Design, chemical synthesis, and in vitro biological evaluation of simplified estradiol-adenosine hybrids as inhibitors of 17β-hydroxysteroid dehydrogenase type 1

Article abstract of DOI:10.1139/V09-083

A series of estradiol (E2) derivatives were designed to interact with, both the substrate- and the cofactor-binding sites of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1). These analogues of potent E2-adenosine hybrid inhibitor EM-1745, where the adenosine moiety was replaced by a more stable benzene derivative, were synthesized from estrone using alkene cross-metathesis and Sonogashira coupling reactions as key steps. In vitro biological evaluation of these steroid derivatives revealed that a spacer of 13 methylenes, between the 16β-position of E2 and the adenosine mimic bearing a carboxylic acid, group, gave the best inhibition of 17β-HSD1.

Full text of DOI:10.1139/V09-083

1180
Design, chemical synthesis, and in vitro biological
evaluation of simplified estradiolÀadenosine
hybrids as inhibitors of 17b-hydroxysteroid
dehydrogenase type 1
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Marie Berube and Donald Poirier
Abstract: A series of estradiol (E2) derivatives were designed to interact with both the substrate- and the cofactor-binding
sites of 17b-hydroxysteroid dehydrogenase type 1 (17b-HSD1). These analogues of potent E2Àadenosine hybrid inhibitor
EM-1745, where the adenosine moiety was replaced by a more stable benzene derivative, were synthesized from estrone
using alkene cross-metathesis and Sonogashira coupling reactions as key steps. In vitro biological evaluation of these ste-
roid derivatives revealed that a spacer of 13 methylenes, between the 16b-position of E2 and the adenosine mimic bearing
a carboxylic acid group, gave the best inhibition of 17b-HSD1.
Key words: 17b-hydroxysteroid dehydrogenase, enzyme, inhibitor, steroid, metathesis, Sonogashira coupling.
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Resume : Plusieurs derives de l’estradiol (E2) ont ete prepares pour interagir avec le site de liaison du substrat et le site
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de liaison du cofacteur de la 17b-hydroxysteroıde deshydrogenase type 1 (17b-HSD1). Ces analogues du puissant inhibi-
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teur EM-1745, un hybride E2Àadenosine dont la partie adenosine a ete remplacee par un derive benzylique plus stable,
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ont ete obtenus a partir de l’estrone en utilisant la metathese de Grubbs et le couplage de Sonogashira comme etapes cle.
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L’evaluation biologique in vitro de ces derives steroıdiens a montre qu’une chaıne ayant 13 groupes methylenes, entre la
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position 16b du steroıde E2 et le noyau benzylique porteur d’un acide carboxylique, provoquait la meilleure inhibition de
l’enzyme 17b-HSD1.
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Mots-cles : 17b-hydroxysteroıde deshydrogenase, enzyme, inhibiteur, steroıde, metathese, couplage de Sonogashira.
fact, E2 concentration has been found to be significantly
Introduction
higher in breast tumours than in normal breast tissue.12,13
Furthermore, the intratumoral E2 levels are high in postme-
nopausal patients with very low serum E2 levels; a study
aimed at explaining this revealed that the expression of
17b-HSD1 and the E2/E1 ratio are higher in breast cancer
tissues of postmenopausal patients than in those of preme-
nopausal ones.14 Such results suggest that the accumulation
of E2 in breast cancer tissue is mainly caused by the intra-
crine activity of 17b-HSD1. In fact, because E2 increases
breast-cancer-cell proliferation, inhibition of 17b-HSD1 ac-
tivity constitutes a good way to reduce tumour estrogen lev-
els, thus promoting tumour regression. Consequently, the
steroidogenic enzyme 17b-HSD1 is an interesting therapeu-
tic target for the treatment of estrogen-sensitive diseases
such as breast cancer.
17b-Hydroxysteroid dehydrogenase type 1 (17b-HSD1) or
human estradiol dehydrogenase [E.C.1.1.1.62] was partially
purified from human placenta by Langer and Engel in
1958.1 It is a 327-amino-acids protein that exists as a homo-
dimer. This enzyme preferentially catalyzes the transforma-
tion of estrone (E1) into estradiol (E2), the most potent
estrogen, using NAD(P)H as cofactor (Fig. 1),2À7 but also
catalyzes the reduction of dehydroepiandrosterone (DHEA)
into 5-androstene-3b,17bdiol (D5-diol).4 17b-HSD1 activities
have been found in many human tissues such as placenta,
ovary, endometrium, liver, and breast.8 In peripheral tissues
such as the breast, enzyme substrates come from circulating
DHEA-sulfate (DHEAS) or E1-sulfate (E1S), which are con-
verted into DHEA and E1 by steroid sulfatase.9À11
E2 and D5-diol have, respectively, high and weak affinity
for the estrogen receptor (ER). The resulting receptorÀestro-
gen complex produces estrogenic effects. Thus, in estro-
gen-dependent breast cancer, activation of ER by E2 is
crucial for the stimulation of growth of tumour cells. In
The design of potent 17b-HSD1 inhibitors has been at-
tempted during the past decades, but progress was not sig-
nificant enough to justify their use in the treatment of
estrogen-sensitive diseases.15À17 However, interesting clini-
Received 6 March 2009. Accepted 5 May 2009. Published on the NRC Research Press Web site at canjchem.nrc.ca on 30 July 2009.
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This article is dedicated to Dr. Robert H. Burnell, a retired professor from the Department of Chemistry at Laval University (Quebec
City, Canada) who passed away in November of 2008, for his creative contribution to the field of natural product synthesis and his
inspiring mentorship.
1
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M. Berube and D. Poirier. Laboratory of Medicinal Chemistry, CHUQ (CHUL)-Research Center and Laval University, Quebec City,
QC G1V 4G2, Canada.
1Corresponding author (e-mail: donald.poirier@crchul.ulaval.ca).
Can. J. Chem. 87: 1180–1199 (2009)
doi:10.1139/V09-083
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Fig. 1. Roles of 17b-HSD1 in the formation of estrogens E2 and D5-diol from precursor steroids E1 and DHEA.
Fig. 2. Main interactions (hydrogen bonds only) of the potent inhi-
bitor EM-1745 (1) with amino acids of 17b-HSD1. E2 and adeno-
sine moieties interact with the substrate- and the cofactor-binding
sites of the enzyme, respectively.
Fig. 3. Proposed simplified hybrid inhibitors of 17b-HSD1 (com-
pounds 2À18) and targeted main interactions with the enzyme.
cal success has been achieved in reducing mammary tu-
mours with inhibitors of aromatase, another enzyme in-
volved in the biosynthesis of E2.18À20 Based on three-
dimensional structures of 17b-HSD1 and on structureÀactiv-
ity relationship (SAR) studies, inhibitors that could interact
with both the substrate- and the cofactor-binding sites were
designed.21À23 EM-1745 (1) (Fig. 2), one of these
E2Àadenosine hybrid compounds, inhibited the transforma-
tion of E1 into E2 by 17b-HSD1 with an IC50 value of
52 nmol/L.22 Furthermore, EM-1745 (1) was crystallized in-
side the enzyme, and X-ray analysis confirmed that it inter-
acts with both the substrate- and the cofactor-binding sites.23
EM-1745 (1) is however a complex molecule. To become
a potential drug, it needs to be simplified to improve its in
vivo stability and enzyme inhibitory activity. Special care
needs to be taken for the ester link and the adenosine moi-
ety.24 With that in mind, simplified hybrid inhibitors were
then designed using the main interaction between EM-1745
(1) and the enzyme (compounds 2À18, Fig. 3). The E2 core
was kept for interacting with His221, Ser142, and Tyr155,
as well as with the hydrophobic residues that recognize the
steroid substrate. Adenosine mimics were designed keeping
in mind that the primary amino group of the adenosine tem-
plate forms a strong interaction with Asp65. Other function-
alities, such as an ester, a carboxylic acid, an alcohol, a
bromide, and a phosphotriester, were added on the aniline
core to potentially interact with Ser11 or other amino acids
in this area of the enzyme. The carboxylic acid and the
phosphotriester were chosen because they should interact in
a way similar to that of a phosphate group. In fact, the co-
factor NADPH has a better affinity than NADH for this en-
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Can. J. Chem. Vol. 87, 2009
Scheme 1. First chemical approach: synthesis of 27. Reagents, conditions, and yields: (a) MsCl, Et3N, DCM, 0 8C, 15 min (97%); (b) 2nd
generation Grubbs’ catalyst, DCM, reflux, 16 h (67%); (c) 10% Pd/C, H2, EtOAc, RT, 16 h; (d) NaI, acetone, reflux, 16 h; (e) DHP, p-TSA,
DCM, RT, 3 h (57% for three steps); (f) (i) CuI, vinylMgBr, THF, À40 8C, 15 min; (ii) HMPA, P(OEt)3, À40 8C to RT, 3 h (89%); (g)
tributyl(vinyl)tin, Pd(PPh3)2Cl2, toluene, 75 8C, 4 h (88%); (h) 2nd generation Grubbs’ catalyst, DCM, reflux, 16 h (34%). Mes: mesityl or
2,4,6-trimethylphenyl; Cy: cyclohexyl.
zyme, and the main structural difference between these two
cofactors is a phosphate group on the C2’ of the adenosine
moiety.25 Furthermore, the ester function in EM-1745 (1)
was removed and the E2 nucleus was joined to the adenosine
mimic by an alkyl chain, the length of which needs to be
optimized (n = 13À17). We report herein two chemical ap-
proaches developed to synthesize compounds 2À18. The in
vitro biological evaluation of compounds 2À18 as inhibitors
of 17b-HSD1 is also reported and discussed.
minal olefins,28 we applied another strategy for the synthesis
of 24. This terminal olefin was easily obtained by addition of
a vinyl cuprate on iodide 23 using hexamethylphosphoramide
(HMPA) and triethyl phosphite as additives.29
Synthesis of aniline derivative 25 was described in our
earlier report.30 Addition of a vinyl group on 25 using Stille
coupling procedure (tributyl(vinyl)tin, Pd(PPh3)2Cl2, toluene,
75 8C)31 afforded 26 in an excellent 88% yield. The next
step was to join by cross-metathesis the side chain of 24 to
the adenosine mimic 26 using the 2nd generation Grubbs’
catalyst. However, the bisubstrate compound 27 was ob-
tained in only 34% yield. Furthermore, the main product re-
covered after the reaction came from the metathesis of two
steroids 24 that was obtained in 47% yield. Because product
27 was only obtained in low yield and synthesis of starting
materials for this reaction necessitated numerous steps (nine
steps for the synthesis of 24 from commercially available es-
trone and six steps for the synthesis of 26 from 2,6-diiodo-4-
nitroaniline), a new synthetic strategy was developed.
This second synthetic approach for the preparation of sim-
plified hybrid inhibitors was reported by us earlier as a short
communication.30 Briefly, an olefinic aldehyde (Scheme 2),
used to elaborate the alkyl side-chain spacer, was joined to
steroid 19 through cross-metathesis (Scheme 3). The alde-
hyde was transformed into an alkyne, and a Sonogashira
coupling was used to link the steroid/spacer to the adenosine
mimic 25 in excellent yields.
Results and discussion
Chemistry
The first approach to synthesize simplified hybrid inhibi-
tors of 17b-HSD1 is presented in Scheme 1. The bisubstrate
compound 27 was obtained by two cross-metathesis reac-
tions26 used as key steps to join E2 and the adenosine mimic
represented by an aniline derivative. Briefly, 9-decenol (20)
was activated as the mesylate 21 using standard conditions
and then linked to diprotected 16b-allyl-E2 (19)27 using the
2nd generation Grubbs’ catalyst in refluxing dichloromethane
(DCM) to afford 22 in 67% yield. Formation of a side prod-
uct resulting from the metathesis of two molecules of steroid
19 was also observed. Hydrogenation of olefin 22 followed
by substitution of the mesylate by an iodide afforded 23.
Partial deprotection of the C17b-OTHP group however ne-
cessitated a subsequent protective step.
For the next step, our first idea was to obtain a terminal
olefin similar to 24 by dehydrohalogenation. However, no ter-
minal olefin was observed when 23 was treated with various
bases (DBU, KOH, and t-BuOK) under different conditions.
In fact, there are few dehydrohalogenation methodologies to
prepare unconjugated terminal olefins in good yields and
mild conditions. Although we later resolved this synthetic
problem by developing a new methodology for obtaining ter-
The first step in this approach was to synthesize spacers
of various lengths as outlined in Scheme 2. Depending on
the spacer length, the synthesis of aldehydes 37À39 started
from the commercially available olefin 33 or the commer-
cially available bromides 31 and 32. However, for the
synthesis of the aldehyde 40, no olefin or bromide with the
appropriate length were available from commercial sources.
Bromide 30 was then synthesized in a good 80% yield from
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Berube and Poirier
1183
Scheme 2. Second chemical approach: synthesis of spacers 37À40. Reagents, conditions, and yields: (a) 2nd generation Grubbs’ catalyst,
DCM, reflux, 72 h (80%); (b) NaI, acetone, reflux, 16 h; (c) (i) CuI, vinylMgBr, THF, À40 8C, 15 min; (ii) HMPA, P(OEt)3, À40 8C to RT,
3 h (60%À71% for two steps); (d) TPAP, NMO, molecular sieves, DCM, RT, 1 h (65%À94%).
the cross-metathesis of 4-bromobutene (28) and 10-undecenol
(29) with the 2nd generation Grubbs’ catalyst. Iodination of
bromides 30À32 using Finkelstein conditions followed by
addition of a vinyl cuprate as described above afforded
olefins 34À36 in 60%À71% yields for two steps. Alcohols
33À36 were next subjected to a tetrapropylammonium
perruthenate (TPAP) oxidation to gain aldehydes 37À40.
Scheme 3. Second chemical approach: synthesis of 49À52. Re-
agents, conditions, and yields: (a) 37À40, 2nd generation Grubbs’
catalyst, DCM, reflux, 16 h; (b) 10% Pd/C, H2, EtOAc, RT, 16 h;
(c) (i) LDA, TMSCHN2 (2.0 mol/L in hexanes), THF, À78 8C, 1 h;
(ii) reflux, 3 h (15%À33% for three steps); (d) 25, CuI, Et3N,
Pd(PPh3)4, benzene, 40 8C, 3 h (91%À95%).
The next steps, as illustrated in Scheme 3, were to join the
three components: the steroid 19, a spacer originating from
aldehydes 37À40, and the adenosine mimic 25. A cross-
metathesis of steroid 1927 and freshly prepared aldehydes
37À40 using the 2nd generation Grubbs’ catalyst provided
compounds 41À44. Hydrogenation of internal olefin(s)
catalyzed by palladium followed by treatment with lithium
diisopropylamide (LDA), (trimethylsilyl)diazomethane, and
heat afforded alkynes 45À48 in 15%À33% yields for three
steps.32 Finally, applying optimized Sonogashira coupling
procedure (Pd(PPh3)4, CuI, Et3N),33 alkynes 45À48 were
added to aryl iodide 25,30 giving 49À52 in excellent yields
(91%À95%).
Molecular diversity was next added on the adenosine
mimic moiety as described in Scheme 4. Depending on the
type of functional group introduced, two synthetic procedures
were applied. The first procedure was used to synthesize
ester, carboxylic acid, and alcohol derivatives. Hydrolysis of
the three protective groups (TBDMS, THP, and BOC) under
acid conditions, followed by hydrogenation of the alkyne,
provided ester derivatives 2À4 and 53 in 41%À66% yield
for two steps. To obtain the salt-free product, the crude pro-
ductÀNH2ÁHCl, resulting from the hydrolysis step, was
treated with a solution of Cs2CO3 in MeOH. Carboxylic acid
derivatives 5À8 and alcohol derivatives 9À11 were, respec-
tively, obtained from esters 2À4 and 53 by acid hydrolysis
or by hydride reduction. The second procedure was used to
prepare bromide and phosphotriester derivatives 12 and 13,
respectively. To avoid formation of side products, the bro-
mide and the phosphotriester needed to be introduced before
removal of protective groups. Thus, reduction of ester 49
with LiAlH4 afforded alcohol 54 in good yield of 80%. From
54, the bromide 55 was obtained by reaction with CBr4 and
PPh3 in DCM. Phosphotriester 56 was obtained by treatment
of alcohol 54 with CBr4 and P(OMe)3 in pyridine.34 Bromide
12 and phosphotriester 13 were finally obtained by hydroly-
sis of the three protective groups and hydrogenation of
alkyne as described above.
As described in the Biological activity section, the best
inhibitor of 17b-HSD1 obtained amongst compounds 2À13
is the carboxylic acid derivative 5 with an alkyl-side-chain
spacer of 13 methylenes. To improve its inhibitory potency,
new inhibitors 14À16 were designed by changing the dis-
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Can. J. Chem. Vol. 87, 2009
Scheme 4. Introduction of molecular diversity: synthesis of 2À13. Reagents, conditions, and yields: (a) (i) HCl (4 mol/L in dioxane), DCM,
MeOH, RT, 16 h; (ii) Cs2CO3, MeOH, 15 min; (b) 10% Pd/C, H2, EtOAc, RT, 16 h (41%À66% for two steps); (c) HCl (1 N in H2O), THF,
reflux, 2 to 5 h (58%À91%); (d) LiAlH4, THF, 0 8C, 4 to 16 h (45%À87%); (e) LiAlH4, THF, 0 8C to RT, 2 to 16 h (80%); (f) CBr4, PPh3,
DCM, 0 8C, 15 min to 4 h (70%); (g) CBr4, P(OMe)3, pyridine, RT, 3 h (82%); (h) HCl (4 mol/L in dioxane), DCM, MeOH, RT, 16 h;
(i) 10% Pd/C, H2, EtOAc, RT, 16 h (Bromide derivative: 25% for two steps); (j) 10% Pd/C, H2, MeOH, RT, 16 h (Phosphotriester deriva-
tive: 35% for two steps).
tance between the carboxylic acid and the aryl group (m =
0À3). Two compounds, 17 and 18, bearing only a carbox-
ylic acid or an amino group were also prepared to comple-
ment our SAR study.
with adenosine mimics 59, 61, and 64À66 afforded 67À71
in 66%À74% yields. Hydrolysis of protective groups
(TBDMS, THP, and BOC) in acid conditions, followed by
hydrogenation of the alkyne, afforded final compound 18
and esters 72À75, which after hydrolysis with LiOH in re-
fluxed acetone provided derivatives 14À17. Control com-
pound 79 that should only interact with the cofactor-binding
site was also prepared as outlined in Scheme 7. This com-
pound was synthesized easily using the same Sonogashira
coupling strategy described above for the preparation of
5À8 and 14À18. Final compounds 2À18 and 79, which
were needed for our SAR study, were carefully purified by
flash column chromatography and fully characterized by IR,
1H NMR, 13C NMR, and HR-MS to validate their structure.
First of all, the new adenosine mimic template had to be
synthesized (Scheme 5). Starting from 57,30 an intermediate
in the synthesis of adenosine mimic 25, ester 59 was syn-
thesized in three steps. Oxidative hydroboration of olefin
57 afforded alcohol 58 in 74% yield. Oxidation with Jones’
reagent, followed by esterification with (trimethylsilyl)dia-
zomethane in MeOH, provided ester 59 in 76% yield for
the two steps. Synthesis of ester 61 began with a cross-
metathesis between olefin 57 and methyl acrylate used in
large excess to obtain 60 in an excellent 87% yield. How-
ever, when hydrogenation catalyzed by Pd/C was used in
EtOAc or MeOH, reduction of the olefin was not observed.
Thus, hydrogenation catalyzed by PtO2 in anhydrous THF
was necessary to reduce the olefin and provide 61 in 93%
yield. Esters 64 (m = 0) and 65 (m = 1, no amino group)
were obtained by esterification of their corresponding car-
boxylic acids 6235,36 and 63.
Biological activity
The inhibition of the transformation of [14C]-E1
(0.1 mmol/L) into [14C]-E2 by compounds 2À18 and 79 was
evaluated with homogenated HEK-293 cells transfected with
a vector encoding for 17b-HSD1.37 NADH (0.1 mmol/L)
was added as cofactor, and the enzymatic assay was per-
formed at pH and temperature close to physiological condi-
tions (pH = 7.4 and T = 37 8C). The first series of simplified
hybrid inhibitors, compounds 2À13, were tested at two con-
centrations: 0.1 and 1 mmol/L, and the results are presented
The synthesis of carboxylic acid derivatives 14À17 and
aniline derivative 18 was achieved using a methodology
similar to that used for the synthesis of 5À8 as summarized
in Scheme 6. Briefly, a Sonogashira coupling of alkyne 45
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Berube and Poirier
1185
Scheme 5. Synthesis of new adenosine mimics 59, 61, 64, and 65.
Reagents, conditions, and yields: (a) (i) BH3ÁTHF, THF, 0 8C, 3 h;
(ii) NaOH (3 N in H2O), H2O2 (30% w/v), 2 h, RT (74%); (b)
Jones’ reagent, acetone, 0 8C, 90 min; (c) TMSCHN2 (2.0 mol/L in
hexanes), MeOH, 0 8C, 10 min (59: 76% for two steps, 64: 93%,
65: 99%); (d) methyl acrylate, 2nd generation Grubbs’ catalyst,
DCM, reflux, 16 h (87%); (e) H2, PtO2, THF, RT, 16 h (93%).
Scheme 6. Synthesis of 14À18. Reagents, conditions, and yields:
(a) CuI, Et3N, Pd(PPh3)4, benzene, 40 8C, 3 h (66%À74%); (b) HCl
(4 mol/L in dioxane), DCM, MeOH, RT, 2 h (for 70) or 16 h (for
67À69); (c) HCl (5% v/v in MeOH), DCM, RT, 1 h (for 71, 81%);
(d) 10% Pd/C, H2, EtOAc, RT, 16 h (72À75: 54%À75% for two
steps, 18: 67%); (e) LiOH (1 mol/L in H2O), acetone, reflux, 1 h
(66%À78%).
in Table 1 as percentage of 17b-HSD1 inhibition or IC50
values (the concentration that inhibited 50% of enzyme ac-
tivity).
Two points must be highlighted in these results. First, car-
boxylic acid derivatives 5À7 are by far the best inhibitors of
that series with percentages of inhibition varying from 88%
to 90% at 1 mmol/L. On the opposite, esters 2À4, alcohols
9À11, bromide 12, and phosphotriester 13 presented almost
no inhibition of 17b-HSD1. Second, varying the length of
the alkyl-side-chain spacer had almost no effect on the inhib-
ition of 17bHSD1. In fact, the inhibition of the enzyme by
carboxylic derivatives 5À7 (from 13 to 15 methylenes) var-
ied only from 88% to 90% at 1 mmol/L. To determine the
optimal spacer length, a compound with a longer side-chain
spacer (8, n = 17 methylenes) was prepared. As presented in
Table 1, compound 5, with an alkyl spacer of 13 methylenes,
is the best inhibitor with an IC50 of 56 nmol/L. Compounds
6À8, with spacers of 14, 15, or 17 methylenes, inhibit the
enzyme slightly less effectively compared with compound 5
with IC50 of 83, 99, and 108 nmol/L, respectively. In fact,
compound 5 is a better inhibitor than compounds 6À8 by
less than twofold. These biological results confirm that the
alkyl-side-chain spacer is very flexible and that its length
may vary to a certain extent. However, shortening it too
much would result in a huge drop of inhibition as was ob-
served in the SAR study carried out to develop hybrid inhib-
itor EM-1745 (1).22 In addition to its better inhibitory
potency, a spacer of 13 methylenes was chosen to continue
the SAR study because the synthesis of the aldehyde spacer
37 required only one synthetic step, as compared with three
steps for the 14- and 15-methylene spacers (38 and 39, re-
spectively) and four steps for the 17-methylene one (40).
As presented above, carboxylic acid derivatives 5À8 give
better inhibition than the other derivatives synthesized. To
explain this result, speculation on the main interactions be-
tween inhibitor 5 and 17b-HSD1 was attempted. Starting
from X-ray analysis of the crystallized EM-1745 (1)/17b-
HSD1 complex,23 we assumed that the E2 moiety of 5 would
form the same interactions with His221, Ser142, and Tyr155
as the E2 moiety of EM-1745 (1) (see Figs. 2 and 3). More-
over, the amino and the carboxylic acid groups of compound
5 should, respectively, interact with Asp65 and Ser11 found
in the cofactor-binding site, as observed for the adenosine
moiety of EM-1745 (1). Furthermore, 17b-HSD1 prefers co-
factor NADPH over NADH,25 and the structural difference
between these two entities is the presence of a phosphate
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Can. J. Chem. Vol. 87, 2009
Scheme 7. Synthesis of monosubstrate compound 79. Reagents, conditions, and yields (a) CuI, Et3N, Pd(PPh3)4, benzene, 40 8C, 1 h (88%);
(b) HCl (1 N in H2O), THF, reflux, 4 h (94%); (c) 10% Pd/C, H2, MeOH, RT, 16 h (75%).
Table 1. Inhibition of 17b-HSD1 by simplified hybrid inhibitors 2À13 and known inhibitors.
Inhibition (%)
Compound
R
n
At 0.1 mmol/L
At 1 mmol/L IC50 (nmol/L)
2
3
4
5
6
7
8
9
10
11
12
13
1
COOMe
COOMe
COOMe
COOH
COOH
COOH
COOH
CH2OH
CH2OH
CH2OH
CH2Br
13
14
15
13
14
15
17
13
14
15
13
13
—
—
—
0
0
14
26
45
88
90
90
Nd
6
Nd
Nd
Nd
56 3
83 8
99 31
108 5
Nd
Nd
Nd
Nd
Nd
12
22
40
42
Nd
6
2
4
22
11
0
95
Nd
19
16
33
12
98
Nd
57
CH2OPO3Me2
—
—
—
7.8 0.2
847 78
239 122
E1
EM-251
Note: Nd: not determined. For the transformation of [14C]-E1 (0.1 mmol/L) into [14C]-E2. The error for
screening assay was 10%. See Experimental section for more details.
group on the adenosine C2’. Accordingly, NADPH was also
To improve the interactions between the adenosine mimic
part of 5 and the enzyme, we next undertook the optimiza-
tion of the distance between the carboxylic acid (COOH)
and the aniline moiety. Compounds 14À16 were then syn-
thesized as described above (Schemes 5 and 6) with a spacer
of 0 to 3 methylenes between the COOH group and the aryl
moiety. Compounds 17 and 18, bearing only one of the
components, respectively, the carboxylic acid and the amino
group, were also prepared. Compounds 5 and 14À18 were
next tested to determine their potential to inhibit the 17b-
HSD1 activity at 0.1 and 1 mmol/L, and the results are pre-
sented in Table 2. Compounds 5 and 14À16 gave almost the
same percentages of inhibition with 22% to 29% and 89% to
crystallized with the enzyme, and X-ray analysis revealed
that the phosphate group forms its main interactions with
three amino acids: Ser11, Arg37, and Lys195.38 The disap-
pointing inhibitory activity obtained with the phosphate
derivative 13 suggests no interaction between this dimethy-
lated phosphate group and one of the three amino acids
identified by X-ray analysis (Ser11, Arg37, and Lys195).
Considering the best inhibition results provided by com-
pounds 5À8, the carboxylic acid probably interacts with at
least one of these amino acids. This hypothesis remains
however to be confirmed by docking studies, or better, by
crystallization of compound 5 with 17b-HSD1.
Published by NRC Research Press

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Berube and Poirier
1187
Table 2. Inhibition of 17b-HSD1 by simplified hybrid inhibitors 5 and 14À18
and known inhibitors.
Inhibition (%)
Compound
R
R’
At 0.1 mmol/L
At 1 mmol/L
5
14
15
16
17
18
79
1
NH2
NH2
NH2
NH2
H
NH2
—
—
CH2COOH
COOH
(CH2)2COOH
(CH2)3COOH
CH2COOHa
27
22
29
22
49
5
—
90
20
91
90
92
89
92
66
30
96
60
H
—
—
—
E1
—
Note: For the transformation of [14C]-E1 (0.1 mmol/L) into [14C]-E2. The screening assay
error was 10%. See experimental section for more details.
aInhibition of compound 17 is statistically different (P < 0.01) from inhibition of
compounds 5 and 14À16.
92% at 0.1 and 1 mmol/L, respectively. Thus, the distance
between the aryl group and the carboxylic acid did not af-
fect the inhibition of 17b-HSD1. Compound 18, which bears
no carboxylic acid, gave, as expected, lower inhibition with,
respectively, 5% and 66% at 0.1 and 1 mmol/L. However,
with only a carboxylic acid on the aryl (no amino group),
compound 17 gave a surprisingly higher inhibition with
49% and 92% at 0.1 and 1 mmol/L, respectively. This last
result reveals that the positions of the carboxylic acid and
the amino group are not optimal and, consequently, opens
the door to additional optimization studies.
However, when simplified hybrid inhibitors are compared
to the activity of EM-1745 (1), the inhibitory potency of car-
boxylic acid derivatives 5 and 14À17 was weaker at
0.1 mmol/L (22%À49% compared with 90% for 1). To ver-
ify that our best simplified hybrid inhibitors, compounds 5
and 14À17, kept their bisubstrate characteristics, compound
79, which should interact only with the cofactor-binding site
of the enzyme, was synthesized (Scheme 7) and tested as in-
hibitor of 17b-HSD1. The monosubstrate compound showed,
as expected, almost no inhibition (30% at 1 mmol/L). When
tested in the same conditions, the monosubstrate 16b-nonyl-
E2 gave the same results (no enzyme inhibition).22 Further-
more, unlabeled E1, which interacts only with the substrate-
binding site, gave lower inhibition (60% at 1 mmol/L) than
compounds 5 and 14À17 (89%À92% at 1 mmol/L). Accord-
ing to IC50 values (Table 1), inhibitor 5 (56 nmol/L) is only
seven times less potent than EM-1745 (7.8 nmol/L), but four
times more potent than EM-251 (239 nmol/L),39 a known in-
hibitor of 17b-HSD1, and 15 times more potent than unla-
beled E1. Thus, 5 and 14À17 cause more interactions with
the enzyme than E1, and they should interact with both the
substrate- and the cofactor-binding site of 17b-HSD1 as
EM-1745 (1).
Conclusion
Two chemical approaches were investigated in the prepara-
tion of simplified hybrid inhibitors of 17b-HSD1 represented
by compounds 2À18. The first approach uses two olefin
cross-metathesis reactions as key steps to join the steroid, the
spacer, and the adenosine mimic, but failed to give satisfac-
tory results because of the very low cross-metathesis yield
for the coupling of the spacer and the adenosine mimic. How-
ever, the second approach, using an olefin cross-metathesis
and a Sonogashira coupling as key steps, was more effective,
giving a better coupling yield of the spacer to the adenosine
mimic. Using this new approach, a series of steroid deriva-
tives (compounds 2À18) were synthesized as simplified
hybrid inhibitors of 17b-HSD1. These compounds featured
ester, carboxylic acid, alcohol, bromide, phosphotriester,
and (or) amine as functional groups to interact with the
cofactor-binding site, and an E2 nucleus to bind in the
substrate-binding site. Inhibitory potency of these compounds
was tested on homogenated HEK-293 cells overexpressing
17b-HSD1 activity. Carboxylic acid derivatives 5À8 and
14À17 were found to be the best bisubstrate inhibitors of
that group. A small effect on the inhibition was observed
when varying the length of the alkyl-side-chain spacer be-
tween the steroid and the adenosine mimic (n = 13, 14, 15,
and 17), but no effect was observed according to the length
of the alkyl chain between the aryl and the carboxylic acid
group (m = 0À3). The inhibition potency of carboxylic acid
derivatives 5À8 and 14À17 was weaker than the potency of
bisubstrate inhibitor EM-1745 (1), but higher than that of
known inhibitors that interact only with one binding site like
unlabeled E1, EM-251, and 79. In addition, even if com-
pounds 5À8 and 14À17 are less potent than EM-1745, they
should be more stable, since they have no ester group and no
Published by NRC Research Press

1188
Can. J. Chem. Vol. 87, 2009
ribose moiety. Furthermore, a better inhibition of 17b-HSD1
was surprisingly observed with compound 17 that contains
only a carboxylic acid group (no amine) on the adenosine
mimic. This result suggests that better hybrid inhibitors than
17 could be obtained with the optimization of the position of
the carboxylic acid and the amino groups on the adenosine
mimic. Docking studies using 3D structure of crystallized
EM-1745/17b-HSD1 complex will be undertaken to achieve
this task.
were added Et3N (1.2 mL, 8.4 mmol) and methanesulfonyl
chloride (0.48 mL, 6.2 mmol). The mixture was stirred for
15 min at 0 8C and extracted with DCM. The organic phase
was washed with an aqueous solution of HCl (1 N) and
brine, dried over MgSO4, filtered, and evaporated to dry-
ness. Purification by flash chromatography (hexanes/EtOAc,
8:2) afforded 21 (1.27 g, 97% yield) as a colourless oil. IR
(neat) n: 3075 (CÀH, alkene), 1640 (C=C, alkene), 1355
1
and 1176 (O=S=O). H NMR (400 MHz, CDCl3) d: 1.34
(m, 5 Â CH2), 1.73 (m, CH2CH2OS), 2.02 (m, CH2CH=CH2),
2.99 (s, CH3), 4.20 (t, J = 6.6 Hz, CH2OS), 4.95
(m, CH=CH2), 5.79 (m, CH=CH2). 13C NMR (100 MHz,
CDCl3) d: 25.3, 28.7, 28.8 (2Â), 29.0, 29.1, 33.6, 37.2,
70.1, 114.1, 139.0.
Experimental
General methods
Reagents were obtained from Sigma-Aldrich Canada Co.
(Oakville, ON, Canada) except for 3-iodophenylacetic acid
purchased from Matrix Scientific (Colombia, SC, USA). The
tricyclohexylphosphine [1,3-bis(2,4,6-trimethylphenyl)-4,5-
dihydroimidazol-2-ylidene] [benzylidine] ruthenium(IV)di-
chloride (2nd generation Grubbs’ catalyst) was purchased
from Strem Chemicals (Newburyport, MA, USA) and Sigma-
Aldrich Canada Co. (Oakville, ON, Canada). Usual solvents
were obtained from Fisher Scientific and VWR (Ville Mont-
Royal, QC, Canada) and were used as received. Anhydrous
solvents were purchased from Aldrich and VWR in SureSeal
bottles, which were kept under positive argon pressure. Tetra-
hydrofuran (THF) was distilled from sodium/benzophenone
under argon. All anhydrous reactions were performed under
positive argon pressure in oven-dried glassware. Thin-layer
chromatography (TLC) was performed on 0.25 mm silica gel
60 F254 plates (Whatman, Maidstone, UK), and compounds
were visualized by exposure to UV light (254 nm) and (or)
with a solution of ammonium molybdate/sulphuric acid/water
(with heating). Flash chromatography was performed on Sili-
cycle 60 (Quebec, QC, Canada) 230À400 mesh silica gel. IR
spectra were obtained neat, from KBr pellets or from a thin
film of the solubilized compound on NaCl pellets (usually in
CH2Cl2). They were recorded on a PerkinElmer series 1600
FTIR spectrophotometer (Norwalk, CT, USA); only signifi-
Synthesis of 9-undecen-11-[3’-(tert-butyldimethylsilyloxy)-
17’b-(tetrahydro-2H-pyran-2-yl-oxy)-estra-1’,3’,5’(10’)-
trien-16’b-yl)-methanesulfonate (22)
A mixture of 16b-allyl-estradiol (19)27 (3.00 g, 5.87 mmol),
9-decenyl methanesulfonate (21) (2.75 g, 11.7 mmol), and
2nd generation Grubbs’ catalyst (500 mg, 0.587 mmol) in
dry DCM (100 mL) was refluxed for 16 h under an argon
atmosphere. The crude mixture was preadsorbed on silica
gel, and a flash chromatography was performed (hexanes/
EtOAc, 9:1 to 8:2) to afford 22 (2.83 g, 67%) as a viscous
yellowish oil. IR (film) n: 1352 and 1174 (O=S=O). 1H
NMR (400 MHz, CDCl3) d: 0.18 (s, Si(CH3)2), 0.79 and
0.84 (2s, 18’-CH3), 0.80 to 2.35 (CH and CH2 of steroid
skeleton and alkyl chain), 0.97 (s, SiC(CH3)3), 2.79 (m, 6’-
CH2), 3.01 (s, CH3SO), 3.51 and 3.95 (2m, OCH2 of THP),
3.72 and 3.79 (2d, J = 10.0 Hz, 17’a-CH), 4.23 (t, J =
6.6 Hz, CH2SO), 4.64 and 4.74 (2m, CH of THP), 5.38 (m,
CH=CH), 6.55 (s, 4’-CH), 6.61 (d, J = 8.2 Hz, 2’-CH), 7.11
(m, 1’-CH). LR-MS calculated for C41H69O6SSi [M + H]+
717.5, found 717.2 m/z.
Synthesis of 13-[3’-(tert-butyldimethylsilyloxy)-17’b-
(tetrahydro-2H-pyran-2-yl-oxy)-estra-1’,3’,5’(10’)-trien-
16’b-yl)-tridecene (24)
1
cant bands are reported (in cmÀ1). H and 13C NMR spectra
were recorded with a Bruker AC/F 300 spectrometer
(Billerica, MA, USA) at 300 and 75 MHz, respectively, and a
Bruker AVANCE 400 spectrometer at 400 (1H) and 100 (13C)
MHz. The chemical shifts (d) are expressed in ppm and refer-
enced to chloroform (7.26 and 77.0 ppm), acetone (2.07 and
Hydrogenation
A suspension of steroid 22 (2.76 g, 3.85 mmol) and 10%
Pd/C (414 mg) in EtOAc (150 mL) was stirred at room tem-
perature under hydrogen atmosphere. After 16 h, the result-
ing suspension was filtered through a pad of Celite, and
EtOAc was evaporated to dryness. H NMR analysis of the
crude mixture revealed the disappearance of alkene protons
that were at 5.38 ppm.
1
206.0 ppm), or methyl sulfoxide (2.51 and 39.5 ppm) for H
1
1
and 13C, respectively. Duplication of H NMR signals was
observed for all THP derivatives (presence of two stereoisom-
ers). Low-resolution mass spectra (LR-MS) were recorded
with an LCQ Finnigan apparatus (San Jose, CA, USA)
equipped with an atmospheric pressure chemical ionization
(APCI) source on positive or negative mode. High-resolution
mass spectra (HR-MS) were provided by the Regional Labo-
Iodination
The crude product (2.72 g, 3.78 mmol) was dissolved in
acetone (50 mL), and NaI (1.98 g, 13.2 mmol) was added.
The reaction mixture was refluxed for 16 h. The product
was then extracted with EtOAc, and the organic phase was
washed with a saturated aqueous solution of Na2S2O3 and
washed with brine, dried over MgSO4, filtered, and evapo-
rated to dryness. Flash chromatography (hexanes/EtOAc,
95:5) afforded a mixture of 23 and 17b-OH form of 23 that
was protected again as a THP derivative (dihydropyran, p-
TSA, DCM, RT, 3 h) using a previously described proce-
dure.27 Then, 23 (2.12 g, 57% yield for three steps) was ob-
tained as a white solid.
´
´
ratory for Instrumental Analysis (Universite de Montreal,
´
Montreal, QC, Canada).
Synthesis of compound 27 using the first chemical
approach
Synthesis of 9-decenyl methanesulfonate (21)
To a solution of 9-decen-1-ol (1.0 mL, 5.6 mmol) in an-
hydrous DCM (30 mL) under an argon atmosphere at 0 8C
Published by NRC Research Press

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Berube and Poirier
1189
Vinylation
(300 MHz, CDCl3) d: 0.18 (s, Si(CH3)2), 0.79 and 0.83 (2s,
18@’-CH3), 0.80 to 2.30 (CH and CH2 of steroid skeleton
and alkyl chain), 0.97 (s, SiC(CH3)3), 1.51 (s, (CH3)3C of N-
BOC), 2.78 (m, 6@’-CH2), 3.52 and 3.96 (2m, OCH2 of THP),
3.57 (s, CH2COOCH3), 3.68 (s, COOCH3), 3.71 and 3.79
(2d, J = 10.2 Hz, 17@’a-CH), 4.62 and 4.75 (2m, CH of
THP), 6.26 (m, CH=CH), 6.48 (s, NH), 6.55 (s, 4@’-CH),
6.60 (d, J = 8.3 Hz, 2@’-CH), 6.93 (s, 6’-CH), 7.12 (m, 1@’-
CH, 2’-CH), 7.29 (s, 4’-CH). LR-MS calculated for
C56H86NO4Si [M À H]À 912.6, found 912.5 m/z.
CuI (484 mg, 2.54 mmol) was suspended in anhydrous
THF (5 mL) under an argon atmosphere. The mixture was
cooled to À40 8C and vinylmagnesium bromide (1 mol/L in
THF) (7.63 mL, 7.63 mmol) was added. The reaction was
stirred for 15 min at À40 8C. Then, HMPA (0.880 mL,
5.08 mmol) and triethyl phosphite (0.880 mL, 5.08 mmol)
were added, and the mixture was stirred for 5 min
at À40 8C. 23 (1.91 g, 2.54 mmol) was added, and the reac-
tion mixture was stirred for 1 h at À40 8C and for 2 h at
room temperature. The reaction was quenched by the addi-
tion of a saturated solution of NH4Cl. The crude product
was extracted with EtOAc, washed with brine, dried over
MgSO4, filtered, and evaporated under reduced pressure.
Purification by flash chromatography (hexanes/EtOAc,
95:5) yielded 24 (1.48 g, 89% yield) as a colourless viscous
Synthesis of spacers 37À40
Synthesis of 13-bromo-10-tridecenol (30)
A mixture of 4-bromobutene (28) (3.0 mL, 29.6 mmol),
10-undecenol (29) (23.7 mL, 118 mmol), and the 2nd genera-
tion Grubbs’ catalyst (1.25 g, 1.48 mmol) in dry DCM
(200 mL) was refluxed for 3 days under an argon atmos-
phere. The crude mixture was purified by flash chromatogra-
phy (hexanes/EtOAc, 8:2) to afford 30 (6.46 g, 80% yield) as
a colourless oil. IR (neat) n: 3365 (OH). 1H NMR (400 MHz,
CDCl3) d: 1.25 (m, 6 Â CH2), 1.53 (m, CH2CH2OH), 1.97
(m, CH2CH2CH=CH), 2.02 (s, OH), 2.51 (q, J = 7.0 Hz,
CH2CH2Br), 3.34 (m, CH2Br), 3.59 (m, CH2OH), 5.35 and
5.50 (2m, CH=CH). 13C NMR (75 MHz, CDCl3) d: 25.6,
28.8, 29.0, 29.2, 29.3, 29.5, 32.4, 32.6, 32.9, 35.9, 62.8,
126.3, 133.9. LR-MS calculated for C13H26BrO [M + H]+
277.1 and 279.1, found 276.9 and 278.9 m/z.
1
oil. IR (film) n: 1640 (C=C, alkene). H NMR (300 MHz,
CDCl3) d: 0.18 (s, Si(CH3)2), 0.79 and 0.83 (2s, 18’-CH3),
0.80 to 2.35 (CH and CH2 of steroid skeleton and alkyl
chain), 0.97 (s, SiC(CH3)3), 2.79 (m, 6’-CH2), 3.52 and 3.95
(2m, OCH2 of THP), 3.72 and 3.79 (2d, J = 9.6 Hz, 17’a-
CH), 4.62 and 4.75 (2m, CH of THP), 4.97 (m, CH2=CH),
5.81 (m, CH=CH2), 6.55 (d, J = 2.2 Hz, 4’-CH), 6.60 (d, J =
8.3 Hz, 2’-CH), 7.11 (m, 1’-CH). LR-MS calculated for
C42H71O3Si [M + H]+ 651.5, found 651.3 m/z.
Synthesis of methyl 2-(3’-tert-butoxycarbonylamino-5’-
vinylphenyl)-acetate (26)
To a solution of 2530 (800 mg, 2.04 mmol) in anhydrous
toluene (68 mL) under an argon atmosphere were added
tributyl(vinyl)tin (1.8 mL, 6.1 mmol) and Pd(PPh3)2Cl2
(143 mg, 0.204 mmol). The reaction was stirred for 4 h at
75 8C and quenched by addition of H2O. The product was
extracted with EtOAc, and the organic phase was washed
with brine, dried over MgSO4, filtered, and evaporated to
dryness. The crude product was purified by flash chromatog-
raphy (hexanes/EtOAc, 9:1) to gain 26 (520 mg, 88% yield)
as a yellowish oil. IR (film) n: 3344 (NH, carbamate), 1728
General procedure to synthesize 34À36
The iodination and vinylation procedures described above
for the synthesis of 24 were used without modification ex-
cept that 5 and 4 equiv. of vinylmagnesium bromide were
used for the synthesis of 35 and 36, respectively, instead of
3 equiv. It is noteworthy that alcohol 33 is commercially
available.
11-Dodecenol (34)
1
(C=O, ester and carbamate). H NMR (300 MHz, acetone-
We reported earlier the synthesis and characterization of
d6) d: 1.50 (s, (CH3)3C of N-BOC), 3.62 (s, CH2COOCH3),
3.65 (s, COOCH3), 5.24 (d, J = 11.1 Hz, 1H of CH=CH2),
5.78 (dd, J1 = 17.6 Hz, J2 = 1.1 Hz, 1H of CH=CH2), 6.72
(dd, J1 = 17.6 Hz, J2 = 10.9 Hz, CH=CH2), 7.05 (s, 6’-CH),
7.45 (s, 2’-CH), 7.59 (s, 4’-CH), 8.44 (s, NH). 13C NMR
(75 MHz, acetone-d6) d: 28.3 (3Â), 41.2, 51.8, 79.8, 114.2,
115.1, 119.3, 122.0, 136.1, 137.5, 138.8, 140.8, 153.5,
171.9. LR-MS calculated for C16H20NO4 [M À H]À 290.1,
found 290.2 m/z.
34.40
12-Tridecenol (35)
Colourless oil (4.24 g, 71% yield for two steps). IR (neat)
n: 3355 (OH), 1641 (C=C, alkene). 1H NMR (400 MHz,
CDCl3) d: 1.26 (m, 8 Â CH2), 1.55 (m, CH2CH2OH), 2.02
(m, CH2CH=CH2), 3.62 (t, J = 6.6 Hz, CH2OH), 4.95 (m,
CH=CH2), 5.80 (m, CH=CH2). 13C NMR (75 MHz, CDCl3)
d: 25.7, 28.9, 29.1, 29.4, 29.5 (4Â), 32.7, 33.8, 63.0, 114.0,
139.2.
Synthesis of methyl 2-{3’-(tert-butoxycarbonylamino)-5’-
[13@-(3’@-(tert-butyldimethylsilyloxy)-17@’b-(tetrahydro-2H-
pyran-2-yl-oxy)-estra-1@’,3’@,5’@(10@’)-trien-16@’b-yl)-
tridecenyl]-phenyl}-acetate (27)
A mixture of steroid 24 (860 mg, 1.32 mmol), adenosine
mimic 26 (500 mg, 1.71 mmol), and 2nd generation Grubbs’
catalyst (112 mg, 0.132 mmol) in dry DCM (20 mL) was re-
fluxed for 16 h under an argon atmosphere. The solvent was
evaporated, and the crude product was purified by flash
chromatography (hexanes/EtOAc, 97:3 to 93:7) to afford 27
(413 mg, 34% yield) as a white foam. IR (film) n: 3364 (NH,
carbamate), 1732 (C=O, ester and carbamate). 1H NMR
Pentadeca-10,14-dien-1-ol (36)
Colourless oil (3.65 g, 70% yield for two steps). IR (neat)
n: 3332 (OH), 1641 (C=C, alkene). 1H NMR (400 MHz,
CDCl3) d: 1.26 (m, 6 Â CH2), 1.54 (m, CH2CH2OH), 1.90
to 2.10 (m, 3 Â CH2CH=CH2 and OH), 3.60 (t, J = 7.4 Hz,
CH2OH), 4.97 (m, CH=CH2), 5.39 (m, CH=CH), 5.80 (m,
CH=CH2). 13C NMR (75 MHz, CDCl3) d: 25.7, 29.1, 29.4
(2Â), 29.5 (2Â), 31.9, 32.5, 32.7, 33.8, 62.8, 114.4, 129.3,
130.8, 138.4. LR-MS calculated for C15H29O [M + H]+
225.2, found 225.1 m/z.
Published by NRC Research Press

1190
Can. J. Chem. Vol. 87, 2009
General procedure to synthesize 37À40
6’-CH2), 3.48 and 3.92 (2m, OCH2 of THP), 3.74 and 3.80
(2d, J = 10.0 Hz, 17’a-CH), 4.63 and 4.73 (2m, CH of
THP), 6.58 (d, J = 2.4 Hz, 4’-CH), 6.64 (d, J = 8.5 Hz, 2’-
CH), 7.15 and 7.17 (2d, J = 5.7 Hz, 1’-CH). LR-MS calcu-
lated for C44H73O3Si [M + H]+ 677.5, found 677.4 m/z.
The TPAP oxidation procedure that we described previ-
ously for the synthesis of 10-undecenal (37)30 was used
without modification.
11-Dodecenal (38)
17-[3’-(tert-butyldimethylsilyloxy)-17’b-(tetrahydro-2H-pyran-
2-yl-oxy)-estra-1’,3’,5’(10’)-trien-16’b-yl]-heptadecyne (48)
Colourless viscous oil (636 mg, 24% yield for three steps).
We reported earlier the synthesis and characterization of
38.40
12-Tridecenal (39)
1
IR (film) n: 3313 (CÀH, alkyne). H NMR (400 MHz, ace-
Colourless oil (2.66 g, 67% yield). IR (neat) n: 2713
(CÀH, aldehyde), 1728 (C=O, aldehyde), 1641 (C=C, al-
tone-d6) d: 0.20 (s, Si(CH3)2), 0.82 and 0.87 (2s, 18’-CH3),
1.00 (s, SiC(CH3)3), 0.90 to 2.35 (CH and CH2 of steroid
skeleton and alkyl chain), 2.18 (m, CH2C:C), 2.33 (t, J =
2.6 Hz, C:CÀH), 2.81 (m, 6’-CH2), 3.48 and 3.92 (2m,
OCH2 of THP), 3.75 and 3.80 (2d, J = 10.0 Hz, 17’a-CH),
4.64 and 4.73 (2m, CH of THP), 6.58 (d, J = 2.3 Hz, 4’-
CH), 6.64 (d, J = 8.4 Hz, 2’-CH), 7.15 and 7.17 (2d, J =
5.6 Hz, 1’-CH). LR-MS calculated for C46H77O3Si [M + H]+
705.6, found 705.5 m/z.
1
kene). H NMR (300 MHz, CDCl3) d: 1.25 (m, 7 Â CH2),
1.60 (m, CH2CH2CHO), 2.02 (m, CH2CH=CH2), 2.41 (dt,
J1 = 7.4 Hz, J2 = 1.6 Hz, CH2CHO), 4.95 (m, CH=CH2),
5.78 (m, CH=CH2), 9.74 (t, J = 1.6 Hz, CHO). 13C NMR
(75 MHz, CDCl3) d: 22.0, 28.9, 29.1 (2Â), 29.3, 29.4 (2Â),
29.5, 33.8, 43.9, 114.1, 139.2, 203.0.
Pentadeca-10,14-dien-1-al (40)
Colourless oil (2.61 g, 74% yield). IR (neat) n: 2715
(CÀH, aldehyde), 1728 (C=O, aldehyde), 1640 (C=C, al-
General procedure to synthesize 49À52
The procedure that we described previously for the synthe-
sis of 4930 was used without modification for the synthesis of
50À52. The chemical synthesis of 25 was also described in
this earlier report.
1
kene). H NMR (400 MHz, CDCl3) d: 1.28 (m, 6 Â CH2),
1.61 (m, CH2CH2CHO), 1.90 to 2.10 (m, 3 Â CH2CH=),
2.41 (dt, J1 = 7.4 Hz, J2 = 1.6 Hz, CH2CHO), 4.97 (m,
CH=CH2), 5.40 (m, CH=CH), 5.80 (m, CH=CH2), 9.75 (t, J
= 1.6 Hz, CHO). 13C NMR (75 MHz, CDCl3) d: 22.0, 29.0,
29.1, 29.2, 29.3, 29.5, 32.0, 32.5, 33.8, 43.9, 114.4, 129.4,
130.8, 138.5, 202.9.
Methyl 2-{3’-(tert-butoxycarbonylamino)-5’-[14@-(3’@-(tert-
butyldimethylsilyloxy)-17@’b-(tetrahydro-2H-pyran-2-yl-
oxy)-estra-1@’,3’@,5’@(10@’)-trien-16@’b-yl)-tetradecynyl]-
phenyl}-acetate (50)
Synthesis of 49À52 using the second chemical approach
Viscous yellowish oil (540 mg, 61% yield (non-opti-
mized)). IR (film) n: 3354 (NH, carbamate), 1732 (C=O, es-
General procedure to synthesize 45À48
1
ter and carbamate). H NMR (400 MHz, acetone-d6) d: 0.20
The procedure that we described previously for the syn-
thesis of 45 from 3730 was used without modification to pre-
pare alkynes 46, 47, and 48, from aldehydes 38, 39, and 40,
respectively.
(s, Si(CH3)2), 0.82 and 0.86 (2s, 18@’-CH3), 1.00 (s,
SiC(CH3)3), 1.50 (s, (CH3)3C of N-BOC), 1.00 to 2.35 (CH
and CH2 of steroid skeleton and alkyl chain), 2.42 (t, J =
6.9 Hz, CH2ÀC:C), 2.81 (m, 6@’-CH2), 3.47 and 3.92 (2m,
OCH2 of THP), 3.60 (s, CH2COOMe), 3.66 (s, COOCH3),
3.74 and 3.79 (2d, J = 10.0 Hz, 17@’a-CH), 4.63 and 4.73
(2m, CH of THP), 6.58 (d, J = 2.0 Hz, 4@’-CH), 6.64 (d, J =
8.4 Hz, 2@’-CH), 6.97 (s, 6’-CH), 7.16 (m, 1@’-CH), 7.45 (s,
2’-CH), 7.58 (s, 4’-CH), 8.46 (s, NH). LR-MS calculated for
C57H86NO7Si [M À H]À 924.6, found 924.5 m/z.
14-[3’-(tert-butyldimethylsilyloxy)-17’b-(tetrahydro-2H-
pyran-2-yl-oxy)-estra-1’,3’,5’(10’)-trien-16’b-yl]-tetradecyne
(46)
Colourless viscous oil (801 mg, 15% yield for three steps).
1
IR (film) n: 3313 (CÀH, alkyne). H NMR (300 MHz, ace-
tone-d6) d: 0.20 (s, Si(CH3)2), 0.82 and 0.86 (2s, 18’-CH3),
0.99 (s, SiC(CH3)3), 0.80 to 2.35 (CH and CH2 of steroid
skeleton and alkyl chain), 2.18 (dt, J1 = 6.8 Hz, J2 = 2.5 Hz,
CH2C:C), 2.33 (t, J = 2.7 Hz, C:CÀH), 2.83 (m, 6’-CH2),
3.47 and 3.95 (2m, OCH2 of THP), 3.74 and 3.79 (2d, J =
10.0 Hz, 17’a-CH), 4.63 and 4.73 (2m, CH of THP), 6.58 (s,
4’-CH), 6.63 (d, J = 8.8 Hz, 2’-CH), 7.15 and 7.17 (2d, J =
8.4 Hz, 1’-CH). LR-MS calculated for C43H71O3Si [M + H]+
663.5, found 663.3 m/z.
Methyl 2-{3’-(tert-butoxycarbonylamino)-5’-[15@-(3’@-(tert-
butyldimethylsilyloxy)-17@’b-(tetrahydro-2H-pyran-2-yl-
oxy)-estra-1@’,3’@,5’@(10@’)-trien-16@’b-yl)-pentadecynyl]-
phenyl}-acetate (51)
White foam (1.41 g, 95% yield). IR (film) n: 3353 (NH,
carbamate), 1732 (C=O, ester and carbamate). 1H NMR
(400 MHz, acetone-d6) d: 0.20 (s, Si(CH3)2), 0.82 and 0.86
(2s, 18@’-CH3), 1.00 (s, SiC(CH3)3), 1.50 (s, (CH3)3C of N-
BOC), 1.00 to 2.35 (CH and CH2 of steroid skeleton and
alkyl chain), 2.42 (t, J = 7.0 Hz, CH2ÀC:C), 2.81 (m, 6@’-
CH2), 3.48 and 3.92 (2m, OCH2 of THP), 3.60
(s, CH2COOMe), 3.66 (s, COOCH3), 3.73 and 3.79 (2d, J =
10.0 Hz, 17@’a-CH), 4.63 and 4.73 (2m, CH of THP), 6.58
(d, J = 2.2 Hz, 4@’-CH), 6.64 (d, J = 8.4 Hz, 2@’-CH), 6.97
(s, 6’-CH), 7.16 (m, 1@’-CH), 7.45 (s, 2’-CH), 7.58 (s, 4’-CH),
8.48 (s, NH). LR-MS calculated for C58H88NO7Si [M À H]À
938.6, found 938.5 m/z.
15-[3’-(tert-butyldimethylsilyloxy)-17’b-(tetrahydro-2H-
pyran-2-yl-oxy)-estra-1’,3’,5’(10’)-trien-16’b-yl]-pentadecyne
(47)
Colourless viscous oil (1.36 g, 33% yield for three steps).
1
IR (film) n: 3312 (CÀH, alkyne). H NMR (400 MHz, ace-
tone-d6) d: 0.20 (s, Si(CH3)2), 0.82 and 0.87 (2s, 18’-CH3),
1.00 (s, SiC(CH3)3), 0.90 to 2.35 (CH and CH2 of steroid
skeleton and alkyl chain), 2.18 (dt, J1 = 6.8 Hz, J2
=
2.6 Hz, CH2C:C), 2.33 (t, J = 2.6 Hz, C:C-H), 2.81 (m,
Published by NRC Research Press

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Berube and Poirier
1191
Methyl 2-{3’-(tert-butoxycarbonylamino)-5’-[17@-(3’@-(tert-
butyldimethylsilyloxy)-17@’b-(tetrahydro-2H-pyran-2-yl-
oxy)-estra-1@’,3’@,5’@(10@’)-trien-16@’b-yl)-heptadecynyl]-
phenyl}-acetate (52)
4.53 (s, NH2), 6.37 (s, 6’-CH), 6.42 (s, 2’-CH and 4’-CH),
6.54 (d, J = 2.6 Hz, 4@’-CH), 6.60 (dd, J1 = 8.4 Hz, J2 =
2.6 Hz, 2@’-CH), 7.11 (d, J = 8.4 Hz, 1@’-CH), 7.97 (s, 3’@-
OH). 13C NMR (100 MHz, acetone-d6) d: 13.0, 27.1, 28.2,
29.1 to 30.3 (12C under solvent peaks), 30.6, 32.0, 32.5,
33.2, 36.4, 38.6, 39.4, 41.2, 41.5, 44.79, 44.82, 49.5, 51.7,
82.2, 113.4, 113.8, 115.8, 118.7, 124.5, 126.8, 132.0, 135.6,
138.2, 144.3, 149.1, 155.7, 172.2. HR-MS calculated for
C42H64NO4 [M + H]+ 646.48299, found 646.48402 m/z.
Yellowish foam (219 mg, 91% yield). IR (film) n: 3356
(NH, carbamate), 1736 (C=O, ester and carbamate). 1H NMR
(400 MHz, acetone-d6) d: 0.20 (s, Si(CH3)2), 0.82 and 0.86
(2s, 18@’-CH3), 1.00 (s, SiC(CH3)3), 1.49 (s, (CH3)3C of N-
BOC), 1.00 to 2.35 (CH and CH2 of steroid skeleton and alkyl
chain), 2.43 (t, J = 6.9 Hz, CH2ÀC:C), 2.81 (m, 6@’-CH2),
3.48 and 3.91 (2m, OCH2 of THP), 3.61 (s, CH2COOMe),
3.66 (s, COOCH3), 3.74 and 3.79 (2d, J = 10.0 Hz, 17@’a-
CH), 4.63 and 4.73 (2m, CH of THP), 6.58 (d, J = 2.3 Hz,
4@’-CH), 6.64 (d, J = 8.5 Hz, 2@’-CH), 6.97 (s, 6’-CH), 7.16
(m, 1@’-CH), 7.45 (s, 2’-CH), 7.58 (s, 4’-CH), 8.49 (s, NH).
LR-MS calculated for C60H92NO7Si [M À H]À 966.7, found
966.5 m/z.
Methyl 2-{3’-amino-5’-[17@-(3’@,17@’b-dihydroxy-estra-
1@’,3’@,5’@(10@’)-trien-16@’b-yl)-heptadecyl]-phenyl}-acetate
(53)
Yellowish viscous oil (61 mg, 41% yield for two steps).
1
IR (film) n: 3376 (OH and NH2), 1732 (C=O, ester). H
NMR (400 MHz, acetone-d6) d: 0.80 (s, 18@’-CH3), 0.95 to
2.35 (CH and CH2 of steroid skeleton and alkyl chain), 2.46
(t, J = 7.5 Hz, 1@-CH2), 2.78 (m, 6@’-CH2), 3.45 (s,
CH2COOMe), 3.54 (d, J = 5.4 Hz, 17@’b-OH), 3.62 (s,
COOCH3), 3.72 (dd, J1 = 9.8 Hz, J2 = 5.5 Hz, 17@’a-CH),
4.54 (s, NH2), 6.37 (s, 6’-CH), 6.41 and 6.42 (2s, 2’-CH and
4’-CH), 6.54 (d, J = 2.5 Hz, 4@’-CH), 6.60 (dd, J1 = 8.4 Hz,
J2 = 2.6 Hz, 2@’-CH), 7.11 (d, J = 8.4 Hz, 1@’-CH), 7.96 (s,
3’@-OH). 13C NMR (100 MHz, acetone-d6) d: 12.9, 27.1,
28.2, 29.1 to 30.3 (14C under solvent peaks), 30.6, 32.1,
32.5, 33.2, 36.4, 38.6, 39.4, 41.2, 41.4, 44.77, 44.81, 49.4,
51.7, 82.1, 113.3, 113.4, 113.8, 115.8, 118.6, 126.9, 132.0,
135.6, 138.3, 144.3, 149.2, 155.7, 172.2. LR-MS calculated
for C44H68NO4 [M + H]+ 674.5, found 674.3 m/z.
Introduction of molecular diversities: synthesis of 2À13
General procedure to synthesize ester derivatives 2À4 and
53
Esters 2À4 and 53 were synthesized in two steps from
49À52. The first step is an acid treatment to remove the
three protective groups, and the second is the hydrogenation
of alkyne catalyzed by palladium. The procedure that we de-
scribed previously for the synthesis of 230 was used without
modification except that 8 equiv. of HCl (4 mol/L) in diox-
anes were used for the synthesis of 3, 4, and 53 instead of
4 equiv.
General procedure to synthesize carboxylic acid derivatives
5À8
Methyl 2-{3’-amino-5’-[14@-(3’@,17@’b-dihydroxy-estra-
1@’,3’@,5’@(10@’)-trien-16@’b-yl)-tetradecyl]-phenyl}-acetate
(3)
The procedure that we described previously for the syn-
thesis of 530 was used without modification for the synthesis
of 5À8. Completion of the reaction was monitored by TLC
(2 to 5 h).
Colourless viscous oil (83 mg, 62% yield for two steps). IR
1
(film) n: 3372 (OH and NH2), 1732 (C=O, ester). H NMR
(400 MHz, acetone-d6) d: 0.80 (s, 18@’-CH3), 0.95 to 2.35
(CH and CH2 of steroid skeleton and alkyl chain), 2.45 (t,
2-{3’-Amino-5’-[14@-(3’@,17@’b-dihydroxy-estra-
1@’,3’@,5’@(10@’)-trien-16@’b-yl)-tetradecyl]-phenyl}-acetic
acid (6)
J
= 7.4 Hz, 1@-CH2), 2.78 (m, 6@’-CH2), 3.44 (s,
CH2COOMe), 3.62 (m, COOCH3, 17@’b-OH), 3.74 (dd, J1 =
9.7 Hz, J2 = 5.7 Hz, 17@’a-CH), 4.53 (s, NH2), 6.37 (s, 6’-
CH), 6.42 (s, 2’-CH and 4’-CH), 6.54 (d, J = 2.5 Hz, 4@’-CH),
6.60 (dd, J1 = 8.4 Hz, J2 = 2.6 Hz, 2@’-CH), 7.10 (d, J =
8.4 Hz, 1@’-CH), 8.06 (s, 3’@-OH). 13C NMR (75 MHz, ace-
tone-d6) d: 12.7, 26.8, 28.0, 28.7 to 30.2 (11C under solvent
peaks), 30.4, 31.8, 32.3, 33.0, 36.2, 38.3, 39.1, 40.9, 41.2,
44.5 (2Â), 49.2, 51.5, 81.9, 113.1 (2Â), 113.6, 115.5, 118.4,
126.6, 131.7, 135.3, 138.0, 144.0, 148.8, 155.5, 172.1. HR-
MS calculated for C41H62NO4 [M + H]+ 632.46734, found
632.46636 m/z.
White foam (31 mg, 91% yield). IR (KBr) n: 3600À2400
(OH, carboxylic acid), 3374 (OH and NH2), 1718 (C=O,
1
carboxylic acid). H NMR (400 MHz, CDCl3 and few drops
of DMSO-d6) d: 0.62 (s, 18@’-CH3), 0.65 to 2.25 (CH and
CH2 of steroid skeleton and alkyl chain), 2.41 (t, J =
7.7 Hz, 1@-CH2), 2.66 (m, 6@’-CH2), 3.40 (s, CH2COOH),
3.58 (d, J = 9.9 Hz, 17@’a-CH), 6.45 (d, J = 2.4 Hz, 4@’-
CH), 6.51 (dd, J1 = 8.4 Hz, J2 = 2.5 Hz, 2@’-CH), 6.71 (m,
2’-CH and 4’-CH, 6’-CH), 6.98 (d, J = 8.4 Hz, 1@’-CH). 13C
NMR (75 MHz, CDCl3 and few drops of DMSO-d6) d: 12.2,
26.0, 27.2, 28.3, 29.1, 29.3 (9Â), 29.6, 31.0, 31.3, 32.1,
35.4, 37.4, 38.1, 38.9 to 40.6 (1C under DMSO peaks),
40.8, 43.66, 43.75, 48.2, 81.8, 112.5, 115.0, 117.6, 117.9,
123.0, 125.9, 131.0, 135.5, 137.5, 139.3, 144.4, 154.5,
Methyl 2-{3’-amino-5’-[15@-(3’@,17@’b-dihydroxy-estra-
1@’,3’@,5’@(10@’)-trien-16@’b-yl)-pentadecyl]-phenyl}-acetate
(4)
173.0. HR-MS calculated for C40H60NO4 [M
+
H]+
Colourless viscous oil (115 mg, 53% yield for two steps).
618.45169, found 618.45187 m/z.
1
IR (film) n: 3376 (OH and NH2), 1734 (C=O, ester). H
2-{3’-Amino-5’-[15@-(3’@,17@’b-dihydroxy-estra-
1@’,3’@,5’@(10@’)-trien-16@’b-yl)-pentadecyl]-phenyl}-acetic
acid (7)
NMR (400 MHz, acetone-d6) d: 0.80 (s, 18@’-CH3), 0.95 to
2.35 (CH and CH2 of steroid skeleton and alkyl chain), 2.46
(t, J = 7.5 Hz, 1@-CH2), 2.78 (m, 6@’-CH2), 3.44 (s,
CH2COOMe), 3.56 (d, J = 5.4 Hz, 17@’b-OH), 3.62 (s,
COOCH3), 3.74 (dd, J1 = 9.8 Hz, J2 = 5.4 Hz, 17@’a-CH),
White foam (43 mg, 75% yield). IR (KBr) n: 3600À2300
(OH, carboxylic acid), 3370 (OH and NH2), 1711 (C=O,
Published by NRC Research Press

1192
Can. J. Chem. Vol. 87, 2009
1
carboxylic acid). H NMR (400 MHz, CDCl3 and few drops
of DMSO-d6) d: 0.67 (s, 18@’-CH3), 0.75 to 2.30 (CH and
CH2 of steroid skeleton and alkyl chain), 2.40 (t, J =
7.6 Hz, 1@-CH2), 2.70 (m, 6@’-CH2), 3.39 (s, CH2COOH),
3.62 (d, J = 10.0 Hz, 17@’a-CH), 6.44 (s, 4@’-CH), 6.49 (sbr,
2-{3’-Amino-5’-[15@-(3’@,17@’b-dihydroxy-estra-
1@’,3’@,5’@(10@’)-trien-16@’b-yl)-pentadecyl]-phenyl}-ethyl
alcohol (11)
Colourless viscous oil (25 mg, 61% yield). IR (film) n:
1
3354 (OH and NH2). H NMR (400 MHz, acetone-d6) d:
2’-CH, 4’-CH, and 6’-CH), 6.55 (dd, J1 = 8.4 Hz, J2
=
0.80 (s, 18@’-CH3), 0.85 to 2.35 (CH and CH2 of steroid
skeleton and alkyl chain), 2.44 (t, J = 7.7 Hz, 1@-CH2), 2.65
(t, J = 7.3 Hz, CH2CH2OH), 2.78 (m, 6@’-CH2), 3.57 (m,
17@’b-OH and CH2OH), 3.71 (m, CH2OH and 17@’a-CH),
4.43 (sbr, NH2), 6.33 (s, 6’-CH), 6.36 (s, 2’-CH and 4’-CH),
6.54 (d, J = 2.5 Hz, 4@’-CH), 6.60 (dd, J1 = 8.4 Hz, J2 =
2.6 Hz, 2@’-CH), 7.11 (d, J = 8.5 Hz, 1@’-CH), 7.97 (s, 3’@-
OH). 13C NMR (100 MHz, acetone-d6) d: 13.0, 27.2, 28.3,
29.1 to 30.3 (11C under solvent peaks), 30.4, 30.7, 32.4,
32.6, 33.3, 36.6, 38.7, 39.5, 40.5, 41.3, 44.88, 44.92, 49.6,
64.1, 82.3, 113.2, 113.4, 113.5, 115.9, 118.7, 126.9, 132.1,
138.3, 140.6, 144.1, 149.0, 155.8. HR-MS calculated for
C41H64NO3 [M + H]+ 618.48807, found 618.48783 m/z.
2.4 Hz, 2@’-CH), 7.02 (d, J = 8.4 Hz, 1@’-CH). 13C NMR
(100 MHz, CDCl3 and few drops of DMSO-d6) d: 12.2,
26.1, 27.3, 28.4, 29.17, 29.24, 29.3, 29.4 (8Â), 29.6, 31.0,
31.3, 32.2, 35.6, 37.6, 38.2, 39.9, 41.1, 43.8, 43.9, 48.4,
82.0, 112.7, 114.4, 114.8, 115.0, 121.0, 125.9, 131.2, 135.3,
137.6, 139.4, 144.2, 154.5, 173.5. HR-MS calculated for
C41H62NO4 [M + H]+ 632.46734, found 632.46688 m/z.
2-{3’-Amino-5’-[17@-(3’@,17@’b-dihydroxy-estra-
1@’,3’@,5’@(10@’)-trien-16@’b-yl)-heptadecyl]-phenyl}-acetic
acid (8)
Yellowish foam (42 mg, 72% yield). IR (KBr) n:
3700À2300 (OH, carboxylic acid), 3369 (OH and NH2),
1
1713 (C=O, carboxylic acid). H NMR (400 MHz, CDCl3
Synthesis of 2-{3’-(tert-butoxycarbonylamino)-5’-[13@-(3’@-
(tert-butyldimethylsilyloxy)-17@’b-(tetrahydro-2H-pyran-2-
yl-oxy)-estra-1@’,3’@,5’@(10@’)-trien-16@’b-yl)-tridecynyl]-
phenyl}-ethyl alcohol (54)
and few drops of DMSO-d6) d: 0.62 (s, 18@’-CH3), 0.80 to
2.25 (CH and CH2 of steroid skeleton and alkyl chain), 2.40
(t, J = 7.6 Hz, 1@-CH2), 2.66 (m, 6@’-CH2), 3.40 (s,
CH2COOH), 3.57 (d, J = 10.0 Hz, 17@’a-CH), 6.43 (d, J =
2.2 Hz, 4@’-CH), 6.50 (dd, J1 = 8.4 Hz, J2 = 2.4 Hz, 2@’-CH),
6.78 (m, 2’-CH, 4’-CH, and 6’-CH), 6.96 (d, J = 8.5 Hz, 1@’-
CH). 13C NMR (75 MHz, CDCl3 and few drops of DMSO-
d6) d: 12.2, 26.0, 27.1, 28.3, 29.0, 29.1, 29.3 (11Â), 29.5,
31.0, 31.3, 32.0, 35.4, 37.4, 38.0, 38.9 to 40.5 (1C under
DMSO peaks), 40.9, 43.6, 43.7, 48.2, 81.7, 112.5, 115.0,
115.3, 115.7, 122.0, 125.8, 131.0, 135.2, 137.4, 142.1, 144.1,
154.5, 173.3. HR-MS calculated for C43H66NO4 [M + H]+
660.49863, found 660.49790 m/z.
The procedure that we described previously for the syn-
thesis of 930 was used without modification except that
1.2 equiv. of LiAlH4 were used instead of 1 equiv. Purifica-
tion by flash chromatography using hexanes/EtOAc (8:2) as
eluent afforded 54 as white foam (491 mg, 80% yield). IR
(film) n: 3340 (OH and NH carbamate), 1718 (C=O, carba-
mate). 1H NMR (400 MHz, acetone-d6) d: 0.21 (s,
Si(CH3)2), 0.82 and 0.86 (2s, 18@’-CH3), 1.00 (s, SiC(CH3)3),
0.95 to 2.35 (CH and CH2 of steroid skeleton and alkyl
chain), 1.49 (s, (CH3)3C of N-BOC), 2.42 (t, J = 7.0 Hz,
CH2ÀC:C), 2.75 (t, J = 6.7 Hz, CH2CH2OH), 2.81 (m,
6@’-CH2), 3.48 and 3.91 (2m, OCH2 of THP), 3.74 (m,
17@’a-CH and CH2OH), 4.63 and 4.73 (2m, CH of THP),
6.58 (d, J = 2.3 Hz, 4@’-CH), 6.64 (d, J = 8.4 Hz, 2@’-CH),
6.92 (s, 6’-CH), 7.16 (2d, J = 5.8 Hz, 1@’-CH), 7.37 (s, 2’-
CH), 7.53 (s, 4’-CH), 8.39 (sbr, NH). LR-MS calculated for
C55H84NO6Si [M À H]À 882.6, found 882.6 m/z.
General procedure to synthesize alcohol derivatives 9À11
The procedure that we described previously for the syn-
thesis of 930 was used without modification for the synthesis
of 9À11 except that 1.1 to 3 equiv. of LiAlH4 were used.
Completion of the reaction was monitored by TLC (4 to
16 h).
2-{3’-Amino-5’-[14@-(3’@,17@’b-dihydroxy-estra-
1@’,3’@,5’@(10@’)-trien-16@’b-yl)-tetradecyl]-phenyl}-ethyl
alcohol (10)
Yellowish foam (15 mg, 45% yield). IR (film) n: 3400
(OH and NH2). H NMR (400 MHz, acetone-d6) d: 0.79 (s,
Synthesize of 2-{3’-(tert-butoxycarbonylamino)-5’-[13@-(3’@-
(tert-butyldimethylsilyloxy)-17@’b-(tetrahydro-2H-pyran-2-
yl-oxy)-estra-1@’,3’@,5’@(10@’)-trien-16@’b-yl)-tridecynyl]-
phenyl}-ethyl bromide (55)
1
To a solution of alcohol 54 (150 mg, 0.172 mmol) in an-
hydrous DCM under an argon atmosphere at 0 8C were
added PPh3 (90 mg, 0.344 mmol) and CBr4 (114 mg,
0.344 mmol). The reaction mixture was stirred for 3.5 h at
0 8C. Then, a saturated aqueous solution of NaHCO3 was
added to quench the reaction, and the product was extracted
with DCM. The organic phases were washed with brine,
dried over MgSO4, filtered, and evaporated to dryness. The
crude product was purified by flash chromatography (hex-
anes/EtOAc, 8:2) to afford a mixture of the desired, depro-
tected form of 55 (113 mg, 70% yield). Desired product 55
was collected with its deprotected form (without THP) as a
minor compound produced during this bromination reaction,
since the next step was to hydrolyze protective groups under
acid conditions. Only the characterization of the major com-
pound 55 is described. Colourless viscous oil (113 mg, 70%
18@’-CH3), 0.75 to 2.35 (CH and CH2 of steroid skeleton and
alkyl chain), 2.44 (t, J = 7.7 Hz, 1@-CH2), 2.65 (t, J = 7.3 Hz,
CH2CH2OH), 2.79 (m, 6@’-CH2), 3.58 (m, 17@’b-OH and
CH2OH), 3.70 (m, CH2OH and 17@’a-CH), 4.44 (sbr, NH2),
6.32 (s, 6’-CH), 6.36 (s, 2’-CH and 4’-CH), 6.54 (d, J =
2.5 Hz, 4@’-CH), 6.60 (dd, J1 = 8.4 Hz, J2 = 2.6 Hz, 2@’-CH),
7.11 (d, J = 8.4 Hz, 1@’-CH), 8.01 (s, 3’@-OH). 13C NMR
(75 MHz, acetone-d6) d: 12.9, 27.1, 28.2, 28.9 to 30.4 (11C
under solvent peaks), 32.1, 32.5, 33.2, 36.3, 36.5, 38.5, 39.3,
40.2, 41.2, 44.8 (2Â), 49.4, 63.8, 82.1, 113.4, 115.7, 117.7,
118.0, 124.2, 126.8, 132.0, 138.2, 140.7, 143.9, 152.6, 155.7.
HR-MS calculated for C40H62NO3 [M + H]+ 604.47242,
found 604.47243 m/z.
Published by NRC Research Press

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Berube and Poirier
1193
1
yield). H NMR (400 MHz, acetone-d6) d: 0.21 (s, Si(CH3)2),
This mixture was stirred for 16 h under a hydrogen atmos-
phere at room temperature. The resulting suspension was fil-
tered through Celite, washed with EtOAc, and the filtrate was
evaporated to dryness. The crude compound was purified by
flash chromatography (hexanes/EtOAc, 8:2 to 7:3) to provide
a mixture of bromide derivative 12 and its salt (NH2ÁHCl).
White foam (12 mg, 25% yield for two steps). IR (film)
0.82 and 0.86 (2s, 18@’-CH3), 0.85 to 2.35 (CH and CH2 of
steroid skeleton and alkyl chain), 1.00 (s, SiC(CH3)3), 1.50
(s, (CH3)3C of N-BOC), 2.43 (t, J = 7.0 Hz, CH2ÀC:C),
2.81 (m, 6@’-CH2), 3.12 (t, J = 7.3 Hz, CH2CH2Br), 3.47 and
3.92 (2m, OCH2 of THP), 3.68 (t, J = 7.4 Hz, CH2Br), 3.73
and 3.79 (2d, J = 9.9 Hz, 17@’a-CH), 4.63 and 4.73 (2m, CH
of THP), 6.58 (d, J = 2.4 Hz, 4@’-CH), 6.64 (d, J = 8.5 Hz,
2@’-CH), 6.97 (s, 6’-CH), 7.15 (m, 1@’-CH), 7.42 (s, 2’-CH),
7.57 (s, 4’-CH), 8.46 (sbr, NH).
1
n: 3356 (OH and NH2). H NMR (400 MHz, acetone-d6)
d: 0.80 (s, 18@’-CH3), 1.00 to 2.35 (CH and CH2 of steroid
skeleton and alkyl chain), 2.46 and 2.58 (2t, J = 7.7 Hz, 1@-
CH2), 2.77 (m, 6@’-CH2), 2.99 and 3.11 (2t, J = 7.6 Hz,
CH2CH2Br), 3.56 (sbr, 17@’b-OH), 3.60 and 3.68 (2t, J =
7.6 Hz, CH2Br), 3.73 (d, J = 9.2 Hz, 17@’a-CH), 6.40 (m, 2’-
CH, 4’-CH and 6’-CH), 6.54 (s, 4@’-CH), 6.60 (d, J = 8.4 Hz,
2@’-CH), 6.83 (s, NH2), 7.11 (d, J = 8.5 Hz, 1@’-CH), 7.98 (s,
3’@-OH). 13C NMR (75 MHz, acetone-d6) d: 12.9, 27.1, 28.2,
28.9 to 30.4 (10C under solvent peaks), 30.5, 32.0, 32.5,
33.2, 34.1, 36.2, 38.5, 39.3, 39.7, 41.2, 44.76, 44.81, 49.4,
82.1, 113.3, 115.7, 117.9, 118.7, 124.2, 126.9, 132.0, 138.3,
140.4, 144.4, 146.4, 155.7. HR-MS calculated for
C39H59BrNO2 [M + H]+ 652.37237, found 652.37128 m/z.
Synthesize of dimethyl 2-{3’-(tert-butoxycarbonylamino)-5’-
[13@-(3’@-(tert-butyldimethylsilyloxy)-17@’b-(tetrahydro-2H-
pyran-2-yl-oxy)-estra-1@’,3’@,5’@(10@’)-trien-16@’b-yl)-
tridecynyl]-phenyl}-ethyl phosphate (56)
In a round-bottom flask, alcohol 54 (300 mg, 0.345 mmol)
and CBr4 (171 mg, 0.517 mmol) were purged under an argon
atmosphere for 10 min. Then, pyridine (2.3 mL) and
P(OMe)3 (163 mL, 1.38 mmol) were added. The reaction
mixture was stirred for 3 h at room temperature. Then, the
product was extracted with EtOAc. The organic phase was
washed with an aqueous solution of CuSO4 1 mol/L and re-
extracted with EtOAc. The combined organic phases were
washed with H2O and brine, dried over MgSO4, filtered, and
evaporated to dryness. Purification by flash chromatography
(hexanes/EtOAc, 6:4) provided 56 (275 mg, 82% yield) as a
yellowish solid. IR (film) n: 3284 (NH carbamate), 1727
Synthesize of dimethyl 2-{3’-amino-5’-[13@-(3’@, 17@’b-
dihydroxy-estra-1@’,3’@,5’@(10@’)-trien-16@’b-yl)-tridecyl]-
phenyl}-ethyl phosphate (13)
The procedure described above for the synthesis of 12
was used without modification except that MeOH was used
as solvent in the hydrogenation step instead of EtOAc. De-
protected form of 56 and phosphotriester derivatives were
purified by flash chromatography using hexanes/EtOAc
(2:8) followed by DCM/MeOH (97:3) as eluent. Compound
13 was obtained as a mixture with its salt (NH2ÁHCl).
Yellowish foam (22 mg, 35% yield for two steps). IR (film)
1
(C=O, carbamate). H NMR (400 MHz, acetone-d6) d: 0.20
(s, Si(CH3)2), 0.82 and 0.86 (2s, 18@’-CH3), 1.00 (s,
SiC(CH3)3), 0.95 to 2.35 (CH and CH2 of steroid skeleton
and alkyl chain), 1.49 (s, (CH3)3C of N-BOC), 2.42 (t, J =
6.9 Hz, CH2ÀC:C), 2.81 (m, 6@’-CH2), 2.95 (t, J = 6.7 Hz,
CH2CH2OP(O)(OMe)2), 3.48 and 3.91 (2m, OCH2 of THP),
3
1
3.67 (d, JPH = 11.1 Hz, 2 Â OMe), 3.74 and 3.79 (2d, J =
n: 3364 (OH and NH2). H NMR (400 MHz, acetone-d6)
9.8 Hz, 17@’a-CH), 4.23 (q, J = 7.0 Hz, CH2CH2O-
P(O)(OMe)2), 4.63 and 4.73 (2m, CH of THP), 6.58 (d, J =
2.1 Hz, 4@’-CH), 6.64 (d, J = 8.4 Hz, 2@’-CH), 6.98 (s, 6’-
CH), 7.16 (m, 1@’-CH), 7.44 (s, 2’-CH), 7.56 (s, 4’-CH), 8.48
(s, NH). LR-MS calculated for C57H90NO9PSiNa [M + Na]+
1014.6, found 1014.5 m/z.
d: 0.80 (s, 18@’-CH3), 0.85 to 2.35 (CH and CH2 of steroid
skeleton and alkyl chain), 2.46 and 2.57 (2t, J = 7.7 Hz, 1@-
CH2), 2.78 (m, 6@’-CH2), 2.83 and 2.94 (2t, J = 6.8 Hz,
3
CH2CH2OP(O)(OMe)2), 3.65 (d, JPH = 11.1 Hz, 2 Â
OMe), 3.72 (d, J = 9.8 Hz, 17@’a-CH), 4.18 and 4.21 (2q,
J = 7.1 Hz, CH2OP(O)(OMe)2), 6.40 (m, 2’-CH, 4’-CH and
6’-CH), 6.54 (s, 4@’-CH), 6.60 (d, J = 8.4 Hz, 2@’-CH), 6.81
(s, NH2), 7.11 (d, J = 8.4 Hz, 1@’-CH). 13C NMR (100 MHz,
acetone-d6) d: 13.0, 27.2, 28.3, 29.1 to 30.3 (10C under
solvent peaks), 30.6, 32.1, 32.6, 33.3, 36.3, 37.2, 38.7, 39.5,
41.3, 44.86, 44.92, 49.6, 54.15, 54.22, 68.5 (68.6), 82.1,
113.4, 115.8, 118.2, 118.4, 124.4, 126.9, 132.1, 138.3,
138.9, 144.4, 151.8, 155.8. HR-MS calculated for
C41H65NO6P [M + H]+ 698.45440, found 698.45359 m/z.
Synthesize of 2-{3’-amino-5’-[13@-(3’@,17@’b-dihydroxy-
estra-1@’,3’@,5’@(10@’)-trien-16@’b-yl)-tridecyl]-phenyl}-ethyl
bromide (12)
Deprotection
A solution of 55 (80 mg, 0.086 mmol) in dry DCM
(0.5 mL) and dry MeOH (0.5 mL) under an argon atmos-
phere was treated with HCl (4 mol/L in dioxane)
(160À255 mL, 0.858 mmol). The resulting mixture was
stirred at room temperature for 16 h. The reaction was
quenched by addition of a saturated aqueous NaHCO3 solu-
tion, and extraction was performed with DCM. The organic
phase was washed with brine, dried over MgSO4, filtered,
and evaporated to dryness under reduced pressure. Purifica-
tion of the crude product by flash chromatography (hexanes/
EtOAc, 8:2) afforded a mixture of the desired products
(NH2) and its salt (NH2ÁHCl).
Synthesis of new adenosine mimics 59, 61, 64, and 65
Synthesis of 3-(3’-tert-butoxycarbonylamino-5’-
iodophenyl)-propanol (58)
To a solution of alkene 5730 (100 mg, 0.278 mmol) in an-
hydrous THF (3 mL) under an argon atmosphere at 0 8C was
added BH3ÁTHF (1.0 mol/L in THF, 0.64 mL, 0.64 mmol).
The mixture was stirred 3 h at 0 8C. Then, 3 N aqueous
NaOH (0.23 mL, 0.695 mmol) and 30% (w/v) hydrogen per-
oxide (0.17 mL, 1.53 mmol) were added. After 2 h at room
temperature, the reaction was quenched with water, and ex-
traction was performed with EtOAc. The organic phase was
Hydrogenation
To a solution of the deprotected form of alkyne 55 (35 mg,
0.054 mmol) in EtOAc (2 mL) was added 10% Pd/C (7 mg).
Published by NRC Research Press

1194
Can. J. Chem. Vol. 87, 2009
washed with brine, dried over MgSO4, filtered, and evapo-
rated to dryness. The crude product was purified by flash
chromatography (hexanes/EtOAc, 6:4) to give 77 mg (74%
yield) of the desired alcohol 58 as a white foam. IR (film)
n: 3316 (OH and NH carbamate), 1700 (C=O carbamate).
1H NMR (400 MHz, acetone-d6) d: 1.49 (s, (CH3)3C of N-
BOC), 1.80 (m, CH2CH2OH), 2.64 (t, J = 7.5 Hz,
CH2CH2CH2OH), 3.58 (m, CH2OH), 3.67 (t, J = 5.4 Hz,
OH), 7.25 (s, 6’-CH), 7.40 (s, 2’-CH), 7.90 (s, 4’-CH), 8.49
(s, NH). 13C NMR (75 MHz, acetone-d6) d: 28.2 (3Â), 32.2,
34.9, 61.3, 80.1, 94.4, 118.2, 124.7, 131.8, 141.6, 146.1,
153.2. LR-MS calculated for C14H19INO3 [M À H]À 376.0,
found 375.9 m/z.
15.5 Hz, J2 = 7.0 Hz, CH2CH=CH), 7.28 (s, 6’-CH), 7.42 (s,
2’-CH), 7.97 (s, 4’-CH), 8.56 (s, NH). 13C NMR (75 MHz,
acetone-d6) d: 28.2 (3Â), 38.0, 51.4, 80.3, 94.6, 118.5,
122.7, 125.5, 132.0, 141.8, 142.0, 147.4, 153.2, 166.6. LR-
MS calculated for C16H19INO4 [M À H]À 416.0, found
415.9 m/z.
Synthesis of methyl 4-(3’-tert-butoxycarbonylamino-5’-
iodophenyl)-butanoate (61)
To a solution of alkene 60 (240 mg, 0.57 mmol) in anhy-
drous THF (6 mL) was added PtO2 (60 mg). This mixture
was stirred for 16 h under a hydrogen atmosphere at room
temperature. The resulting suspension was filtered through
Celite, and THF was evaporated under reduced pressure. Pu-
rification by flash chromatography (hexanes/EtOAc, 85:15)
afforded 61 (222 mg, 93% yield) as a white solid. IR (film)
n: 3342 (NH, carbamate), 1720 (C=O, ester and carbamate).
1H NMR (400 MHz, acetone-d6) d: 1.49 (s, (CH3)3C of N-
BOC), 1.89 (dt, J1 = 15.2 Hz, J2 = 7.5 Hz, CH2CH2COOMe),
2.35 (t, J = 7.4 Hz, CH2COOMe), 2.60 (t, J = 7.5 Hz,
CH2CH2CH2COOMe), 3.63 (s, COOCH3), 7.26 (s, 6’-CH),
7.39 (s, 2’-CH), 7.93 (s, 4’-CH), 8.49 (s, NH). 13C NMR
(75 MHz, acetone-d6) d: 27.0, 28.3 (3Â), 33.4, 35.0, 51.4,
80.2, 94.5, 118.3, 125.1, 131.8, 141.8, 145.4, 153.3, 173.6.
LR-MS calculated for C16H21INO4 [M À H]À 418.0, found
417.9 m/z.
Synthesis of methyl 3-(3’-tert-butoxycarbonylamino-5’-
iodophenyl)-propanoate (59)
Oxidation
To a solution of alcohol 58 (329 mg, 0.872 mmol) in ace-
tone (44 mL) was added dropwise Jones’ reagent (0.48 mL
of a CrO3ÁH2SO4, 2.7 mol/L solution) at 0 8C, and the reac-
tion mixture was stirred for 90 min. Then, 2-propanol (few
drops) and brine were added. Acetone was partially evapo-
rated under reduced pressure, the mixture was extracted
with EtOAc. The organic phase was dried over MgSO4, fil-
tered, and evaporated to dryness.
Esterification
Synthesis of methyl 3-tert-butoxycarbonylamino-5-
iodophenyl)-benzoate (64)
The crude acid was dissolved in anhydrous MeOH (9 mL)
under an argon atmosphere at 0 8C. TMSCHN2 (2.0 mol/L
in hexanes, 2.6 mL, 5.2 mmol) was added slowly, and the
mixture was stirred for 10 min at 0 8C. Then, MeOH was
removed under reduced pressure. The crude product was pu-
rified by flash chromatography (hexanes/EtOAc, 9:1) to af-
ford ester 59 (269 mg, 76% yield for two steps) as a white
foam. IR (film) n: 3339 (NH, carbamate), 1725 (C=O, ester
The starting material 62 was obtained according to an al-
ready described procedure.35,36 The esterification procedure
described above for the synthesis of 59 was used without
modification. Purification by flash chromatography (hex-
anes/EtOAc, 9:1) yielded ester 64 (962 mg, 93% yield) as a
white solid. IR (film) n: 3343 (NH, carbamate), 1710 (C=O,
1
ester and carbamate). H NMR (400 MHz, CDCl3) d: 1.52 (s,
1
and carbamate). H NMR (400 MHz, acetone-d6) d: 1.49 (s,
(CH3)3C of N-BOC), 3.90 (s, COOCH3), 6.70 (s, NH), 7.86
(dd, J1 = 2.0 Hz, J2 = 1.5 Hz, 6-CH), 8.02 (t, J = 1.5 Hz, 4-
CH), 8.16 (s, 2-CH). 13C NMR (75 MHz, CDCl3) d: 28.2
(3Â), 52.5, 81.3, 94.0, 118.6, 131.0, 132.1, 132.7, 139.6,
152.2, 165.4. LR-MS calculated for C13H15INO4 [M À H]À
376.0, found 376.0 m/z.
(CH3)3C of N-BOC), 2.63 (t, J = 7.6 Hz, CH2COOMe), 2.86
(t, J = 7.6 Hz, CH2CH2COOMe), 3.63 (s, COOCH3), 7.29
(s, 6’-CH), 7.40 (s, 2’-CH), 7.93 (s, 4’-CH), 8.49 (s, NH).
13C NMR (75 MHz, acetone-d6) d: 28.3 (3Â), 30.9, 35.5,
51.5, 80.3, 94.5, 118.2, 125.3, 131.8, 141.8, 144.5, 153.3,
173.0. LR-MS calculated for C15H19INO4 [M À H]À 404.0,
found 403.9 m/z.
Synthesis of methyl 2-(3’-iodophenyl)-acetate (65)
The procedure described above for the synthesis of 59
was used without modification. Purification by flash chro-
matography (hexanes/EtOAc, 95:5) afforded ester 65
(263 mg, 99% yield) as a colourless oil. IR (film) n: 1738
Synthesis of methyl 4-(3’-tert-butoxycarbonylamino-5’-
iodophenyl)-2-butenoate (60)
Alkene 5730 (500 mg, 1.39 mmol) was dissolved in anhy-
drous DCM (23 mL) under an argon atmosphere. Methyl
acrylate (2.5 mL, 27.8 mmol) and the 2nd generation
Grubbs’ catalyst (118 mg, 0.139 mmol) were added. The re-
action mixture was refluxed for 16 h. Then, DCM was re-
moved under reduced pressure, and the crude product was
purified by flash chromatography (hexanes/EtOAc, 9:1) to
yield 60 (505 mg, 87% yield, 36:1 E/Z as determined by in-
1
(C=O, ester). H NMR (400 MHz, CDCl3) d: 3.57 (s, CH2),
3.70 (s, COOCH3), 7.06 (t, J = 7.8 Hz, 5’-CH), 7.25 (d, J =
7.4 Hz, 6’-CH), 7.61 (d, J = 7.9 Hz, 4’-CH), 7.64 (s, 2’-CH).
13C NMR (75 MHz, CDCl3) d: 40.5, 52.2, 94.4, 128.6,
130.2, 136.1, 136.2, 138.2, 171.4. LR-MS calculated for
C9H10IO2 [M + H]+ 277.0, found 276.8 m/z.
1
tegration of peaks at 5.91 and 6.02 ppm in the H NMR
Synthesis of 14À18 using the second chemical approach
spectrum) as a white amorphous solid. IR (film) n: 3335
General procedure to synthesize 67À71
(NH, carbamate), 1700 (C=O, conjugated ester and carba-
mate), 1654 (C=C, conjugated alkene). H NMR (400 MHz,
acetone-d6) d: 1.49 (s, (CH3)3C of N-BOC), 3.53 (dd, J1 =
7.0 Hz, J2 = 1.1 Hz, CH2CH=), 3.68 (s, COOCH3), 5.91 (dt,
J1 = 15.6 Hz, J2 = 1.6 Hz, CH=CHÀCOOMe), 7.03 (dt, J1 =
1
The Sonogashira coupling procedure described previ-
ously30 for the synthesis of 49 was used without modifica-
tion except for the synthesis of 71 where 2 equiv. of 3-
iodoaniline were used instead of 1 equiv.
Published by NRC Research Press

´
´
Berube and Poirier
1195
Methyl 3-tert-butoxycarbonylamino-5-[13’-(3@-(tert-
butyldimethylsilyloxy)-17@b-(tetrahydro-2H-pyran-2-yl-oxy)-
estra-1@,3@,5@(10@)-trien-16@b-yl)-tridecynyl]-benzoate (67)
Yellowish foam (249 mg, 72% yield). IR (film) n: 3355
(NH, carbamate), 1732 (C=O, ester), 1712 (C=O, carbamate).
1H NMR (400 MHz, acetone-d6) d: 0.20 (s, Si(CH3)2), 0.81
and 0.85 (2s, 18@-CH3), 1.00 (s, SiC(CH3)3), 1.00 to 2.40
(CH and CH2 of steroid skeleton and alkyl chain), 1.51 (s,
(CH3)3C of N-BOC), 2.45 (t, J = 6.9 Hz, CH2-C:C), 2.80
(m, 6@-CH2), 3.48 and 3.94 (2m, OCH2 of THP), 3.72 and
3.78 (2d, J = 10.0 Hz, 17@a-CH), 3.89 (s, COOCH3), 4.62
and 4.72 (2m, CH of THP), 6.57 (d, J = 1.8 Hz, 4@-CH),
6.63 (d, J = 8.4 Hz, 2@-CH), 7.15 (m, 1@-CH), 7.63 (d, J =
1.4 Hz, 6-CH), 7.89 (s, 4-CH), 8.20 (d, J = 1.5 Hz, 2-CH),
8.71 (s, NH). LR-MS calculated for C55H87N2O7Si [M +
NH4]+ 915.6, found 915.8 m/z.
SiC(CH3)3), 1.00 to 2.35 (CH and CH2 of steroid skeleton
and alkyl chain), 2.43 (t, J = 6.9 Hz, CH2ÀC:C), 2.81 (m,
6@’-CH2), 3.47 and 3.92 (2m, OCH2 of THP), 3.66 (s,
CH2COOCH3), 3.74 and 3.79 (2d, J = 10.0 Hz, 17@’a-CH),
4.63 and 4.73 (2m, CH of THP), 6.58 (s, 4@’-CH), 6.64 (d,
J = 8.4 Hz, 2@’-CH), 7.15 (m, 1@’-CH), 7.30 (m, 2’-CH, 4’-
CH, 5’-CH and 6’-CH). LR-MS calculated for C51H80N2O5Si
[M + NH4]+ 814.6, found 814.8 m/z.
3-[13’-(3@-(tert-butyldimethylsilyloxy)-17@b-(tetrahydro-2H-
pyran-2-yl-oxy)-estra-1@,3@,5@(10@)-trien-16@b-yl)-
tridecynyl]-aniline (71)
Yellowish viscous oil (151 mg, 71% yield). IR (film) n:
1
3460 and 3354 (NH2). H NMR (400 MHz, acetone-d6) d:
0.21 (s, Si(CH3)2), 0.82 and 0.86 (2s, 18@-CH3), 1.00 (s,
SiC(CH3)3), 1.00 to 2.35 (CH and CH2 of steroid skeleton
and alkyl chain), 2.39 (t, J = 6.9 Hz, CH2ÀC:C), 2.81 (m,
6@-CH2), 3.49 and 3.92 (2m, OCH2 of THP), 3.74 and 3.79
(2d, J = 9.7 Hz, 17@a-CH), 4.69 (m, CH of THP and NH2),
6.58 (d, J = 2.1 Hz, 4@-CH), 6.62 (m, 2@-CH, 4-CH and 6-
CH), 6.71 (d, J = 1.7 Hz, 2-CH), 7.00 (t, J = 7.8 Hz, 5-CH)
7.16 (m, 1@-CH). LR-MS calculated for C48H74NO3Si [M +
H]+ 740.5, found 740.6 m/z.
Methyl 3-{3’-tert-butoxycarbonylamino-5’-[13@-(3’@-(tert-
butyldimethylsilyloxy)-17@’b-(tetrahydro-2H-pyran-2-yl-
oxy)-estra-1@’,3’@,5’@(10@’)-trien-16@’b-yl)-tridecynyl]-
phenyl}-propanoate (68)
Yellowish foam (263 mg, 74% yield). IR (film) n: 3354
(NH, carbamate), 1732 (C=O, ester and carbamate). 1H
NMR (400 MHz, acetone-d6) d: 0.20 (s, Si(CH3)2), 0.82 and
0.86 (2s, 18@’-CH3), 1.00 (s, SiC(CH3)3), 1.00 to 2.35 (CH
and CH2 of steroid skeleton and alkyl chain), 1.49 (s,
(CH3)3C of N-BOC), 2.42 (t, J = 6.9 Hz, CH2ÀC:C), 2.62
(t, J = 7.6 Hz, CH2COOMe), 2.81 (m, 6@’-CH2), 2.86 (t, J =
7.7 Hz, CH2CH2COOMe), 3.47 and 3.92 (2m, OCH2 of
THP), 3.63 (s, COOCH3), 3.73 and 3.78 (2d, J = 10.0 Hz,
17@’a-CH), 4.63 and 4.72 (2m, CH of THP), 6.58 (s, 4@’-
CH), 6.63 (d, J = 8.4 Hz, 2@’-CH), 6.92 (s, 6’-CH), 7.16 (m,
1@’-CH), 7.37 (s, 2’-CH), 7.54 (s, 4’-CH), 8.43 (s, NH). LR-
MS calculated for C57H91N2O7Si [M + NH4]+ 943.7, found
943.8 m/z.
General procedure to synthesize 72À75
The same procedure described above for the preparation
of 12 was used without modification except that 15% w/w
of 10% Pd/C was used instead of 20% w/w in the hydroge-
nation step. The salt product (NH2ÁHCl) was not observed
for compounds 72À75.
Methyl 3-amino-5-[13’-(3@, 17@b- dihydroxy-estra-
1@,3@,5@(10@)-trien-16@b-yl)-tridecyl]-benzoate (72)
White solid (98 mg, 63% yield for two steps). IR (film) n:
3368 (OH and NH2), 1704 (C=O, ester). 1H NMR (400 MHz,
acetone-d6) d: 0.79 (s, 18@-CH3), 0.95 to 2.35 (CH and CH2
of steroid skeleton and alkyl chain), 2.54 (t, J = 7.5 Hz, 1’-
CH2), 2.78 (m, 6@-CH2), 3.56 (d, J = 5.4 Hz, 17@bOH), 3.74
(dd, J1 = 9.8 Hz, J2 = 5.5 Hz, 17@a-CH), 3.82 (s, COOCH3),
4.84 (s, NH2), 6.54 (d, J = 2.5 Hz, 4@-CH), 6.60 (dd, J1 =
8.4 Hz, J2 = 2.6 Hz, 2@-CH), 6.77 (s, 4-CH), 7.13 (m, 1@-
CH, 2-CH and 6-CH), 7.97 (s, 3@-OH). 13C NMR
(100 MHz, acetone-d6) d: 12.9, 27.1, 28.2, 29.1 to 30.2 (9C
under solvent peaks), 30.3, 30.6, 32.0, 32.6, 33.2, 36.3, 38.5,
39.3, 41.2, 44.7, 44.8, 49.4, 51.8, 82.1, 113.2, 113.4, 115.8,
118.5, 119.4, 126.9, 131.6, 132.0, 138.3, 144.5, 149.3,
155.7, 167.6. LR-MS calculated for C39H58NO4 [M + H]+
604.4, found 604.6 m/z.
Methyl 4-{3’-tert-butoxycarbonylamino-5’-[13@-(3’@-(tert-
butyldimethylsilyloxy)-17@’b-(tetrahydro-2H-pyran-2-yl-
oxy)-estra-1@’,3’@,5’@(10@’)-trien-16@’b-yl)-tridecynyl]-
phenyl}-butanoate (69)
Yellowish viscous oil (238 mg, 66% yield). IR (film) n:
1
3342 (NH, carbamate), 1734 (C=O, ester and carbamate). H
NMR (400 MHz, acetone-d6) d: 0.20 (s, Si(CH3)2), 0.82 and
0.86 (2s, 18@’-CH3), 1.00 (s, SiC(CH3)3), 1.00 to 2.30 (CH
and CH2 of steroid skeleton, alkyl chain and
CH2CH2COOMe), 1.49 (s, (CH3)3C of N-BOC), 2.34 (t, J =
7.4 Hz, CH2COOMe), 2.42 (t, J = 7.0 Hz, CH2ÀC:C), 2.60
(t, J = 7.6 Hz, CH2CH2CH2COOMe), 2.81 (m, 6@’-CH2), 3.46
and 3.91 (2m, OCH2 of THP), 3.63 (s, COOCH3), 3.73 and
3.79 (2d, J = 10.0 Hz, 17@’a-CH), 4.63 and 4.72 (2m, CH of
THP), 6.58 (d, J = 2.2 Hz, 4@’-CH), 6.64 (dd, J1 = 6.7 Hz,
J2 = 2.2 Hz, 2@’-CH), 6.89 (s, 6’-CH), 7.16 (m, 1@’-CH), 7.37
(s, 2’-CH), 7.53 (s, 4’-CH), 8.42 (s, NH). LR-MS calculated
for C58H90NO7Si [M + H]+ 940.6, found 940.4 m/z.
Methyl 3-{3’-amino-5’-[13@-(3’@, 17@’b- dihydroxy-estra-
1@’,3’@,5’@(10@’)-trien-16@’b-yl)-tridecyl]-phenyl}-propanoate
(73)
Yellowish viscous oil (97 mg, 63% yield for two steps).
1
IR (film) n: 3366 (OH and NH2), 1734 (C=O, ester). H
NMR (400 MHz, acetone-d6) d: 0.80 (s, 18@’-CH3), 0.95 to
2.35 (CH and CH2 of steroid skeleton and alkyl chain), 2.44
(t, J = 7.5 Hz, 1@-CH2), 2.55 (t, J = 7.8 Hz, CH2COOMe),
2.76 (m, 6@’-CH2, CH2CH2COOMe), 3.54 (d, J = 5.4 Hz,
17@’b-OH), 3.62 (s, COOCH3), 3.73 (dd, J1 = 9.8 Hz, J2 =
5.6 Hz, 17@’a-CH), 4.46 (s, NH2), 6.32 (s, 6’-CH), 6.35 (s,
2’-CH and 4’-CH), 6.54 (d, J = 2.4 Hz, 4@’-CH), 6.60 (dd,
J1 = 8.4 Hz, J2 = 2.6 Hz, 2@’-CH), 7.11 (d, J = 8.4 Hz, 1@’-
Methyl 2-{3’-[13@-(3’@-(tert-butyldimethylsilyloxy)-17@’b-
(tetrahydro-2H-pyran-2-yl-oxy)-estra-1@’,3’@,5’@(10@’)-trien-
16@’b-yl)-tridecynyl]-phenyl}-acetate (70)
Yellowish viscous oil (220 mg, 72% yield). IR (film) n:
1
1742 (C=O, ester). H NMR (400 MHz, acetone-d6) d: 0.21
(s, Si(CH3)2), 0.82 and 0.86 (2s, 18@’-CH3), 1.00 (s,
Published by NRC Research Press

1196
Can. J. Chem. Vol. 87, 2009
CH), 7.96 (s, 3@’-OH). 13C NMR (100 MHz, acetone-d6) d:
12.9, 27.1, 28.2, 29.1 to 30.2 (8C under solvent peaks),
30.3, 30.5, 30.6, 31.5, 32.1, 32.5, 33.2, 36.1, 36.5, 38.6,
39.4, 41.2, 44.7, 44.8, 49.4, 51.4, 82.1, 112.5, 113.2, 113.4,
115.8, 117.7, 126.8, 132.0, 138.3, 142.0, 144.2, 149.1,
155.7, 173.8. LR-MS calculated for C41H62NO4 [M + H]+
632.5, found 632.6 m/z.
phy (DCM/MeOH, 97:3 to 90:10) to provide carboxylic
acids 14À17 (26À44 mg, 66%À78% yield).
3-amino-5-[13’-(3@,17@b-dihydroxy-estra-1@,3@,5@(10@)-trien-
16@b-yl)-tridecyl]-benzoic acid (14)
Yellowish foam (26 mg, 66% yield). IR (KBr) n:
3600À2300 (OH, carboxylic acid), 3415 (OH and NH2),
1
1694 (C=O, carboxylic acid). H NMR (400 MHz, DMSO-
d6) d: 0.65 (s, 18@-CH3), 0.80 to 2.25 (CH and CH2 of ste-
roid skeleton and alkyl chain), 2.45 (t, J = 7.4 Hz, 1’-CH2),
2.69 (m, 6@-CH2), 3.55 (d, J1 = 9.4 Hz, 17@a-CH), 4.39 (sbr,
17@b-OH), 5.21 (sbr, NH2), 6.43 (d, J = 2.2 Hz, 4@-CH), 6.50
(dd, J1 = 8.3 Hz, J2 = 2.4 Hz, 2@-CH), 6.58 (s, 4-CH), 6.92
(s, 2-CH), 6.98 (d, J = 1.7 Hz, 6-CH), 7.03 (d, J = 8.5 Hz,
1@-CH), 9.03 (sbr, COOH). 13C NMR (75 MHz, DMSO-d6)
d: 12.7, 26.1, 27.2, 28.4, 28.7, 28.9, 29.1 (7Â), 29.5, 30.9,
31.7, 32.3, 35.2, 37.5, 38.2, 38.7 to 40.3 (1C under solvent
peaks), 43.6, 43.7, 48.2, 80.5, 112.3, 112.7, 114.9, 116.9,
117.9, 126.0, 130.5, 131.6, 137.2, 142.9, 148.7, 154.9,
Methyl 4-{3’-amino-5’-[13@-(3’@, 17@’b- dihydroxy-estra-
1@’,3’@,5’@(10@’)-trien-16@’b-yl)-tridecyl]-phenyl}-butanoate
(74)
Yellowish viscous oil (81 mg, 64% yield for two steps).
1
IR (film) n: 3377 (OH and NH2), 1732 (C=O, ester). H
NMR (400 MHz, acetone-d6) d: 0.80 (s, 18@’-CH3), 0.95 to
2.35 (CH and CH2 of steroid skeleton, alkyl chain and
CH2CH2COOMe), 2.32 (m, CH2COOMe), 2.46 (m, 1@-
CH2), 2.58 (m, J = 7.8 Hz, CH2CH2CH2COOMe), 2.78 (m,
6@’-CH2), 3.56 (m, 17@’b-OH), 3.62 (s, COOCH3), 3.73 (dd,
J1 = 9.8 Hz, J2 = 5.9 Hz, 17@’a-CH), 4.45 (s, NH2), 6.33 (m,
2’-CH, 4’-CH and 6’-CH), 6.54 (d, J = 2.2 Hz, 4@’-CH), 6.59
(d, J = 8.4 Hz, 2@’-CH), 7.11 (d, J = 8.5 Hz, 1@’-CH), 7.97
(s, 3’’’-OH). 13C NMR (75 MHz, acetone-d6) d: 12.9, 27.0,
27.2, 28.2, 28.9, 29.1 to 30.2 (7C under solvent peaks),
30.3, 30.4, 30.6, 32.1, 32.5, 33.2, 33.5, 35.6, 36.5, 38.5,
39.3, 41.1, 44.8 (2Â), 49.4, 51.3, 82.1, 112.7, 113.0, 113.3,
115.7, 118.0, 126.8, 131.9, 138.2, 142.8, 144.0, 149.0,
155.7, 173.8. LR-MS calculated for C42H64NO4 [M + H]+
646.5, found 646.3 m/z.
168.1. HR-MS calculated for C38H56NO4 [M
590.42039, found 590.42017 m/z.
+
H]+
3-{3’-amino-5’-[13@-(3’@,17@’b-dihydroxy-estra-
1@’,3’@,5’@(10@’)-trien-16@’b-yl)-tridecyl]-phenyl}-propanoic
acid (15)
Yellowish foam (42 mg, 78% yield). IR (KBr) n:
3600À2400 (OH, carboxylic acid), 3378 (OH and NH2),
1
1708 (C=O, carboxylic acid). H NMR (400 MHz, DMSO-
d6) d: 0.65 (s, 18@’-CH3), 0.80 to 2.30 (CH and CH2 of ste-
roid skeleton and alkyl chain), 2.35 (t, J = 7.5 Hz, 1@-CH2),
2.42 (t, J = 7.9 Hz, CH2COOH), 2.61 (t, J = 7.5 Hz,
CH2CH2COOH), 2.70 (m, 6@’-CH2), 3.55 (d, J1 = 9.5 Hz,
17@’a-CH), 4.85 (sbr, NH2), 6.18 (s, 6-CH), 6.20 (s, 2’-CH
and 4’-CH), 6.43 (d, J = 2.2 Hz, 4@’-CH), 6.49 (dd, J1 =
8.4 Hz, J2 = 2.4 Hz, 2@’-CH), 7.03 (d, J = 8.5 Hz, 1@’-CH).
13C NMR (75 MHz, DMSO-d6) d: 12.7, 26.1, 27.2, 28.3,
28.8, 29.0, 29.1 (7Â), 29.5, 30.6, 31.0, 31.7, 32.3, 35.4,
35.6, 37.5, 38.2, 38.7 to 40.3 (1C under solvent peaks), 43.6
(2Â), 48.2, 80.5, 111.3, 111.8, 112.7, 114.9, 116.0, 126.0,
130.5, 137.1, 141.2, 142.8, 148.4, 154.9, 174.0. HR-MS cal-
Methyl 2-{3’-[13@-(3’@, 17@’b- dihydroxy-estra-
1@’,3’@,5’@(10@’)-trien-16@’b-yl)-tridecyl]-phenyl}-acetate (75)
The deprotection step was carried out in 2 h instead of
16 h. Purification by flash chromatography (hexanes/EtOAc,
8:2) afforded 75 (105 mg, 75% yield for two steps) as a
1
white foam. IR (film) n: 3400 (OH), 1732 (C=O, ester). H
NMR (400 MHz, acetone-d6) d: 0.80 (s, 18@’-CH3), 0.95 to
2.35 (CH and CH2 of steroid skeleton and alkyl chain), 2.61
(t, J = 7.6 Hz, 1@-CH2), 2.78 (m, 6@’-CH2), 3.55 (d, J =
5.4 Hz, 17@’b-OH), 3.62 (s, CH2COOCH3), 3.65 (s,
COOCH3) 3.73 (dd, J1 = 9.7 Hz, J2 = 5.6 Hz 17@’a-CH),
6.54 (d, J = 2.4 Hz, 4@’-CH), 6.60 (dd, J1 = 8.4 Hz, J2 =
2.6 Hz, 2@’-CH), 7.12 (m, 1@’-CH, 2’-CH, 4’-CH and 6’-CH),
7.23 (t, J = 7.4 Hz, 5’-CH), 7.97 (s, 3@’-OH). 13C NMR
(75 MHz, acetone-d6) d: 12.9, 27.0, 28.2, 28.9, 29.1 to 30.1
(8C under solvent peaks), 30.4, 30.6, 32.1, 32.5, 33.2, 36.2,
38.5, 39.3, 41.1, 41.2, 44.8 (2Â), 49.4, 51.7, 82.1, 113.3,
115.7, 126.8, 127.2, 127.5, 128.9, 130.0, 131.9, 135.1,
138.2, 143.6, 155.7, 172.1. LR-MS calculated for
C40H62NO4 [M+NH4]+ 620.5, found 620.6 m/z.
culated for C40H60NO4 [M
618.45148 m/z.
+
H]+ 618.45169, found
4-{3’-amino-5’-[13@-(3’@,17@’b-dihydroxy-estra-
1@’,3’@,5’@(10@’)-trien-16@’b-yl)-tridecyl]-phenyl}-butanoic
acid (16)
Yellowish foam (43 mg, 66% yield). IR (KBr) n:
3600À2300 (OH, carboxylic acid), 3384 (OH and NH2),
1
1707 (C=O, carboxylic acid). H NMR (400 MHz, DMSO-
d6) d: 0.65 (s, 18@’-CH3), 0.80 to 2.10 (CH and CH2 of ste-
roid skeleton, alkyl chain and CH2CH2COOH), 2.18 (t, J =
7.3 Hz, CH2COOH), 2.36 (m, 1@-CH2, CH2CH2CH2COOH),
2.69 (m, 6@’-CH2), 3.55 (d, J1 = 9.5 Hz, 17@’a-CH), 4.41 (sbr,
17@b-OH), 4.87 (sbr, NH2), 6.14 (s, 6’-CH), 6.19 (s, 2’-CH
and 4’-CH), 6.43 (d, J = 2.2 Hz, 4@’-CH), 6.50 (dd, J1 =
8.3 Hz, J2 = 2.3 Hz, 2@’-CH), 7.03 (d, J = 8.5 Hz, 1@’-CH).
13C NMR (100 MHz, DMSO-d6) d: 12.7, 26.1, 26.3, 27.2,
28.4, 28.8, 29.0, 29.1 (5Â), 29.2, 29.3, 29.5, 31.0, 31.8,
32.3, 33.2, 34.7, 35.4, 37.6, 38.2, 38.9 to 40.2 (1C under sol-
vent peaks), 43.6, 43.7, 48.2, 80.5, 111.5, 111.7, 112.7,
115.0, 116.4, 126.1, 130.6, 137.2, 141.9, 142.7, 148.5,
General procedure to synthesize 14À17
To
a
solution of esters 72À75 (40À68 mg,
0.066À0.113 mmol) in acetone (2.0À3.5 mL) was added
LiOH (1 mol/L in H2O, 2.0À3.5 mL). The mixture was re-
fluxed for 1 h and quenched by addition of an aqueous sol-
ution of HCl (1 N) until it reached pH 7. Then, a saturated
NaHCO3 aqueous solution was added, and extraction was
performed with EtOAc. The organic phase was washed with
brine, dried over MgSO4, filtered, and evaporated to dry-
ness. The crude product was purified by flash chromatogra-
Published by NRC Research Press

´
´
Berube and Poirier
1197
154.9, 174.5. HR-MS calculated for C41H62NO4 [M + H]+
632.46734, found 632.46672 m/z.
1.2 equiv., and the reaction mixture was stirred for 1 h at
40 8C instead of 3 h. Purification by flash chromatography
(hexanes/EtOAc, 7:3) gained 77 (156 mg, 88% yield) as col-
1
2-{3’-[13@-(3’@,17@’b-dihydroxy-estra-1@’,3’@,5’@(10@’)-trien-
16@’b-yl)-tridecyl]-phenyl}-acetic acid (17)
ourless oil. IR (film) n: 3390 (OH), 1732 (C=O, ester). H
NMR (400 MHz, acetone-d6) d: 1.69 (m, 2 Â CH2), 2.46
(m, CH2ÀC:C), 3.54 (m, OH), 3.61 (m, CH2OH), 3.66 (s,
CH2COOCH3), 7.29 (m, 2’-CH, 4’-CH, 5’-CH, and 6’-CH).
13C NMR (100 MHz, acetone-d6) d: 19.5, 26.0, 32.8, 40.8,
52.0, 61.9, 81.1, 91.0, 125.0, 129.2, 129.5, 130.6, 133.1,
135.7, 171.5. LR-MS calculated for C15H19O3 [M + H]+
247.1, found 247.1 m/z.
White gummy solid (44 mg, 66% yield). IR (KBr)
n: 3600À2400 (OH, carboxylic acid), 3400 (OH), 1708
1
(C=O, carboxylic acid). H NMR (400 MHz, DMSO-d6) d:
0.65 (s, 18@’-CH3), 0.80 to 2.30 (CH and CH2 of steroid skel-
eton and alkyl chain), 2.51 (m, 1@-CH2 under solvent peaks),
2.70 (m, 6@’-CH2), 3.50 (s, CH2COOH), 3.55 (d, J1 = 9.5 Hz,
17@’a-CH), 6.43 (d, J = 2.3 Hz, 4@’-CH), 6.50 (dd, J1
=
Synthesis of 2-[3’-(6@-hydroxyhex-1@-ynyl)-phenyl]-acetic
acid (78)
8.4 Hz, J2 = 2.4 Hz, 2@’-CH), 7.06 (m, 1@’-CH, 2’-CH, 4’-CH
and 6’-CH), 7.19 (t, J = 8.0 Hz, 5’-CH). 13C NMR (75 MHz,
DMSO-d6) d: 12.7, 26.1, 27.2, 28.3, 28.7, 28.9, 29.0 (4Â),
29.1, 29.2, 29.5, 29.6, 31.1, 31.7, 32.3, 35.1, 37.5, 38.2, 38.7
to 40.3 (1C under solvent peaks), 41.0, 43.59, 43.63, 48.2,
80.5, 112.7, 114.9, 126.0, 126.4, 126.7, 128.1, 129.3, 130.5,
135.1, 137.1, 142.2, 154.9, 172.8. HR-MS calculated for
C39H57O4 [M + H]+ 589.4179, found 589.4202 m/z.
The procedure that we described previously30 for the syn-
thesis of 5 was used without modification except that the re-
action mixture was stirred at reflux for 4 h instead of 5 h.
Purification by flash chromatography (DCM/MeOH, 97:3 to
70:30) afforded 78 (128 mg, 94% yield) as a white viscous
oil. IR (film) n: 3700À2200 (OH, carboxylic acid), 3322
1
(OH), 3420 (OH), 1711 (C=O, carboxylic acid). H NMR
Synthesis of 3-[13’-(3@, 17@b- dihydroxy-estra-1@,3@,5@(10@)-
trien-16@b-yl)-tridecyl]-aniline (18)
(400 MHz, CDCl3 and few drops of MeOH-d4) d: 1.67 (m,
2 Â CH2), 2.41 (t, J = 6.7 Hz, CH2ÀC:C), 3.54 (s,
CH2COOH), 3.64 (t, J = 6.1 Hz, CH2OH), 7.22 (m, 2’-CH,
4’-CH, 5’-CH, and 6’-CH). 13C NMR (100 MHz, acetone-d6)
d: 19.1, 24.9, 31.7, 40.9, 62.1, 80.7, 90.1, 124.1, 128.4,
128.6, 130.1, 132.4, 134.2, 174.2. LR-MS calculated for
C14H15O3 [M À H]À 231.1, found 231.0 m/z.
Compound 71 (133 mg, 0.180 mmol) was dissolved in a
minimum amount of DCM (~0.5 mL). Then, a solution of
concentrated HCl in MeOH (5% v/v, 3.6 mL) was added.
The mixture was stirred for 1 h at room temperature. The re-
action was quenched by addition of H2O, followed by evap-
oration of MeOH. Then, a saturated solution of NaHCO3 was
added, and extraction was performed with EtOAc. The or-
ganic phase was washed with brine, dried over MgSO4, fil-
tered, and evaporated to dryness. Purification by flash
chromatography (hexanes/EtOAc, 8:2 to 7:3) afforded the
deprotected product (79 mg, 81%) as a white foam. This
compound was then hydrogenated, using the hydrogenation
procedure described previously for the synthesis of 12, ex-
cept that 15% w/w of 10% Pd/C were used instead of 20%
w/w, to afford 18 (49 mg, 67% yield) as a white foam. IR
Synthesis of 2-[3’-(6@-hydroxyhexyl)-phenyl]-acetic acid
(79)
The general hydrogenation procedure described for the
synthesis of 13 was used without modification. Purification
by flash chromatography (DCM/MeOH, 95:5 to 70:30) pro-
vided 79 (72 mg, 75% yield) as a white solid. IR (film) n:
3700À2400 (OH, carboxylic acid), 3398 (OH), 1711 (C=O,
1
carboxylic acid). H NMR (400 MHz, CDCl3 and few drops
of MeOH-d4) d: 1.32 (m, 2 Â CH2), 1.52 (t, J = 6.9 Hz.
CH2CH2OH), 1.60 (t, J = 7.1 Hz, 5@-CH2), 2.58 (t, J =
7.6 Hz, 6@-CH2), 3.57 (t, J = 6.6 Hz, CH2OH), 3.58 (s,
CH2COOH), 7.11 (m, 2’-CH, 4’-CH, and 6’-CH), 7.22 (t, J =
7.4 Hz, 5’-CH). 13C NMR (100 MHz, acetone-d6) d: 25.4,
28.8, 31.1, 32.4, 35.6, 41.1, 62.7, 126.4, 127.2, 128.4, 129.4,
133.8, 143.0, 175.2. HR-MS calculated for C14H21O3 [M +
H]+ 237.14852, found 237.14839 m/z.
1
(film) n: 3366 (OH and NH2). H NMR (400 MHz, acetone-
d6) d: 0.80 (s, 18@-CH3), 0.95 to 2.35 (CH and CH2 of steroid
skeleton and alkyl chain), 2.47 (t, J = 7.5 Hz, 1’-CH2), 2.78
(m, 6@-CH2), 3.58 (d, J = 5.4 Hz, 17@b-OH), 3.73 (dd, J1 =
9.7 Hz, J2 = 5.4 Hz, 17@a-CH), 4.50 (s, NH2), 6.43 (d, J =
7.4 Hz, 6-CH), 6.47 (dd, J1 = 7.9 Hz, J2 = 1.4 Hz, 4-CH),
6.52 (s, 2-CH), 6.54 (d, J = 2.5 Hz, 4@-CH), 6.60 (dd, J1 =
8.4 Hz, J2 = 2.6 Hz, 2@-CH), 6.95 (t, J = 7.7 Hz, 5-CH),
7.11 (d, J = 8.4 Hz, 1@-CH), 8.00 (s, 3@-OH). 13C NMR
(75 MHz, acetone-d6) d: 12.9, 27.0, 28.1, 28.9, 29.1 to 30.2
(8C under solvent peaks), 30.4, 30.6, 32.1, 32.5, 33.1, 36.4,
38.5, 39.3, 41.1, 44.7 (2Â), 49.3, 82.1, 112.4, 113.3, 115.1,
115.7, 117.5, 126.8, 129.4, 131.9, 138.2, 144.0, 148.9,
Enzymatic assay: inhibition of 17b-HSD1 in
homogenated cells
This enzymatic assay was performed as previously
described.37 Briefly, HEK-293 cells transfected with a 17b-
HSD1 cDNA fragment were sonicated in 50 mmol/L sodium
phosphate buffer (pH 7.4), containing 20% glycerol and
1 mmol/L EDTA to obtain cellular fragmentation. The cyto-
sol fraction containing the enzyme was isolated as the super-
natant after centrifugation (100 000g, 5 min, 4 8C). The
enzymatic reaction was performed at 37 8C for 2 h in 1 mL
of a solution, which included 870 mL of 50 mmol/L sodium
phosphate buffer (pH 7.4, 20% glycerol and 1 mmol/L
EDTA), 100 mL of 1 mmol/L NADH in phosphate buffer,
10 mL of 10 mmol/L [14C]-estrone in ethanol (54 mCi/mmol,
American Radiolabeled Chemicals Inc., St-Louis, MO, USA),
155.7. HR-MS calculated for C37H56NO2 [M
+
H]+
546.43056, found 546.43092 m/z.
Synthesis of monosubstrate 79
Synthesis of methyl 2-[3’-(6@-hydroxyhex-1@-ynyl)-phenyl]-
acetate (77)
The Sonogashira coupling procedure described previ-
ously30 for the synthesis of 49 was used without modifica-
tion except that 2 equiv. of 76 were used instead of
Published by NRC Research Press

1198
Can. J. Chem. Vol. 87, 2009
(6) Moeller, G.; Adamski, J. Mol. Cell. Endocrinol. 2006, 248 (1-
2), 47À55. doi:10.1016/j.mce.2005.11.031. PMID:16413960.
(7) Poirier, D. Drug Dev. Res. 2008, 69 (6), 304À318. doi:10.
1002/ddr.20263.
10 mL of the indicated inhibitor dissolved in ethanol, and
10 mL of diluted enzymatic source in phosphate buffer.
Each inhibitor was assessed in duplicate (Table 1) or in
triplicate (Table 2). To obtain the IC50 values, each inhibi-
tor was assessed in duplicate at 0.001, 0.01, 0.03, 0.06,
0.1, 0.3, 0.6, 1, 10, and 30 mmol/L. Afterward, radiolabeled
steroids were extracted twice from the reaction mixture by
1 mL of diethyl ether. The organic phases were pooled and
evaporated to dryness with nitrogen. Residues were
dissolved in 50 mL of DCM, applied on silica gel 60 F254
thin-layer chromatography plates (EMD Chemicals Inc.,
Gibbstown, NJ, USA), and eluted with a mixture of toluene/
acetone (4:1). Substrate ([14C]-E1) and metabolite ([14C]-E2)
were identified by comparison with reference steroids and
quantified using the Storm 860 system (Molecular Dynam-
ics, Sunnyvale, CA, USA). The percentage of transformation
of [14C]-E1 into [14C]-E2 was calculated as follows: % trans-
formation = 100 Â [[14C]-E2 / ([14C]-E2 + [14C]-E1)], and
subsequently, % inhibition = 100 Â [(% transformation with-
out inhibitor À % transformation with inhibitor) / % trans-
formation without inhibitor]. The IC50 values were
calculated using an unweighted iterative least-squares
method for four-parameters logistic curve fitting (DE50 pro-
´
¨
(8) Martel, C.; Rheaume, E.; Takahashi, M.; Trudel, C.; Couet,
J.; Luu-The, V.; Simard, J.; Labrie, F. J. Steroid Biochem.
Mol. Biol. 1992, 41 (3-8), 597À603. doi:10.1016/0960-
0760(92)90390-5. PMID:1314080.
(9) Poirier, D.; Ciobanu, L. C.; Maltais, R. Exp. Opin. Ther. Pa-
tents 1999, 9 (8), 1083À1099. doi:10.1517/13543776.9.8.
1083.
(10) Hanson, S. R.; Best, M. D.; Wong, C. H. Angew. Chem. Int.
Ed. 2004, 43 (43), 5736À5763. doi:10.1002/anie.200300632.
(11) Reed, M. J.; Purohit, A.; Woo, L. W. L.; Newman, S. P.;
Potter, B. V. L. Endocr. Rev. 2004, 26 (2), 171À202.
doi:10.1210/er.2004-0003. PMID:15561802.
(12) Vermeulen, A.; Deslypere, J. P.; Paridaens, R.; Leclercq, G.;
Roy, F.; Heuson, J. C. Eur. J. Cancer Clin. Oncol. 1986, 22
(4), 515À525. doi:10.1016/0277-5379(86)90121-5. PMID:
3015631.
(13) Geisler, J.; Berntsen, H.; Lonning, P. E. J. Steroid Biochem.
Mol. Biol. 2000, 72 (5), 259À264. doi:10.1016/S0960-
0760(00)00036-4. PMID:10822015.
(14) Miyoshi, Y.; Ando, A.; Shiba, E.; Taguchi, T.; Tamaki, Y.;
Noguchi, S. Int. J. Cancer 2001, 94 (5), 685À689. doi:10.
1002/ijc.1525. PMID:11745463.
´
gram, CHUL Research Center, Quebec, QC, Canada).
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Supplementary data
12570693.
Supplementary data (1H NMR spectra and 13C NMR spec-
tra of compounds tested as inhibitors of 17b-HSD1) for this
article are available on the journal Web site (canjchem.nrc.
ca) or may be purchased from the Depository of Unpub-
lished Data, Document Delivery, CISTI, National Research
Council Canada, Ottawa, ON K1A 0R6, Canada. DUD
3985. For more information on obtaining material, refer to
cisti-icist.nrc-cnrc.gc.ca/cms/unpub_e.shtml.
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Acknowledgements
This work was supported by the Canadian Institutes of
Health Research (CIHR) (D.P. for an operating grant, and
M.B. for a scholarship). We thank Dr. Van Luu-The for pro-
viding HEK-293 cells overexpressing 17b-HSD1. We are
grateful to Fatima Kamal for chemical synthesis of inter-
mediate compound 19 and to Julie Lefebvre for performing
some of the enzymatic assays. We also thank Ms. Sylvie
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