Synthetic silvestrol analogues as potent and selective protein synthesis inhibitors

Article abstract of DOI:10.1021/jm3011542

Misregulation of protein translation plays a critical role in human cancer pathogenesis at many levels. Silvestrol, a cyclopenta[b]benzofuran natural product, blocks translation at the initiation step by interfering with assembly of the eIF4F translation complex. Silvestrol has a complex chemical structure whose functional group requirements have not been systematically investigated. Moreover, silvestrol has limited development potential due to poor druglike properties. Herein, we sought to develop a practical synthesis of key intermediates of silvestrol and explore structure-activity relationships around the C6 position. The ability of silvestrol and analogues to selectively inhibit the translation of proteins with high requirement on the translation-initiation machinery (i.e., complex 5′-untranslated region UTR) relative to simple 5′UTR was determined by a cellular reporter assay. Simplified analogues of silvestrol such as compounds 74 and 76 were shown to have similar cytotoxic potency and better ADME characteristics relative to those of silvestrol.

Full text of DOI:10.1021/jm3011542

Article
pubs.acs.org/jmc
Synthetic Silvestrol Analogues as Potent and Selective Protein
Synthesis Inhibitors
Tao Liu,† Somarajan J. Nair,† Andre Lescarbeau, Jitendra Belani,‡ Stephane Peluso, James Conley,
́
́
Bonnie Tillotson, Patrick O’Hearn, Sherri Smith, Kelly Slocum,§ Kip West, Joseph Helble, Mark Douglas,
Adilah Bahadoor, Janid Ali, Karen McGovern, Christian Fritz, Vito J. Palombella, Andrew Wylie,§
Alfredo C. Castro, and Martin R. Tremblay*
Infinity Pharmaceuticals, Inc., 780 Memorial Drive, Cambridge, Massachusetts 02139, United States
S
* Supporting Information
ABSTRACT: Misregulation of protein translation plays a critical role in human cancer pathogenesis at many levels. Silvestrol, a
cyclopenta[b]benzofuran natural product, blocks translation at the initiation step by interfering with assembly of the eIF4F
translation complex. Silvestrol has a complex chemical structure whose functional group requirements have not been
systematically investigated. Moreover, silvestrol has limited development potential due to poor druglike properties. Herein, we
sought to develop a practical synthesis of key intermediates of silvestrol and explore structure−activity relationships around the
C6 position. The ability of silvestrol and analogues to selectively inhibit the translation of proteins with high requirement on the
translation−initiation machinery (i.e., complex 5′-untranslated region UTR) relative to simple 5′UTR was determined by a
cellular reporter assay. Simplified analogues of silvestrol such as compounds 74 and 76 were shown to have similar cytotoxic
potency and better ADME characteristics relative to those of silvestrol.
the tumor suppressor programmed cell death protein 4
(PDCD4) binds eIF4A and blocks protein translation
initiation.10 Loss or reduced expression of PDCD4 has been
implicated in the growth of human cancers.11
INTRODUCTION
■
Misregulation of protein translation plays a critical role in
human cancer pathogenesis at many levels.1,2 Aberrant
activation of oncogenic pathways such as PI3K/Akt/mTOR
and Ras/Raf/MEK enhance the availability and/or the activity
of translation initiation factors, increasing the efficiency of
protein synthesis.3 Among all initiation factors, eIF4E has been
the most studied for its role in cancer in the initiation and
progression of the disease.4,5 Overexpression of eIF4E is
commonly observed in human cancer.6 In experiments with
tumor cells, overexpression of eIF4E results in rapid cell
proliferation, suppression of apoptosis, and malignant trans-
formation.7 These phenotypic changes are caused by enhance-
ment of translation of mRNAs that code for proteins involved
in cell survival and growth (c-myc, cyclin D1), angiogenesis
(VEGF, FGF2), and invasion and metastasis (MMP-9,
heparanase). The expression and phosphorylation state of
eIF4E’s natural repressor 4E-BP play a major role in integrating
oncogenic pathways and translation initiation.8,9 While the role
of eIF4G and eIF4A in cancer has not been studied extensively,
In addition to the tight control exerted on the translation
machinery components, the efficiency of protein synthesis can
also be regulated at the mRNA level. The degree of complexity
of 5′-untranslated region (5′-UTR) of the mRNA correlates
with the requirement for helicase activity of eIF4A.12 Long and
structured 5′UTRs are typically stable secondary structures that
impede the ribosomal scanning process. It is estimated that
around 10% of all mRNAs contain atypically long and complex
5′-UTR. Several of these encode oncoproteins, growth factors,
transcription factors, signal transduction proteins, and a variety
of receptors.13
The involvement of translation initiation in the etiology of
malignant diseases, as well as the differential translation
Received: August 6, 2012
Published: October 1, 2012
© 2012 American Chemical Society
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Figure 1. Structures of silvestrol (1) and synthetic flavagine analogues 2 and 3.
Figure 2. General synthetic approach to silvestrol analogues.
a
Scheme 1
a
Reagents and conditions: (a) (i) BnBr, K2CO3, acetone; (ii) Me2SO4; (b) KOH/pyridine, diethylene glycol; (c) (i) TBSOTf, Et3N, CH2Cl2; (ii) m-
CPBA, NaHCO3, CH2Cl2; (iii) p-TSA, THF/H2O; (d) (i) (p-OMe)BzCl, Et3N, DMAP, CH2Cl2; (ii) LiHMDS, THF; (iii) H2SO4, AcOH; (e)
NaOH, EtOH/water; (f) (i) methyl cinnamate, CH2Cl2/CH3CN/MeOH, UV lamp; (ii) NaOMe/MeOH; (iii) Me4NBH(OAc)3, AcOH, CH3CN;
(g) H2, Pd/C; (h) chiral supercritical fluid chromatography.
efficiency based on the mRNA secondary structure, provides a
rationale to target protein synthesis for the treatment of
proliferating diseases such as cancer.14 Recently, small-molecule
inhibitors of translation initiation with various modes of action
have been identified.15 Among them, plant-derived silvestrol (1,
Figure 1) and structurally simplified flavaglines were shown to
affect the composition of the eIF4F complex by acting on
eIF4A, the ATP-dependent RNA-helicase subunit of the eIF4F
complex.16,17 Moreover, silvestrol has shown in vivo antitumor
activity in a variety of murine models of solid tumors17−19 and
hematological cancers.16,20,21
Silvestrol, isolated from Aglaia lepthanta19 or Aglaia
foveolata18 species, belongs to the family of cyclopenta[b]-
benzofuran natural products such as rocaglates and rocagla-
mides. Naturally occurring cyclopenta[b]benzofurans from a
variety of species (also known as flavaglines) display a wide
range of biological effects.22−24 Slightly over 60 cyclopenta[b]-
benzofuran natural products have been isolated from different
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a
Scheme 2
a
Reagents and conditions: (a) Bu2SnO, Bu4NI, BrCH2COOEt; (b) DIBALH, toluene; (c) Bu2SnO, Bu4NI, BrCH(OMe)COOEt.
a
Scheme 3
a
Reagents and conditions: (a) BrMgCH2CH2CHCH2, Li2CuCl4; (b) (i) O3, NaHCO3; (ii) PPh3; (c) (i) NH2CH2CHCH2; (ii) Cbz-Cl.
Aglaia species, but silvestrol and a small number of related
epimeric structures have a unique feature with the presence of a
dioxanyloxy moiety at the C6 position.23 While most
cyclopenta[b]benzofuran rocaglate displays cytostatic effects
on cells,25,26 silvestrol and C4‴-episilvestrol are cytotoxic in
their mode of action, suggesting the importance of the
dioxanyloxy group for this effect.19 Several studies with either
synthetic analogues27−30 or naturally occurring epimers of
silvestrol18,31 suggested a crucial role of stereochemistry at the
C1‴ and C2‴ on the biological activity. While silvestrol’s mode
of action has been studied,17,32,33 the nature of the interaction
of the dioxanyloxy moiety with eIF4A, as well as its
contribution to the cytotoxic action of this compound, is not
well understood. This unusual chemical moiety contributes
considerably to the synthetic complexity of the molecule34,35
and negatively impacts druglike properties of silvestrol. Of note,
We set out to generate selective oncoprotein translation
inhibitors based on the silvestrol scaffold. Consequently,
systematic exploration of structure−activity relationships
around the dioxanyl moiety and the cyclopenta[b]benzofuran
core was warranted to identify simpler and better analogues of
silvestrol. Herein we report explorative studies that led to the
discovery of rocaglate analogues with improved pharmaceutical
properties and similar potencies relative to those of silvestrol.
CHEMISTRY
■
Similar to total syntheses of silvestrol described previously by
Porco’s group27 and Rizzacasa’s group,28,29 a convergent
approach was elaborated that features a late-stage glycosylation
by a Mitsunobu reaction to assemble the flavagline and the
dioxanyl moiety. This approach enabled access to a diverse set
of silvestrol analogues by varying the dioxanyl moiety (Figure
2).
Des
́
aubry et al.36 and Porco et al.37 have recently unveiled
In order to support this late-stage diversification strategy, a
robust, multigram synthesis of 6-phenolic flavagline 4 was
implemented (Scheme 1). As pioneered by Porco’s group,27,38
hydroxyflavones such as 10 form oxidopyryliums upon UV light
flavagline compounds 2 and 3 (Figure 1) with similar potency
relative to that of silvestrol, supporting the need for further
understanding the structure−activity relationship of silvestrol.
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a
Scheme 4. Coupling of Flavagline 4 with Lactols
a
Reagents and conditions: (a) DMEAD, PPh3, toluene; (b) H2, Pd(OH)2/C; (c) TBAF, THF; (d) H2, Pd/C; (e) formaldehyde, NaBH(OAc)3,
THF.
a
Scheme 5
a
Reagents and conditions: (a) TMSCHN2, MeOH, toluene; (b) Tf2O, i-Pr2EtN, CH2Cl2. (c) For 49: HCOOH, Et3N, Pd(OAc)2, PPh3. For 50:
Pd(OAc)2, Cs2CO3, MeB(OH)2. For 51: Pd(OAc)2, Ph2CNH, then NH2OH. For 52: Pd(OAc)2, P(t-Bu)3, Cs2CO3, MeNH2. For 53: Pd(OAc)2,
P(t-Bu)3, Cs2CO3, HNMe2. For 61: Pd2(dba)3, dppf, Zn(CN)2. For 62: PdCl2(dppf)2 bis(pinacolato)diboron, then CuCl2; (d) BtMs, Et3N, DMAP;
(e) Pd(OAc)2, dppf, CO; (f) Pd(OAc)2, PPh3, Bu3Sn((CCH2)OEt); (g) EDCI, HOBt, NH4OAc; (h) EDCI, HOBt, MeNH2; (i) EDCI, HOBt,
HNMe2.
activation and are very productive partners in [3 + 2]
cycloaddition with dipolarophiles like cinnamates to give the
flavagline cores. To access large quantities of hydroxyflavone
10, we devised a synthetic route that could (1) offer flexibility
in the substitution pattern at the C6 and C4′ of the flavagline,
(2) generate the hydroxyflavone under mild conditions, and (3)
give opportunities for crystalline intermediates. Accordingly,
selective benzylation of chrysin 5 followed by methylation gave
protected chrysin 6. Base-mediated fragmentation of 6 yielded
the acetophenone 7, which was converted to the hydroxyketone
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8 through a Rubottom oxidation.39 Condensation of 8 with p-
methoxybenzoyl chloride and Baker−Venkataraman rearrange-
ment40,41 gave intermediate 9, which generated the hydroxy-
flavone 10 upon basic treatment. Hydroxyflavone 10 was
submitted to a known sequence of photocycloaddition, ketol
rearrangement, and directed stereoselective reduction to give a
separable diastereoisomeric mixture (∼3/1) of flavagline rac-11
and rac-12. Notably, the throughput and robustness of the
photoaddition reaction were increased by flow chemistry (see
Experimental Section) in such a way that we could produce
tens of grams of rac-11 from hydroxyflavone 10 in a few hours.
With large quantities of this racemic mixture in hand, chiral
supercritical fluid chromatography (SFC) performed on the
phenol rac-13 gave multigram amounts of optically pure
flavagline 4.
The syntheses of dioxanyl building blocks were performed by
a tandem stannylene acetal alkylation−lactonization sequence27
involving various chiral diols with α-bromoesters (Schemes 2
and 3). Glycosylation reactions with flavagline 4 were
performed using a modified diazodicarboxylate42 to directly
give final compounds or access them through deprotection of
penultimate intermediates (Scheme 4).
In addition to silvestrol analogues, flavagline 4 was used as a
synthetic precursor to produce C6-modified rocaglate ana-
logues (Scheme 5). The triflate 48 served as an intermediate in
various Pd-mediated cross-coupling reactions (Suzuki, amina-
tion, and carbonylation) that provided access to C-or N-
substituted analogues at the C6-position (49−62). Likewise, a
small set of C4′-analogues (Scheme 6) were synthesized from
4′-bromo precursor 63 prepared according to a method similar
to that for 4 (Supporting Information). Suzuki cross-coupling
followed by a deprotection−protection sequence yielded the 4′-
methyl analogue 66. Palladium-mediated cyanation provided
intermediate 67 which was converted to the 6-methyl ether
analogue 69.
RESULTS AND DISCUSSION
■
Identification of Simplified Silvestrol Analogues.
Silvestrol inhibits protein translation initiation by targeting
the ATP-dependent RNA helicase eIF4A in the eIF4F
complex.17 This helicase unwinds the secondary structure of
the mRNA and facilitates scanning of the 43S preinitiation
complex through the 5′ untranslated region (5′UTR) for the
AUG start codon. As a consequence, the translation of mRNAs
flanked with long and highly structured 5′UTR should be more
sensitive to silvestrol relative to mRNAs with short and
unstructured 5′UTRs. To evaluate the potency of our new
silvestrol analogues as inhibitors of protein translation
initiation, two reporter assays were developed in human
MDA-MB-231 breast cancer cells (Figure 3). These assays
take advantage of the differential translation of luciferase
reporters flanked by the highly structured 5′UTR of c-myc
(myc-LUC) versus the short 5′UTR of tubulin (tub-LUC). The
inhibitory effect on luciferase reporter protein translation was
measured after a 24 h treatment with compounds, whereas
proliferation of MDA-MB-231 cells was measured with the
MTS assay after 72 h. Generally, synthetic analogues of
silvestrol and rocaglates were shown to be selective inhibitors of
complex mRNA protein translation with tub-LUC/myc-LUC
EC50 ratios ranging from 5 to 50. In contrast and as expected, a
ratio of 1 was consistently observed with the translational
elongation inhibitor cycloheximide (Figure 3) or the
polypeptide chain initiation inhibitor verrucarin A (data not
shown). Data for silvestrol analogues and rocaglate analogues
are summarized in Tables 1 and 2, respectively. A good
correlation was observed between translation inhibition and
growth inhibition, together suggesting that the observed effect
on cells is due to inhibition of protein translation initiation.
Interestingly, flavagline 4 retains potency despite the absence of
the dioxanyl moiety, albeit approximately 10-fold weaker than
silvestrol.
a
Scheme 6
A convergent synthetic approach allowed us to systematically
probe the importance of functional groups and stereochemistry
of the dioxanyl moiety on the potency of silvestrol analogues.
Naturally occurring 2‴-episilvestrol was reported to be
significantly less cytotoxic than silvestrol (>3000-fold) on the
HT-29 human colon cancer cell line.31 Likewise, synthetic
2‴,5‴-diepisilvestrol was also shown to be considerably less
active than the corresponding 5‴-episilvestrol.30 Consequently,
it was anticipated that altering this portion of the molecule
would have a dramatic effect on activity. However, deletion of
the 2‴-MeO group does not have merely the same influence as
the inversion of configuration, since 34 was only ∼3-fold less
potent than 1. Presumably, changing the 2‴-MeO group from
an axial to equatorial configuration may either influence the
overall conformational equilibrium of the dioxanyl fragment
(Figure S1, Supporting Information)43 or bring significant bulk
near the flavagline portion, thereby disrupting binding to
eIF4A. Deletion of the 5‴-hydroxy group (35) or 5‴-
hydroxymethyl group (39) or deletion of the 2‴-MeO group
(43) has little influence on activity. The latter observation
correlates well with the reported data on 5‴-episilvestrol, which
is essentially equipotent to silvestrol. However, inversion
configuration at the 1‴ and/or 2‴ position on these latter
compounds resulted in significant loss of activity (36−38 and
40−42), which is also consistent with the previous report on
a
Reagents and conditions: (a) Pd(OAc)2, Cs2CO3, MeB(OH)2; (b)
H2, Pd/C; (c) TMSCHN2, MeOH, toluene; (d) Pd2(dba)3, dppf,
Zn(CN)2.
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Figure 3. Differential translation reporter assay in intact cells. MBA-MB-231 cell lines were stably transfected by luciferase reporters flanked by either
the highly structured 5′UTR of c-myc (myc-LUC) or the short 5′UTR of tubulin (tub-LUC). Silvestrol potently inhibits the translation of luciferase
in myc-LUC cells relative to tub-LUC cells. In contrast, cycloheximide does not differentiate between the two assay mRNA constructs.
Table 1. Translation Initiation Inhibitory and Antiproliferative Effects of Silvestrol Analogues
myc-LUC
EC50, nM (N)
tub-LUC
EC50, nM (N)
tub-LUC/myc-LUC
ratio
growth inh
EC50, nM (N)
compd
R1
R2
X
1‴
1
HOCH2(R-CHOH)(R-CH)-
(R)-OMe
O
S
0.8 0.5 (19)
10 7 (11)
7
7 (19)
∼9
∼20
∼5
∼5
∼18
∼8
3
2 (19)
20 10 (11)
5 (4)
4
200 100 (11)
14 5 (3)
34
35
36
37
38
39
40
41
42
43
44
45
46
HOCH2(R-CHOH)(R-CH)-
HOCH2CH2(S-CH)-
HOCH2CH2(S-CH)-
HOCH2CH2(S-CH)-
HOCH2CH2(S-CH)-
HOCH2(S-CH)-
H
O
O
S
S
R
S
R
S
R
S
R
S
S
S
S
3
1
2 (3)
9
(R)-OMe
(R)-OMe
(S)-OMe
(S)-OMe
(R)-OMe
(R)-OMe
(S)-OMe
(S)-OMe
H
0.5 (3)
5
5 (3)
1.5 0.5 (2)
>500 (2)
O
>200 (2)
35 (1)
>3500 (2)
O
275 (1)
70 (1)
O
120 30 (2)
>2000 (2)
nd
300 200 (2)
15 1 (2)
60 40 (2)
>100 (2)
O
3
2 (2)
60 10 (2)
240 120 (2)
>2500 (2)
∼20
∼7
nd
HOCH2(S-CH)-
O
35 25 (2)
170 150 (2)
>2000
HOCH2(S-CH)-
O
HOCH2(S-CH)-
O
>5000
nd
>5000
HOCH2 (S-CH)-
HOCH2 (S-CH)-
HOCH2(S-CH)-
O
3
2 (4)
40 20 (3)
2000 1000 (4)
3500 1000 (3)
>5000
∼13
∼40
∼43
nd
9
5 (4)
H
CH2
NH
NMe
50 30 (4)
80 40 (3)
>5000
200 100 (4)
350 100 (4)
>5000
H
HOCH2(S-CH)-
H
2‴-episilvestrol. With compound 43 being a considerably
simpler and potent starting point, we next probed the
importance of the dioxanyl ring system. Replacement of
dioxanyl by tetrahydropyranyl (44) or morpholine (45)
reduced the potency by 15-fold and 25-fold, respectively,
while the activity was abolished by N-methylation of the
morpholine (46).
Our initial SAR study suggested that the structure of
silvestrol can be simplified dramatically while maintaining very
respectable potencies as protein translation inhibitors and
antiproliferative agents. Indeed, an approximately 3-fold
potency difference exists between flavagline 4 and dioxanyl-
containing 5. These data, together with the synthetic
complexity and the pharmacological liabilities associated to
the dioxanyl moiety, prompted us to expand our SAR at the
C6-position with simpler functional groups. Data are
summarized in Table 2. Generally, synthetic rocaglates were
shown to be less potent than silvestrol with a few notable
exceptions bearing electron-withdrawing group at the 6-
position (56, COOMe; 60, C(O)Me; 61, CN; 62, Cl).
These encouraging data prompted us to explore the 4′-position,
rationalizing that replacement of the methoxy with a different
functional group could enhance potency and metabolic
́
stability. In this regard, disclosure of compound 2 by Desaubry
and co-workers36,44 reinforced our hypothesis. Once again, the
addition of the electron-withdrawing nitrile group (69) resulted
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Table 2. Translation Initiation Inhibitory and Antiproliferative Effects of Rocaglate Analogues
myc-LUC
EC50, nM (N)
tub-LUC
EC50, nM (N)
tub-LUC/myc-LUC
ratio
growth inh
EC50, nM (N)
compd
R1
R2
1
HOCH2(R-CHOH)(R-CH)dioxanyloxy-
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
Me
0.8 0.5 (19)
10 7 (11)
10 3 (6)
>2000 (3)
>5000
7
7 (19)
200 100 (11)
90 40 (6)
>5000 (3)
>5000
∼9
∼20
∼9
3
2 (19)
4
OH
20 10 (11)
16 9 (6)
90 (1)
47
49
50
51
52
53
54
55
56
57
58
59
60
61
62
66
69
OMe
H
nd
Me
nd
>5000
NH2
40 20 (5)
11 9 (4)
>2000 (3)
>5000
>2000
nd
50 30 (5)
120 60 (4)
>4000 (1)
>5000
NHMe
NMe2
NHSO2Me
COOH
COOMe
CONH2
CONHMe
CONMe2
C(O)Me
CN
700 300 (4)
>5000 (3)
>5000
∼63
nd
nd
>1000
>5000
nd
>5000
0.2 0.1 (3)
29 2 (2)
35 5 (2)
>5000
4
2 (3)
∼20
∼16
nd
0.3 0.2 (3)
80 10 (2)
100 50 (2)
>5000
450 100 (2)
>2000
>5000
nd
1
0.5 (2)
30 20 (2)
15 10 (7)
70 50 (4)
>100
∼30
∼17
∼18
>20
∼9
1
0.5 (2)
1.8 0.9 (7)
2 (3)
32 5 (2)
1 (2)
0.9 0.7 (7)
Cl
4
5
2
2 (4)
2 (2)
1 (2)
5
OMe
OMe
CN
17 10 (2)
3
a
Scheme 7. Synthesis of Dimethylamide Analogues
a
Reagents and conditions: (a) LiOH; (b) EDCI, HOBt, HNMe2; (c) H2, Pd(OH)2, THF.
in a notable increase in potency, whereas the 4′-methyl
analogue 66 showed similar potency as the 4′-OMe (47).
Among the rocaglate analogues, 6-cyano 61, 6-chloro 62, and
4′-cyano 69 analogues were chosen for further evaluation
alongside the simplified dioxanyl-containing silvestrol analogue
43, since they had potencies very similar to that of silvestrol.
Optimization of Simplified Silvestrol Analogues. In
addition to its structural complexity, druglike properties of
silvestrol are suboptimal. Silvestrol has poor metabolic stability,
resulting in high plasma clearance in mice.45 Interestingly, the
hydrolysis product silvestric acid was found in equal amount as
the parent in plasma, 1 h after intravenous administration.
Moreover, silvestrol is a Pgp-substrate and expression of these
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Table 3. In Vitro Potency, Metabolic Stability, Permeability, and PK Properties of Optimized Silvestrol Analogues
plasma stability
microsome stability
Caco-2
A−B
myc-LUC
compd EC50, nM (N)
tub-LUC
EC50, nM (N)
growth inh
EC50, nM (N)
efflux
ratio
t1/2
(h)
AUClast
(ng·h/mL)
a
a
a
a
d
M (min) H (min) M (min) H (min) permeability
b
1
0.8 0.5 (19)
7
7 (19)
3
3
9
5
2 (19)
1 (6)
5 (4)
160
>240
48
>240
>240
>240
>240
>240
80
97
0.05
>100
>100
62
0.7
1.3
87
e
b
71
43
73
61
74
62
75
69
76
3
3
3
1 (6)
2 (4)
1 (2)
19 7 (6)
40 20 (3)
23 3 (2)
15 10 (7)
35 15 (2)
70 50 (4)
15 10 (2)
17 10 (2)
40 10 (2)
>180
>180
>180
75
160
>180
>180
32
ND
255
0.26
e
>240
92
ND
2.8
3.5
1.8
15.5
5.2
2.9
>100
2.3
0.9 0.7 (7)
0.8 0.2 (2)
1.8 0.9 (7)
2
7.3
2
184
108
4
2 (4)
0.6 0.4 (2)
1 (2)
1.2 0.2 (2)
5
2 (3)
1.4
0.4
3
2.0
0.7
2
1 (2)
1.7
c
7
5.6
1.2
174
a
b
c
d
e
M: mouse. H: human. Dose is 1 mg/kg, iv. Dose is 1 mg/kg, ip. Permeability × 10−6 cm/s ND: not detected, very low permeability.
transporters confers resistance to this agent.46 Consequently,
we aimed at improving druglike properties and drug delivery to
the target by reducing potential metabolic sites and reducing its
interaction with P-glycoprotein.
The synthesis of dimethylamide derivatives of the most
promising analogues (1, 43, 61, 62, and 69) was performed to
modulate hydrolysis in vivo (Scheme 7). Generally, this
transformation resulted in a marginal decrease in potency and
an improvement in metabolic stability (Table 3, 1 vs 71; 43 vs
73). While rocaglate analogues have similar PK characteristics
relative to silvestrol analogues, removal of the dioxanyl moiety
dramatically reduces their interaction with Pgp. The cellular
efflux ratio as measured by the bidirectional transport from
basolateral to apical (B/A) divided by apical to basolateral (A/
B) in Caco-2 cell monolayers was consistently higher with
silvestrol analogues relative to the rocaglate analogues (Table
3). An efflux ratio greater than 2 indicates the compound is a
Pgp substrate. In addition, the A to B permeability was very low
with silvestrol analogues (1 and 43) and low to moderate with
rocaglate analogues (e.g., 75). Finally, concentration−time PK
profiles in mice were similar for all these compounds after
intravenous administration, with newer compounds having
slightly better plasma exposure than silvestrol.
Pharmacology and Tolerability. Having potent silvestrol
analogues with acceptable pharmacokinetic profile, we next
evaluated whether these compounds would demonstrate
antiproliferative activity in vivo. After screening of a large
panel of cell lines, the human pancreatic cancer cell line L3.6pl
and human myeloma cell line RPMI-8226 were found to be
very sensitive to silvestrol (Figure S2, Supporting Information).
Notably, loss of viability was observed by 7-AAD exclusion in
MDA-MB-231, L3.6pl, and RPMI-8226 cells tested with
silvestrol and 76. Similar to MDA-MB-231,17 treatment of
these cell lines with silvestrol in culture rapidly reduces the
protein levels of c-myc, while the levels of the housekeeping
protein β-actin remain unchanged (Figure 4). This is consistent
with our reporter assay and could provide a pharmacodynamic
measurement of target modulation in vivo. For these reasons,
L3.6pl and RPMI-8226 subcutaneous xenograft models were
selected for the evaluation of silvestrol and various analogues in
vivo .
Figure 4. Inhibition of c-myc protein levels in pancreatic L3.6pl cells
(A) and myeloma RMPI-8226 cells (B) in culture after treatment with
silvestrol (100 nM).
days. Likewise, a larger bolus of 2.5 mg/kg (ip) could be
administered on a twice-a-week (b.i.w.) regimen for the same
period of time. Conversely, tolerability to silvestrol treatment in
NOD/SCID mice (used for RPMI-8226 xenografts) was lower
and the maximum tolerated dose was determined at 0.2 mg/kg
q.d. × 5 days (ip) in this strain.
Although silvestrol is very potent (cytotoxicity LC50 ≈ 4 nM;
CellTiter-Glo) in L3.6pl cells in culture, it failed to demonstrate
any tumor growth delay in L3.6 tumor-bearing mice when
administered at the maximum tolerated dose. No difference was
observed between the vehicle and the treatment arms regardless
of dosing regimens (1 mg/kg q.d. × 5 days × 2 weeks or 2.5
mg/kg b.i.w. × 2 weeks). In a PK−PD experiment performed in
L3.6 tumor-bearing mice treated with silvestrol (1 mg/kg, ip),
total drug concentrations were measured at 100 and 15 ng of
silvestrol per gram of tumor tissues after 6 and 24 h postdose,
but no modulation of c-myc level was observed in the tumor at
these time-points. Furthermore, despite the fact that silvestrol
and analogues 74−76 were extremely cytotoxic (LC50 ≈ 0.06
nM; CellTiter-Glo) to RMPI-8226 cells in culture, no effect
was observed on the growth of RMPI-8226 xenografts in
NOD/SCID mice after daily intraperitoneal administration of
silvestrol or rocaglate compound 76 when dosed at their
maximum tolerated doses (0.2 mg/kg) for 3 weeks (q.d. × 5
In order to inform dose selection for the efficacy studies,
tolerability to silvestrol treatment was assessed in L3.6pl-
bearing BALB/c nude mice. In this strain, daily intraperitoneal
(ip) dosing (q.d.) of 1 mg/kg silvestrol (formulated in 5.2%
PEG-400, 5.2% Tween 80, 2% DMSO) was tolerated over 5
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days × 3 weeks). Dose-limiting toxicities led to insufficient
target coverage, as little to no modulation of the c-myc protein
levels were observed in the RPMI-8226 tumors (not shown). In
an attempt to understand whether this narrow therapeutic
window was compound- or target-related, C1‴-episilvestrol was
used as a probe. This analogue, isolated as a side product of
silvestrol synthesis, was inactive in the translation reporter
assays and had similar PK profile as silvestrol. Unlike silvestrol,
C1‴-episilvestrol was well tolerated in tumor-bearing mice (5
mg/kg ip, q.d. × 5) with no change in body weight. This result
suggests that the toxicity of silvestrol is related to the inhibition
of translation in vivo, albeit different off-target or metabolite
profiles for these compounds cannot be totally ruled out.
Nevertheless, our studies show that the therapeutic window of
silvestrol and analogues that inhibit translation is too narrow to
warrant further optimization without a better understanding of
silvestrol pharmacology.
of silvestrol in many cancer cell lines in culture seemed to
indicate that this would indeed be the case. The lack of
correlation between silvestrol sensitivity in vitro and in vivo is
confounding. For instance, silvestrol was shown to significantly
reduce the growth of prostate cancer PC-3 and breast cancer
MDA-MB-231 cell lines grown as xenografts.17,48 However, in
culture, silvestrol had modest activity in MDA-MB-231 relative
to L3.6pl and RPMI-8226 cells, which were chosen for in vivo
evaluation in this study. It was expected that doses of the drugs
required to affect growth of L3.6pl and RPMI-8226 tumors
would be well tolerated. However, the growth of L3.6pl and
RPMI-8226 tumors in vivo was unaffected by silvestrol
treatment at the MTD in contrast to the MDA-MB-231
xenografts reported in the literature and reproduced in our
laboratories.48 It has been reported that silvestrol is ineffective
as a single agent in PTEN Eμ-myc and Eμ-myc/eIF4E
lymphoma models but acts as sensitizer to doxorubicin.16 It is
plausible that the degree of addiction to altered mRNA
translation rates dictates the sensitivity of these tumor cells to
silvestrol, and dose-limiting toxicities are often observed before
these mechanisms can be affected to produce a therapeutic
benefit.
Our results bring the drug potential of eIF4A inhibitors into
question because of the narrow therapeutic window observed
with silvestrol and compound 76. In recent literature, a protein
toxin from Burkholderia pseudomallei was found to inhibit the
helicase activity of eIF4A and was lethal to BALB/c mice.49 By
contrast, some results with other alternative eIF4A inhibitors
appear more encouraging. Hippuristanol, a polyoxygenated
steroid with a similar mode of action as that of silvestrol, is
highly effective and well-tolerated at 7.5 mg/kg q.d. × 28 days
in a model of T-cell leukemia.50
CONCLUSION
■
We postulated that translation initiation factors could be good
targets for cancer therapy, since the protein translation of many
oncogenes is dependent on these factors in cells. In vitro
studies with compounds such as the natural product silvestrol
indicate that it may be feasible to inhibit such factors. However,
the druglike properties of silvestrol are suboptimal, limiting the
ability to evaluate its therapeutic potential. In addition, sources
of silvestrol are not readily available, which further complicates
comprehensive preclinical studies. In this study, efficient and
versatile synthetic routes were devised to access simplified,
potent, and selective protein translation inhibitors based on the
silvestrol scaffold. As an example, compound 76 displays very
similar in vitro potency and PK characteristics compared to
silvestrol. However, simplified analogues exemplified by
compound 76 also show a similar toxicity profile despite the
absence of the dioxanyl moiety of the molecule. Arguments for
on-target based toxicity can be offered to explain the narrow
therapeutic window that limits the single agent activity of
silvestrol analogues. For all analogues tested in vitro, tub-LUC/
myc-LUC IC50 ratios range from 5 to 50. It is noteworthy that
c-myc and tubulin 5′UTR structures represent extreme cases
with melting energy (ΔG) of approximately −164 and −5.5
kcal/mol, respectively.47 Consequently, the translation of other
proteins essential for cell viability could be affected at
concentrations and amplitudes similar to those of highly
structured 5′UTR-containing oncoproteins. Moreover, the lack
of toxicity observed with the inactive C1‴-episilvestrol provides
additional evidence that the toxicity could be related to the
inhibition of translation in vivo. We considered whether target
modulation by silvestrol within the tumor was insufficient to
provide a therapeutic benefit. While silvestrol produces a toxic
effect in normal cells, we speculated that low permeability and/
or Pgp mediated efflux46 in tumor cells may result in
intracellular drug concentrations too low to achieve target
modulation and reduction of tumor growth. However,
compound 76 did not perform better than silvestrol despite
improved permeability and efflux ratio relative to silvestrol.
Therefore, it seems unlikely that reduced permeability was a
significant contributor to the lack of efficacy in the two models
described.
Although more work is required to understand the optimal
settings for the use of these translation inhibitors as single
agents, or in combination with other agents, our results
underscore the possibility of using these simplified silvestrol
analogues to modulate the level of oncoproteins otherwise
difficult to target with conventional therapies. Therefore, these
compounds represent a unique class of agents with anticancer
therapeutic potential worthy of further investigation.
EXPERIMENTAL SECTION
■
Chemical Synthesis. General Methods. Silvestrol was prepared
from 4 using procedure described by Porco et al.27 All physical data
(Supporting Information) match an authentic sample (Prof. Mark A.
Rizzacasa). Commercial reagents and solvents were used as received
without further purification or drying. All experiments involving water-
sensitive compounds were carried out under argon and scrupulously
dry conditions, using commercially available anhydrous solvents. Thin-
layer chromatography was performed on glass-backed precoated
Merck silica gel (60 F254) plates, and compounds were visualized
using UV light, ceric ammonium molybdate, or 2% aqueous potassium
permanganate solution. Silica gel column chromatography was carried
out using Merck silica gel 60. Flash chromatography was run using
silica gel (200−400 mesh) from Sorbent Technologies. The purity of
tested compounds was determined by analytical liquid chromatog-
raphy performed by methods A, B, or C. Method A consisted of the
following: Agilent 1100 HPLC system equipped with an XBridge C18
column (2.5 μm, 4.6 mm × 50 mm). The mobile phases were (A) 10
mM ammonium formate buffer, pH 3.8, and (B) 0.05% formic acid in
CH3CN. After injection the gradient holds were at A/B (25%/75%)
for 2 min followed by a gradient to A/B (5%/95%) over 16 min, a 2
min organic flush at 5%/95% (A/B), and a 2 min re-equilibration at a
flow rate of 1.0 mL/min, column temperature at 40 °C, and detection
wavelength at 215 nm. Method B consisted of the following: Waters
It was originally thought that approaches to target eIF4A
would result in broad-acting therapeutics due to the
convergence of many oncogenic pathways that result in
enhanced mRNA translation. The broad and potent activity
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Alliance 2795XE LC with a Waters 2996 photodiode array detector
equipped with a Waters Symmetry C18 column (3.5 μm, 2.1 mm × 50
mm). The mobile phases were (A) 0.1% formic acid in water and (B)
0.1% formic acid in acetonitrile. After injection the gradient holds were
at A/B (75%/15%) for 0.5 min followed by a gradient to A/B (0%/
100%) over 5 min, a 1 min flush at 0%/100% (A/B), and a 2 min re-
equilibration at A/B (75%/15%) at a flow rate of 1.5 mL/min and
column temperature at 40 °C. Method C consisted of the following:
Waters Alliance 2795XE LC with a Waters 2996 photodiode array
detector equipped with an Waters Symmetry C18 column (3.5 μm, 2.1
mm × 30 mm). The mobile phases were (A) 0.1% formic acid in water
and (B) 0.1% formic acid in acetonitrile. After injection the gradient
change was from A/B (95%/5%) to A/B (0%/100%) over 3 min, with
a 0.5 min flush at 0%/100% (A/B) and a 0.5 min re-equilibration at A/
B (95%/5%) at a flow rate of 1.5 mL/min and column temperature at
40 °C. 1H NMR spectra were recorded on a Bruker 400 spectrometer
(400 MHz). Chemical shifts are reported in ppm with the solvent
resonance as the internal standard (CHCl3, δ 7.26). Data are reported
as follows: chemical shift, integration, multiplicity (s = singlet, d =
doublet, dd = doublet of doublet, ddd = doublet of doublet of doublet,
t = triplet, q = quartet, br = broad, m = multiplet), and coupling
constants (Hz). 13C NMR spectra were recorded on a Bruker 400
spectrometer (100 MHz) with complete proton decoupling. Chemical
shifts are reported in ppm with the solvent as the internal reference
(CDCl3, δ 77.16). Electrospray positive ionization mass spectrometry
(ESI-MS) was performed on samples using a reversed phase HPLC−
MS system. The system was equipped with a diode array UV detector
and a single quadrupole mass spectrometer.
Preparation of Compounds in Scheme 1. 7-(Benzyloxy)-5-
methoxy-2-phenyl-4H-chromen-4-one (6). A suspension of chrysin 5
(255 g, 1.0 mol, 1 equiv) in acetone (3 L) was treated with potassium
carbonate (415 g, 3.0 mol, 3 equiv) and benzyl bromide (129 mL, 1.0
mol, 1 equiv). The mixture was heated to 60 °C and stirred for 3 h.
The mixture was cooled to 40 °C, treated with dimethyl sulfate (287
mL, 3.0 mol, 3 equiv), and stirred for 3 days. The mixture was cooled
to room temperature and the solid removed by filtration on a Buchner
funnel. The cake was partitioned in dichloromethane/water and
combined with the filtrate that was previously concentrated to dryness.
The pH of the aqueous phase was adjusted to 2 with a 2 N aqueous
hydrochloric acid solution, and the aqueous phase was extracted with
dichloromethane. The solvent was removed under reduced pressure
and coevaporated with ethyl acetate. The residue was suspended in
ethyl acetate and heated to reflux for 1 h. The mixture was cooled to
room temperature, filtered on a Buchner funnel and the solid washed
with ethyl acetate. The solid was air-dried to constant weight to give
the desired protected chrysin 6 (303 g, 0.85 mol, 84%). NMR δH (400
MHz, CDCl3) 7.9−7.8 (m, 2H), 7.5−7.35 (m, 8H), 6.69 (s, 1H), 6.66
(d, J = 2.4 Hz, 1H), 6.47 (d, J = 2.4 Hz, 1H), 5.16 (s, 2H), 3.96 (s,
3H); NMR δC (100 MHz, CDCl3) 177.59, 163.57, 161.36, 160.96,
159.92, 135.63, 131.51, 131.21, 129.01, 128.83, 128.53, 127.67, 126.12,
108.94, 108.43, 97.01, 93.85, 70.65, 56.49; ESI-MS m/z = 359.3 [M +
H]+.
167.57, 165.21, 162.99, 135.97, 128.75, 128.37, 127.68, 106.15, 94.49,
91.27, 70.23, 55.58, 32.97; ESI-MS m/z = 273.3 [M + H]+.
1-(4-(Benzyloxy)-2-hydroxy-6-methoxyphenyl)-2-hydroxyetha-
none (8). A solution of acetophenone 7 (53.5 g, 197 mmol, 1 equiv) in
dichloromethane (500 mL) was cooled to 0 °C, treated with
triethylamine (69 mL, 492 mmol, 2.5 equiv) and tert-butyldimethylsilyl
trifluoromethanesulfonate (104 mL, 452 mmol, 2.3 equiv). The
mixture was stirred at 0 °C for 1 h. The solution was charged with
saturated aqueous sodium bicarbonate solution and warmed to room
temperature. The organic phase was separated, dried with sodium
sulfate and the solvent removed under reduced pressure. The residue
was coevaporated with dichloromethane and used directly for the next
step. The residue, dissolved in dichloromethane (200 mL), was added
to a cooled (0 °C) mixture of anhydrous m-chloroperbenzoic acid (76
g, 310 mmol, 1.6 equiv) and sodium bicarbonate (40.7 g, 484 mmol,
2.5 equiv) in dichloromethane (800 mL). The mixture was warmed to
room temperature and vigorously stirred for 4 h. The mixture was
diluted with dichloromethane and washed successively with a saturated
aqueous sodium bicarbonate solution and water. The solvent was
removed under reduced pressure and the residue used directly for the
next step. A suspension of crude product in tetrahydrofuran (1 L) and
water (0.1 L) was treated with p-toluenesulfonic acid (3.7 g, 19 mmol,
0.1 equiv). The solution was heated to reflux and stirred for 6 h. The
mixture was cooled to room temperature and partitioned between
ethyl acetate and a saturated aqueous sodium bicarbonate solution.
The organic phase was dried with sodium sulfate and the solvent
removed under reduced pressure. The residue was suspended in
ethanol and heated at reflux for 1 h. The solution was cooled to room
temperature. The precipitated solid was filtered on a Buchner funnel
and the solid washed with cold ethanol. The solid was air-dried to
constant weight to give the desired hydroxyacetophenone 8 (36 g, 126
mmol, 65% over three steps). NMR δH (400 MHz, CDCl3) 13.19 (s,
1H), 7.4−7.3 (m, 5H), 6.18 (d, J = 2.4 Hz, 1H), 6.00 (d, J = 2.4 Hz,
1H), 5.07 (s, 2H), 4.70 (s, 2H), 3.84 (s, 3H); NMR δC (100 MHz,
CDCl3) 167.19, 166.15, 163.21, 135.73, 128.78, 128.46, 127.68,
103.49, 94.73, 91.46, 70.41, 68.65, 55.71; ESI-MS m/z = 289.16 [M +
H]+.
7-(Benzyloxy)-5-methoxy-2-(4-methoxyphenyl)-4-oxo-4H-chro-
men-3-yl 4-methoxybenzoate (9). A solution of hydroxyacetophe-
none 8 (36.2 g, 126 mmol, 1 equiv) in dichloromethane (350 mL) was
treated with dimethylaminopyridine (0.767 g, 6.3 mmol, 0.05 equiv)
and triethylamine (52.5 mL, 377 mmol, 3 equiv). The mixture was
cooled to 0 °C, treated with 4-methoxybenzoyl chloride (43.3 g, 254
mmol, 2 equiv), warmed to room temperature, and stirred for 3 h. The
solution was quenched with 1 N aqueous hydrochloric acid, and the
phases were separated. The aqueous phase was back-extracted with
dichloromethane, and the combined organic phases were dried with
sodium sulfate and concentrated under reduced pressure. The desired
bisbenzoate residue was used directly for the next step. A solution of
the crude bisbenzoate in tetrahydrofuran (700 mL) was cooled to −70
°C and was treated with a 1 M solution of lithium hexamethyldisi-
lazane in tetrahydrofuran (377 mL, 377 mmol, 3 equiv). The mixture
was stirred below −60 °C for 1 h and then warmed to −20 °C for 1 h.
The solution was quenched with a saturated aqueous ammonium
chloride solution and warmed to room temperature. The aqueous
phase was extracted with ethyl acetate, and the combined organic
phases were dried with sodium sulfate and concentrated under
reduced pressure. The desired phenol residue was used directly for the
next step. A suspension of crude phenol in acetic acid (1.5 L) was
treated with sulfuric acid (33.5 mL, 628 mmol, 5 equiv) and stirred at
room temperature for 20 h. The reaction mixture was poured over
water/ice and stirred for 15 min. The mixture was filtered on a
Buchner funnel and the cake washed with water. The wet cake was
suspended in ethanol and heated to reflux for 1 h. The mixture was
cooled to room temperature, filtered on a Buchner funnel, and washed
with cold ethanol. The solid was air-dried to constant weight to give
the desired protected 3-hydroxyflavone 9 (62.3 g, 116 mmol, 92% over
three steps). NMR δH (400 MHz, CDCl3) 8.17 (d, J = 9.2 Hz, 2H),
7.86 (d, J = 9.2 Hz, 2H), 7.41−7.32 (m, 5H), 6.96 (d, J = 9.2 Hz, 2H),
6.94 (d, J = 9.2 Hz, 2H), 6.63 (d, J = 2.4 Hz, 1H), 6.45 (d, J = 2.4 Hz,
1-(4-(Benzyloxy)-2-hydroxy-6-methoxyphenyl)ethanone (7). Pro-
tected chrysin 6 (150 g, 419 mmol, 1 equiv) was added to a mixture of
50% aqueous sodium hydroxide solution (1172 g, 14.6 mol, 35 equiv)
and pyridine (677 mL, 8.3 mol, 20 equiv). The mixture was vigorously
stirred and treated with diethylene glycol (800 mL, 8.3 mol, 20 equiv).
The mixture was heated to 100 °C and stirred for 2 h. The mixture was
cooled to <20 °C, and the pH was adjusted to 1 with a 12 N aqueous
hydrochloric acid solution. The aqueous portion was extracted with
ethyl acetate. The combined organic phase was washed with saturated
aqueous sodium bicarbonate, dried with sodium sulfate, and the
solvent was removed under reduced pressure. The residue was
suspended in methanol and heated to reflux for 1 h. The solution was
cooled to room temperature, filtered on a Buchner funnel, and the
crystals were washed with cold methanol. The solid was air-dried to
constant weight to give the desired acetophenone 7 (79 g, 290 mol,
70%). NMR δH (400 MHz, CDCl3) 13.99 (s, 1H), 7.41−7.32 (m,
5H), 6.13 (d, J = 2.4 Hz, 1H), 5.99 (d, J = 2.4 Hz, 1H), 5.05 (s, 2H),
3.83 (s, 3H), 2.60 (s, 3H); NMR δC (100 MHz, CDCl3) 203.19,
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1H), 5.16 (s, 2H), 3.91 (s, 3H), 3.89 (s, 3H), 3.83 (s, 3H); NMR δC
(100 MHz, CDCl3) 170.72, 163.96, 163.79, 163.31, 161.53, 161.18,
159.06, 153.25, 135.79, 133.88, 132.72, 129.67, 128.76, 128.41, 127.68,
122.28, 121.45, 114.07, 113.83, 108.95, 96.53, 93.59, 70.51, 56.27,
55.52, 55.37; ESI-MS m/z = 539.42 [M + H]+.
Hz, 2H), 4.32 (d, J = 15 Hz, 1H), 3.90 (dd, J = 15, 7 Hz, 2H), 3.83 (s,
3H), 3.70 (br s, 1H) 3.66 (s, 3H), 3.65 (s, 3H); NMR δC (100 MHz,
CDCl3) 170.63, 163.25, 160.88, 158.76, 157.13, 137.08, 136.60,
129.05, 128.79, 128.27, 127.93, 127.80, 127.67, 126.64, 126.57, 112.79,
108.10, 101.94, 93.77, 93.44, 90.58, 79.66, 70.57, 55.84, 55.14, 55.08,
52.05, 50.57; ESI-MS m/z = 569.22 [M + H]+.
7-(Benzyloxy)-3-hydroxy-5-methoxy-2-(4-methoxyphenyl)-4H-
chromen-4-one (10). A suspension of benzoate 9 (31 g, 57.6 mmol, 1
equiv) in ethanol (715 mL) was treated with a 5% aqueous sodium
hydroxide solution (86 g, 108 mmol, 1.875 equiv). The suspension
was heated to 80 °C and stirred for 1 h. The reaction mixture was
cooled to room temperature and treated with a 1 N aqueous
hydrochloric acid solution (108 mL, 108 mmol, 1.875 equiv). The
resulting suspension was filtered on a Buchner funnel and washed with
ethanol. The solid was air-dried to constant weight to give the desired
3-hydroxyflavone 10 (22 g, 54.4 mmol, 95%). NMR δH (400 MHz,
CDCl3) 8.16 (d, J = 9.2 Hz, 2H), 7.5−7.35 (m, 5H), 7.02 (d, J = 9.2
Hz, 2H), 6.64 (d, J = 2.4 Hz, 1H), 6.44 (d, J = 2.4 Hz, 1H), 5.15 (s,
2H), 3.97 (s, 3H), 3.88 (s, 3H); NMR δC (100 MHz, CDCl3) 171.92,
163.41, 160.70, 160.59, 158.79, 142.32, 137.52, 135.75, 128.95, 128.88,
128.58, 127.78, 123.61, 114.06, 106.40, 96.21, 93.38, 70.64, 56.47,
55.45; ESI-MS m/z = 405.31 [M + H]+.
(1R,2R,3S,3aR,8bS)-Methyl 1,6,8b-Trihydroxy-8-methoxy-3a-(4-
methoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]-
cyclopenta[d]furan-2-carboxylate (4). A solution of benzyl ether rac-
11 (6.40 g, 11.26 mmol, 1 equiv) and 5% palladium on carbon (1.25 g,
20 wt %) in tetrahydrofuran (120 mL) was placed under hydrogen
atmosphere and stirred for 18 h at room temperature. The reaction
mixture was filtered on Celite and the filtrate concentrated to dryness
to give the desired phenol rac-13 (5.10 g, 10.66 mmol, 95%). This
material was purified by chiral supercritical fluid chromatography
(column, 3.0 cm × 25.0 cm ChiralPak AD-H (Chiral Technologies);
isocratic CO2/EtOH (35%) at 80 mL/min; 100 bar) to obtain
enantiomerically enriched (>95% ee) phenol 4. NMR δH (400 MHz,
CDCl3) 7.09−7.02 (m, 5H), 6.90−6.87 (m, 2H), 6.30 (d, J = 9 Hz,
2H), 6.17 (d, J = 1 Hz, 1H), 5.97 (s, 1H), 5.03 (d, J = 7 Hz, 1H), 4.30
(d, J = 14 Hz, 1H), 3.90 (dd, J = 14, 6 Hz, 1H), 3.76 (s, 3H), 3.65 (s,
3H), 3.64 (s, 3H), 3.20 (s, 1H); NMR δC (100 MHz, CDCl3) 171.28,
160.88, 160.66, 158.76, 157.14, 136.89, 129.07, 128.06, 127.91, 126.78,
126.62, 112.87, 107.20, 101.79, 93.78, 93.18, 92.33, 79.30, 55.84,
55.22, 52.31, 50.59, 27.07; ESI-MS m/z = 479.19 [M + H]+.
Preparation of Compounds in Scheme 4. General Procedure
for Coupling Lactols to Phenolic Intermediate of Rocaglate. A
solution of phenol (1 equiv), lactol (2−3 equiv), and triphenylphos-
phine (2−3 equiv) in dry toluene was stirred at 0 °C for 10 min.
Molecular sieves, 3 Å, were added, and then a solution of di-2-
methoxyethyl azodicarboxylate (DMEAD) (2−3 equiv) in dry toluene
is added. The mixture is stirred until acceptable conversion is
observed. The reaction mixture is then concentrated, and the residue is
purified by silica gel column chromatography.
(1R,2R,3S,3aR,8bS)-Methyl 6-((2S,6R)-6-((R)-1,2-Dihydroxyethyl)-
1,4-dioxan-2-yloxy)-1,8b-dihydroxy-8-methoxy-3a-(4-methoxy-
phenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]cyclopenta[d]-
furan-2-carboxylate (34). A mixture of phenol 4 (35.6 mg, 0.074
mmol, 1.0 equiv), 3 Å molecular sieves (101 mg), the lactol 16 (67
mg, 0.19 mmol, 2.6 equiv), and triphenylphosphine (50 mg, 0.19
mmol, 2.6 equiv) in 1.8 mL of toluene was stirred for 30 min at room
temperature, cooled to 0−5 °C, and treated dropwise with a solution
of di-2-methoxyethyl azodicarboxylate (DMEAD) (50 mg, 0.21 mmol,
2.8 equiv) in 0.16 mL of toluene over a period of 6 min. The mixture
was stirred for 19 h. The molecular sieves were removed by filtration
though a pad of Celite. The Celite pad was washed with EtOAc (2 ×
10 mL). The combined filtrates were washed with water (2 × 10 mL)
and dried (Na2SO4). The drying agent was filtered off, and the
solvents were removed in vacuo. The residue was purified by
chromatography on silica gel (4 g). The product containing fractions
were concentrated in vacuo. The residue was further purified on silica
gel by preparative TLC using 10% EtOAc in DCM to give 18 mg
(0.022 mol, 30%) of bis-benzyl ether as a white foam (ESI-MS m/z =
805.3 [M + H]+). A mixture of bis-benzyl ether (18.0 mg, 0.022 mmol,
1.0 equiv) and 5% palladium hydroxide on carbon (14.0 mg, 0.005
mmol, 0.26 equiv) in 3 mL of THF was evacuated and filled with
hydrogen four times and stirred under hydrogen for 40 min. The
hydrogen supply was disconnected, flushed with nitrogen, and the
catalyst was filtered off. The filtrate was concentrated in vacuo and the
residue was purified by preparative TLC on silica gel (40% acetone in
DCM) and lyophilized to give 7.8 mg (0.012 mmol, 65%) of 34. NMR
δH (400 MHz, CDCl3) 7.12−7.05 (m, 5H), 6.87−6.84 (m, 2H), 6.70−
6.67 (m, 2H), 6.46 (d, J = 1.6 Hz, 1H), 6.31 (d, J = 2.0 Hz, 1H), 5.46
(brs, 1H), 5.04 (dd, J = 6.8, 1.6 Hz, 1H), 5.04 (dd, J = 6.8, 1.6 Hz,
1H), 4.29 (d, J = 14.4 Hz, 1H), 4.25−4.21 (m, 1H), 4.00 (d, J = 14.4
Hz, 1H), 3.92 (m, 1H), 3.89 (brs, 1H), 3.88 (s, 3H), 3.85−3.79 (m,
2H), 3.74 (brs, 1H), 3.72 (s, 3H), 3.67−3.60 (m, 3H), 3.65 (s, 3H);
ESI-MS m/z = 625.2 [M + H]+. Purity (method A): 98% AUC.
(1R,2R,3S,3aR,8bS)-Methyl 1,8b-Dihydroxy-6-((2S,3R,6S)-6-(2-hy-
droxyethyl)-3-methoxy-1,4-dioxan-2-yloxy)-8-methoxy-3a-(4-me-
thoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]-
(1,2,3,3a,8b)-Methyl 6-(Benzyloxy)-1,8b-dihydroxy-8-methoxy-
3a-(4-methoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]-
cyclopenta[d]furan-2-carboxylate (rac-11). A solution of 3-hydroxy-
flavone 10 (15.9 g, 39.3 mmol, 1 equiv) and methyl cinnamate (73.3 g,
452 mmol, 11.5 equiv) in dichloromethane (800 mL), acetonitrile
(320 mL), and methanol (320 mL) was placed in a jacketed flask
(temperature controlled) equipped with a recirculation pump. The
reaction mixture was pumped (∼50−100 mL/min) through a coil
wrapped around a jacketed glass cylinder containing a UV lamp. The
jacket temperature was adjusted so that the internal reaction
temperature was 0−5 °C while the UV light was turned on. The
reaction mixture was irradiated for 10 h and then warmed to room
temperature. The solvent was removed under reduced pressure and
the residue purified using a plug of silica gel (EtOAc/hexanes) to
remove excess cinnamate (low polarity). The remaining material was
concentrated under reduced pressure and used directly for the next
step. The crude residue was suspended in methanol (300 mL) and
treated with a 25% methanolic sodium methoxide solution (22 g, 102
mmol, 2.6 equiv). The mixture was heated to 60 °C and stirred for 1 h.
The mixture was cooled to room temperature, and approximately half
of the methanol was removed under reduced pressure. The resulting
mixture was filtered on the a Buchner funnel. The solids were washed
with cold methanol and air-dried to constant weight. The cake was
then partitioned between ethyl acetate and a saturated aqueous
ammonium chloride solution. The organic phase was separated, dried
with sodium sulfate, and concentrated under reduced pressure to give
the desired keto ester in a 3/1 ratio along with the undesired
regioisomer (8.46 g, 15.76 mmol, 40%). Additional material can be
obtained by processing the mother liquor as follows: residue from
concentrated mother liquor (∼9.5 g) was partitioned between ethyl
acetate and a saturated aqueous ammonium chloride solution. The
organic phase was separated, dried with sodium sulfate, and
concentrated under reduced pressure. The residue was purified using
silica gel chromatography (EtOAc/hexanes). The product containing
fractions were pooled, concentrated, and crystallized from ethanol to
give the additional desired keto ester as a single regioisomer (1.5 g,
2.65 mmol, 7%). A regioisomeric mixture (3/1) of keto ester (8.93 g,
15.76 mmol, 1 equiv) was charged to a solution of tetramethylammo-
nium triacetoxyborohydride (24.9 g, 95 mmol, 6 equiv) in acetonitrile
(300 mL) and acetic acid (9 mL, 158 mmol, 10 equiv). The resulting
mixture was stirred for 48 h and then partitioned between a saturated
aqueous sodium bicarbonate solution and dichloromethane. The
organic layer was separated, dried over sodium sulfate, and
concentrated. The residue was purified using silica gel chromatography
(EtOAc/hexanes) to give the desired alcohol rac-11 (6.4 g, 11.3 mmol,
72%) and the undesired regioisomer rac-12 (2.1 g, 3.63 mmol, 23%).
Data for rac-11: NMR δH (400 MHz, CDCl3) 7.50−7.035 (m, 5H),
7.12−7.05 (m, 5H), 6.90−6.86 (m, 2H), 6.65 (d, J = 9 Hz, 2H), 6.37
(d, J = 2 Hz, 1H), 6.21 (d, J = 2 Hz, 1H), 5.08 (s, 2H), 5.03 (d, J = 7
8869
dx.doi.org/10.1021/jm3011542 | J. Med. Chem. 2012, 55, 8859−8878

Journal of Medicinal Chemistry
Article
cyclopenta[d]furan-2-carboxylate (35) and (1R,2R,3S,3aR,8bS)-
Methyl 1,8b-Dihydroxy-6-((2R,3R,6S)-6-(2-hydroxyethyl)-3-me-
thoxy-1,4-dioxan-2-yloxy)-8-methoxy-3a-(4-methoxyphenyl)-3-
phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]cyclopenta[d]furan-2-car-
boxylate (36). A solution of phenol 4 (15 mg, 0.031 mmol), lactol 20
(21 mg, 0.07 mmol), and triphenylphosphine (21 mg, 0.078 mmol) in
toluene (3 mL) was stirred at 0 °C for 10 min. Molecular sieves, 3 Å
(25 mg), were added, and then a solution of di-2-methoxyethyl
azodicarboxylate (21 mg, 0.088 mmol) in toluene (0.5 mL) was added
over a period of 5 min. The mixture was stirred for an additional 2 h.
The reaction mixture was then concentrated, and the residue was
purified by silica gel column chromatography (4 g) and eluted using
30−50% EtOAc−hexanes to provide the desired compound benzyl
ether (8 mg) and its corresponding 1‴ epimer (7 mg). Both epimers
were independently subjected to hydrogenolysis and HPLC
purification to give compounds 35 (4 mg) and 36 (4 mg). Compound
35: NMR δH (400 MHz, CDCl3) 7.10 (d, J = 8.8 Hz, 2H), 7.07−7.05
(m, 3H), 6.89−6.87 (m, 2H), 6.68 (d, J = 8.8 Hz, 2H), 6.47 (d, J = 1.6
Hz, 1H), 6.32 (d, J = 1.6 Hz, 1H), 5.30 (brs, 1H), 5.03 (d, J = 6.4 Hz,
1H), 4.61 (s, 1H), 4.40−4.35 (m, 1H), 4.32 (d, J = 14 Hz, 1H), 3.92
(dd, J = 6.4, 14 Hz, 1H), 3.87 (s, 3H), 3.75−3.70 (m, 2H), 3.71 (s,
3H), 3.65 (s, 3H), 3.56−3.54 (m, 1H), 3.50 (s, 3H), 1.90 (brs, 1H),
1.70−1.60 (m, 2H); ESI-MS m/z = 639.4 [M + H]+. Purity (method
B): >95% AUC. Compound 36: NMR δH (400 MHz, CDCl3) 7.10 (d,
J = 8.8 Hz, 2H), 7.07−7.05 (m, 3H), 6.88−6.85 (m, 2H), 6.68 (d, J =
8.8 Hz, 2H), 6.40 (d, J = 1.6 Hz, 1H), 6.30 (d, J = 1.6 Hz, 1H), 5.27
(d, J = 1.6 Hz, 1H), 5.03 (dd, J = 1.6, 6.8 Hz, 1H), 4.30 (d, J = 14.4
Hz, 1H), 4.250−4.18 (m, 1H), 3.90 (dd, J = 6.4, 14.4 Hz, 1H), 3.86 (s,
3H), 3.85−3.75 (m, 3H), 3.71 (s, 3H), 3.65 (s, 3H), 3.57 (s, 3H), 3.49
(dd, J = 2.8, 11.2 Hz, 1H), 1.90 (brs, 1H), 1.89−1.81 (m, 1H), 1.78−
1.69 (m, 2H); ESI-MS m/z = 639.4 [M + H]+. Purity (method B):
>95% AUC.
(1R,2R,3S,3aR,8bS)-Methyl 1,8b-Dihydroxy-6-((2S,3S,6S)-6-(2-hy-
droxyethyl)-3-methoxy-1,4-dioxan-2-yloxy)-8-methoxy-3a-(4-me-
thoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]-
cyclopenta[d]furan-2-carboxylate (37) and (1R,2R,3S,3aR,8bS)-
Methyl 1,8b-Dihydroxy-6-((2R,3S,6S)-6-(2-hydroxyethyl)-3-me-
thoxy-1,4-dioxan-2-yloxy)-8-methoxy-3a-(4-methoxyphenyl)-3-
phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]cyclopenta[d]furan-2-car-
boxylate (38). A solution of phenol 4 (11 mg, 0.023 mmol), lactol 21
(15 mg, 0.057 mmol), and triphenylphosphine (15 mg, 0.057 mmol)
in toluene (3 mL) was stirred at 0 °C for 10 min. Molecular sieves, 3 Å
(25 mg), were added, and then a solution of di-2-methoxyethyl
azodicarboxylate (15 mg, 0.064 mmol) in toluene (0.5 mL) was added
over a period of 5 min. The mixture was stirred for an additional 2 h.
The reaction mixture was then concentrated and the residue was
purified by silica gel column chromatography (4 g) and eluted using
30−50% EtOAc−hexanes to provide the desired compound benzyl
ether (7 mg) and its corresponding 1‴ epimer (6 mg). Both epimers
were independently subjected to hydrogenolysis and HPLC
purification to give compound 37 (4 mg) and compound 38 (5
mg). Compound 37: NMR δH (400 MHz, CDCl3) 7.10 (d, J = 9.2 Hz,
2H), 7.07−7.05 (m, 3H), 6.89−6.87 (m, 2H), 6.67 (d, J = 9.2 Hz,
2H), 6.42 (d, J = 1.6 Hz, 1H), 6.26 (d, J = 1.6 Hz, 1H), 5.04 (d, J = 5.2
Hz, 1H), 5.01 (d, J = 1.6 Hz, 1H), 4.46 (d, J = 4.8 Hz, 1H), 4.30 (d, J
= 14.4 Hz, 1H), 4.11−4.06 (m, 1H), 3.98 (dd, J = 2.8, 11.6 Hz, 1H),
3.91 (dd, J = 6.4, 14.4 Hz, 1H), 3.88 (s, 3H), 3.81−3.75 (m, 2H), 3.71
(s, 3H), 3.65 (s, 3H), 3.64−3.62 (m, 1H), 3.58 (s, 3H), 1.89 (brs, 1H),
1.88−1.78 (m, 2H), 1.67 (brs, 1H); ESI-MS m/z = 639.4 [M + H]+.
Purity (method B): >95% AUC. Compound 38: NMR δH (400 MHz,
CDCl3) 7.09 (d, J = 8.8 Hz, 2H), 7.06−7.04 (m, 3H), 6.87−6.85 (m,
2H), 6.67 (d, J = 8.8 Hz, 2H), 6.50 (d, J = 1.6 Hz, 1H), 6.40 (d, J = 1.6
Hz, 1H), 5.32 (brs, 1H), 5.30 (s, 1H),5.03 (d, J = 6.8 Hz, 1H), 4.29 (d,
J = 14 Hz, 1H), 4.27−4.25 (m, 1H), 4.01 (dd, J = 2.4, 11.6 Hz, 1H),
3.89 (dd, J = 6.8, 14.4 Hz, 1H), 3.86 (s, 3H), 3.71 (s, 3H), 3.65 (s,
3H), 3.61 (s, 3H), 1.98 (brs, 1H), 1.68−1.64 (m, 2H); ESI-MS m/z =
639.4 [M + H]+. Purity (method B): >95% AUC.
Methyl 1,8b-Dihydroxy-6-((2R,3R,6S)-6-(hydroxymethyl)-3-me-
thoxy-1,4-dioxan-2-yloxy)-8-methoxy-3a-(4-methoxyphenyl)-3-
phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]cyclopenta[d]furan-2-car-
boxylate (40). A solution of phenol 4 (15 mg, 0.031 mmol), lactol 26
(20 mg, 0.078 mmol), and triphenylphosphine (21 mg, 0.078 mmol)
in toluene (3 mL) was stirred at 0 °C for 10 min. Molecular sieves, 3 Å
(25 mg), were added, and then a solution of di-2-methoxyethyl
azodicarboxylate (21 mg, 0.088 mmol) in toluene (0.5 mL) was added
over a period of 5 min. The mixture was stirred for an additional 2 h.
The reaction mixture was then concentrated, and the residue was
purified by silica gel column chromatography (4 g) and eluted using
30−50% EtOAc−hexanes to provide the desired compound benzyl
ether (14 mg) and its corresponding 1‴ epimer (2 mg). Both epimers
were independently subjected to hydrogenolysis and HPLC
purification provide the desired compound 39 (4 mg) and compound
40 (1 mg). Compound 39: NMR δH (400 MHz, CDCl3) 7.11 (d, J =
8.8 Hz, 2H), 7.06−7.04 (m, 3H), 6.86−6.80 (m, 2H), 6.69 (d, J = 8.8
Hz, 2H), 6.49 (d, J = 1.6 Hz, 1H), 6.28 (d, J = 1.6 Hz, 1H), 5.24 (s,
1H), 5.04 (d, J = 6.4 Hz, 1H), 4.59 (s, 1H), 4.31 (d, J = 14.4 Hz, 1H),
4.26−4.22 (m, 1H), 4.10 (dd, J = 11.2, 11.2 Hz, 1H), 3.91 (dd, J = 6.4,
14 Hz, 1H), 3.87 (s, 3H), 3.71 (s, 3H), 3.68 (brs, 1H), 3.60 (s, 3H),
3.63−3.561 (m, 2H), 3.50 (s, 3H), 1.90 (brs, 1H), 1.8−1.7 (m, 1H);
ESI-MS m/z = 625.4 [M + H]+. Purity (method B): 92% AUC.
Compound 40: NMR δH (400 MHz, CDCl3) 7.11 (d, J = 8.8 Hz, 2H),
7.07−7.06 (m, 3H), 6.86−6.80 (m, 2H), 6.69 (d, J = 8.8 Hz, 2H), 6.43
(d, J = 1.6 Hz, 1H), 6.31 (d, J = 1.6 Hz, 1H), 5.32 (s, 1H), 5.03 (d, J =
6.8 Hz, 1H), 4.66 (s, 1H), 4.29 (d, J = 14.4 Hz, 1H), 4.13−4.10 (m,
1H), 3.97−3.94 (m, 1H), 3.86 (s, 3H), 3.78−3.75 (m, 1H), 3.71 (s,
3H), 3.69 (brs, 1H), 3.65 (s, 3H), 3.58 (s, 3H), 3.55−3.51 (m, 1H),
2−1.9 (m, 1H), 1.81 (brs, 1H); ESI-MS m/z = 625.4 [M + H]+. Purity
(method B): 97% AUC.
(1R,2R,3S,3aR,8bS)-Methyl 1,8b-Dihydroxy-6-((2S,3S,6S)-6-(hy-
droxymethyl)-3-methoxy-1,4-dioxan-2-yloxy)-8-methoxy-3a-(4-me-
thoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]-
cyclopenta[d]furan-2-carboxylate (41) and (1R,2R,3S,3aR,8bS)-
Methyl 1,8b-Dihydroxy-6-((2R,3S,6S)-6-(hydroxymethyl)-3-me-
thoxy-1,4-dioxan-2-yloxy)-8-methoxy-3a-(4-methoxyphenyl)-3-
phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]cyclopenta[d]furan-2-car-
boxylate (42). A solution of phenol 4 (15 mg, 0.031 mmol), lactol 27
(20 mg, 0.078 mmol), and triphenylphosphine (21 mg, 0.078 mmol)
in toluene (3 mL) was stirred at 0 °C for 10 min. Molecular sieves, 3 Å
(25 mg), were added, and then a solution of di-2-methoxyethyl
azodicarboxylate (21 mg, 0.088 mmol) in toluene (0.5 mL) was added
over a period of 5 min. The mixture was stirred for an additional 2 h.
The reaction mixture was then concentrated and the residue was
purified by silica gel column chromatography (4 g) and eluted using
30−50% EtOAc−hexanes to provide the desired benzyl ether (12 mg)
and its corresponding 1‴ epimer (2 mg). Both epimers were
independently subjected to hydrogenolysis and HPLC purification to
provide the desired compound 41 (5 mg) and compound 42 (1 mg).
Compound 41: NMR δH (400 MHz, CDCl3) 7.11 (d, J = 8.8 Hz, 2H),
7.06−7.05 (m, 3H), 6.88−6.85 (m, 2H), 6.67 (d, J = 8.8 Hz, 2H), 6.47
(d, J = 1.6 Hz, 1H), 6.26 (d, J = 1.6 Hz, 1H), 5.15 (d, J = 3.2 Hz, 1H),
5.02 (dd, J = 1.6, 6.8 Hz, 1H), 4.52 (d, J = 3.2 Hz, 1H), 4.29 (d, J = 14
Hz, 1H), 4.10 (dd, J = 3.6, 12 Hz, 1H), 3.95−3.91 (m, 3H), 3.88 (s,
3H), 3.83−3.76 (m, 2H), 3.71 (s, 3H), 3.69 (d, J = 5.6 Hz, 1H), 3.65
(s, 3H), 3.56 (s, 3H), 1.84 (d, J = 14 Hz, 1H); ESI-MS m/z = 625.4
[M + H]+. Purity (method C): 86% AUC. Compound 42: NMR δH
(400 MHz, CDCl3) 7.11 (d, J = 8.8 Hz, 2H), 7.06−7.05 (m, 3H),
6.88−6.85 (m, 2H), 6.69 (d, J = 8.8 Hz, 2H), 6.53 (d, J = 1.6 Hz, 1H),
6.39 (d, J = 1.6 Hz, 1H), 5.35 (s, 1H), 5.04 (d, J = 5.6 Hz, 1H), 4.63
(s, 1H), 4.29 (d, J = 14 Hz, 1H), 4.23−4.19 (m, 1H), 4.08 (dd, J = 2.8,
12 Hz, 1H), 3.95−3.91 (m, 2H), 3.88 (s, 3H), 3.81−3.77 (m, 1H),
3.72 (s, 3H), 3.71−3.66 (m, 1H), 3.65 (s, 3H), 3.62 (s, 3H), 1.81 (s,
1H) 1.71−1.69 (m, 1H); ESI-MS m/z = 625.4 [M + H]+. Purity
(method C): 79% AUC.
(1R,2R,3S,3aR,8bS)-Methyl 1,8b-Dihydroxy-6-((2S,6S)-6-(hydroxy-
methyl)-1,4-dioxan-2-yloxy)-8-methoxy-3a-(4-methoxyphenyl)-3-
phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]cyclopenta[d]furan-2-car-
boxylate (43). A solution of phenol 4 (40 mg, 0.084 mmol), lactol 23
(47 mg, 0.21 mmol), and triphenylphosphine (55 mg, 0.21 mmol) in
(1R,2R,3S,3aR,8bS)-Methyl 1,8b-Dihydroxy-6-((2S,3R,6S)-6-(hy-
droxymethyl)-3-methoxy-1,4-dioxan-2-yloxy)-8-methoxy-3a-(4-me-
thoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]-
cyclopenta[d]furan-2-carboxylate (39) and (1R,2R,3S,3aR,8bS)-
8870
dx.doi.org/10.1021/jm3011542 | J. Med. Chem. 2012, 55, 8859−8878

Journal of Medicinal Chemistry
Article
toluene (3 mL) was stirred at 0 °C for 10 min. Molecular sieves, 3 Å
(25 mg), were added, and then a solution of di-2-methoxyethyl
azodicarboxylate (55 mg, 0.23 mmol) in toluene (0.5 mL) was added
over a period of 5 min. The mixture was stirred for an additional 2 h.
The reaction mixture was then concentrated and the residue was
purified by silica gel column chromatography (4 g) and eluted using
30−50% EtOAc-CH2Cl2 to provide the desired benzyl ether (46 mg).
The benzyl ether was hydrogenated as described for compound 46 to
generate the desired compound 43 (5 mg) after purification by HPLC.
NMR δH (400 MHz, CDCl3) 7.11 (d, J = 8.8 Hz, 2H), 7.06−7.05 (m,
3H), 6.88−6.85 (m, 2H), 6.68 (d, J = 8.8 Hz, 2H), 6.52 (d, J = 2 Hz,
1H), 6.32 (d, J = 2 Hz, 1H), 5.40 (s, 1H), 4.31 (d, J = 6.8 Hz, 1H),
4.28−4.21 (m, 1H), 3.97 (dd, J = 12, 12 Hz, 2H), 3.94−3.91 (m, 1H),
3.88 (s, 3H), 3.88−3.86 (m, 1H), 3.78 (dd, J = 2, 12 Hz, 1H), 3.76 (d,
J = 12 Hz, 1H), 3.71 (s, 3H), 3.69 (brs, 1H), 3.65 (s, 3H), 1.86 (brs,
1H) 1.71−1.65 (m, 1H); ESI-MS m/z = 595.2 [M + H]+. Purity
(method C): 92% AUC.
(1R,2R,3S,3aR,8bS)-Methyl 1,8b-Dihydroxy-6-((2S,6R)-6-
(hydroxymethyl)tetrahydro-2H-pyran-2-yloxy)-8-methoxy-3a-(4-
methoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]-
cyclopenta[d]furan-2-carboxylate (44). A solution of phenol 4 (25
mg, 0.052 mmol), lactol 30 (32 mg, 0.13 mmol), and triphenylphos-
phine (34 mg, 0.13 mmol) in anhydrous toluene (3 mL) was stirred at
0 °C for 10 min. Molecular sieves, 3 Å (50 mg), were added, and then
a solution of di-2-methoxyethyl azodicarboxylate (34 mg, 0.15 mmol)
in toluene (0.5 mL) was added over a period of 5 min. The mixture
was stirred for an additional 10 h. The reaction mixture was then
concentrated, and the residue was purified by silica gel column
chromatography and eluted using 10% EtOAc−hexanes to provide the
desired TBS ether adduct (29 mg). To a solution of TBS ether (10
mg, 0.014 mmol) in THF (1 mL) was added a solution of TBAF (56
uL, 1 M) in THF at 0 °C. The mixture was stirred at room
temperature for 1 h before the solvent was removed under reduced
pressure. The residue was dissolved into DCM, and purification by
flash chromatography with EtOAc as eluent gave 7 mg of the desired
product 44. NMR δH (400 MHz, CDCl3) 7.12 (d, J = 8.8 Hz, 2H),
7.06−7.04 (m, 3H), 6.87−6.85 (m, 2H), 6.67 (d, J = 8.8 Hz, 2H), 6.50
(d, J = 2 Hz, 1H), 6.26 (d, J = 2 Hz, 1H), 5.53 (brs, 1H), 5.04 (dd, J =
1.6, 6.8 Hz, 1H), 4.30 (d, J = 14 Hz, 1H), 3.90 (d, J = 8.4 Hz, 1H),
3.93−3.91 (m, 1H), 3.88 (s, 3H), 3.71 (s, 3H), 3.65 (s, 3H), 3.54−
3.49 (m, 1H), 2.1−2.0 (m, 1H), 1.91 (brs, 1H), 1.92−1.89 (m, 1H),
1.82−1.71 (m, 3H), 1.65−1.56 (m, 2H); ESI-MS m/z = 593.3 [M +
H]+. Purity (method B): 90% AUC
(1R,2R,3S,3aR,8bS)-Methyl 1,8b-Dihydroxy-6-((2S,6R)-6-
(hydroxymethyl)morpholin-2-yloxy)-8-methoxy-3a-(4-methoxy-
phenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]cyclopenta[d]-
furan-2-carboxylate (45). A suspension of phenol 4 (100 mg, 0.21
mmol), lactol 33 (200 mg, 0.52 mmol), and triphenylphosphine (137
mg, 0.52 mmol) with molecular sieves, 3 Å (35 mg), in toluene (6 mL)
was stirred at 0 °C for 10 min. A solution of di-2-methoxyethyl
azodicarboxylate (137 mg, 0.59 mmol) in toluene (0.8 mL) was added
over a period of 5 min. The mixture was stirred for an additional 6 h at
0 °C. The mixture was filtered and diluted with EtOAc and washed
with water and brine and dried over Na2SO4, filtered, and
concentrated. Purification on silica gel (30−40% EtOAc in hexane)
gave the desired Cbz-protected adduct (140 mg). To a solution of
Cbz-protected material (15 mg, 0.018 mmol) in MeOH (3 mL) was
added (10%) Pd on carbon (10 mg). After three purge−fill cycles, the
vessel was placed under 1 atm of hydrogen. After completion of the
reaction, the vessel was purged and catalyst was filtered off from Celite.
The Celite was washed with THF (3 × 5 mL), and the combined
filtrate was concentrated and purified on HPLC to give 10 mg of the
desired morpholine. To a solution of TBS ether (5 mg, 0.007 mmol)
in THF (2 mL) was added a solution of TBAF (22 μL, 1 M) in THF
at 0 °C. The mixture was stirred at room temperature for 1 h before
the solvent was removed under reduced pressure. The residue was
purified by HPLC to give 3 mg of the desired product 45. NMR δH
(400 MHz, CDCl3) 7.11 (d, J = 8.8 Hz, 2H), 7.06−7.05 (m, 3H),
6.87−6.85 (m, 2H), 6.67 (d, J = 8.8 Hz, 2H), 6.51 (d, J = 1.6 Hz, 1H),
6.30 (d, J = 1.6 Hz, 1H), 5.39 (brs, 1H), 5.03 (d, J = 6.4 Hz, 1H), 4.30
(d, J = 14 Hz, 1H), 4.05−3.97 (m, 1H), 3.92 (d, J = 7.2 Hz, 1H), 3.88
(s, 3H), 3.88−3.87 (m, 1H), 3.71 (s, 3H), 3.68 (d, J = 1.6 Hz, 1H),
3.65 (s, 3H), 3.65−3.64 (m, 1H), 3.57 (dd, J = 4.8, 12 Hz, 1H), 3.06
(d, J = 6 Hz, 1H), 2.93−2.91 (m, 2H); ESI-MS m/z = 594.3 [M+ +
H]. Purity (method C): 87% AUC.
(1R,2R,3S,3aR,8bS)-Methyl 1,8b-Dihydroxy-6-((2R,6R)-6-(hydrox-
ymethyl)-4-methylmorpholin-2-yloxy)-8-methoxy-3a-(4-methoxy-
phenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]cyclopenta[d]-
furan-2-carboxylate (46). To a solution of the morpholine made
during the synthesis of compound 57 (5 mg, 0.007 mmol) in THF
(1.5 mL)/MeOH (0.5 mL) were added formaldehyde (37% in water,
21 μL, 0.28 mmol), acetic acid (16 μL, 0.28 mmol), and sodium
triacetoxyborohydride (15 mg, 0.07 mmol). The mixture was stirred at
room temperature for 3 h. The mixture was purified on HPLC to give
4.5 mg of the desired methylmorpholine derivative. To a solution of
TBS ether (4.5 mg, 0.006 mmol) in THF (2 mL) was added a solution
of TBAF (34 μL, 1 M) in THF at 0 °C. The mixture was stirred at
room temperature for 1 h and worked up with dilution of DCM,
washed with sodium bicarbonate, dried over Na2SO4, filtered, and
concentrated. Flash chromatography on silica gel (DCM, 1−2%
MeOH in DCM, 5% MeOH, 5% Et3N in DCM) gave product 46 (3
mg). NMR δH (400 MHz, CDCl3) 7.11 (d, J = 8.8 Hz, 2H), 7.06−7.04
(m, 3H), 6.87−6.85 (m, 2H), 6.68 (d, J = 8.8 Hz, 2H), 6.54 (d, J = 1.6
Hz, 1H), 6.36 (d, J = 1.6 Hz, 1H), 5.51 (brs, 1H), 5.04 (dd, J = 1.2, 6.4
Hz, 1H), 4.30 (d, J = 14 Hz, 1H), 4.24−4.16 (m, 1H), 3.90 (dd, J =
6.8, 14 Hz, 1H), 3.85 (s, 3H), 3.75−3.72 (m, 2H), 3.71 (s, 3H), 3.64
(s, 3H), 3.65−3.62 (m, 1H), 3.03 (d, J = 12 Hz, 1H), 2.37 (s, 3H),
2.34 (dd, J = 2.4, 12 Hz, 1H), 2.22 (dd, J = 11.2, 11.2 Hz, 1H), 1.95−
1.91 (m, 1H); ESI-MS m/z = 608.3 [M + H]+. Purity (method C):
92% AUC
Preparation of Compounds in Scheme 5. (1R,2R,3S,3aR,8bS)-
Methyl 1,8b-Dihydroxy-6,8-dimethoxy-3a-(4-methoxyphenyl)-3-
phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]cyclopenta[d]furan-2-car-
boxylate (47). A solution of phenol 4 (15 mg, 0.031 mmol, 1 equiv) in
toluene (1 mL) and methanol (1 mL) was treated with
trimethylsilyldiazomethane (0.470 mL, 0.500 mmol, 30 equiv) and
stirred for 3 h at 25 °C. Solvent was removed under vacuum. The
residue was purified using silica gel chromatography (40% EtOAc/
hexanes) to give the desired rocaglate 47 (14 mg, 90%). All physical
data matched the natural product from a commercially available
source.
(1R,2R,3S,3aR,8bS)-Methyl 1,8b-Dihydroxy-8-methoxy-3a-(4-me-
thoxyphenyl)-3-phenyl-6-(trifluoromethylsulfonyloxy)-2,3,3a,8b-tet-
rahydro-1H-benzo[b]cyclopenta[d]furan-2-carboxylate (48). To a
solution of phenol 4 (506 mg, 1.06 mmol, 1.0 equiv) and
diisopropylethylamine (0.3 mL, 1.7 mmol, 1.7 equiv) in 11 mL of
DCM at −10 °C was added trifluoromethanesulfonic anhydride (0.21
mL, 1.22 mmol, 1.15 equiv) dropwise over a period of 4 min. The
mixture was aged for 45 min and partitioned between 25 mL of
saturated sodium bicarbonate. The lower organic layer was collected.
The aqueous layer was extracted with DCM (20 mL). The combined
organic layers were washed successively with 10% citric acid (20 mL),
water (20 mL), brine (20 mL) and dried (Na2SO4). The drying agent
was filtered off. The filtrate was concentrated in vacuo, and the residue
was chromatographed on silica gel (4g) using 0% → 10% ethyl acetate
in DCM to give the desired triflate 48 (515 mg, 1.06 mmol, 76%) as a
white powder. NMR δH (400 MHz, CDCl3) 7.08−7.10 (m, 5H),
6.90−6.92 (m, 2H), 6.66−6.70 (m, 3H), 6.46 (d, J = 1.0 Hz, 1H), 5.03
(d, J = 6.0 Hz, 1H), 4.38 (d, J = 14.0 Hz, 1H), 3.96 (dd, J = 14.0, 6.0
Hz, 1H), 3.94 (s, 3H), 3.71 (s, 3H), 3.68 (s, 3H), 3.48 (brs, 1H), 2.02
(brs, 1H), 1.59 (brs, 1H); NMR δC (100 MHz, CDCl3) 170.56,
160.22, 158.97, 157.25, 152.15, 136.28, 128.78, 127.84, 127.77, 126.78,
125.52, 120.30, 117.12, 115.41, 112.90, 102.71, 98.67, 98.14, 93.38,
79.43, 56.38, 55.49, 55.10, 52.16, 50.42; ESI-MS m/z 633.1 [M +
Na]+. Purity (method A): 95% AUC.
(1R,2R,3S,3aR,8bS)-Methyl 1,8b-Dihydroxy-8-methoxy-3a-(4-me-
thoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]-
cyclopenta[d]furan-2-carboxylate (49). To a solution of the triflate
48 (20 mg, 0.033 mmol) in dimethylformamide (2 mL) were added
triphenylphosphine (9 mg, 0.033 mmol), palladium acetate (3.6 mg,
0.016 mmol), triethylamine (10 mg, 0.1 mmol), and formic acid (3
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mg, 0.066 mmol). The mixture was slowly warmed to 60 °C and
stirred for an additional 2 h at that temperature. The reaction was
quenched by adding brine (5 mL), and the aqueous portion was
extracted using EtOAc (20 mL). Combined organic layers were dried
over Na2SO4, filtered, and concentrated to provide a residue that was
further purified by a preparatory TLC plate using 50% EtOAc−
hexanes as an eluent. The central band was removed and further
purified using preparative HPLC. The desired compound 49 was
obtained as a white solid after lyophilization (2.1 mg, 14%). NMR δH
(400 MHz, CDCl3) 7.34 (t, J = 8.0 Hz), 7.15 (dd, J = 8.0, 2.0 Hz, 2H),
7.07−7.10 (m, 3H), 6.89−6.92 (m, 2H), 6.74 (d, J = 8.0 Hz, 1H), 6.70
(dd, J = 8.0, 2.0 Hz, 2H), 6.57 (d, J = 8.0 Hz, 1H), 5.09 (d, J = 6.0 Hz,
1H), 4.35 (d, J = 14.0 Hz, 1H), 3.96 (dd, J = 14.0, 6.0 Hz, 1H), 3.94 (s,
3H), 3.77 (brs, 1H), 3.72 (s, 3H), 3.67 (s, 3H), 1.98 (brs, 1H), 1.59
(brs, 1H); NMR δC (100 MHz, CDCl3) 170.51, 15.87, 158.72, 156.56,
136.84, 132.44, 128.96, 127.69, 126.53, 126.30, 115.26, 112.70, 105.15,
103.59, 101.10, 93.75, 79.77, 55.78, 55.06, 51.97, 50.41; ESI-MS m/z
463.1 [M + H]+. Purity (method A): 99% AUC.
(1R,2R,3S,3aR,8bS)-Methyl 1,8b-Dihydroxy-8-methoxy-3a-(4-me-
thoxyphenyl)-6-methyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo-
[b]cyclopenta[d]furan-2-carboxylate (50). To a mixture of triflate 48
(20 mg, 0.033 mmol), palladium acetate (1.8 mg, 8.19 μmol), cesium
carbonate (32 mg, 0.098 mmol), X-phos (7.8 mg, 0.016 mmol), and
methylboronic acid (5.9 mg, 0.098 mmol) was added toluene (2 mL),
and the mixture was heated at 100 °C overnight . The reaction mixture
was filtered through a 0.2 μm filter, concentrated, and then redissolved
in CH2Cl2 (2 mL). Purification by preparative TLC using 20%
EtOAc−hexanes provided the desired product 50, which was isolated
as a white solid after lyophilization (1.8 mg, 12%). NMR δH (400
MHz, CDCl3) 7.14 (d, J = 8.8 Hz, 2H), 7.07−7.08 (m, 3H), 6.89−6.92
(m, 2H), 6.70 (d, J = 8.8 Hz, 1H), 6.58 (s, 1H), 6.38 (s, 1H), 5.07 (d, J
= 6.0 Hz, 1H), 4.33 (d, J = 14.4 Hz, 1H), 3.95 (dd, J = 14.0, 6.0 Hz,
1H), 3.94 (s, 3H), 3.78 (brs, 1H), 3.72 (s, 3H), 3.66 (s, 3H), 2.40 (s,
3H), 1.87 (brs, 1H), 1.59 (brs, 1H); NMR δC (100 MHz, CDCl3)
170.56, 159.99, 158.73, 156.18, 143.42, 136.95, 128.99, 127.83, 127.70,
126.53, 126.44, 112.72, 112.53, 105.66, 104.72, 101.30, 93.67, 79.70,
55.70, 5.10, 55.00, 51.98, 50.44, 22.22; ESI-MS m/z 477.2 [M + H]+.
Purity (method A): 93% AUC.
(1R,2R,3S,3aR,8bS)-Methyl 6-Amino-1,8b-dihydroxy-8-methoxy-
3a-(4-methoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]-
cyclopenta[d]furan-2-carboxylate (51). A mixture of triflate 48 (263
mg, 0.431 mmol, 1 equiv), 2,2′-bis(diphenylphosphino)-1,1′-binaphth-
yl (BINAP) (148 mg, 0.237 mmol, 0.55 equiv), diphenylimine (625
mg, 3.45 mmol, 8 equiv), palladium acetate (24 mg, 0.108 mmol, 0.25
equiv), and cesium carbonate (332 mg, 1.728 mmol, 4 equiv) was
suspended in toluene (25 mL) and heated to 100 °C for 20 h. The
resulting mixture was cooled and partitioned between ethyl acetate and
a saturated aqueous sodium chloride solution. The organic layer was
separated, dried over sodium sulfate, and concentrated. The residue
was dissolved in methanol (25 mL) and treated with sodium acetate
(1.0g, 12.0 mmol, 28 equiv) and hydroxylamine hydrochloride (800
mg, 12.0 mmol, 28 equiv). The mixture was stirred for 3 h at room
temperature. The mixture was partitioned between ethyl acetate and
water. The organic layer was separated, dried over sodium sulfate, and
concentrated. The residue was purified using silica gel chromatography
(EtOAc/hexanes) to give the desired aniline 51 (153 mg, 0.320 mmol,
74%). NMR δH (400 MHz, CDCl3) 7.12 (d, J = 8.0, 2H), 7.04−7.07
(m, 3H), 6.85−6.87 (m, 2H), 6.68 (d, J = 8.0 Hz, 1H), 5.99 (s, 1H),
5.83 (s, 1H), 5.02 (d, J = 6.0 Hz, 1H), 4.30 (d, J = 14.0 Hz, 1H), 3.91
(dd, J = 14.0, 6.0 Hz, 1H), 3.83 (s, 3H), 3.71 (brs, 1H), 3.64 (s, 3H),
3.67 (s, 3H), 2.01 (brs, 1H), 1.61 (brs, 2H); NMR δC (100 MHz,
CDCl3) 170.59, 161.11, 158.65, 157.25, 150.91, 137.10, 129.02,
127.84, 127.66, 126.77, 126.47, 112.65, 105.66, 101.56, 93.64, 91.83,
91.20, 79.53, 55.58, 55.10, 54.84, 51.93, 50.50; ESI-MS m/z 478.1 [M
+ H]+. Purity (method C): >95% AUC
μmol), tert-butylphosphine (4 mg, 9.01 μmol), and cesium carbonate
(18 mg, 0.054 mmol). The mixture was heated at 100 °C for 12 h in a
sealed tube. The reaction mixture was cooled to room temperature,
and CH2Cl2 (4 mL) was added. The mixture was filtered using a 0.45
μm HPLC filter, and the filtrate was concentrated. The residue was
redissolved in acetonitrile (2 mL) and purified by preparative HPLC to
provide the desired 52 (4 mg, 45%). NMR δH (400 MHz, CDCl3)
7.12 (d, J = 8.0, 2H), 7.07−7.10 (m, 3H), 6.89−6.92 (m, 2H), 6.74 (d,
J = 8.0 Hz, 1H), 5.99 (s, 1H), 5.80 (s, 1H), 5.02 (d, J = 6.0 Hz, 1H),
4.34 (d, J = 14.0 Hz, 1H), 3.96 (dd, J = 14.0, 6.0 Hz, 1H), 3.86 (s, 3H),
3.76 (brs, 1H), 3.74 (s, 3H), 3.67 (s, 3H), 2.79 (s, 3H), 1.80 (brs, 1H),
1.59 (brs, 2H); NMR δC (100 MHz, CDCl3) 170.66, 161.21, 158.62,
157.02, 154.85, 137.22, 129.02, 127.86, 127.66, 126.84, 126.43, 112.66,
103.48, 101.44, 93.83, 89.21, 88.50, 79.53, 55.4, 55.09, 54.73, 51.92,
50.53, 40.79; ESI-MS m/z 492.1 [M + H]+. Purity (method A): 95%
AUC.
(1R,2R,3S,3aR,8bS)-Methyl 6-(Dimethylamino)-1,8b-dihydroxy-8-
methoxy-3a-(4-methoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-
benzo[b]cyclopenta[d]furan-2-carboxylate (53). A solution of the
triflate 48 (15 mg, 0.025 mmol) in toluene (2 mL) was treated with
dimethylamine (0.123 mL, 0.246 mmol, 2 M in THF), palladium
acetate (1.4 mg, 6.14 μmol), tert-butylphosphine (5 mg, 0.012 mmol),
and cesium carbonate (24 mg, 0.074 mmol). The mixture was heated
at 100 °C for 12 h in a sealed tube. The reaction mixture was filtered
using a 0.45 μm HPLC filter and then purified by preparative TLC
plate using 50% EtOAc−hexanes. The desired product 53 was
obtained as a white solid after lyophilization (4 mg, 31%). NMR δH
(400 MHz, CDCl3) 7.15 (d, J = 8.0, 2H), 7.05−7.09 (m, 3H), 6.88−
6.90 (m, 2H), 6.67 (d, J = 8.0 Hz, 1H), 6.07 (s, 1H), 5.88 (s, 1H), 5.05
(d, J = 6.0 Hz, 1H), 4.33 (d, J = 14.0 Hz, 1H), 3.92 (dd, J = 14.0, 6.0
Hz, 1H), 3.89 (s, 3H), 3.78 (brs, 1H), 3.71 (s, 3H), 3.65 (s, 3H), 3.01
(s, 6H), 2.05 (brs, 1H), 1.85 (brs, 1H); NMR δC (100 MHz, CDCl3)
170.61, 161.20, 158.61, 157.02, 154.83, 137.25, 129.04, 127.87, 127.65,
126.88, 126.42, 112.64, 101.43, 103.82, 93.82, 89.25, 88.54, 79.54,
76.71, 55.4, 55.09, 54.73, 51.92, 50.53, 40.79; ESI-MS m/z 506.2 [M +
H]+. Purity (method A): 93% AUC.
(1R,2R,3S,3aR,8bS)-Methyl 1,8b-Dihydroxy-8-methoxy-3a-(4-me-
thoxyphenyl)-6-(methylsulfonamido)-3-phenyl-2,3,3a,8b-tetrahy-
dro-1H-benzo[b]cyclopenta[d]furan-2-carboxylate (54). To a sol-
ution of aniline 51 (10 mg, 0.021 mmol) in DMF (2 mL) were added
DMAP (2.6 mg, 0.021 mmol), 1-(methylsulfonyl)-1H-benzo[d]-
[1,2,3]triazole (9 mg, 0.042 mmol), and Et3N (11 mg, 0.105
mmol). The reaction mixture was stirred for 4 h at room temperature.
Brine (10 mL) was added, and the aqueous portion was extracted with
EtOAc (20 mL). The organic layer was separated, dried, and
concentrated. The residue was purified by preparative HPLC to
provide the sulfonamide 54 as a white solid after lyophilization (8 mg,
68%). NMR δH (400 MHz, CDCl3) 7.16 (d, J = 8.0, 2H), 7.01−7.07
(m, 3H), 6.93−6.95 (m, 2H), 6.67 (d, J = 8.0 Hz, 1H), 6.63 (s, 1H),
6.52 (s, 1H), 5.11 (d, J = 6.0 Hz, 1H), 4.37 (d, J = 14.0 Hz, 1H), 3.98
(dd, J = 14.0, 6.0 Hz, 1H), 3.93 (s, 3H), 3.74 (brs, 1H), 3.69 (s, 3H),
3.05 (s, 3H), 1.91(brs, 1H), 1.58 (brs, 2H); NMR δC (100 MHz,
CDCl3) 170.78, 160.33, 159.07, 157.21, 141.10, 136.37, 128.95,
127.88, 126.90, 125.88, 112.99, 112.40, 102.10, 97.13, 93.71, 91.23,
79.85, 56.18, 55.64, 55.20, 52.22, 50.40, 39.34; ESI-MS m/z 556.2 [M
+ H]+. Purity (method A): 94% AUC.
(1R,2R,3S,3aR,8bS)-1,8b-Dihydroxy-8-methoxy-2-(methoxycar-
bonyl)-3a-(4-methoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-
benzo[b]cyclopenta[d]furan-6-carboxylic Acid (55). Carbon mon-
oxide was bubbled through a mixture of the triflate 48 (102 mg, 0.17
mmol, 1.0 equiv), DPPF (18.6 mg, 0.034 mmol, 0.2 equiv), potassium
carbonate (116 mg, 0.838 mmol, 5.0 equiv), and palladium acetate (3.8
mg, 0.017 mmol, 0.1 equiv) in 2.0 mL of DMF for 15 min. The
resulting reddish orange solution was heated at 100 °C for 2 h, cooled,
diluted with brine (5 mL), acidified with 1 N HCl to pH 1−2, and
partitioned between EtOAc (10 mL). The aqueous layer was extracted
with EtOAc (2 × 10 mL), and the combined organic layers were
washed with water (10 mL) and brine (10 mL). The mixture was dried
(Na2SO4). The drying agent was filtered off. The solvents were
evaporated in vacuo and the residue was purified by preparative TLC
(1R,2R,3S,3aR,8bS)-Methyl 1,8b-Dihydroxy-8-methoxy-3a-(4-me-
thoxyphenyl)-6-(methylamino)-3-phenyl-2,3,3a,8b-tetrahydro-1H-
benzo[b]cyclopenta[d]furan-2-carboxylate (52). A solution of the
triflate 48 (11 mg, 0.018 mmol) in toluene (2 mL) was treated with
methylamine (0.090 mL, 0.180 mmol), palladium acetate (1 mg, 4.50
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using 5% MeOH in DCM as the developing solvent to give the desired
acid which was then taken up in 2 mL of 90% tert-butanol, frozen, and
lyophilized to give 63 mg of 55 (0.125 mmol, 74%) as an off white
solid. NMR δH (400 MHz, CDCl3) 7.47 (brs, 1H), 7.29 (brs, 1H),
7.13−7.07 (m, 5H), 6.92 (d, J = 7.6 Hz, 2H), 6.68 (d, J = 8.8 Hz, 2H),
5.06 (d, J = 6.4 Hz, 1H), 4.37 (d, J = 14 Hz, 1H), 3.98−3.93 (m, 4H),
3.71 (s, 3H), 3.67 (s, 3H); NMR δC (100 MHz, CDCl3) 170.66,
169.91, 159.81, 158.88, 156.46, 136.49, 133.51, 128.87, 127.81, 126.69,
125.87, 120.58, 112.85, 107.13, 105.55, 101.84, 93.46, 79.76, 56.14,
55.46, 55.10, 52.13, 50.38; ESI-MS m/z 507.1 [M + H]+. Purity
(method A): >99% AUC.
(1R,2R,3S,3aR,8bS)-Dimethyl 1,8b-Dihydroxy-8-methoxy-3a-(4-
methoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]-
cyclopenta[d]furan-2,6-dicarboxylate (56). To a solution of the acid
55 (12 mg, 0.024 mmol, 1.0 equiv) in 1 mL of methanol at 22 °C was
added a 1.0 M solution of trimethylsilyldiazomethane (0.25 mL, 0.237
mmol, 10 equiv), and the mixture was stirred for 1 h. The mixture was
concentrated in vacuo and the residue was purified by preparative TLC
(10% EtOAc in DCM) to give 8.0 mg (0.015 mmol, 63%) of 56. NMR
δH (400 MHz, CDCl3) 7.41 (d, J = 1 Hz, 1H), 7.25 (d, J = 1 Hz, 1H),
7.12−7.06 (m, 5 H), 6.91−6.89 (m, 2H), 6.69−6.66 (m, 2H), 5.05 (d,
J = 6.4 Hz, 1H), 4.34 (d, J = 14.0 Hz, 1H), 3.98 (s, 3H), 3.96−3.92
(m,1H), 3.95 (s, 3H) 3.71 (s, 3H), 3.66 (s, 3H), 2.00 (s, 1H). ESI-MS
m/z 521.1 [M + H]+. Purity (method A): 93.6% AUC.
0.147 mmol, 6.6 equiv), and EDCI (22 mg, 0.11 mmol, 5.3 equiv) in 1
mL of DCM at 22 °C was added diisopropylethylamine (40 μL, 0.22
mmol, 10.6 equiv). The mixture was stirred for 1 h before diluting with
DCM (2 mL) and 10% citric acid (2 mL). The organic layer was
collected. The aqueous layer was extracted with DCM (5 mL). The
combined organic layers were washed with saturated NaHCO3 (2
mL), water (2 mL) and dried (Na2SO4). The drying agent was filtered
off and the filtrate was concentrated in vacuo and the crude was
purified by preparative HPLC (20% → 85% acetonitrile−water with
0.05% formic acid) to give 5.3 mg (0.099 mmol, 45%) of 59 after
lyophilization. NMR δH (400 MHz, CDCl3) 7.12−7.06 (m, 5H),
6.89−6.87 (m, 2H), 6.74 (brs, 1H), 6.69−6.66 (m, 2H), 6.60 (brs,
1H), 5.06 (dd, J = 6.4, 1 Hz, 1H), 4.34 (d, J = 14.4 Hz, 1H), 3.94 (dd,
J = 14.4, 6.4 Hz, 1H), 3.93 (s, 3H), 3.71 (s, 3H), 3.66 (brs, 4H), 3.13
(s, 3H), 3.05 (s, 3H), 1.92 (s, 1H); ESI-MS m/z = 534.2 [M + H]+.
Purity (method A): 97.5% AUC.
(1R,2R,3S,3aR,8bS)-Methyl 6-Acetyl-1,8b-dihydroxy-8-methoxy-
3a-(4-methoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]-
cyclopenta[d]furan-2-carboxylate (60). Degassed DMF (2 mL) was
added to a 25 mL dry flask. Subsequently, aryl triflate 48 (20 mg, 0.033
mmol), tributyl(1-ethoxyvinyl)stannane (12 mg, 0.033 mmol),
palladium acetate (4 mg, 0.016 mmol), and triphenylphosphine (13
mg, 0.049 mmol) were added under an atmosphere of argon. The
mixture was stirred for 5 min at room temperature and then heated to
100 °C. The stirring was continued for an additional 3 h. The
compound was purified by silica gel chromatography using 50%
EtOAc−hexanes to provide the acetophenone 60 (11 mg, 66%). NMR
δH (400 MHz, CDCl3) 7.29 (s, 1H), 7.17 (s, 1H), 7.13 (d, J = 8.0,
2H), 7.07−7.09 (m, 3H), 6.91−6.93 (m, 2H), 6.66 (d, J = 8.0 Hz,
1H), 5.05 (d, J = 6.0 Hz, 1H), 4.38 (d, J = 14.0 Hz, 1H), 3.97 (s, 3H),
3.94 (dd, J = 14.0, 6.0 Hz, 1H), 3.70 (s, 3H), 3.66 (s, 3H), 2.62 (s,
3H), 2.21 (brs, 1H), 1.62 (brs, 2H); NMR δC (100 MHz, CDCl3)
197.22, 170.60, 159.98, 158.87, 156.71, 141.23, 136.55, 128.85, 127.81,
126.69, 125.94, 120.05, 112.84, 105.86, 103.20, 101.92, 93.47, 79.71,
56.10, 55.48, 55.10, 52.10, 50.40, 26.85; ESI-MS m/z = 527.1 [M +
Na]+. Purity (method A): 99% AUC
(1R,2R,3S,3aR,8bS)-Methyl 6-Cyano-1,8b-dihydroxy-8-methoxy-
3a-(4-methoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]-
cyclopenta[d]furan-2-carboxylate (61). A mixture of the triflate 48
(147 mg, 0.241 mmol, 1.0 equiv), zinc cyanide (62.7 mg, 0.53 mol, 2.2
equiv), DPPF (28.2 mg, 0.05 mmol, 0.21 equiv), and Pd2(dba)3 (24.8
mg, 0.27 mmol 0.11 equiv) in 6.0 mL of N-methylpyrrolidinone
(previously degassed) was degassed for 5 min and heated at 98−104
°C for 20 h. The mixture was cooled and diluted with EtOAc (20 mL)
and water (20 mL). The organic layer was collected. The aqueous
layer was extracted with EtOAc (2 × 15 mL). The combined organic
layers were washed with water (20 mL), 20 mL of brine (20 mL) and
dried (Na2SO4), and the solvent was evaporated in vacuo. The residue
was chromatographed on silica gel (12 g) using 0% → 20% EtOAc in
DCM to give 107 mg (0.21 mmol, 87%) of 61. NMR δH (400 MHz,
CDCl3) 7.11−7.05 (m, 5H), 7.02 (d, J = 1.0 Hz, 1H), 6.93−6.91 (m,
2H), 6.80 (d, J = 1.0 Hz, 1H), 6.90−6.65 (m, 2H), 4.99 (d, J = 6.0 Hz,
1H), 4.37 (d, J = 14.0 Hz, 1H), 3.96 (dd, J = 14.0, 6.0 Hz, 1H), 3.94 (s,
3H), 3.70 (s, 3H), 3.67 (s, 3H), 3.47 (brs, 1H), 2.02 (brs, 1H); NMR
δC (100 MHz, CDCl3) 170.76, 159.92, 158.99, 157.13, 136.17, 128.70,
127.85, 127.77, 126.78, 125.44, 120.42, 118.42, 115.23, 112.91, 108.89,
107.60, 102.20, 93.39, 79.42, 56.31, 55.70, 55.07, 52.19, 50.38. ESI-MS
m/z = 510.0 [M + Na]+. Purity (method A): 97.6% AUC.
(1R,2R,3S,3aR,8bS)-Methyl 6-Chloro-1,8b-dihydroxy-8-methoxy-
3a-(4-methoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]-
cyclopenta[d]furan-2-carboxylate (62). The aryl triflate 48 (105 mg,
0.17 mmol), bis(pinacolato)diboron (217 mg, 0.86 mmol),
PdCl2(dppf) (13 mg, 0.02 mo1%), and Et3N (120 μL, 0.86 mmol)
were added to a flask. Anhydrous dioxane (10 mL) was added to the
mixture. The flask was put under vacuum and then refilled with
nitrogen. This process was repeated three times before the mixture was
heated at reflux for 14 h. The reaction was quenched with water (10
mL), and extraction was with CH2Cl2 (3 × 25 mL). After drying
(Na2SO4), the solvent was removed to give pinacol boronate as a
brown solid. To a solution of the boronate ester (90 mg, 0.15 mmol)
(1R,2R,3S,3aR,8bS)-Methyl 6-Carbamoyl-1,8b-dihydroxy-8-me-
thoxy-3a-(4-methoxyphenyl)-3-phenyl-2,3,3a,8b-tetrahydro-1H-
benzo[b]cyclopenta[d]furan-2-carboxylate (57). To a mixture of the
acid 55 (10 mg, 0.02 mmol, 1.0 equiv), HOBt (16 mg, 0.13 mmol, 6.5
equiv), ammonium acetate (12.7 mg, 0.16 mmol, 8 equiv), and EDCI
(21 mg, 0.1 mmol, 5.0 equiv) in DCM at 22 °C was added
diisopropylethylamine (35 μL, 0.2 mmol, 10.0 equiv) before diluting
with DCM (2 mL) and 10% citric acid (2 mL). The organic layer was
collected. The aqueous layer was extracted with DCM (5 mL). The
combined organic layers were washed with saturated NaHCO3 (2
mL), water (2 mL) and dried (Na2SO4). The drying agent was filtered
off. The filtrate was concentrated in vacuo and the crude was purified
by preparative TLC (5% MeOH in DCM) to give 5.9 mg (0.012
mmol, 59%) of 57. NMR δH (400 MHz, CDCl3) 7.11−7.03 (m, 7H),
6.91−6.88 (m, 2H), 6.69−6.66 (m, 2 H), 6.10 (brs, 1H), 5.64 (brs,
1H), 5.04 (d, J = 6.4 Hz, 1H), 4.34 (d, J = 14.4 Hz, 1H), 3.96 (s, 3H),
3.94 (dd, J = 14.4, 6.4 Hz, 1H), 3.71 (s, 3H), 3.66 (s, 3H), 3.48 (s,
+
2H). ESI-MS m/z = 528.2 [M + Na] . Purity (method A): >99%
AUC.
(1R,2R,3S,3aR,8bS)-Methyl 1,8b-Dihydroxy-8-methoxy-3a-(4-me-
thoxyphenyl)-6-(methylcarbamoyl)-3-phenyl-2,3,3a,8b-tetrahydro-
1H-benzo[b]cyclopenta[d]furan-2-carboxylate (58). To a mixture of
the acid 55 (10 mg, 0.02 mmol, 1.0 equiv), HOBt (16 mg, 0.13 mmol,
6.5 equiv), methylamine hydrochloride (9 mg, 0.133 mmol, 6.6 equiv),
and EDCI (20 mg, 0.1 mmol, 5.2 equiv) in 1 mL of DCM at 22 °C
was added diisopropylethylamine (35 μL, 0.2 mmol, 10.0 equiv), and
the mixture was stirred for 1 h before diluting with DCM (2 mL) and
10% citric acid (2 mL). The organic layer was collected. The aqueous
layer was extracted with DCM (5 mL), and the combined organic
layers were washed with saturated NaHCO3 (2 mL), water (2 mL)
and dried (Na2SO4). The drying agent was filtered off and the filtrate
was concentrated in vacuo and the crude was purified by preparative
TLC (5% MeOH in DCM) to give 7.8 mg (0.015 mmol, 76%) of 58.
NMR δH (400 MHz, CDCl3) 7.11 (m, 6H), 6.97 (d, J = 1.2 Hz, 1H),
6.89−6.87 (m, 2H), 6.16 (br m, 1H), 5.04 (d, J = 6.8 Hz, 1H), 4.32 (d,
J = 14.0 Hz, 1H), 3.95 (s, 3H), 3.91 (d, J = 6.4 Hz, 1H), 3.71 (s, 3H),
3.65 (s, 3H), 3.48 (s, 3H), 3.02 (d, J = 4.8 Hz, 3H), 2.10 (brs, 1H).
NMR δC (100 MHz, CDCl3) 170.60, 167.72, 159.78, 158.79, 156.68,
138.84, 136.60, 128.87, 127.77, 127.75, 126.64, 126.01, 118.36, 112.76,
103.44, 103.04, 101.79, 93.43, 79.72, 76.69, 56.07, 55.25, 55.07, 52.06,
50.34, 26.97; ESI-MS m/z = 520.2 [M + H]+. Purity (method A):
>99% AUC.
(1R,2R,3S,3aR,8bS)-Methyl 6-(Dimethylcarbamoyl)-1,8b-dihy-
droxy-8-methoxy-3a-(4-methoxyphenyl)-3-phenyl-2,3,3a,8b-tetra-
hydro-1H-benzo[b]cyclopenta[d]furan-2-carboxylate (59). To a
mixture of the acid 55 (11 mg, 0.022 mmol, 1.0 equiv), HOBt (16
mg, 0.11 mmol, 5.3 equiv), dimethylamine hydrochloride (12 mg,
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in 3 mL of MeOH was added a water (3 mL) solution of copper(II)
chloride (62 mg, 0.46 mmol). The mixture was stirred at reflux for 3 h.
The reaction mixture was cooled to room temperature. Then water
(10 mL) was added and the residue was extracted with CH2Cl2 (3 ×
25 mL). After drying (Na2SO4) and filtration, the solvent was removed
to give a brown solid, which was purified by HPLC to give 35 mg of
the desired product 62. NMR δH (400 MHz, CDCl3) 7.10−7.04 (m,
5H), 6.91−6.87 (m, 2H), 6.76 (d, J = 1.6 Hz, 1H), 6.67 (d, J = 9.2 Hz,
2H), 6.54 (d, J = 1.6 Hz, 1H), 5.01 (dd, J = 2, 6 Hz, 1H), 4.32 (d, J =
14.4 Hz, 1H), 3.92 (dd, J = 6.4, 14.4 Hz, 1H), 3.90 (s, 3H), 3.70 (s,
3H), 3.66 (s, 3H), 3.52 (d, J = 1.6 Hz, 1H), 1.87 (s, 1H); NMR δC
(100 MHz, CDCl3) 170.75, 160.38, 158.97, 156.99, 137.75, 136.75,
129.0, 127.96, 127.94, 126.82, 126.13, 114.13, 112.95, 105.89, 105.11,
102.22, 93.61, 79.64, 56.28, 55.45, 55.24, 55.24, 52.24, 50.62; ESI-MS
m/z = 497.2 [M + H]+. Purity (method B): >95% AUC.
Preparation of Compounds in Scheme 6. (1R,2R,3S,3aR,8bS)-
Methyl 1,8b-Dihydroxy-6,8-dimethoxy-3-phenyl-3a-p-tolyl-
2,3,3a,8b-tetrahydro-1H-benzo[b]cyclopenta[d]furan-2-carboxy-
late (66). To a mixture of bromide 63 (10 mg, 0.016 mmol),
palladium acetate (0.3 mg, 1.6 μmol), cesium carbonate (21 mg, 0.065
mmol), X-phos (3.6 mg, 0.008 mmol), and methylboronic acid (2 mg,
0.032 mmol) was added toluene (2 mL), and the mixture was heated
at 100 °C overnight . The volatiles were removed and purification by
normal phase column, eluting with 20−60% EtOAc−hexanes,
provided the desired product 64 (6 mg). A solution of compound
64 (25 mg, 0.045 mmol, 1 equiv) and 5% palladium on carbon (5 mg)
in tetrahydrofuran (3 mL) was placed under hydrogen atmosphere and
stirred for 18 h at room temperature. The reaction mixture was filtered
on Celite and the filtrate concentrated to dryness. The residue was
purified using silica gel chromatography (40−60% EtOAc/hexanes) to
give the desired phenol 65 (15 mg). A solution of phenol 65 (13 mg,
0.028 mmol, 1 equiv) in toluene (1 mL) and methanol was treated
with trimethylsilyldiazomethane (0.422 mL, 0.84 mmol, 30 equiv) and
stirred for 4 h. The mixture was treated with acetic acid (5 μL) and
partitioned between ethyl acetate and a saturated aqueous sodium
chloride solution. The organic layer was separated, dried over sodium
sulfate, and concentrated. The residue was purified using silica gel
chromatography (20−60% EtOAc/hexanes) to give the desired
compound 66 (7 mg). NMR δH (400 MHz, CDCl3) 7.06−7.04 (m,
5H), 6.96−6.88 (m, 4H), 6.29 (d, J = 2 Hz, 1H), 6.12 (d, J = 2 Hz,
1H), 5.03 (d, J = 7 Hz, 2H), 4.35 (d, J = 15 Hz, 2H), 4.00 (d, J = 15
Hz, 2H), 3.97 (d, J = 7 Hz, 2H), 3.91 (s, 3H), 3.84 (s, 3H), 3.66 (s,
3H); NMR δC (100 MHz, CDCl3) 170.79, 164.30, 161.12, 157.20,
137.35, 137.14, 131.46, 128.27, 128.02, 127.87, 127.75, 126.70, 107.82,
102.26, 93.93, 92.83, 89.68, 79.80, 55.95, 55.91, 55.17, 52.18, 50.70,
31.43, 21.18; ESI-MS m/z = 476.99 [M + H]+. Purity (method A):
95.8% AUC.
(1R,2R,3S,3aR,8bS)-Methyl 3a-(4-Cyanophenyl)-1,8b-dihydroxy-
6,8-dimethoxy-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]-
cyclopenta[d]furan-2-carboxylate (69). A mixture of bromide 63 (30
mg, 0.049 mmol, 1 equiv), DPPF (5.4 mg, 9.7 μmol, 0.2 equiv), zinc
cyanide (11.4 mg, 0.097 mmol, 2 equiv), and Pd2(dba)3 (4.5 mg, 4.8
μmol, 0.1 equiv) was suspended in toluene (25 mL) and degassed for
15 min. The mixture was heated to 100 °C for 20 h. The resulting
mixture was cooled and partitioned between ethyl acetate and a
saturated aqueous sodium chloride solution. The organic layer was
separated, dried over sodium sulfate, and concentrated. The residue
was purified using silica gel chromatography (EtOAc/hexanes) to give
the desired cyanide 67 (25 mg, 0.045 mmol, 94%). A solution of
compound 67 (20 mg, 0.035 mmol, 1 equiv) and 15% palladium on
carbon (20 mg) in tetrahydrofuran (2 mL) was placed under hydrogen
atmosphere and stirred for 18 h at room temperature. The reaction
mixture was filtered on Celite and the filtrate concentrated to dryness.
The residue was purified using silica gel chromatography (EtOAc/
hexanes) to give the desired phenol 68 (12 mg, 0.025 mmol, 71%).
ESI-MS m/z = 474.00 [M + H]+. A solution of phenol 68 (8 mg, 0.017
mmol, 1 equiv) in toluene (1 mL) and methanol was treated with
trimethylsilyldiazomethane (0.250 mL, 0.500 mmol, 30 equiv) and
stirred for 4 h. The mixture was treated with acetic acid (5 μL) and
partitioned between ethyl acetate and a saturated aqueous sodium
chloride solution. The organic layer was separated, dried over sodium
sulfate, and concentrated. The residue was purified using silica gel
chromatography (EtOAc/hexanes) to give the desired nitrile 69 (5
mg, 10.2 μmol, 60%). NMR δH (400 MHz, CDCl3) 7.40 (d, J = 2.4
Hz, 2H), 7.31 (d, J = 2.4 Hz, 2H), 7.04−7.06 (m, 3H), 6.88−6.85 (m
2H), 6.30 (d, J = 2 Hz, 1H), 6.14 (d, J = 2 Hz, 1H), 5.02 (dd, J = 6, 2
Hz, 1H), 4.40 (d, J = 14 Hz, 1H), 3.92 (dd, J = 14, 6 Hz, 1H), 3.88 (s,
3H), 3.85 (s, 3H), 3.67 (s, 3H), 3.51 (d, J = 1 Hz, 1H), 1.96 (s, 1H);
NMR δC (100 MHz, CDCl3) 170.43, 164.62, 160.76, 157.06, 140.71,
136.24, 130.96, 128.70, 128.20, 127.69, 127.20, 118.89, 111.25, 107.06,
101.87, 94.11, 93.10, 89.68, 79.88, 55.98, 55.51, 52.35, 50.48, 31.43;
ESI-MS m/z = 488.01 [M + H]+. Purity (method A): 98.3% AUC.
Preparation of Compounds in Scheme 7. (1R,2R,3S,3aR,8bS)-
6-((2S,3R,6R)-6-((S)-1,2-Dihydroxyethyl)-3-methoxy-1,4-dioxan-2-
yloxy)-1,8b-dihydroxy-8-methoxy-3a-(4-methoxyphenyl)-N,N-di-
methyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]cyclopenta[d]-
furan-2-carboxamide (71). A mixture of the O,O-dibenzylsilvestrol
7027 (35 mg, 0.042 mmol, 1.0 equiv) and lithium hydroxide (24 mg,
1.0 mmol, 24 equiv) in 4 mL of 3:1 methanol−water was stirred at 23
°C for 17 h and then heated at 50 C for 4 h. The resulting mixture was
cooled and acidfied with 1.0 M HCl to pH 5−6, and extraction was
with DCM (3 × 5 mL). The combined DCM layers were dried over
sodium sulfate. The solvents were evaporated in vacuo to give the acid
(32.3 mg, 0.039 mmol) as a white solid. To a mixture of the acid (20.1
mg, 0.024 mmol, 1.0 equiv), HOBt (9.7 mg, 0.072 mmol, 3.0 equiv),
dimethylamine hydrochloride (9.1 mg, 0.111 mmol, 4.65 equiv), and
EDCI (12.9 mg, 0.067 mmol, 2.8 equiv) in 1 mL DCM at 22 °C was
added diisopropylethylamine (42 μL, 0.288 mmol, 12.0 equiv) for 45
min before diluting with DCM (2 mL) and 10% citric acid (2 mL).
The organic layer was collected. The aqueous layer was extracted with
DCM (5 mL). The combined organic layers were washed with
saturated NaHCO3 (2 mL), water (2 mL) and dried (Na2SO4). The
drying agent was filtered off and the filtrate was concentrated in vacuo
and the crude was purified by silica gel chromatography (4 g, ISCO)
using 0% → 5% MeOH in DCM to give the amide (20.2 mg, 0.023
mmol) of as a solid. A mixture of the O,O-dibenzylamide (19.0 mg,
0.022 mmol, 1.0 equiv) and 5% palladium hydroxide on carbon (21.8
mg, 0.0077 mmol, 0.35 equiv) in 4 mL of THF was evacuated and
filled with hydrogen four times and stirred under hydrogen for 1.5 h.
The hydrogen supply was disconnected, flushed with nitrogen, and the
catalyst was filtered off. The filtrate was concentrated in vacuo and the
residue was purified on silica gel (4 g, ISCO), using 0% → 6% MeOH
in DCM, and lyophilized to give 12.6 mg (0.019 mmol, 84%) of 71 as
a white powder. NMR δH (400 MHz, CDCl3) 7.10−7.01 (m, 5H),
6.84−6.82 (m, 2H), 6.70−6.67 (m, 2H), 6.43 (d, J = 2.0 Hz, 1H), 6.26
(d, J = 2.0 Hz, 1H), 5.29 (brs, 1H), 4.96 (d, J = 7.0 Hz, 1H), 4.57 (brs,
1H), 4.49 (d, J = 13.4 Hz, 1H), 4.19−4.09 (m, 3H), 4.02 (dd, J = 13.4,
6.8 Hz, 1H), 3.85 (s, 3H), 3.71 (s, 3H), 3.55−3.45 (m, 3H), 3.48 (s,
3H), 3.29 (s, 3H), 2.92 (s, 3H), 2.13 (brs, 1H). ESI-MS m/z = 668.33
[M + H]+. Purity (method A): 98.4% AUC.
(1R,2R,3S,3aR,8bS)-1,8b-Dihydroxy-6-((2S,6S)-6-(hydroxymeth-
yl)-1,4-dioxan-2-yloxy)-8-methoxy-3a-(4-methoxyphenyl)-N,N-di-
methyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo[b]cyclopenta[d]-
furan-2-carboxamide (73). To a solution of ester 72 (46 mg, 0.067
mmol) in MeOH (4 mL) was added 2 mL of 1 M LiOH. This solution
was stirred for 16 h at 45 °C, cooled to room, and quenched with 1 N
HCl (1 mL). The aqueous solution was extracted with DCM (30 mL).
The organic layer was dried over MgSO4, filtered, and concentrated in
vacuo to afford the crude acid (45 mg) as a white solid. A solution of
the crude carboxylic acid (45 mg, 0.0067 mmol), EDCI (31 mg, 0.20
mmol), HOBt (27 mg, 0.020 mmol), and dimethylamine HCl salt (16
mg, 0.20 mmol) in 2 mL of anhydrous CH2Cl2 was chilled at 0 °C.
After 5 min, Hunig's base (119 μL, 0.67 mmol) was added dropwise.
After 10 min at 0 °C, the solution was stirred for an additional 12 h at
room temperature. The solution was concentrated and the material
was purified by flash chromatography and then by HPLC to give 35
mg of desired amide. To the latter (35 mg, 0.050 mmol) in THF (3
mL) was added (10%) Pd(OH)2 on carbon (5 mg). After three
purge−fill cycles, the vessel was placed under 1 atm of hydrogen. After
completion of the reaction, the vessel was purged and catalyst was
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filtered off from Celite. The Celite was washed with THF (3 × 5 mL)
and the combined filtrate was concentrated and purified on silica gel to
give 12 mg of the desired product 73. NMR δH (400 MHz, CDCl3)
7.13 (d, J = 8.8 Hz, 2H), 7.02−7.0 (m, 3H), 6.89−6.87 (m, 2H), 6.66
(d, J = 8.8 Hz, 2H), 6.54 (d, J = 2 Hz, 1H), 6.28 (d, J = 2 Hz, 1H),
5.33 (brs, 1H), 4.95 (dd, J = 1.6, 6 Hz, 1H), 4.57 (d, J = 13.6 Hz, 1H),
4.18−4.12 (m, 1H), 4.10 (brs, 1H), 4.08 (dd, J = 13.6, 6 Hz, 1H), 3.94
(d, J = 12 Hz, 1H), 3.89−3.85 (m, 1H), 3.85 (s, 3H), 3.77−3.72 (m,
3H), 3.69 (s, 3H), 3.56−3.53 (m, 2H), 3.32 (s, 3H), 2.94 (s, 3H),
1.89−1.82 (m, 1H); ESI-MS m/z = 608.4 [M + H]+. Purity (method
B): 93.4% AUC.
(1R,2R,3S,3aR,8bS)-3a-(4-Cyanophenyl)-1,8b-dihydroxy-6,8-di-
methoxy-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-benzo-
[b]cyclopenta[d]furan-2-carboxamide (76). To a solution of the
ester 69 (25 mg, 0.051 mmol) in MeOH (3 mL) was added 0.13 mL
of 2 M LiOH. This solution was stirred for 16 h at 45 °C, cooled to
room temperature, and quenched with 1 N HCl (1 mL). The aqueous
solution was extracted with DCM (30 mL). The organic layer was
dried over Na2SO4, filtered, and concentrated in vacuo to afford the
crude acid as a white solid. A solution of the crude carboxylic acid (23
mg, 0.049 mmol), EDCI (28 mg, 0.15 mmol), HOBt (20 mg, 0.15
mmol), and dimethylamine HCl salt (20 mg, 0.24 mmol) in 2 mL of
anhydrous CH2Cl2 was chilled at 0 °C. After 5 min, Hunig's base (51
uL, 0.29 mmol) was added dropwise. After 10 min at 0 °C, the
solution was stirred for an additional 12 h at room temperature. The
solution was concentrated and the material was purified by normal
phase column to give 9 mg of the desired product 76. NMR δH (400
MHz, CDCl3) 7.37 (d, J = 8 Hz, 2H), 7.29 (d, J = 8 Hz, 1H), 7.02−
7.00 (m, 3H), 6.83−6.81 (m, 2H), 6.27 (s, 1H), 6.10 (d, J = 7 Hz,
1H), 4.87 (d, J = 6 Hz, 1H), 4.62 (d, J = 14 Hz, 1H), 3.90 (dd, J = 14,
6 Hz, 1H), 3.82 (s, 6H), 3.31 (s, 3H), 2.91 (s, 3H); NMR δC (100
MHz, CDCl3) 169.44, 164.32, 161.0, 157.5, 141.51, 136.89, 130.86,
128.62, 128.13, 127.61, 126.89, 119.4, 110.90, 107.04, 101.61, 94.50,
92.9, 89.43, 78.88, 56.8, 55.87, 47.7, 37.5, 36.2; ESI-MS m/z = 501.03
[M + H]+. Purity (method A): 98.3% AUC.
Biological Evaluation. Stable Cell Line Generation. The c-Myc
and tubulin luciferase constructs (see Supporting Information) were
subcloned into the pLenti6/R4R2/V5-DEST lentiviral expression
vector following the Invitrogen Gateway cloning protocol (A11145).
Lentiviral particles were generated in 293FT cells using Invitrogen’s
ViraPower lentiviral expression kit. MDA-MB-231 (ATCC HTB-26)
cells were transduced with lentiviral supernatant according to the
manufacturer’s protocol. The cells were then selected with 10 μg/mL
blasticidin for 5 days to select for positive clones. These stables were
then maintained normally in RPMI-1640 medium supplemented with
10% fetal bovine serum.
Differential Translation Assay with c-myc 5′-UTR-Luciferase and
Tubulin 5′UTR-Luciferase in MDA-MB-231 Cells. MDA-MB-231 Myc-
luciferase and MDA-MB-231 tubulin-luciferase cells stably express
luciferase constructs and have been selected with 10 μg/mL blasticidin.
On day 1, cells were plated as follows: in a T162 flask, cells were
trypsinize with 3 mL of warm trypsin until cells were detached. Then 7
mL of warm medium (RPMI 1640 + 10% FBS + Pen/Strep) was
added to neutralize trypsin and cells were counted. Cells were diluted
to 100 000 cells/mL and then plated 100 μL/well for a final count of
10 000 cells/well in to CoStar 3903 TC-treated 96-well white-walled
polystyrene plate. Cells were incubated at 37 °C (5% CO2). On day 2,
compound dilution plate was prepared by diluting compounds from 10
mM DMSO stocks (stored at room temperaturein the dark), 1:5 in
DMSO to 200× final concentration. Intermediate plate was prepared
by adding 2 μL of compound stock plate into a fresh 96-well
polypropylene plate. Compounds were diluted 1:20 with medium
(RPMI 1640 + 10% FBS + Pen/Strep) by adding 40 μL with mixing.
Compound from the dilution plate (10 uL) was added to the cells,
which were then incubated at 37 °C (5% CO2). On day 3 (luciferase
assay), reconstituted Steady-Glo luciferase assay buffer with substrate
(Promega E2520 Steady-Glo luciferase assay kit) (100 μL) was added
to the cells, which were incubated at room temperature in the dark for
10 min. Then a white 96-well plate seal (Perkin-Elmer 6005199) was
added to the bottom of the plate to prevent leakage of signal. Plates
were read on an Envision 0.5s luminescence instrument. Signal is
proportional to luciferase expression. On day 5 (72h MTS cell growth
assay), the CellTiter 96 AQueous One solution cell proliferation assay
(MTS, Promega) was used to measure the metabolic activity of cells
by measuring the conversion of the MTS tetrazolium compound to a
colored formazan product that is soluble in tissue culture medium. The
assay can be used to measure both cell viability and growth. At the end
of the incubation period, formazan absorbance is measured at 490 nm
according to manufacturer’s instructions. The percent inhibition with
respect to compound concentration was plotted using Prism graphing
(1R,2R,3S,3aR,8bS)-6-Cyano-1,8b-dihydroxy-8-methoxy-3a-(4-
methoxyphenyl)-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-
benzo[b]cyclopenta[d]furan-2-carboxamide (74). A solution of the
ester 61 (95 mg, 0.195 mol, 1.0 equiv) and 0.5 mL of 2.0 M lithium
hydroxide (1.0 mmol, 5.1 equiv) in 3 mL of methanol was heated at 50
°C for 2 h. The solution was cooled, acidified with 1.0 M HCl to pH
1−2, and diluted with DCM (10 mL) and water (10 mL). The organic
layer was collected. The aqueous layer was extracted with DCM(2 ×
10 mL). The combined organic layers were dried (Na2SO4). The
drying agent was filtered off and the solvent was evaporated in vacuo
to give 88.1 mg (0.186 mmol, 95%) of the crude acid. Then 10 mg of
the crude was purified by preparative TLC on silica gel (7% MeOH in
DCM) to give 6.1 mg of pure acid (ESI-MS m/z 474.1 [M + H]+). To
a mixture of the acid (15 mg, 0.032 mmol, 1.0 equiv), HOBt (14.5 mg,
0.10 mmol, 3.1 equiv), dimethylamine hydrochloride (15 mg, 0.18
mmol, 6.0 equiv), and EDCI (22 mg, 0.11 mmol, 3.4 equiv) in 1 mL of
DCM at 22 °C was added diisopropylethylamine (34 μL, 0.19 mmol,
6.0 equiv) before diluting with DCM (2 mL) and 10% citric acid (2
mL). The organic layer was collected. The aqueous layer was extracted
with DCM (5 mL). The combined organic layers were washed with
saturated NaHCO3 (2 mL), water (2 mL) and dried (Na2SO4). The
drying agent was filtered off. The filtrate was concentrated in vacuo.
The crude was purified by preparative TLC on silica gel (5% MeOH in
DCM) and the desired product was dissolved in 2 mL of 90% tert-
butanol, lyophilized to give 9.7 mg (0.019 mmol, 61%) of 74. NMR δH
(400 MHz, CDCl3) 7.09−7.03 (m, 5H), 7.00 (d, J = 0.8 Hz, 1H), 6.90
(d, J = 6.8 Hz, 2H), 6.77 (d, J = 0.8 Hz, 1H), 6.67 (d, J = 9.2 Hz, 2H),
4.77 (dd, J = 5.2, 2.3 Hz, 1H), 4.67 (d, J = 13.4 Hz, 1H), 4.20 (dd, J =
13.4, 5.2 Hz, 1H), 3.91 (s, 3H), 3.69 (s, 3H), 3.36 (s, 3H), 2.97 (s,
3H), 2.09 (s, 1H); NMR δC (100 MHz, CDCl3); 170.18, 160.28,
158.78, 157.45, 136.78, 128.60, 127.87, 127.70, 126.55, 126.23, 120.20,
118.61, 114.83, 112.85, 108.25, 107.55, 102.00, 93.72, 78.17, 77.31,
56.98, 56.14, 55.02, 46.81; ESI-MS m/z = 501.1 [M + H]+. Purity
(method A): 91% AUC.
(1R,2R,3S,3aR,8bS)-6-Chloro-1,8b-dihydroxy-8-methoxy-3a-(4-
methoxyphenyl)-N,N-dimethyl-3-phenyl-2,3,3a,8b-tetrahydro-1H-
benzo[b]cyclopenta[d]furan-2-carboxamide (75). To a solution of
the ester 62 (30 mg, 0.06 mmol) in MeOH (6 mL) was added 1 mL of
1 M LiOH. This solution was stirred for 16 h at 45 °C, cooled to room
temperature, and quenched with 1 N HCl (1 mL). The aqueous
solution was extracted with DCM (30 mL). The organic layer was
dried over MgSO4, filtered, and concentrated in vacuo to afford the
crude acid (29 mg) as a white solid. A solution of the crude carboxylic
acid (29 mg, 0.06 mmol), EDCI (33 mg, 0.22 mmol), HOBt (29 mg,
0.022 mmol), and dimethylamine HCl salt (17 mg, 0.22 mmol) in 2
mL of anhydrous CH2Cl2 was chilled at 0 °C. After 5 min, Hunig's
base (129 uL, 0.72 mmol) was added dropwise. After 10 min at 0 °C,
the solution was stirred for an additional 12 h at room temperature.
The solution was concentrated and the material was purified by HPLC
to give 24 mg of the desired product 75. NMR δH (400 MHz, CDCl3)
7.09−7.03 (m, 5H), 6.87 (brd, J = 7.2 Hz, 2H), 6.74 (d, J = 1.6 Hz,
1H), 6.67 (d, J = 8.8 Hz, 2H), 6.51 (d, J = 1.6 Hz, 1H), 4.85 (dd, J =
1.6, 5.6 Hz, 1H), 4.60 (d, J = 13.6 Hz, 1H), 3.92 (dd, J = 6, 13.6 Hz,
1H), 4.04 (d, J = 2.4 Hz, 1H), 3.87 (s, 3H), 3.70 (s, 3H), 3.33 (s, 3H),
2.95 (s, 3H), 1.95 (s, 1H). NMR δC (100 MHz, CDCl3) 169.88,
160.58, 158.71, 157.15, 137.33, 137.22, 128.78, 127.82, 127.77, 126.67,
126.45, 113.75, 112.83, 105.32, 104.87, 101.92, 93.84, 78.36, 56.51,
56.04, 55.09, 47.26. ESI-MS m/z = 510.1 [M + H]+. Purity (method
C): >95%.
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software on a semilog plot, and EC50 values were determined by
nonlinear regression analysis with a four-parameter logistic equation.
Cell Viability Assay on L3.6pl and RPMI-8226. L3.6pl cells were
maintained in DMEM medium with 10% FBS. RPMI-8226 (ATCC
CCL-155) cells were maintained in RPMI-1640 medium with 10%
FBS. Cells were treated with compound for 4−24 h at 37 °C and 5%
CO2 in 10 cm tissue culture treated Petri dishes.
The CellTiter-Glo luminescent cell viability assay (Promega)
measures the number of viable cells based on the cellular ATP levels.
The luminescent signal is proportional to the amount of ATP present
which correlates to the number of cells present. The assay is run
according to the manufacturer’s instructions. Briefly, the cells are
plated in 96-well plates and treated with compounds as described
above. After the incubation period, the combined lysis and luciferin
reagent is added and luciferase is measured on a luminometer. In order
to distinguish cell growth inhibition from loss of cell viability, a
separate plate was taken for a time zero read that indicates starting cell
number to be used to determine level of cell killing at the end of the
incubation period.
EDTA, 3 mM of MgCl2, and 0.5 mg/mL human liver microsomes.
Compounds were added to a v-bottom 96-well plate (Costar, Corning,
NY). Reaction solution was preincubated at 37 °C for 5 min. Reaction
mixture was added to each 96-well plate. To start the reaction,
NADPH was added to each plate at a final concentration of 1 mg/mL.
Reaction plates were incubated at 37 °C. Reaction was stopped at
designated time points (0, 5, 15, 30, 45, and 60 min) by adding two
volumes of acetonitrile containing internal standard. Samples were
placed in plate centrifuge (Eppendorf) and spun at 1550 rcf for 10
min. The supernatant was taken and diluted with 1 volume of water in
a 96-well autosampler plate with inert glass inserts (MicroLiter).
Samples were analyzed by LC−MS/MS. A 2.0 mm × 25 mm, 5 μm
Phenomenex Gemini C18 column (Torrance, CA) was used, and
samples were analyzed using an Applied Biosystems 4000QTrap mass
spectrometer. Half-lives were determined by the slope of the line for
percent remaining parent compound over time course.
Pharmacokinetic Studies. Dosing. Each compound dissolved in
aqueous formulation containing 5.2% PEG-400, 5.2% Tween 80, 2%
DMSO was administered intravenously or intraperitoneally to CD-1
mice. At 0.083, 0.25, 0.5, 1, 2, 4, 6, and 24 h postdose, blood was
collected and processed to plasma by centrifugation and stored at −80
°C until analysis.
The 7-aminoactinomycin D (7-AAD) (Millipore) exclusion assay
was used to measure cell death. 7-AAD is a cell impermeable nucleic
acid dye that is excluded from viable cells but penetrates dead or
damaged cells to label DNA. Cells were treated for 72 h with
compound followed by incubation with 7-AAD according to the
manufacturer’s instructions.
Bioanalytical Analysis. Plasma samples were thawed at room
temperature and processed by protein precipitation with two volumes
of acetonitrile containing an internal standard. After centrifugation, the
supernatants of the precipitated plasma samples were diluted 1:1 with
water and analyzed by LC−MS/MS. A standard curve was prepared in
plasma (Bioreclamation LLC) from 0.5 to 1000 ng/mL and processed
in the same way with internal standard solution as the samples. Sample
analysis was performed on an Agilent 1200 series (Foster City, CA)
with an API4000 QTrap mass spectrometer from Applied Biosystems
(Foster City, CA). Samples were injected on an analytical column (2.0
mm × 25 mm, 5 μm Phenomenex Gemini C18 column (Torrance,
CA)), and compounds eluted from the analytical column with a 5 min
gradient from 30% to 95% acetonitrile in H2O, 5 mM ammonium
bicarbonate. Mass spectrometric detection of the drug as well as the
internal standard was performed by MRM in negative mode. The data
were acquired and processed using the software Analyst 1.4 (Applied
Biosystems, Foster City, CA). The pharmacokinetics of each
compound were analyzed by WinNonlin 5.0.1 (Pharsight, Mountain
View, CA) via noncompartmental analysis.
Western Blot Analysis. RPMI-8226 (ATCC CCL-155) cells were
maintained in RPMI-1640 medium with 10% FBS. L3.6pl cells were
maintained in DMEM medium with 10% FBS. Cells were treated with
compound for 4−24 h at 37 °C and 5% CO2 in 10 cm tissue culture
treated Petri dishes. After incubation, cells were washed in PBS twice
and collected by centrifugation. Cells were lysed in RIPA buffer
containing protease inhibitor cocktail (∼100 μL) and incubated for 30
min on ice followed by centrifugation at 20000g. Protein
concentrations were directly quantified using BCA protein assay kit
(Pierce no. 23227) according to the manufacturer’s instructions.
Samples were diluted in LD loading buffer with reducing agent to a
uniform concentration. Samples were denatured by boiling for 5 min.
For Western blotting analysis, equal protein quantities were loaded
onto a 4−12% Bis-Tris SDS−PAGE gel, and electrophoresis
separation was followed by protein transfer onto PVDF membranes
(Invitrogen no. LC2005). Membranes were probed with appropriate
primary antibodies in BSA saturation buffer (PBS/0.1% Tween, 5%
BSA) for anti-c-myc (from rabbit, diluted 1:1000, CST 5605) and
antiactin (from rabbit, diluted 1:5000, Santa Cruz, CA). The
secondary antibodies corresponding to anti-rabbit were coupled to
HRP (from goat, diluted 1:5000, Amersham). The signal was
enhanced by chemiluminescence using SuperSignal West Pico kit
(Pierce no. 34080) and detected by camera (Biorad).
In Vitro Metabolism Assays and Mouse Pharmacokinetic
Studies. Plasma Stability Assay. Test compounds and controls
(propranolol) were tested at 1000 nM in frozen mouse/human plasma
(Bioreclamation LLC, Westbury, NY). Compounds were spiked into
mouse/human plasma preincubated to 37 °C in a heated plate shaker
(Eppendorf Thermomixer R). Reaction was stopped at designated
time points (0, 5, 15, 30, 45, 60, 90, and 120 min) by adding three
volumes of acetonitrile containing internal standard. Samples were
transferred to a 96-well filter plate (Millipore Multiscreen Solvinert
0.45 μM hydrophobic PTFE) and spun at 1550 rcf for 5 min
(Eppendorf centrifuge 5804). The samples were taken and diluted
with one volume of water in a 96-well autosampler plate with inert
glass inserts (MicroLiter). Samples were analyzed by LC−MS/MS
using a 2.0 mm × 25 mm, 5 μm Phenomenex Gemini C18 column
(Torrance, CA), and samples were analyzed using an Applied
Biosystems 4000QTrap mass spectrometer. Half-lives were deter-
mined by the slope of the line for the percent remaining parent
compound over time course.
ASSOCIATED CONTENT
* Supporting Information
■
S
Description of the procedure and physical characterization for
synthetic intermediates made in Schemes 2, 3, and 6; PDF
copies of physical data (1H and 13C NMR spectra, mass
spectrometry, HPLC) for tested compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 617-453-1268. Fax: 617-682-1418. E-mail: Martin.
Tremblay@infi.com.
■
Present Addresses
‡Jefferson School of Pharmacy, Philadelphia, PA 19107.
§Novartis Institutes for Biomedical Research, Cambridge, MA
02139.
Author Contributions
†These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
Microsomes Stability Assay. Test compounds and controls
(propranolol) were tested at 500 nM in pooled mouse and human
liver microsomes (BD Biosciences, Woburn, MA). An incubation
solution was made in 100 mM phosphate buffer containing 1 mM
ACKNOWLEDGMENTS
The authors thank Prof. Dr. Mark A. Rizzacasa for providing an
authentic sample of silvestrol. The authors thank Elizabeth
■
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Article
Jr.; Pelletier, J. Therapeutic suppression of translation initiation
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Clarke, Alice Lim, and Cassandra Coleman for preliminary
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́
(17) Cencic, R.; Carrier, M.; Galicia-Vazquez, G.; Bordeleau, M.-E.;
ABBREVIATIONS USED
■
Sukarieh, R.; Bourdeau, A.; Brem, B.; Teodoro, J. G.; Greger, H.;
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4E-BP, eIF4E binding protein; Akt, protein kinase B; AUG,
adenine−uracil−guanine; BIW, twice a week; c-myc, myelocy-
tomatosis oncogene; eIF4E, eukaryotic initiation factor 4E;
eIF4F, eukaryotic initiation factor 4F; LUC, luciferase; mTOR,
mammalian target of rapamycin; MEK, mitogen-activated
protein kinase kinase; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium;
PDCD4, programmed cell death protein 4; PEG, polyethylene
glycol; PTEN, phosphatase and tensin homologue; q.d., daily
dosing; rac, racemic; Raf, rapidly accelerated fibrosarcoma
kinase; Ras, rat sarcoma; UTR, untranslated region
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