Expanding the repertoire of small molecule transcriptional activation domains

Article abstract of DOI:10.1016/j.bmc.2008.02.045

Molecules that can reconstitute the function of transcriptional activators hold enormous potential as therapeutic agents and as mechanistic probes. Previously we described an isoxazolidine bearing functional groups similar to natural transcriptional activ

Full text of DOI:10.1016/j.bmc.2008.02.045

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 17 (2009) 1034–1043
Expanding the repertoire of small molecule
transcriptional activation domains
Ryan J. Casey,a, Jean-Paul Desaulniers,a, Jonas W. Hojfeldtb and Anna K. Mappa,b,
*
aDepartment of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA
bProgram in Chemical Biology, University of Michigan, Ann Arbor, MI 48109, USA
Received 11 December 2007; revised 8 February 2008; accepted 13 February 2008
Available online 16 February 2008
We dedicate this paper to Professor Jonathan Ellman in honor of the 2006 Tetrahedron Young Investigator Award in recognition of his many
seminal contributions to Bio-organic Chemistry.
Abstract—Molecules that can reconstitute the function of transcriptional activators hold enormous potential as therapeutic agents
and as mechanistic probes. Previously we described an isoxazolidine bearing functional groups similar to natural transcriptional
activators that up-regulates transcription 80-fold at 1 lM in cell culture. In this study, we analyze analogs of this molecule to define
key characteristics of small molecules that function as transcriptional activation domains in cells. Conformational rigidity is an
important contributor to function as is an overall amphipathic substitution pattern. Using these criteria, we identified additional
molecular scaffolds with excellent (ꢀ60-fold) activity as transcriptional activation domains. These results point the way for the cre-
ation of new generations of small molecules with this function.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
minimally consists of a DNA binding domain (DBD)
and a transcriptional activation domain (TAD) that
are linked by either covalent or non-covalent interac-
tions.4 These domains operate largely independently,
meaning that the DNA binding domain of one activator
can be coupled to the transcriptional activation domain
of a second activator without losing function.5 Thus, a
modular replacement strategy can be used to construct
an activator ATF, replacing one or both of the two
key domains with non-natural counterparts. Although
many different classes of DBDs have been developed
and used for activator ATF construction,4 one of the
prevailing challenges has been the identification of small
molecules that can be used as transcriptional activation
Molecules that reconstitute the function of transcrip-
tional activators, activator artificial transcription factors
(activator ATFs), have proven to be powerful tools for
parsing the details of transcriptional regulation.1 Activa-
tor ATFs also have outstanding potential as therapeutic
agents targeting diseases ranging from cancer to meta-
bolic disorders.2 In one example, a Sangamo protein-
based activator ATF that up-regulates the gene for vas-
cular endothelial growth factor is currently in Phase II
clinical trials for diabetic neuropathy.3 As this example
illustrates, protein-based activator ATFs are at the fore-
front of therapeutic development. There is, however,
considerable interest in the development of small mole-
cule activator ATFs with improved stability, delivery,
and immunogenicity characteristics.4
domains, with only two examples reported to date6,7
;
peptidomimetic TADs have also been described.8
Amphipathic isoxazolidine 1 was the first example, ini-
tially reported in 2004.6 The early studies of this mole-
cule were in cell free systems and the data from these
studies suggested that the molecule functions mechanis-
tically like a natural TAD.6,9 More recently, we dis-
closed that this molecule also functions well in human
cell culture, with a maximal activity of 80-fold activation
at a concentration of 1 lM.10 In contrast, a more hydro-
phobic version, compound 2, shows no detectable activ-
ity over the same concentration range. Here, we describe
the synthesis and activity of analogs of 1 that define
On the surface, the design of an activator ATF is
straightforward. A natural transcriptional activator
Keywords: Transcription; Transcriptional activation domain; Isoxa-
zolidine; Santonin; Hydrastine.
*
Corresponding author. Tel.: +1 734 615 6862; fax: +1 734 615
8553; e-mail: amapp@umich.edu
These authors contributed equally to this work.
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.045

R. J. Casey et al. / Bioorg. Med. Chem. 17 (2009) 1034–1043
1035
general characteristics of a small molecule transcrip-
tional activation domain with activity in cells. In addi-
tion, we show that using these criteria, new molecular
scaffolds with TAD function can be identified.
vator function.15–17 Thus, 1 was designed to contain
amphipathic functional groups commonly found in
endogenous TADs arrayed on a conformationally con-
strained scaffold, the isoxazolidine ring. Analogs 3 and
4 were designed to examine the importance of confor-
mational constraint in activity. Compound 3 is a ring-
opened analog of 1 in which the N–O bond has been
cleaved whereas 4 is a D-peptide containing the same
functional groups as 1.18,19 Isoxazolidines 5–13 were
designed to assess the tolerance limit of hydrophobic
and polar substituents at N2 and C3, respectively (see
Fig. 1).
2. Results and discussion
Isoxazolidine 1 was originally designed as a generic mi-
mic of the amphipathic class of transcriptional activa-
tion domains, a group defined by the characteristic
pattern of hydrophobic amino acids interspersed with
more polar amino acid residues.1 While typically lacking
structure when in an unbound state, the current model is
that they fold into an amphipathic helix when bound to
their transcriptional machinery binding partners,
although other secondary structures may also play a
role.11–14 The hydrophobic residues are generally the
strongest contributors to binding and ultimately to acti-
The analogs of 1 were prepared in a straightforward
manner from a common intermediate, isoxazolidine 14
(Schemes 1 and 2).6 Alkylation under microwave-accel-
erated conditions was used to install the functional
groups at N2, and this was followed by the removal of
the silyl protecting group under basic conditions. For-
2
N
O
H
N
HO
AEEA-OxDex
3
H
1
HO
O
O
H
H
N
N
H2N
N
H
AEEA-OxDex
N
O
H
H
N
NH OH
H
N
HO
O
AEEA-OxDex
AEEA-OxDex
4
3
2
R
R
2
2
N
O
H
N
N
O
H
H
N
HO
HO
HO
N
3
O
H
N
3
AEEA-OxDex
3
AEEA-OxDex
R
H
AEEA-OxDex
H
5 R =
6 R =
CH3
8 R =
9 R =
H3CO2C
11 R =
12 R =
13 R =
HO2C
7 R =
10 R =
H2NOC
OH
O
O
O
F
O
NH2
HO
H
HO
O
HO
AEEA
OxDex
Figure 1. Structures of isoxazolidine analogs.

1036
R. J. Casey et al. / Bioorg. Med. Chem. 17 (2009) 1034–1043
3
e,g,f
R
R
R
2
HN
O
H
N
O
H
N
3
O
H
N
3
O
H
a
b
c,d
OTBS
HO
N3
3
OH
N3
3
14
23 R = Ph
24 R =
15 R = Ph
16 R =
19 R = Ph
20 R = p-tolyl (62%)
p
-tolyl (51%)
p
-tolyl (54%)
25 R = 2-naphthyl (65%)
26 R = 3-biphenyl (59%)
17 R = 2-naphthyl (61%)
18 R = 3-biphenyl (66%)
21 R = 2-naphthyl (61%)
22 R = 3-biphenyl (71%)
c,e,f
e,f
8-10
5-7
Scheme 1. Reagents and conditions: (a) i—RBr, i-Pr2NEt, DMF, microwave; ii—TBAF, THF, rt; (b) i—Ms-Cl, Et3N, DCM, rt; ii—NaN3, DMF,
85 ꢁC; (c) OsO4, NMO, t-BuOH, THF, H2O, rt; (d) i—NaIO4, CH3CN, H2O, rt; ii—NaBH4, MeOH, 0 ꢁC–rt; (e) i—PPh3, H2O, THF, reflux; ii—
Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-AEEA), HOBt, HBTU, Et3N, NMP, rt; iii—20% piperidine, DMF, rt; (f) OxDex, HOBt, HBTU, i-
Pr2NEt, 2,6-lutidine, NMP, rt; (g) Zn, AcOH, rt.
11 R = Me
12 R = OH
13 R = NH2
a,b,c
f
d,e
N
O
H
19
g
MeOOC
N3
72% yield
21% yield
27
10:1, E:Z
Scheme 2. Reagents and conditions: (a) OsO4, NMO, t-BuOH, THF, H2O, rt; (b) NaIO4, CH3CN, H2O, rt; (c) methyl (triphenylphosphoranylidene)
acetate, toluene, 60 ꢁC; (d) i—PPh3, H2O, THF, reflux; ii—Fmoc-AEEA, HOBt, HBTU, Et3N, NMP, rt; iii—20% piperidine, DMF, rt; (e) OxDex,
HOBt, HBTU, i-Pr2NEt, 2,6-lutidine, NMP, rt; (f) NaOH, EtOH, rt, (31%); (g) NH4HCO3, Boc2O, pyr, CH3CN, rt, (33%).
mation of a mesylate and its subsequent displacement by
azide anion provided isoxazolidines 19–22. Oxidation of
the C3 allyl group enabled installation of a hydroxyl (5–
7), a diol (8–10), an ester (11), a carboxylic acid (12), and
amide (13) at this position. The ring-opened variant of 1,
analog 3, was produced by treatment of isoxazolidine 23
with Zn(0) in acidic media. For each analog, the final
reaction sequence installed the DNA targeting moiety
using the C5 azide handle to produce a primary amine.
This was subsequently coupled to a short polar linker
(Fmoc-8-amino-3,6-dioxaoctanoic acid, Fmoc-AEEA)
and then to an oxidized form of dexamethasone (Ox-
Dex). The OxDex moiety is used to target the isoxazoli-
dines to DNA as the cells in which activity assays are
run express a fusion protein, Gal4(1–147) + GR, con-
sisting of the DNA binding domain of Gal4 linked to
the minimal ligand binding domain of the glucocorticoid
receptor (GR), a domain with which OxDex interacts
strongly.20
Gal4(1–147) + GR. Firefly luciferase activity thus pro-
vides a direct readout of the efficiency with which the
molecules function as TADs. The cells also contain a
Renilla luciferase reporter lacking Gal4 sites and this
serves as an internal control to assess impact on gen-
eral transcription. In each experiment, the normalized
luciferase activity observed at a given concentration of
small molecule is compared to that obtained with
AEEA + OxDex alone, giving rise to the fold activa-
tion values shown. As illustrated in Figure 2, both 3
and 4 exhibited attenuated activity relative to 1 (max-
imal activity of 80-fold at 1 lM). Diol 3 had low
activity at 1 lM (ꢀ11-fold) that increased to 40.9-fold
at 10 lM and an EC50 of 2.5 lM, corresponding to an
approximately 100-fold increase relative to 1 (33 nM).
Tripeptide 4 was even less active, with 24-fold up-reg-
ulation at 10 lM and an EC50 of 1.86 lM. Of the
two, 4 has the greater conformational flexibility, with
rotatable bonds connecting each of the three side
chains. In contrast, compound 3, although more flex-
ible than 1, still has the three key side chains affixed
in a radial array and it is possible that an internal
hydrogen bond between the C3–OH and the amine
could limit conformational flexibility. Thus, although
conformational constraint is an important contributor
to overall function, a ring is not required for activity.
The ability of the molecules to function as transcrip-
tional activation domains was assessed using a stan-
dard luciferase assay in HeLa cells.10,20 Briefly,
OxDex conjugates are incubated with cells containing
a firefly luciferase reporter under the control of five
Gal4 binding sites in addition to a plasmid expressing

R. J. Casey et al. / Bioorg. Med. Chem. 17 (2009) 1034–1043
1037
Figure 2. Results from luciferase assays in HeLa cell culture. (a) Activity of OxDex-conjugates is expressed as fold activation relative to OxDex-
AEEA alone. Briefly, HeLa cells were transfected with a plasmid expressing the Gal4(1–147) + GR fusion protein, a second plasmid bearing 5 Gal4-
binding sites upstream of a firefly luciferase reporter gene, and a third plasmid expressing Renilla luciferase as a transfection control, as has been
previously described.9,12 Compounds were added to the cells as a 1 or 10 mM DMSO solution 3 h after transfection such that the final concentration
of DMSO in all wells was 1% (v/v). The final concentration of each compound was 10 lM for all compounds (1 lM data is shown as Fig. S1 in the
Supporting Information). The firefly and Renilla luciferase activities were measured 40 h after compound addition. Fold activation was determined at
each concentration by first dividing the firefly luciferase activity by that of Renilla luciferase. This value was then divided by the amount of activity
observed with OxDex + AEEA alone. Each value is the average of at least three independent experiments with the indicated error (SDOM). (b)
Results from competitive inhibition experiments with 28 and 29. Luciferase assays were carried as described for (a) with 100 nM of 28 or 1 lM of 29.
Increasing concentrations of either hydrastine (for compound 28) or santonin (for compound 29) were added to the cells to assess if the DNA-bound
and free forms of the molecules were targeting the same binding sites.
As described earlier, the second key aspect of the design
of 1 was its amphipathic character. More specifically,
the functional groups at N2 and C3 were designed to mi-
mic amino acids often identified as functionally impor-
tant in natural TADs: phenylalanine and leucine;
serines are also common constituents.17,21,22 As illus-
trated in Figure 2, alteration of the N2 and C3 func-
tional groups on the isoxazolidine scaffold most often
lead to functional molecules, but maximal activity was
achieved only at higher concentrations relative to 1
(10 lM). More specifically, the N2 benzyl group (Phe
mimic) can be replaced with a larger substituent such
as tolyl (5, 31-fold up-regulation at 10 lM) but larger
hydrophobic groups result in significant diminuation
of activity (6, 5-fold; 7, 1.9-fold).
function modestly as transcriptional activators despite
exhibiting good affinity for the transcriptional machin-
ery; there is some evidence that this is due to increased
non-specific binding events leading to sequestration
and/or degradation.11,23,24 Further, this effect can be
ameliorated through the incorporation of masking inter-
actions that effectively decrease the hydrophobic surface
area.24 In the case of the isoxazolidines, we observed a
similar trend upon incorporation of a more polar group
at C3. For 9 and 10, a diol moiety at C3 restored signif-
icant activity, with these molecules up-regulating
approximately 20-fold at 10 lM. In addition, a hydro-
gen bond donor is not required to be the polar group
at C3, as the activity of methyl ester 11 demonstrates
(41-fold activity). Increasing the polarity of the substitu-
ent as in amide 13 and acid 12, however, does result in a
further loss of activity (9- and 4-fold, respectively).
Given the many different molecular recognition events
that TADs participate in both extra- and intracellularly
as part of transcriptional activation and that these inter-
actions are poorly characterized,1 it is difficult to pin-
point the origin of the decreased activity observed with
isoxazolidines 6 and 7. It has, however, been demon-
strated that hydrophobic peptides and small molecules
Many different amphipathic peptide sequences function
as transcriptional activation domains,25 and it seemed
likely that more than one amphipathic small molecule
scaffold should also function as a transcriptional activa-
tion domain. To narrow the search, we sought structures

1038
R. J. Casey et al. / Bioorg. Med. Chem. 17 (2009) 1034–1043
CH3
HO
CH3
H
N
N
N
O
O
H
CH3
R
CH3
a,b
a,b
O
H
HO
O
O
O
O
O
O
H
H3C
H3C
O
R
NH
O
OCH3
OCH3
O
O
H3CO
CH3
28
29
(+/-)-hydrastine
santonin
H
N
O
= R
O
O
O
OH
HO
H
F
H
O
Scheme 3. Reagents and conditions: (a) 4,7,10-trioxa-1,13-tridecanediamine, DMAP, i-Pr2NEt, DMF, rt; (b) OxDex, HOBt, HBTU, i-Pr2NEt, 2,6-
lutidine, NMP, rt.
that displayed some degree of conformational rigidity
and were amphipathic. With regard to the latter crite-
rion, we noted that the most active isoxazolidines fell
within a relatively narrow logP range (2.5–3.1, calcu-
lated without the AEEA–OxDex moiety using Osiris
Properties calculator26) and used this as an approximate
assessment of amphipathic character. Two structures
that fell into this category were the commercially avail-
able natural products santonin and hydrastine (Scheme
3). Santonin is an anti-parasitic agent commonly em-
ployed as a synthetic intermediate and hydrastine is a
GABA receptor inhibitor often used for the study of
GABA-linked signaling.27–29 In addition to having logP
values similar to that of 1 and containing distinct hydro-
phobic surfaces, both structures contain a lactone used
for installation of the OxDex moiety (Scheme 3).
Remarkably, both structures showed good activity, with
59- and 63-fold up-regulation at 10 lM, respectively
(Fig. 2a). In both cases, competitive inhibition experi-
ments with the original natural product verified that it
was the DNA-bound form of the molecule that was
functioning as a transcriptional activation domain
(Fig. 2b). In addition, neither molecule had any effect
on a reporter gene that did not have DNA binding sites
for Gal4(1–147) + GR. Taken together, these data sug-
gest that the molecules function similar to natural TADs
and the isoxazolidine TADs. Further, the general TAD
descriptors used to identify santonin and hydrastine
should enable to the discovery of an even wider array
of small molecule TADs.
binding partners in the transcriptional machinery are
not known, rendering detailed thermodynamic and
structural characterization of activator complexes chal-
lenging. In this regard, this class of small molecules
may prove to be powerful tools to investigate these ques-
tions, as modified variants containing photo-activatable
groups suitable for cross-linking are synthetically readily
accessible.
3. Experimental
3.1. General
Unless otherwise noted, starting materials were obtained
from commercial suppliers and used without further
purification. CH2Cl2 and THF were dried by passage
through activated alumina columns. NMP and DMF
were used as purchased without further purification.
Et3N was distilled from CaH2. Purification by flash
chromatography was carried out with E. Merck Silica
1
Gel 60 (230–400 mesh). H and 13C NMR spectra were
recording in CDCl3 at 400 and 100 MHz, respectively,
unless otherwise specified. Reverse-phase HPLC purifi-
cations were performed on a Varian ProStar 210
equipped with Rainin Dynamax UV-D II detector
(k = 254 nm and k = 214) using a C18 (8 · 100 mm) Ra-
dial-PakTM cartridge with a gradient of 0.1% TFA in
H2O and CH3CN as the mobile phase. Isoxazolidines
15, 19, and 23 were prepared as previously reported.9,12
The structures of intermediates not shown in Schemes
1–3 can be found in Supporting Information (Scheme
S1) and are numbered S1–S6.
2.1. Future directions
In conclusion, we have identified several new isoxazoli-
dine-based transcriptional activation domains as well
as two unique TAD scaffolds, thus significantly expand-
ing the repertoire of small molecule transcriptional acti-
vation domains for the targeted control of gene
expression. The design criteria are simple and should
facilitate the discovery of future classes of novel TADs.
However, analogous to the natural system, it is not yet
possible a priori to predict how well a given small mol-
ecule TAD will function. This is a reflection of the
dearth of detailed mechanistic information about how
TADs, natural and unnatural, function.1 For example,
for most natural activators the physiologically relevant
3.1.1. OxDex + AEEA + D-LSF (4). The tripeptide D-
LeuSerPhe was prepared using solid phase peptide syn-
thesis in accordance with standard procedures with the
AEEA linker and OxDex added on solid support at
the conclusion of the synthesis.20,30 The crude product
isolated after resin cleavage was purified by reverse
phase HPLC to provide 3.5 mg of 4 (15% yield) as a
white solid. HRMS (ESI) calcd for [C49H68FN3O10
+
Na]+: 892.4484, found: 892.4493.
3.1.2. [3-Allyl-3-isobutyl-2-(4-methyl-benzyl)-isoxazoli-
din-5-yl]-methanol (16). To a solution of isoxazolidine

R. J. Casey et al. / Bioorg. Med. Chem. 17 (2009) 1034–1043
1039
146 (0.50 g, 1.6 mmol, 1.0 equiv; 6:1 mixture of diaste-
reomers) in DMF (7.5 mL) were added a-bromo-p-xy-
lene (1.5 g, 8.0 mmol, 5.0 equiv) and i-Pr2NEt (1.4 mL,
8.0 mmol, 5.0 equiv). The reaction mixture was irradi-
ated in a 1000 W microwave oven (6 · 20 s) at 20%
power with mixing between each interval. Once the reac-
tion was complete as judged by TLC analysis, the
reaction mixture was diluted with water (60 mL) and ex-
tracted with EtOAc (3· 30 mL). The combined organic
extracts were washed with water (1· 30 mL), dried over
Na2SO4, filtered, and concentrated in vacuo to a yellow
oil. Following N-alkylation, the crude reaction mixture
was dissolved in THF (4.5 mL) and TBAF was added
(3.0 mL of a 1 M solution in THF, 3.0 mmol, 1.8 equiv
based on starting material 14). The mixture was allowed
to stir for 6 h at which time the reaction mixture was di-
luted with water (60 mL) and extracted with EtOAc (3·
30 mL). The combined organic extracts were dried over
Na2SO4, filtered, and concentrated in vacuo to a yellow
oil. The crude material was purified by flash chromatog-
raphy (85:15 hexanes/EtOAc) to provide 0.26 g (54%
yield) of 16 as a colorless oil. The major diastereomer
was isolated by normal-phase HPLC and used in subse-
quent steps. 1H NMR: d 0.98 (d, 3H, J = 3.4), 1.01
(d, 3H, J = 3.4), 1.40 (dd, 1H, J = 14.7, 7.0), 1.63
(dd, 1H, J = 14.4, 4.7), 1.83–1.98 (m, 1H), 2.05 (dd,
1H, J = 12.3, 5.9), 2.24–2.51 (m, 7H), 3.51–3.65
(m, 2H), 3.77–3.91 (m, 2H), 4.02–4.12 (m, 1H), 5.08–
5.18 (m, 2H), 5.86–6.02 (m, 1H), 7.10–7.16 (m, 2H),
7.22–7.28 (m, 2H); 13C NMR: d 21.09, 24.10, 24.48,
25.29, 38.62, 38.78, 43.92, 53.18, 65.61, 68.55, 75.38,
117.7, 127.8, 129.1, 135.1, 135.7, 136.5; HRMS
(ESI) calcd for [C19H29NO2 + H]+: 304.2277, found:
304.2274.
3H, J = 6.8), 1.38 (dd, 1H, J = 15.0, 6.8), 1.61 (dd, 1H,
J = 14.4, 4.8), 1.86–1.92 (m, 1H), 2.01–2.05 (m, 1H),
2.21 (m, 2H), 2.28 (dd, 1H, J = 8.2, 4.6), 2.44 (dd, 1H,
J = 14.0, 7.2), 3.53–3.63 (m, 2H), 3.85 (d, 1H,
J = 14.4), 3.92 (d, 1H, J = 14.4), 4.04–4.09 (m, 1H),
5.08–5.12 (m, 2H), 5.88–5.98 (m, 1H), 7.28–7.31 (m,
4H), 7.37–7.42 (m, 1H), 7.51–7.56 (m, 4H); 13C NMR
d 24.09, 24.48, 25.26, 38.64, 38.77, 43.29, 53.14, 65.58,
68.51, 75.45, 117.7, 127.0, 127.2, 128.2, 128.7, 133.9,
135.1, 137.9, 139.8, 141.0; HRMS (ESI) calcd for
[C24H31NO2 + Na]+: 388.2252, found: 388.2253.
3.1.5. 3-Allyl-5-azidomethyl-3-isobutyl-2-(4-methyl-ben-
zyl)-isoxazolidine (20). To a solution of isoxazolidine
16 (80 mg, 0.26 mmol, 1.0 equiv), in CH2Cl2 (2.6 mL)
was added Et3N (70 lL, 0.53 mmol, 2.0 equiv). To this
solution was added methanesulfonyl chloride (40 lL,
0.53 mmol, 2.0 equiv). The solution was stirred at ambi-
ent temperature for 4 h at which time the reaction was
complete as judged by ESI-MS analysis. The reaction
mixture was concentrated in vacuo and the crude resi-
due was dissolved in EtOAc (20 mL) and water
(40 mL). The aqueous and organic layers were separated
and the water layer was extracted with EtOAc (3·
20 mL). The combined organic extracts were washed
with water (1· 30 mL), dried over Na2SO4, filtered,
and concentrated in vacuo to a yellow-orange oil. The
crude material was dissolved in DMF (2.6 mL) and so-
dium azide was added (0.17 g, 2.6 mmol, 10 equiv based
on starting material 16) to the solution. The solution
was heated to 85 ꢁC and was allowed to stir for 8 h at
which time the reaction was complete as judged by
TLC analysis. The reaction mixture was diluted with
water (15 mL) and the aqueous layer was extracted with
EtOAc (3· 40 mL). The combined organic fractions
were washed with water (1· 30 mL), dried over Na2SO4,
filtered, and concentrated in vacuo to a yellow oil. The
crude material was purified by flash chromatography
(97:3 hexanes/EtOAc) to provide 53 mg (62% yield) of
3.1.3. [3-Allyl-3-isobutyl-2-(naphthalen-2-ylmethyl)isox-
azolidin-5-yl]methanol (17). Preparation of 17 was
accomplished under conditions identical to those used
for 16 using 2-(bromomethyl)-naphthalene in place of
a-bromo-p-xylene. Purification by flash chromatography
(9:1 hexanes/EtOAc) afforded 0.22 g of 17 as a colorless
1
20 as a colorless oil. H NMR: d 0.98 (d, 3H, J = 2.6),
1.01 (d, 3H, J = 2.3), 1.40 (dd, 1H, J = 14.7, 7.0), 1.40
(dd, 1H, J = 14.7, 6.5), 1.62 (dd, 1H, J = 14.7, 5.0),
1.8–1.95 (m, 2H), 2.23–2.50 (m, 7H), 3.11 (dd, 1H,
J = 12.6, 4.4), 3.45 (dd, 1H, J = 12.6, 7.0), 3.76–3.92
(m, 2H), 4.06–4.17 (m, 1H), 5.07–5.19 (m, 2H), 5.86–
6.02 (m, 1H), 7.10–7.16 (m, 2H), 7.23–7.30 (m, 2H);
13C NMR: d 21.09, 24.15, 24.67, 25.25, 38.74, 40.31,
45.60, 53.10, 54.45, 68.36, 74.42, 117.8, 128.0, 128.9,
135.0, 135.6, 136.3; HRMS (ESI) calcd for
[C19H28N4O2 + H]+: 329.2341, found: 329.2336.
1
oil in 61% yield. H NMR: d 0.98 (d, 3H, J = 6.8), 0.99
(d, 3H, J = 6.8), 1.41 (dd, 1H, J = 14.4, 6.8), 1.64 (dd,
1H, J = 14.4, 4.8), 1.88–1.95 (m, 1H), 2.01–2.06 (m,
1H), 2.21 (m, 2H), 2.28 (dd, 1H, J = 12.5, 8.8), 2.46
(dd, 1H, J = 14.0, 7.0), 3.49–3.56 (m, 2H), 3.95–4.08
(m, 3H), 5.10–5.14 (m, 2H), 5.91–6.01 (m, 1H), 7.40–
7.43 (m, 2H), 7.50–7.52 (m, 2H), 7.75–7.79 (m, 3H);
13C NMR: d 24.14, 24.48, 25.27, 38.68, 38.88, 44.00,
53.66, 65.53, 68.54, 75.49, 117.7, 125.5, 125.9, 126.2,
126.5, 127.6, 127.7, 128.0, 132.7, 133.4, 135.1, 136.4;
HRMS (ESI) calcd for [C22H29NO2 + Na]+: 362.2096,
found: 362.2084.
3.1.6. 3-Allyl-5-(azidomethyl)-3-isobutyl-2-(naphthalen-2-
ylmethyl)isoxazolidine (21). Preparation of 21 was
accomplished under conditions identical to those used
for 20 starting with 0.93 mmol of 17. Purification by
flash chromatography (9:1 hexanes/EtOAc) afforded
3.1.4. (3-Allyl-2-(biphenyl-4-ylmethyl)-3-isobutylisoxaz-
olidin-5-yl)methanol (18). Preparation of 18 was accom-
plished under conditions identical to those used for 16
using 4-(bromomethyl)biphenyl in place of a-bromo-p-
xylene. Purification by flash chromatography (7:3 hex-
anes/EtOAc) provided 0.39 g of 18 in 66% yield as a col-
orless oil. The major diastereomer was isolated by
normal-phase HPLC and used for subsequent steps.
1H NMR (400 MHz): d 0.96 (d, 3H, J = 6.4), 0.97 (d,
1
0.22 g of 21 as a colorless oil in 61% yield. H NMR:
d 0.98 (d, 3H, J = 6.8), 1.0 (d, 3H, J = 6.8), 1.41 (dd,
1H, J = 14.4, 6.4), 1.63 (dd, 1H, J = 14.4, 4.8), 1.84–
1.93 (m, 2H), 2.27–2.48 (m, 5H), 3.09 (d, 1H, J = 8.4),
3.43 (d, 1H, J = 12.8, 7.2), 4.09–4.15 (m, 1H), 5.09–
5.12 (m, 2H), 5.91–5.99 (m, 1H), 7.37–7.43 (m, 2H),
7.50–7.52 (m, 2H), 7.75–7.77 (m, 3H); 13C NMR d

1040
R. J. Casey et al. / Bioorg. Med. Chem. 17 (2009) 1034–1043
24.18, 24.63, 25.24, 29.67, 38.75, 40.33, 43.66, 53.60,
54.39, 68.47, 74.54, 117.9, 125.3, 125.7, 126.5, 126.6,
127.6, 127.8, 132.7, 133.4, 134.9, 136.2; HRMS (ESI)
calcd for [C22H28N4O + Na]+: 387.2008, found:
387.2005.
organic extracts were dried over Na2SO4, filtered, and
concentrated in vacuo. The crude material was purified
by flash chromatography (1:1 hexanes/EtOAc) to pro-
vide 27 mg of the title compound (24) in 51% yield (from
1
20) as a colorless oil. H NMR: d 0.97–1.02 (m, 6H),
1.46–1.57 (m, 1H), 1.59–1.79 (m, 4H), 1.80–1.91 (m,
1H), 1.98–2.07 (m, 1H), 2.23–2.36 (m, 4H), 3.25–3.48
(m, 2H), 3.71–3.96 (m, 4H), 4.16–4.27 (m, 1H), 7.06–
7.22 (m, 4H); 13C NMR: d 21.09, 24.29, 24.86, 25.21,
35.49, 39.97, 42.80, 53.59, 54.24, 59.64, 70.65, 73.22,
128.6, 129.0, 134.5, 136.8; HRMS (ESI) calcd for
[C18H28N4O2 + Na]+: 355.2110, found: 355.2105.
3.1.7. 3-Allyl-5-(azidomethyl)-2-(biphenyl-4-ylmethyl)-3-
isobutylisoxazolidine (22). Preparation of 22 was accom-
plished under conditions identical to those used for 20
starting with 0.96 mmol of 18. Purification by flash
chromatography (9:1 hexanes/EtOAc) to afford 0.28 g
1
of 22 as a colorless oil in 71% yield. H NMR: d 0.96
(d, 3H, J = 6.6), 0.97 (d, 3H, J = 6.6), 1.38 (dd, 1H,
J = 14.4, 6.2), 1.60 (dd, 1H, J = 14.4, 4.8), 1.84–1.91
(m, 2H), 2.24–2.33 (m, 2H), 3.10 (dd, 1H, J = 11.0,
4.0), 3.44 (dd, 2H, J = 12.8, 7.2), 3.89 (s, 2H), 4.08–
4.14 (m, 1H), 5.08–5.12 (m, 2H), 5.87–5.97 (m, 1H),
7.22–7.31 (m, 4H), 7.37–7.42 (m, 1H), 7.50–7.56 (m,
4H); 13C NMR: d 24.13, 24.64, 25.22, 38.74, 40.28,
53.04, 54.43, 68.38, 74.49, 117.9, 126.9, 127.1, 128.4,
128.6, 134.9, 137.7, 139.7, 141.2; HRMS (ESI) calcd
for [C24H30N4O + Na]+: 413.2317, found: 413.2312.
3.1.9. 2-(-5-(Azidomethyl)-3-isobutyl-2-(naphthalen-2-yl-
methyl)isoxazolidin-3-yl)ethanol: (25). Preparation of 25
was accomplished under conditions identical to those
used for 24 using 0.16 mmol of 21. Purification by flash
chromatography (1:1 hexanes/EtOAc) provided 53 mg
1
of 25 in 65% yield as a colorless oil. H NMR: d 0.89–
0.90 (m, 6H), 1.41–1.48 (m, 1H), 1.60–1.79 (m, 4H),
1.96–2.00 (m, 2H), 2.21–2.26 (m, 1H), 3.24–3.33 (m,
2H), 3.75–3.85 (m, 4H), 3.98–4.12 (m, 1H), 7.11 (m
2H), 7.29–7.35 (m, 2H), 7.61–7.70 (m, 3H). HRMS
(ESI) calcd for [C21H28N4O2 + Na]+: 391.2110, found:
391.2105.
3.1.8. 2-(5-Azidomethyl-2-(4-methyl-benzyl)-3-isobutyl-isox-
azolidin-3-yl)-ethanol (24). To a solution of 20 (53 mg,
0.16 mmol, 1.0 equiv) dissolved in t-BuOH (1.1 mL),
THF (0.40 mL), and H2O (0.080 mL) was added
NMO (26 mg, 0.22 mmol, 1.4 equiv). To this solution
was added OsO4 (0.16 mL of a 2.5 wt% solution in t-
BuOH, 0.016 mmol, 0.10 equiv). The reaction mixture
was allowed to stir at ambient temperature for 5 h at
which time the reaction was complete as judged by
TLC analysis (4:6 hexanes/EtOAc). Excess reagents
were quenched by the addition of Na2S2O3 (1.0 mL of
10% solution w/v in water) and allowed to stir for 3 h.
The mixture was then poured into a biphasic mixture
of H2O (40 mL) and EtOAc (40 mL). The organic layer
was separated and the aqueous layer was extracted with
EtOAc (3· 30 mL). The combined organic layers were
dried over Na2SO4, filtered, and concentrated in vacuo
to provide the crude material as a tan oil. The crude
product was dissolved in CH3CN (800 lL) and H2O
(800 lL) and added NaIO4 (68 mg, 0.32 mmol, 2.0 equiv
based on starting material 20). The mixture was allowed
to stir at rt until the reaction was complete as judged by
TLC analysis (9:1 hexanes/EtOAc). The reaction mix-
ture was poured into a biphasic solution of H2O
(60 mL) and EtOAc (35 mL). Brine (15 mL) was added
to separate the emulsion. The organic layer was sepa-
rated and the aqueous layer was extracted with EtOAc
(2· 35 mL). The combined organic layers were dried
over Na2SO4, filtered, and concentrated in vacuo to pro-
vide the crude material as a tan oil. Crude aldehyde was
dissolved in methanol (1.6 mL) and the solution was
cooled in an ice–H2O bath. NaBH4 (14 mg, 0.40 mmol,
2.5 equiv based on starting material 20) was added and
the mixture was then allowed to stir until the reaction
was complete as judged by TLC analysis (0.5 h, 1:1 hex-
anes/EtOAc). Excess reagents were quenched with H2O
(2 mL) and the reaction solution was poured into a bi-
phasic mixture of EtOAc (25 mL) and H2O (25 mL).
The organic layer was separated and the aqueous phase
was extracted with EtOAc (2· 25 mL). The combined
3.1.10. 2-(-5-(Azidomethyl)-2-(biphenyl-4-ylmethyl)-3-iso-
butylisoxazolidin-3-yl)ethanol (26). Preparation of 26
was accomplished under conditions identical to those
used for 25 using 0.16 mmol of 22. Purification by flash
chromatography (1:1 hexanes/EtOAc) provided 50 mg
1
of 26 in 59% yield as a colorless oil. H NMR: 0.98–
1.0 (m, 6H), 1.52–1.60 (m, 1H), 1.67–1.87 (m, 4H),
2.04–2.09 (m, 2H), 2.31–2.36 (m, 1H), 3.39 (m, 2H),
3.80–4.00 (m, 4H), 4.20–4.24 (m, 1H), 7.27–7.55 (9H).
HRMS (ESI) calcd for [C23H30N4O2 + Na]+: 417.2266,
found: 417.2258.
3.1.11. 9-Fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-
6,7,8,9,11,12,14,15,16,17-dodecahydro-3H-cyclopenta[a]-
phenanthrene-17-carboxylic acid [2-(2-{[(2-(4-methyl-ben-
zyl-3-(2-hydroxy-ethyl)-3-isobutyl-isoxazolidin-5-ylmethyl)-
carbamoyl]-methoxy}-ethoxy)-ethyl]-amide (5). To
a
solution of azide 24 (5.0 mg, 0.015 mmol, 1.0 equiv) in
THF (0.17 mL) were added triphenylphosphine
(8.0 mg, 0.030 mmol, 2.0 equiv) and H2O (2.4 lL,
0.15 mmol, 10 equiv). The mixture was allowed to stir
at reflux for 2 h. H2O (0.17 mL) was added to the reac-
tion and the mixture was allowed to stir in a 70 ꢁC-oil
bath for an additional 20 min. The mixture was allowed
to cool to room temperature and was then transferred
into a biphasic mixture of 1 M HCl (10 mL) and ether
(10 mL). The layers were partitioned and the organic
layer was extracted with 1 M HCl (2· 10 mL). The com-
bined aqueous layers were basified with 3 M NaOH (un-
til pH 10 or greater). The aqueous mixture was then
extracted with CH2Cl2 (3· 15 mL) and the combined or-
ganic extracts were dried over Na2SO4, filtered, and con-
centrated in vacuo. To
a
solution of 8-(9-
fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic
acid (18 mg, 0.045 mmol, 3.0 equiv based on starting
material 24) dissolved in NMP (0.067 mL) were added
HOBt (6.3 mg, 0.045 mmol, 3.0 equiv based on starting

R. J. Casey et al. / Bioorg. Med. Chem. 17 (2009) 1034–1043
1041
material 24) and HBTU (17 mg, 0.045 mmol, 3.0 equiv
based on starting material 24). This solution was agi-
tated for 15 min. The solution of activated ester was
added to the crude amine dissolved in NMP
(0.067 mL) and the resulting mixture was allowed to stir
for 12 h at which time the reaction was complete as
judged by ESI-MS analysis. Excess reagents were
quenched by the addition of 1M HCl (10 mL) and
EtOAc (10 mL). The reaction vessel was washed with
EtOAc (2· 2.0 mL) to remove all residues. The resulting
biphasic mixture was separated and the aqueous layer
was extracted with EtOAc (3· 10 mL). The combined
organic fractions were dried over Na2SO4, filtered, and
concentrated in vacuo. The resulting oil was dissolved
in a solution of 20% piperidine in DMF (0.037 mL,
0.076 mmol piperidine, 5.0 equiv based on starting
material 24) and was allowed to stir for 30 minutes.
The resulting solution was diluted with 0.1% TFA
H2O (0.50 mL) and CH3CN (0.50 mL) and partially
purified by reverse-phase HPLC to remove Fmoc
byproducts. The partially purified amine was used
immediately in subsequent steps. To a solution of Ox-
Dex20 (17 mg, 0.045 mmol, 3.0 equiv) dissolved in
NMP (0.15 mL) were added HOBt (6.2 mg, 0.045 mmol,
3.0 equiv) and HBTU (17 mg, 0.045 mmol, 3.0 equiv)
and the resulting mixture was agitated for 15 min. To
this solution were added the amine dissolved in NMP
(0.15 mL), 2,6-lutidine (35 lL, 0.30 mmol, 6.7 equiv),
and i-Pr2NEt (49 lL, 0.30 mmol, 6.7 equiv). The result-
ing mixture was stirred for 12 h at ambient temperature.
The product was isolated by reverse-phase HPLC
purification to provide 5 as a white solid (7% yield
from 24; 0.8 mg). The purity of compound 5 was
confirmed by analytical reverse-phase HPLC analysis.
The identity was verified by mass spectral analysis
of the isolated compound. HRMS (ESI) calcd for
[C45H64FN3O8 + H]+: 812.4861, found: 812.4885.
benzylamino-2-hydroxy-4-(2-hydroxy-ethyl)-6-methyl-
heptylcarbamoyl]-methoxy}-ethoxy)-ethyl]-amide (3).
The conversion of azide 23 to amine S6 via reduction
of the azide, coupling to Fmoc-AEEA, and removal of
the Fmoc protecting group has been previously de-
scribed.10 Amine S6 (2.0 mg, 4.6 lmol, 1.0 equiv) was
dissolved in AcOH (0.10 mL) and to the solution was
added zinc (1.5 mg, 23 lmol, 5.1 equiv) to reduce the
isoxazolidine N–O bond. The reaction mixture was al-
lowed to stir at room temperature for 4 h until the reac-
tion was complete as judged by ESI-MS. The reaction
mixture was then diluted with 0.1% TFA H2O
(0.50 mL) and CH3CN (0.50 mL). The resulting mixture
was filtered through a syringe filter and purified by re-
verse phase HPLC to provide 1.1 mg (54% yield) of a
colorless oil. HRMS (ESI) calcd for [C23H41N3O6 +
Na]+: 462.2944, found: 462.2945. The intermediate was
conjugated to OxDex under the conditions described
for 5 yielded 0.70 mg of 3 (23% yield) as a white solid
after reverse phase HPLC purification. HRMS (ESI)
calcd for [C44H66FN3O9 + H]+: 800.4861, found:
800.4887.
3.1.15. 9-Fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-
oxo-6,7,8,9,11,12,14,15,16,17-dodecahydro-3H-cyclo-
penta[a]phenanthrene-17-carboxylic acid [2-(2-{[(2-ben-
zyl-3-(2,3-dihydroxy-propyl)-3-isobutyl-isoxazolidin-5-
ylmethyl)-carbamoyl]-methoxy}-ethoxy)-ethyl]-amide (8).
Dihydroxylation of alkene 19 was accomplished as pre-
viously reported.9 Briefly, to a solution of alkene 19
(21 mg, 64 lmol, 1.0 equiv) dissolved in t-BuOH
(430 lL), THF (140 lL), and H2O (35 lL) was added
NMO (9.8 mg, 86 lmol, 1.4 equiv). To this solution
was added OsO4 (61 lL of a 2.5 wt% solution in t-
BuOH, 6.1 lmol, 0.10 equiv). The reaction mixture
was allowed to stir at ambient temperature until the
reaction was complete as judged by TLC analysis (typi-
cally 5 h). Excess reagents were quenched by the addi-
tion of Na2S2O3 (1.0 mL of 10% solution w/v in water)
and allowed to stir for 3 h. The mixture was then trans-
ferred into biphasic mixture H2O (40 mL) and EtOAc
(40 mL). The organic layer was separated and the aque-
ous layer was extracted EtOAc (3· 25 mL). The com-
bined organic layers were dried over Na2SO4, filtered,
and concentrated in vacuo. The crude product was sub-
jected to flash chromatography (4:6 hexanes/EtOAc) to
provide 19 mg of diol S1 which was conjugated to
AEEA and OxDex using the conditions described for
5. Purification of the crude product thus obtained by re-
verse phase HPLC providing 4.3 mg of 8 (8.0% yield) as
3.1.12. 9-Fluoro-11,17-dihydroxy-N-(2-(2-(2-(((5R)-3-(2-
hydroxyethyl)-3-isobutyl-2-(naphthalen-2-ylmethyl)isox-
azolidin-5-yl)methylamino)-2-oxoethoxy)ethoxy)ethyl)-
8,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-
dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbox-
amide (6). Preparation of was accomplished under
conditions identical to those used for 5. Purification
by reverse phase HPLC providing 0.8 mg of 6 (6%
yield) as a white solid. HRMS (ESI) calcd for
[C48H66FN3O9 + H]+: 848.4861, found: 848.4865.
3.1.13. N-(2-(2-(2-((2-(Biphenyl-4-ylmethyl)-3-(2-hydroxy-
ethyl)-3-isobutylisoxazolidin-5-yl)methylamino)-2-oxo-
ethoxy)ethoxy)ethyl)-9-fluoro-11,17-dihydroxy-8,13,16-
trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodeca-
hydro-3H-cyclopenta[a]phenanthrene-17-carboxamide (7).
Preparation of was accomplished under conditions iden-
tical to those used for 5. Purification by reverse phase
HPLC providing 1.0 mg of 7 (8% yield) as a white solid.
HRMS (ESI) calcd for [C50H68FN3O9 + H]+: 874.5018,
found: 874.5001.
a white solid. HRMS (ESI) calcd for [C45H66FN3O10
+
Na]+: 850.4630, found: 850.4810.
3.1.16. N-(2-(2-(2-((3-(2,3-Dihydroxypropyl)-3-isobutyl-
2-(naphthalen-2-ylmethyl)isoxazolidin-5-yl)methylamino)-2-
oxoethoxy)ethoxy)ethyl)-9-fluoro-11,17-dihydroxy-8,13,16-
trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodeca-
hydro-3H-cyclopenta[a]phenanthrene-17-carboxamide (9).
To a solution of 21 (62 mg, 0.17 mmol, 1.0 equiv) in t-
BuOH (1.2 mL) and THF (0.32 mL) were added NMO
(32 mg, 0.26 mmol, 1.5 equiv) and H2O (0.13 mL). To
this solution was added OsO4 (0.17 mL of a 2.5 wt%
solution in t-BuOH, 0.02 mmol, 0.10 equiv). The result-
3.1.14. 9-Fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-
oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-
cyclopenta[a]phenanthrene-17-carboxylic acid [2-(2-{[4-

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R. J. Casey et al. / Bioorg. Med. Chem. 17 (2009) 1034–1043
ing mixture was stirred for 8 h at which time TLC anal-
ysis indicated consumption of 21. Excess reagents were
quenched by the addition of Na2S2O3 (50 mg) followed
by stirring at ambient temperature for 1 h. To this mix-
ture was added 20 mL of H2O and the resulting mixture
was partitioned with EtOAc (20 mL). The aqueous layer
was further extracted with EtOAc (1· 20 mL). The com-
bined organic extracts were dried over Na2SO4, filtered,
and concentrated under reduced pressure. The crude
product thus obtained was purified by flash chromatog-
raphy (1:1 hexanes/EtOAc) to yield 57 mg of a diol S2 as
a 1:1 mixture of diasteromers at the newly formed 2ꢁ OH
in 84% yield. 1H NMR d 0.98–1.04 (6H), 1.34–1.50 (2H),
1.58–1.75 (3H), 1.90–2.41 (4H), 3.43–3.64 (3H), 3.78–4.3
(5H), 7.27–7.55 (7H). HRMS (ESI) calcd for
[C22H30N4O3 + H]+: 399.2396, found: 399.2392. This
material was then conjugated to AEEA and OxDex
using the method described for 5. Purification by reverse
phase HPLC providing 0.4 mg of 9 (3% yield) as a white
solid. HRMS (ESI) calcd for [C49H68FN3O10 + H]:
878.4967, found: 878.4972.
E isomer: 1H NMR: d 0.92–1.01 (m, 6H), 1.36–1.42
(m, 1H), 1.52–1.63 (m, 2H), 1.74–1.87 (m, 1H), 1.91–
1.98 (m, 1H), 2.19–2.29 (m, 1H), 2.30–2.40 (m, 1H),
2.48–2.58 (m, 1H), 2.98–3.09 (m, 1H), 3.36–3.45 (m,
1H), 3.66–3.89 (m, 4H), 4.02–4.11 (m, 1H), 5.85 (d,
J = 14.3, 1H), 7.03 (quintet of doublets, J = 8.0, 2.7,
1H), 7.14–7.38 (m, 5H); 13C NMR: d 24.22, 24.56,
25.23, 37.21, 40.45, 42.56, 51.56, 53.34, 54.36, 68.53,
74.49, 119.5, 126.9, 128.0, 128.3, 138.1, 145.6, 166.6;
HRMS (ESI) calcd for [C20H28N4O3 + H]+: 373.2240,
found: 373.2236.
3.1.19. 4-(2-Benzyl-5-{[2-(2-{2-[(9-fluoro-11,17-dihydroxy-
10,13,16-trimethyl-3-oxo-6,7,8,9,11,12,14,15,16,17-dodeca-
hydro-3H-cyclopenta[a]phenanthrene-17-carbonyl)-ami-
no]-ethoxy}-ethoxy)-acetylamino]-methyl}-3-isobutyl-isox-
azolidin-3-yl)-but-2-enoic acid methyl ester (11).
Preparation of 11 was accomplished under conditions
identical to those used for 5 starting with 0.038 mmol
of 27. Purification by reverse phase HPLC providing
6.8 mg of 11 (21% yield) as a white solid. HRMS
(ESI) calcd for [C47H66FN3O10 + H]+: 852.4810, found:
852.4810.
3.1.17. N-(2-(2-(2-((2-(Biphenyl-4-ylmethyl)-3-(2,3-dihydr-
oxypropyl)-3-isobutylisoxazolidin-5-yl)methylamino)-2-
oxoethoxy)ethoxy)ethyl)-9-fluoro-11,17-dihydroxy-8,13,16-
trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodeca-
hydro-3H-cyclopenta[a]phenanthrene-17-carboxamide (10).
Preparation of 10 was accomplished under conditions
identical to those used for 9 starting with 0.17 mmol
of 22. Purification by reverse phase HPLC providing
0.7 mg of 10 (5% yield) as a white solid. HRMS (ESI)
calcd for [C51H70FN3O10 + H]+: 904.5135, found:
904.5123.
3.1.20. 4-(2-Benzyl-5-{[2-(2-{2-[(9-Fluoro-11,17-dihydroxy-
10,13,16-trimethyl-3-oxo-6,7,8,9,11,12,14,15,16,17-dodeca-
hydro-3H-cyclopenta[a]phenanthrene-17-carbonyl)-amino]-
ethoxy}-ethoxy)-acetylamino]-methyl}-3-isobutyl-isoxaz-
olidin-3-yl)-but-2-enoic acid (12). To compound 11
(6.0 mg, 12 lmol, 1.0 equiv) was added ethanol
(0.13 mL) then sodium hydroxide (1.3 mg, 30 lmol,
2.5 equiv). The mixture was allowed to stir at rt for
8 h until the reaction was complete as judged by ESI-
MS. 0.1% TFA H2O (1.5 mL) and CH3CN (1.5 mL)
were added to the reaction. The resulting mixture was
filtered through a syringe filter and purified by reverse
phase HPLC providing 3.1 mg of 12 (31% yield) as a
3.1.18. 4-(5-Azidomethyl-2-benzyl-3-isobutyl-isoxazoli-
din-3-yl)-but-2-enoic acid methyl ester (27). Alkene 19
was dihydroxylated exactly as described for 8 to pro-
vided S1. The oxidative cleavage of diol S1 to provide
aldehyde S4 has been previously reported.10 Briefly, to
a solution of diol S1 (26 mg, 0.076 mmol, 1.0 equiv) in
CH3CN (0.36 mL) and H2O (0.36 mL) was added
NaIO4 (32 mg, 0.15 mmol, 2.0 equiv). The mixture was
allowed to stir at room temperature until the reaction
was complete as judged by TLC analysis. The reaction
mixture was poured into a biphasic solution of H2O
(20 mL) and EtOAc (20 mL). The organic layer was sep-
arated and the aqueous layer was extracted with EtOAc
(2· 20 mL). The combined organic layers were dried
over Na2SO4, filtered, and concentrated in vacuo. The
crude material was partially purified on a short silica
gel column (9:1 hexanes/EtOAc) and the resulting oil,
21 mg after concentration in vacuo, was used immedi-
ately without further purification (86% approximate
yield). To a solution of aldehyde S4 (21 mg, 0.066 mmol,
1.0 equiv) dissolved in toluene (0.66 mL) was added
methyl (triphenylphosphoranylidene) acetate (90 mg,
0.26 mmol, 4.0 equiv). The mixture was allowed to stir
at 50 ꢁC for 6 h at which time the reaction was complete
as judged by TLC analysis (8:2 hexanes/EtOAc). The
solvent was removed in vacuo and the crude material
was purified by flash chromatography (85:15 hexanes/
EtOAc) to provide 24 mg of the title compound (27)
as a colorless oil (10:1 E/Z mixture). Spectral data for
white
solid.
HRMS
(ESI)
calcd
for
[C46H64FN3O10 + H]+: 838.4654, found: 838.4675.
3.1.21. 4-(2-Benzyl-5-{[2-(2-{2-[(9-Fluoro-11,17-dihydroxy-
10,13,16-trimethyl-3-oxo-6,7,8,9,11,12,14,15,16,17-dodeca-
hydro-3H-cyclopenta[a]phenanthrene-17-carbonyl)-ami-
no]-ethoxy}-ethoxy)-acetylamino]-methyl}-3-isobutyl-isox-
azolidin-3-yl)-but-2-enoic acid amide (13). To a solution
of compound 12 (2.1 mg, 2.5 lmol, 1.0 equiv) in
CH3CN (0.030 mL) and pyridine (1.0 lL, 13 lmol,
5.0 equiv) were added Boc2O (1.0 mg, 4.6 lmol,
1.8 equiv) and NH4HCO3 (1.0 mg, 13 lmol, 5.0 equiv).
The mixture was allowed to stir at rt for 24 h until the
reaction was complete as judged by ESI mass spectrom-
etry. About 0.1% TFA H2O (0.80 mL) and CH3CN
(0.70 mL) were added to the reaction. The resulting
mixture was filtered through a syringe filter and purified
by reverse phase HPLC providing 0.70 mg of 13
(33% yield) as a white solid. ESI-MS calcd for
[C46H65FN4O9 + H]+: 837.5, found: 837.5.
3.1.22. 9-Fluoro-11,17-dihydroxy-N-(1-(6-(hydroxy(6-
methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-
5-yl)methyl)-2,3-dimethoxyphenyl)-1-oxo-6,9,12-trioxa-
2-azapentadecan-15-yl)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,
11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phen-

R. J. Casey et al. / Bioorg. Med. Chem. 17 (2009) 1034–1043
1043
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6, 765.
anthrene-17-carboxamide (28). Beta-(+/À)-hydrastine
hydrochloride (10 mg, 0.024 mmol, 1.0 equiv) was dis-
solved in 5.0 mL of DMF, followed by the addition of
i-Pr2NEt (0.034 mL, 0.24 mmol, 10 equiv) and DMAP
(3.0 mg, 0.024 mmol, 1.0 equiv). To this solution was
5. Ptashne, M.; Gann, A. Genes & Signals; Cold Spring
Harbor Laboratory: New York, 2001.
added
4,7,10-trioxa-1,13-tridecanediamine
(0.11 g,
0.48 mmol, 20 equiv) and the reaction mixture was stir-
red for 12 h. The reaction mixture was diluted with
water (10 mL) and extracted with EtOAc (3· 10 mL).
The organic extract was washed with water (2·
10 mL), dried over Na2SO4, and concentrated in vacuo.
To this crude mixture were added HOBt (8.0 mg,
0.060 mmol, 2.5 equiv), HBTU (23 mg, 0.060 mmol,
2.5 equiv), i-Pr2NEt (8.0 lL, 0.060 mmol, 2.5 equiv),
and OxDex (23 mg, 0.060 mmol, 2.5 equiv) and the reac-
tion mixture was stirred for 12 h. The reaction progress
was monitored by ESI-MS. The purification was accom-
plished by reverse-phase HPLC to yield 1.1 mg of 28 in
4.6% yield as a white solid. HRMS (ESI) calcd for
[C52H70FN3O13 + Na]+: 986.4790, found: 986.4823.
6. Minter, A. R.; Brennan, B. B.; Mapp, A. K. J. Am. Chem.
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K. J. Am. Chem. Soc. 2005, 127, 12456.
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3.1.23. 9-Fluoro-11,17-dihydroxy-N-(16-(1-hydroxy-4a,8-
dimethyl-7-oxo-1,2,3,4,4a,7-hexahydronaphthalen-2-yl)-
15-oxo-4,7,10-trioxa-14-azaheptadecyl)-10,13,16-trimethyl-
3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-
cyclopenta[a]phenanthrene-17-carboxamide (29). Prepa-
ration of was accomplished under conditions identical
to those used for 28, except that (À)-a-santonin was
used as the starting material. Purification by reverse
phase HPLC provided 0.7 mg (3% yield) of 29 as a white
solid. HRMS (ESI) calcd for [C46H67FN2O10 + Na]+:
849.4677, found: 849.4660.
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The assay was carried out using the method described by
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Acknowledgments
A.K.M. is grateful for financial support from the Amer-
ican Cancer Society (RSG 05-195-01-CDD), the NSF
(CAREER award), the Alfred P. Sloan Foundation,
Amgen, and GSK. J.P.D. was supported by the Ameri-
can Cancer Society (PF-06-116-01).
22. Gill, G.; Sadowski, I.; Ptashne, M. Proc. Natl. Acad. Sci.
U.S.A. 1990, 87, 2127.
23. Lu, Z.; Rowe, S. P.; Brennan, B. B.; Davis, S. E.; Metzler,
R. E.; Nau, J. J.; Majmudar, C. Y.; Mapp, A. K.; Ansari,
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24. Lum, J. K.; Majmudar, C. Y.; Ansari, A. Z.; Mapp, A. K.
ACS Chem. Biol. 2006, 1, 639.
25. Ruden, D. M.; Ma, J.; Li, Y.; Wood, K.; Ptashne, M.
Nature 1991, 350.
Supplementary data
26. Osiris properties calculator was used to calculate the
clogP values. http://www.organic-chemistry.org/prog/peo/
.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2008.02.045.
´
27. Blay, G.; Cardona, L.; Collado, A. M.; Garcıa, B.;
Morcillo, V.; Pedro, J. R. J. Org. Chem. 2004, 69,
7294.
28. Birladeanu, L. Angew. Chem., Int. Ed. 2003, 42, 1202.
29. Huang, J. H.; Johnston, G. A. Br. J. Pharmacol. 1990, 99,
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30. Wu, Z.; Belanger, G.; Brennan, B. B.; Lum, J. K.; Minter,
A. R.; Rowe, S. P.; Plachetka, A.; Majmudar, C. Y.;
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References and notes
1. Mapp, A. K.; Ansari, A. Z. ACS Chem. Biol. 2007, 2, 62,
and references cited therein.
2. Majmudar, C. Y.; Mapp, A. K. Curr. Opin. Chem. Biol.
2005, 5, 467, and references therein.