Synthesis of new analogs of tetraiodothyroacetic acid (tetrac) as novel angiogenesis inhibitors for treatment of cancer

Article abstract of DOI:10.1016/j.bmcl.2018.02.045

In the angiogenesis process, integrins, which are members of a family of cell surface transmembrane receptors, play a critical role particularly in blood vessel formation and the local release of vascular growth factors. Thyroid hormones such as L-thyroxine (T₄) and 3,5,3′-triiodo-L-thyronine (T₃) promote angiogenesis and tumor cell proliferation via integrin αvβ3 receptor. At or near an arginine-glycine-aspartate (RGD) recognition site on the binding pocket of integrin α_vβ₃, tetraiodothyroacetic acid (tetrac, a deaminated derivative of T₄) is a thyrointegrin receptor antagonist and blocks the actions of T₃ and T₄ as well as different growth factors-mediated angiogenesis. In this study, we synthesized novel tetrac analogs by modifying the phenolic moiety of tetrac and tested them for their anti-angiogenesis activity using a Matrigel plug model for angiogenesis in mice. Pharmacological activity results showed that tetrac can accommodate numerous modifications and maintain its anti-angiogenesis activity.

Full text of DOI:10.1016/j.bmcl.2018.02.045

Accepted Manuscript
Synthesis of new analogs of tetraiodothyroacetic acid (tetrac) as novel angio-
genesis inhibitors for treatment of cancer
Mehdi Rajabi, Murat Yalcin, Shaker A. Mousa
PII:
S0960-894X(18)30150-1
https://doi.org/10.1016/j.bmcl.2018.02.045
BMCL 25641
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 November 2017
23 February 2018
24 February 2018
Please cite this article as: Rajabi, M., Yalcin, M., Mousa, S.A., Synthesis of new analogs of tetraiodothyroacetic
acid (tetrac) as novel angiogenesis inhibitors for treatment of cancer, Bioorganic & Medicinal Chemistry Letters
(2018), doi: https://doi.org/10.1016/j.bmcl.2018.02.045
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1
Synthesis of new analogs of tetraiodothyroacetic acid
(tetrac) as novel angiogenesis inhibitors for treatment
of cancer
Mehdi Rajabia, Murat Yalcina,b, Shaker A. Mousaa
aThe Pharmaceutical Research Institute, Albany College of Pharmacy and Health
Sciences, Rensselaer, NY, USA
bDepartment of Physiology, Veterinary Medicine Faculty, Uludag University,
Bursa, Turkey
* Corresponding author:
Shaker A. Mousa, PhD, MBA, FACC, FACB
The Pharmaceutical Research Institute
Albany College of Pharmacy and Health Sciences
1 Discovery Drive, Rensselaer, NY 12144
Phone: 518-694-7397
Fax: 518-694-7567
shaker.mousa@acphs.edu

2
ABSTRACT
In the angiogenesis process, integrins, which are members of a family of cell surface
transmembrane receptors, play a critical role particularly in blood vessel formation and the local
release of vascular growth factors. Thyroid hormones such as L-thyroxine (T4) and 3,5,3’-
triiodo-L-thyronine (T3) promote angiogenesis and tumor cell proliferation via integrin αvβ3
receptor. At or near an arginine-glycine-aspartate (RGD) recognition site on the binding pocket
of integrin αvβ3, tetraiodothyroacetic acid (tetrac, a deaminated derivative of T4) is a
thyrointegrin receptor antagonist and blocks the actions of T3 and T4 as well as different growth
factors-mediated angiogenesis. In this study, we synthesized novel tetrac analogs by modifying
the phenolic moiety of tetrac and tested them for their anti-angiogenesis activity using a Matrigel
plug model for angiogenesis in mice. Pharmacological activity results showed that tetrac can
accommodate numerous modifications and maintain its anti-angiogenesis activity.
KEYWORDS:
Angiogenesis, thyroid hormones, tetraiodothyroacetic acid, tetrac, integrin

3
Tumor angiogenesis is the formation of new blood vessel growth from the existing
vasculature by cell adhesion to the extra cellular matrix (ECM), which results in tumor
progression.1,2 Rapid tumor cell proliferation produces environmental stresses such as a hypoxic,
glucose-deprived environment that begins the angiogenic switch whereby tumor cells produce
angiogenic activators including angiogenin, transforming growth factor (TGF)-α, TGF-β, tumor
necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), granulocyte colony-
stimulating factor, basic fibroblast growth factor (bFGF, FGF2), platelet-derived endothelial
growth factor, and hepatocyte growth factor.3
Integrins are important transmembrane receptors that play a critical role in the angiogenesis
process, particularly in blood formation and local release of vascular growth factors. They are
members of a family of cell surface receptors that are immunoglobulin superfamily molecules or
ECM proteins,4 and consist of α and β chain units. Integrins αvβ3 and αvβ5 are the main receptor
types involved in the angiogenesis process, especially in binding with angiogenesis modulators
containing an arginine-glycine-aspartate (RGD) recognition site that binds to the integrin
receptor.5 There are several natural RGD-containing proteins such as fibronectin, fibrinogen, λ-
receptor on E.coli, sindbis coat protein, and α-lytic protease protein6 as well as cyclic RGD
tripeptide (c-RGD) and c-RGD peptidomimetics7 that show high binding affinity to the integrin
recognition site.
Many studies have reported the effects of thyroid hormone analogs like L-thyroxine (T4),
which is a prohormone antecedent to 3,5,3’-triiodo-L-thyronine (T3), on thyroid hormone
receptor (TR) via binding to integrin αvβ3 and the effects of the angiogenesis activity of T4 and
T3 with an interaction site that is located at or near the RGD recognition site of integrins (Figure
1A). Tetraiodothyroacetic acid (tetrac), a deaminated derivative of T4, inhibited the pro-
angiogenesis response of thyroid hormone by inhibiting the cell surface-initiated actions of T4
and T3 in the chick chorioallantoic membrane (CAM) and many other angiogenesis models.8,9
Anti-proliferative activity of tetrac against other cancer cell lines such as human non-small cell
lung cancer10 have been investigated both in vitro and in xenografts and results showed anti-
angiogenic activity at the integrin αvβ3 receptor-binding site. For example, Yoshida et al. found
tetrac to be an effective inhibitor of retinal angiogenesis and of the pro-angiogenic effect of both
erythropoietin (EPO) and VEGF on retinal endothelial cells; this suggested that tetrac (and

4
antagonism of integrin αvβ3) is a viable therapeutic strategy for proliferative diabetic
retinopathy.11
Figure 1. a) Pro-angiogenic activity of thyroid hormones T4 and T3 on vascular smooth and
endothelial cells is initiated at the cell surface receptor (integrin) for the hormone on the
extracellular domain of integrin αvβ3. Tetrac, a thyroid hormone analog, is inhibitory at the αvβ3
integrin receptor and is anti-angiogenic. b) Conjugation of tetrac to PLGA nanoparticle results
in tetrac-nanoparticle that has shown anti-angiogenic activity.
In order to limit tetrac to the cell surface thyroid hormone receptor and to provide optimized
exposure of the biphenyl structure and acetic acid side chain of its inner ring to the receptor site
on αvβ3, our group has conjugated tetrac to the polymeric nanoparticle (NP) poly (lactic-co-
glycolic acid) (PLGA) via covalent binding (an amide bond formation) between tetrac’s outer
ring hydroxyl to NHS-modified PLGA, resulting in tetrac NP (Nano-diamino-tetrac; NDAT;
Nanotetrac). This NP showed anti-angiogenic activity, confirming the role of the integrin
receptor in the results explained above (Figure 1B).8 Results from both in vitro and in vivo
experiments for the treatment of drug-resistant breast cancer showed that NDAT is not able to
enter into the cell nucleus but will enhance inhibition of tumor proliferation at a low-dose
equivalent of free tetrac.12 It has been concluded that NDAT has high potential as an anticancer
agent,13-15 with possible applications in the treatment of drug-resistant cancer. NDAT also
inhibits the PI3-K and MAPK pathways16,17 and blocks the expression of a panel of genes critical
to cancer cell survival pathways and the epidermal growth factor receptor (EGF-R) gene.18,19 We
also designed and synthesized a novel derivative of tetrac that showed pro-angiogenic activity

5
rather than anti-angiogenic activity by mimicking the action of the iodothyronine deiodinases
(the enzymes that convert T4 to T3). In this regard, the phenolic OH group of tetrac was initially
deprotonated, and subsequent anion extraction of a proton formed a tautomeric dienone. Finally,
nucleophilic attack on the iodine atom resulted in a deiodinated product of tetrac, designated
MR-49.20
Knowing that the angiogenesis activity of tetrac is stimulated by FGF or VEGF without
influencing the pre-existing blood vessels, we synthesized new tetrac analogs by modifying the
phenolic moiety of tetrac and studied the structure-activity relationship. The phenolic hydroxyl
group (-OH) of thyroid analogs is an important site for their modification and is a target site for
converting tetrac to an integrin antagonist without any changes to the carboxylic acid moiety of
tetrac. We previously synthesized diamino-tetrac, which showed anticancer/anti-angiogenic
activity and was able to target the thyroid hormone-tetrac receptor on the extracellular domain of
integrin αvβ3. Wu et al.21 showed that conjugation of the phenolic hydroxy group (4’-OH) of T4
with sulfate or glucuronic acid yielded the corresponding sulfated (T4S) and glucuronidated
(T4G) hormone22; sulfate and glucuronic acid conjugations are useful to enhance the water
solubility of many hydrophobic drugs and to enhance their excretion through urine and/or bile.23
Similar to T4S and T4G, tetrac also undergoes sulfonation and glucuronidation, thus suggesting
that the body can metabolize tetrac.22
As shown in Scheme 1, a large scale of compound 7, tetrac, was synthesized according to
the method of Wikinson24 and optimized by our group.25 The scheme starts with nitration of p-
hydroxyphenylacetic acid 1 in the presence of nitric acid and acetic acid at 45°C to yield the first
intermediate, dinitrophenylacetic acid, in 81% yield (m.p. 175-176°C). This was then converted
to its ethyl ester 2 in 73% yield (m.p. 70-71°C) by dissolving in dry chloroform containing
ethanol and p-toluene sulfonic acid under reflux conditions. Ethyl 3,5-dinitro-4-(4'-
methoxyphenoxy)phenyl acetate 2 was mixed with p-toluene sulfonyl chloride in anhydrous
pyridine and heated at 95-100°C for 30 min, and then p-methoxyphenol (temperature raised to
125°C for 2.5 h under nitrogen) was added to give the diphenyl ether derivative 3 in 63% yield.
The two nitro groups of 3 were reduced and converted to amine 4 using a catalytic amount of 5%
palladium, H2, and calcium carbonate in ethanol as solvent. Compound 4 was then reacted with
sodium iodide to convert the amine groups to iodine, resulting in compound 5 in 69 % yield,
followed by hydrolysis and deprotection under reflux conditions using hydro-iodic acid (HI) as a

6
strong cleaving agent for facile removal of both the ester and O-methyl groups to form 6. Acetic
acid was used to avoid decomposition of HI into iodine by sulfuric acid, thereby avoiding the
generation of unwanted side products in the reaction medium. Finally, tetrac 7 was obtained from
the di-iodo compound 6 using iodine in methanolic-NH3 with near quantitative yields.
Regioselective iodination of the ring in compound 6 was facilitated by the transformation of the
methyl ether group of compound 5 into phenol 6. This was done to allow activation of the ortho
position in compound 6 through inductive and resonance effects.
Scheme 1. Synthetic route for the preparation of tetrac, 7.
The phenolic hydroxyl group of tetrac 7 was selected as the target site for converting tetrac to
an integrin antagonist without any changes to its carboxylic acid moiety. In order to increase
reaction yields, we initially protected the carboxylic acid moiety of 7, which was deprotected in
the last step because the carboxylic acid moiety is crucial for the thyroid antagonist activity-
related bindings and for initial receptor and metal binding as observed in molecular
simulations.25 We then evaluated anticancer activity of those analogs containing carboxylic acids
that remained intact.
An outline of the synthetic route for the tetrac analogs is shown in Scheme 2. The phenyl
acetic acid end of tetrac 7 was readily protected to methyl ester using a

7
(
) was
Further derivatization was
provided
OH group of 8 with different alkyl bromides in the presence of
potassium carbonate and acetone under reflux conditions to give compounds 9a-9e with yields
ranging from 62-98%.26 The use of potassium carbonate as mild base was critical because amine
bases resulted in loss of the iodine in the ortho position of the phenol ring of 8 (outer-ring).20 A
3-fold excess of alkyl bromides was used in reaction of 8 to 9, except propargyl bromide (PB),
where an excess of PB resulted in transesterification. A 1:1 ratio of PB to 8 yielded 72% of 9a.
Eighty-two percent of 9b and 62% of 9c were obtained by reaction of 8 with commercially
available 1-bromo-3-phenylpropane and 3-(bromomethyl)pyridine, respectively. It was
surprising that more than 95% of 9d and 9e was obtained by reaction of 8 with N-(2-bromoethyl)
phthalimide 12a and N-(2-bromopropyl) phthalimide 12b, respectively. Although 12a and 12b
are commercially available, we synthesized them from ethylene dibromide 11a and propylene
dibromide 11b, respectively, in reaction with phthalimide potassium in DMF and yielded 65-
75%. Compounds 9a-9e were finally deprotected in the presence of potassium hydroxide (2 M)
and tetrahydrofuran and methanol (1:1) as a co-solvent, yielding the compounds 10a-10e in
yields ranging from 66-87% (Scheme 2, Table 1).

8
Scheme 2. Synthetic route for the preparation of tetrac analogs.

9
Table 1. Target tetrac analogs
Compound
R
Yield (%)
H
91
76
7 (tetrac)
10a
69
66
10b
10c
N
O
N
83
87
10d
10e
O
O
N
O
To investigate the structure-activity relationships, compounds 10a-10e were tested for their
ability to block angiogenesis in comparison to tetrac. Inhibition of angiogenesis was measured in
an in vivo study with an assay that exposed test angiogenesis-inducing and inhibitory compounds
to cold liquid Matrigel® Matrix (Corning, New York, NY), a mix of several proteins that, after
subcutaneous injection into mice, solidified and permitted the formation of new blood vessels.
Female C57/B6 mice aged 5-6 weeks, with body weights of 20 g, were purchased from
Taconic Farms (Hudson, NY). All animal studies were conducted at the animal facility of the
Veteran Affairs Medical Center (Albany, NY) in accordance with and approved by the IACUC
of the Veterans Affairs Medical Center and their current institutional guidelines for humane
animal treatment. Animals were maintained under specific pathogen-free conditions and housed
4 animals per cage, under controlled conditions of temperature (20-24°C) and humidity (60-
70%) and a 12 h light/dark cycle. Water and food were provided ad libitum. Mice were allowed
to acclimatize for 5 d prior to the start of experiments. Mice were dived into 7 groups (3 mice per
group): control, tetrac, and 5 tetrac derivatives.
Compounds were added at 10 µg/10 µL to 200 µL Matrigel, which contains murine vascular
growth factors, and injected subcutaneously in the mice, and the control group received Matrigel

10
only at 200 µL+ 10 µL vehicle/implant. There were four subcutaneous implants per mouse (2
implants on the right and 2 implants on left backsides of the animal). The Matrigel liquid plug at
4°C became a solid matrix at 37°C for the sustained release of the compounds over 14 days. At
day 14 post-Matrigel implant, all animals were sacrificed and vessel formation was quantified by
measuring the hemoglobin concentration in the Matrigel plug according to the Drabkin method27
using spectrophotometry. Briefly, Matrigel plugs were placed into a 0.5 mL tube containing
double distilled water and then homogenized for 5-10 min. The samples were centrifuged at
1700 x g for 10 min and the supernatants collected. Fifty μL of supernatant was mixed with 50
μL of Drabkin’s reagent and allowed to sit at room temperature for 15-30 min, after which it was
placed in a 96-well plate and absorbance measured at 540 nm with a Microplate Manager ELISA
reader. Hb concentration was expressed as mg/dL based on comparison with a standard curve.
As shown in Figure 2, in the Matrigel plug experiment, tetrac and the 5 derivatives
significantly inhibited angiogenesis and results are presented as mean ± standard error means
(S.E.M.) or ± standard deviation (S.D.) as given in Figure legends. Control and experimental
groups were compared and statistically analyzed with ANOVA and student’s t-test using
SigmaPlot 10.0 software. Differences between control and experimental end points were
considered statistically significant if P <0.01. Tetrac analog compounds also blocked the FGF-
induced angiogenesis in a comparable fashion to tetrac. Any modulations on either or both sides
of tetrac did not greatly affect the antagonist effect of the parent compound. Anti-angiogenesis
activity of 10a might be related to the presence of the alkyne function group on its structure,
which may be modified in our further structure-activity studies. Also, anti-angiogenesis activity
of compounds 10d and 10e were very interesting because of the presence of the phthalimide
portion of their structures, which made them similar to thalidomide (Figure 3), a known anti-
angiogenesis inhibitor that can inhibit angiogenesis induced by angiogenic cytokines such as
basic fibroblast growth factor (bFGF) in the rabbit cornea and VEGF in the corneas of mice.28-30

11
Figure 2. Matrigel plug assay of angiogenesis (anti-angiogenesis effect of
tetrac and its analogs). Values are expressed as mean ± SEM versus control
vehicle (PBS).
Figure 3. Phthalimide ring of thalidomide is responsible for its activity.
In summary, to find new angiogenesis inhibitors we synthesized new tetrac analogs by
modifying the phenolic moiety of tetrac. We analyzed their structure-activity relationships and
tested for anti-angiogenesis activity using a mouse Matrigel model for angiogenesis. Design and
development of novel angiogenesis inhibitors has been validated as a target in several tumor
types. Our results showed that phthalimide-modified tetrac analogs 10d and 10e have higher
anti-angiogenesis activity than the other analogs generated and this might be due to the presence
of the phthalimide portion of their structure, similar to thalidomide. Our further studies will

12
require additional bioanalytical studies (LC/MS/MS) to see the exact contribution of the
phthalimide portion to the in vivo activity in the tetrac combination. In order to increase the
water solubility of these new materials, we also plan to conjugate these anti-angiogenesis
molecules to PLGA-based NPs.
Declarations of interest
S.A.M. holds stock in NanoPharmaceuticals LLC, which is developing anticancer drugs. All
other authors declare no competing financial interest.
Acknowledgments
We appreciate the excellent editing by Dr. Kelly A. Keating, Pharmaceutical Research
Institute (PRI) and the technical support by members of the PRI. Thanks to Dr. Scott A.
McCallum at the Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic
Institute (Troy, NY) for NMR analysis, and thanks to Dr. Qishan Lin (SUNY Albany) for the
LC/MS/MS analysis. Thanks to Dr. Jay Jayaraman (PRI) for his help and constant support.
Funding
This work was supported in part by a grant from NanoPharmaceuticals LLC and in part by
The Pharmaceutical Research Institute at the Albany College of Pharmacy and Health Sciences.
Supplementary data
Supplementary data contains information on the synthetic protocols and analytical
characterizations of all molecules. Also included are the NMR spectra of some of the synthesized
compounds.

13
References
1. Folkman J. Role of angiogenesis in tumor growth and metastasis. Semin Oncol. 2002;29:15.
2. Folkman J. A new family of mediators of tumor angiogenesis. Cancer Invest. 2001;19:754.
3. Rajabi M, Mousa SA. The role of angiogenesis in cancer treatment. Biomedicines.
2017;5:[Epub] June 22.
4. Avraamides CJ, Garmy-Susini B, Varner JA. Integrins in angiogenesis and
lymphangiogenesis. Nat Rev Cancer. 2008;8:604.
5. Hynes RO. A reevaluation of integrins as regulators of angiogenesis. Nat Med. 2002;8:918.
6. Jaracz S, Chen J, Kuznetsova LV, Ojima I. Recent advances in tumor-targeting anticancer
drug conjugates. Bioorg Med Chem. 2005;13:5043.
7. 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.
8. Yalcin M, Dyskin E, Lansing L, Bharali DJ, Mousa SS, Bridoux A, Hercbergs AH, Lin HY,
Davis FB, Glinsky GV, Glinskii A, Ma J, Davis PJ, Mousa SA. Tetraiodothyroacetic acid
(tetrac) and nanoparticulate tetrac arrest growth of medullary carcinoma of the thyroid. J
Clin Endocrinol Metab. 2010;95:1972.
9. 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.
10. Mousa SA, Yalcin M, Bharali DJ, Meng R, Tang HY, Lin HY, Davis FB, Davis PJ.
Tetraiodothyroacetic acid and its nanoformulation inhibit thyroid hormone stimulation of
non-small cell lung cancer cells in vitro and its growth in xenografts. Lung Cancer.
2012;76:39.
11. Yoshida T, Gong J, Xu Z, Wei Y, Duh EJ. Inhibition of pathological retinal angiogenesis by
the integrin αvβ3 antagonist tetraiodothyroacetic acid (tetrac). Exp Eye Res. 2012;94:41.
12. Bharali DJ, Yalcin M, Davis PJ, Mousa SA. Tetraiodothyroacetic acid-conjugated PLGA
nanoparticles: A nanomedicine approach to treat drug-resistant breast cancer. Nanomedicine
(Lond). 2013;8:1943.
13. Bharali DJ, Rajabi M, Mousa SA. Application of nanotechnology to target tumor
angiogenesis in cancer therapeutics. In: Mousa SA, Davis PJ, eds. Anti-Angiogenesis
Strategies in Cancer Therapeutics. Amsterdam: Academic Press; 2017:165.
14. Srinivasan M, Rajabi M, Mousa SA. Nanobiomaterials in cancer therapy. In: Grumezescu A,
ed. Nanobiomaterials in Cancer Therapy: Applications of Nanobiomaterials. Amsterdam:
Elsevier; 2016:57.
15. Rajabi M, Srinivasan M, Mousa SA. Nanobiomaterials in drug delivery. In: Grumezescu A,
ed. Nanobiomaterials in Drug Delivery: Applications of Nanobiomaterials. Amsterdam:
Elsevier; 2016:1.
16. Davis PJ, Davis FB, Mousa SA, Luidens MK, Lin HY. Membrane receptor for thyroid
hormone: Physiologic and pharmacologic implications. Annu Rev Pharmacol Toxicol.
2011;51:99.
17. Lin HY, Sun M, Tang HY, Lin C, Luidens MK, Mousa SA, Incerpi S, Drusano GL, Davis
FB, Davis PJ. L-thyroxine vs. 3,5,3'-triiodo-L-thyronine and cell proliferation: Activation of
mitogen-activated protein kinase and phosphatidylinositol 3-kinase. Am J Physiol Cell
Physiol. 2009;296:C980.
18. Lin HY, Chin YT, Nana AW, Shih YJ, Lai HY, Tang HY, Leinung M, Mousa SA, Davis PJ.
Actions of L-thyroxine and nano-diamino-tetrac (nanotetrac) on PD-L1 in cancer cells.
Steroids. 2016;114:59.

14
19. Davis PJ, Glinsky GV, Lin HY, Leith JT, Hercbergs A, Tang HY, Ashur-Fabian O, Incerpi
S, Mousa SA. Cancer cell gene expression modulated from plasma membrane integrin αvβ3
by thyroid hormone and nanoparticulate tetrac. Front Endocrinol (Lausanne). 2014;5:240.
20. Rajabi M, Sudha T, Darwish NH, Davis PJ, Mousa SA. Synthesis of MR-49, a deiodinated
analog of tetraiodothyroacetic acid (tetrac), as a novel pro-angiogenesis modulator. Bioorg
Med Chem Lett. 2016;26:4112.
21. Wu SY, Green WL, Huang WS, Hays MT, Chopra IJ. Alternate pathways of thyroid
hormone metabolism. Thyroid. 2005;15:943.
22. Mondal S, Raja K, Schweizer U, Mugesh G. Chemistry and biology in the biosynthesis and
action of thyroid hormones. Angew Chem Int Ed Engl. 2016;55:7606.
23. Rikke BA, Roy AK. Structural relationships among members of the mammalian
sulfotransferase gene family. Biochim Biophys Acta. 1996;1307:331.
24. Wilkinson JH. Synthesis of some possible metabolites of thyroxine and triiodothyronine.
Biochem J. 1956;63:601.
25. Bridoux A, Khan RA, Chen C, Cheve G, Cui H, Dyskin E, Yasri A, Mousa SA. Design,
synthesis, and biological evaluation of bifunctional thyrointegrin inhibitors: New anti-
angiogenesis analogs. J Enzyme Inhib Med Chem. 2011;26:871.
26. General procedure for preparation of 9a-9n: A mixture of methyl protected tetrac 2 (1 eq) and
alkyl bromide (2-3 eq) and potassium carbonate in (5 eq) in 25 mL acetone was heated at
reflux for 24 h. Reaction was filtered, concentrated, and then crude purified with flash
column chromatography over silica gel using n-hexane and ethyl acetate (9:1 to 7:3) to give
3a-3n with 60-80% yield. N-hexane and ethyl acetate (9:1 to 7:3) were used as solvents for
TLC.
27. Nuti E, Cantelmo AR, Gallo C, Bruno A, Bassani B, Camodeca C, Tuccinardi T, Vera L,
Orlandini E, Nencetti S, Stura EA, Martinelli A, Dive V, Albini A, Rossello A. N-O-
isopropyl sulfonamido-based hydroxamates as matrix metalloproteinase inhibitors: Hit
selection and in vivo antiangiogenic activity. J Med Chem. 2015;58:7224.
28. Perino S, Contino-Pepin C, Satchi-Fainaro R, Butterfield C, Pucci B. Inhibition of
angiogenesis by THAM-derived cotelomers endowed with thalidomide moieties. Bioorg
Med Chem Lett. 2004;14:421.
29. Kenyon BM, Browne F, D'Amato RJ. Effects of thalidomide and related metabolites in a
mouse corneal model of neovascularization. Exp Eye Res. 1997;64:971.
30. D'Amato RJ, Loughnan MS, Flynn E, Folkman J. Thalidomide is an inhibitor of
angiogenesis. Proc Natl Acad Sci U S A. 1994;91:4082.

15
Graphical Abstract
The phenolic hydroxyl group (-OH) of tetrac is selected as its modification site and as a target
site for converting tetrac to an integrin antagonist without any changes to the carboxylic acid
moiety of tetrac.