Bioreductively activatable prodrug conjugates of phenstatin designed to target tumor hypoxia

Article abstract of DOI:10.1016/j.bmcl.2016.11.093

A variety of solid tumor cancers contain significant regions of hypoxia, which provide unique challenges for targeting by potent anticancer agents. Bioreductively activatable prodrug conjugates (BAPCs) represent a promising strategy for therapeutic intervention. BAPCs are designed to be biologically inert until they come into contact with low oxygen tension, at which point reductase enzyme mediated cleavage releases the parent anticancer agent in a tumor-specific manner. Phenstatin is a potent inhibitor of tubulin polymerization, mimicking the chemical structure and biological activity of the natural product combretastatin A-4. Synthetic approaches have been established for nitrobenzyl, nitroimidazole, nitrofuranyl, and nitrothienyl prodrugs of phenstatin incorporating nor-methyl, mono-methyl, and gem-dimethyl variants of the attached nitro compounds. A series of BAPCs based on phenstatin have been prepared by chemical synthesis and evaluated against the tubulin-microtubule protein system. In a preliminary study using anaerobic conditions, the gem-dimethyl nitrothiophene and gem-dimethyl nitrofuran analogues were shown to undergo efficient enzymatic cleavage in the presence of NADPH cytochrome P450 oxidoreductase. Each of the eleven BAPCs evaluated in this study demonstrated significantly reduced inhibitory activity against tubulin in comparison to the parent anti-cancer agent phenstatin (IC₅₀?=?1.0?μM). In fact, the majority of the BAPCs (seven of the eleven analogues) were not inhibitors of tubulin polymerization (IC₅₀?>?20?μM), which represents an anticipated (and desirable) attribute for these prodrugs, since they are intended to be biologically inactive prior to enzyme-mediated cleavage to release phenstatin.

Full text of DOI:10.1016/j.bmcl.2016.11.093

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Bioreductively activatable prodrug conjugates of phenstatin designed to
target tumor hypoxia
a
a
a
a
a
a
Blake A. Winn , Zhe Shi , Graham J. Carlson , Yifan Wang , Benson L. Nguyen , Evan M. Kelly ,
a
b
a,c
a,
⇑
a,
⇑
R. David Ross IV , Ernest Hamel , David J. Chaplin , Mary L. Trawick , Kevin G. Pinney
a
Department of Chemistry and Biochemistry, Baylor University, One Bear Place #97348, Waco, TX 76798-7348, United States
Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Frederick National Laboratory
for Cancer Research, National Institutes of Health, Frederick, MD 21702, United States
b
c
Mateon Therapeutics, Inc., 701 Gateway Boulevard, Suite 210, South San Francisco, CA 94080, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A variety of solid tumor cancers contain significant regions of hypoxia, which provide unique challenges
for targeting by potent anticancer agents. Bioreductively activatable prodrug conjugates (BAPCs) repre-
sent a promising strategy for therapeutic intervention. BAPCs are designed to be biologically inert until
they come into contact with low oxygen tension, at which point reductase enzyme mediated cleavage
releases the parent anticancer agent in a tumor-specific manner. Phenstatin is a potent inhibitor of tubu-
lin polymerization, mimicking the chemical structure and biological activity of the natural product com-
bretastatin A-4. Synthetic approaches have been established for nitrobenzyl, nitroimidazole, nitrofuranyl,
and nitrothienyl prodrugs of phenstatin incorporating nor-methyl, mono-methyl, and gem-dimethyl vari-
ants of the attached nitro compounds. A series of BAPCs based on phenstatin have been prepared by
chemical synthesis and evaluated against the tubulin-microtubule protein system. In a preliminary study
using anaerobic conditions, the gem-dimethyl nitrothiophene and gem-dimethyl nitrofuran analogues
were shown to undergo efficient enzymatic cleavage in the presence of NADPH cytochrome P450 oxi-
doreductase. Each of the eleven BAPCs evaluated in this study demonstrated significantly reduced inhi-
Received 3 October 2016
Revised 30 November 2016
Accepted 30 November 2016
Available online xxxx
Keywords:
Bioreductively activatable prodrug
conjugates (BAPCs)
Phenstatin
Prodrug synthesis
Tumor-associated hypoxia
Inhibitors of tubulin polymerization
bitory activity against tubulin in comparison to the parent anti-cancer agent phenstatin (IC50 = 1.0
In fact, the majority of the BAPCs (seven of the eleven analogues) were not inhibitors of tubulin polymer-
ization (IC50 > 20 M), which represents an anticipated (and desirable) attribute for these prodrugs, since
lM).
l
they are intended to be biologically inactive prior to enzyme-mediated cleavage to release phenstatin.
Ó 2016 Published by Elsevier Ltd.
Tumor-associated vasculature has emerged as a promising tar-
tumors.1–10 The rapid growth of tumor-associated vasculature
results in compromised structural integrity, which is characterized
by weakened vessel walls, increased interstitial pressure, blind
get for anti-cancer therapies due to its marked differences from
vasculature feeding healthy tissue.1
–10
Vasculature associated with
1
–10
healthy tissue forms a well-organized delivery network for oxygen
ends, and bulges.
Tumor-associated vasculature is generated
and nutrients to cells.1
–10
In contrast, tumor-associated vascula-
rapidly, leading to groups of vessels spaced far apart from each
other, and this results in regions of hypoxia that develop when this
distance is greater than the diffusion distance of oxygen.1
ture is forced to develop rapidly to meet the enhanced demand
for significant amounts of nutrients and oxygen required by
–10
One promising therapeutic option for targeting tumor-associ-
ated vasculature involves treatment with vascular targeting agents
(VTAs), which include angiogenesis-inhibiting agents (AIAs) and
Abbreviations: BAPC, bioreductively activatable prodrug conjugate; CA1, com-
bretastatin A-1; CA4, combretastatin A-4; Et
mance liquid chromatography; HRMS, high resolution mass spectrometry; MeOH,
methanol; NaBH , sodium borohydride; TLC, thin layer chromatography; THF,
3
N, triethylamine; HPLC, high perfor-
1
–10
vascular disrupting agents (VDAs).
AIAs, which represent a
4
fairly well investigated therapeutic strategy, act by inhibiting
angiogenesis, the formation of new tumor-associated vasculature,
tetrahydrofuran; EtOAc, ethyl acetate; POR, NADPH cytochrome P450 oxidoreduc-
tase; DMP, Dess-Martin periodinane; DEAD, diethylazodicarboxylate; DIAD, diiso-
4
,11,12
0
while leaving existing vessels intact.
Inhibition of angiogene-
propyl azodicarboxylate; ADDP, 1,1 -(azodicarbonyl)dipiperidine.
⇑
Corresponding authors.
sis limits tumor growth and also leads to increased blood flow in
the remaining vasculature, allowing for increased delivery of
E-mail addresses:
Mary_Lynn_Trawick@baylor.edu
(M.L.
Trawick),
Kevin_Pinney@baylor.edu (K.G. Pinney).
http://dx.doi.org/10.1016/j.bmcl.2016.11.093
0
960-894X/Ó 2016 Published by Elsevier Ltd.
Please cite this article in press as: Winn B.A., et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.11.093

2
B.A. Winn et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
H CO
3
H
3
CO
R1
O
NHCOCH
O
3
H
3
CO
CO
R2
OCH
H
3
CO
H CO
H CO
3
3
OCH
3
R
OCH3
H
3
3
OCH
3
OCH3
Colchicine
Combretastatin A-4 and its Prodrug
Phenstatinsand their Prodrugs
Hydroxyphenstatin:R1 = R = OH
2
CombretastatinA-4 (CA4) : R = OH
CombretastatinA-4P (CA4P) : R = OPO
3
Na
2
HydroxyphenstatinDiphosphate: R = R2 = OPO Na2
1
3
Phenstatin:R1 = H; R2 = OH
PhenstatinPhosphate:R = H; R2 = OPO
1
Na
3
2
Fig. 1. Colchicine, phenstatin, and combretastatin natural products and their corresponding phosphate salts.
chemotherapy and potentially enhanced tumor damage from
radiotherapy to an otherwise difficult therapeutic target due to
releasing
manner.
a
potent anti-cancer agent in
a
tumor-specific
8
,25
4
,11,12
hypoxia.
VDAs impact tumor vasculature from a mechanistic
A series of BAPCs utilizing CA4 as the parent anticancer agent
approach that is distinct from AIAs. One subset of VDAs is com-
prised of small-molecule inhibitors of tubulin polymerization that
target existing tumor-associated vasculature by causing rapid mor-
and incorporating nor-, mono-, and gem-dimethyl nitrothiophene
triggers was reported in 2006 by Davis and co-workers.3
7,38
The
CA4-BAPC bearing the gem-dimethyl nitrothiophene trigger proved
to be the most active of the trio, demonstrating the greatest resis-
tance to cleavage under normoxic conditions in vitro, in effect dis-
phology changes (flat to round) of the endothelial cells lining these
vessels.1
–10
This leads to irreversible vessel damage and tumor
1
–10
37
necrosis.
playing a high selectivity for low-oxygen tumor environments.
A representative clinically relevant small-molecule VDA that
disrupts microtubule formation is the natural product combretas-
tatin A-4 (CA4) (Fig. 1). First isolated from the African bush willow
tree Combretum caffrum Kuntze by the Pettit group, CA4 is a potent
inhibitor of tubulin polymerization, functioning through a binding
interaction at the colchicine site on tubulin.13–17 Its corresponding
water-soluble phosphate prodrug salt [combretastatin A-4P
While the nor-methyl nitrothiophene BAPC was only activated at
very low oxygen concentrations (<0.01% O
2
), the bioreductive trig-
gers for mono-methyl and gem-dimethyl BAPCs were cleaved over
3
7
a greater range of oxygen concentrations. The gem-dimethyl
CA4-BAPC was significantly more effective in hypoxic environ-
ments in vitro compared to the nor- and mono-methyl CA4-BAPCs,
releasing approximately 50% CA4 at 0.5% O
2
with the aid of POR.37
(
CA4P)] has reached advanced clinical trials as a promising
The mechanism of cleavage (under hypoxia) for the nitrothio-
phene trigger begins with a one electron reductase such as POR
1
8–22
VDA.
However, no small-molecule VDA has yet been approved
3
7
by the FDA.
Phenstatin, originally synthesized by the Pettit group in 1998, is
another potent inhibitor of tubulin polymerization with pro-
nounced anti-cancer activity against human cancer cell lines
reducing the nitro group on the trigger (Fig. 2). Once the nitro
group on the trigger has been reduced, an electron cascade through
the thiophene ring leads to trigger detachment, releasing the active
3
7
VDA (CA4 in this example). In comparison, under normal oxygen
tension, the species obtained after the initial one-electron reduc-
tion is simply re-oxidized (by molecular oxygen) and thus does
2
3,24
(Fig. 1).
Phenstatin was discovered serendipitously by the Pet-
tit group during an attempt to prepare a CA4 analog bearing an
2
3
37
epoxide moiety as a replacement for the ethylene bridge. The
ketone functionality of phenstatin was the surprising result of a
Jacobsen epoxidation reaction intended to form an epoxide from
the corresponding olefin. Phenstatin mirrors the biological mech-
anism of action of CA4, disrupting microtubule assembly through a
binding interaction with the colchicine site on tubulin.2 Phen-
statin and its corresponding water-soluble phosphate salt prodrug
counterpart demonstrate pronounced cytotoxicity against human
cancer cell lines. While hydroxyphenstatin, the diol analog of
phenstatin, displays potent inhibition of tubulin polymerization,
its diphosphate salt analog is less active against in vitro cancer cell
lines and as an inhibitor of tubulin polymerization than its phen-
statin phosphate counterpart.24
not lead to cleavage.
Although no BAPC has yet been approved by the FDA, two
BAPCs that have reached advanced clinical trials are TH-302 and
PR-104. TH-302 (from Threshold Pharmaceuticals) is a 2-nitroimi-
2
3
dazole based BAPC attached to the DNA alkylating agent bromo-
4
39,40
isophosphoramide (Fig. 3).
The prodrug is activated by one
electron reductases such as POR, reducing the nitro group on the
nitroimidazole trigger in a similar mechanistic pathway to the
2
4
8
nitrothienyl trigger cleavage. Highly effective in in vitro studies
and early in vivo studies in mice with hypoxia cytotoxicity ratios
(HCR) as high as 600, TH-302 has advanced to Phase III clinical tri-
als after successful Phase I and II studies, although the results of
41–43
the first Phase III trial were not statistically significant.
PR-
Solid tumors represent inherently challenging therapeutic tar-
gets due, in part, to the significant differences between vasculature
feeding healthy tissue versus tumor-associated vasculature, which
is a contributing factor to the profound regions of hypoxia that
often characterize tumors.3,8,25–36 A combination of blind ends,
leaky vessel walls, occlusions, and kinked vessels, along with the
increased average distance between capillaries, leads to hypoxic
104 (Fig. 3), synthesized and biologically evaluated by Wilson
et al. at the University of Auckland, is a nitroaromatic preprodrug
of PR-104A, eventually being reduced to the active forms PR104H
8
,44–46
and PR-104M.
The cytotoxicity of PR-104M derives from its
8
ability to form interstrand DNA crosslinks. PR-104 was taken into
Phase I and II clinical trials, yielding promising results in Phase I
trials but stalled at Phase II due to dose-limiting toxicity and over-
8
,47,48
regions where many common radiotherapies and chemotherapeu-
all efficacy issues.
tic options are less effective.8
,25–36
The challenges created by
Intrigued by the concept of targeting tumor hypoxia with
BAPCs, a series of such prodrugs were prepared by chemical syn-
thesis based on the unique tubulin-active anticancer agent, phen-
statin. Utilizing a combination of synthetic pathways previously
described in the literature along with our modifications designed
to improve yield and reaction efficiency, a selected subset of
nor-, mono-, and gem-dimethyl nitrothiophene, nitrobenzyl,
hypoxia and large diffusion distances offer a unique opportunity
for targeted therapeutic intervention.8 Bioreductively activatable
prodrug conjugates (BAPCs) represent a possible hypoxia-activated
,25
8
,25
treatment method.
BAPCs are designed to be activated by
reductase enzymes, such as NADPH cytochrome P450 oxidoreduc-
tase (POR), in regions of hypoxia in the tumor microenvironment,
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B.A. Winn et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
3
H CO
3
H CO
3
H
3
CO
H
3
C CH3
3
H CO
H C
3
CH
3
Cyt P450 Reductase
OCH
3
S
NO
2
OCH
3
S
O
3
O
3
NHOH
OCH
OCH
H
3
C
H CO
3
S
H
3
C
NHOH
H
3
CO
(after protonation)
OCH
3
OH
OCH
3
Fig. 2. gem-Dimethyl nitrothienyl trigger release from CA4.37
NO
2
NO
2
NHOH
NH
2
2
-
PO
3
OH
OH
OH
Br
H
N
H
N
H
N
H
N
Br
O
2
N
O
2
N
O
2
N
2
O N
NH
O
CH
3
N
HN
N
O
N
O
N
O
N
O
P
O
NO2
N
Br
OSO
2
CH
3
Br
OSO
2
CH
3
Br
OSO
2
CH
3
Br
OSO
2 3
CH
TH-302
PR104
PR104A
PR104H
Fig. 3. TH-302, PR104, PR104A, PR104H, and PR104M.8
PR104M
nitroimidazole, and nitrofuran triggers were synthesized and
trigger 11. Methylation conditions described by Reetz et al. pro-
vided an improved synthetic route that generated both mono-
and gem-dimethyl nitrothiophene triggers from aldehyde 7 in good
linked to phenstatin.3
7,38
Preliminary biological assessment of
these phenstatin BAPCs evaluated their ability to inhibit tubulin
polymerization, and a subset were evaluated for their suitability
as substrates for enzymatic-mediated cleavage to release the par-
ent anticancer agent, phenstatin.
5
0
yield. Mono-methyl trigger 9 was synthesized through methyla-
tion of aldehyde 7 with methyllithium and titanium tetrachlo-
5
0
ride. Oxidation with Dess-Martin periodinane (DMP) generated
While each of the phenstatin-based BAPCs represents a new
chemical entity, phenstatin (Scheme 1) and the prodrug triggers
ketone 10 in high yield, and further methylation of ketone 10 fur-
5
0,51
nished the gem-dimethyl trigger 11 (Scheme 2).
Further inves-
2
3,49
were synthesized utilizing methodology previously described.
Isovanillin was protected as its corresponding tert-
tigation determined that trimethyl aluminum was a more effective
methylation agent for the conversion of aldehyde 7 and ketone 10
to their corresponding mono- and gem- triggers 9 and 11, respec-
tively, in comparison to the methyllithium/titanium tetrachloride
method (see Supplementary data).
butyldimethylsilyl ether, aldehyde 2.2
3,49
Halogen-metal exchange
of aryl bromide 3, followed by the introduction of aldehyde 2,
afforded the secondary alcohol 4, which, upon oxidation, generated
2
3,49
phenstatin precursor 5.
yielded phenstatin 6.
Removal of the TBS protecting group
A Mitsunobu reaction was utilized to conjugate the bioreduc-
tive triggers to phenstatin to generate the requisite BAPCs
2
3,49
3
7,49,52,53
The nor-methyl nitrothiophene trigger 8 was generated in
excellent yield through reduction of aldehyde 7, as reported by
Davis et al. (Scheme 2).37 The Davis route to the gem-dimethyl
nitrothiophene trigger 11 proved less effective in our hands, gener-
ating the product but only in low yield. This motivated us to con-
sider a modified synthetic methodology toward the gem-dimethyl
(Scheme 3).
Depending on the reactivity of the bioreduc-
tive triggers involved in each reaction, a combination of either
diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate
0
(DIAD), or 1,1 -(azodicarbonyl)dipiperidine (ADDP) and triph-
enylphosphine or tributylphosphine were employed to generate
3
7,52,53
the ether linkage.
Synthesis of the phenstatin nor-methyl
O
H
1
2
R = H
TBSCl, Et
3
N, DMAP,
R = TBS
CH Cl , 90%
2
2
OR
OCH
3
Br
OH
O
1
) n-BuLi, THF,
H
H
3
CO
H
3
CO
o
-78 C, 30 min
PCC, K
rt, 18 h, 60%
2 2
CO3, CH Cl2,
H
3
CO
OCH
3
2) cmpd 2, 5 h,
CO
OCH
OTBS
H
3
CO
OCH
3
3
3
OCH
3
3
o
-78 C to rt, 62%
OCH
OCH
R = TBS
R = H
3
OR
3
4
5
6
TBAF 3H O,
2
THF, 87%
Phenstatin
Scheme 1. Synthesis of Phenstatin 6.23,49
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4
B.A. Winn et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
HO
S
NaBH
THF, 0 C to rt,
4
, MeOH,
o
NO
2
O
1
h, 92%
8
H
S
NO2
MeLi, TiCl
4
OH
O
H C CH
HO
3
3
7
MeLi, TiCl
78 oC to rt,
S
4
78 oC to rt,
H
3
C
S
DMP, CH2Cl2
H
3
C
S
-
NO2
NO
2
NO
2
-
Et O, 12 h, 90%
rt, 1 h, 90%
2
Et O, 12 h, 45%
2
1
9
10
1
Scheme 2. Synthesis of nitrothiophene triggers.37,50,51
O
O
H
3
CO
CO
H
3
CO
CO
A
H
3
OCH
3
R1 R2
H
3
OCH
3
OCH
3
OH
S
8
9
1
: R1 = R2 = H
R
1
NO2
OCH
3
O
HO
1
2
: R = H, R = CH
3
R2
S
1
2
1: R = R = CH
3
6
12-14
8,9,11
(
A) cmpd 12: DIAD, P(Ph)
cmpd 13: DIAD, P(Ph) , CH
cmpd 14: ADDP, P(Bu)
, CH
3
, CH
Cl
Cl
2
Cl
, cmpd 9, rt, 2 d, 30% (R
, cmpd 11, rt, 2 d, 7% (R
2
, cmpd 8, rt, 2 d, 34% (R
= H, R
= R = CH
1
= R
2
2
= H);
= CH
);
NO
2
3
2
2
1
3
);
3
2
2
1
2
3
Scheme 3. Synthesis of the phenstatin nitrothiophene BAPCs.37,49,52,53
O
H
O
O
NaBH
0
8
4
, MeOH,
C, 2.5 h
0%
HO
O
NO
2
o
NO
2
3
3
3
4
O
CH3
OH
H C
HO
3
H
O
O
O
Al(CH
3
)
3
O
MeLi, TiCl
4
H
3
C
DMP, CH
2
Cl
2
3
H C
NO
2
NO2
o
NO
2
NO
2
CH Cl , 0 C,
o
-78 C to rt,
2
2
1
h, 76%
Et
2
O, 12 h, 81%
1.5 h, 83%
3
3
36
37
3
5
Scheme 4. Synthesis of the nitrofuran triggers.37,50,51,64
nitrothiophene BAPC 12 and its corresponding mono-methyl BAPC
data).51,54,64 Despite several attempts directed towards methyla-
tion of the nitroimidazole ketone, the gem-dimethyl nitroimidazole
trigger was not successfully synthesized in our hands (Scheme S5,
1
3 involved the reaction of phenstatin with nitrothiophene 8 (or 9),
37,49
DIAD, and triphenylphosphine.
The phenstatin gem-dimethyl
5
0,51,55–63
BAPC 14 was synthesized in a Mitsunobu reaction utilizing ADDP,
Supplementary data).
3
7,49
tributylphosphine, phenstatin, and nitrothiophene trigger 11.
The synthetic route for the nitrofuran bioreductive triggers
(Scheme 4) was based on the new route to the nitrothiophene trig-
The phenstatin nitrobenzyl BAPCs were synthesized in a similar
fashion to the phenstatin nitrothiophene BAPCs, utilizing a Mit-
3
7,50,51,64
gers shown in Scheme 2.
The nor-methyl nitrofuran trigger
sunobu reaction to generate the critical ether linkage
34 was generated through the reduction of aldehyde 33.37 Alde-
hyde 33 was methylated to yield mono-methyl nitrofuran trigger
7,49,52,53
(
Scheme S1, Supplementary data).3
Synthesis of the nor-
5
0,51
methyl nitrobenzyl BAPC 20 and the mono-methyl nitrobenzyl
BAPC 21 was achieved through the reaction of phenstatin, DIAD,
35.
The synthesis of the gem-dimethyl nitrofuran trigger 37
was achieved in high yield by oxidation of mono-methyl trigger
35 to its corresponding ketone 36, followed by methylation to gen-
and triphenylphosphine, with the appropriate trigger (4-nitroben-
zyl alcohol 19 or mono-methyl trigger 16, respectively).3
7,49
A reac-
erate the gem-dimethyl nitrofuran trigger 37.
50,51
tion of tributylphosphine, ADDP, phenstatin, and the gem-dimethyl
nitrobenzyl trigger 18 furnished the gem-dimethyl nitrobenzyl
Mitsunobu chemistry was once again employed to form the
requisite ether linkage between the nitrofuran triggers and phen-
3
7,49
37,49,54,64
BAPC 22.
The synthetic route reported by Reetz and co-workers
statin, generating the nitrofuran BAPCs (Scheme 5).
The
was utilized for the synthesis of the nitrobenzyl triggers 16 and 18
nor-methyl nitrofuran BAPC 38 and the mono-methyl nitrofuran
BAPC 39 were generated through the reaction of phenstatin, DIAD,
and triphenylphosphine with the appropriate trigger (nor-methyl
nitrofuran 34 or mono-methyl nitrofuran 35, respectively).3
The gem-dimethyl nitrofuran BAPC 40 was likewise synthesized
50
(Scheme S2, Supplementary data).
The nitroimidazole phenstatin BAPCs (Scheme S3, Supplemen-
7,49
tary data) were generated through a Mitsunobu reaction analogous
to the chemistry described previously for the nitrobenzyl and
3
7,49,51–54
37,49
nitrothiophene BAPCs.
The nor-methyl nitroimidazole
via a Mitsunobu reaction with nitrofuran 37.
BAPC 31 was synthesized utilizing a Mitsunobu reaction with
phenstatin, DIAD, triphenylphosphine, and nitroimidazole 28.
The phenstatin BAPCs (Fig. 4), as well as phenstatin, were
evaluated for their ability to inhibit tubulin polymerization and
compete for the colchicine binding site (Table 1). In addition to
the mono-methyl nitrothiophene BAPC 13 and the gem-dimethyl
nitrofuran BAPC 40, the entire nitrobenzyl series (20, 21, 22) and
the nitroimidazole series (31, 32) all proved to be inactive
3
7,49
Triphenylphosphine, DIAD, phenstatin, and mono-methyl nitroim-
idazole 30 reacted to yield the mono-methyl nitroimidazole BAPC
3
7,49
3
2.
The synthesis of the nitroimidazole triggers followed a
route developed by Conway et al. (Scheme S4, Supplementary
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B.A. Winn et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
5
O
O
H
3
CO
CO
H
3
CO
CO
a
H
3
OCH
3
R1 R2
H
3
OCH
3
1
1
OCH
3
OH
34: R = R2 = H
R
O
OCH
3
O
HO
NO
2
1
2
R2
O
3
5: R = H, R = CH
3
1
2
6
37: R = R = CH
3
38-40
34,35,37
1
2
NO
2
(
a) cmpd 38: DIAD, P(Ph)
cmpd 39: DIAD, P(Ph)
, CH
cmpd 40: ADDP, P(Bu) , CH
3
, CH
Cl
Cl
2
Cl
, cmpd 35, rt, 2 d, 47% (R = H, R = CH
3
);
2
, cmpd 34, rt, 2 d, 41% (R = R = H);
1
2
3
2
2
1
2
3
2
2 3
, cmpd 37, rt, 2 d, 8% (R = R = CH );
Scheme 5. Synthesis of the phenstatin nitrofuran BAPCs.37,49,54,64
O
O
O
O
H
3
CO
CO
H
3
CO
H CO
3
H CO
3
H
3
OCH
3
H CO
OCH
H CO
OCH
H CO
OCH
3
3
1
3
3
3
3
1
OCH3
20-22
O
R
1
1
OCH
3
O
R
OCH3
31,32
O
R
OCH3
38-40
O
R
R2
R2
S
R2
R2
12-14
N
CH
3
O
N
NO
2
NO
2
NO
2
NO
2
cmpd 12,20,31,38 : R = R2 = H
1
1
2
cmpd 13,21,32,39 : R = H, R = CH
3
cmpd 14,22,40: R = R2 = CH
1
3
Fig. 4. Phenstatin BAPCs prepared by chemical synthesis.
Table 1
Inhibition of tubulin polymerization and percent inhibition of colchicine binding.
Table 2
Bioreductive trigger hydrolysis (untreated) and cleavage of POR-Treated BAPCs.
Compd
Inhibition of tubulin
assembly IC50
Inhibition of colchicine binding
% inhibition M ± SD
5 lM
Compd Hydrolysis percentage in pH 7.4
phosphate buffer for 24 h
Cleavage percentage of
l
POR-treated for 24 ha
(l
M)±SD
14
22
40
0
0
0
100
0
100
4
2
0.73 ± 0.04
1.0 ± 0.2a
98 ± 0.1
CA4
6
Phenstatin
85 ± 2
a
Anoxic conditions.
1
1
1
2
2
2
3
3
3
3
4
2
3
4
0
1
2
1
2
8
9
0
16 ± 0.6
>20
9.0 ± 1
>20
>20
>20
>20
>20
15 ± 1
12 ± 0.5
>20
19 ± 2
15 ± 0.09
16 ± 3
11 ± 4
analogues), the three phenstatin-based gem-dimethyl BAPCs pre-
pared in this study were subjected to further initial evaluation. A
stability study carried out in pH 7.4 potassium phosphate buffer
solution on the three gem-dimethyl trigger BAPCs (14, 22, 40)
demonstrated promising results, with each BAPC remaining struc-
turally intact over a 24 h period with no observable (by HPLC anal-
ysis) cleavage or degradation. These same three BAPCs (14, 22, 40)
were treated (in separate experiments) with NADPH cytochrome
P450 oxidoreductase (POR) to evaluate them as substrates for this
enzyme under anoxic conditions. The gem-dimethyl furan and
thiophene compounds (14 and 40, respectively) were fully cleaved
over the course of 24 h, while, interestingly, the gem-dimethyl ben-
zyl compound 22 did not undergo cleavage in the 24 h assay
(Table 2). The reduction potential for the nitrobenzyl trigger is less
than the reduction potential (less electron-philic) than that of the
nitrofuran, nitroimidazole, and nitrothiophene triggers, possibly
explaining its resistance to cleavage by POR under these assay
5.8 ± 5
8.8 ± 5
7.1 ± 0.01
11 ± 5
17 ± 0.01
16 ± 3
13 ± 5
a
Data from reference 23.
(
IC50 > 20 lM) as inhibitors of tubulin polymerization. This is an
important and desirable attribute for these phenstatin-based
BAPCs, since they are designed to be biologically inactive until
enzyme-mediated prodrug cleavage releases the active anticancer
agent (phenstatin), which itself is a potent inhibitor of tubulin
polymerization (IC50 = 1.0
9) evaluated in this study demonstrated significantly reduced
IC50 range of 9–16 M) inhibition of tubulin polymerization, in
comparison to phenstatin. The limited activity of these four BAPCs
might be attributed to partial trigger cleavage under the assay
conditions, leading to generation of the tubulin-active parent
anticancer agent phenstatin. This hypothesis of partial cleavage
has not been confirmed or further investigated to date.
Based on the pioneering work by Davis and co-workers with
CA4-BAPCs, which demonstrated the potency of the gem-dimethyl
variant (in comparison to the corresponding nor- and mono-methyl
lM). The other four BAPCs (12, 14, 38,
6
4
3
(
conditions.
l
In conclusion, a series of eleven promising phenstatin-based
BAPCs were prepared by chemical synthesis and had little or no
activity as inhibitors of tubulin assembly or binding of colchicine
to tubulin, in comparison to the parent anticancer agent phen-
statin, a potent tubulin inhibitor. In preliminary studies, the three
phenstatin-based gem-dimethyl BAPCs (14, 22, 40) demonstrated
aqueous solution stability (over 24 h), and two of the BAPCs (14,
40) were suitable substrates for POR. These BAPCs have the poten-
tial to be therapeutic agents that target hypoxic tumor cells.
Please cite this article in press as: Winn B.A., et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.11.093

6
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The authors are grateful to the Cancer Prevention and Research
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of the authors and does not necessarily reflect the official views of
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Please cite this article in press as: Winn B.A., et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.11.093