Design, synthesis, and biological evaluation of novel bifunctional thyrointegrin antagonists for neuroblastoma

Article abstract of DOI:10.1016/j.bmc.2021.116250

Receptor-mediated cancer therapy has received much attention in the last few decades. Neuroblastoma and other cancers of the sympathetic nervous system highly express norepinephrine transporter (NET) and cell plasma membrane integrin αvβ3. Dual targeting

Full text of DOI:10.1016/j.bmc.2021.116250

Bioorg. Med. Chem. 42 (2021) 116250
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of novel bifunctional
thyrointegrin antagonists for neuroblastoma
Ozlem Ozen Karakus, Kavitha Godugu, Kazutoshi Fujioka, Shaker A. Mousa*
Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Rensselaer, NY 12144, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
Receptor-mediated cancer therapy has received much attention in the last few decades. Neuroblastoma and other
cancers of the sympathetic nervous system highly express norepinephrine transporter (NET) and cell plasma
Dual targeting
Drug-drug Conjugate
membrane integrin αvβ3. Dual targeting of the NET and integrin αvβ3 receptors using a Drug-Drug Conjugate
Integrin αvβ3
(DDC) might provide effective treatment strategy in the fight against neuroblastoma and other neuroendocrine
tumors. In this work, we synthesized three dual-targeting BG-P400-TAT derivatives, dI-BG-P400-TAT, dM-BG-P400
TAT, and BG-P400-PAT containing di-iodobenzene, di-methoxybenzene, and piperazine groups, respectively.
Norepinephrine transporter (NET)
Neuroblastoma
-
Thyrointegrin antagonist
These derivatives utilize to norepinephrine transporter (NET) and the integrin αvβ3 receptor to simultaneously
modulate both targets based on evaluation in a neuroblastoma animal model using the neuroblastoma SK-N-F1
cell line. Among the three synthesized agents, the piperazine substituted BG-P400-PAT exhibited potent integrin
α
vβ3 antagonism and reduced neuroblastoma tumor growth and cancer cell viability by >90%. In conclusion,
BG-P400-PAT and derivatives represent a potential therapeutic approach in the management of neuroblastoma.
1. Introduction
stable norepinephrine structural analogue9,10, which can be taken up by
NET, and accumulate in the cytoplasm and mitochondria11,12. Because
Neuroblastoma is the most common extracranial solid tumor in ba-
bies and the third-most common cancer in children after leukemia and
brain cancer1. It most frequently (65% of cases) starts from the abdomen
and arises in the medulla of the adrenal gland. However, it can develop
in the chest (20%), pelvis (3%), or neck (2%)2,3. Several chemothera-
peutic agents are used to treat neuroblastoma such as cisplatin,
vincristine, rapamycin, carboplatin, etoposide, cyclophosphamide and
13-cis-retinoic acid4,5. However, drug resistance and toxicity often cause
treatment failure6–8. Therefore, the development of new anticancer
strategies is crucial to overcome drug resistance and safety issues for
better management of neuroblastoma.
BG competes with norepinephrine for uptake and storage by neuroen-
docrine cells, it can be used as both a targeting agent and as treatment
for neouroblastomas12,13
.
Another family of proteins, the integrins, regulate the growth, in-
vasion and metastatic process of neuroblastomas and other tumors.
Integrins are a family of heterodimeric cell surface receptors that consist
of 24 subunits designated
α and β. Integrins can mediate cell adhesion,
proliferation, and survival by interacting with proteins of the extracel-
lular matrix14. Abnormal regulation of integrins on cancer cells and
surrounding tissues compared to normal cells plays a key role in tumor
progression and pathological angiogenesis —angiogenesis is an essential
process for oxygen and nutrient supply for tumor growth15. Integrin
Neuroblastoma cells are derived from neural crest and they show
high norepinephrine transporter (NET) expression, which is a trans-
membrane protein encoded by the SLC6A2 gene. NET actively transports
norepinephrine, a catecholamine hormone made by the adrenal glands,
from the synapse into presynaptic neurons. Norepinephrine structural
analogues of neuro-adrenergic blocking agents guanethidine and bre-
tylium compete with norepinephrine for uptake and storage by neuro-
endocrine cells. Benzylguanidine (BG), is known as a metabolically
α
vβ3, a plasma membrane integrin that has a receptor for thyroid hor-
mones, is upregulated in both tumor cells and angiogenic endothelial
cells, thus blocking upregulation of integrin vβ3 via integrin antago-
α
nists can be an approach to combat tumor progression and metastasis for
neuroblastomas16,17
.
The most common integrin recognition motif, tripeptide Arg-Gly-Asp
(RGD), is responsible for cell adhesion to the extracellular matrix protein
* Corresponding author at: The Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, 1 Discovery Drive (Room 238), Rensselaer, NY
12144, USA.
E-mail address: shaker.mousa@acphs.edu (S.A. Mousa).
https://doi.org/10.1016/j.bmc.2021.116250
Received 26 March 2021; Received in revised form 10 May 2021; Accepted 28 May 2021
Available online 6 June 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

O.O. Karakus et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116250
(ECM). Integrin receptors recognize the RGD sequence and RGD can
bind to many types of integrins. Therefore, RGD-containing peptides
(PAT). So, here we synthesized three different derivatives of BG-P-TAT
(Fig. 1), dI-BG-P400-TAT (7a), dM-BG-P400-TAT (7b), and BG-P400-PAT
have been taken advantage of to design effective integrin
αvβ3 antago-
(15) and evaluated their αvβ3 binding affinity and anticancer efficacy in
nists18–20. Recent studies stated that in addition to integrin antagonists,
neuroblastoma xenograft mice model.
thyroid hormone antagonists can also regulate angiogenesis21–23. The
We are the first group to develop thyrointegrin αvβ3 antagonist that
effects of thyroid hormone analogs on the integrin
α
vβ3 thyroid hor-
contain tetrac, a thyromimetic that is very effective against various
cancers but with limited efficacy against neuroendocrine cancers
because of its limited access and transport into neuroendocrine tumors
mone receptor and membrane Na+/H+ anti-porter ion pumps were also
investigated and a complex relationship was found between thyroid
hormone and angiogenesis24. The thyroid hormone agonist analogs have
been shown to induce angiogenesis, while the metabolic degradation
product of thyroid hormone, tetraiodothyroacetic acid (tetrac), inhibits
angiogenesis25,26. Tetrac is a deaminated derivative of thyroid hormone
L-thyroxine (T4) and functions as a thyroid hormone antagonist by
blocking the actions of triiodothyronine (T3) and T4 at the thyroid hor-
such as neuroblastoma despite their
αvβ3 high affinity. However,
optimal anticancer efficacy was achieved upon conjugation of different
thyrointegrin αvβ3 antagonists via polyethylene glycol to MIBG deriv-
ative (Benzyl Guanidine), a known NET transporter utilized for targeted
radiation delivery and imagining of neuroendocrine cancers.
mone receptor site on integrin
α
vβ325,27,28. Thus, we called this specific
2. Results and discussion
type of ligand that blocks both thyroid hormones and integrin αvβ3 re-
ceptor sites a “thyrointegrin
α
vβ3” antagonist29
.
2.1. Design and strategy
In the past decade, our group developed a series of high affinity
thyrointegrin
α
vβ3 antagonists30–32. One of these studies was the design
We have elsewhere demonstrated that BG-P400-TAT exhibited 6-fold
of a polymeric antagonist containing two tetrac groups with a triazole
ring (triazole tetrac, TAT), called P-bi-TAT, which was then investigated
for its anticancer activities in glioblastoma tumors33. In another study,
we designed and synthesized a dual Drug-Drug Conjugate (DDC) tar-
geting ligand and named it BG-P400-TAT; it contains polyethylene glycol
(PEG) with about 400 g/mol molar mass and we investigated its anti-
cancer activity for neuroblastoma34,35. In that case, we used BG for NET
higher binding affinity towards integrin αvβ3 with lower 50% inhibitory
concentration IC50 = 10.32 nM versus tetrac35. That study also showed
that para substitution of BG did not change its therapeutic activity on
neuroblastoma tumor cells.
Here, we examined the effect of meta substitution on BG for targeting
NET transporter. Iodo and methoxy groups were chosen as meta sub-
stituents on the BG aromatic ring. The iodo group withdraws electrons
through an inductive effect from the aromatic ring, and methoxy do-
nates its lone pairs on the oxygen atom through a resonance effect to the
aromatic ring. The binding sites of both targeted proteins, NET, and
targeting and triazole tetrac (TAT) for integrin
α
vβ3 targeting. Based on
the promising results from that study, for enhanced
α
vβ3 integrin
binding affinity of BG-P400-TAT derivatives to neuroblastoma cells.
Thus, we chose two substituents, iodo and methoxy, for the BG aromatic
unit to change the hydrophilicity of the aromatic ring for interaction
with target protein. We also examined the effect of a basic linker,
piperazine by replacing the triazole linker to form piperazine tetrac
integrin αvβ3, consist of several hydrophobic residues. Increased hy-
drophilicity of the BG-P400-TAT with iodo groups may enhance its
binding affinity via van der Waals interactions. We also tried methoxy
group as substituent to make a more electron rich aromatic ring than the
non-substituted BG, and this substitution not only increased π-cation
interactions but also increased binding affinity of the compound towards
the targeted proteins. Other polar interactions such as dipole–dipole and
hydrogen bonding also play a role in increasing binding affinity9.
Therefore, we replaced the neutral triazole linker with basic piperazine
for attaching tetrac and PEG units to study the effect of polar in-
teractions on the binding. Br and N3 functionalized PEG6 and PEG7 used
for conjugation in this report.
2.2. Chemistry
The synthesis of dI-BG-P400-TAT (7a) and dM-BG-P400-TAT (7b) was
accomplished as described in Scheme 1. Amine groups of iodo and
methoxy substituted 4-hydroxy benzyl amine were protected with di-
tert-butyl di-carbonate. Compounds 2a and 2b were characterized with
1H NMR (Figs. S1 and S3). The peak observed at 1.49 ppm was assigned
to tert-butyloxycarbonyl (Boc) protons. In the next reaction, compounds
2a and 2b were reacted with commercially available Br-PEG6-N3 in the
presence of K2CO3 and ACN under reflux conditions to get compound 3a
and 3b with 90% and 85% yields, respectively. The 1H NMR spectra of
compounds 3a and 3b (Figs. S5 and S7) exhibited peaks of PEG protons
between 3.40 and 3.97 ppm. Then, we deprotected amino groups in 4 N
HCl (in dioxane) and confirmed the product by disappearance of Boc-
proton signals at 1.48 and 1.49 ppm in the 1H NMR spectra of 4a and
4b (Figs. S9 and S11). In the next step, N,N′-di-Boc-1H-pyrazole-1-car-
boxamidine was reacted with compounds 4a and 4b to acquire Boc-
protected guanidine compounds 5a and 5b. The 1H NMR spectra of
compounds 5a and 5b (Figs. S13 and S15) clearly showed peaks at
1.49–1.52 and 150–1.52 ppm, respectively, which can be assigned to
two separate Boc groups’ protons.
Then, azide-containing compounds 5a and 5b were conjugated with
propargylated tetrac, (PGT)36, which is terminal alkyne-containing tet-
rac, in a click reaction by forming a triazole ring to get compounds 6a
Fig. 1. Chemical structures of BG-P400-TAT, dI-BG-P400-TAT, dM-BG-P400-TAT,
and BG-P400-PAT (BG, benzyl guanidine; TAT, triazole tetrac; PAT, pipera-
zine tetrac).
2

O.O. Karakus et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116250
Scheme 1. Synthesis diagram of compounds 7a and 7b. (a) Boc2O, 6 h; (b) K2CO3, ACN, Br-PEG6-N3, reflux, 24 h; (c) HCl (4 N in dioxane), rt, 4 h; (d) DCM, TEA, N,
N′-di-Boc-1H-pyrazole-1-carboxamidine, rt, 12 h; (e) PGT, THF:water 4:1, CuSO4, Na Ascorbate, rt, 24 h; (f) HCl (4 N in dioxane), rt, 24 h.
and 6b. CuSO4/Na Ascorbate (0.3 eq:0.6 eq) in THF :water (4:1) was
used to generate Cu+ in situ at room temperature. The characteristic
singlet peak of triazole ring protons appeared at 8.59 and 8.60 ppm in
the 1H NMR spectra of compounds 6a and 6b, respectively. (Figs. S17,
S19). Lastly, protecting Boc groups were removed in 4 N HCl (in
dioxane), and the resulting product was purified with reverse phase
column chromatography with MeOH:water (70:30) to get compounds
7a and 7b. The 1H NMR (Figs. S21, S23), 13C NMR (Figs. S22, S24), and
mass spectra (Figs. S29, S31) of compounds 7a and 7b confirmed their
structure.
Aromatic protons of tetrac were observed at 7.32 and 8.04 ppm and
piperazine protons were observed at 2.77 and 2.94 ppm.
Compound 19 was introduced (Scheme 2) to a PEG unit after the
tosylation reaction of PEG-OH 10 in the presence of K2CO3 and ACN to
give compound 12. The 1H NMR spectrum of 12 (Fig. S40) confirmed the
structure by observing tetrac and N-Boc benzylamine aromatic proton
peaks at 7.18–7.79 and 6.88–7.20, respectively. After N-Boc depro-
tection of compound 12, free amine of 13 was used with N,N′-di-Boc-1H-
pyrazole-1-carboxamidine in DCM and TEA as a base to introduce Boc-
protected guanidine group and afforded compound 14. Finally, methyl
ester and Boc protection groups were hydrolyzed with conc. HCl in
dioxane :water to give desired compound 15. 1H NMR (Fig. S46) and the
mass spectrum (Fig. S53) of 15 confirmed its structure. Purities of final
synthesized products 7a, 7b, and 15 were confirmed to be > 95% by
HPLC (Figs. S55–S57).
The synthesis of BG-P400-PAT 15 was accomplished as described in
Scheme 2. First, the amino group of 4-hydroxybenzyl amine 8 was
protected with Boc group. Then, Br-PEG7-OH was reacted with the
phenolic OH group of 9 in the presence of K2CO3 and ACN at reflux
temperature to get 10, and it was characterized with 1H NMR by
observing PEG proton peaks at 3.6–3.8 ppm (Fig. S34).
A different method was used to introduce a tetrac unit on the PEG
(Scheme 3). First, carboxylic acid group of tetrac 16 was converted to
methyl ester in MeOH and SOCl2 to get 17. Then it was reacted with tert-
butyl 4-(3-(methanesulfonyloxy)propyl)piperazine-1-carboxylate hy-
drochloride 18 and Cs2CO3 as a base in ACN, followed by treatment with
HCl (4 N in dioxane) solution to deprotect the Boc group. The structure
of resulting compound 19 was characterized with 1H NMR (Fig. S38).
2.3. Integrin binding affinity
DDC Compounds dI-BG-P400-TAT (7a), dM-BG-P400-TAT (7b), and
BG-P400-PAT (15) showed relatively higher binding affinity towards
purified integrin αvβ3 receptor with lower IC50 values 1.1 nM, 0.5 nM,
and 0.3 nM, respectively, compared to 10.3 nM for BG-P400-TAT (Fig. 2)
by introducing m-substituents on the BG ring (7a and 7b). This is most
Scheme 2. Synthesis diagram of compound 15. (a) Boc2O, 6 h; (b) K2CO3, ACN, Br-PEG7-OH reflux, 24 h; (c) Tos-Cl, DCM, TEA, 0 ◦C-rt, 2 h; (d) compound 19 (see
Scheme 3), ACN, K2CO3 60 ◦C, 18 h; (e) HCl (4 N in dioxane), rt, 4 h; (f) DCM, TEA, N,N′-di-Boc-1H-pyrazole-1-carboxamidine, rt, 12 h; (g) dioxane:water, conc. HCl,
rt, 24 h.
3

O.O. Karakus et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116250
Scheme 3. Synthesis diagram of compound 19. (a) SOCl2, MeOH; (b) 17, ACN, Cs2CO3, 60 ◦C, 18 h; (c) 4 N HCl in dioxane, 2 h.
2.4. In vitro cellular uptake
MIBG, BG-P400-TAT35, dI-BG-P400-TAT (7a), dM-BG-P400-TAT (7b),
BG-P400-PAT (15) and at N-Boc-Benzylamine-P400-TAT (20) 1uM were
incubated with SK-N-F1 neuroblastoma cells (~1M) for 15, 30, and 60
min (Fig. 3). 20 was included in this test as a negative control since it has
a moiety of integrin αvβ3 binding (TAT) but lacks a moiety of NET
binding pocket. Substrates in cells were extracted and quantified with
LC-MS/MS. The uptake of MIBG was the highest at 60 min, followed by
BG-P400-TAT, 7a, 7b, and 20 (Fig. 3a). The uptake of BG-P400-TAT and
derivatives were comparable to each other and more than 20 (Fig. 3b).
The increased uptake of BG-P400-TAT and derivatives compared with 20
suggest that they are actively transported with facilitation of NET.
2.5. Molecular docking
Molecular docking studies were carried out with 7a, 7b, and 15 to
Fig. 2. Binding affinities of dI-BG-P400-TAT, dM-BG-P400-TAT, and BG-P400
-
understand the mode of binding and type of interaction with the active
PAT compared to BG-P400-TAT towards purified integrin αvβ3 receptor.
site of integrin αvβ3 (PDB:1L5G) by using auto dock tools (Figs. S58,
S59). The molecular docking results show a bent structure of the mol-
ecules at the binding site. The interaction and docking analysis revealed
that 15 has the best interaction rate with high binding energy ꢀ 14.4
kcal/mol and forms 9 hydrogen bonds with integrin β3 subunit (Fig. 4).
7a and 7b had binding energies of ꢀ 6.1 kcal/mol and ꢀ 7.8 kcal/mol,
likely due to increased van der Waals and π-cation interactions between
receptor and ligand with newly added iodine and methoxy groups. On
the other hand, keeping the BG ring and replacing the triazole linker
with piperazine increased the binding affinity towards integrin αvβ3
receptor. This phenomenon can be explained with various factors such
as newly formed hydrogen bonds between basic piperazine and integrin
respectively, and 7a formed 6 hydrogen bonds (1 with
α
v domain and 5
v domain, 4
with β3 domain) and 7b formed 6 hydrogen bonds (1 with
α
α
vβ3 receptor. Also, linker chain length might affect accommodation of
with β3 domain and 1 with Mn atom). Energy values for 7a, 7b, and 15
the molecule in the αvβ3 binding pocket side because BG-P-PAT (15),
with binding energies and residues involved in interactions are listed in
with a slightly longer linker chain, has a 30-fold increase in binding
affinity relative to BG-P-TAT.
Table 1. The 30-fold higher αvβ3 binding affinity of 15 versus the close
analog BG-P400-TAT may be due to the additional hydrogen bonds of the
Fig. 3. (a) The cellular uptake of MIPG, BG-P400-TAT, BG-P400-PAT (15), dI-BG-P400-TAT (7a), dM-BG-P400-TAT in neuroblastoma cells (7b) and 20 in SK-N-F1
neuroblastoma cells incubated at a concentration of 10 uM for 15, 30 and 60 min. (b) The uptake of MIPG, BG-P400-TAT and derivatives in SK-N-F1 neuroblas-
toma cells (~1M), concentration of 10 uM at 60 min. *: P-value <0.05 compared with BG-P400-TAT. ***: P-value <0.001 compared with BG-P400-TAT.
4

O.O. Karakus et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116250
Fig. 4. Binding mode of BG-P-PAT (15) with the crystal structure of integrin
show hydrogen bonds, integrin subunit β3 is shown in green.
α
vβ3 (left) (PDB ID 1L5G) and structure of compound 15 (right). Dashed yellow lines
BG portion of 15 in with Asp-127 and Asp-126. This may be a result of
the longer linker chain in BG-P400-PAT, allowing the BG portion easier
access to this domain than the BG in BG-P400-TAT. Additional hydrogen
bonding of the piperazine nitrogen may also be responsible for some of
Table 1
Binding energies of the compounds with integrin
α
vβ3.
Compound
Docking Score
(kcal/mol)
Interacting Residues
Bond
Distance (Å)
the increased αvβ3 binding affinity of BG-P400-PAT.
A-chain (
α
v-
B-chain (β3-
subunit)
subunit)
2.6. In vivo anticancer efficacy
dI-BG-P-TAT
(7a)
ꢀ 6.1
Tyr 178
Tyr 178
2.9
3
Tyr-166
Arg 214
Ser 334
Ser 337
Lys 125
2.8
2.5
2.5
3.7
dI-BG-P400-TAT (7a), dM-BG-P400-TAT (7b), and BG-P400-PAT (15)
at 3 mg kgꢀ 1 were administered subcutaneously (s.c.) daily for 20 days
to nude mice implanted with the SK-N-F1 neuroblastoma cell line for
comparative anticancer activities. The tumor volumes significantly
decreased compared to control (Fig. 5A). Compound 15 treatment
resulted in significant decrease in tumor weight by 90%, and compounds
7b and 7a resulted in 86% and 67% decreases in tumor weight,
respectively (Fig. 5B). For compound 7a, the treatment was dis-
continued, and animals were terminated for after 7 days due to skin
irritation of the mice.
dM-BG-P-
TAT (7b)
ꢀ 7.8
3.2
Arg 216
Ser 334
Mn
2.2, 2.2, 4.5
2.4
4.8
BG-P-PAT
(15)
ꢀ 14.4
Asp 126
Asp 127
Arg 214
Asn 215
Ala 218
Asp 251
Lys 253
Thr 311
Asn 313
3.5
3.5, 4.2
4.1
3.8
3.2
2.7. Histopathology
3.5, 3.4
4.5
3.4
To compare the histopathological changes in tumors of untreated
and treated groups, tumors were harvested, fixed, and stained with
hematoxylin and eosin (H&E). Necrosis at low magnification of tumors
3.3
Fig. 5. Comparative (A) tumor volumes and (B) tumor weights of dI-BG-P400-TAT, dM-BG-P400-TAT, and BG-P400-PAT at treatment dose of 3 mg kgꢀ 1 body weight
once a day subcutaneously for 20 days compared to vehicle control (PBS). The treatment with dI-BG-P400-TAT was discontinued after 7 days because of skin
irritation at the injection site that caused pain and distress to those animals.
5

O.O. Karakus et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116250
from animals treated with compounds 7a, 7b, and 15 versus control is
seen clearly (Fig. 6). The staining showed large areas of necrosis,
fibrosis, and cell debris with approximately 98% (15), 85% (7b), and
70% (7a). On the other hand, tumors from the untreated group had
P400-TAT derivatives (compounds 7b and 15) improve upon the original
compound and may be promising new drug candidates for the treatment
of neuroblastoma.
mostly viable tumor cells. At higher magnification (40×) of BG-P400
-
4. Methods
PAT (15), there were large areas of necrosis replaced with normal tissue.
It is important to note that compound 7a was administered for only 7
days versus 20 days for the other two compounds.
4.1. Chemicals and Analysis
4-Hydroxybenzylamine, di-tert-butyl dicarbonate, HCl (4
N in
3. Conclusions
dioxane), N,N′-Di-Boc-1H-pyrazole-1-carboxamidine, TEA, sodium
ascorbate, and copper sulfate were purchased from Sigma-Aldrich. 4-Hy-
droxy-3,5-dimethoxy-benzylamine, 4-hydroxy-3,5-diiodo-benzylamine,
and tert-butyl 4-(3-(methanesulfonyloxy)propyl)piperazine-1-carbox-
ylate hydrochloride were purchased from Chem-Space, Labseeker, and
Synthonix, respectively. Br-PEG6-N3 and OH-PEG7-N3 were purchased
from Broad Pharm. Propargylated tetrac (PGT) and methyl ester of tet-
rac were synthesized according to a published method36. All commer-
cially available chemicals were used without further purification. All
solvents were dried, and anhydrous solvents were obtained using acti-
vated molecular sieves (0.3 or 0.4 nm depending on solvent type). All
reactions (if not specifically containing water as reactant, solvent, or co-
solvent) were performed under N2 atmosphere in oven-dried glassware.
Chemical structures of all synthesized compounds were confirmed with
1H NMR, 13C NMR, and mass spectrometry. The NMR experiments were
performed on a Bruker Advance II 800 MHz spectrometer at the Center
for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic
Institute. Thin layer chromatography was performed on silica gel plates
with fluorescent indicator. Visualization was accomplished with UV
light (254 and/or 365 nm). High resolution mass spectral analysis was
performed on either an Applied Biosystems API4000 LC/MS/MS or
Advion Expression CMCL. Purity of all final compounds was ≥95% as
determined by HPLC, Waters 2695 system equipped with a Pursuit XRs
C18 column (150 × 4.6 mm) with a flow rate of 1 mL/min using a
Recent data have highlighted that the BG-P400-TAT ligand dually
targeting NET and integrin αvβ3 shows more effective results in the
treatment of neuroblastoma than administration of BG and TAT sepa-
rately34,35. The current study developed the anticancer activities of dual
targeting BG-P400-TAT derivatives for neuroblastoma. To enhance the
potency towards target proteins NET and integrin αvβ3 receptor, com-
pound BG-P400-TAT35 was modified by adding new substituents iodo (dI-
BG-P400-TAT, 7a) and methoxy (dM-BG-P400-TAT, 7b) and replacing the
triazole group with piperazine (BG-P400-PAT, 15) as novel dual-
targeting molecules. Compounds 7a and 7b demonstrated 67% and
86% tumor shrinkage, respectively, and compound 15 showed 90%
shrinkage with maximal loss of cancer cell viability. These results for
compounds 7b and 15 are higher than those for previously synthesized
BG-P400-TAT, which showed 80% tumor shrinkage.
In the vivo studies of these derivatives (7a, 7b, and 15), results can
be explained by their relatively higher binding affinity towards purified
integrin
αvβ3 receptor with lower IC50 values compared to parent
compound BG-P400-TAT. Histopathology results of compounds 15 and
7b showed 98% and 85% necrosis of cancer cells, respectively, and all
these experimental results strongly support using these compounds for
neuroblastoma therapy development, which has less than 40% overall
patient survival. Hence, our dual NET thyrointegrin αvβ3 antagonist BG-
Fig. 6. Histology images showing the effect of compound 15 (BG-P-PAT), 7a (dI-BG-P-TAT), and 7b (dM-BG-P-TAT) after 20 days of daily treatment on SK-N-F1
neuroblastoma xenografts in mice at low power view (4X and 10X) and high-power view (40X) in histologic sections from untreated and treated animals. Sec-
tions form untreated animals showed complete infiltration with malignant cells (~100%) whereas sections from treated animals revealed complete necrosis, fibrosis,
and inflammation.
6

O.O. Karakus et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116250
(2H, s, CH2 ), 6.51 (2H, s, ArH). 13C NMR (150 MHz, CDCl3): 28.4,
44.9, 50.7, 56.1, 61.7, 70.0, 70.2, 70.3, 70.5, 70.7, 72.3, 72.6, 79.9,
104.4, 134.7, 136.2, 153.4, 155.9. MS (ESI+): C28H48N4O11 + Na+
Calcd, 639.3 found: 639.7. Anal. Calcd. (%): C, 54.53; H, 7.85; N, 9.08.
Found (%): C, 54.81; H, 7.35; N, 9.44.
– –
gradient of MeOH and water (0.1% TFA) and UV detection at 227 and
254 nm.
4.2. Synthesis of tert-butyl 4-hydroxy-3,5-diiodobenzylcarbamate (2a)
4-Hydroxy-3,5-diodo-benzylamine (750 mg, 1.8 mmol, 1 eq) was
slowly added with stirring to a solution of di-tert-butyl dicarbonate (500
mg, 1.1 mmol, 1.1 eq) and NaHCO3 (400 mg, 4.5 mmol, 2.5 eq) in
MeOH:THF:water (1:2:2) mixture at room temperature. After the reac-
tion mixture was stirred for 6 h, the solvent mixture was evaporated. The
residue was dissolved in 50 mL DCM and washed with 1% HCl (25 mL)
and brine (25 mL) and then dried (MgSO4) (Yield: 0.78 g, 90%). 1H NMR
(600 MHz, CDCl3): 1.49 (9H, s, t-butyl), 4.20 (2H, s, CH2), 4.87 (1H, s,
OH), 5.77 (1H, s, NH), 7.62 (2H, s, ArH). 13C NMR (150 MHz, CDCl3):
28.4, 42.6, 79.9, 82.2, 135.1, 137.6, 138.2, 152.3, 155.2. MS (ESI+):
4.6. Synthesis of (4-((20-azido-3,6,9,12,15,18-hexaoxaicosyl)oxy)-3,5-
diiodophenyl) methanamine (4a)
Compound 3a (500 mg, 0.62 mmol) was dissolved in 3 mL anhy-
drous 1,4-dioxane and 3 mL HCl (4 N in dioxane) was added to it and
stirred at room temperature. After 4 h, the solvent was removed under
reduced pressure, and the oily residue was precipitated with stirring in
diethyl ether to afford 4a as a white solid (Yield: 394 mg, 90%). 1H NMR
– –
(600 MHz, CDCl3): 3.44 (2H, t, CH2 N3), 3.57 (2H, t, PEG), 3.61–3.75
C
12H15I2NO3 -H- Calcd, 473.9 found: 473.9. Anal. Calcd. (%): C, 30.34;
(20H, s, PEG), 3.80 (2H, s, PEG), 3.96 (2H, s, PEG), 4.10 (2H, s,
CH2 ), 8.06 (2H, s, ArH). 13C NMR (150 MHz, CDCl3): 41.6, 50.7,
– –
H, 3.18; N, 2.95. Found (%): C, 30.25; H, 3.25; N, 2.88.
70.0, 70.2, 70.3, 70.4, 70.5, 70.6, 70.7, 72.2, 90.9, 133.2, 141.4, 158.0.
MS (ESI+): C21H34I2N4O7 + H+ Calcd, 709.1 found: 709.0. Anal. Calcd.
(%): C, 35.61; H, 4.84; N, 7.91. Found (%): C, 35.87; H, 4.33; N, 7.41.
4.3. Synthesis of tert-butyl 4-hydroxy-3,5-dimethoxybenzylcarbamate
(2b)
4-Hydroxy-3,5-dimethoxy-benzylamine (750 mg, 3.4 mmol, 1 eq)
was slowly added with stirring to a solution of di-tert-butyl di-carbonate
(820 mg, 3.7 mmol, 1.1 eq) and NaHCO3 (720 mg, 8.5 mmol, 2.5 eq) in
MeOH:THF:water (1:2:2) mixture at room temperature. After the reac-
tion mixture was stirred for 6 h, the solvent mixture was evaporated. The
residue was dissolved in 50 mL DCM and washed with 1% HCl (25 mL)
and brine (25 mL) and then dried (MgSO4) (Yield: 0.87 g, 90%). 1H NMR
(600 MHz, CDCl3): 1.49 (9H, s, t-butyl), 3.90 (6H, s, OCH3), 5.52 (1H, s,
OH), 6.54 (2H, s, ArH). 13C NMR (150 MHz, CDCl3): 28.4, 45.0, 56.4,
4.7. Synthesis of (4-((20-azido-3,6,9,12,15,18-hexaoxaicosyl)oxy)-3,5-
dimehoxyphenyl) methanamine (4b)
Compound 3b (500 mg, 0.81 mmol) was dissolved in 3 mL anhy-
drous 1,4-dioxane and 3 mL HCl (4 N in dioxane) was added to it and
stirred at room temperature. After 4 h, the solvent was removed under
reduced pressure, and the oily residue was precipitated with stirring in
diethyl ether to afford 4b as a white solid that was used for the next step
without further purification (Yield: 377 mg, 90%). 1H NMR (600 MHz,
– –
98.4 79.5, 104.3, 130.1, 134.0, 147.1, 156.0. MS (ESI+): C14H21NO15
+
CDCl3): 2.11 (2H, s, NH2), 3.40 (2H, t, CH2 N3), 3.63–3.71 (20H, m,
Na+ Calcd, 306.1. found: 305.9 Anal. Calcd. (%): C, 59.35; H, 7.47; N,
4.94. Found (%): C, 59.26; H, 7.35; N, 4.89.
PEG), 3.76 (2H, s, PEG), 3.79 (2H, s, PEG), 3.84 (6H, s, OCH3), 4.01 (2H,
s, PEG), 4.05 (2H, s, CH2 ), 6.83 (2H, s, ArH). 13C NMR (150 MHz,
– –
CDCl3): 44.2, 50.7, 56.3, 70.0, 70.2, 70.1, 70.4, 70.5, 70.6, 71.8, 106.4,
129.4, 135.3, 152.6. MS (ESI+): C23H40N4O9 + H+ Calcd. 517.2. Anal.
Calcd. (%): C, 55.08; H, 7.14; N, 9.33. Found (%): C, 54.81; H, 7.35; N,
9.44.
4.4. Synthesis of tert-butyl 4-((20-azido-3,6,9,12,15,18-hexaoxaicosyl)
oxy)3,5diiodo benzyl carbamate (3a)
K2CO3 (580 mg, 4.2 mmol, 2 eq) was added with stirring to a solution
of 2a (1000 mg, 2.1 mmol, 1 eq) in ACN (150 mL) at room temperature.
After the reaction mixture was stirred at 80 ◦C for 30 min, Br-PEG6-N3
(870 mg, 2.1 mmol, 1.05 eq) was added to the mixture and reaction
mixture was refluxed for 24 h at 80 ◦C. It was filtered to remove K2CO3.
Solvents were removed under reduced pressure, and the oily residue was
purified with column chromatography [SiO2: DCM :MeOH (95:5)] to
afford 3a as a colorless oil (Yield: 1.45 g, 85%). 1H NMR (600 MHz,
4.8. Synthesis of N,N′diboc-1–4-((20-azido-3,6,9,12,15,18-
hexaoxaicosyl)oxy)-3,5-diiodobenzyl)guanidine (5a)
Compound 4a (1000 mg, 2.7 mmol, 1 eq), N,N′-Di-Boc-1H-pyrazole-
1-carboxamidine (840 mg, 2.7 mmol, 1 eq) was dissolved in 10 mL
anhydrous DCM and then TEA (0.75 mL 5.4 mmol, 2 eq) was added to
the solution. The reaction mixture was stirred at room temperature for
12 h. After completion of the reaction the solvent was removed under
reduced pressure and the residue dissolved in DCM (50 mL). The organic
phase was washed with 1% HCl (25 mL) and brine (25 mL) and then
dried (MgSO4). The solvent was removed under reduced pressure to
yield 5a, which was purified with column chromatography [SiO2:
EtOAc/hexanes (2:8)] (Yield: 1140 mg, 85%). 1H NMR (600 MHz,
CDCl3): 1.50 (9H, s, t-butyl), 1.52 (9H, s, t-butyl), 3.40 (2H, s,
– –
CDCl3): 1.48 (9H, s, t-butyl), 3.40 (2H, t, CH2 N3), 3.67–3.73 (20H,
m, PEG), 3.81 (2H, s, PEG), 3.98 (2H, s, PEG), 4.15 (2H, s, PEG), 4.21
(2H, s, CH2 ), 7.68 (2H, s, ArH). 13C NMR (150 MHz, CDCl3): 28.4,
– –
42.6, 50.7, 70.0, 70.1, 70.6, 70.7, 70.8, 70.9, 72.2, 79.9, 90.8, 138.2,
155.3, 156.1. MS (ESI+): C26H42I2N4O9 + H+ Calcd, 809.1. Anal. Calcd.
(%): C, 38.63; H, 5.24; N, 6.93. Found (%): C, 38.70; H, 5.35; N, 6.85.
– –
CH2 N3), 3.67–3-73 (20H, m, PEG), 3.80 (2H, m, PEG), 3.99 (2H, M,
4.5. Synthesis of tert-butyl 4-((20-azido-3,6,9,12,15,18-hexaoxaicosyl)
oxy)3,5dimethoxy benzyl carbamate (3b)
–
–
–
CH2-PEG), 4.16 (2H, s, CH2-PEG), 4.50 (2H, s, CH2-Guanidine),
7.73 (2H, s, ArH), 8.57 (1H, s, NH). 13C NMR (150 MHz, CDCl3): 28.1,
28.3, 42.6, 50.7, 70.1, 70.6, 70.9, 72.2, 79.5, 83.4, 90.8, 137.45, 139.5,
153.6, 156.1, 157.2, 163.4. MS (ESI+): C32H52I2N6O11 + H+ Calcd,
951.2 found: 951.0. Anal. Calcd. (%): C, 40.43; H, 5.51; N, 8.84. Found
(%): C, 40.81; H, 5.38; N, 8.47.
K2CO3 (700 mg, 5.1 mmol, 3 eq) was added with stirring to a solution
of tert-butoxycarbonyl-4-hydroxy-3,5-dimethoxybenzylamine (2b)
(500 mg, 1.7 mmol, 1 eq) in ACN (150 mL) at room temperature. After
the reaction mixture was stirred at 80 ◦C for 30 min, Br-PEG6-N3 (730
mg, 1.8 mmol, 1.05 eq) was added to the mixture and reaction mixture
was refluxed for 24 h at 80 ◦C. It was filtered to remove K2CO3. Solvents
were removed under reduced pressure, and the oily residue was purified
with column chromatography [SiO2: DCM:MeOH (95:5)] to afford 3b as
a colorless oil (Yield: 0.7 g, 65%). 1H NMR (600 MHz, CDCl3): 1.49 (9H,
4.9. Synthesis of N,N′diboc-1–4-(20-azido-3,6,9,12,15,18-
hexaoxaicosyl)oxy)-3,5-dimetoxybenzyl)guanidine (5b)
Compound 4b (1000 mg, 2.7 mmol, 1 eq), N,N′-Di-Boc-1H-pyrazole-
1-carboxamidine (840 mg, 2.7 mmol, 1 eq) was dissolved in 10 mL
anhydrous DCM and then TEA (0.75 mL 5.4 mmol, 2 eq) was added to
– –
s, t-butyl), 3.41 (2H, t, CH2 N3), 3.63 (2H, s, PEG), 3.67–3.71 (20H,
m, PEG), 3.74 (2H, m, PEG), 3.85 (6H, s, OCH3), 4.14 (2H, m, PEG), 4.26
7

O.O. Karakus et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116250
the solution. The reaction mixture was stirred at room temperature for
12 h. After completion of the reaction the solvent was removed under
reduced pressure and the residue dissolved in DCM (50 mL). The organic
phase was washed with 1% HCl (25 mL) and brine (25 mL) and then
dried (MgSO4). The solvent was removed under reduced pressure to
yield 5b, which was purified with column chromatography [SiO2:
EtOAc/hexanes (2:8)] (Yield: 92 mg, 80%). 1H -NMR (600 MHz, CDCl3):
4.12. Synthesis of 2-(4-(4-((1-(20-(4-(guanidinomethyl)-2,6-
diiodophenoxy)-3,6,9,12,15,18-hexaoxaicosyl)-1H-1,2,3-triazol-4-yl)
methoxy)-3,5-diiodophenoxy)-3,5-diiodophenyl)acetic acid (7a)
Compound 6a (100 mg) was dissolved in 3 mL anhydrous 1,4-
dioxane and 9 mL HCl (4 N in dioxane) was added to it and stirred at
rt. After 24 h the solvent was removed under reduced pressure, and the
oily residue was precipitated with diethyl ether to afford 7a as a white
powder which was purified with RP column chromatography. [C18:
MeOH/Water (70:30)] (Yield: 60 mg, 70%). Purity > 98%, tR = 17.1
min [analytical HPLC/gradient: 50–95% MeOH in H2O (0.1% TFA), 50
min, flow rate 1 mL/min, Pursuit XRs C18 column (150 × 4.6 mm)]. 1H
– –
1.49 (9H, s, t-butyl), 1.52 (9H, s, t-butyl), 3.39 (2H, s, CH2 N3),
–
3.66–3.73 (20H, m, PEG), 3.80 (2H, m, PEG), 3.84 (2H, s, CH2-PEG),
–
–
–
3.92 (6H, s, OCH3),4.13 (2H, s, CH2-PEG), 4.54 (2H, s, CH2-
Guanidine), 6.56 (2H, s, ArH), 8.57 (1H, s, NH). 13C NMR (150 MHz,
CDCl3): 28.1, 28.3, 45.3, 50.7, 56.1, 70.1, 70.3, 70.6, 72.2, 77.0, 79.4,
83.2, 103.8, 105.2, 106.7, 132.9, 136.4, 153.4. 156.2, 163.6. MS (ESI+):
–
NMR (600 MHz, DMSO): 3.51 (2H, s, CH2-PEG), 3.60–3.68 (20H, s,
34H58N6O13 + H+ Calcd, 759.4. Anal. Calcd. (%): C, 53.81; H, 7.70; N,
CH2-PEG), 3.76 (2H, s, CH2 COOH), 3.91 (2H, s, CH2-PEG), 3.95
–
–
C
– –
(2H, s, CH2-PEG), 4.27 (2H, s, CH2-PEG), 4.60 (2H, s, Ar-CH2-
11.07. Found (%): C, 54.10; H, 7.44; N, 11.73.
– –
Guanidine), 5.14 (2H, s, Triazole CH2 O), 7.60 (2H, s, ArH Guani-
4.10. Synthesis of 2-(4-(4-((1-(20-(4-((2,3-bis(tert-butoxycarbonyl)
guanidino)methyl)-2,6-diiodophenoxy)-3,6,9,12,15,18-hexaoxaicosyl)-
1H-1,2,3-triazol-4-yl)methoxy)-3,5-diiodophenoxy)-3,5-diiodophenyl)
acetic acid (6a)
dine), 7.76 (2H, s, ArCH-TAT), 7.84 (2H, s, ArCH-TAT), 8.09 (1H, s, NH),
8.24 (1H, s, Triazole CH), 9.45 (1H, s, COOH). 13C NMR (150 MHz,
CDCl3): 39.9, 42.1, 50.0, 66.4, 69.1, 69.7, 70.3, 71.9, 90.1, 90.4, 90.8,
124.8, 126.1, 137.1, 138.4, 140.8, 142.7, 150.9, 152.1, 152.8, 156.6,
157.7, 164.3, 174.7. MS (ESI+): C39H46I6N6O11 + H+ Calcd, 1536.2
found: 1535.6. Anal. Calcd. (%): C, 30.49; H, 3.02; N, 5.47. Found (%):
C, 30.11; H, 3.48; N, 5.79.
Compound 5a (100 mg, 1 eq) and 1 eq of PGT were dissolved in 20
mL THF and stirred for 5 min, then 0.5 eq of NaAscorbate and 0.5 eq of
Cu2SO4 in 2 mL water were added to the mixture and stirred for 24 h at
rt. After 24 h, the solvents were removed under reduced pressure to yield
6a, which was purified with column chromatography. [SiO2: DCM/
MeOH (95:5)] (Yield: 138 mg, 65%). 1H NMR (600 MHz, CDCl3): 1.50
4.13. Synthesis of 2-(4-(4-((1-(20-(4-(guanidinomethyl)-2,6-
diiodophenoxy)-3,6,9,12,15,18-hexaoxaicosyl)-1H-1,2,3-triazol-4-yl)
methoxy)-3,5-diiodophenoxy)-3,5-dimetoxyphenyl) acetic acid (7b)
–
(9H, s, t-butyl), 1.52 (9H, s, t-butyl), 3.60 (2H, s, CH2-PEG), 3.63 (20H,
– –
– –
m, PEG), 3.72 (2H, s, CH2 ), 3.81 (2H, s, CH2 ), 3.90 (2H, s,
Compound 6b (100 mg) was dissolved in 3 mL anhydrous 1,4-
dioxane and 9 mL HCl (4 N in dioxane) was added to it and stirred at
rt. After 24 h the solvent was removed under reduced pressure, and the
oily residue was precipitated with diethyl ether to afford 7b as a white
powder, which was purified with RP column chromatography. [C18:
MeOH/Water (70:30)] (Yield: 61 mg, 70%). Purity > 98%, tR = 13.5
min [analytical HPLC/gradient: 50–95% MeOH in H2O (0.1% TFA), 50
min, flow rate 1 mL/min, Pursuit XRs C18 column (150 × 4.6 mm)]. 1H
– –
–
–
CH2 ), 3.99 (2H, s, CH2 COOH), 4.49 (2H, s, CH2-Guanidine),
– –
– –
4.60 (2H, s, CH ), 5.20 (2H, s, Triazole CH2 O), 7.22 (2H, s, ArCH)
7.72 (2H, d, ArH), 7.82 (2H, d, ArCH), 8.04 (1H, s, CH), 8.59 (1H, s, NH),
11.51 (1H, s, COOH). 13C NMR (150 MHz, CDCl3): 28.1, 28.3, 39.1,
42.6, 50.4, 66.6, 69.5, 70.1, 70.5, 70.6, 70.9, 72.2, 77.7, 79.7, 83.5,
90.6, 90.9, 124.9, 126.5, 135.3, 137.2, 139.5, 141.2, 143.2, 152.5,
156.1, 157.2, 163.3, 172.6. MS (ESI+): C49H62I6N6O15 + H+ Calcd,
1736.9 found: 1737.1. Anal. Calcd. (%): C, 33.89; H, 3.60; N, 4.84.
Found (%): C, 33.11; H, 3.22; N, 5.17.
–
–
NMR (600 MHz, DMSO): 3.36 (2H, s, CH2-PEG), 3.50 (2H, s, CH2-
–
–
PEG), 3.58–3.67 (20H, s, CH2-PEG), 3.74 (2H, s, CH2-PEG), 3.82
–
–
–
(6H, s, OCH3), 3.90 (2H, s, CH2-PEG), 4.08 (2H, s, CH2 COOH),
–
– –
4.11. Synthesis of 2-(4-(4-((1-(20-(4-((2,3-bis(tert-butoxycarbonyl)
guanidino)methyl)-2,6-dimethoxyphenoxy)-3,6,9,12,15,18-
hexaoxaicosyl)-1H-1,2,3-triazol-4-yl)methoxy)-3,5-diiodophenoxy)-3,5-
diiodophenyl)acetic acid (6b)
4.60 (2H, s, CH2-Guanidine), 5.40 (2H, s, Triazole CH2 O), 6.59
(2H, s, ArCH) 7.63 (2H, s, ArH), 7.83 (2H, d, ArCH), 8.09 (1H, s, Triazole
CH), 8.25 (1H, s, NH). 9.28 (1H, s, COOH). 13C NMR (150 MHz, CDCl3):
44.8, 47.1, 49.6, 50.0, 58.5, 60.9, 71.3, 74.1, 75.2, 76.8, 82.6, 95.0,
95.4, 109.2, 129.8, 131.1, 137.6, 140.8, 144.0, 145.8, 147.7, 155.9,
157.1, 157.8, 162.9, 169.3, 179.7. MS (ESI+): C41H52I4N6O13 + H+
Calcd, 1345.0, found 1344.7. Anal. Calcd. (%): C, 36.63; H, 3.90; N,
6.25. Found (%): C, 36.18; H, 4.48; N, 6.48.
Compound 5b (100 mg, 1 eq) and 1 eq of PGT were dissolved in 20
mL THF and stirred for 5 min, then 0.5 eq of NaAscorbate and 0.5 eq of
Cu2SO4 in 2 mL water were added to the mixture and stirred for 24 h at
rt. After 24 h, the solvents were removed under reduced pressure to yield
6b, which was purified with column chromatography. [SiO2: DCM/
MeOH (95:5)] (Yield: 142 mg, 70%). 1H NMR (600 MHz, CDCl3): 1.49
4.14. Synthesis of tert-butoxycarbonyl-4-hydroxybenzylamine (9)
–
(9H, s, t-butyl), 1.52 (9H, s, t-butyl), 3.59 (2H, s, CH2-PEG), 3.62–3.74
4-Hydroxybenzylamine (8) (0.62 g, 5 mmol) was slowly added with
stirring to di-tert-butyl di-carbonate (1.2 g, 5.1 mmol) at rt. After the
reaction mixture was stirred for 4 h, the oily residue was purified with
column chromatography [SiO2: EtOAc/hexanes (1:4)] to afford 0.82 g
of N-Boc-4-hydroxybenzylamine (9) as a colorless oil (Yield: 53 mg,
71%). 1H NMR (600 MHz, CDCl3): 1.49 (9H, s, t-butyl), 4.26 (2H, s,
–
– –
–
(20H, m, CH2-PEG), 3.81 (2H, s, CH2 PEG), 3.84 (6H, s, OCH3),
– –
CH2 COOH), 4.59 (2H, s,
– –
3.90 (2H, s, CH2 PEG), 4.14 (2H, s, CH2 PEG), 4.52 (2H, s,
–
–
CH2-Guanidine), 5.20 (2H, s, Tri-
– –
azole CH2 O), 6.56 (2H, s, ArCH) 7.22 (2H, s, ArH), 7.82 (2H, d,
ArCH), 8.03 (1H, s, Triazole CH), 8.60 (1H, s, NH). 13C NMR (150 MHz,
CDCl3): 28.1, 28.3, 39.3, 45.3, 50.4, 56.1, 66.7, 69.5, 70.6, 72.2, 77.7,
79.5, 83.3, 90.6, 105.2, 124.9, 126.5, 132.9, 135.7, 136.4, 141.2, 143.3,
–
–
CH2), 4.89 (1H, s, NH), 5.76 (1H, s, OH), 6.79 (2H, d, ArH), 7.14
(2H, d, ArH). 13C NMR (150 MHz, CDCl3): 22.7, 28.4, 44.2, 45.5, 79.7,
115.5, 121.4, 128.9, 130.6, 155.2, 156.0. MS (ESI+): C12H17NO3 Calcd
223.1. Anal. Calcd. (%): C, 64.55; H, 7.67; N, 6.20. Found (%): C, 64.71;
H, 7.51; N, 5.99.
152.3, 152.9, 153.4, 156.1, 163.4, 172.5. MS (ESI+): C51H68I4N6O17
+
H+ Calcd, 1545.1. Anal. Calcd. (%): C, 39.65; H, 4.44; N, 5.44. Found
(%): C, 39.10; H, 4.12; N, 5.88.
4.15. Synthesis of tert-butyl 4-((23-hydroxy-3,6,9,12,15,18,21-
heptaoxatricosyl)oxy) benzyl carbamate (10)
K2CO3 (478 mg, 3.5 mmol, 3 eq) was added with stirring to a solution
8

O.O. Karakus et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116250
of tert-butoxycarbonyl-4-hydroxybenzylamine (9) (280 mg, 1.2 mmol, 1
eq) in ACN (25 mL) at rt. After the reaction mixture was stirred for 30
min, Br-PEG7-OH (500 mg, 1.2 mmol, 1 eq) was added to mixture and
then refluxed for 24 h. It was filtered to remove excess K2CO3. Solvents
were removed under reduced pressure, and the oily residue was purified
with column chromatography [SiO2:DCM:MeOH (95:5)] to afford 10 as
a yellow oil (Yield: 530 mg, 80%). 1H NMR (600 MHz, CDCl3): 1.48 (9H,
residue was purified with reverse phase chromatography. [C18: MeOH/
Water (55:45)] (Yield: 118 mg, 60%). 1H NMR (600 MHz, DMSO): 1.47
– –
(9H, s, t-butyl), 2.12 (2H, t, CH2) 2.63–2.67 (8H, m, N-CH2 CH2 N),
– –
– –
– –
3.59 (2H, s, CH2 ), 3.61–3.70 (24H, m, PEG CH2 ), 3.73 (2H, s,
– –
– –
–
CH2 ), 3.76 (3H, s, OCH3), 3.86 (2H, s, CH2 ), 4.02 (2H, s,
– –
– –
CH2 ), 4.12 (2H, s, CH2 ), 4.24 (2H, s, CH2 ), 6.88 (2H, d,
–
ArH), 7.18 (2H, s, ArH Tetrac), 7.20 (2H, d, ArH), 7.79 (2H, s, ArH-
– –
– ––
s, t-butyl), 3.62 (2H, s, CH2 OH), 3.66–3.70 (24H, m, CH2
CH2-
PEG), 3.74 (2H, s, CH2-PEG), 3.87 (2H, s, CH2-PEG), 4.13 (2H, s,
Tetrac). 13C NMR (150 MHz, CDCl3): 27.3, 28.4, 39.2, 44.2, 52.5,
52.9, 53.5, 55.0, 57.7, 67.5, 68.8, 69.8, 70.4, 70.5, 70.8, 71.7, 90.3,
90.6, 114.7, 126.4, 128.8, 135.2, 141.1, 152.5, 153.2, 155.9, 158.1
170.7. MS (ESI+): C50H71I4N3O14 + H+ Calcd, 1446.1 found: 1445.9.
Anal. Calcd. (%): C, 41.54; H, 4.95; N, 2.91. Found (%): C, 41.14; H,
4.55; N, 2.21.
–
–
–
– –
CH2-PEG), 4.26 (2H, s, CH2 ), 4.84 (1H, s, NH), 6.89 (2H, d, ArCH),
7.21 (2H, d, ArCH). 13C NMR (150 MHz, CDCl3): 28.4, 44.2, 61.7, 67.4,
69.7, 70.3, 70.5, 70.6, 70.8, 72.6, 114.7, 128.3, 131.2, 155.9, 158.3. MS
(ESI+): C28H49NO11 + NH+4 Calcd 593.3, found 593.2. Anal. Calcd. (%):
C, 58.42; H, 8.58; N, 2.43. Found (%): C, 58.81; H, 8.11; N, 2.91.
Synthesis of 23-(4-(((tert-butoxycarbonyl)amino)methyl)phenoxy)-
3,6,9,12,15,18,21-heptaoxatricosyl 4-methylbenzenesulfonate (11)
To a solution of the 10 (150 mg, 0.26 mmol) in DCM (20 mL) at 0 ◦C
was added TEA (0.15 mL, 1.04 mmol) followed by TosCl (150 mg, 0.78
mmol). The reaction mixture was stirred at 0 ◦C for 30 min, then was
allowed to warm to rt over 2 h. The mixture was quenched with H2O (10
mL) and extracted with DCM (2 × 50 mL). The combined organics were
washed with H2O (50 mL), brine (50 mL), dried (Na2SO4), and
concentrated. The resulting material was purified with column chro-
matography [SiO2: DCM:MeOH (95:5)] to provide 11 as a white powder
(Yield: 150 mg, 79%). 1H NMR (600 MHz, CDCl3): 1.47 (9H, s, t-butyl),
4.18. Synthesis of methyl 2-(4-(4-(3-(4-(23–(4-(aminomethyl)
phenoxy)-3,6,9,12,15,18,21-heptaoxatricosyl)piperazin-1-yl)propoxy)-
3,5-diiodophenoxy)-3,5-diiodophenyl) acetate (13)
Compound 12 (100 mg) was dissolved in 3 mL anhydrous 1,4-
dioxane and 6 mL HCl (4 N in dioxane) was added to it and stirred at
rt. After 24 h the solvent was removed under reduced pressure, and the
oily residue was precipitated with diethyl ether to afford compound 13
as a white powder that was used for the next step without further pu-
rification (Yield: 90 mg, 96%). 1H NMR (600 MHz, DMSO): 1.98 (2H, s,
– –
–
–
CH2 ), 3.52–3.55 (28H, m, CH2-PEG), 3.57 (3H, s, OCH3), 3.58
–
–
–
– – – –
(8H, m, N CH2 CH2 N), 3.65 (2H, s, CH2 COOCH3), 3.75 (2H, s,
2.46 (3H, s, CH3), 3.62 (2H, s, CH2-PEG), 3.66–3.73 (24H, s, CH2-
–
–
– –
– –
– –
PEG), 3.86 (2H, s, CH2-PEG), 4.12 (2H, s, CH2-PEG), 4.17 (2H, s,
CH2 ), 3.84 (2H, s, Ar CH2 ), 3.95 (2H, m, CH2 ), 6.99 (2H, s,
ArH Guanidine), 7.17 (2H, s, ArCH-TAT), 7.42 (2H, s, ArH Guanidine),
7.88 (2H, s, ArCH-TAT), 8.34 (1H, s, NH). 13C NMR (150 MHz, CDCl3):
38.74, 52.6, 56.9, 67.8, 69.5, 81.6, 83.31, 83.38, 84.1, 106.1, 128.9,
139.9, 140.4, 144.9, 155.4, 165.9, 167.2, 172.9, 186.4. MS (ESI+):
–
– –
CH2-PEG), 4.25 (2H, s, CH2 ), 6.87 (2H, d, ArCH), 7.20 (2H, d,
ArH), 7.35 (2H, d, ArCH), 7.80 (2H, d, ArH). 13C NMR (150 MHz,
CDCl3): 21.6, 28.4, 43.4, 44.1, 61.5, 67.4, 68.6, 69.2, 69.7, 70.4, 70.6,
70.7, 70.8, 72.6, 90.6, 91.2, 114.7, 126.3, 127.9, 128.8, 129.8, 131.2,
131.9, 139.6, 144.8, 155.8, 158.1, 190.8. MS (ESI+): C35H55NO13S +
NH+4 Calcd, 747.4 found: 747.8. Anal. Calcd. (%): C, 57.60; H, 7.60; N,
1.92. Found (%): C, 57.12; H, 7.32; N, 1.66.
C
45H63I4N3O12 + H+ Calcd, 1346.1 found: 1345.7. Anal. Calcd. (%): C,
40.17; H, 4.72; N, 3.12 Found (%): C, 40.56; H, 5.12; N, 3.51.
4.19. Synthesis of methyl 2-(4-(4-(3-(4-(23–(4-((2,3-bis(tert-
butoxycarbonyl)guanidino)methyl) phenoxy)-3,6,9,12,15,18,21-
heptaoxatricosyl)piperazin-1-yl)propoxy)-3,5-diiodophenoxy)-3,5-
diiodophenyl) acetate (14)
4.16. Synthesis of methyl 2-(4-(3,5-diiodo-4-(3-(piperazin-1-yl)
propoxy)phenoxy)-3,5-diiodophenyl) acetate (19)
In a 2-neck flask, the methyl ester of tetrac (1 g, 1.3 mmol) and
Cs2CO3 (1 g, 3.25 mmol) were stirred in 50 mL ACN at 60 ◦C. After 30
min tert-butyl 4-(3-(methanesulfonyloxy)propyl)piperazine-1-carbox-
ylate hydrochloride (18) (0.47 g, 1.3 mmol) was added to the flask and
refluxed for 18 h. After completion of the reaction, it was filtered to
remove the Cs2CO3. Solvents were removed under reduced pressure. The
residue was dissolved in 5 mL anhydrous 1,4-dioxane and 5 mL HCl (4 N
in dioxane) was added to it and stirred at rt. After 2 h the solvent was
removed under reduced pressure, and the residue was precipitated with
diethyl ether to afford 19 as a white powder that was used for the next
step without further purification (Yield: 860 mg, 75%). 1H NMR (600
– – –
Compound 13 (120 mg, 0.09 mmol, 1 eq), N,N′-Di-Boc 1H-pyrazole-
1-carboxamidine (33 mg, 0.11 mmol, 1.2 eq) was dissolved in 5 mL
anhydrous DCM and then TEA (25 µL, 0.18 mmol, 2 eq) was added to the
solution. The reaction mixture was stirred at room temperature for 18 h.
After completion of the reaction the solvent was removed under reduced
pressure and the residue dissolved in DCM (30 mL). The organic phase
washed with 1% HCl (25 mL) and brine (25 mL) and then dried
(MgSO4). The solvent was removed under reduced pressure to yield 14,
which was purified with column chromatography [SiO2: DCM:MeOH
(95:5)] (Yield: 120 mg, 85%). 1H NMR (600 MHz, DMSO): 1.39 (9H, s, t-
– –– –
butyl), 1.52 (9H, s, t-butyl), 1.97 (2H, s, CH2
CH2 ), 2.51–2.67
MHz, DMSO): 2.77 (4H, m,
N CH2 CH2 N), 2.94 (4H, m,
– – –
–
– – –
– – –
–
OCH3),
(8H, m, N CH2 CH2 N), 3.49–3.51 (24H, m, CH2-PEG), 3.54 (2H,
N
CH2 CH2 N), 3.59 (2H, s, CH2 CH2
– – ), 3.62 (3–H, s, –
– –
– –
– –
–
–
s, CH2 ), 3.57 (2H, s, CH2 ), 3.64 (3H, s, OCH3), 3.72 (2H, m,
3.73 (2H, s, ArCH2 ), 3.86 (2H, s, CH2 ), 4.13 (2H, s, CH2 ), 7.32
(2H, s, ArCH-TAT), 8.04 (2H, s, ArCH-TAT). 13C NMR (150 MHz, CDCl3):
29.3, 34.3, 34.4, 37.9, 48.1, 51.7, 53.9, 70.3, 90.6, 91.2, 136.9, 141.7,
152.2, 153.2, 161.9 171.2. MS (ESI+): C22H24I4N2O4 + H+ Calcd, 888.8
found: 888.8. Anal. Calcd. (%): C, 29.75; H, 2.72; N, 3.15. Found (%): C,
29.63; H, 2.59; N, 3.76.
– –
–
CH2 ), 3.74 (2H, m, CH2 ), 3.90 (2H, s, CH2-PEG), 4.06 (2H, s,
–
CH2 COOCH3), 4.43 (2H, s, Ar-CH2-Guanidine), 6.92 (2H, s, ArH
Guanidine), 7.14 (2H, s, ArCH-TAT), 7.21 (2H, s, ArH Guanidine), 7.88
(2H, s, ArCH-TAT), 8.54 (1H, s, NH). 13C NMR (150 MHz, CDCl3): 28.3,
42.9, 44.5, 44.9, 52.3, 52.9, 54.8, 57.4, 67.4, 68.4, 69.7, 70.3, 70.5,
70.8, 71.4, 79.3, 83.9, 90.2, 114.8, 126.4, 129.1, 141.9, 151.1, 155.9,
158.3, 164.6, 175.9. MS (ESI+): C56H81 4
I
N5O16 + H+ Calcd, 1588.2
4.17. Synthesis of methyl 2-(4-(4-(3-(4-(23–(4-(((tert-butoxycarbonyl)
amino)methyl)phenoxy)-3,6,9,12,15,18,21-heptaoxatricosyl)piperazin-
1-yl)propoxy)-3,5-diiodophenoxy)-3,5-diiodophenyl)acetate (12)
found: 1588.2. Anal. Calcd. (%): C, 42.36; H, 5.14; N, 4.41. Found (%):
C, 42.24; H, 5.22; N, 4.48.
Compound 11 (100 mg, 0.14 mmol, 1 eq), 17 (120 mg, 0.14 mmol, 1
eq) and K2CO3 (90 mg, 0.55 mmol, 1 eq) were refluxed with 30 mL ACN
for 18 h. After completion of the reaction, it was filtered to remove the
K2CO3. Solvents were removed under reduced pressure, and the oily
9

O.O. Karakus et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116250
4.20. Synthesis of 2-(4-(4-(3-(4-(23-(4-(guanidinomethyl)phenoxy)-
3,6,9,12,15,18,21-heptaoxatricosyl)piperazin-1-yl)propoxy)-3,5-
diiodophenoxy)-3,5-diiodophenyl)acetic acid (15)
with a hand homogenizer. After centrifugation, solvent was evaporated
under a nitrogen stream at 50 ◦C. Extracts were reconstituted with 50
μL
of acetonitrile/water (80/20 v/v). Samples were immediately injected
into LC-MS/MS for quantification of analytes on the same day.
Compound 14 (100 mg) was dissolved in 3 mL 1,4-dioxane:water
(3:1) and 3 mL conc. HCl added to it and stirred at rt. After 24 h the
solvent was removed under reduced pressure, and the oily residue was
precipitated with diethyl ether to afford compound 15 as a white pow-
der, which was purified with reverse phase column chromatography.
[C18: MeOH/Water (55:45)] (Yield: 60 mg, 70%). Purity > 98%, tR =
27.5 min [analytical HPLC/gradient: 50–95% MeOH in H2O (0.1%
6.1. Cell culture
Neuroblastoma cells SK-N-F1 (ATCC) were grown with IMDM sup-
plemented with 10% FBS, 10% penicillin, and 1% streptomycin. Cells
were cultured at 37 ◦C to subconfluence and treated with 0.25% (w/v)
trypsin/ethylenediaminetetraacetic acid (EDTA) to induce cell release
from flask. The cells were washed with culture medium that was free of
phenol red and fetal bovine serum and counted.
TFA), 50 min, flow rate 1 mL/min, Pursuit XRs C18 column (150 × 4.6
1
– –– –
mm)]. H NMR (600 MHz, DMSO): 2.09 (2H, m, CH2
CH2 ), 2.58
– – –
– – –
(2H, m, CH2 CH2 ), 2.65 (8H, m, N CH2 CH2 N), 3.20 (2H, s,
– – –
– –
–
CH2 CH2 ), 3.51 (2H, s, CH2 COOH), 3.55–3.62 (24H, m,
6.2. Competitive binding
–
–
CH2 PEG), 3.69 (2H, s, CH2-PEG), 3.83 (2H, s, CH2-PEG), 3.99
– –
–
– –
(2H, s, CH2 PEGCH2 PEG), 4.10 (2H, s, CH2 PEG), 4.26 (2H, s,
The binding affinities of dI-BG-P400-TAT (7a), dM-BG-P400-TAT (7b),
– –
Ar CH2 Guanidine), 6.89 (2H, s, ArH Guanidine), 7.15 (2H, s, ArH
BG-P400-PAT (15) and BG-P400-TAT to purified
αvβ3 were measured
using previously described methods with slight modifications37. The
–
–
Guanidine), 7.58 (2H, s, ArCH TAT), 7.81 (2H, s, ArCH TAT), 8.23
(1H, s, NH), 8.73 (1H, s, COOH). 13C NMR (150 MHz, CDCl3): 26.7, 39.9,
41.2, 44.1, 52.2, 52.7, 54.4, 57.1, 67.2, 68.2, 69.3, 70.2, 71.3, 90.1,
114.6, 126.1, 128.4, 138.1, 140.9, 151.1, 152.7, 157.7, 158.1, 164.4,
173.9. MS(ESI+): C45H63I4N5O12 + H+ Calcd, 1373.9 found:1373.5.
Anal. Calcd. (%): C, 39.35; H, 4.62; N, 5.10. Found (%): C, 39.12; H,
4.89; N, 5.67.
purified αvβ3 (1 µg/mL) was coated to polystyrene micro-titer plate
wells at 4 ◦C overnight, and then the wells were blocked with 3% BSA for
2 h at room temperature. Wells were washed with buffer A (50 mM Tris/
HCl, 100 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1% BSA), and anti-αvβ3
(conjugated with biotin 1:1000 in buffer A) was added and incubated for
1 h at room temperature. Increasing concentrations of 7a, 7b, and 15
were added in the presence or absence of fibrinogen and incubated for 2
h at room temperature, and then wells were washed 3 times with buffer
A and incubated with a streptavidin HRP conjugate (1:1000 in buffer A)
for 1 h at room temperature. Finally, wells were washed 3 times with
buffer A and 100 µL peroxidase substrate 3,3′,5,5′-tetramethylbenzidine
(TMB) was added, and the reaction was terminated after 30 min with 50
µL of 450 nm stop solution for TMB. Absorbance was determined at 450
nm with a plate reader. The best-fit IC50 values for the compounds were
calculated by fitting the data with nonlinear regression using GraphPad
4.21. Biological Materials
Bovine serum albumin (BSA) and fetal bovine serum (FBS) were
purchased from Sigma-Aldrich, anti-αvβ3 was purchased from Bioss Inc.,
streptavidin HRP conjugate was from ThermoFischer Scientific, and
Iscove’s Modified Dulbecco’s Medium (IMDM) was from Hyclon.
GraphPad Prism software was used for calculation of IC50 values.
Prism38
.
5. LC-MS/MS instrumentation
6.3. Cellular uptake of BG-P-TAT derivatives
An API-4000 mass spectrometer (Sciex, Framingham, MA) equipped
with a Shimadzu UPLC system (Kyoto, Japan) was used for LC-MS/MS
SK-N-FI cells were treated with MIBG, BG-P400-TAT, 7a, 7b, 15 and
20 at 1 µM concentration. After 15 min, 30 min and 60 min incubation
then cells were collected and washed with PBS followed by separating
cells from media by centrifugation at 5,000g for 10 min.
analyses. A Kinetex 2.6 μm Biphenyl 100 LS column (50 × 2.1 mm,
Phenomenex, Torrance, CA) was used for reversed-phase separation.
Mobile phases were (A) water containing 0.1% formic acid and 5%
acetonitrile and (B) acetonitrile. The flow rate was 0.35 mL/min, and the
gradient was linear from 25% B to 95% B for 2–4 min. The oven tem-
perature was 40 ◦C and the injection volume was 5
μL. Electro-spray
6.4. Animals
ionization (ESI) was used in positive MRM mode. Mass transitions for
analytes were: Q1/Q3: 1285.0/133.0 (BG-P400-TAT); 1535.0/291.1
(7a); 1344.9/139.2 (7b); 1374.3/89.0 (15); 1343.3/1225.9 (20);
275.09/275.5 (MIBG). The operative parameters of the mass spec-
trometer were as follows: declustering potentials (DP): 75 V; entrance
potentials (EP): 10; collision energies (CE): 90 eV; collision cell exit
potential (CXP), 8 V; curtain gas (CUR), 30 psi; gas 1 (GS1, nebulizer
gas) 30 psi; gas 2 (GS2, heater gas) 30 psi; ion spray voltage (IS), 5000 V;
temperature (TEM), 500 ◦C; collision activate dissociation (CAD) gas: 12
psi; dwell time: 150 ms. Nitrogen was used for the gases. Standard
curves were obtained using standard solutions of the analytes with
concentrations of 1000, 300, 100, 300, 100, 30, 10, 3, and 1 ng/mL. The
regression curves of the LC-MS/MS method for standard solutions was
linear or quadratic (r = 0.99 or more) from a concentration of 1 to 1000
ng/mL for each analyte. The LOQ was estimated to be 10 ng/mL or less
under the current conditions for each analyte. Accuracy ranged
90–100%; and recovery was more than 90% or more for each analyte.
Immunodeficient female NCr nude homozygous mice aged 5–6
weeks and weighing 20–25 g were purchased from Taconic Biosciences,
Inc. All animal studies were conducted at the animal facility of the
Veteran Affairs (VA) Medical Center in accordance with institutional
guidelines for humane animal treatment and approved by the VA
IACUUC. Four mice in each group were maintained under specific
pathogen-free conditions and housed under controlled conditions of
temperature (20–24 ◦C) and humidity (60–70%) and 12 h light/dark
cycle with ad libitum access to water and food. Mice were allowed to
acclimatize for 5 days and injected with 2 million cells for each implant.
The tumor volumes were allowed to reach 500 mm3 and then com-
pounds were administrated subcutaneously (s.c.) daily for 21 days. At
the end of the study, animals were sacrificed, and tumors were collected.
6.5. Histopathology analysis
Collected tumors were fixed in 10% formalin for at least 48 h. The
fixed samples were placed in plastic cassettes and dehydrated using an
automated tissue processor. The processed tissues were embedded in
paraffin wax and blocks trimmed and sectioned to about 5 × 4 µm size
using a microtome. The tissue sections were mounted on glass slides
6. Sample preparation of cell samples with liquid–liquid
extraction
Cell samples were thawed and homogenized with acetonitrile (1 mL)
10

O.O. Karakus et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116250
8
9
Piskareva O, Harvey H, Nolan J, et al. The development of cisplatin resistance in
neuroblastoma is accompanied by epithelial to mesenchymal transition in vitro.
Cancer Lett. 2015;364:142–155.
using a hot plate and subsequently treated in the order of 100%, 90%
and 70% ethanol for 2 min each. Finally, the tissue sections were rinsed
in tap water, stained with the Harris’s hematoxylin and eosin (H&E),
and examined under a light microscope.
Schlessinger A, Geier E, Fan H, et al. Structure-based discovery of prescription drugs
that interact with the norepinephrine transporter, NET. Proc Natl Acad Sci U S A.
2011;108:15810–15815.
10 Streby KA, Shah N, Ranalli MA, Kunkler A, Cripe TP. Nothing but NET: a review of
norepinephrine transporter expression and efficacy of 131I-mIBG therapy. Pediatr
Blood Cancer. 2015;62:5–11.
6.6. Preparation of ligands for docking
11 Sharp SE, Trout AT, Weiss BD, Gelfand MJ. MIBG in Neuroblastoma Diagnostic
Imaging and Therapy. Radiographics. 2016;36:258–278.
All compounds (7a, 7b, 15) were drawn using the 2D and 3D options
of Chem Draw 12.0 and saved in mol2 format. The created .pdbfile was
submitted to AutoDock Vina (version 1.1.2) to set number of torsion and
for .pdbqt file construction.
12 Zhang H, Xie F, Cheng M, Peng F. Novel Meta-iodobenzylguanidine-Based Copper
Thiosemicarbazide-1-guanidinomethylbenzyl Anticancer Compounds Targeting
Norepinephrine Transporter in Neuroblastoma. J Med Chem. 2019;62:6985–6991.
13 Kayano D, Kinuya S. Iodine-131 metaiodobenzylguanidine therapy for
neuroblastoma: reports so far and future perspective. Sci World J. 2015;2015,
189135.
6.7. Target selection and preparation
14 Holtke C. isoDGR-Peptides for Integrin Targeting: Is the Time Up for RGD? J Med
Chem. 2018;61:7471–7473.
The X-ray crystal structure of integrin αvβ3 (PDB:1L5G) with 3.20 Å
15 Neubauer S, Rechenmacher F, Brimioulle R, et al. Pharmacophoric modifications
lead to superpotent alphavbeta3 integrin ligands with suppressed alpha5beta1
activity. J Med Chem. 2014;57:3410–3417.
resolution was obtained in .pdb format from the Protein Data Bank.
Before performing the docking calculation, all water molecules were
removed from the crystal structure, all hydrogen atoms and Gasteiger
charges were added to every atom of the protein, and rotatable bonds
were selected using AutoDock Tools-1.5.6. Auto grid box was generated
with dimensions 50 × 50 × 50 Å with grid spacing 0.375 Å. The grid box
is based on the inhibitory site residue used for the docking calculation.
16 Desgrosellier JS, Cheresh DA. Integrins in cancer: biological implications and
therapeutic opportunities. Nat Rev Cancer. 2010;10:9–22.
17 Klim JR, Fowler AJ, Courtney AH, et al. Small-molecule-modified surfaces engage
cells through the alphavbeta3 integrin. ACS Chem Biol. 2012;7:518–525.
18 Kapp TG, Rechenmacher F, Neubauer S, et al. A Comprehensive Evaluation of the
Activity and Selectivity Profile of Ligands for RGD-binding Integrins. Sci Rep. 2017;7:
39805.
19 Mas-Moruno C, Rechenmacher F, Kessler H. Cilengitide: the first anti-angiogenic
small molecule drug candidate design, synthesis and clinical evaluation. Anticancer
Agents Med Chem. 2010;10:753–768.
6.8. Molecular docking
20 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.
21 Davis PJ, Davis FB, Mousa SA, Luidens MK, Lin HY. Membrane receptor for thyroid
hormone: physiologic and pharmacologic implications. Annu Rev Pharmacol Toxicol.
2011;51:99–115.
After running docking calculations, nine conformers were generated
for each compound and all the conformers were ranked in a log file
based on their docking score. Molecular docking interactions were
analyzed using PyMOL (The PyMOL Molecular Graphics System,
Version 1.7.4, Schrodinger LLC).
22 Davis PJ, Lin HY, Sudha T, et al. Nanotetrac targets integrin alphavbeta3 on tumor
cells to disorder cell defense pathways and block angiogenesis. Onco Targets Ther.
2014;7:1619–1624.
23 Lin HY, Mousa SA, Davis PJ. Demonstration of the Receptor Site for Thyroid
Hormone on Integrin alphavbeta3. Methods Mol Biol. 2018;1801:61–65.
24 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.
25 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.
Declaration of Competing Interest
The authors declare the following financial interests/personal re-
lationships which may be considered as potential competing interests:
‘S.A.M. and O.O.K are inventors on patents related to BG-P-TAT and S.A.
M. is a founder of Nanopharmaceutical LLC, which is developing anti-
cancer drugs. The remaining author K.G., K.F. declares no conflict of
interest’.
26 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.
27 Luidens MK, Mousa SA, Davis FB, Lin HY, Davis PJ. Thyroid hormone and
angiogenesis. Vascul Pharmacol. 2010;52:142–145.
Acknowledgements
28 Mousa SA. Compositions of dual thyrointegrin antagonists and use in vascular-
associated disorders US; 2017.
29 Bridoux A, Khan RA, Chen C, et al. Design, synthesis, and biological evaluation of
bifunctional thyrointegrin inhibitors: new anti-angiogenesis analogs. J Enzyme Inhib
Med Chem. 2011;26:871–882.
Funding was received from both NanoPharmaceuticals LLC,
Rensselaer, NY and from the Pharmaceutical Research Institute (PRI) at
Albany College of Pharmacy and Health Sciences. We thank Dr. Keating,
Medical Editor/Writer at PRI for her excellent editorial review of the
manuscript.
30 Glinskii AB, Glinsky GV, Lin HY, et al. Modification of survival pathway gene
expression in human breast cancer cells by tetraiodothyroacetic acid (tetrac). Cell
Cycle. 2009;8:3562–3570.
31 Mousa SA, Bergh JJ, Dier E, 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.
32 Yalcin M, Dyskin E, Lansing L, et al. Tetraiodothyroacetic acid (tetrac) and
nanoparticulate tetrac arrest growth of medullary carcinoma of the thyroid. J Clin
Endocrinol Metab. 2010;95:1972–1980.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116250.
33 Rajabi M, Godugu K, Sudha T, Bharali DJ, Mousa SA. Triazole Modified
Tetraiodothyroacetic Acid Conjugated to Polyethylene Glycol: High Affinity
Thyrointegrin alphavbeta3 Antagonist with Potent Anticancer Activities in
Glioblastoma Multiforme. Bioconjug Chem. 2019;30:3087–3097.
34 Mousa SA, Rajabi M, Karakus OO. Composition and method for dual targeting in
treatment of neuroendocrine tumors, US; 2019.
References
1
Maris JM, Hogarty MD, Bagatell R, Cohn SL. Neuroblastoma. Lancet. 2007;369:
2106–2120.
35 Karakus OO, Godugu K, Rajabi M, Mousa SA. Dual Targeting of Norepinephrine
Transporter (NET) Function and Thyrointegrin alphavbeta3 Receptors in the
Treatment of Neuroblastoma. J Med Chem. 2020;63:7653–7662.
2
3
Colon NC, Chung DH. Neuroblastoma. Adv Pediatr. 2011;58:297–311.
Friedman GK, Castleberry RP. Changing trends of research and treatment in infant
neuroblastoma. Pediatr Blood Cancer. 2007;49:1060–1065.
36 Mousa SA, Rajabi M. Non-Cleavable Polymer Conjugated with
Antagonists, US; 2019.
αVβ3 Integrin Thyroid
4
5
Brodeur GM, Iyer R, Croucher JL, Zhuang T, Higashi M, Kolla V. Therapeutic targets
for neuroblastomas. Expert Opin Ther Targets. 2014;18:277–292.
Morgenstern DA, Baruchel S, Irwin MS. Current and future strategies for relapsed
neuroblastoma: challenges on the road to precision therapy. J Pediatr Hematol Oncol.
2013;35:337–347.
37 Kouns WC, Kirchhofer D, Hadvary P, et al. Reversible conformational changes
induced in glycoprotein IIb-IIIa by a potent and selective peptidomimetic inhibitor.
Blood. 1992;80:2539–2547.
38 Wilkinson JH. Synthesis of some possible metabolites of thyroxine and
triiodothyronine. Biochem J. 1956;63:601–605.
6
7
Fruci D, Cho WC, Nobili V, Locatelli F, Alisi A. Drug Transporters and Multiple Drug
Resistance in Pediatric Solid Tumors. Curr Drug Metab. 2016;17:308–316.
Li C, Yang C, Wei G. Vandetanib inhibits cisplatinresistant neuroblastoma tumor
growth and invasion. Oncol Rep. 2018;39:1757–1764.
11