Photoresponsive azo-combretastatin A-4 analogues

Article abstract of DOI:10.1016/j.ejmech.2017.11.012

Colchicine analogues in which an azo group is incorporated into a molecule containing the key pharmacophore of colchicine, have found particular utility as switchable tubulin binding chemotherapeutics. Combretastatin is a related compound containing a stilbene fragment that shows different bioactivity for the cis and trans isomers. We have performed cell assays on 17 new compounds structurally related to a previously reported azo-analogue of combretastatin. One of these compounds showed enhanced potency against HeLa (IC₅₀ = 0.11 μM) and H157 cells (IC₅₀ = 0.20 μM) for cell studies under 400 nm irradiation and the highest photoactivity (IC₅₀ with irradiation/IC₅₀ in dark = 550). We have performed docking and physicochemical studies of this new compound (7). Kinetic studies in water reveal a longer half-life for the cis isomer of 7 which may be one factor responsible for the better IC₅₀ values in cell assays and the improved photoresponsive behavior.

Full text of DOI:10.1016/j.ejmech.2017.11.012

Accepted Manuscript
Photoresponsive azo-combretastatin A-4 analogues
Shiva K. Rastogi, Zhenze Zhao, Scott L. Barrett, Spencer D. Shelton, Martina
Zafferani, Hailee E. Anderson, Madeleine O. Blumenthal, Lindsey R. Jones, Lei
Wang, Xiaopeng Li, Craig N. Streu, Liqin Du, William J. Brittain
PII:
S0223-5234(17)30905-4
10.1016/j.ejmech.2017.11.012
EJMECH 9887
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 August 2017
Revised Date: 25 October 2017
Accepted Date: 4 November 2017
Please cite this article as: S.K. Rastogi, Z. Zhao, S.L. Barrett, S.D. Shelton, M. Zafferani, H.E. Anderson,
M.O. Blumenthal, L.R. Jones, L. Wang, X. Li, C.N. Streu, L. Du, W.J. Brittain, Photoresponsive
azo-combretastatin A-4 analogues, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.11.012.
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ACCEPTED MANUSCRIPT

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Photoresponsive Azo-Combretastatin A-4 Analogues
†
*
†
†
†
§
Shiva K. Rastogi, Zhenze Zhao, Scott L. Barrett, Spencer D. Shelton, Martina Zafferani , Hailee E.
†
†
†
‡
‡
§
Anderson, Madeleine O. Blumenthal, Lindsey R. Jones, Lei Wang, Xiaopeng Li, Craig N. Streu,
†
†*
Liqin Du, and William J. Brittain
†
Department of Chemistry and Biochemistry, 601 University Drive, Texas State University
‡
San Marcos, TX - 78666, USA, Chemistry Department, University of South Florida, Tampa, FL –
3620, USA, Department of Chemistry, Albion College, 611 E. Porter Str. Albion, MI - 49224, USA
§
3
Abstract
Colchicine analogues in which an azo group is incorporated into a molecule containing the key
pharmacophore of colchicine, have found particular utility as switchable tubulin binding
chemotherapeutics. Combretastatin is a related compound containing a stilbene fragment that shows
different bioactivity for the cis and trans isomers. We have performed cell assays on 17 new compounds
structurally related to a previously reported azo-analogue of combretastatin. One of these compounds
showed enhanced potency against HeLa (IC = 0.11 ꢀM) and H157 cells (IC = 0.20 ꢀM) for cell studies
50
50
under 400 nm irradiation and the highest photoactivity (IC50 with irradiation/IC50 in dark = 550). We
have performed docking and physicochemical studies of this new compound (7). Kinetic studies in water
reveal a longer half-life for the cis isomer of 7 which may be one factor responsible for the better IC50
values in cell assays and the improved photoresponsive behavior.
Introduction
Photopharmacology is a unique and potentially powerful tool of use for creating more selective
cancer-targeting compounds. Biological functions can be controlled by localized application of light that
selectively excites photoswitches.[1-3] Light is a non-invasive control element where deleterious toxic
effects can be minimized in a qualitative and quantitative manner by adjusting wavelength and intensity
respectively.[4-7] One of the chief advantages of light is that it can be supplied with very high spatial and
temporal accuracy in biological systems and thus overcome the poor specificity of drug activity.
While this type of dynamic molecular system has great pharmaceutical potential, there are a host
of additional considerations inherent to such a system that must be considered when evaluating a potential
new therapy. For example, for photopharmaceuticals it is the difference between the biological activity of
the excited and unexcited photoswitch that is fundamental to obtaining target selectivity, rather than the
absolute potency of the excited photoswitch. In addition, proper evaluation of new photopharmaceuticals
requires physicochemical studies to evaluate relative solubilities of active and inactive isomers, or the
half-life of the excited state. Both of these properties can impact the observed biological activity and
utility of a potential photopharmaceutical.
[
[
Figure 1]
Figure 2]
One promising area for application of photopharmacology is tubulin polymerization inhibitors.[8]
Microtubules are dynamic heterodimeric cytoskeletal structures that are made by self-assembly of and
-tubulin protein and participate in many cellular functions.[9] The key role of microtubules is in the
formation of mitotic spindles during mitotic cell division. Tubulin is the most validated protein target for
α
β
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small molecules as anti-proliferative and anticancer ligands, which interact with tubulin and disturb
microtubule dynamics.[10] In anticancer drug discovery, these ligands are classified into two major
categories, first are those that inhibit the polymerization of the mitotic spindle such as vinca alkaloids[11]
and colchicine [12,13] (1, Figure 1) and second are those that inhibit the depolymerization of the mitotic
spindle once it has formed, such as paclitaxel.[14] Combretastatin A-4 (CA-4), a stilbene natural product
isolated from the stem wood of the South African tree Combretum caffrum (2, Figure 1),[15] has been
investigated extensively for its tubulin polymerization inhibitor activity and was found to possess potent
anticancer activity against a number of human cancer cell lines including multidrug resistance cancer cell
lines.[16] The X-ray structure of a tubulin complex bound by CA-4 shows that this molecule binds to the
colchicine site in tubulin.[17] One disadvantage of CA-4 is its low solubility (0.35 mM),[18] which has
prompted the synthesis of the water-soluble phosphate prodrug, combretastatin A-4-phosphate (3
fosbretabulin disodium, Figure 1)[19,20] and amino acid prodrug (4 ombrabulin, Figure 1).[21] Both 3
and 4 are in clinical trials.
CA-4 (2) and its prodrugs (3 and 4) have been found to be effective anti-angiogenesis agents that
inhibit new vascular system development within solid tumors.[22,23] The anti-angiogenic activity of 2
has been widely reported and this activity has been demonstrated to arise from binding to the same site in
β
-tubulin as colchicine.[22,24] However, stilbene compounds are well-known to photoisomerize, and
while the trans isomer is more stable, the cis isomer of CA-4 is orders of magnitude more potent for
anticancer activity.[16,25,26] This isomerization limits the stability and shelf-life of CA-4, so much
effort has been dedicated to developing conformationally restricted analogues of 2, in which the cis-
double bond of 2 is substituted with a heterocyclic ring to prevent isomerization. Some examples of these
compounds are azetidinone,[27] β-lactum,[28] heteroarylcumarin,[29] benzoxepine,[30] aroylindole,[31]
furazane,[32] imidazole,[33] pyrazole,[34] and 1,3-dioxolane.[35] Although these non-isomerizable
molecules show better activity against cancer cells growth, drug specificity problems persist because
these ligands are also toxic to both normal and healthy cells.
To address issues of tissue selectivity, a photopharmacological azologue of CA-4 in which the
stilbene unit of CA-4 is replaced by azobenzene has been reported by Borowjak et al.[36] (photostatins),
Engdahl et al.[37] (azo-combretastatin A4), and later by Sheldon et al.[38] (Figure 2). Both groups
utilized a photopharmacology approach to control the bioactivity of 5 in spatiotemporal manner by
substituting azobenzene for the stilbene fragment in CA-4 (substitution of N=N bond for C=C).
Compared to stilbene photochemistry, azobenzene has negligible side-reactions during
photoisomerization, has lower isomerization activation energy, and abundant literature exists on structural
modifications to tune photochemical response. Cellular media are irradiated with ultraviolet light (UV,
3
95-400 nm) to convert the inactive trans isomer of 5 into the bioactive cis-5.
These promising results prompted our group to investigate these systems, and their
physiochemical properties in more detail. Many structure-activity studies on CA-4 have been
reported.[31,32] We note that there are recent reports on pharmacophore features for the colchicine
binding site[39] and properties of isocombretaquinolines as tubulin inhibitors.[40] It has been
demonstrated that the 3,4,5-trimethoxy substituted A-ring of colchicine is required for bioactivity.[26]
The principle focus of this study is therefore substituent changes to the B-ring. A key functional group in
the B-ring is either a hydrogen bond donating alcohol or amine at the 3-position which gives potent
bioactivity and good water solubility.[25] We have synthesized and tested a library of 17 derivatives
against two cancer cell lines (HeLa-cervical cancer and H157-lung cancer) in the dark and in presence of
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UV light. For the subset with the most promising inhibitory activity, we used a combination of
physicochemical analysis and ligand docking simulations to investigate inhibitory differences.
Results and Discussion
Synthesis
We synthesized 17 compounds (6-22) for biological testing (see Table 1 and Figure 3 for
structures). Of these, only compounds 21 and 22 have structural changes to the A-ring. These compounds
were synthesized by a combination of three principle reactions: 1) azo-coupling of the diazonium salt
from either 3,4,5-triemthoxyaniline or 3,4-dimethoxyanline, 2) alkylation of phenol groups, and 3) Pd-
catalyzed deprotection of allyl ether protecting group. Details of all synthetic procedures and full
1
13
characterization ( H NMR, C NMR, IR, mp, ESI-HRMS) of all new compounds are available in the
supplementary material.
[
Figure 3.]
Photochemistry
The photoresponsiveness of all new compounds was studied by UV-visible spectroscopy prior to
cellular assay. The photoisomerization of azobenzene compounds is easily monitored by changes in the n-
* and * transitions. UV irradiation converted the trans isomer to a photostationary state (PSS)
π
π-π
within 3-7 min for all compounds. Representative UV-vis spectra are shown in Figures 4 and S1. All
compounds showed spectral changes consistent with trans-to-cis isomerization as evidenced by distinct
spectra for the two isomers. Compounds 6, 16, 20 and 21 do not display spectral changes upon irradiation
because they fall into a class of azobenzenes known as “psedostilbenes.”[41] These four compounds all
possess a para-phenol substituent that participates in a tautomeric equilibrium characterized by extremely
fast thermal relaxation of the photoinduced cis isomer.[42]
[
Figure 4]
Cancer Cell Viability Assays
Seventeen azo-analogues of Azo-CA-4 (5) were tested for their antiproliferative activities on
HeLa cells as a model for human cervical adenocarcinoma and H157 cells as a model for lung cancer.
Series dilutions (from 125 µM to 0.1 nM) of individual azo-compounds were incubated with HeLa and
H157 cells for four days in a 96-well plate. The incubated cells in one plate were grown in the dark and in
another plate grown in the presence of UV light (390-400 nm, 10 sec irradiation every 34 min). An
automated UV light switch system uploaded with html codes on an Arduino board (Figure S2) was used
for the experiment. The UV light switch setup (Arduino board, 51 LED flash light, bread board, electrical
components and power source) was developed as earlier reported.[36]
The activity of azo-compounds on cancer cells was measured via a colorimetric MTT assay
(
Figure 5). The intensity of color correlates to the viability of remaining HeLa and H157 cells; the
relative cell viability versus concentration of test compounds were plotted and IC50 values determined by
curve fitting and statistical analysis (Figure 6). Many results have relatively large standard deviations of
the mean value. For every MTT assay a new batch of cells (HeLa or H157) was grown and treated with
the compounds at the same culture conditions, including cell culture medium, temperature and treatment
time. Therefore, the replicates of our MTT assays represent the true biological replicates. Given the
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intrinsic heterogeneity of cell populations widely known in cancer cell lines, the large variations in
response to compound treatment in different and independent batches of cells are expected, even though
the culture condition was strictly controlled. This is an explanation for large standard deviations. The
observed variations, however, is of particular importance since it can be used as an estimation of the
possible minimum and maximum responses of the cancer line to the compounds that may occur in real
biological environments. After analysis of the IC50 values of 17 compounds (see Table 1) we observed a
good structure-activity-relationship (SAR) for compounds 5, 7 and 8. Only compounds that displayed
activity are included in Table 1. Figure 3 displays the structure of tested compounds that displayed no
detectable activity. Compound 7 with the ethoxy group is the most active ligand in the series and shows
6
00-fold and 550-fold greater activity in the presence of UV light for HeLa and H157 cells respectively.
This is an improvement upon the previously reported azo-CA-4 (5) that displays 250-fold and 5-fold
enhanced activity following UV light irradiation. Borowjak et al.[36] reported 74-fold enhanced activity
for 5 for HeLa cell assays, which is comparable to our result. These assay results are supported by white
light microscopy of compounds 5, 7 and 8 (Figure 7). Similar results were observed for HeLa and H157
cells where a lower cell count is observed at 200 and 40 nM for compounds 5 and 7 under UV irradiation.
[
[
[
[
Table 1]
Figure 5]
Figure 6]
Figure 7]
Physicochemical and Docking Studies
We performed docking and physicochemical studies to investigate the origin of increased potency
and increased photochemical effect; i.e., increased ratio of IC (dark)/ IC (light). We hypothesized that
50
50
several factors may be playing a role in explaining the difference between 5 and 7: 1) stronger binding to
the tubulin active site, 2) differential solubility of cis and trans isomers and/or 3) different half-lives of cis
isomer.
To address the question of tubulin binding, we performed ligand binding simulations using
AutoDock Vina (version 4.2).[43] The tubulin-combretastatin complex (PBD accession number: 5LYJ(
[
17] were converted to required PDBQT files using MGL Tools (version 1.5.6).[44] The default search
box size was calculated using the protocol outlined by the authors of Vina. The radius of gyration for
ligands cis-5, 7 and 8 was calculated from optimized structures from density functional theory (B3LYP/6-
3
11+G(d) as implemented in Gaussian 09[45]) and used to estimate box size. Figure 8 shows an overlay
of 2 in the bound state and the lowest energy binding conformations of 5 and 7. Azo compounds 5 and 7
have nearly identical binding conformations and closely overlap the X-ray binding structure of
combretastatin. The RMSD for comparison of docking pose of combretastatin versus the X-ray structure
was 0.14 nm. The distance between the stilbene double bond and the azo group is 0.1 nm for best
docking poses of 2 versus 5 and 7. One difference between combrestastatin A4 and azo compound 5 and
7
is the orientation of the para-methoxy group on the A-ring. The calculated binding affinity obtained via
Vina was -8.1, -7.6, and -7.1 kcal/mol for 5, 7 and 8 for the lowest energy conformer. Visualization of
the binding conformations and binding energies do not indicate a significant difference in the binding
affinity of 5 versus 7.
[
Figure 8]
4

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The aqueous solubility of azobenzene compounds is generally sub-mM and the cis isomer can be
0-times more soluble than trans.[46] Aqueous solubility will decrease with an increasing number of
5
carbon atoms so it is expected that S (water solubility) will decrease in the order 5 > 7 > 8. However, we
w
could not predict cis/trans differential solubility nor the half-lives of the cis isomers of 5, 7 and 8. Table
2
w
contains kinetic data on the thermal cis-to-trans isomerization and S values for cis and trans isomers.
Both of these physicochemical characteristics could potentially affect the effective concentration of the
bioactive cis isomer. The values for S (cis) correspond to photostationary states (PSS) and thus
w
represent lower limits for S . To put the data for 5, 7 and 8 into perspective, several literature examples
w
are included in the Table 2. From the data in Table 2, we observed the expected order of aqueous
solubility where 5 possesses the highest S for both isomers. The modest difference in cis/trans solubility
w
for 5, 7 and 8 is not general for less substituted azobenzenes and can be understood by comparison of
calculated dipole moments. We calculated dipole moments (Table S2, supplementary material) using
w
density functional theory and have observed that there is a correlation between ꢁꢀ and ꢁS as
exemplified by 4-trifluoroazobenzene where ꢁꢀ is negative and this is reflected in equivalent solubility
for the geometric isomers. The activity of these compounds is related to concentration of active species
and binding affinity to tubulin. Sparingly soluble compounds in water potentially have stronger
hydrophobic interactions with the protein so it can be argued the lower solubility of 7 relative to 5 may be
associated with stronger binding to active site.
1
We studied the half-life for the cis isomer of compounds 5 and 7 via 500 MHz H NMR. We
attempted to use UV-Vis spectroscopy to measure the kinetics of the thermal cis-to-trans isomerization.
However, we obtained inconsistent results due to precipitation of these sparingly soluble compounds. We
have previously studied cis/trans ratios of azobenzene compounds in water,[46] and the low solubility of
such derivatives requires long acquisition times making these experiments difficult and prone to trace
impurities. Kinetics studies in methanol-d revealed nearly identical isomerization rates for 5, 7 and 8.
4
Kinetics studies in water were replicated and limited to 5 and 7. The first-order rate plots are provided in
the supplementary material (Tables S5 and S6). Surprisingly, the rate of isomerization for 5 is 3-fold
greater than 7 when measured in water, which corresponds to half-lives of 138 and 46 h, respectively for
5
and 7. This rate difference is unexpected given the minor and remote structural difference between 5
and 7. Density functional theory (Table S3) and rate measurements in methanol-d support equivalent
4
reactivity for all three compounds. The half-lives for 5 and 7 may be a reflection of differential solubility
for the cis and trans isomers. This effect has been reported recently and is pronounced for the parent
azobenzene compound where S (cis)/S (trans) = 40 leading to longer-lived cis populations. Regardless
w
w
of the origin of the experimentally observed rate difference, the resulting difference in half-lives is one
potential factor that explains the different potency and photoresponsive behavior of compounds 5 and 7.
Conclusion
In summary, we have synthesized a library of photoswitchable anologues of the known tubulin-
binding photoswitch 5, measured their photoisomerization, and evaluated their light induced cytotoxicity
in two human cancer cell lines. These studies yielded a novel compound (7) that demonstrates a greater
biological activity and relative improvement in activity upon irradiation than the previously reported
compound (5) in both HeLa and H157 cells. Molecular modeling suggests that this compound binds
within the colchicine binding pocket of tubulin. To characterize the physiochemical behavior of 7, and in
an attempt to explain its improved bioactivity upon irradiation, the thermal cis-to-trans half-life, as well
5

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as the dipole moments and water solubility of the cis and trans isomers were measured. The reduced
water solubility of cis-7 with respect to cis-5 may explain its improved potency, as desolvation upon
binding tubulin would be predicted to be energetically favored for the less water soluble compound.
Additionally, an experimentally observed longer half-life for cis-7 relative to cis-5 has been identified as
one potential factor for the observed difference in activity.
Acknowledgment
Albion College Faculty Development Fund and the Foundation for Undergraduate Research, Scholarship,
and Creative Activity (FURSCA) for funding. We thank the National Science Foundation (PREM Center
for Interfaces, DMR-1205670) for financial support of this research. The authors also acknowledge the
support of the National Science Foundation for NMR instrumentation (CR1IF:MU-0946998 and MRI-
0
821254).
References
[
[
[
[
1] Fehrentz, T.; Schonberger, M.; Trauner, D. Optochemical Genetics Angew. Chem. Int. Ed. 2011,
0, 12156-12182.
2] Velema, W. A.; Szymanski,W.; Feringa, B. L. Photopharmacology: Beyond Proof of Principle J.
Am. Chem. Soc. 2104, 135, 2178-2191.
3] Broichhagen, J.; Frank, J. A.; Trauner, D. A Roadmap to Success in Photopharmacology, Acc.
Chem. Res. 2015, 48(7), 1947-1960.
4] Syzmanski, W.; Beierle, J. M.; Kistemaker, H. A. V.; Velma W. A.; Feringa, B. L. Reversible
Photocontrol of Biological Systems by the Incorporation of Molecular Photoswitches, Chem. Rev.
5
2
013, 113, 6114-6178.
[
[
[
[
5] Brieke, C.; Rohrbach, F.; Gottschalk, A.; Mayer, G.; Heckel, A. Light-Controlled Tools Angew.
Chem. Int. Ed. 2012, 51, 8446-8476.
6] Beharry, A. A.; Woolley, G. A. Azobenzene Photoswitches for Biomolecules Chem. Soc. Rev.
2
011, 40, 4422-4437.
7] Mayer, G.; Heckel, A. Biologically Active Molecules with a “Light Switch”, Angew. Chem. Int. Ed.
006, 45, 4900-4921.
2
8] Borowiak, M.; Nahaboo, W.; Reynders, M.; Nekolla, K.; Jalinot, P; Hasserodt, J.; Rehburg, M.;
Delatree, M.; Zahler, S.; Vollamar, A.; Trauner, D. Photoswitchable Inhibitors of Microtubule
Dynamics Optically Control Mitosis and Cell Death Cell 2015, 162(2), 403-411.
[
[
[
[
[
[
[
[
9] Downing, K. H.; Nogales, E. Tubulin and Microtubulin Structure. Curr. Opin. Cell Biol. 1998,
1
0(1), 16-22.
10] Jordan, M. A. Mechanism of Action of Antitumor Drugs that Interact with Microtubules and
Tubulin Current Medicinal Chemistry-Anti-Cancer Agents 2, 2002, 2(1), 1-17.
11] Hardfield, J. A.; Ducki, S.; Hirst, N.; McGown, A. T. Tubulin and Microtubules Agents for
Anticancer Drugs Progress in Cell Cycle Research 2003, 5, 309-325.
12] Bhattacharyya, B.; Panda, D.; Gupta, S.; Banerjee, M. Anti-Mitotic Activity of Colchicine and the
Structural Basis for its Interaction with Tubulin Med. Res. Rev. 2008, 28(1), 155-183.
13] Margolis, R. L.; Rauch, C. T.; Wilson, L. Mechanism of Colchicine-Dimer Addition to Microtubule
Ends: Implication for the Microtubules Polymerization Mechanism Biochem. 1980, 19, 5550-5557.
14] Dumontet, C.; Jordan, M. A. Microtubule-Binding Agents: A Dynamic Field of Cancer
Therapeutics Nat. Rev. Drug Discov. 2010, 9, 790.
15] Watt, J. M.; Gerdina, M. The Medicinal and Poisonous Plants of Southern and Eastern Africa, E. &
S. Livingston Ltd.: Edenburg and London, U.K., 1962.
16] Pettit, G. R.; Rhodes, M. R.; Herald, D. L.; Hamel, E.; Schemidt, J. M.; Pettit, R. K. Antineoplastic
Agent. 445. Synthesis and Evaluation of Structural Modification of of (Z)- and (E)-Combretastatin
A-41, J. Med. Chem. 2005, 48, 4087-4099.
6

ACCEPTED MANUSCRIPT
[
[
17] Gaspari, R.; Prota, A. E.; Bargsten, K.; Cavalli, A.; Steinmetz, M.O. Structural Basis of cis-and
trans-Combretastatin Binding to Tubulin Chem. 2017, 2(1), 102-113.
18] Lee, M.; Brockway, O.; Dandavati, A.; Tzou, S.; Sjoholm, R.; Nickols, A.; Babu, B.; Chavda, S.;
Satam, V.; Hartley, R. M.; Westbrook, C. Design and Synthesis of Novel Enhanced Water Soluble
Hydroxyethyl Analogs of Combretastatin A-4. Bioorg. Med. Chem. Lett. 2011, 21(7), 2087-2091.
19] Cooney, M. M.; Ortiz, J.; Bukowski, R. M.; Remick, S. C. Novel Vascular Targeting/Disrupting
Agents: Combretastatin A4 Phospate and Related Compounds. Curr. Oncol. Rep. 2005, 7(2), 90-95.
20] https://clinicaltrials.gov/ct2/show/NCT00060242?term=NCT00060242&rank=1.
21] Ohsumi, K.; Hatanaka, T.; Nakagawa, R.; Fukuda, Y.; Morinaga, Y.; Suga, Y.; Nihei, Y.; Ohishi,
K.; Akiyama, Y.; Tsuji, T.; Synthesis and Antitumor Activities of Amino Acid Prodrugs of Amino-
Combretastatins. Anti-Cancer Drug Design 1999, 14, 539-548.
[
[
[
[
[
22] Dark, G. G.; Hill, S. A.; Prise, V. E.; Tozer, G. M.; Pettit, G. R.; Chaplin, D. J.; Combretastatine A-
4
1
and Agent That Displays Potent and Selective Toxicity Towards Tumor Vasculature Cancer Res.,
997, 57 (10), 1829-1834.
23] Sessa, C.; Lorusso, P.; Tolcher, A.; Farace, F.; Lassau, N.; Delmonte, A.; Braghetti, A.; Bahleda,
R.; Cohen, P.; Hospitel, M.; Veyrat-Follet, C.; Soria, J. C. Phase I Safety, Pharmacokinetic and
Pharmacodynamic Evaluation of the Vascular Disrupting Agent Ombrabulin (AVE8062) in
Patients With Advanced Solid Tumors Clin. Cancer Res., 2013, 19, 4832-4842.
[
24] Prota A. E.; Danel, E.; Bachmann, F.; Bargstem, K.; Buey, R. M.; Pohlmann, J.; Reinelt, S.; Lane,
H.; Steimetz, M. O. The Novel Microtubule-Destabilizing Drug BAL27862 Bind to the Colchicine
Site of Tubulin with Distinct Effects on Microtubule Organization. J. Mol. Biol. 2014, 426, 1848-
1
860.
[
[
[
25] Ohsumi, K.; Hatanaka, T.; Fujita, K.; Nakagawa,R.; Fukuda,Y.; Nihei, Y.; Suga, Y.; Morinaga, Y.;
Akiyama, Y.; Tsuji, T. Syntheses and Antitumor Activity of Cis-Restricted Combretastatins: 5-
Membered Heterocyclic Analogues. Bioorg. Med. Chem. Lett. 1998, 8 (22), 3153-3158.
26] Cushman, M.; He, H. M.; Lin, C. M.; Hamel, E. Synthesis and Evaluation of a Series of
Benzylaniline Hydrochlorides as Potential Cytotoxic and Antimitotic Agents Acting by Inhibition
of Tubulin Polymerization. J. Med. Chem. 1993, 36 (19), 2817-2821.
27] O’Boyle, N. M.; Carr, M.; Greene, L. M.; Bergin, O.; Nathwani, S. M.; McCabe, T.; Lloyd, D. G.;
Zisterer, D. M. and Meegan, M. J. Synthesis and Evaluation of Azetidinone Analogues of
Combretastatin A-4 as Tubulin Targeting Agents, J. Med. Chem. 2010, 53, 8569–8584.
28] Banik, I.; Becker, F. F.; Banik, B. K. Stereoselective Synthesis of Beta-Lactams with Polyaromatic
Imines: Entry to New and Novel Anticancer Agents. J. Med. Chem. 2003, 46 (1), 12-15.
29] Ganina, O. G.; Daras, E.; Bourgarel-Rey, V.; Peyrot, V.; Andresyuk, A. N.; Finet, J. P.; Fedorov, A.
Y.; Beletskaya, I. P.; Combes, S. Synthesis and Biological Evaluation of Polymethoxylated 4-
Heteroarylcoumarins as Tubulin Assembly Inhibitor. Bioorg. Med. Chem. 2008, 16 (19), 8806-
[
[
8
812.
[
[
30] Barrett, I.; Carr, M.; O’Boyle, N.; Greene, L. M.; Knox, A., J. S.; Lloyd, D. G.; Zisterer, D. M.;
Meegan, M. J. Lead Identification of Conformationally Restricted Benzoxepin Type Combretastatin
Analogs: Synthesis, Antiproliferative Activity, and Tubulin Effects J. Enzym. Inhib. Med. Ch. 2010,
2
5 (2), 180-194.
31] Liou, J. P.; Chang, Y. L.; Kuo, F. M.; Chang, C. W.; Tseng, H. Y.; Wang, C. C.; Yang, Y. N.;
Chang, J. Y.; Lee, S. J.; Hsieh, H. P. Concise Synthesis and Structure-Activity Relationships of
Combretastatin A-4 Analogues,1-Aroylindoles and 3-Aroylindoles, as Novel Classes of Potent
Antitubulin Agents. J. Med. Chem. 2004, 47 (17), 4247-4257.
[
[
32] Tron, G. C.; Pirali, T.; Sorba,G.; Pagliai, F.; Busacca,S.; Genazzani, A. A. Medicinal Chemistry of
Combretastatin A4: Present and Future Directions. J. Med. Chem. 2006, 49 (11), 3033-3044.
33] Wang, L.; Woods, K. W.; Li, Q.; Barr, K. J.; McCroskey, R. W.; Hannick, S. M.; Gherke, L.;
Credo, R. B.; Hui, Y. H.; Marsh, K.; Warner, R.; Lee, J. Y.; Zielinski-Mozng, N.; Frost, D.;
Rosenberg, S. H.; Sham, H. L. Potent, Orally Active Heterocycle-Based Combretastatin A-4
7

ACCEPTED MANUSCRIPT
Analogues: Synthesis, Structure-Activity Relationship, Pharmacokinetics, and In Vivo Antitumor
Activity Evaluation. J. Med. Chem. 2002, 45, 1697-1711.
[
[
[
[
[
[
[
34] Ohsumi, K.; Hatanaka, T.; Fujita, K.; Nakagawa, R.; Fukuda,Y.; Nihei, Y.; Suga, Y.; Morinaga, Y.;
Akiyama, Y.; Tsuji, T. Syntheses and Antitumor Activity of Cis-Restricted Combretastatins: 5-
Membered Heterocyclic Analogues. Bioorg. Med. Chem. Lett. 1998, 8 (22), 3153-3158.
35] Shirai, R.; Takayama, H.; Nishikawa, A.; Koiso, Y.; Hashimoto, Y. Asymmetric Synthesis of
Antimitotic Combretadioxolane with Potent Antitumor Activity Against Multi-Drug Resistant Cells
Bioorg. Med. Chem. Lett. 1998, 8 (15), 1997-2000.
36] Borowiak, M.; Nahaboo, W.; Reynders, M.; Nekolla, K.; Jalinot, P.; Hasserodt, J.; Rehberg, M.;
Delattre, M.; Zahler, S.; Vollmar, A.; Trauner, D.; Thorn-Seshold, O. Photoswitchable Inhibitors of
Microtubule Dynamics Optically Control Mitosis and Cell Death. Cell 2015,162 (2), 403-411.
37] Engdahl, A. J.; Torres, E. A.; Lock, S. E.; Engdahl, T. B.; Mertz, P. S.; Streu, C. N. Synthesis,
Characterization, and Bioactivity of the Photoisomerizable Tubulin Polymerization Inhibitor azo-
Combretastatin A4, Org. Lett. 2015, 17, 4546−4549.
38] Sheldon, J. E.; Dcona, M. M.; Lyons, C. E.; Hackett, J. C.; Hartman, M.C. Photoswitchable
Anticancer Activity via Trans–Cis Isomerization of a Combretastatin A-4 Analog Org. Biomol.
Chem. 2016, 14(1), 40-49.
39] Mangiatordi, G. F.; Trisciuzzi, D.; Alberga, D.; Denora, N.; Iacobazzi, R. M.; Gadaleta, D.; Catto,
M.; Nicolotti, O. Novel Chemotypes Trageting Tubulin at the Colchicene Binging Site and
Unbiasing P-gycloprotein Eur. J. Med. Chem. 2017, 139, 792-803.
40] Khelifi, I.; Naret, T.; Renko, D.; Hamze, A.; Bernadat, G.; Bignon, J.; Lenoir, C.; Dubois, J.; Brion,
J.-D.; Revot, O. Design, Synthesis and Anticancer Properties of IsoCombretaQuinolines as Potent
Tubulin Assembly Inhibitors Eur. J. Med. Chem. 2017, 127, 1025-1034.
[
[
[
41] Dhammika Bandara, H. M.; Burdette, S. C. Photoisomerization in Different Classes of Azobenzene
Chem. Soc. Rev., 2012, 41, 1809-1825.
42] Gabor, G.; Fischer, E. Spectra and Cis-Trans Isomerism in Highly Bipolar Derivatives of
Azobenzene J. Phys. Chem. 1971, 75, 581-583.
43] Trott, O.; Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new
scoring function, efficient optimization, and multithreading Trott, O. and Olson, A.J. J. Comput.
Chem. 2010, 31, 455-461.
[
[
44] Morris, G.M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J.
AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J.
Comput. Chem. 2009, 30, 2785-2791.
45] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G.
Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P.
Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota,
R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A.
Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N.
Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J.
Tomasi, M. Cossi, N. Rega, N. J. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo,
J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W.
Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J.
Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, D. J.
Fox, D. J. Gaussian 09, Revision A.1; Gaussian, Inc.: Wallingford, CT, 2009.
[
[
46] Brown, C.; Rastogi, S. K.; Barrett, S. L.; Anderson, H. E.; Twichell, E.; Gralinski, S.; McDonald,
A.; Brittain, W. J. Differential Azobenzene Solubility Increases Equilibrium Cis/Trans Ratio in
Water J. Photoch. Photobio. A 2017, 336, 140-145.
47] Nakano, M.; Uematsu, Y.; Arita, T. Solution and Partition Behaviors of Colchicine Chemical and
Pharmaceutical Bulletin 1977, 25(5), 1109-1112.
8

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FIGURE CAPTIONS
Figure 1. Natural product and synthetic tubulin polymerization inhibitors: colchicine (1), cis- and trans-
forms of combretastatin A-4 (2) and related compounds, CA-4 phosphate (3) and amino-acid derivative of
CA-4 (4).
Figure 2. Tubulin polymerization inhibition profile for azo-combretastatin A-4 (5) and analogues 6, 7, 8.
Figure 3. Structures of compounds that showed no detectable activity in MMT cell assays.
Figure 4. UV-Visible photo-isomerization of compounds 5 (a), 6 (b), 7 (c) and 8 (d) in DMSO at
0
.01mg/mL concentration.
Figure 5. MTT assay of HeLa (left) and H157 (right) cells in the presence of 200 nM (a) and 40 nM (b) of
compounds 5, 7 and 8 in dark and in the presence of 400 nm isomerizing light.
Figure 6. Relative cell viability vs. concentration graphs of Azo-CA4 5 and analogues 6, 7, and 8 for two
cancer cell lines, HeLa (left column) and H157 cells (right column).
Figure 7. White light microscopy (phase contrast images using confluence mask of gold color) of HeLa
cells incubated 72 h with DMSO and compounds 5, 7 and 8 at concentrations of (a) 200 nM and (b) 40
nM. Images are shown for samples in the dark versus 400 nm irradiation.
Figure 8. AutoDock 4.2 Vina results for the most stable binding conformation of combretastatin A4 (a),
5
(b) and 7 (c).
9

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a
Table 1. IC50 values (µM) and dark vs UV ratio of IC50 of Azo-CA4 and analogues that display
bioactivity in dark and/or in UV (395 nm) from MTT assay (ND=not detected).
b
Structure
HeLa Cells
H157 Cells
UV
Entry
ArB
Dark
UV
Dark/UV
Dark
50 ± 40
Dark/UV
5
50 ± 20 0.16 ± 0.07 310 ± 20
30 ± 30 0.34 ± 0.16 80 ± 30
60 ± 30 0.11 ± 0.03 550 ± 30
0.16 ± 0.03 310 ± 40
6
7
10 ± 2
1.9 ± 1.4
0.2 ± 0.1
5 ± 2
110 ± 60
550 ± 60
8
9
ND
3 ± 2
ND
ND
2 ± 2
ND
c
23 ± 16
36 ± 25
~ 1
-
-
c
1
3
ND
ND
90 ± 60
100 ± 70
~ 1
1
1
2
6
7
0
60 ± 20
ND
ND
-
-
ND
ND
ND
ND
-
-
53 ± 14
12 ± 5
c
c
14 ± 7
~ 1
18 ± 1
5.3 ± 0.3
3 ± 1
2
2
1
2
21 ± 15
15 ± 10
13 ± 3
~ 1
~ 1
ND
ND
-
c
c
9 ± 7
10 ± 10
3.6 ± 0.3
~ 1
1
Colchicene
0.003 ± 0.001 (dark)
0.010 ± 0.003 (dark)
a
Reported numbers are mean values ± standard deviation. b Ar
Ar ) = 3,4,5-trimethoxyphenyl. Ratio can only be expressed as an order of magnitude because standard
A
A
-N=N-Ar
B
where structure of the A-Ring
c
(
deviation was larger than the mean.

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Table 2. Solubility and half-life data for azobenzene compounds in water at 23°C.
w
S (cis)/
Compound
Sw, mM
Trans
Sw(trans
τ1/2, h
Cis
2
2
0.02
14
0.07
.04
.02
a
Azobenzene
0.05
0.09
0.02
0.3
0.03
.02
40
22
1
47
2
48
6
4
4
-methoxyazobenzene
-trifluoromethylazobenzene
d
nd
a
Azobenzene-L-Leu-L-Pro
24 e
46 (7)
5
7
8
e
2
2
138 (8)
d
e
.01
nd (9)
b
Colchicine
41.6
c
Combretastatin 2
0.35
a
b
c
d
e
Reference 46. Reference 47. Reference 18. Not determined. Half-life in methanol.

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Highlights
•
Seventeen analogs of azocombretastatin A-4 were tested for photopharmacological activity
against HeLa and H157 cell lines
•
•
•
Compound 7 displayed ~2x improved activity relative to previously reported A-4 (compound 5)
The structural difference between 7 and 5 is an ethoxy substitution for methoxy on the A-ring
The hypothesis to account for this improved activity is based on differential aqueous solubility of
cis and trans isomers