Design, synthesis, and biological evaluation of bifunctional thyrointegrin inhibitors: New anti-angiogenesis analogs

Article abstract of DOI:10.3109/14756366.2011.557023

Context: Inhibition of pathological angiogenesis. Objective: Obtaining new transactivator, bifunctional, thyroid antagonist, non-toxic anti-angiogenic compounds. Materials and methods: In silico drug design, synthesis in bulk and biological evaluation in chick chorioallantoic membrane (CAM) model. Results: Significant inhibition (range 6573%) at 0.252.0 g/ml doses. Discussion and conclusion: The synthesis of compounds (9), (10), and (11) incorporating long-chain moieties guanidine, urea, methyl amine and, propyl amine substitutions, respectively, into the core molecular framework of tetrac (tetraiodothyroacetic acid) were undertaken. The evaluation of the anti-angiogenic bioactivity of these compounds in the CAM model revealed no loss of activity in comparison with tetrac and XT199, which showed nearly 86% inhibition at dose levels of 1 and 0.5 g/ml, respectively, and validated the concept.

Full text of DOI:10.3109/14756366.2011.557023

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(6): 871–882
© 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.557023
OrIgInal artIcle
Design, synthesis, and biological evaluation of bifunctional
thyrointegrin inhibitors: new anti-angiogenesis analogs
Alexandre Bridoux1,2, Riaz A. Khan1,3, Celei Chen1, Gwenaël Chevé4, Huadong Cui1, Evgeny Dyskin1,
Aziz Yasri4, and Shaker A. Mousa1,2,5
1Pharmaceutical Research Institute, Rensselaer, NY, USA, 2Vascular Vision Pharmaceuticals, Rensselaer, NY,
USA, 3Department of Chemistry, Manav Rachna International University (MRIU), Faridabad, Haryana, India,
4NOVADECISION, Rond point Benjamin Franklin–C539521, 34950 Montpellier Cedex 2, France, and 5King Saud
University, Riyadh, Kingdom of Saudi Arabia
abstract
Context: Inhibition of pathological angiogenesis.
Objective: Obtaining new transactivator, bifunctional, thyroid antagonist, non-toxic anti-angiogenic compounds.
Materials and methods: In silico drug design, synthesis in bulk and biological evaluation in chick chorioallantoic
membrane (CAM) model.
Results: Significant inhibition (range 65–73%) at 0.25–2.0 µg/ml doses.
Discussion and conclusion: The synthesis of compounds (9), (10), and (11) incorporating long-chain moieties
guanidine, urea, methyl amine and, propyl amine substitutions, respectively, into the core molecular framework
of tetrac (tetraiodothyroacetic acid) were undertaken. The evaluation of the anti-angiogenic bioactivity of these
compounds in the CAM model revealed no loss of activity in comparison with tetrac and XT199, which showed nearly
86% inhibition at dose levels of 1 and 0.5 µg/ml, respectively, and validated the concept.
Keywords: Antiangiogenesis, XT199, dual thyrointegrin antagonist, molecular modelling, chick chorioallantoic
membrane assay
Introduction
of biological processes, including cell differentiation,
wound-healing, and apoptosis9–12. e integrin receptors
play an important role in cell–cell signalling by mediating
interactions between cells and, attachment of cells to the
extracellular matrix, thus, helping to define the cellular
shape, cell motility regulation, and modulation of cell
cycle progressions13–16. Integrins also play a critical role in
angiogenesis, a complex biological process of new blood
vessel formation that involves local release of vascular
growth factors, interactions between adjacent and far-off
cells, the extracellular matrix, and modulation of bio-
chemical and other metabolic processes17. e integrins
αVβ3 and αVβ5 are the main receptor types involved in
angiogenesis18,19. One of the ways that tumours respond to
Integrins are transmembrane, obligate, and heterodi-
meric receptor proteins consisting of α and β chain
units1–4. On binding of a ligand to a specific recognition
site in the extracellular domains of the integrin receptor
αVβ3, identified as Arginine-Glycine-Asparagine (RGD)
recognition site, the multicomponent signalling com-
plexes are activated via specific protein–protein interac-
tions of the cytoplasmic tail of the receptor with various
signal transduction molecules. e action of mitogen-ac-
tivated protein kinase (MAPK) signalling cascade is one of
the earliest events following ligand–receptor binding5–8
.
rough the activation of diverse signalling pathways,
including MAPK, the integrins also regulate a variety
Address for Correspondence: Shaker A. Mousa, Ph.D., MBA, FACC, FACB, Professor of Pharmacology and Executive Vice President,
Chairman of the Pharmaceutical Research Institute at Albany, Albany College of Pharmacy and Health Sciences, 1 Discovery Drive
(Room 238), Rensselaer, NY 12144. E-mail: Shaker.Mousa@acphs.edu
(Received 09 September 2010; revised 04 January 2011; accepted 18 January 2011)
871

872 A. Bridoux et al.
damage induced by chemotherapy or other means is by
expressing and secreting biochemical factors that protect
the tumour vasculature, including integrin receptors20. In
addition, tumour growth, resistance to chemotherapy,
and metastasis, all involve integrin-mediated cellular and
extracellular signalling pathways, wherein the up-reg-
ulation of integrin αVβ3 expression serves as an escape
mechanism for tumours21,22. One of the ways to control
the tumour progression has been to control angiogenesis
via integrin antagonists23–27. e integrin antagonists that
function effectively as anti-angiogenic agents were iden-
tified earlier28. e arginine-glycine-aspartate (RGD),
cyclic RGD tripeptide (c-RGD) (Figure 1) and c-RGD
peptidomimetics have been shown to be effective inte-
grin αVβ3 antagonists as well29,30. e c-RGD exhibited
enhanced antiangiogenic and antitumour activities in
inhibit the activation of MAPK, an early signalling event
that is required for angiogenesis. ese results indicate
that the thyroid hormone interaction site is located at or
near the RGD recognition site on integrin αVβ3 receptor.
us, ligands that block this site would function both as
a thyroid and an αVβ3 integrin receptor antagonist. e
examples of blocking of iodothyronine binding to the
integrin αVβ3 receptor site by tetrac results in the growth
arrest of glioma cells46–51 and human breast cancer cells
in vitro18,52–55 that has led to infer that desirable neovas-
cularization could be promoted with local application
of thyroid hormone analogues during wound healing,
and the undesirable angiogenesis that supports tumour
growth, could be arrested with tetrac or its structurally
related derivatives.
Tetrac and tetrac analogs, through their activity at the
integrin αVβ3 receptor binding site for thyroid hormone,
inhibit angiogenesis and have been shown56 to modulate
proliferation of drug-sensitive and drug-resistant cells, e.g.,
human neuroblastoma SKN-SH cells, osteogenic sarcoma
SaOS2 cells, and human breast adenocarcinoma MCF7
cells. Because of its ability to reverse the poor response
of cancer cells to chemotherapy and reduce the prolifera-
tion of drug-resistant tumours, tetrac represents not only
a biological tool for modulating thyroid hormone action
but also a prospective anti-cancer agent that functions as
a dual action thyroid hormone and αVβ3 integrin receptor
antagonist. Herein, rational drug design was used to gen-
erate compounds that were capable of suppressing angio-
genesis and, thereby potentially inhibiting tumour growth
by suppressing undesirable angiogenesis in the tumour
environment and also function as thyroid antagonists. e
structural analogs of tetrac with bifunctional biological
activity as transactivated thyroid and integrin αVβ3 antag-
onists were designed by molecular modelling57,58 program
FlexX v3.1.2 BioSolveIT GmbH (Sankt Augustin, Germany)
at the thyroid and integrin sites identified by respective
ligands, i.e., tetrac and XT199 (Figure 1) in silico at the
radiation and chemotherapy led studies in tumours31,32
.
Results of recent studies33,34 suggest that in addition to
integrin antagonists, the thyroid antagonists could also
modulate angiogenesis. Several other reports35–38 have
described the effects of thyroid hormone analogs on inte-
grin αVβ3, thyroid hormone receptor, and membrane
Na+/H+ anti-porter ion pumps, revealing a complex rela-
tionship between thyroid hormone and nuclear events
that underlie important cellular and tissue processes,
including angiogenesis and tumour cell proliferation. e
thyroidhormoneanalogs,2-ammonium-3-[4-(4-hydroxy-
3-iodophenoxy)-3,5-diiodophenyl]-propionate (T3) and
2-ammonium-3-[4-(4-hydroxy-3,5-diiodophenoxy)-3-
,5-diiodophenyl] propionate (T4), have been shown to
induce angiogenesis39,40, while the metabolic degrada-
tion product of thyroid hormone, tetra-iodothyroacetic
acid (tetrac), inhibits angiogenesis in the chick cho-
rioallantoic membrane (CAM) and other angiogenesis
models (Figure 1)41–45. us, tetrac, which is a deami-
nated derivative of T4, serves as a thyroid antagonist by
blocking the actions of T3 and T4. Interestingly, c-RGD
and other small molecule c-RGD peptidomimetics also
I
I
COO−
NH3+
HO
I
I
COO−
NH3+
HO
I
I
HO
I
I
O
OH
O
O
O
I
I
I
T4
T3
Tetrac
Me
Me
O
H
N
O
HN
OH
O
NH
HN
HN
HN
H
N
HN
O
O
N
O
HN
NH2
NH
O
NH
N
O
O
O
OH
XT-199
Figure 1. Structures of T4, T3, Tetrac, XT199, and cyclic RGD tripeptide.
Journal of Enzyme Inhibition and Medicinal Chemistry
cyclic RGD peptide

Dual thyrointegrin inhibitors 873
macromolecular structure site. e DOCK and Autodock
that modulates and characterize ligand–integrin inter-
play at the atomic level were used wherein Autodock was
programmed to allow torsional flexibility in ligands to
optimize the ligand binding, whereas DOCK was pre-set
with a different set of algorithm to match common points
located within the binding site and incoming ligands.
e simulated docking of known ligand RGD, tetrac, and
its antagonistic analogue XT199 to integrin αVβ3 recep-
tor was performed using FlexX version 3.1.2 (BioSolveIT
GmbH). e three-dimensional crystal structure of the
extracellular segment of integrin αVβ3 in conjunction
with the known cyclic peptide ligand RGD was obtained
from the Protein Database (PDB: 1L5G).
integrin αVβ3 receptor site. e simulated in silico binding
of designed compounds and their virtual anti-angiogenic
binding profiles in live biological systems corroborated the
bifunctional nature of the RGD and c-RGD led receptor
sites59. Several theoretical compounds were generated in
the in silico molecular modelling using tetrac and the selec-
tive αVβ3 integrin antagonist XT199 as starting templates.
e compounds with similar binding characteristics and
nearly identical structures and physico-chemical proper-
ties were synthesized. e target compounds exhibited
pronounced anti-angiogenic activity in the CAM model of
angiogenesis in the bioevaluation process.
Methods
e active site was identified as a pocket consisting
of all amino acids residual atoms within 15 Å radius of
the cyclic peptide ligand. e peptide ligand was then
removed but three locations of manganese ions were
retained, as they participate in the test-template bind-
ings. e receptor was not further modified and treated
as a rigid entity during all docking experiments for all
test templates. Since the FlexX (v3.1.2) uses incremen-
tal construction, each test compound was before-hand
fragmented into smaller components and reconstructed
sequentially within receptor cavity for docking. e qual-
itative interactions for the first fragment were identified
followed by estimation for the target compound by join-
ing the remaining components in a step-wise manner.
After addition of each component, the new interactions
were defined, and scoring function was used to select the
best partial solution. is was repeated until reconstruc-
tion of the original ligand was fully achieved. Of the 30
different solutions generated by FlexX, the best one was
chosen based on FlexX score and best optimal position
within the active site.
All commercially available chemicals were used without
further purification. All solvents were dried, and mois-
ture-sensitive reactions were performed under dry nitro-
gen. Analytical thin layer chromatography (TLC) was
performed on precoated Kieselgel 60F254 plates Merck
(Gibbstown, NJ). Analytical Reverse Phase in Layer
Chromatography (RPTLC) was performed on precoated
Kieselgel plates (Merck). Compounds were visualised
by ultraviolet (UV) and/or with iodine. Column chro-
matography was performed with silica gel Kieselgel Si
60, 0.040–0.063 mm (Merck). Melting points were deter-
mined on an Electrothermal MEL-TEMP® melting point
apparatus and then on a omas Hoover Uni-melt capil-
lary melting point apparatus and were not corrected. e
structures of all compounds were supported by infrared
spectra recorded on a ermo Electron Nicolet Avatar™
330 Fourier transform infrared apparatus. UV spectra
were obtained from a Shimadzu UV-1650 (PC) UV–vis
1
spectrophotometer. H NMR data were obtained using
a Varian Inova™ 500 MHz spectrometer and referenced
to CDCl3 (δ= 7.27 ppm) or DMSO-d6 (δ= 2.50 ppm).
High-resolution mass spectral analyses were obtained
on either Applied Biosystems API4000™ LC/MS/MS or
Synthesis of compounds
[4-(4-Hydroxy-3, 5-diiodophenoxy)-3, 5-diiodophenyl] acetic
acid (6)
1
Applied Biosystems QSTAR® XL mass spectrometers. H
e title compound was prepared as described by
NMR chemical shifts were reported in parts per million
downfield from tetramethylsilane, J values were in Hertz
and the splitting patterns were designated as follows:
s, singlet; d, doublet; dd, doublet of doublets; t, triplet;
q, quartet; m, multiplet; b, broad. High pressure liquid
chromatography (HPLC) experiments were carried out at
a flow rate of 1.1 mL/min with a Waters™ 2695 HPLC appa-
ratus (120 vials) and a Phenomenex Luna® 5u NH2 100A
or a Waters µBondapak® C18 10 µm 125A column oper-
ated at 40°C and atmospheric pressure with UV detection
between 210 and 400 nm. Refractive index was measured
with an Abbe 3L Refractometer with bromonaphtalene as
the contact liquid. Combustion analyses were performed
by Intertek, Inc, Whitehouse, NJ. e yields quoted in this
study were recrystallization yields.
Wilkinson60.
Methyl [4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]
acetate (7)
[4-(4-Hydroxy-3, 5-diiodophenoxy)-3,5-diiodophenyl]
acetic acid (5.0 g, 6.7 mmol, 1.0 equiv.) was dissolved in
200 mL of dried MeOH and thionyl chloride (485 µL, 6.7
mmol, 1.0 equiv.) was added dropwise. e reaction was
refluxed for 2 days. After the completion of the reaction,
water was added (200 mL) and concentrated to ½ (or till
precipitate appeared) which was collected by filtration
and dried. e solid thus obtained was crystallized from
EtOH to yield the desired product as white powder. Yield:
100%; TLC: Rf = 0.81 (DCM); RPTLC: Rf = 0.49 (AcOH/H2O
90/10); mp = 163°C; IR (cm−1): 1719 (characteristic peak);
UV (DMSO): λmax 225 nm; HPLC (µBondapak C18): rt 3.2
Molecular modelling
1
Lock and key model57 was used to investigate ligand-
binding model behaviour at αVβ3 integrin receptor
minutes (MeOH/H2O 65/35); H NMR (CDCl3): 7.78 (s,
2H, ArH), 7.12 (s, 2H, ArH), 3.75 (s, 3H, CH3), 3.58 (s, 2H,
© 2011 Informa UK, Ltd.

874 A. Bridoux et al.
CH2); 13C NMR (CDCl3): 152.8, 150.2, 149.6, 142.3, 135.2,
126.9, 125.3, 91.0, 81.9, 52.3, 39.3; HRMS (APCI) m/z:
760.8904 [(M−H)−, 5%].
thus obtained was filtered, dried, and crystallized from
hot EtOH as light brown powder. Yields: 50%; TLC: Rf
0.77 (CH2Cl2); RPTLC: Rf 0.5 (AcOH); mp 267°C; IR (cm−1):
1735; UV (DMSO): λmax 256 nm; HPLC (µBondapak C18):
1
Methyl {4-[4-(2-chloroethoxy)-3,5-diiodophenoxy]-3,5-diiodo
phenyl}-acetate (8)
rt 3.85 minutes (MeOH/H2O 60/40); H NMR (CD3OD):
7.88 (s, 2H, ArH), 7.19 (s, 2H, ArH), 4.19 (t, J= 5.5 Hz,
2H, CH2), 3.95 (t, J= 5.5 Hz, 2H, CH2), 3.45 (s, 2H, CH2);
HRMS (APCI) m/z: 834.5700 [(M+H)+, 34%]; Elem. Anal.
(C17H14I4N2O5) C: calcd, 24.48; found, 24.57; H: calcd, 1.69;
found, 1.59.
Methyl [4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodo-
phenyl]acetate (7, 2.4 g, 3.2 mmol, 1.0 equiv.) was dis-
solved in anhydrous acetone, cesium carbonate (522 mg,
1.6 mmol, 0.5 equiv.), followed by 1-bromo-2- chloro-
ethane (275 µL, 3.2 mmol, 1.0 equiv.) were added drop-
wise and after stirring over 3 days at room temperature
(RT), the reaction medium was Celite-assisted filtered
and filtrate evaporated to give a yellow solid that was
crystallized from EtOH to give white product. Yields:
100%, TLC: Rf 0.76 (CH2Cl2); mp 119°C; IR (cm−1): 1732
(characteristic peak); UV (DMSO): max = 256 nm; HPLC
(µBondapak C18): rt 4.7 min (MeOH/H2O 70/30); 1H
NMR (CDCl3): 7.75 (s, 2H, ArH), 7.15 (s, 2H, ArH), 4.19
(t, J= 5.5 Hz, 2H, CH2), 3.91 (t, J= 5.5 Hz, 2H, CH2), 3.72
(s, 3H, CH3), 3.55 (s, 2H, CH2); HRMS (APCI) m/z: 823.6
[(M)+, 100%]; Elem. Anal. (C17H13ClO4I4) C: calcd, 24.77;
found, 24.98, H: calcd, 1.59; found, 1.52.
{4-[4-(3-Aminopropoxy)-3,5-diiodophenoxy]-3,5-diiodo
phenyl} acetic acid (11)
To a solution of methyl {4-[4-(2-chloroethoxy)-3,5-
diiodophenoxy]-3,5-diiodophenyl}acetate (8, 2.5 g, 3.3
mmol, 1.0 equiv.) in cyclopentyl methyl ether (20.0 mL),
Et3N (460 µL, 3.3 mmol, 1.0 equiv.), chloropropylam-
ine (427 mg, 3.3 mmol, 1.0 equiv.) was added and RM
was refluxed for 3 h, followed by solvent evaporation to
a minimum volume. Water was added to the residual
material, which was further treated with LiOH solution
(1.6 g, 66.0 mmol, 20.0 equiv.) to give a precipitate that
was collected after filtration and dried to give a powdery
solid. e obtained powder was washed under vacuuo
with warm MeCN followed by warm EtOH and dried to
give a solid that was further dissolved in anhydrous EtOH
(5.0 mL) and a solution of HCl 0.1 N (5.0 mL) was added
dropwise to give a precipitate, which after filtration was
obtained as a white powder (668 mg) to which NaOH
(280 mg, 6.6 mmol, 2.0 equiv.) as aqueous solution was
added under stirring, this solid suspension was filtered,
dried, and crystallised in EtOH to give a white powder.
Yield: 47%,mp 250°C; IR (cm−1): 1697; UV (DMSO):
λmax 259 nm; HPLC (µBondapak C18): rt 17.5 minutes
(ammonium acetate 25mM pH4/MeOH 50/50); 1H NMR
(Acetone-d6): 7.99 (s, 2H, ArH), 7.30 (s, 2H, ArH), 4.27 (t,
J= 5.5 Hz, 2H, CH2), 3.95 (t, J= 5.5 Hz, 2H, CH2), 3.75 (s,
2H, CH2), 3.57 (q, J= 14.0 Hz, J= 7.0 Hz, 2H, CH2); HRMS
(APCI) m/z: 832.6368 [(M−2H+Li+Na)+, 100%]; HRMS
(APCI) m/z: 764.7010 [(MCOOH+Li)−, 100]; ‘Elem. Anal.
(C17H14I4LiNO4) C: calcd, 25.18; found, 24.87; H: calcd,
1.74; found, 1.53.
Lithium [4-(4-{2-[(diaminomethylene) amino]-ethoxy}-3,5-
diiodophenoxy)-3,5-diiodophenyl] acetate (9)
To
a solution of methyl {4-[4-(2-chloroethoxy)-3,5-
diiodophenoxy]-3,5-diiodophenyl}acetate (8, 200mg,
0.2mmol,1.0equiv.)inanhydrousN,N’-dimethylformamide
(DMF) (5.0mL) was added sodium hydride (60% disper-
sion in mineral oil; 19mg, 0.5 mmol, 2.0 equiv.) in por-
tions followed by 10% guanidine hydrochloride (46mg, 0.5
mmol, 2.0 equiv.) in dry DMF (5.0mL) and RM stirred for
12h at RT. After the completion of reaction monitored by
TLC, the RM was treated with LiOH solution (115mg, 4.9
mmol, 20.0 equiv.) and precipitate obtained was filtered,
dried, and crystallized from EtOH to give white powder.
Yield: 50%, TLC: Rf 0.12 (EtOH); mp 133°C; IR (cm−1): 1736
(characteristic peak); UV (DMSO): λmax 256nm; HPLC
1
(µBondapak C18): rt 6.1 minutes (MeOH/H2O 65/35); H
NMR (CD3OD): 7.88 (s, 2H, ArH), 7.19 (s, 2H, ArH), 4.20 (t,
J=5.5 Hz, 2H, CH2), 3.91 (t, J=5.5 Hz, 2H, CH2), 3.45 (s, 2H,
CH2); 13C NMR (CDCl3): 206.9, 171.5, 153.2, 152.7, 151.8,
141.5, 136.7, 126.0, 92.0, 91.8, 72.9, 52.3, 43.8, 38.1; HRMS
(APCI) m/z: 841.7599 [(M+3H+Li)+, 100%].
2-Isopropyl anisole (13)
e title compound was prepared with minor variations
from the protocol described by Baxter et al.61. Yield: 60%,
1
(4-{4-[2-(Carbamoylamino)ethoxy]-3,5-diiodophenoxy}-3,5-
diiodophenyl) acetic acid (10)
yellow oil; TLC: Rf 0.65 (cyclohexane-EtOAc (95:5)); H
NMR (CDCl3): 7.32–7.30 (dd, J= 8.0 Hz, 2.5 Hz, 1H, ArH),
7.27–7.23 (dt, J= 9.5 Hz, 2.0 Hz, 1H, ArH), 7.04–7.01 (dt,
J= 7.5 Hz, 1.0 Hz, 1H, ArH), 6.94–6.92 (dd, J= 8.0 Hz, 1.0
Hz, 1H, ArH), 3.90 (s, 3H, CH3), 3.47–3.41 (m, 1H, CH),
1.30 (s, 3H, CH3), 1.20 (s, 3H, CH3); n[D]20 1.5069.
To a solution of methyl {4-[4-(2-chloroethoxy)-3,5-
diiodophenoxy]-3,5-diiodophenyl}acetate (8, 1.5 g,
1.8 mmol, 1.0 equiv.) in anhydrous DMF (15.0 mL) was
added sodium hydroxide (285 mg, 7.1 mmol, 4.0 equiv.)
by portions followed by urea solution (107 mg, 1.8 mmol,
1.0 equiv.) in anhydrous DMF (5.0 mL) and RM was
stirred for 10 h at RT. After the completion of reaction
monitored by TLC, the RM was treated with a solution
of LiOH (504 mg, 21.0 mmol, 12.0 equiv.) and precipitate
bis[4-methoxy-3-(propan-2-yl)phenyl]iodonium
tetrafluoroborate (14)
e title compound was prepared as described by
Yokoyama et al.62
Journal of Enzyme Inhibition and Medicinal Chemistry

Dual thyrointegrin inhibitors 875
and (11) of 1.0mg/mL and 1.2 µM were diluted to a final
concentration of 0.25mg/mL or 0.3 µM and the two con-
centrations for each compound were tested. XT199 (10.0
µM), a synthetic avβ3 antagonist developed at DuPont
Pharmaceuticals, and tetrac (1.0 µM) were tested as refer-
ences and compared with synthesized designed products.
Ten microliters of each test solution was added to the fil-
ter disks pretreated with FGF as described earlier. All the
compounds exhibited strong anti-angiogenic activity with
an average of 63–75% inhibition at 0.3 µM dose levels.
Methyl (3, 5-di-tert-butyl-4-hydroxyphenyl) acetate (16)
To 2,6-di-tert-butyl-4-hydroxyphenylacetic acid (15.0 g,
56.7 mmol; 1.0 equiv.) dissolved in 100mL of dried
MeOH was added thionyl chloride (4.1 mL; 56.7 mmol;
1.0 equiv.) in a dropwise manner and RM refluxed for
50 h. After completion of the reaction, monitored by TLC,
water was added (200 mL) and RM was concentrated to
one-third volume to give a precipitate that was collected
by filtration and extracted from water with the EtOAc
organic phase after evaporation and drying of the solvent
(total 20 g). e solid was crystallised in EtOH, filtered,
and washed with cold EtOH to give transparent crystals.
Yield: 100%; mp: 88°C; TLC: Rf 0.81 (CH2Cl2); RPTLC: Rf
0.78 (AcOH); IR (cm−1): 1722; UV (DMSO) λmax 276 nm;
HPLC (µBondapak C18): rt 4.0 min (MeOH/H2O 65/35);
1H NMR (CDCl3): 7.14 (s, 2H, ArH), 3.75 (s, 2H, CH2), 3.60
(s, 2H, CH2), 1.50 (s, 18H, CH3); 13C NMR (CDCl3) 172.9,
153.2, 136.2, 126.9, 125.3, 124.9, 53.3, 51.7, 51.0, 41.2, 40.1,
34.5, 32.4, 31.2, 29.9, 28.5; Elem. Anal. (C17H26O3) C: calcd,
73.34; found, 73.39; H: calcd, 9.41; found, 9.27.
Statistical methods
Statistical analyses were performed using ANOVA,
software Statview (Adept Scientific, Acton, MA) and
SigmaPlot (version 10.0, Systat software Inc., Chicago,
IL). e mean SEM of branch points and the concentra-
tion of haemoglobin from each experimental group were
compared with their respective controls. e statistical
significances was defined as P< 0.01. In the CAM studies,
the angiogenesis index for each treatment group were
compared with the corresponding control groups.
Biological assays
e compounds were tested in the CAM model43–45
wherein 10 day old fertilized chicken eggs, eight per treat-
ment (Sunrise Farms Inc., Catskill, NY) were incubated at
37°C with 55% relative humidity. In dark, with the help of
a candling lamp, a small hole was punctured in the area
of the shell covering egg-air sac with the help of a hypo-
dermic needle. A second hole was punctured on the wider
side of the egg above a vascular area of the embryonic
membrane. An artificial air sac was created below the sec-
ond hole by gently applying vacuum to the first hole using
a small rubber squeeze bulb. e vacuum was caused by
the separation of CAM from the egg shell. A small window
of approximately 1cm2 was cut in the egg shell with the
help of a mini drill. e underlying CAM was accessed
through this window. Filter disks were cut (filter paper
#1; Whatman Ltd (Maidstone, Kent, UK)) and soaked in
3.0mg/mL cortisone acetate solution (in 95% ethanol)
and air-dried under sterile conditions. To induce angio-
genesis, sterile filter disks were saturated with Fibroblast
Growth Factor (FGF) and placed on the CAM window. For
control, disks were saturated with PBS without calcium or
magnesium. e window was sealed with Highland Brand
transparent tape. After an hour, 10 µL of the compound
was applied topically to the FGF-stimulated CAM. After a
period of 48h, the CAM tissue beneath the filter disk was
harvested for evaluation of angiogenesis. e affected
CAM in petri dishes were examined using a SV6 stereomi-
croscope (Carl Zeiss MicroImaging, Inc.,(ornwood, NY)
and Media Cybernetics, Inc. (Bethesda, MD)) at 50× mag-
nification and the images were captured using a 3-CCD
colour digital video camera (Toshiba America Inc., New
York, NY). e images were analyzed using Image-Pro Plus
program (Media Cybernetics, Inc). e numbers of branch
points in samples within a circular region superimposed
on the area of the filter disk were counted for each treat-
ment condition. Stock solutions of compounds (9), (10),
results and discussion
e integrin αVβ3 receptor transduces signals through
intracellular activation, which induces conformational
changes in the targeted receptor that allows extracellu-
lar binding of ligand63. Cocrystallization studies64 have
revealed the details of the binding interactions of natural
ligands to integrin αVβ3 at the molecular levels. ese
studies reveal that the interface of the propeller region
of the αV subunit and the β3A region (the head of the
receptor protein) are key regions that participate in the
receptor–ligand interaction.
e structures of T4, T3, tetrac, XT199, and the cyclic
RGD tripeptide are shown in Figure 1. Analysis of the
integrin receptor cocrystallized with the cyclic RGD trip-
eptide provided the starting point for the design process,
and templates were based on the molecular character-
istics of the α and β units of the receptor and ligand–
receptor interaction areas (Supporting information).
Structural mapping has provided details of the molecular
architecture of the RGD tripeptide bound at the interface
of the propeller region of αV and β3A subunits in the
head region of integrin αVβ3 receptor65. Other model-
ling studies have confirmed that there is sufficient space
in the binding cavity for thyroid hormone constituents
to bind. Since thyroid hormone analogs (T3 and T4) and
tetrac are smaller in overall length than the cyclic RGD
tripeptide, they do not interact with the Arg214 recogni-
tion site in the propeller domain of αV subunit; most of
the interactions are confined to the βA subunit domain of
the αVβ3 receptor66.
e linear RGD tripeptide, length-modified RGD as
RGDF tetrapeptide and antagonist XT199 occupied a
shallow crevice between the αV propeller and β3A sub-
units located in the integrin upper region on the αV pro-
peller (head) and βA domains exclusively contacting Arg
© 2011 Informa UK, Ltd.

876 A. Bridoux et al.
and Asp amino acids (AA) residues while Asp chelated
the metal ion located at a distance of 2.6 Å in the receptor
cavity.
would have bifunctional activity as integrin inhibitors
and thyroid antagonists60,67–73
.
e in silico binding of the designed compounds and
superimposition of their binding orientations with pep-
tide ligands, tetrac, and XT199 antagonist again showed
the effective geometry of the compounds 9, 10, and 11 to
be shorter than those in peptides and XT199 due to the
perpendicular twisted geometry of the adjacent phenyl
rings linked and manoeuvrable through an ether bond.
e fact that the ligands and other molecules with known
integrin and thyroid antagonist action bind to different
amino acid residues in the same receptor cavity of αVβ3
integrin again suggested an interlinked role for the inte-
grin receptor in modulating angiogenesis and thyroid
action.
To design tetrac derivatives that would function as
true integrin αVβ3 antagonists, the structure of tetrac
was modified using molecular spacers to achieve the
required molecular length for in silico binding to the αV
and βA receptor-binding sites. e structural compari-
sons of non-peptide integrin antagonists that are active
in the nanomolar dose levels61,74–76 were also evaluated
using FlexX to derive compounds 9, 10, 11, and 17. e
functional groups that were altered/affixed to tetrac were
selected based on their ability to reach and interact with
the Arg214 recognition site in the propeller region of the
αV subunit. e distance between the carboxylic acid
and the amino functional groups was found to be critical
for in silico binding; thus, we designed molecules with
substitutions wherein the distance between these two
functional groups was close enough to retain binding
to the desired location in the receptor cavity and main-
tain integrin antagonist activity (Figure 3). e distance
between the amino acid and carboxylic acid groups was
in agreement with average interpoint atomic distance
defined according to a three-point pharmacophore pat-
ternforallincomingtemplatesattheintegrinreceptorsite
in conjunction with the observation of the ligand length
and size of the receptor cavity. e interpoint atomic dis-
tance was estimated by a three-point model delineating
15.00 Å for two sides and a maximum of 7.00 Å for the
third and extreme left arm of the triangle. Optimization
of structures that were close to the molecular length and
geometry of tetrac were carried out to obtain improved
e molecular details of the interaction of the cyclic
RGD tripeptide were used to dock the primary ligand
tetrac (Figure 2A and 2C), XT199 (Figure 2B) and newly
designed compounds (Supporting information) to the
integrin aVβ3 receptor. For each compound, the carbox-
ylic acid moiety was responsible for the ligand–receptor
interaction and was involved in metal ion binding (2.6 Å
from the metal ion), which occurs only in the active state
of the integrin receptor. For thyroid antagonist XT199,
the phenyl sulphonamide moiety stabilized the metal
binding through an interaction with Arg214, whereas tet-
rac interacted with the manganese metal ion and was
stabilized by electrostatic interaction with the vicinal
Asn215 together with hydrogen bonding with the OH of
phenol of Tyr178. Of all the compounds examined, XT199
was the only entity with sufficient molecular length to
interact with the Arg214 recognition site in the propeller
region of αV, thus the constituting imidazolyl group in
XT199 was found as a best fit between Asp150 and Asp218
amino acid residues within the binding pocket and pav-
ing the way for lengthening of the ligand beyond 13.51
Å as in the case of RGDF. e superimposition of struc-
tures of XT199 and the RGD tripeptide showed that the
spatial orientation of the entire XT199 molecule was
nearly identical to the structure of the RGD tripeptide
(Figure 3). e RGDF peptide extends the length of the
RGD tripeptide beyond 14.48 Å. e lengths of RGDF and
compounds 9, 10, and 11 were in the effective binding
range lengths of 15.00 Å for the adopted conformations
during binding. e adopted geometry of the templates
(tetrac and XT199) and the designed compounds 9,
10, and 11 contributed positively in binding to the αV/
βA region of the integrin receptor. e role of different
amino acids in providing hydrogen bonding and inter-
actions within the propeller region of αV subunit for
larger length molecules was clearly different than for
shorter length molecules, that is, tetrac. is was a fur-
ther indication of a dual role of the integrin receptor, via
both integrin ligand and thyroid binding domains, as a
thyrointegrin receptor with transactivation capabilities,
and a strong indication that the designed compounds
A
B
C
Figure 2. (A) Cyclic RGD tripeptide (white) bound at the interface of αV (yellow) and β3A (purple). e blue ball represents a manganese
ion; yellow doted lines represent H-bond interactions (low visibility here). (B) XT199 (green) bound at the interface of the αV and β3A
subunits of the αVβ3 integrin; (C) tetrac (green) bound at the interface of the αV and β3A subunits.
Journal of Enzyme Inhibition and Medicinal Chemistry

Dual thyrointegrin inhibitors 877
A
B
C
D
E
F
Figure 3. (A) Superimposition of the RGDF tetrapeptide and compound 9; (B) superimposition of RGDF and XT199; (C) superimposition
of XT199 (green) and compound 9 (yellow) revealed slight geometric agreement at CO2H terminal of compound (9) in binding mode; (D)
superposition of RGD and compound (9) revealed slight geometric agreement in binding mode only at CO2H terminal of compound (9);
(E) the RGD (white/blue) tripeptide superimposed at common coordinates with XT199 (green) in the bound state at the integrin receptor;
(F) superposition of XT199, RGDF and tetrac.
alternatives. On the basis of effective in silico binding of
the tetrac-derived molecules in the receptor cavity that
satisfied all the requirements of thyroid and integrin sites
interactions by binding at both the αV and βA domains
due to its effective molecular length and physico-chemi-
cal characteristics, both iodinated and deiodinated ana-
logs have been generated. e fact that both the thyroid
antagonist ligand and integrin receptor-binding (block-
ing) compounds contained geometric and electronic
specifications for binding prompted us also to consider
adding high-to-moderate and low electron-rich molecu-
lar chain substitutions to tetrac. Due to the presence of
the carboxylic acid moiety, responsible for the thyroid
antagonist activity-related bindings as observed in the
ongoing simulations and which was shown to be crucial
for initial receptor and metal binding in the undergoing
molecular simulations the OH of phenol of tetrac was
selected as the target site for converting tetrac to an inte-
grin antagonist. In line with the observation of binding of
different peptides and XT199 ligands in silico, the other
side (carboxyl end) of the molecule was not modified so
as to preserve partial antithyroidal activity.
Estimates of polarizability, refractive index, log P,
and molecular volume as well as surface area approxi-
mations of tetrac, XT199, cyclic RGD, and compounds
9, 10, 11, and 17 were carried out using Hyperchem
(v7.5, Hypercube Inc., Gainesville, FL). Estimated values
were nearly identical and in proportion to the designed
molecules in accordance with template characteris-
tics (Supporting Information, Table 1). Electrostatic
potential, charge density, and distribution in molecules
exhibited identical patterns for tetrac, XT 199, RGD, and
compounds 9, 10, and 11, as depicted by 2D contour
plots and 3D renderings (Figures 4 and 5 and Supporting
Information). Estimates of molecular characteristics and
analysis of ligand binding to the αVβ3 integrin receptor
binding site suggested that the biological activity of deio-
dinated compound 17 may be in the same range of that of
the other compounds (9, 10, and 11) with only limitation
in the geometry of phenyl ring that is hindered from tak-
ing a perpendicularly twisted orientation at the receptor
site in compound (17).
Tetrac (6) was synthesized in sufficient quantities
for subsequent modification as per synthetic Scheme 1.
Appropriate starting material (1) was obtained in a two-
step process. Commercially available p-hydroxy pheny-
lacetic acid was first converted into a dinitro-ester. e
nitration reaction, despite the hydroxyl group being
favoured for oxidation, occurred in dilute HNO3 in AcOH
with minimum side reactions, contrary to the classical
oxidation approach77 using H2SO4, with a moderate yield.
Acid-catalyzed esterification of the resulting compound
gave the desired intermediate (72% yield), which was
then reacted with para-toluenesulphonyl chloride and
methoxyphenol in pyridine to give the diphenylether
derivative (2) at a yield of 75%. Catalytic reduction using
H2, 5% Pd and CaCO3 in ethanol as solvent gave product
(3), which could be tetrazotized in ethanol at 0°C. e
in situ tetrazolium salt was reacted with NaI to give the
3,5-diiodo derivative (4) with a 69% yield78. Given the
difficulties encountered with aromatic ether hydrolysis
of substrate (4), complete acidolysis under reflux con-
ditions using hydroiodic acid (HI) as a strong cleaving
agent was carried out for facile removal of both the ester
© 2011 Informa UK, Ltd.

878 A. Bridoux et al.
and O-methyl groups from product79,80 (4). Acetic acid
was used to avoid decomposition of HI into I2 by H2SO4,
thereby avoiding the generation of unwanted side prod-
ucts in the reaction medium78. Tetrac target molecule (6)
was obtained from the di-iodo compound (5) by reaction
with iodine. In the reaction, iodo-deprotonation was car-
ried out using I2 in methanolic-NH3 with near quantitative
yields. Regioselective iodination of the ring in compound
(5) was facilitated by the transformation of the methyl
ether group of compound (4) into an alcohol in substrate
(5). is was carried out to allow activation of the ortho
position in compound (5) through inductive and reso-
nance effects. Tetrac (6) was then esterified in thionyl
chloride-MeOH under reflux for 2 days for >60% yield.
e hydroxyl group in the resulting compound (7) was
substituted by 1-chloro-2-bromoethylene C2H4BrCl, and
the obtained key intermediate (8) was then transformed
to give the thyrointegrin antagonist compounds (9), (10)
Table 1. Inhibition of angiogenesis by XT199.
Series no.
Treatment
PBS
Branch pointsa % Inhibitionb
1
2
3
74
8
FGF
183 11*
FGF+XT199
89 10
86 9
aData represent the means SEM, n=8.
bAs compared with FGF alone.
*P<0.001; for series no. 2, dose level=1.0 µg/ml; for Series no. 3,
dose level=0.5µg/ml.
A
B
C
D
E
Figure 4. Two-dimensional contour plots of electrostatic potential for XT199 (A), tetrac (B) and compounds (9) (C), (10) (D) and (11) (E).
A B
C
D
E
Figure 5. Two-dimensional contour plots of charge density distribution for XT199 (A), tetrac (B) and compounds (9) (C), (10) (D) and (11) (E).
Journal of Enzyme Inhibition and Medicinal Chemistry

Dual thyrointegrin inhibitors 879
1. pCH3C6H4SO2Cl (1.0 equiv.)
C5H5N (4.0equiv.)
O2N
O
Et
O
O2N
HO
O2N
O
Et
H2(g), 5% Pd-CaCO3 cat.
O
95–100°C,30min.
O
CH3CH2OH
Quant.
2. pCH3OC6H4OH
125°C, 2hrs, N2(g)
1
2
O2N
75%
MeO
1. NaNO2 (2.0 equiv.)
H2SO4/CH3CO2H (v/v)
0°C, 1 hr 45 min
O
Et
I
I
O
Et
O
H2N
O
O
O
2. NaI (4.0 equiv.) + I2 (1.0 equiv.)
urea (2.0 equiv.)
H2O/CHCl3 (3/1)
r.t., 1hr
4
3
H2N
MeO
MeO
69%
I
O
OH
I2 excess
NH3 excess
HI (excess)
Red P (1.0 equiv.)
O
CH3CO2H, reflux, 1 hr
CH3OH
I
5
95%
91%
HO
I
O
I
I
O
OH
OMe
C2H4BrCl (1.0 equiv.)
Cs2CO3 (0.5 equiv.)
O
O
SOCl2 (1.0 equiv.)
7
6
CH3OH, reflux,
2 d, N2(g)
I
I
I
C3H6O, r.t., 3 d, N2 (g)
Quant.
HO
I
HO
I
59%
I
O
1. CH5N3, HCl (2.0 equiv.)
NaH 60% (2.0 equiv.)
(CH3)2 NCHO
OLi
OLi
O
I
r.t.,1 day, N2 (g)
9
I
I
I
O
2. LiOH (20 equiv.), H2O
OMe
O
50%
HN
O
I
HN
NH2
8
I
I
I
O
1. NH2CONH2 (1.2 equiv.)
NaOH (4.0 equiv.)
(CH3)2NCHO
O
O
I
Cl
r.t.,1 day, N2 (g)
10
I
I
2. LiOH (20 equiv.), H2O
O
50%
HN
O
NH2
I
O
1. NH2C3H6Cl, HCl (1.0 equiv.)
N(CH2CH3)3 (1.0 equiv.)
C5H9OCH3
I
O
OH
OMe
O
I
O
I
reflux, 3hrs, N2 (g)
11
I
I
7
I
I
2. LiOH (20 equiv.), H2O
3. HCl 0.1N
O
HO
50%
H2N
Scheme 1. Preparation of compounds (9), (10) and (11).
and (11). Compound (8) was reacted with guanidine and
urea to yield compounds (9) and (10), respectively82,83
LiOH, while compound (11) was reacted with aqueous
NaOH to produce the corresponding salts.
.
Compound (11) was obtained in 47% yield from interme-
diate (7) by refluxing with chloro-propylamine in cyclo-
pentyl-methyl ether as the solvent. Finally, compounds
(9) and (10) were reacted with an excess of aqueous
e critical step in the synthesis of deiodinated prod-
uct (17) (Supporting Information) was the condensation
of compounds (14) and (16). e bis-aryliodonium tri-
flate (14) was prepared from compound (13) according
© 2011 Informa UK, Ltd.

880 A. Bridoux et al.
requirements for selective binding with integrin αVβ3
were set and then validated compounds were synthe-
sized. e biological activities of these tetrac analogues
also indicated that tetrac interaction with αVβ3 integrin, a
non-genomic receptor, is not dependent on free phenolic
OH groups in the thyroid components.
Table 2. Inhibition of angiogenesis by compounds 9, 10, and 11.
Series no.
Treatment
PBS
Branch pointsa
% Inhibitionb
1
2
3
4
5
6
7
8
9
41
5
—
FGF
—
108 8**
57 10
FGF + tetrac
FGF + 9
FGF + 9
FGF + 10
FGF + 10
FGF + 11
FGF + 11
85 14
64 13
75 16
77 13
74 13
88 11
65
8
58 10
56
58
55
66
9
9
8
2
Declaration of interest
e authors report Vascular Vision Pharmaceuticals Co
(VVP Co.) for financial support.
63
3
aData represent means SEM, n=8.
bAs compared with FGF alone.
references
**P<0.001; for series no. 2, dose level=2.0 µg/mL; for series
nos. 3, 4, 6 and 8, dose=1.0 µg/mL; for series nos. 5, 7 and 9,
dose=0.25 µg/mL.
1. Giancotti FG, Ruoslahti E. Integrin signaling. Science
1999;285:1028–1032.
2. Schwartz MA. Integrin signaling revisited. Trends Cell Biol
2001;11:466–470.
3. Hynes RO. Integrins: bidirectional, allosteric signaling machines.
Cell 2002;110:673–687.
4. Kinbara K, Goldfinger LE, Hansen M, Chou FL, Ginsberg MH.
Ras GTPases: integrins’ friends or foes? Nat Rev Mol Cell Biol
2003;4:767–776.
5. Boudreau NJ, Jones PL. Extracellular matrix and integrin signalling:
the shape of things to come. Biochem J 1999;339 (Pt 3):481–488.
6. Brakebusch C, Fässler R. e integrin-actin connection, an eternal
love affair. Embo J 2003;22:2324–2333.
to the aryliodonium formation method described by
Yokoyama et al.62. Compound (13) was prepared on both
small and high scales by the refluxing methyletherifica-
tion procedure of 2-isopropylphenol (12) using cesium
carbonate as a proton trap. In parallel, compound (16)
was obtained by the methylesterification of commercially
available 3,5-di-tert-butyl-4-hydroxybenzoic acid (15).
However, likely due to steric hindrance, the coupling of
compounds (14) and (16) by tetrafluoroborate treatment
failed to yield the desired product.
Compounds (9), (10), and (11) exhibited angiogenesis
antagonist activity in the CAM model system, with inhi-
bition rates of 63–75% at a dose of 0.25 µg/mL compared
with thyroid antagonist XT 199 showing 86% angiogenesis
inhibition in same test models at somewhat higher dose
levels (Table 2). is dose is not high, considering the
designed dual action potency of these characteristically
intermediate derivatives of tetrac and XT199. However,
these putative dual-action thyrointegrin modulators pro-
vide an important tool and foundation for further studies
into the extra-cellular biochemical cascades initiated
by the thyroid hormone receptor and integrin receptor
binding in terms of the signalling pathways activated and
the resultant induction of angiogenesis in cancer cells.
It is important to note that the interactions predicted by
FlexX adhered to strict requirements defined by the pat-
terns exhibited by the αVβ3 antagonists.
7. Burridge K, Fath K, Kelly T, Nuckolls G, Turner C. Focal adhesions:
transmembrane junctions between the extracellular matrix and
the cytoskeleton. Annu Rev Cell Biol 1988;4:487–525.
8. Ingber DE. Extracellular matrix as
a solid-state regulator in
angiogenesis: identification of new targets for anti-cancer therapy.
Semin Cancer Biol 1992;3:57–63.
9. Sun HW, Li CJ, Chen HQ, Lin HL, Lv HX, Zhang Y et al. Involvement
of integrins, MAPK, and NF-kappaB in regulation of the shear
stress-induced MMP-9 expression in endothelial cells. Biochem
Biophys Res Commun 2007;353:152–158.
10. Brooks PC, Montgomery AM, Rosenfeld M, Reisfeld RA, Hu T,
Klier G et al. Integrin alpha v beta 3 antagonists promote tumor
regression by inducing apoptosis of angiogenic blood vessels. Cell
1994;79:1157–1164.
11. Lewis JM, Truong TN, Schwartz MA. Integrins regulate the
apoptotic response to DNA damage through modulation of p53.
Proc Natl Acad Sci USA 2002;99:3627–3632.
12. Wang XQ, Sun P, Paller AS. Inhibition of integrin-linked kinase/
protein kinase B/Akt signaling: mechanism for ganglioside-
induced apoptosis. J Biol Chem 2001;276:44504–44511.
13. GiancottiFG.Integrinsignaling:specificityandcontrolofcellsurvival
and cell cycle progression. Curr Opin Cell Biol 1997;9:691–700.
14. Zheng DQ, Fornaro M, Bofetiado CJ, Tallini G, Bosari S, Languino
LR. Modulation of cell proliferation by the integrin cytoplasmic
domain. Kidney Int 1997;51:1434–1440.
15. Watt FM. Role of integrins in regulating epidermal adhesion,
growth and differentiation. Embo J 2002;21:3919–3926.
16. Yamada KM, Even-Ram S. Integrin regulation of growth factor
receptors. Nat Cell Biol 2002;4:E75–E76.
17. Ruoslahti E, Engvall E. Integrins and vascular extracellular matrix
assembly. J Clin Invest 1997;99:1149–1152.
18. Mousa S A, Davis F B, Davis P J. Hormone-integrin cross talk and
angiogenesis modulation. Immunol Endocr Metab Agents Med
Chem 2009;9:75–83.
conclusion
Novel dual thyrointegrin modulators were designed and
synthesized based on the molecular structures of the thy-
roid and integrin antagonists. ese newly designed com-
poundshaveenhancedtheunderstandingofthebiological
activity of tetrac, a molecule that inhibits tumour growth
and metastatic angiogenesis and counteracts resistance
to chemotherapy in numerous biological models. Despite
the fact that tetrac does not fulfil the requirements for a
selective integrin antagonist, it has been observed experi-
mentally to inhibit angiogenesis stimulated by FGF, VEGF,
T4, and T3. In the current design process, strict molecular
19. Gottschalk KE, Günther R, Kessler H. A three-state mechanism of
integrin activation and signal transduction for integrin alpha(v)
beta(3). Chembiochem 2002;3:470–473.
20. Truong T, Sun G, Doorly M, Wang JY, Schwartz MA. Modulation
of DNA damage-induced apoptosis by cell adhesion is
Journal of Enzyme Inhibition and Medicinal Chemistry

Dual thyrointegrin inhibitors 881
independently mediated by p53 and c-Abl. Proc Natl Acad Sci USA
2003;100:10281–10286.
42. Rebbaa A, Chu F, Davis FB, Davis PJ, Mousa SA. Novel function
of the thyroid hormone analog tetraiodothyroacetic acid:
a
21. Puigvert JC, Huveneers S, Fredriksson L, op het Veld M, van de
Water B, Danen EH. Cross-talk between integrins and oncogenes
modulates chemosensitivity. Mol Pharmacol 2009;75:947–955.
22. Brozovic A, Majhen D, Roje V, Mikac N, Jakopec S, Fritz G et al.
alpha(v)beta(3) Integrin-mediated drug resistance in human
laryngeal carcinoma cells is caused by glutathione-dependent
elimination of drug-induced reactive oxidative species. Mol
Pharmacol 2008;74:298–306.
cancer chemosensitizing and anti-cancer agent. Angiogenesis
2008;11:269–276.
43. Maas JW, Le Noble FA, Dunselman GA, de Goeij AF, Struyker
Boudier HA, Evers JL. e chick embryo chorioallantoic membrane
as a model to investigate the angiogenic properties of human
endometrium. Gynecol Obstet Invest 1999;48:108–112.
44. Akhtar N, Dickerson EB, Auerbach R. e sponge/Matrigel
angiogenesis assay. Angiogenesis 2002;5:75–80.
23. Luna J, Tobe T, Mousa SA, Reilly TM, Campochiaro PA. Antagonists
of integrin alpha v beta 3 inhibit retinal neovascularization in a
murine model. Lab Invest 1996;75:563–573.
45. Baffour R, Garb JL, Kaufman J, Berman J, Rhee SW, Norris MA et al.
Angiogenic therapy for the chronically ischemic lower limb in a
rabbit model. J Surg Res 2000;93:219–229.
24. Mousa SA, Mousa AS. Angiogenesis inhibitors: current & future
directions. Curr Pharm Des 2004;10:1–9.
25. Wary KK. Molecular targets for anti-angiogenic therapy. Curr Opin
Mol er 2004;6:54–70.
46. Mousa SA. Cell adhesion molecules: potential therapeutic &
diagnostic implications. Mol Biotechnol 2008;38:33–40.
47. Gadbois DM, Bradbury EM, Lehnert BE. Control of radiation-
induced G1 arrest by cell-substratum interactions. Cancer Res
1997;57:1151–1156.
48. Wild-Bode C, Weller M, Rimner A, Dichgans J, Wick W. Sublethal
irradiation promotes migration and invasiveness of glioma cells:
implications for radiotherapy of human glioblastoma. Cancer Res
2001;61:2744–2750.
26. Mousa SA. Anti-integrins as a potential therapeutic target in
angiogenesis. Expert Opin er Patents 1999;9:1237–1248.
27. Mousa SA. Angiogenesis Inhibitors and Stimulators: Potential
erapeutic Implications. Texas: Landes Bioscience, 2000.
28. Giannis A, Rübsam F. Integrin antagonists and other low molecular
weight compounds as inhibitors of angiogenesis: new drugs in
cancer therapy. Angew Chem Int Ed Engl 1997;36:588–590.
29. Zitzmann S, Ehemann V, Schwab M. Arginine-glycine-aspartic
acid (RGD)-peptide binds to both tumor and tumor-endothelial
cells in vivo. Cancer Res 2002;62:5139–5143.
49. Lisiak E, Cordes N. Integrins and the cellular radiation response.
Cancer er 2004;2:271–278.
50. Garcia-Barros M, Paris F, Cordon-Cardo C, Lyden D,
Rafii S, Haimovitz-Friedman
A et al. Tumor response to
radiotherapy regulated by endothelial cell apoptosis. Science
2003;300:1155–1159.
30. Weber WA, Haubner R, Vabuliene E, Kuhnast B, Wester HJ,
Schwaiger M. Tumor angiogenesis targeting using imaging agents.
Q J Nucl Med 2001;45:179–182.
51. Dimitrijevic-Bussod M, Balzaretti-Maggi VS, Gadbois DM.
Extracellular matrix and radiation G1 cell cycle arrest in human
fibroblasts. Cancer Res 1999;59:4843–4847.
52. Sloan EK, Pouliot N, Stanley KL, Chia J, Moseley JM, Hards DK
et al. Tumor-specific expression of alphavbeta3 integrin promotes
spontaneous metastasis of breast cancer to bone. Breast Cancer
Res 2006;8:R20.
53. Felding-Habermann B, O’Toole TE, Smith JW, Fransvea E, Ruggeri
ZM, Ginsberg MH et al. Integrin activation controls metastasis in
human breast cancer. Proc Natl Acad Sci USA 2001;98:1853–1858.
54. Cordes N, Meineke V. Cell adhesion-mediated radioresistance
(CAM-RR). Extracellular matrix-dependent improvement of cell
survival in human tumor and normal cells in vitro. Strahlenther
Onkol 2003;179:337–344.
55. Hallahan D, Geng L, Qu S, Scarfone C, Giorgio T, Donnelly E et al.
Integrin-mediated targeting of drug delivery to irradiated tumor
blood vessels. Cancer Cell 2003;3:63–74.
31. Urbinati C, Mitola S, Tanghetti E, Kumar C, Waltenberger J, Ribatti
D et al. Integrin alphavbeta3 as a target for blocking HIV-1 Tat-
induced endothelial cell activation in vitro and angiogenesis in
vivo. Arterioscler romb Vasc Biol 2005;25:2315–2320.
32. Temming K, Schiffelers RM, Molema G, Kok RJ. RGD-based
strategies for selective delivery of therapeutics and imaging agents
to the tumour vasculature. Drug Resist Updat 2005;8:381–402.
33. Ramsden JD. Angiogenesis in the thyroid gland. j Endocrinol
2000;166:475–480.
34. Mousa SA, Bergh JJ, Dier E, Rebbaa A, O’Connor LJ, Yalcin M et al.
Tetraiodothyroacetic acid, a small molecule integrin ligand, blocks
angiogenesis induced by vascular endothelial growth factor and
basic fibroblast growth factor. Angiogenesis 2008;11:183–190.
35. Davis PJ, Davis FB, Lin HY, Mousa SA, Zhou M, Luidens MK.
Translational implications of nongenomic actions of thyroid
hormone initiated at its integrin receptor. Am J Physiol Endocrinol
Metab 2009;297:E1238–E1246.
36. Lei J, Nowbar S, Mariash CN, Ingbar DH. yroid hormone
stimulates Na-K-ATPase activity and its plasma membrane
insertion in rat alveolar epithelial cells. Am J Physiol Lung Cell Mol
Physiol 2003;285:L762–L772.
37. Lei J, Mariash CN, Bhargava M, Wattenberg EV, Ingbar DH. T3
increases Na-K-ATPase activity via a MAPK/ERK1/2-dependent
pathway in rat adult alveolar epithelial cells. Am J Physiol Lung
Cell Mol Physiol 2008;294:L749–L754.
56. Damiano JS, Cress AE, Hazlehurst LA, Shtil AA, Dalton WS. Cell
adhesion mediated drug resistance (CAM-DR): role of integrins
and resistance to apoptosis in human myeloma cell lines. Blood
1999;93:1658–1667.
57. Gardiner EJ, Willett P, Artymiuk PJ. Graph-theoretic techniques
for macromolecular docking.
2000;40:273–279.
J
Chem Inf Comput Sci
58. Moitessier N, Henry C, Maigret B, Chapleur Y. Combining
pharmacophore search, automated docking, and molecular
dynamics simulations as a novel strategy for flexible docking.
Proof of concept: docking of arginine-glycine-aspartic acid-
like compounds into the alphavbeta3 binding site. j Med Chem
2004;47:4178–4187.
38. Scarlett A, Parsons MP, Hanson PL, Sidhu KK, Milligan TP, Burrin
JM. yroid hormone stimulation of extracellular signal-regulated
kinase and cell proliferation in human osteoblast-like cells is
initiated at integrin alphaVbeta3. J Endocrinol 2008;196:509–517.
39. Mousa SA, Davis FB, Mohamed S, Davis PJ, Feng X. Pro-
angiogenesis action of thyroid hormone and analogs in a three-
dimensional in vitro microvascular endothelial sprouting model.
Int Angiol 2006;25:407–413.
59. Cody V, Davis PJ, Davis FB. Molecular modeling of the thyroid
hormone interactions with alpha
2007;72:165–170.
v beta 3 integrin. Steroids
60. Wilkinson JH. Synthesis of some possible metabolites of thyroxine
and triiodothyronine. Biochem j 1956;63:601–605.
40. Mousa SA, O’Connor L, Davis FB, Davis PJ. Proangiogenesis action
of the thyroid hormone analog 3,5-diiodothyropropionic acid
(DITPA) is initiated at the cell surface and is integrin mediated.
Endocrinology 2006;147:1602–1607.
41. Yalcin M, Lansing L, Bharali D, Bridoux A, Davis FB, Lin HY, Rebbaa
A, Davis PJ, Mousa SA. 79th Annual Meeting of e American
yroid Association. Chicago, IL: Abstract 110, 2008.
61. Baxter JD, Goede P, Apriletti JW, West BL, Feng W, Mellstrom K
et al. Structure-based design and synthesis of a thyroid hormone
receptor (TR) antagonist. Endocrinology 2002;143:517–524.
62. Yokoyama N, Walker GN, Main AJ, Stanton JL, Morrissey MM,
Boehm C et al. Synthesis and structure-activity relationships of
oxamic acid and acetic acid derivatives related to L-thyronine. J
Med Chem 1995;38:695–707.
© 2011 Informa UK, Ltd.

882 A. Bridoux et al.
63. Xiong JP, Stehle T, Goodman SL, Arnaout MA. New insights into the
structural basis of integrin activation. Blood 2003;102:1155–1159.
64. Xiong JP, Stehle T, Zhang R, Joachimiak A, Frech M, Goodman
SL et al. Crystal structure of the extracellular segment of integrin
alpha Vbeta3 in complex with an Arg-Gly-Asp ligand. Science
2002;296:151–155.
factor-dependent and is initiated at the cell surface. Circ Res
2004;94:1500–1506.
73. Bergh JJ, Lin HY, Lansing L, Mohamed SN, Davis FB, Mousa S
et al. Integrin alphaVbeta3 contains a cell surface receptor site for
thyroid hormone that is linked to activation of mitogen-activated
protein kinase and induction of angiogenesis. Endocrinology
2005;146:2864–2871.
74. Davis PJ, Davis FB, Cody V. Membrane receptors mediating thyroid
hormone action. Trends Endocrinol Metab 2005;16:429–435.
75. Tucker GC. Inhibitors of integrins. Curr Opin Pharmacol
2002;2:394–402.
65. Marinelli L, Lavecchia A, Gottschalk KE, Novellino E, Kessler H.
Docking studies on alphavbeta3 integrin ligands: pharmacophore
refinement and implications for drug design.
2003;46:4393–4404.
J Med Chem
66. Henry C, Moitessier N, Chapleur Y. Vitronectin receptor alpha(V)
beta(3) integrin antagonists: chemical and structural requirements
for activity and selectivity. Mini Rev Med Chem 2002;2:531–542.
67. Mousa SA. alphav Vitronectin receptors in vascular-mediated
disorders. Med Res Rev 2003;23:190–199.
68. Mousa SA. Anti-integrin as novel drug-discovery targets: potential
therapeutic and diagnostic implications. Curr Opin Chem Biol
2002;6:534–541.
69. Mousa SA. Angiogenesis promoters and inhibitors: potential
therapeutic implications. Mol Med Today 1996;2:140–142.
70. Kerr JS, Slee AM, Mousa SA. e alpha v integrin antagonists as
novel anticancer agents: an update. Expert Opin Investig Drugs
2002;11:1765–1774.
71. Bridoux A, Cui H, Dyskin E, Yalcin M, Mousa SA. Semisynthesis
and pharmacological activities of Tetrac analogs: angiogenesis
modulators. Bioorg Med Chem Lett 2009;19:3259–3263.
72. Davis FB, Mousa SA, O’Connor L, Mohamed S, Lin HY, Cao HJ
et al. Proangiogenic action of thyroid hormone is fibroblast growth
76. Tucker GC. Alpha v integrin inhibitors and cancer therapy. Curr
Opin Investig Drugs 2003;4:722–731.
77. Crater WdeC. Nitration. Ind Eng Chem 1951;43:1987–1990.
78. Clugston M, Flemming R (eds). Advanced Chemistry (Advanced
Science). USA: Oxford University Press, 2000, p. 326.
79. Skinner HF. Metamphetamine synthesis via hydriodic acid/
red phosphorus reduction of ephedrine. Forensic Sci Int
1990;48:123–134.
80. McCullagh JV, Daggett KA. Synthesis of triarylmethane and
xanthenes dyes using electrophilic aromatic substitution reactions.
J Chem Educ 2007;84:1799.
81. Vaidyanathan G, Shankar S, Affleck DJ, Alston K, Norman J, Welsh
P et al. Meta-iodobenzylguanidine derivatives containing a second
guanidine moiety. Bioorg Med Chem 2004;12:1649–1656.
82. Sham HL, Zhao C, Stewart KD, Betebenner DA, Lin S, Park CH
et al. A novel, picomolar inhibitor of human immunodeficiency
virus type 1 protease. J Med Chem 1996;39:392–397.
Journal of Enzyme Inhibition and Medicinal Chemistry