Design and synthesis of iodocarborane-containing ligands with high affinity and selectivity toward ERβ

Article abstract of DOI:10.1016/j.bmcl.2017.07.053

The selectivity and the binding affinity of previously reported carborane-containing ligands 2 and 3 toward ERβ remains to be optimized. To improve their biological profiles, a series of iodinated carboranyl phenol derivatives (4–6) were designed and synthesized as prospective ERβ-selective ligands with high affinity. Several iodinated carboranyl phenols showed high relative binding affinity (RBA) values for both ERs, and especially for ERβ, due to suitable hydrophobic interactions of the iodine atoms with the hydrophobic amino acid residues of the ERβ ligand-binding domains. Among these derivatives, 9,10-diiodo-m-carborane 5f exhibited a more than 100% increase of the RBA values toward ERβ, a 14-fold increased selectivity for ERβ over ERα, and ER-agonistic activity in MCF-7 cell proliferation assays.

Full text of DOI:10.1016/j.bmcl.2017.07.053

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of iodocarborane-containing ligands with high
affinity and selectivity toward ERb
⇑
Kiminori Ohta , Takumi Ogawa, Yasuyuki Endo
Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The selectivity and the binding affinity of previously reported carborane-containing ligands 2 and 3
toward ERb remains to be optimized. To improve their biological profiles, a series of iodinated carboranyl
phenol derivatives (4–6) were designed and synthesized as prospective ERb-selective ligands with high
affinity. Several iodinated carboranyl phenols showed high relative binding affinity (RBA) values for both
ERs, and especially for ERb, due to suitable hydrophobic interactions of the iodine atoms with the
hydrophobic amino acid residues of the ERb ligand-binding domains. Among these derivatives, 9,10-
diiodo-m-carborane 5f exhibited a more than 100% increase of the RBA values toward ERb, a 14-fold
Received 21 June 2017
Revised 19 July 2017
Accepted 20 July 2017
Available online xxxx
Keywords:
Carborane
Estrogen receptor
ERb
increased selectivity for ERb over ER
a
, and ER-agonistic activity in MCF-7 cell proliferation assays.
Ó 2017 Elsevier Ltd. All rights reserved.
Subtype selectivity
Estrogen receptor b (ERb)1 was initially cloned from a rat ven-
tral prostate cDNA library as a second form of ERa 2
and has since
shown quite different biological functions compared to ERa 3
high RBA values and high selectivity toward ERb, respectively
(Fig. 1). Although 3 showed much better ERb selectivity than 2, it
is still not sufficient for the development of drug candidates or bio-
logical tools on account of the low binding affinity toward ERb. An
SAR study on the ERb selectivity of 2 revealed that the iodine atom
on the carbon atom of the m-carborane cage in 2 increases the
binding affinity toward ERb. Thus, we designed novel iodinated
carborane-containing ERb-selective ligands with high affinity
toward ERb, using 2 as a lead compound. In this paper, we describe
the syntheses of several iodinated carboranyl phenols (4–6) and
the evaluation of their ER-binding affinity and selectivity.
The synthesis of iodinated-o-carborane 4 is summarized in
Scheme 1. 3-Iodo-o-carborane 7, which was prepared from the o-
carborane-reconstructing reaction of nido-7,8-C2B9HÀ12 with BI3,15
was treated with 4-methoxyphenylmagnesium bromide in the
presence of CuI under Pd-catalyzed coupling conditions to afford
3-(4-methoxyphenyl)-o-carborane 8 in 98% yield.16 Two iodine
atoms were introduced on two carbon atoms of the o-carborane
cage of 8 by consecutive exposure to n-BuLi and I2, which afforded
9 in 91% yield. Subsequently, 9 was treated with BBr3 to furnish
1,2-diiodo-o-carboranylphenol 4 in 96% yield.
,
.
Endogenous estrogen 17b-estradiol (E2, 1) modulates various
physiological processes, including the development and function
of the female reproductive system, as well as the maintenance of
bone mineral density.4 However, the therapeutic use of 1 is limited
by an increased risk of breast cancer, which has been linked to the
activation of ER
a
(Fig. 1).5 Nevertheless, the activation of ERb
induces an anti-proliferative effect on breast cancer.6 ERb-selective
agonists down-regulate the expression of the androgen receptor
(AR),7 inhibit the proliferation and migration of prostate cancer cell
lines,8 and delay the progression of Alzheimer’s disease by includ-
ing the degradation of Ab aggregates.9 Thus, ERb-selective ligands
are of interest as potential therapeutic agents for Alzheimer’s
disease and several other types of cancer, and as probes for ERb-
related molecular biology.10
The development of ER-subtype-selective ligands remains a
major challenge, as there are only two different amino acid resi-
dues in the hydrophobic pocket of the ER-ligand-binding domains
(LBDs),11 i.e., Leu384 and Met421 in ER
a are substituted in ERb by
Met336 and Ile373, respectively.11 Most ERb-selective ligands
obtain their subtype selectivity through electronic, hydrophobic,
or steric interactions with these key amino acid residues.12
Based on the above theory, we have recently developed carbo-
rane-containing ERb-selective ligands 213 and 3,14 which showed
Iodinated m-carboranes 5a–5e were synthesized from 11
(Scheme 2), which was obtained in 28% yield from the iodination
of m-carborane 10 with ICl in the presence of AlCl3,17 followed
by a transformation into a copper salt and an Ullman coupling.18
In this coupling reaction, 7,9-diiodo derivative 12 was also
obtained unexpectedly in 23% yield, which suggests that the cop-
per salt of 11 reacts with the iodine liberated during the reaction.
The iodinated m-carboranes 11 and 12 were treated with BBr3 to
⇑
Corresponding author.
E-mail address: k-ohta@tohoku-mpu.ac.jp (K. Ohta).
http://dx.doi.org/10.1016/j.bmcl.2017.07.053
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
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2
K. Ohta et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
afford 9-iodo-m-carboranylphenol 5a and 7,9-diiodo-m-carbo-
ranylphenol 5b in 90% and quantitative yield, respectively. Allyl,
n-butyl, and benzyl groups were introduced into the C–H moiety
of the m-carborane cage of 11 using the corresponding halide
reagents in 68–83% yield, which were treated with BBr3 to afford
the corresponding 9-iodophenol derivatives 5c, 5d, and 5e in
68%, 75%, and 93% yield, respectively. Scheme 2 also includes the
synthesis of 9,10-diiodo-m-carboranylphenol 5f. 9,10-Diiodo-m-
carborane 13,19 prepared from the iodination of the corresponding
m-carborane, was transformed into 14 (65%) under Cu-mediated
coupling conditions, followed by a deprotection of the methyl
group with BBr3 to afford 5f in 61% yield.
Fig. 1. Structures of 17b-estradiol and carborane-containing ERb-selective ligands.
Scheme 3 summarizes the synthesis of iodinated p-carboranyl
phenols 6a–6c. Compound 16, which was prepared by the Cu-
mediated coupling reaction of p-carborane 15 with 4-iodoanisole,
was consecutively treated with n-BuLi and I2, before a demethyla-
tion with BBr3 afforded 12-iodo-p-carboranylphenol 6a in 33%
overall yield. A Pd-catalyzed B-arylation of 2-iodo-p-carborane
18, which was obtained from the iodination of p-carborane with
4-methoxymagnesium bromide, afforded 19 in 90% yield.16 Com-
pound 19 was subsequently treated with 2 equivalents of n-BuLi,
quenched with I2, and demethylated to afford 6b in 73% overall
yield. One of the less hindered carbon atoms of the p-carborane
cage of 19 was protected with a triphenylsilyl group,20 while
another carbon atom was iodinated to afford 22 in 20% overall
yield. The two protecting groups, i.e., the triphenylsilyl and methyl
groups, were separately removed with TBAF and BBr3, respectively,
to afford 6c in 90% overall yield.
Scheme 1. Synthesis of iodinated o-carborane 4. Reagents and conditions: (a)
4-methoxyphenylmagnesium bromide, PdCl2(PPh3)2, CuI, THF, reflux; (b) (i) n-BuLi,
Et2O, 0 °C; (ii) I2, rt; (c) BBr3, CH2Cl2, 0 °C.
The relative binding affinity (RBA) values toward ERa, ERb, and
the ER-subtype selectivity of the compounds synthesized in this
study are summarized in Table 1.21 Interestingly, several deriva-
tives showed RBA values of more than 100% toward ERb. Although
o-carborane 4 showed a high ERb-binding affinity, the ERb selectiv-
ity was moderate due to the potent binding affinity toward ERa.
We found that iodinated m-carborane 5a is an ERb-selective ligand
with high binding affinity toward ERb. The introduction of an
iodine atom onto the carbon atom of the m-carborane cage
reduced the RBA values toward ERa and ERb, but the ERb selectiv-
ity of 5b was almost similar to that of 5a. On the other hand, the
introduction of an alkyl substituent, such as an allyl (5c), n-butyl
(5d), or benzyl (5e) group, enhanced the ERa-binding affinity
under concomitant decrease of the ERb selectivity. Benzyl deriva-
tive 5e exhibited a remarkable decrease of the binding affinity
Scheme 2. Synthesis of iodinated m-carborane derivatives 5a–f. Reagents and
conditions: (a) (i) n-BuLi, DME, 0 °C; (ii) CuCl, rt; (iii) 4-iodoanisole, pyridine, 100 °C;
(b) (i) n-BuLi, Et2O, 0 °C; (ii) R-X, rt; (c) BBr3, CH2Cl2, 0 °C.
Scheme 3. Synthesis of iodinated p-carboranes 6a–c. Reagents and conditions: (a) (i) n-BuLi, DME, 0 °C; (ii) CuCl, rt; (iii) 4-iodoanisole, pyridine, 100 °C; (b) (i) n-BuLi, Et2O,
0 °C; (ii) I2, rt; (c) BBr3, CH2Cl2, 0 °C; (d) 4-methoxyphenylmagnesium bromide, PdCl2(PPh3)2, CuI, THF, reflux; (e) (i) n-BuLi, Et2O, 0 °C; (ii) Ph3SiCl, rt; (f) TBAF, THF, rt.
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K. Ohta et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Table 1
RBA values toward ERa
and ERb, and ER-subtype selectivity of the iodinated carboranyl phenols 7–9.a
Compound
Carborane
R
RBAb
Selectivityc
ER /ERb
ER
a
ERb
a
ERb/ERa
4
o-Carborane
m-Carborane
–
H
I
42
13
8.4
17
29
28
8.0
574
18
242
129
81
62
157
19
111
888
107
38
0.17
0.10
0.10
0.27
0.18
1.50
0.07
0.65
0.17
0.26
5.8
9.9
9.6
3.6
5.4
0.68
14
1.5
5.9
3.8
5a
5b
5c
5d
5e
5f
6a
6b
6c
Allyl
n-Butyl
Benzyl
–
–
I
p-Carborane
H
10
a
In all binding assays, which were performed in triplicate (n = 3), the test compounds (0.4 nM–4
l
M) were examined in the presence of [2,4,6,7-3H]17b-estradiol (4 nM).
RBA values were calculated from the IC50 values of E2 and the test compounds, whereby that of E2 was set to 100%.
The ER-subtype selectivity was estimated from the RBA values toward ER and ERb.
b
c
a
using the MCF-7 cell line, which shows estrogen-dependent
growth (Fig. 2).21 5f promoted the MCF-7 cell proliferation in a
concentration-dependent manner and acted as an ER agonist.
In conclusion, we designed and synthesized various iodinated
carboranyl phenols containing o-, m-, and p-carborane cages as
prospective ligands with high selectivity and affinity toward ERb.
A competitive binding assay revealed that the introduction of
iodine atoms enhanced their ERb selectivity, which might be
caused by a suitable hydrophobic interaction of the iodine atom
with the amino acid residues of ERb LBD. 5f showed the highest
ERb selectivity, potent ERb binding affinity, and ER agonistic activ-
ity in an MCF-7 cell proliferation assay.
Acknowledgments
This research was supported by
a grant-in-aid from the
Strategic Research Program for Private Universities (2015–2019)
and Grants-in-Aid for Scientific Research (C) (26460151 and
15K08029) from the Japanese Ministry of Education, Culture,
Sports, Science, and Technology (MEXT).
Fig. 2. Concentration-dependent MCF-7 cell proliferation induced by 5f. MCF-7
cells were incubated with 5f (100 nM–1 nM) for 5 days; the results are shown as
the fold cell number, whereby the value for 0.1 nM E2 was set to 100%; assays were
performed in triplicate (n = 3).
References
1. Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson J-A. Proc Natl
Acad Sci USA. 1996;93:5925.
toward ERb, and the RBA value of 5e toward ER
ERb. 9,10-Diiodo-m-carborane 5f showed a 14-fold higher selectiv-
ity toward ERb than toward ER with an RBA value of more than
100% for ERb. Surprisingly, the RBA value of C-iodo-p-carboranyl
phenol 6a toward ERb was more than 800%. Unfortunately, the
a surpassed that of
2. Green S, Walter P, Kumar V, et al. Nature. 1986;320:134.
3. Heldring N, Pike A, Andersson S, et al. Physiol Rev. 2007;87:905.
4. (a) Lanyon L, Armstrong V, Ong D, Zaman G, Price J. J Endocrinol. 2004;182:183;
(b) Pfaff D, Waters E, Khan Q, Zhang X, Numan M. Endocrinology.
2011;152:1209;
(c) Ribeiro JR, Freiman RN. J Steroid Biochem Mol Biol. 2014;143:160.
5. Piu F, Cheevers C, Hyldtoft L, et al. Eur J Pharmacol. 2008;590:423.
6. Haldosen L-A, Zhao C, Dahlman-Wright K. Mol Cell Endocrinol. 2014;382:665.
7. Wu W-F, Maneix L, Insunza J, et al. Proc Natl Acad Sci USA. 2017;114:E3816.
8. (a) Pravettoni A, Mornati O, Martini PGV, et al. Mol Cell Endocrinol. 2007;263:46;
(b) Kim IY, Kim B-C, Seong DH, et al. Cancer Res. 2002;62:5365.
9. George S, Petit GH, Gouras GK, Brundin P, Olsson R. ACS Chem Neurosci.
2013;4:1537.
a
RBA value toward ER
a was also more than 500%, resulting in an
overall poor ERb selectivity. These results suggest that the rectilin-
ear C-iodo-p-carboranyl phenol structure is particularly suitable
for the hydrophobic pocket of both ERs. p-Carboranes 6b and 6c
exhibit a bent core structure comparable to that of 4 and 5, and
showed 5.9- and 3.8-fold selectivity toward ERb, respectively.
The biological profiles, as well as the agonistic and antagonistic
activity of 5f were evaluated by means of a cell proliferation assay
10. Warner M, Huang B, Gustafsson J-A. Trends Pharmacol Sci. 2017;38:92.
11. (a) Paech K, Webb P, Kuiper GGJM, et al. Science. 1997;277:1508;
(b) Brzozowski AM, Pike AC, Dauter Z, et al. Nature. 1997;389:753.
Please cite this article in press as: Ohta K., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.07.053

4
K. Ohta et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
12. (a) Sun W, Cama LD, Birzin ET, et al. Bioorg Med Chem Lett. 2006;16:1468;
(b) Blizzard TE, Gude C, Morgan II JD, et al. Bioorg Med Chem Lett. 2006;16:834;
(c) Wilkening RR, Ratcliffe RW, Tynebor EC, et al. Bioorg Med Chem Lett.
2006;16:3489.
13. Ohta K, Ogawa T, Kaise A, Endo Y. Bioorg Med Chem Lett. 2013;23:6555.
14. Ohta K, Ogawa T, Oda A, Kaise A, Endo Y. Bioorg Med Chem Lett. 2015;25:4174.
15. (a) Viñas C, Barberà G, Oliva JM, Teixidor F, Welch AJ, Rosair GM. Inorg Chem.
2001;40:6555;
16. Ohta K, Yamazaki H, Endo Y. J Organomet Chem. 2009;694:1646.
17. Andrews JS, Zayas J, Jones Jr M. Inorg Chem. 1985;24:3715.
18. (a) Ohta K, Goto T, Endo Y. Inorg Chem. 2005;44:8569;
(b) Coult R, Fox MA, Gill WR, Herbertson PL, MacBride JAH, Wade K. J Organomet
Chem. 1993;462:19.
19. Ohta K, Ogawa T, Kaise A, Endo Y. Bioorg Med Chem. 2014;22:3508.
20. Douglass AG, Pakhomov S, Reeves B, Janousek Z, Kaszynski P. J Org Chem.
2000;65:1434.
(b) Barberà G, Viñas C, Teixidor F, Rosair GM, Welch AJ. J Chem Soc, Dalton Trans.
2002;3647.
21. Ohta K, Chiba Y, Ogawa T, Endo Y. Bioorg Med Chem Lett. 2008;18:5050.
Please cite this article in press as: Ohta K., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.07.053