Biologically active biotin derivatives of schweinfurthin F

Article abstract of DOI:10.1016/j.bmcl.2010.08.143

As a prelude to efforts to identify schweinfurthin binding proteins, an ester conjugate and an amide conjugate of schweinfurthin F and biotin have been prepared by chemical synthesis. These compounds maintain activity in SF-295 cells comparable to the parent system, and display the lower potency in A549 cells that is a characteristic of the schweinfurthin pattern of activity.

Full text of DOI:10.1016/j.bmcl.2010.08.143

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6716–6720
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Biologically active biotin derivatives of schweinfurthin F
Natalie C. Ulrich a, Craig H. Kuder b,c, Raymond J. Hohl b,c, David F. Wiemer a,b,
⇑
a Department of Chemistry, University of Iowa, Iowa City, IA 52242-1294, United States
b Department of Pharmacology, University of Iowa, Iowa City, IA 52242-1294, United States
c Department of Internal Medicine, University of Iowa, Iowa City, IA 52242-1294, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
As a prelude to efforts to identify schweinfurthin binding proteins, an ester conjugate and an amide con-
jugate of schweinfurthin F and biotin have been prepared by chemical synthesis. These compounds main-
tain activity in SF-295 cells comparable to the parent system, and display the lower potency in A549 cells
that is a characteristic of the schweinfurthin pattern of activity.
Received 31 July 2010
Revised 28 August 2010
Accepted 31 August 2010
Available online 6 September 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Schweinfurthin
Biotin
Conjugate
Synthesis
Anti-proliferative
The identification of a drug’s molecular target is very desirable,
both to elucidate the underlying biological mechanism(s) of action
furthins toward specific cell lines in this large panel. A compound’s
pattern of activity in the NCI’s 60-cell line assay can be analyzed
by the COMPARE algorithm to determine whether it shows a signif-
icant correlation to any known agents in terms of mechanism of ac-
tion.21 Importantly, the pattern displayed in the 60-cell line screen
by the schweinfurthins shows no correlation to any clinically used
anti-neoplastic agent, indicating that this family of compounds
probably acts via a novel mechanism or at a novel target. The COM-
PARE analysis of the schweinfurthins at the GI50 and TGI levels of re-
sponse20 does show significant correlations to several structurally
unrelated natural products (Fig. 1), including the stellettins (e.g.,
stellettin A, 4),22–25 the cephalostatins (e.g., cephalostatin 1, 5),26–
28 and OSW-1 (6).29 Unfortunately, at this time none of these corre-
lated natural products has a known molecular target or a defined
mechanism of action. It has been determined that the schweinfur-
thin-sensitive cell lines possess no shared biochemical features
and differ substantially in DNA repair phenotype, in vitro doubling
time, and MDR status.20 Discovery of the mechanism of action of
the schweinfurthins may aid in similar determination of some or
all of the correlated natural products, and in turn increase under-
standing of a new class of prospective chemotherapeutic agents.
In order to elucidate the basis of schweinfurthin toxicity, we
have initiated an effort to identify the intracellular binding part-
ner(s). Although numerous kinds of interacting molecules are pos-
sible, based on the schweinfurthins’ reactivity, chemical structure,
and overall pattern of cytotoxicity, we hypothesize that these com-
pounds bind to protein(s). Herein we report chemical modifica-
tions of a potent natural schweinfurthin to yield biotinylated
schweinfurthins that might be used to probe potential protein
and to guide the rational design of more effective therapeutics.1
A
wide variety of techniques, including yeast three-hybrid systems,2
phage display,3 and protein microarrays4 has been employed for
the deconvolution of different small-molecule targets. Recently,
affinity-based chromatography coupled with mass spectrometry
(MS) has become increasingly popular due to the remarkable
sensitivity and speed this technique offers.1,5–7
Identifying a molecular target using affinity-based chromatog-
raphy typically requires the synthesis of an appropriate molecular
probe amenable to immobilization on a solid support.1 Biotinyla-
tion commonly has been used for the purification/identification
of small molecule targets8–14 (e.g., the identification of trapoxin
as a histone deacetylase inhibitor).15 This methodology requires
derivatization of the molecule in question with biotin and subse-
quently relies on the extremely tight binding of biotin to streptavi-
din (kd = 10À15 M).1
One unique family of natural products with an as yet unknown
molecular target is the schweinfurthins (e.g., schweinfurthin A, F,
and 3-deoxyschweinfurthin B, 1–3, Fig. 1). These compounds were
isolated from the African plant Macaranga schweinfurthii,16–20 and
all known members of this family that possess a hexahydroxanthene
system exhibit potent and differential antineoplastic activity in the
National Cancer Institute’s (NCI) 60-cell line anti-cancer screen. Of
particularinterestisthepatternofactivityexhibitedbytheschwein-
⇑
Corresponding author. Tel.: +1 319 335 1365; fax: +1 319 335 1270.
E-mail address: david-wiemer@uiowa.edu (D.F. Wiemer).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.143

N. C. Ulrich et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6716–6720
6717
OH
1) is essential for activity, and that extensive modifications in this
region would not be well-tolerated. Therefore we have pursued
alterations to the D-ring geranyl tail to minimize interference in
binding to potential protein targets. Finally, recent studies have
demonstrated that methylation of one of the D-ring phenols pro-
vides additional stability to the oxidation-prone resorcinol moiety.
These results suggested that analogues such as compound 7
(Scheme 1) may serve as probes, and the synthesis and bioassay
of compounds bearing these design elements are reported here.
A retrosynthetic analysis of a biotin derivative (7) inspired by
schweinfurthin F (2) and 3-deoxyschweinfurthin B (3) is detailed
HO
HO
O
OH
OH
H
H
1 Schweinfurthin A
OH
OH
OCH3
O
HO
in Scheme 1. Late-stage esterification of D-biotin (9) and the schwein-
furthin analogue 8 followed by removal of the phenolic protecting
group would provide the indicated target. Disconnection of the
stilbene olefin could afford the Horner–Wadsworth–Emmons cou-
pling partners known aldehyde 1034 and phosphonate 11. In turn
phosphonate 11 could be obtained from the benzylic alcohol 12,
which could result from alkylation of the corresponding benzyl
alcohol 13 under standard conditions for formation and alkylation
of a dianion.39
n
2 n = 0, Schweinfurthin F
3 n = 1, 3-Deoxyschweinfurthin B
O
H
O
Synthesis of arene 12 began with the known benzyl alcohol 1338
(Scheme 2). Regioselective formation of the lithiate, transmetalla-
tion to the cuprate, and subsequent alkylation with readily pre-
pared bromide 1440,41 gave the C-alkylated product 12 in modest
yield. Subsequent conversion to the phosphonate was accom-
plished via standard reaction conditions, and fluoride-mediated si-
lyl group removal afforded phosphonate 11. When the
phosphonate 11 was allowed to react with the tricyclic aldehyde
10 in the presence of NaH and 15-crown-5, the protected stilbene
8 was obtained in good yield. A final EDC-mediated esterification of
H
4 Stellettin A
O
OH
O
O
O
HO
OH
H
OH
N
N
H
H
H
H
stilbene 8 and D-biotin (9) afforded the biotinylated analogue 15 in
H
modest yield (48%), along with a substantial amount of recovered
starting material (35%), but this reaction gave sufficient material
to allow biological analysis. Attempted hydrolysis of the MOM ace-
tal of compound 15 under various acidic conditions proved unsuc-
cessful and resulted instead in cleavage of the allylic ester to return
alcohol 8. Chemical hydrolysis of the MOM groups would not be
essential if compound 15 displays biological activity, but the facile
hydrolysis of the allylic ester prompted concerns about its meta-
bolic stability.
5 Cephalostatin 1
O
O
O
O
OH
OH
H
O
O
AcO
H
O
OH
H
O
HO
HO
In an effort to increase the stability of the allylic linkage,42–45
a
HO
more stable amide group was envisioned (Scheme 3). Treatment of
analogue 8 with phthalimide under standard Mitsunobu condi-
tions46 gave the desired compound 16. Treatment of phthalimide
16 with hydrazine hydrate followed by EDC-mediated condensa-
p-MeOC6H4CO2
6 OSW-1
Figure 1. Some schweinfurthins (1–3), stellettin A (4), cephalostatin 1 (5), and
OSW-1 (6).
tion with D-biotin (9) afforded the desired biotinylated schweinfur-
thin 17. After preparation of this compound, both the biotin amide
17 and the ester 15 were advanced to assays to gauge their biolog-
ical activity.
interactions. For these analogues to have utility as probes for pro-
tein interactions, it would be at least desirable and perhaps essen-
tial for them to retain the characteristic schweinfurthin activity.
Here we demonstrate that two specific biotinylated schweinfur-
thin analogues display schweinfurthin-like activity in a recently re-
ported, two-cell line screen.30
To date, we have reported not only the total syntheses of several
natural schweinfurthins,31–36 but also the preparation of numerous
synthetic analogues for structure–activity relationship (SAR) stud-
ies.30,37,38 During the course of these studies, a schweinfurthin ana-
logue with a terminal allylic alcohol37 was synthesized and found
to exhibit significant and differential cytotoxicity in the distinctive
As described above, maintaining schweinfurthin-like activity
may be essential to the utility of these biotinylated compounds.
To address whether these analogues possess schweinfurthin-like
activity, compounds 15 and 17 were tested in a relevant two-cell
line screen.30 This screen measures the cytotoxicity of schweinfur-
thin analogues in the human-derived glioblastoma multiforme cell
line SF-295, which is highly sensitive to schweinfurthin treatment,
and the human-derived lung adenocarcinoma cell line A549, which
is relatively insensitive. Results from these assays are indicative of
overall schweinfurthin-like activity, as demonstrated in a recent
report.30
schweinfurthin pattern (mean GI50 = 1.0
l
M in the 60-cell line
Biotinylated analogues 15 and 17 were tested against SF-295
and A549 cell lines under standard conditions (Fig. 2). Both com-
pounds exhibited activity against SF-295 cells (EC50 = 2.7 and
assay), indicating that structural modifications at the distal end
of the geranyl chain do not diminish significantly the biological
activity. Conversely, the limited activity of schweinfurthin C, which
possesses an undecorated geranyl left-half, suggests that the hexa-
hydroxanthene core of the schweinfurthins (e.g., schweinfurthin A,
1.1
creased in comparison to the schweinfurthin analogue 3-deoxy-
schweinfurthin B (3, EC50 = 0.5 M in this assay), these results
lM, respectively). Although this activity is somewhat de-
l

6718
N. C. Ulrich et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6716–6720
OCH3
O
OH
O
HO
O
HN
H
H
NH
O
OCH3
S
H
7
OCH3
O
OMOM
O
HO
O
HN
H
S
H
+
8
NH
OH
HO
OCH3
H
9
O
OCH3
OH
P(OEt)2
O
OMOM
OMOM
+
O
HO
OTBS
OH
H
H
OCH3
OCH3
10
11
12
Scheme 1. Retrosynthesis of the biotin–schweinfurthin conjugate 7.
1) TMEDA, nBuLi
2) CuBr DMS
3) 14
OMOM
OMOM
HO
R
OR'
34%
OCH3
13
OCH3
1) MsCl, TEA
2) NaI, Acetone
3) P(OEt)3
12 R = OH, R' = TBS
11 R = P(O)(OEt)2, R' = H
Br
4) TBAF
69%
OCH3
O
OTBS
14
O
71%
HO
H
OCH3
10
H
O
NaH, 15-crown-5
OMOM
HO
H
OH
HN
8
OCH3
OCH3
D-biotin (9), EDC HCl
HOBt, TEA
O
48%
OR
O
HO
O
O
H
H
NH
OCH3
S
H
15 R = MOM
R = H
Various acids
7
X
Scheme 2. Attempted synthesis of allylic ester 7.
indicate that biotinylation of these compounds does not substan-
tially diminish activity. As with the natural schweinfurthins,
analogues 15 and 17 also are relatively inactive against the A549
cells (EC50 >10 and 5.0
thin B (EC50 4.9 M). Taken together, these data suggest overall
schweinfurthin-like activity and indicate that attachment of biotin
lM, respectively), as is 3-deoxyschweinfur-
l

N. C. Ulrich et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6716–6720
6719
OCH3
O
Phthalimide
PPh3, DIAD
OMOM
HO
H
O
8
58%
N
16
OCH3
O
H
1) NH2NH2 H2O
2) D-biotin (9), EDC HCl
OCH3
58%
O
OMOM
O
HO
O
HN
H
NH
NH
17
OCH3
S
H
Scheme 3. Preparation of amide 17.
120
100
80
60
40
20
0
0
2
4
6
8
10
12
Concentration (uM)
15 (A549 cells)
17 (A549 cells)
15 (SF-295 cells)
17 (SF-295 cells)
Figure 2. Concentration–response curves of compounds 15 and 17 in SF-295 and A549 cells. Values shown are the mean of quadruplicate treatments the standard
deviation.
at this position does not affect significantly the interaction be-
tween the compounds and their presumed target.
Supplementary data
In conclusion, two biotinylated schweinfurthin analogues
have been prepared. After ester analogue 15 proved to be acid-
labile, the amide analogue 17 was prepared in order to increase
the chemical/metabolic stability of the biotin linkage. Both ana-
logues exhibit schweinfurthin-like activity in SF-295 and A549
cancer cell lines, indicating that biotinylation at the right-half
of the schweinfurthins does not significantly affect cytotoxicity.
Pull-down experiments utilizing these probes are currently
underway, and the results of these studies will be reported in
due course.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.08.143.
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