Improved Procedures for Gram-Scale Synthesis of Galeterone 3β-Imidazole and Galeterone 3β-Pyridine Methoxylate, Potent Androgen Receptor/Mnk Degrading Agents

Article abstract of DOI:10.1021/acs.oprd.6b00217

Galeterone (1) and its C-3 analogs are of substantial interest because of their multitarget anticancer activities, including AR and Mnk degrading activities. Here, we describe improved and efficient procedures for the gram-scale synthesis of 3β-(1H-imidazole-1-yl)-17-(1H-benzimidazole-1-yl)-androsta-5,16-diene (galeterone 3β-imidazole, 2) and 3β-(pyridine-4-ylmethoxy)-17-(1H-benzimidazol-1-yl)androsta-5,16-diene (galeterone 3β-pyridine methoxylate, 3). Whereas compound 2 was synthesized in 63% overall yield from galeterone (1) over four steps, via key intermediate, 3β-azido galeterone (8); compound 3 was synthesized in 61% overall yield from 1 in one step. This article also reports on the facile synthesis of other potential AR/Mnk degrading agents (ARDAs/MNKDAs), including galeterone 3α-imidazole (5) and galeterone 3β-amine (10), both in excellent overall yields. Notably, except for the one-step synthesis of compound 3 which required purification by flash column chromatography, none of the intermediates and target compound 2 required extensive chromatographic purifications or multiple crystallizations.

Full text of DOI:10.1021/acs.oprd.6b00217

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Full Paper
Improved Procedures for Gram-Scale Synthesis of Galeterone
3#-Imidazole and Galeterone 3#-Pyridine Methoxylate,
Potent Androgen Receptor/Mnk Degrading Agents
Puranik Purushottamachar, Francis N Murigi, and Vincent C. O. Njar
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.6b00217 • Publication Date (Web): 10 Aug 2016
Downloaded from http://pubs.acs.org on August 12, 2016
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Improved Procedures for GramꢀScale Synthesis
of Galeterone 3βꢀImidazole and Galeterone 3βꢀ
Pyridine Methoxylate, Potent Androgen
Receptor/Mnk Degrading Agents
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Puranik Purushottamachar 1,2, Francis N. Murigi1,2, and Vincent C. O. Njar1,2,3, *
2
3
1Department of Pharmacology, Center for Biomolecular Therapeutics, and Marlene and
Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of
Medicine, 685 West Baltimore Street, Baltimore, MD 21201ꢀ1559, USA
Keywords: C3ꢀimidazole/pyridyl methoxylate synthesis, androgen receptor/Mnk degrading
agents, aminosteroids, anticancer agents.
*Corresponding author: Dr. Vincent C. O. Njar, Department of Pharmacology, University of
Maryland School of Medicine, 685 West Baltimore Street, HSF1, Suite 580E, Baltimore, MD
21201, USA. Tel: (410) 706 6364; Fax: (410) 706 0032; Eꢀmail: vnjar@som.umaryland.edu
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12%
Discovery Route of Synthesis Yield
11%
N
12%
N
84%
MeSO O
2
O
Prep HPLC 11%
61%HO
N
VNPP414
Galeterone
100%
N
MeSO O
N
H N
2
3
2
93%
90%
75%
N
VNPP433ꢀ3
β
61%
Novel Process Development Yield
63%
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ABSTRACT
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Galeterone (1) and its Cꢀ3 analogs are of substantial interest because of their multiꢀtarget
anticancer activities, including AR and Mnk degrading activities. Here, we describe improved
and efficient procedures for the gramꢀscale synthesis of 3βꢀ(1Hꢀimidazoleꢀ1ꢀyl)ꢀ17ꢀ(1Hꢀ
benzimidazoleꢀ1ꢀyl)ꢀandrostaꢀ5,16ꢀdiene (galeterone 3βꢀimidazole, 2) and 3βꢀ(pyridineꢀ4ꢀ
ylmethoxy)ꢀ17ꢀ(1Hꢀbenzimidazolꢀ1ꢀyl)androstaꢀ5,16ꢀdiene (galeterone 3βꢀpyridine methoxylate,
3). Whereas compound 2 was synthesized in 63% overall yield from galeterone (1) over four
steps, via key intermediate, 3βꢀazido galeterone (8); compound 3 was synthesized in 61% overall
yield from 1 in one step. This article also reports on the facile synthesis of other potential
AR/Mnk degrading agents (ARDAs/MNKDAs), including galeterone 3αꢀimidazole (5) and
galeterone 3βꢀamine (10), both in excellent overall yields. Notably, except for the oneꢀstep
synthesis of compound 3 which required purification by flash column chromatography, none of
the intermediates and target compound 2 required extensive chromatographic purifications or
multiple crystallizations.
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INTRODUCTION
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In the course of studies to develop potent androgen receptor degrading agents (ARDAs),1
using our phase 3 clinical candidate, galeterone (1) (Figure 1)2, 3 as lead, to modulate AR
signaling in prostate cancer models,1 we discovered that these novel ARDAs also effectively
target oncogenic eukaryotic protein translation, via modulation of MnkꢀeIF4E axis. Since we
determined that these agents also suppress oncogenic peIF4E via degradation of Mnk1 and 2,
they are also referred to as Mnk degrading agents (MNKDAs). We note that these targets have
been implicated in the development, progression, metastasis and drug resistance of a variety of
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cancers, including prostate4ꢀ8 and pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC).9ꢀ
12
These studies enabled us to synthesize and identify 3βꢀ(1Hꢀimidazoleꢀ1ꢀyl)ꢀ17ꢀ(1Hꢀ
benzimidazoleꢀ1ꢀyl)ꢀandrostaꢀ5,16ꢀdiene (galeterone 3βꢀimidazole, 2) (Figure 1) and 3βꢀ
(pyridineꢀ4ꢀylmethoxy)ꢀ17ꢀ(1Hꢀbenzimidazolꢀ1ꢀyl)androstaꢀ5,16ꢀdiene (galeterone 3βꢀpyridine
methoxylate, 3) (Figure 1) as promising new leads with superior antiꢀprostate cancer activities
compared to galeterone (1).13, 14 However, these two promising lead compounds, 2 and 3, were
obtained in very low discouraging overall yields of 11 and 12%, respectively.
N
N
N
N
17
N
N
16
1
HO
N
5
O
3
N
N
3
1
2
Figure 1: Structure of galeterone (1) and new analogs 2 and 3.
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Our strategy to improved and efficient procedures for the gramꢀscale synthesis of
compounds 2 and 3, was based on critical analyses of our prior synthetic procedures, including
the byꢀproducts that were obtained. First, the treatment of 3βꢀmesyl galeterone (4) with
imidazole in refluxing toluene afforded the desired compound 2 (11%), stereo 3αꢀisomer (5,
3%), positional 6βꢀisomer of 3α, 5αꢀcycloandrostane (6, 35%) and uncharacterized elimination
products (Scheme 1).13, 14 We note that these three products were isolated following tedious flash
column chromatography and preparative HPLC procedures. Similar products were also obtained
when the reaction was conducted in pyridine at 85 oC.
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N
N
i
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O
S
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O
(3%)
5
(11%)
2
6
N
N
4
ii
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Elimination
products
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O
S
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O
N
3
(35%)
7
ꢀ
N3
Scheme 1: Influence of nucleophiles on reaction of ꢁ5 steroidsa
aReagents and conditions: (i) imidazole, PhCH3, reflux, 12 h or pyridine/DMF, 85 oC; (ii) DMF,
NaN3, 15ꢀcrownꢀ5, 80 oC, 18 h.
Based on literature precedence,15ꢀ19 the basis for the formation of stereo/positional
isomers in our previous reported synthetic method for 2 is depicted in Scheme 1.13, 14 Formation
of the 6βꢀsubstituted imidazole (6) as major product is probably a result of ionization of mesylate
prior to the attack of nucleophile at C3 position (SN1 mechanism), which further forms
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homoallylic hybrid carbonium ion intermediate due to the participation of C5 double bond as
previously reported by other investigators.15ꢀ19 Attack of nucleophile on hybrid carbonium ion
has been demonstrated to be faster at C6 position than at C3 due to the difference in their
reactivity.20 However, strong nucleophile, such as, azide predominantly follows SN2 mechanism
to yield stereo inverted C3ꢀazide (7).21 From these observations, it is clear that the nature of
nucleophile has influence on mechanism of SN reaction of ꢀ5 steroids. Weak nucleophile such as
imidazole predominantly follow SN1 mechanism while reactive nucleophiles follow SN2
mechanism. Therefore, direct installation of imidazole unit at C3 position in the presence of C5
double bond may be impractical. Thus, synthesis of imidazole ring starting from azide by
functional group modification provides an attractive route as described below.
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Second, for the synthesis of the pyridyl methoxylate (3), the strategy was to improve on
our reported procedure of Williamson’s etherification following treatment of galeterone (1) with
4ꢀ(bromomethyl)pyridine hydrobromide in the presence of sodium hydride as base in DMF at 65
oC.13, 14 Specifically, we explored using different solvents and salt effects. This strategy proved
successful as described below.
RESULTS AND DISCUSSION
Synthesis of galeterone 3βꢀimidazole (2): We first envisioned and designed a new synthetic
route that would enable the tethering of the imidazole moiety to C3 (3α/βꢀisomers) of galeterone
(12/1) as depicted in Figure 2A. Retroꢀsynthetically, the 3βꢀimidazole ring can be constructed
from 3βꢀamine (10) by applying Heinrich Debus cyclization method,22, 23 where the 3βꢀamine
(10) would be obtained from selective reduction of key intermediate, 3βꢀazide (8), without
affecting the double bonds in rings B and D.24 Next, we envisioned two possible routes for the
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synthesis of 3βꢀazide (8). The first method involves conversion of galeterone (1) to the 3βꢀ
mesylate (4) followed by treatment with trimethylsilyl azide (TMSN3) and boron trifluroride
etherate (BF3•OEt2) in dichroromethane (DCM). This procedure is termed, Lewis acid mediated
iꢀsteroid/ retroꢀiꢀsteroid rearrangement method (Figure 2B).24, 25 Alternatively, 8 can also be
obtained from epiꢀgaleterone (12) either by Mitsunobu method of using hydrogen azide or via
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treatment of
αꢀmesyl derivative (13) with NaN3 (Figure 2B). Both methods involving epiꢀ
galaterone (12) are known to produce the azide as major product, with inversion of
configuration.21, 26
N
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N
H
H
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O
+
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H N
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H
H
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H
H
N
O
+
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: 3 ꢀimidazole
: 3 ꢀamine
: 3 ꢀazide
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: 3 ꢀimidazole
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HN3
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Figure 2A & B: Retrosynthetic procedure for synthesis of compounds 2 and 5 and key
intermediate 8.
The Heinrich Debus method for the synthesis of imidazole ring from primary amines is
widely reported in the literature.22, 27, 28 However, its applicability to aminosteroids has not been
reported.
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Before initiating any efforts towards the stereospecific synthesis of our key intermediate,
5
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7
3β
ꢀazide (8), we considered it prudent to first evaluated the synthetic feasibility of imidazole ring
8
9
from amine at C3 position of steroid using easily accessible 3
our group.21 In addition, the resultant galeterone 3
αꢀazide (7), previously reported by
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α
ꢀimidazole (5), a possible metabolite of
galeterone 3βꢀimidazole (2) would be valuable during pharmacokinetics (PK) and other in vivo
studies.
Accordingly, our experimentation began with the mesylation of galaterone (Scheme 2).
Although we have reported the milligram scale synthesis of 3βꢀmesyl galeterone (4), in 84%
yield, using pyridine as solvent and base, its application in gram scale yield intractable mixture
of products.1 Therefore, we modified the procedure, by replacing DCM as solvent and using
triethyl amine (TEA) as base.24 This modification gave 4, in 100% yield after simple solvent
removal, water wash of residue solids, filtration and drying under vacuum. The conversion of 4
to 3αꢀazide (7) was achieved in relatively shorter time (18 h) in comparison to our previous
method (48 h, 60% yield),21 by conducting the reaction in the presence of catalytic amount of 15ꢀ
crownꢀ5 (yield 60%).29 For the reduction of 3αꢀazide 7 to 3αꢀamine 9, a reported LiAlH4 in ether
method a gave good yield (60 %).24 Considering the requirement of anhydrous condition and
difficulties in handling pyrophoric reagent in scaleꢀup, we looked for a nonhazardous reduction
method. Application of Staudinger method of azide reduction, using PPh3 in THF/MeOH/water
(4:4:1) at 60 °C smoothly converted 7 into 3
α
ꢀamine, 9 within 10 h.30 The product, in 67% yield,
was isolated by simple acidꢀbase workup. The cyclization of 9 into 3
αꢀimidazole (5) was
achieved by reacting with aq. ammonia, formaldehyde and glyoxal at 70 °C for 5 h.27 As in the
case of amine, the imidazole product was also isolated by acid base workup and further purified
by passing through a plug of silica using 1ꢀ5% metanol in ethyl acetate. The pure product
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obtained was reprocessed with acidꢀbase to obtain solvent free product in 71% yield. This route
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7
of synthesis gave 28% overall yield of galeterone 3
αꢀimidazole (5) starting from 4 in three steps
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(β
ꢀmesyl → ꢀN3 → ꢀNH2 → αꢀimidazole). By these test reactions using 3α
α
α
ꢀazide (7), we not
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only establish the viability of our intended synthetic strategy, but also established reaction
conditions and purification procedures which were extended to the synthesis of the desired
galeterone 3
β
ꢀimidazole (2).
iia
iv
iii
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N
H N
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N
N
60%
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67%
71%
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9
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90%
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18%
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NO
2
Scheme 2: Stereoꢀelective synthesis of galeterone 3βꢀimidazole (2) and galetrone 3αꢀ
imidazole (5)a.
aReagents and conditions: (i) anhydrous DCM, TEA, 4 ºC, Mesyl chloride, 30 min, ~22 ºC, 16
h; (iia) anhydrous DMF, NaN3, 15ꢀCrownꢀ5, 80 ºC, 18 h; (iib) anhydrous DCM, TMSN3,
BF3.OEt2, ~22 ºC, 5 h, 2M aq. NaOH, ~22 ºC, 6 h; (iic) anhydrous DCM, TMSꢀimidazole,
BF3.OEt2, ~22 ºC, 12ꢀ48 h, 2M aq. NaOH, ~22 ºC, 5ꢀ20 h; (iii) PPh3, THF/MeOH/water (4:4:1),
60 ºC, 3ꢀ10 h; (iv) NH3 (aq. 25%), dist. H2O, MeOH, glyoxal trimer dihydrate, HCHO (aq. 36%),
70 ºC, 5 h;( v) pꢀnitrobenzoicacid, PPh3, THF, 4 ºC, DEAD (40% in Toluene), ~22 ºC, 12 h. vi)
THF/MeOH (2:1), aq. NaOH (1.0 N), ~22 ºC 2.5 h.
Having demonstrated the working synthetic strategy, we directed our efforts towards the
synthesis of key intermediate 3βꢀazide (8). Recently, Sun et al. reported a practical synthesis for
the conversion of cholestꢀ5ꢀenꢀ3βꢀol methanesulfonate to 3βꢀaminoꢀ5ꢀcholestene with retention
of configuration in 93% yield).24 The method involves use of TMSN3 in the presence of Lewis
acid which led to C3 steroidal azidation with complete stereoꢀretention.
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Thus, we attempted this method on 3βꢀmesylate (4) using the reported ratio of reagents
and also doubled the quantity of reagents with longer duration of reaction time. Neither of these
reaction conditions indicated formation of the expected 3βꢀazido product, 8. We therefore
pursued the epiꢀgalaterone (12) route for the synthesis of 3βꢀazido (8) (Scheme 2). The desired
epiꢀgalaterone (12) was obtained from galeterone (1) by Mitsunobu stereoꢀinversion method.31
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This involves the formation of nitrobenzoicacid 3αꢀepiester (11) and its hydrolysis with aq.
NaOH to obtain epiꢀgalaterone (12). This stereoꢀinversion method required cumbersome
purification procedure due to the polar nature of substrate, product, and byꢀproducts, such as,
PPh3O, diethyl 1,2ꢀhydrazinedicarboxylate of reagents PPh3, DEAD. As a result, it provided only
17% yield of 12 from galeterone (1).
For the conversion of epiꢀgalaterone (12) to desire 3βꢀazide (8) we only attempted
mesylation route (Figure 2B, Scheme 2) to avoid hazardous nature of hydrogen azide in
Mitsunobu method. Unfortunately, our attempts to synthesize 3
elimination products along with intractable adducts.
αꢀmesyl (13) resulted in
This unexpected difficulty in functional group modifications inspired us to revisit the iꢀ
steroid and retroꢀiꢀsteroid rearrangement method for steroidal 3 ꢀazide synthesis. As stated
earlier, Sun et al reported that, the method worked efficiently on 3 ꢀcholesteryl mesylate while
β
β
using 1 equivalent of TMSN3 and 2 equivalents BF3.OEt3 in DCM.24 They also stated that, no
reaction was observed when solvents bearing heteroatoms that function as Lewis base were
used.24 Based on these data, we reasoned that the failure in our earlier efforts to synthesize 3
azide (8) from 3 ꢀmesylate (4) could be due to presence of heteroatoms in the substrate, 4
(i.e.,benzimidazole heteroꢀnitrogen atoms). Thus, we hypothesized that the amount of BF3•OEt2
βꢀ
β
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used in the earlier reactions could be consumed by nitrogen atoms of benzimidazole ring of
compound 4. To evaluate this possibility, we setup a reaction using 10 equivalents of BF3•OEt2
which indicated very little progress in the reaction as evidenced by TLC and was incomplete
even after longer duration of reaction time (48 h). To determine the optimum conditions required
for this azidation reaction, we evaluated various ratio of TMSN3/BF3.OEt3. We eventually found
that the best yield (93%), was obtained when 15 and 27 equivalents of TMSN3 and BF3.OEt2,
respectively, were used for 1 equivalent of 4 in anhydrous DCM at ~22 ºC for 5 h. Pure Lewis
acid salt free 3βꢀazido product (8) was isolated by filtration after neutralizing the reaction
mixture (exothermic) as well as breaking the complex of product with Lewis acid with aqueous
NaOH for 6 h.
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Finally, reduction of 3βꢀazide (8) to 3βꢀamine (10, 90%) followed by its cyclization, as
described about for 3αꢀamine (7) gave the desired 3βꢀimidazole (2) in 75% yield. Surprisingly,
both Staudinger azide reduction and Heinrich Debus method of imidazole synthesis gave better
yield in shorter time in the case of βꢀepimer in comparison to αꢀepimer. This may be due to easy
accessibility of equatorial functions in comparison to axial on steroid scaffold.32 Functional
group modification (βꢀmesyl → βꢀN3 → βꢀNH2 → βꢀimidazole) proceeded rapidly and in high
yield (overall yield 63%) with complete retention of configuration. The whole process was
repeated twice to confirm the yields with standard deviation of ~2%.
We also evaluated the possibility of extension of this iꢀsteroid and retoꢀiꢀsteroid method
with TMSꢀimidazole which could provide the desired compound 2 from 4 in one step (Scheme
2). Unfortunately, the method was unsuccessful even with various ratios of TMSꢀimidazole and
BF3•OEt3; as it gave small amount elimination product and majority of unreacted 4
.
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Synthesis of galeterone 3βꢀpyridine methoxylate (3): To improve on our reported low yield
synthesis of compound 3,13, 14 we initially attempted the synthesis following the procedure
reported by Jilka et al.33 where galeterone (1) in THF was treated with sodium hydride (NaH) at
0 °C and stirred at room temperature for 10 minutes followed by addition of 4ꢀ
(bromomethyl)pyridine hydrobromide solution [14; THF/DMF (1:1), TEA]. The product was
isolated in low yields of 24% although it was slightly higher than our previously 12% yield
reported in our recent reports.13, 14 In an effort to improve the product yield, DMF and DMSO
were investigated as alternative reaction solvents to THF/DMF mixture, as well as addition of
phase transfer catalyst (tetrabutylmmonium iodide), but insignificant product formation
improvement was observed by TLC analysis and isolated product yields.
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Salts are known to affect the transition state of SN2 reactions positively.34 In attempt to
investigate salt effect in our substrate reaction, we incorporated lithium carbonate (Li2CO3)
which concurrently eliminated neutralization of 14 with TEA. The procedure that we developed
is depicted in Scheme 3.
N
N
N
N
i
61%
HO
O
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Scheme 3: Synthesis of galeterone 3βꢀpyridine methoxylate (3)a
aReagents and conditions: (i) NaH, Li2CO3, DMF/THF, 0 oC, 4ꢀ(bromomethyl)pyridine
hydrobromide (14), rt, 18 h.
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Briefly, galeterone (1) in THF was treated with NaH followed by addition of DMF after
one minute. Li2CO3 followed by reagent 13 were then added to give the product in good yields
(61ꢀ64%) and unreacted compound 1 was recovered. Several reactions conditions were explored
in an attempt to drive the Li2CO3 reaction to completion without success. Although we obtained
an excellent yield of the desired compound 3, it is unclear at this time why the reaction could not
be driven to completion.
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Overall, several important observations were noted. (1) The reaction done with DMF as
the solvent produced a green/blue reaction mixture color and no product formed irrespective of
the reaction duration while THF revealed low product formation as analyzed by TLC. (2) The
appropriate ratio of DMF to THF should not be > 1:2 a ratio of 1:1 gave no product. (3) The
optimized DMF to THF ratio of 1:16 was applicable to both milligram (≥ 250 mg) and gram (1ꢀ5
g) scales reactions. (4) DMSO and THF (1:2) solvent mixture showed no product formation. (5)
Cesium carbonate salt gave 50% of product. (6) The color of the reaction mixture after addition
of all reactants should remain colorless for about one hour and gradually change to dark red after
18 h. (7) The developed reaction procedure was conducted at 50 °C as an attempt to optimize the
yield but the isolated yield was 45% which was significantly lower compared to 61% as reported
at room temperature. In summary, the order of the reagent/solvent addition as well as
temperature control during addition was found to be crucial to attaining the desired product.
CONCLUSION
In conclusion, we have developed efficient fourꢀstep synthesis of galeterone 3βꢀimidazole
(2) and a oneꢀstep synthesis of galeterone 3βꢀpyridine methoxylate (3) in 63 and 61% overall
yields, respectively, both from galeterone (1). We also developed an interesting stereospecific
synthesis of galeterone 3αꢀimidazole (5), which could be useful in pharmacokinetics studies of
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compound 2. In addition, the penultimate precursor of compound 2, 3βꢀamino galeterone (10),
obtained in 84% overall yield from galeterone (1) is also proving to be a potent
ARDA/MNKDA. These new and improved procedure for the synthesis of compound 2 do not
require column chromatography or multiple crystallizations and both procedures for target
compounds 2 and 3 are amendable to commercial productions. The efficient production of these
new and improved lead ARDAs/MNKDAs will undoubtedly facilitate our ongoing antiꢀtumor
efficacy studies.
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EXPERIMENTAL SECTION
General: Galeterone used in this process development was provided by Tokai Pharmaceutical
Inc. (TPI). All other reagents were obtained from SigmaꢀAldrich or Acros and were used without
further purification. Room temperature means ~22 ºC. Reactions were conducted using oven
dried glassware under a positive pressure of argon. Anhydrous tetrahydrofuran (THF, 99.8%,
sigma), triethylamine (TEA, 99.5%, sigma), anhydrous N,Nꢀdimethylformamide (DMF, 99.8%,
Acros), anhydrous Dimethyl sulfoxide (DMSO, 99.8%, sigma) were used as supplied. Reactions
were monitored by analytical thinꢀlayer chromatography on Silica plate TLC aluminum baked
plates coated with 200 µm silica gel, indicator F254 and Flash Column Chromatography (FCC)
was performed using silica gel (230ꢀ400 mesh, 60 Å). Melting points were recorded on Fisherꢀ
Johns melting point apparatus and are uncorrected. NMR spectra were recorded on a Bruker
Ascend 400 spectrometer, and chemical shifts δ are expressed in ppm relative to TMS as internal
reference (1H and 13C). 'H and C NMR data’s analyzed and reports were generated by using
ACD/NMR Processor Academic Edition. Highꢀresolution mass spectrometry was obtained on
Bruker 12T APEXꢀQe FTICRꢀMS instrument by positive ion ESI mode by Isaiah Ruhl, Interim
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Facility Director, College of Sciences Major instrumentation cluster, Old Dominion University,
Norfolk, VA. Purity of intermediates and final compound were determined by HPLC method.
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Purity check of compounds used for biological activity (HPLC Chromatograms). The purity
of compounds determined by reverse phase on LC system of Waters Acquity Preparative HPLC
2535 Quaternary Gradient Module coupled with a Waters 2489 UV/visible photodiode array
detector operated at 254 nm using Novapak C18 4 ꢁl, 3.9 X 150 mm column as the stationary
phase at room temperature. Mobile phaseꢀA comprised of Water/MeOH/CH3CN (20: 50:30,
v/v/v + 1 mL of TEA) and maintained isocratically at the flow rate of 2.5 mL/min for mesyl (4),
imidazoles derivatives (2 and 5) and epiꢀgaleterone (12) compound. Where purity of azide (7ꢀ8)
and amines (9ꢀ10) were determined by applying gradient method of using above Mobile phaseꢀ
A and Mobile PhaseꢀB contained methanol with flow rate of1.5 mL/min (see chromatograms for
detail in SI). Similarly, a gradient method applied for compound 3 by using Mobile phaseꢀC
comprised of 10 mM ammonium acetate (NH4OAc) buffer solution and Mobile phaseꢀD
comprised of Water/MeOH/CH3CN (35: 35:30, v/v/v + 20 ꢁL of TEA + 77 mg of NH4OAc)
with flow rate of 0.8 mL/min. Purity of all compounds are > 96.5%.
3βꢀ(1Hꢀimidazolꢀ1ꢀyl)ꢀ17ꢀ(1Hꢀbenzimidazolꢀ1ꢀyl)androstaꢀ5,16ꢀdiene (2). A mixture of
amine 10 (6.50 g, 16.8 mmol), ammonia (25% aq., 3.90 mL, 48.3 mmol), distilled water (5 mL)
and MeOH (120 mL) at ~22 ºC was added glyoxal trimer dihydrate (5.50 g, 26.2 mmol) and
formaldehyde (37% aq., 2.14 mL, 29.3 mmol) simultaneously. The reaction mixture was
immediately taken to 70 ºC (pre heated oil bath) and stirred for 2 h, and then additional half more
quantity of ammonia and formaldehyde added and continued. When 10 was consumed as
evidenced by TLC (~ 3 h), reaction mixture evaporated under vacuum at 60 ºC, reconstituted
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with 120 mL of DCM and extracted with 1N HCl (60 mL x 2). Aqueous phase collected washed
with DCM and neutralized with 1N aq. NaOH and resulting precipitate is extracted with ethyl
acetate (60 mL x 2). Biphasic solution passed through a plug of celite, organic phase collected,
washed with water and evaporated to obtain yellowish red sticky crude product. Sticky mass
absorbed on silica (1.5 w/w) and passed through a short bed of silica using 1ꢀ5% methanol in
ethyl acetate to obtain a cream solid 2. Product is reprocessed with acidꢀbase and collected by
filtration to obtain solvent free 2 (5.50 g, 75%), mp 180ꢀ184 ºC; Rf = 0.21 (DCM/MeOH/TEA,
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10/0.5/0.025); H NMR (400 MHz, CDCl3) δ 1.04 (s, 3 H, 18ꢀCH3), 1.14 (s, 3 H, 19ꢀCH3), 3.87 ꢀ
4.00 (m, 1 H, 3αꢀH), 5.50 (d, J=5.14 Hz, 1 H, 6ꢀH), 5.96 ꢀ 6.03 (m, 1 H, 16ꢀH), 7.01 (s, 1 H, Ar'ꢀ4ꢀH),
7.10 (s, 1 H, Ar'ꢀ5ꢀH), 7.27 ꢀ 7.35 (m, 2 H, 6ꢀHs), 7.46 ꢀ 7.54 (m, 1 H, Arꢀ7ꢀH), 7.67 (br. s., 1 H, Ar'ꢀ2ꢀ
13C NMR (101 MHz, CDCl3) δ 147.1,
H), 7.79 ꢀ 7.86 (m, 1 H, Arꢀ4ꢀH), 7.97 (s, 1 H, Arꢀ2ꢀH);
143.2, 141.6, 140.0, 135.2, 134.5, 129.0, 124.0, 123.4, 122.5, 122.2, 120.2, 116.8, 111.1, 57.4,
55.8, 50.5, 47.2, 40.5, 37.8, 36.9, 34.8, 31.0, 30.2, 30.2, 29.9, 20.6, 19.3, 16.0; HPLC: tR 2.26
min 97.58%; HRMS calcd 339.2856 (C26H34N4H+), found 339.3856.
3βꢀ(pyridineꢀ4ꢀylmethoxy)ꢀ17ꢀ(1Hꢀbenzimidazolꢀ1ꢀyl)androstaꢀ5,16ꢀdiene
(3):
Sodium
hydride {3.00 g, 125 mmol, 10 eq. (5.00 g of 60% NaH in oil)} was added to a solution of
galeterone (1, 5.00 g, 12.9 mmol, 1 eq.) in THF (80 mL) at 0 °C under argon atmosphere. A
white precipitate formed and after stirring for one minute, DMF (5.0 mL) was added and the
reaction mixture was allowed to stir for 10 minutes at room temperature. The reaction mixture
was placed at 0 °C and lithium carbonate (5.00 g, 67.7 mmol, 5 eq.) was added, followed
immediately by 4ꢀ(bromomethyl)pyridine hydrobromide (13, 10.0 g, 39.5 mmol, 3 eq.) and the
reaction mixture was allowed to stir at 0 °C for 30 minutes. The reaction mixture was removed
from 0 °C and stirring continued at ambient temperature for 18 h under argon atmosphere. The
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color of the reaction mixture changed from clear to dark red during the 18 h period. After 18 h,
the reaction mixture was placed at 0 °C, water (30 mL) was added to quench unreacted NaH, and
the mixture was stirred for 10 minute. Volatile (THF) were removed in vacuo and water (70 mL)
was added to the residue aqueous phase. The aqueous phase was extracted with ethyl acetate
(EtOAc) (150 mL x 3). Ethyl acetate extract was washed with brine (80 mL x 2), dried with
anhydrous Na2SO4 and concentrated in vacuo to give a dark red crude product. Purification by
flash chromatography using 3% MeOH/EtOAc as eluent afforded compound 3 as a white solid
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(3.78 g, 7.88 mmol, 61%), mp 177ꢀ179 °C. Rf = 0.31 (5% MeOH/EtOAc). H NMR (400 MHz,
CDCl3) δ 1.02 (s, 3 H, 18ꢀCH3), 1.09 (s, 3 H, 19ꢀCH3), 3.35 ꢀ 3.21 (m, 1 H, 3αꢀH), 4.59 (s, 2 H,
2″ꢀCH2 ), 5.42 (d, J = 5.0 Hz, 1 H, 6ꢀH), 5.96 ꢀ 6.00 (m, 1 H, 16ꢀH), 7.27ꢀ7.31 (m, 4 H,
aromatic and pyridinyl ꢀHs), 7.48ꢀ7.50 (m, 1 H, aromaticꢀH), 7.80ꢀ7.83 (m, 1 H, aromaticꢀH),
7.96 (s, 1 H, 2′ꢀH), 8.57 (d, J = 5.4 Hz, 2 H, pyridinylꢀHs). 13C NMR (100 MHz, CDCl3) δ 19.3,
20.6, 28.3, 30.2, 30.3, 31.1, 34.8, 37.0, 37.1, 39.0, 47.2, 50.5, 55.8, 68.3, 79.1, 111.1, 120.2,
121.2, 121.7, 122.4, 123.3, 124.0, 134.5, 141.0, 141.6, 143.2, 147.1, 148.2, 149.8. HPLC: tR
3.721 min 100%; HRMS calcd 502.2828 (C32H37N3O2Na+, found 502.2834.
3βꢀMesyloxyꢀ17ꢀ(1Hꢀbenzimidazoleꢀ1ꢀyl)androstaꢀ5,16ꢀdiene (4). To a solution of galeterone
(12.0 g, 30.9 mmol) in anhydrous DCM (100 mL) at 4 ºC was added TEA (6.46 mL, 46.4 mmol),
followed by the addition of a solution of mesyl chloride (2.87 mL, 37.1 mmol) in anhydrous
DCM (30 mL) drop wise. The reaction was continued at 4 ºC for 30 min and then stirred at ~22
ºC for total of 16 h. The reaction mixture was concentrated in vacuo, residue solid treated with
water, filtered and dried to afford 4 (14.4 g, 100%) as a white solid, mp 172ꢀ174 ºC; Rf = 0.4
1
(DCM/ EtOH/TEA, 10:0.25:0.025); H NMR (400 MHz, CDCl3) δ 1.02 (s, 3 H, 18ꢀCH3), 1.08
(s, 3 H, 19ꢀCH3), 3.02 (s, 3 H, MesylꢀCH3), 4.49 ꢀ 4.61 (m, 1 H, 3
αꢀH), 5.49 (d, J=4.89 Hz, 1 H,
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6ꢀH), 5.99 (m, 1 H, 16ꢀH), 7.28 ꢀ 7.34 (m, 2 H, Arꢀ5, 6ꢀHs), 7.47 ꢀ 7.52 (m, 1 H, Arꢀ7ꢀH), 7.79 ꢀ
7.85 (m, 1 H, Arꢀ4ꢀH), 7.95 (s, 1 H, Arꢀ2ꢀH); 147.1, 143.2, 141.6, 139.1, 134.5, 124.0, 123.4,
123.1, 122.4, 120.2, 111.1, 81.6, 55.7, 50.2, 47.2, 39.1, 38.8, 36.7, 36.6, 34.7, 31.0, 30.2, 28.8,
20.6, 19.1, 16.0; HPLC: tR 1.62 min 97.69%; HRMS calcd 955.4472 (C27H34N2O4S)2Na+ (note:
dimer formation), found 955.4468.
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3αꢀ(1Hꢀimidazolꢀ1ꢀyl)ꢀ17ꢀ(1Hꢀbenzimidazolꢀ1ꢀyl)androstaꢀ5,16ꢀdiene (5). A mixture of
amine 9 (0.20 g, 0.52 mmol), ammonia (25% aq., 0.12 mL, 1.76 mmol), distilled water (0.15
mL) and MeOH (7.5 mL) at ~22 ºC was added glyoxal trimer dihydrate (0.17 g, 0.81 mmol) and
formaldehyde (37% aq., 0.07 mL, 0.85 mmol) simultaneously. The reaction mixture immediately
taken to 70 ºC (pre heated oil bath) and stirred for 3 h before the addition of one more portion of
glyoxal trimer dihydrate (0.16g), formaldehyde (0.067 mL) and ammonia (0.12 mL), and
continued stirring at 70 ºC for 2 h. When 9 was consumed as evidenced by TLC, reaction
mixture allowed to cool, filtered through celite, residue washed with MeOH (5 mL), and
combined methanolic solutions evaporated. Resulting sticky crude product was reconstituted
with DCM (20 mL) and washed with water, then extracted with 1N HCl solution (10 mL x 2).
Combined acid extracts were washed with DCM (7.5 mL x 2), and then aqueous layer basified to
neutralization with saturated NaHCO3 solution to obtain yellow solid. Colored product was
dissolved in DCM, absorbed on silica (1.5 eq. w/w) and passed through a short bed of silica
using 1ꢀ5% methanol in ethyl acetate to obtain a cream solid, which reprocessed with acidꢀ
basification to obtain solvent free 5 (0.16 g, 71%), mp 207ꢀ209 ºC; Rf = 0.2 (DCM/MeOH/ TEA,
1
10/0.5/0.025); H NMR (400 MHz, CDCl3) δ 1.02 (s, 3 H, 18ꢀCH3), 1.14 (s, 3 H, 19ꢀCH3), 4.41
(br. s., 1 H, 3βꢀH ), 5.56 (d, J=3.18 Hz, 1 H, 6ꢀH), 5.94 ꢀ 6.06 (m, 1 H, 16ꢀH), 7.03 (d, J=3.67
Hz, 2 H, Ar'ꢀ4, 5ꢀHs ), 7.28 ꢀ 7.37 (m, 2 H, Arꢀ5, 6ꢀHs), 7.45 ꢀ 7.54 (m, 1 H, Arꢀ7ꢀH), 7.73 (s, 1
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H, Ar'ꢀ2ꢀH), 7.77 ꢀ 7.86 (m, 1 H, Arꢀ4ꢀH), 7.95 (s, 1 H, Arꢀ2ꢀH); 13C NMR (101 MHz, CDCl3) δ
147.0, 143.2, 141.6, 139.1, 136.7, 134.5, 128.5, 124.1, 123.4, 123.3, 122.4, 120.2, 118.6, 111.1,
55.7, 52.9, 50.1, 47.2, 37.2, 35.9, 34.7, 32.2, 31.1, 30.2, 30.1, 28.3, 20.2, 19.3, 16.0; HPLC: tR
1.83 min 98.83%; HRMS calcd 439.2856 (C26H34N4H+), found 439.3856.
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3αꢀAzidoꢀ17ꢀ(1Hꢀbenzimidazoleꢀ1ꢀyl)androstaꢀ5,16ꢀdiene (7). NaN3 (0.42 g, 6.43 mmol) was
added to a stirred solution of mesylate 4 (1.00 g, 2.14 mmol) and 15ꢀcrownꢀ5 (0.005 g, 0.210
mmol) in anhydrous DMF (7.5 mL). The mixture was heated at 80 ºC for 18 h. After cooling, the
reaction mixture was poured into ice water mixture, stirred for 30 min, filtered and dried to
obtain 0.86g of crude product. Pure product was obtained after crystallization from ethyl acetate
and petroleum ether (0.53 g, 60%), mp 164ꢀ165 ºC; Rf = 0.21 (petroleum ether : ethyl acetate,
2:1) ; 1H NMR (400 MHz, CDCl3) δ 1.01 (s, 3 H, 18ꢀCH3), 1.06 (s, 3 H, 19ꢀCH3), 3.91 (t, J=2.81
Hz, 1 H, 3βꢀH), 5.46 (d, J=5.14 Hz, 1 H, 6ꢀH), 5.93 ꢀ 6.01 (m, 1 H, 16ꢀH), 7.27 ꢀ 7.34 (m, 2 H,
Arꢀ5, 6ꢀHs), 7.46 ꢀ 7.52 (m, 1 H, Arꢀ7ꢀH), 7.78 ꢀ 7.84 (m, 1 H, Arꢀ4ꢀH), 7.97 (s, 1 H, Arꢀ2ꢀH);
13C NMR (101 MHz, CDCl3) δ 147.1, 143.2, 141.6, 138.5, 134.5, 124.1, 123.3, 122.5, 122.4,
120.2, 111.1, 58.1, 55.7, 50.2, 47.2, 37.3, 36.0, 34.8, 33.5, 31.0, 30.2, 30.2, 26.0, 20.3, 18.9,
16.0; HPLC: tR 7.02 min 98.16%; HRMS calcd 414.2652 (C26H31N5H+), found 414.2652.
3βꢀAzidoꢀ17ꢀ(1Hꢀbenzimidazoleꢀ1ꢀyl)androstaꢀ5,16ꢀdiene (8). To a solution of mesylate 4
(13.0 g, 27.8 mmol) in anhydrous DCM (100 mL) was added TMSN3 (55.5 mL, 418 mmol),
followed by BF3.OEt2 (71.2 mL, 753 mmol). The reaction was stirred at ~22 ºC for 5 h. When
starting material was completely consumed as evidenced by TLC (petroleum ether : ethyl
acetate, 2:1; Rf = 0.44 for product and Lewis acid complex), reaction was slowly poured into RB
flask on ice bath containing aqueous 2M NaOH (750 ml) and stirred at ~22 ºC for 6 h. When
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product is completely free of Lewis acid salt as evidenced by TLC, DCM from aqueous
suspension was evaporated. The resulting precipitate containing product and inorganic salts were
filtered and washed with water. The dry crude product then stirred with 75 mL chloroform and
filtered, residue washed with chloroform (75 mL). Combined filtrates evaporated to obtain pure
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product 8 (10.7 g, 93%), mp 136ꢀ137 ºC; Rf = 0.21 (petroleum ether : ethyl acetate, 2:1); H
NMR (400 MHz, CDCl3) δ 1.05 (s, 3 H, 18ꢀCH3), 1.08 (s, 3 H, 19ꢀCH3), 3.19 ꢀ 3.32 (m, 1 H, 3αꢀH),
5.48 (d, J=5.38 Hz, 1 H, 6ꢀH), 6.01 (dd, J=3.18, 1.71 Hz, 1 H, 16ꢀH), 7.29 ꢀ 7.37 (m, 2 H, Arꢀ5, 6ꢀHs),
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7.47 ꢀ 7.56 (m, 1 H, Arꢀ7ꢀH), 7.81 ꢀ 7.88 (m, 1 H, Arꢀ4ꢀH), 7.98 (s, 1 H, Arꢀ2ꢀH
)
; C NMR (101
MHz, CDCl3) δ 147.1, 143.2, 141.6, 140.2, 134.5, 124.0, 123.3, 122.4, 121.8, 120.2, 111.1, 61.0,
55.8, 50.4, 47.2, 38.1, 37.4, 36.8, 34.8, 31.0, 30.2, 27.8, 20.5, 19.2, 16.0; HPLC: tR 7.86 min
100%; HRMS calcd 414.2652 (C26H31N5H+), found 414.2653.
3αꢀAminoꢀ17ꢀ(1Hꢀbenzimidazoleꢀ1ꢀyl)androstaꢀ5,16ꢀdiene (9). A solution of compound 6a
(0.40 g, 0.97 mmol) and PPh3 (1.00 g, 3.97 mmol) in THF/MeOH/water (4/4/1 mL) was stirred
at 60 ºC for 10 h. Solvents evaporated under vacuo, residue reconstituted with DCM (25 mL) and
extracted with 1N HCl (20 mL x 2). Combined aqueous extracts washed with DCM, then
aqueous layer neutralized with 1N NaOH solution. Resulting solid filtered, washed with water
and dried under vacuo to obtain pure 9 (0.25 g, 67%), mp 104ꢀ106 ºC; Rf = 0.2
1
(DCM:MeOH:TEA,10:1:0.05); H NMR (400 MHz, CDCl3) δ 1.02 (s, 3 H, 18ꢀCH3), 1.07 (s, 3
H, 19ꢀCH3), 3.31 (br. s., 1 H, 3βꢀH), 5.47 (d, J=4.40 Hz, 1 H, 6ꢀH), 5.93 (br. s., 1 H, 16ꢀH), 7.26
ꢀ 7.31 (m, 2 H, Arꢀ5, 6ꢀHs), 7.45 ꢀ 7.51 (m, 1 H, Arꢀ7ꢀH), 7.76 ꢀ 7.83 (m, 1 H, Arꢀ4ꢀH), 7.95 (s, 1
H, Arꢀ2ꢀH); 13C NMR (101 MHz, CDCl3) δ 147.1, 143.2, 141.5, 138.4, 134.5, 123.8, 123.4,
123.3, 122.4, 120.1, 111.1, 55.7, 50.4, 47.2, 47.1, 38.8, 37.5, 34.8, 32.8, 31.1, 30.3, 30.2, 28.2,
18.8, 16.0; HPLC: tR 8.18 min 96.72%; HRMS calcd 388.2747 (C26H33N3H+), found 388.2747.
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ꢀAminoꢀ17ꢀ(1Hꢀbenzimidazoleꢀ1ꢀyl)androstaꢀ5,16ꢀdiene (10). A solution of compound 8
5
(9.00 g, 21.8 mmol) and PPh3 (22.9 g, 87.1 mmol) in THF/MeOH/water (145/145/36 mL) was
stirred at 60 ºC for 3 h. Solvents evaporated under vacuo, residue reconstituted with DCM (200
mL). To this solution under stirring added 1N HCl (100 mL) and stirred for 5 min to obtain
precipitate which collected by filtration (filtrate discarded). Solid product on Buchner funnel
made slurry with DCM (20 mL), filtered and suck dried. The second filtrate collected and
separately processed for second crop. The above solid product suspended in water (100 mL) and
neutralized with aq. NaOH solution (1.25 g in 25 mL). The product filtered, washed with water
(50 mL x 3), suck dried and further dried under oven at 45ꢀ50 ºC (7.36 g, 87%). For the second
crop, second filtrate containing DCM and aqueous phase was treated with fresh 1N HCl (10 mL).
The precipitate collected by filtration, solids treated with aq. NaOH solution, filtered and washed
with water, and suck dried (0.28g, 3%). Altogether 90% yield. mp 155ꢀ156 ºC; Rf = 0.2
(DCM:MeOH:TEA,10:1:0.05); 1H NMR (400 MHz, CDCl3) δ 1.02 (s, 3 H, 18ꢀC H3), 1.05 (s, 3 H,
6
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19ꢀCH3), 2.58 ꢀ 2.69 (m, 1 H,
3
αꢀH), 5.38 (d, J=4.89 Hz, 1 H, 6ꢀH), 5.98 (dd, J=2.93, 1.71 Hz, 1 H, 16ꢀ
13
H), 7.47 ꢀ 7.52 (m, 1 H, 7ꢀH), 7.78 ꢀ 7.85 (m, 1 H, Arꢀ4ꢀH), 7.96 (s, 1 H, , Arꢀ2ꢀH
)
; C NMR (101
MHz, CDCl3) δ 147.2, 143.2, 142.1, 141.6, 134.5, 124.0, 123.3, 122.4, 120.1, 120.0, 111.1, 55.9,
51.9, 50.6, 47.2, 43.2, 38.0, 36.7, 34.9, 32.4, 31.0, 30.3, 30.3, 20.6, 19.3, 16.0; HPLC: tR 8.26
min 97.47%; HRMS calcd 388.2747 (C26H33N3H+), found 388.2747.
3αꢀ(pꢀNitrophenylcarbonyloxy)ꢀ17ꢀ(1Hꢀbenzimidazoleꢀ1ꢀyl)androstaꢀ5,16ꢀdiene (11). To a
two neck flask was added glaterone (1, 2.00 g, 5.15 mmol), 0.95 g (5.66 mmol) of pꢀ
nitrobenzoicacid, 1.50 g (5.66 mmol) of PPh3 and 30 mL anhydrous THF. The mixture was
stirred until all solids dissolved and then cooled to 4 ºC in an iceꢀwater bath. A solution of 40%
DEAD 2.46 mL (5.66 mmol) in anhydrous toluene was added drop wise, allowed to attain room
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temperature, stirred for 12 h. The reaction mixture concentrated in vacuo and the resulting sticky
residue was suspended in 5 mL of ethyl acetate, stirred, cooled and filtered. Mother liquor
collected, stirred vigorously and 30 mL of petroleum ether added slowly. Resulting sticky
suspension filtered, washed with 10% ethyl acetate in petroleum ether (25 mL) and dried under
vacuo. The sticky solid made slurry on Buckner funnel with ether (7.5 mL x 2), filtered and dried
under vacuo. Free solids (0.8 g) obtained was a mixture of product 11, traces of
tripheylphosphinoxide and diethyl 1,2ꢀhydrazinedicarboxylate. The crude product dissolved in
hot IPA, allowed to cool to room temperature, resulting precipitate filtered and dried under vacuo
to obtain a white solid (0.50 g, 18%) of pure 11, mp 193ꢀ194 ºC; Rf = 0.4 (3% acetone in DCM);
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1H NMR (400 MHz, CDCl3) δ 1.05 (s, 3 H, 18ꢀCH3), 1.13 (s, 3 H, 19ꢀCH3), 5.31 (br. s., 1 H, 3
βꢀ
H), 5.39 (d, J=4.89 Hz, 1 H, 6ꢀH), 6.00 (br. s., 1 H, 16ꢀH), 7.28 ꢀ 7.34 (m, 2 H, Arꢀ5, 6ꢀHs), 7.48
ꢀ 7.52 (m, 1 H, Arꢀ7ꢀH), 7.80 ꢀ 7.85 (m, 1 H, Arꢀ4ꢀH), 7.97 (s, 1 H, Arꢀ2ꢀH), 8.17 (m, J=8.80 Hz,
2 H, Ar'ꢀ2, 6ꢀHs), 8.30 (m, J=8.80 Hz, 2 H, Ar'ꢀ3, 5ꢀHs); 13C NMR (101 MHz, CDCl3) δ 164.0,
150.4, 147.1, 141.6, 138.6, 136.4, 130.5, 124.1, 123.5, 123.4, 122.5, 122.0, 120.2, 111.1, 72.3,
55.8, 50.7, 47.2, 37.3, 36.4, 34.8, 33.9, 31.1, 30.3, 30.2, 26.2, 25.3, 20.4, 18.8, 16.0.
3αꢀHydroxyꢀ17ꢀ(1Hꢀbenzimidazoleꢀ1ꢀyl)androstaꢀ5,16ꢀdiene (12). Ester 11 (0.50 g, 0.93
mmol) was dissolved in THF/MeOH solvent mixture (2:1, 7.5 mL), and the resulting solution
was treated with 1N aq. NaOH solution (1.25 mL). The mixture was stirred at ~22 ºC for 2.5 h
and then solvents evaporated under vacuo at ~40 ºC. The residue solid treated with water,
filtered, washed with water and dried to afford 12 (0.33 g, 91%) as a white solid, mp 203 ºC; Rf
= 0.34 (DCM/ MEOH/TEA, 10:0.5:0.025); 1H NMR (400 MHz, CDCl3) δ 1.02 (s, 3 H, 18ꢀCH3),
1.07 (s, 3 H, 19ꢀCH3), 4.05 (br. s., 1 H, 3βꢀH), 5.47 (d, J=4.89 Hz, 1 H, 6ꢀH), 5.99 (br. s., 1 H,
16ꢀH), 7.28 ꢀ 7.38 (m, 2 H, Arꢀ5, 6ꢀHs), 7.50 (d, J=5.38 Hz, 1 H, Arꢀ7ꢀH), 7.77 ꢀ 7.88 (m, 1 H,
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Arꢀ4ꢀH), 7.97 (s, 1 H, Arꢀ2ꢀH); 13C NMR (101 MHz, CDCl3) δ 147.2, 143.2, 141.6, 139.1,
134.5, 124.1, 123.3, 123.1; 122.4, 120.2, 111.1, 67.0, 55.8, 50.6, 47.2, 39.8, 37.5, 34.8, 33.1,
31.1, 30.3, 30.2, 28.8, 20.3, 18.6, 16.0; HPLC: tR 1.89 min 99.28%; HRMS calcd 429.2587
(C26H32N2OH+), found 429.2587.
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ASSOCIATED CONTENT
Supporting Information
The Supporting is available free of charge on the ACS Publications website at DOI:
10.1021/acs.oprd.5b00394.
Full characterization (1H, 13C NMR and HRMS) for all compounds (intermediate and
final products) and HPLC chromatograms for final compounds (PDF).
AUTHOR INFORMATION
*Corresponding author: Dr. Vincent C. O. Njar, Eꢀmail: vnjar@som.umaryland.edu
Notes
Vincent C. O. Njar is the lead inventor of galeterone and new analogs, patents and technologies
thereof owned by the University of Maryland, Baltimore, and licensed to Tokai Pharmaceuticals,
Inc. Vincent Njar and Puranik Purushottamachar are coꢀinventors of all compounds described in
this report. A patent application to protect these novel improved synthetic procedures has been
filed, and the authors of this manuscript are coꢀinventors of the patent application.
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ACKNOWLEDGEMENTS
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This work was supported in part by a grant from NIH and NCI (R21CA195694) and startꢀup
funds from University of Maryland School of Medicine and the Center for Biomolecular
Therapeutics (CBT), and Marlene and Stewart Greenbaum Comprehensive Cancer Center
(Philanthropic Funds), Baltimore, USA and Sponsored Research grant from Tokai
Pharmaceuticals Inc., Boston, MA, USA to Professor Vincent C. O. Njar.
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