Synthesis and preliminary evaluation steroidal antiestrogen-geldanamycin conjugates

Article abstract of DOI:10.1016/j.bmcl.2013.03.116

Three novel steroidal antiestrogen-geldanamycin conjugates were prepared using a convergent strategy. The antiestrogenic component utilized the 11β-(4-functionalized-oxyphenyl) estradiol scaffold, while the geldanamycin component was derived by replacement of the 17-methoxy group with an appropriately functionalized amine. Ligation was achieved in high yield using azide alkyne cyclization reactions. Evaluation of the products against two breast cancer cell lines indicated that the conjugates retained significant antiproliferative activity.

Full text of DOI:10.1016/j.bmcl.2013.03.116

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3635–3639
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and preliminary evaluation steroidal antiestrogen–gel-
danamycin conjugates
b
b
b
J. Adam Hendricks a, Robert N. Hanson a, , Michael Amolins , John M. Mihelcic , Brian S. Blagg
⇑
a Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, United States
b Department of Medicinal Chemistry, The University of Kansas, Lawrence, KS, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Three novel steroidal antiestrogen–geldanamycin conjugates were prepared using a convergent strategy.
The antiestrogenic component utilized the 11b-(4-functionalized-oxyphenyl) estradiol scaffold, while the
geldanamycin component was derived by replacement of the 17-methoxy group with an appropriately
functionalized amine. Ligation was achieved in high yield using azide alkyne cyclization reactions. Eval-
uation of the products against two breast cancer cell lines indicated that the conjugates retained signif-
icant antiproliferative activity.
Received 13 February 2013
Revised 22 March 2013
Accepted 27 March 2013
Available online 4 April 2013
Keywords:
Ó 2013 Published by Elsevier Ltd.
Anti-estrogen geldanamycin conjugates
Targeted drug delivery
Breast cancer
Click chemistry
Introduction
lack of synergy may have involved the properties of the linker.
Unfortunately, issues regarding the availability mitomycin C pre-
Breast cancer is the most prevalent form of cancer in women
and the well-established association between the human estrogen
receptor (ER) and cell proliferation provided the basis for endo-
crine (antihormonal) therapy.1,2 However, prolonged treatment
with antiestrogens often results in the development of hormonal
resistance, leading to recurrence of the disease and the use of more
potent, but nonselective, therapeutic agents.3–5 One strategy that
attempts to circumvent the effects of resistance is the use of drug
conjugates in which two therapeutic agents are combined into a
single entity.6–8
As part of our program in breast cancer research, we have fo-
cused on using ER as a targeting mechanism for which the steroidal
anti-estrogenic component may also provide a beneficial therapeu-
tic response. The choice of the therapeutic component is also crit-
ical as it should not only be active within the same concentration
range as the hormonal component but exert a complementary or
synergistic effect. The ER-targeting component was developed in
our initial work with the 11b-(4-substituted-oxyphenyl) estradi-
ols.9,10 Based on the affinity of the steroids for the ER and their
antiestrogenic activity, we prepared a steroidal antiestrogen–mito-
mycin C conjugate to test our concept.11 Although the compound
retained high ER affinity and antiestrogenic properties, it was no
more active than mitomycin C and displayed no selectivity toward
ER-expressing breast cancer cells. One possible explanation for the
cluded further studies with this conjugate. Therefore we elected
to evaluate the effect of linker length and conformational flexibility
using the Hsp90 N-terminal inhibitor, geldanamycin (GDA), as the
therapeutic component (Fig. 1).
Heat shock proteins (HSP) are molecular chaperones that are
critical for the maintenance of cellular homeostasis through regu-
lation of protein transport, conformational folding and matura-
tion.12 Hsp90 is a 90 kDa protein that is often overexpressed in
breast cancer, as well as other cancers, and, as a result of these in-
creased levels, is responsible for maintaining high levels of active
oncogenic proteins.13–15 One of these proteins is ER
dormant, is confined to the nucleus in an Hsp90 complex.16 Disrup-
tion of the Hsp90-ER complex leads to improper folding of ER
a which, when
a
a
and its subsequent degradation, resulting in down-regulation of
its corresponding pathways, such as transcription. Therefore, dis-
ruption of Hsp90-mediated responses provides an alternative tar-
get for breast cancer therapy, and has led to the use of
geldanamycin (GDA) and its derivatives as therapeutic agents.
The geldanamycin component was developed based upon our
work with chaperone inhibiting agents. Structure–activity rela-
tionship studies demonstrated that modification at the 17-position
not only generates GDA derivatives that exhibit reduced toxicity,
but this position is also substituent tolerant as groups at this posi-
tion of GDA exit the Hsp90 binding pocket and thus do not signif-
icantly affect inhibitory activity.17 Other 17-GDA derivatives have
been synthesized that exhibit improved solubility and lower toxic-
ity than GDA, but are still hepatotoxic.18,19 Therefore we planned to
⇑
Corresponding author.
E-mail address: r.hanson@neu.edu (R.N. Hanson).
0960-894X/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2013.03.116

3636
J. Adam Hendricks et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3635–3639
NH2
O
O
N
O
O
O
O
N
N
O
H
N
OH
H3CO
H3CN
N
CH3
HO
Antiestrogen-Mitomycin C conjugate
O
O
O
NH
linker
linker
O
OH
NH
H3C
O
"Click"
ligation
CH3
HO
O
O
O
H2N
Antiestrogen
derivative
Geldanamycin
derivative
Figure 1. Proposed extension of research from the antiestrogen–mitomycin C conjugate to the antiestrogen–geldanamycin conjugates.
introduce modifications at the 17-postion that will permit conju-
gation to the steroidal derivatives.
11b-(4-substituted oxyphenyl) estradiols.9,10 Scheme 1 deltenone
3-ethylene ketal 1 was converted initially to the 11b-(4-hydroxy-
phenyl) estra-4,9-diene-3,17-dione 2. This compound then served
as the intermediate for the preparation of the requisite 11b-(4-azi-
doethoxyphenyl)estradiol 4a and 11b-(4-N-propargyl-N-methyla-
minoethoxyphenyl) estradiol components 4b. For the propargyl
derivative, we prepared the 2-(N-propargyl-N-methylamino)etha-
nol which was then coupled to the 11b-(4-hydroxyphenyl) estra-
4,9-diene-3,17-dione 2 using the Mitsunobu reaction to give 3b.
Aromatization with acetic anhydride-acetyl bromide followed by
reduction–saponification gave the desired product 4b. Overall
yields for the two compounds were 28% (eight steps) and 19% (seven
steps), respectively. We had previously characterized the azido
derivative 4a, determined its binding affinity (RBA = 39%) and
We chose a convergent approach in which each component
contained a side chain that is terminally substituted with a reactive
functionality. The final step then involves a ligation reaction under
mild conditions. The reaction selected for this study was the
Huisgen [3+2] cycloaddition reaction between a terminal azide
and a terminal alkyne to generate a chemically stable triazole moi-
ety.20–22 The reaction has the advantage of being chemoselective
and allowing the reactive groups to reside on either component.
In this study we chose to use different lengths of the linker to
investigate what effect, if any, it exerts on the biological activity
of the final conjugate. The overall synthetic strategy for our conju-
gates is shown in Figure 2.
showed that it was a full antagonist of ERa. The N-propargyl-N-
methyl derivative 4b is a close analog of the RU39411 for which
we had determined ER affinity (RBA = 39%) and efficacy (full antag-
onism). Having demonstrated that additional substituents distal to
the nitrogen in the side chain did not adversely affect either binding
Results
The synthesis of the steroidal antiestrogen component was
accomplished using a strategy similar to one described for our
GDA Component
N3
O
O
O
H
N
O
O
O
O
CH3
N
H
N
H
O
H
O
CH3
O
CH3
O
N
O
O
N
N
HO
OH
HO
O
CH3
N
N
H
CH3
"Click"
O
O
O
CH3
O
O
HO
O
H2N
O
CH3
HO
H2N
Antiestrogen Component
O
O
HO
H2N
O
OH
OH
N3
Target AE-GDA Conjugate
HO
HO
Figure 2. Approach for synthesis of individual components and assembly as AE–GDA conjugates.

J. Adam Hendricks et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3635–3639
3637
or efficacy, we felt that the steroidal components were appropriate
substrates for subsequent ligation reactions10 (see Schemes 1–3).
The geldanamycin components were prepared using variations
of methods previously described for 17-amino derivatives.15,23
of the conjugate was comparable to that of the parent mitomycin
C.11 In this study, the two conjugates 7a and 7b having the least
sterically constrained linkers were also the most potent com-
pounds. The conjugate 7c, having the cyclooctyl triazole closest
to the 11b position of estradiol was most likely to produce signif-
icant steric interactions with the estrogen receptor which would
compromise the targeting toward ER-expressing cells. The results
suggest that the accessibility of the antiestrogenic component for
the target membrane ER may influence the overall potency. The
least sterically demanding conjugate 7b is an order of magnitude
more potent than the conjugate with the shorter linker 7a which
is an order of magnitude more potent than the sterically compro-
mised conjugate 7c.
The linker component may also affect the therapeutic activity.
Previous studies indicated that geldanamycin forms a stable com-
plex with Hsp90 via a complex set of interactions that are modu-
lated by substituents at the 17-position. With the 17-amino-17-
desmethoxy derivatives, the exit site for this group corresponds
to the heteroatom and therefore the length of the group would
be expected to affect the biological response. In this study, two
conjugates 7a and 7b display sub-micromolar activity against ER-
expressing cells, although, both compounds are more active
against the SKBr3 breast cancer cells that do not express ER. In
those cells, conjugate 7b, having the longer linker, while less po-
tent than geldanamycin alone, is more than an order of magnitude
more potent than 7a, the conjugate with the shorter linker. Activity
of the more complex conjugate 7c was not determined, but the re-
sults suggest that the longer, more conformationally flexible link-
ers are favored at the 17-position.
The results suggest that ER-targeting was not the major factor
underlying the biological effectiveness of the conjugates. If ER-tar-
geting were the major component, one would expect thet cytotox-
icity to be greater in MCF-7 cells as opposed to the SKBr3 cells. This
response pattern was observed with our steroidal antiestrogen–
mitomycin C conjugate in which ER-based selectivity was not
achieved, even though the ER binding affinity for the conjugate
was relatively high.25 For the two most active conjugates 7a and
7b, activity was greater in the SKBr3 cells than in the MCF-7, a pat-
tern that was similar to geldanamycin alone. Therefore it appears
that the overall antiproliferative responses were modulated by
the presence of the steroidal components, but did not enhance
the overall effect compared to geldanamycin. It should be noted
that the desired response pattern was observed for our doxorubi-
cin–antiestrogen conjugate that we recently described in which
MCF-7 antiproliferative activity was enhanced compared to doxo-
rubicin alone and almost sevenfold greater than that observed in
MDA-MB-231 cells which are ER-negative.26
Scheme 2 treatment of geldanamycin 5 with either propargyl
9
x
amine or
-azido pentaethylene glycol amine in dichloromethane
gave the corresponding 17-amino geldanamycin components 6a
and 6b in 80% and 68% yields, respectively. For the third geldana-
mycin component, a two step procedure was used, similar to that
employed in our previous preparation of the biotinylated deriva-
tive. Geldanamycin 5 was initially treated with a fivefold excess
of 1,5-pentanediamine in dichloromethane. Purification by column
chromatography gave the 17-(5-aminopentyl)amino geldanamy-
cin 6c in a 95% yield. Bertozzi’s difluoro-cyclooctyne carboxylic
acid24 was converted to the corresponding acyl chloride and imme-
diately reacted with 17-(5-aminopentyl)amino geldanamycin 6c to
form the corresponding amide 6d. The product was isolated in a
42% yield following column chromatography.
Ligation to form the final antiestrogen–geldanamycin conju-
gates used two versions of the ‘‘click’’ reaction. Scheme 3 in the
conventional version, we used the 17-propargylamino geldanamy-
cin 6a and the 11b-(4-azidoethoxyphenyl) estradiol 4a as coupling
partners to give the 1,2,3-triazole conjugate 7a with a short linker
in a 46% isolated yield. Coupling the 17-(azidopentaethylene glyco-
lamino) geldanamycin 6b with 11b-(4-N-propargyl-N-methylami-
noethoxyphenyl) estradiol 4b under the same conditions gave
the triazole conjugate 7b with a longer linker in 47% isolated yield.
The third conjugate was prepared from the cyclooctynylated amino
geldanamycin 6d and 11b-(4-azidoethoxyphenyl) estradiol 4a in
which the copperless-method gave the corresponding annulated
triazole 7c in a 73% isolated yield.
The three new conjugates and geldanamycin were evaluated for
antiproliferative activity against MCF-7 and SKBr3 breast cancer
cell lines (Table 1). In this assay, the antiproliferative activity of
geldanamycin 5 in the two cell lines was determined to be 9.8
and 8.5 nM, respectively. Conjugate 7a with the shortest linker
group manifested an IC50 of 1150 90 nM in MCF-7 and
710 160 nM in SKBr3 cells. Conjugate 7b with the longer linker
was more potent with IC50 values of 102 4.6 nM and 41 4.6 in
the respective cell lines. Conjugate 7c that incorporated the bulkier
Bertozzi linker had an IC50 value of 15200 3000 nM in MCF-7
cells and was not therefore evaluated in the SKBr3 cell line. The re-
sults indicated that while all of the new conjugates retained signif-
icant antiproliferative activity, however, the potency was clearly
modulated by the additional linker and antiestrogen components.
The objectives of this study were to evaluate the effects of the
linker on the antiproliferative activity of the antiestrogen-drug
conjugate. We had observed in our initial study with a antiestro-
gen–mitomycin C conjugate that a long, linear oligoethylene glyo-
col linker retained high ER binding affinity (RBA = 7%), similar to
the effects observed previously by Essigmann and co-workers with
One of the significant differences between our doxorubicin–
antiestrogen conjugate and the current series of geldamycin conju-
gates is that the former contain a component that allows the drug
to dissociate within cancer cells. As with the mitomycin C conju-
gate, the synthetic strategy used in this study did not incorporate
their 7a
-derivatives.25 In that study, the antiproliferative activity
HO
O
O
O
O
R
R
O
O
O
OH
iv,v
or vi
a. R = N3,
b. R =
i,ii,iii
vii,viii,ix
N
CH3
O
O
HO
4a
4b
1
2
3a
3b
Scheme 1. Synthesis of steroidal antiestrogen component. Reagents and conditions: (a) CF3COCF3ÁH2O, H2O2 (50%), C5H5N, CH2Cl2, 0 °C, 18 h; (b) TMSiOC6H4MgBr, CuI, THF;
16 h; (c) HOAc–H2O (7:3), 1.5 h; (d) TsOCH2CH2OTs, Cs2CO3, CH3CN, 13 h; (e) NaN3, EtOH, 4 h; (f) (HCCHCH2)(CH3)NCH2CH2OH, DEAD, PS-PPh3, CH3CN, 16 h; (g) Ac2O, AcBr,
CH2Cl2, 16 h; (h) NaBH4, MeOH, 1 h; (i) NaOH, MeOH, 16 h.

3638
J. Adam Hendricks et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3635–3639
F
F
O
O
R
N
H
N
R
O
O
O
O
N
O
O
O
H
O
O
CH3
N
H
ii,iii
N
H
N
H
i
H3C
O
O
6c
CH3
HO
O
CH3
O
HO
O
CH3
HO
O
CH3
CH3
O
O
O
O
O
O
H2N
H2N
H2N
6a R=
6b R=
6c R=
5
O
N3
4
NH2
Scheme 2. Synthesis of geldanamycin components. Reagents and conditions: (a) amine, CH2Cl2, rt, 24 h; (b) substituted benzoic acid, SOCl2, toluene, 70 °C, 2 h; (c) CH2Cl2,
TEA, 0 °C–rt, 2 h.
O
O
HN
NH
O
CH3
N
N
OH
O
i
O
N
4a + 6a
HO
O
H2N
O
O
H3C
O
HO
O
HN
CH3
NH
O
i
N
CH3
O
OH
N
O
4b + 6b
O
N
HO
N
3
O
H2N
O
O
H3C
HO
O
CH3
OH
HN
O
O
O
O
H2N
NH
O
O
H3C
NH
ii
O
4a + 6d
O
N
N
OH
F
N
F
HO
Scheme 3. Ligation of steroidal antiestrogen and geldanamycin components using ‘click’ chemistry. Reagents and conditions: (a) CuSO4–5ÁH2O, sodium ascorbate, t-BuOH–
H2O, rt, 18–70 h; (b) t-BuOH–H2O, rt, 24 h.

J. Adam Hendricks et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3635–3639
3639
Table 1
(J.A.H.). We gratefully acknowledge the contribution of Professor
John A. Katzenellenbogen, whose research group carried out the
ER binding assays for the parent steroidal anti-estrogen.
Anti-proliferation activity of ateroidal antiestrogen–geldanamycin (AE–GDA) conju-
gates 7a–7c
Compd
MCF-7 (IC50
)
SKBr3 (IC50)
9.8 0.1a nM
1150 90 nM
102 4.6 nM
8.5 1.1a nM
710 160 nM
41 4.6 nM
N.D.
Supplementary data
5 (GDA)
7a
7b
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
03.116.
7c
15200 3000 nM
IC50 = concentration needed to produce 50% inhibition.
a
Ref. 15. ND = not determined.
References and notes
that property. It is possible that for these conjugates that cellular
uptake may be mediated via the membrane estrogen receptor
but that effective intracellular distribution requires dissociation
of the therapeutic component from the antiestrogen targeting
group. Continued association with the antiestrogen component
may reduce the effectiveness of the drug from accessing its site
of action, even if elevated intracellular concentrations are ob-
tained. Oligoethylene glycol linkes, such as those used in 7b and
the doxorubicin–anitestrogen conjugate, may also contribute
physicochemical properties that enhance cellular uptake. Because
of the potent antiproliferative activity observed for 7b, incorpoara-
tion of a linker that can impart both properties may generate the
desired biological effect.
1. Garcia, M.; Jemal, A.; Ward, E. M.; Center, M. M.; Hao, Y.; Siegel, R. L. Global
Cancer Facts and Figures 2007; American Cancer Society: Atlanta, Georgia, 2007.
2. Langan Fahey, S. M.; Jordan, V. C.; Fritz, N. F.; Robinson, S. P.; Waters, D.;
Tormey, D. C. Long-Term Tamoxifen Treatment for Breast Cancer In Jordan, V.
C., Ed.; University of Wisconsin Press: Madison, WI, 1994; pp 27–56.
3. Madaio, R. A.; Spalletta, G.; Cravello, L.; Ceci, M.; Repetto, L.; Naso, G. Curr.
Cancer Drug Targets 2010, 10, 519–528.
4. Dodwell, D.; Williamson, D. Cancer Treat. Rev. 2008, 34, 137–144.
5. Lo, S. S.; Pritchard, K. I.; Robinson, P.; Albain, K. S. Cancer Treat. Res. 2009, 147,
95–123.
6. Meunier, B. Acc. Chem. Res. 2008, 41, 69–77.
7. Kummar, S.; Chen, H. X.; Wright, J.; Holbeck, S.; Millin, M. D.; Tomaszewski, J.;
Zweibel, J.; Collins, J.; Doroshow, J. H. Nat. Rev. Drug Disc. 2010, 9, 843–856.
8. Katzenellenbogen, J. A. Chem. Biol. 2005, 12, 719–721.
9. Hendricks, J. A.; Adam, J.; Gulla, S. V.; Budil, D. E.; Hanson, R. N. Bioorg. Med.
Chem. Lett. 2012, 22, 1743–1746.
10. Hanson, R. N.; Hua, E.; Hendricks, J. A.; Labaree, D.; Hochberg, R. B. Bioorg. Med.
Chem. 2012, 20, 3368–3380.
In conclusion, we have described a convergent strategy for the
preparation of a novel series of novel steroidal antiestrogen-drug
conjugates. This approach has distinct advantages in preparing
and evaluating combinations of targeting groups, therapeutic
drugs and linkers. The conjugates in this study were obtained in
good overall yields and demonstrated significant activity against
two breast cancer cell lines. Although one of the compounds (7b)
demonstrated significant antiproliferative activity, it did not, how-
ever, demonstrate enhanced potency compared to the parent drug
or selectivity for ER-expressing cells as compared to non-express-
ing cells. The results suggest that further modifications in both ER-
targeting strategies and linking groups are needed in order to
achieve greater potency and selectivity in therapeutic drug deliv-
ery. The effects of different linkers on both ER binding and Hsp90
warrant further evaluation as well. Those studies are in progress
and will be described in future publications.
11. Hanson, R. N.; Hua, E.; Larabee, D.; Hochberg, R. B.; Essigmann, J. M.; Croy, R. G.
Org. Biomol. Chem. 2012, 10, 8501–8508.
12. Rutherford, S. L.; Lindquist, S. Nature (Lond.) 1998, 396, 336–342.
13. Hartmann, F.; Horak, E. M.; Cho, C.; Lupu, R.; Bolen, J. B.; Stetler-Stevenson, M.
A.; Pfreundschuh, M.; Waldmann, T. A.; Horak, I. D. Int. J. Cancer 1997, 70, 221–
229.
14. Miller, P.; Schnur, R. C.; Barbacci, E.; Moyer, M. P.; Moyer, J. D. Biochem. Biophys.
Res. Commun. 1994, 201, 1313–1319.
15. Clevenger, R. C.; Raibel, J. M.; Peck, A. M.; Blagg, B. S. J. J. Org. Chem. 2004, 69,
4375–4380.
16. McDonnell, D. P.; Norris, J. D. Science 2002, 296, 1642–1644.
17. Stebbins, C. E.; Russo, A. A.; Schneider, C.; Rosen, N.; Hartl, F. U.; Pavletich, N. P.
Cell 1997, 89, 239–250.
18. Ruden, D. M.; Xiao, L.; Lu, X. Mini-Rev. Med. Chem. 2006, 6, 1137–1143.
19. Schnur, R. C.; Corman, M. L.; Gallaschun, R. J.; Cooper, B. A.; Dee, M. F.; Doty, J.
L.; Muzzi, M. L.; Moyer, J. D.; DiOrio, C. I. J. Med. Chem. 2002, 38, 3806–3812.
20. Hein, J. E.; Fokin, V. V. Chem. Soc. Rev. 2010, 39, 1302–1315.
21. Remzi Becer, C.; Hoogenboom, R.; Schubert, U. S. Angew. Chem., Int. Ed. 2009,
48, 4900–4908.
22. Tron, G. C.; Pirali, T.; Billington, R. A.; Canonico, P. L.; Sorba, G.; Genazzani, A. A.
Med. Res. Rev. 2008, 28, 278–308.
23. Shen, G.; Blagg, B. S. J. Org. Lett. 2005, 7, 2157–2160.
24. Codelli, J. A.; Baskin, J. M.; Agard, N. J.; Bertozzi, C. R. J. Am. Chem. Soc. 2008, 130,
11486–11493.
Acknowledgments
25. Mitra, K.; Marquis, J. C.; Hillier, S. M.; Rye, P. T.; Zayas, B.; Lee, A. S.; Essigmann,
J. M.; Croy, R. G. J. Am. Chem. Soc. 2002, 124, 1862.
26. Dao, K.-L.; Sawant, R. R.; Hendricks, J. A.; Ronga, V.; Vladimir, P.; Torchilin, V. P.;
Hanson, R. N. Bioconjugate Chem. 2012, 23, 785–795.
This work was supported by Public Health Service award 1R01
CA109265 (B.S.J.B), department of Defense BCRP award
W81XWH06-1-0551 (R.N.H.) and NSF-IGERT award NSF-0504331