Preparation of 16β-estradiol derivative libraries as bisubstrate inhibitors of 17β-hydroxysteroid dehydrogenase type 1 using the multidetachable sulfamate linker

Article abstract of DOI:10.3390/molecules15031590

Combinatorial chemistry is a powerful tool used to rapidly generate a large number of potentially biologically active compounds. In our goal to develop bisubstrate inhibitors of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) that interact with both th

Full text of DOI:10.3390/molecules15031590

Molecules 2010, 15, 1590-1631; doi:10.3390/molecules15031590
OPEN ACCESS
molecules
ISSN 1420–3049
www.mdpi.com/journal/molecules
Article
Preparation of 16β-Estradiol Derivative Libraries as Bisubstrate
Inhibitors of 17β-Hydroxysteroid Dehydrogenase Type 1 Using the
Multidetachable Sulfamate Linker
Marie Bérubé, Florian Delagoutte and Donald Poirier *
Laboratory of Medicinal Chemistry, CHUQ (CHUL), Research Center and Laval University, Quebec,
G1V 4G2, Canada
* Author to whom correspondence should be addressed; E-Mail: donald.poirier@crchul.ulaval.ca.
Received: 26 January 2010; in revised form: 8 February 2010 / Accepted: 3 March 2010 /
Published: 10 March 2010
Abstract: Combinatorial chemistry is a powerful tool used to rapidly generate a large number
of potentially biologically active compounds. In our goal to develop bisubstrate inhibitors of
17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) that interact with both the substrate
(estrone or estradiol) and the cofactor (NAD(P)H) binding sites, we used parallel solid-phase
synthesis to prepare three libraries of 16β-estradiol derivatives with two or three levels of
molecular diversity. From estrone, we first synthesized a sulfamate precursor that we loaded
on trityl chloride resin using the efficient multidetachable sulfamate linker strategy recently
developed in our laboratory. We then introduced molecular diversity [one or two amino
acid(s) followed by a carboxylic acid] on steroid nucleus by Fmoc peptide chemistry. Finally,
after a nucleophilic cleavage, libraries of 30, 63 and 25 estradiol derivatives were provided. A
library of 30 sulfamoylated estradiol derivatives was also generated by acidic cleavage and its
members were screened for inhibition of steroid sulfatase. Biological evaluation on
homogenated HEK-293 cells overexpressing 17β-HSD1 of the estradiol derivatives carrying
different oligoamide-type chains at C-16 first revealed that three levels of molecular diversity
(a spacer of two amino acids) were necessary to interact with the adenosine part of the
cofactor binding site. Second, the best inhibition was obtained when hydrophobic residues
(phenylalanine) were used as building blocks.

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Keywords: Solid-phase synthesis; sulfamate linker; steroid; inhibitor; 17β-HSD
1. Introduction
17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1), also called human estradiol dehydrogenase
[E.C.1.1.1.62], is a protein comprised of 327 amino acids that exists as a homodimer of 35 kDa [1]. This
very important steroidogenic enzyme catalyzes the last step in the biosynthesis of estradiol (E2), the most
potent estrogen [2–4]. For this purpose, this enzyme uses the cofactor NAD(P)H to stereoselectively
reduce the C17 ketone of estrone (E1) into a 17β-alcohol, as illustrated in Figure 1. Because E2
concentration has been found to be significantly higher in breast tumours than in normal breast tissue [5,6]
17β-HSD1 is an interesting therapeutic target for estrogen-sensitive diseases such as breast cancer.
Figure 1. 17β-HSD1 uses the cofactor NAD(P)H to transform estrone (E1) into estradiol (E2), the
most potent estrogen.
Estrogenic
activity
H
N
NH2
N
H
O
OH
OH
HO
O
N
O
N
HO
HO
O
H
O
O
H
H
17β-HSD1
N
O
N
P
P
O
H
H
H
+
O
H
H
HO
OR
ER
HO
HO
E1
E2
Cofactor NADH (R = H)
Cofactor NADPH (R = PO3H2)
Although 17β-HSD1 activity was reported 50 years ago [7,8], no significant progress was made to
obtain active inhibitors as drugs [9–14]. Among the known inhibitors [9] the bisubstrate category is
promising because these inhibitors interact with both the substrate (E1) and the cofactor (NAD(P)H)
binding sites. In collaboration with Dr. S.X. Lin, our research group developed the first bisubstrate
inhibitors of 17β-HSD1 by linking E2 and adenosine with an alkyl side-chain spacer. With a spacer of
eight methylenes, EM-1745 (1a, Figure 2) was found to be the best bisubstrate inhibitor of that series
[15,16]. EM-1745 (1a) was also co-crystallized with the enzyme and X-ray analysis revealed that 1a
interacts with both the substrate (E2) and the cofactor (adenosine part of NADH) enzyme binding sites
[17]. The amine and the alcohol groups on the adenosine moiety of 1a form hydrogen bonds with Asp65
and Ser11, respectively while the steroid nucleus forms hydrogen bonds with His221, Ser142 and Tyr155.
Simplified bisubstrate inhibitors were also developed by our research group in order to improve the
stability and the bioavailability of 1a [18,19]. The carboxylic acid derivatives 1b were found to be the best
inhibitors of that series. In order to explain the high potential of compound 1b as inhibitors of 17β-HSD1,
we hypothesized that, as a bioisostere of a phosphate, the carboxylic acid functional group should form
hydrogen bonds with one or more of the same amino acids interacting with the phosphate of NADPH
(Ser11, Arg37 and Lys195) [19–21]. More recently, a new generation of hybrid inhibitors of 17β-HSD1
was reported by Sterix [22–24] and by us [25]. Lawrence et al. [22–24] have reported potent inhibitors of

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Molecules 2010, 15
17β-HSD1 structurally composed of a pyridyl moiety linked by an amide at C16 position of E1
(compounds 2a). Using the GOLD docking program, they determine that the steroid moiety interacts with
the substrate-binding site while the pyridyl moiety forms interactions with the nicotinamide part of the
cofactor. On the other hand, we reported a new family of 16β-E2 derivatives (compounds 2b) [25] having
a m-carbamoylbenzyl moiety that interacts with the enzyme as demonstrated by the 3D analysis of the
ternary (17β-HSD1/2b/NADP) complex [26]. Taken together, the results discussed above indicate that the
enzyme contains important amino acids, which could be targeted with an appropriate side-chain added at
position 16β of E2.
Figure 2. Bisubstrate hybrid inhibitors 1a and 1b, simplified inhibitors 2a and 2b, and general
structure 3 of proposed inhibitors of 17β-HSD1.
In order to improve the affinity of the bisubstrate inhibitors 1a and 1b for 17β-HSD1, a series of E2
derivatives was designed. Thus, the 16β-methylene side-chain of bisubstrate inhibitor 1 was replaced by
functional groups that potentially interact with the left part (nicotinamide and spacer) of the cofactor-

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Molecules 2010, 15
binding site. On the other hand, an amine and/or a carboxylic acid as functional group to form interactions
with the right part (adenosine) of the cofactor-binding site was added at the spacer end. In fact, the amine
group on the adenosine moiety of 1a and the carboxylic acid function on 1b were found to be important
for the interactions with the cofactor-binding site of this enzyme. To rapidly provide a large number of
16β-E2 derivatives of general structure 3 for structure activity relationship (SAR) study, parallel solid-
phase synthesis was used. Among the resins and the linkers known for preparing libraries of E2 derivatives
by solid-phase chemistry [27] the linker sulfamate on trityl chloride resin developed by our research group
was chosen [28,29]. This multidetachable type linker can generate a phenol or a sulfamate derivative
depending on cleavage conditions as presented in Figure 3. It could be used for the synthesis of steroidal
and non-steroidal compounds, thus adding to its potential. Furthermore, this linker has already been used
to successfully prepare several libraries of E2 derivatives, sulfamoylated or not [30–32]. The members of
these libraries provided 17β-HSD1 inhibitors or steroid sulfatase potential inhibitors, respectively [32].
We report herein the preparation of one library of sulfamoylated E2 derivatives (library A: 30 members)
and three libraries of E2 derivatives (libraries B, C and D: 30, 63 and 25 members, respectively).
Biological evaluation of members of libraries B and C on homogenated HEK-293 cells overexpressing
17β-HSD1 will also be presented and discussed. Finally, the chemical problems met during the
preparation of library D will be discussed and solutions will be proposed.
Figure 3. Versatility of the multidetachable sulfamate linker for generating sulfamate and phenol
derivatives by acidic or nucleophilic cleavage, respectively.
Sulfamate derivatives
Rx
as prodrugs, inhibitors of
steroid sulfatase and
carbonic anhydrase
O
S
H2N
O
O
1) Loading
on trityl resin
Rx
O
S
R
O
S
N
H
O
H2N
O
Phenol derivatives as
inhibitors of 17β-HSDs,
ER antagonists and other
therapeutic targets
2) Introduction
of molecular
diversity
O
O
Rx
HO
2. Results and Discussion
New bisubstrate inhibitors of 17β-HSD1 that should interact with the substrate binding site and the left
(nicotinamide and spacer) and the right (adenosine) part of the cofactor binding site were designed and
prepared by parallel solid-phase synthesis. One or two amino acid building blocks were introduced at
position C16β of the E2 scaffold linked on solid support by the sulfamate linker, followed by the addition
of a carboxylic acid building block. After removal of the protective group and nucleophilic cleavage,
peptidosteroids with the general structure 3 were generated. The preparation of peptidosteroids was
chosen since side chain of natural and non-natural amino acids provides an interesting pool of
functionalities. Furthermore, amino acid coupling procedures are well known for solid-phase synthesis
and the Fmoc coupling strategy could be easily used with the sulfamate linker.

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Molecules 2010, 15
2.1. Synthesis of steroidal solid-phase precursors 6a and 6b in solution (Scheme 1)
Before the preparation of libraries A-D, compounds 6a and 6b were synthesized in solution as
presented in Scheme 1. Azide intermediates 4a and 4b were prepared as reported previously in 8 and 9
steps, respectively, from commercially available E1 [33]. Since amino acids will be introduced using the
Fmoc-coupling procedure, a Fmoc on the aminopropyl side chain at position C16β was selected as an
appropriate protective group. For this purpose, azides 4a and 4b were first reduced using hydrogen
catalyzed by Pd/C. The C3 silylated protective group was next removed using TBAF in THF. Compounds
5a and 5b were subsequently obtained by selective protection of the amine with a Fmoc group. Treatment
of 5a and 5b in DCM with sulfamoyl chloride [34] in the presence of a non-nucleophilic base, 2,6-di-t-
butyl-4-methylpyridine, provided solid-phase precursors 6a and 6b in 4 and 3 steps (82% and 52% overall
yield, respectively) from 4.
Scheme 1. Synthesis of solid-phase precursors 6a and 6b.
OR
OR
N3
E1
R'O
PGO
4a (R = Ac, R' = TBDMS);
4b (R = THP, R' = H)
a, c, d (for 5a)
b, c, d (for 5b)
OR
OR
NHFmoc
NHFmoc
e
O
H2N S O
O
HO
6a (R = Ac); 6b (R = THP)
5a (R = Ac); 5b (R = THP)
Reagents and conditions: (a) H2, 5% Pd/C, EtOAc, MeOH, rt, 2 h (for 4a); (b) H2, 5% Pd/C, MeOH, rt, 3 h
(for 4b); (c) TBAF, THF, 0 °C, 30 min (for 4a); (d) Fmoc-OSu, NaHCO3, THF/H2O (3:1, v/v), 0 °C, 1 h (for
5a and 5b); (e) NH2SO2Cl, 2,6-di-t-butyl-4-methylpyridine, DCM, rt, 16–24 h (6a: 82% for 4 steps; 6b: 52%
for 3 steps).
2.2. Synthesis of carboxylic acid building blocks 14–18 (Scheme 2)
For library C members, capping with an amine functional group was chosen to interact with the
cofactor (adenosine) binding site. For this purpose, aniline derivatives were chosen. In order to obtain
optimal interactions with the cofactor-binding site of the enzyme, carboxylic acid with several alkyl

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Molecules 2010, 15
spacer lengths (n = 0 to 3 methylenes) was chosen. Aniline derivatives 9–10 with a spacer of two
methylenes were not commercially available, but were prepared easily in one step from 4-aminocinnamic
acid or 3-nitrocinnamic acid, respectively, as previously reported [35]. In order to avoid polymerisation
during the capping coupling step on solid-phase organic synthesis, free anilines 7–11 were protected as
Fmoc using FmocOSu and NaHCO3 in a mixture THF/H2O (5:1) to provide 14–18 in good yields
(51–93%). It is noteworthy to mention that Fmoc-aniline derivatives 12–13 were commercially available.
Scheme 2. Synthesis of N-Fmoc protected carboxylic acid building blocks 14–18.
H2N
NHFmoc
a
(CH2)nCOOH
7 (4'-NH2, n = 1)
8 (3'-NH2, n = 1)
9 (4'-NH2, n = 2)
10 (3'-NH2, n = 2)
11 (4'-NH2, n = 3)
(CH2)nCOOH
12 (4'-NHFmoc, n = 0)
13 (3'-NHFmoc, n = 0)
14 (4'-NHFmoc, n = 1)
15 (3'-NHFmoc, n = 1)
16 (4'-NHFmoc, n = 2)
17 (3'-NHFmoc, n = 2)
18 (4'-NHFmoc, n = 3)
Commercially
available
Reagents and conditions: (a) Fmoc-OSu, NaHCO3, THF/H2O (5:1, v/v), rt, 16 h (51–93%).
2.3. Solid-phase synthesis of libraries A, B and C (Scheme 3)
A library of 30 sulfamoylated E2 derivatives (A), a library of 30 E2 derivatives (B), and a library of 63
E2 derivatives (C) were prepared by parallel solid-phase synthesis using the multidetachable linker
sulfamate. Precursor 6a was first loaded on trityl chloride resin. For this reaction, trityl chloride resin was
swelled in dry DCM and treated with 6a and diisopropylethylamine (DIPEA) in a peptide flask. After 16 h
of shaking, the reaction mixture was filtered and washed with DCM and MeOH to obtain resin 19. The
loading yield of 19 was calculated by the increase of the resin weight. This yield was 70% for libraries A
and B and 42% for library C. A lower loading yield was obtained for library C because 1 equivalent of 6a
was used for 2 equivalents of trityl chloride resin instead of 1 equivalent of resin used in the preparation of
libraries A and B. On a model library with a loading of 75%, completion of the coupling reaction was very
difficult for the introduction of the third level of molecular diversity. It was hypothesized that steric
hindrance could be responsible for the lower reactivity of the amine on the steroid. Therefore, less
precursor 6a was loaded on resin when more than two levels of molecular diversity needed to be
introduced on the steroid. In the next step, resin 19 was treated for 1 h with a freshly prepared solution of
20% piperidine in DCM to remove the Fmoc protective group and to free the amine for the next step. It is
noteworthy to mention that after each solid-phase organic step, the resin was washed with the appropriate
solvent and dried under a vacuum. Furthermore, the solid-phase reactions were monitored by a mini-
cleavage test of a random sampling of resin with 5% TFA in DCM.

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Molecules 2010, 15
The resin 20 was next split into 30 equal portions for libraries A and B and 63 equal portions for library
C. The resins were then placed in bottom fritted reaction vessels of a 96 solid-phase reaction block of an
ACT-Labtech semi-automated synthesizer. The first level of molecular diversity (Ri) was introduced on
each resin 20 with one of a selection of Fmoc-protected amino acids from L series. Fmoc-L-Phe-OH,
Fmoc-L-Ile-OH, Fmoc-L-Pro-OH, Fmoc-L-Ala-OH and Fmoc-Gly-OH were used as building blocks for
libraries A and B, and Fmoc-L-Phe-OH, Fmoc-L-4-NO2-Phe-OH and Fmoc-Gly-OH were chosen as
building blocks for library C. The coupling reaction was performed for 2 to 3 h with DIPEA in DMF using
PyBOP and HOBt as coupling reagents. The Fmoc protective group was then removed as described above
to afford resins 22 from 21. Introduction of a second level of molecular diversity (n = 2) on resins 22
using the same procedure and Fmoc-L-amino acids (Phe, 4-NO2-Phe and Gly) was carried on for library
C. Removal of Fmoc group then provided resins 24 from 23. For members of both libraries, a second or a
third level of molecular diversity (Rj) was introduced using a selection of carboxylic acids as building
blocks (4-pyridylacetic acid, benzoic acid, acid 14, 2-pyrazinecarboxylic acid, 3-aminopyrazine-2-
carboxylic acid, and indole-2-carboxylic acid for libraries A and B, and carboxylic acids 12–18 for library
C). These carboxylic acids were preactivated with PyBrOP and HOBt. The reaction was carried on for 2
to 3 h with DIPEA in DMF. Then, resins 25 (n = 1 or 2) that were prepared with carboxylic acids 12–18
as building blocks were treated with a solution of 20% piperidine in DCM in order to remove the Fmoc
protective group. Finally, the C17-acetate protective group of resins 26 and 27 was removed by treatment
with MeONa in MeOH and THF to afford resins 28.
The last step was to release the phenolic and the sulfamate derivatives from the solid support by either
a nucleophilic or an acid treatment, respectively. The nucleophilic cleavage was done first because the
phenolic derivatives are not totally released from the solid support under these conditions. Furthermore, E2
derivatives are preferred since building blocks were chosen to target 17β-HSD1. Thus, resins 28 were
treated with a solution of 30% diethylamine (DEA) in THF at room temperature for 24 to 66 h to release
E2 derivatives 59–88 (library B) and 89–151 (library C). After the nucleophilic treatment, the resin was
washed with acetone or THF and MeOH for libraries B and C, respectively. The filtrate was evaporated.
The crude product was dissolved in EtOAc or THF and evaporated again. This procedure was done twice
in order to provide DEA-free E2 derivatives. The acid cleavage was performed on resins 28 that were
previously treated by a nucleophilic cleavage for 24 h to generate library B. In fact, there were still
steroids loaded on resins 28 after the nucleophilic cleavage. Thus, these resins 28 were treated with a
solution of 30% hexafluoroisopropanol (HFIP) in DCM for 6 h at room temperature. Evaporation of the
solvent afforded sulfamoylated E2 derivatives 29–58 (library A). The acid cleavage was not carried on
with 5% TFA in DCM to avoid the formation of C17 trifluoroacetate derivative as previously reported in
the preparation of a 17α-substituted E2 sulfamate library [31].

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Molecules 2010, 15
Scheme 3. Preparation of libraries A, B and C.
Libraries A and B
CH3
H
e
Ri*
Rj
a
b
c**
h
d
N
N
H2N
N
N
NH2
N
H
N
f
g
j
k
i
*
Synthesis from NH-Fmoc protected L-amino acids; ** Residue of proline.
Library C
Ri*
NO2
H
l
m
n
NH2
NH2
NH2
NH2
NH2
NH2
NH2
Rj**
o
p
q
r
s
t
u
*
Synthesis from NH-Fmoc protected L-amino acids; ** Synthesis from NH-Fmoc protected carboxylic acids (12-18).
Reagents and conditions: (a) Trityl chloride resin, DIPEA, DCM, rt, 12–16 h (libraries A and B: loading of
70%, library C: loading of 42%); (b) 20% piperidine/DCM (v/v), rt, 1 h; (c) PyBOP, HOBt,
RiCH(NHFmoc)COOH, DIPEA, DMF, rt, 2–3 h; (d) PyBrOP, HOBt, RjCOOH or 14 (libraries A and B), 12–
18 (library C), DIPEA, DMF, rt, 2–3 h; (e) 1M MeONa/MeOH : THF (25/75, v/v) rt, 48–100 h; (f) 30%
DEA/THF v/v, rt, 24–66 h (library B: 59–88, 10–63% for 6 or 7 solid-phase steps, library C: 89–151, 6–41%
for 9 solid-phase steps); (g) 30% HFIP/DCM, rt, 6 h (library A: 29–58, 35–53% for 6 or 7 solid-phase steps).

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Molecules 2010, 15
2.4. Characterization of libraries A, B and C (Tables 1–3)
Sulfamoylated E2 derivatives of library A and E2 derivatives of libraries B and C were submitted to a
random sampling after we confirmed the presence of a major compound by TLC analysis. Six members of
library A, six members of library B and 14 members of library C were then characterized by 1H-NMR and
LRMS. In addition, the purity of the sampling of libraries A and B was determined by HPLC analysis.
1
Expected masses matched for all these compounds. Furthermore, H-NMR analysis confirmed the
structure of the library members. The HPLC-purity of final compounds ranged from 89 to 91% and 85 to
95% for libraries A and B, respectively. The average HPLC-purity was 90% for both libraries. Results
were very satisfactory because no purification step was performed after the final cleavage. The excess of
reagent (HFIP and DEA) and solvent were simply removed by evaporation. The average overall yields for
the solid-phase sequence of reactions (6 or 7 steps for libraries A and B, and 9 steps for library C) were
46%, 27% and 31%, respectively. The average overall yield was calculated after the loading step of
precursor 6a or 6b on trityl chloride resin. The mass of phenols released from solid-support by
nucleophilic cleavage to provide library B was not removed in the calculation of the overall yield of
1
library A. The purity of library C members was estimated to be around 70–75% by TLC and H-NMR
analysis. The lower purity of library C members could be explained by the final deprotection step on
solid-phase. In fact, resins 27 were treated with a solution of MeONa in MeOH and THF for
approximately 100 h instead of 48 h in order to complete the reaction. However, this long treatment
resulted in degradation of part of the product. Nevertheless, the purity of library C members was found
sufficiently satisfactory for screening assay on 17β-HSD1.
Table 1. Characterization of members from library A (sulfamoylated E2 derivatives 29–58).
OH
O
H
N
Rj
N
H
Ri
O
O
H2N S O
O
LRMS [M + H]+ (m/z)
Overall yield
HPLC purity
(%)
Compounds Ria Rja
calculated
measured
(%)b
50
45
46
53
46
45
45
42
39
50
47
39
47
53
29
30
31
32
33
34
35
36
37
38
39
40
41
42
a
a
a
a
a
a
b
b
b
b
b
b
c
c
f
g
h
i
j
k
f
g
h
i
j
k
f
660.3
660.4
89
628.3
625.3
628.4
625.4
90
89
g

1599
Molecules 2010, 15
Table 1. Cont.
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
c
c
c
c
d
d
d
d
d
d
e
e
e
e
e
e
h
i
j
k
f
g
h
i
j
k
f
g
h
i
47
49
51
53
51
49
38
50
47
43
49
42
42
42
35
41
613.3
601.3
613.3
601.3
91
90
j
k
609.3
609.2
89
a See Scheme 3 for the chemical structure of R building blocks; Crude overall yields
b
calculated for the solid-phase sequence of 6 or 7 steps.
Table 2. Characterization of members from library B (E2 derivatives 59–88).
OH
O
H
N
Rj
N
H
Ri
O
HO
LRMS [M + H]+ (m/z)
Overall yield
HPLC purity
(%)
Compounds
Ria Rjb
calculated
measured
(%)b
50
35
26
63
35
39
23
26
42
18
22
15
41
37
46
43
12
47
29
21
14
13
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
a
a
a
a
a
a
b
b
b
b
b
b
c
c
c
c
c
c
d
d
d
d
f
g
h
i
j
k
f
g
h
i
j
k
f
g
h
i
j
k
f
g
h
i
610.4
610.4
85
564.4
564.5
95
533.3
505.3
533.4
505.5
91
90

1600
Molecules 2010, 15
Table 2. Cont.
81
82
83
84
85
86
87
88
d
d
e
e
e
e
e
e
j
k
f
g
h
i
16
13
28
22
12
13
10
14
544.3
506.3
544.5
506.5
89
88
j
k
a See Scheme 3 for the chemical structure of R building blocks; b Crude overall yields calculated
for the solid-phase sequence of 6 or 7 steps.
Table 3A. Characterization of members from library C (E2 derivatives 89–123)
OH
O
Ri''
O
H
N
N
N
Rj
H
H
Ri'
O
HO
LRMS [M + H]+ (m/z)
Overall yield
Compounds
Ri' Ri'' Rja
calculated
measured
(%)b
27
28
22
25
25
26
28
36
38
30
28
33
30
30
18
30
27
26
30
28
32
34
40
23
32
35
35
37
39
41
89
90
91
92
93
94
95
96
97
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
L
L
L
L
L
L
L
M
M
M
M
M
M
M
N
N
N
n
o
p
q
r
s
t
u
o
p
q
r
563.3
563.3
589.3c
589.5c
98
99
665.4c
698.3
665.5c
698.2
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
s
t
u
o
p
q
r
l
l
n
s
l
n
t
l
n
l
l
l
l
l
l
l
u
o
p
q
r
s
t
u
o
p
740.4
653.4
740.5
653.2
m
m
m
m
m
m
m
m
m
681.4
743.4
681.5
743.2
m
m

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Molecules 2010, 15
Table 3A. Cont.
119
120
121
122
123
m
m
m
m
m
m
m
m
m
m
q
r
s
t
31
35
35
38
38
771.4
771.6
u
a See Scheme 3 for the chemical structure of R building blocks. b Crude overall yields calculated for the
solid-phase sequence of 9 steps. c [M-H]-.
Table 3B. Characterization of members from library C (E2 derivatives 124–151).
OH
O
Ri''
O
H
N
N
N
Rj
H
H
Ri'
O
HO
LRMS [M + H]+ (m/z)
Overall yield
Compounds
Ri' Ri'' Rja
calculated
measured
(%)b
38
31
32
33
34
34
37
31
40
26
28
30
28
34
34
36
29
32
28
33
36
33
6
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
m
m
m
m
m
m
m
n
n
n
n
n
n
n
n
l
o
p
q
r
s
t
u
o
p
q
r
802.4
802.4
n
n
n
l
l
l
712.4
712.5
n
l
s
n
l
t
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
l
u
o
p
q
r
m
m
m
m
m
m
m
n
n
n
n
n
n
n
s
t
816.4
816.4
u
o
p
q
r
s
t
u
828.4c
828.3c
26
30
28
28
31
847.4
847.3
a See Scheme 3 for the chemical structure of R building blocks.b Crude overall yields calculated for the
solid-phase sequence (9 steps). c [M-H]-.

1602
Molecules 2010, 15
2.5. Chemical synthesis of compound 156 used in the preparation of library D (Scheme 4)
The capping groups of members from library D have a carboxylic acid as functional group to interact
with the cofactor (adenosine) binding site of 17β-HSD1. In fact, among the simplified bisubstrate
compounds prepared to inhibit this enzyme, carboxylic acid derivatives were found to be by far the best
inhibitors of that series [36]. The introduction of the capping building block on solid-phase was done by a
reaction between an amine and a carboxylic acid. Indeed, the capping synthon 156 is a dicarboxylic acid
and one of the COOH group has to be appropriately protected. The tert-butyl ester was chosen as
protective group because it could be easily removed in acid conditions after the nucleophilic cleavage.
Thus, starting from commercially available 2-(3-bromophenyl)-acetic acid (152), the carboxylic acid was
protected with di-tert-butyl dicarbonate and dimethylaminopyridine (DMAP) in tert-butanol to afford tert-
butyl ester 153 in an excellent 94% yield [37]. A vinyl was next added on aryl bromide 153 by a Stille
reaction using mild conditions (tributylvinyl tin, Pd2(dba)3, P(t-Bu)3 in toluene) to afford 154 in 88% yield
[38]. Hydroboration of vinyl 154 using standard condition provided alcohol 155. This alcohol was
oxidized under Jones’ conditions in order to give carboxylic acid 156. However, the reaction conditions
were too drastic and afforded the dicarboxylic acid product instead of 156. A 2-step procedure was then
necessary to avoid hydrolysis of the tert-butyl ester. Thus, alcohol 155 was first oxidized with Dess-
Martin periodinane to provide the aldehyde, which was subsequently oxidized in carboxylic acid using
mild conditions (NaClO2, NaH2PO4 and 2-methyl-2-butene in t-BuOH). Thus, carboxylic acid 156 was
synthesized in five steps and 26% overall yield.
Scheme 4. Synthesis of carboxylic acid building block 156.
a
b
COOH
COOtBu
COOtBu
Br
Br
152
153
154
c
d, e
COOtBu
HOOC
COOtBu
HO
156
155
Reagents and conditions: (a) BOC2O, DMAP, t-BuOH, rt, 24 h (94%); (b) tributyl(vinyl)tin, Pd2(dba)3,
P(t-Bu)3 (10% w/v in hexanes), toluene, rt, 16 h (88%); (c) i. BH3.THF, THF, 0 °C, 3 h; ii. NaOAc
(4M), H2O2 (30% w/v), 0 °C, 2 h (45%); (d) Dess-Martin periodinane, DCM, rt, 1 h (78%); (e)
NaClO2, NaH2PO4, H2O, 2-methyl-2-butene, t-BuOH, rt, 15 min (88%).

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Molecules 2010, 15
2.6. Solid-phase synthesis of library D (Scheme 5)
The preparation of library D required a series of Fmoc-protected amino acids with a functionalised side
chain as building blocks instead of a hydrophobic one. Therefore, these amino acids needed an appropriate
protective group. A tert-butyl ester and a trityl group were chosen as protective groups of the amino acid
side chain since they could be easily removed in acid conditions as for the tert-butyl ester found on the
capping building block. Furthermore, the molecular diversity was limited to the introduction of two amino
acids. The same carboxylic acid 156 was introduced on each member of library D. Thus, focus was put on
the interactions with the left part (nicotinamide and the spacer) of the cofactor-binding site. Library D was
prepared as previously described for libraries B and C. Briefly, sulfamate precursor 6b was loaded on
trityl chloride resin. The loading yield of 6b on the resin was 40%. Since three levels of diversity were
introduced on the steroid scaffold, 1 equivalent of precursor 6b was used for 2 equivalents of trityl
chloride resin as for library C. The two levels of molecular diversity were then introduced by coupling
Fmoc-protected amino acids on resin 158. Fmoc-L-Phe-OH, Fmoc-L-4-Pal-OH or Fmoc-L-Ala-OH,
Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Ser(Trt)-OH and Fmoc-L-Tyr(2-Cl-Trt)-OH were selected as building
blocks for both levels. The carboxylic acid 156 was introduced as a capping group on resins 162 in DMF
and DIPEA, using PyBrOP and HOBt as coupling reagent. Removal of the C17-THP and the trityl
protective group in one step on resin 163 was tried but without success. Thus, the trityl and the 2-chloro-
trityl were removed by two 10-minute treatments with a solution of TFA/TIS(triisopropylsilane)/ DCM
(2:1:97). The C17-THP protective group was next taken off with a solution of p-TSA in DCM and t-
BuOH. Phenols were then released from the solid support under nucleophilic conditions (30% DEA/THF)
for 65 h. Next, tert-butyl ester of compounds 165 was hydrolysed under acid conditions. Reaction of
esters 165 with a solution of HCl (4M) in dioxane for 3 h was found to give the best conditions of
hydrolysis. Evaporation of the reaction mixture provided carboxylic acids 166–190. However, these
carboxylic acids were not pure enough for screening assay on 17β-HSD1. For this purpose, crude
carboxylic acids (dried for at least 16 h under a vacuum) were dissolved in MeOH and preadsorbed on C-
18 silica gel. They were subsequently purified by flash chromatography on C18-silica gel (reverse phase)
with a mixture of H2O and MeOH as eluent.
2.7. Characterization of library D (Table 4) and discussion
1
All products were characterized by LRMS and H-NMR and the results are presented in Table 4. The
molecular ion peak was found in LRMS for all expected compounds (except for compounds 174 and 181)
1
and H-NMR analysis reveals the presence of the steroid nucleus and the molecular diversity. However,
only six compounds were obtained as carboxylic acid derivatives. In fact, the presence of numerous peaks
in the region of 3.4 to 3.7 ppm in the 1H-NMR revealed that methyl ester contaminated the carboxylic acid
products in different proportions for several compounds. Furthermore, a peak corresponding to
[M+H+14]+ or [M-H+14]- was found in almost all LRMS spectra. We found that the methyl ester was
produced during the preabsorption process on silica gel using MeOH as solvant. Even if the carboxylic

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Molecules 2010, 15
acids were dried under a vacuum for 16 h after the hydrolysis step, traces of HCl trapped in the product
seem to be enough to promote the formation of the methyl ester. Unfortunately, the carboxylic acid
function was very important for the interactions with the cofactor (adenosine) - binding site. For this
reason, carboxylic acids 166–190 were not tested as inhibitors of 17β-HSD1.
The average overall yield for the preparation of library D (9 solid-phase steps and 1 solution-phase
step) was 16% after purification. The lower overall yield was generally obtained with serine derivatives,
which seem to be less tolerant under acid conditions during the final deprotection step. In fact, an
important proportion of a less polar compound was observed but could not be identified.
Scheme 5. Preparation of library D.
OTHP
O
OTHP
NHR
N
H
NHR
R1
a
6b
O
S
c
O
S
N
H
O
N
H
O
159 (R = Fmoc)
160 (R= H)
b
O
O
157 (R = Fmoc)
158 (R = H)
b
c
OTHP
O
R2
H
N
OTHP
O
R2
O
H
N
N
H
NH
R
COOtBu
N
H
N
H
O
R1
O
R1
O
S
d
O
S
163
N
H
O
161 (R = Fmoc)
162 (R= H)
N
H
O
b
O
O
e, f
OH
O
R2
O
H
N
COOtBu
N
H
N
H
OH
O
R2
O
H
N
O
R1
COOtBu
g
N
H
N
H
O
164
O
N
H
S
O
R1
O
165
HO
h
OH
R1* and R2*
CH3
N
OH
O
R2
O
H
N
COOH
OH
COOH
N
H
N
H
O
R1
a
b2***
c
e
b1**
d
HO
*
Synthesis from NH-Fmoc protected L-amino acids building blocks;
166-190 (Library D: 5 X 5 = 25 phenols)
** Only for R1 ; *** Only for R2
Reagents and conditions: (a) Trityl chloride resin, DIPEA, DCM, rt, 16 h (loading: 40%); (b) 20%
piperidine/DCM (v/v), rt, 1 h; (c) PyBrOP, HOBt, R1CH(NHFmoc)COOH, DIPEA, DMF, rt, 4 h; (d)
PyBrOP, HOBt, 156, DIPEA, DMF, rt, 4 h; (e) TFA/TIS/DCM (2:1:97), rt, 2 × 10 min; (f) p-TSA, t-
BuOH, DCM, rt, 24 h; (g) 30% DEA/THF v/v, rt, 65 h; (g) HCl (4M in dioxane), rt, 3 h (2–33% for 8
solid-phase steps and 1 solution-phase step).
The main problem during the preparation of library D occurred with the hydrolysis of the tert-butyl
ester. After the nucleophilic cleavage step, the products were obtained with a good purity estimated to be
1
higher than 80% by TLC analysis of all members and H-NMR analysis of two compounds (165: R1 = a
and R2 = e; 165: R1 = c and R2 = d). The choice of protective groups for this library was based on the fact

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Molecules 2010, 15
that trityl and tert-butyl ester could be easily removed with TFA/H2O/TIS (95:2.5:2.5) and the product
obtained should precipitate in diethyl ether as described in the note from the NovaBiochem catalogue.
Even if this procedure works well for peptide synthesis, the acid conditions were too drastic for our steroid
derivatives, resulting in decomposition of the product. Knowing this fact, a new chemical strategy could
be used for the preparation of library D. The main difference would be the structure of the building block
for the capping. Instead of using a tert-butyl ester to protect the carboxylic acid, a methyl ester could be
used. It is obvious that aspartic acid used as a building block should also be protected as a methyl ester.
Thus, the solid-phase synthesis would be very similar to the preparation of library D and the methyl ester
would be hydrolysed at the last solid-phase step, using a solution of NaOH in THF. This procedure was
previously described for the hydrolysis of an amino triflate loaded on solid support by the sulfamate
linker.31 Using this method, or alternatively a benzyl ester as protective group, library D members should
be obtained in good purity without any purification.
Table 4. Characterization of members from library D (E2 derivatives 166–190).
OH
O
R2
O
H
N
COOH
N
N
H
H
O
R1
HO
Overall
yield
Note on
LRMS [M + H]+ (m/z)
1H-NMR analysis
a
a
Compounds R1
R2
calculated
800.4
measured
(%)b
18
26
4
7
17
29
166
167
168
169
170
a
a
a
a
a
a
b2
c
d
e
a
814.3c
ester
722.4d
766.4d
738.4d
816.4
722.4d
mix of ester and acid
766.7d
acid
mix of ester and acid
acid
mix of ester and acid
mix of ester + acid +
impurities
ester
738.4d + 752.4c
816.3
171
b1
801.4
723.4d
801.3 + 815.4c
172
b1
b2
723.5d + 737.6c
26
173
174
175
176
177
178
b1
b1
b1
c
c
c
c
d
e
a
b2
c
767.4
741.4
817.4
766.4d
690.4d
734.3d
781.5c
not found
831.4c
22
0
16
26
2
impurities
ester
780.5c
ester
690.4d
mix of acid + impurities
acid
734.4d
28
mix of ester + acid +
impurities
ester
179
c
d
708.3
708.1 + 722.1c
796.5c
not found
676.4c
10
180
181
182
c
d
d
e
a
b2
782.4d
740.4
662.4d
20
0
4
impurities
ester
mix of acid +
impurities
acid
183
184
d
d
c
706.3d
678.3d
706.3d
678.3d
19
3
d

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Molecules 2010, 15
Table 4. Cont.
754.4d + 768.4c
816.3 + 830.3c
738.3d + 752.4c
782.5d + 796.4c
185
186
187
188
d
e
e
e
e
754.4d
816.4
5
mix of ester and acid
mix of ester and acid
mix of ester and acid
mix of ester and acid
a
21
33
31
b2
c
738.4d
782.4d
189
190
e
e
d
e
754.4d
830.4d
754.3d
7
18
acid
acid
830.4d
a See Scheme 5 for the chemical structure of R building blocks. b Overall yields calculated for the solid
phase sequence of nine steps after C-18 silica gel purification. c M+14 ; d LRMS [M-H]-
2.8. Inhibition of steroid sulfatase by sulfamoylated E2 derivatives from library A
The inhibitory potential of the members of library A on the steroid sulfatase was tested and the results
were presented in our early report [30]. A sulfamate on position C3 of the steroid is an essential functional
group to obtain a potent steroid sulfatase inhibition [39]. Among the 30 sulfamoylated E2 derivatives of
this library, compound 34 gave a better inhibition than estrone sulfamate (EMATE), a well known and
potent inhibitor of steroid sulfatase [40]. The polar building blocks were however chosen to target 17β-
HSD1 instead of steroid sulfatase, the inhibitory result could thus be better when using hydrophobic
groups as elements of molecular diversity. In fact, potent inhibitors of steroid sulfatase were obtained
when hydrophobic groups were added on the C16- or C17-position of E2 sulfamate [30–32].
2.9. Inhibition of 17β-HSD1 by E2 derivatives from libraries B and C
Inhibition of the transformation of [14C]-E1 into [14C]-E2 by members from libraries B and C was tested
on homogenated human embryonic kidney (HEK)-293 cells transfected with a vector encoding for 17β-
HSD1 according to a previously reported procedure [15,41]. Briefly, the enzymatic assay was conducted
for 2 h at 37 °C in a sodium phosphate buffer (pH = 7.4) with NADH as cofactor.
The screening assay on 17β-HSD1 for members of library B (Table 2) is presented in Figure 4. There
are two levels of molecular diversity in this library. Hydrophobic amino acids were mainly chosen for the
first level to avoid the protective group on amino acid side chains. Pyridine, phenyl, aniline, pyrazine, 3-
aminopyrazine and indole nucleus were selected for the second level of molecular diversity. These
capping building blocks were chosen to mimic the adenosine moiety found in EM-1745 (1), one of the
most potent inhibitors of 17β-HSD1 [20]. For library B, the best inhibitions were obtained when
phenylalanine was used as a building block for the first level with 16 to 57% at 1 µM for compounds 59–
64. In general, lower inhibitions were obtained with isoleucine and proline (compounds 65–76) and almost
no inhibition was observed when alanine and glycine were used as building blocks for the first level
(compounds 77–88). For the second level of molecular diversity, better inhibitions were usually obtained
with pyrazine, 2-aminopyrazine and indole moieties. However, these compounds weren’t more potent

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Molecules 2010, 15
inhibitors than unlabeled-E1 (59% at 1 µM) and EM-251 (52% at 1 µM), a known inhibitor of 17β-HSD1
used as reference compound [42]. They were also no more potent than EM-1745 (1, 97% at 1 µM).
Figure 4. Inhibition of the transformation of [14C]-E1 (0.1 µM) into [14C]-E2 in homogenated HEK-
293 cells overexpressing 17β-HSD1 by E2 derivatives of library B (compounds 59–88) at 1 µM. The
letters a-k represent the residue (Ri and Rj) on side chain (see Scheme 3). See experimental section
for more details on this enzymatic assay.
OH
O
H
100
N
Rj
N
H
Ri
O
HO
90
80
70
60
50
40
30
20
10
k
j
i
h
Rj
0
g
a
b
f
c
d
e
Ri
Therefore, in order to improve the inhibitory potency of library B members, another library was
prepared. The members of library C have longer spaces between the C16β E2 and the polar capping
building block. This way, the capping was able to interact with the cofactor (adenosine) binding site.
Fmoc-L-Phe-OH and Fmoc-L-4-NO2-Phe-OH were chosen as building blocks for the first and second
level of molecular diversity because members of library B with a phenylalanine as building block gave the
best inhibitors. Glycine was also chosen but should give less potent inhibitors. Aniline derivatives
(compounds 12–18) were selected as building blocks for the third level of molecular diversity. In fact, the
aniline should form interactions with Asp65 such as the amine of the adenosine moiety of EM-1745 does
in the cofactor-binding site [21]. Members of library C were tested to determine their potential to inhibit
the enzyme activity at 1 µM and the results are presented in Figure 5. Three points can be highlighted
from these results. First, better inhibitions were generally obtained with three levels of molecular diversity
(library C) than with two levels (library B). In fact, the average percentage of inhibition for library B is

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Molecules 2010, 15
26% while for library C it is 56%. Second, the best inhibitors were obtained when phenylalanine was used
as a building block for the first level of molecular diversity (Ri’ = m, compounds 110–130) with more than
half of the compounds giving better inhibition than EM-251 (59% at 1 µM) and eight compounds giving a
better inhibition than unlabeled-E1 (73% at 1 µM). Good inhibitions were also obtained with 4-nitro-
phenylalanine used as building block for the first or the second level of molecular diversity. However, as
expected, lower inhibition was obtained with glycine. Third, similar inhibitions were found regardless of
which aniline building blocks were selected for the third level. Globally, 26 of the 63 members of library
C gave better inhibition than EM-251, and eight compounds (117, 119, 120, 121, 124, 126, 128 and 147)
gave a better inhibition than E1.
Figure 5. Inhibition of the transformation of [14C]-E1 (0.1 µM) into [14C]-E2 in homogenated HEK-
293 cells overexpressing 17β-HSD1 by E2 derivatives of library C (compounds 89–151) at 1 µM.
The letters l-u represent the residue (Ri’, Ri’’ and Rj) on side chain (see Scheme 3). See the
experimental section for more details on this enzymatic assay.
3. Conclusions
We have successfully used the sulfamate linker to prepare one library of sulfamoylated E2 derivatives
and two libraries of E2 derivatives by parallel solid-phase synthesis. Precursor 6a was loaded on trityl

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Molecules 2010, 15
chloride resin via the sulfamate linker and two or three levels of molecular diversity were introduced using
Fmoc-protected amino acids or carboxylic acids as building blocks. Acid (HFIP/THF) or nucleophilic
(DEA/THF) cleavage respectively released the sulfamoylated E2 derivatives (library A) or the E2
derivatives (library B) from the resin in excellent HPLC purity (90% for libraries A and B). However, E2
derivatives from library C were obtained with a lower purity (around 70–75%) estimated by TLC and 1H-
NMR analysis. Members of libraries B and C were also screened on homogenated HEK-293 cells
overexpressing 17β-HSD1. Compounds of library C, with three levels of molecular diversity, gave better
inhibition than E2 derivatives of library B. Furthermore, 26 compounds gave a better inhibition than EM-
251, a known inhibitor of 17β-HSD1 and 8 of these compounds gave a better inhibition than E1, used as
an inhibitor. In both libraries, better inhibitors where those bearing a phenylalanine as building block. The
eight top inhibitors of library C should be evaluated as pure compounds on 17β-HSD1 in order to confirm
the potential of these inhibitors. However, none of the members of both libraries gave a better inhibition
than EM-1745, one of the most potent inhibitors of this enzyme. In order to obtain more potent inhibitors,
a third library of E2 derivatives (library D) was also prepared as described above. However, Fmoc-
protected amino acids with functionalised side-chain and a protected carboxylic acid were used as
building blocks. Unfortunately, the final hydrolysis of tert-butyl ester did not work as well as expected
and methyl ester contaminated the final carboxylic acid products. The use of a methyl or a benzyl ester as
protective group instead of a tert-butyl ester is suggested as a mean to avoid the final purification step. It
is also expected that the E2 derivatives reported in library D will more closely mimic the EM-1745/17β-
HSD1 interactions than those reported in libraries A-C, thus resulting in better 17β-HSD1 inhibition.
4. Experimental
4.1. General
Trityl chloride resin, Fmoc-OSu, coupling reagents and Fmoc amino acids were supplied by EMD
Biosciences (Novabiochem, La Jolla, CA, USA) or Advanced ChemTech (Louisville, KY, USA). Other
reagents were obtained from Sigma-Aldrich Canada Co. (Oakville, ON, Canada). Usual solvents were
obtained from Fisher Scientific (Montréal, QC, Canada) 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. Tetrahydrofuran (THF) was distilled from
sodium/benzophenone under argon. Solution-phase reactions were performed under positive argon
pressure in oven-dried glassware with magnetic stirring bars. Fritted peptide synthesis vessels (peptide
flasks, 25 or 50 mL) equipped for vacuum filtration (ChemGlass Inc, Vineland, NJ) were used for the
solid-phase synthesis on a Burrell wrist-action shaker model 75 (Burrell, Pittsburgh, PA). The libraries of
steroid derivatives were realized with ACT LabTech manual synthesizer (Advanced ChemTech,
Louisville, KY, USA) using either a 40 or a 96 solid-phase reaction block. Thin-layer chromatography
(TLC) was performed on 0.25-mm silica gel 60 F254 plates (Whatman, Maidstone, England), and
compounds were visualized by exposure to UV light (254 nm), with a solution of ammonium

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Molecules 2010, 15
molybdate/sulphuric acid/water (with heating) or with a solution of p-anisaldehyde (with heating). Flash
chromatography was performed on Silicycle 60 (Québec, QC, Canada) 230–400 mesh silica gel. The
purity of a random sampling of libraries A and B members was determined by HPLC (Waters Associates,
Milford, MA, USA) using a NovaPak C18 reversed-phase column (150 × 3.9 mm id) and an ultra-violet
detector (205 or 210 nm). The eluent for HPLC was a mixture of 50% of MeOH/H2O (90:10) and 50% of
H2O, both containing 20 mM of NH4OAc. IR spectra were obtained neat, from KBr pellet or from a thin
film of the solubilized compound on NaCl pellet (usually in CH2Cl2). They were recorded on a Perkin-
Elmer series 1600 FT-IR spectrometer (Norwalk, CT, USA); only significant bands are reported (in cm-1).
13
1H- and C-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 (δ) are expressed in ppm and referenced to chloroform (7.26 and 77.0 ppm), acetone
(2.07 and 206.0 ppm), methyl sulfoxide (2.51 and 39.5 ppm) or methanol (3.33 and 49.0 ppm) for 1H and
13C respectively. Duplication of NMR signals was generally recorded for THP derivatives (presence of 2
stereoisomers). These 2 stereoisomers increased the complexity of 13C-NMR spectra, and additional peaks
are written in parentheses. Assignment of NMR signals was made easier using literature data [43]. Low-
resolution mass spectra (LRMS) were recorded with an LCQ Finnigan apparatus (San Jose, CA, USA)
equipped with an atmospheric pressure chemical ionisation (APCI) source on positive or negative mode.
4.2. Chemical synthesis
4.2.1. 3-Sulfamoyloxy-16β-[3-(9H-fluoren-9-ylmethoxycarbonylamino)-propyl]-17β-acetoxy-estra-
1,3,5(10)-triene (6a)
The starting material 3-(3-tert-butyldimethylsilyloxy-17β-acetoxy-estra-1,3,5(10)-trien-16β-yl)-azido-
propane (4a) was synthesized as previously reported [33]. A suspension of 4a (680 mg, 1.32 mmol) and
5% Pd/C (136 mg) in EtOAc (15 mL) and MeOH (160 mL) was stirred under a hydrogen atmosphere at
room temperature. After 2 h, the resulting suspension was filtered through Celite, washed with MeOH and
evaporated to dryness to afford the amine (620 mg, 96%) in the form of a viscous colourless oil in good
purities without purification. To a solution of the crude amine (620 mg) in anhydrous THF (40 mL) under
an argon atmosphere at 0 °C was added a solution of TBAF (1M in THF, 1.53 mL, 1.53 mmol). After
30 min, the reaction was quenched by addition of a saturated aqueous solution of NaHCO3. The crude
product was extracted with EtOAc and the organic phase was washed with brine, dried over MgSO4 and
evaporated to dryness to afford the phenol (585 mg) in the form of a yellowish solid. The crude phenol
(500 mg) was dissolved in THF (120 mL) and H2O (40 mL). At 0 °C, NaHCO3 (1M in H2O, 1.84 mL,
1.84 mmol) and Fmoc-OSu (570 mg, 1.69 mmol) were added and the reaction was stirred for 1 h at 0 °C.
Then, the reaction mixture was quenched by addition of H2O and the crude product was extracted with
EtOAc and DCM. Each organic phase was washed with brine, dried over MgSO4, and evaporated to
dryness to provide 5a (1.05 g) in the form of a white foam. To a solution of crude phenol 5a (1.05 g) in
dry DCM (120 mL) under an argon atmosphere were added 2,6-di-t-butyl-4-methylpyridine (933 mg,

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Molecules 2010, 15
4.55 mmol) and NH2SO2Cl [34] (421 mg, 3.64 mmol). The reaction was stirred for 24 h at room
temperature and quenched by the addition of ice water. The crude product was extracted with DCM and
the organic phase was washed with brine, dried over MgSO4, and evaporated to dryness. Purification by
flash chromatography (hexanes/EtOAc, 6:4) gained precursor 6a (604 mg, 82% yield for 4 steps) in the
form of a white foam. IR (film) 3393 (NH and NH2), 1716 (C=O, ester and carbamate), 1374 and 1188
1
(S=O, sulfamate); H-NMR (300 MHz, CDCl3) δ 0.80 (s, 18-CH3), 1.00 to 2.35 (m, CH and CH2 of
steroid skeleton and alkyl chain), 2.07 (s, CH3CO), 2.84 (m, 6-CH2), 3.18 (m, CH2NH), 4.20 (t, J = 6.8
Hz, CH2CH of Fmoc), 4.38 (d, J = 6.9 Hz, CH2CH of Fmoc), 4.73 (d, J = 10.0 Hz, 17α-CH), 4.76 (m,
NHFmoc), 5.01 (sbr, NH2), 7.01 (s, 4-CH), 7.05 (d, J1 = 8.6 Hz, J2 = 2.3 Hz, 2-CH), 7.28 (m, 1-CH and 2 ×
CH of Fmoc), 7.38 (t, J = 7.4 Hz, 2 × CH of Fmoc), 7.57 (d, J = 7.4 Hz, 2 × CH of Fmoc), 7.75 (d, J = 7.4
13
Hz, 2 × CH of Fmoc); C-NMR (75 MHz, CDCl3) δ 13.3, 21.0, 25.9 (2×), 27.0, 28.8, 29.5, 32.1, 37.5,
37.6, 38.2, 41.1, 43.4, 43.9, 47.3, 48.7, 66.6, 83.2, 118.9, 120.0 (2×), 121.9, 125.0 (2×), 126.8, 127.0 (2×),
127.7 (2×), 138.8, 139.5, 141.3 (2×), 144.0 (2×), 147.9 156.4, 171.2; LRMS calculated for C38H43N2O7S
[M-H]- 671.3, found 671.1 m/z.
4.2.2. 3-Sulfamoyloxy-16β-[3-(9H-fluoren-9-ylmethoxycarbonylamino)-propyl]-17β-(tetrahydro-2H-
pyran-2-yl-oxy)-estra-1,3,5(10)-triene (6b)
3-(3-Hydroxy-17β-(tetrahydro-2H-pyran-2-yl-oxy)-estra-1,3,5(10)-trien-16β-yl)-azidopropane
(4b)
was synthesized as previously reported [33]. A suspension of 4b (2.35 g, 5.35 mmol) and 5% Pd/C (470
mg) in MeOH (535 mL) was stirred under hydrogen atmosphere at room temperature. After 3 h, the
resulting suspension was filtered through celite, washed with MeOH and evaporated to dryness to afford
the amine (2.00 g) in good purities without purification. The crude amine (2.00 g) was dissolved in THF
(360 mL) and H2O (120 mL). At 0 °C, NaHCO3 (1M in H2O, 6.3 mL, 6.3 mmol) and Fmoc-OSu (1.96 g,
5.81 mmol) were added and the reaction was stirred for 1 h at 0 °C. The reaction mixture was then
quenched by addition of H2O and the crude product was extracted with EtOAc and DCM. Each organic
phase was washed with brine, dried over MgSO4, and evaporated to dryness to provide 5b (3.62 g) in the
form of a white foam. To a solution of crude phenol 5b (3.62 g) in dry DCM (280 mL) under an argon
atmosphere were added di-tert-butyl-4-methylpyridine (2.92 g, 14.2 mmol) and NH2SO2Cl [34] (1.31 g,
11.4 mmol). The reaction was stirred for 16 h at room temperature and quenched by addition of icewater.
The crude product was extracted with DCM and the organic phase was washed with brine, dried over
MgSO4 and evaporated to dryness. Purification by flash chromatography (hexanes/EtOAc, 8:2 to 6:4)
provided precursor 6b (2.00 g, 52% yield for 3 steps) as a white foam. The 17β-unprotected precursor
(1.27 g) was also obtained. This product was reprotected with a THP using this procedure. The 17β-
hydroxy precursor (1.27 g, 2.07 mmol) was dissolved in a minimum of dry THF (~2 mL) and DCM (40
mL) under an argon atmosphere at 0 °C. Then, 3,4-dihydro-2H-pyran (187 µL, 2.07 mmol) and p-TSA (39
mg, 0.21 mmol) were added and the reaction was stirred for 10 min. The reaction was quenched by
addition of saturated aqueous solution of NaHCO3 and the crude precursor was extracted with DCM. The
organic phase was washed with brine, dried over MgSO4 and evaporated under reduced pressure.

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Purification by flash chromatography (hexanes/EtOAc, 7:3 to 6:4) afforded precursor 6b (1.09 g, 74%
yield). IR (film) 3392 (NH and NH2), 1698 (C=O, carbamate), 1386 and 1187 (S=O, sulfamate); 1H-NMR
(400 MHz, acetone-d6) δ 0.83 and 0.87 (2s, 18-CH3), 1.00 to 2.40 (m, CH and CH2 of steroid skeleton and
alkyl chain), 2.87 (m, 6-CH2), 3.19 (m, CH2NH), 3.47 and 3.92 (2m, OCH2 of THP), 3.76 and 3.80 (2d, J
= 10.0 Hz, 17α-CH), 4.23 (t, J = 7.1 Hz, CH2CH of Fmoc), 4.33 (d, J = 7.2 Hz, CH2CH of Fmoc), 4.64
and 4.71 (2m, CH of THP), 6.52 (NH), 7.08 (m, 2-CH, 4-CH and NH2), 7.34 (m, 1-CH and 2 × CH of
Fmoc), 7.42 (t, J = 7.4 Hz, 2 × CH of Fmoc), 7.71 (d, J = 7.5 Hz, 2 × CH of Fmoc), 7.88 (d, J = 7.5 Hz,
13
2 × CH of Fmoc); C-NMR (100 MHz, acetone-d6) δ 13.5 (14.3), 20.0 (20.5), 26.2 (26.3), 26.77, 26.84,
27.7, 29.1, 29.3 to 30.2 (1C under solvent peaks), 31.3 (31.6), 32.78 (32.82), 38.6, 38.70 (38.74), 39.0
(40.2), 41.5 (41.6), 44.2 (44.7), 44.6 (44.8), 48.0, 49.3 (49.5), 62.0 (63.0), 66.5, 86.1 (86.6), 98.2 (99.7),
120.0, 120.6 (2×), 122.8, 125.9 (2×), 127.2, 127.7 (2×), 128.5 (2×), 139.0, 139.6, 141.9 (2×), 145.0 (2×),
149.1, 156.9; LRMS calculated for C41H49N2O7S [M-H]- 713.3, found 713.3 m/z.
4.2.3. General procedure to synthesize Fmoc-NH protected carboxylic acid building blocks 14–18
Amines 9 and 10 were prepared as previously reported [35]. Amines 7–11 (0.90–1.15 g,
5.45–6.93 mmol) were dissolved in THF (50 mL) and H2O (10 mL). Then, NaHCO3 (1M in H2O, 13.6–
17.3 mL, 13.6–17.3 mmol) and Fmoc-OSu (2.76–3.51 g, 8.17–10.4 mmol) were added. The reaction was
stirred for 16 h at room temperature. The reaction mixture was then preabsorbed on silica gel and purified
by flash chromatography (DCM/MeOH, 95:5 to 85:15) to afford 14–18 (1.35–2.00 g, 51–93% yield).
2-[4’-(9H-Fluoren-9-ylmethoxycarbonylamino)-phenyl]-acetic acid (14). White solid (1.72 g, 70% yield);
1
IR (KBr) 3700–2200 (OH, COOH), 3336 (NH), 1708 (C=O, acid and carbamate); H-NMR (400 MHz,
DMSO-d6) δ 3.47 (s, CH2COOH), 4.31 (t, J = 6.5 Hz, CH2CH of Fmoc), 4.49 (d, J = 6.2 Hz, CH2CH of
Fmoc), 7.16 (d, J = 7.8 Hz, 2’-CH and 6’-CH), 7.39 (m, 3’-CH, 5’-CH and 4 × CH of Fmoc), 7.76 (d,
J = 7.3 Hz, 2 × CH of Fmoc), 7.91 (d, J = 7.6 Hz, 2 × CH of Fmoc), 9.69 (sbr, NH); 13C-NMR (75 MHz,
DMSO-d6) δ 40.3, 46.7, 65.5, 118.2 (2×), 120.2 (2×), 125.2 (2×), 127.1 (2×), 127.7 (2×), 129.5, 129.7
(2×), 137.5, 140.8 (2×), 143.8 (2×), 153.5, 173.2; LRMS calculated for C23H18NO4 [M-H]- 372.1, found
372.2 m/z.
2-[3’-(9H-Fluoren-9-ylmethoxycarbonylamino)-phenyl]-acetic acid (15). White solid (2.00 g, 81% yield);
1
IR (KBr) 3600–2200 (OH, COOH), 3287 (NH), 1702 (C=O, acid and carbamate); H-NMR (400 MHz,
DMSO-d6) δ 3.51 (s, CH2COOH), 4.32 (t, J = 6.7 Hz, CH2CH of Fmoc), 4.47 (d, J = 6.6 Hz, CH2CH of
Fmoc), 6.90 (d, J = 7.5 Hz, 6’-CH), 7.21 (t, J = 7.7 Hz, 5’-CH), 7.40 (m, 2’-CH, 4’-CH and 4 × CH of
Fmoc), 7.77 (d, J = 7.4 Hz, 2 × CH of Fmoc), 7.92 (d, J = 7.4 Hz, 2 × CH of Fmoc), 9.74 (sbr, NH), 12.37
(sbr,COOH); 13C-NMR (75 MHz, DMSO-d6) δ 41.0, 46.6, 65.6, 116.7, 119.2, 120.2 (2×), 123.6, 125.2
(2×), 127.2 (2×), 127.7 (2×), 128.6, 135.6, 139.0, 140.8 (2×), 143.8 (2×), 153.4, 172.6; LRMS calculated
for C23H20NO4 [M+H]+ 374.1, found 374.1 m/z.

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3-[4’-(9H-Fluoren-9-ylmethoxycarbonylamino)-phenyl]-propionic acid (16). White solid (1.35 g, 51%
1
yield); IR (KBr) 3500–2000 (OH, COOH), 3331 (NH), 1702 (C=O, acid and carbamate); H-NMR (400
MHz, DMSO-d6) δ 2.50 (t, J = 7.5 Hz, CH2COOH), 2.76 (t, J = 7.5 Hz, CH2CH2COOH), 4.31 (t, J = 6.5
Hz, CH2CH of Fmoc), 4.48 (d, J = 6.3 Hz, CH2CH of Fmoc), 7.13 (d, J = 7.9 Hz, 2’-CH and 6’-CH), 7.39
(m, 3’-CH, 5’-CH and 4 × CH of Fmoc), 7.76 (d, J = 7.4 Hz, 2 × CH of Fmoc), 7.91 (d, J = 7.4 Hz, 2 ×
CH of Fmoc), 9.65 (sbr, NH), 12.13 (sbr, COOH); 13C-NMR (75 MHz, DMSO-d6) δ 29.7, 35.4, 46.6, 65.5,
118.4 (2×), 120.2 (2×), 125.1 (2×), 127.1 (2×), 127.7 (2×), 128.5 (2×), 134.9, 137.0, 140.8 (2×), 143.8
(2×), 153.4, 173.8; LRMS calculated for C24H22NO4 [M+H]+ 388.1, found 388.0 m/z.
3-[3’-(9H-Fluoren-9-ylmethoxycarbonylamino)-phenyl]-propionic acid (17). White solid (1.96 g, 93%
1
yield); IR (KBr) 3500–2000 (OH, COOH), 3307 (NH), 1696 (C=O, acid and carbamate); H-NMR (400
MHz, DMSO-d6) δ 2.52 (t, J = 7.5 Hz, CH2COOH), 2.79 (t, J = 7.4 Hz, CH2CH2COOH), 4.32 (t, J = 6.6
Hz, CH2CH of Fmoc), 4.48 (d, J = 6.3 Hz, CH2CH of Fmoc), 6.87 (d, J = 7.5 Hz, 6’-CH), 7.18 (t, J = 7.7
Hz, 5’-CH), 7.40 (m, 2’-CH, 4’-CH and 4 × CH of Fmoc), 7.77 (d, J = 7.4 Hz, 2 × CH of Fmoc), 7.91 (d,
J = 7.5 Hz, 2 × CH of Fmoc), 9.70 (sbr, NH), 12.18 (sbr, COOH); 13C-NMR (75 MHz, DMSO-d6) δ 30.5,
35.2, 46.7, 65.6, 116.2, 118.2, 120.2 (2×), 122.4, 125.2 (2×), 127.2 (2×), 127.7 (2×), 128.7, 139.1, 140.8
(2×), 141.5, 143.8 (2×), 153.4, 173.7; LRMS calculated for C24H22NO4 [M+H]+ 388.1, found 388.0 m/z.
4-[4’-(9H-Fluoren-9-ylmethoxycarbonylamino)-phenyl]-butanoic acid (18). White solid (1.59 g, 68%
1
yield); IR (KBr) 3500–2000 (OH, COOH), 3345 (NH), 1708 (C=O, acid and carbamate); H-NMR (400
MHz, DMSO-d6) δ 1.77 (quintuplet, J = 7.5 Hz, CH2CH2COOH), 2.20 (t, J = 7.4 Hz, CH2COOH), 2.52
(m, CH2CH2CH2COOH), 4.31 (t, J = 6.5 Hz, CH2CH of Fmoc), 4.48 (d, J = 6.2 Hz, CH2CH of Fmoc),
7.09 (d, J = 7.9 Hz, 2’-CH and 6’-CH), 7.39 (m, 3’-CH, 5’-CH and 4 × CH of Fmoc), 7.76 (d, J = 7.4 Hz,
2 × CH of Fmoc), 7.91 (d, J = 7.5 Hz, 2 × CH of Fmoc), 9.65 (sbr, NH), 12.07 (sbr, COOH); 13C-NMR (75
MHz, DMSO-d6) δ 26.4, 33.0, 33.7, 46.7, 65.5, 118.4 (2×), 120.2 (2×), 125.2 (2×), 127.1 (2×), 127.7 (2×),
128.6 (2×), 135.6, 136.9, 140.8 (2×), 143.8 (2×), 153.5, 174.3; LRMS calculated for C25H24NO4 [M+H]+
402.2, found 402.0 m/z.
4.2.4. Synthesis of resin 20 for preparation of libraries A and B
The loading reaction was run in two 25 mL peptide flasks using the same amount of resin. In each
peptide flask, a solution of precursor 6a (1.07 g, 1.62 mmol) in dry DCM (10 mL) was added to trityl
chloride resin (1.09 g, 1.24 mmol/g theoretical loading) under an argon atmosphere. After 5 min,
diisopropylethylamine (DIPEA, 2.16 mL) was added and the mixture was shaken for 12 h at room
temperature. It is noteworthy to mention that to avoid total evaporation of DCM during the loading step,
the argon inlet was removed after 30 min of reaction. The resin was filtered and washed with DCM (3×),
MeOH (3×), and again with DCM, then dried overnight under a vacuum to afford 3.34 g of resin 19
globally. The loading yield, calculated by the mass increase, was 70%. The deprotection step was also run
in two 25 mL peptide flasks using the same amount of resin. Resin 19 (1.67 g) was swollen in 15 mL of a

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Molecules 2010, 15
solution of piperidine (20%) in DCM and shaken for 90 min at room temperature. Then, the resin was
filtered and washed with DCM (3×), MeOH (3×), and again with DCM, and dried under a vacuum for
16 h. Acidic mini-cleavage (5% TFA/DCM, 10 min) of a sample of resin 20 and TLC analysis confirmed
the complete deprotection of the secondary amine. The two batches of resin 20 prepared as described
above were mixed before the next step. Then, 30 bottom fritted reaction vessels of the 96 solid-phase
reaction block of ACT LabTech manual synthesizer were loaded with 80 mg of resin 20, which
correspond to 0.052 mmol of compound 6a (without the Fmoc protective group).
4.2.5. Introduction of two levels of molecular diversity (libraries A and B)
Five stock solutions, each containing 2 equivalents (6×) of Fmoc-protected amino acid [Fmoc-L-Phe-
OH (259 mg, 0.672 mmol), Fmoc-L-Ile-OH (236 mg, 0.672 mmol), Fmoc-L-Pro-OH (226 mg,
0.672 mmol), Fmoc-L-Ala-OH (208 mg, 0.672 mmol) or Fmoc-Gly-OH (199 mg, 0.672 mmol)],
2 equivalents (6×) of benzotriazole-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyBOP)
(348 mg, 0.672 mmol) and 2 equivalents (6×) of hydroxybenzotriazole (HOBt) (91 mg, 0.672 mmol),
were prepared in dry DMF (5.0 mL). Prior to addition to the resin, 4 equivalents (6×) of DIPEA (230 µL,
1.34 mmol) were added to each stock solution. To each of the 30 resins 20 was added 0.8 mL of the
appropriate stock solution. The resins were shaken under argon for 3 h at room temperature, then filtered,
washed with DMF (2×), MeOH (3×) and DCM (3×) and dried under a vacuum. TLC analysis after a mini-
cleavage test (5% TFA/DCM, 10 min) with samples of resins 21 revealed that the coupling reactions were
not completed. Thus, the coupling step was repeated using the same procedure as described above. This
time, TLC analysis after a mini-cleavage with sample of resins 21 indicated the completion of the
coupling reaction. Thus, the resins 21 were reacted 1 h with 1.0 mL of a solution of piperidine (20%) in
DCM to remove the Fmoc protective group. The resins were then filtered, washed with DCM (3×), MeOH
(3×) and DCM (1×), and dried under a vacuum to give 5 groups of resin of general structure 22. The
second level of molecular diversity was introduced by a similar coupling procedure described for the
introduction of the first level. Thus, 6 stock solutions, each containing 2 equivalents (5x) of carboxylic
acid building blocks [4-pyridyl-acetic acid (89 mg, 0.515 mmol), benzoic acid (63 mg, 0.515 mmol), 14
(192 mg, 0.515 mmol), 2-pyrazinecarboxylic acid (64 mg, 0.515 mmol), 3-aminopyrazine-2-carboxylic
acid (72 mg, 0.515 mmol) or indole-2-carboxylic acid (83 mg, 0.515 mmol)], 2 equivalents (5x) of bromo-
tris-pyrrolidino-phosphonium hexafluorophosphate (PyBrOP) (240 mg, 0.515 mmol) and 2 equivalents
(5x) of HOBt (70 mg, 0.515 mmol), were prepared in 4 mL of dry DMF and 4 equivalents (5x) of DIPEA
(180 µL, 1.03 mmol) was added. To each of the 30 resins 22 was added 0.8 mL of the appropriate stock
solution. The resins were shaken under an argon atmosphere for 3 h at room temperature, then filtered,
washed with MeOH (1×), DMF (2×), MeOH (3×) and DCM (2×) and dried under a vacuum. The
completion of the reaction was monitored by TLC analysis of acidic mini-cleavage of samples of resins 25
(when Rj contains a Fmoc protecting group) and 27 (when Rj contains any Fmoc protecting group). As for
the introduction of the first level of molecular diversity, the acylation step was repeated (2 times) as
described above to afford completion.

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4.2.6. Removal of protective groups (libraries A and B)
Resins 25, containing the carboxylic acid 14 as building block were treated with 1 mL of a solution of
piperidine (20%) in DCM to remove the Fmoc protective group. The mixtures were shaken for 1 h at
room temperature and then filtered, washed with DCM (2×) and MeOH (2×), and dried under a vacuum.
Completion of the reactions was confirmed by TLC analysis after a sampling mini-cleavage (5%
TFA/DCM, 10 min) of resins 26. The acetate protective group of resins 26 or 27 was removed with 1 mL
of a solution of 1 M MeONa in MeOH (25%) in THF. The mixtures were shaken for 48 h at room
temperature and then, filtered, washed with MeOH (3×) and DCM (2×), and dried under a vacuum. TLC
analysis of acidic mini-cleavage of sampling of resins 28 confirmed the complete deprotection of C17β-
alcohol.
4.2.7. Generation of E2 derivatives (library B) by nucleophilic cleavage
To each of the 30 resins 28 was added a solution of diethylamine (DEA, 30% in THF, 1.5 mL). The
mixtures were shaken for 24 h at room temperature, then filtered and washed with acetone (3×). The
filtrates were collected in preweighed tubes and evaporated in a Speedvac apparatus. Each product was
dissolved in EtOAc, evaporated twice, and dried under a vacuum pump in order to obtain the DEA-free
product. The phenol derivatives 59–88 (2.6–18.8 mg, 10–63% overall yield from 19, Table 2) were
obtained in high average HPLC purity (90%) according to a random sampling of 6 library members,
compounds 61, 69, 74, 78, 82 and 83.
16β-(N-4-Amino-phenylacetyl-L-phenylalanine-aminopropyl)-3,17β-dihydroxy-estra-1,3,5(10)-triene
1
(61). Yellowish solid (8.2 mg, 26% yield); IR (film) 3292 (OH, NH and NH2), 1641 (C=O, amide); H-
NMR (400 MHz, methanol-d4) δ 0.78 (s, 18-CH3), 0.80 to 2.35 (m, CH and CH2 of steroid skeleton and
alkyl chain), 2.77 (m, 6-CH2), 2.91 and 3.05 (2m, CHCH2Ph), 3.13 (t, J = 6.5 Hz, CH2NH), 3.37 (s,
CH2PhNH2), 3.70 (d, J = 9.9 Hz, 17α-CH), 4.55 (m, COCHNH), 6.49 (d, J = 2.5 Hz, 4-CH), 6.55 (dd,
J1 = 8.4 Hz, J2 = 2.6 Hz, 2-CH), 6.65 (d, J = 8.4 Hz, 2 × CH of aminophenyl), 6.90 (d, J = 8.4 Hz, 2 × CH
of aminophenyl), 7.08 (d, J = 8.4 Hz, 1-CH), 7.20 (m, 5 × CH of phenyl); LRMS calculated for
C38H48N3O4 [M+H]+ 610.4, found 610.4 m/z; HPLC purity = 85%.
16β-(N-3-Amino-2-pyrazinoyl-L-isoleucine-aminopropyl)-3,17β-dihydroxy-estra-1,3,5(10)-triene
(69).
Light yellow solid (6.4 mg, 22% yield); IR (film) 3312 (OH, NH and NH2), 1649 (C=O, amide); 1H-NMR
(400 MHz, methanol-d4) δ 0.74 (s, 18-CH3), 0.90 to 2.30 (m, CH and CH2 of steroid skeleton and alkyl
chain), 0.96 (t, J = 7.4 Hz, CH2CH3), 1.00 (d, J = 6.8 Hz, CHCH3), 2.77 (m, 6-CH2), 3.24 (m, CH2NH and
CHCH3), 3.69 (d, J = 9.9 Hz, 17α-CH), 4.39 (d, J = 7.6 Hz, COCHNH), 6.49 (d, J = 2.6 Hz, 4-CH), 6.55
(dd, J1 = 8.4 Hz, J2 = 2.6 Hz, 2-CH), 7.08 (d, J = 8.5 Hz, 1-CH), 7.84 (d, J = 2.4 Hz, 1 × CH of pyrazine),
8.10 (d, J = 2.4 Hz, 1 × CH of pyrazine); LRMS calculated for C32H46N5O4 [M+H]+ 564.4, found 564.5
m/z; HPLC purity = 95%.

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16β-(N-2-Pyrazinoyl-L-proline-aminopropyl)-3,17β-dihydroxy-estra-1,3,5(10)-triene (74). Yellowish
1
solid (11.7 mg, 43% yield); IR (film) 3312 (OH and NH), 1631 (C=O, amide); H-NMR (400 MHz,
methanol-d4) δ 0.70 to 2.40 (m, CH and CH2 of steroid skeleton and alkyl chain), 0.78 and 0.79 (2s,
18-CH3), 2.79 (m, 6-CH2), 3.05 and 3.23 (2m, CH2NH), 3.71 to 3.95 (m, CH2N of proline and 17α-CH),
4.60 and 5.05 (2m, CHN of proline), 6.49 (s, 4-CH), 6.55 (dd, J1 = 8.4 Hz, J2 = 2.6 Hz, 2-CH), 7.08 (d,
J = 8.5 Hz, 1-CH), 8.69 (m, 2 × CH of pyrazine), 9.05 (dd, J1 = 2.5 Hz, J2 = 1.5 Hz, 1 × CH of pyrazine);
LRMS calculated for C31H41N4O4 [M+H]+ 533.3, found 533.4 m/z; HPLC purity = 91%.
16β-(N-Benzoyl-L-alanine-aminopropyl)-3,17β-dihydroxy-estra-1,3,5(10)-triene (78). Light yellow solid
(5.4 mg, 21% yield). HPLC purity = 90% [50% of MeOH/H2O (90:10) and 50% of H2O, both containing
20 mM of NH4OAc]. The characterization of compound 78 was reported earlier by us [30].
16β-(N-1H-Indole-2-carbonyl-L-alanine-aminopropyl)-3,17β-dihydroxy-estra-1,3,5(10)-triene (82). Light
yellow solid (3.7 mg, 13% yield); IR (film) 3292 (OH and NH), 1650 (C=O, amide); 1H -NMR (400 MHz,
methanol-d4) δ 0.72 (s, 18-CH3), 0.85 to 2.30 (m, CH and CH2 of steroid skeleton and alkyl chain), 1.50
(d, J = 7.2 Hz, CHCH3), 2.75 (m, 6-CH2), 3.22 (m, CH2NH), 3.69 (d, J = 9.9 Hz, 17α-CH), 4.59 (q,
J = 7.2 Hz, CHCH3), 6.48 (d, J = 2.6 Hz, 4-CH), 6.55 (d, J = 8.4 Hz, 2-CH), 7.06 (m, 1-CH and 1 × CH of
indole), 7.20 (m, 2 × CH of indole), 7.43 (dd, J1 = 8.3 Hz, J2 = 0.8 Hz, 1 × CH of indole), 7.61 (d, J = 8.1
Hz, 1 × CH of indole); LRMS calculated for C33H42N3O4 [M+H]+ 544.3, found 544.5 m/z;
HPLC purity = 89%.
16β-(N-4-Pyridylacetyl-glycine-aminopropyl)-3,17β-dihydroxy-estra-1,3,5(10)-triene (83). White solid
1
(7.3 mg, 28% yield); IR (film) 3284 (OH and NH), 1649 (C=O, amide); H-NMR (400 MHz, methanol-
d4) δ 0.79 (s, 18-CH3), 0.80 to 2.30 (m, CH and CH2 of steroid skeleton and alkyl chain), 2.79 (m, 6-CH2),
3.23 (m, CH2NH), 3.69 (s, CH2pyridine), 3.71 (d, J = 10.0 Hz, 17α-CH), 3.86 (s, COCH2NH), 6.49 (d,
J = 2.5 Hz, 4-CH), 6.55 (dd, J1 = 8.4 Hz, J2 = 2.6 Hz , 2-CH), 7.09 (d, J = 8.6 Hz, 1-CH), 7.43 (dd,
J1 = 4.6 Hz, J2 = 1.4 Hz, 2’-CH and 6’-CH of pyridine), 8.48 (dd, J1 = 4.6 Hz, J2 = 1.6 Hz, 3’-CH and 5’-
CH of pyridine); LRMS calculated for C30H40N3O4 [M+H]+ 506.3, found 506.5 m/z; HPLC purity = 88%.
4.2.8. Generation of sulfamoylated E2 derivatives (library A) by acidic cleavage
To each of the 30 resins 28 which have already been treated by nucleophilic cleavage conditions, was
added 1 mL of a solution of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (30%) in DCM. The mixtures were
shaken for 6 h at room temperature, then filtered and washed with acetone (2×). The filtrates were
collected in preweighed tubes, evaporated in a Speedvac apparatus and dried under a vacuum pump. The
sulfamate derivatives 29–58 (10.5–18.2 mg, 35–53% overall yield from 19, Table 1) were obtained in
high average HPLC purity (90%) according to a random sampling of six library members, compounds 30,
38, 41, 49, 51 and 58.

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3-O-Sulfamate-16β-(N-benzoyl-L-phenylalanine-aminopropyl)-17β-hydroxy-estra-1,3,5(10)-triene (30).
Light yellow solid (15.4 mg, 45% yield); IR (film) 3307 (OH, NH and NH2), 1636 (C=O, amide), 1374
1
and 1184 (S=O, sulfamate); H-NMR (400 MHz, methanol-d4) δ 0.77 (s, 18-CH3), 0.95 to 2.40 (m, CH
and CH2 of steroid skeleton and alkyl chain), 2.88 (m, 6-CH2), 3.05 and 3.19 (2m, CHCH2Ph and
CH2NH), 3.72 (d, J = 9.8 Hz, 17α-CH), 4.80 (m, COCHNH), 7.02 (d, J = 2.4 Hz, 4-CH), 7.06 (dd,
J1 = 8.4 Hz, J2 = 2.6 Hz, 2-CH), 7.20 to 7.55 (m, 1-CH, 5 × CH of phenyl, and 3’-CH, 4’-CH and 5’-CH of
benzoyl), 7.76 (dd, J1 = 8.5 Hz, J2 = 1.4 Hz, 2’-CH and 6’-CH of benzoyl); LRMS calculated for
C37H46N3O6S [M+H]+ 660.3, found 660.4 m/z; HPLC purity = 89%.
3-O-Sulfamate-16β-(N-2-pyrazinoyl-L-isoleucine-aminopropyl)-17β-hydroxy-estra-1,3,5(10)-triene (38).
Light yellow solid (16.2 mg, 50% yield). HPLC purity = 90%. The characterization of compound 38 was
reported earlier by us [30].
3-O-Sulfamate-16β-(N-4-pyridylacetyl-L-proline-aminopropyl)-17β-hydroxy-estra-1,3,5(10)-triene (41).
Light yellow solid (15.0 mg, 47% yield); IR (film) 3292 (OH, NH and NH2), 1641 (C=O, amide), 1374
1
and 1184 (S=O, sulfamate); H-NMR (400 MHz, methanol-d4) δ 0.78 (s, 18-CH3), 0.80 to 2.40 (m, CH
and CH2 of steroid skeleton and alkyl chain), 2.89 (m, 6-CH2), 3.22 (t, J = 7.1 Hz, CH2NH), 3.33
(COCH2pyridine under solvent peaks), 3.70 (m, 17α-CH and CH2N of proline), 4.40 (m, CHN of proline),
7.03 (d, J = 2.4 Hz, 4-CH), 7.06 (dd, J1 = 8.5 Hz, J2 = 2.5 Hz, 2-CH), 7.34 (d, J = 6.6 Hz, 1-CH), 7.40 (dd,
J1 = 4.6 Hz, J2 = 1.6 Hz, 2’-CH and 6’-CH of pyridine), 8.47 (dd, J1 = 4.5 Hz, J2 = 1.6 Hz, 3’-CH and 5’-
CH of pyridine); LRMS calculated for C33H45N4O6S [M+H]+ 625.3, found 625.4 m/z;
HPLC purity = 89%.
3-O-Sulfamate-16β-(N-4-amino-phenylacetyl-L-alanine-aminopropyl)-17β-hydroxy-estra-1,3,5(10)-triene
(49). Light yellow solid (12.1 mg, 38% yield); IR (film) 3308 (OH, NH and NH2), 1652 (C=O, amide),
1
1374 and 1184 (S=O, sulfamate); H-NMR (400 MHz, methanol-d4) δ 0.79 (s, 18-CH3), 0.90 to 2.40 (m,
CH and CH2 of steroid skeleton and alkyl chain), 1.33 (d, J = 7.1 Hz, CHCH3), 2.89 (m, 6-CH2), 3.18 (t, J
= 7.1 Hz, CH2NH), 3.43 (s, CH2PhNH2), 3.73 (d, J = 9.8 Hz, 17α-CH), 4.30 (q, J = 7.2 Hz, CHCH3), 6.70
(dd, J1 = 6.4 Hz, J2 = 2.0 Hz, 2 × CH of aminophenyl), 7.04 (m, 2-CH, 4-CH and 2 × CH of
aminophenyl), 7.35 (d, J = 8.6 Hz, 1-CH); LRMS calculated for C32H45N4O6S [M+H]+ 613.3, found 613.3
m/z; HPLC purity = 91%.
3-O-Sulfamate-16β-(N-3-amino-pyrazinoyl-L-alanine-aminopropyl)-17β-hydroxy-estra-1,3,5(10)-triene
(51). Light yellow solid (14.5 mg, 47% yield); IR (film) 3346 (OH, NH and NH2), 1646 (C=O, amide),
1
1375 and 1186 (S=O, sulfamate); H-NMR (400 MHz, methanol-d4) δ 0.77 (s, 18-CH3), 0.95 to 2.40 (m,
CH and CH2 of steroid skeleton and alkyl chain), 1.47 (d, J = 7.1 Hz, CHCH3), 2.88 (m, 6-CH2), 3.23 (m,
CH2NH), 3.72 (d, J = 9.8 Hz, 17α-CH), 4.54 (q, J = 7.1 Hz, CHCH3), 7.02 (d, J = 2.4 Hz, 4-CH), 7.06
(dd, J1 = 8.6 Hz, J2 = 2.5 Hz, 2-CH), 7.33 (d, J = 8.6 Hz, 1-CH), 7.86 (d, J = 2.4 Hz, 1 × CH of pyrazine),

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Molecules 2010, 15
8.13 (d, J = 2.4 Hz, 1 × CH of pyrazine); LRMS calculated for C29H41N6O6S [M+H]+ 601.3, found 601.3
m/z; HPLC purity = 90%.
3-O-Sulfamate-16β-(N-1H-indole-2-carbonyl-glycine-aminopropyl)-17β-hydroxy-estra-1,3,5(10)-triene
58). White solid (13.0 mg, 41% yield); IR (film) 3292 (OH, NH and NH2), 1636 (C=O, amide), 1374 and
1
1186 (S=O, sulfamate); H-NMR (400 MHz, methanol-d4) δ 0.75 (s, 18-CH3), 0.95 to 2.35 (m, CH and
CH2 of steroid skeleton and alkyl chain), 2.88 (m, 6-CH2), 3.26 (m, CH2NH), 3.71 (d, J = 9.9 Hz, 17α-
CH), 4.05 (s, COCH2NH), 7.02 (d, J = 2.5 Hz, 4-CH), 7.06 (m, 2-CH and 1 × CH of indole), 7.14 (d,
J = 0.8 Hz, 1 × CH of indole), 7.22 (dt, J1 = 8.0 Hz, J2 = 1.2 Hz, 1 × CH of indole), 7.33 (d, J = 8.4 Hz, 1-
CH), 7.44 (dd, J1 = 8.3 Hz, J2 = 0.8 Hz, 1 × CH of indole), 7.62 (d, J = 8.1 Hz, 1 × CH of indole); LRMS
calculated for C3 2H4 1 N4 O6 S [M+H]+ 609.3, found 609.2 m/z; HPLC purity = 89%.
4.2.9. Synthesis of resin 20 for preparation of library C
The loading procedure described above for the preparation of libraries A and B was used without any
significant modifications. Briefly, trityl chloride resin (10.1 g, 1.50 mmol/g theoretical loading) and
precursor 6a (5.11 g, 7.6 mmol) were put in a round 250 mL dry flask equipped with a septum and an
argon inlet. DCM (100 mL) was added and the reaction mixture was stirred for 3 min. Then, DIPEA
(24.3 mL) was added and the mixture was shaken at room temperature. After 30 min of reaction, the argon
inlet was removed. After 16 h, the mixture was filtered, washed with DCM (3×) and MeOH (3×), and
dried under a vacuum to afford 14.1 g of resin 19. The loading yield, calculated by the mass increase, was
42%. It is noteworthy to mention that the loading could not be higher than 50% because 2 equivalents of
trityl chloride resin were used for 1 equivalent of precursor 6a. Then 63 fritted bottom reaction vessels of
the 96 solid-phase reaction block of ACT LabTech manual synthesizer were loaded with 200 mg of resin
19. A solution of piperidine (20%) in DCM was added to resins 19 and the mixtures were shaken for 1 h at
room temperature to remove the Fmoc protective group. Then, the resin was filtered and washed with
DCM (2×), MeOH (3×), and dried under a vacuum for 16 h. The completion of the deprotection was
proven by acidic mini-cleavage as described above. Thus, each reaction vessel contains resins loading
0.109 mmol of compound 6a (without the Fmoc protective group).
4.2.10. Introduction of three levels of molecular diversity (library C)
The levels of molecular diversity were introduced using a similar procedure as the one described above
for the preparation of libraries A and B. To introduce the first level of molecular diversity, three stock
solutions, each containing 2 equivalents (21×) of Fmoc-protected amino acid [Fmoc-Gly-OH (1.36 g,
4.56 mmol), Fmoc-L-Phe-OH (1.77 g, 4.56 mmol) or Fmoc-L-4-NO2-Phe-OH (1.98 g, 4.56 mmol)], 2
equivalents (21×) of PyBOP (2.37 g, 4.56 mmol) and 2 equivalents (21×) of HOBt (616 mg, 4.56 mmol),
were prepared in dry DMF (42 mL). Prior to addition to the resin, 4 equivalents (21×) of DIPEA
(1.59 mL, 9.12 mmol) were added to each stock solution. To each of the 63 resins 20 was added 2.0 mL of

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Molecules 2010, 15
the appropriate stock solution. The resins were shaken under an argon atmosphere for 2 h at room
temperature, then filtered, washed with DMF (2×), DCM (2×) and MeOH (3×) and dried under a vacuum.
This procedure was repeated (3 times) until completion of the reaction was afforded and verified by acidic
mini-cleavage as described above. The cleavage of Fmoc group of resins 21 was then performed by a 1 h
treatment with 2.0 mL of a solution of piperidine (20%) in DCM. The resins were then filtered, washed
with DCM (3×), MeOH (3×), and dried under a vacuum to give three groups of resins of general structure
22. The second level of molecular diversity was introduced using the same building blocks and the same
procedure described above for the introduction of the first level of molecular diversity. This time, the
coupling procedure was repeated twice to afford completion of the reaction by TLC analysis after acidic
mini-cleavage. Resins of general structure 23 were then treated with a solution of piperidine (20%) in
DCM for 1 h at room temperature. The resins were filtered, washed with DCM (2×) and MeOH (2×), and
dried to afford resins of general structure 24. The third level of molecular diversity was introduced by
coupling resins 24 with carboxylic acid building blocks 12–18. Seven stock solutions, each containing 2
equivalents (9x) of carboxylic acid building blocks 12–18 [12 (701 mg, 1.95 mmol), 13 (701 mg,
1.95 mmol), 14 (729 mg, 1.95 mmol), 15 (729 mg, 1.95 mmol), 16 (756 mg, 1.95 mmol), 17 (756 mg,
1.95 mmol) or 18 (784 mg, 1.95 mmol)], 2 equivalents (9x) of PyBrOP (911 mg, 1.95 mmol) and 2
equivalents of HOBt (264 mg, 1.95 mmol), were prepared in dry DMF (18 mL) and 4 equivalents (9x) of
DIPEA (681 µL, 3.90 mmol) were added. To each of the 63 resins 24 was added 2.0 mL of the
appropriate stock solution. The resins were shaken under an argon atmosphere for 2 h at room
temperature, then filtered, washed with DMF (2×), DCM (2×) and MeOH (3×) and dried under a vacuum
to afford resins of general structure 25 (n = 2). The completion of the coupling step was proven by acidic
mini-cleavage and TLC analysis of samples of resins 25.
4.2.11. Removal of protective groups (library C)
The Fmoc protective group from carboxylic acid building blocks on resins 25 (n = 2) was removed
with the same procedure described above for the preparation of libraries A and B. The acetate protective
group of resins 26 (n = 2) was removed using a similar procedure described above for the preparation of
libraries A and B. However, the mixtures were shaken for 100 h instead of 48 h in order to obtain
complete deprotection of the steroidal C17β-hydroxy by TLC analysis after acidic mini-cleavage of
samples of resins 28 (n = 2).
4.2.12. Generation of E2 derivatives (library C) by nucleophilic cleavage
To each of the 63 resins 28 (n = 2) was added 1.5 mL of a solution of DEA (30%) in THF. The
mixtures were shaken for 50 h at room temperature. Then 1.0 mL of a solution of DEA (30%) in THF was
added to each of the 63 resins and the mixtures were shaken for an additional 16 h. The mixtures were
next filtered and washed with THF (1×) and MeOH (3×). The filtrates were collected in preweighed tubes
and evaporated in a Speedvac apparatus. Each product was dissolved in THF, evaporated and dried under