Design, synthesis, and biological evaluation of water-soluble amino acid prodrug conjugates derived from combretastatin, dihydronaphthalene, and benzosuberene-based parent vascular disrupting agents

Article abstract of DOI:10.1016/j.bmc.2016.01.007

Targeting tumor vasculature represents an intriguing therapeutic strategy in the treatment of cancer. In an effort to discover new vascular disrupting agents with improved water solubility and potentially greater bioavailability, various amino acid prodru

Full text of DOI:10.1016/j.bmc.2016.01.007

Bioorganic & Medicinal Chemistry 24 (2016) 938–956
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of water-soluble amino
acid prodrug conjugates derived from combretastatin,
dihydronaphthalene, and benzosuberene-based parent vascular
disrupting agents
Laxman Devkota a,y, Chen-Ming Lin a,y, Tracy E. Strecker a, Yifan Wang a, Justin K. Tidmore a, Zhi Chen a,
Rajsekhar Guddneppanavar a, Christopher J. Jelinek a, Ramona Lopez b, Li Liu b, Ernest Hamel c,
a,
Ralph P. Mason b, David J. Chaplin a,d, Mary Lynn Trawick a, , Kevin G. Pinney
⇑
⇑
a Department of Chemistry and Biochemistry, Baylor University, One Bear Place #97348, Waco, TX 76798-7348, United States
b Department of Radiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9058, United States
c 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
d OXiGENE 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:
Targeting tumor vasculature represents an intriguing therapeutic strategy in the treatment of cancer. In
an effort to discover new vascular disrupting agents with improved water solubility and potentially
greater bioavailability, various amino acid prodrug conjugates (AAPCs) of potent amino combretastatin,
amino dihydronaphthalene, and amino benzosuberene analogs were synthesized along with their corre-
sponding water-soluble hydrochloride salts. These compounds were evaluated for their ability to inhibit
tubulin polymerization and for their cytotoxicity against selected human cancer cell lines. The amino-
based parent anticancer agents 7, 8, 32 (also referred to as KGP05) and 33 (also referred to as KGP156)
demonstrated potent cytotoxicity (GI50 = 0.11–40 nM) across all evaluated cell lines, and they were
Received 21 October 2015
Revised 27 December 2015
Accepted 5 January 2016
Available online 6 January 2016
Keywords:
Small-molecule synthesis
Inhibitors of tubulin polymerization
Vascular disrupting agents
Amino acid prodrug salts
Anti-cancer agents
Combretastatin analogs
Benzosuberene analogs
Dihydronaphthalene analogs
strong inhibitors of tubulin polymerization (IC50 = 0.62–1.5 lM). The various prodrug conjugates and
their corresponding salts were investigated for cleavage by the enzyme leucine aminopeptidase (LAP).
Four of the glycine water-soluble AAPCs (16, 18, 44 and 45) showed quantitative cleavage by LAP, result-
ing in the release of the highly cytotoxic parent drug, whereas partial cleavage (<10–90%) was observed
for other prodrugs (15, 17, 24, 38 and 39). Eight of the nineteen AAPCs (13–16, 42–45) showed significant
cytotoxicity against selected human cancer cell lines. The previously reported CA1-diamine analog and its
corresponding hydrochloride salt (8 and 10, respectively) caused extensive disruption (at a concentration
of 1.0 lM) of human umbilical vein endothelial cells growing in a two-dimensional tubular network on
matrigel. In addition, compound 10 exhibited pronounced reduction in bioluminescence (greater than
95% compared to saline control) in a tumor bearing (MDA-MB-231-luc) SCID mouse model 2 h post treat-
ment (80 mg/kg), with similar results observed upon treatment (15 mg/kg) with the glycine amino-dihy-
dronaphthalene AAPC (compound 44). Collectively, these results support the further pre-clinical
development of the most active members of this structurally diverse collection of water-soluble prodrugs
as promising anticancer agents functioning through a mechanism involving vascular disruption.
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
disorganized proliferation of endothelial cells.1–5 Both small-
molecules and biologics that specifically interact with tumor
Tumor vasculature is as an attractive target for the treatment of
cancer due, in part, to its distinct characteristics, such as rapid and
vasculature are referred to as vascular targeting agents (VTAs),
which are further sub-classified as angiogenesis-inhibiting agents
(AIAs) and vascular disrupting agents (VDAs).6,7 AIAs are typically
represented by compounds that target vascular endothelial growth
factors to prevent the development of new tumor vasculature.7
Conversely, VDAs are comprised of compounds that disrupt and
directly damage established tumor vasculature, by affecting
⇑
Corresponding authors. Tel.: +1 254 710 6857 (M.L.T.), +1 254 710 4117 (K.G.P.).
E-mail addresses: Mery_Lynn_Trawick@baylor.edu (M.L. Trawick), Kevin_Pin-
ney@baylor.edu (K.G. Pinney).
y
Authors contributed equally.
http://dx.doi.org/10.1016/j.bmc.2016.01.007
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

L. Devkota et al. / Bioorg. Med. Chem. 24 (2016) 938–956
939
H3CO
H3CO
H3CO
rapidly growing endothelial cells, to suppress tumor blood flow.
One class of VDAs interacts with the colchicine (Fig. 1) site located
on the ,b-heterodimer of tubulin, causing cell retraction, round-
NHCOCH3
O
A
R1
R2
H3CO
H3CO
B
a
OCH3
ing, and ultimately detachment from the aggregated sheet of
OCH3
cells.1,4,8,9
OCH3
Combretastatin Natural Products and Analogues
Combretastatin A-4 (CA4) : R1 = H; R2 = OH
Colchicine
The natural products combretastatin A-4 (CA4) and combretas-
tatin A-1 (CA1) (Fig. 1), originally isolated by Pettit and co-workers
(Arizona State University) from the South African bush-willow
tree, Combretum caffrum Kuntze, are among the colchicine site
class of tubulin binding VDAs.10–13 This family of natural products,
along with a number of synthetic derivatives and analogs,6,14–17
significantly inhibit microtubule assembly in endothelial cells lin-
ing tumor-feeding vasculature, leading to a series of cell signaling
events that ultimately result in endothelial cell morphology
changes and blood flow reduction.1,7,17 The inhibition of tubulin
polymerization results in activation of RhoA, an intracellular coor-
dinator of the cytoskeletal rearrangement of microtubules and
actin, and leads to rapid vascular collapse.15,18,19 Administration
of these VDAs in amounts significantly lower than their maximum
tolerated doses has resulted in tumor necrosis in treated laboratory
mice.3,12
Combretastatin A-4P (CA4P) : R1 = H; R2 = OPO3Na2
Combretastatin A-1 (CA1) : R1 = R2 = OH
Combretastatin A-1P (OXi4503) : R1 = R2 = OPO3Na2
2' CA4-amine: R1 = NH2; R2 = H
CA1-diamine: R1 = R2 = NH2
3' CA4-amine: R1 = H; R2 = NH2
n
H3CO
H3CO
R3
H3CO
OCH3
OCH3
H3CO
O
Fused-Ring Analogues
OCH3
N
H
OH
Dihydronaphthalene phenol (OXi6196): n = 1; R3 = OH
Dihydronaphthalene amine (KGP05): n = 1; R3 = NH2
Benzosuberene phenol (KGP18): n = 2; R3 = OH
Benzosuberene amine (KGP156): n = 2; R3 = NH2
OCH3
R4
3' CA4-L-serinamide: R4 = NH2
AVE8062: R4 = NH2 . HCl
Figure 1. Colchicine and natural and synthetic combretastatin, dihydronaph-
thalene, and benzosuberene analogs.
The combretastatin water-soluble phosphate prodrugs, com-
bretastatin A-4 phosphate (CA4P also known as ZybrestatTM,
Fig. 1) and combretastatin A-1 diphosphate (CA1P also known as
OXi4503, Fig. 1) are among a group of VDAs which have demon-
strated promising efficacy in human clinical trials.18–27 They
undergo enzyme-mediated dephosphorylation and, as their parent
compounds (CA4 and CA1), they bind to tubulin and interfere with
the tubulin-microtubule protein system. This causes pronounced
morphological effects in the tumor vasculature.8,22–28 Pre-clinical
and pharmacokinetic evaluations of these combretastatin prodrugs
in patients bearing advanced tumors indicate their efficacy as
VDAs.22,29–31 Interestingly, CA1P has dual mechanistic capability,
functioning as both a VDA and as a cytotoxic agent based on its
(sub-nanomolar to picomolar GI50 values) against selected human
cancer cell lines and strong tubulin inhibitory activities.45–47 Cer-
tain of these dihydronaphthalene analogs have subsequently been
reported by another group.49 In our previous work,46 we also
demonstrated robust tubule disruption [in a human umbilical vein
endothelial cell (HUVEC) tube disruption assay] and cell rounding
capability of KGP156, which is one of the parent compounds for
several of the prodrugs designed and synthesized in this study.
The issue of limited water solubility associated with these ani-
line based anti-cancer agents can be addressed through prodrug
strategies.50,51 Amino acid prodrugs, like glycine and serine, with
shorter hydrocarbon or polar side chains, are reported to be more
readily water soluble and more likely to be cleaved because of their
structural simplicity.50 Another advantage of using amino acids is
that they are capable of undergoing quantitative cleavage by
hydrolytic enzymes, likely an aminopeptidase.51,52 The synthesis
and biological evaluation of a series of water-soluble amino acid
in vivo mediated conversion to
a highly reactive ortho-
quinone.18,29–31 The relative structural simplicity of the combretas-
tatins has motivated synthetic chemists to develop libraries of
structurally inspired analogs through alteration of the A-ring, the
B-ring, and the ethylene bridge.6,15,16,32–35
prodrugs
of
amino-combretastatin
were
previously
Incorporation of the NH2 substituent within either ring A or ring
B in the combretastatin family resulted in new analogs that exhib-
ited important biological activity.15,36,37 In 2006, we reported the
initial design and synthesis of the 20 CA4-amine (Fig. 1) and
described its potent inhibition of tubulin assembly and its activity
as a VDA.15 Later work by others confirmed the potency of this 20
CA4-amine analog and related compounds.34 Subsequently, our
studies showed that the di-amino variant of combretastatin A-1
(CA1-diamine, Fig. 1)16 was strongly cytotoxic against human can-
cer cell lines (average GI50 = 13.9 nM) and also demonstrated
potent activity in regard to inhibition of tubulin assembly
reported.15,35,53 A water-soluble serinamide prodrug of 30 CA4-
amine (Fig. 1) known as AVE8062 (Ombrabulin, synthesis36 is
described in Supplementary data), showed significant promise as
a VDA in phase III human clinical trials54,55 and enhanced antitu-
mor activity and decreased toxicity (for normal cells) in both
in vitro and in vivo models.36,53,55–57 The parent drug is generated
after amide bond cleavage by a hydrolytic enzyme.51,53 Leucine
aminopeptidase (LAP)58–60 is one of a widely distributed group of
aminopeptidases that exhibit broad specificity61–63 and that cat-
alyzes the hydrolysis of amino acids from the amino terminus of
polypeptide chains.64 There are also a number of literature reports
of amino acid prodrugs cleaved by LAP.50,53 In humans, LAP is
found primarily in the cytosol of liver cells, which makes the serum
leucine aminopeptidase a marker of hepatic disorders.65,66 The
increase of LAP in human sera can also be diagnostically indicative
of a number of cancers such as carcinoma of the pancreas and head
and neck cancer.67–69
(IC50 = 2.8
l
M).16 It is fairly common for compounds that interact
M range (cell free assay) to demonstrate
with tubulin in the low
l
nM cytotoxicity against human cancer cell lines; a variety of
factors are postulated to influence this activity differential.38 The
trimethoxyphenyl moiety, the p-methoxyphenyl moiety, the Z-
configuration of the two aryl rings, and the optimal 4–5 Å aryl–aryl
distance all proved important for enhanced tubulin binding activ-
ity of the combretastatin analogs.17,39–41 Inspired, in part, by the
SAR studies associated with the combretastatins and their close
structural analogs, we were the first to report the synthesis and
biological activity of related dihydronaphthalene tubulin-binding
agents [for example, OXi6196 and KGP05, Fig. 1],42–44 followed
by the discovery of a phenolic-based benzosuberene (KGP18,
Fig. 1) analog and its corresponding amino congener (KGP156,
Fig. 1).40,45–48 These compounds have emerged as potential pre-
clinical candidates due to their robust in vitro cytotoxicity
Inspired by these developments, herein we report the synthesis
of eight glycine and serine AAPCs of highly potent amino-bearing
structural variants incorporated within the combretastatin, dihy-
dronaphthalene, and benzosuberene molecular scaffolds.42,44,70,71
Furthermore, in order to enhance water solubility (and potentially
bioavailability)72 of these newly synthesized AAPCs, their eleven
hydrochloride salts were synthesized.51,53 The synthesis of com-
pounds 13, 15, 37, 39, 30 CA4-
L-serinamide and AVE8062 were pre-
viously reported,15,36,47,53 and they were re-synthesized as a part of
our ongoing biological studies. Each of the parent amino-based

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L. Devkota et al. / Bioorg. Med. Chem. 24 (2016) 938–956
anticancer agents and their corresponding AAPCs were evaluated
for their cytotoxicity against selected human cancer cell lines
and for their ability to inhibit tubulin polymerization. In addition,
these amino acid prodrug hydrochloride salts and two selected
serinamide prodrugs (non-salts) were evaluated for their ability
to undergo amide bond cleavage by LAP.53
conditions,73 the respective HCl salts of glycine and serine amino
acid prodrug conjugates (15 and 16, Scheme 2) were synthesized
from compounds 12 and 13. The water-soluble hydrochloride salt
of the glycine variant of CA1-diamine (24, Scheme 3) was obtained
using a similar synthetic strategy with only about 10% isomeriza-
tion to the E-isomer. Our initial attempts to prepare the hydrochlo-
ride salt of compound 22 were unsuccessful. However, treatment
of compound 20 (Scheme 3) with NaOH (2 M) followed by HCl
(4 N) yielded the desired CA1-diamine-based water-soluble amino
acid prodrug conjugate (23) in a single step.
2. Results and discussion
2.1. Synthesis
2.1.2. Synthesis of amino acid prodrug conjugates of amino
dihydronaphthalene and amino benzosuberene
Twenty six compounds (including amino combretastatin, dihy-
dronaphthalene and benzosuberene parent compounds, amino
acid prodrugs, and their corresponding water soluble hydrochlo-
ride salts) were synthesized for this study. Among these, com-
pounds 7–10, 13, 15,15,16 32,42,43 33, 37, 39,46,47 30 CA4-amine, 30
2.1.2.1. Synthesis of dihydronaphthalene and benzosuberene
amines.
The synthesis of amino dihydronaphthalene (32)42,44
and amino benzosuberene (33),45–47 as previously reported by our
group, is illustrated in Scheme 4. Initially, 6-methoxy-1-tetralone
was nitrated to form two constitutional isomers, with one isomer
being the desired product, 5-nitro-6-methoxy-1-tetralone (25).
Compound 32 was obtained by reaction of 5-bromo-1,2,3-
trimethoxybenzene with n-butyllithium, followed by the addition
of compound 25. The reaction of compound 30 with Zn in the pres-
ence of AcOH resulted in reduction of the nitro group, and, follow-
ing a subsequent condensation reaction, the desired product (32)
was obtained in a 53% yield (over these two steps). The synthesis
of amino-benzosuberene KGP156 (33), initially reported by us in
2012,46 utilized a sequential Wittig olefination, selective reduction
with 1,4-cyclohexadiene, Eaton’s reagent mediated cyclization,
1,2-addition of the appropriately functionalized aryl ring, followed
by condensation and reduction.
CA4-L
-serinamide and AVE806236,53 were re-synthesized for the
purpose of further biological evaluation.
2.1.1. Synthesis of amino acid prodrug conjugates of amino
combretastatins
2.1.1.1. Synthesis of combretastatin amines.
Our previous
synthesis of 20 CA4-amine 7 involved a Wittig reaction between
(4-methoxy-2-nitrobenzyl)triphenylphosphonium bromide and
3,4,5-trimethoxybenzaldehyde, followed by a reduction of the
nitro group to form the corresponding amine using Na2S2O4.15,32
Later, this compound was synthesized and reported by another
group as well.34 Switching the Wittig reaction partners to 3,4,5-
trimethoxybenzyltriphenylphosphoniumbromide 4 and 4-meth-
oxy-2-dinitrobenzaldehyde (Scheme 1), followed by the separation
of Z and E-isomers (1:0.4 ratio) and reduction of Z-isomer 5a to
amine 6a using zinc35 in acetic acid (AcOH) increased the overall
yield for compound 7 about 4-fold over two steps. The synthesis
of CA1-diamine (8, Scheme 1) was achieved as reported earlier,16
involving a key Wittig reaction between 3,4,5-trimethoxybenzylt-
riphenylphosphonium bromide 4 and 4-methoxy-2,3-dinitroben-
zaldehyde 1, followed by reduction of the nitro group of
Z-isomer 6a to achieve the desired target compound (CA1-diamine
8). Upon treatment with HCl (4 N), these combretastatin-based
amines (7 and 8) were converted to their corresponding
hydrochloride salts (9 and 10).
2.1.2.2. Synthesis of hydrochloride salt of amino acid prodrug
conjugates of amino dihydronaphthalene and benzo-
suberene.
amino dihydronaphthalene (32), Fmoc-
To obtain the desired amino acid prodrugs of
-ser(Ac)-OH or Fmoc-gly-
L
OH were reacted utilizing general peptide synthetic methodology.
Compound 32 was treated with T3P, Et3N, and the appropriate
Fmoc-amino acid to obtain N-Fmoc protected amino acid amides
(34) and (40), which upon deprotection resulted in the desired
amino acid prodrugs (36) and (42). The AAPCs 37 and 39 (previ-
ously reported) were re-synthesized for this study.47 Similarly,
the hydrochloride salts (38, 44, and 45) of amino acids (36, 42,
and 43, respectively), were obtained upon treatment with a 4 N
HCl in dioxane solution as shown in Scheme 5.
2.1.1.2. Synthesis of amino acid prodrug conjugates.
The
serine and glycine amino acid prodrug conjugates (13 and 14,
Scheme 2) of 20 CA4-amine 7 were prepared by coupling with
Fmoc-L-ser(Ac)-OH and Boc-glycine-OH, respectively, in the pres-
2.2. Biological evaluation
ence of Et3N and the peptide coupling reagent propylphosphonic
anhydride (T3P), followed by deprotection. This procedure is
reminiscent of the synthetic strategy employed by Ohsumi and
co-workers53 for the synthesis of water-soluble amino acid pro-
drug salts of 30 CA4-amine56 and our previous studies,15,16 but this
modified synthetic route has benefits in terms of higher yield and
ease of purification from the use of T3P. Similarly, the serine and
glycine amino acid prodrug conjugates (19–21, Scheme 3) of
CA1-diamine (8) were synthesized using a standard peptide cou-
pling procedure (Scheme 2).
Each of the twenty six compounds synthesized for this study
(including parent drugs, amino acid prodrugs, and their corre-
sponding water soluble hydrochloride salts; compiled in Fig. 2)
were evaluated for their cytotoxicity against selected human can-
cer cell lines [SK-OV-3 (ovarian), NCI-H460 (lung), and DU-145
(prostate), Table 1]. Furthermore, these compounds were evalu-
ated biologically for their ability to inhibit tubulin polymerization
(cell free assay) and colchicine binding (Table 2). In addition, thir-
teen water soluble AAPCs (including two as their non-salt forms)
were evaluated for enzymatic cleavage by LAP (Table 3 and
Fig. 3).37,42–47 An endothelial tube disruption assay for CA1-dia-
mine (compound 8, Fig. 2) was carried out utilizing HUVECs
(Fig. 4). The hydrochloride salts of CA1-diamine (compound 10,
Fig. 5) and dihydronaphthalene glycinamide (compound 44,
Fig. 2) were initially evaluated for their in vivo ability to disrupt
tumor blood flow utilizing dynamic bioluminescence imaging
(BLI, Figs. 6 and 7) studies.
2.1.1.3. Synthesis of hydrochloride salts of amino acid prodrug
conjugates.
The synthesis of compound 15 is described in our
earlier publication.15 Our initial efforts to re-synthesize compound
15 and compound 16 (new compound) at room temperature in the
presence of solvent resulted in the complete isomerization from Z
to E, affording compounds 17 and 18. These E-isomers were used as
a model system to evaluate LAP mediated cleavage of their corre-
sponding amino acid prodrugs. Utilizing solvent free reaction

L. Devkota et al. / Bioorg. Med. Chem. 24 (2016) 938–956
941
H
O
H
O
H
O
NO2
NO2
NO2
NO2
HNO3, H2SO4
+
H2SO4, 0 oC, 10 min
O2N
OCH3
1
OCH3
2
OCH3
OH
Br
PPh3Br
OCH3
PBr3, CH2Cl2
0 oC, 1 h
PPh3, acetone
rt, 5 h
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
H3CO
OCH3
3
4
H3CO
H3CO
H3CO
H3CO
H3CO
1. NaH, CH2Cl2 0 o
C
NO2
R
OCH3
5a. (Z): R = H
NH2 4 N HCl-dioxane
NH2 . HCl
R
OCH3
9. R = H
10. R = NH2 . HCl
Zn/AcOH
rt, 3 h
Compound 4
H3CO
CH2Cl2, rt, 5-12 h
R
2. 4-methoxy-2-
OCH3
OCH3
OCH3
nitrobenzaldehyde
1
or compound
OCH3
7. R = H (from 5a)
6a
5b.
(E): R = H
8
. R = NH2 (from
)
6a. (Z): R = NO2
6b. (E): R = NO2
Scheme 1. Synthesis of combretastatin amines.15,16
H3CO
H3CO
H3CO
NH2 . HCl
H
NH2 . HCl
OH
H
N
N
H3CO
OCH3
O
OCH3
O
OCH3
13
From compound
OCH3
From compound
16.
12
15.
4 N HCl-dioxane
oC, 30 min
4 N HCl-dioxane
0
0
oC, 30 min
H3CO
H3CO
H3CO
H3CO
R2
R1
H3CO
H3CO
HN
NH2
R1
H
N
H
N
c or d
NH2
a or b
rt, 30 min
Et3N, T3P, CH2Cl2, rt, 3-8 h
OCH3
7
OCH3
O
OCH3
O
OCH3
1 = CH2OAc, R2 = Fmoc
12. R1 = H2, R2 = Boc
OCH3
OCH3
c = 2 M NaOH, CH2Cl2:MeOH (1:1)
d = TFA, CH2Cl2
a = Fmoc-L-Ser(Ac)-OH
b = Boc-Gly
11.
13.
R1 = CH2OH
R
14. R1 = H2
4 N HCl-dioxane
CH2Cl2, rt, 4 h
4 N HCl-dioxane
CH2Cl2, rt, 4 h
OCH3
NH2 . HCl
OCH3
H3CO
H3CO
H3CO
H3CO
NH2 . HCl
OH
HN
HN
OCH3
18.
O
OCH3
17.
O
12
13
From compound
From compound
Scheme 2. Synthesis of amino acid prodrug conjugates of 20-combretastatin amines.15,53
2.2.1. Cytotoxicity, inhibition of tubulin polymerization, and
percent inhibition of colchicine binding
corresponding serinamide AAPC 17. Negligible cytotoxicity was
observed for the bis-substituted AAPCs (22–24). The serinamide
AAPCs (36–39) of the parent compounds 32 (KGP05) and 33
(KGP156) demonstrated limited cytotoxicity against these cancer
cell lines, while the glycinamide analogs (42–45) were more active.
Pronounced inhibition of tubulin polymerization and inhibition
of colchicine binding were observed for amino-dihydronaph-
Cytotoxicity studies carried out with all twenty six compounds
against selected human cancer cell lines [SK-OV-3 (ovarian), NCI-
H460 (lung), and DU-145 (prostate), Table 1] indicated that each
of the parent drugs (compounds 7,15 8,16 3242,43 and 3346,47) were
highly potent with GI50 values ranging from sub-nanomolar to
nanomolar. The amino dihydronaphthalene 32 was found to be
especially potent (GI50 = 1–3 nM across all three of the human can-
cer cell lines evaluated in this study) and was comparable to the
clinically relevant agent CA4.35 Both the serinamide and glyci-
namide AAPCs (compounds 15 and 16, respectively) of the 20
CA4-amine 7 demonstrated significant cytotoxicity against each
of the cancer cell lines. When comparing the E-isomers 17 and
18, the glycinamide AAPC 18 was more cytotoxic than the
thalene 32 (assembly IC50 = 0.62
colchicine binding at 5 M, respectively, Table 2). Similarly, the
other parent compounds 7, 8 and 33 proved highly potent in these
tubulin assays (ranging from 1.1 M to 1.5 M and 76% to 88% at
M, respectively). Uniformly, the AAPCs (compounds 13–18,
22–24, 36–39 and 42–45) proved to be inactive (IC50 >20 M) as
lM and 92% inhibition of
l
l
l
5
l
l
inhibitors of tubulin assembly, which was anticipated in this type
of cell free assay.

942
L. Devkota et al. / Bioorg. Med. Chem. 24 (2016) 938–956
NH2 . HCl
H3CO
H3CO
O
OH
NH
O
OCH3
OH
NH2 . HCl
N
H
OCH3
23.
20
From compound
1. 2 M NaOH, CH2Cl2:MeOH (1:1)
rt, 30 min
2. 4 N HCl-dioxane
CH2Cl2, rt, 2 h
R2
HN
NH2
H3CO
H3CO
O
H3CO
H3CO
O
OH
H3CO
H3CO
R1
NH
NH
NH2
NH2
2 M NaOH
O
e or f or g
O
OCH3
R1 CH2Cl2/MeOH (1:1)
OCH3
OCH3
8
Et3N, T3P, CH2Cl2, rt, 3-8 h
N
N
H
OH
rt, 30 min
H
OCH3
HN
OCH3
NH2
OCH3
R2
e = Fmoc-L-Ser(Ac)-OH
19. R1 = CH2OAc, R2 = Fmoc (from e)
22.
19
From compound
f = Boc-L-Ser(Ac)-OH. DCHA
20.
R
21
1 = CH2OAc, R2 = Boc (from f)
. R1 = H2, R2 = Boc (from g)
g = Boc-Gly
4 N HCl-dioxane
CH2Cl2, rt, 2 h
NH2 . HCl
O
H3CO
H3CO
NH
O
OCH3
N
OCH3
H
NH2 . HCl
24. From compound 21
Scheme 3. Synthesis of amino acid prodrug conjugates of combretastatin diamine.15,16,53
O
O
O
HNO3/AcOH (1:1)
acetic anhydride
0
O2N
oC, 20 h
H3CO
H3CO
H3CO
NO2
25
26
O
O
COOH
COOH
Eaton's reagent
rt, 17 h
H3CO
H
Ph3P(CH2)3COOH
10% Pd/C, EtOH
1,4-cyclohexadiene
rt, 3 h
KOtBu, THF
NO2
NO2
OCH3
NO2
OCH3
28
0 o
C
rt, 20 h
OCH3
NO2
29
27
OCH3
OCH3
H3CO
Br
H3CO
OCH3
OCH3
1. g or h
Zn, AcOH
rt, 6 h
O
HO
H3CO
OCH3
OCH3
2.
,i or j
H3CO
H3CO
H3CO
n
n
n
NH2
32
NO2
NO2
n = 1, g = n-BuLi, THF, -78 oC, 1 h
30
. n = 1
. n = 1
33. n = 2
i = -78 oC
rt, 18 h
n = 2, h = n-BuLi, THF, -78 oC, 1 h
j = -78 oC
rt, 16 h
31. n = 2
Scheme 4. Synthesis of dihydronaphthalene42,44 and benzosuberene amines.42,45–47
2.2.2. Enzymatic assay
LAP, the amount of CA1 diamine (compound 8) detected increased
to 19% and 24% respectively. The mono-serinamide prodrugs of
both the Z and E-isomers (15 and 17, respectively) of 20 CA4-amine
were effectively cleaved by LAP, as was the mono-serinamide
derivative of 30 CA4-amine and its hydrochloride salt (AVE8062),
used as positive controls.53 Only a partial release (less than 10%)
was achieved with excess LAP (3.6 units, 24 h) for the mono-seri-
namide analogs of the amino dihydronaphthalene 38 and amino
benzosuberene 39 compounds. Treatment of the bis-serinamide
prodrug 22 of CA1-diamine or its dihydrochloride salt 23 with a
Preliminary studies were carried out to determine the ability of
LAP to cleave individual amino acid prodrug conjugates (Table 3).
The mono-glycinamide prodrugs 16, 18, 44, 45 were quantitatively
cleaved to their parent compounds. Treatment of the bis-glyci-
namide prodrug (24) with LAP (0.12 units) resulted in disappear-
ance of greater than 95% of the prodrug by 25 h, but only 9% of
the parent CA1-diamine (compound 8) was formed. Two additional
peaks, presumably the mono-glycinamide intermediates, were
observed in the HPLC chromatogram. Using 1.5 and 3.7 units of

L. Devkota et al. / Bioorg. Med. Chem. 24 (2016) 938–956
943
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H3CO
H3CO
H3CO
H3CO
2 M NaOH,
CH2Cl2/MeOH (1:1)
T3P, Et3N
Fmoc-L-Ser(Ac)-OH
4 N HCl-dioxane
CH2Cl2, rt, 1h
CH2Cl2, rt
rt, 18 h
H3CO
H3CO
H3CO
H3CO
n
n
Fmoc
n
n
NH2
O
NH
O
NH
O
NH
AcO
HO
HO
.
N
H
NH2
NH2 HCl
32.
36. n = 1
37.
n = 1
38. n = 1
34. n = 1
35
33. n = 2
n = 2
39.
n = 2
. n = 2
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H3CO
H3CO
H3CO
H3CO
2 M NaOH
MeOH/CH2Cl2 (1:1)
rt, 13 h
T3P, Et3N
Fmoc-Gly-OH
CH2Cl2, rt, 12 h
4 N HCl-dioxane
CH2Cl2, rt, 1 h
H3CO
H3CO
H3CO
H3CO
n
n
n
n
NH2
HN
Fmoc
HN
HN
N
NH2
NH2.HCl
H
O
O
O
32. n = 1
33.
42. n = 1
40. n = 1
44. n = 1
43.
n = 2
41
n = 2
45.
. n = 2
n = 2
Scheme 5. Synthesis of amino acid prodrug conjugates of amino dihydronaphthalene and benzosuberene.45,47[Note: A portion of Scheme 5 was reproduced from Ref. 47 with
permission from Elsevier.]
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
R1
R2
R1
R2
R1
R2
R1
R2
OCH3
OCH3
OCH3
OCH3
OCH3
7. R1 = NH2; R2 = H
OCH3
OCH3
R1 = NH-L-Ser; R2 = H
14. R1 = NH-Gly; R2 = H
3' CA4-L-serinamide. R1 = H;
2 = NH-L-Ser
OCH3
13.
15.
R1 = NH-L-Ser . HCl; R2 = H
9. R1 = NH2 . HCl; R2 = H
10. R1 = R2 = NH2 . HCl
8
16. R1 = NH-Gly . HCl; R2 = H
. R1 = R = NH2
3' CA4-amine. R1 = H; R2 = NH2
AVE8062 = R1 = H; R = NH-L-Ser . HCl
R
OCH3
NH2 . HCl
R1
NH2
H3CO
H3CO
O
H3CO
O
OH
H3CO
H3CO
NH2 . HCl
R1
NH
NH
HN
O
H3CO
OCH3
O
R1
NH2 . HCl
OCH3
OCH3
O
N
H
N
H
OH
OCH3
OCH3
NH2
17.
R1 = CH2OH
22
23.
18. R1 = H2
R1 = CH2OH
24. R1 = H2
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H3CO
OCH3
H3CO
H3CO
H3CO
OCH3
H3CO
H3CO
H3CO
O
n
n
H3CO
H3CO
H3CO
n
n
n
O
NH
NH
HN
NH2
HN
NH2 . HCl
NH2
HO
HO
32
. n = 1
. n = 2
NH2
36.
NH2 . HCl
O
O
33
44.
42. n = 1
n = 1
45. n = 2
n = 1
37. n = 2
38. n = 1
43.
n = 2
39.
n = 2
Figure 2. Compilation of parent drugs and their amino-acid prodrug conjugates evaluated in this study.
large amount (1.5 units) of LAP did not result in any observable
cleavage. For AAPCs that produced more than 50% of the final pro-
duct upon cleavage with LAP in preliminary experiments, rate
studies were conducted. The glycinamide prodrug of the Z-isomer
amine 18, which was cleaved at a rate more than 100 times faster
than its serinamide analog 17, as determined by the relative rates
of cleavage of each prodrug. Differences were also found in the rate
of cleavage of the glycinamide prodrugs of amino dihydronaph-
20 CA4-amine 16 was cleaved at a rate of 180
l
M/min/enzyme unit
(Fig. 3A, B), 150 times faster than its corresponding serinamide
analog 15 (1.2 M/min/enzyme unit). similar result was
observed for the glycinamide prodrug of the E-isomer 20 CA4-
thalene 44 (15.5
zosuberene 45 (0.12
l
M/min/enzyme unit, Fig. 3C, D) and amino ben-
M/min/enzyme unit).
l
l
A
Despite a significantly faster cleavage of the glycinamide pro-
drug 16 versus the serinamide 15 by LAP, the cytotoxicity toward

944
L. Devkota et al. / Bioorg. Med. Chem. 24 (2016) 938–956
Table 1
Table 3
Cytotoxicity data for the amino-based combretastatin, dihydronaphthalene, and
benzosuberene analogs against human cancer cell lines [SK-OV-3 (ovarian), NCI-H460
(lung), and DU-145 (prostate)]
Activity of LAP toward amino acid prodrug conjugates
Compound
Prodrug type % Cleavagea Specific activity of LAP
M/min/enzyme unit)
(
l
Compound
GI50 (nM) SRB assaya
NCI-H460
15
16
17
18
22
23
24
38
39
44
45
Serinamide
Glycinamide
Serinamide
Glycinamide
Serinamide
Serinamide
Glycinamide
Serinamide
Serinamide
Glycinamide
Glycinamide
62%
100%
90%
100%
NC
1.2
180
Ce
SK-OV-3
DU-145
CA1b
38.4 24.2
5.06 0.145
4.45 0.870
15.3 15.8
5.00 0.359
4.49 0.894
24.1 2.00
3.87 0.297
40.1 17.1
35.9 21.8
26.3 16.1
124 116
33.3 19.0
53.2 5.23
31.7 4.87
29.2 5.67
2240 1450
418 137 (D)
32.6 17.3
6.02 0.661
3.30 0.281
16.4 7.29
3.34 0.261
33.1 21.4
39.1 26.5
28.5 23.2
205 241
34.7 8.27
43.6 2.43
48.4 3.36
44.7 3.53
4550 2700
630 306 (D)
Ce
CA4c
ND
ND
ND
ND
ND
15.5
0.12
ND
ND
30 CA4-amined
NC
30 CA4- -serinamided 9.02 4.81
L
Cb
AVE8062d
3.96 0.912
26.6 22.6
23.5 23.4
7.35 3.50
22.8 15.2
39.5 21.1
32.3 6.38
51.6 6.88
48.5 8.53
2290 1170
427 111 (D)
426 94.1 (W)
25900 2550
>86600
Cc,<10%
Cc,<10%
100%
100%
Cd, 95%
Cd, 95%
7e
8f
9
10f
13e
14
15e
16
17
18
30 CA4-
AVE8062
L
-serinamide Serinamide
Serinamide
Cb, disappearance of the bis-glycinamide prodrug 24 was observed, but less than
50% of the expected final product 8 was detected. Cc, the serinamide prodrug was
cleaved, but less than 10% of the expected product was obtained.
Cd, used as positive controls.52 Ce, the prodrug was readily cleaved, but the absolute
rate could not be determined because the parent compound (E-isomer of 20 CA4-
amine) was not available since isomerization (Z to E took place as a byproduct
during salt formation after installation of the amino acid prodrug). ND: Not
determined.
380 60.2 (W) 674 255 (W)
24400 4420
>86600
22
23
24
33600 2600
>86600
39300 13500 (D) >68500 (D)
>64700 (D)
>90400 (W)
0.111 0.114
2.26 1.58
40600 32300
>81300
26000 15700
69400 31300
50.1 4.80
507 188
a
>56400 (W)
0.308 0.402
0.137 0.0596
13700 9700
44900 4100
11600 10200
47000 21400
13.8 7.54
>96600 (W)
0.290 0.126
1.85 1.87
31100 9940
45900 1790
18600 7670
53800 864
36.5 0.899
566 38.2
Percentage of cleavage is calculated based on the area ratio of the parent drug
32g
33h
36
37
38
39
42i
43
44
45
and prodrug at a specific wavelength. NC: No cleavage observed using 1.5 units LAP.
C: Cleaved (a rate study was not carried out).
cancer cell lines over a 48 h treatment was very similar for both
prodrugs and the parent Z-isomer 20 CA4-amine 7 (Table 1), indi-
cating that release of compound 7 from both prodrugs in the pres-
ence of these cancer cell lines was efficient. The slower rate of
cleavage for the E-isomer series resulted in a differential cytotoxi-
city for the serinamide 17 and glycinamide 18 prodrugs. The lack of
cleavage by LAP of the bis-serinamide prodrugs 22 and 23 and the
very low production of parent CA1-diamine (compound 8) from
the bis-glycinamide 24 was reflected in the lack of cytotoxicity
for all three compounds (22–24). This may indicate steric hin-
drance in the interaction of these compounds with LAP. There is
a marked difference in the LAP cleavage rates between glycinamide
prodrugs (compounds 44 and 45) that was reflected in their differ-
ential cytotoxicity in cancer cell lines, and their cytotoxicity was
significantly less than that of either of the corresponding parent
compounds (KGP05, KGP156, respectively).
195 31.9
48.5 32.5
203 29.4
42.0 3.72
567 56.6
58.5 12.0
773 302
D = compound dissolved in DMSO. W = compound dissolved in water.
a
Average of n P 3 independent determinations.
Data from Ref. 74, see Ref. 14 for additional data.
For additional data, see Ref. 14.
For additional data, see Refs. 15,36,37,53.
For additional data, see Ref. 15.
For additional data, see Ref. 16.
For additional data, see Refs. 42,43.
For additional data, see Refs. 46,47.
b
c
d
e
f
g
h
i
For additional data, see Ref. 47.
Table 2
Inhibition of tubulin polymerization and percent inhibition of colchicine binding
Compound
Inhibition of tubulin polymerization
Inhibition of colchicine binding
(% inhibition SD)
IC50
(lM) SD
1
l
M
5 lM
CA1
1.9a
ND
99.6 0.7b
98 0.10
97 1.9
ND
CA4
1.2 0.049
1.2 0.056c
14 1.1d
84 1.1
84 1.8
ND
30 CA4-aminec
30 CA4- -serinamidec
L
AVE8062c
13 0.28d
1.1 0.057d
1.5 0.13e
2.5 0.16
ND
ND
7
8
9
10
32f
33
55 3.8
55 0.14
ND
46 2.3
65 3.6
ND
88 0.93
87 0.19
75 0.90
86 3.8
92 0.16
82 2.4
2.3 0.21e
0.62 0.019
1.3 0.11g
ND = Not determined for this study.
a
Data from Ref. 16, see Ref. 14 for additional data.
Data from Ref. 14.
b
c
d
e
f
For additional data, see Refs. 15,37.
For additional data, see Ref. 15.
For additional data, see Ref. 16.
For additional data, see Refs. 42,43.
For additional data, see Refs. 46,47.
g

L. Devkota et al. / Bioorg. Med. Chem. 24 (2016) 938–956
945
Figure 3. Cleavage of the glycine conjugates of 20 CA4-amine 16 and the amino dihydronaphthalene 44 by LAP. (A) Prodrug 16 was treated with 0.5 units of LAP for 40 h. A
single peak corresponding to the product 20 CA4 amine 7 (tR = 31.29 min) was observed (HPLC chromatogram mobile phase: 26% acetonitrile/74% water containing 0.05%
TFA). Inset: Control, compound 16 was incubated for 40 h without LAP. (B) Rate study for the cleavage of prodrug 16 to form 20 CA4 amine 7. Inset: Calibration curve for 20 CA4
monoamine 7. (C) Prodrug 44 was treated with 0.05 units of LAP for 2 h. A single peak corresponding to the product amino dihydronaphthalene 32 (tR = 12.10 min) was
observed (HPLC chromatogram mobile phase: 28% acetonitrile/72% water containing 0.05% TFA). Inset: Control, compound 44 was incubated for 2 h without LAP. (D) Rate
study for the cleavage of prodrug 44 to form amino dihydronaphthalene 32. Inset: Calibration curve for amino dihydronaphthalene 32.
Control (DMSO)
Compound 8 (0.1 µM)
Compound 10 (0.1 µM)
Compound 10 (1 µM)
Compound 8 (1 µM)
Control (DMSO)
Figure 4. HUVEC tubule disruption at various inhibitor concentrations.
Overall, the cancer cell line cytotoxicity mirrored the cleavage
by LAP of the amino acid prodrug conjugates. In amino acid amides
steric factors and stereospecificity (L vs D) in the position adjacent
to the N-terminal amino acid that is observed in peptide substrates
and in some non-peptidic amino acid conjugates.75,76 For example,
Pettit et al. reported that the serinamide prodrug of 30 CA2-amine
and peptides, an N-terminal
L-serine residue is preferred over gly-
cine, but there is clearly a contribution of binding interactions,

946
L. Devkota et al. / Bioorg. Med. Chem. 24 (2016) 938–956
0hr
2hr
4hr
24hr
Dose: 80 mg/kg
Baseline 2hr
4hr
24hr
2.50E+10
2.00E+10
1.50E+10
1.00E+10
5.00E+09
Radiance
(p/Sec/Cm2/Sr
Color Scale
0.00E+00
1
6
11
16
21
26
Min = 1.56e8
Max = 2.82e9
Time [min]
Figure 5. Dynamic BLI of tumor in response to treatment with compound 10.
Dose: 15 mg/kg
Cage 3, Mouse 1
0hr
4hr
24hr
5E+10
4E+10
3E+10
2E+10
1E+10
0E+00
0hr
4hr
24hr
1
3
5
7
9
11 13 15 17 19 21 23 25 27 29
Time [min]
Dose: 10 mg/kg
Cage 1, Mouse 1
0hr
0hr
4hr
4hr
24hr
24hr
5E+10
4E+10
3E+10
2E+10
1E+10
0E+00
0hr
4hr
24hr
1
3
5
7
9
11 13 15 17 19 21 23 25 27 29
Time [min]
5E+10
4E+10
3E+10
2E+10
1E+10
0E+00
Control (Saline)
Cage 1, Mouse 2
0hr
4hr
24hr
1
3
5
7
9
11 13 15 17 19 21 23 25 27 29
Time [min]
Figure 6. Dynamic BLI of tumor in response to treatment with compound 44.
displayed significantly less activity compared to the glycine deriva-
tive in a cancer cell line panel.35
effect for compound 33 (KGP156) at 0.1
greatly enhanced at a 1
l
M, and this effect was
l
M concentration.46
2.2.3. Endothelial tube disruption assay77
2.2.4. Dynamic bioluminescence imaging77
Rapidly growing HUVECs can be induced to form a capillary-like
network of tubules, which serves as a model for evaluating VDAs.
CA1-diamine (compound 8) and its hydrochloride salt (compound
10) demonstrated significant disruption of a capillary-like network
Bioluminescence Imaging (BLI) represents a valuable indirect
in vivo technique for the assessment of tumor-specific vascular
damage in response to treatment with VDAs.9,78,79 A decrease in
light flux emission at various time points post VDA administration
presumably results from an inability of the injected luciferin to
reach the tumor (grown from luciferase expressing transfected
of tubules (from HUVECs) at a concentration of 0.1
lM. Our previ-
ous work shows a significant tubule disruption and cell rounding

L. Devkota et al. / Bioorg. Med. Chem. 24 (2016) 938–956
947
1.2
1.0
0.8
0.6
0.4
0.2
0.0
caused significant disruption to a network of HUVECs growing on
matrigel, and both CA1-diamine salt 10 and dihydronaphthalene
glycinamide 44 demonstrated a significant reduction in light emis-
sion in a BLI study in SCID mice bearing the luciferase expressing
MDA-MB-231-luc human breast cancer cell line induced tumor.
These results demonstrate that these compounds can function as
water-soluble VDAs.
CA4P (120 mg/kg)
Control
44 (5 mg/kg)
44 (10 mg/kg)
44 (15 mg/kg)
44 (20 mg/kg)
4. Experimental section
4.1. Chemistry
0 h
4 h
24 h
Figure 7. Dose escalation data for BLI evaluation of compound 44. CA4P and
compound 44 (at 10 or 15 mg/kg) each gave significantly less signal at both 4 h and
24 h compared with baseline. Signal from the saline group was significantly
reduced at the 4 h time point. At the 4 h time point compound 44 (5 mg/kg) was not
significantly different from saline control, but was significantly different (p <0.05)
from compound 44 (10 mg/kg) and compound 44 (15 mg/kg). Similarly compound
44 (15 mg/kg) was significantly different from saline and compound 44 (5 mg/kg),
but not compound 44 (10 mg/kg). In general it appeared that a dose of 10, but not
5 mg/kg, generated a significantly greater response, as judged by reduced light
emission. CA4P was significantly different from saline, compound 44 (5 mg/kg), and
compound 44 (10 mg/kg), but not compound 44 (15 mg/kg) or compound 44
(20 mg/kg).
4.1.1. General materials and methods
AcOH, acetic anhydride, acetonitrile, CH2Cl2, dimethylfor-
mamide (DMF), ethanol, methanol, HNO3, H2SO4, and tetrahydro-
furan (THF) were used in their anhydrous forms or as obtained
from the chemical suppliers. Reactions were performed under N2.
Thin-layer chromatography (TLC) plates (precoated glass plates
with silica gel 60 F254, 0.25 mm thickness) were used to monitor
reactions. Water was used to quench and wash the reaction mix-
ture as appropriate. Purification of intermediates and products
was carried out with a Biotage Isolera flash purification system
using silica gel (200–400 mesh, 60 Å) or RP-18 pre-packed columns
or manually in glass columns. Intermediates and products synthe-
cancer cells) due to VDA-induced damage of the vasculature feed-
ing the tumor. Treatment (Fig. 5) of an MDA-MB-231-luc human
breast tumor induced in a SCID mouse model with the diamino-
CA1 salt (compound 10) at a dose of 80 mg/kg resulted in approx-
imately 95% reduction in light emission [at 17 min time-point
(midpoint)] at 2 h post VDA treatment relative to baseline. This
reduction was still maintained (94% decrease) at the 4 h time point
(fresh luciferin injection but no further VDA). There was some
recovery (79% reduction, compared to baseline) at 24 h, which is
typical due to the so-called viable rim.9 The glycinamide dihydron-
aphthalene AAPC (44) demonstrated (Fig. 6) similarly promising
results (97% and 90% decrease in BLI signal at 4 and 24 h, respec-
tively) when administered at a dose of 15 mg/kg, while the signal
reduction at a dose of 10 mg/kg was 70% at 4 h and 55% at 24 h.
In this experiment, the control mouse (saline administration)
demonstrated a 22% decrease in signal at 4 h and 24% reduction
at 24 h. These results, which are comparable to the clinically rele-
vant VDA, CA4P (Fig. 7), provide further preliminary evidence sug-
gesting that compounds 10 and 44 function as water-soluble VDAs
and that glycinamide 44 is capable of being converted in vivo (pre-
sumably through the action of LAP) to its corresponding tubulin-
active parent compound KGP05 (compound 32), which then causes
the tumor-specific vascular damage.
sized during this study were characterized on the basis of their 1
NMR (600 or 500 MHz), 13C NMR (150, 125 or 90 MHz) and 31P
NMR (240 MHz) spectroscopic data using Varian VNMRS
H
a
500 MHz or a Bruker DRX 600 MHz or a Bruker DPX 360 MHz
instrument. Melting point ranges were determined (single experi-
ment) using a Thomas Hoover capillary melting point apparatus
and are uncorrected. Spectral data were recorded in CDCl3, D2O,
(CD3)2CO, DMSO-d6, or CD3OD. All chemical shifts are expressed
in ppm (d), coupling constants (J) are presented in Hz, and peak
patterns are reported as broad singlet (bs), singlet (s), doublet
(d), triplet (t), quartet (q), pentet (p), double doublet (dd), triplet
of doublets (td), doublet of triplets (dt) and multiplet (m).
Purity of the final compounds was further analyzed at 25 °C
using an Agilent 1200 HPLC system with a diode-array detector
(k = 190–400 nm), a Zorbax XDB-C18 HPLC column (4.6 mm Å–
150 mm, 5 lm), and a Zorbax reliance cartridge guard-column;
method A: solvent A, acetonitrile, solvent B, 0.1% TFA in H2O; or
method B: solvent A, acetonitrile, solvent B, H2O; gradient, 10%
A/90% B to 100% A/0% B over 0–40 min; post-time 10 min; or
method C: solvent A, acetonitrile, solvent B, 0.1% TFA in H2O; iso-
cratic, 10% A/90% B over 0–5 min, 10% A/90% B to 100% A/0% B over
5–25 min; post-time 5 min, flow rate 1.0 mL/min; injection volume
20 lL; monitored at wavelengths of 210, 254, 230, 280, and
3. Conclusions
360 nm. Mass spectrometry was carried out under positive or neg-
ative electrospray ionization (ESI) using a Thermo Scientific LTQ
Orbitrap Discovery instrument. Compound numbering for the
combretastatin molecular scaffold followed this protocol: the tri-
methoxy A ring was numbered 1–6, and the B ring was numbered
10–60. The ethylene bridging atoms were numbered as 1a and 1a0
respectively for the carbons connected to the A ring and B ring,
respectively.
Eleven water-soluble amino acid prodrug conjugates (AAPCs)
derived from parent amino combretastatin, dihydronaphthalene,
and benzosuberene derivatives (7, 8, 32 and 33) were designed
and synthesized. Each of the parent compounds (7, 8, 32 and 33)
were potent inhibitors of tubulin assembly binding to the colchi-
cine site and were strongly cytotoxic against human cancer cell
lines [SK-OV-3 (ovarian), NCI-H460 (lung), and DU-145 (prostate)].
Without exception, the glycinamide AAPCs based on the dihydron-
aphthalene and benzosuberene molecular scaffolds demonstrated
efficient cleavage by LAP and were superior in this regard to their
serinamide counterparts. Cytotoxicity mirrored the relative cleav-
age of these agents by LAP. The story was slightly different for
the AAPCs derived from the combretastatins in that both the glyci-
namide and serinamde AAPCs (15–18) were efficiently cleaved by
LAP for both the 20 and 30 amino variants, but the diamine-CA1
derived glycinamide (24) and serinamide (22 and 23) were resis-
tant to LAP mediated cleavage to regenerate the parent compound
(8). CA1-diamine (8) and its corresponding hydrochloride salt (10)
4.1.2. Synthesis of amino acid prodrug conjugates of amino
combretastatins
4.1.2.1. Nitration of aldehyde16
.
4-Methoxy-2-nitroben-
zaldehyde (1.53 g, 8.45 mmol) was dissolved in concentrated
H2SO4 (25 mL). To this solution, a pre-cooled mixture of HNO3
and H2SO4 (3.85 mL/2.85 mL) was added dropwise. The reaction
mixture was stirred for 10 min, and then the resulting solution
was added dropwise into ice-water (100 mL). After stirring for
2 h at 0 °C, the solution was filtered, and the solid containing the
desired product was rinsed with ice-water (10 mL). Purification

948
L. Devkota et al. / Bioorg. Med. Chem. 24 (2016) 938–956
by flash column chromatography (40% EtOAc/hexanes) yielded
4.1.2.3.2. (E)-1,2,3-Trimethoxy-5-(4-methoxy-2-nitrostyryl)ben-
zene (5b). This compound was isolated as an orange solid
(0.239 g, 0.692 mmol, 25%). 1H NMR (CDCl3, 600 MHz): d 7.67
(1H, d, J = 8.8 Hz), 7.47 (1H, d, J = 2.7 Hz), 7.44 (1H, d, J = 16.0 Hz),
7.16 (1H, dd, J = 8.7, 2.7 Hz), 6.92 (1H, d, J = 16.0 Hz), 6.74 (2H, s),
3.91 (6H, s), 3.90 (3H, s), 3.87 (3H, s). 13C NMR (150 MHz, CDCl3):
d 159.1, 153.4, 148.3, 138.4, 132.5, 132.2, 129.1, 125.5, 122.8,
120.4, 108.9, 103.9, 61.0, 56.2, 55.9.
regio-isomers 1 and 2.
4.1.2.1.1.
4-Methoxy-2,3-dinitrobenzaldehyde
(1)16
.
This
compound was isolated as a yellow solid (0.879 g, 3.89 mmol,
46%). 1H NMR (500 MHz, CDCl3): d 9.95 (1H, s), 8.15 (1H, d,
J = 8.9 Hz), 7.40 (1H, d, J = 8.9 Hz), 4.09 (3H, s), 13C NMR
(125 MHz, CDCl3):
115.92, 114.53, 57.88.
4.1.2.1.2. 4-Methoxy-2,5-dinitrobenzaldehyde (2)16
d 184.05, 155.69, 133.11, 128.96, 121.04,
.
This com-
pound was isolated as a colorless solid (0.725 g, 3.20 mmol, 38%).
1H NMR (500 MHz, CDCl3): d 10.30 (1H, s) 8.43 (1H, s), 7.74 (1H,
s), 4.15 (3H, s). 13C NMR (125 MHz, CDCl3): d 184.63, 155.99,
127.12, 123.78, 123.04, 109.63, 57.89.
4.1.2.4. Procedure for the synthesis of Z- and E-stilbenes (6a and
6b)16
.
These compounds were synthesized using the proce-
dure as described for compounds 5a and 5b. Starting from NaH
(1.57 g, 65.4 mmol), 3,4,5-trimethoxybenzylphosphonium bro-
mide (5.71 g, 1.09 mmol), and 4-methoxy-2,3-dinitrobenzalde-
hyde (2.24 g, 9.90 mmol) in CH2Cl2 (150 mL), the desired Z and E-
isomers were isolated after flash column chromatography (50%
EtOAc/hexanes).
4.1.2.2. Synthesis of Wittig salt80
benzyl bromide (3)80
The mixture of 3,4,5-trimethoxybenzyl
.
4.1.2.2.1. 3,4,5-Trimethoxy-
.
alcohol (20.1 g, 101 mmol) and PBr3 (4.80 mL, 50.7 mmol) in anhy-
drous CH2Cl2 (100 mL) was stirred for 1 h at 0 °C. Water (10 mL)
was added, and the mixture was extracted with CH2Cl2
(2 ꢀ 100 mL). The combined organic phase was washed with brine,
dried over Na2SO4, filtered, and removed by evaporation under
reduced pressure. After the recrystallization of the crude solid with
4.1.2.4.1.
(Z)-2-(40-Methoxy-20,30-dinitrophenyl)-1-(3,4,5-tri-
methoxyphenyl)ethane (6a). This compound was isolated as a yel-
low solid (0.818 g, 2.09 mmol, 52%). 1H NMR (500 MHz, CDCl3): d
7.35 (1H, d, J = 8.9 Hz), 7.08 (1H, d, J = 8.9 Hz), 6.77 (1H, d,
J = 11.8 Hz), 6.50 (1H, d, J = 11.8 Hz), 6.30 (2H, s), 3.95 (3H, s),
3.83 (3H, s), 3.69 (6H, s). 13C NMR (150 MHz, CDCl3): d 153.1,
150.9, 143.0, 137.9, 135.2, 134.5, 134.3, 130.7, 124.5, 121.6,
115.9, 106.0, 60.9, 57.4, 56.0.
EtOAc/hexanes, the bromide
3 (23.6 g, 90.3 mmol, 89%) was
obtained as an off-white solid. 1H NMR (500 MHz, CDCl3): d 6.62
(2H, s) 4.47 (2H, s), 3.87 (6H, s), 3.85 (3H, s). 13C NMR (125 MHz,
CDCl3): d 153.3, 138.2, 133.2, 106.1, 60.9, 56.1, 34.3.
4.1.2.4.2.
(E)-2-(40-Methoxy-20,30-dinitrophenyl)-1-(3,4,5-tri-
4.1.2.2.2. 3,4,5-Trimethoxybenzyltriphenylphosphonium bromide
methoxyphenyl)ethane (6b). This compound was isolated as an
orange solid (0.630 g, 1.61 mmol, 40%). 1H NMR (500 MHz, CDCl3):
d 7.84 (1H, d, J = 9.0 Hz), 7.25 (1H, d, J = 9 Hz), 6.96 (2H, dd, J = 38.2,
16.0 Hz), 6.69 (2H, s), 4.01 (3H, s), 3.91 (6H, s), 3.88 (3H, s). 13C
NMR (125 MHz, CDCl3): d 153.7, 151.0, 142.3, 139.3, 135.0, 134.6,
131.5, 130.0, 124.3, 118.8, 116.3, 104.4, 61.1, 57.5, 56.4.
(4)80
.
A mixture of bromide 3 (11.0 g, 42.1 mmol) and PPh3
(12.1 g, 46.3 mmol) in anhydrous acetone (100 mL) was stirred
for 5 h. The resulting suspension was filtered through a Buchner
funnel, and the solid was washed with acetone (100 mL) followed
by hexanes (50 mL) to afford an off-white solid. The solid was dried
in vacuo to obtain the phosphonium salt 4 (20.3 g, 38.2 mmol, 92%)
as a colorless solid. 1H NMR (600 MHz, CDCl3): d 7.74–7.64 (9H, m),
7.58–7.50 (6H, m), 6.43 (2H, d, J = 2.6 Hz), 5.29 (2H, d, J = 14.1 Hz),
3.70 (3H, d, J = 3.4 Hz), 3.43 (6H, d, J = 3.7 Hz). 13C NMR (150 MHz,
CDCl3): d 152.91 (d, J = 3.6 Hz), 137.49 d, J = 3.7 Hz), 134.84 (d,
J = 2.9 Hz), 134.58 (d, J = 9.8 Hz), 129.99 (d, J = 12.5 Hz), 122.44
(d, J = 8.9 Hz), 117.76 (d, J = 85.7 Hz), 108.76 (d, J = 5.2 Hz), 60.83
(d, J = 2.3 Hz), 56.16 (s), 30.72 (d, J = 46.7 Hz). 31P NMR (240 MHz,
CDCl3): d 23.2.
4.1.2.5. General procedure for the reduction of nitro groups to
amines15,16
.
4.1.2.5.1.
(Z)-2-(20-Amino-40-methoxyphenyl)-1-
(3,4,5-trimethoxyphenyl)ethene (7)15
.
Zinc (8.36 g, 128 mmol)
was added slowly to a well stirred solution of Z-stilbene (0.884 g,
2.56 mmol) in glacial AcOH (75 mL). The resulting suspension
was stirred for 3 h at room temperature. At this point, the reaction
mixture was filtered through CeliteÒ, and the CeliteÒ was washed
with ethyl acetate. The filtrate was concentrated under reduced
pressure. The desired amine was purified by flash column chro-
matography using a pre-packed 50 g silica column [solvent A:
EtOAc; solvent B: hexanes; gradient: 20%A/80%B (1 CV), 20%
A/80%B ? 75%A/25%B (10 CV), 75%A/25%B (2 CV); flow rate:
40 mL/min; monitored at 254 and 280 nm] to afford the amine 7
(0.615 g, 1.95 mmol, 76%, melting point range 95–98 °C) as a color-
less solid. 1H NMR (500 MHz, CDCl3): d 7.04 (1H, d, J = 8.4 Hz), 6.52
(2H, s), 6.51 (1H, d, J = 12 Hz), 6.43 (1H, d, J = 11.9 Hz), 6.30 (1H, dd,
J = 8.4, 2.5 Hz), 6.26 (1H, d, J = 2.5 Hz), 3.81 (3H, s), 3.76 (3H, s), 3.74
(2H, b), 3.65 (6H, s). 13C NMR (125 MHz, CDCl3): d 160.1, 152.7,
144.9, 137.3, 132.3, 131.0, 130.6, 125.7, 116.1, 105.8, 104.3,
100.7, 60.8, 55.8, 55.2. HRMS: m/z: obsd 316.1544 [M+H]+, calcd
for C18H22NO+4, 316.1543. HPLC (Method B): 13.153 min.
4.1.2.3. Procedure for the synthesis of Z- and E-stilbenes (5a and
5b)15,16
.
At 0 °C, a NaH suspension (0.478 g, 19.9 mmol) in dry
CH2Cl2 (50 mL) was stirred for 10 min. The previously prepared
solution of 3,4,5-trimethoxybenzylphosphonium bromide (1.73 g,
3.30 mmol) was added dropwise. After stirring for 20 min, 4-meth-
oxy-2-nitrobenzaldehyde (0.501 g, 2.76 mmol) was added. The
resulting mixture was stirred for 5 h. At this point, ice-water was
added carefully until H2 evolution stopped. Workup of the reaction
mixture was carried out by extraction with CH2Cl2 (2 ꢀ 25 mL), fol-
lowed by washing the combined organic layers with water (twice)
and brine and finally drying over Na2SO4. The Z- and E-isomers
were isolated after flash column chromatography (40% EtOAc/hex-
anes) and re-crystallization of the Z and E-isomer mixture from
EtOAc and hexanes.
4.1.2.5.2.
(Z)-2-(20,30-Diamino-40-methoxyphenyl)-1-(3,4,5-tri-
This compound was synthesized
methoxyphenyl)ethene (8)16
.
4.1.2.3.1. (Z)-2-(40-Methoxy-20-nitrophenyl)-1-(3,4,5-trimethoxy-
phenyl)ethene (5a). This compound was isolated as a yellow solid
(0.690 g, 1.99 mmol, 72%). 1H NMR (500 MHz, CDCl3): d 7.59 (1H, d,
J = 2.7 Hz), 7.24 (1H, d, J = 8.6 Hz), 7.01 (1H, dd, J = 8.6, 2.6 Hz), 6.80
(1H, d, J = 11.9 Hz), 6.62 (1H, d, J = 11.9 Hz), 6.29 (2H, s), 3.87 (3H,
s), 3.81 (3H, s), 3.62 (6H, s). 13C NMR (125 MHz, CDCl3): d 159.0,
152.9, 148.6, 137.4, 133.4, 131.5, 131.2, 125.9, 125.7, 120.0,
108.7, 106.3, 60.9, 55.9, 55.8.
using the procedure as described for compound 7. Starting from
zinc (13.4 g, 205 mmol) and Z-stilbene (0.797 g, 2.04 mmol) in gla-
cial AcOH (72 mL), the desired product 8 (0.403 g, 1.22 mmol, 60%)
was obtained as a brown oil. 1H NMR (500 MHz, CDCl3): d 6.66 (1H,
d, J = 8.4 Hz), 6.52 (1H, d, J = 12 Hz), 6.50 (1H, d, J = 12 Hz), 6.48
(2H, s), 6.39 (1H, d, J = 8.4 Hz), 3.83 (3H, s), 3.81 (3H, s), 3.62 (6H,
s), 3.49 (4H, bs). 13C NMR (125 MHz, CDCl3): d 152.68, 147.72,
137.31, 133.00, 132.22, 131.26, 126.14, 123.31, 119.53, 117.99,

L. Devkota et al. / Bioorg. Med. Chem. 24 (2016) 938–956
949
105.90, 102.23, 60.84, 55.82, 55.73. HRMS: m/z: obsd 331.1656 [M
+H]+, calcd for C18H23N2O+4, 331.1658. HPLC (Method B):
11.603 min.
After the evaporation of solvent, the resulting oil was purified by
normal phase preparative TLC (95% CH2Cl2/MeOH) to obtain 20
CA4-L-serinamide 13 (0.125 g, 0.311 mmol, 71%) as a colorless
solid. 1H NMR (500 MHz, CDCl3): d 9.65 (1H, s), 8.04 (1H, d,
J = 2.6 Hz), 7.16 (1H, dd, J = 8.5, 0.9 Hz), 6.69 (1H, dd, J = 8.5,
2.6 Hz), 6.61 (1H, d, J = 12.0 Hz), 6.50 (1H, d, J = 12.0 Hz), 6.42
(2H, s), 3.82 (3H, s), 3.80 (3H, s), 3.76 (1H, dd, J = 10.8, 5.2 Hz),
3.61 (7H, s), 3.37 (1H, t, J = 5.4 Hz). 13C NMR (90 MHz, CDCl3): d
171.7, 159.5, 152.8, 137.9, 135.8, 132.6, 131.8, 130.1, 124.3,
120.1, 110.8, 106.1, 105.6, 64.8, 60.8, 56.8, 55.9, 55.4. HRMS: m/z:
obsd 403.1866 [M+H]+, calcd for C21H27N2O6+, 403.1864. HPLC
(Method A): 12.97 min.
4.1.2.6. Preparation of hydrochloride salts of amino combre-
tastatin analogs15,16
.
4.1.2.6.1. (Z)-2-(20-Methoxy-40-aminophe-
nyl)-1-(3,4,5-trimethoxyphenyl)ethene hydrochloride (9)15
.
To a
solution of 20 CA4-amine 7 (0.0550 g, 0.175 mmol) in anhydrous
CH2Cl2 (8 mL) was added a HCl solution (4 N in dioxane, 3 equiv)
at 0 °C. The reaction was monitored by TLC and stirred for 12 h.
After evaporating the solvents under reduced pressure, the final
hydrochloride salt 9 (0.0440 g, 0.125 mmol, 72%, melting point
range 143–146 °C) was obtained as a green solid after recrystalliza-
tion with ethanol/diethyl ether solution. 1H NMR (500 MHz, CD3
OD): d 7.27 (1H, d, J = 8.3 Hz), 6.97–6.91 (2H, m), 6.78 (1H, d,
J = 11.9 Hz), 6.55 (1H, d, J = 11.8 Hz), 6.46 (2H, s), 3.83 (3H, s),
3.71 (3H, s), 3.60 (6H, s). 13C NMR (125 MHz, CD3OD): d 160.11,
152.83, 152.82, 133.86, 132.04, 131.52, 130.51, 123.86, 122.08,
113.51, 108.42, 106.21, 59.66, 54.89, 54.83. HRMS: m/z: obsd
316.1544 [MꢁCl]+, calcd for C18H22NO+4, 316.1543. HPLC (Method
B): 13.02 min.
4.1.2.7.3.
methoxystyryl)phenyl)propanamide hydrochloride (17)53
stirred solution of 20 CA4-
-serinamide 13 (0.0950 g, 0.236 mmol)
(S,E)-2-Amino-3-hydroxy-N-(5-methoxy-2-(3,4,5-tri-
.
To a well
L
in CH2Cl2 (10 mL) was added a HCl solution (4 N in dioxane,
0.8 mmol) at room temperature. After completion of the reaction
(monitored by TLC), diethyl ether (20 mL) was added to the reac-
tion mixture, producing a colorless solid that was recovered by fil-
tration through a membrane filter. After the solid was washed with
diethyl ether (10 mL), the water soluble 20 CA4-
L-serinamide 17
4.1.2.6.2. (Z)-2-(20,30-Diamine hydrochloride-40-methoxyphenyl)-
1-(3,4,5-trimethoxyphenyl)ethene (10). To a solution of CA1-dia-
mine 8 (0.320 g, 0.970 mmol) in anhydrous CH2Cl2 (50 mL) was
added a HCl solution (4 N in dioxane, 5 equiv), and the reaction
mixture was stirred for 5 h. The resulting crystals were filtered
and re-crystallized from anhydrous methanol at ꢁ20 °C. The brown
crystals thus obtained were washed with dry CH2Cl2 to obtain the
salt 10 (0.130 g, 0.320 mmol, 30%, melting point range 184–
186 °C). 1H NMR (600 MHz, CD3OD): d 6.98 (1H, d, J = 8.5 Hz),
6.60 (1H, d, J = 11.9 Hz), 6.47 (1H, d, J = 8.6 Hz), 6.42 (2H, s), 6.37
(1H, d, J = 11.8 Hz), 3.83 (3H, s), 3.62 (3H, s), 3.52 (6H, s). 13C
NMR (150 MHz, CD3OD): d 152.7, 152.3, 137.1, 137.0, 132.6,
132.2, 129.2, 124.0, 119.0, 107.4, 106.0, 101.4, 59.7, 55.4, 54.9.
HRMS: m/z: obsd 353.1476 [Mꢁ2HCl+Na]+, calcd for C18H22N2O4
Na+, 353.1472. HPLC (Method C): 13.06 min.
(0.0780 g, 0.178 mmol, 75%, melting point range 208–212 °C) was
obtained as a colorless solid. 1H NMR (600 MHz, CD3OD): d 7.72
(1H, d, J = 8.8 Hz), 7.23 (1H, d, J = 16.2 Hz), 7.09 (1H, d, J = 2.6 Hz),
7.01 (1H, d, J = 16.1 Hz), 6.92 (1H, dd, J = 8.7, 2.6 Hz), 6.89 (2H, s),
4.26 (1H, t, J = 5.2 Hz), 4.12 (2H, d, J = 5.2 Hz), 3.91 (6H, s), 3.84
(3H, s), 3.79 (3H, s). 13C NMR (150 MHz, CD3OD): d 166.2, 159.5,
153.2, 137.4, 134.5, 133.9, 128.6, 126.5, 124.9, 122.4, 112.8,
111.0, 103.5, 60.5, 59.8, 55.3, 55.1, 54.5. HRMS: m/z: obsd
403.1873 [MꢁCl]+, calcd for C21H27N2O+6 403.1864. HPLC (Method
C): 13.23 min.
4.1.2.7.4.
methoxystyryl)phenyl)propanamide hydrochloride (15). To 20 CA4-
-serinamide 13 (0.0500 g, 0.124 mmol) was added a HCl solution
(S,Z)-2-Amino-3-hydroxy-N-(5-methoxy-2-(3,4,5-tri-
L
(4 N in dioxane, 0.36 mmol) at 0 °C. The resulting reaction mixture
was stirred for 3 h, followed by evaporation of the solvent under
reduced pressure at 30 °C. The resulting green oil was washed with
anhydrous diethyl ether to furnish the water soluble HCl salt of 20
4.1.2.7. Preparation of amino acid prodrug conjugates of 20
combretastatin A-4 amines15,53
.
4.1.2.7.1. (S,Z)-2-((((9H-Fluo-
CA4-L-serinamide 15 (0.0360 g, 0.0820 mmol, 66%, melting point
ren-9-yl)methoxy)carbonyl)amino)-3-((5-methoxy-2-(3,4,5-tri-
range 55–61 °C) as a dark green solid. 1H NMR (600 MHz, CD3OD):
d 7.37 (1H, d, J = 2.4 Hz), 7.12 (1H, dd, J = 8.5, 2.5 Hz), 6.75 (1H, d,
J = 7.8 Hz), 6.56 (1H, d, J = 11.8 Hz), 6.47 (1H, d, J = 11.9 Hz), 6.42
(2H, s), 3.98 (1H, t, J = 5.9 Hz), 3.75 (3H, s), 3.72 (1H, d,
J = 4.5 Hz), 3.67 (3H, s), 3.61 (1H, m), 3.55 (6H, s). 13C NMR
(150 MHz, CD3OD): d 165.35, 159.38, 152.70, 137.08, 135.26,
132.31, 131.57, 130.38, 124.80, 123.57, 111.27, 109.52, 105.93,
60.09, 59.70, 55.08, 54.87, 54.52. HRMS: m/z: obsd 403.1885 [M
+H]+, calcd for C21H27N2O6+, 403.1884. HPLC (Method C): 13.05 min.
4.1.2.7.5. tert-Butyl (Z)-(2-((5-methoxy-2-(3,4,5-trimethoxystyryl)
phenyl)amino)-2-oxoethyl) carbamate (12). To a solution of 20
CA4-amine 7 (0.363 g, 0.700 mmol) in anhydrous CH2Cl2 (75 mL)
was added Et3N (0.300 mL, 1.72 mmol), peptide coupling reagent
T3P (50% in EtOAc, 1.70 mL, 2.30 mmol) and Boc-glycine-OH
(0.311 g, 1.44 mmol). The reaction mixture was stirred for 3 h at
room temperature. Water (25 mL) was added, and the organic sol-
vent was removed by evaporation under reduced pressure. The
resulting crude mixture was extracted with EtOAc, and the organic
extract was washed with brine, dried over Na2SO4, and concen-
trated under reduced pressure. The residue was purified using flash
column chromatography (50% EtOAc/hexanes) to afford the desired
Boc-protected glycinamide 12 (0.490 g, 1.04 mmol, 90%) as a color-
less solid. 1H NMR (500 MHz, CDCl3): d 7.95 (1H, s), 7.93 (1H, bs),
7.15 (1H, d, J = 8.5 Hz), 6.68 (1H, d, J = 7.7 Hz), 6.61 (1H, d,
J = 12.0 Hz), 6.46 (1H, d, J = 12.0 Hz), 6.41 (2H, s), 4.80 (1H, s),
3.81 (3H, s), 3.80 (3H, s), 3.67 (1H, s), 3.61 (6H, d, J = 1.4 Hz), 1.46
(9H, s). 13C NMR (125 MHz, CDCl3): d 167.26, 159.51, 155.67,
methoxystyryl)phenyl)amino)-3-oxopropyl acetate (11)15
.
To a
solution of 20 CA4-amine 7 (0.220 g, 0.700 mmol) in anhydrous CH2
Cl2 (30 mL) was added Et3N (0.110 mL, 0.760 mmol), peptide
coupling reagent T3P (50% in EtOAc, 0.830 mL, 1.40 mmol) and
Fmoc-L-ser(Ac)-OH (0.284 g, 0.760 mmol). The reaction mixture
was stirred for 8 h at room temperature. H2O (20 mL) was added,
and the reaction mixture was extracted with CH2Cl2. The organic
extract was washed with brine, dried over Na2SO4 and concentrated
under reduced pressure, and the residue was purified using flash col-
umn chromatography (50% EtOAc/hexanes) to afford the desired
Fmoc-L-serinamide acetate 11 (0.388 g, 0.580 mmol, 83%) as a color-
less solid. 1H NMR (500 MHz, CDCl3): d 8.23 (1H, b), 7.93 (2H, d,
J = 7.5), 7.60 (2H, d, J = 7.5), 7.60 (2H, d, J = 7.5), 7.40 (2H, dd,
J = 7.5, 7.0), 7.33 (2H, dd, J = 7.5, 7.0), 6.98 (1H, d, J = 8.5), 6.65 (1H,
d, J = 9.0), 6.56 (1H, s), 6.00 (1H, t, J = 4.5), 5.75 (1H, d, J = 6.5), 4.47
(4H, m), 4.23 (1H, d, J = 11.5), 3.89 (3H, s), 3.84 (6H, s), 3.79 (3H, s),
2.67 (2H, t, J = 7.5), 2.30 (2H, td, J = 7.5, 4.5), 2.11 (3H, s). 13C NMR
(150 MHz, CDCl3): d 171.2, 166.6, 159.8, 153.2, 143.9, 141.7, 138.1,
135.3, 133.2, 131.5, 130.2, 128.2, 127.5, 127.5, 125.2, 125.1, 124.2,
120.4, 120.4, 111.7, 105.8, 105.8, 67.2, 63.8, 61.1, 56.43, 56.0, 55.8,
47.4, 20.8.
4.1.2.7.2.
(S,Z)-2-Amino-3-hydroxy-N-(5-methoxy-2-(3,4,5-tri-
-serinamide acet-
methoxystyryl)phenyl)propanamide (13). Fmoc-
L
ate 11 (0.290 g, 0.435 mmol) was dissolved in a CH2Cl2/MeOH
mixture (10 mL, 1:1 ratio), a NaOH solution (2 M, 0.650 mL,
1.30 mmol) was added, and the mixture was stirred for 30 min.

950
L. Devkota et al. / Bioorg. Med. Chem. 24 (2016) 938–956
152.86, 137.83, 135.32, 132.51, 131.43, 130.06, 124.34, 119.50,
110.91, 109.99, 105.67, 105.64, 60.91, 55.80, 55.46, 45.10, 28.29.
4.1.2.7.6. (Z)-2-Amino-N-(5-methoxy-2-(3,4,5-trimethoxystyryl)
ture was washed with water (3 ꢀ 50 mL) and brine and dried over
Na2SO4. The organic solvent was removed under reduced pressure,
and the crude product was purified by flash column chromatogra-
phenyl)acetamide (14)81
.
The Boc- protected glycinamide 12
phy (50% EtOAc/hexanes) to afford the Fmoc-L-diserinamide acet-
(0.128 g, 0.270 mmol) in anhydrous CH2Cl2 (2 mL) was reacted
with TFA (0.650 mL, 1.30 mmol) and stirred for 30 min.81 After
evaporation of the solvent, the resulting oily residue was diluted
with water (2 mL) and washed with ether (2 ꢀ 2 mL). The aqueous
phase was treated with NaHCO3 (1 N) until pH 7–8 and extracted
with CH2Cl2 (3 ꢀ 5 mL). The combined organic phase was dried
over Na2SO4, concentrated under reduced pressure and purified
using flash column chromatography (50% EtOAc/hexanes) to
obtain 20 CA4 glycinamide prodrug 14 (0.0680 g, 0.183 mmol,
71%) as a brown oil. 1H NMR (500 MHz, CDCl3): d 9.52 (1H, s),
8.08 (1H, d, J = 2.6 Hz), 7.14 (1H, d, J = 8.5 Hz), 6.65 (1H, dd,
J = 8.6, 2.5 Hz), 6.60 (1H, d, J = 12.0 Hz), 6.50 (1H, d, J = 12.1 Hz),
6.41 (2H, s), 3.82 (3H, s), 3.80 (3H, s), 3.60 (6H, s), 3.28 (2H, s).
13C NMR (125 MHz, CDCl3): d 170.62, 159.52, 152.73, 137.63,
136.05, 132.44, 131.77, 130.01, 124.43, 119.59, 110.51, 105.85,
105.21, 60.89, 55.82, 55.45, 45.42. HRMS: m/z: obsd 373.1759 [M
+H]+, calcd for C20H25N2O5+, 373.1758. HPLC (Method A): 8.14 min.
4.1.2.7.7. (E)-2-Amino-N-(5-methoxy-2-(3,4,5-trimethoxystyryl)
ate 19 (1.31 g, 1.30 mmol, 55%) as a colorless solid. 1H NMR
(500 MHz, CDCl3): d 8.35 (1H, s), 8.05 (1H, s), 7.71 (2H, d,
J = 6.6 Hz), 7.66 (2H, s), 7.49 (4H, d, J = 6.0 Hz), 7.33 (4H, m),
7.23–7.15 (4H, m), 7.18 (1H, d, J = 9.0 Hz), 6.78 (1H, d, J = 8.0 Hz),
6.46 (1H, d, J = 12.0 Hz), 6.43 (2H, s), 6.39 (1H, d, J = 12.0 Hz),
6.09 (1H, s), 5.84 (1H, s), 4.68–4.02 (12H, m), 3.79 (6H, s), 3.62
(6H, s), 2.04 (6H, s). 13C NMR (150 MHz, CDCl3): d 171.0, 171.0,
168.1, 167.5, 156.6, 156.4, 152.9, 152.5, 143.6, 143.6, 143.5,
143.5, 141.3, 141.2, 137.3, 132.1, 131.0, 130.4, 128.8, 127.8,
127.8, 127.1, 127.1, 125.9, 125.1, 125.1, 125.0, 120.0, 109.6,
105.9, 67.6, 67.5, 63.7, 63.6, 60.9, 56.2, 55.9, 54.8, 46.9, 34.7,
29.1, 25.3, 20.8.
4.1.2.8.2.
tyryl)-1,2-phenylene)bis(2-amino-3-hydroxypropanamide)
(22). Fmoc- -diserinamide acetate 19 (0.501 g, 0.485 mmol) was
(2S,20S)-N,N0-(3-Methoxy-6-((Z)-3,4,5-trimethoxys-
L
dissolved in a CH2Cl2/MeOH mixture (20 mL, 1:1 ratio) to which a
NaOH solution (2 M, 0.970 mL, 1.98 mmol) was added, followed by
stirring (30 min). After the evaporation of solvent, the resulting oil
was purified by normal phase preparative TLC (90% CH2Cl2/MeOH.
phenyl)acetamide hydrochloride (18)53
of Boc-protected 20 CA4 glycinamide 12 (0.146 g, 0.310 mmol) in
CH2Cl2 (10 mL) was added HCl solution (4 N in dioxane,
.
To a well stirred solution
0.2% Et3N) to obtain CA1-L-diserinamide prodrug 22 (0.0650 g,
a
0.128 mmol, 27%, melting point range 75–80 °C) as a colorless
solid. 1H NMR (600 MHz, CD3OD): d 7.17 (1H, d, J = 8.6 Hz), 6.98
(1H, d, J = 8.7 Hz), 6.54 (1H, d, J = 12 Hz), 6.49 (2H, s), 6.48 (1H, d,
J = 13.3 Hz), 3.90 (2H, d, J = 5.7 Hz), 3.89–3.87 (1H, m), 3.86 (3H,
s), 3.85–3.80 (2H, m), 3.76 (1H, m), 3.71 (3H, s), 3.61 (6H, s), 1.95
(4H, s). 13C NMR (150 MHz, CD3OD): d 180.70, 173.77, 155.83,
154.95, 139.05, 134.89, 133.67, 132.82, 130.62, 130.45, 128.38,
124.07, 111.92, 108.28, 65.55, 65.50, 61.94, 58.85, 58.54, 57.51,
57.20. HRMS: m/z: obsd 505.2289 [M+H]+, calcd for C24H33N4O8+,
505.2293. HPLC (Method C): 10.94 min.
1.20 mmol) at room temperature. After completion of the reaction
(monitored by TLC), diethyl ether (25 mL) was added to the reac-
tion mixture, producing a colorless solid that was filtered through
a membrane filter. After washing the product on the membrane
with diethyl ether (10 mL), the HCl salt of the E-isomer of 20 CA4
glycinamide 18 (0.109 g, 0.226 mmol, 79%, melting point range
240–243 °C) was obtained as a colorless solid. 1H NMR (500 MHz,
CD3OD): d 7.66 (1H, d, J = 8.8 Hz), 7.20 (1H, d, J = 15 Hz), 7.19
(1H, d, J = 2.7 Hz), 6.98 (1H, d, J = 16.1 Hz), 6.88 (1H, d, J = 2.5 Hz),
6.87 (2H, s), 3.98 (2H, s), 3.88 (6H, s), 3.81 (3H, s), 3.77 (3H, s).
13C NMR (125 MHz, CD3OD): d 165.04, 159.47, 153.25, 137.58,
134.59, 133.83, 129.04, 126.75, 124.24, 122.22, 112.35, 110.48,
103.75, 59.76, 55.31, 54.46, 40.66. HRMS: m/z: obsd 373.1769
[MꢁCl]+, calcd for C20H25N2O5+ 373.1758. HPLC (Method B):
6.661 min.
4.1.2.8.3. (2S,20S)-((3-Methoxy-6-((Z)-3,4,5-trimethoxystyryl)-1,2-
phenylene)bis(azanediyl))bis(2-((tert-butoxycarbonyl)amino)-3-oxo-
propane-3,1-diyl) diacetate (20). To a solution of CA1 diamine 8
(0.270 g, 0.817 mmol) in anhydrous CH2Cl2 (50 mL) was added
Boc-serine (Ac)-OHꢂDCHA (0.875 g, 2.04 mmol), Et3N (0.290 mL,
2.04 mmol), and T3P (50% in EtOAc, 2.10 mL, 3.27 mmol). The
resulting solution was stirred for 8 h at room temperature. At this
point, the reaction mixture was washed with water (3 ꢀ 20 mL)
and brine and dried over Na2SO4. The organic solvent was removed
under reduced pressure, and the crude reaction mixture was puri-
fied by flash column chromatography (50% EtOAc/hexanes) to
4.1.2.7.8. (Z)-2-Amino-N-(5-methoxy-2-(3,4,5-trimethoxystyryl)
phenyl)acetamide hydrochloride (16)73
.
To Boc-protected 20 CA4
glycinamide 12 (0.0898 g, 0.190 mmol) was added a HCl solution
(4 N in dioxane, 0.270 mmol) at 0 °C. The resulting reaction mix-
ture was stirred for 30 min., followed by evaporation of the solvent
under reduced pressure at 30 °C. The resulting brownish oil, on
washing with anhydrous diethyl ether, furnished the water soluble
HCl salt of 20 CA4 glycinamide 16 (0.0691 g, 0.171 mmol, 90%,
melting point range 59–63 °C) as a colorless solid. 1H NMR
afford the Boc-protected-L-serinamide acetate 20 (0.251 g,
0.318 mmol, 39%) as a colorless solid. 1H NMR (500 MHz, CDCl3):
d 8.40 (1H, s), 8.21 (1H, s), 7.14 (1H, d, J = 8.6 Hz), 6.75 (1H, d,
J = 8.6 Hz), 6.45 (1H, d, J = 12.0 Hz), 6.41 (2H, s), 6.38 (1H, d,
J = 11.9 Hz), 5.66 (1H, s), 5.48–5.41 (1H, m), 4.62 (1H, s), 4.48
(2H, d, J = 5.2 Hz), 4.40 (2H, d, J = 7.5 Hz), 4.30 (1H, dd, J = 7.0 Hz),
3.80 (3H, s, 1 Hz), 3.79 (3H, s), 3.62 (6H, s), 2.05 (3H, s), 2.02 (3H,
s), 1.46 (9H, s), 1.43 (9H, s). 13C NMR (125 MHz, CDCl3): d 170.97,
170.80, 168.40, 167.67, 155.56, 155.33, 152.82, 152.40, 137.27,
132.00, 130.91, 130.42, 128.50, 128.36, 125.95, 121.24, 109.53,
105.98, 64.00, 63.92, 60.80, 60.38, 56.13, 55.83, 54.05, 28.31,
28.28, 20.66, 20.65.
(600 MHz, CD3OD):
d 7.37 (1H, d, J = 2.6 Hz), 7.12 (1H, d,
J = 8.4 Hz), 6.73 (1H, dd, J = 8.3, 2.4 Hz), 6.57 (1H, d, J = 11.8 Hz),
6.48 (1H, d, J = 11.9 Hz), 6.43 (2H, s), 3.75 (3H, s), 3.67 (3H, d,
J = 1.6 Hz), 3.63 (2H, s), 3.56 (6H, s). 13C NMR (150 MHz, CD3OD):
d 164.38, 159.37, 152.65, 136.96, 135.29, 132.57, 131.37, 130.58,
124.93, 123.22, 111.06, 109.45, 105.84, 59.75, 54.89, 54.51, 40.59.
HRMS: m/z: obsd 373.1759 [MꢁCl]+, calcd for C20H25N2O5+,
373.1759. HPLC (Method C): 12.94 min.
4.1.2.8.4.
tyryl)-1,2-phenylene)bis(2-amino-3-hydroxypropanamide)
drochloride (23). Boc- -serinamide acetate 20
(2S,20S)-N,N0-(3-Methoxy-6-((Z)-3,4,5-trimethoxys-
4.1.2.8. Preparation of amino acid prodrug conjugates of
dihy-
combretastatin A-1 diamines15,16,53
.
4.1.2.8.1. (Z)-2-(20,30-Dia-
L
(0.203 g,
mino-40-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)ethene-Fmoc-
serinamide (19). To solution of CA1-diamine (0.780 g,
2.37 mmol) in dry CH2Cl2 (150 mL) was added Fmoc- -serine
L
-
0.258 mmol) was dissolved in a CH2Cl2/MeOH mixture (20 mL,
1:1 ratio) to which a NaOH solution (2 M, 0.650 mL, 1.30 mmol)
was added. The reaction mixture was stirred for 30 min. After the
evaporation of solvent, the resulting colorless solid (0.130 g,
0.184 mmol) in CH2Cl2 (15 mL) was reacted with a HCl solution
(4 N in dioxane, 2.27 mmol) at room temperature for 2 h. Following
a
8
L
(Ac)-OH (2.40 g, 7.11 mmol), Et3N (1.00 mL, 7.11 mmol), and T3P
(50% in EtOAc, 5.62 mL, 9.48 mmol). The resulting solution was
stirred for 8 h at room temperature. At this point, the reaction mix-

L. Devkota et al. / Bioorg. Med. Chem. 24 (2016) 938–956
951
evaporation of solvent under reduced pressure, the solid was puri-
fied by reversed phase column chromatography (RP-18 silica
column, acetonitrile/water) to afford compound 20 (0.0590 g,
0.102 mmol, 40%) as an off-white solid. 1H NMR (500 MHz, CD3-
OD): d 7.16 (1H, d, J = 8.7 Hz), 6.99 (1H, d, J = 8.7 Hz), 6.55 (1H, d,
J = 12.0 Hz), 6.49 (2H, s), 6.48 (1H, d, J = 12.0 Hz), 4.32 (1H, dd,
J = 5.5, 4.3 Hz), 4.27 (1H, dd, J = 6.1, 4.8 Hz), 4.12 (1H, dd, J = 11.5,
4.5 Hz), 4.03 (1H, dd, J = 11.5, 4.5 Hz), 4.00 (1H, dd, J = 12.0,
8.5 Hz), 3.89 (1H, dd, J = 12.0, 8.5 Hz), 3.85 (3H, s), 3.71 (3H, s),
3.61 (6H, s). 13C NMR (150 MHz, CD3OD): d 166.1, 165.6, 153.9,
152.6, 136.8, 132.4, 131.4, 131.0, 129.0, 128.7, 125.9, 121.7,
110.3, 106.2, 60.4, 59.7, 55.4, 55.1, 55.1, 55.0, 55.0. HRMS: m/z:
obsd 527.2102 [MꢁHCl+Na]+, calcd for C24H32N4O8Na+, 527.2112.
HPLC (Method C): 11.17 min.
NMR (600 MHz, CDCl3): d 8.18 (1H, d, J = 8.8 Hz), 7.02 (1H, d,
J = 8.8 Hz), 3.97 (3H, s), 2.86 (2H, t, J = 6.5 Hz), 2.65 (2H, t,
J = 6.5 Hz), 2.15 (2H, p, J = 6.5 Hz).13C NMR (150 MHz, CDCl3): d
195.5, 154.4, 140.3, 137.3, 131.2, 126.1, 110.7, 56.8, 38.2, 24.7,
22.3.
4.1.3.2. 2-Methoxy-1-nitro-5-(3,4,5-trimethoxyphenyl)-6,7,8,9-
tetrahydro-5H-benzo[7]annulen-5-ol (30).
To a solution of
5-bromo-1,2,3-trimethoxybenzene (1.11 g, 4.49 mmol) in THF
(20 mL) at ꢁ78 °C, n-BuLi (1.80 mL, 2.50 M) was added, and the
reaction mixture was stirred for 1 h. Compound 25 (0.500 g,
2.30 mmol) in THF (3 mL) was added to the reaction mixture drop-
wise. The reaction mixture was stirred for 18 h and was allowed to
warm to room temperature. The reaction mixture was quenched
with H2O (20 mL) and extracted with CH2Cl2 (3 ꢀ 25 mL). The
organic extract was dried over Na2SO4, filtered, and concentrated
under reduced pressure, and the residue was purified by flash col-
umn chromatography (40% EtOAc/hexanes) to yield alcohol 30
(0.521 g, 1.34 mmol, 59% yield) as a light yellow solid. 1H NMR
4.1.2.8.5. Di-tert-butyl (((3-methoxy-6-(3,4,5-trimethoxystyryl)-
1,2-phenylene)bis(azanediyl))bis(2-oxoethane-2,1-diyl))(Z)-dicarba-
mate (21). To a solution of CA1-diamine 8 (0.300 g, 0.908 mmol)
in anhydrous CH2Cl2 (50 mL) was added Boc-glycine-OH (0.398 g,
2.27 mmol), Et3N (0.270 mL, 1.91 mmol), and T3P (50% in EtOAc,
2.25 mL, 3.63 mmol). The resulting solution was stirred for 3 h at
room temperature. At this point, the reaction mixture was washed
with water (3 ꢀ 20 mL) and brine and dried over Na2SO4. The
organic solvent was removed under reduced pressure, and the
crude product was purified by flash column chromatography
(50% EtOAc/hexanes) to afford the Boc-protected CA1-diglyci-
namide 21 (0.503 g, 0.780 mmol, 86%) as a colorless solid. 1H
NMR (600 MHz, CDCl3): d 8.21 (1H, bs), 7.96 (1H, bs), 7.14 (1H, d,
J = 8.6 Hz), 6.74 (1H, d, J = 8.6 Hz), 6.49 (1H, d, J = 12.0 Hz), 6.44
(2H, s), 6.43 (1H, d, J = 11.6 Hz), 5.54 (1H, bs), 5.38 (1H, bs), 3.96
(2H, d, J = 5.6 Hz), 3.83 (2H, d, J = 5.8 Hz), 3.80 (6H, s), 3.63 (6H,
s), 1.47 (9H, s), 1.45 (9H, s). 13C NMR (150 MHz, CDCl3): d 169.2,
168.8, 156.6, 156.4, 153.1, 152.8, 137.4, 132.6, 131.2, 128.9,
128.7, 126.5, 121.7, 109.8, 106.2, 61.2, 56.4, 56.2, 45.2, 44.8, 28.7,
28.7.
(500 MHz, CDCl3):
d 7.18 (1H, d, J = 8.8 Hz), 6.85 (1H, d,
J = 8.8 Hz), 6.54 (2H, s), 3.87 (3H, s), 3.85 (3H, s), 3.81 (6H, s),
2.79 (2H, t, J = 7.4 Hz), 2.12 (1H, s), 2.10 (2H, m), 2.04 (1H, m),
1.86 (1H, m). 13C NMR (125 MHz, CDCl3): d 152.9, 150.0, 143.7,
137.0, 135.3, 132.1, 130.6, 110.8, 103.8, 74.9, 61.0, 56.5, 56.4,
40.6, 24.6, 18.6.
4.1.3.3. 2-Methoxy-5-(3,4,5-trimethoxyphenyl)-7,8-dihydron-
aphthalen-1-amine (32).
Alcohol 30 (0.521 g, 1.34 mmol)
was dissolved in AcOH (18 mL) in a round-bottom reaction flask,
followed by the addition of Zn (1.75 g, 26.8 mmol). The reaction
mixture was stirred for 6 h at room temperature and then filtered
through CeliteÒ, which was washed with CH2Cl2. The filtrate was
concentrated under reduced pressure and the residue was purified
by flash column chromatography (30% EtOAc/hexanes) to give the
desired amino-dihydronaphthalene (32) (0.410 g, 1.27 mmol, 89%
yield, melting point range 183–185 °C) as a dark brown solid. 1H
NMR (500 MHz, CDCl3): d 6.59 (1H, d, J = 8.5 Hz), d 6.56 (2H, s), d
6.51 (1H, d, J = 8.5 Hz), d 5.92 (1H, t, J = 4.5 Hz), d 3.89 (3H, s), d
3.85 (3H, s), d 3.83 (6H, s), d 2.67 (2H, t, J = 7.5 Hz), d 2.41 (2H,
td, J = 7.5, 4.5 Hz). 13C NMR (125 MHz, DMSO-d6): d 152.9, 146.7,
140.0, 137.1, 136.9, 134.0, 127.8, 124.2, 120.3, 115.1, 107.6,
106.2, 60.4, 56.2, 55.7, 23.0, 21.7. HRMS: m/z: obsd 342.1698 [M
+H]+, calcd for C20H24NO+4, 342.1705. HPLC (Method B): 14.18 min.
4.1.2.8.6.
(Z)-N,N0-(3-Methoxy-6-(3,4,5-trimethoxystyryl)-1,2-
phenylene)bis(2-aminoacetamide) (24). To a solution of Boc-pro-
tected CA1-diglycinamide 21 (0.355 g, 0.550 mmol) in CH2Cl2
(50 mL) was added a HCl solution (4 N in dioxane, 2.75 mmol) at
room temperature, and the mixture was stirred for 3 h. On adding
diethyl ether (25 mL) to the reaction mixture, a colorless solid
formed, and it was filtered through a membrane filter. After wash-
ing the solid with diethyl ether (20 mL), CA1-diglycinamide 24
(0.142 g, 0.275 mmol, 50%, melting point range 81–84 °C) was
obtained as a colorless solid. 1H NMR (600 MHz, CD3OD): d 7.18
(1H, d, J = 8.3 Hz), 7.01 (1H, d, J = 8.4 Hz), 6.59 (1H, d, J = 11.8 Hz),
6.53 (1H, d, J = 12 Hz), 6.51 (2H, s), 4.02 (4H, bs, NH2, CH2), 3.93
(2H, bs, NH2), 3.92 (2H, bs, CH2), 3.87 (3H, s), 3.73 (3H, s), 3.63
(6H, s). 13C NMR (150 MHz, CD3OD): d 164.9, 164.7, 153.9, 152.6,
136.8, 132.5, 131.4, 131.0, 128.9, 128.7, 126.0, 121.7, 110.2,
106.1, 59.7, 55.3, 55.1, 40.7, 40.6. HRMS: m/z: obsd 467.1897
[MꢁHCl+Na]+, calcd for C22H28N4O6Na+, 467.1901. HPLC (Method
A): 6.06 min.
4.1.3.4.
(27).
5-(30-Methoxy-20-nitrophenyl)pent-4-enoic
To a well-stirred solution of 3-(carboxypropyl)triph-
acid
enylphosphonium bromide (21.5 g, 50.1 mmol) in THF (400 mL),
potassium tert-butoxide (13.4 g, 119 mmol) was added, and the
reaction mixture was stirred for 1 h at ambient temperature. The
reaction mixture was cooled to 0 °C, and 3-methoxy-2-nitroben-
zaldehyde (9.00 g, 47.7 mmol) in THF (100 mL) was added dropwise.
After the reaction mixture was stirred for 16 h, warming from 0 °C to
room temperature, the reaction was quenched with HCl (2 M,
200 mL). The organic solvent was removed by evaporation under
reduced pressure, and the residue was extracted with EtOAc
(4 ꢀ 300 mL). The combined organic phase was washed with brine,
dried over Na2SO4, filtered, and evaporated under reduced pressure.
The resulting organic crude product was purified by flash column
chromatography using a pre-packed 100 g silica column [solvent
A: EtOAc; solvent B: hexanes; gradient: 12%A/88%B (1 CV), 12%
A/88%B ? 100%A/0%B (15 CV), 100%A/0%B (4 CV); flow rate:
40 mL/min; monitored at 254 and 280 nm] affording a mixture of
E and Z-isomers 27 (8.78 g, 34.9 mmol, 73%) as a red solid. 1H NMR
(500 MHz, CDCl3): d 7.38 (1H, t, J = 8.1 Hz), 7.33 (1H, t, J = 8.2 Hz),
7.12 (1H, d, J = 7.8 Hz), 6.95 (1H, d, J = 8.3 Hz), 6.89 (2H, d,
J = 7.7 Hz), 6.37 (1H, d, J = 11.3 Hz), 6.31 (2H, m), 5.83 (1H, dt,
4.1.3. Synthesis of amino acid prodrug conjugates of amino
dihydronaphthalene and amino benzosuberene42,43,46
4.1.3.1. 6-Methoxy-5-nitro-1-tetralone (25).
To a well-stir-
red solution of 6-methoxy-1-tetralone (20.0 g, 114 mmol) in acetic
anhydride (200 mL), a solution of HNO3 (10 mL) and AcOH (10 mL)
was slowly added over the course of 1 h at 0 °C. The reaction was
stirred for another 20 h and was allowed to reach room tempera-
ture. Water (20 mL) was added to the reaction mixture, and the
crude product was extracted with CH2Cl2 (3 ꢀ 50 mL). The organic
phase was dried with Na2SO4 and concentrated under reduced
pressure. The residue was purified by flash column chromatogra-
phy (20% EtOAc/hexanes) to give the desired 6-methoxy-5-nitro-
tetralone 25 (8.28 g, 37.5 mmol, 33%) as a light yellow solid. 1H

952
L. Devkota et al. / Bioorg. Med. Chem. 24 (2016) 938–956
J = 11.5, 7.0 Hz), 3.89 (3H, s), 3.87 (3H, s), 2.53 (4H, m), 2.45 (4H, m).
13C NMR (125 MHz, CDCl3): d 178.5, 178.4, 150.9, 150.9, 134.9,
134.5, 130.8, 130.7, 123.7, 123.6, 121.7, 118.2, 111.4, 111.0, 56.5,
56.5, 33.6, 33.3, 28.1, 23.9.
133.6, 129.4, 109.3, 104.1, 79.7, 60.9, 56.3, 56.2, 40.9, 28.7, 26.4,
26.0.
4.1.3.8. 1-Amino-2-methoxy-5-(30,40,50-trimethoxyphenyl)-ben-
zosuber-5-ene (33).
To a solution of alcohol 31 (0.498 g,
4.1.3.5.
(28).
5-(30-Methoxy-20-nitrophenyl)pentanoic
Anhydrous ethanol (18 mL) was added to a flask con-
acid
1.16 mmol) in AcOH (15 mL) was added Zn dust (1.52 g,
23.2 mmol), and the reaction was stirred for 6 h at ambient tem-
perature. The reaction mixture was filtered through CeliteÒ, which
was washed with CH2Cl2, and the filtrate was concentrated under
reduced pressure. The concentrated residue was neutralized with
NaHCO3 and extracted with EtOAc (3 ꢀ 20 mL). The combined
extracts were washed with brine, dried over Na2SO4, filtered, and
removed by evaporation under reduced pressure. The crude
product was purified by flash column chromatography using a
pre-packed 25 g silica column [solvent A: EtOAc; solvent B: hex-
anes; gradient: 5%A/95%B (1 CV), 5%A/95%B ? 60%A/40%B (13
CV), 60%A/40%B (2 CV); flow rate: 40 mL/min; monitored at 254
and 280 nm] to afford benzosuberene 33 (KGP156, 0.350 g,
0.980 mmol, 85%, melting point range 140–145 °C) as a colorless
solid. 1H NMR (500 MHz, CDCl3): d 6.67 (1H, d, J = 8.4 Hz), 6.52
(2H, s), 6.49 (1H, d, J = 8.4 Hz), 6.30 (1H, t, J = 7.3 Hz), 3.88 (3H,
s), 3.86 (3H, s), 3.80 (6H, s), 2.59 (2H, t, J = 6.9 Hz), 2.12 (2H, p,
J = 7.0 Hz), 1.95 (2H, q, J = 7.2 Hz). 13C NMR (125 MHz, CDCl3): d
152.9, 146.5, 143.7, 138.7, 137.4, 133.7, 132.6, 126.9, 126.4,
120.0, 107.7, 105.4, 61.1, 56.3, 55.7, 33.4, 25.8, 25.4. HRMS: m/z:
obsd 356.1863 [M+H]+, calcd for C21H26NO+4, 356.1862. HPLC
(Method B): 15.34 min.
taining pentenoic acid 27 (0.750 g, 2.98 mmol) and Pd/C (10%,
0.830 g), and the solution was stirred for 10 min at ambient tem-
perature. 1,4-Cyclohexadiene (8.90 mL, 93.8 mmol) was added to
the solution, which was then stirred for another 3 h. The reaction
mixture was filtered through CeliteÒ, and the CeliteÒ was washed
with EtOAc (3 ꢀ 20 mL). The combined filtrate was removed by
evaporation under reduced pressure, and the residue was purified
by flash column chromatography using a pre-packed 100 g silica
column [solvent A: EtOAc; solvent B: hexanes; gradient: 12%
A/88%B (1 CV), 12%A/88%B ? 100%A/0%B (12 CV), 100%A/0%B (2
CV); flow rate: 40 mL/min; monitored at 254 and 280 nm] to afford
pentanoic acid analog 28 (0.490 g, 1.93 mmol, 65%) as a tan solid.
1H NMR (500 MHz, CDCl3): d 7.33 (1H, t, J = 8.1 z), 6.87 (2H, dd,
J = 8.1, 3.7 Hz), 3.87 (3H, s), 2.58 (2H, m), 2.36 (2H, m), 1.67 (4H,
m). 13C NMR (125 MHz, CDCl3): d 179.3, 150.8, 142.0, 134.8,
130.8, 121.7, 110.3, 56.5, 33.7, 30.8, 29.9, 24.4.
4.1.3.6. 2-Methoxy-1-nitro-benzosuber-5-one (29).
Pen-
tanoic acid analog 28 (0.490 g, 1.93 mmol) was dissolved in Eaton’s
reagent (14.5 mL, 7.7% P2O5 in CH3SO3H) under N2, and the reac-
tion mixture was stirred for 16 h at ambient temperature. To the
solution was added ice, which allowed to melt, and the reaction
mixture was neutralized slowly with NaHCO3 (aq.) and extracted
with EtOAc (3 ꢀ 20 mL). The combined organic phase was dried
over Na2SO4, filtered, and removed by evaporation under reduced
pressure, and the residue was purified by flash chromatography
using a pre-packed 50 g silica column [solvent A: EtOAc; solvent
B: hexanes; gradient: 7%A/93%B (1 CV), 7%A/93%B ? 60%A/40%B
(13 CV), 60%A/40%B (1 CV); flow rate: 25 mL/min; monitored at
254 and 280 nm] to afford benzosuberone analog 29 (0.340 g,
1.45 mmol, 75%) as a colorless solid. 1H NMR (500 MHz, CDCl3): d
7.84 (1H, d, J = 8.8 Hz), 6.98 (1H, d, J = 8.8 Hz), 3.95 (3H, s), 2.78
(2H, m), 2.71 (2H, m), 1.91 (2H, m), 1.81 (2H, m).13C NMR
(125 MHz, CDCl3): d 203.2, 153.4, 141.5, 134.1, 132.3, 131.9,
110.4, 56.7, 40.4, 26.3, 24.5, 20.3.
4.1.3.9. (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-
((2-methoxy-5-(3,4,5-trimethoxyphenyl)-7,8-dihydronaph-
thalen-1-yl)amino)-3-oxopropyl acetate (34).
stirred solution of amino compound 32 (0.500 g, 1.45 mmol) in
CH2Cl2 (30 mL), Fmoc- -ser(Ac)-OH (0.648 g, 2.18 mmol), T3P
(50% in EtOAc, 2.60 mL, 4.35 mmol), and Et3N (0.31 mL, 2.18 mmol)
were added, and the reaction mixture was stirred for 17 h at room
temperature. Water (30 mL) was added, and the reaction mixture
was extracted with EtOAc (3 ꢀ 30 mL). The organic phase was
rinsed with brine, dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by flash column chro-
To a well-
L
matography (35% EtOAc/hexanes) to afford the desired Fmoc-L-
serinamide acetate 34 (0.695 g, 1.12 mmol, 77%) as a yellow solid.
1H NMR (500 MHz, CDCl3): d 7.77 (2H, d, J = 7.5 Hz), 7.60 (2H, d,
J = 7.5 Hz), 7.60 (1H, s), 7.40 (2H, dd, J = 7.5, 7.0 Hz), 7.33 (2H, dd,
J = 7.5, 7.0 Hz), 6.98 (1H, d, J = 8.5 Hz), 6.65 (1H, d, J = 9.0 Hz),
6.56 (2H, s), 6.00 (1H, t, J = 4.5 Hz), 5.75 (1H, d, J = 6.5 Hz), 4.75
(1H, t, J = 11.5 Hz), 4.47 (4H, m), 4.23 (1H, t, J = 11.5 Hz), 3.89
(3H, s), 3.84 (6H, s), 3.79 (3H, s), 2.67 (2H, t, J = 7.5 Hz), 2.30 (2H,
td, J = 7.5, 4.5 Hz), 2.11 (3H, s). 13C NMR (125 MHz, CDCl3): d
170.77, 167.42, 152.96, 143.62, 143.55, 141.32, 139.47, 137.11,
136.55, 135.32, 128.81, 127.80, 127.11, 125.30, 125.16, 124.98,
121.79, 120.03, 120.01, 107.62, 105.78, 67.27, 64.47, 60.92, 56.15,
55.67, 54.36, 47.15, 23.99, 22.92, 20.78. HRMS: m/z: obsd
715.2625 [M+Na]+, calcd for C40H40N2O9Na+, 715.2630.
4.1.3.7. 2-Methoxy-1-nitro-5-(30,40,50-trimethoxyphenyl)-benzo-
suber-5-ol (31).
To a well-stirred solution of 3,4,5-trimethox-
yphenyl bromide (0.680 g, 2.75 mmol) in THF (25 mL) at ꢁ78 °C,
n-BuLi (1.00 mL, 2.50 M in hexanes) was added, and the reaction
mixture was stirred for 1 h. Benzosuberone analog 29 (0.390 g,
1.66 mmol) in THF (5 mL) was added dropwise into the reaction
mixture, which was then stirred for 18 h and allowed to warm
from ꢁ78 °C to room temperature. The reaction was quenched
with H2O (20 mL), and the solvent was removed under reduced
pressure. The mixture was extracted with EtOAc (3 ꢀ 25 mL), and
the combined organic phase was washed with brine, dried over
Na2SO4, filtered, and concentrated under reduced pressure. The
residue was purified by flash column chromatography using a
pre-packed 50 g silica column [solvent A: EtOAc; solvent B: hex-
anes; gradient: 5%A/95%B (1 CV), 5%A/95%B ? 100%A/0%B (13
CV), 100%A/0%B (2 CV); flow rate: 40 mL/min; monitored at 254
and 280 nm] to afford alcohol 31 (0.468 g, 1.16 mmol, 70%) as a
4.1.3.10. (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-
((3-methoxy-9-(3,4,5-trimethoxyphenyl)-6,7-dihydro-5H-benzo
[7]annulen-4-yl)amino)-3-oxopropyl acetate (35)47
.
This
compound was synthesized using the procedure as described for
compound 34. Starting from amino compound 33 (0.355 g,
1.00 mmol), Fmoc-L-ser(Ac)-OH (0.553 g, 1.50 mmol), T3P (50% in
EtOAc, 1.50 mL, 2.50 mmol), and Et3N (0.210 mL, 1.50 mmol) in
CH2Cl2 (25 mL), the desired product 35 (0.393 g, 0.556 mmol,
56%) was obtained as a colorless solid. 1H NMR (500 MHz, CDCl3):
d 7.74 (2H, d, J = 7 Hz), 7.65 (1H, s), 7.57 (2H, d, J = 7.5 Hz), 7.38 (2H,
dd, J = 7.5, 7.0 Hz), 7.27 (2H, dd, J = 7.5, 7.0 Hz), 6.97 (1H, d,
colorless solid. 1H NMR (500 MHz, CDCl3):
d
7.68 (1H, d,
J = 8.7 Hz), 6.88 (1H, d, J = 8.7 Hz), 6.46 (2H, s), 3.90 (3H, s), 3.84
(3H, s), 3.76 (6H, s), 2.60 (2H, m), 2.41 (1H, m), 2.37 (1H, s), 2.11
(1H, m), 1.95 (1H, m), 1.79 (2H, m), 1.53 (1H, m). 13C NMR
(125 MHz, CDCl3): d 153.3, 149.4, 142.4, 140.5, 138.6, 137.6,

L. Devkota et al. / Bioorg. Med. Chem. 24 (2016) 938–956
953
J = 8.5 Hz), 6.73 (1H, d, J = 8.5 Hz), 6.49 (2H, s), 6.36 (1H, t,
J = 7.5 Hz), 5.94 (1H, d, J = 6.5 Hz), 4.82 (1H, bs), 4.49 (4H, m),
4.23 (1H, t, J = 6.5 Hz), 3.86 (3H, s), 3.79 (6H, s), 3.74 (3H, s), 2.59
(2H, t, J = 7.5 Hz), 2.14 (2H, m,), 2.10 (3H, s). 1.95 (2H, m). 13C
NMR (125 MHz, CDCl3): d 170.90, 168.51, 156.24, 153.31, 152.94,
143.69, 143.60, 142.46, 141.32, 140.74, 138.38, 137.42, 133.53,
129.45, 127.84, 127.15, 125.04, 122.19, 120.04, 108.42, 105.36,
67.32, 64.61, 60.95, 56.20, 55.71, 54.26, 47.14, 33.91, 27.09,
25.63, 20.82. [Note: A portion of the experimental data for this
compound was reproduced from Ref. 47 with permission from
Elsevier.]
J = 11, 4.0 Hz), 3.97 (1H, dd, J = 11, 4.0 Hz), 3.82 (3H, s), 3.80 (6H,
s), 3.79 (3H, s), 2.67 (2H, m), 2.30 (2H, m). 13C NMR (125 MHz, CD3
OD): d 166.1, 153.7, 152.9, 139.4, 136.9, 136.8, 135.6, 128.3, 125.3,
124.6, 121.6, 107.8, 105.7, 60.6, 59.7, 55.1, 55.1, 54.8, 23.2, 22.4.
HRMS: m/z: obsd 429.2063 [MꢁCl]+, calcd for C23H29N2O6+,
429.2026. HPLC (Method A): 7.94 min.
4.1.3.14.
methoxyphenyl)-6,7-dihydro-5H-benzo[7]annulen-4-yl)propa-
namide hydrochloride (39)47
This compound was
(S)-2-Amino-3-hydroxy-N-(3-methoxy-9-(3,4,5-tri-
.
synthesized using the procedure as described for compound 38.
Starting from serinamide 37 (0.10 g, 0.23 mmol) and HCl (4 N in
dioxane, 0.28 mL, 1.1 mmol) in CH2Cl2/MeOH (6 mL, 1:1 ratio),
the desired product 39 (0.074 g, 0.15 mmol, 67%, melting point
range 260–263 °C) was obtained as a colorless solid. 1H NMR
4.1.3.11.
methoxyphenyl)-7,8-dihydronaphthalen-1-yl)propanamide
(36). To a stirred solution of CH2Cl2/MeOH (6 mL, 1:1 ratio)
were added Fmoc- -serinamide acetate 34 (0.30 g, 0.43 mmol)
(S)-2-Amino-3-hydroxy-N-(2-methoxy-5-(3,4,5-tri-
(500 MHz, CD3OD):
d 6.96 (1H, d, J = 8.5 Hz), 6.92 (1H, d,
L
J = 8.5 Hz), 6.53 (2H, s), 6.39 (1H, t, J = 7.5 Hz), 4.26 (1H, dd,
J = 7.5, 4.5 Hz), 4.19 (1H, dd, J = 11, 4.0 Hz), 3.99 (1H, dd, J = 11,
7.5 Hz), 3.82 (3H, s), 3.76 (6H, s), 3.75 (3H, s), 2.62 (2H, m), 2.16
(2H, m), 1.92 (2H, m). 13C NMR (125 MHz, CD3OD): d 166.9,
153.9, 152.8, 142.5, 140.7, 138.4, 137.1, 133.3, 129.3, 126.9,
121.7, 108.5, 105.1, 60.7, 59.7, 55.1, 55.1, 54.8, 33.5, 26.2, 24.9.
HRMS: m/z: obsd 443.2214 [MꢁCl]+, calcd for C24H31N2O6+,
443.2177. HPLC (Method A): 8.32 min. [Note: A portion of the
experimental data for this compound was reproduced from Ref.
47 with permission from Elsevier.]
and a NaOH solution (2 M, 0.48 mL, 0.97 mmol). After stirring for
18 h at room temperature, the solvent was evaporated under
reduced pressure, and water (6 mL) was added. The solution was
extracted with EtOAc (3 ꢀ 10 mL), and then the combined organic
phase were rinsed with brine, dried with Na2SO4, and concentrated
under reduced pressure. The residue was purified by flash column
chromatography (5% MeOH/ CH2Cl2) to afford the desired seri-
namide 36 (0.12 g, 0.28 mmol, 65%, melting point range 176–
178 °C) as a yellow solid. 1H NMR (500 MHz, CDCl3): d 8.91 (1H,
s), 6.98 (1H, d, J = 8.5 Hz), 6.65 (1H, d, J = 9.0 Hz), 6.56 (2H, s),
6.00 (1H, t, J = 4.5 Hz), 4.00 (1H, dd, J = 11, 4.5 Hz), 3.88 (3H, s),
3.84 (6H, s), 3.81 (3H, s), 3.81 (1H, dd, J = 11, 4.5 Hz), 3.69 (1H, t,
J = 4.5 Hz), 2.67 (2H, t, J = 7.5 Hz), 2.30 (2H, td, J = 7.5, 4.5 Hz). 13C
NMR (125 MHz, CDCl3): d 172.6, 152.9, 152.8, 139.4, 137.0, 136.6,
135.2, 128.7, 125.1, 125.0, 122.3, 107.7, 105.7, 65.3, 60.8, 56.6,
56.1, 55.7, 23.9, 22.9. HRMS: m/z: obsd 429.2041 [M+H]+, calcd
for C23H29N2O+6, 429.2026. HPLC (Method A): 7.80 min.
4.1.3.15. (9H-Fluoren-9-yl)methyl (2-((2-methoxy-5-(3,4,5-tri-
methoxyphenyl)-7,8-dihydronaphthalen-1-yl)amino)-2-
oxoethyl)carbamate (40).
This compound was synthesized
using the procedure as described for compound 34. Starting from
amino compound 32 (0.500 g, 1.45 mmol), Fmoc-glycine-OH
(0.648 g, 2.18 mmol), T3P (50% in EtOAc, 2.60 mL, 4.35 mmol),
and Et3N (0.310 mL, 2.18 mmol) in CH2Cl2 (30 mL) with 12 h stir-
ring, the desired product 40 (0.678 g, 1.10 mmol, 75%) was
obtained as a yellow solid. 1H NMR (500 MHz, CDCl3): d 7.75 (2H,
d, J = 7.5 Hz), 7.60 (2H, d, J = 7.5 Hz), 7.46 (1H, s), 7.38 (2H, t,
J = 7.5 Hz), 7.29 (2H, t, J = 7.5 Hz), 6.97 (1H, d, J = 8.5 Hz), 6.65
(1H, d, J = 9.0 Hz), 6.57 (2H, s), 5.99 (1H, t, J = 4.5 Hz), 5.61 (1H,
s), 4.48 (2H, d, J = 7 Hz), 4.24 (1H, t, J = 7 Hz), 4.12 (2H, s), 3.88
(3H, s), 3.84 (6H, s), 3.76 (3H, s), 2.67 (2H, t, J = 7.5 Hz), 2.30 (2H,
td, J = 7.5, 4.5 Hz). 13C NMR (125 MHz, CDCl3): d 167.71, 156.57,
152.94, 152.63, 143.84, 143.66, 141.31, 139.47, 137.09, 136.61,
135.38, 128.80, 127.76, 127.07, 125.19, 125.00, 121.92, 120.00,
107.57, 105.80, 67.19, 60.92, 56.16, 55.65, 47.17, 44.86, 24.05,
22.94.
4.1.3.12.
methoxyphenyl)-6,7-dihydro-5H-benzo[7]annulen-4-yl)propa-
namide (37)47
This compound was synthesized using the
procedure as described for compound 36. Starting from Fmoc-
(S)-2-Amino-3-hydroxy-N-(3-methoxy-9-(3,4,5-tri-
.
L
-
serinamide acetate 35 (0.393 g, 0.556 mmol) and a NaOH solution
(2 N, 0.630 mL, 1.26 mmol) in CH2Cl2/MeOH (6 mL, 1:1 ratio), the
desired product 37 (0.185 g, 0.418 mmol, 75%, melting point range
234–235 °C) was obtained as a colorless solid. 1H NMR (500 MHz,
CDCl3): d 8.73 (1H, s), 6.97 (1H, d, J = 8.5 Hz), 6.78 (1H, d,
J = 8.5 Hz), 6.49 (2H, s), 6.36 (1H, t, J = 7.5 Hz), 4.04 (1H, dd,
J = 11, 4.5 Hz), 3.86 (3H, s), 3.84 (6H, s), 3.80 (3H, s), 3.80 (1H, dd,
J = 11, 4.5 Hz), 3.71 (1H, t, J = 4.5 Hz), 2.62 (2H, t, J = 6.5 Hz), 2.16
(2H, m), 1.98 (2H, q, J = 7.0 Hz). 13C NMR (125 MHz, CDCl3): d
173.5, 153.1, 152.8, 142.5, 140.6, 138.3, 133.5, 129.1, 127.6,
122.5, 109.9, 108.4, 105.3, 65.9, 60.9, 56.3, 56.1, 55.8, 34.0, 27.0,
25.5. HRMS: m/z: obsd 443.2190 [M+H]+, calcd for C24H31N2O6+,
443.2177. HPLC (Method A): 8.23 min. [Note: A portion of the
experimental data for this compound was reproduced from Ref.
47 with permission from Elsevier.]
4.1.3.16. (9H-Fluoren-9-yl)methyl (2-((3-methoxy-9-(3,4,5-tri-
methoxyphenyl)-6,7-dihydro-5H-benzo[7]annulen-4-yl)
amino)-2-oxoethyl)carbamate (41).
This compound was
synthesized using the procedure as described for compound 34.
Starting from amino compound 33 (0.200 g, 0.563 mmol), Fmoc-
glycine-OH (0.250 g, 0.841 mmol), T3P (50% in EtOAc, 1.07 mL,
1.68 mmol), and Et3N (0.118 mL, 0.841 mmol) in CH2Cl2 (10 mL)
with 12 h stirring, the desired product 41 (0.350 g, 0.552 mmol,
98%) was obtained as a colorless solid. 1H NMR (500 MHz, CDCl3):
d 7.73 (2H, d, J = 7.04 Hz), 7.58 (2H, d, J = 7.04 Hz), 7.42 (1H, s), 7.35
(2H, t, J = 6.80 Hz), 7.27 (2H, t, J = 6.80), 6.96 (1H, d, J = 8.20 Hz),
6.73 (1H, d, J = 7.87 Hz), 6.49 (2H, s), 6.35 (1H, t, J = 6.45 Hz), 5.80
(1H, s), 4.45 (2H, m), 4.22 (1H, t, J = 6.93 Hz), 4.13 (2H, s), 3.86
(3H, s), 3.79 (6H, s), 3.76 (3H, s), 2.58 (2H, t, J = 6.93 Hz), 2.14
(2H, td, J = 6.93, 7.32 Hz), 1.95 (2H, q, J = 7.32 Hz). 13C NMR
4.1.3.13.
methoxyphenyl)-7,8-dihydronaphthalen-1-yl)propanamide
hydrochloride (38). Serinamide 36 (0.055 g, 0.13 mmol) was
(S)-2-Amino-3-hydroxy-N-(2-methoxy-5-(3,4,5-tri-
dissolved in CH2Cl2 (0.8 mL), and HCl (4 N in dioxane, 0.080 mL,
0.32 mmol) was added to the solution. After stirring for 1 h, the
mixture was removed by evaporation to dryness, and the crude
solid was re-crystallized using EtOAc/MeOH to afford desired seri-
namide salt 38 (0.015 g, 0.032 mmol, 25%, melting point range
241–242 °C) as a light yellow solid 1H NMR (500 MHz, CD3OD): d
6.98 (1H, d, J = 8.5 Hz), 6.83 (1H, d, J = 8.5 Hz), 6.58 (2H, s), 6.01
(1H, t, J = 4.5 Hz), 4.20 (1H, dd, J = 7.0, 4.0 Hz), 4.15 (1H, dd,
(125 MHz, CDCl3):
d 168.93, 156.84, 153.43, 152.97, 143.76,
142.50, 141.36, 140.86, 138.45, 137.46, 133.53, 129.40, 127.89,
127.82, 127.14, 125.10, 122.34, 120.05, 108.44, 105.41, 67.26,
60.99, 56.26, 55.75, 47.23, 44.93, 33.97, 27.18, 25.69.

954
L. Devkota et al. / Bioorg. Med. Chem. 24 (2016) 938–956
4.1.3.17. 2-Amino-N-(2-methoxy-5-(3,4,5-trimethoxyphenyl)-
7,8-dihydronaphthalen-1-yl)acetamide (42). This com-
4.2. Biological evaluations
pound was synthesized using the procedure as described for com-
pound 36. Starting from Fmoc-glycinamide 40 (0.477 g,
0.768 mmol) and a NaOH solution (2 M, 0.770 mL, 1.54 mmol) in
CH2Cl2/MeOH (9 mL/1:1 ratio) with 13 h stirring, the desired pro-
duct 42 (0.285 g, 0.715 mmol, 93%, melting point range 180–
181 °C) was obtained as a tan solid. 1H NMR (500 MHz, CDCl3): d
8.84 (1H, s), 6.93 (1H, d, J = 8.5 Hz), 6.63 (1H, d, J = 9.0 Hz), 6.55
(2H, s Hz), 5.95 (1H, t, J = 4.5 Hz), 3.85 (3H, s), 3.80 (6H, s), 3.77
(3H, s), 3.55 (2H, s), 2.67 (2H, t, J = 7.5 Hz), 2.26 (2H, td, J = 7.5,
4.5 Hz), 2.02 (2H, s). 13C NMR (125 MHz, CDCl3): d 171.5, 152.8,
152.8, 139.5, 136.9, 136.7, 135.1, 128.6, 125.0, 124.7, 122.5,
107.6, 105.7, 60.8, 56.1, 55.6, 45.0, 24.1, 22.9. HRMS: m/z: obsd
399.1930 [M+H]+, calcd for C22H27N2O+5, 399.1914. HPLC (Method
A): 8.10 min.
4.2.1. Cell culture and SRB cytotoxicity assay16,82–84
Three cancer cell lines (DU-145, prostate; SK-OV-3, ovarian; and
NCI-H460, lung cancer) were grown and passaged using DMEM
media supplemented with 10% FBS (Gibco One ShotÒ) and 1% gen-
tamycin sulfate (Teknova, Hollister, CA). Cells were maintained at
37 °C in a humidified atmosphere of 5% CO2, up to passage 15 for
use in these experiments. The sulforhodamine B (SRB) assay was
used to assess inhibition of human cell line growth as previously
described.16,82–84 Briefly, cancer cells were plated at 7500 cells/
well using DMEM supplemented with 5% FBS and 1% gentamycin
sulfate in 96-well plates and incubated for 24 h. Subsequently,
10-fold serial dilutions of the compounds to be tested were then
added to the wells. After 48 h, the cells were fixed with 10% tri-
chloroacetic acid (final concentration), stained with sulforho-
damine B (Acid Red 52) (TKI, Tokyo), read at 540 nm, and
normalized at 630 nm with an automated Biotek Elx800 plate
reader (Biotek, Winooski, VT). A growth inhibition of 50% (GI50 or
the drug concentration causing a 50% reduction in the net protein
staining relative to controls) was calculated from optical density
data with Excel software.
4.1.3.18. 2-Amino-N-(3-methoxy-9-(3,4,5-trimethoxyphenyl)-
6,7-dihydro-5H-benzo[7]annulen-4-yl)acetamide (43).
This
compound was synthesized using the procedure as described for
compound 36. Starting from Fmoc-glycinamide 41 (0.350 g,
0.552 mmol) and a NaOH solution (2 M, 0.560 mL, 1.12 mmol) in
CH2Cl2/MeOH (6 mL, 1:1 ratio) with 13 h stirring, the desired pro-
duct 43 (0.193 g, 0.468 mmol, 83%, melting point range 123–
128 °C) was obtained as a colorless solid. 1H NMR (500 MHz, CD3
OD): d 6.92 (1H, d, J = 8.58 Hz), 6.88 (1H, d, J = 8.61 Hz), 6.53 (2H,
s), 6.37 (1H, t, J = 7.31 Hz), 3.80 (3H, s), 3.76 (3H, s), 3.74 (6H, s),
3.53 (2H, s), 2.60 (2H, t, J = 6.81 Hz), 2.13 (2H, p, J = 6.88 Hz), 1.93
4.2.2. Inhibition of tubulin polymerization85
Tubulin assembly experiments were performed using 0.25 mL
reaction mixtures (final volume),85 which contained 1.0 mg/mL
(10 lM) purified bovine brain tubulin, 0.8 M monosodium gluta-
mate (pH 6.6 in a 2 M stock solution) 4% (v/v) dimethyl sulfoxide,
0.4 mM GTP, and varying compound concentrations. Initially, all
components except GTP were preincuabted for 15 min at 30 °C in
(2H, q, J = 7.11 Hz). 13C NMR (125 MHz, CD3OD):
d 175.20,
155.54, 154.20, 144.12, 142.12, 139.97, 138.54, 134.61, 130.37,
128.23, 123.91, 109.79, 106.61, 61.15, 56.57, 56.19, 45.06, 34.96,
27.69, 26.38. HRMS: m/z: obsd 413.2074 [M+H]+, calcd for
0.24 mL. After chilling the mixtures on ice, 10 lL of 10 mM GTP
was added. The reaction mixtures were placed in cuvettes held at
0 °C in Beckman DU-7400 and DU-7500 spectrophotometers
equipped with electronic temperature controllers. The tempera-
ture was jumped to 30 °C over about 30 s, and polymerization
was followed turbidimetrically at 350 nM for 20 min. Each reaction
set included a reaction mixture without compound, and the IC50
was defined as the compound concentration that inhibited extent
of assembly by 50% after 20 min at 30 °C.
C
23H29N2O+5, 413.2071. HPLC (Method A): 8.45 min.
4.1.3.19. 2-Amino-N-(2-methoxy-5-(3,4,5-trimethoxyphenyl)-
7,8-dihydronaphthalen-1-yl)acetamide hydrochloride
(44). This compound was synthesized using the procedure
as described for compound 38. Starting from glycinamide 42
(0.274 g, 0.688 mmol) and HCl (4 N in dioxane, 0.510 mL,
2.04 mmol) in MeOH (5 mL), the desired product 44 (0.171 g,
0.393 mmol, 57%, melting point range 238–239 °C) was obtained
as a light yellow solid. 1H NMR (500 MHz, CD3OD): d 6.97 (1H, d,
J = 8.5 Hz), 6.82 (1H, d, J = 8.5 Hz), 6.58 (2H, s), 6.01 (1H, t,
J = 4.5 Hz), 3.97 (2H, s), 3.81 (3H, s), 3.80 (6H, s), 3.79 (3H, s),
2.69 (2H, m), 2.31 (2H, m). 13C NMR (125 MHz, CD3OD): d 165.1,
153.7, 152.9, 139.5, 136.9, 136.8, 135.6, 128.3, 125.3, 124.6,
121.6, 107.8, 105.7, 59.7, 55.1, 54.7, 40.2, 33.2, 22.47. HRMS: m/z:
obsd 399.1919 [MꢁCl]+, calcd for C22H27N2O5+, 399.1914. HPLC
(Method A): 8.03 min.
4.2.3. Colchicine binding assay86
Inhibition of [3H]colchicine binding to tubulin was determined
using 100
5.0
M [3H]colchicine (from Perkin-Elmer), 5% (v/v) dimethyl sul-
foxide, potential inhibitors at 1.0 or 5.0 M and components
lL reaction mixtures, each containing 1.0 lM tubulin,
l
l
demonstrated to stabilize the colchicine binding activity of tubu-
lin86 (1.0 M monosodium glutamate [adjusted to pH 6.6 with HCl
in a 2.0 M stock solution], 0.5 mg/mL bovine serum albumin,
0.1 M glucose-1-phosphate, 1.0 mM MgCl2, and 1.0 mM GTP). Incu-
bation was for 10 min at 37 °C, a time point at which the binding
reaction in the control is 40–60% complete. Reactions were stopped
by adding 2.0 mL of ice-cold water and placing the samples on ice.
Each sample was poured onto a stack of two DEAE-cellulose filters,
followed immediately by 6 mL of ice-cold water, and the water was
aspirated under reduced vacuum. The filters were washed three
times with 2 mL of water and, following removal of excess water
under a strong vacuum, placed into vials containing 5 mL of Biosafe
II scintillation cocktail. Samples were counted the next day in a
Beckman scintillation counter. Samples with potential inhibitors
were compared to controls with no inhibitor to determine percent
inhibition. All samples were corrected for the amount of colchicine
that bound to the filters in the absence of tubulin.
4.1.3.20. 2-Amino-N-(3-methoxy-9-(3,4,5-trimethoxyphenyl)-
6,7-dihydro-5H-benzo[7]annulen-4-yl)acetamide
hydrochlo-
ride (45). This compound was synthesized using the proce-
dure as described for compound 38. Starting from glycinamide
43 (0.150 g, 0.364 mmol) and HCl (4 N in dioxane, 0.450 mL,
1.80 mmol) in MeOH/CH2Cl2 (4 mL, 1:1 ratio), the desired product
45 (0.0847 g, 0.189 mmol, 53%, melting point range 274–276 °C)
was obtained as a light yellow solid. 1H NMR (500 MHz, CD3OD):
d 6.98 (1H, d, J = 8.59 Hz), 6.94 (1H, d, J = 8.62 Hz), 6.54 (2H, s),
6.41 (1H, t, J = 7.31 Hz), 3.99 (2H, s), 3.84 (3H, s), 3.77 (3H, s),
3.77 (6H, s), 2.64 (2H, t, J = 6.81 Hz), 2.16 (2H, p, J = 6.72 Hz), 1.95
(2H, q, J = 7.13 Hz). 13C NMR (125 MHz, CD3OD):
d 167.39,
155.48, 154.25, 144.05, 142.07, 139.85, 138.63, 134.75, 130.80,
128.30, 123.13, 109.93, 106.63, 61.17, 56.59, 56.24, 41.63, 35.01,
27.70, 26.35. HRMS: m/z: obsd 413.2074 [MꢁCl]+, calcd for
4.2.4. In vitro LAP cleavage assay
LAP (from porcine kidney microsomes) was purchased from
Sigma–Aldrich or Calzyme Laboratories Inc, and its activity
C
23H29N2O+5, 413.2071. HPLC (Method A): 8.47 min.

L. Devkota et al. / Bioorg. Med. Chem. 24 (2016) 938–956
955
(enzyme units) was determined with 24 mM
L
-leucine p-nitroani-
assessed using Fisher’s PLSD (protected least squares difference)
lide as substrate in 50 mM sodium phosphate buffer (pH 7.2) at
37 °C.5 In preliminary experiments, the extent of cleavage of indi-
vidual amino acid prodrug conjugates were determined. In a gen-
eral procedure, LAP (appropriate amount of units) was pre-
incubated in 50 mM sodium phosphate buffer (pH 7.2) at 37 °C.
The enzymatic reaction was initiated by the addition of substrate.
After varying periods of incubation, a small portion of the reaction
mixture was quenched by the addition of acetonitrile on ice and
centrifuged. The resulting supernatant was diluted by HPLC solvent
using Statview software with p <0.05 considered significant.
Acknowledgements
The authors are grateful to the National Cancer Institute of the
National Institutes of Health (Grant No. 5R01CA140674 to K.G.P.,
M.L.T., and R.P.M.), the Cancer Prevention and Research Institute
of Texas (CPRIT, Grant No. RP140399 to K.G.P., M.L.T., and R.P.M.),
and OXiGENE, Inc. (Grant to K.G.P. and M.L.T.) for their financial
support of this project. The content is solely the responsibility of
the authors and does not necessarily reflect the official views of
the National Institutes of Health. The authors also thank Dr.
Michelle Nemec (Director) for the use of the shared Molecular Bio-
sciences Center at Baylor University and Dr. Alejandro Ramirez
(Mass Spectrometry Core Facility, Baylor University). The authors
are grateful to Mr. Jarrod Tunnell (Baylor University) for his contri-
butions to the synthesis of an advanced intermediate, and to Jeni
Gerberich (UTSW) for valuable technical assistance. Imaging (at
UTSW) was facilitated with the assistance of Resources of the Har-
old C. Simmons Cancer Center supported through an National Insti-
tutes of Health National Cancer Institute Cancer Center Support
Grant [Grant 1P30 CA142543], specifically, the Southwestern Small
Animal Imaging Resource, and Live Cell Imaging Resource. The IVIS
Spectrum was purchased with support of 1S10RR024757.
(acetonitrile/water) and filtered through a 0.2
ter and analyzed by HPLC (Agilent Technologies 1200 series) with
an Agilent ZORBAX Eclipse XDB-18 column (4.6 ꢀ 150 mm, 5 m).
lm nylon syringe fil-
l
HPLC operating conditions were: mobile phase, acetonitrile/water
containing 0.05% TFA, flow rate, 1.0 mL/min; column temperature,
22 °C; diode array UV detector. For rate studies, aliquots were
withdrawn from incubation mixtures at different time points (1–
15 min), and they were immediately quenched with acetonitrile
and analyzed by HPLC (see supplemental data for more informa-
tion). Standard calibration curves were used to determine the con-
centration of individual amino acid prodrug conjugates and their
parent compounds.87
4.2.5. In vitro HUVEC tubule disruption assay
A layer of MatrigelTM (9.5 mg/mL) was plated manually into each
well of a 24-well plate, which was then placed into an incubator at
37 °C for 60 min prior to cell plating. HUVECs (124,000 cells sus-
Supplementary data
pended in 300 lL of M200 growth factors) were plated into 24
wells and allowed to incubate at 37 °C for 16 h. The incubated
HUVECs were treated with varying concentrations of inhibitors
(0.01 lM, 0.1 lM, 1.0 lM), and a photographic record was
obtained (9 fields/well, 40ꢀ objective).
Supplementary data (the synthesis of AVE8062, characteriza-
tion data (1H NMR, 13C NMR, 31P NMR, HPLC, HRMS) for final com-
pounds and intermediates (1H NMR, 13C NMR), HPLC
chromatograms, calibration curves and rate studies related to
LAP enzymatic assays, and aqueous solubility data for representa-
tive compounds) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmc.2016.01.007.
4.2.6. In vivo tumor model78
Human breast cancer cells, MDA-MB-231(ATCC), were trans-
fected with a lentivirus containing a firefly luciferase reporter.
Induction of tumors was carried out by injecting 106 cells mixed
with 30% MatrigelTM (BD Biosciences, San Jose, CA) into the mam-
mary fat pads of female SCID mice (UTSW breeding colony).
Tumors were allowed to grow to about 5 mm in diameter, deter-
mined by calipers, before BLI and assessment of VDAs. All animal
procedures were approved by the University of Texas Southwest-
ern Medical Center Institutional Animal Care and Use Committee.
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