δ: 151.5, 147.4,
147.3, 134.2, 133.1, 129.3, 123.5, 123.1, 66.3, 57.5, 54.5, 47.5, 39.3, 36.2, 35.3, 34.0, 31.8, 31.7,
29.0, 28.4, 20.8, 18.3, 16.5. Measured m/z for [M+H]= 350.2481 and predicted m/z = 350.2478.
2.3. Enzymology studies
2.3.1. Spectral assay for ligand binding
Page 9 of 30

Spectral binding titrations with Abi and related compounds were carried out with purified
CYP17A1 from E coli at room temperature using a UV-visible scanning spectrophotometer (UV-
2600; Shimadzu Scientific Instruments, Columbia, MD). The CYP17A1 protein was diluted to
200 nM in 100 mM potassium phosphate buffer, pH 7.4, containing 20% glycerol. The diluted
protein was equally divided between two 1.0 mL quartz cuvettes (1-cm path length), then
aliquots of the steroidal ligand dissolved in 100% methanol were added to the sample cuvette,
and an equal volume of methanol was added to the reference cuvette. After the solution was
mixed, spectra were recorded at 350-490 nm with a 1 min interval until no further increase in the
Δ(A426 - A392) value was observed (typically 3–5 min). During the spectral titrations, the total
amount of methanol added did not exceed 1.5% by volume. GraphPad Prism 6.02 software
(GraphPad Software Inc., San Diego, CA) was used to calculate spectral binding constants (Ks)
by fitting spectral data into one of the following equations:
∆A=∆Amax× (2[E])-1×[Ks+ [E] + [L] - {(Ks+ [E] + [L])2 – 4[E][L])}1/2]
(1)
or
∆A=(∆Amax[L])/(Ks+[L])
(2)
Where ΔA is the spectral response at different ligand concentrations [L], ΔAmax is the maximal
amplitude of the spectral response, and [E] is the total enzyme concentration. Equation 1 was
applied when the value of Ks was lower than the enzyme concentration, and equation 2 was used
when Ks was similar or higher than the enzyme concentration, assuming 1:1 binding
stoichiometry.
2.3.2. CYP17A1 activity assays
Page 10 of 30

The hydroxylase activity of CYP17A1 was determined by measuring the conversion of
progesterone to 17-hydroxyprogesterone and 16α-hydroxyprogesterone. Microsomes were
prepared from an yeast strain that expresses human CYP17A1 and human P450-oxidoreductase
(POR), according to the methods described [10]. The test inhibitor (Scheme 1, 5–80 nM,
delivered in <5 μL methanol) and microsomes (0.5–1.5 μL, 5-15 pmol P450) were pre-incubated
in 0.25 mL 50 mM potassium phosphate (pH 7.4) at 37 °C for 3 min. Progesterone (0.4-40 µM)
and [3H]-progesterone (0.1 µCi/µL) were added to the mixture, which was incubated at 37 °C for
3 min. NADPH (1 mM) was added, and an additional incubation was carried out at 37 °C for 20
min. The reaction was stopped by the addition of 1 mL of dichloromethane, and the organic
phase was collected and dried under a nitrogen flow. The steroids were analyzed using an
Agilent 1260 Infinity HPLC system with UV detector and a β-RAM4 in-line scintillation counter
(LabLogic, Brandon, FL). Steroids were dissolved in 20 µL of methanol, and 5 µL injections
were resolved with a 50 mm x 2.1 mm, 26 µm, C8 Kinetex column (Phenomenex, Torrence, CA)
equipped with a guard column at a flow of 0.4 mL/min. Aqueous methanol linear gradients were
employed (27% methanol from 0 to 0.5 min, jump to 39% methanol, and gradient from 39 to
75% methanol over 30 min). Products were identified by retention times of external standards
chromatographed at the beginning of the experiment using radiochemical detection of [3H]-
labeled steroids with Bio-safe II scintillation cocktail (Research Products International, Mount
Prospect, IL) at a flow rate of 1.2 mL/min. The data were processed with Laura 4 (LabLogic)
program. Inhibitory constants Ki were determined by fitting v vs [S] using Origin 7.0 (OriginLab,
Northampton, MA) to determine Km and Vmax constants and with Lineweaver-Burk (v-1 vs [S]-1)
plots to determine the mode of inhibition. Ki values were calculated for competitive and
noncompetitive inhibition using the following equations:
Page 11 of 30

Competitive inhibition: Ki = Km [I] / (K′m–Km)
(3)
(4)
Noncompetitive inhibition: Ki = [I] / [(Vmax / V′max) -1]
Where K′m is the apparent Km in the presence of inhibitor at [I], and V′max is the apparent Vmax in
the presence of inhibitor at [I].
2.3.3. Dissociation kinetics
Purified CYP17A1 from E coli (800 nM) was incubated with increasing amount of Abi (20-900
nM) to achieve complete binding of the inhibitor to the enzyme. The enzyme-Abi complex was
then rapidly diluted 5-fold into 100 mM potassium phosphate buffer (pH 7.4) containing 20 µM
of allopregnanolone. The loss of Abi binding was followed by a recording spectra as a function
of time over 300 min after dilution to monitor conversion from type II to type I binding and to
estimate the rate constant (koff) for Abi-CYP17A1 dissociation.
3. Results
3.1. Synthesis of compounds
The synthetic approach to novel 16-dehydro-17-(3-pyridyl) steroidal derivatives are outlined in
Scheme 1. Our strategy employed Suzuki coupling of diethyl-(3-pyridyl)borane with a steroidal
vinyl triflate or iodide, similar to the synthesis of Abi. The commercially available steroidal
starting materials, androsterone and dehydroepiandrosterone facilitated the synthesis of the final
products—compounds 5, 6, 11 and 13—using standard reactions such as: oxidation, reduction
and protecting group removal.
3.2. Inhibitor binding spectra and Ks determinations
Page 12 of 30

The binding of the steroidal inhibitors to the heme iron of CYP17A1 was investigated using UV-
visible difference spectroscopy. Compounds 5, 6, 11, and 13, as well as Abi and ketoconazole,
each induced a type II difference spectrum as shown in Figure 2, indicating coordination of
pyridine-ring nitrogen to the heme iron of CYP17A1, with formation of low-spin iron. The Soret
peak maximum was centered at 426 nm for the enzyme complex with Abi and for compounds 5,
6, and 11, while for compound 13 and ketoconazole, it was shifted to 422 and 429 nm,
respectively. These spectral binding constants (Ks) for compounds 5, 6, and 11 were in the
subnanomolar-to-low nanomolar range and similar to that of Abi (0.1-2.6 nM, Table 1). In fact,
these inhibitors bind to CYP17A1 with such high affinity that the population of free inhibitor
molecules is significantly depleted by the formation of the enzyme-inhibitor complex. For these
tight binding inhibitors, the steady state approximations are no longer valid, and an alternative
method as described in the experimental procedures was used for data analysis to correctly
calculate the binding constants Ks for the enzyme-inhibitor complexes. Surprisingly, the Ks for
compound 13 (1660 nM) is over 3 orders of magnitude higher than that for the related
compounds 5 and 11, which differ only by the oxidation state and stereochemistry at carbon 3 or
the saturation at the A-B ring junction of carbons 4 and 5. By comparison, the reference
compound ketoconazole gave an intermediate Ks value of 140 nM. These data demonstrated that
the 3β-hydroxyl group of Abi is not necessary for a tight binding to CYP17A1 and that other
substitution patterns might yield better inhibitors, with the exception of the combination of a Δ4-
double bond with a 3α-hydroxyl group at carbon-3.
3.3. CYP17A1 inhibition
Page 13 of 30

All synthesized compounds were found to inhibit CYP17A1 17- and 16α-hydroxylase activities
(Figure 3). The Ki values are presented with comparison to ketoconazole and Abi in Table 1. All
3-keto compounds (detailed structures in Schemes 1) were strong inhibitors (Ki< 22 nM) with 3-
keto-5α-Abi (compound 6, Ki=7 nM) being the most potent. Compound 5, 3α-hydroxy-5α-Abi
remained highly potent (Ki =14 nM) in inhibiting CYP17A1 hydroxylation as compared to Abi
(Ki = 27 nM), despite the opposite stereochemistry of the 3-hydroxyl group. In contrast,
compound 13, 3α-hydroxy-Δ4-Abi, was a less potent inhibitor (Ki=80 nM) than the other
steroidal azoles but similar to ketoconazole (Ki=98 nM). For compound 13, the discrepancy
between its Ki and Ks values suggests that its mode of inhibition is largely not active-site
directed. Consistent with this model, compound 13 showed primarily non-competitive inhibition
at higher inhibitor concentrations, whereas all other compounds showed mixed but primarily
competitive inhibition (Figure 3). Inhibition of 17,20-lyase activity was similar to that of
hydroxylase activities (not shown). The mode of inhibition was confirmed after analyzing the
data using Cornish-Bowden plots. This analysis showed that Abi, ketoconazole, and compounds
5, 6, and 11 all act as mixed-type inhibitors of CYP17A1, whereas 3α-hydroxy-Δ4-abi
(compound 13) is a non-competitive inhibitor (Figure 4).
3.4. Abi-CYP17A1 dissociation rate
To determine the rate of ligand dissociation, the equilibrated CYP17A1-Abi complex was
rapidly diluted with a competing substrate (allopregnanolone), and the amount of residual bound
Abi was then spectroscopically measured as a function of time. The combination of the larger
reduction in Abi concentration and the high concentration of the competing substrate caused by
the jump-dilution results in conditions, which strongly disfavor Abi rebinding after dissociation.
Page 14 of 30

As shown in Figure 5, upon addition of allopregnanolone, 55% of the type II complex remained
intact after 5 h. A plot of residual bound Abi as a function of time was then fitted to one phase
decay equation and the koff was estimated to be 0.0093 min-1 or t1/2 of 1.8 h.
4. Discussion
In the present study, we synthesized four Abi analogs with various A-B ring functional groups
and found that all except the 3α-hydroxy-Δ4 Abi derivative retained very high potency, based on
both Ki values for native CYP17A1 in yeast microsomes and Ks values for purified, modified
CYP17A1 from E coli. These compounds were designed to mimic the naturally occurring
pregnane substrates pregnenolone (3β-hydroxy-Δ5-Abi), progesterone (3-keto-Δ4-Abi), 5α-
dihydroprogesterone (3-keto-5α-Abi), and allopregnanolone (3α-hydroxy-5α-Abi). The very
high potency of compound 5 (3α-hydroxy-5α-Abi) reflects the high affinity of CYP17A1 for
steroids with this A-ring stereochemistry [11]. In fact, 5α-pregnan-3α,17α-diol-20-one (17-
hydroxyallopregnanolone), which has the same A/B-ring structure as 3α-hydroxy-5α-Abi, is the
best known substrate for the 17,20-lyase activity of human CYP17A1 [11]. Our results
demonstrated also that the stereochemistry at C-3 of Abi (3β-hydroxy) is not a major contributor
to its very high affinity for CYP17A1. The binding affinities of CYP17A1 for Abi and
compounds 5, 6, and 11 were so enhanced that their spectral binding constants Ks could only be
calculated using equation 1, since the Ks values were much lower than the enzyme concentration.
The koff for Abi was estimated to be 0.0093 min-1, which is consistent with extremely tight
binding, and more than half of the CYP17A1-Abi complex remained intact after 5 hours. In
essence, every molecule of Abi or one of our potent analogs binds to and inhibits a molecule of
CYP17A1 enzyme over a wide range of concentrations.
Page 15 of 30

Surprisingly, compound 13 had much poorer affinity than the other steroidal azoles and a
largely noncompetitive mode of inhibition. To our knowledge, steroids with similar A-B ring
modifications have not been tested as substrates, but the substrate-binding pocket of CYP17A1
appears not to favor this geometry and functionality. A comprehensive understanding of which
inhibitors will form a tight complex with the active site of CYP17A1 enzyme will require a more
detailed structure-activity study. This work might not only provide insight to human
biochemistry but could also lead to more potent CYP17A1 inhibitors, which might be useful in
the treatment of androgen-dependent disorders such as prostate cancer and androgen excess
states.
5. Acknowledgments
This work was supported by grant R01-GM086596 from the National Institutes of Health.
Mariana Garrido thanks CONACYT (a funding agency in Mexico) and DGAPA project
IN211312 for the scholarship, which permitted the realization of this study.
Page 16 of 30

6. References
[1] C. Huggins, Effect of orchiectomy and irradiation on cancer of the prostate, Ann Surg 115
(1942) 1192-1200.
[2] D.L. Trump, K.H. Havlin, E.M. Messing, K.B. Cummings, P.H. Lange, V.C. Jordan, High-
dose ketoconazole in advanced hormone-refractory prostate cancer: endocrinologic and
clinical effects, J Clin Oncol 7 (1989) 1093-1098.
[3] W.L. Miller, R.J. Auchus, The molecular biology, biochemistry, and physiology of human
steroidogenesis and its disorders, Endocr Rev 32 (2011) 81-151.
[4] G. Attard, A.H. Reid, T.A. Yap, F. Raynaud, M. Dowsett, S. Settatree, M. Barrett, C. Parker,
V. Martins, E. Folkerd, J. Clark, C.S. Cooper, S.B. Kaye, D. Dearnaley, G. Lee, J.S. de
Bono, Phase I clinical trial of a selective inhibitor of CYP17, abiraterone acetate, confirms
that castration-resistant prostate cancer commonly remains hormone driven, J Clin Oncol 26
(2008) 4563-4571.
[5] E.A. Mostaghel, P.S. Nelson, Intracrine androgen metabolism in prostate cancer progression:
mechanisms of castration resistance and therapeutic implications, Best Pract Res Clin
Endocrinol Metab 22 (2008) 243-258.
[6] G.A. Potter, S.E. Barrie, M. Jarman, M.G. Rowlands, Novel steroidal inhibitors of human
cytochrome P45017α (17α-hydroxylase-C17,20-lyase): potential agents for the treatment of
prostatic cancer, J Med Chem 38 (1995) 2463-2471.
[7] N.M. DeVore, E.E. Scott, Structures of cytochrome P450 17A1 with prostate cancer drugs
abiraterone and TOK-001, Nature 482 (2012) 116-119.
[8] H.M. Peng, R.J. Auchus, The action of cytochrome b5 on CYP2E1 and CYP2C19 activities
requires anionic residues D58 and D65, Biochemistry 52 (2013) 210-220.
Page 17 of 30

[9] R. Li, K. Evaul, K.K. Sharma, K.H. Chang, J. Yoshimoto, J. Liu, R.J. Auchus, N. Sharifi,
Abiraterone inhibits 3β-hydroxysteroid dehydrogenase: a rationale for increasing drug
exposure in castration-resistant prostate cancer, Clin Cancer Res 18 (2012) 3571-3579.
[10] D.P. Sherbet, D. Tiosano, K.M. Kwist, Z. Hochberg, R.J. Auchus, CYP17 mutation E305G
causes isolated 17,20-lyase deficiency by selectively altering substrate binding, J Biol Chem
278 (2003) 48563-48569.
[11] M.K. Gupta, O.L. Guryev, R.J. Auchus, 5α-reduced C21 steroids are substrates for human
cytochrome P450c17, Arch Biochem Biophys 418 (2003) 151-160.
Page 18 of 30

OTf
O
O
H
Ac2O
Pyridine
1)THF/KHMDS/ -78 °C
2)PhNTf2/ -78 °C
O
H
H
H
H
O
H
H
O
H
H
H
3
O
BEt2
HO
H
H
1
N
2
N
Pd(PPh3)2Cl2
N
THF/Na2CO3 2M
N
KOH 10%
MeOH
H
Acetone
Chromic acid 10 %
O
O
H
H
H
H
H
H
H
H
H
4
HO
O
H
H
5
6
I
N
NH2
O
Hydrazine hydrate
Hydrazine sulfate
(CH3CH2)3N, I2
H
H
H
H
H
H
H
H
H
HO
HO
HO
9
8
N-methylpiperidone
aluminium isopropoxide
7
BEt2
N
I
I
N
NaOH/MeOH/H2O
NaBH4, pyridine
Pd(PPh3)2Cl2
H
THF/Na2CO3 2M
H
H
H
H
H
H
H
H
O
10
HO
BEt2
HO
12
13
N
Pd(PPh3)2Cl2
N
THF/Na2CO3 2M
H
H
H
O
11
Scheme 1. Synthetic pathway for abiraterone derivatives
Page 19 of 30

Table 1. Inhibition of CYP17A1 Hydroxylase Activity by Abiraterone and Related Compounds
Inhibitor
Abiraterone
Ks(nM)
2.6±3.2
0.6±1.3
0.7±2.8
0.1±0.4
1660 ± 286
140 ± 3
Ki(nM)
27
Typea
M
3α-hydroxy-5α-Abi (cpd 5)
3-keto-5α-Abi (cpd 6)
3-keto-Δ4-Abi (cpd 11)
3α-hydroxy-Δ4-Abi (cpd 13)
Ketoconazole
14
M
7
M
22
M
80
N
98
M
CYP17A1 activities were determined by using progesterone as a substrate
aType of inhibition: M, mixed competitive-noncompetitive inhibition; N, primarily
noncompetitive inhibition.
Data are the means (± SE) of 2-4 experiments.
Page 20 of 30

Figure legends
Figure 1. Compounds studied as CYP17A1 inhibitors herein.
Figure 2. Spectrophotometric equilibrium binding titrations of CYP17A1 with abiraterone and
related inhibitors. Difference spectra were obtained upon titration of 200 nM CYP17A1 in 0.1 M
potassium phosphate buffer, pH 7.4, containing 20% glycerol (v/v) with inhibitors at 20-600 nM.
CYP17A1 titration with (A) abiraterone, (B) compound 5, (C) compound 6, (D) compound 11,
(E) compound 13, and (F) ketoconazole. Progressive shifts in the UV-visible difference spectra
indicate nitrogen binding to heme iron. Plots of Δ(A426 - A392) versus concentration of inhibitor
werefit to a equations (1) or (2) using GraphPad Prism (see “Experimental Procedures”). The
resulting Ks values were 2.6 nM for abiraterone, less than 1 nM for compounds 5, 6, and 11,
1660 nM for compound 13, and 140 nM for ketoconazole.
Figure 3. Lineweaver-Burke plots and v versus [S] plots of human CYP17A1 17-hydroxylase
activity in the presence of inhibitors (A) abiraterone, (B) compound 5, (C) compound 6, (D)
compound 11, (E) compound 13, and (F) ketoconazole. Yeast microsomes containing human
CYP17A1 and POR were incubated with 0.4–40 μM progesterone in the absence or presence of
5-80 nM inhibitors. The resulting Ki values are 27 nM for abiraterone, 14 nM for compound 5, 7
nM for compound 6, and 22 nM for compound 11, using inhibitor concentrations of 10-20 nM.
The Ki value is 80 nM for compound 13 using inhibitor concentrations of 40-60 nM. The Ki
value for ketoconazole is 98 nM using inhibitor concentration of 1000 nM. These plots show
mixed competitive-noncompetitive inhibition for abiraterone, compounds 5, 6, and 11 (A-D),
Page 21 of 30

and ketoconazole (F). In contrast, compound 13 inhibition of CYP17A1 is primarily
noncompetitive (E). Data points are the means of 2-4 experiments.
Figure 4. Cornish-Bowden plots illustrating the mode of inhibition of CYP17A1-mediated
progesterone hydroxylation by abiraterone and related compounds. V, initial rate (velocity) of
the reaction; S, substrate concentration.
Figure 5. A jump-dilution experiment to measure the dissociation rate (koff) of the
CYP17A1•abiraterone complex. Initially, abiraterone and the enzyme were allowed to bind
until equilibrium was reached. At that point, dissociation was initiated by introduction of excess
allopregnanolone, a CYP17A1substrate with high affinity (Ks = 1.2 nM, not shown). Binding
decreased over the first 3 h incubation. A koff value of 0.0093 min-1 was obtained from fitting
the data to a single-phase exponential decay equation (Prism 6.02; GraphPad Software).
Page 22 of 30

N
N
N
N
O
O
H
H
N
N
O
O
H
H
H
H
HO
Cl
Cl
HO
H
abiraterone
ketoconazole
5
N
N
N
H
H
H
H
H
H
H
H
H
HO
O
O
H
13
11
6
Figure 1
Page 23 of 30

(A)
(B)
0.010
0.005
0.000
0
200
400
600
800
abiraterone (nM)
(C)
(D)
0.010
0.010
0.005
0.000
0.005
0.000
0
200
400
600
800
0
200
400
600
800
3-keto-Δ4-abiraterone (nM)
3-keto-5α-abiraterone (nM)
(E)
(F)
0.00759
0.00500
0.00410
0.00200
.
s
b
A
0.00000
.
s
b
A
0.00000
-0.00500
-0.00749
-0.00200
-0.00352
350.00
400.00
450.00
500.00
350.00
400.00
450.00
500.00
nm.
nm.
Figure 2
Page 24 of 30

(A)
(B)
(C)
Page 25 of 30

(D)
(E)
Figure 3
Page 26 of 30

(A)
(B)
(C)
(D)
(E)
(F)
Figure 4
Page 27 of 30

Figure 5
Page 28 of 30

Highlights
•
•
•
•
Abiraterone analogs with modified A/B-rings retain nanomolar affinity for CYP17A1
CYP17A1 inhibition by abiraterone and analogs is mixed competitive/noncompetitive
Dissociation of abiraterone from CYP17A1 is slow (koff < 0.01 min-1) and incomplete
The 3β-hydroxyl Δ5-system of abiraterone is not required for high CYP17A1 affinity
Page 29 of 30