Unexpected stereochemical tolerance for the biological activity of tyroscherin

Article abstract of DOI:10.1016/j.bmc.2011.01.027

Here we describe the concise syntheses of the 15 diastereomers and key analogs of the natural product tyroscherin. While systematic analysis of the analogs clearly demonstrated that the hydrocarbon tail is important for biological activity, structure-activity relationship studies of the complete tyroscherin diastereoarray revealed a surprisingly expansive stereochemical tolerance for the cytotoxic activity. Our results represent a departure from the tenet that biological activity is constrained to a narrow pharmacophore, and highlight the recently emerging appreciation for stereochemical flexibility in defining the essential structural elements of biologically active small molecules.

Full text of DOI:10.1016/j.bmc.2011.01.027

Bioorganic & Medicinal Chemistry 19 (2011) 1708–1713
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Unexpected stereochemical tolerance for the biological activity of tyroscherin
Hyun Seop Tae a, John Hines a, Ashley R. Schneekloth b, Craig M. Crews a,b,c,
⇑
a Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06511, USA
b Department of Chemistry, Yale University, New Haven, CT 06511, USA
c Department of Pharmacology, Yale University, New Haven, CT 06511, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Here we describe the concise syntheses of the 15 diastereomers and key analogs of the natural product
tyroscherin. While systematic analysis of the analogs clearly demonstrated that the hydrocarbon tail is
important for biological activity, structure–activity relationship studies of the complete tyroscherin dia-
stereoarray revealed a surprisingly expansive stereochemical tolerance for the cytotoxic activity. Our
results represent a departure from the tenet that biological activity is constrained to a narrow pharma-
cophore, and highlight the recently emerging appreciation for stereochemical flexibility in defining the
essential structural elements of biologically active small molecules.
Received 2 December 2010
Revised 7 January 2011
Accepted 13 January 2011
Available online 19 January 2011
Keywords:
Tyroscherin
Total synthesis
Ó 2011 Elsevier Ltd. All rights reserved.
Structure–activity relationship
Stereochemical tolerance
1. Introduction
with racemic mixtures, and could also have an impact on frag-
ment-based drug discovery.7–9
It is widely believed that the pharmacological properties of a
biologically active small molecule are inextricably linked to both
its pendant functional groupings and their relative orientations in
space.1,2 Thus, systematic alteration of structural elements and/or
their stereochemistry would be expected to significantly impact
the biological activity of a natural product, thereby providing use-
ful information about its pharmacophore. Today, most new drugs
require only a single enantiomer,3 since typically only one enantio-
mer of a racemic mixture binds to a target protein; the other enan-
tiomer often plays a different role, ranging from significantly
reduced binding affinity for the same target protein to interactions
with other non-target proteins. Nonetheless, Schreiber and
coworkers have reported some tolerance for the binding of diaste-
reomeric ligands to a common target.4 Their study focused on pep-
tide binding to the SH3 domain of the tyrosine kinase Src, where
stereochemical variation of a single stereocenter attached to the
C-terminus of a poly-proline hexamer resulted in nominal change
to the dissociation constant. More recently, Breinbauer and
coworkers have discovered the first example of the simultaneous
(i.e., non-competitive) binding of both enantiomers of a chiral mol-
ecule to the active site of an enzyme.5,6 Although the consequence
of these stereochemical changes beyond binding affinity was not
determined in these cases (i.e., their impact on the functional re-
sponse was not determined), these findings could have ramifica-
tions for drug screening studies, which are commonly performed
Tyroscherin was originally reported to inhibit insulin-like
growth factor-1 (IGF-1)-induced growth of MCF-7 human breast
cancer cells more potently than bovine serum (FBS)-induced
growth.10,11 The reported highly selective inhibition of IGF-1 in-
duced growth of MCF-7 cancer cells made this molecule an inter-
esting tool for chemical biology studies, with the only activity
analyses of this natural product having been performed by the
groups that originally isolated it.11
Two total syntheses of tyroscherin have been communicated by
the Watanabe group and the Maier group.11–13 Recently, we also
reported the total synthesis and biological evaluation of tyroscher-
in utilizing a cross metathesis approach (Fig. 1).14 Our aim was to
correct the initially reported tyroscherin structure and to investi-
gate its putative IGF-specificity, the latter of which we failed to
confirm.
Herein, we report a concise and convergent synthesis of the 15
diastereomers and key analogs of tyroscherin. Fascinatingly, an
SAR study of these tyroscherin diastereomers uncovered an unex-
pected and intriguing stereochemical tolerance for the shared cyto-
toxicity. This observation may find relevance in drug discovery
programs involving the screening of optically active molecules.
2. Results and discussion
2.1. Syntheses and biological characterization of tyroscherin
diastereomers
To establish extensive SAR studies and to determine the correct
structure of tyroscherin, we synthesized all possible stereoisomers
⇑
Corresponding author. Tel.: +1 203 432 9364; fax: +1 203 432 6161.
E-mail address: craig.crews@yale.edu (C.M. Crews).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.027

H. S. Tae et al. / Bioorg. Med. Chem. 19 (2011) 1708–1713
1709
Figure 1. Structure of reported tyroscherin 1 and revised tyroscherin 2.
Figure 2. Synthesis of stereoarrayed 16 tyroscherin stereoisomers. TS, TyroScherin.
of 1 using a cross metathesis reaction we had employed to gener-
ate tyroscherin 1 (Fig. 2).14 As shown in Figure 2, this was achieved
with eight stereochemically different fragments: 3, 4, 5, 6, 7, 8, 9,
and 10. As previously reported, TS8 matched in all spectral and
physical data to the published natural product.14
All diastereomers were examined for their ability to inhibit ser-
um-driven mitogenesis versus IGF-driven mitogenesis. Surpris-
ingly, despite the diversity of the stereochemical alterations
implemented, all diastereomers had virtually identical biological
activities (Table 1). Thus, while tyroscherin is a potent cytotoxic
agent, we were unable to validate the claim of its enhanced po-
tency to inhibit IGF signaling. Instead, our data support our previ-
ous findings in that tyroscherin acts as a more generalized anti-
proliferative agent against many growth factors.
This implies that all tyroscherin diastereomers not only bind
their target with uniform affinity, but also retain the same degree
of activity—that is, none of the bound diastereomers elicited a neu-
tralized or reversed pharmacodynamic effect. Thus, the syntheses
and biological characterization of all possible tyroscherin diaste-
reomers revealed an unexpected and expansive stereochemical tol-
erance for the observed cytotoxic activity. While there were
occasional minor differences in relative potency of inhibition be-
tween serum and IGF-driven proliferation, none of them were of
the magnitude reported in the original isolation or recent struc-
tural revision report.11,12
Table 1
Inhibitory activity of 16 TS stereoisomers using MCF-7 cellsa
Compound
10 ng/mL IGF
IC50
(
lM)
10% FBS
100 ng/mL IGF
TS1 (2R,3R,8S,10S)
TS2 (2R,3R,8R,10R)
TS3 (2S,3S,8S,10S)
TS4 (2S,3S,8R,10R)
TS5 (2R,3R,8S,10R)
TS6 (2S,3R,8S,10S)
TS7 (2R,3S,8R,10R)
TS8 (2S,3R,8R,10R)
TS9 (2S,3S,8S,10R)
TS10 (2R,3S,8S,10R)
TS11 (2S,3R,8S,10R)
TS12 (2R,3R,8R,10S)
TS13 (2S,3S,8R,10S)
TS14 (2S,3R,8R,10S)
TS15 (2R,3S,8R,10S)
TS16 (2R,3S,8S,10S)
Reported tyroscherin
2.07 0.40
2.00 0.69
1.77 0.25
1.90 0.44
2.07 0.23
1.83 0.49
3.63 2.39
2.23 0.74
3.10 1.74
2.90 1.68
2.00 0.53
2.50 1.04
2.76 0.60
2.77 1.55
2.90 1.93
1.87 0.31
2.27 0.31
1.87 0.46
1.83 0.25
2.27 1.07
1.97 0.65
2.20 0.70
2.33 0.25
3.07 0.67
3.13 1.29
2.03 0.23
2.30 0.36
3.07 0.90
2.23 0.35
2.76 1.65
2.90 0.98
2.73 1.46
4.60 0.79
4.23 1.00
5.40 3.15
5.97 2.15
4.83 2.23
4.47 2.90
4.93 4.14
7.13 2.78
6.47 2.10
4.80 2.34
7.03 5.70
5.70 6.38
7.37 5.24
6.07 5.19
7.67 0.76
8.87 1.30
2.2. Syntheses and biological characterization of tyroscherin
analogs
The surprising results concerning the conserved biological
activity among the diastereomers led us to question whether the
hydrocarbon tail might be unimportant for biological activity.
Thus, in the next phase of our tyroscherin structure–activity rela-
tionship studies, we synthesized five structurally distinct tyrosch-
erin analogs: 12, 15, 17, 19, and 23 according to our reported
methods (Schemes 1–3).14
30 nM (in serum-free medium containing IGF-1)
The truncated analog 12 was prepared by the treatment of 11
with TFA. Analog 15 was obtained via a four-step sequence from
a
3H-Thymidine incorporation assay.

1710
H. S. Tae et al. / Bioorg. Med. Chem. 19 (2011) 1708–1713
Scheme 1. Synthesis of tyroscherin analogs 12 and 15.
Scheme 2. Synthesis of tyroscherin analogs 17 and 19.
Scheme 3. Synthesis of tyroscherin analog 23.
alcohol 11. Protection of hydroxyl group as an acetate and cross
metathesis with 1-heptene in the presence of 5 mol % Grubbs cat-
alyst 13 afforded exclusively the E-isomer of the adduct 14.15,16
Deprotection of acetyl in 14 with DIBAL followed by deprotection
of Boc and tert-butyl groups with TFA gave analog 15.
ether 16 via a five-step sequence. Treatment of ether 16 with TBAF
afforded the alcohol, and oxidation of the alcohol with PDC and
DMF provided acid 18. Coupling reaction of acid 18 and amino
diphenylmethane using EDCI, deprotection of acetate with DIBAL,
and treatment of TFA gave analog 19.
Analog 17 was achieved from the known intermediate 16. Three
sequential deprotections of 16 with DIBAL, TBAF, and 4 N-HCl pro-
duced analog 17 having a terminal hydroxyl at the right-side. Ana-
log 19 with a bulky benzhydryl amide was obtained from TBDPS
Scheme 3 summarizes the synthesis of analog 23 that removed
the hydroxyl group from the tyrosine fragment. Cross metathesis
of fragment 20 with fragment 7 produced the E-isomer of the ad-
duct 21. Treatment of TBDPS ether 21 with TBAF gave the alcohol,

H. S. Tae et al. / Bioorg. Med. Chem. 19 (2011) 1708–1713
1711
Table 2
Our results obtained with a functional readout are a noteworthy
advance over previous demonstrations of stereochemical toler-
ance, which were limited to measuring binding affinities without
determining the biological impact of the stereochemical altera-
tions. Moreover, this study sheds new light on the stereochemical
requirements (or potential lack thereof) of biologically active nat-
ural products, and provides proof of a new type of enantiomer
behavior that may have important implications for drug discovery.
Efforts to identify the in vivo biological target of tyroscherin and
elucidate its mode of action are currently underway.
Growth inhibitory activity of TS analogs using MCF-7 cellsa
Compound
10 ng/mL IGF
IC50
(l
M)
10% FBS
100 ng/mL IGF
12
15
17
19
23
TS8
>100
>100
>100
18.2 6.33
149 26.7
19.1 4.95
2.97 1.34
2.23 0.74
24.0 6.68
76.5 16.3
28.6 6.78
2.63 0.87
3.07 0.67
19.8 6.86
92.6 18.6
13.7 3.32
5.17 4.89
7.13 2.78
Reported tyroscherin
30 nM (in a serum-free medium containing IGF-1)
a
3H-Thymidine incorporation assay.
4. Materials and methods
and mesylation of this primary alcohol followed by treatment with
MeMgBr and CuI resulted in the addition of the terminal methyl
group. Finally, deprotection using 4 N-HCl in 1,4-dioxane afforded
analog 23.
Eight key fragments and tyroscherin stereoisomers (TS1–TS16)
were synthesized by the reported procedure (Org. Lett. 2010, 12,
4308–4311). See Supplementary data for the detailed experimental
procedure.
These analogs were then tested in the same MCF-7 cell anti-
proliferation assays used to analyze tyroscherin diastereomers
TS1 through TS16 (Table 2). Paradoxically, these analogs clearly
demonstrated that the hydrocarbon tail was important for biolog-
ical activity even though its stereochemistry was found to be
unimportant. First, truncation of the hydrocarbon tail led to a bio-
logically inactive molecule 12, despite the fact that it retained the
olefin and all hydrogen-bond donors/acceptors. Similarly, removal
of the methyl groups at the C8 and C10 positions of 1 or the addi-
tion of a polar hydroxyl group at the right terminal led to signifi-
cant reductions in the activity.
The results of analogs 12, 15, and 17 led to speculation that the
length and/or overall hydrophobicity of the tail is important for
biological activity of tyroscherin. Thus, we introduced a bulky
benzhydryl amide group (19) to the right end of the tail: despite
the steric bulk of the additional group, the hydrocarbon tail of 19
is arguably closer in length and hydrophobicity to that of TS8 than
were those of analogs 12 and 17. Interestingly, while the activity of
analog 19 was somewhat less than TS8 itself, analog 19 was indeed
far more potent than analogs 12 and 17. Tangential to this, removal
of the aromatic hydroxyl group (23) had no impact on activity.
While it had been tempting to speculate that phosphorylation of
tyroscherin at this position by a tyrosine kinase might have been
key for its biological activity, the finding with analog 23 indicates
this mechanism is unlikely. Taken together, the data obtained indi-
cates that the hydrocarbon tail vis-à-vis it’s overall length or de-
gree of hydrophobicity is essential for biological activity. Further
studies are therefore needed to investigate SAR studies of the re-
vised tyroscherin.
4.1. Tyroscherin diastereomers
TS1: 1H NMR (500 MHz, CD3OD) d 7.05 (d, J = 8.5 Hz, 2H), 6.72
(d, J = 8.5 Hz, 2H), 5.32 (dt, J = 15.3, 6.7 Hz, 1H), 5.17 (dd, J = 15.3,
8.2 Hz, 1H), 3.53 (dt, J = 9.0, 3.9 Hz, 1H), 2.77–2.72 (m, 1H), 2.72
(dd, J = 11.8, 5.6 Hz, 1H), 2.67 (dd, J = 11.8, 5.3 Hz, 1H), 2.40 (s,
3H), 2.15–2.06 (m, 2H), 2.02–1.96 (m, 1H), 1.59–1.54 (m, 1H),
1.50–1.44 (m, 1H), 1.35–1.25 (m, 2H), 1.22 (ddd, J = 13.9, 9.6,
4.6 Hz, 1H), 1.15–1.09 (m, 1H), 0.97 (ddd, J = 13.8, 9.0, 5.1 Hz,
1H), 0.90 (d, J = 6.7 Hz, 3H), 0.84 (t, J = 7.3 Hz, 3H), 0.81 (d,
J = 6.4 Hz, 3H). 13C NMR (125 MHz, CD3OD) d 157.1, 138.2, 131.2,
130.5, 129.0, 116.5, 71.2, 66.8, 45.6, 36.0, 35.7, 34.7, 34.2, 33.2,
31.1, 28.8, 22.3, 19.3, 11.6. FTIR (neat, cmꢀ1) 3239 (br), 2957,
2923, 2873, 1615, 1515, 1457, 1263, 1225. LRMS (ES+) [M+H]+
334.69, [M+Na]+ 356.64. HRMS (ES+) calculated for C21H36NO2
[M+H]+ 334.2746, found 334.2741. ½a 2D0
ꢁ
+21.5 (c 0.346, MeOH).
TLC (10% CH3OH in CH2Cl2), Rf 0.16 (UV, CAM).
TS2: 1H NMR (500 MHz, CD3OD) d 7.10 (d, J = 8.5 Hz, 2H), 6.76
(d, J = 8.5 Hz, 2H), 5.27 (dt, J = 15.2, 6.5 Hz, 1H), 5.16 (dd, J = 15.3,
8.2 Hz, 1H), 3.62 (dt, J = 8.0, 4.7 Hz, 1H), 3.14 (ddd, J = 8.6, 8.6,
2.9 Hz, 1H), 2.93 (dd, J = 14.1, 5.9 Hz, 1H), 2.85 (dd, J = 14.1,
8.4 Hz, 1H), 2.64 (s, 3H), 2.12–2.07 (m, 1H), 2.07–1.97 (m, 2H),
1.55–1.50 (m, 1H), 1.30–1.24 (m, 1H), 1.30–1.24 (m, 2H), 1.14–
1.07 (m, 1H), 1.20 (ddd, J = 13.7, 9.6, 4.3 Hz, 1H), 0.96 (ddd,
J = 13.9, 8.7, 5.1 Hz, 1H), 0.89 (d, J = 6.7 Hz, 3H), 0.84 (t, J = 7.3 Hz,
3H), 0.81 (d, J = 6.3 Hz, 3H). 13C NMR (125 MHz, CD3OD) d 157.8,
138.6, 131.3, 128.4, 127.9, 116.8, 68.9, 66.1, 45.5, 35.7, 35.3, 34.3,
33.1, 32.1, 31.1, 29.3, 22.3, 19.3, 11.6. LRMS (ES+) [M+H]+ 334.25,
[M+Na]+ 356.21, ½a 2D0
ꢀ20.1 (c 0.6, MeOH). TLC (10% CH3OH in
ꢁ
3. Conclusions
CH2Cl2), Rf 0.18 (UV, CAM).
TS3: 1H NMR (500 MHz, CD3OD) d 7.10 (d, J = 8.5 Hz, 2H), 6.76
(d, J = 8.5 Hz, 2H), 5.27 (dt, J = 15.3, 6.7 Hz, 1H), 5.15 (dd, J = 15.3,
8.2 Hz, 1H), 3.62 (dt, J = 8.0, 4.6 Hz, 1H), 3.16 (ddd, J = 8.5, 8.5,
5.6 Hz, 1H), 2.94 (dd, J = 14.1, 6.0 Hz, 1H), 2.86 (dd, J = 14.1,
8.5 Hz, 1H), 2.65 (s), 2.13–2.08 (m, 1H), 2.07–1.95 (m, 2H), 1.56–
1.50 (m, 2H), 1.31–1.24 (m, 2H), 1.14–1.08 (m, 1H), 1.20 (ddd,
J = 13.7, 9.6, 4.3 Hz, 1H), 0.95 (ddd, J = 13.6, 9.6, 5.1 Hz, 1H), 0.88
(d, J = 6.6 Hz, 3H), 0.84 (t, J = 7.3 Hz, 3H), 0.80 (d, J = 6.4 Hz, 3H).
13C NMR (125 MHz, CD3OD) d 157.8, 138.6, 131.3, 128.4, 127.8,
116.8, 68.8, 66.1, 45.5, 35.7, 35.3, 34.2, 33.1, 32.0, 31.1, 29.3,
In conclusion, our concise, unified syntheses of the 15 diaste-
reomers and key analogs of tyroscherin afforded a comprehensive
biological analysis of this natural product. Surprisingly, all diaste-
reomers possess similar anti-proliferative activity in cell prolifera-
tion assays irrespective of whether the cells were stimulated with
IGF or serum. We are able to show that this stereochemical toler-
ance does not extend to structural alterations in the hydrocarbon
tail, indicating a role for this segment of the natural product in
mediating its biological activity. The degree of tolerated stereo-
chemical divergence shown with tyroscherin involves all four inde-
pendent stereocenters, comprising the majority of the molecular
structure and demonstrating a remarkable stereochemical toler-
ance. The extent to which removal of specific functional groups
or reduction of hydrocarbon tail hydrophobicity or length can have
a negative effect on activity provides strong evidence that the ob-
served similarity in potency of all 16 diastereomers is due to a
common target, rather than non-specific effects.
22.3, 19.3, 11.6. LRMS (ES+) [M+H]+ 334.10, [M+Na]+ 356.21, ½a 2D0
ꢁ
+19.6 (c 0.75, MeOH). TLC (10% CH3OH in CH2Cl2), Rf 0.19 (UV,
CAM).
TS4: 1H NMR (500 MHz, CDCl3) d 7.11 (d, J = 8.5 Hz, 2H), 6.76 (d,
J = 8.5 Hz, 2H), 5.27 (dt, J = 15.3, 6.6 Hz, 1H), 5.15 (dd, J = 15.3,
8.2 Hz, 1H), 3.63 (dt, J = 11.0, 6.3 Hz, 1H), 3.21 (ddd, J = 8.6, 8.6,
5.5 Hz, 1H), 2.96 (dd, J = 14.2, 5.8 Hz, 1H), 2.87 (dd, J = 14.2,

1712
H. S. Tae et al. / Bioorg. Med. Chem. 19 (2011) 1708–1713
8.5 Hz, 1H), 2.67 (s, 3H), 2.15–2.07 (m, 1H), 2.06–1.95 (m, 2H),
1.56–1.51 (m, 2H), 1.31–1.24 (m, 2H), 1.15–1.07 (m, 1H), 1.20
(ddd, J = 13.9, 9.5, 4.6 Hz, 1H), 0.96 (ddd, J = 13.8, 8.8, 4.8 Hz, 1H),
0.89 (d, J = 6.7 Hz, 3H), 0.84 (t, J = 7.4 Hz, 3H), 0.81 (d, J = 6.4 Hz,
3H). 13C NMR (125 MHz, CD3OD) d 157.9, 138.5, 131.4, 128.3,
127.6, 116.8, 68.7, 65.9, 44.5, 35.7, 35.4, 34.1, 33.1, 31.8, 31.1,
139.1, 131.4, 127.9, 127.8, 116.8, 68.7, 66.0, 45.5, 35.4, 35.3, 34.2,
33.0, 32.0, 30.2, 29.3, 21.2, 19.8, 11.5. ½a D20
ꢁ
+7.9 (c 0.88, MeOH).
TLC (10% CH3OH in CH2Cl2), Rf 0.18 (UV, CAM).
TS10: 1H NMR (500 MHz, CD3OD) d 7.10 (d, J = 8.3 Hz, 2H), 6.76
(d, J = 8.2 Hz, 2H), 5.34 (dt, J = 15.3, 6.3 Hz, 1H), 5.28 (dd, J = 15.3,
7.2 Hz, 1H), 3.80 (dt, J = 9.3, 3.1 Hz, 1H), 3.24 (ddd, J = 7.2, 7.2,
2.6 Hz, 1H), 2.87 (dd, J = 14.7, 7.9 Hz, 1H), 2.82 (dd, J = 14.6,
6.9 Hz, 1H), 2.58 (s, 3H), 2.23–2.12 (m, 2H), 2.03–1.95 (m, 1H),
1.59–1.45 (m, 2H), 1.39–1.32 (m, 2H), 1.12–1.08 (m, 1H), 1.17
(ddd, J = 13.5, 6.7, 6.7 Hz, 1H), 1.08–1.02 (m, 1H), 0.89 (d,
J = 6.7 Hz, 3H), 0.85 (t, J = 7.3 Hz, 3H), 0.83 (d, J = 6.1 Hz, 3H). 13C
NMR (125 MHz, CD3OD) d 157.8, 139.2, 131.2, 128.2, 128.0,
116.8, 69.0, 66.8, 45.6, 35.5, 33.1, 33.0, 32.8, 32.7, 30.2, 29.9,
29.3, 22.2, 19.3, 11.6. ½a D20
ꢀ18.6 (c 0.32, MeOH). TLC (10% CH3OH
ꢁ
in CH2Cl2), Rf 0.18 (UV, CAM).
TS5: 1H NMR (500 MHz, CD3OD) d 7.10 (d, J = 8.4 Hz, 2H), 6.76
(d, J = 8.4 Hz, 2H), 5.28 (dt, J = 15.3, 6.4 Hz, 1H), 5.21 (dd, J = 15.4,
7.2 Hz, 1H), 3.62 (dt, J = 11.0, 6.4 Hz, 1H), 3.17 (ddd, J = 8.2, 8.2,
5.6 Hz, 1H), 2.94 (dd, J = 14.2, 5.9 Hz, 1H), 2.86 (dd, J = 14.2,
8.4 Hz, 1H), 2.65 (s, 3H), 2.13–2.07 (m, 1H), 2.06–1.97 (m, 2H),
1.56–1.51 (m, 2H), 1.37–1.30 (m, 2H), 1.17–1.12 (m, 1H), 1.10
(ddd, J = 13.3, 7.2, 5.8 Hz, 1H), 1.03 (ddd, J = 13.9, 7.1, 5.8 Hz, 1H),
0.87 (d, J = 6.7 Hz, 3H), 0.84 (t, J = 7.3 Hz, 3H), 0.82 (d, J = 6.5 Hz,
3H). 13C NMR (125 MHz, CD3OD) d 157.8, 139.1, 131.4, 127.9,
127.8, 116.8, 68.8, 66.0, 45.5, 35.4, 35.3, 34.2, 33.0, 32.0, 30.2,
21.2, 19.8, 11.5. ½a D20
ꢁ
+3.0 (c 0.307, MeOH). TLC (10% CH3OH in
CH2Cl2), Rf 0.18 (UV, CAM).
TS11: 1H NMR (500 MHz, CD3OD) d 7.09 (d, J = 8.5 Hz, 2H), 6.76
(d, J = 8.5 Hz, 2H), 5.35 (dt, J = 15.3, 6.3 Hz, 1H), 5.28 (dd, J = 15.3,
7.1 Hz, 1H), 3.79 (dt, J = 9.0, 3.3 Hz, 1H), 3.19 (ddd, J = 7.5, 7.5,
2.7 Hz, 1H), 2.87 (dd, J = 14.7, 6.7 Hz, 1H), 2.79 (dd, J = 14.7,
7.8 Hz, 1H), 2.55 (s, 3H), 2.23–2.11 (m, 2H), 2.04–1.95 (m, 1H),
1.60–1.45 (m, 2H), 1.39–1.31 (m, 2H), 1.11–1.07 (m, 1H), 1.17
(ddd, J = 13.4, 13.4, 6.8 Hz, 1H), 1.07–1.02 (m, 1H), 0.91 (d,
J = 6.7 Hz, 3H), 0.85 (t, J = 7.3 Hz, 3H), 0.84 (d, J = 6.5 Hz, 3H). 13C
NMR (125 MHz, CD3OD) d 157.7, 139.1, 131.2, 128.4, 128.0,
116.8, 69.2, 66.8, 45.5, 35.5, 33.1, 33.0, 33.0, 32.9, 30.2, 29.9,
29.3, 21.2, 19.8, 11.5. ½a D20
ꢀ2.3 (c 0.9, MeOH). TLC (10% CH3OH
ꢁ
in CH2Cl2), Rf 0.16 (UV, CAM).
TS6: 1H NMR (500 MHz, CD3OD) d 7.11 (d, J = 8.4 Hz, 2H), 6.77
(d, J = 8.4 Hz, 2H), 5.35 (dt, J = 15.1, 6.8 Hz, 1H), 5.22 (dd,
J = 15.2 Hz, 8.1, 1H), 3.84 (dt, J = 8.9, 3.7 Hz, 1H), 3.35 (ddd,
J = 7.6, 7.6, 3.5 Hz, 1H), 2.93 (dd, J = 14.7, 6.7 Hz, 1H), 2.85 (dd,
J = 14.7, 6.7 Hz, 1H), 2.62 (s, 3H), 2.22–2.11 (m, 2H), 2.03–1.95
(m, 1H), 1.57–1.48 (m, 2H), 1.35–1.27 (m, 2H), 1.15–1.09 (m,
1H), 1.23 (ddd, J = 14.0, 9.5, 4.6 Hz, 1H), 0.99 (ddd, J = 13.9, 8.8,
4.8 Hz, 1H), 0.92 (d, J = 6.7 Hz, 3H), 0.85 (t, J = 7.3 Hz, 3H), 0.81
(d, J = 6.4 Hz, 3H). 13C NMR (125 MHz, CD3OD) d 157.9, 138.6,
131.2, 128.4, 127.6, 116.9, 68.7, 66.6, 45.5, 35.7, 33.2, 33.1, 32.4,
21.2, 19.8, 11.5. ½a D20
ꢁ
+0.4 (c 0.32, MeOH). TLC (10% CH3OH in
CH2Cl2), Rf 0.17 (UV, CAM).
TS12: 1H NMR (500 MHz, CD3OD) d 7.10 (d, J = 8.4 Hz, 2H), 6.76
(d, J = 8.4 Hz, 2H), 5.26 (dt, J = 15.3, 6.3 Hz, 1H), 5.21 (dd, J = 15.2,
7.2 Hz, 1H), 3.62 (dt, J = 8.0, 4.7 Hz, 1H), 3.16 (ddd, J = 8.6, 8.6,
5.3 Hz, 1H), 2.94 (dd, J = 14.2, 5.8 Hz, 1H), 2.86 (dd, J = 14.2,
8.5 Hz, 1H), 2.66 (s, 3H), 2.13–2.07 (m, 1H), 2.07–2.02 (m, 1H),
2.03–1.95 (m, 1H), 1.56–1.50 (m, 2H), 1.38–1.31 (m, 2H), 1.11–
1.05 (m, 1H), 1.15 (ddd, J = 13.6, 6.9, 6.9 Hz, 1H), 1.02 (ddd,
J = 13.7, 7.0, 7.0 Hz, 1H), 0.87 (d, J = 6.8 Hz, 3H), 0.84 (t, J = 7.1 Hz,
3H), 0.82 (d, J = 6.4 Hz, 3H). 13C NMR (125 MHz, CD3OD) d 157.8,
139.1, 131.4, 127.9, 127.8, 116.8, 68.7, 66.0, 45.5, 35.4, 35.3, 34.2,
32.3, 31.1, 29.8, 22.3, 19.3, 11.6. ½a D20
ꢁ
+19.7 (c 0.76, MeOH). TLC
(10% CH3OH in CH2Cl2), Rf 0.18 (UV, CAM).
TS7: 1H NMR (400 MHz, CD3OD) d 7.10 (d, J = 8.5 Hz, 2H), 6.76
(d, J = 8.5 Hz, 2H), 5.35 (dt, J = 15.2, 6.8 Hz, 1H), 5.22 (dd, J = 15.2,
8.1 Hz, 1H), 3.82 (dt, J = 8.8, 3.1 Hz, 1H), 3.27 (ddd, J = 7.1, 7.1,
2.8 Hz, 1H), 2.90 (dd, J = 14.6, 6.7 Hz, 1H), 2.82 (dd, J = 14.6,
6.7 Hz, 1H), 2.58 (s, 3H), 2.24–2.12 (m, 2H), 2.04–1.94 (m, 1H),
1.57–1.48 (m, 2H), 1.34–1.27 (m, 2H), 1.16–1.09 (m, 1H), 1.23
(ddd, J = 14.0, 9.4, 4.6 Hz, 1H), 0.99 (ddd, J = 13.8, 8.8, 4.5 Hz, 1H),
0.92 (d, J = 6.7 Hz, 3H), 0.85 (t, J = 7.3 Hz, 3H), 0.81 (d, J = 6.4 Hz,
3H). 13C NMR (100 MHz, CD3OD) d 157.8, 138.6, 131.2, 128.5,
128.0, 116.8, 69.0, 66.7, 45.5, 35.7, 33.2, 33.1, 32.7, 32.6, 31.1,
33.0, 32.0, 30.2, 29.3, 21.2, 19.8, 11.5. ½a D20
ꢀ8.3 (c 0.38, MeOH).
ꢁ
TLC (10% CH3OH in CH2Cl2), Rf 0.17 (UV, CAM).
TS13: 1H NMR (500 MHz, CD3OD) d 7.10 (d, J = 8.4 Hz, 2H), 6.76
(d, J = 8.4 Hz, 2H), 5.27 (dt, J = 15.3, 6.3 Hz, 1H), 5.21 (dd, J = 15.4,
7.3 Hz, 1H), 3.61 (dt, J = 11.1, 6.3 Hz, 1H), 3.14 (ddd, J = 7.5, 7.5,
5.4 Hz, 1H), 2.93 (dd, J = 14.1, 5.9 Hz, 1H), 2.85 (dd, J = 14.1,
8.4 Hz, 1H), 2.64 (s, 3H), 2.13–2.07 (m, 1H), 2.06–1.95 (m, 2H),
1.56–1.51 (m, 2H), 1.37–1.30 (m, 2H), 1.17–1.12 (m, 1H), 1.10
(ddd, J = 13.3, 7.2, 5.8 Hz, 1H), 1.03 (ddd, J = 13.9, 7.1, 7.1 Hz, 1H),
0.87 (d, J = 6.7 Hz, 3H), 0.84 (t, J = 7.3 Hz, 3H), 0.82 (d, J = 6.5 Hz,
3H). 13C NMR (125 MHz, CD3OD) d 157.8, 139.1, 131.4, 128.0,
127.9, 116.8, 69.0, 66.0, 45.5, 35.4, 35.3, 34.3, 33.0, 32.1, 30.2,
29.9, 22.3, 19.3, 11.7. ½a D20
ꢀ19.2 (c 0.48, MeOH). TLC (10% CH3OH
ꢁ
in CH2Cl2), Rf 0.18 (UV, CAM).
TS8: 1H NMR (500 MHz, CD3OD) d 7.11 (d, J = 8.3 Hz, 2H), 6.77
(d, J = 8.3 Hz, 2H), 5.33 (dt, J = 15.3, 6.7 Hz, 1H), 5.21 (dd, J = 15.3,
8.2 Hz, 1H), 3.83 (dt, J = 9.2, 3.1 Hz, 1H), 3.34 (ddd, J = 7.5, 7.5,
2.6 Hz, 1H), 2.91 (dd, J = 14.7, 7.0 Hz, 1H), 2.86 (dd, J = 14.7,
7.7 Hz, 1H), 2.62 (s, 3H), 2.23–2.12 (m, 2H), 1.99 (m, 1H), 1.59–
1.45 (m, 2H), 1.22 (ddd, J = 13.8, 9.5, 4.5 Hz, 1H), 1.12 (m, 1H),
0.99 (ddd, J = 13.6, 8.9, 4.9 Hz, 1H), 0.91 (d, J = 6.6 Hz, 3H), 0.85 (t,
J = 7.4 Hz, 3H), 0.82 (d, J = 6.4 Hz, 3H). 13C NMR (125 MHz, CD3OD)
d 157.9, 138.8, 131.2, 128.4, 127.6, 116.9, 68.7, 66.7, 45.5, 35.8,
33.2, 33.0, 32.4, 32.3, 31.1, 29.8, 22.3, 19.3, 11.7. LRMS (ES+)
[M+H]+ 334.67, [M+Na]+ 356.62. HRMS (ES+) calculated for
29.3, 21.2, 19.8, 11.5. ½a D20
ꢁ
+2.1 (c 0.5, MeOH). TLC (10% CH3OH in
CH2Cl2), Rf 0.18 (UV, CAM).
TS14: 1H NMR (500 MHz, CD3OD) d 7.11 (d, J = 8.3 Hz, 2H), 6.77
(d, J = 8.4 Hz, 2H), 5.34 (dt, J = 15.2, 6.4 Hz, 1H), 5.28 (dd, J = 15.2,
7.2 Hz, 1H), 3.83 (dt, J = 9.1, 3.1 Hz, 1H), 3.35 (ddd, J = 7.3, 7.3,
2.7 Hz, 1H), 2.92 (dd, J = 14.6, 7.0 Hz, 1H), 2.86 (dd, J = 14.6,
7.0 Hz, 1H), 2.62 (s, 3H), 2.22–2.12 (m, 2H), 2.03–1.95 (m, 1H),
1.58–1.45 (m, 2H), 1.39–1.32 (m, 2H), 1.12–1.08 (m, 1H), 1.17
(ddd, J = 13.6, 6.8, 6.8 Hz, 1H), 1.08–1.02 (m, 1H), 0.89 (d,
J = 6.7 Hz, 3H), 0.85 (t, J = 7.3 Hz, 3H), 0.83 (d, J = 6.2 Hz, 3H). 13C
NMR (125 MHz, CD3OD) d 157.9, 139.2, 131.2, 127.9, 127.7,
116.8, 68.7, 66.7, 45.5, 35.5, 33.1, 33.0, 32.5, 32.4, 30.2, 29.8,
C
21H36NO2 [M+H]+ 334.2748, found 334.2742.
½
a 2D0
ꢁ
ꢀ19.7 (c
0.483, MeOH). TLC (10% CH3OH in CH2Cl2), Rf 0.17 (UV, CAM).
TS9: 1H NMR (500 MHz, CD3OD) d 7.10 (d, J = 8.2 Hz, 2H), 6.76
(d, J = 8.1 Hz, 2H), 5.26 (dt, J = 15.0, 6.3 Hz, 1H), 5.21 (dd, J = 15.1,
7.1 Hz, 1H), 3.62 (dt, J = 8.6, 3.8 Hz, 1H), 3.17 (ddd, J = 8.7, 8.7,
5.1 Hz, 1H), 2.94 (dd, J = 14.2, 5.8 Hz, 1H), 2.86 (dd, J = 14.2,
8.5 Hz, 1H), 2.66 (s, 3H), 2.13–2.07 (m, 1H), 2.07–2.02 (m, 1H),
2.03–1.95 (m, 1H), 1.56–1.50 (m, 2H), 1.38–1.32 (m, 2H), 1.11–
1.06 (m, 1H), 1.15 (ddd, J = 13.6, 6.9, 6.9 Hz, 1H), 1.02 (ddd,
J = 13.9, 6.8, 6.8 Hz, 1H), 0.87 (d, J = 6.8 Hz, 3H), 0.84 (t, J = 7.1 Hz,
3H), 0.82 (d, J = 6.2 Hz, 3H). 13C NMR (125 MHz, CD3OD) d 157.8,
21.2, 19.8, 11.5. ½a D20
ꢀ3.1 (c 1.0, MeOH). TLC (10% CH3OH in
ꢁ
CH2Cl2), Rf 0.19 (UV, CAM).
TS15: 1H NMR (500 MHz, CD3OD) d 7.11 (d, J = 8.4 Hz, 2H), 6.77
(d, J = 8.4 Hz, 2H), 5.35 (dt, J = 15.4, 6.3 Hz, 1H), 5.28 (dd, J = 15.4,
7.3 Hz, 1H), 3.84 (dt, J = 8.9, 3.5 Hz, 1H), 3.35 (ddd, J = 7.1, 7.1,

H. S. Tae et al. / Bioorg. Med. Chem. 19 (2011) 1708–1713
1713
2.5 Hz, 1H), 2.93 (dd, J = 14.7, 6.7 Hz, 1H), 2.85 (dd, J = 14.7, 7.8 Hz,
1H), 2.62 (s, 3H), 2.21–2.12 (m, 2H), 2.03–1.95 (m, 1H), 1.58–1.46
(m, 2H), 1.40–1.31 (m, 2H), 1.12–1.08 (m, 1H), 1.18 (ddd, J = 13.5,
6.7, 6.7 Hz, 1H), 1.07–1.02 (m, 1H), 0.91 (d, J = 6.7 Hz, 3H), 0.85
(t, J = 7.3 Hz, 3H), 0.84 (d, J = 6.4 Hz, 3H). 13C NMR (125 MHz,
CD3OD) d 157.9, 139.2, 131.2, 127.9, 127.7, 116.8, 68.7, 66.6,
harvested from the wells and passed through glass fiber filters
using a Ska-Tron cell harvester (Molecular Devices, Sunnyvale,
CA). The filters were transferred to vials, scintillant was added
and the amount of radioactivity incorporated into the cells in the
filters was quantified by scintillation counting. The resulting data
was analyzed using PRISM software (GRAPHPAD Software, San Diego,
CA).
45.5, 35.5, 33.2, 33.0, 32.4, 32.3, 30.2, 29.8, 21.2, 19.8, 11.5. ½a D20
ꢁ
ꢀ0.2 (c 1.0, MeOH). TLC (10% CH3OH in CH2Cl2), Rf 0.18 (UV, CAM).
TS16: 1H NMR (500 MHz, CD3OD) d 7.10 (d, J = 8.4 Hz, 2H), 6.76
(d, J = 8.3 Hz, 2H), 5.34 (dt, J = 15.2, 6.7 Hz, 1H), 5.21 (dd, J = 15.3,
8.1 Hz, 1H), 3.81 (dt, J = 9.4, 3.0 Hz, 1H), 3.27 (ddd, J = 7.2, 7.2,
2.5 Hz, 1H), 2.89 (dd, J = 14.6, 6.9 Hz, 1H), 2.83 (dd, J = 14.6,
7.8 Hz, 1H), 2.59 (s, 3H), 2.23–2.12 (m, 2H), 2.03–1.95 (m, 1H),
1.59–1.52 (m, 1H), 1.51–1.45 (m, 1H), 1.34–1.26 (m, 2H), 1.15–
1.09 (m, 1H), 1.23 (ddd, J = 14.0, 9.5, 4.6 Hz, 1H), 0.99 (ddd,
J = 13.6, 8.7, 4.9 Hz, 1H), 0.91 (d, J = 6.6 Hz, 3H), 0.85 (t, J = 7.4 Hz,
3H), 0.82 (d, J = 6.3 Hz, 3H). 13C NMR (125 MHz, CD3OD) d 157.8,
138.7, 131.2, 128.5, 128.0, 116.8, 68.9, 66.9, 45.5, 35.8, 33.2, 33.0,
Acknowledgements
The authors gratefully acknowledge members of the Crews lab
for helpful discussions throughout the preparation of this manu-
script. This work was supported by a Korea Research Foundation
Grant (KRF-2005-214-C00218) (H.S.T.), the National Science Foun-
dation (A.R.S.) and the National Institutes of Health (GM062120)
(C.M.C.).
Supplementary data
32.7, 32.6, 31.1, 29.9, 22.3, 19.3, 11.6. ½a D20
ꢁ
+19.8 (c 0.42, MeOH).
TLC (10% CH3OH in CH2Cl2), Rf 0.17 (UV, CAM).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.01.027.
4.2. Cell culture
MCF-7 cells were a generous gift from Anton Bennett (Yale
University, New Haven, CT) and were routinely grown in RPMI
1640 medium supplemented with 10% heat-inactivated fetal
bovine serum. All culture medium was supplemented with
100 units/mL penicillin G and 100 lg/mL streptomycin sulfate.
Cells were grown at 37 °C in an atmosphere of 5% CO2.
References and notes
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MCF-7 cells were seeded into 96 well plates in reduced serum
(1%) medium at a density of 10,000 cells/well. After an overnight
incubation to allow for cell attachment, the growth medium was
removed and replaced with fresh serum-free medium. After at
least 48 h of serum starvation to growth arrest the cells, each well
was overlaid with an equal volume of medium containing double-
strength mitogen and/or diastereomer. The cells were incubated
another 24 h at which point each well of cells received another
20
(Perkin–Elmer, Boston, MA). Following another 4 h, the cells were
lL of (serum-free) medium containing 2 l
Ci of [3H]-thymidine
16. Connon, S. J.; Blechert, S. Angew. Chem., Int. Ed. 2003, 42, 1900.