17α-ethynylestradiol peptide labeling by 'click' chemistry

Article abstract of DOI:10.1055/s-0032-1316702

A synthesis of 17α-ethynylestradiol-labeled native peptides is reported. The peptide moiety is tethered to the steroid hormone by a 1,2,3-triazole bridge formed by a CuAAC reaction in which the azido group of the peptide combines with the terminal acetylenic moiety of ethynylestradiol to link the two bioactive molecules. Thus bioconjugates containing the hormone moiety at three positions within the peptide molecule could be useful targets for hormono-enzyme interaction studies. Georg Thieme Verlag Stuttgart ? New York.

Full text of DOI:10.1055/s-0032-1316702

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PAPER
p1a7peαr -Ethynylestradiol Peptide Labeling by ‘Click’ Chemistry
17α-Ethynylestradiol Peptide Labeling by ‘Click’ Chemistry
Oleg I. Bol’shakov,a Iryna O. Lebedyeva,a Alan R. Katritzky*a,b
a
Center for Heterocyclic Compounds, Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, USA
Fax +1(352)3929199; E-mail: katritzky@chem.ufl.edu
b
Chemistry Department, King Abdulaziz University, Jeddah, 21589, Saudi Arabia
Received: 27.04.2012; Accepted after revision: 14.06.2012
gates through CuAAC-catalyzed reactions of native pep-
tides with 17EE. We believe that successful and selective
combination of tetrapeptide scaffolds with 17EE by a
1,2,3-triazole linkage should allow the generation of nov-
Abstract: A synthesis of 17α-ethynylestradiol-labeled native pep-
tides is reported. The peptide moiety is tethered to the steroid hor-
mone by a 1,2,3-triazole bridge formed by a CuAAC reaction in
which the azido group of the peptide combines with the terminal
acetylenic moiety of ethynylestradiol to link the two bioactive mol- el bioactive peptidomimetics ligands tailored for specific
ecules. Thus bioconjugates containing the hormone moiety at three
positions within the peptide molecule could be useful targets for
hormono–enzyme interaction studies.
site-selective interactions with corresponding receptors.
We planned to label 17EE with azido-containing peptides
I–III containing the azido group in diverse positions with-
in the peptide moiety to access to a set of three 17EE ste-
roid-enzyme conjugates 1–3 (Scheme 1).
Key words: cyclization, azides, alkynes, peptides, drugs, click
chemistry
Since organic azides are excellent reaction partners for a
variety of transformations, numerous approaches have
been developed to access peptides with an azido moiety
ready for further functionalization.8 The major routes as
prior steps for introduction of azide into the molecule are:
(i) nucleophilic substitution of a leaving group with an
azide anion9a,b and (ii) diazo transfer from primary
amines.9c,d The approach (i) was implemented to convert
2-chloroethylamine into the corresponding azide, which
was then engaged in the synthesis of target I.10 Target II
was synthesized by route (ii) α-Boc-protected lysine 14
was converted into an ε-azide derivative by diazo trans-
fer.11 Enantiopure azidoalanine 23 for target III was syn-
thesized from L-alanine using diazo transfer methodology
developed earlier in our group.11
The steroidal estrogen (17R)-17-ethynylestradiol (17EE),
a mechanism-based inactivator of cytochrome P450s
(P450s) 2B1 and 2B6,1 is useful for the identification of
the active sites of proteins and enzymes.2 The introduction
of a labeled 17EE molecule into a cell could help deter-
mine if modification of the P450 apoprotein by a reactive
17EE intermediate is accompanied by loss of activity in
the 2B enzyme.2a,d Although the relationship between
membrane, cytosolic, and nuclear actions of 17EE has
been investigated, the interaction between estrogen recep-
tor mediated genomic and non-genomic pathways needs
further study.3 Literature synthetic and biochemical stud-
ies of 17EE labeled glycine peptoids,2b,c,4 S-linked side-
chain 17EE peptide conjugates,2a and ‘miktoarm’ core-
crosslinked star copolymers5a have assisted the identifica-
tion of the P450s enzymatic activity. Macromolecular de-
rivatives of estradiol,5b,c including 17EE bovine serum
albumin,5d cyclodextrin,5e and dendrimer conjugates,5f
have previously assisted studies of the activation of the
membrane-associated estrogen receptors,3a but the highly
variable stability and activity of 17EE–macromolecule
complexes requires further development of flexible and
stable 17EE bioconjugates.
Click chemistry6a is very useful for the synthesis of pepti-
domimetics,2b,c,4b and bioconjugates6b,c and for the label-
ing of recombinant and membrane proteins in living
bacteria.6d–f The high chemoselectivity,7a,b speed, green
reaction conditions, and excellent yields7c of copper(I)-
catalyzed azide–alkyne cycloaddition (CuAAC) reactions
have encouraged multiple applications in chemical biolo-
gy.7d,e We now combine two biologically active molecules
capable of cell recognition to prepare nontoxic bioconju-
Conjugate 1 was synthesized by solution-phase coupling–
deprotection methodology,12a starting from 2-azidoethyl-
amine (5) (Scheme 2). Further reaction of 5 with Boc-pro-
tected tryptophan employing benzotriazole as a coupling
additive12b generated 6. A sequence of successive steps of
Boc-amino acid couplings and the Boc group deprotec-
tions resulted in adding valine, phenylalanine, and alanine
units to the peptide chain to afford azidopeptide 12. Tetra-
peptide 12 was then labeled with 17EE in the presence of
copper(I) by application of the Sharpless and Fokin proce-
dure,7c affording 13 which was deprotected under a posi-
tive pressure of hydrogen, using palladium on carbon as a
catalyst, to give target conjugate 1.
Synthesis of bioconjugate 2 required the introduction of ε-
azido lysine 15 into a peptide chain. Further N,N′-dicyclo-
hexylcarbodiimide-mediated coupling of azide 15 with L-
phenylalanine methyl ester followed by the deprotection
of 16 gave hydrochloride salt 17. Stepwise solution-phase
coupling–deprotection methodology12b was then success-
fully applied to convert 17 into 18, 19, and finally tetra-
peptide 20. Subsequent reaction of 20 with 17EE (Scheme
3) gave the Boc-protected conjugate 21. Final deprotec-
SYNTHESIS 2012, 44, 2926–2932
Advanced online publication: 20.08.2012
0
0
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7
8
8
1
1
4
3
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2
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DOI: 10.1055/s-0032-1316702; Art ID: SS-2012-M0399-OP
© Georg Thieme Verlag Stuttgart · New York

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17α-Ethynylestradiol Peptide Labeling by ‘Click’ Chemistry
2927
OH
peptide
OH
N
N
Cu (I)
N
H
+
N3
peptide
H
H
H
H
H
HO
HO
1–3
17EE
O
O
H
H
N
N
N3
N3
peptide
H2N
N
N
H
H
O
O
N
I
H
N3
O
O
O
O
H
N
H
H
H
N
N
N
H2N
N
H
OMe
N3
N
H
OMe
O
O
O
O
II
III
Scheme 1 General scheme for native peptide labeling with 17EE
i (100%)
ii (82%)
ii (66%)
H2N(CH2)2N3
H2N(CH2)2Cl
4
5
iii (100%)
Boc-Trp-HN(CH2)2N3
HCl*H-Trp-HN(CH2)2N3
6
7
ii (50%)
ii (68%)
iii (100%)
Boc-Val-Trp-HN(CH2)2N3
HCl*H-Val-Trp-HN(CH2)2N3
8
9
iii (100%)
Boc-Phe-Val-Trp-HN(CH2)2N3
HCl*H-Phe-Val-Trp-HN(CH2)2N3
10
11
N
N
iv (56%)
17EE
N
Z-Ala-Phe-Val-Trp-HN(CH2)2N3
Z-Ala-Phe-Val-Trp
N
H
17EE
12
13
N
N
N
17EE
v (95%)
H-Ala-Phe-Val-Trp
N
H
1
Scheme 2 Synthesis of conjugate 1. Reagents and conditions: (i) NaN3, DMF, r.t., 12 h; (ii) PG-AA-PH, DCC, BtH, DIPEA, MeCN, 0 °C to
r.t.; (iii) HCl, MeOH, r.t., 2 h; (iv) Cu(I), t-BuOH, r.t., 72 h; (v) H2, Pd, MeOH, r.t., 12 h.
tion resulted in target 2 with the peptide tethered to the ste- (28) peptide chain was effected by coupling 28 with azi-
roid.
doalanine 23 (Scheme 4). Reaction of the 29 with 17EE
was performed using a routine ‘click’ procedure7c which
required four days to complete the reaction as monitored
by TLC. The target peptide 3 was obtained in 75% yield
and the enantiopurity of the product was confirmed by
HPLC (99.3%).
Azido-terminus peptide 29 was prepared by utilizing a
systematic stepwise coupling–deprotection strategy.12b In
this a way, leucine and glycine units were added to the
starting L-phenylalanine methyl ester 24. Introduction of
the azido acid residue to the HCl·H-Gly-Leu-Phe-OMe
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2012, 44, 2926–2932

2928
O. I. Bol’shakov et al.
PAPER
i (63%)
ii (73%)
Table 1 HPLC and HRMS Data of Targets 1–3
α-Boc-Lys(N3)-OH
α-Boc-Lys-OH
14
Puritya (%)
HRMS [M + Na]+
15
Target
Found
Calcd
iii (100%)
iii (100%)
iv (63%)
iv (78%)
HCl*H-Lys-Phe-OMe
Boc-Lys-Phe-OMe
1
2
3
100.00
99.322
99.422
908.4778
864.4986
765.3927
908.4794
864.4994
765.3946
N3
N3
16
17
Boc-Leu-Lys-Phe-OMe
HCl*H-Leu-Lys-Phe-OMe
a Purity based on analytical HPLC (Phenomenex, mobile phase 100%
MeOH, 0.5 mL/min–1).
N3
19
N3
18
v (42%)
17EE
iii (100%)
Boc-Val-Leu-Lys-Phe-OMe
Boc-Val-Leu-Lys-Phe-OMe
N
N
N3
conjugation is found to be stable under both Boc- and
Cbz-deprotection conditions.
20
N
17EE
21
HCl*H-Val-Leu-Lys-Phe-OMe
Melting points were determined on a capillary point apparatus
equipped with a digital thermometer and are uncorrected. 1H NMR
spectra were recorded at 300 MHz and 13C NMR spectra were re-
corded at 75 MHz on Gemini or Varian spectrometers at r.t. relative
to TMS as internal standard (1H NMR) or to the residual solvent
peak (13C NMR). Elemental analysis was performed on a Carlo
Erba-1106 instrument. HRMS were recorded using Thermo Scien-
tific LCQ Ion Trap. Column chromatography was performed on sil-
ica gel (230–400 mesh). HPLC analysis was performed on
Shimadzu liquid chromatographic system equipped with UV detec-
tor. Detection was done at 254 nm using Phenomenex Luna C18 re-
versed-phase column (250 × 4.6 mm id) 5 micron.
N
N
N
17EE
2
Scheme 3 Synthesis of conjugate 2. Reagents and conditions: (i)
BtSO2N3, Et3N, MeCN–H2O, r.t., 6 h; (ii) HCl·H-Phe-OMe, DIPEA,
DCC, BtH, MeCN, 0 °C to r.t.; (iii) HCl, MeOH, r.t., 2 h; (iv) Boc-
AA-OH, DIPEA, DCC, BtH, MeCN, 0 °C to r.t.; (v) Cu(I), t-BuOH,
r.t., 72 h.
N-Boc- and N-Cbz-protected L-amino acids, L-alanine (22), and L-
phenylalanine methyl ester (24) were purchased from Peptides In-
ternational Inc. All commercially available substrates were used as
received without further purification.
i (60%)
N3-Ala-OH
H-Ala-OH
2-Azidoethylamine (5) was synthesized according to Inverarity et
al.10 Azido-alanine 23 was prepared by our previously reported pro-
cedure.11
22
23
iii (100%)
iii (100%)
ii (82%)
ii (68%)
Boc-Leu-Phe-OMe
H-Phe-OMe
24
25
(S)-6-Azido-2-[(tert-butoxycarbonyl)amino]hexanoic Acid (15)
α-Boc-Lys-OH (2.46 g, 10.0 mmol) was dissolved in a mixture of
H2O–MeCN (1:1, 40 mL) and Et3N (2.02 g, 20 mmol). Then
BtSO2N3 (2.23 g, 10.0 mmol) was added as a solid at r.t. and the
mixture was stirred overnight. The organic solvent was evaporated
and residue was acidified with 0.33 M citric acid (60 mL) and ex-
tracted with EtOAc (2 × 30 mL). The organic layers were combined,
dried (anhyd Na2SO4), and evaporated. The residue was then puri-
fied by column chromatography (EtOAc–hexanes, 1:2) to give 15
(1.71 g, 6.3 mmol, 63%) as a pale yellow oil. NMR spectra were in
good compliance with previously reported spectra,13 (mixture of
two rotamers).
Boc-Gly-Leu-Phe-OMe
HCl*H-Leu-Phe-OMe
26
27
iv (75%)
17EE
ii (75%)
N3-Ala-Gly-Leu-Phe-OMe
HCl*H-Gly-Leu-Phe-OMe
23
28
29
N
N
N
17EE
Ala-Gly-Leu-Phe-OMe
3
Scheme 4 Synthesis of conjugate 3. Reagents and conditions: (i)
BtSO2N3, Et3N, MeCN–H2O, r.t., 6 h; (ii) PG-AA-OH, DIPEA, DCC,
BtH, MeCN, 0 °C to r.t.; (iii) HCl, MeOH, r.t., 2 h; (iv) Cu(I), t-
BuOH, r.t., 72 h.
1H NMR (300 MHz, CDCl3): δ = 10.65 (s, 1 H), 6.75 (br s, 0.3 H),
5.13 (d, J = 7.8 Hz, 0.7 H), 4.40–4.08 (m, 1 H), 3.29 (t, J = 6.6 Hz,
2 H), 1.98–1.81 (m, 1 H), 1.78–1.57 (m, 3 H), 1.54–1.34 (m, 11 H).
13C NMR (75 MHz, CDCl3): δ = 177.3, 176.7, 157.1, 155.8, 82.1,
80.5, 54.5, 53.3, 51.3, 32.2, 31.1, 28.6, 28.5, 22.7.
All the target tethered peptides were characterized by
HPLC and HRMS revealing purity not lower than 99.3%
for 2 (see the Supporting information) (Table 1).
Anal. Calcd for C11H20N4O4: C, 48.52; H, 7.40; N, 20.57. Found: C,
48.85; H, 7.97; N, 20.25.
Synthesis of 6, 8, 10, 12, 16, 18, 20, 25, 27, and 29; General
Procedure
Conjugation of enantiopure versatile azido-enhanced oli-
gopeptides with alkyne hormones is reported for the first
time. We also provide different approaches for the intro-
duction of azido moiety under solution-phase peptide syn-
thesis conditions. The triazole bridge after ‘click’
Equimolar amounts of amine or amine hydrochloride, N-protected
L-amino acid, and benzotriazole together with DIPEA (1 equiv; 2
equiv in the case of the hydrochloride) were dissolved in MeCN and
the mixture was cooled to 0 °C. DCC (1 equiv) was added and the
mixture was stirred overnight at r.t. Then the solvent was evaporat-
Synthesis 2012, 44, 2926–2932
© Georg Thieme Verlag Stuttgart · New York

PAPER
17α-Ethynylestradiol Peptide Labeling by ‘Click’ Chemistry
2929
ed, the residue was diluted with EtOAc and washed with 0.33 M cit-
ric acid soln (1 ×) and sat. Na2CO3 soln (2 ×). The organic layer was
dried (anhyd Na2SO4), evaporated, and the residue was separated by
column chromatography (EtOAc–hexanes, 1:4).
13C NMR (75 MHz, DMSO-d6): δ = 172.3, 171.5, 170.7, 170.4,
155.5, 137.7, 136.0, 136.0, 129.3, 128.3, 127.9, 127.7, 127.3, 126.1,
123.5, 120.8, 118.4, 118.2, 111.3, 109.8, 65.4, 57.6, 53.5, 53.3,
50.2, 49.8, 41.6, 38.7, 38.2, 37.1, 30.8, 27.9, 19.1, 18.2, 18.0.
Anal. Calcd for C38H45N9O6: C, 63.06; H, 6.27; N, 17.42. Found: C,
61.76; H, 6.94; N, 16.65.
tert-Butyl {(S)-1-[(2-Azidoethyl)amino]-3-(1H-indol-3-yl)-1-
oxopropan-2-yl}carbamate (6)
Light-yellow solid (3.05 g, 8.20 mmol, 82%); mp 140.0–141.8 °C.
Methyl (S)-2-{(S)-6-Azido-2-[(tert-butoxycarbonyl)ami-
no]hexanamido}-3-phenylpropanoate (16)
Pale yellow solid (1.99 g, 4.60 mmol, 73%); mp 80.7–82.0 °C.
1H NMR (300 MHz, CDCl3): δ = 8.64 (br s, 1 H), 7.62 (d, J = 7.5
Hz, 1 H), 7.36 (dd, J = 8.1, 0.9 Hz, 1 H), 7.22–7.16 (m, 1 H), 7.13–
7.08 (m, 1 H), 7.00 (s, 1 H), 6.28 (s, 1 H), 5.30 (s, 1 H), 4.43 (s, 1
H), 3.25–3.00 (m, 6 H), 1.43 (s, 9 H).
13C NMR (75 MHz, CDCl3): δ = 172.4, 155.7, 136.4, 127.6, 123.4,
122.4, 119.8, 119.0, 111.5, 110.5, 80.5, 55.6, 50.6, 38.8, 28.7, 28.5.
1H NMR (300 MHz, CDCl3): δ = 7.32–7.20 (m, 3 H), 7.12–7.09 (m,
2 H), 6.56 (d, J = 7.5 Hz, 1 H), 5.04 (d, J = 8.1 Hz, 1 H), 4.85 (dd,
J = 13.5, 6.3 Hz, 1 H), 4.09–4.04 (m, 1 H), 3.71 (s, 3 H), 3.24 (t, J =
6.9 Hz, 2 H), 3.19–3.03 (m, 2 H), 1.79–1.69 (m, 2 H), 1.60–1.50 (m,
4 H), 1.44 (br s, 9 H).
13C NMR (75 MHz, CDCl3): δ = 171.8, 171.7, 155.9, 135.8, 129.4,
128.7, 127.3, 80.3, 54.4, 53.3, 52.5, 51.3, 38.0, 32.2, 28.6, 28.4,
22.8.
Anal. Calcd for C18H24N6O3: C, 58.05; H, 6.50; N, 22.57. Found: C,
59.48; H, 7.05; N, 21.14.
Product 6 was isolated as a light-yellow solid and used crude as an
intermediate for the preparation of 7 (see below).
Anal. Calcd for C21H31N5O5: C, 58.18; H, 7.21; N, 16.16. Found: C,
58.24; H, 7.60; N, 16.25.
tert-Butyl [(S)-1-({(S)-1-[(2-Azidoethyl)amino]-3-(1H-indol-3-
yl)-1-oxopropan-2-yl}amino)-3-methyl-1-oxobutan-2-yl]carba-
mate (8)
Methyl (2S,5S,8S)-5-(4-Azidobutyl)-2-benzyl-8-[(tert-butoxy-
carbonyl)amino]-10-methyl-4,7-dioxo-3,6-diazaundecanoate
(18)
White solid (2.33 g, 4.95 mmol, 66%); mp 175.1–176.8 °C.
1H NMR (300 MHz, CDCl3): δ = 8.54 (s, 1 H), 7.60 (d, J = 7.2 Hz,
1 H), 7.35 (d, J = 8.1 Hz, 1 H), 7.21–7.09 (m, 2 H), 7.01 (s, 1 H),
6.86 (d, J = 7.2 Hz, 1 H), 6.71 (br s, 1 H), 4.94 (d, J = 5.7 Hz, 1 H),
4.75 (q, J = 6.9 Hz, 1 H), 3.91 (t, J = 5.4 Hz, 1 H), 3.41–3.11 (m, 6
H), 2.17–2.11 (m, 1 H), 1.32 (s, 9 H), 0.91 (d, J = 6.6 Hz, 3 H), 0.81
(d, J = 6.9 Hz, 3 H).
13C NMR (75 MHz, CDCl3): δ = 171.6, 171.4, 156.2, 136.2, 127.4,
123.3, 122.3, 119.7, 118.4, 111.4, 109.9, 80.4, 60.5, 53.9, 50.1,
38.7, 30.1, 28.0, 27.5, 19.2, 17.3.
Pale yellow oil (1.38 g, 2.52 mmol, 63%).
1H NMR (300 MHz, CDCl3): δ = 7.22–7.12 (m, 3 H), 7.02 (d, J =
6.3 Hz, 1 H), 6.85–6.81 (m, 1 H), 5.08–5.03 (m, 1 H), 4.74–4.68 (m,
1 H), 4.40–4.34 (m, 1 H), 4.05–3.95 (m, 1 H), 3.60 (s, 3 H), 3.19–
3.06 (m, 2 H), 3.02–2.96 (m, 2 H), 1.85–1.13 (m, 20 H), 0.83 (d, J =
6.3 Hz, 6 H).
13C NMR (75 MHz, CDCl3): δ = 172.8, 171.7, 171.1, 155.8, 135.8,
129.2, 128.6, 127.1, 80.0, 53.4, 52.8, 52.3, 51.1, 41.2, 37.9, 32.0,
28.5, 28.4, 25.6, 24.8, 23.1, 22.5, 21.9.
Anal. Calcd for C23H33N7O4: C, 58.58; H, 7.05; N, 20.79. Found: C,
58.30; H, 7.45; N, 20.63.
Anal. Calcd for C27H42N6O6: C, 59.32; H, 7.74; N, 15.37. Found: C,
60.52; H, 8.69; N, 15.49.
tert-Butyl ((S)-1-{[(S)-1-({(S)-1-[(2-Azidoethyl)amino]-3-(1H-
indol-3-yl)-1-oxopropan-2-yl}amino)-3-methyl-1-oxobutan-2-
yl]amino}-1-oxo-3-phenylpropan-2-yl)carbamate (10)
Product 18 was isolated as a pale yellow solid and used crude as in-
termediate for the preparation of 19 (see below).
Light-yellow solid (1.33 g, 2.15 mmol, 50%); mp 128.1–130.6 °C.
Methyl (2S,5S,8S,11S)-5-(4-Azidobutyl)-2-benzyl-11-[(tert-but-
oxycarbonyl)amino]-8-isobutyl-12-methyl-4,7,10-trioxo-3,6,9-
triazatridecanoate (20)
1H NMR (300 MHz, CDCl3): δ = 8.21 (s, 1 H), 7.54 (d, J = 8.1 Hz,
1 H), 7.30–7.05 (m, 7 H), 6.98 (s, 1 H), 6.90–6.58 (m, 3 H), 4.94–
4.86 (m, 1 H), 4.80 (q, J = 6.9 Hz, 1 H), 4.28–4.10 (m, 2 H), 3.42–
3.23 (m, 5 H), 2.97–2.90 (m, 2 H), 2.16 (br s, 1 H), 1.90 (d, J = 10.2
Hz, 1 H), 1.75–1.62 (m, 1 H), 1.59 (d, J = 9.6 Hz, 1 H), 1.39–1.20
(m, 10 H), 0.94–0.80 (m, 3 H), 0.69 (d, J = 6.6 Hz, 1 H).
13C NMR (75 MHz, CDCl3): δ = 172.3, 171.5, 155.9, 136.7, 136.2,
129.4, 128.6, 127.5, 126.9, 123.3, 122.1, 119.5, 118.7, 111.4, 110.3,
80.3, 58.8, 55.7, 53.9, 50.5, 49.5, 38.9, 33.9, 31.4, 28.4, 25.7, 25.1,
21.0, 19.3, 18.2.
White solid (1.27 g, 1.97 mmol, 78%); mp 174.1–174.8 °C.
1H NMR (300 MHz, DMSO-d6): δ = 8.32 (d, J = 7.5 Hz, 1 H), 8.00–
7.75 (m, 3 H), 7.26–7.17 (m, 5 H), 6.73 (d, J = 9.0 Hz, 1 H), 4.51–
4.27 (m, 4 H), 3.79–3.73 (m, 1 H), 3.56 (s, 3 H), 3.28–3.23 (m, 2 H),
3.06–2.91 (m, 2 H), 2.00–1.82 (m, 1 H), 1.75–1.20 (m, 18 H), 0.92–
0.80 (m, 12 H).
13C NMR (75 MHz, DMSO-d6): δ = 171.7, 171.6, 171.4, 171.1,
155.4, 137.0, 129.0, 128.2, 126.5, 78.0, 59.9, 54.8, 53.4, 51.8, 50.7,
50.6, 40.9, 36.6, 31.8, 30.3, 28.2, 27.9, 24.0, 23.1, 22.2, 21.5, 19.2,
18.2.
Anal. Calcd for C32H42N8O5: C, 62.12; H, 6.84; N, 18.11. Found: C,
62.00; H, 7.54; N, 16.87.
Product 10 was isolated as light-yellow solid and used crude as in-
termediate for the preparation of 11 (see below).
Anal. Calcd for C32H51N7O7: C, 59.51; H, 7.96; N, 15.18. Found: C,
59.70; H, 8.67; N, 15.02.
Benzyl {(5S,8S,11S,14S)-1-Azido-11-benzyl-5-[(1H-indol-3-
yl)methyl]-8-isopropyl-4,7,10,13-tetraoxo-3,6,9,12-tetraaza-
pentadecan-14-yl}carbamate (12)
Methyl (S)-2-{(S)-2-[(tert-Butoxycarbonyl)amino]-4-methyl-
pentanamido}-3-phenylpropanoate (25)
NMR spectra are in good compliance with those previously report-
ed.14 Pale yellow solid (2.86 g, 7.30 mmol, 82%); mp 82.4–83.2 °C
(Lit.14 83.5–84.0 °C).
1H NMR (300 MHz, CDCl3): δ = 7.31–7.20 (m, 3 H), 7.11 (d, J =
6.0 Hz, 2 H), 6.61 (d, J = 7.5 Hz, 1 H), 4.94–4.81 (m, 2 H), 4.13–
4.08 (m, 2 H), 3.70 (s, 3 H), 3.20–3.00 (m, 2 H), 1.72–1.50 (m, 2 H),
1.44 (s, 9 H), 0.98–0.89 (m, 6 H).
Light-brown solid (1.06 g, 1.46 mmol, 68%); mp 187.1–188.8 °C.
1H NMR (300 MHz, DMSO-d6): δ = 10.84 (s, 1 H), 8.16 (s, 1 H),
8.09 (d, J = 7.8 Hz, 1 H), 7.99 (d, J = 7.8 Hz, 1 H), 7.83 (d, J = 8.4
Hz, 1 H), 7.56 (d, J = 7.8 Hz, 1 H), 7.42 (d, J = 7.2 Hz, 1 H), 7.32–
6.92 (m, 10 H), 4.98 (s, 2 H), 4.54 (br s, 2 H), 4.19 (t, J = 6.6 Hz, 1
H), 4.00 (t, J = 7.2 Hz, 1 H), 3.57–3.54 (m, 1 H), 3.37–3.31 (m, 2
H), 3.08–2.81 (m, 5 H), 2.10–1.86 (m, 2 H), 1.38–1.05 (m, 6 H),
0.95–0.70 (m, 6 H).
13C NMR (75 MHz, CDCl3): δ = 172.3, 171.8, 155.6, 135.9, 129.4,
128.7, 127.2, 80.2, 55.3, 52.5, 41.4, 38.1, 28.5, 24.8, 23.1, 22.1.
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2012, 44, 2926–2932

2930
O. I. Bol’shakov et al.
PAPER
Anal. Calcd for C21H32N2O5: C, 64.26; H, 8.22; N, 7.14. Found: C,
64.50; H, 8.82; N, 7.52.
13C NMR (75 MHz, DMSO-d6): δ = 168.8, 136.3, 127.2, 124.9,
121.1, 118.6, 118.4, 111.5, 107.0, 52.9, 49.7, 38.2, 27.2.
Product 7 was used crude as an intermediate for the synthesis of 8
(see above); full spectral characterization is provided for 8.
Methyl (2S,5S)-2-Benzyl-8-[(tert-butoxycarbonyl)amino]-5-iso-
butyl-4,7-dioxo-3,6-diazaoctanoate (27)
White solid (2.14g, 4.76 mmol, 68%); mp 143.4–144.5 °C.
(S)-2-Amino-N-{(S)-1-[(2-azidoethyl)amino]-3-(1H-indol-3-yl)-
1-oxopropan-2-yl}-3-methylbutanamide (9)
White solid (1.84 g, 4.50 mmol, 100%); mp 178.6–179.3 °C.
1H NMR (300 MHz, CDCl3): δ = 7.29–7.21 (m, 3 H), 7.12–7.09 (m,
2 H), 6.89 (d, J = 7.8 Hz, 1 H), 6.78 (d, J = 7.2 Hz, 1 H), 5.38–5.33
(m, 1 H), 4.87–4.79 (m, 1 H), 4.50–4.47 (m, 1 H), 3.80–3.63 (m, 5
H), 3.17–3.00 (m, 2 H), 1.65–1.48 (m, 3 H), 1.45 (s, 9 H), 0.89 (t,
J = 5.7 Hz, 6 H).
13C NMR (75 MHz, CDCl3): δ = 171.9, 171.8, 169.7, 156.2, 136.0,
129.5, 128.7, 127.2, 80.4, 53.4, 52.5, 51.7, 44.4, 41.1, 38.0, 28.5,
24.8, 23.1, 22.2.
1H NMR (300 MHz, CDCl3): δ = 10.94 (s, 1 H), 8.72 (d, J = 7.8 Hz,
1 H), 8.41 (t, J = 5.4 Hz, 1 H), 8.35–8.15 (m, 2 H), 7.62 (d, J = 7.5
Hz, 1 H), 7.33 (d, J = 8.1 Hz, 1 H), 7.22 (d, J = 1.8 Hz, 1 H), 7.08–
6.95 (m, 2 H), 4.60–4.52 (m, 1 H), 4.14 (br s, 2 H), 3.66–3.61 (m, 1
H), 3.39–2.97 (m, 4 H), 2.15–2.08 (m, 1 H), 1.23–0.89 (m, 6 H).
13C NMR (75 MHz, CDCl3): δ = 171.3, 167.7, 136.1, 127.3, 123.9,
120.9, 118.4, 118.2, 111.4, 109.6, 57.1, 53.8, 49.8, 46.8, 38.2, 33.4,
29.9, 28.6, 27.8, 25.3, 24.5, 23.7, 18.4, 17.6.
Anal. Calcd for C23H35N3O6: C, 61.45; H, 7.85; N, 9.35. Found: C,
61.22; H, 7.85; N, 11.44.
Anal. Calcd for C18H26ClN7O2: C, 53.00; H, 6.42; N, 24.04. Found:
C, 53.09; H, 6.16; N, 24.67.
Methyl (S)-2-((S)-2-{2-[(S)-2-Azidopropanamido]acetamido}-
4-methylpentanamido)-3-phenylpropanoate (29)
White solid (1.34 g, 3 mmol, 75%); mp 149.7–150.2 °C.
(S)-2-[(S)-2-Amino-3-phenylpropanamido]-N-{(S)-1-[(2-azido-
ethyl)amino}-3-(1H-indol-3-yl)-1-oxopropan-2-yl}-3-methylbu-
tanamide Hydrochloride (11)
Isolated as white solid (1.19 g, 2.15 mmol, 100%) and used crude as
an intermediate for the preparation of 12 (see above); full spectral
characterization is provided for 12.
1H NMR (300 MHz, CDCl3): δ = 7.52 (t, J = 4.8 Hz, 1 H), 7.45–7.38
(m, 2 H), 7.29–7.18 (m, 3 H), 7.11 (d, J = 7.5 Hz, 2 H), 4.86 (q, J =
6.8 Hz, 1 H), 4.68 (q, J = 7.4 Hz, 1 H), 4.08–4.00 (m, 2 H), 3.90–
3.83 (m, 1 H), 3.71 (s, 3 H), 3.17–3.02 (m, 2 H), 1.69–1.55 (m, 3 H),
1.53 (d, J = 7.2 Hz, 3 H), 0.91 (d, J = 5.7 Hz, 3 H), 0.89 (d, J = 5.7
Hz, 3 H).
13C NMR (75 MHz, CDCl3): δ = 172.1, 172.0, 170.8, 168.5, 136.0,
129.4, 128.6, 127.2, 58.5, 53.5, 52.5, 51.8, 42.9, 41.8, 38.0, 24.8,
22.9, 22.4, 17.2.
Methyl (S)-2-[(S)-2-Amino-6-azidohexanamido]-3-phenylpro-
panoate Hydrochloride (17)
Pale yellow oil (1.59 g, 4.31 mmol, 100%).
1H NMR (300 MHz, CD3OD): δ = 8.21 (br s, 1 H), 7.23–7.10 (m, 5
H), 4.64–4.58 (m, 1 H), 3.82–3.75 (m, 1 H), 3.60 (s, 3 H), 3.26–3.16
(m, 2 H), 3.14–3.07 (m, 1 H), 2.99– 2.92 (m, 1 H), 1.81–1.74 (m, 2
H), 1.55–1.47 (m, 2 H), 1.43–1.33 (m, 2 H).
Anal. Calcd for C21H30N6O5: C, 56.49; H, 6.77; N, 18.82. Found: C,
56.74; H, 7.18; N, 18.72.
Synthesis of 2, 7, 9, 11, 17, 19, 26, and 28; General Procedure
Boc-protected peptide was dissolved in sat. HCl soln in MeOH and
stirred at r.t. for 2 h. The mixture was then evaporated and dried un-
der high vacuum.
13C NMR (75 MHz, CD3OD): δ = 173.1, 170.3, 138.1, 130.3, 129.7,
128.1, 55.7, 54.2, 53.0, 52.2, 38.1, 32.3, 29.6, 22.9.
Anal. Calcd for C16H24ClN5O3: C, 51.96; H, 6.54; N, 18.94. Found:
C, 53.03; H, 7.46; N, 18.41.
Methyl (S)-2-((S)-2-{(S)-2-[(S)-2-Amino-3-methylbutanamido]-
4-methylpentanamido}-6-{4-[(8R,9S,13S,14S,17S)-3,17-dihy-
droxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cy-
clopenta[a]phenanthren-17-yl]-1H-1,2,3-triazol-1-yl}hexan-
amido)-3-phenylpropanoate Hydrochloride (2)
Methyl (S)-2-{(S)-2-[(S)-2-Amino-4-methylpentanamido]-6-azi-
dohexanamido}-3-phenylpropanoate Hydrochloride (19)
Isolated as white solid and used crude as an intermediate for the
preparation of 20; full spectral characterization is provided for 20.
Yellow solid (0.18 g, 0.20 mmol, 100%); mp 163.9–164.8 °C.
1H NMR (300 MHz, CD3OD): δ = 8.29 (br s, 1 H), 8.05–7.96 (m, 1
H), 7.06–6.93 (m, 6 H), 6.73 (d, J = 8.4 Hz, 1 H), 6.29–6.24 (m, 2
H), 4.43–4.12 (m, 5 H), 3.54 (d, J = 5.1 Hz, 1 H), 3.07 (p, J = 1.5
Hz, 1 H), 2.89 (dd, J = 13.8, 5.4 Hz, 1 H), 2.75 (dd, J = 13.8, 8.4 Hz,
1 H), 2.50 (br s, 2 H), 2.18–2.06 (m, 1 H), 2.04–1.91 (m, 4 H), 1.83–
1.73 (m, 3 H), 1.64 (br s, 3 H), 1.46–1.44 (m, 4 H), 1.39–1.29 (m, 3
H), 1.24–1.10 (m, 8 H), 0.81–0.76 (m, 10 H), 0.70 (q, J = 6.6 Hz, 8
H), 0.34 (m, 1 H).
13C NMR (75 MHz, CD3OD): δ = 174.4, 173.9, 173.6, 169.9, 156.3,
153.3, 139.1, 138.3, 132.5, 130.7, 129.9, 128.3, 127.6, 127.1, 116.5,
114.2, 83.1, 59.8, 55.6, 54.4, 53.9, 53.8, 53.3, 45.0, 42.1, 41.3, 39.5,
38.7, 34.7, 33.0, 32.1, 31.1, 30.5, 29.0, 27.8, 26.2, 24.8, 23.9, 23.8,
22.6, 19.6, 18.4, 15.1.
Methyl (S)-2-[(S)-2-Amino-4-methylpentanamido]-3-phenyl-
propanoate Hydrochloride (26)
White solid (2.3 g, 7.0 mmol, 100%); mp 163.2–166.0 °C (Lit.15
162.3–163.0 °C).
Methyl (S)-2-[(S)-2-(2-Aminoacetamido)-4-methylpentanami-
do]-3-phenylpropanoate (28)
White solid (1.74 g, 4.49 mmol, 100%); mp 127.3–128.2 °C.
1H NMR (300 MHz, CD3OD): δ = 7.30–7.20 (m, 5 H), 4.98–4.92
(m, 1 H), 4.68–4.62 (m, 1 H), 4.44 (t, J = 7.5 Hz, 1 H), 3.71 (s, 3 H),
3.31 (t, J = 1.8 Hz, 1 H), 3.15 (dd, J = 14.0, 5.6 Hz, 1 H), 3.02 (dd,
J = 13.4, 8.7 Hz, 1 H), 1.70–1.59 (m, 1 H), 1.51 (d, J = 7.2 Hz, 2 H),
0.99–0.88 (m, 6 H).
13C NMR (75 MHz, CD3OD): δ = 174.5, 173.4, 167.2, 138.2, 130.4,
129.6, 128.0, 55.4, 53.3, 52.9, 42.2, 41.6, 38.3, 25.9, 23.5, 22.1.
HRMS (ESI): m/z [M + Na]+ calcd for C47H67N7O7Na: 864.4994;
found: 864.4986.
Anal. Calcd for C18H28ClN3O4: C, 56.03; H, 7.31; N, 10.89. Found:
C, 55.62; H, 8.00; N, 10.46.
(S)-2-Amino-N-(2-azidoethyl)-3-(1H-indol-3-yl)propanamide
Hydrochloride (7)
Yellow oil (2.47g, 8.00 mmol, 100%).
Synthesis of 13, 21, and 3; General Procedure
17α-Ethynylestradiol (150 mg, 0.50 mmol) and azido-peptide (0.50
mmol) were suspended in t-BuOH–H2O (3:1, 5 mL). A 1 M sodium
ascorbate soln (0.1 mL) was added, followed by CuSO4·5H2O (3
mg) dissolved in H2O (0.30 mL). The mixture was stirred for 3 d for
13 and 21, and 4 d for 3 at r.t., solvent was evaporated and the resi-
due was separated by column chromatography (EtOAc–hexanes).
1H NMR (300 MHz, DMSO-d6): δ = 11.14 (s, 1 H), 9.00 (s, 1 H),
8.36 (br s, 3 H), 7.71 (d, J = 7.8 Hz, 1 H), 3.37 (d, J = 7.8 Hz, 1 H),
7.25 (s, 1 H), 7.10–6.96 (m, 2 H), 4.40 (br s, 2 H), 4.00–3.97 (m, 1
H), 3.33–3.16 (m, 4 H).
Synthesis 2012, 44, 2926–2932
© Georg Thieme Verlag Stuttgart · New York

PAPER
17α-Ethynylestradiol Peptide Labeling by ‘Click’ Chemistry
2931
Benzyl ((5S,8S,11S,14S)-11-Benzyl-1-{4-[(8R,9S,13S,14S,17S)-
3,17-dihydroxy-13-methyl-7,8,9,11,12,13,14,15,16,17-deca-
hydro-6H-cyclopenta[a]phenanthren-17-yl]-1H-1,2,3-triazol-1-
yl}-5-[(1H-indol-3-yl)methyl]-8-isopropyl-4,7,10,13-tetraoxo-
3,6,9,12-tetraazapentadecan-14-yl)carbamate (13)
(S)-2-{(S)-2-[(S)-2-Aminopropanamido]-3-phenylpropanami-
do}-N-{(S)-1-[(2-{4-[(8R,9S,13S,14S,17S)-3,17-dihydroxy-13-
methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopen-
ta[a]phenanthren-17-yl]-1H-1,2,3-triazol-1-yl}ethyl)amino]-3-
(1H-indol-3-yl)-1-oxopropan-2-yl}-3-methylbutanamide (1)
Conjugate 13 (0.1 g, 0.09 mmol) was dissolved in MeOH (20 mL),
and Pd/C (20 mg) was added. The mixture was kept under positive
pressure of H2 for 12 h. Then the suspension was filtered through
Celite, and evaporated to give 1 as a white solid (86 mg, 0.09 mmol,
95%); mp 186.0–188.2 °C.
White solid (285 mg, 0.28 mmol, 56%); mp 158.5–160.3 °C.
1H NMR (300 MHz, CD3OD): δ = 8.00–7.72 (m, 3 H), 7.53 (d, J =
7.8 Hz, 1 H), 7.30–6.80 (m, 11 H), 4.46–6.40 (m, 2 H), 4.95 (s, 2 H),
4.62–4.56 (m, 2 H), 4.37–4.33 (m, 2 H), 4.12–4.00 (m, 2 H), 3.70–
3.50 (m, 2 H), 3.47 (q, J = 7.2 Hz, 1 H), 3.10–2.90 (m, 2 H), 2.75–
2.60 (m, 2 H), 2.50–2.37 (m, 1 H), 2.15–1.70 (m, 6 H), 1.65–1.10
(m, 9 H), 0.99 (s, 2 H), 0.95–0.87 (m, 2 H), 0.80–0.60 (m, 4 H).
13C NMR (75 MHz, CD3OD): δ = 176.0, 174.5, 174.1, 173.6, 158.6,
155.9, 155.5, 138.9, 138.1, 138.0, 132.7, 130.5, 129.6, 129.2, 128.9,
128.7, 128.0, 127.3, 124.8, 124.7, 122.6, 120.0, 119.5, 116.1, 113.8,
112.5, 111.0, 99.5, 83.4, 68.0, 56.3, 56.1, 52.8, 50.2, 50.0, 44.8,
41.1, 40.8, 38.7, 38.2, 34.4, 31.6, 30.8, 29.0, 28.8, 27.6, 24.8, 19.7,
19.1, 18.1, 17.7, 15.6, 15.1.
1H NMR (300 MHz, CD3OD): δ = 7.91–7.74 (m, 1 H), 7.62–7.60
(m, 1 H), 7.43 (d, J = 7.8 Hz, 1 H), 7.21–7.06 (m, 8 H), 6.99–6.80
(m, 3 H), 6.76 (d, J = 8.4 Hz, 1 H), 6.39–6.32 (m, 2 H), 4.44 (t, J =
6.3 Hz, 2 H), 4.27–4.22 (m, 2 H), 4.01–3.94 (m, 1 H), 3.58–3.38 (m,
3 H), 3.24 (s, 1 H), 3.16–2.80 (m, 3 H), 2.56 (br s, 2 H), 2.32 (br s,
1 H), 1.96–1.83 (m, 4 H), 1.80 (s, 2 H), 1.73–1.66 (m, 2 H), 1.55–
1.30 (m, 3 H), 1.28–1.06 (m, 5 H), 0.90 (s, 3 H), 0.84–0.60 (m, 7 H).
13C NMR (75 MHz, CD3OD): δ = 174.7, 173.5, 172.4, 156.2, 155.7,
139.2, 133.0, 132.9, 130.8, 130.5, 130.3, 130.0, 129.6, 129.3, 129.1,
128.4, 127.7, 125.1, 123.0, 120.4, 119.9, 116.5, 114.1, 112.9, 111.1,
83.7, 61.2, 56.9, 56.3, 50.5, 45.2, 41.5, 41.1, 39.0, 34.7, 31.2, 29.2,
28.0, 25.1, 24.3, 20.2, 19.3, 16.8, 16.6, 15.4.
Isolated as white solid and used crude as an intermediate for the
preparation of 1 (see below); full spectral characterization is provid-
ed for 1.
HRMS (ESI): m/z [M + Na]+ calcd for C50H63N9O6Na: 908.4794:
found: 908.4778.
Methyl (2S,5S,8S,11S)-2-Benzyl-11-[(tert-butoxycarbonyl)ami-
no]-5-(4-{4-[(8R,9S,13S,14S,17S)-3,17-dihydroxy-13-methyl-
7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenan-
thren-17-yl]-1H-1,2,3-triazol-1-yl}butyl)-8-isobutyl-12-methyl-
4,7,10-trioxo-3,6,9-triazatridecanoate (21)
Acknowledgment
White solid (198 mg, 0.21 mmol, 42%); mp 158.2–159.0 °C.
1H NMR (300 MHz, CD3OD): δ = 8.19 (d, J = 7.5 Hz, 1 H), 8.01 (t,
J = 7.5 Hz, 2 H), 7.71 (s, 2 H), 7.19–7.07 (m, 5 H), 6.89 (d, J = 8.7
Hz, 1 H), 6.54–6.39 (m, 3 H), 4.59 (q, J = 5.7 Hz, 1 H), 4.39–4.25
(m, 4 H), 3.82 (t, J = 7.2 Hz, 1 H), 3.58 (s, 3 H), 3.04 (dd, J = 13.9,
5.7 Hz, 1 H), 2.91 (dd, J = 13.9, 8.3 Hz, 1 H), 2.66–2.64 (m, 2 H),
2.44–2.30 (m, 2 H), 2.02–1.20 (m, 31 H), 0.95 (s, 3 H), 0.90–0.79
(m, 10 H), 0.60 (t, J = 9.9 Hz, 2 H).
13C NMR (75 MHz, CD3OD): δ = 174.6, 174.4, 173.7, 173.3, 158.1,
155.9, 155.4, 138.8, 137.9, 132.5, 130.3, 129.6, 127.9, 127.2, 124.0,
116.2, 113.8, 83.3, 80.6, 61.7, 55.2, 54.1, 53.2, 52.9, 51.0, 50.0,
44.9, 41.8, 41.1, 38.5, 34.4, 32.7, 32.0, 30.9, 28.9, 27.6, 25.8, 24.8,
23.7, 22.2, 20.1, 18.9, 15.1.
We thank the University of Florida, The Kenan Foundation and
King Abdulaziz University, Jeddah, Saudi Arabia for financial sup-
port.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synthesis. HPLC,
MS, 1H and 13C NMR data of compounds 1–29 are included.SunpIgfopi
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References
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Anal. Calcd for C52H75N7O9: C, 66.29; H, 8.02; N, 10.41. Found: C,
65.94; H, 8.48; N, 10.32.
Methyl (S)-2-{(S)-2-[2-((S)-2-{4-[(8R,9S,13S,14S,17S)-3,17-Di-
hydroxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-
cyclopenta[a]phenanthren-17-yl]-1H-1,2,3-triazol-1-yl}propan-
amido)acetamido]-4-methylpentanamido}-3-phenylpropano-
ate (3)
(2) (a) Kent, U. M.; Lin, H.-L.; Mills, D. E.; Regal, K. A.;
Hollenberg, P. F. Chem. Res. Toxicol. 2006, 19, 279.
(b) Holub, J. M.; Garabedian, M. J.; Kirshenbaum, K. QSAR
Comb. Sci. 2007, 26, 1175. (c) Holub, J. M.; Jang, H.;
Kirshenbaum, K. Org. Biomol. Chem. 2006, 4, 1497.
(d) Kent, U. M.; Sridar, Ch.; Spahlinger, C.; Hollenberg, P.
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(3) (a) Bjornstrom, L.; Sjoberg, M. Mol. Endocrinol. 2005, 19,
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Sandra, P.; Madder, A. Eur. J. Org. Chem. 2009, 1796.
(4) (a) Fowler, S. A.; Blackwell, H. E. Org. Biomol. Chem.
2009, 7, 1508. (b) Holub, J. M.; Garabedian, M. J.;
Kirshenbaum, K. Mol. BioSyst. 2011, 7, 337.
(5) (a) Javakhishvili, I.; Hvilsted, S. Polym. Chem. 2010, 1,
1650. (b) Kiplin, K. J.; Gavey, E.; McAdam, C. D.;
Anderson, Ch. B.; Lind, S. J.; Keep, C. C.; Gordon, K. C.;
Crowley, J. D. Inorg. Chem. 2011, 50, 6334. (c) Lipshutz, B.
H.; Taft, B. R. Angew. Chem. Int. Ed. 2006, 45, 8235.
(d) Stevis, P. E.; Deecher, D. C.; Suhadolnik, L.; Mallis, L.
M.; Frail, D. E. Endocrinology 1999, 140, 5455. (e) Kim, H.
Y.; Sohn, L.; Wijewickrama, G. T.; Edirisinghe, P.;
Gherezghiher, T.; Hemachandra, M.; Lu, P. Y.;
White solid (282 mg, 0.38 mmol, 75%); mp 140.0–141.8 °C.
1H NMR (300 MHz, DMSO-d6): δ = 11.90 (br s, 1 H), 8.98 (br s, 1
H), 8.62 (t, J = 5.7 Hz, 1 H), 8.40 (d, J = 7.5 Hz, 1 H), 8.01 (d, J =
8.4 Hz, 1 H), 7.91 (s, 1 H), 7.28–7.17 (m, 4 H), 6.95 (d, J = 8.4 Hz,
1 H), 6.49–6.42 (m, 2 H), 5.55–5.45 (m, 1 H), 5.10 (br s, 1 H), 4.49–
4.32 (m, 2H), 3.78 (d, J = 5.4 Hz, 2 H), 3.56 (s, 3 H), 3.44 (br s, 2
H), 3.04–2.95 (m, 2 H), 2.70 (br s, 2 H), 2.45–2.30 (m, 1 H), 2.12–
1.21 (m, 14 H), 0.94–0.82 (m, 6 H), 0.70–0.52 (m, 1 H).
13C NMR (75 MHz, DMSO-d6): δ = 172.0, 171.8, 169.1, 167.9,
154.9, 154.1, 137.2, 137.1, 130.4, 129.1, 128.3, 126.6, 126.1, 121.6,
114.9, 112.7, 81.3, 57.9, 53.6, 51.8, 50.7, 47.6, 46.8, 43.2, 41.9,
41.1, 39.4, 37.3, 36.5, 32.6, 29.4, 27.3, 26.2, 24.1, 23.6, 23.0, 21.8,
21.1, 18.3, 14.5.
Anal. Calcd for C41H54N6O7: C, 66.29; H, 7.33; N, 11.31. Found: C,
65.19; H, 7.90; N, 10.55..
HRMS (ESI): m/z [M + Na]+ calcd for C41H54N6O7Na: 765.3946;
found: 765.3927.
Chandrasena, R. E.; Molloy, M. E.; Tonetti, D. A.; Thatcher,
G. R. Bioorg. Med. Chem. 2010, 18, 809. (f) Harrington, W.
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2012, 44, 2926–2932

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