Chemical synthesis of hormone receptor probes: High affinity photoactivated enediyne-estrogens

Article abstract of DOI:10.1016/S0040-4039(01)01902-5

A family of enediyne-estrogens has been prepared, and evaluated for affinity to hER. The optimal ligand has nM affinity, and undergoes photoBergman cycloaromatization.

Full text of DOI:10.1016/S0040-4039(01)01902-5

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 8579–8582
Chemical synthesis of hormone receptor probes: high affinity
photoactivated enediyne-estrogens
Ajay Purohit,a Justin Wyatt,a George Hynd,a Justin Wright,a Ahmed El-Shafey,a Narashima Swamy,b
Rahul Rayb and Graham B. Jonesa,*
aDepartment of Chemistry, Northeastern University, 360 Huntington Ave., Boston, MA 02115, USA
bDepartment of Biochemistry, Boston University Medical Center, Boston, MA 02118, USA
Received 19 September 2001; accepted 9 October 2001
Abstract—A family of enediyne-estrogens has been prepared, and evaluated for affinity to hER. The optimal ligand has nM
affinity, and undergoes photoBergman cycloaromatization. © 2001 Elsevier Science Ltd. All rights reserved.
Steroid hormone receptors are members of a superfam-
ily of nuclear transcription factors which modulate gene
activity by both hormone dependent and hormone
independent mechanisms.1 Hormonal response is medi-
ated by the interaction of the ligand with its receptor,
which then initiates the binding of the complex to
specific regulatory sequences at target genes, referred to
as hormone response elements (HRE’s). In the case of
the human estrogen receptor (hER a/b) distinct subdo-
mains have been characterized including those for
transactivation (A, B) DNA binding (C) and hormone
binding (E) (Fig. 1), which recognizes the endogenous
ligand b-estradiol with nanomolar affinity.2 Estrogen-
mediated pathways are of paramount importance in
endocrinology and have a strong correlation with the
progression of mammary tumors, and continue to be
the focus of numerous studies.3 On the basis of QSAR
analysis, selective antagonists of b-estradiol have been
developed including tamoxifen and raloxifene, both of
which are competitive antagonists and show efficacy
against human breast cancer.4 A complimentary strat-
egy is the disruption of estrogen biosynthesis, as exem-
pioneered by Covey and Robinson.5 Our own efforts
have been directed towards agents with the ability to
selectively interfere with the transcriptional activation
process, and we recently developed a class of enediyne-
estrogens exemplified by the thermally labile ligand 2.6
Enediynes and related ene–yne–allenes are thermally
activated to produce diyl radicals by a processes
referred to as the Bergman or Myers-Saito cyclizations,
respectively (Scheme 1).7 These high energy species are
capable of abstracting hydrogen atoms from DNA
(causing strand scission) and from proteins (leading to
degradation and/or agglomeration). In the case of 2,
though this molecule was effective in inducing tempera-
ture dependent degradation of hERa, and of modulat-
ing transcription, its affinity for hER was only 10−6
M
presumably due to the hydrophilic bridging group link-
ing the enediyne to the alkynyl-steroid.6 Encouraged by
this observation, we have been actively pursuing sys-
tems with the nM affinity of the endogenous estradiol,
and that are thermally stable but can be activated on
demand. Our strategy was to use affinity [hERa] driven
synthesis of either 17a substituted steroidal ene–yne–
allenes or enediynes to determine the minimal linker
unit coupling the warhead and steroid, avoiding
hydrophilic groups which are known to reduce binding
affinity. A central feature was to incorporate enediyne
building blocks which have been made available
through our carbenoid route to linear and cyclic
plified by the ketosteroid isomerase inhibitor
1
hormone
binding
domain
1
37
180 250 302
552 595
enediynes.8
A
B
F
C
D
O
E
OH
O
O
O
Figure 1. Human ER binding domains.
O
C
C C
HO
2
1
H
H
* Corresponding author.
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01902-5

8580
A. Purohit et al. / Tetrahedron Letters 42 (2001) 8579–8582
estrogen
estrogen
estrogen
estrogen
C
H
Bergman
C
Myers-Saito
H
C
H
Scheme 1. Possible pathways for generation of proteolytic diradical-estrogen conjugates.
findings in the design of photoproteases, selected a
carbocyclic enediyne.10 Specifically, enediyne 11 which
is available via an efficient carbenoid coupling reac-
tion,10 was coupled to 3 via its organocerium derivative,
then unmasked to give 12 (Scheme 3). Unfortunately
this compound, which had an RBA of >100 nM,
proved stable at physiological temperature, and did not
undergo the expected photo-Bergman cycloaromatiza-
tion even on prolonged irradiation.11 Following the
strategy adopted previously, we elected to insert a
spacer unit in the system, and pursued m-disubstituted
ligand 15. Coupling of 11 with iodoalkyne 13 followed
by 1,2 addition and deprotection gave 15 in good yield.
To our surprise, the affinity of 15 was greatly superior
to 12, supporting the notion that the LBD of hERa has
considerable tolerance for lipophilic functionality at the
Our first target was enediyne 5, which was available by
coupling the acetylide of monoprotected linear enediyne
48 with estrone derivative 3, followed by desilylation
(Scheme 2). Though compound 5 had enhanced affinity
for the estrogen receptor (Table 1), it failed to undergo
thermal Bergman cycloaromatization even at elevated
temperatures (ꢀ100°C). Prolonged thermolysis resulted
in isomerization to the corresponding E enediyne,
prompting us to search for an alternate diyl progenitor.
Accordingly, the ene–yne–allene
7 was pursued.
Estrone 3 was coupled with THP ether 6 via its
organocerium derivative,8 the propargylic alcohol
exposed and then converted to the corresponding
allene,9 and finally the phenol unmasked to give 7 in
moderate yield. As with 5, this ligand had appreciable
affinity for hERa, (Table 1) but also proved stable even
after prolonged incubation at physiological temperature
and even in refluxing THF. Suspecting that the steroid
core may inhibit the (reversible) cycloaromatization
process, we opted to install a spacing linker between the
two entities and prepared 10. Coupling of the pre-
assembled alkynyl enediyne to 3 proved impossible,
warranting synthesis via intermediate iodoalkyne 8,
which was then coupled with enediyne 98 then the
allene moiety elaborated. As was the case with 7,
cycloaromatization of 10 could not be induced even
under forcing conditions. Intriguingly, prompted by
earlier investigations, photochemical cycloaromatiza-
tion of 10 was attempted, resulting in trace (>10%)
conversion to the expected arene product. Encouraged
by this finding, we sought to couple a more efficient
photoactivated group to the steroid, and based on prior
Table 1. Relative binding affinity of steroid–enediynes for
hERa
Entry
Substrate
RBA (nM)
1
2
3
4
5
6
7
2
5
7
10
12
15
21
510
8
45
75
103
14
12
a Candidate+3H b-estradiol incubated with cytosol at 4°C. Unbound
agents removed (DCC), and bound 3H b-estradiol measured by
scintillation counter. RBA corresponds to concentration required to
reduce hER bound 3H b-estradiol by 50%.12,15
THPO
1.
TES
HO
6
HO
O
LiHMDS, CeCl3, THF
2. MgBr2, Et2O
1.
4
C
H
n-BuLi, CeCl3,THF
C
H
C
3. MesCl, Et3N, CH2Cl2
4. NH2NH2, MTAD, Et2O
5. TBAF, THF
2. TBAF, THF
H
H
3
5
HO
7
TBSO
HO
43%
29%
Li
TMS
1.
CeCl3, THF
2. TBDMSOTf, 2,6-lutidine, CH2Cl2
3. AgNO3, NaCN, EtOH, H2O
4. BuLi, I2, THF
HO
9
I
TBDMSO
HO
1.
PdCl2(PPh3)2,
C
H
C
CuI, Et3N, CH2Cl2
H
C
2. MesCl, Et3N, CH2Cl2
3. NH2NH2, MTAD, Et 2O
H
10
8
4. TBAF, THF
TBDMSO
56%
HO
35%
Scheme 2. Alkynyl linked estrogen-enediynes and estrogen–eneyne–allenes.

A. Purohit et al. / Tetrahedron Letters 42 (2001) 8579–8582
8581
13
1. TBAF, THF 0 oC
OH
I
2. BuLi, THF, -78 o
C
11
TBS
TBS
Ph
then CeCl3, 3
3. TBAF, THF 0 oC
Pd(PPh3)2Cl2,
CuI, Et3N
Ph
Ph
14
HO
80%
15
72%
1. BuLi, THF, -78 o
C
hν / iPrOH
then CeCl3, 3
2. TBAF, THF 0 oC
75%
OH
OH
OH
Ph
Ph
Ph
<5%
HO
17
HO
12
HO
16
Scheme 3. High affinity carbocyclic enediyne-estrogens.
H
OH
OH
TMS
Ph
H
1.
1. K2CO3, MeOH
2. 1,3 diiodobenzene
Pd(PPh3)2Cl2, CuI,
nBuNH2
I
Pd(PPh3)2Cl2,
CuI, nBuNH2
+
Ph
TBSO
TBSO
19
18
20
2.
70%
TMS
H
1. Pd(PPh3)2Cl2,
CuI, Et3N
2. TBAF, THF 0 oC
63%
Pd(PPh3)2Cl2,
CuI, Et3N
3.
TBAF, THF
65%
OH
OH
I
hν, iPrOH
I
Ph
HO
HO
22
21
Scheme 4. High affinity photoactivated enediyne-estrogens.
steroidal 17a position.12 Though the binding affinity of
15 was spectacular, to our disappointment its photo-
Bergman cycloaromatization profile was poor, giving
only trace amounts of the expected product 17 even
after prolonged irradiation. We elected to improve the
efficiency of the critical process by extending the conju-
gation of the ligand, and pursued 21 as a refined target.
Though this required complete re-synthesis, an efficient
process was developed via pre-assembly of enediyne
template 20, in turn available by sequential coupling to
1,2-diiodobenzene (Scheme 4). This was coupled in turn
to iodoarene 19, produced by arylation of the
unmasked alkyne derived from 18, which could be
produced either from 3, or the commercially available
17a-ethynyl estradiol. Though the binding affinity of 21
did not differ markedly from 15, this ligand underwent
smooth photocycloaromatization to give complete con-
version to product 22 within 3 h.13 We have thus
succeeded in designing an enediyne-estrogen with nM
affinity, and which can be activated on irradiation.14
Given the promising data obtained with the mM binder
2, numerous applications of this molecular probe can
be envisioned, and we are actively pursuing experiments
designed to maximize its potential.
In summary, the convergent synthesis of a photoacti-
vated enediyne-estrogen conjugate has been accom-
plished. The ligand binds to the ligand binding domain
of the human estrogen receptor with low nM affinity,
and more importantly, undergoes efficient photo-
Bergman cycloaromatization. We anticipate this ligand
will serve as a useful probe for the receptor, and may
permit site selective modification via diyl radical atom
transfer chemistry.16
References
1. (a) O’Malley, B. W. Mol. Endocrinol. 1990, 4, 363; (b)
Evans, R. M. Science 1988, 240, 889; (c) Kumar, V.;
Green, S.; Stack, G.; Berry, M.; Jin, J.-R.; Chambon, P.
Cell 1987, 51, 941.
2. (a) Chen, H.; Lin, R. J.; Xie, W.; Wilpitz, D.; Evans, R.
M. Cell 1999, 98, 675; (b) Ing, N. H.; Beekman, J. M.;
Tsai, S. Y.; Tsai, M.-J.; O’Malley, B. W. J. Biol. Chem.
1992, 267, 17617.
3. Korach, K. S. In Molecular Structure and Biological
Activities of Steroids; Bohl, W.; Duax, W. L., Eds.; CRC
Press, 1992; p. 210.

8582
A. Purohit et al. / Tetrahedron Letters 42 (2001) 8579–8582
9. Myers, A. G.; Parrish, C. A. Bioconjugate Chem. 1996, 7,
4. Kym, P. R.; Anstead, G. M.; Pinney, K. G.; Wilson, S.
322.
R.; Katzenellenbogen, J. A. J. Med. Chem. 1993, 36,
10. Jones, G. B.; Wright, J. M.; Plourde, G. W.; Wyatt, J.
K.; Hynd, G.; Fouad, F.; Purohit, A. J. Am. Chem. Soc.
2000, 122, 9872.
3910.
5. Covey, D. F.; Robinson, C. H. J. Am. Chem. Soc. 1976,
98, 5038.
11. Recent reports on photo-Bergman cycloaromatization:
(a) Clark, A. E.; Davidson, E. R.; Zaleski, J. M. J. Am.
Chem. Soc. 2001, 123, 2650; (b) Choy, N.; Blanco, B.;
Wen, J.; Krishan, A.; Russell, K. C. Org. Lett. 2000, 2,
3761; (c) Funk, R. L.; Young, E. R. R.; Williams, R. M.;
Flanagan, M. F.; Cecil, T. L. J. Am. Chem. Soc. 1996,
118, 3291; (d) Turro, N. J.; Evenzahav, A.; Nicolaou, K.
C. Tetrahedron Lett. 1994, 35, 8089.
6. Jones, G. B.; Wright, J. M.; Hynd, G.; Wyatt, J. K.;
Yancisin, M.; Brown, M. A. Org. Lett. 2000, 2, 1863.
7. Recent reviews: (a) Smith, A. L.; Nicolaou, K. C. J. Med.
Chem. 1996, 39, 2103; (b) Xi, Z.; Goldberg, I. In Compre-
hensive Natural Products Chemistry; Barton, D. H. R.;
Nakanishi, K., Eds. DNA Damaging enediyne com-
pounds. Pergamon: Oxford, 1999; Vol. 7, p. 553. Recent
enediyne publications: (c) Jones, G. B.; Warner, P. M. J.
Am. Chem. Soc. 2001, 132, 2134; (d) Lin, C. F.; Hsieh, P.
C.; Lu, W. D.; Chiu, H. F.; Wu, M. J. Bioorg. Med.
Chem. 2001, 9, 1707; (e) Basak, A.; Bdour, H. M.; Shain,
J. C.; Mandal, S.; Rudra, K. R.; Nag, S. Bioorg. Med.
Chem. Lett. 2000, 10, 1321; (f) David, W. M.; Kumar, D.;
Kerwin, S. M. Bioorg. Med. Chem. Lett. 2000, 10, 2509;
(g) Jones, G. B.; Plourde, G. W. Org. Lett. 2000, 2, 1757;
(h) Jones, G. B.; Plourde, G. W.; Wright, J. M. Org. Lett.
2000, 2, 811; (i) Hirama, M.; Akiyama, K.; Tanaka, T.;
Noda, T.; Iida, K.; Sato, I.; Hanaishi, R.; Fukuda-
Ishisaka, S.; Ishiguro, M.; Otani, T.; Leet, J. E. J. Am.
Chem. Soc. 2000, 122, 720; (j) Thorson, J. S.; Sievers, E.
L.; Ahlert, J.; Shepard, E.; Whitwam, R. E.; Onwueme,
K. C.; Ruppen, M. Curr. Pharm. Des. 2000, 6, 1841.
8. Jones, G. B.; Wright, J. M.; Plourde, G. W.; Hynd, G.;
Huber, R. S.; Mathews, J. E. J. Am. Chem. Soc. 2000,
122, 1937.
12. Gao, H.; Katzenellenbogen, J. A.; Garg, R.; Hansch, C.
Chem. Rev. 1999, 99, 723.
13. Hanovia 450 W medium pressure lamp, degassed iPrOH,
−1
Pyrex filter. UV–vis (21) m311=2.1×104
M
cm−1; m275
=5.9×104 M−1 cm−1. Prolonged irradiation results in
polymerization and decomposition.
14. Previous work on enediyne-estrogens: (a) Jones, G. B.;
Hynd, G.; Wright, J. M.; Purohit, A.; Plourde, II, G. W.;
Huber, R. S.; Mathews, J. E.; Li, A.; Kilgore, M. W.;
Bubley, G. J.; Yancisin, M.; Brown, M. J. Org. Chem.
2001, 66, 3688; (b) Py, S.; Harwig, C. W.; Banerjee, S.;
Brown, D. L.; Fallis, A. G. Tetrahedron Lett. 1998, 39,
6139; (c) Jones, G. B.; Huber, R. S.; Mathews, J. E.; Li,
A. Tetrahedron Lett. 1996, 37, 3643; (d) Wang, J.;
DeClercq, P. J. Angew. Chem., Int. Ed. 1995, 34, 1749.
15. Scatchard, G. Ann. N.Y. Acad. Sci. 1949, 51, 660.
16. This work was supported by the National Institutes of
Health [R01GM57123].