Boron Accumulation in Brain Tumor Cells through Boc-Protected Tryptophan as a Carrier for Boron Neutron Capture Therapy

Article abstract of DOI:10.1021/acsmedchemlett.0c00064

Boron neutron capture therapy (BNCT) is a binary therapeutic approach. Nonradioactive boron-10 atoms accumulated in tumor cells combining with the neutron beams produce two highly energetic particles that could eradicate the cell that takes it and the neighboring cells. Small molecules that carry boron atom, e.g. 5- and 6-boronated and 2,7-diboronated tryptophans, were assessed for their boron accumulation in U87-MG, LN229, and 3T3 for BNCT. TriBoc tryptophan, TB-6-BT, shows boron-10 at 300 ppm in both types of tumor cells with a tumor to normal ratio (T/N) of 5.19-5.25 (4 h). TB-5-BT and DBA-5-BT show boron-10 at 300 ppm (2 h) in U87-MG cells. TB-5-BT exerts a T/N of >9.66 (1 h) in LN229 compared with the current clinical boronophenyl alanine with a highest T/N of 2.3 (1 h) and accumulation concentration of <50 ppm. TB-5-BT and TB-6-BT warrant further animal study.

Full text of DOI:10.1021/acsmedchemlett.0c00064

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Letter
Boron accumulation in brain tumor cells through Boc-protected
tryptophan as a carrier for boron neutron capture therapy
Chun-Ming Chio, Ying-Cheng Huang, You-Cheng Chou, Fu-Chun Hsu, Yen-Buo Lai, and Chung-Shan Yu
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00064 • Publication Date (Web): 16 Mar 2020
Downloaded from pubs.acs.org on March 16, 2020
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Boron accumulation in brain tumor cells through Boc-protected
tryptophan as a carrier for boron neutron capture therapy
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a,†
b,†
a
a
a
Chun-Ming Chio, Ying-Cheng Huang, You-Cheng Chou, Fu-Chun Hsu , Yen-Buo Lai and
Chung-Shan Yu
a,c*
aDepartment of Biomedical Engineering and Environmental Sciences, National Tsinghua University, Hsinchu 300, Taiwan
bDepartment of Neurosurgery, Chang-Gung Memorial Hospital at Linkou, Chang Gung University, Taiwan.
cInstitute of Nuclear Engineering and Science, National Tsing-Hua University, Hsinchu, 300, Taiwan.
KEYWORDS BNCT, tryptophan, boronodehalogenation, tumor to normal, U87-MG, protecting group
ABSTRACT: Boron neutron capture therapy (BNCT) is a binary therapeutic approach. Nonradioactive boron-10 atom accumulated
in tumor cells combining with the neutron beams produce two high energetic particles that could eradicate the cell that takes it and
the neighboring cells. Small molecules that carry boron atom e.g. 5- and 6-boronated and 2,7-diboronated tryptophans were assessed
for their boron accumulation in U87-MG, LN229 and 3T3 for BNCT. TriBoc tryptophan, TB-6-BT, shows boron-10 at 300 ppm in
both types of tumor cells with a tumor to normal ratio (T/N) of 5.19-5.25 (4 h). TB-5-BT and DBA-5-BT show boron-10 at 300 ppm
(2 h) in U87-MG cells. TB-5-BT exerts a T/N of > 9.66 (1 h) in LN229 compared with the current clinical boronophenyl alanine with
a highest T/N of 2.3 (1 h) and accumulation concentration of < 50 ppm. TB-5-BT and TB-6-BT warrant a further animal study.
Introduction
the transport of large neutral amino acids through the plasma
4
,
5
membrane.
LAT1 exhibits high affinity for several
As it is the most common type of primary brain tumor in
adults, glioblastoma multiforme (GBM) is classified as a World
Health Organization grade IV astrocytoma. Its estimated
incidence in North America is 3.0 per 100,000 people according
to age-adjusted statistics. Due to its aggressive malignancy,
GBM has an estimated 2-year survival rate of 8.7%. Even after
maximum treatment, the median duration of survival is 12–18
nutritionally essential amino acids such as leucine, isoleucine,
3
valine, phenylalanine, tryptophan and methionine. Thus, one
such amino acid analog of phenylalanine bearing a boron-10
atom can be triggered to release energy upon irradiation with
neutron beams.
Boron neutron capture therapy (BNCT) is a targeted radiation
therapy that significantly increases the therapeutic ratio relative
to conventional radiotherapeutic modalities (Fig. 1). BNCT is a
1
months. Whereas the tumor pathogenesis has been well
deciphered, the median overall survival of patients has
increased by only 3.3 months (from 11.3 months to 14.6
months) over the past 25 years. This disappointing prognosis is
mostly associated with the prevalent recurrence of tumors after
initial treatment with the maximum safe surgical resection,
1
0
binary approach: a boron-10 ( B)-labeled compound is
1
0
administered that delivers high concentrations of B to the
target tumor relative to surrounding normal tissues. This is
followed by irradiation with thermal neutrons or epithermal
neutrons which become thermalized at a depth in tissues. The
1
radiotherapy, and chemotherapy. Moreover, the cause of death
7
of glioma patients is not a distant metastasis but the failure of
local control of the tumor. When the tumor repeatedly happens,
additional systemic and local therapies as well as repeat surgery
are commonly considered. While each of these therapies has
potential benefits, each also carries associated risks, particularly
short range (5-9 um) of the  and Li particles released from the
1
0
7
B(n, ) Li neutron capture reaction make the
1
0
6
microdistribution of B of critical importance in therapy.
Thermal neutrons
2
surgery.
The invasive nature of glioma often prevents total surgical
excision, hence adjuvant therapy for the residual tumor is
10B
Cancer cell death from nuclear fission
essential.
However,
conventional
chemotherapy,
7Li
4He
immunotherapy, and radiotherapy have been proven to be of
3
limited value. The highly proliferating malignant tumor cells
may require significant substrates such as sugars and amino
acids. If a specific upregulation of the amino acid transport
system in malignant tumor cells does exist, it could be a
Tumor cell
Normal cell
3
molecular target for therapy.
Figure 1. Illustration of the basic principles of boron neutron
capture therapy (BNCT).
The leucine-type amino acid transporter (LAT1) is a Na-
independent neutral amino acid transport agent and essential for
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O
O
One of the advantages of a binary system is the potential that
O
O
O
O
O
B
*
O
B
*
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
each component can be manipulated independently. Only when
the timing is correct for achieving the maximal cytotoxic effect
upon malignant cells, together with the maximum tolerated
dose to contiguous normal cells, are the two components
juxtaposed. If this approach is to succeed, it is essential that at
least one of the components be confined rather specifically to
tumor cells, while the second component is exposed to all cells
O
B
NBoc
Ac
NHAc
NBoc2
O
O
O
4
2
N
N
N
Boc
H
Boc
N-acetyl-5-boronopinacol
tryptophan (NA-5-BT) 3
diboc acetyl-5-boronopinacol
tryptophan (DBA-5-BT) 2
triboc-5-boronopinacol
tryptophan (TB-5-BT) 1
O
O
O
O
N3
O
O
NBoc2
O
B
O
B
*
N
O
NH2
H
O
N
O
B
O
B
7
Boc
in a particular area.
O
O
N
H
The potential for developing NCT rests upon the fact that the
various normal elemental constituents of tissue have very low
capture cross-section values for thermal neutrons compared
with boron-10 (3838 barns). The ideal boron agents should have
the following features: (1) low intrinsic toxicity; (2) high tumor
triboc-6-boronopinacol
tryptophan (TB-6-BT) 5
5
-boronopinacol
azido-2,7-diboronopinacol
tryptophan (AZ-2,7-DBT) 6
tryptophan (5-BT) 4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
OH
HO
NH2
B
OH
10
uptake (~ 20–50 μg B) and low normal tissue uptake, ideally
Boron phenylalanine (BPA)
with tumor to normal tissue and tumor to blood boron
concentration ratios of > 3:1; and (3) relatively rapid clearance
from the blood and normal tissues along with persistence in the
Figure 2. Boronopinacoltryptophan derivatives described in this
work.
8
tumor for at least several hours during neutron irradiation. The
Electrophilic substitution on the indole-based tryptophan is
apt to take place at the pyrrole ring or the 5- or 6- position of
the benzene ring. However, borylation on 5- and 6- positions
via regioselective control over a common intermediate remains
difficult. In general, the reactivity of the two fused rings has
been speculated (Fig. 3). The intermediates formed from 4- or
7-substitution lead to the entire loss of aromaticity of both rings.
Experimental data and theoretical calculation predicts that
the reactivity under the catalysis of Pd(0) is in an order of C-4>
C-6 > C-5 > C-7. Furthermore, Pd(0)-catalyzed oxidative
addition for the boronopinacol group at the six-membered ring
moiety suffers from other disadvantages. For example, Pd(0) is
present boron-bearing compounds used for BNCT are briefly
divided to two types: small molecules e.g. boronophenylalanine
(
BPA) and cage-like boron clusters e.g. sodium borocaptate
1
0
8
2 12
(Na B H11SH, BSH). The clinically studied amino acid
1
6
analog BPA exhibits acceptable in vivo tumor targeting and has
proven to be sufficient in the treatment of brain tumors in
9
humans. Although BPA and BSH remain unsatisfactory
1
7
18
according to the above criteria, these two agents are the
clinically used drugs. Other amino acid derivatives of
1
1
11
tryptophan such as - C-methyl-L-tryptophan ( C-AMT), are
amino acid PET tracers that can measure tryptophan
metabolism via the immunomodulatory kynurenine pathway.
It has been shown that C-AMT accumulates because of both
transport and metabolism in both untreated and recurrent World
1
0
1
1
2
deactivated by electron donors, such as -NH through its
coordination to Pd, thereby rendering the metal insertion into
R-X inefficient. The bulky electron-withdrawing Boc as the
Health Organization (WHO) grade II–IV gliomas.
A
2
protecting group for –NH not only blocks its capacity for
standardized uptake value (SUV) of 2.68 is distributed over the
brain tumor along with a lesion to cortex ratio of 2.14 from a
clinical study for 21 adults.
coordination but also weakens the electron delocalization to the
halogenated carbon, thereby retaining the polarizability of the
C-Br bond (Scheme 1 (A)).
Although liposomal and nanoparticle compounds have been
intensively developed as boron carriers, the accumulated boron
X
11, 12
in brain tumors remains low, i.e. < 0.4 % injection dose/g.
X
+
X
10
Satisfactorily high concentrations of 50 ppm B have been
achieved recently. However, the preparation is relatively
N
N
N
N
1
3
H
H
H
H
X
uncommon to organic chemists, i.e. via grinding of a micron-
2
- or 3-substitution 5- or 6-substitution
4-substitution 7-substitution
(b)
1
0
sized B-enriched rock-hard powder to give the particle size of
5 nm. Hence, we are encouraged to prepare single-boron
(a)
8
Figure 3. Electrophilic substitution prefers the route via a cation
intermediate (a) over that of a cation (b).
carried small molecule via the common covalent-bond
formation with the hope of improving tumoral accumulation.
With respect to the similar features to those of BPA such as the
transportation mechanism via LAT-1, essential nutrition source
and insignificant cytotoxicity, we intend to prepare the amino
acid-like boron derivatives of tryptophan (Fig. 2). The
pseudotryptophan structure can be regarded as a prodrug that
can diffuse through the BBB layer via LAT-1 mediated
O
O
NR'2
Br
O
*
O
NR2
HN
Br
RO
O
B
b
*
O
N
N
O
c
H
O
R'
N
O
4:1, d,l-mixture
R
O
Br
O
TB-5-BT 1 R = Boc
5-BT 4 R = H
1
4
5-bromotryptophan
transportation.
This is similar to the concept of
a
HN
R=H, R'=H
a
N
H
pseudonutrient that has been applied by DOPA, a clinically
7
R=CH3, R'=Boc
1
5
used prodrug of dopamine. Hence, the protecting groups are
optionally attached to examine their effects on assisting the
penetration across the cell membrane for accumulation. The
present study is aimed to prepare boronopinacol tryptophan
derivatives with protecting groups for assessing their potencies
as BNCT agents. The relevant in-vitro assay includes the
accumulation, cytotoxicity and the eradication effects through
neutron irradiation.
Pd(0) catalyst
(B)
(A)
Scheme 1. (A) Deactivating Pd(0) through electron delocalization.
B) TriBoc protected 5-boronopinacol tryptophan analog TB-5-BT
and its deprotection leading to 5-BT 4. Reaction conditions a:
SOCl , CH OH; Boc O, DMAP, CH CN, 50% over two steps; b:
diborono pinacol, Ferocene PdCl , DMSO, KOAc, 100 C, 2 h,
90%. c. As listed in Table 1.
(
1
2
3
2
3
o
2
2
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O
O
O
NR
NR
O
1
2
3
4
5
6
7
8
9
Br
NH
c
O
e
O
B
*
O
Results and Discussion
Chemical Synthesis
Br
d
+
N
O
O
O
H
O
N
N
5-boromoindole
R
R
9
Starting from
a
d,l-mixture of the commercial 5-
10 R = H
NA-5-BT 3 R = H, from 10
b
O
bromotryptophan, the subsequent Miyamura borylation
proceeded smoothly when an appropriate solvent was chosen,
e.g., DMSO (Scheme 1 (A)). After several batches of
optimization, the boronopinacol tryptophan TB-5-BT 119 was
obtained in a greater than 90% yield.
11 R = Boc
OH
DBA-5-BT 2 R = Boc, from 11
O
O
a
NH
+
O
O
HO
NH2
8
Scheme 2. 5-boronopinacol tryptophan analog DBA-5-BT 2 and
NA-5-BT 3 prepared from an indole analog. Reaction conditions a:
Whereas t-Boc in peptide synthesis is typically removed by
an acid such as trifluoroacetic acid (pKa = 0.30),
trimethylsilyl iodide (TMSI)
deprotection of boronopinacol tryptophan derivatives. The
inherent instability of boron groups associated with the indole
ring prevents the use of acidic conditions. In our hands, a milder
3 4 a
acid H PO (pK = 2.15) also failed. Only under the modified
conditions of TMSI at 0 °C can the three Boc groups be
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
0, 21
only
o
2
2, 23
toluene, reflux, Soxhlet, 55%. b. CH
3
I, K
, 0 C, 85%. d. Boc
9%. e: diborono pinacol, Pd(fer) Cl , DMSO, KOAc, 100 C, 2h,
4% for DBA-5-BT 2; 65% for NA-5-BT 3 from a consecutive
2
CO
3
, acetone, 60-65 C,
has been reported for the
o
93%. c. EtAlCl
8
6
3
, CH
2
Cl
2
2 3
O, DMAP, CH CN,
o
2
2
repeated reaction.
removed (Table 1). Whereas the in-situ generation of TMSI
Another synthetic route to prepare a constitutional isomer of
(entry 1 and 2) can eliminate the moisture disturbance and
the above TB-5-BT 1, i.e., TB-6-BT 5, is illustrated in Scheme
3
3
should be capable of increasing the yield, the mud-like mixture
3. Starting from the nitration reaction of the tryptophan, the
desired nitro tryptophan was obtained as two constitutional
isomers with a ratio ofp-12 : o-12 = 3 : 1. Due to the significant
yield advantage by tri-Boc protected iodotryptophan, i.e. 70 -
90 % vs. 30 - 60 % yield (triBoc vs. diBoc), the tri-Boc
+
-
[
3 3 3
CH CN Si(CH ) )I was difficult to quantitatively assess for
equivalence control. On the other hand, the commercial solution
of TMSI was straightforward but required the reaction to be
terminated in time to obtain an acceptable yield.
22,
tryptophan precursor 14 was chosen for the next preparation.
Table 1. Conditions for the removal of triBoc moieties of TB-5-BT
34, 35
1
to give 5-BT 4.
HO
NBoc2
NHBoc
TMSI
Reaction
time (h)
HO
O
Entry
Reagent
Temperature
Solvent
CHCl
CN CN
Yield
R1
R1
R1
O
O
O
O
O
(eq)
a
b
NH2
Lit.
1
TMSI
TMSCl/NaI
TMSCl/NaI
TMSI
rt
rt
3
8
2
2
81%22
16%
27%
42%
68%
NH2
+
N
R2
N
R2
N
R2
Boc
N
H
H
Boc
3
8
4
4
1
2 (mixture)
o-14 R1 = NO , R2 = H
p-14 R1 = H, R2 = NO2
o-13 R1 = NO2, R2 = H
p-13 R1 = H, R2 = NO2
2
2
rt
CHCl
CHCl
CHCl
3
3
3
1
l-tryptophan
o-12 R1 = NO2, R2 = H
p-12 R1 = H, R2 = NO2
3
rt
0.5
1
c
para : ortho = 3 : 1
4
TMSI
0℃
4 + 2
NBoc2
NBoc2
R1
NBoc2
O
R1
For expanding the structural variation and pursuing an
inexpensive 5-bromo tryptophan source, an attempt was made
O
e
d
O
O
O
O
R2
N
R2
N
Boc
to assemble 5-bromoindole and an in-situ generated compound
O
B
O
N
Boc
Boc
o-15 + p-15
2
4
o-16+ p-16
8
(Scheme 2). However, we were unable to reproduce the
o-15 R1 = NH2, R2 = H
p-15 R1 = H, R2 = NH2
TB-6-BT 5
isomer was not found
o-16 R1 = I, R2 = H
p-16 R1 = H, R2 = I
results and only found oligomerized byproducts. Compound 8
is typically obtained through the condensation between
acetamide and pyruvic acid using the Deanstark setup.
Scheme 3. TriBoc protected 6-boronopinacol tryptophan analog
TB-6-BT 5 prepared from tryptophan. Reaction conditions a:
2
5-28
In
our hands, Soxhlet extraction also proceeded smoothly.
Compound 10 obtained from Friedel Crafts alkylation was
identified as a d,l-mixture in a ratio of 3:1 from HPLC analysis.
The final borylation afforded a similar yield of products DBA-
HNO
3 and 30% for 14 from individual preparation; c: Zn, AcOH, 92%;
d: NaNO , HCl (aq), KI, I , THF, 51%; e: diborono pinacol,
Pd(fer) Cl , DMSO, KOAc, 100 C, 1h, 63%.
3 2 3 2
, AcOH, 41%; b: SOCl , CH OH; Boc O, DMAP; 70% for
1
2
2
o
2
2
2
9
5-BT 2 and NA-5-BT 3 from the full-Boc-protected bromo
tryptophan 11 and partial protected compound 10, respectively.
As discussed above, the partial protected 10 can deactivate the
Pd(0) catalysis thereby rendering the reaction incomplete. The
mixture of 10 and NA-5-BT 3 is inseparable through
chromatography but can be completely transformed to NA-5-
BT 3 by repeating the reaction. Although, as expected, 5-
Introduction of the three Bocs did not proceed smoothly.
Partially protected 13 and fully protected 14 were obtained in a
36-38
ratio of 1:3.
Incomplete protection was due to the
the cocrystallized salt of
O·HNO from the previous nitration step.
Hence, neutralization with Na CO is a prerequisite before
disturbance
of
nitrotryptophan·H
2
3
2
3
30
bromoindole is capable of producing 5-boronopinacol indole,
the subsequent Friedel Crafts coupling with compound 9
failed to give any desired product. Thus, the pathway of Scheme
2 is adopted throughout this study.
esterification. The reduction reaction using Zn powder
generated the amino derivative 15 in a quantitative yield.
However, in some experimental batches, the reaction was
retarded due to the incomplete removal of the residual base
from the previous chromatography. An additional one
equivalent of Zn powder and optional solvent can drive the
reaction toward completeness. Followed by a subsequent
31, 32
3
9
Sandmeyer reaction , the desired iodo tryptophan analog 16
was obtained in a 51% yield which contained an unseparated
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deiodinated byproduct (~ 10%) even after HPLC purification.
After borylation, the 6-isomer TB-6-BT 5 was obtained as the
sole product from column chromatography.
derivatives with Boc groups including TB-5-BT 1, DBA-5-BT 2
and TB-6-BT 5 showed better accumulation than the partial
protected derivatives 5-BT 4 and NA-5-BT 3 (Fig. 4). Both the
triBoc-protected TB-5-BT 1 and TB-6-BT 5 accumulated more
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The third target compound A,Z-2,7-DBT 6 with two boron atoms
is thought to be more useful than the above single boron tryptophan
derivatives (Scheme 4). Ir-assisted direct borylation on tryptophan
1
0
than 100 ppm of B after 1 h incubation in LN229 cell. Moreover,
TB-5-BT 1 and DBA-5-BT 2 reach a high level of 300 ppm after 2
h incubation in U87-MG cells, and TB-5-BT 1 exhibited an even
higher accumulation level of 450 ppm post 1 h of incubation in
LN229 cell. By comparison with the 6-boronopinacol derivative,
5-boronopinacol derivatives accumulated preferentially in U87-
MG cells. Setting the 3T3 cells as a control, accumulation
selectivity indices defined by the tumor vs. normal (T/N) ratio of
TB-6-BT 6 are >5 (T/N) 4 h post addition in both types of tumor
cells (Fig. 5). TB-5-BT 1 showed an average T/N >2 and an
impressive T/N > 9 in LN229 cells before 1 h. Thus, the most
highly accumulated and selective TB-5-BT 1 warrants further study.
Furthermore, to serve as a clinically useful BNCT agent, plenty of
dosages (>1g/kg) are commonly given to patients over two hours
prior to neutron irradiation. Based on this rationale, the 6-
boronopinacol derivative TB-6-BT 5 is potent because of its
increasing accumulation trend. Due to the slight toxicity of 100 uM
of some tryptophan derivatives (Table 2), half concentration (50
uM) was employed for neutron irradiation experiments. The results
of the BNCT were assessed using WST-1 assay. After neutron
irradiation, more cells treated with the most accumulated TB-5-BT
4
0-42
has been thoroughly documented in several reports.
However,
intrinsic instability is associated with the diboronopinacol moieties,
4
1
especially when oxidizing using H
2
O
2
.
Whereas Ir-involved
borylation did not produce a satisfactory yield of the diboron
product AZ-2,7-DBT 6, the quantity was enough for the in-vitro
biological analysis.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
R'O
O
O
N3
O
c
N3
O
R
B
O
N
B
O
H
N
N
H
B
H
O
O
tryptophan,
R = NH2, R' = H
2
-BAT 18
a, b
AZ-2,7-DBT 6
1
7, R = N , R' = CH3
3
Scheme 4. 2,7-Diboronopinacol tryptophan analog AZ-2,7-DBT 6
prepared from tryptophan. Reaction conditions a: SOCl , CH OH;
b: TfN , CuSO , K CO , CH OH, rt, over 2 steps, 69%; c. h:
Ir(COD)OMe] , (CH phen, B pin , THF, 80 °C, 11% forAZ-2,7-
2
3
3
4
2
3
3
1
, DBA-5-BT 2 and TB-6-BT 5 were rendered nonviable than those
[
2
3
)
4
2
2
treated with BPA (Fig. 6). Unlike TB-6-BT 1 and DBA-5-BT 2
both showed enhanced cancerous eradication, TB-5-BT 5 in U87-
MG cell is somewhat inferior probably due to the bias as indicated
by the internal standard of DMSO with increasing survival rate of
greater than 100%.
DBT 6 and 18, respectively.
Cytotoxicity
The cytotoxicities of these tryptophan derivatives were assessed in
three cell lines including two tumor cell lines: U87-MG and LN-
229 from human and one control cell line: 3T3 fibroblast cell from
mice. The reduction potential of mitochondrial was evaluated by
the formation of a dark yellow precipitate when treating WST-1
reagents with aliquots of compounds in a series of dilutions from 1
nM to 100 M. The cell viability was thus obtained as shown in
Supp. Fig. 22 and Table 2. To prepare the samples as a solution
phase, compounds were first dissolved in 100% DMSO to give 10
mM as a stock for the following dilutions. The most concentrated
aliquots (100 uM) contains 1% DMSO. Except for the control 3T3
cells, both tumor cells tolerate the presence of 1% DMSO well.
Furthermore, the Boc-protected derivatives, e.g., TB-6-BT 5, TB-
500
U87-MG
4
3
00
00
TB-5-BT
TB-6-BT
DBA-5-BT
AZ-27-DBT
200
5-BT
1
00
0
NA-5-BT
BPA
5
-BT 1 and DBA-5-BT 2, are slightly toxic against 3T3 cells but
0
1
2
3
4
5
Time (h)
LN229
insignificant when compared with the control compound BPA.
4
3-48
BPA was generally regarded as nontoxic.
6
00
Table 2. Compound toxicity (100 M) against tumor (U87-MG
and LN-229) and normal (3T3) cells
500
4
00
Cell viability (%)
TB-5-BT
TB-6-BT
DBA-5-BT
AZ-27-DBT
5-BT
TB-6-BT TB-5-BT DBA-5-BT NA-5- 5-BT AZ-2,7-DBT BPA
BT
DMSO
(1%)
Cell lines
U87
300
200
100
0
60.1 ±
.9
53.2 ±
.0
67.4 ± 70.2 ± 0.1 66.2 ± 65.8 ±
1.6 3.2 3.3
60.7 ± 70.8 ± 1.4 69.9 ± 67.3 ±
61.3 ± 5.0
57.2 ± 6.2
67.0 ±
2.0
79.6 ±
3.6
97
91
1
LN229
2
5.2
2.9
42.9 ± 47.6 ±
0.7 1.5
1.9
NA-5-BT
BPA
6
3.6 ±
.9
60.2 ±
2.3
53.0 ±
1.6
3T3
66.6 ± 1.8
109.9 ± 1.1
57
0
Results were tabulated from the most concentrated (100 uM) aliquots in 1% DMSO after 24-h
treatment. Data were obtained in quadruplicate and were expressed as meanꢀ±ꢀSD. GraphPad Prism
0
1
2
3
4
5
Time (h)
5
software was used.
In-vitro accumulation
The cellular accumulation of boron tryptophan derivatives was
evaluated by determining the boron concentration using induced
coupled mass spectrometry (ICP-MS). The three tryptophan
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2
1
1
50
00
50
00
50
0
1
20
120
100
80
60
40
20
0
1
2
3
4
5
6
7
8
9
3
T3
100
8
6
0
0
TB-5-BT
TB-6-BT
DBA-5-BT
AZ-27-DBT
40
20
5-BT
NA-5-BT
BPA
0
T
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
7
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
9
7
3
9
7
3
7
9
3
9
7
3
9
(3T
2
8
(3T
2
8
(3T (U
8
2
(3T
2
8
(3T
2
8
0
1
2
3
4
5
2
(U
2
(U
2
2
(U
2
(U
(LN
(LN
(LN
(LN
(LN
T
T
T
T
T
T
T
O
O
Time (h)
5-B
T
-B
-B
T
-B
-B
-B
T
DB
T
B
S
O
S
S
-
5-B
-5
-6 6-B
-6
-5
-5 5-B 27-
B
A
-D
M
M
B
B
B
B
A
A
-D
27
0
D
M
D
T
-
T
T
-
B
T
B
B
-
-
-27
-
%
D
%
B
D
D
A
Z
Z
.33
3
3
T
T
B
A
Z
33%
Figure 4. Schematic comparison of boron accumulation by
boronotryptophan derivatives in tumor (U87-MG and LN229) and
normal brain cells (3T3).
D
A
.
0
.
0
(A) Less polar tryptophan derivatives
9
8
120
100
120
100
80
60
40
20
0
U87-MG T/N ratio
7
8
0
6
TB-5-BT
60
40
5
TB-6-BT
4
3
2
1
0
DBA-5-BT
AZ-27-DBT
5-BT
2
0
NA-5-BT
BPA
)
)
)
)
)
)
)
)
)
)
)
)
7
0
T
3
9
7
3
9
7
3
9
7
3
9
T
2
8
(3T
2
8
(3T
2
8
(3T
2
8
U
(
3
(LN
2
(
U
(LN
2
(
U
(LN
2
(
U
(LN
2
(
T
T
T
A
A
O
O
-
B
T
5-B
-B
T
5-B
P
A
P
S
O
S
5
B
-5
B
B
B
M
M
-
A
-5-
-
P
B
D
S
D
5
NA
M
N
%
3
D
33%
A
0
1
2
3
4
5
N
3
%
3
.
.
0
0
0.3
Time (h)
(
B) More polar tryptophan derivatives
Figure 6. Comparison of the cell survival ratio from the treatment
of boron agents (50 uM) with neutron irradiation (A and continued
part B). Data were collected from quadruplicate of three sets and
two sets of experiments for cancer cells (LN229 and U87-MG) and
normal cell 3T3, respectively.
2
2
0
8
6
4
2
0
8
6
4
2
0
2
1
1
1
1
1
TB-5-BT
TB-6-BT
LAT-1 expression assay
LN229 T/N ratio
It is necessary to examine whether these tryptophan derivatives can
affect the expression of LAT-1. Although no direct binding assay
was performed, the protein expression by Western blot can
generate a qualitative comparison (Fig. 7). In general, no
significant inhibition of LAT-1 was observed over the 2-h time
course except 3T3 cell. Especially, the two more T/N selective TB-
5-BT 1 and TB-6-BT 5 can be due to the differential expression of
LAT-1. In addition, the preferential accumulation of these
tryptophan derivatives in tumor cells can be accounted for by two
reasons. First, the hydrophobic characteristics of these compounds
can facilitate diffusion across the cell membrane. Second, tumor
cells tend to ingest more tryptophan derivatives as nutrition sources
than normal cells do.
DBA-5-BT
AZ-27-DBT
5-BT
NA-5-BT
BPA
0
1
2
3
4
5
Time (h)
Figure 5. Schematic comparison of boron accumulation ratio (T/N)
in tumor (U87-MG and LN229) vs normal brain cells (3T3).
Figure 7. Western blot of the LAT-1 expression level of the three
cell lines after 2 h incubation of the selected tryptophan derivatives.
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Primary antibody LAT-1 (1:2500, Cell Signaling Tech) and α-
tubulin (1:5000, GeneTex).
2. Park, J. K.; Hodges, T.; Arko, L.; Shen, M.; Dello Iacono, D.;
McNabb, A.; Bailey, N. O.; Kreisl, T. N.; Iwamoto, F. M.; Sul, J.;
Auh, S.; Park, G. E.; Fine, H. A.; Black, P. M., Scale to Predict
Survival After Surgery for Recurrent Glioblastoma Multiforme.
Journal of Clinical Oncology 2010, 28 (24), 3838-3843.
3. Nawashiro, H.; Otani, N.; Shinomiya, N.; Fukui, S.; Ooigawa, H.;
Shima, K.; Matsuo, H.; Kanai, Y.; Endou, H., L-type amino acid
transporter 1 as a potential molecular target in human astrocytic
tumors. Int J Cancer 2006, 119 (3), 484-92.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Conclusion
The three Boc-protected tryptophan derivatives TB-5-BT 1, DBA-
5-BT 2 and TB-6-BT 5 are not soluble in aqueous solution, and
hence, the question now rests on the formulation optimization for
injection in an animal study. In brief, TB-5-BT 1 and DBA-5-BT 2
as well as TB-6-BT 5, showed better tumor accumulation and
selectivity, and their in vivo application should occur in due course.
4. Habermeier, A.; Graf, J.; Sandhofer, B. F.; Boissel, J. P.; Roesch,
F.; Closs, E. I., System L amino acid transporter LAT1 accumulates
O-(2-fluoroethyl)-L-tyrosine (FET). Amino Acids 2015, 47 (2), 335-
3
5
44.
0
1
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6
7
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
. Fuchs, B. C.; Bode, B. P., Amino acid transporters ASCT2 and
LAT1 in cancer: partners in crime? Semin Cancer Biol 2005, 15 (4),
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54-66.
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neutron capture therapy. Radiation Research 1999, 151 (1), 1-18.
. Soloway, A. H.; Tjarks, W.; Barnum, B. A.; Rong, F. G.; Barth,
7
TB-5-BT TB-6-BT DBA-5-BT AZ-2,7-DBT 5-BT
NA-5-BT
BPA
R. F.; Codogni, I. M.; Wilson, J. G., The chemistry of neutron
capture therapy (vol 98, pg 1515, 1998). Chemical Reviews 1998, 98
Figure 8. Aqueous solubility test for the tryptophan derivatives (1
mM, 20% EtOH in saline).
(6), 2389-2389.
8. Barth, R. F.; Mi, P.; Yang, W. L., Boron delivery agents for
neutron capture therapy of cancer. Cancer Communications 2018, 38,
1
5.
ASSOCIATED CONTENT
Supporting Information
9. Barth, R. F.; Coderre, J. A.; Vicente, M. G. H.; Blue, T. E., Boron
neutron capture therapy of cancer: Current status and future prospects.
Clinical Cancer Research 2005, 11 (11), 3987-4002.
10. Alkonyi, B.; Barger, G. R.; Mittal, S.; Muzik, O.; Chugani, D.
C.; Bahl, G.; Robinette, N. L.; Kupsky, W. J.; Chakraborty, P. K.;
Juhasz, C., Accurate Differentiation of Recurrent Gliomas from
Radiation Injury by Kinetic Analysis of alpha-C-11-Methyl-L-
Tryptophan PET. Journal of Nuclear Medicine 2012, 53 (7), 1058-
1064.
11. Peura, L.; Malmioja, K.; Huttunen, K.; Leppanen, J.;
Hamalainen, M.; Forsberg, M. M.; Rautio, J.; Laine, K., Design,
Synthesis and Brain Uptake of LAT1-Targeted Amino Acid Prodrugs
of Dopamine. Pharmaceutical Research 2013, 30 (10), 2523-2537.
12. Gomes, P.; Soares-da-Silva, P., L-DOPA transport properties in
an immortalised cell line of rat capillary cerebral endothelial cells,
RBE 4. Brain Research 1999, 829 (1-2), 143-150.
The Supporting Information is available free of charge on the
ACS Publications website. Experimental procedures for the
preparation of both described tryptophan derivatives and
biological data including the cell survival assay, cellular
accumulation assay, neutron irradiation and LAT-1 blotting are
1
13
provided. Figures 1-21, H-NMR, C-DEPT-135 NMR, LC-
ESI-HRMS and chromatogram; Figure 22, Cytotoxicity of
boronopincaol tryptophan derivatives in various concentrations
ranging from 1 nM to 100 M (PDF).
AUTHOR INFORMATION
Corresponding Author
* To whom correspondence should be addressed.
csyu@mx.nthu.edu.tw; Tel: 886-3-5715131ext35582.
1
3. Fan, C. H.; Wang, T. W.; Hsieh, Y. K.; Wang, C. F.; Gao, Z.
Y.; Kim, A.; Nagasaki, Y.; Yeh, C. K., Enhancing Boron Uptake in
Brain Glioma by a Boron-Polymer/Microbubble Complex with
Focused Ultrasound. ACS Appl. Mater. Interfaces 2019, 11 (12),
1
1
1144-11156.
Author Contributions
†These authors contributed equally.
4. Shi, Y. X.; Li, J. Y.; Zhang, Z. Z.; Duan, D. B.; Zhang, Z. C.;
Liu, H.; Liu, T.; Liu, Z. B., Tracing Boron with Fluorescence and
Positron Emission Tomography Imaging of Boronated Porphyrin
Nanocomplex for Imaging-Guided Boron Neutron Capture Therapy.
ACS Appl. Mater. Interfaces 2018, 10 (50), 43387-43395.
ACKNOWLEDGMENT
We are grateful to the Ministry of Science and Technology of
Taiwan and the Chang-Gung Medical Research Project for
providing financial support via the following grants: MOST-107-
2113-M-007-025-, MOST-106-2113-M-007-012-, MOST-108-
1
5. Kuthala, N.; Vankayala, R.; Li, Y. N.; Chiang, C. S.; Hwang, K.
C., Engineering Novel Targeted Boron-10-Enriched Theranostic
Nanomedicine to Combat against Murine Brain Tumors via MR
Imaging-Guided Boron Neutron Capture Therapy. Adv. Mater. 2017,
29 (31), 10.
16. Huang, Z.; Kwon, O.; Huang, H. Y.; Fadli, A.; Marat, X.;
Moreau, M.; Lumb, J. P., A Bioinspired Synthesis of Polyfunctional
Indoles. Angewandte Chemie-International Edition 2018, 57 (37),
2
113-M-007-023-,
MOST-106-2314B-182A-058-MY2,
NMRPG3G612, CMRPG3F1511.
ABBREVIATIONS
BNCT, boron neutron capture therapy; LAT1, leucine-type
amino acid transporter 1; T/N, tumor to normal accumulation ratio;
BPA, boronophenyl alanine.
1
1963-11967.
17. Katritzky, A. R.; Ramsden, C. A.; Joule, J. A.; Zhdankin, V. V.,
Handbook of heterocyclic chemistry. 3rd ed.; Elsevier: Amsterdam,
2
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010.
8. Leitch, J. A.; Bhonoah, Y.; Frost, C. G., Beyond C2 and C3:
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