Synthesis and biological evaluation of 2-alkoxycarbonylallyl esters as potential anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2017.01.037

The reaction of carboxylic acids with Baylis-Hillman reaction derived α-bromomethyl acrylic esters readily provide 2-(alkoxycarbonyl)allyl esters in good to excellent yields. These functionalized allyl esters have been evaluated for their cell proliferation inhibition properties against breast cancer (MDA-MB-231 and 4T1) and pancreatic cancer (MIAPaCa-2) cell lines to explore their potential as anticancer agents. Several of the synthesized derivatives exhibit good potency against all three cancer cell lines. Our structure activity relationship (SAR) studies on 2-carboxycarbonyl allyl esters indicate that substituted aromatic carboxylic acids provide enhanced activity compared to substituted aliphatic carboxylic acid analogs. Di- and tri-allyl esters derived from di-and tri-carboxylic acids exhibit higher inhibition of cell proliferation than mono esters. Further SAR studies indicate that the double bond in the 2-(alkoxycarbonyl)allyl ester is required for its activity, and there is no increase in activity with increased chain length of the alkoxy group. Two lead candidate compounds have been identified from the cell proliferation inhibition studies and their preliminary mechanism of action as DNA damaging agents has been evaluated using epifluorescence and western blot analysis. One of the lead compounds has been further evaluated for its systemic toxicity in healthy CD-1 mice followed by anticancer efficacy in a triple negative breast cancer MDA-MB-231 xenograft model in NOD-SCID mice. These two in vivo studies indicate that the lead compound is well tolerated in healthy CD-1 mice and exhibits good tumor growth inhibition compared to breast cancer drug doxorubicin.

Full text of DOI:10.1016/j.bmcl.2017.01.037

Accepted Manuscript
Synthesis and biological evaluation of 2-alkoxycarbonylallyl esters as potential
anticancer agents
Conor T. Ronayne, Lucas N. Solano, Grady L. Nelson, Erica A. Lueth, Skyler
L. Hubbard, Tanner J. Schumacher, Zachary S. Gardner, Sravan K
Jonnalagadda, Shirisha Gurrapu, Jon Holy, Venkatram R. Mereddy
PII:
S0960-894X(17)30048-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.01.037
BMCL 24613
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 December 2016
11 January 2017
12 January 2017
Please cite this article as: Ronayne, C.T., Solano, L.N., Nelson, G.L., Lueth, E.A., Hubbard, S.L., Schumacher, T.J.,
Gardner, Z.S., Jonnalagadda, S.K., Gurrapu, S., Holy, J., Mereddy, V.R., Synthesis and biological evaluation of 2-
alkoxycarbonylallyl esters as potential anticancer agents, Bioorganic & Medicinal Chemistry Letters (2017), doi:
http://dx.doi.org/10.1016/j.bmcl.2017.01.037
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Synthesis and biological evaluation of 2-
alkoxycarbonylallyl esters as potential
anticancer agents
Leave this area blank for abstract info.
Conor T. Ronayne, Lucas N. Solano, Grady L. Nelson, Erica A. Lueth, Skyler L. Hubbard, Tanner J.
Schumacher, Zachary S. Gardner, Sravan K Jonnalagadda, Shirisha Gurrapu, Jon Holy, and Venkatram R.
Mereddy

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Synthesis and biological evaluation of 2-alkoxycarbonylallyl esters as potential
anticancer agents
Conor T. Ronaynea, Lucas N. Solanob, Grady L. Nelsonb, Erica A. Luetha, Skyler L. Hubbarda, Tanner J.
Schumachera, Zachary S. Gardnera, Sravan K Jonnalagaddab, Shirisha Gurrapub, Jon Holyc, and Venkatram
R. Mereddya,b,d ∗
aDepartment of Chemistry and Biochemistry,, University of Minnesota Duluth,, Duluth, MN 55812
bIntegrated Biosciences Graduate Program, University of Minnesota, Duluth, MN 55812
cDepartment of Biomedical Sciences, Medical School Duluth, University of Minnesota Duluth, MN 55812
dDepartment of Pharmacy Practice & Pharmaceutical Sciences, University of Minnesota, Duluth, MN 55812
ARTICLE INFO
ABSTRACT
Article history:
Received
The reaction of carboxylic acids with Baylis-Hillman reaction derived α-bromomethyl acrylic
esters readily provide 2-(alkoxycarbonyl)allyl esters in good to excellent yields. These
functionalized allyl esters have been evaluated for their cell proliferation inhibition properties
against breast cancer (MDA-MB-231 and 4T1) and pancreatic cancer (MIAPaCa-2) cell lines to
explore their potential as anticancer agents. Several of the synthesized derivatives exhibit good
potency against all three cancer cell lines. Our structure activity relationship (SAR) studies on 2-
carboxycarbonyl allyl esters indicate that substituted aromatic carboxylic acids provide
enhanced activity compared to substituted aliphatic carboxylic acid analogs. Di- and tri-allyl
esters derived from di-and tri-carboxylic acids exhibit higher inhibition of cell proliferation than
mono esters. Further SAR studies indicate that the double bond in the 2-(alkoxycarbonyl)allyl
ester is required for its activity, and there is no increase in activity with increased chain length of
the alkoxy group. Two lead candidate compounds have been identified from the cell
proliferation inhibition studies and their preliminary mechanism of action as DNA damaging
agents has been evaluated using epifluorescence and western blot analysis. One of the lead
compounds has been further evaluated for its systemic toxicity in healthy CD-1 mice followed
by anticancer efficacy in a triple negative breast cancer MDA-MB-231 xenograft model in
NOD-SCID mice. These two in vivo studies indicate that the lead compound is well tolerated in
healthy CD-1 mice and exhibits good tumor growth inhibition compared to breast cancer drug
doxorubicin.
Revised
Accepted
Available online
Keywords:
Baylis-Hillman, bromomethyl acrylate, 2-
alkoxycarbonylallyl esters, DNA alkylation, breast
cancer, pancreatic cancer, Karyorrhexis, γ-H2AX,
PARP-1 activation
2009 Elsevier Ltd. All rights reserved.
The Baylis-Hillman (BH) reaction is an important C-C bond
forming reaction in organic synthesis that provides functionalized
allyl alcohols and amines in one step.1-11 The BH reaction has
attracted a lot of attention in recent years owing to the simple
reaction conditions and versatility of the structural synthons that
can be readily obtained utilizing this reaction. The acetates of the
product BH-alcohols undergo facile allylic rearrangement in
predominantly SN2’ fashion with a wide variety of nucleophiles
including C, S, N, and O to afford tri-substituted α, β-unsaturated
olefins stereoselectively.1-11 Several applications in medicinal
chemistry have been reported based on BH reaction as a critical
step.12-18 Our interest in the discovery of novel anticancer agents
has prompted us to explore BH reaction as a pharmacological
tool to discover new therapeutics for cancer treatment.19-22
In the present work, we envisioned that bromomethyl
acrylates 1-4 derived from BH reaction would interact with
nucleophilic intracellular components and/or DNA in an SN2
and/or SN2’ or 1,4-addition fashion. However, these bromides are
relatively unstable upon long storage at room temperature,
limiting their usefulness as anticancer agents. We also envisaged
that 1-4 being reactive allyl bromides, may also have limited
metabolic stability to survive the systemic circulation to be useful
as therapeutic agents.
———
∗ Corresponding author. Tel.: +1-218-726-6776; fax: +1-218-726-7394; e-mail: vmereddy@d.umn.edu

Scheme-1: a) Schematic diagram of potential intracellular reactivity of 2-alkoxycarbonylallyl esters; b) Scheme for the synthesis of 2-
alkoxycarbonylallyl esters 5-14 and 16-24; c) Scheme for the synthesis of dihydroxy derivative 15.
Table-1: IC50* (µM) values of synthesized compounds in MDA-MB-231, MIAPaCa-2 and 4T1 cell lines
Sl.
Sl.
No
MDA-MB-231
MIAPaCa-2 4T1
MDA-MB-231 MIAPaCa-2
4T1
No
39.32 5.89
13.68 2.18
12.21 1.05
20.98 1.32
7.70 0.36
8.14 0.88
11.42 1.23
17.56 0.63
11.82 1.3
>100
12.26 1.09
>100
22.38 1.05
29.66 4.27
41.47 4.35
45.32 0.50
17.43 4.00
3.85 0.48
4.11 1.16
68.74 1.50
>100
72.53 2.90
64.36 4.27
>100
22.50 3.22
36.00 4.00
52.33 1.90
14.75 1.50
5.51 1.15
4.36 0.64
49.81 6.71
>100
1
18
24.70 1.10
19.36 2.43
21.96 1.45
18.92 1.04
13.36 1.74
13.36 1.35
25.12 6.85
16.84 0.94
>100
2
19
33.55 5.38
59.59 6.00
28.5 4.12
22.14 3.34
42.6 2.09
24.07 3.34
28.68 3.30
>100
5
20
13.89 2.21
3.35 1.27
4.63 0.45
83.44 2.56
>100
6
21
7
22
8
23
9
24
10
11
12
13
Chlorambucil
26.79 0.63
94.80 1.18
75.3 8.99
22.96 1.32
14.26 0.82
66.87 3.92
24.53 2.55
39.32 0.47
>100
25.99 1.89
14
25
26
>100
>100
>100
>100
15
16
18.00 1.38
29.53 3.63
>100
>100
28.65 1.96
72.58 8.40
43.13 11.7
17
* average SEM, minimum of three separate experimental results
In this regard, we hypothesized that 2-(alkoxycarbonyl)allyl
Compound 5 exhibited similar or higher cell proliferation
inhibition to that of bromo congener 1, so we then carried out
structure activity relationship (SAR) studies of 2-
(alkoxycarbonyl)allyl esters derived from various carboxylic
acids and BH bromides. Initially keeping BH bromide 1 as a
constant we synthesized its corresponding esters from various
substituted aromatic carboxylic acids. For electron donating
substituents we utilized 4-N,N-dimethyl, 4-methoxy, 3,4,5-
trimethoxy, and piperonyl substituted carboxylic acids. For
electron withdrawing groups we employed 4-nitro and 4-cyano
esters obtained from BH reaction derived bromomethyl acrylate 1
would have the necessary chemical and metabolic stability to
react with nucleophilic cellular components in SN2 and/or SN2’ or
1,4-addition fashion (Scheme 1a), resulting in cellular damage
and death. To test this hypothesis we evaluated the cell
proliferation inhibition of 2-(methoxycarbonyl)allyl benzoate 5
derived from benzoic acid and BH reaction derived bromomethyl
acrylate 1 (Scheme 1b).

substituted carboxylic acids. The reaction of these carboxylic
acids with BH bromide 1 under basic conditions followed by
silica gel column chromatography readily provided the
corresponding 2-(methoxycarbonyl)allyl esters 6-11 in good to
excellent yields (Scheme 1b).
utilized di and tri-carboxylic acids to synthesize di and tri-
functionalized allyl esters to test whether these additions could
further enhance potency. For this purpose, carboxycarbonyl allyl
esters 21-23 were synthesized starting from terephthalic acid,
2,6-pyridinedicarboxylic acid, and trimesic acid, respectively
(Scheme 1b). Cell proliferation inhibition of 21-23 indicated
higher potency compared to monoesters of aromatic carboxylic
acids (Table 1).
We then carried out cell proliferation inhibition studies of
5-11 against triple negative breast cancer MDA-MB-231 cells,
metastatic murine breast cancer cell line 4T1, and pancreatic
cancer cell line MIAPaCa-2. These cancers are highly aggressive
in nature with limited treatment options and poor patient survival.
For these studies, we employed the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) cell viability assay.19
Cell cultures in 96-well plates were incubated with the
compounds for 72 hours, and MTT values expressed as percent
of vehicle-only (control) wells. The IC50 value was calculated for
each compound as the dose required to suppress the MTT signal
to 50% of control values. In general, both electron donating and
electron withdrawing carboxylic acid-derived α-carboxy allyl
esters 5-11 exhibited similar cell proliferation inhibition
properties (Table 1). We further evaluated the SAR of these α-
carboxy allyl esters by taking benzoic acid as a representative
example. Replacing the ester group and substituting with simple
allyl group resulted in allylbenzoate 12 and it did not show any
cell proliferation inhibition properties even up to 100µM
concentration.
As a demonstration of activity enhancing carboxycarbonyl
allyl esters from BH-bromide 1, we have synthesized the prodrug
24 of the clinically important anticancer agent chlorambucil,
which is used for chronic lymphatic leukemia, Hodgkin's disease,
and other types of lymphomas. Although chlorambucil exhibits
good activity against leukemia and lymphoma, it does not exhibit
strong potency against several solid tumor cell lines. For
example, chlorambucil does not exhibit any significant
cytotoxicity at 100µM concentration against MDA-MB-231,
MIAPaCa-2, and 4T1 cells. Carboxylic acid in chlorambucil was
converted into its carboxycarbonyl allyl ester 24 by treatment
with BH-bromide 1 under basic conditions (Scheme 1b). 24
exhibited enhanced cell proliferation inhibition with IC50 values
in the range from 49-83µM against all three cell lines compared
to its parent drug chlorambucil, which has no activity even at
100µM (Table 1).
It is interesting to note that parent BH alcohols 26 and
carboxylic acids (ex. 4-methoxybenzoic acid and 2,6-pyridine
dicarboxylic acid) did not exhibit any appreciable biological
activity up to 100µM concentrations. However, carboxycarbonyl
allyl benzoate 25 obtained from BH alcohol 26 and benzoyl
chloride exhibited cell proliferation inhibition properties albeit in
lower potency than some of the 2-carboxycarbonyl allyl esters
(Table 1). The biological activity of 25 could be attributed to the
leaving group capacity of allyl benzoate in the presence of
cellular nucleophiles.
The structural requirement of the ester moiety for
biological activity was tested by substituting methyl with ethyl
and butyl substituents. The corresponding BH bromides 3 and 4
were synthesized by reacting formaldehyde with ethyl and butyl
acrylates in the presence of DABCO followed by bromination
with HBr and H2SO4. The reaction of benzoic acid with bromides
3 and 4 in Na2CO3/DMSO followed by silica gel column
chromatography purification resulted in the corresponding
carboxycarbonyl allyl esters 13 and 14 respectively (Scheme 1b).
Cell proliferation inhibition evaluation of these compounds did
not result in any enhanced activity, and the methyl ester was
found to be the optimal structural unit for inhibiting cell
proliferation. We then determined the importance of the double
bond for biological activity, since our hypothesis involved the
reaction of nucleophilic cellular components with the candidate
compounds in an SN2’ and/or 1,4-addition fashion (Scheme 1a).
Dihydroxylation of the double bond in 5 with catalytic OsO4 and
NMO provided the corresponding diol 15 in good yield (Scheme
1c). As expected, diol 15 did not show any cell proliferation
inhibition properties against all three cell lines even at 100µM
concentration, indicating the importance of strong nucleophile
acceptance capability of the carboxycarbonyl double bond in
providing the biological activity. Subsequently, the SAR of β-
From these results, two lead compounds 7 and 22 derived
from 4-methoxybenzoic acid and 2,6-pyridine dicarboxylic acid
were selected for further mechanistic studies due to their higher
potency in mono and di-substitution. For this purpose,
concentrations of 7 and 22 equivalent to their 1X, 2X, and 4X
values of their corresponding IC50 of cell proliferation on MDA-
MB-231 were utilized, which corresponded to 8, 16, and 32 µM
for 7, and 4, 8, and 16 µM for 22, respectively. First, overall cell
morphology was examined by phase contrast microscopy after
both short (6 hr.) and longer (48 hr.) treatment times. For both
compounds, little change was evident at either time point at the 4
and 8 µM concentrations. For 7, cell rounding became noticeable
by 6 hrs of treatment with 16 and 32 µM, which by 48 hrs
advanced to clustering of cells into masses that detached from the
culture plate (Figure 1A). For 22 the highest concentration
tested (16 µM) led to noticeable cell rounding, and clustering was
evident at 48 hrs, although most cells remained attached to the
culture dish. Alkylator-induced cell rounding, caused by
perturbations of integrins, cytoskeletal organization, and cadherin
expression, has been reported previously.23
substitution on
5 was evaluated via synthesis of the
carboxycarbonyl ester 16 from the reaction of benzoic acid with
methyl (Z)-2-(bromomethyl)-3-phenylacrylate 2. β-Substituted
16 exhibited decreased activity compared to β-unsubstituted 5
(Table 1).
We then studied the SAR of expanded aromatic rings such
as naphthalene and anthracene with carboxylic acid functional
groups. 1-naphthoic acid and 9-anthracenecarboxylic acid were
reacted with BH bromide 1 under basic conditions to provide the
corresponding 2-carboxycarbonyl allyl esters 17 and 18
respectively (Scheme 1b). Evaluation of 17 and 18 did not
provide any improved cell proliferation inhibition compared to
the benzoic acid derivatives. Carboxycarbonylallyl esters 19 and
20 derived from cinnamic acid and 4-phenylbutyric acid,
respectively, also did not show any elevated biological activity
compared to aromatic carboxylic acids (Table 1). We then
The nucleophilic character of DNA renders it a highly
susceptible target for alkylation. Alkylating agents can react with
ring nitrogens and exocyclic oxygens to produce a variety of
adducts.24,25 Both monofunctional and bifunctional alkylating
agents can result in a range of complex effects, including DNA
strand breakage, cell cycle arrest, and activation of programmed
cell death pathways.25,26

Figure-1: A) Phase contrast imaging of MDA-MB-231 cells after treatment with differing concentrations of compound 7 and 22. All
images were captured using the same magnification (see scale bar). Note cell rounding at the higher compound concentrations. B)
MDA-MB-231 cells treated with vehicle (DMSO) or compound 22 for 24 hours, and subsequently pulsed with BrdU to identify
replicating (S-phase) cells, and counterstained with Hoechst dye to label all nuclei. Hoechst fluorescence is shown in blue, and BrdU
antibody labeling is shown in red. Inset: higher magnification of fragmented nuclear bodies indicative of karyorrhexis in a cell treated
with 22. Scale bar equals 50µm for all images except for inset where it equals 20µm. C) Western blot analysis of MDA-MB-231 whole
cell lysates treated with differing concentrations of compounds 7, 22, DMSO (vehicle control) or Mitomycin C (MC) as an example of a
well-characterized alkylating compound. γ-H2AX was probed after six hours whereas PARP-1 and Cyclin-D1 were probed after 48
hours of treatment with 7 or 22.
Because of the sensitivity of DNA to alkylation, effects of 7
and 22 on nuclear organization, DNA damage response, and cell
cycle regulation were examined. Cells plated on coverslips were
treated with 7 or 22 for 24 hrs, and then fixed with 2%
paraformaldehyde and stained with Hoechst dye to label nuclei.
In some experiments, cells were also pulsed with BrdU to
visualize cells in S-phase of the cell cycle. In control cultures
(vehicle only treated), most nuclei present a typical appearance,
although occasional multinucleated cells, as well cells containing
one or two small micronuclei, can be present. Treatment with 4
µM 22 for 24 hrs resulted in the appearance of cells displaying
multiple irregular nuclear bodies, indicative of karyorrhexis
(Figure 1B). More modest nuclear fragmentation resulted from
treatment with 7 (data not shown). Previous studies have shown
that alkylating agents, including thiotepa, cisplatin, carboplatin,
ifosfamide, and N-methyl-N-nitrosourea can also induce
karyorrhexis in a number of different cell types.26-28
caspase activation and apoptosis. Western blotting shows an
initial increase in PARP signal after exposure to lower
concentrations of 7 and 22, followed by a reduction of the PARP
signal. These results suggest an initial activation of PARP by
these compounds, and subsequent triggering of an apoptotic
response at higher drug concentrations (Figure 1B).
Effects on the cell cycle were examined by looking for
changes in cyclin D1 expression, and by examining S-phase
perturbation via BrdU uptake experiments. Western blots show
that 7 and 22 at 16µM cause an increase in cyclin D1 quantity,
which is blocked or reversed by a higher concentration of 7
(Figure 1C). Cyclin D1 quantity fluctuates throughout the cell
cycle, and proteolysis is an important mechanism in regulating
cyclin D1 quantity following DNA damage and during
apoptosis.33,34 The initial increase in cyclin D1 quantity observed
with both compounds may reflect cell cycle arrest during phases
of higher D1 expression, and the decrease at
a higher
A commonly used indicator of DNA damage and repair
pathway activation is histone H2AX phosphorylation, forming γ-
H2AX. Initially reported to be strongly activated following DNA
double-strand breaks29, H2AX also plays critical roles in cell
responses to alkylating agents.30 The ability of compounds 7 and
22 to upregulate γ-H2AX was examined using Western blots
with a phospho-specific antibody. MDA-MB-231 cells are
concentration could be the result of triggering apoptosis, which is
supported by the reduction in the quantity of full-length PARP
(Figure 1C). The effect of 7 and 22 on DNA synthesis was
tested by evaluating BrdU pulse experiments. After 24 hours of
drug treatment, DNA synthesis were visualized by pulsing cells
with BrdU for one hour, followed by fixation and
immunofluorescence labeling of incorporated BrdU. At early
time points, relatively little change in the numbers of BrdU-
positive (S-phase) cells were observed (data not shown), but at
longer time points a noticeable decrease in the numbers of BrdU-
positive cells became apparent (Figure 1B). In particular,
karyorrhectic cells with fragmented nuclei were rarely BrdU-
positive, indicating an arrest or cessation of cycling. Although we
carried out preliminary in vitro mechanism of action of candidate
compounds as alkylators of DNA, we do not rule out other
potential mechanisms such as reaction with cytosolic or
mitochondrial nucleophilic components.
genetically unstable, and thus display
a relatively high
endogenous baseline level of γ-H2AX in control cultures.
Nevertheless, a clear increase in the γ-H2AX signal was noted for
both 7 and 22 after 6 hrs of treatment and is comparable to the
signal when treated with DNA alkylating agent mitomycin C
(MC) (Figure 1C). In addition to H2AX activation, poly (ADP-
ribose) polymerase (PARP) plays important roles in DNA repair,
and is activated by DNA alkylating agents.31 Depending on the
extent of damage and other cellular factors, initial PARP
activation can be followed by necrosis or apoptosis, with the
latter leading to caspase-induced PARP cleavage.32 In fact,
cleavage of full-length PARP is frequently used as a marker of

Figure-2: A) Systemic toxicity study of compound 22 in CD-1 mice; B) Tumor growth inhibition study of compound 22 in a MDA-
MB-231; xenograft model.
Based on these results, compound 22 was selected as a lead
derivative for in vivo studies of chemotherapeutic efficacy, based
on its potency in the assays described above. Initially, we carried
out systemic toxicity studies with 22 in healthy CD-1 mice. 12
mice were divided into two groups (n = 6): Group-1 was
administered with compound 22 (20mg/kg) whereas group-2 was
administered with vehicle only (control) intraperitoneally, once
daily, six days a week for a total of 14 days (Figure 2A). This
study indicated that compound 22 is generally well tolerated at
accepted therapeutic doses without causing any apparent side
effects as evidenced by normal body weight gains of the treated
animals compared to vehicle group.
consistent with alkylation-induced DNA damage, activation of a
DNA repair response, karyorrhexis, and programmed cell death.
One of the lead candidate compounds (22) exhibited an efficient
tumor growth inhibition in a TNBC MDA-MB-231 tumor
xenograft model comparable to, or slightly exceeding that of
doxorubicin, and this compound was well tolerated as evidenced
by normal body weight gains in healthy CD-1 mice. Owing to the
ease of compound synthesis, high potential for product diversity,
structural tunability, novelty of mechanism, and encouraging in
vitro and in vivo biological activity make the compounds
reported in this manuscript highly promising anticancer agents.
Thus, bromomethylacrylates derived from BH reaction could also
be utilized as cytotoxicity enhancing hydrophobic prodrugs of
carboxylic acids.
We then evaluated the anticancer efficacy of compound 22
in an in vivo triple negative breast cancer MDA-MB-231
xenograft model in female NOD SCID mice. Tumor cells were
inoculated into the right flank of mice and the treatment was
initiated when the average tumor volume reached ~150mm3. The
mice were randomly assigned into three groups (n = 7) based on
average tumor volume. Group-1 was treated with compound 22
at 20 mg/Kg, ip, 3 times/week and group-2 was administered
with clinical breast cancer drug doxorubicin at 0.5 mg/Kg, 5
times/week whereas group-3 was injected with vehicle only.
Tumor volumes and body weights were measured every 3 days
and at the end of the study, mice were euthanized according to
the IACUC guidelines. Compound 22 exhibited 55% tumor
growth inhibition based on the tumor volume and 41% tumor
growth inhibition based on tumor weight compared to the control
group. Similarly, doxorubicin group showed 49% tumor growth
inhibition based on the tumor volume and 28% tumor growth
inhibition based on tumor weight compared to the control group
(Figure 2B).
Acknowledgments
This work was supported by Whiteside Clinical Research
Institute, Department of Chemistry & Biochemistry, College of
Pharmacy and Medical School, University of Minnesota Duluth.
Supplementary Material
Materials and methods, synthesis and compound
characterization, in vitro cytotoxicity, immunofluorescence,
western blotting, other in vivo studies.
References
1. Bharadwaj, C. K. RSC Advances. 2015; 5, 75923-46.
2. Wei, Y.; Shi, M. Organocatalysis. 2013; 3, 899-939.
3. Wei, Y.; Shi, M. Chem. Rev. 2013; 113, 6659-90.
4. Sasai, H.; Takizawa, S. Compr. Chirality. 2012; 6, 234-63.
5. Basavaiah, D.; Veeraraghavaiah, G. Chem. Soc. Rev. 2012; 41, 68-78.
6. Wang, F.; Wei, Y.; Shi, M. RSC Catalysis Series. 2011; Chapter 5, 485-
551.
7. Zhong, W.; Liu, Y.; Wang, G.; Hong, L.; Chen, Y.; Chen, X.; Zheng, Y.;
Zhang, W.; Ma, W.; Shen, Y.; Yao, Y. Org. Prep. Proced. Int.
2011; 43, 1-66.
In
conclusion,
we
synthesized
several
2-
(alkoxycarbonyl)allyl esters from the reaction of carboxylic acids
with α-bromomethyl acrylic esters. These functionalized allyl
esters were evaluated for their cell proliferation inhibition
properties against three cancer cell lines MDA-MB-231, 4T1,
and MIAPaCa-2. Several of the synthesized derivatives exhibited
potency against all three cell lines at low µM concentrations. In
general, carboxycarbonyl allyl esters derived from aromatic
carboxylic acids provided good activity when compared to
aliphatic carboxylic acids. SAR studies indicated that the double
bond was an essential component along with the α-ester group in
the 2-(alkoxycarbonyl)allyl ester for biological activity, and that
the methyl ester and β-unsubstitution enhanced biological
activity. The biological effects of two lead compounds were
8. Basavaiah, D.; Reddy, B. S.; Badsara, S. S. Chem. Rev. 2010; 110, 5447-
674.
9. Ma, G.; Jiang, J.; Shi, M.; Wei, Y. Chem. Comm. 2009; 37, 5496-514.
10. Declerck, V.; Martinez, J.; Lamaty, F. Chem. Rev. 2009; 109, 1-48.
11. Basavaiah, D.; Rao, K. V.; Reddy, R. J. Chem. Soc. Rev. 2007; 36, 1581-
88.
12. da Silva, W. A.; Rodrigues, D. C.; de Oliveira, R. G.; Mendes, R. K.;
Olegario, T. R.; Rocha, J. C.; Keesen, T. S.; Lima-Junior, C. G.;
Vasconcellos, M. L. Bioorg. Med. Chem. Lett. 2016, 26, 4523-4526.

13. Hou, W.; Wang, Z.; Peng, C.; Lin, J.; Liu, X.; Chang, Y.; Xu, J.; Jiang,
R.; Lin, H.; Sun, P.; Chen, W. M. Eur. J. Med. Chem. 2016, 122, 149-
163.
23. Sourdeval, M.; Boisvieux-Ulrich, E.; Gendron, M. C.; Marano, F. Front.
Biosci. (Landmark Ed). 2009; 14, 1917-31.
24. Beranek, D. T. Mutat. Res. 1990; 231, 11-30.
14. Manyeruke, M. H.; Olomola, T. O.; Majumder, S.; Abrahams, S.; Isaacs,
M.; Mautsa, N.; Mosebi, S.; Mnkandhla, D.; Hewer, R.; Hoppe, H. C.;
Klein, R.; Kaye, P.T. Bioorg. Med. Chem. 2015, 23, 7521-7528.
15. Kumar, T.; Verma, D.; Menna-Barreto, R. F. S.; Valenca, W. O.; da
Silva, E. N.; Namboothiri, I. N. N. Org. Biomol. Chem. 2015, 13, 1996-
2000.
16. Bhowmik, S.; Batra S. Curr. Org. Chem. 2014; 18, 3078-119.
17. Kłossowski, S.; Muchowicz, A.; Firczuk, M.; Świech, M.; Redzej, A.;
Golab, J.; Ostaszewski, R. J. Med. Chem 2012, 55, 55−67
18. Lima-Junior, C.G.; Vasconcellos, M. L. Bioorg. Med. Chem. 2012; 20,
3954-71.
25. Drablos, F.; Feyzi, E.; Aas, P. A.; Vaagbo, C. B., Kavli, B.; Bratlie, M.
S.; Peña-Diaz, J.; Otterlei, M.; Slupphaug, G.; Krokan, H. E. DNA Repair
(Amst). 2004; 3, 1389-407.
26. Rasmussen, K., Peterson, B. L.; Jacobo, E.; Penick, G. D.; Sall, J. Acta.
Cytol. 1980; 3, 237-43.
27. Torres-Bugarin, O.; Ventura-Aguilar, A.; Zamora-Perez, A.; Gomez-
Meda, B.C.; Ramos-Ibarra, M. L.; Morga-Villela, G.; Gutierrez-Franco,
A.; Zuniga-Gonzolaz, G. Mutat. Res. 2004; 565, 91-101.
28. Kinoshita, J.; Iwata, N.; Maejima, T.; Imaoka, M.; Kimotsuki, T.;
Yasuda, M. Invest. Opthalmol. Vis. Sci. 2015; 56, 7146-58.
29. Rogakou, E. P.; Pilch, D. R.; Orr, A. H.; Ivanova, V. S.; Bonner, W. M.
J. Biol. Chem. 1998; 273: 5858-68.
19. Solano, L. N.; Nelson, G. L.; Ronayne, C. T.; Lueth, E. A.; Foxley, M.
A.; Jonnalagadda, S. K.; Gurrapu, S.; Mereddy, V. R. Bioorg. Med.
Chem. Lett. 2015; 25, 5777-80.
30. Meador, J. A.; Zhao, M.; Su, Y.; Narayan, G.; Geard, C. R.; Balajee, A.
S. Oncogene. 2008; 27, 5662-71.
20. Tekkam, S.; Alam, M. A.; Jonnalagadda, S. C.; Mereddy, V. R. Chem.
Comm. 2011; 47, 3219-21.
21. Tekkam, S.; Alam, M. A.; Just, M. J.; Berry, S. M.; Johnson, J. L.;
Jonnalagadda, S. C.; Merddy, V. R. Anticancer Agents Med. Chem. 2013;
13, 1514-30.
31. Debiak, M.; Kehe, K.; Burkle, A. Toxicology. 2009; 263, 20-5.
32. Baptiste-Okoh, N.; Barsotti, A. M.; Prives, C. Proc. Natl. Acad. Sci. USA
2008; 105, 1937-42.
33. Yang, K.; Hitomi, M.; Stacey, D. W. Cell. Div. 2006; 1, 32.
34. Jia, L. and Sun, Y. Pigment. Cell. Melanoma. Res. 2009; 22, 518–19.
22. Tekkam, S.; Johnson, J. L.; Jonnalagadda, S. C.; Mereddy V. R. J. Het.
Chem. 2013; 50, 969-72.