Chemical synthesis of 7-oxygenated 12α-hydroxy steroid derivatives to enable the biochemical characterization of cytochrome P450 8B1, the oxysterol 12α-hydroxylase enzyme implicated in cardiovascular health and obesity

Article abstract of DOI:10.1016/j.steroids.2018.10.010

Cholic acid is the endogenous 12α-hydroxylated bile acid, which possesses enhanced cholesterol absorption properties compared to its 12-desoxy counterpart, chenodeoxycholic acid. The oxysterol 12α-hydroxylase enzyme is cytochrome P450 8B1 (P450 8B1), which regioselectively and stereoselectively incorporates the 12α-hydroxy group in 7α-hydroxycholest-4-en-3-one, the biosynthetic precursor of cholic acid. Despite the vital role of P450 8B1 activity in cardiovascular health, research studies of other 12α-hydroxy steroid derivatives are rare. A synthetic route to incorporate a C12α-hydroxy group into the C12-methylene (–CH₂–) in dehydroepiandrosterone derivatives is disclosed. The incorporation of the C12-oxygen was accomplished through a copper mediated Sch?necker oxidation of an imino-pyridine intermediate, introducing the 12β-hydroxy group. The resulting 12β-hydroxy steroid derivative was oxidized to the C12-ketone, which was stereoselectively reduced with lithium tri-sec-butylborohydride to afford the 12α-hydroxy stereochemistry. The C7-position was oxidized to yield the various 7-keto, 7β-hydroxy, and 7α-hydroxy derivatives. Furthermore, 7-ketodehydroepiandrosterone and 12 α-hydroxy-7-ketodehydroepiandrosterone both displayed NMDA receptor antagonistic activities at 10 μM concentrations. These C12α-hydroxy steroids will be used as tools to identify new biochemical properties of the enzymatic products of P450 8B1, the oxysterol 12α-hydroxylase.

Full text of DOI:10.1016/j.steroids.2018.10.010

Accepted Manuscript
Chemical Synthesis of 7-Oxygenated 12α-Hydroxy Steroid Derivatives to En-
able the Biochemical Characterization of Cytochrome P450 8B1, the Oxysterol
1
2α-Hydroxylase Enzyme Implicated in Cardiovascular Health and Obesity
Samuel D. Offei, Hadi D. Arman, Mirza Oais Baig, Lazaro S. Chavez, Carlos
A. Paladini, Francis K. Yoshimoto
PII:
S0039-128X(18)30197-1
https://doi.org/10.1016/j.steroids.2018.10.010
STE 8332
DOI:
Reference:
To appear in:
Steroids
Received Date:
Revised Date:
Accepted Date:
31 July 2018
10 October 2018
19 October 2018
Please cite this article as: Offei, S.D., Arman, H.D., Baig, M.O., Chavez, L.S., Paladini, C.A., Yoshimoto, F.K.,
Chemical Synthesis of 7-Oxygenated 12α-Hydroxy Steroid Derivatives to Enable the Biochemical Characterization
of Cytochrome P450 8B1, the Oxysterol 12α-Hydroxylase Enzyme Implicated in Cardiovascular Health and
Obesity, Steroids (2018), doi: https://doi.org/10.1016/j.steroids.2018.10.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Chemical Synthesis of 7-Oxygenated 12α-Hydroxy Steroid Derivatives to Enable the
Biochemical Characterization of Cytochrome P450 8B1, the Oxysterol 12α-Hydroxylase
Enzyme Implicated in Cardiovascular Health and Obesity
1
1
2
2
2
Samuel D. Offei, Hadi D. Arman, Mirza Oais Baig, Lazaro S. Chavez, Carlos A. Paladini,
1
and Francis K. Yoshimoto *
(
1) Department of Chemistry, The University of Texas at San Antonio, San Antonio, TX, 78249-
698, United States
2) Department of Biology, UTSA Neurosciences Institute, The University of Texas at San
0
(
Antonio, San Antonio, TX, 78249-0698, United States
ABSTRACT
Cholic acid is the endogenous 12α-hydroxylated bile acid, which possesses enhanced cholesterol
absorption properties compared to its 12-desoxy counterpart, chenodeoxycholic acid. The
oxysterol 12α-hydroxylase enzyme is cytochrome P450 8B1 (P450 8B1), which regioselectively
and stereoselectively incorporates the 12α-hydroxy group in 7α-hydroxycholest-4-en-3-one, the
biosynthetic precursor of cholic acid. Despite the vital role of P450 8B1 activity in
cardiovascular health, research studies of other 12α-hydroxy steroid derivatives are rare. A
synthetic route to incorporate a C12α-hydroxy group into the C12-methylene (-CH -) in
2
dehydroepiandrosterone derivatives is disclosed. The incorporation of the C12-oxygen was
accomplished through a copper mediated Schönecker oxidation of an imino-pyridine
intermediate, introducing the 12-hydroxy group. The resulting 12-hydroxy steroid derivative
was oxidized to the C12-ketone, which was stereoselectively reduced with lithium tri-sec-

butylborohydride to afford the 12α-hydroxy stereochemistry. The C7-position was oxidized to
yield the various 7-keto, 7-hydroxy, and 7α-hydroxy derivatives. Furthermore, 7-
ketodehydroepiandrosterone and 12-hydroxy-7-ketodehydroepiandrosterone both displayed
NMDA receptor antagonistic activities at 10 M concentrations. These C12α-hydroxy steroids
will be used as tools to identify new biochemical properties of the enzymatic products of P450
8
B1, the oxysterol 12α-hydroxylase.
Abstract Figure:
H
H
O
O
1
. ketalization
O
Schönecker
Oxidation
OH
O
2. [O] at C12
1
2
O
12
12
HO
DHEA
Stereoselective
[
H] at C12
OH
O
OH
O
12
R = R = H (1)
1
2
O
R = R = O (2)
12
1
2
R = H, R = OH (3)
R1 = OH, R2 = H (4)
1
2
7
R2
R1
HO
Introduction
Cytochrome P450 8B1 is the oxysterol 12-hydroxylase enzyme, which oxidizes 7-hydroxy-
cholest-4-en-3-one to form 7-,12-dihydroxycholest-4-en-3-one [1, 2] (Figure 1A), the
biosynthetic precursor of cholic acid, the 12-hydroxylated bile acid (Figure 1B). Bile acids
form micelles, which regulate the intestinal absorption of lipids [3]. In particular, cholic acid
supplementation has been shown to enhance the absorption of cholesterol [4]. Moreover, mice

lacking the CYP8B1 gene encoding for P450 8B1, showed a resistance to weight gain when fed a
high-cholesterol diet [5].
Furthermore, the isolation of other steroids with a 12α-hydroxy group has been reported.
For instance, a 12α-hydroxypregnenolone derivative, named menarandroside A (Figure 1C), has
been recently isolated from plants (Cynanchum marnierianum) [6]. This novel 12α-hydroxylated
steroid was identified from a bio-guided fractionation assay that detected the stimulation of
glucagon-like peptide 1 (GLP-1) secretion, suggesting anti-diabetic activity. Other 12α-
hydroxylated sterol plant natural products have been previously isolated in plants [7].
2
7
O
2
1
2
3
25
26
O
(
A)
OH18
OH
(B)
(C)
OH
2
2
4
OH
12
1
2
20
17
22
1
1
1
1
9
5
13
1
H
16
9
H
H
2
8H14 15
7 OH
OH
1
0
H
H
H
H
H
3
O
O
HO
HO
4
6
O
HO
OH
7
-hydroxycholest-4-en-3-one
H
OH
menarandroside A
cholic acid
Figure 1. Examples of naturally occurring 12-hydroxylated steroids: (A) 7-hydroxycholest-4-
en-3-one, (B) cholic acid, and (C) menarandroside A.
Moreover, although it is an endogenous steroid hormone, 7-ketodehydroepiandrosterone
and its 3-acetate form have been used to treat certain health conditions. For example, 7-keto
dehydroepiandrosterone-3-acetate (7-keto DHEA-3OAc) has been shown to reduce post-
traumatic stress disorder (PTSD) symptoms [8] and also to reverse the resting metabolic rate
associated with dieting [9]. 7-Keto DHEA has also decreased voluntary intake of ethanol in male
rats [10]. The various 7-oxygenated DHEA forms (7-hydroxy-, 7-hydroxy, and 7-keto DHEA)
are interconverted by liver 11-hydroxysteroid dehydrogenase 1 [11]. In addition, 7-keto DHEA

has been shown to be effective at enhancing weight loss [12]. Due to the steroid backbone of 7-
keto DHEA, it is reasonable to hypothesize that P450 8B1 would recognize 7-keto DHEA as a
substrate and incorporate a 12-hydroxy group into 7-keto DHEA.
Access to 12-hydroxylated steroid structures would be necessary to explore their
biochemical properties and facilitate the identification of new bioactive 12-hydroxy steroid
products arising from P450 8B1 activity. However, there is no known report to introduce a 12-
hydroxy group from a C12-desoxy steroid. Therefore, a synthetic route to introduce a C12-
hydroxy group from the methylene (-CH -) in the steroid backbone was developed. This report
2
focuses on the syntheses of 12-hydroxy dehydroepiandrosterone (Figure 2, 1) and its 7-
oxygenated derivatives (i.e. 7-keto-, 7-hydroxy-, and 7-hydroxy) (Figure 2, 2, 3, and 4)
beginning with dehydroepiandrosterone (DHEA). We further tested the hypothesis that 7-
ketodehydroepiandrosterone is an N-methyl-D-asparatate (NMDA) receptor antagonist and
found that both 7-ketodehydroepiandrosterone and 12-hydroxy-7-ketodehydroepiandrosterone
(2) had antagonistic effects towards the NMDA receptor.
H
OH
H
O
O
12
This Report
12
R = R = H (1)
1
2
R = R = O (2)
1
2
R = H, R = OH (3)
R1 = OH, R2 = H (4)
1
2
7
R2
R1
HO
HO
DHEA
Figure 2. 12-Hydroxy-dehydroepiandrosterone (1), 12-hydroxy-7-keto-
dehydroepiandrosterone (2), 7-,12-dihydroxy-dehydroepiandrosterone (3), and 7-,12-
dihydroxy-dehydroepiandrosterone (4) were synthesized from dehydroepiandrosterone (DHEA).

Synthesis of 12-Hydroxy Dehydroepiandrosterone (1)
The incorporation of a 12-hydroxy group from a DHEA-imine derivative through a copper
mediated oxygenation has been reported [13-15]. For the synthesis of 12-hydroxy DHEA
(
compound 1), dehydroepiandrosterone (DHEA, Scheme 1) was protected as the DHEA-3-O-
tert-butyldimethylsilyl ether 5, which was converted to the pyridine-imine derivative at C17
Scheme 1, 6). The Schönecker protocol through a copper mediated oxidation resulted in the
introduction of the 12-hydroxy group to yield 12-hydroxy DHEA-3-OTBDMS ether 7 [13-
5]. The resulting C17-ketone would be protected as ketal 8. The C12-hydroxy group of ketal 8
(
1
was oxidized to the C12-ketone 9. Subsequent stereoselective reduction at the C12-position gave
the 12-hydroxy compound 10. Various reducing conditions were explored to find the optimal
hydride reagent as summarized in Table 1. In general, a sterically hindered hydride (i.e. lithium
triethylborohydride and lithium tri-sec-butylborohydride) source gave the 12-hydroxy epimer
as the major diastereomer. Deprotection of the TBDMS group and the C17-ketal of intermediate
1
0 with dilute HCl (aqueous) in THF yielded 12-hydroxy DHEA (1).

O
N
O
N
TBDMSCl
imidazole
NH2
H
N
H
H
CH CN
3
pTsOH
toluene,
HO
95%
TBDMSO
HO
DHEA
5
90%
6
TBDMSO
1
.
O2
OH
O
OH
O
O
Cu(OTf)2
O
OH
PCC
O
O
pTsOH
toluene,
2
. Na EDTA
CH2Cl2
4
48%
48%
20%
TBDMSO
TBDMSO
TBDMSO
7
8
9
OH
O
OH
O
L-Selectride
HCl
1
2
O
THF
THF
o
-
78 C to rt
68%
80%
HO
TBDMSO
1
10
Scheme 1. Synthesis of 12-hydroxy DHEA (1) from DHEA.
Stereoselective Reduction of the C12-Ketone (Compound 9) with Various Reducing Agents
a
a
Entry Hydride Source
12-Hydroxy (8)
12-Hydroxy (10)
b
1
2
3
4
5
6
DIBAL
NaBH4
2.9
9.8
12
1.0
1.0
1.0
1.0
10
c
c
b
b
b
NaBH /CeCl
4
3
LiAlH4
14
Lithium triethylborohydride
1.0
1.0
Lithium tri-sec-butylborohydride
9.2
Table 1. Summary of stereoselectivity of the different reduction conditions tested to yield the
C12-hydroxy epimer 10 or the C12-hydroxy epimer 8 from C12-ketone 9. Excess hydride
source (5 mol equivalents) was used and the reaction was run for 3 hours in test tubes.

a. The ratios of the 12-hydroxy and 12-hydroxy products were determined by integration of
1
the C18-methyl peak of the H NMR spectra of the crude reaction material ( 0.86 ppm for the
1
2-hydroxy epimer, and  0.92 ppm for the 12-hydroxy epimer).
b. THF (5 ml) was the solvent, the reaction was run at -78 C with 30 mg of starting material
Compound 9).
c. CH OH (5 ml) was the solvent, the reaction was run at 0 C with 30 mg of starting material
(
3
(Compound 9).
Synthesis of 12-Hydroxy-7-Keto-Dehydroepiandrosterone (2)
The synthesis of 12-hydroxy-7-keto dehydroepiandrosterone (2) commenced with 12-hydroxy
DHEA (Scheme 2). The dihydroxy compound (1) was diacetylated with pyridine and acetic
anhydride as the solvent (1:10, v/v) using experimental conditions previously employed [15].
The C7-position was oxidized to the C7-keto group using CrO and 3,5-dimethylpyrazole in
3
CH Cl (Scheme 2). The resulting diacetate (12) was deprotected with K CO in CH OH to yield
2
2
2
3
3
1
2-hydroxy-7-keto DHEA (2) (Figure 3: crystal structure of compound 2).

OH
O
OAc
O
pyridine
Ac O
2
69%
HO
AcO
1
11
CrO3
,5-dimethylpyrazole
CH Cl2
3
2
45%
OH
O
OAc
O
K CO
2
3
1
2
CH OH
3
22%
2
HO
7 O
AcO
O
12
Scheme 2. Synthesis of 12-hydroxy-7-keto-dehydroepiandrosterone (2) from 12-hydroxy
DHEA (1).
Figure 3. Crystal structure of 12-hydroxy-7-keto DHEA (compound 2).

Synthesis of 7-,12-Dihydroxy-Dehydroepiandrosterone (3)
In order to efficiently access 7-,12-dihydroxy DHEA (3), we hypothesized that a 7-,12-diketo
steroid intermediate (e.g. Scheme 3, 13) could be stereoselectively reduced by lithium tri-sec-
butylborohydride (L-Selectride) to furnish the -hydroxy stereochemistry at both the C7- and
C12- positions. This stereoselective reduction of the diketone was based on the observation that
L-Selectride results in the delivery of the hydride onto the -face of C7-keto steroid backbones
[
16] and C12-keto backbones (vide supra, Scheme 1, 9 to 10).
The synthesis of the 7-,12-dihydroxy DHEA (3) commenced with the oxidation of the C7-
allylic position of 12-keto C17-ketal 9 with CrO to furnish diketone 13. Gratifyingly, diketone
3
1
3 was reduced with L-Selectride to afford the 7-,12-dihydroxy ketal 14. Treating ketal 14
with dilute HCl in THF resulted in the deprotection of both the C17-ketal and the C3-tert-
butyldimethylsilyl ether to afford 7-,12-dihydroxy DHEA (3) (Scheme 3) (Figure 4: crystal
structure of compound 3).
O
O
O
O
CrO3
,5-dimethylpyrazole
O
3
O
CH Cl2
2
8
9%
TBDMSO
TBDMSO
O
9
13
L-Selectride
THF
o
-
78 C to rt
42%
OH
OH
O
O
HCl
H O/THF
O
1
2
2
3
41%
OH14
HO
7 OH
TBDMSO

Scheme 3. Synthesis of 7α-,12α-dihydroxy DHEA (3).
Figure 4. Crystal structure of 7-,12-dihydroxy DHEA (compound 3).
Synthesis of 7-,12α-Dihydroxy-Dehydroepiandrosterone (4)
Among the four 12α-hydroxylated DHEA derivatives (1-4), 7-,12α-dihydroxy-
dehydroepiandrosterone (4) was the most challenging due to the opposing stereochemistry of the
hydroxy groups at the C7- and C12- positions (i.e. C7-hydroxy and C12α-hydroxy, cf. Figure 1,
4
).
The 12-hydroxy group of ketal 8 was protected as the acetate with pyridine and acetic
anhydride to yield the 12-acetate 15 (Scheme 4). The C7-allylic position was oxidized with
CrO and 3,5-dimethylpyrazole to furnish enone 16. The C7-ketone was stereoselectively
3
reduced with NaBH to yield a crude mixture of 7-hydroxy and 7-hydroxy epimers in a 4:1
4
1
ratio (determined by H NMR, Supporting Information). This mixture was purified by silica gel

column chromatography, giving the 7- to 7- epimeric mixture, which would be resolved over
the subsequent five steps towards the final product, 4. The 7-hydroxy group of alcohol 17 was
protected as the tert-butyldimethylsilyl diether 18 by refluxing with TBDMSCl and imidazole in
CH CN. The acetate was methanolyzed with K CO in CH OH to yield alcohol 19. Alcohol 19
3
2
3
3
was oxidized with pyridinium chlorochromate (PCC) to yield C12-ketone 20, which was
stereoselectively reduced with L-Selectride to give the 12-hydroxy epimer 21. Deprotection of
the two tert-butyldimethylsilyl groups and the C17-ketal with HCl in water and THF to furnish
7-,12-dihydroxy DHEA 4.
OH
O
OAc
OAc
O
O
O
CrO3
,5-dimethylpyrazole
O
O
pyridine
Ac O
3
2
CH Cl
2
2
47%
TBDMSO
TBDMS
40%
8
16
15
TBDMSO
O
O
OAc
O
OAc
OH
O
TBDMSCl
imidazole
O
NaBH4
O
O
O
K CO
2
3
CH OH
3
CH CN
, 60%
3
80%
CH OH
3
7
1
8
7
75%
7
19
TBDMSO
OH1
7
TBDMSO
OTBDMS
:7 , 2:1
TBDMSO
OTBDMS
7
:7 , 4:1
7
7
7 :7 , 3:1
O
OH
O
O
OH
O
PCC
L-Selectride
O
HCl
12
O
CH Cl
THF
2
2
o
H2O/THF
-
78 C to rt
4
9%
TBDMSO
7
2
0
4
65%
7
21
40%
OTBDMS
HO
7
OH
7
:7 , 5:1
TBDMSO
OTBDMS
:7 , 20:1
Scheme 4. Synthesis of 7-,12-dihydroxy DHEA (4). For compounds 17-21, the ratio of 7- to
1
5
7
- epimers were determined from H NMR spectrum by integration of the  proton signals at

5.3 ppm and  5.5 ppm, respectively (See Supporting Information).
Synthesis of 12-Hydroxy-Dehydroepiandrosterone (22)

1
2-Hydroxydehydroepiandrosterone (22) was accessed through deprotection of the TBDMS
group of Schoenecker product (compound 7) with HCl and CH OH/CH Cl (Figure 5: crystal
3
2
2
structure of compound 22).
Figure 5. Crystal structure of 12-hydroxy DHEA (compound 22).
1
H NMR Analysis of C7- and C12- Oxygenated Steroid Derivatives – Diagnostic Signals
5
,6
Intriguingly, when the C7-methylene position is converted to the C7-keto- -steroid scaffold,
5
the  -proton shifts downfield from  5.39 ppm (apparent doublet (J = 6 Hz) of compound 1,
Figure 6A) to  5.75 ppm as a broad singlet (Figure 6B, compound 2). On the other hand, when
5
the C7-position bears a C7-hydroxy intermediate (compound 3) the  -proton appears as a

doublet (J = 6 Hz) at  5.67 ppm (Figure 6C). However, when the C7-hydroxy group is in the -
5
orientation (compound 4), the  -proton has an upfield shift and appears as a singlet at  5.33
ppm (Figure 6D).
Furthermore, the C12-proton of the C12-oxygenated steroids had a diagnostic peak depending on
whether the hydroxy group was in the 12- or the 12-orientation. For example, the C12-
proton (axial) of 12-hydroxy DHEA (compound 22) had a chemical shift of  3.80 ppm, which
appeared as a doublet of doublets (J = 11 and 4.6 Hz) (Figure 6E). In contrast, the C12-proton
(equatorial) of 12-hydroxy DHEA (compound 1) appeared as a broad singlet at  4.14 ppm.
OH
O
(
A)
B)
C)
D)
E)
1
2
1
7
HO
HO
5
OH
O
1
2
(
9
2
O
7
X (CH OH)
3
3
5
OH
O
1
2
(
(
(
X (CH Cl )
2
2
3
OH
7
HO
HO
5
OH
O
1
2
4
OH
7
5
OH
O
1
2
22
7
HO
5

1
Figure 6. H NMR spectral overlay of Compounds 1, 2, 3, 4, and 22 (500 MHz,  2.65-6.00 ppm
range). (A) 12-hydroxy DHEA (compound 1), (B) 12-hydroxy-7-keto DHEA (compound 2),
(
C) 12-,7-dihydroxy DHEA (compound 3), (D) 12-,7-dihydroxy DHEA (compound 4), and
E) 12-hydroxy DHEA (compound 22).
(
Effect of 7-Ketodehydroepiandrosterone and 12-Hydroxy-7-ketodehydroepiandrosterone on the
NMDA Receptor
7
-Ketodehydroepiandrosterone has been shown to reduce symptoms of post-traumatic stress
disorder (PTSD) [8]. Therefore, we hypothesized that this compound can act as an N-methyl-D-
aspartate (NMDA) receptor antagonist, which has been the activity associated with ketamine,
another compound used to treat PTSD [17]. Furthermore, with access to the 12-hydroxylated
derivative (12-hydroxy-7-ketodehydroepiandrosterone, compound 3), the effect of the 12-
hydroxy group in the steroid scaffold was tested. Interestingly, both compounds were found to
have antagonistic activity towards the NMDA receptor at 10 M concentration (Figure 7). Other
steroid derivatives have shown biological activity towards the NMDA receptor [18, 19],
confirming steroids as potential ligands to modulate NMDA receptor activity.

Figure 7. 7-Ketodehydroepiandrosterone and 12-hydroxy-7-ketodehydroepiandrosterone (2)
reduce the NMDA receptor-mediated current amplitude recorded from midbrain dopamine
neurons. (A, B). Single pulse electrical stimulation of the slice elicits an outward current under
baseline conditions (blue trace). When either (A) 7-ketodehydroepiandrosterone or (B) 12-
hydroxy-7-ketodehydroepiandrosterone (compound 2) are bath applied to the horizontal brain
slice, the peak current amplitude in response to the same electrical stimulation is decreased,
indicating the effect of these compounds on NMDA receptors in the brain. (C, D). Summarized
data of all recordings for each compound tested (C) 7-keto DHEA, and (D) 12-hydroxy-7-
ketodehydroepiandrosterone (compound 2).
Conclusion
In summary, a synthetic route to introduce a 12-hydroxy group from the C12-methylene in
steroid derivatives was established. The key feature of this route involved three transformations:
(1) the copper-mediated Schönecker oxidation to introduce the 12-hydroxy group, (2) oxidation
of the 12-hydroxy to the C12-keto moiety, and (3) stereoselective reduction to the 12-hydroxy
group using lithium tri-sec-butylborohydride (i.e. L-Selectride). Both 7-
ketodehydroepiandrosterone and 12-hydroxy-7-ketodehydroepiandrosterone (3) were found to
have NMDA receptor antagonist properties at 10 M concentrations. These novel 12-hydroxy
steroids and sterols will be used as chemical tools to explore the biochemistry of cytochrome
P450 8B1, the oxysterol 12-hydroxylase enzyme implicated in cardiovascular health.
Experimental Section

Materials. Reagents and solvents were purchased from commercial sources. TLC plates (silica
gel) with 254 nm fluorescent indicator (Sigma, St. Louis, MO) were used to monitor reactions.
Both a UV lamp and ceric ammonium molybdate staining were used to visualize compounds on
TLC. Silica gel (Silicycle (Quebec, Canada), 40-63 m, 60 Å) was used for flash column
separations. A WXG-4 optical polarimeter (Bante Instruments, Ltd, Shanghai, China) was used
to measure optical rotations with a 10 cm long cell. A melting point apparatus (Global Medical
and Lab Solutions, India) was used to measure the melting points of each compound. A Bruker
5
00 Advance III High Definition (HD) NMR spectrometer (Billerica, MA) was used to record
1
13
the spectra of synthesized intermediates at 500 MHz for H and 125 MHz for C NMR. CDCl
3
(Cambridge Isotope Laboratories, Tewksbury, MA) was used to take NMR spectra. The
1
13
chloroform peaks were referenced to δ 7.26 ppm and δ 77.16 ppm for the H NMR and C
NMR, respectively. Infrared (IR) spectra were recorded using a Nicolet iS50 FT-IR Spectrometer
(
Thermo Fisher Scientific, Waltham, MA) equipped with the Attenuated Total Reflectance
ATR) module. OMNIC software (version 9.3.32) was used to analyze the IR data (Thermo
(
Fisher Scientific, Waltham, MA).
X-Ray Crystallaography.
Single crystals of C H O (7-,12-dihydroxy DHEA, compound 3) and C H O (12-
3
8
58
9
19 30
6
hydroxy-7-keto DHEA, compound 2) were prepared by slow evaporation of 1:1 mixture of
methanol and dichloromethane. Suitable colorless plate-like crystals for compounds 3 and 2,
with dimensions of 0.33 mm × 0.30 mm × 0.03 mm and 0.33 mm x 0.27 mm x 0.13 mm, were
mounted in paratone oil onto a nylon loop. Single crystals of C H O (12-hydroxy DHEA,
1
9
28
3
compound 22) were prepared by slow evaporation of a 1:1 mixture of diethyl ether and acetone.

A suitable colorless plank-like crystal for compound 22 with dimensions of 0.50 mm × 0.20 mm
0.10 mm, was mounted in paratone oil onto a nylon loop. All data were collected at 98(2) K,
×
using a Rigaku AFC12 / Saturn 724 CCD fitted with MoKα radiation (λ = 0.71075 Å). Data
collection and unit cell refinement were performed using CrysAlisPro software [20]. The total
number of data were measured in the range 4.7° < 2θ < 52.0°, 4.7° < 2θ < 52.0° and 5.0° < 2θ <
5
1.0° for compounds 3, 2, and 22, respectively, using ω scans. Data processing and absorption
correction, giving minimum and maximum transmission factors (0.9144, 1.000 for compound 3,
.8617, 1.000 for compound 2, 0.8958, 1.000 for compound 22) were accomplished with
0
CrysAlisPro [20] and SCALE3 ABSPACK [21], respectively. The structure, using Olex2 [22],
was solved with the ShelXT [23] structure solution program using direct methods and refined (on
2
F ) with the ShelXL [22] refinement package using full-matrix, least-squares techniques. All
non-hydrogen atoms were refined with anisotropic displacement parameters. All carbon bound
hydrogen atom positions were determined by geometry and refined by a riding model. All
oxygen bound hydrogen atom positions, on compound 3, were determined by electron density
plots, except for H6 hydrogen atom which was placed on a calculated position. All solvent bound
hydrogen atom positions, on compound 2, were determined by electron density plots. Non-
solvent oxygen bound hydrogen atom positions were determined by geometry and refined by a
riding model, except for hydrogen atom, H4A, which was determined by electron density plot.
All oxygen bound hydrogen atom positions, on compound 22, were determined by electron
density plots. Crystal structures were submitted to the Cambridge Structural Database (CSD)
with CSD numbers: 1870940, 1870939, and 1870941 for compounds 2, 3, and 22.
Brain Slice Preparation and Electrophysiology

Male and female C57BL/6J mice were used. All procedures were approved by the University of
Texas at San Antonio Institutional Animal care and Use Committees in accordance with the
National Institutes of Health guidelines. Mice were deeply anesthetized with isoflurane followed
by rapid decapitation. The brain was quickly removed and chilled in ice cold cutting solution
containing (in mM): 110 cholineCl, 2.5 KCl, 1.25 NaH PO , 7 MgCl , 0.5 CaCl , 10 dextrose, 25
2
4
2
2
NaHCO , and oxygenated with 95% O -5%CO . Horizontal brain slices (250 µm) containing
3
2
2
VTA were incubated for 30 minutes at 35 ºC in a chamber filled with artificial cerebrospinal
fluid (aCSF) and stored at room temperature for the remainder of the day. Slices were
transferred to a recording chamber where they continuously received aCSF (33-35º C) at a rate of
2
1
5
ml per minute by a gravity-feed system. aCSF was composed of (in mM): 126 NaCl, 2.5 KCl,
.25 NaH PO , 2 MgCl , 2 CaCl , 10 dextrose, 25 NaHCO , was equilibrated using 95% O -
2
4
2
2
3
2
%CO and had a pH of 7.2.
2
Midbrain neurons were visualized using gradient contrast illumination through a 40X water-
immersion lens attached to an Olympus BX51 upright microscope. Neurons were considered
dopaminergic based on the following electrophysiological criteria: have a hyperpolarization-
activated inward current, slow spontaneous firing frequency ≤ 5 Hz and an action potential half-
width ≥ 1.5 ms. Dopamine (TH-positive) neurons filled with biocytin were further identified
immunocytochemically.
Whole cell recording pipettes with a tip resistance between 4-6 MOhm were pulled from
borosilicate glass using a P-97 Flaming/Brown electrode puller (Sutter Instruments) and filled
with internal solution containing (in mm): 110 d-gluconic acid, 110 CsOH, 10 CsCl , 1 EGTA,
2

1
0 HEPES, 1 ATP, 10 mm phosphocreatine.
Postsynaptic currents (PSC) were evoked by placing a 250 µm bipolar stimulating electrode
within 500 µm of the neuron being recorded and applying a single pulse (0.05 ms). Whole cell
recordings were made using a Multiclamp 700B amplifier (Molecular Devices). Experiments
were conducted in the presence of the AMPA receptor antagonist, NBQX (25 µM), to isolate
NMDA receptor-mediated currents. Signals were digitized at 50 kHz and saved to a hard drive
for analysis using Axograph X (Axograph). Access resistance was monitored and measured post
hoc to ensure access resistance had not changed by less than 20%.
Statistics: A student’s two-tailed t-test was used when comparing between two groups. A P-
value < 0.05 was used as our significance level for all tests.
Chemical Synthesis.
3-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]-androst-5-en-17-one
(Compound
5):
tert-
Butyldimethylsilyl chloride (9.05 g, 60 mmol, 3 eq) and Imidazole (6.81 g, 100 mmol, 5 eq)
were added to a solution of Dehydroepiandrosterone (DHEA) (6.00 g, 20 mmol, 1 eq) in
acetonitrile (100 ml). The reaction was stirred at room temperature for 6 h. The reaction mixture
was diluted with water (50 ml) and extracted three times with ethyl acetate (3 x 50 ml). The
organic layer was concentrated under reduced pressure to afford DHEA-3β-tert-
butyldimethylsilyl ether (5) (7.70 g, 19 mmol, 95%) as a white solid. No further purification was
done; R : 0.86 (hexanes:ethyl acetate, 4:1, v/v); IR (neat) 2926.42, 2889.06, 2856.05, 1738.49,
f

-1 1
1
462.61, 1379.73, 1250.67, 1076.66, 1059.33 cm ; H NMR (500 MHz, CDCl ) δ 5.34 (d, J = 6
3
Hz, 1H), 3.48 (m, 1H), 2.45 (dd, J = 19, 8 Hz, 1H), 2.27 (m, 1H), 2.19 (m, 1H), 2.08 (m, 2H),
.94, (m, 1H), 1.82 (m, 2H), 1.74 – 1.42 (m, 7H), 1.29 - 1.27 (m, 2H), 1.01 (s, 3H), 1.09 -0.96
1
1
3
(
m, 2H), 0.88 (s, 12H), 0.05 (s, 6H); C NMR (125 MHz, CDCl ) δ 141.94, 120.54, 72.59,
3
5
1.95, 50.47, 47.70, 42.93, 37.45, 36.87, 35.99, 32.16, 31.67, 31.61, 30.97, 26.06, 22.03, 20.50,
+
1
9.60, 18.39, 13.68, -4.42; HRMS (m/z) calculated for C H O SiNa [M + Na] , 425.2846;
2
5
42
2
found, 425.2839 (Δ 1.64 ppm).
3
β-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]-17-[(2-pyridinylmethyl)imino]-androst-5-ene
Compound 6): To a solution of DHEA-3β-tert-butyldimethylsilyl ether 5 (3.00 g, 7.5 mmol, 1
eq) in toluene (100 ml) was added 2-aminomethyl pyridine (2.20 g, 23 mmol, 3 eq) and (50 mg,
(
0
.2 mmol, 0.03 eq) of para-toluenesulfonic acid. The solution was refluxed for 4 h with a Dean
1
Stark apparatus. The progress of the reaction was monitored by H NMR. The reaction was
cooled to room temperature. The reaction mixture was diluted with ethyl acetate and
subsequently washed three times with saturated NaHCO (200 ml), dried with MgSO and
3
4
concentrated under reduced pressure to afford the imine 6 as a white solid (3.1904 g, 6.8 mmol,
9
1
0%). No further purification was done. IR (neat) 2940.26, 2927.23, 2890.02, 2856.88, 1745.74,
-1
1
678.36, 1590.54, 1567.92, 1471.49, 1435.35, 1382.00, 1254.46, 1083.24 cm ; H NMR (500
MHz, CDCl ) δ 8.52 (d, J = 5 Hz, 1H), 7.65 (td, J = 8, 2 Hz, 1H), 7.42 (dt, J = 8 Hz, 1H) , 7.14 –
3
7
1
4
0
.09 (m, 1H), 5.33 (d, J = 6 Hz, 1H), 4.58 (ABq, JAB = 16.7 Hz, 2H), 3.48 (M, 1H), 2.45 (dd, J =
7, 9 Hz, 1H), 2.34 – 2.13 (m, 3H), 2.25 – 1.98 (m, 2H), 1.92 – 1.79 (m, 2H), 1.75 – 1.58 (m,
H), 1.55 – 1.30 (m, 4H), 1.20 – 1.12 (m, 1H), 1.11 – 0.96 (m, 2H), 1.06 (s, 3H), 0.91 (s, 3H),
1
3
.88 (s, 9H), 0.05 (s, 6H) ; C NMR (125 MHz, CDCl ) δ 186.19, 160.62, 149.07, 141.92,
3

1
4
36.65, 128.87, 126.07, 122.35, 121.66, 120.66, 120.73, 120.51, 72.63, 58.10, 53.43, 50.71,
2.93, 37.48, 36.48, 34.19, 32.17, 31.45, 28.16, 26.05, 23.47, 20.85, 19.67, 18.37, 16.34, -4.46;
+
HRMS (m/z) calculated for C H N OSi [M + H] , 493.3609; found, 493.3611 (Δ -0.48 ppm).
3
1
49
2
1
2β-Hydroxy-3β-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-androst-5-en-17-one (Compound 7):
Copper triflate, (2.11 g, 5.84 mmol, 1.3 eq) and sodium-(L)-ascorbate (1.78 g, 8.98 mmol, 2 eq)
were added to the imine 6 (2.12 g, 4.4 mmol, 1 eq) in an oven baked round bottom flask
backfilled with N . Acetone (18 ml) and methanol (18 ml) were added to the mixture and stirred
2
for 10 minutes. The reaction solution turned brown after stirring for 5 minutes. The round bottom
flask containing the reaction mixture was sealed with a rubber stopper and degassed. The
reaction solution was bubbled with O gas with the aid of a balloon and an exit needle until the
2
°
color of the solution changed from brown to green. The reaction mixture was heated at 50 C
under an atmosphere of O for 2 h. The reaction was cooled to room temperature and then
2
quenched by adding saturated Na EDTA (30 ml, pH = 5) and ethyl acetate (15 ml). The resulting
4
solution was stirred for 1 h and then transferred to a separatory funnel and allowed to stand for 5
minutes to differentiate the organic layer from the aqueous layer. The organic layer was
separated from the aqueous layer. The aqueous layer was extracted with ethyl acetate (150 ml).
The combined organic layers were concentrated under reduced pressure to afford a crude brown
oil. The crude brown oil was purified by flash column chromatography (100% hexanes to 50%
ethyl acetate in hexanes) to afford the alcohol 7 as a white solid (0.9 g, 2.1 mmol, 48%). An
analytical sample of compound 7 was recrystallized with hexane/ethyl acetate (1:1) to afford a
°
20
D
°
white amorphous solid: mp: 175 – 177 C; R : 0.79 (hexanes:ethyl acetate, 1:1, v/v); [α] -25
f

[
0.02% in CHCl ]; IR (neat) 3545.58, 2949.63, 2928.84, 2890.64, 1733.18, 1470.79, 1403.66,
3
-1
1
1
359.09, 1291.15, 1270.45, 1111.92, 1047.42, 1027.11, 1018.23 cm ; H NMR (500 MHz,
CDCl ) δ 5.34 (d, J = 6 Hz, 1H), 3.8 (dd, J = 11.3, 4.9 Hz, 1H), 3.47 (m, 1H), 3.06 (s, 1H), 2.47
3
(dd, J = 19, 10 Hz, 1H), 2.31 – 2.16 (m, 2H), 2.17 – 2.04 (m, 2H), 2.02 – 1.95 (m, 1H), 1.86 –
1
1
3
5
8
.77 (dt, J = 13, 5 Hz, 2H), 1.76 – 1.70 (m, 1H), 1.69 – 1.57 (m, 3H), 1.57 – 1.50 (m, 1H), 1.49 –
.37 (m, 1H), 1.29 - 1.19 (m, 1H), 1.16 – 1.06 (m, 2H), 1.06 – 0.99 (m, 1H), 1.03 (s, 3H), 0.95 (s,
1
3
H), 0.88 (s, 9H), 0.07 (s, 6H); C NMR (125 MHz, CDCl ) δ 141.94, 120.35, 77.82, 77.51,
3
1.54, 49.72, 49.35, 42.83, 37.40, 37.00, 35.90, 32.10, 30.74, 30.55, 26.05, 21/84, 19.51, 18.38,
+
.19, -4.45; HRMS (m/z) calculated for C H O SiNa [M + Na] , 441.2795; found, 441.2795 (Δ
2
5
42
3
-0.01 ppm).
.
1
7-,17-Ethylenedioxy-3β-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-androst-5-en-12β-ol
(Compound 8): Ethylene glycol (2 ml, 35.9 mmol, 16 mol eq), para-toluenesulfonic acid (0.103
g, 0.59 mmol, 0.28 eq) were added to the ketone 7 (0.89 g, 2.13 mmol, 1 eq) in toluene (100 ml).
The reaction was refluxed with a Dean Stark apparatus for 4 h. Upon completion of the reaction
as judged by TLC, the reaction was cooled to room temperature. The reaction mixture was
diluted with ethyl acetate (150 ml), washed three times with saturated aqueous NaHCO (3 x 100
3
ml) and dried with magnesium sulfate. The resulting solution was concentrated under reduced
pressure to form a crude yellowish-brown oil, which was the purified by silica gel column
chromatography (100% hexanes to 50% ethyl acetate in hexanes) to afford the ketal 8 as a white
°
solid (0.2064 g, 0.44 mmol, 20%); mp: 140 – 143 C; R : 0.15 (hexanes:ethyl acetate, 4:1, v/v);
f

2
0
°
[
α]D -12.5 [0.12% in CHCl ]; IR (neat) 3574.56, 3545.91, 2929.47, 2891.15, 2854.47, 1733.91,
3
-1
1
1
470.18, 1434.08, 1381.34, 1371.54, 1248.77, 1217.62, 1080.94, 1048.63, 1033.95 cm ; H
NMR (500 MHz, CDCl ) δ 5.31 (d, J = 5 Hz, 1H), 4.03 – 3.95 (m, 4H), 3.91 – 3.85 (m, 1H), 3.47
3
(m, 1H), 2.30 – 2.22 (m, 1H), 2.21 – 2.14 (m, 1H), 2.05 – 1.96 (m, 1H), 1.94 -1.86 (m, 1H), 1.86
–
1.77 (m, 2H), 1.76 – 1.68 (m, 2H), 1.67 – 1.59 (m, 2H), 1.57 – 1.45 (m, 2H), 1.50 – 1.41 (m,
H), 1.42 – 1.36 (m, 3H), 1.10 - 0.99 (m, 2H), 1.02 (s, 3H), 0.93 (s, 3H), 0.88 (s, 9H), 0.05 (s,
2
1
3
6
H); C NMR (125 MHz, CDCl ) δ 141.65, 120.89, 119.25, 72.59, 71.51, 49.41, 49.25, 48.87,
3
4
2.84, 37.48, 36.84, 32.10, 35.95, 32.15, 31.38, 30.97, 29.53, 26.07, 22.47, 19.55, 18.37, 9.01, -
+
4
.41; HRMS (m/z) calculated for C H O SiNa [M + Na] , 485.3058; found, 485.3051 (Δ 1.42
2
7
46
4
ppm).
1
7-,17-Ethylenedioxy-3-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-androst-5-en-12-one
(Compound 9): Pyridinium chlorochromate (0.90 g, 19 mmol, 1 eq) was added to a solution of
the ketal-alcohol 8 (0.90 g, 19 mmol, 1 eq) in dichloromethane (50 ml). The solution was stirred
at room temperature for 6 h. The reaction mixture was washed with 5% NaOH (3×50 ml) and
then concentrated under reduced pressure to obtain a crude yellow solid residue. The crude
residue was purified by silica gel column chromatography (100% hexanes to 10% ethyl acetate
in hexanes) to afford ketone 9 as a white solid (0.419 g, 0.909 mmol, 48%). An analytical sample
of compound 9 was recrystallized from 100% acetone to afford colorless needlelike crystals: mp
°
20
D
°
1
43 – 145 C; R : 0.77 (hexanes:ethyl acetate, 4:1, v/v); [α]
+33.3 [0.03% in CHCl ]; IR
f
3
(neat) 3545.09, 2934.26, 2893.37, 2854.03, 1714.58, 1460.25, 1434.19, 1381.52, 1359.66,
-1 1
1
246.17, 1178.01, 1074.15, 1050.79, 1025.94 cm ; H NMR (500 MHz, CDCl ) δ 5.34 (d, J = 6
3
Hz, 1H), 4.28 – 4.15 (m, 1H), 4.15 – 4.08 (m, 1H), 4.01 – 3.90 (m, 1H), 3.90 – 3.75 (m, 1H),

3
.47 (m, 1H), 2.42 (dd, J = 15, 11 Hz, 1H), 2.31 – 2.19 (m, 2H), 2.19 – 2.12 (m, 1H), 2.05-1.91
(m, 1H), 1.84 – 1.75 (m, 2H), 1.68 – 1.60 (m, 1H), 1.65 – 1.55 (m, 1H), 1.54 – 1.37 (m, 2H),
1
.33 – 1.16 (m, 5H), 1.11 (s, 3H), 1.07 - 0.98 (m, 1H), 1.05 (s, 3H), 0.85 (s, 9H), 0.04 (s, 6H);
1
3
C NMR (125 MHz, CDCl ) δ 210.90, 141.29, 120.81, 116.92, 72.28, 65.59, 65.02, 57.73,
3
5
0.83, 49.96, 42.77, 38.64, 37.09, 37.04, 34.58, 31.92, 31.36, 30.79, 26.06, 20.92, 19.21, 18.37, -
+
4
.45; HRMS (m/z) calculated for C H O SiNa [M + Na] , 483.2901; found, 483.2903 (Δ -0.42
2
7
44
4
ppm).
1
7-,17-Ethylenedioxy-3-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-androst-5-en-12α-ol
(Compound 10): L- Selectride (6.0 ml of a 1.0 M solution in tetrahydrofuran, 6.0 mmol, 1.8 eq),
°
was added to ketone 9 (1.5 g, 3.3 mmol, 1.0 eq) in THF (50 ml) at -78 C under an N
2
atmosphere for 12 h. The reaction was quenched with the addition of water (20 ml) dropwise at -
°
7
8 C, allowed to warm to room temperature and then extracted with ethyl acetate (3×50 ml).
The organic layer was concentrated under reduced pressure and purified by silica gel column
chromatography to afford the 12α-hydroxylated compound (10) as a white waxy solid (1.20 g,
2
.59 mmol, 80%). An analytical sample of compound 10 was recrystallized from 100% acetone
°
to afford colorless needlelike crystals: mp 128 – 130 C; R : 0.29 (hexanes:ethyl acetate, 4:1, v/v);
f
2
0
°
[
α]D +100 [0.01% in CHCl ]; IR (neat) 3485.67, 3209.96, 2956.47, 2926.19, 2887.82, 2854.42,
3
1
1
3
470.61, 1460.75, 1404.53, 1380.79, 1279.49, 1249.29, 1181.18, 1154.53, 1085.57, 1042.45,
-1 1
007.23, 1029.96 cm ; H NMR (500 MHz, CDCl ) 5.32 (d, J = 6 Hz, 1H), 4.70 (s, 1H), 4.05 –
3
.84 (m, 5H), 3.47 (m, 1H), 2.29 – 2.21 (m, 1H), 2.19 – 2.08 (m, 2H), 2.06 – 1.98 (m, 1H), 1.96
-1.87 (m, 1H), 1.86 – 1.63 (m, 6H), 1.62 – 1.46 (m, 4H), 1.38 – 1.23 (m, 2H), 0.99 (s, 3H), 0.88
1
3
(
s, 9H), 0.85 (s, 3H), 0.05 (s, 6H); C NMR (125 MHz, CDCl ) δ 141.88, 121.73, 120.94, 72.72,
3

7
2
1.79, 64.87, 63.53, 43.76, 43.18, 43.00, 42.61, 37.33, 36.52, 34.97, 32.76, 32.18, 39.49, 28.17,
6.08, 22.77, 19.41, 18.40, 15.71, 1.16, -4.43; HRMS (m/z) calculated for C H O SiNa [M +
2
7
46
4
+
Na] , 485.3058; found, 485.3051 (Δ 1.33 ppm).
3
β-,12α-Dihydroxy-andros-5-en-17-one (Compound 1): Hydrochloric acid (4.0 ml of a 2.0 M
solution, 8.0 mmol, 37 mol eq) was added to a solution of ketal 10 (100 mg, 0.22 mmol, 1.0 eq)
in THF (10 ml). The reaction was stirred for 4 h and quenched with saturated NaHCO (aqueous)
3
and extracted with CH Cl (50 ml). The CH Cl extract was concentrated under reduced pressure
2
2
2
2
and purified by silica gel column chromatography (70% hexanes to 50% hexanes in ethyl
acetate) to afford the diol 1 as a white solid (45 mg, 0.15 mmol, 68%). An analytical sample of
compound 1 was recrystallized from acetone/diethylether (1:1) to afford white crystals: mp 178 –
°
20
°
1
3
79 C; R : 0.17 (hexanes: ethyl acetate, 1:1, v/v); [α]
-22.7 [0.02% in CHCl ]; IR (neat)
f
D
3
446.37, 3350.12, 2963.99, 2933.57, 2885.49, 1724.51, 1463.65, 1431.58, 1406.30, 1375.51,
-
1
357.81, 1309.33, 1259.72, 1191.52, 1132.46, 1089.15, 1054.47, 1037.11, 1024.17, 1007.20 cm
1
1
;
H NMR (500 MHz, CDCl ) 5.39 (d, J = 6 Hz, 1H), 4.14 (s, 1H), 3.52 (m, 1H), 2.45 (dd, J =
3
1
4
9, 9 Hz, 1H), 2.36 – 2.28 (m, 1H), 2.27 – 2.18 (m, 1H), 2.15 - 1.93 (m, 4H), 1.87 – 1.65 (m,
1
3
H), 1.55-1.40 (m, 3H), 1.17 – 1.08 (m, 1H), 1.00 (s, 3H), 0.99 (s, 3H), 0.87 (s, 3H) ; C NMR
(
125 MHz, CDCl ) 141.03, 121.11, 71.60, 69.65, 53.04, 43.37, 42.33, 37.11, 36.40, 36.26, 31.64,
3
3
1.38, 30.63, 27.41, 25.77, 21.16 19.35, 14.31, 13.86; HRMS (m/z) calculated for C H O Na
19 28 3
+
[
M + Na] , 327.1931; found, 327.1934 (Δ -0.94 ppm).

3
1
β-,12α-Diacetoxy-androst-5-en-17-one (Compound 11): Acetic anhydride (10.0 ml, 106 mmol,
7.9 eq) and pyridine (1.20 ml, 14.8 mmol, 2.5 eq) were added to diol 1 (1.8 g, 5.9 mmol, 1 eq).
The reaction was stirred at room temperature for 12 h. The reaction mixture was diluted with 50
ml of water and extracted with ethyl acetate (150 ml). The organic layer was concentrated under
reduced pressure and purified by silica gel chromatography (90% hexanes to 70% hexanes in
°
ethyl acetate) to afford diacetate 11 as a white solid (1.69 g, 4.3 mmol, 72%); mp: 189 – 192 C;
20
D
°
R : 0.29 (hexanes:ethyl acetate, 4:1, v/v); [α]
f
-7.5 [0.2% in CHCl ]; IR (neat) 3450.22,
3
2
1
960.86, 2944.30, 2905.91, 2826.58, 2249.42, 1731.33, 1556.73, 1463.17, 1438.07, 1438.07,
-1 1
373.99, 1357.81, 1239.62, 1134.04, 1114.17, 1022.49, cm ; H NMR (500 MHz, CDCl ) 5.41
3
(d, J = 1 Hz, 1H), 5.19 (s, 1H), 4.57 (m, 1H), 2.45 – 2.25 (m, 3H), 2.17 – 2.05 (m, 2H), 2.01 (s,
3
1
H), 2.00 – 1.96 (m, 2H), 1.98 – 1.94 (s, 3H), 1.90 – 1.80 (m, 2H), 1.79 – 1.50 (m, 6H), 1.33 –
1
3
.18 (m, 1H), 1.12 – 1.05 (td, J = 14, 3 Hz, 1H), 0.98 (s, 3H), 0.90 (s, 3H); C NMR (125 MHz,
CDCl ) 216.69, 170.60, 170.20, 139.97, 121.94, 73.66, 71.77, 50.70, 45.48, 44.84, 38.17, 36.95,
3
3
6.44, 36.32, 30.99, 30.64, 27.68, 25.45, 21.50, 21.36, 21.27, 19.26, 13.94; HRMS (m/z)
+
calculated for C H O Na [M + Na] , 411.2142; found, 411.2144 (Δ -0.6 ppm)
2
3
32
5
3
β-,12α-Diacetoxy-7-oxo-DHEA (Compound 12): Chromium trioxide (1.235 g, 12.35 mmol, 6
°
eq) was stirred in CH Cl (50 ml) at -78 C for 15 minutes. 3,5-dimethylpyrazole (1.188 g, 12.35
2
2
mmol, 6 eq) was added to the reaction and stirred for another 20 minutes. Diacetate 11 (0.600 g,
.059 mmol, 1 eq) in CH Cl (10 ml) was added to the mixture and stirred for 24 h. The mixture
2
2
2
was concentrated under reduced pressure to obtain a crude black mixture which was bounded to
silica gel and introduced onto an already packed silica gel column and purified (80% hexanes to

5
0% hexanes in ethyl acetate) to afford diketone 12 as a white solid (0.370 g, 0.919 mmol, 45%);
°
20
D
°
mp: 169 – 172 C; R : 0.48 (hexanes:ethyl acetate, 1:1, v/v); [α] +3.57 [0.28% in CHCl ]; IR
f
3
(neat) 3450.95, 3348.12, 2943.86, 2935.27, 1736.78, 1668.34, 1468.59, 1439.60, 1375.50,
-
1 1
1
5
7
3
3
7
1
364.22, 1229.63, 1134.41, 1035.68, 1025.14, cm ; H NMR (500 MHz, CDCl ) 5.78 (s, 1H),
3
.18 (s, 1H), 4.7 (m, 1H), 2.87 (m, 1H), 2.61 (m, 1H), 2.52 – 2.38 (s, 3H), 2.37 – 2.30 (td, J = 12,
Hz, 1H), 2.20 – 2.10 (m, 1H), 2.04 (s, 3H), 2.03 – 1.97 (m, 1H), 1.98 – 1.94 (m, 1H), 1.96 (s,
H), 1.94 – 1.79 (m, 3H), 1.78 – 1.70 (m, 2H), 1.70 – 1.62 (m, 1H), 1.22 -1.17 (m, 4H), 0.91 (s,
1
3
H); C NMR (125 MHz, CDCl ), 215.98, 200.37, 170.39, 170.16, 164.99, 126.68, 71.94,
3
0.71, 51.08, 45.14, 43.86, 38.19, 38.01, 36.11, 36.07, 27.32, 25.60, 23.63, 21.35, 21.21, 17.33,
+
4.10; HRMS (m/z) calculated for C H O [M + H] , 403.2115; found, 403.2115 (Δ 0.16 ppm).
2
3
31
6
3
β-,12α-Dihydroxy-andros-5-en-7,17-dione (Compound 2): To a stirring solution of diketone 12
(0.35 g, 0.87 mmol, 1 eq) in methanol (30 ml) was added potassium carbonate (120 mg, 0.87
mmol, 1 eq). The reaction was stirred at room temperature for 24 h. The resulting reaction was
diluted with water (30 ml) and extracted with ethyl acetate (100 ml). The ethyl acetate extract
was concentrated under reduced pressure and then purified by silica gel column chromatography
(50% hexanes to 100% ethyl acetate) to afford 12α-hydroxy-7-oxo DHEA (2) as a pale yellow
°
solid (60 mg, 0.19 mmol, 22%); mp: 178 – 181 C; R : 0.78 (ethyl acetate:methanol, 4:1, v/v);
f
2
0
°
[
α]D -8.33 [0.18% in CHCl ]; IR (neat) 3619.00, 3357.93, 3296.12, 3296.12, 3131.07, 3109.09,
3
3
1
1
1
035.64, 2979.73, 2938.52, 2875.31, 2846.05, 1738.61, 1722.26, 1670.31, 1654.82, 1627.01,
442.62, 1434.02, 1382.14, 1328.99, 1297.17, 1258.99, 1186.50, 1164.83, 1050.61, 1037.49,
-1 1
028.92, cm ; H NMR (500 MHz, CDCl ) 5.75 (s, 1H), 4.12 (s, 1H), 3.68 (m, 1H), 2.81 (m,
3
H), 2.58 – 2.50 (m, 1H), 2.48 – 2.34 (m, 4H), 2.17 – 2.06 (m, 2H), 2.06 – 1.96 (m, 2H), 1.96 –

1
1
6
.87 (m, 2H), 1.85 – 1.76 (m, 3H), 1.76 – 1.68 (m, 2H), 1.66 – 1.58 (m, 2H), 1.32 – 1.22 (m,
1
3
H), 1.18 (s, 3H), 0.88 (s, 3H); C NMR (125 MHz, CDCl ) 200.83, 166.42, 126.15, 70.21,
3
8.70, 53.51, 44.46, 44.06, 42.00, 38.05, 37.82, 36.21, 36.04, 31.19, 27.50, 23.42. 17.41, 13.99;
+
HRMS (m/z) calculated for C H O [M + H] , 319.1904; found, 319.1904 (Δ -0.48 ppm). 12-
19
27
4
Hydroxy-7-oxo DHEA (40 mg) was dissolved in 10 ml of 1:1, methanol:dichloromethane (v/v)
in a 40 ml screw cap vial. The solvent was slowly evaporated in the fume hood over 2 days to
afford crystals, which were used to solve the crystal structure.
1
7-,17-Ethylenedioxy-3-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-androst-5-en-7,12-dione
(Compound 13): Chromium trioxide (0.69 g, 6.51 mmol, 6 eq) was added to a stirring solution of
°
CH Cl (50 ml) in a round bottom flask at -78 C and left to stir for 15 minutes. 3,5-
2
2
Dimethylpyrazole (0.63 g, 6.51 mmol, 6 eq) was added to the reaction and was stirred for 25
minutes. Ketone 9 (0.50 g, 1.09 mmol, 1 eq) was dissolved in CH Cl (10 ml) and added to the
2
2
reaction mixture and stirred for 12 h. In a separate round bottom flask A, a stirring solution of
°
CH Cl (50 ml) was cooled to -78 C and chromium trioxide (0.69 g, 6.51 mmol, 6 eq) and 3,5-
2
2
dimethylpyrazole (0.63 g, 6.51 mmol, 6 eq) were added sequentially at a 15 minute-interval.
°
After 1 h, the existing reaction was transferred to reaction flask A at -78 C and stirred for
another 12 h. A crude black solution was obtained, bound to silica gel and loaded onto an already
packed column and purified (hexane 100% to 80% hexanes in ethyl acetate) to afford diketone
1
3 as a pale-yellow solid (0.46 g, 0.97 mmol, 89%). An analytical sample of compound 13 was
°
recrystallized from hexanes/ethyl acetate (1:1) to give yellow crystals: mp 268 – 270 C; R : 0.33
f
20
°
(
hexanes:ethyl acetate, 4:1, v/v); [α]D -12.19 [0.04% in CHCl ] IR (neat) 2977.36, 2948.37,
3

2
1
1
3
2
0
7
1
4
929.25, 2894.92, 1737.84, 1710.55, 1655.69, 1621.96, 1470.41, 1458.15, 1428.66, 1417.83,
382.15, 1373.73, 1354.75, 1296.51, 1257.83, 1184.39, 1168.27, 1138.89, 1099.14, 1090.54,
-1 1
035.79, 1030.28, cm ; H NMR (500 MHz, CDCl ) 5.73 (s, 1H), 4.18 (m, 1H), 4.07 (m, 1H),
3
.96 (m, 1H), 3.86 (m, 1H), 3.60 (m, 1H), 2.57 (m, 3H), 2.43 (d, J = 8, 2 Hz, 1H), 2.31 (m, 2H),
.01 (m, 2H), 1.80 (m, 3H), 1.72 – 1.56 (m, 4H), 1.23 (s, 3H), 1.19 – 1.09 (m, 1H), 1.14 (s, 3H),
1
3
.88 (s, 9H), 0.06 (s, 6H); C NMR (125 MHz, CDCl ) 208.80, 199.01, 166.09, 126.04, 116.20,
3
1.04, 65.99, 65.51, 57.91, 49.83, 44.58, 44.21, 42.62, 38.27, 36.10, 34.56, 31.58, 25.94, 23.28,
+
8.25, 17.07, 15.48, -4.49; HRMS (m/z) calculated for C H O Si [M + H] , 475.2874; found,
27
43
5
75.2874 (Δ 0.07 ppm). *In subsequent procedures to oxidize the C7-allylic position using CrO3
and 3,5-dimethylpyrazole, we found that increasing the mol equivalents of both CrO and 3,5-
3
dimethylpyrazole to 12 mol equivalents resulted in higher yields to afford the C7-keto steroid
product.
1
7,17-Ethylenedioxy-3-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-androst-5-en-7α,12α-diol
(Compound 14): L-Selectride (15 ml of 1.0 M solution in THF, 15 mmol, 16 eq) was added to a
°
solution of diketone 13 (0.4613 g, 0.9717 mmol, 1 eq) in THF (30 ml) dropwise at -78 C under
an atmosphere of N . The reaction was stirred for 12 h and then quenched by adding water (20
2
°
ml) to reaction solution at 78 C. The mixture was warmed to room temperature and then
extracted with ethyl acetate (3×50 ml). The ethyl acetate extract was concentrated under reduced
pressure and purified by silica gel column chromatography (silica gel, 100% hexanes to 50 %
hexanes in ethyl acetate) to afford diol 14 as a white solid (0.20 g, 0.42 mmol, 42%). An
analytical sample of compound 14 was recrystallized with hexane/ethyl acetate (1:1) to afford
°
20
°
white amorphous solid: mp: 120 – 122 C; [α]D + 7.4 [0.07% in CHCl ]. R : 0.11
3
f
(hexanes:ethyl acetate, 4:1, v/v); IR (neat) 3578.41, 3416.56, 3357.55, 3207.67, 2953.08,