Design, Synthesis, and Biological Evaluation of Boron-Containing Macrocyclic Polyamines and Their Zinc(II) Complexes for Boron Neutron Capture Therapy

pubs.acs.org/jmc

Article

Design, Synthesis, and Biological Evaluation of Boron-Containing

Macrocyclic Polyamines and Their Zinc(II) Complexes for Boron

Neutron Capture Therapy

Hiroki Ueda, Minoru Suzuki, Reiko Kuroda, Tomohiro Tanaka, and Shin Aoki*

Cite This: J. Med. Chem. 2021, 64, 8523 −8544

Read Online

sı

*

Metrics & More

Article Recommendations

Supporting Information

ACCESS

ABSTRACT: Boron neutron capture therapy (BNCT) is a binary

therapeutic method for cancer treatment based on the use of a

combination of a cancer-speciﬁc drug containing boron-10 (¹⁰B)

and thermal neutron irradiation. For successful BNCT, ¹⁰B-

containing molecules need to accumulate speciﬁcally in cancer

cells, because destructive eﬀect of the generated heavy particles is

limited basically to boron-containing cells. Herein, we report on

the design and synthesis of boron compounds that are function-

alized with 9-, 12-, and 15-membered macrocyclic polyamines and

their Zn²⁺complexes. Their cytotoxicity, intracellular uptake

activity into cancer cells and normal cells, and BNCT eﬀect are

also reported. The experimental data suggest that mono- and/or

diprotonated forms of metal-free [12]aneN₄- and [15]aneN₅-type ligands are uptaken into cancer cells, and their complexes with

intracellular metals such as Zn²⁺would induce cell death upon thermal neutron irradiation, possibly via interactions with DNA.

Scheme 1. Structures of Representative BNCT Agents

INTRODUCTION

■

Boron neutron capture therapy (BNCT) is a potential

radiotherapy based on the nuclear reaction between boron-

10 (¹⁰B) atoms and thermal neutrons (¹n). The neutron

capture reaction [¹⁰B(n, α)⁷Li] generates high linear energy

transfer (LET) α particles and lithium ions that have

destructive eﬀects and short path lengths in the 5−9 μm

range. Therefore, it is expected that cancer cells containing ¹⁰B

species would be selectively destroyed with minimal eﬀects on

healthy tissues.¹

For successful BNCT, a high level of accumulation and

selective delivery of ¹⁰B into cancer cells are required. The

design of eﬀective BNCT agents requires the following criteria:

(1) low systemic toxicity and higher uptake in tumor tissue

than in normal tissue [tumor to blood (T/B) ratios should be

greater than 3]; (2) ¹⁰B must be retained in the tumor tissue

but also be rapidly cleared from blood and normal tissues; and

(3) the concentration of boron inside or near tumor cells must

be ≥10^{9 10}B atoms/cell (20−35 μg/gram of tumor tissue).²In

this context, only two compounds, sodium mercaptoborate

(BSH) 1³and L-4-boronophenylalanine (BPA) 2⁴(used as a

complex with ^D-fructose) have been used for the clinical

treatment of cancers such as malignant glioma, malignant

melanoma, and recurrent head and neck cancer, which are not

enough for treatment of multiple tumor types (Scheme 1).⁵

To date, numerous boron-containing analogues including

amino acids,⁶biochemical precursors of nucleic acids,⁷

carbohydrates,⁸amines,⁹porphyrins,¹⁰peptides,¹¹liposomes,¹²

and monoclonal antibodies have been developed.¹³However,

most of them do not satisfy the above criteria for clinical

applications. Therefore, more potent boron agents are highly

required in order to improve the therapeutic eﬀect and to

apply to various tumor types such as breast, lung, and

pancreatic cancer.

For the aforementioned purpose, we previously reported on

the design and synthesis of sulfoquinovosyl acyl glycerol

(SQAG) derivatives and 2-boryl-1,2-dideoxy-^D-glucose deriv-

atives, which were possibly transferred into cancer cells

Received: March 11, 2021

Published: June 2, 2021

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

8523

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

through the glucose transporter 1 (GLUT1),^14,15because large

amounts of ^D-glucose are consumed by anaerobic glycolysis

during the rapid proliferation of cancer cells, which is known as

the Warburg eﬀect.¹⁶However, their eﬀect on BNCT was not

satisfying, despite the moderate intracellular uptake of these

agents.

It is also known that polyamines including spermidine 3 and

spermine 4 are essential for numerous cellular functions such

as DNA replication and protein synthesis.¹⁷The increase in

polyamine concentrations in cancer cells is associated with the

activation of cell proliferation and regulated by the promoted

polyamine transport system (PTS) and biosynthesis.¹⁸There-

fore, polyamine derivatives could serve as potentially useful

scaﬀolds for the delivery of boron-containing drugs into cancer

cells, as represented by the spermidine derivatives 5 and 6

(Scheme 2).^9,19To the best of our knowledge, however, the

use of these derivatives in BNCT has not been reported.

ca. 20 ppm, which corresponds to B(OH)₃. In addition, we

also found that 7 was eﬃciently transferred into cancer cell

lines (Jurkat, A549 and HeLa S3 cells).^15,20In subsequent

studies, the decomposition of ortho-carborane-polyamine

conjugates upon metal complexation was discovered and

applied to the magnetic resonance imaging (MRI) of Cu²⁺in

solutions.²¹

The aforementioned background and the high intracellular

uptake of 7 in cancer cells prompted us to examine the

development of boron carriers equipped with macrocyclic

polyamine scaﬀolds such as [9]aneN₃(1,4,7-triazacyclono-

nane) 9, [12]aneN₄(cyclen) 10, and [15]aneN₅(1,4,7,10,13-

pentaazacyclopentadecane) 11 (Scheme 4). In this work, we

designed and synthesized the phenylboronic acid-pendant

macrocyclic polyamines 12−14, their corresponding boronic

acid ester analogues 15−17, and Zn²⁺complexes 18−20. It

was expected that the cationic charge of 15−17 due to the

Scheme 2. Structures of Polyamines and Boron-Containing

Spermidine Derivatives 5 and 6

Scheme 4. Structures of Macrocyclic Polyamine Derivatives

and Their Zn²⁺Complexes Synthesized in This Work

We previously reported on the design and synthesis of

phenylboronic acid-pendant cyclen (1,4,7,10-tetraazacyclodo-

decane, [12]aneN₄) 7 for the sensing of metal cations such as

zinc (Zn²⁺), iron (Fe²⁺), copper (Cu²⁺), and cobalt (Co²⁺)

(Scheme 3).²⁰It was found that the carbon−boron bond at the

o-position of the (2-boronophenyl)methyl side chain in 7 is

hydrolyzed upon complexation with these metal ions to give 8,

resulting in a shift of the ¹¹B NMR signal from ca. 30 ppm to

Scheme 3. Hydrolytic Cleavage of C−B Bond of 7

8524

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

protonation of macrocyclic polyamine groups (15−17·nH⁺, n

= 1 or 2) would facilitate their intracellular uptake.^18,22,23We

hypothesized that the protonated form of these boron−

polyamine conjugates (15−17) would be restricted to mono-

or dicationic forms (n = 1, 2) (15a,b·nH⁺, 16a,b·nH⁺, and

17a−c·nH⁺forms in Scheme 4) due to the deprotonation

constants of the macrocyclic polyamines, 9,²⁴10,²⁵and 11,²⁶

as described below.

Scheme 6. Synthesis of 12a,b, 15a,b, and 18a,b

It is also well known that macrocyclic polyamines form

stable complexes with intracellular metal ions such as Zn²⁺,

Cu²⁺, and Ni²⁺in aqueous solutions at physiological pH

(Scheme 4),²⁷⁻²⁹and these complexes are much more stable

than Zn²⁺complexes of linear polyamines such as spermidine 3

and spermine 4. In addition, it was reported that the

cytotoxicity of macrocyclic polyamines is reduced by the

complexation with Zn²⁺.³⁰It is well established that Zn²⁺−

cyclen complexes such as 21 bind to thymidines (dT) in DNA

to form stable complexes 22 through the coordination bonding

between the deprotonated imide moiety of dT (dT⁻) and Zn²⁺

in aqueous solution at neutral pH (Scheme 5).³¹Therefore, we

Scheme 5. Complexation of Zn²⁺−Cyclen 21 with the

Deprotonated dT⁻in Aqueous Solution at Neutral pH

Scheme 7. Synthesis of 13a,b, 16a,b, 7, and 19a,b

expected that the neutron irradiation of 18−20 when located

in close proximity to DNA would eﬀectively induce DNA

damage. In this study, we report on the cytotoxicity and

intracellular uptake activity of 12−17 and the corresponding

Zn²⁺complexes 18−20 in several cancer cell lines. These

agents were ﬁrst prepared as ligands containing boron in a

natural abundance ratio (¹⁰B/¹¹B = 19.9/80.1). After the

biological assessment of these ¹⁰B/¹¹B agents, three promising

compounds were chosen among them and the corresponding

¹⁰B-enriched compounds and their Zn²⁺complexes were

synthesized and used in BNCT experiments.

RESULTS AND DISCUSSION

■

Synthesis of Boron-Containing Macrocyclic Poly-

amine Derivatives and the X-ray Single Crystal

Structure Analysis of 19a. The synthesis of the macrocyclic

polyamine derivatives is shown in Schemes 6−8. The boron-

containing BNCT agents were initially synthesized using

naturally abundant ratio of boron (¹⁰B/¹¹B = 19.9/80.1), in

order to evaluate their intracellular uptake, from which more

potent candidates were selected and the corresponding ¹⁰B-

enriched compounds were synthesized for use in BNCT

experiments.

2TFA salt, the reaction of 12a with bicyclohexyl-1,1′-diol 26³⁵

gave 15a. The synthesis of the m-isomer 15b was carried out in

a similar manner.³⁶The o-isomers of 12 and 15 (12c and 15c)

were not obtained, due to the cleavage of their C−B bonds in

aqueous solution even in the absence of metal ions. The

complexation of 15a and 15b with Zn²⁺was conducted in situ

before the biological evaluation.

The 9-membered macrocyclic polyamine 9 ([9]aneN₃)³²

was treated with (Boc)₂O to give 23,³³which was then reacted

with 4-(bromomethyl)phenylboronic acid 24a³⁴to aﬀord 25a

(Scheme 6). After removing the Boc groups of 25a by

treatment with triﬂuoroacetic acid (TFA) to give 12a as the

The synthesis of the 12-membered tetraamine (cyclen)

([12]aneN₄) derivatives 16a,b and the 15-membered penta-

8525

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Scheme 8. Synthesis of 14a−c, 17a−c, and 20a−c

−

Figure 1. ORTEP drawing of 19a with a Zn²⁺bound NO₃.

Selected bond lengths: Zn(1)−N(1) 2.059 Å, Zn(1)−N(2) 2.167 Å,

Zn(1)−N(3) 2.091 Å, Zn(1)−N(4) 2.962 Å, Zn(1)−O(3) 1.999 Å,

C(13)−B(1) 1.561 Å, B(1)−O(1) 1.363 Å, and B(1)−O(2) 1.366 Å.

One external nitrate anion, ethanol, and hydrogen atoms were

omitted for clarity.

16a, and 17a are more toxic than 12a, 13a, and 14a, possibly

due to the hydrophobicity of the boronic acid ester group. It

should be noted that the cytotoxicity of Zn²⁺complex 19b,

20b, and 20c is lower than the corresponding Zn²⁺-free ligands

16b, 17b, and 17c, while the Zn²⁺-free ligands 15a, 15b, 16a,

17a, and their Zn²⁺complexes 18a, 18b, 19a, and 20a have a

similar toxicity. The similar toxicity between 15a and 18a and

15b and 18b would be due to the weak Zn²⁺complexation of

15a and 15b, which have only three nitrogen atoms in the

[9]aneN₃ring group.

Intracellular Uptake of Boron-Containing Macrocyclic

Polyamine Derivatives into HeLa S3, A549, and IMR-90

Cells, as Determined by Inductively Coupled Plasma

Mass Spectrometry. The intracellular uptake of the boron

compounds into HeLa S3, A549, and IMR-90 cells was

evaluated by inductively coupled plasma mass spectrometry

(ICP−MS), as shown in Scheme 9. The cells (5 × 10⁵cells/

well) were seeded on 6-well plates and incubated in culture

medium containing 10% FBS for 1 day at 37 °C in a 5% CO₂

environment (20% O₂) and then treated with boron

compounds 1, 2, 7, and 12−20 (30 μM) under same

conditions. This concentration (30 μM) of 1, 2, 7, and 12−

20 (lower concentrations are better to reduce their toxicity)

was carefully determined based on the consideration of a

balance between their IC₅₀values (toxicity) and intracellular

uptake values that are listed in Table 1 and Figure 2. After

incubating the cells for 24 h, they were washed with PBS and

broken down with nitric acid overnight, and the amount of

boron atoms (total amount of ¹⁰B and ¹¹B) was quantitatively

determined by ICP−MS and normalized as the amount of per

cell because some compounds have weak toxicity.

amine ([15]aneN₅) derivatives 17a−c was carried out, as

shown in Schemes 7 and 8.³²⁻³⁹The deprotection of 28a and

30a−c with TFA aﬀorded 13a and 14a−c 2TFA and 3TFA

salts, respectively, as determined by elemental analysis.

The Zn²⁺complexes of 16a and 16b (19a and 19b) were

isolated and those of 17a−c (20a−c) were prepared in situ for

use in biological experiments. The structure of 19a was

conﬁrmed by a single-crystal X-ray structure analysis, as shown

in Figure 1. The Zn²⁺complex of the o-form 7 was not

obtained due to the carbon−boron bond cleavage that

occurred upon complexation with Zn²⁺, as previously

described.²⁰In contrast, the C−B bond in 20c (Zn²⁺−17c

complex) was hydrolyzed very slowly (approximate half-life is

24 h) as observed by ¹¹B-NMR, possibly due to the higher pK_a

value of the Zn²⁺-bound water in the Zn²⁺−[15]aneN₅

complex than that of 19c, which is a Zn²⁺complex of the

[12]aneN₄-type ligand 7.³⁹

Evaluation of the Cytotoxicity of Boron-Containing

Macrocyclic Polyamine Derivatives against HeLa S3,

A549, and IMR-90 Cells. The cytotoxicity of the boron-

containing macrocyclic polyamine derivatives 7, 12−17, and

their corresponding Zn²⁺complexes 18−20 against HeLa S3

(human cervical carcinoma), A549 (human caucasian lung

carcinoma), and IMR-90 (normal human ﬁbroblast) cells was

examined by an MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphe-

nyltetrazolium bromide) assay in comparison with those of

BSH (1) and BPA−^D-fructose complex (2). The cells (1 × 10⁴

cells/well) were incubated with boron compounds 1, 2, 7, and

12−20 (0−200 μM) in culture medium containing 10% fetal

bovine serum (FBS) for 24 h at 37 °C under 5% CO₂and then

treated with the MTT reagent to evaluate cell viability.

The results are presented in Figures S1−S3 in the

Supporting Information, and the IC₅₀values of these agents

are summarized in Table 1. The ﬁndings indicated that 7 and

12−20 are somewhat more toxic than 1 and 2, and that 15a,

As shown in Figure 2a, the intracellular uptake of 7 and 15−

17 is higher than that of reference compounds BSH 1

comprised of twelve ¹⁰B and BPA 2, possibly because cell-

membrane permeability is improved by their boronic acid ester

group. In addition, it was found that intracellular uptake of the

8526

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 1. IC₅₀Values of Boron Compounds 1, 2, 7, and 12−20 [0−200 μM] against HeLa S3, A549, and IMR-90 Cells after the

Treatment for 24 h

compound

HeLa S3

A549

>200

IMR-90

compound (Zn²⁺complexes)

HeLa S3

A549

IMR-90

1

2

7

>200

162

100

9

7

108

5

12a

13a

14a

15a

15b

16a

16b

17a

17b

17c

>200

131 11

>200

112

163

>200

22

>200

151

>200

128

>200

34

117 10

>200

83

187 14

94

135

151

18

3

9

5

18a

18b

19a

19b

20a

20b

20c

112

>200

148

>200

71

3

155

>200

139 11

>200

2

130

162

95 16

>200

129 16

32

83

9

5

4

1

6

2

8

3

1

2

1

6

5

8

65

35

138

197 21

Scheme 9. Typical Procedure Used for Measuring the Intracellular Uptake of Boron Compounds in Living Cells

9-membered triamine derivatives 15a,b and 18a,b into A549

cells was higher than 16a,b and 17a−c, and their Zn²⁺

complexes 19a,b and 20a−c exhibited a lower intracellular

uptake (Figure 2b), suggesting that the 9-membered triamine

group in 15a and 15b is better for the intracellular uptake into

A549 cells.

The tumor/normal cell (T/N) ratios with respect to the

intracellular uptake of the boron compounds (1, 2, 7, and 12−

20) were calculated using eq 1, and their intracellular boron

uptake (in HeLa S3 cells and A549 cells)-T/N ratio proﬁles

are shown in Figure 3a,c. The boron uptake-IC₅₀value

(indicating the toxicity) proﬁles are plotted in Figure 3b (HeLa

S3 cells) and 3d (A549 cells). These data suggest that 17a has

a higher boron uptake (>2.5 fmol/cell) and T/N selectivity

(ca. 4) and a rather low toxicity against HeLa S3 cells (Figure

3a,b) and that 15b, 16b, and 17a exhibit better boron uptake,

higher T/N ratios (over 3), and lower toxicity against A549

cells and normal cells (IC₅₀> 100 μM) (Figure 3c,d), although

the reasons for their selective uptake to cancer cells are yet to

be studied.

cyclic polyamine conjugates, the deprotonation constants (pK_a

values) of unmodiﬁed macrocyclic polyamines 9, 10, and 11

are summarized in Scheme 10.²⁴⁻²⁶It is likely that the major

forms of 9 (the amine moieties of 15a,b) at neutral pH are

diprotonated (9·2H⁺) and monoprotonated (9·H⁺) forms, and

those of 10 (the amine moieties of 16a,b) and 11 (the amine

moieties of 17a−c) are diprotonated forms (10·2H⁺and 11·

2H⁺, respectively). These ﬁndings regarding the intracellular

uptake of 15a,b, 16a,b, and 17a−c (Figure 2) suggest that the

diprotonated and/or monoprotonated forms of these boron−

polyamine conjugates are preferable for eﬀective intracellular

uptake and that the monoprotonated form might be more

favorable. The intrinsic stability constants (log K_ZnL) of the

Zn²⁺complexes 31−33 are also described in Scheme 10. The

similar intracellular uptake of [9]aneN₃-type 15a,b and 18a,b

in A549 cells and higher uptake of 18a,b than that of 19a,b and

20a−c (Figure 2) can be explained by a smaller log K_ZnLvalue

for 31 (Zn²⁺−9 complex) than those for 32 (Zn²⁺−10

complex) and 33 (Zn²⁺−11 complex) (less stability of 31 than

32 and 33), although the reasons for higher intracellular

uptake of 18a,b than 15a,b in HeLa S3 cells and IMR-90 cells

are yet to be studied. The relationship of these complexation

properties and the results of BNCT experiments of 15, 16, and

17 will be discussed below.

T/N ratio

[B(¹⁰B and¹¹B) uptake in HeLa S3 or A549 cells (fmol/cell)]

=

[B(¹⁰B and ¹¹B) uptake in IMR‐90 cells (fmol/cell)]

(1)

Consideration of protonation/deprotonation situations in

Scheme 10 suggest that [9]aneN₃(9) and [15]aneN₅(11)

would exist as 9·2H⁺and 11·3H⁺forms as well as 9·H⁺and 11·

Concerning the relationship of these data and the

protonation properties of the aforementioned boron-macro-

8527

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 2. Comparison of intracellular boron atoms against HeLa S3 (open bars), A549 (shaded bars), and IMR-90 (closed bars) cells as

determined by ICP−MS. All cells were treated with boron compounds 1, 2, 7, 12−17 (a), and 18−20 (b) (30 μM) in culture medium at 37 °C for

24 h. Data represent the mean standard deviation (SD) of at least three replicates.

2H⁺forms, respectively, under (slightly) acidic conditions in

cancer cells, so that exclusion of these drugs form the cells

through the hydrophobic cell membrane would be somewhat

disturbed. This point might be one of advantages of these

boron−polyamine agents. It is unlikely that 9, 10, and 11 exist

as 9·3H⁺, 10·3H⁺, 10·4H⁺, 11·4H⁺, and 11·5H⁺forms,

respectively, under physiological conditions, because their

pK_a1values (and pK_a2values for 10 and 11) are very low (less

than 2).

Eﬀect of Temperature and Inhibitors on the Intra-

cellular Uptake of Boron Compounds. The mechanism

responsible for the intracellular uptake of 15a, 16a, and 17a

into HeLa S3 and A549 cells was examined. As shown in

Figure 4a,b, the intracellular uptake of 15a, 16a, and 17a was

inhibited to a considerable extent at 4 °C, suggesting that the

transfer of 15a, 16a, and 17a into the cells is due to an energy-

dependent process.

It is known that the PTS in mammalian cells is associated

with the endocytosis pathway of linear polyamines such as

spermidine 3 and spermine 4 (Scheme 2).¹⁸In addition,

methyl-beta-cyclodextrin (MβCD) was reported to inhibit the

caveola-endocytosis pathway due to the depletion of

cholesterol,⁴⁰and dynasore and amiloride are used as

inhibitors of clathrin-endocytosis⁴¹and micropinocytosis,⁴²

respectively (the chemical structures of these inhibitors are

shown in Scheme S1 of the Supporting Information). As

shown in Figure 5, the intracellular uptake of 17a into HeLa S3

cells is inhibited to a considerable extent by dynasore and

8528

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 3. Intracellular boron uptake-T/N selectivity proﬁles (a,c) and intracellular boron uptake-IC₅₀value against normal cell proﬁles (b,d) of

boron compounds 1, 2, 7, and 12−20. (a,c) Selectivity (T/N ratio) to HeLa S3 cells (a) and A549 cells (c) were calculated from the results for the

intracellular uptake of the boron compounds into HeLa S3 and A549 cells in comparison to the uptake into IMR-90 cells, respectively. (b,d) IC₅₀

values (μM) of boron compounds 1, 2, 7, and 12−20 against IMR-90 cells and boron uptake (fmol/cell) into HeLa S3 cells (b) and A549 cells (d).

spermidine 3, suggesting that 17a is transferred into the cells

via the clathrin-endocytosis pathway, possibly including PTS.¹⁸

A similar inhibitory eﬀect of spermidine on the intracellular

uptake of 17a into A549 cells was observed, as shown in Figure

S4 in the Supporting Information. We assume that the weak

inhibition of the uptake of 2 and 17a by MβCD is due to the

inclusion of these boron compounds in the inner cavity of

MβCD. It is reported that amiloride inhibits the Na⁺/H⁺

exchanger and hence lower the intracellular Na⁺concentration.

It is assumed that this Na⁺deﬁciency would be compensated

by the Na⁺uptake via sodium-dependent amino acid

transporters such as ATB^0,+(amino acid transporter system

B^0,+) that had been reported to mediate the co-transport of

Na⁺with phenylalanine analogue 2.^4cThis assumption may

explain the increased intracellular uptake of 2 in the presence

of amiloride.

decided to choose 15b, 16b, and 17a for the BNCT, in which

¹⁰B and ¹¹B are contained in a natural abundance ratio

(¹⁰B/¹¹B = 19.9/80.1) and synthesized the corresponding ¹⁰B-

enriched compounds ¹⁰B-15b, ¹⁰B-16b and ¹⁰B-17a, as shown

in Scheme 11.

The ¹⁰B-enriched forms of 24a and 24b (¹⁰B-24a and ¹⁰B-

24b) were prepared by the reaction of 34a,b with ¹⁰B-enriched

trimethyl borate (>99.5% of ¹⁰B), followed by hydrolysis with

aqueous HCl to give ¹⁰B-35a and ¹⁰B-35b and bromination

with N-bromosuccinimide (NBS). The reaction of ¹⁰B-24a and

¹⁰B-24b with 23, 27, and 29 and the following conversions

were conducted as described in Schemes 6−8 to obtain ¹⁰B-

15b, ¹⁰B-16b, and ¹⁰B-17a, respectively.

BNCT experiments using A549 cells in the presence of the

aforementioned B-containing drugs (¹⁰B/¹¹B and ¹⁰B-enriched

compounds) were conducted at the Institute for Integrated

Radiation and Nuclear Science, Kyoto University (KURNS).

As shown in Scheme 12, A549 cells were incubated with the

boron compounds (30 μM) for 24 h and suspensions (5 × 10⁴

Evaluation of the Anti-tumor Eﬀect of the Selected

Boron-Containing Macrocyclic Polyamine Derivatives

with Thermal Neutron Irradiation by a Colony

Formation Assay. Based on the aforementioned results, we

8529

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Scheme 10. Reported Deprotonation Constants (pK_a) of Macrocyclic Polyamines 9−11²⁴⁻²⁶and Stability Constants (log

K

_ZnL) of Their Zn²⁺Complexes 31−33 in Aqueous Solution at 25 °C²⁷

The irradiated cells were seeded on the 12 well plate (3 × 10³

cells/well), incubated for 7 days, ﬁxed with EtOH, and stained

with crystal violet to produce visualizable images (Figure S5 in

the Supporting Information). The surviving fractions were

calculated as the stained colony area using the “ImageJ-plugin

Colony Area”⁴³software and normalized by comparing the

results with those for non-irradiated cell samples (Figure 6).

The results for the anti-tumor eﬀect of boron compounds

against A549 cells are summarized in Figure 6, which suggests

the following points:

(1) The cytotoxic activity of ¹⁰B-15b, ¹⁰B-16b, and ¹⁰B-17a

against A549 cells is higher than that for 1 (¹⁰B-BSH)

and 2 (¹⁰B-BPA).

(2) The cytotoxic activity of the ¹⁰B-enriched analogues is

more potent than that of the ¹⁰B/¹¹B derivatives (¹⁰B-

15b vs 15b, ¹⁰B-16b vs 16b, and ¹⁰B-17a vs 17a)

apparently due to the enrichment of ¹⁰B.

(3) The BNCT activity of ¹⁰B-18b, ¹⁰B-19b, and ¹⁰B-20a,

which are Zn²⁺complexes of ¹⁰B-15b, ¹⁰B-16b, and ¹⁰B-

17a, is also displayed in Figure 6. It was found that

metal-free ¹⁰B-16b and ¹⁰B-17a exhibit a higher BNCT

eﬀect than ¹⁰B-19b and ¹⁰B-20a, possibly because of

their higher intracellular uptake than that of stable ¹⁰B-

19b and ¹⁰B-20a, which are very stable (see Figure 2 and

Scheme 10).

(4) The BNCT eﬀect of ¹⁰B-15b and its Zn²⁺complex ¹⁰B-

18b were nearly the same, possibly due to rather low

stability of ¹⁰B-18b, as indicated by a relatively small log

K_ZnLvalue (11.3) for 31 (Zn²⁺−9) in Scheme 10.

(5) The relationship between the intracellular boron uptake

(from Figure 2) and the BNCT eﬀect (from Figure 6) is

summarized in Figure 7. The BNCT eﬀect of ¹⁰B-16b

and ¹⁰B-17a was more potent than that of ¹⁰B-15b,

while the intracellular uptake of the metal-free ¹⁰B-16b

and ¹⁰B-17a was lower than that of ¹⁰B-15b. It was

Figure 4. Eﬀect of low temperature on the intracellular uptake of

boron compounds 2 and 15a−17a (30 μM) into HeLa S3 (a) and

A549 cells (b) at 37 °C (open bars) or 4 °C (closed bars) for 1 h.

Data represent the mean SD of at least three replicates.

cells/mL) of these cells were irradiated with thermal neutrons

[average thermal neutron ﬂux: (1.5 0.1) × 10⁹n/cm²·s] at

room temperature for various times (0, 15, 30, and 45 min).

8530

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

complexes ¹⁰B-18b, ¹⁰B-19b, and ¹⁰B-20a (37 and 38 in

Scheme 13), because it is very likely that these B-containing

macrocyclic polyamines would form complexes with metal

cations contained in the media and/or in living cells.

As described in the Introduction (Scheme 5), we expected

that the Zn²⁺complexes ¹⁰B-18b, ¹⁰B-19b, and ¹⁰B-20a would

interact with deprotonated dT (dT⁻) in DNA and that the

DNA would be eﬃciently damaged upon thermal neutron

irradiation (38 in Scheme 13) (it had been reported that Cu²⁺,

Ni²⁺, and Fe²⁺complexes of cyclen negligibly interact with

DNA).^31cThese data allow us to consider that the metal-free

¹⁰B-15b, ¹⁰B-16b, and ¹⁰B-17a (possibly the diprotonated

form, as speculated in Scheme 10) are transferred into cancer

cells eﬃciently and form complexes with intracellular Zn²⁺and

recognize dT in DNA, resulting in an eﬃcient BNCT eﬀect.

In order to obtain experimental data for this hypothesis, we

measured the melting temperature (T_m) of the double-

stranded calf-thymus DNA (ctDNA) (50 μM in phosphate)

in the presence of 16b, 19b, 17a, 20a, and 3 (for the

reference).^31d,e,45As shown in Table 2 and Figure S6 in the

Supporting Information, the T_mvalue of ctDNA was raised by

16b, 17a, and 3 (ΔT_m= +6 and +8 °C for 16b and 17a,

respectively, at r = 5.0 and ΔT_m= +12 °C for 3 at r = 0.2,

where r = [16b, 17a, or 3]/[ctDNA(P)]) due to stabilization

of the double-stranded structure of ctDNA (36 in Scheme 13).

On the other hand, the T_mvalue was lowered in the presence

of 19b (ΔT_m= −6 °C at r = 1.0), possibly due to the

destabilization of ctDNA by the interaction of its Zn²⁺−

[12]aneN₄complex part with dT units in DNA (Scheme 5 and

38 in Scheme 13).

An interesting ﬁnding was that the T_mvalue was raised by

20a (ΔT_m= +6 °C at r = 1.0), suggesting that 19b and 20a

interact with ctDNA in diﬀerent modes. It should be noted

that coordination sites of Zn²⁺, whose general coordination

number is 4−6 (or 7),^29b,39,46would be almost occupied by the

coordination of ﬁve nitrogens from its [15]aneN₅ring unit of

20a and hence Lewis acidity of the Zn²⁺ion would be

considerably reduced. Therefore, it is considered that these

factors would hamper the coordination of 20a with dT⁻sites

in DNA and that 20a interacts with ctDNA mainly by the

electrostatic interaction to stabilize the DNA double-strand, as

shown in 37 of Scheme 13.^47,48These data support the

eﬃcient DNA damage induced by ¹⁰B-16b, ¹⁰B-19b, ¹⁰B-17a,

and ¹⁰B-20a at the close position upon neutron irradiation, as

proposed in Scheme 13.

Figure 5. Relative uptake of 2 and 17a (30 μM) into HeLa S3 cells in

the absence (open bars) and presence of inhibitors (closed bars), 1.5

mM of MβCD (a), 80 μM of dynasore (b), 2 mM of amiloride (c),

and 2 mM of spermidine 3 (d). After pretreatment with the inhibitors

for 1 h, the cells were incubated with 2 and 17a at 37 °C for 1 h in the

presence of inhibitors. Data represent the mean SD of at least three

replicates.

conﬁrmed that the intracellular uptake of ¹⁰B/¹¹B forms

and ¹⁰B-enriched forms of 15b, 16b, and 17a was almost

the same, respectively (data are not shown).

(6) As presented in Figures 4 and 5, intracellular uptake of

boron-containing macrocyclic polyamines is consider-

ably inhibited at 4 °C and in the presence of endocytosis

inhibitor and spermidine. Besides, the BNCT eﬀect of

¹⁰B-enriched agents is not parallel to their intracellular

uptake, as shown in Figure 7, which suggests their close

interaction with DNA in living cells. Therefore, it is

likely that B-macrocycles are transferred into living cells

via an energy-dependent process such as endocytosis

and then make a close contact to DNA, resulting in an

eﬃcient BNCT eﬀect, although the possibility of the

partial distribution of these boron agents in the cell

membrane cannot be denied.

CONCLUSIONS

■

In conclusion, we report on the design and synthesis of boron-

containing macrocyclic polyamine derivatives as novel boron

delivery agents for BNCT. The results of biological studies

suggest that the intracellular uptake of 15−17, especially, the

9-membered triamine derivatives 15a and 15b, into cancer

cells is higher than that of 1 and 2, and that 15b, 16b, and 17a

are selectively transferred into A549 cells. The results of

BNCT experiments using A549 cells in the presence of 15b,

16b, and 17a including boron in a natural abundance ¹⁰B/¹¹B

ratio and their ¹⁰B-enriched derivatives ¹⁰B-15b, ¹⁰B-16b, and

¹⁰B-17a suggest that metal-free forms of these boron carriers

inhibit the proliferation of A549 cells to a considerable extent

after irradiation with thermal neutrons and that this inhibition

is stronger than that for 1 and 2. In addition, it was suggested

that [12]aneN₄-type ¹⁰B-16b and [15]aneN₅-type ¹⁰B-17a

These experimental data allow us to propose two

possibilities for the BNCT eﬀect of ¹⁰B-15b, ¹⁰B-16b, and

¹⁰B-17a, as presented in Scheme 13. One possible explanation

would be that cytotoxicity is dependent on the close

interaction of metal-free macrocyclic polyamines with DNA

via the ionic interaction (36 in Scheme 13) and the amount of

4

double-strand breaks in DNA by He and/or ⁷Li generated by

the [¹⁰B(n, α)⁷Li] reaction.⁴⁴More plausible possibility would

be the breakdown of DNA via the interaction with metal

8531

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Scheme 11. Synthesis of ¹⁰B-Enriched 15b, 16b, and 17a (¹⁰B-15b, ¹⁰B-16b, and ¹⁰B-17a)

Scheme 12. Evaluation of the Anti-tumor Eﬀect of Boron Compounds in an In Vitro BNCT Study

exhibit a higher BNCT eﬀect than [9]aneN₃-type derivatives,

possibly due to the formation of the corresponding Zn²⁺

complexes (¹⁰B-19b and ¹⁰B-20a) in cancer cells that would

interact with DNA, although ¹⁰B-19b and ¹⁰B-20a interact

with DNA in diﬀerent modes. It should also be noted that

macrocycles containing boron in a natural abundance ¹⁰B/¹¹B

ratio are capable of inducing cell death in BNCT to some

extent and hence can be used as ¹¹B MRI probes to detect their

distribution in living bodies as well as BNCT agents.

design and synthesis of more eﬃcient and safer (high T/N

ratio) boron carriers are currently underway in our laboratory.

EXPERIMENTAL SECTION

■

General Information. All reagents and solvents were purchased

at the highest commercial quality and were used without further

puriﬁcation. MTT was purchased from Dojindo Laboratories.

Spermidine was purchased from WAKO Pure Chemical Industries

Ltd. MβCD, amiloride, and ctDNA were purchased from Sigma-

Aldrich. Dynasore was purchased from Tokyo Chemical Industry. ¹⁰B-

B(OMe)₃was purchased from Katchem Ltd. Anhydrous tetrahy-

drofuran (THF) was prepared by distillation from sodium and

benzophenone. All aqueous solutions were prepared using deionized

We believe that these ﬁndings provide useful information for

the further development of BNCT for cancer treatment. The

8532

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 7. Relationship between the intracellular uptake of boron

10

◇

□

■

○

△

)

compounds 1 ( ), 2 ( ), B-15b ( ), B-16b ( ), B-17a (

●

(30 μM) and control (in the absence of boron compound) ( ) into

A549 cells after incubation for 24 h and their BNCT eﬀect (survival

fractions after irradiation with thermal neutrons for 45 min; thermal

neutron ﬂuence: 4.1 0.1 × 10¹²n/cm²).

(PerkinElmer, Massachusetts, USA) to determine the purity (>95%)

of all compounds. Isotopic purity of ¹⁰B were determined on ICP−

MS (NexION300S, PerkinElmer, Waltham, Massachusetts, USA).

Thin-layer chromatography (TLC) and silica gel column chromatog-

raphy were performed using Merck Silica gel 60 F₂₅₄plate (Merck

KGaA, Darmstadt, Germany) and Fuji Silysia Chemical FL-100D

(Fuji Silysia Chemical, Aichi, Japan), Fuji Silysia Chromatorex NH-

DM1020 silica gel for chromatography (Fuji Silysia Chemical, Aichi,

Japan), respectively.

1-[(4-Boronopheny)methyl]-4,7-bis(tert-butoxycarbonyl)-1,4,7-

triazacyclononane (25a). To a solution of 4-(bromomethyl)-

phenylboronic acid 24a³⁴(66.1 mg, 0.308 mmol, 1.2 equiv) in

MeCN (2.5 mL), 2Boc-tacn 23³³(84.7 mg, 0.257 mmol) and

potassium carbonate (44.0 mg, 0.318 mmol, 1.2 equiv) were added

and the resulting mixture was stirred at reﬂux for 4 h. After adding

H₂O, the reaction mixture was extracted with CHCl₃. The organic

layer was washed with brine, dried over Na₂SO₄, and concentrated

under reduced pressure. The resulting residue was puriﬁed by silica

gel column chromatography (CHCl₃/MeOH = 50/1) to aﬀord 25a

(122.3 mg, 0.264 mmol, quant.) as a colorless amorphous solid: mp

106−110 °C; ¹H NMR (400 MHz, acetone-d₆, TMS): δ 1.44 (s, 9H),

1.51 (d, J = 5.2 Hz, 9H), 2.64−2.72 (m, 4H), 3.18−3.31 (m, 4H),

3.47−3.54 (m, 4H), 3.66−3.74 (m, 2H), 7.07−7.10 (m, 2H), 7.39−

7.42 (m, 2H), 7.76−7.86 (m, 2H) ppm; ¹³C NMR (100 MHz,

acetone-d₆, TMS): δ 27.86, 48.29−49.84 (m), 50.15−51.53 (m),

52.89−54.34 (m), 60.39−60.58 (m), 78.6−78.69 (m), 128.02−

128.41 (m), 133.97−134.16 (m), 142.53, 155.02, 155.20 ppm; ¹¹B

NMR (128 MHz, acetone-d₆, BF₃·OEt₂): δ 29.3 (br s) ppm; IR

(ATR) ν: 3407, 2974, 1668, 1462, 1410, 1365, 1247, 1143, 999, 856,

752, 648, 532, 491, 458, 436 cm⁻¹; HRMS (ESI⁺) m/z: calcd for [M

+ H]⁺C₂₃H₃₉¹⁰BN₃O₆, 463.2963; found, 463.2976; Anal. Calcd (%)

for C₂₃H₃₈BN₃O₆·0.2CHCl₃: C, 57.19; H, 7.90; N, 8.62. Found: C,

57.32; H, 7.66; N, 8.42.

Figure 6. Anti-tumor eﬀect of boron compounds 1, 2, 15b, ¹⁰B-15b,

16b, ¹⁰B-16b, 17a, ¹⁰B-17a, ¹⁰B-18b, ¹⁰B-19b, and ¹⁰B-20a (30 μM)

against A549 cells was examined by a colony formation assay: (a)

◇

○

●

control (in the absence of boron compound) ( ), 1 ( ), 2 ( ), 15b

10

), B-15b ( ), and B-18b ( ). (b) Control ( ), 16b ( ), ¹⁰B-

□ ■

○ ●

◆

(

16b ( ), 17a ( ), and B-17a ( ), and B-19b ( ), and ¹⁰B-20a

(×). After treatment with the boron compound for 24 h, the cells

were irradiated with thermal neutrons for 0, 15, 30, and 45 min and

then incubated without neutron irradiation for 7 days. Averaged

thermal neutron ﬂux was 1.4 × 10⁹n/cm²·s for control (in the

absence of boron compound), 1, 2, 15b, 16b, ¹⁰B-16b, 17a, and ¹⁰B-

17a and 1.6 × 10⁹n/cm²·s for ¹⁰B-15b, ¹⁰B-18b, ¹⁰B-19b, and ¹⁰B-

20a, respectively. The survival fraction was determined by ImageJ-

plugin Colony Area. Data represent the mean SD of at least three

replicates.

10

◇

◆

□

■

water. ¹H (300 and 400 MHz), ¹³C (100 MHz), and ¹¹B (128 MHz)

NMR spectra were recorded on a JEOL Always 300 (JEOL, Tokyo,

Japan) and a JEOL LAMDA 400 (JEOL, Tokyo, Japan) spectrometer.

Tetramethylsilane (TMS) was used as an internal reference (0 ppm)

1

for H and ¹³C NMR measurements in CDCl₃and acetone-d₆and

DMSO-d₆. 3-(Trimethylsilyl)propionic-2,2,3,3-d₄acid sodium (TSP)

was used as an internal reference (0 ppm) for ¹H NMR measurements

in D₂O. 1,4-Dioxane was used as an internal reference (67.19 ppm)

for ¹³C NMR measurements in D₂O. ¹¹B NMR spectra were

measured in quartz NMR tubes using boron triﬂuoride diethyl ether

complex (BF₃·OEt₂) in CDCl₃as an internal reference (0 ppm). IR

spectra were recorded on Perkin-Elmer FTIR Spectrum 100 (ATR)

(PerkinElmer, Massachusetts, USA). Melting points were measured

on a Yanaco micro melting point apparatus and are uncorrected. MS

measurements were performed on a Sciex X500R QTOF (AB SCIEX,

Framingham, Massachusetts, USA) and Varian 910-MS (Varian

Medical Systems, California, USA) spectrometer. Elemental analyses

were performed on a 2400 series II CHNS elemental analyzer

1-[(3-Boronopheny)methyl]-4,7-bis(tert-butoxycarbonyl)-1,4,7-

triazacyclononane (25b). To a solution of 3-(bromomethyl)-

phenylboronic acid 24b³⁶(70.1 mg, 0.326 mmol, 1.2 equiv) in

MeCN (2.5 mL), 2Boc-tacn 23³³(90.1 mg, 0.273 mmol) and

potassium carbonate (46.0 mg, 0.333 mmol, 1.2 equiv) were added

and the mixture was stirred at reﬂux for 26 h. After adding H₂O, the

reaction mixture was extracted with CHCl₃. The organic layer was

washed with brine, dried over Na₂SO₄, and concentrated under

reduced pressure. The resulting residue was puriﬁed by silica gel

column chromatography (hexanes/AcOEt = 1/1 to CHCl₃/MeOH =

50/1) to aﬀord 25b (120.8 mg, 0.261 mmol, 95%) as a colorless

1

amorphous solid: mp 95−97 °C; H NMR (400 MHz, acetone-d₆,

TMS): δ 1.43 (J = 3.6 Hz, 9H), 1.50 (J = 3.6 Hz, 9H), 2.67−2.74 (m,

8533

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Scheme 13. Proposed Scheme for the BNCT Eﬀect of ¹⁰B-15b, ¹⁰B-16b, ¹⁰B-17a, and Their Zn²⁺Complexes

found, 463.2963; Anal. Calcd (%) for C₂₃H₃₈BN₃O₆: C, 59.62; H,

8.27; N, 9.07. Found: C, 59.93; H, 8.24; N, 8.81.

Table 2. T_mValues of ctDNA in the Presence of 3, 16b, 19b,

17a, and 20a (r = [3, 16b, 19b, 17a, or 20a]/[ctDNA(P)])

([ctDNA(P)] = 50 μM in Phosphate)

1-[(4-Boronopheny)methyl]-1,4,7-triazacyclononane TFA Salt

(2TFA) (12a). TFA (1.5 mL) was added to a solution of 25a (122.3

mg, 0.264 mmol) in CH₂Cl₂(1.5 mL), and the resulting mixture was

stirred at room temperature for 1 h. After evaporation, the resulting

residue was dissolved in MeCN and reprecipitated with Et₂O to aﬀord

12a (95.7 mg, 0.195 mmol, 74%) as colorless powder, which was

determined to be the 2TFA salt by elemental analysis: mp 138−141

°C; ¹H NMR (400 MHz, D₂O, TSP): δ 3.06 (t, J = 5.6 Hz, 4H), 3.24

(t, J = 5.6 Hz, 4H), 3.64 (s, 4H), 3.95 (s, 2H), 7.49 (d, J = 7.6 Hz,

2H), 7.83 (d, J = 7.6 Hz, 2H) ppm; ¹³C NMR (100 MHz, D₂O, 1,4-

dioxane): δ 42.84, 44.32, 48.35, 59.57, 116.93, 130.33, 134.65, 138.81,

163.43 ppm; ¹¹B NMR (128 MHz, D₂O, BF₃·OEt₂): δ 29.0 (br s)

ppm; IR (ATR) ν: 3005, 2774, 1665, 1610, 1485, 1420, 1397, 1384,

1353, 1183, 1130, 1083, 1055, 1004, 875, 841, 795, 723, 696, 654,

518, 410 cm⁻¹; HRMS (ESI⁺) m/z: calcd for [M + H]⁺

C₁₃H₂₃¹⁰BN₃O₂, 263.1920; found, 263.1914; Anal. Calcd (%) for

C₁₃H₂₂BN₃O₂·2TFA: C, 41.57; H, 4.93; N, 8.55. Found: C, 41.72; H,

4.85; N, 8.54.

additive

r

T_m(°C)

ΔT_m(°C)

none

3

66

73

78

66

67

72

64

62

60

69

74

71

72

0.1

0.2

0.1

0.5

1.0

5.0

0.1

0.5

1.0

5.0

0.5

1.0

+7

+12

16b

19b

+1

+6

−2

−4

−6

+3

+8

+5

+6

17a

20a

1-[(3-Boronopheny)methyl]-1,4,7-triazacyclononane TFA Salt

(2TFA) (12b). TFA (1.0 mL) was added to a solution of 25b (81.4

mg, 0.174 mmol) in CH₂Cl₂(1.0 mL), and the mixture was stirred at

room temperature for 1 h. After evaporation, the resulting residue was

dissolved in MeCN and reprecipitated with Et₂O to aﬀord 12b (60.9

mg, 0.124 mmol, 71%) as colorless powder, which was determined to

4H), 3.21−3.26 (m, 4H), 3.47−3.51 (m, 4H), 3.66−3.76 (m, 2H),

7.13 (s, 2H), 7.26−7.34 (m, 1H), 7.49−7.57 (m, 1H), 7.73−7.86 (m,

2H) ppm; ¹³C NMR (100 MHz, acetone-d₆, TMS): δ 28.70, 49.38−

50.24 (m), 50.70−51.33 (m), 52.18, 53.75−53.98 (m), 55.09−55.25

(m), 61.89−61.98 (m), 79.54−79.68 (m), 128.11−128.19 (m),

131.96−132.13 (m), 133.62, 135.77−135.90 (m), 139.92, 155.88−

156.13 (m) ppm; ¹¹B NMR (128 MHz, acetone-d₆, BF₃·OEt₂): δ 29.0

(br s) ppm; IR (ATR) ν: 3407, 2974, 2931, 1669, 1460, 1413, 1364,

1246, 1142, 1093, 997, 856, 751, 710, 665, 621, 527, 459, 436 cm⁻¹;

HRMS (ESI⁺) m/z: calcd for [M + H]⁺C₂₃H₃₉¹⁰BN₃O₆, 463.2968;

1

be the 2TFA salt by elemental analysis: mp 136−138 °C; H NMR

(400 MHz, D₂O, TSP): δ 3.00 (t, J = 6.0 Hz, 4H), 3.15 (br s, 4H),

3.51 (br s, 4H), 3.94 (s, 2H), 7.51 (t, J = 8.0 Hz, 1H), 7.57 (d, J = 8.0

Hz, 1H), 7.79−7.80 (m, 2H) ppm; ¹³C NMR (100 MHz, D₂O, 1,4-

dioxane): δ 42.84, 44.17, 48.42, 59.82, 129.02, 116.94, 133.21, 133.94,

135.85, 136.01 ppm; ¹¹B NMR (128 MHz, D₂O, BF₃·OEt₂): δ 29.3

(br s) ppm; IR (ATR) ν: 2810, 1667, 1429, 1337, 1180, 1126, 1010,

8534

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

834, 797, 719, 582, 515, 441, 414 cm⁻¹; HRMS (ESI⁺) m/z: calcd for

[M + H]⁺C₁₃H₂₃¹⁰BN₃O₂, 263.1920; found, 263.1914; Anal. Calcd

(%) for C₁₃H₂₂BN₃O₂·2TFA−0.8H₂O: C, 42.83; H, 4.74; N, 8.81.

Found: C, 42.88; H, 5.04; N, 8.81.

H₂O, the reaction mixture was extracted with CHCl₃. The organic

layer was washed with brine, dried over Na₂SO₄, and concentrated

under reduced pressure. The resulting residue was puriﬁed by silica

gel column chromatography (CHCl₃/MeOH = 100/1) to aﬀord 28b

(121.8 mg, 0.201 mmol, 95%) as a colorless amorphous solid; the ¹H

and ¹³C and ¹¹B NMR spectra of product were identical to previously

reported data.²⁰

1-[4-(13,15-Dioxa-15-boradispiro[5.0.5.3]pentadec-14-yl)-

phenyl]methyl-1,4,7-triazacyclononane (15a). A mixture of 12a

(30.0 mg, 0.0611 mmol) and bicyclohexyl-1,1′-diol 26³⁵(15.7 mg,

0.0792 mmol, 1.3 equiv) in EtOH (0.8 mL) was reﬂuxed for 6 h. After

evaporation, the resulting residue was puriﬁed by NH silica gel

column chromatography (CHCl₃/MeOH = 20/1) to aﬀord 15a (24.8

mg, 0.0583 mmol, 95%) as a colorless amorphous solid: mp 65−67

°C; ¹H NMR (400 MHz, CDCl₃, TMS): δ 1.13−1.32 (m, 6H), 1.63−

1.81 (m, 14H), 2.61−2.67 (m, 8H), 2.78 (s, 4H), 3.73 (s, 2H), 7.34

(d, J = 8.0 Hz, 2H), 7.82 (d, J = 7.6 Hz, 2H) ppm; ¹³C NMR (100

MHz, CDCl₃, TMS): δ 22.32, 25.81, 32.47, 46.40, 46.54, 52.76, 61.71,

84.64, 128.33, 134.92, 142.61 ppm; ¹¹B NMR (128 MHz, CDCl₃,

BF₃·OEt₂): δ 31.5 (br s) ppm; IR (ATR) ν: 2929, 2851, 1611, 1449,

1398, 1356, 1284, 1238, 1131, 1087, 1018, 937, 822, 750, 652, 506,

418 cm⁻¹; HRMS (ESI⁺) m/z: calcd for [M + H]⁺C₂₅H₄₁¹⁰BN₃O₂,

425.3328; found, 425.3322; Anal. Calcd (%) for C₂₅H₄₀BN₃O₂·

0.2CHCl₃·0.6MeOH: C, 66.14; H, 9.17; N, 8.97. Found: C, 66.05; H,

8.98; N, 8.70.

1-[3-(13,15-Dioxa-15-boradispiro[5.0.5.3]pentadec-14-yl)-

phenyl]methyl-1,4,7-triazacyclononane (15b). A mixture of 12b

(20.0 mg, 0.041 mmol) and bicyclohexyl-1,1′-diol 26³⁵(10.5 mg,

0.053 mmol, 1.3 equiv) in EtOH (0.5 mL) was reﬂuxed for 6 h. After

evaporation, the resulting residue was puriﬁed by NH silica gel

column chromatography (CHCl₃/MeOH = 20/1) to aﬀord 15b (11.8

mg, 0.028 mmol, 68%) as a colorless amorphous solid: mp 57−58 °C;

¹H NMR (400 MHz, CDCl₃, TMS): δ 1.14−1.33 (m, 6H), 1.63−1.84

1-[(4-Boronopheny)methyl]-1,4,7,10-tetraazacyclododecane

TFA Salt (2TFA) (13a). TFA (1.0 mL) was added to a solution of 28a

(94.2 mg, 0.155 mmol) in CH₂Cl₂(1.0 mL), and the mixture was

stirred at room temperature for 1 h. After evaporation, the resulting

residue was dissolved in AcOEt and reprecipitated with hexanes to

aﬀord 13a (72.5 mg, 0.136 mmol, 88%) as colorless powder, which

were determined to be the 2TFA salt by elemental analysis: mp 172−

1

175 °C; H NMR (400 MHz, D₂O, TSP): δ 2.93−3.01 (m, 8H),

3.17−3.25 (m, 8H), 3.88 (s, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.83 (d, J

= 8.0 Hz, 2H) ppm; ¹³C NMR (100 MHz, D₂O, 1,4-dioxane): δ

42.31, 42.46, 44.78, 48.38, 57.18, 129.96, 134.87, 138.74 ppm; ¹¹B

NMR (128 MHz, D₂O, BF₃·OEt₂): δ 29.05 (br s) ppm; IR (ATR) ν:

3301, 3088, 2856, 1668, 1610, 1454, 1409, 1343, 1196, 1176, 1120,

1052, 1017, 829, 796, 719, 693, 651, 596, 516, 435, 414 cm⁻¹; HRMS

(ESI⁺) m/z: calcd for [M + H]⁺C₁₅H₂₈¹⁰BN₄O₂, 306.2342; found,

306.2336; Anal. Calcd (%) for C₁₅H₂₇BN₄O₂·2TFA: C, 42.71; H,

5.47; N, 10.49. Found: C, 42.75; H, 5.38; N, 10.44.

1-[(3-Boronopheny)methyl]-1,4,7,10-tetraazacyclododecane

(13b).²⁰TFA (2.0 mL) was added to a solution of 28b (110.2 mg,

0.182 mmol) in CH₂Cl₂(2.0 mL), and the mixture was stirred at

room temperature for 1 h. After evaporation, the resulting residue was

puriﬁed by NH silica gel column chromatography (CHCl₃/MeOH =

20/1) to aﬀord 13b (50.4 mg, 0.165 mmol, 91%) as a colorless

amorphous solid. The ¹H, ¹³C and ¹¹B NMR spectra of product were

identical to previously reported data.²⁰

(m, 14H), 2.65−2.69 (m, 8H), 2.83 (s, 4H), 3.74 (s, 2H), 7.34 (t, J =

7.6 Hz 1H), 7.46 (d, J = 8.0 Hz 1H), 7.75 (d, J = 7.6 Hz 1H), 7.79 (s,

1H) ppm; ¹³C NMR (100 MHz, CDCl₃, TMS): δ 22.34, 25.81, 32.48,

46.58, 46.86, 52.95, 61.54, 84.71, 127.75, 131,66, 133.64, 135.24,

138.84 ppm; ¹¹B NMR (128 MHz, CDCl₃, BF₃·OEt₂): δ 30.8 (br s)

ppm; IR (ATR) ν: 2928, 2852, 1448, 1353, 1285, 1239, 1200, 1145,

1131, 1076, 1040, 939, 779, 751, 708, 615, 507, 409 cm⁻¹; HRMS

(ESI⁺) m/z: calcd for [M + H]⁺C₂₅H₄₁¹⁰BN₃O₂, 425.3323; found,

425.3327; Anal. Calcd (%) for C₂₅H₄₀BN₃O₂·0.1CHCl₃·MeOH: C,

66.78; H, 9.47; N, 8.95. Found: C, 66.88; H, 9.29; N, 8.57.

1-[4-(13,15-Dioxa-15-boradispiro[5.0.5.3]pentadec-14-yl)-

phenyl]methyl-1,4,7,10-tetraazacyclododecane (16a). A mixture of

13a (40.0 mg, 0.0748 mmol) and bicyclohexyl-1,1′-diol 26³⁵(15.2

mg, 0.0766 mmol, 1.0 equiv) in EtOH (1.0 mL) was reﬂuxed for 6 h.

After evaporation, the resulting residue was dissolved in EtOH and

reprecipitated with hexanes, and the resulting precipitate was puriﬁed

by NH silica gel column chromatography (CHCl₃/MeOH = 20/1) to

aﬀord 16a (34.5 mg, 0.0736 mmol, 98%) as a colorless solid: mp

1

129−131 °C; H NMR (300 MHz, CDCl₃, TMS): δ 1.13−1.31 (m,

1-[(4-Boronopheny)methyl]-4,7,10-tris(tert-butoxycarbonyl)-

1,4,7,10-tetraazacyclododecane (28a). To a solution of 4-

(bromomethyl)phenylboronic acid 24a³⁴(66.4 mg, 0.309 mmol, 1.5

equiv) in MeCN (2.0 mL), 3Boc-cyclen 27³⁸(100 mg, 0.212 mmol)

and potassium carbonate (58.1 mg, 0.420 mmol, 2.0 equiv) were

added and the mixture was stirred at reﬂux for 3 h. After adding H₂O,

the reaction mixture was extracted with CHCl₃. The organic layer was

washed with brine, dried over Na₂SO₄, and concentrated under

reduced pressure. The resulting residue was puriﬁed by silica gel

column chromatography (CHCl₃/MeOH = 100/1) to aﬀord 28a

(108.3 mg, 0.179 mmol, 84%) as a colorless amorphous solid: mp

6H), 1.62−1.83 (m, 14H), 2.57 (t, J = 5.2 Hz, 8H), 2.67 (t, J = 5.2

Hz, 4H), 2.81 (t, J = 5.6 Hz, 4H), 3.63 (s, 2H), 7.31 (d, J = 8.0 Hz,

2H), 7.81 (d, J = 8.0 Hz, 2H) ppm; ¹³C NMR (100 MHz, CDCl₃,

TMS): δ 22.31, 25.81, 32.45, 45.09, 46.34, 47.14, 51.26, 59.33, 84.51,

128.36, 134.95, 141.93 ppm; ¹¹B NMR (128 MHz, CDCl₃, BF₃·

OEt₂): δ 31.1 (br s) ppm; IR (ATR) ν: 2933, 2856, 2811, 1610, 1450,

1403, 1356, 1319, 1284, 1272, 1239, 1131, 1088, 1041, 1020, 937,

911, 822, 801, 746, 725, 653, 540, 507 cm⁻¹; HRMS (ESI⁺) m/z:

calcd for [M + H]⁺C₂₇H₄₆¹⁰BN₄O₂, 468.3750; found, 468.3745; Anal.

Calcd (%) for C₂₇H₄₅BN₄O₂·0.5MeOH: C, 68.17; H, 9.78; N, 11.56.

Found: C, 68.35; H, 9.79; N, 11.26.

1

112−115 °C; H NMR (300 MHz, CDCl₃, TMS): δ 1.44−1.49 (m,

1-[3-(13,15-Dioxa-15-boradispiro[5.0.5.3]pentadec-14-yl)-

phenyl]methyl-1,4,7,10-tetraazacyclododecane (16b). A mixture of

13b (27.0 mg, 0.0882 mmol) and bicyclohexyl-1,1′-diol 26³⁵(17.5

mg, 0.0882 mmol, 1.0 equiv) in EtOH (0.9 mL) was reﬂuxed for 3 h.

After evaporation, the resulting residue was puriﬁed by NH silica gel

column chromatography (CHCl₃/MeOH = 20/1) to aﬀord 16b (33.5

mg, 0.0714 mmol, 81%) as a colorless amorphous solid: mp 46−48

°C; ¹H NMR (400 MHz, CDCl₃, TMS): δ 1.22−1.32 (m, 6H), 1.63−

1.83 (m, 14H), 2.56−2.59 (m, 8H), 2.67 (t, J = 4.4 Hz, 4H), 2.81 (t, J

= 4.4 Hz, 4H), 3.64 (s, 2H), 7.32 (t, J = 7.6 Hz, 1H), 7.41 (d, J = 8.0

Hz, 1H), 7.74 (d, J = 7.2 Hz, 1H), 7.77 (s, 1H) ppm; ¹³C NMR (100

MHz, CDCl₃, TMS): δ 22.31, 25.80, 32.45, 45.34, 46.40, 47.39, 51.38,

59.26, 84.57, 127.71, 131.79, 133.80, 135.43, 138.04 ppm; ¹¹B NMR

(128 MHz, CDCl₃, BF₃·OEt₂): δ 30.8 (br s) ppm; IR (ATR) ν: 2929,

2851, 1604, 1448, 1429, 1392, 1352, 1320, 1284, 1272, 1253, 1239,

1200, 1146, 1131, 1114, 1077, 1039, 939, 909, 834, 803, 747, 707,

681, 658, 541, 507 cm⁻¹; HRMS (ESI⁺) m/z: calcd for [M + H]⁺

C₂₇H₄₆¹⁰BN₄O₂, 468.3750; found, 468.3745; Anal. Calcd (%) for

27H), 2.71 (br s, 4H), 3.30−3.40 (m, 8H), 3.59 (br s, 4H), 3.70−3.74

(m, 2H), 7.29 (d, J = 8.0 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H), 7.70 (d, J

= 8.0 Hz, 1H), 8.18 (d, J = 7.6 Hz, 1H) ppm; ¹³C NMR (100 MHz,

CDCl₃, TMS): δ 18.45, 28.49, 28.72, 2872, 29.70, 47.39, 49.82, 58.50,

79.68, 129.46, 129.97, 133.59, 135.61, 139.38, 155.34, 156.18, 158.78,

163.43, 196.70, 209.38, 210.84 ppm; ¹¹B NMR (128 MHz, CDCl₃,

BF₃·OEt₂): δ 29.1 (br s) ppm; IR (ATR) ν: 3397, 2975, 2931, 1682,

1668, 1611, 1478, 1457, 1412, 1364, 1341, 1249, 1151, 1109, 1019,

979, 856, 770, 754, 734, 649, 555, 516, 458 cm⁻¹; HRMS (ESI⁺) m/z:

calcd for [M + H]⁺C₃₀H₅₂¹⁰BN₄O₈, 606.3909; found, 606.3917; Anal.

Calcd (%) for C₃₀H₅₁BN₄O₈·0.3CHCl₃: C, 56.65; H, 8.05; N, 8.72.

Found: C, 56.73; H, 8.10; N, 8.66.

1-[(3-Boronopheny)methyl]-4,7,10-tris(tert-butoxycarbonyl)-

1,4,7,10-tetraazacyclododecane (28b).²⁰To a solution of 3-

(bromomethyl)phenylboronic acid 24b³⁶(67.8 mg, 0.316 mmol,

1.5 equiv) in MeCN (2.0 mL), 3Boc-cyclen 27³⁸(100 mg, 0.212

mmol) and potassium carbonate (58.4 mg, 0.423 mmol, 2.0 equiv)

were added and the mixture was stirred at reﬂux for 3 h. After adding

8535

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

C₂₇H₄₅BN₄O₂·0.1CHCl₃·2.4MeOH: C, 63.58; H, 9.89; N, 10.05.

Found: C, 63.90; H, 9.63; N, 9.65.

1-[(4-Boronopheny)methyl]-1,4,7,10,13-pentaazacyclopentade-

cane TFA Salt (3TFA) (14a). TFA (1.5 mL) was added to a solution

of 30a (111.0 mg, 0.148 mmol) in CH₂Cl₂(1.5 mL), and the mixture

was stirred at room temperature for 30 min. After evaporation, the

resulting residue was dissolved in MeCN and reprecipitated with Et₂O

to aﬀord 14a (90.0 mg, 0.130 mmol, 88%) as colorless powder, which

was determined to be the 3TFA salt by elemental analysis: mp 136−

137 °C; ¹H NMR (400 MHz, D₂O, TSP): δ 3.01 (t, J = 6.0 Hz, 4H),

3.17 (s, 4H), 3.26−3.33 (m, 8H), 3.38 (t, J = 6.0 Hz, 4H), 3.95 (s,

2H), 7.43 (d, J = 8.0 Hz, 2H), 7.83 (d, J = 8.0 Hz, 2H) ppm; ¹³C

NMR (100 MHz, D₂O, 1,4-dioxane): δ 44.31, 45.30, 45.81, 50.24,

56.73, 116.92, 130.56, 134.66, 137.03, 163.59 ppm; ¹¹B NMR (128

MHz, D₂O, BF₃·OEt₂): δ 29.2 (br s) ppm; IR (ATR) ν: 3029, 1668,

1410, 1382, 1359, 1178, 1129, 1055, 1018, 888, 838, 798, 720, 698,

654, 517 cm⁻¹; HRMS (ESI⁺) m/z: calcd for [M + H]⁺

C₁₇H₃₃¹⁰BN₅O₂, 349.2764; found, 349.2770; Anal. Calcd (%) for

C₁₇H₃₂BN₅O₂·3TFA: C, 39.96; H, 5.10; N, 10.13. Found: C, 39.97;

H, 4.98; N, 10.05.

1-[(3-Boronopheny)methyl]-1,4,7,10,13-pentaazacyclopentade-

cane TFA Salt (3TFA) (14b). TFA (1.5 mL) was added to a solution

of 30b (127.2 mg, 0.170 mmol) in CH₂Cl₂(1.5 mL), and the mixture

was stirred at room temperature for 1 h. After evaporation, the

resulting residue was dissolved in MeCN and reprecipitated with Et₂O

to aﬀord 14b (107.1 mg, 0.155 mmol, 91%) as colorless powder,

which was determined to be the 3TFA salt by elemental analysis: mp

106−108 °C; ¹H NMR (400 MHz, D₂O, TSP): δ 3.03 (t, J = 5.2 Hz

4H), 3.18 (s, 4H), 3.27−3.29 (m, 4H), 3.33 (t, J = 5.2 Hz 4H), 3.37−

3.40 (m, 4H), 3.98 (s, 2H), 7.48−7.55 (m, 2H), 7.73 (s, 1H), 7.80−

7.83 (m, 1H) ppm; ¹³C NMR (100 MHz, D₂O, 1,4-dioxane): δ 44.34,

45.21, 45.49, 45.79, 50.27, 56.94, 116.92, 129.09, 133.53, 133.62,

134.35, 136.20, 163.58 ppm; ¹¹B NMR (128 MHz, D₂O, BF₃·OEt₂): δ

28.6 (br s) ppm; IR (ATR) ν: 3030, 2856, 1666, 1426, 1337, 1179,

1123, 834, 797, 719, 597, 517 cm⁻¹; HRMS (ESI⁺) m/z: calcd for [M

+ H]⁺C₁₇H₃₃¹⁰BN₅O₂, 349.2764; found, 349.2758; Anal. Calcd (%)

for C₁₇H₃₂BN₅O₂·3.4TFA: C, 38.79; H, 4.82; N, 9.43. Found: C,

38.66; H, 4.95; N, 9.33.

1-[(4-Boronopheny)methyl]-4,7,10,13-tetra(tert-butoxycarbon-

yl)-1,4,7,10,13-pentaazacyclopentadecane (30a). To a solution of

4-(bromomethyl)phenylboronic acid 24a³⁴(48.3 mg, 0.225 mmol,

1.2 equiv) in MeCN (1.5 mL), 29³⁹(113.9 mg, 0.185 mmol) and

potassium carbonate (38.1 mg, 0.276 mmol, 1.5 equiv) were added

and the mixture was stirred at reﬂux for 2 h. After adding H₂O, the

reaction mixture was extracted with CHCl₃. The organic layer was

washed with brine, dried over Na₂SO₄, and concentrated under

reduced pressure. The resulting residue was puriﬁed by silica gel

column chromatography (CHCl₃/MeOH = 100/1) to aﬀord 30a

(115.0 mg, 0.153 mmol, 83%) as a colorless amorphous solid: mp

1

94−96 °C; H NMR (400 MHz, acetone-d₆, TMS): δ 1.30 (s, 9H),

1.44 (s, 9H), 1.48 (s, 18H), 2.74 (s, 4H), 3.47 (s, 16H), 3.67 (s, 2H),

7.09 (s, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.83 (d, J = 8.0 Hz, 2H) ppm;

¹³C NMR (100 MHz, acetone-d₆, TMS): δ 28.63, 31.99, 47.35, 53.23,

55.49, 60.21, 69.71, 79.56, 79.89, 128.59, 135.05, 142.72, 155.59,

155.72 ppm; ¹¹B NMR (128 MHz, acetone-d₆, BF₃·OEt₂): δ 29.7 (br

s) ppm; IR (ATR) ν: 3420, 2976, 1682, 1465, 1411, 1365, 1245,

1155, 1018, 858, 753, 648, 559, 458 cm⁻¹; HRMS (ESI⁺) m/z: calcd

for [M + H]⁺C₃₇H₆₅¹⁰BN₅O₁₀, 749.4861; found, 749.4855; Anal.

Calcd (%) for C₃₇H₆₄BN₅O₁₀0.3H₂O: C, 58.85; H, 8.62; N, 9.27.

Found: C, 58.88; H, 8.56; N, 9.06.

1-[(3-Boronopheny)methyl]-4,7,10,13-tetra(tert-butoxycarbon-

yl)-1,4,7,10,13-pentaazacyclopentadecane (30b). To a solution of

3-(bromomethyl)phenylboronic acid 24b³⁶(51.6 mg, 0.240 mmol,

1.2 equiv) in MeCN (2.0 mL), 29³⁹(121.0 mg, 0.196 mmol) and

potassium carbonate (40.6 mg, 0.294 mmol, 1.5 equiv) were added

and the mixture was stirred at reﬂux for 4 h. After adding H₂O, the

reaction mixture was extracted with CHCl₃. The organic layer was

washed with brine, dried over Na₂SO₄, and concentrated under

reduced pressure. The resulting residue was puriﬁed by silica gel

column chromatography (CHCl₃/MeOH = 100/1) to aﬀord 30b

(127.4 mg, 0.170 mmol, 87%) as a colorless amorphous solid: mp

106−110 °C; ¹H NMR (400 MHz, acetone-d₆, TMS): δ 1.29 (s, 9H),

1.45 (s, 9H), 1.47 (s, 18H), 2.74 (s, 4H), 3.47 (s, 16H), 3.68 (s, 2H),

7.15 (s, 2H), 7.29 (t, J = 7.2 Hz, 1H), 7.40 (d, J = 6.8 Hz, 1H), 7.72

(d, J = 5.6 Hz, 1H), 7.82 (s, 1H) ppm; ¹³C NMR (100 MHz, acetone-

d₆, TMS): δ 28.61, 31.99, 46.54, 47.34, 53.22, 55.44, 60.19, 79.65,

79.89, 128.26, 131.44, 133.58, 135.15, 155.74 ppm; ¹¹B NMR (128

MHz, acetone-d₆, BF₃·OEt₂): δ 30.0 (br s) ppm; IR (ATR) ν: 3419,

2976, 1682, 1464, 1413, 1365, 1244, 1155, 1043, 860, 770, 710, 558,

462, 418 cm⁻¹; HRMS (ESI⁺) m/z: calcd for [M + H]⁺

C₃₇H₆₅¹⁰BN₅O₁₀, 749.4861; found, 749.4855; Anal. Calcd (%) for

C₃₇H₆₄BN₅O₁₀0.25CHCl₃: C, 57.39; H, 8.31; N, 8.92. Found: C,

57.67; H, 8.21; N, 8.72.

1-[(2-Boronopheny)methyl]-1,4,7,10,13-pentaazacyclopentade-

cane TFA Salt (3TFA) (14c). TFA (1.5 mL) was added to a solution of

30c (115.7 mg, 0.154 mmol) in CH₂Cl₂(1.5 mL), and the mixture

was stirred at room temperature for 1 h. After evaporation, the

resulting residue was dissolved in AcOEt and reprecipitated with

hexanes to aﬀord 14c (94.8 mg, 0.137 mmol, 89%) as colorless

powder, which was determined to be the 3TFA salt by elemental

1

analysis: mp 118−120 °C; H NMR (400 MHz, D₂O, TSP): δ 2.99

(t, J = 4.8 Hz 4H), 3.11 (m, J = 5.6 Hz 4H), 3.17 (s, 12H), 4.02 (s,

2H), 7.40 (d, J = 6.8 Hz 1H), 7.47−7.51 (m, 2H), 7.70 (d, J = 7.2 Hz

1H) ppm; ¹³C NMR (100 MHz, D₂O, 1,4-dioxane): δ 44.60, 44.86,

45.50, 51.06, 60.39, 116.91, 128.88, 130.75, 131.36, 134.06, 138.21,

163.60 ppm; ¹¹B NMR (128 MHz, D₂O, BF₃·OEt₂): δ 29.7 (br s)

ppm; IR (ATR) ν: 3023, 2843, 1668, 1440, 1359, 1193, 1147, 1126,

1050, 1032, 836, 797, 767, 719, 624, 588, 517, 443, 420 cm⁻¹; HRMS

(ESI⁺) m/z: calcd for [M + H]⁺C₁₇H₃₃¹⁰BN₅O₂, 349.2764; found,

349.2769; Anal. Calcd (%) for C₁₇H₃₂BN₅O₂·3TFA: C, 39.96; H,

5.10; N, 10.13. Found: C, 39.67; H, 4.87; N, 9.84.

1-[(2-Boronopheny)methyl]-4,7,10,13-tetra(tert-butoxycarbon-

yl)-1,4,7,10,13-pentaazacyclopentadecane (30c). To a solution of

2-(bromomethyl)phenylboronic acid 24c³⁷(55.1 mg, 0.256 mmol,

1.2 equiv) in MeCN (2.0 mL), 29³⁹(129.8 mg, 0.211 mmol) and

potassium carbonate (44.3 mg, 0.32 mmol, 1.5 equiv) were added and

the mixture was stirred at reﬂux for 10 h. After adding H₂O, the

reaction mixture was extracted with CHCl₃. The organic layer was

washed with brine, dried over Na₂SO₄, and concentrated under

reduced pressure. The resulting residue was puriﬁed by silica gel

column chromatography (CHCl₃/MeOH = 100/1) to aﬀord 30c

(115.7 mg, 0.154 mmol, 73%) as a colorless amorphous solid: mp

1-[4-(13,15-Dioxa-15-boradispiro[5.0.5.3]pentadec-14-yl)-

phenyl]methyl-1,4,7,10,13-pentaazacyclopentadecane (17a). A

mixture of 14a (30.4 mg, 0.044 mmol) and bicyclohexyl-1,1′-diol

26³⁵(9.4 mg, 0.0474 mmol, 1.1 equiv) in EtOH (0.8 mL) was

reﬂuxed for 2 h. After evaporation, the resulting residue was puriﬁed

by NH silica gel column chromatography (CHCl₃/MeOH = 100/1)

to aﬀord 17a (21.4 mg, 0.0419 mmol, 88%) as a colorless amorphous

1

105−109 °C; H NMR (400 MHz, acetone-d₆, TMS): δ 1.47 (s,

36H), 2.82 (s, 4H), 3.45 (s, 16H), 3.82 (s, 2H), 7.25−7.36 (m, 4H),

7.87 (br, 1H), 8.78 (br, 1H) ppm; ¹³C NMR (100 MHz, acetone-d₆,

TMS): δ 28.39, 28.63, 47.12, 50.94, 51.42, 55.49, 62.29, 79.77, 79.94,

127.94, 130.61, 131.96, 137.26, 142.60, 155.44, 155.62 ppm; ¹¹B

NMR (128 MHz, acetone-d₆, BF₃·OEt₂): δ 29.7 (br s) ppm; IR

(ATR) ν: 2975, 2932, 1692, 1463, 1412, 1391, 1365, 1309, 1244,

1156, 1032, 947, 894, 860, 770, 653, 550, 460 cm⁻¹; HRMS (ESI⁺)

m/z: calcd for [M + H]⁺C₃₇H₆₅¹⁰BN₅O₁₀, 749.4861; found,

749.4864; Anal. Calcd (%) for C₃₇H₆₄BN₅O₁₀1.5MeOH: C, 57.96;

H, 8.84; N, 8.78. Found: C, 57.84; H, 9.06; N, 9.07.

1

solid: mp 43−44 °C; H NMR (400 MHz, CDCl₃, TMS): δ 1.16−

1.31 (m, 6H), 1.73−1.80 (m, 14H), 2.65 (s, 12H), 2.79 (s, 8H), 3.62

(s, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.81 (d, J = 7.6 Hz, 2H) ppm; ¹³C

NMR (100 MHz, CDCl₃, TMS): δ 22.31, 25.81, 32.47, 47.29, 47.79,

48.24, 49.00, 54.55, 59.62, 84.68, 128.36, 134.93, 142.52 ppm; ¹¹B

NMR (128 MHz, CDCl₃, BF₃·OEt₂): δ 30.3 (br s) ppm; IR (ATR) ν:

3287, 2930, 2849, 1610, 1449, 1399, 1356, 1284, 1238, 1130, 1087,

937, 823, 727, 652, 507 cm⁻¹; HRMS (ESI⁺) m/z: calcd for [M +

Na]⁺, C₂₉H₅₀¹⁰BN₅O₂Na, 533.3986; found, 533.4010; Anal. Calcd

8536

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(%) for C₂₉H₅₀BN₅O₂·CHCl₃: C, 57.11; H, 8.15; N, 11.10. Found: C,

57.46; H, 8.13; N, 10.79.

1495, 1432, 1349, 1283, 1238, 1204, 1131, 1092, 961, 937, 909, 808,

708, 694, 641, 614, 565, 502 cm⁻¹; HRMS (ESI⁺) m/z: calcd for

[M]²⁺C₂₇H₄₅¹⁰BN₄O₂⁶⁴Zn, 265.6476; found, 265.6475; Anal. Calcd

(%) for C₂₇H₄₅BN₆O₈Zn·0.5H₂O: C, 48.63; H, 6.95; N, 12.60.

Found: C, 48.73; H, 7.11; N, 12.37.

1-[3-(13,15-Dioxa-15-boradispiro[5.0.5.3]pentadec-14-yl)-

phenyl]methyl-1,4,7,10,13-pentaazacyclopentadecane (17b). A

mixture of 14b (30.2 mg, 0.0437 mmol) and bicyclohexyl-1,1′-diol

26³⁵(8.7 mg, 0.0439 mmol, 1.0 equiv) in EtOH (0.6 mL) was

reﬂuxed for 5 h. After evaporation, the resulting residue was puriﬁed

by NH silica gel column chromatography (CHCl₃/MeOH = 20/1) to

aﬀord 17b (15.6 mg, 0.0305 mmol, 70%) as a colorless amorphous

Synthesis of ¹⁰B-Enriched Compounds. 4-(Bromomethyl)-

phenylboronic Acid (¹⁰B-24a).³⁴To a solution of 4-bromotoluene

34a (904 mg, 5.28 mmol, 1.3 equiv) in THF (10 mL), 1.6 N of n-

butyllithium (n-BuLi) in hexanes (3.3 mL, 5.28 mmol, 1.3 equiv) was

added at −78 °C and the reaction mixture was stirred at the same

temperature for 1 h, after which, ¹⁰B-enriched trimethyl borate

(>99.5% of ¹⁰B) (450 μL, 4.06 mmol, 1.0 equiv) was slowly added.

After stirring at −78 °C to room temperature overnight, 2 N aqueous

HCl was added to the reaction mixture, which was further stirred at 0

°C for 3 h. After extraction with CHCl₃, the organic layer was washed

with brine, dried over Na₂SO₄, and concentrated under reduced

pressure. The resulting residue was recrystallized from hexanes to

aﬀord ¹⁰B-35a (279 mg, 2.06 mmol, 51%) as a colorless needle

crystal.

1

solid: H NMR (400 MHz, CDCl₃, TMS): δ 1.17−1.33 (m, 6H),

1.76−1.80 (m, 14H), 2.62−2.80 (m, 20H), 3.60 (s, 2H), 7.33 (t, J =

7.6 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.68 (s, 1H), 7.76 (d, J = 7.6

Hz, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃, TMS): δ 22.33, 25.78,

32.48, 47.30, 47.85, 48.33, 48.96, 54.84, 59.63, 84.79, 127.70, 132.04,

133.90, 135.45, 138.83 ppm; ¹¹B NMR (128 MHz, CDCl₃, BF₃·

OEt₂): δ 30.2 (br s) ppm; IR (ATR) ν: 3281, 2929, 2849, 1550, 1449,

1353, 1272, 1238, 1201, 1130, 1077, 1041, 939, 910, 807, 760, 708,

611, 540, 509 cm⁻¹; HRMS (ESI⁺) m/z: calcd for [M + Na]⁺,

C₂₉H₅₀¹⁰BN₅O₂Na, 533.3986; found, 533.4010; Anal. Calcd (%) for

C₂₉H₅₀BN₅O₂: C, 68.09; H, 9.85; N, 13.69. Found: C, 68.06; H,

10.15; N, 13.77.

A mixture of ¹⁰B-35a (270 mg, 2.00 mmol), NBS (390 mg, 2.19

mmol, 1.1 equiv), and benzoyl peroxide (BPO) (16 mg, 0.066 mmol,

0.03 equiv) in CCl₄(13 mL) was reﬂuxed for 6 h and then diluted

with CHCl₃. The reaction mixture was washed with H₂O and brine,

dried over Na₂SO₄, and evaporated. The resulting residue was

recrystallized from hexanes/AcOEt to give ¹⁰B-24a (281 mg, 1.31

mmol, 66%) as a colorless solid: isotopic purity of ¹⁰B: 98.5 0.3%;

1-[2-(13,15-Dioxa-15-boradispiro[5.0.5.3]pentadec-14-yl)-

phenyl]methyl-1,4,7,10,13-pentaazacyclopentadecane (17c). A

mixture of 14c (26.5 mg, 0.0383 mmol) and bicyclohexyl-1,1′-diol

26³⁵(7.6 mg, 0.0383 mmol, 1.0 equiv) in EtOH (0.4 mL) was

reﬂuxed for 6 h. After evaporation, the resulting residue was puriﬁed

by NH silica gel column chromatography (CHCl₃/MeOH = 30/1) to

aﬀord 17c (15 mg, 0.0293 mmol, 77%) as a colorless solid: mp 78−80

°C; ¹H NMR (400 MHz, CDCl₃, TMS): δ 1.14−1.33 (m, 6H), 1.64−

1.81 (m, 14H), 2.58−2.77 (m, 20H), 3.94 (s, 2H), 7.23 (t, J = 7.2 Hz,

1H), 7.42 (td, J = 7.6, 1.2 Hz, 1H), 7.59 (d, J = 7.2 Hz, 1H), 7.82 (dd,

J = 7.2, 1.6 Hz, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃, TMS): δ

22.52, 25.78, 32.48, 47.57, 48.03, 48.52, 49.16, 55.40, 57.57, 84.76,

125.97, 129.34, 130.78, 135.91, 147.03 ppm; ¹¹B NMR (128 MHz,

CDCl₃, BF₃·OEt₂): δ 30.4 (br s) ppm; IR (ATR) ν: 3285, 2932, 2814,

1598, 1568, 1439, 1345, 1311, 1284, 1272, 1236, 1132, 1110, 1064,

1039, 938, 805, 749, 733, 655, 545, 507 cm⁻¹; HRMS (ESI⁺) m/z:

calcd for [M + H]⁺C₂₉H₅₁¹⁰BN₅O₂, 511.4167; found, 511.4173; Anal.

Calcd (%) for C₂₉H₅₀BN₅O₂·0.25CHCl₃: C, 64.89; H, 9.36; N, 12.94.

Found: C, 65.04; H, 9.60; N, 12.82.

1

mp 159−162 °C; H NMR (400 MHz, DMSO-d₆, TMS): δ 4.69 (s,

2H), 7.40 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 7.6 Hz, 2H), 8.09 (s, 2H)

ppm; ¹³C NMR (100 MHz, DMSO-d₆, TMS): δ 34.53, 125.42,

128.26, 134.39, 139.62 ppm; IR (ATR) ν: 3268, 1613, 1518, 1398,

1373, 1230, 1178, 1110, 1094, 1028, 1014, 844, 803, 744, 691, 659,

633, 601, 500, 442 cm⁻¹; HRMS (ESI⁺) m/z: calcd for [M + Na]⁺

C₇H₈¹⁰BBrO₂Na, 235.9729; found, 235.9735; Anal. Calcd (%) for

C₇H₈¹⁰BBrO₂·0.1H₂O: C, 38.95; H, 3.83. Found: C, 38.56; H, 3.52.

3-(Bromomethyl)phenylboronic Acid (¹⁰B-24b).³⁶To a solution

of 3-bromotoluene 34b (916 mg, 5.36 mmol, 1.7 equiv) in THF (6

mL), 1.6 N of n-BuLi in hexanes (3.5 mL, 5.6 mmol, 1.8 equiv) was

added at −78 °C and the reaction mixture was stirred at the same

temperature for 1 h, after which, ¹⁰B-enriched trimethyl borate

(>99.5% of ¹⁰B) (350 μL, 3.16 mmol, 1.0 equiv) was slowly added.

After stirring at −78 °C to room temperature overnight, 2 N aqueous

HCl was added to the reaction mixture, which was further stirred at 0

°C for 3 h. After extraction with CHCl₃, the organic layer was washed

with brine, dried over Na₂SO₄, and concentrated under reduced

pressure. The resulting residue was recrystallized from hexanes to

aﬀord ¹⁰B-35b (103 mg, 0.762 mmol, 24%) as a colorless needle

crystal.

Complexation of 16a with Zn²⁺(19a). To a solution of 16a (10.8

mg, 0.023 mmol) in EtOH (0.3 mL), Zn(NO₃)₂·6H₂O (6.9 mg,

0.0232 mmol, 1.0 equiv) in EtOH (0.2 mL) was added at room

temperature. After evaporation, the resulting residue was recrystallized

from EtOH (0.1 mL) to provide colorless crystals of 19a (10.1 mg,

1

0.015 mmol, 67%): mp 257−259 °C; H NMR (400 MHz, D₂O,

TSP): δ 1.44−1.83 (m, 20H), 2.74 (br, 2H), 2.87 (br, 8H), 3.00 (br,

4H), 3.26 (br, 2H), 3.83 (br, 1H), 4.04−4.11 (m, 4H), 7.52−7.57 (m,

2H), 7.74−7.92 (m, 2H) ppm; ¹³C NMR (100 MHz, D₂O, 1,4-

dioxane): δ 22.44, 25.51, 32.00, 42.75, 44.17, 45.09, 49.73, 56.24,

87.52, 131.42, 134.44, 135.30 ppm; ¹¹B NMR (128 MHz, D₂O, BF₃·

OEt₂): δ 29.3 (br s) ppm; IR (ATR) ν: 3243, 2928, 1611, 1482, 1449,

1354, 1284, 1238, 1131, 1088, 992, 935, 856, 819, 730, 702, 673, 644,

538, 473 cm^{− 1}; HRMS (ESI⁺) m/z: calcd for [M]²⁺

C₂₇H₄₅¹⁰BN₄O₂⁶⁴Zn, 265.6476; found, 265.6474; Anal. Calcd (%)

for C₂₇H₄₅BN₆O₈Zn·0.5H₂O: C, 48.63; H, 6.95; N, 12.60. Found: C,

48.58; H, 6.88; N, 12.47.

A mixture of ¹⁰B-35b (98 mg, 0.721 mmol), NBS (140 mg, 0.787

mmol, 1.1 equiv), and BPO (6.0 mg, 0.025 mmol, 0.03 equiv) in CCl₄

(4 mL) was reﬂuxed for 7 h and then diluted with CHCl₃. The

reaction mixture was washed with H₂O and brine, dried over Na₂SO₄,

and evaporated. The resulting residue was recrystallized from

hexanes/AcOEt to give ¹⁰B-24b (110.2 mg, 0.515 mmol, 71%) as a

colorless solid: isotopic purity of ¹⁰B: 98.8 0.1%; mp 208−211 °C;

¹H NMR (400 MHz, DMSO-d₆, TMS): δ 4.70 (s, 2H), 7.33 (t, J =

8.0 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.84

(s, 1H), 8.12 (s, 2H) ppm; ¹³C NMR (100 MHz, DMSO-d₆, TMS): δ

34.90, 127.63, 130.81, 133.92, 134.95, 136.79 ppm; IR (ATR) ν:

3054, 1693, 1604, 1486, 1434, 1370, 1331, 1222, 1200, 1079, 999,

926, 806, 739, 696, 612, 597, 553, 431 cm⁻¹; HRMS (ESI⁺) m/z:

calcd for [M + Na]⁺C₇H₈¹⁰BBrO₂Na, 235.9729; found, 235.9739;

Anal. Calcd (%) for C₇H₈¹⁰BBrO₂·0.3AcOEt·3H₂O: C, 43.39; H,

3.35. Found: C, 43.66; H, 3.01.

1-[(3-Boronopheny)methyl]-1,4,7-triazacyclononane TFA Salt

(2TFA) (¹⁰B-12b). A mixture of 3-(bromomethyl)phenylboronic acid

¹⁰B-24b³⁶(35 mg, 0.164 mmol, 1.1 equiv), 2Boc-tacn 23³³(50 mg,

0.152 mmol), and potassium carbonate (25.1 mg, 0.182 mmol, 1.2

equiv) in MeCN (1.5 mL) were reﬂuxed for 16 h. After adding H₂O,

the reaction mixture was extracted with CHCl₃. The organic layer was

washed with brine, dried over Na₂SO₄, and concentrated under

Complexation of 16b with Zn²⁺(19b). To a solution of 16b (15.5

mg, 0.0331 mmol) in EtOH (0.3 mL), Zn(NO₃)₂·6H₂O (9.8 mg,

0.033 mmol, 1.0 equiv) in EtOH (0.2 mL) was added at room

temperature. The generated crystalline sample of 19b was recrystal-

lized from EtOH (0.5 mL) and Et₂O (0.5 mL) to provide colorless

1

crystals (10.6 mg, 0.016 mmol, 49%): mp 208−210 °C; H NMR

(400 MHz, D₂O, TSP): δ 1.26 (br, 2H), 1.49 (br, 4H), 1.66−1.81

(m, 14H), 2.73 (br, 2H), 2.86 (br, 8H), 2.99 (br, 4H), 3.24 (br, 2H),

3.84 (br, 1H), 4.05 (s, 2H), 4.10 (br s, 2H), 7.46 (d, J = 7.6 Hz, 2H),

7.90 (d, J = 7.6 Hz, 2H) ppm; ¹³C NMR (100 MHz, D₂O, 1,4-

dioxane): δ 21.90, 25.84, 30.08, 42.77, 44.17, 45.09, 49.67, 56.33,

77.05, 128.85, 131.42, 134.24, 134.52, 136.86 ppm; ¹¹B NMR (128

MHz, D₂O, BF₃·OEt₂): δ 28.3 (br s) ppm; IR (ATR) ν: 3211, 2927,

8537

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

reduced pressure. The resulting residue was puriﬁed by silica gel

column chromatography (CHCl₃/MeOH = 50/1) to aﬀord ¹⁰B-25b

(62.1 mg) as a colorless amorphous solid.

NMR (100 MHz, CDCl₃, TMS): δ 22.31, 25.81, 32.44, 45.30, 46.43,

47.37, 51.38, 59.18, 84.57, 127.69, 131.77, 133.77, 135.43, 138.06

ppm; IR (ATR) ν: 2930, 2851, 1579, 1435, 1392, 1371, 1347, 1272,

1243, 1201, 1131, 1076, 1040, 939, 808, 748, 713, 660, 618, 507, 412

cm⁻¹; HRMS (ESI⁺) m/z: calcd for [M + H]⁺C₂₇H₄₆¹⁰BN₄O₂,

468.3745; found, 468.3741; Anal. Calcd (%) for C₂₇H₄₅¹⁰BN₄O₂·

0.5CHCl₃·MeOH: C, 61.19; H, 8.92; N, 10.02. Found: C, 61.27; H,

9.08; N, 9.64.

TFA (1.0 mL) was added to a solution of ¹⁰B-25b in CH₂Cl₂(1.0

mL), and the mixture was stirred at room temperature for 1 h. After

evaporation, the resulting residue was dissolved in MeCN and

reprecipitated with Et₂O to aﬀord ¹⁰B-12b (55.6 mg, 0.113 mmol,

1

74%) as colorless powder: mp 134−136 °C; H NMR (400 MHz,

D₂O, TSP): δ 3.03 (t, J = 6.0 Hz, 4H), 3.19 (br s, 4H), 3.57 (br s,

4H), 3.95 (s, 2H), 7.51 (t, J = 7.6 Hz, 1H), 7.57 (d, J = 7.6 Hz, 1H),

7.78−7.80 (m, 2H) ppm; ¹³C NMR (100 MHz, D₂O, 1,4-dioxane): δ

42.77, 44.17, 48.29, 59.74, 116.92, 129.03, 133.26, 134.00, 135.70,

135.91, 163.61 ppm; IR (ATR) ν: 2808, 1667, 1489, 1439, 1366,

1313, 1180, 1126, 1012, 834, 797, 719, 591, 517, 417 cm⁻¹; HRMS

(ESI⁺) m/z: calcd for [M + H]⁺C₁₃H₂₃¹⁰BN₃O₂, 263.1914; found,

263.1922; Anal. Calcd (%) for C₁₃H₂₂¹⁰BN₃O₂·2TFA: C, 41.64; H,

4.93; N, 8.57. Found: C, 41.94; H, 5.02; N, 8.50.

1-[(4-Boronopheny)methyl]-1,4,7,10,13-pentaazacyclopentade-

cane TFA Salt (3TFA) (¹⁰B-14a). A mixture of 4-(bromomethyl)-

phenylboronic acid ¹⁰B-24a³⁴(38.2 mg, 0.178 mmol, 1.2 equiv), 29³⁹

(92.1 mg, 0.150 mmol), and potassium carbonate (25.5 mg, 0.184

mmol, 1.2 equiv) in MeCN (1.5 mL) was reﬂuxed for 11 h. After

adding H₂O, the reaction mixture was extracted with CHCl₃. The

organic layer was washed with brine, dried over Na₂SO₄and

concentrated under reduced pressure. The resulting residue was

puriﬁed by silica gel column chromatography (CHCl₃/MeOH = 50/

1) to aﬀord ¹⁰B-30a (112.2 mg) as a colorless amorphous solid.

TFA (1.5 mL) was added to a solution of ¹⁰B-30a in CH₂Cl₂(1.5

mL), and the mixture was stirred at room temperature for 1 h. After

evaporation, the resulting residue was dissolved in MeCN and

reprecipitated with Et₂O to aﬀord ¹⁰B-14a (79.1 mg, 0.115 mmol,

1-[3-(13,15-Dioxa-15-boradispiro[5.0.5.3]pentadec-14-yl)-

phenyl]methyl-1,4,7-triazacyclononane (¹⁰B-15b). A mixture of

¹⁰B-12b (24 mg, 0.049 mmol) and bicyclohexyl-1,1′-diol 26³⁵(10.1

mg, 0.043 mmol, 1.0 equiv) in EtOH (0.7 mL) was reﬂuxed for 13 h.

After evaporation, the resulting residue was puriﬁed by NH silica gel

column chromatography (CHCl₃/MeOH = 20/1) to aﬀord ¹⁰B-15b

(18.7 mg, 0.051 mmol, 90%) as a colorless amorphous solid: mp 58−

1

77%) as colorless powder: mp 136−140 °C; H NMR (400 MHz,

D₂O, TSP): δ 2.99 (t, J = 5.2 Hz, 4H), 3.16 (s, 4H), 3.24−3.33 (m,

12H), 3.95 (s, 2H), 7.45 (d, J = 8.0 Hz, 2H), 7.84 (d, J = 7.6 Hz, 2H)

ppm; ¹³C NMR (100 MHz, D₂O, 1,4-dioxane): δ 44.29, 45.29, 45.78,

50.21, 56.67, 116.90, 130.55, 134.65, 137.01, 163.59 ppm; IR (ATR)

ν: 3045, 2852, 1671, 1610, 1421, 1391, 1200, 1177, 1126, 1060, 1016,

836, 799, 735, 720, 699, 666, 518, 503, 413 cm⁻¹; HRMS (ESI⁺) m/z:

calcd for [M + H]⁺C₁₇H₃₃¹⁰BN₅O₂, 349.2758; found, 349.2754; Anal.

Calcd (%) for C₁₇H₃₂¹⁰BN₅O₂·3TFA: C, 40.00; H, 5.11; N, 10.14.

Found: C, 40.13; H, 5.03; N, 10.07.

1

59 °C; H NMR (400 MHz, CDCl₃, TMS): δ 1.16−1.33 (m, 6H),

1.71−1.84 (m, 14H), 2.63−2.69 (m, 8H), 2.81 (s, 4H), 3.74 (s, 2H),

7.33 (t, J = 7.6 Hz 1H), 7.46 (d, J = 7.6 Hz 1H), 7.74 (d, J = 7.2 Hz

1H), 7.80 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃, TMS): δ 22.33,

25.80, 32.45, 46.51, 46.86, 52.95, 61.54, 84.69, 127.73, 131.66, 133.59,

135.22, 138.86 ppm; IR (ATR) ν: 2930, 2854, 1612, 1449, 1397,

1372, 1284, 1242, 1146, 1132, 1091, 1020, 937, 823, 750, 727, 678,

662, 616, 495, 404 cm⁻¹; HRMS (ESI⁺) m/z: calcd for [M + H]⁺

C₂₅H₄₁¹⁰BN₃O₂, 425.3323; found, 425.3323; Anal. Calcd (%) for

C₂₅H₄₀¹⁰BN₃O₂·0.4CHCl₃·1.6MeOH: C, 61.93; H, 9.01; N, 8.02.

Found: C, 61.66; H, 8.65; N, 7.65.

1-[4-(13,15-Dioxa-15-boradispiro[5.0.5.3]pentadec-14-yl)-

phenyl]methyl-1,4,7,10,13-pentaazacyclopentadecane (¹⁰B-17a).

A mixture of ¹⁰B-14a (32.5 mg, 0.047 mmol) and bicyclohexyl-1,1′-

diol 26³⁵(9.7 mg, 0.049 mmol, 1.0 equiv) in EtOH (0.7 mL) was

reﬂuxed for 7 h. After evaporation, the resulting residue was puriﬁed

by NH silica gel column chromatography (CHCl₃/MeOH = 20/1) to

aﬀord ¹⁰B-17a (20.9 mg, 0.041 mmol, 87%) as a colorless amorphous

1-[(3-Boronopheny)methyl]-1,4,7,10-tetraazacyclododecane

TFA Salt (2TFA) (¹⁰B-13b).²⁰A mixture of 3-(bromomethyl)-

phenylboronic acid ¹⁰B-24b³⁶(25.7 mg, 0.12 mmol, 1.1 equiv),

3Boc-cyclen 27³⁸(51.2 mg, 0.108 mmol), and potassium carbonate

(18.5 mg, 0.134 mmol, 1.2 equiv) in MeCN (1.5 mL) was reﬂuxed for

10 h. After adding H₂O, the reaction mixture was extracted with

CHCl₃. The organic layer was washed with brine, dried over Na₂SO₄,

and concentrated under reduced pressure. The resulting residue was

puriﬁed by silica gel column chromatography (CHCl₃/MeOH = 50/

1) to aﬀord ¹⁰B-28b (83.2 mg) as a colorless amorphous solid.

TFA (1.0 mL) was added to a solution of ¹⁰B-28b in CH₂Cl₂(1.0

mL), and the mixture was stirred at room temperature for 1 h. After

evaporation, the resulting residue was dissolved in MeCN and

reprecipitated with Et₂O to aﬀord ¹⁰B-13b (54.4 mg, 0.102 mmol,

1

solid: mp 47−49 °C; H NMR (400 MHz, CDCl₃, TMS): δ 1.16−

1.32 (m, 6H), 1.62−1.83 (m, 14H), 2.64 (s, 12H), 2.78 (s, 8H), 3.61

(s, 2H), 7.33 (d, J = 7.6 Hz, 2H), 7.80 (d, J = 8.0 Hz, 2H) ppm; ¹³C

NMR (100 MHz, CDCl₃, TMS): δ 22.29, 25.80, 32.45, 47.34, 47.91,

48.41, 49.14, 54.66, 59.54, 84.61, 128.30, 134.90, 142.52 ppm; IR

(ATR) ν: 2929, 2849, 1612, 1517, 1448, 1396, 1373, 1243, 1131,

1091, 1040, 1020, 937, 823, 747, 730, 677, 661, 507 cm⁻¹; HRMS

(ESI⁺) m/z: calcd for [M + H]⁺C₂₉H₅₁¹⁰BN₅O₂, 511.4167; found,

511.4166; Anal. Calcd (%) for C₂₉H₅₀¹⁰BN₅O₂·0.3CHCl₃·2MeOH:

C, 61.56; H, 9.62; N, 11.47. Found: C, 61.77; H, 9.25; N, 11.15.

X-ray Data Collection and Reﬁnement. The crystals of 19a

were suitable for a single-crystal X-ray structure analysis, which were

performed on a Bruker APEX CCD diﬀractometer equipped with a

Rigaku Instruments low-temperature attachment. Data were collected

at 93 K using monochromated Mo Kα radiation (λ = 0.71073 Å). The

frames were indexed, integrated, and scaled using the SMART and

SAINT software packages. An empirical absorption correction was

applied to the collection reﬂections with SADABS using XPREP. The

structure was solved by the direct method and reﬁned on F₂by the

full-matrix least squares technique using the SHELX-2015 program

package. All non-hydrogen atoms were reﬁned anisotropically. The

crystal data in this manuscript can be obtained free of change from

The Cambridge Crystallographic Data Centre via www.cccdc.cam.ac.

uk/data_request/cif. Crystal data for 19a C₂₉H₅₁BN₆O₉Zn, M_r=

703.93, orthorhombic, P 2₁2₁2₁, a = 10.008 (4), b = 10.967 (4), c =

30.483 (12) Å, V = 3346 (2) Å³, Z = 4, ρ_calc= 1.397 g·cm⁻³, R =

0.0553 (7083 reﬂections), R_w= 0.1229 (7656 reﬂections), GOF =

1.195. CCDC 2058200 contains the supplementary crystallographic

data for the paper.

1

94%) as colorless powder: mp 113−117 °C; H NMR (400 MHz,

D₂O, TSP): δ 2.93−3.02 (m, 8H), 3.16−3.23 (m, 8H), 3.89 (s, 2H),

7.51−7.53 (m, 2H), 7.75 (s, 1H), 7.79−7.81 (m, 1H) ppm; ¹³C NMR

(100 MHz, D₂O, 1,4-dioxane): δ 42.33, 42.45, 44.75, 48.37, 57.23,

116.91, 129.22, 132.98, 134.07, 135.47, 135.53, 163.60 ppm; IR

(ATR) ν: 3018, 2859, 1668, 1404, 1175, 1123, 1064, 830, 796, 718,

629, 593, 517, 411 cm⁻¹; HRMS (ESI⁺) m/z: calcd for [M + H]⁺

C₁₅H₂₈¹⁰BN₄O₂, 306.2336; found, 306.2334; Anal. Calcd (%) for

C₁₅H₂₇¹⁰BN₄O₂·2.4TFA: C, 41.07; H, 5.12; N, 9.68. Found: C, 40.84;

H, 5.32; N, 9.79.

1-[3-(13,15-Dioxa-15-boradispiro[5.0.5.3]pentadec-14-yl)-

phenyl]methyl-1,4,7,10-tetraazacyclododecane (¹⁰B-16b). A mix-

ture of ¹⁰B-13b (26 mg, 0.049 mmol) and bicyclohexyl-1,1′-diol 26³⁵

(10.1 mg, 0.051 mmol, 1.0 equiv) in EtOH (1.0 mL) was reﬂuxed for

8 h. After evaporation, the resulting residue was puriﬁed by NH silica

gel column chromatography (CHCl₃/MeOH = 20/1) to aﬀord ¹⁰B-

16b (16.1 mg, 0.034 mmol, 71%) as a colorless amorphous solid: mp

1

56−58 °C; H NMR (400 MHz, CDCl₃, TMS): δ 1.13−1.32 (m,

6H), 1.62−1.80 (m, 14H), 2.56−2.59 (m, 8H), 2.67 (t, J = 4.8, 4H),

2.81 (t, J = 4.8 Hz, 4H), 3.65 (s, 2H), 7.32 (t, J = 8.0 Hz, 1H), 7.40

(d, J = 8.0 Hz, 1H), 7.74 (d, J = 7.2 Hz, 1H), 7.77 (s, 1H) ppm; ¹³C

Cell Cultures. HeLa S3 cells (human cervical carcinoma) were

cultured in Minimum essential medium (MEM) containing 10% FBS,

8538

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

penicillin, and streptomycin. A549 cells (human caucasian lung

carcinoma) and IMR-90 cells (normal human ﬁbroblast) were

cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM) with

10% FBS, penicillin, and streptomycin. All cells were cultured at 37

°C in a humidiﬁed atmosphere containing 5% CO₂.

Irradiation (Colony Formation Assay). A549 cells (5 × 10⁵cells/

well) were seeded on 6-well plates (TrueLine, USA) and incubated in

cell culture medium at 37 °C under 5% CO₂for 1 day. After removing

the cell culture medium, the cells were washed gently with PBS. Cell

culture medium containing 30 μM of boron compounds (2 mL) was

added to the wells, which was incubated for 24 h under same

conditions. After removing the medium, the cells were washed twice

with PBS (1.0 mL) and collected by trypsinization. After

centrifugation, the supernatant was removed, and cell culture medium

was added to prepare a cell suspension (5 × 10⁴cells/mL). The cells

(5 × 10⁴cells/mL, 1 mL) in 1.5 mL tubes were irradiated with

thermal neutrons (Institute for Integrated Radiation and Nuclear

Science, Kyoto University, Osaka, Japan) for 0, 15, 30, and 45 min,

respectively. The thermal neutron ﬂux (1.5 × 10⁹n/cm²·s) was

measured by two gold foils which were attached to the surface of the

1.5 mL tube. To evaluate the cell proliferation, the irradiated cells (3

× 10³cells/well, 1.0 mL) were seeded on 12-well plates (TrueLine,

USA) and incubated for 7 days at 37 °C under 5% CO₂in cell culture

medium. After removing the medium, the attached cells were washed

gently with PBS, ﬁxed with EtOH, stained by 0.1% crystal violet, and

washed with PBS three times.

For analyzing the cell proliferation, images of the stained colony

were acquired using a Bio-Rad Chemidoc MP Imaging System (Bio-

Rad, Hercules, CA, USA), which were automatically examined by

ImageJ-plugin Colony Area to determine the percentage of colony

area of each wells.⁴³The surviving fractions were calculated as the

colony area and normalized by the result for non-irradiated condition.

Eﬀect of Boron-Containing Macrocyclic Polyamine Deriva-

tives on the Melting Temperature of ctDNA. Thermal

denaturation experiments of ctDNA (50 μM in phosphate) in 10

mM HEPES buﬀer (pH 7.4) with I = 0.02 (NaNO₃) were performed

on a JASCO V-550 UV/vis spectrophotometer (JASCO, Tokyo,

Japan) equipped with a thermoelectric temperature controller ( 0.5

°C), a stirring unit, and a 10 mm quartz cuvette. All aqueous solutions

were made with puriﬁed water. The concentration of ctDNA was

determined by UV absorption spectroscopy based on its molar

extinction coeﬃcient at 253 nm (ε₂₅₃= 6.6 × 10³).^31d,45Thermal

melting curves for ctDNA with and without additives (16b, 19b, 17a,

20a, and 3) were obtained by following the absorption change at 260

nm as a function of the temperature (the temperature was raised at

the rate of 1 °C/min). The T_mvalue was graphically determined from

the spectral data, and the ΔT_mvalue for each condition was calculated

from the results in the presence and absence of additives.

MTT Assays. HeLa S3, A549, and IMR-90 cells (1 × 10⁴cells/

well) were seeded on 96-well plates (Watson) in cell culture medium.

After incubation overnight at 37 °C under 5% CO_2., the cells were

treated with ¹⁰B-BSH 1 (Stella Chemifa, Japan, ¹⁰B-enrichment ≥

95%), BPA 2 (Fluka, USA)-^D-fructose complex, 7 and 12−20 (0−200

μM) in cell culture medium under same conditions for 24 h, and then,

0.5% MTT reagent in PBS (10 μL) was added to each well. After

incubation for 4 h, a formazan lysis solution (10% sodium dodecyl

sulfate in 0.01 N HCl aq) (100 μL) was added and the resulting

solution was incubated under same conditions overnight. The

absorbance at λ = 570 nm was measured with a microplate reader

(Bio-Rad).

Measurement of Intracellular Uptake of Boron Compounds

into HeLa S3, A549, and IMR-90 Cells Evaluated by ICP−MS.

HeLa S3, A549, and IMR-90 cells (5 × 10⁵cells/well) were seeded on

6-well plates (TrueLine, USA) in cell culture medium. After

incubation overnight at 37 °C under 5% CO₂and 18−20% O₂

(normoxic conditions), the cells were washed gently with PBS (1 mL)

and treated with the boron compounds 1, 2, 7, and 12−20 (30 μM)

in cell culture medium (2 mL) under same conditions for 24 h (n =

4). To count the number of cells after treatment with the boron

compounds, the cells (n = 1) were washed with PBS, detached by

trypsin, and counted with a hemocytometer. For measurement of

boron uptake, the cells (n = 3) were washed with PBS (1 mL ×3) and

digested with 60% HNO₃aq (0.5 mL) at room temperature for 24 h,

which were transferred to 15 mL centrifuge tubes with Milli-Q water

(3.5 mL). These tubes were centrifuged at 3000 rpm and 4 °C for 10

min, and the resulting sample solutions were ﬁltered. The

concentration of boron atoms was determined by ICP−MS

(NexION300S, PerkinElmer, Waltham, Massachusetts, USA).

Active Energy-Dependent Uptake of Boron-Containing

Macrocyclic Polyamine Derivatives into HeLa S3 and A549

Cells. HeLa S3 and A549 cells (5 × 10⁵cells/well) were seeded on 6-

well plates (TrueLine, USA) and incubated in cell culture medium at

37 °C under 5% CO₂(n = 4). After incubation for 2 days, the cells

were washed with PBS (1 mL) and treated with boron compounds 2

and 15a−17a (30 μM) in cell culture medium (2 mL) at 37 °C or 4

°C for 1 h (n = 4). To count the number of cells after treatment with

the boron compounds, the cells (n = 1) were washed with PBS,

detached by trypsin, and counted with a hemocytometer. For

measurement of boron uptake, the cells (n = 3) were washed with

PBS (1 mL ×3) and digested with 60% HNO₃aq (0.5 mL) at room

temperature for 24 h and then transferred to 15 mL centrifuge tubes

with Milli-Q water (3.5 mL). These tubes were centrifuged at 3000

rpm and 4 °C for 10 min and then sample solution was ﬁltered. The

concentration of boron atoms was determined by ICP−MS

(NexION300S, PerkinElmer, Waltham, Massachusetts, USA).

Eﬀect of Inhibitors on the Intracellular Uptake of 17a. HeLa

S3 and A549 cells (5 × 10⁵cells/well) were seeded on 6-well plates

(TrueLine, USA) and incubated in cell culture medium at 37 °C

under 5% CO₂for 2 days (n = 4). After preincubation with inhibitors

in cell culture medium (2 mL) at 37 °C for 1 h, the cells were treated

with boron compounds 2 and 17a (30 μM) in the presence of

inhibitors at 37 °C for 1 h. To count the number of cells after

treatment with the boron compounds, the cells (n = 1) were washed

with PBS, detached by trypsin, and counted with a hemocytometer.

For measurement of boron uptake, the cells (n = 3) were washed with

PBS (1 mL ×3) and digested with 60% HNO₃aq (0.5 mL) at room

temperature for 24 h, which were transferred to 15 mL centrifuge

tubes with Milli-Q water (3.5 mL). These tubes were centrifuged at

3000 rpm and 4 °C for 10 min and then sample solutions were

ﬁltered. The concentration of boron atoms was determined by ICP−

MS (NexION300S, PerkinElmer, Waltham, Massachusetts, USA).

Evaluation of the Anti-tumor Eﬀect of Boron-Containing

Macrocyclic Polyamine Derivatives with Thermal Neutron

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00445.

Structures of endocytosis inhibitors (MβCD, dynasore,

and amiloride), results of MTT assay of 1, 2, 7, and 12−

20 against HeLa S3, A549, and IMR-90 cells, intra-

cellular uptake of 2 and 17a into A549 cells in the

presence and absence of spermidine (3), images of

colony formation assay of A549 cells after thermal

neutron irradiation, and results of thermal denaturation

experiments of ctDNA (PDF)

Crystallographic data of boron uptake of HeLa S3, A549,

and IMR-90 cells (CSV)

AUTHOR INFORMATION

■

Corresponding Author

Shin Aoki − Faculty of Pharmaceutical Sciences, Tokyo

University of Science, Chiba 278-8510, Japan; Research

Institute for Science and Technology and Research Institute

for Biomedical Sciences, Tokyo University of Science, Noda,

8539

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Chiba 278-8510, Japan; orcid.org/0000-0002-4287-

6487; Email: shinaoki@rs.tus.ac.jp

[12]aneN₄, 1,4,7,10-tetraazacyclododecane (cyclen); [15]-

aneN₅, 1,4,7,10,13-pentaazacyclopentadecane

Authors

REFERENCES

■

Hiroki Ueda − Faculty of Pharmaceutical Sciences, Tokyo

University of Science, Chiba 278-8510, Japan

Minoru Suzuki − Institute for Integrated Radiation and

Nuclear Science, Kyoto University, Kumatori, Osaka 590-

0494, Japan

Reiko Kuroda − Research Institute for Science and

Technology, Tokyo University of Science, Noda, Chiba 278-

8510, Japan

(1) (a) Barth, R. F.; Soloway, A. H.; Fairchild, R. G. Boron Neutron

Capture Therapy of Cancer. Cancer Res. 1990, 50, 1061−1070.

(b) Salt, C.; Lennox, A. J.; Takagaki, M.; Maguire, J. A.; Hosmane, N.

S. Boron and Gadolinium Neutron Capture Therapy. Russ. Chem.

Bull. 2004, 53, 1871−1888. (c) Ali, F.; S Hosmane, N.; Zhu, Y. Boron

Chemistry for Medical Applications. Molecules 2020, 25, 828.

(2) (a) Hawthorne, M. F. The Role of Chemistry in the

Development of Boron Neutron Capture Therapy of Cancer.

Angew. Chem., Int. Ed. Engl. 1993, 32, 950−984. (b) Morin, C. The

Chemistry of Boron Analogues of Biomolecules. Tetrahedron 1994,

50, 12521−12569. (c) Soloway, A. H.; Tjarks, W.; Barnum, B. A.;

Rong, F.-G.; Barth, R. F.; Codogni, I. M.; Wilson, J. G. The Chemistry

of Neutron Capture Therapy. Chem. Rev. 1998, 98, 1515−1562.

(d) Barth, R. F.; Coderre, J. A.; Vicente, M. G. H.; Blue, T. E. Boron

Neutron Capture Therapy of Cancer: Current Status and Future

Prospects. Clin. Cancer Res. 2005, 11, 3987−4002. (e) Luderer, M. J.;

de la Puente, P.; Azab, A. K. Advancements in Tumor Targeting

Strategies for Boron Neutron Capture Therapy. Pharm. Res. 2015, 32,

2824−2836. (f) Barth, R. F.; Mi, P.; Yang, W. Boron Delivery Agents

for Boron Neutron Capture Therapy of Cancer. Canc. Commun. 2018,

38, 35. (g) Cerecetto, H.; Couto, M. Medicinal Chemistry of Boron-

Bearing Compounds for BNCT-Glioma Treatment: Current

Challenges and Perspectives. In GliomaContemporary Diagnostic

Tomohiro Tanaka − Faculty of Pharmaceutical Sciences,

Tokyo University of Science, Chiba 278-8510, Japan

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00445

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by grants-in-aid from the Ministry of

Education, Culture, Sports, Science and Technology (MEXT)

of Japan (nos. 17K08225, 18F18412, and 20K05712 for S.A.),

research grant from Uehara Memorial Foundation, research

grant from Tokyo Ohka Foundation for the Promotion of

Science and Technology, Kanagawa, Japan, and research grant

from Tokyo Biochemical Research Foundation, Tokyo, Japan.

We wish to acknowledge Prof. Yoshinori Sakurai (Institute for

Integrated Radiation and Nuclear Science, Kyoto University)

for the in vitro BNCT experiments, Prof. Toshiyuki Kaji and

Dr. Eiko Yoshida (Faculty of Pharmaceutical Sciences, Tokyo

University of Science) for their helpful support for the

measurement of the boron concentration in cells using ICP−

MS, Fukiko Hasegawa and Dr. Yayoi Yoshimura (Faculty of

Pharmaceutical Sciences, Tokyo University of Science) for

collecting and interpreting the mass spectral data, Noriko

Sawabe and Dr. Satoru Matsuda for the NMR measurements,

and Satoko Nakamura and Dr. Hiroki Kuramochi (Research

Institute for Science and Technology, Tokyo University of

Science) for the elemental analyses.

̌

́

and Therapeutic Approaches; Omerhodzic, I., Arnautovic, K., Eds.;

IntechOpen: U.K., 2018.

(3) Soloway, A. H.; Hatanaka, H.; Davis, M. A. Penetration of Brain

and Brain Tumor. VII. Tumor-Binding Sulfhydryl Boron Compounds.

J. Med. Chem. 1967, 10, 714−717.

(4) (a) Snyder, H. R.; Reedy, A. J.; Lennarz, W. J. Synthesis of

Aromatic Boronic Acids. Aldehydo Boronic Acids and a Boronic Acid

Analog of Tyrosine. J. Am. Chem. Soc. 1958, 80, 835−838. (b) Shull,

B. K.; Spielvogel, D. E.; Head, G.; Gopalaswamy, R.; Sankar, S.;

Devito, K. Studies on the Structure of the Complex of the Boron

Neutron Capture Therapy Drug, L-p -Boronophenylalanine, with

Evidence for the β -D-Fructofuranose 2,3,6-(p -Phenylalanylorthobor-

onate) Structure. J. Pharm. Sci. 2000, 89, 215−222. (c) Wongthai, P.;

Hagiwara, K.; Miyoshi, Y.; Wiriyasermkul, P.; Wei, L.; Ohgaki, R.;

Kato, I.; Hamase, K.; Nagamori, S.; Kanai, Y. Boronophenylalanine, a

Boron Delivery agent for Boron Neutron Capture Therapy, is

Transported by ATB^o,+, LAT1 and LAT2. Cancer Sci. 2015, 106,

279−286. (d) Watanabe, T.; Hattori, Y.; Ohta, Y.; Ishimura, M.;

Nakagawa, Y.; Sanada, Y.; Tanaka, H.; Fukutani, S.; Masunaga, S.;

Hiraoka, M.; Ono, K.; Suzuki, M.; Kirihata, M. Comparison of the

Pharmacokinetics Between L-BPA and L-FBPA Using the Same

Administration Dose and Protocol: a Validation Study for the

Theranostic Approach Using [¹⁸F]-L-BPA Positron Emission

Tomography in Boron Neutron Capture Therapy. BMC Canc.

2016, 16, 859.

(5) (a) Barth, R. F.; Vicente, M. G. H.; Harling, O. K.; Kinger, W. S.,

III; Riley, K. J.; Binns, P. J.; Wagner, F. M.; Suzuki, M.; Aihara, T.;

Kato, I.; Kawabata, S. Current Status of Boron Neutron Capture

Therapy of High Grade Gliomas and Recurrent Head and Neck

Cancer. Radiat. Oncol. 2012, 7, 146. (b) Suzuki, M.; Kato, I.; Aihara,

T.; Hiratsuka, J.; Yoshimura, K.; Niimi, M.; Kimura, Y.; Ariyoshi, Y.;

Haginomori, S.-i.; Sakurai, Y.; Kinashi, Y.; Masunaga, S.-i.; Fukushima,

M.; Ono, K.; Maruhashi, A. Boron Neutron Capture Therapy

Outcomes for Advanced or Recurrent Head and Neck Cancer. J.

Radiat. Res. 2014, 55, 146−153. (c) Barth, R. F.; Zhang, Z.; Liu, T. A

Realistic Appraisal of Boron Neutron Capture Therapy as a Cancer

Treatment Nodality. Canc. Commun. 2018, 38, 36. (d) Suzuki, M.

Boron Neutron Capture Therapy (BNCT): a Unique Role in

Radiotherapy with a View to Entering the Accelerator-based BNCT

era. Int. J. Clin. Oncol. 2020, 25, 43−50.

ABBREVIATIONS

■

ATB^0,+, amino acid transporter system B^0,+; ATR, attenuated

total reﬂection; A549 cells, human caucasian lung carcinoma;

brs, broad signal; BNCT, boron neutron capture therapy; BPA,

L-4-boronophenylalanine; BPO, benzoyl peroxide; BSH,

sodium mercaptoborate; n-BuLi, n-butyllithium; ctDNA, calf

thymus DNA; dT, thymidine; DMEM, Dulbecco’s modiﬁed

Eagle medium; FBS, fetal bovine serum; GLUT, glucose

transporter; HeLa S3 cells, human cervical carcinoma; IMR-90

cells, normal human ﬁbroblast; ICP−MS, inductively coupled

plasma mass spectrometry; Jurkat cells, human T lymphocyte

cells; KURNS, The Institute for Integrated Radiation and

Nuclear Science, Kyoto University; LAT, L-type amino acid

transporter; LET, linear energy transfer; MTT, 3-(4,5-

dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide;

MβCD, methyl-beta-cyclodextrin; MEM, minimum essential

medium; PTS, polyamine transport system; quant, quantita-

tive; SD, standard deviation; SQAG, sulfoquinovosyl acyl

glycerol; T/B, tumor to blood; T/N, tumor to normal cell;

THF, tetrahydrofuran; TSP, 3-(trimethylsilyl)propionic-

2,2,3,3-d₄acid sodium; [9]aneN₃, 1,4,7-triazacyclononane;

8540

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(6) (a) Wyzlic, I. M.; Soloway, A. H. A General, Convenient Way to

Carborane-Containing Amino Acids for Boron Neutron Capture

Therapy. Tetrahedron Lett. 1992, 33, 7489−7490. (b) Nemoto, H.;

Iwamoto, S.; Nakamura, H.; Yamamoto, Y. A New Water-soluble p-

Boronophenylalanine Derivatives for Neutron Capture Therapy.

Chem. Lett. 1993, 22, 465−468. (c) Prashar, J. K.; Moore, D. E.

Synthesis of Carboranyl Phenylalanine for Potential Use in Neutron

Capture Therapy of Melanoma. J. Chem. Soc., Perkin Trans. 1 1993,

1051−1053. (d) Karnbrock, W.; Musiol, H.-J.; Moroder, L.

Enantioselective Synthesis of S-o -Carboranylalanine via Methylated

Bislactim Ethers of 2,5-Diketopiperazines. Tetrahedron 1995, 51,

1187−1196. (e) Denniel, V.; Bauchat, P.; Danion, D.; Danion-

Bougot, R. Hydroboration of Vinylglycine and Allylglycine as a Route

to Boron-derivatives of α -Amino Acids. Tetrahedron Lett. 1996, 37,

5111−5114. (f) Radel, P. A.; Kahl, S. B. Enantioselective Synthesis of

L- and D-Carboranylalanine. J. Org. Chem. 1996, 61, 4582−4588.

(g) Kabalka, G. W.; Yao, M.-L.; Marepally, S. R.; Chandra, S.

Biological Evaluation of Boronated Unnatural Amino Acids as New

Boron Carriers. Appl. Radiat. Isot. 2009, 67, S374−S379. (h) Hattori,

Y.; Kusaka, S.; Mukumoto, M.; Uehara, K.; Asano, T.; Suzuki, M.;

Masunaga, S.-i.; Ono, K.; Tanimori, S.; Kirihata, M. Biological

Evaluation of Dodecaborate-Containing L-Amino Acids for Boron

Neutron Capture Therapy. J. Med. Chem. 2012, 55, 6980−6984.

(i) Hattori, Y.; Kusaka, S.; Mukumoto, M.; Ishimura, M.; Ohta, Y.;

Takenaka, H.; Uehara, K.; Asano, T.; Suzuki, M.; Masunaga, S.-i.;

Ono, K.; Tanimori, S.; Kirihata, M. Synthesis and in Vitro Evaluation

of Thiododecaborated α , α -Cycloalkylamino Acids for the Treatment

of Malignant Brain Tumors by Boron Neutron Capture Therapy.

Amino Acids 2014, 46, 2715−2720. (j) Futamura, G.; Kawabata, S.;

Nonoguchi, N.; Hiramatsu, R.; Toho, T.; Tanaka, H.; Masunaga, S.;

Hattori, Y.; Kirihata, M.; Ono, K.; Kuroiwa, T. Evaluation of a Novel

Sodium Borocaptate-Containing Unnatural Amino Acid as a Boron

Delivery Agent for Neutron Capture Therapy of the F98 Rat Glioma.

Radiat. Oncol. 2017, 12, 26. (k) Li, R.; Zhang, J.; Guo, J.; Xu, Y.;

Duan, K.; Zheng, J.; Wan, H.; Yuan, Z.; Chen, H. Application of

Nitroimidazole −Carborane-Modified Phenylalanine Derivatives as

Dual-Target Boron Carriers in Boron Neutron Capture Therapy.

Mol. Pharmaceutics 2020, 17, 202−211.

(N5-2OH), a New Boron Delivery Agent for Boron Neutron Capture

Therapy of Brain Tumors. J. Med. Chem. 2006, 49, 5513−5523.

́

(k) Nizioł, J.; Zielinski, Z.; Les, A.; Dąbrowska, M.; Rode, W.; Ruman,

T. Synthesis, Reactivity and Biological Activity of N(4)-Boronated

Derivatives of 2 ’-Deoxycytidine. Bioorg. Med. Chem. 2014, 22, 3906−

3912. (l) Nizioł, J.; Uram, Ł.; Szuster, M.; Sekuła, J.; Ruman, T.

Biological Activity of N(4)-boronated Derivatives of 2 ’-Deoxycyti-

dine, Potential Agents for Boron Neutron Capture Therapy. Bioorg.

Med. Chem. 2015, 23, 6297−6304.

(8) (a) Giovenzana, G. B.; Lay, L.; Monti, D.; Palmisano, G.; Panza,

L. Synthesis of Carboranyl Derivatives of Alkynyl Glycosides as

Potential BNCT Agents. Tetrahedron 1999, 55, 14123−14136.

(b) Tietze, L. F.; Bothe, U.; Schuberth, I. Preparation of a New

Carboranyl Lactoside for the Treatment of Cancer by Boron Neutron

Capture Therapy: Synthesis and Toxicity of Fluoro Carboranyl

Glycosides for in Vivo ¹⁹F-NMR Spectroscopy. Chem.Eur. J. 2000,

6, 836−842. (c) Tietze, L. F.; Bothe, U.; Griesbach, U.; Nakaichi, M.;

Hasegawa, T.; Nakamura, H.; Yamamoto, Y. Carboranyl Bisglycosides

for the Treatment of Cancer by Boron Neutron Capture Therapy.

ChemBioChem 2001, 2, 326−334. (d) Tietze, L. F.; Bothe, U.;

Griesbach, U.; Nakaichi, M.; Hasegawa, T.; Nakamura, H.;

Yamamoto, Y. ortho -Carboranyl Glycosides for the Treatment of

Cancer by Boron Neutron Capture Therapy. Bioorg. Med. Chem.

2001, 9, 1747−1752. (e) Thimon, C.; Panza, L.; Morin, C. Synthesis

of a Glycosylated ortho -Carboranyl Amino Acid. Synlett 2003, 34,

1399−1402. (f) Tietze, L. F.; Griesbach, U.; Schuberth, I.; Bothe, U.;

Marra, A.; Dondoni, A. Novel Carboranyl C -Glycosides for the

Treatment of Cancer by Boron Neutron Capture Therapy. Chem.

Eur. J. 2003, 9, 1296−1302. (g) Ronchi, S.; Prosperi, D.;

Compostella, F.; Panza, L. Synthesis of Novel Carborane-hybrids

Based on a Trizine Scaffold for Boron Neutron Capture Therapy.

Synlett 2004, 1007−1010. (h) Ronchi, S.; Prosperi, D.; Thimon, C.;

Morin, C.; Panza, L. Synthesis of Mono- and Bisglucuronoylated

Carboranes. Tetrahedron Asymmetry 2005, 16, 39−44. (i) Lechten-

berg, B.; Gabel, D. Synthesis of (B₁₂H₁₁S)^2‑Containing Glucurono-

side as Potential Prodrug for BNCT. J. Organomet. Chem. 2005, 690,

2780−2782. (j) Orlova, A. V.; Kononov, L. O.; Kimel, B. G.; Sivaev, I.

B.; Bregadze, V. I. Conjugates of Polyhedral Boron Compounds with

Carbohydrates. 4. Hydrolytic Stability of Carborane-Lactose Con-

jugates Depends on the Structure of a Spacer between Carborane

Cage and Sugar Moiety. Appl. Organomet. Chem. 2006, 20, 416−420.

(k) Genady, A. R.; El-Zaria, M. E. Novel Glycosylated Carbor-

anylquinazolines for Boron Neutron Capture Therapy of Tumors:

Synthesis, Characterization, and in Vitro Toxicity Studies. Appl.

Organomet. Chem. 2008, 22, 227−232. (l) Satapathy, R.; Dash, B. P.;

Bode, B. P.; Byczynski, E. A.; Hosmane, S. N.; Bux, S.; Hosmane, N.

S. New Classes of Carborane-appended 5-Thio-D-glucopyranose

Derivatives. Dalton Trans. 2012, 41, 8982−8988. (m) Imperio, D.;

Del Grosso, E.; Fallarini, S.; Lombardi, G.; Panza, L. Synthesis of

Suger-Boronic Acid Derivatives: A Class of Potential Agents for

Boron Neutron Capture Therapy. Org. Lett. 2017, 19, 1678−1681.

(n) Imperio, D.; Muz, B.; Azab, A. K.; Fallarini, S.; Lombardi, G.;

Panza, L. A Short and Convenient Synthesis of closo -Dodecaborate

Sugar Conjugates. Eur. J. Org. Chem. 2019, 7228−7232. (o) Imperio,

D.; Del Grosso, E.; Fallarini, S.; Lombardi, G.; Panza, L. Anomeric

Sugar Boronic Acid Analogues as Potential Agents for Boron Neutron

Capture Therapy. Beilstein J. Org. Chem. 2019, 15, 1355−1359.

(p) Tsurubuchi, T.; Shirakawa, M.; Kurosawa, W.; Matsumoto, K.;

Ubagai, R.; Umishio, H.; Suga, Y.; Yamazaki, J.; Arakawa, A.;

Maruyama, Y.; Seki, T.; Shibui, Y.; Yoshida, F.; Zaboronok, A.;

Suzuki, M.; Sakurai, Y.; Tanaka, H.; Nakai, K.; Ishikawa, E.;

Matsumura, A. Evaluation of a Novel Boron-Containing α -D-

Mannopyranoside for BNCT. Cells 2020, 9, 1277.

(7) (a) Liao, T. K.; Podrebarac, E. G.; Cheng, C. C. Boron-

Substituted Pyrimidines. J. Am. Chem. Soc. 1964, 86, 1869−1870.

(b) Schinazi, R. F.; Prusoff, W. H. Synthesis and Properties of Boron

and Silicon Substituted Uracil or 2 ’-Deoxyuridine. Tetrahedron Lett.

1978, 19, 4981−4984. (c) Schinazi, R. F.; Prusoff, W. H. Synthesis of

́

5-(Dihydroxyboryl)-2-deoxyuridine and Related Boron-Containing

Pyrimidines. J. Org. Chem. 1985, 50, 841−847. (d) Reynolds, R. C.;

Trask, T. W.; Sedwick, W. D. 2,4-Dichloro-5-(1-o -carboranylmethyl)-

6-methylpyrimidine:

A P otential Synthon for 5-(1-o -

carboranylmethyl)pyrimidines. J. Org. Chem. 1991, 56, 2391−2395.

(e) Tjarks, W.; Anisuzzaman, A. K. M.; Liu, L.; Soloway, A. H.; Barth,

R. F.; Perkins, D. J.; Adams, D. M. Synthesis and in Vitro Evaluation

of Boronated Uridine and Glucose Derivatives for Boron Neutron

Capture Therapy. J. Med. Chem. 1992, 35, 1628−1633. (f) Wyzlic, I.

M.; Tjarks, W.; Soloway, A. H.; Anisuzzaman, A. K. M.; Rong, F.-G.;

Barth, R. F. Strategies for the Design and Synthesis of Boronated

Nucleic Acid and Protein Components as Potential Delivery Agents

for Neutron Capture Therapy. Int. J. Radiat. Oncol., Biol., Phys. 1994,

28, 1203−1213. (g) Goudgaon, N. M.; El-Kattan, G. F.; Schinazi, R.

F. Boron Containing Pyrimidines, Nucleosides, and Oligonucleotides

for Neutron Capture Therapy. Nucleosides Nucleotides 1994, 13, 849−

880. (h) Barth, R. F.; Yang, W.; Al-Madhoun, A. S.; Johnsamuel, J.;

Byun, Y.; Chandra, S.; Smith, D. R.; Tjarks, W.; Eriksson, S. Boron

Containing Nucleosides as Potential Delivery Agents for Neutron

Capture Therapy of Brain Tumors. Cancer Res. 2004, 64, 6287−6295.

(i) Al-Madhoun, A. S.; Tjarks, W.; Eriksson, S. The Role of

Thymidine Kinases in the Activation of Pyrimidine Nucleoside

Analogues. Mini-Rev. Med. Chem. 2004, 4, 341−350. (j) Byun, Y.;

Thirumamagal, B. T. S.; Yang, W.; Eriksson, S.; Barth, R. F.; Tjarks,

W. Preparation and Biological Evaluation of ¹⁰B-Enriched 3-[5-{2-

(2,3-dihydroxyprop-1-yl)-o-carboran-1-yl}pentan-1-yl]thymidine

(9) (a) Cai, J.; Soloway, A. H. Synthesis of Carboranyl Polyamines

for DNA Targeting. Tetrahedron Lett. 1996, 37, 9283−9286. (b) Cai,

J.; Soloway, A. H.; Barth, R. F.; Adams, D. M.; Hariharan, J. R.;

Wyzlic, I. M.; Radcliffe, K. Boron-Containing Polyamines as DNA

Targeting Agents for Neutron Capture Therapy of Brain Tumors:

Synthesis and Biological Evaluation. J. Med. Chem. 1997, 40, 3887−

8541

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

3896. (c) Ghaneolhosseini, H.; Tjarks, W.; Sjöberg, S. Synthesis of

Novel Boronated Acridines- and Spermidines as Possible Agents for

BNCT. Tetrahedron 1998, 54, 3877−3884. (d) Zhuo, J.-C.; Cai, J.;

Soloway, A. H.; Barth, R. F.; Adams, D. M.; Ji, W.; Tjarks, W.

Synthesis and Biological Evaluation of Boron-Containing Polyamines

as Potential Agents for Neutron Capture Therapy of Brain Tumors. J.

Med. Chem. 1999, 42, 1282−1292. (e) Martin, B.; Possémé, F.; Le

Barbier, C.; Carreaux, F.; Carboni, B.; Seiler, N.; Moulinoux, J.-P.;

Delcros, J.-G. N -Benzylpolyamines as Vectors of Boron and Fluorine

for Cancer Therapy and Imaging: Synthesis and Biological Evaluation.

J. Med. Chem. 2001, 44, 3653−3664. (f) Pan, X. Q.; Wang, H.; Shukla,

S.; Sekido, M.; Adams, D. M.; Tjarks, W.; Barth, R. F.; Lee, R. J.

Boron-Containing Folate Receptor-Targeted Liposomes as Potential

Delivery Agents for Neutron Capture Therapy. Bioconjugate Chem.

2002, 13, 435−442. (g) El-Zaria, M. E.; Dörfler, U.; Gabel, D.

Synthesis of (Aminoalkylamine)-N -aminoalkyl)azanonaborane(11)-

Derivatives for Boron Neutron Capture Therapy. J. Med. Chem. 2002,

45, 5817−5819. (h) Lee, J.-D.; Lee, Y.-J.; Jeong, H.-J.; Lee, J. S.; Lee,

C.-H.; Ko, J.; Kang, S. O. Practical Synthesis of Aminoethyl-o -

carboranes. Organometallics 2003, 22, 445−449. (i) El-Zaria, M. E.

Synthesis and Biological Evaluation of Novel Azanonaboranes as

Potential Agents for Boron Neutron Capture Therapy. Appl.

Organomet. Chem. 2005, 19, 683−689. (j) El-Zaria, M. E.; Genady,

A. R.; Gabel, D. Azanonaboranes Containing Imidazole Derivatives

for Boron Neutron Capture Therapy: Synthesis, Cheracterization, and

In Vitro Toxicity Evaluation. Chem.Eur. J. 2006, 12, 8084−8089.

(10) (a) Woodburn, K.; Phadke, A. S.; Morgan, A. R. An in Vitro

Study of Boronated Porphyrins for Potential Use in Boron Neutron

Capture Therapy. Bioorg. Med. Chem. Lett. 1993, 3, 2017−2022.

(b) Toi, H.; Nagai, Y.; Aoyama, Y.; Kawabe, H.; Aizawa, K.; Ogoshi,

H. Preparation of Porphyrins Having Phenylboronic Acid Groups.

Chem. Lett. 1993, 22, 1043−1046. (c) Miura, M.; Micca, P. L.; Fisher,

C. D.; Heinrichs, J. C.; Donaldson, J. A.; Finkel, G. C.; Slatkin, D. N.

Synthesis of a Nickel Tetracarboranylphenylporphyrin for Boron

Neutron Capture Therapy: Biodistribution and Toxicity in Tumor-

bearing Mice. Int. J. Cancer 1996, 68, 114−119. (d) Chayer, S.;

Jaquinod, L.; Smith, K. M.; Vicente, M. G. H. Synthesis of

Carboranylpyrroles. Tetrahedron Lett. 2001, 42, 7759−7761. (e) Si-

brian-Vazquez, M.; Hao, E.; Jensen, T. J.; Vicente, M. G. H. Enhanced

Cellular Uptake with a Cobaltacarborane-Porphyrin-HIV-1 Tat 48 −

60 Conjugate. Bioconjugate Chem. 2006, 17, 928−934. (f) Koo, M.-S.;

Ozawa, T.; Santos, R. A.; Lamborn, K. R.; Bollen, A. W.; Deen, D. F.;

Kahl, S. B. Synthesis and Comparative Toxicology of a Series of

Polyhedral Borane Anion-Substituted Tetraphenyl Porphyrins. J. Med.

Chem. 2007, 50, 820−827. (g) El-Zaria, M. E.; Ban, H. S.; Nakamura,

H. Boron-Containing Protoporphyrin IX Derivatives and Their

Modification for Boron Neutron Capture Therapy: Synthesis,

Characterization, and Comparative In Vitro Toxicity Evaluation.

Chem.Eur. J. 2010, 16, 1543−1552. (h) Bhupathiraju, N. V. S. D.

K.; Vicente, M. G. H. Synthesis and Cellular Studies of Polyamine

Conjugates of a Mercaptomethyl-carboranylporphyrin. Bioorg. Med.

Chem. 2013, 21, 485−495.

Carriers for Neutron Capture Therapy. Mol. Pharmaceutics 2020, 17,

3740−3747.

(12) (a) Miyajima, Y.; Nakamura, H.; Kuwata, Y.; Lee, J.-D.;

Masunaga, S.; Ono, K.; Maruyama, K. Transferrin-Loaded nido -

Carborane Liposomes: Tumor-Targeting Boron Delivery System for

Neutron Capture Therapy. Bioconjugate Chem. 2006, 17, 1314−1320.

(b) Nakamura, H. Liposomal Boron Delivery for Neutron Capture

Therapy. Methods Enzymol. 2009, 465, 179−208. (c) Ueno, M.; Ban,

H. S.; Nakai, K.; Inomata, R.; Kaneda, Y.; Matsumura, A.; Nakamura,

H. Dodecaborate Lipid Liposomes as New Vehicles for Boron

Delivery System of Neutron Capture Therapy. Bioorg. Med. Chem.

2010, 18, 3059−3065. (d) Koganei, H.; Tachikawa, S.; El-Zaria, M.

E.; Nakamura, H. Synthesis of Oligo-closo -dodecaborates by Huisgen

̈

Click Reaction as Encapsulated Agents for the Preparation of High-

boron-content Liposomes for Neutron Capture Therapy. New J.

Chem. 2015, 39, 6388−6394. (e) Luderer, M. J.; Muz, B.; Alhallak, K.;

Sun, J.; Wasden, K.; Guenther, N.; Puente, P.; Federico, C.; Azab, A.

K. Thermal Sensitive Liposomes Improve Delivery of Boronated

Agents for Boron Neutron Capture Therapy. Pharm. Res. 2019, 36,

144.

(13) (a) Paxton, R. J.; Beatty, B. G.; Varadarajan, A.; Hawthorne, M.

F. Carboranyl Peptide-Antibody Conjugates for Neutron Capture

Therapy: Preparation, Characterization, and in Vivo Evaluation.

Bioconjugate Chem. 1992, 3, 241−247. (b) Barth, R. F.; Adams, D. M.;

Soloway, A. H.; Alam, F.; Darby, M. V. Boronated Starburst

Dendrimer-Monoclonal Antibody Immunoconjugates: Evaluation as

a Potential Delivery System for Neutron Capture Therapy.

Bioconjugate Chem. 1994, 5, 58−66. (c) Wu, G.; Barth, R. F.; Yang,

W.; Chatterjee, M.; Tjarks, W.; Ciesielski, M. J.; Fenstermaker, R. A.

Site-Specific Conjugation of Boron-Containing Dendrimers to Anti-

EGF Receptor Monoclonal Antibody Cetuximab (IMC-C225) and Its

Evaluation as a Potential Delivery Agent for Neutron Capture

Therapy. Bioconjugate Chem. 2004, 15, 185−194.

(14) Tanaka, T.; Sawamoto, Y.; Aoki, S. Concise and Versatile

Synthesis of Sulfoquinovosyl Acyl Glycerol Derivatives for Biological

Applications. Chem. Pharm. Bull. 2017, 65, 566−572.

(15) Itoh, T.; Tamura, K.; Ueda, H.; Tanaka, T.; Sato, K.; Kuroda,

R.; Aoki, S. Design and Synthesis of Boron Containing Mono-

saccharides by the Hydroboration of D-Glucal for Use in Boron

Neutron Capture Therapy (BNCT). Bioorg. Med. Chem. 2018, 26,

5922−5933.

(16) (a) Warburg, O. On the Origin of Cancer Cells. Science 1956,

123, 309−314. (b) Heiden, M. G. V.; Cantley, L. C.; Thompson, C.

B. Understanding the Warburg Effect: The Metabolic Requirements

of Cell Proliferation. Science 2009, 324, 1029−1033.

(17) (a) Gerner, E. W.; Meyskens, F. L. Polyamines and Cancer:

Old Molecules, New Understanding. Nat. Rev. Cancer 2004, 4, 781−

792. (b) Miller-Fleming, L.; Olin-Sandoval, V.; Campbell, K.; Ralser,

M. Remaining Mysteries of Molecular Biology: The Role of

Polyamines in the Cell. J. Mol. Biol. 2015, 427, 3389−3406.

(18) (a) Palmer, A. J.; Wallace, H. M. The Polyamine Transport

System as a Target for Anticancer Drug Development. Amino Acids

2010, 38, 415−422. (b) Nowotarski, S. L.; Woster, P. M.; Casero, R.

A. Polyamines and Cancer: Implications for Chemoprevention and

Chemotherapy. Expert Rev. Mol. Med. 2013, 15, No. e3. (c) Murray-

Stewart, T. R.; Woster, P. M.; Casero, R. A. Targeting Polyamine

Metabolism for Cancer Therapy and Prevention. Biochem. J. 2016,

473, 2937−2953.

(19) (a) Edwards, M. L.; Snyder, R. D.; Stemerick, D. M. Synthesis

and DNA-Binding Properties of Polyamine Analogues. J. Med. Chem.

1991, 34, 2414−2420. (b) Bergeron, R. J.; McManis, J. S.; Liu, C. Z.;

Feng, Y.; Weimar, W. R.; Luchetta, G. R.; Wu, Q.; Ortiz-Ocasio, J.;

Vinson, J. R. T.; Kramer, D.; Porter, C. Antiproliferative Properties of

Polyamine Analogues: A Structure −Activity Study. J. Med. Chem.

1994, 37, 3464−3476. (c) Casero, R. A.; Woster, P. M. Terminally

Alkylated Polyamine Analogues as Chemotherapeutic Agents. J. Med.

Chem. 2001, 44, 1−26. (d) Casero, R. A.; Marton, L. J. Targeting

Polyamine Metabolism and Function in Cancer and Other Hyper-

proliferative Diseases. Nat. Rev. Drug Discovery 2007, 6, 373−390.

(11) (a) Ahrens, V. M.; Frank, R.; Boehnke, S.; Schutz, C. L.;

̈

Hampel, G.; Iffland, D. S.; Bings, N. H.; Hey-Hawkins, E.; Beck-

Sickinger, A. G. Receptor-Mediated Uptake of Boron-Rich Neuro-

peptide Y Analogues for Boron Neutron Capture Therapy.

ChemMedChem 2015, 10, 164−172. (b) Isono, A.; Tsuji, M.;

Sanada, Y.; Matsushita, A.; Masunaga, S.; Hirayama, T.; Nagasawa,

H. Design, Synthesis, and Evaluation of Lipopeptide Conjugates of

Mercaptoundecahydrododecaborate for Boron Neutron Capture

Therapy. ChemMedChem 2019, 14, 823−832. (c) Nakase, I.;

Katayama, M.; Hattori, Y.; Ishimura, M.; Inaura, S.; Fujiwara, D.;

Takatani-Nakase, T.; Fujii, I.; Futaki, S.; Kirihata, M. Intracellular

Target Delivery of Cell-penetrating Peptide-conjugated Dodecaborate

for Boron Neutron Capture Therapy (BNCT). Chem. Commun. 2019,

55, 13955−13958. (d) Kawai, K.; Nishimura, K.; Okada, S.; Sato, S.;

Suzuki, M.; Takata, T.; Nakamura, H. Cyclic RGD-Functionalized

closo -Dodecaborate Albumine Conjugates as Integrin Targeting Boron

8542

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(e) Casero, R. A.; Woster, P. M. Recent Advances in the

Development of Polyamine Analogues as Antitumor Agents. J. Med.

Chem. 2009, 52, 4551−4573.

(26) Kodama, M.; Kimura, E. Effects of Cyclization and Ring Size on

Complex Formation Between Penta-amine Ligands and Copper(II). J.

Chem. Soc., Dalton Trans. 1978, 104−110.

(20) Kitamura, M.; Suzuki, T.; Abe, R.; Ueno, T.; Aoki, S. ¹¹B NMR

Sensing of d-Block Metal Ions in Vitro and in Cells Based on the

Carbon-Boron Bond Cleavage of Phenylboronic Acid-Pendant Cyclen

(Cyclen = 1,4,7,10-Tertaazacyclododecane). Inorg. Chem. 2011, 50,

11568−11580.

(27) (a) Kodama, M.; Kimura, E. Equilibria of Complex Formation

Between Several Bivalent Metal Ions and Macrocyclic Tri- and Penta-

amines. J. Chem. Soc., Dalton Trans. 1978, 1081−1085. (b) Kimura,

E.; Yatsunami, T. Synthesis of Some Cyclic Derivatives of Spermidine

and Spermine. Chem. Pharm. Bull. 1980, 28, 994−997. (c) Diez-

Castellnou, M.; Salassa, G.; Mancin, F.; Scrimin, P. The Zn(II)-1,4,7-

Trimethyl-1,4,7-Triazacyclononane Complex: A Monometallic Cata-

lyst Active in Two Protonation States. Front. Chem. 2019, 7, 469.

(d) Savastano, M.; Fiaschi, M.; Ferraro, G.; Gratteri, P.; Mariani, P.;

Bianchi, A.; Bazzicalupi, C. Sensing Zn²⁺in Aqueous Solution with a

Fluorescent Scorpiand Macrocyclic Ligand Decorated with an

Anthracene Bearing Tail. Molecules 2020, 25, 1355.

(21) (a) Tanaka, T.; Nishiura, Y.; Araki, R.; Saido, T.; Abe, R.; Aoki,

S. ¹¹B NMR Probes of Copper(II): Finding and Implications of the

Cu²⁺-Promoted Decomposition of ortho -Carborane Derivatives. Eur.

J. Inorg. Chem. 2016, 1819−1834. (b) Tanaka, T.; Araki, R.; Saido, T.;

Abe, R.; Aoki, S. ¹¹B NMR/MRI Sensing of Copper(II) Ions In Vitro

by the Decomposition of a Hybrid Compound of a nido-o -Carborane

and a Metal Chelator. Eur. J. Inorg. Chem. 2016, 3330−3337.

(22) (a) Mislick, K. A.; Baldeschwieler, J. D. Evidence for the Role of

Proteoglycans in Cation-Mediated Gene Transfer. Proc. Natl. Acad.

Sci. U.S.A. 1996, 93, 12349−12354. (b) Belting, M.; Persson, S.;

Fransson, L.-Å. Proteoglycan Involvement in Polyamine Uptake.

Biochem. J. 1999, 338, 317−323. (c) Belting, M.; Mani, K.; Jönsson,

M.; Cheng, F.; Sandgren, S.; Jonsson, S.; Ding, K.; Delcros, J.-G.;

Fransson, L.-Å. Glypican-1 Is a Vehicle for Polyamine Uptake in

Mammalian Cells. J. Biol. Chem. 2003, 278, 47181−47189. (d) Welch,

J. E.; Bengtson, P.; Svensson, K.; Wittrup, A.; Jenniskens, G. J.; Ten

Dam, G. B.; Van Kuppevelt, T. H.; Belting, M. Single Chain Fragment

Anti-heparan Sulfate Antibody Targets the Polyamine Transport

System and Attenuates Polyamine-Dependent Cell Proliferation. Int. J.

Oncol. 2008, 32, 749−756. (e) Uemura, T.; Stringer, D. E.; Blohm-

Mangone, K. A.; Gerner, E. W. Polyamine Transport is Mediated by

Both Endocytic and Solute Carrier Transport Mechanisms in the

Gastrointestinal Tract. Am. J. Physiol.: Gastrointest. Liver Physiol. 2010,

299, G517−G522. (f) Christianson, H. C.; Belting, M. Heparin

Sulfate Proteoglycan as a Cell-surface Endocytosis Receptor. Matrix

Biol. 2014, 35, 51−55. (g) Abdulhussein, A. A.; Wallace, H. M.

Polyamines and Membrane Transporters. Amino Acids 2014, 46,

655−660. (h) Nikitovic, D.; Berdiaki, A.; Spyridaki, I.; Krasanakis, T.;

Tsatsakis, A.; Tzanakakis, G. N. Proteoglycans −Biomarkers and

Targets in Cancer Therapy. Front. Endocrinol. 2018, 9, 69.

(23) It is reported that macrocyclic polyamine-modiﬁed lipids and

chitosan are utilized for gene transfection. See: (a) Li, C.; Tian, H.;

Rong, N.; Liu, K.; Liu, F.; Zhu, Y.; Qiao, R.; Jiang, Y. Chitosan

Grafted with Macrocyclic Polyamines on C-2 and C-6 Positions as

Nonviral Gene Vectors: Preparation, Characterization, and In Vitro

Transfection Studies. Biomacromolecules 2011, 12, 298−305. (b) Li,

L.; Zhao, F.; Zhao, B.; Zhang, J.; Li, C.; Qiao, R. Chitosan Grafted

with Phosphorylcholine and Macrocyclic Polyamine as an Effective

Gene Delivery Vector: Preparation, Characterization, and In Vitro

Transfection. Macromol. Biosci. 2015, 15, 912−926. (c) Chang, D.-C.;

Zhang, Y.-M.; Zhang, J.; Liu, Y.-H.; Yu, X.-Q. Cationic Lipids with a

Cyclen Headgroup: Synthesis and Structure −Activity Relationship

Studies as Non-viral Gene Vectors. RSC Adv. 2017, 7, 18681−18689.

(24) (a) Yang, R.; Zompa, L. J. Metal Complexes of Cyclic

Triamines. 1. Complexes of 1,4,7-Triazacyclononane ([9]aneN₃) with

Nickel(II), Copper(II), and Zinc(II). Inorg. Chem. 1976, 15, 1499−

1502. (b) Kimura, E. Macrocyclic Polyamines as Biological Cation

and Anion ComplexonesAn Application to Calculi Dissolution.

Biomimetic and Bioorganic Chemistry; Topics in Current Chemistry;

Springer, 1985; Vol. 128, pp 113−141.

(28) (a) Kimura, E. Model Studies for Molecular Recognition of

Carbonic Anhydrase and Carboxypeptidase. Acc. Chem. Res. 2001, 34,

171−179. (b) Aoki, S.; Kagata, D.; Shiro, M.; Takeda, K.; Kimura, E.

Metal Chelation-Controlled Twisted Intramolecular Charge Transfer

and Its Application to Fluorescent Sensing of Metal Ions and Anions.

J. Am. Chem. Soc. 2004, 126, 13377−13390. (c) Aoki, S.; Zulkefeli,

M.; Kitamura, M.; Hisamatsu, Y. Supramolecular Host and Catalysts

Formed by the Synergistic Molecular Assembly of Multinuclear

Zinc(II) Complexes in Aqueous Solution. In Synergy in Supra-

molecular Chemistry; Nabeshima, T., Ed.; CRC: Boca Raton, FL, USA,

2015; pp 33−56. (d) Kimura, E.; Koike, T.; Aoki, S. Evolution of

ZnII−Macrocyclic Polyamines to Biological Probes and Supra-

molecular Assembly. In Macrocyclic and Supramolecular Chemistry:

How Izatt-Christensen Award Winners Shaped the Field; Izatt, R. M.,

Ed.; John Wiley & Sons: Hoboken, NJ, USA, 2016; pp 417−445.

(29) (a) Kimura, E. Evolution of Macrocyclic Polyamines from

Molecular Science to Supramolecular Science. Bull. Jpn. Soc. Coord.

Chem. 2012, 59, 26−47. (b) Itoh, S.; Sonoike, S.; Kitamura, M.; Aoki,

S. Design and Synthesis of Chiral Zn²⁺Complexes Mimicking Natural

Aldolases for Catalytic C −C Bond Forming Reactions in Aqueous

Solution. Int. J. Mol. Sci. 2014, 15, 2087−2118.

(30) (a) Inouye, Y.; Kanamori, T.; Yoshida, T.; Bu, X.; Shionoya,

M.; Koike, T.; Kimura, E. Inhibition of Human Immunodeficiency

Virus Proliferation by Macrocyclic Polyamines and Their Metal

Complexes. Biol. Pharm. Bull. 1994, 17, 243−250. (b) Inouye, Y.;

Kanamori, T.; Yoshida, T.; Koike, T.; Shionoya, M.; Fujioka, H.;

Kimura, E. Differential Contribution of Metal Complexation and

Dimerization to the Chemotherapeutic Potential of Bicyclen-Zn^II

2

Complex against Human Immunodeficiency Virus. Biol. Pharm. Bull.

1996, 19, 456−458.

(31) (a) Shionoya, M.; Kimura, E.; Shiro, M. A New Ternary

Zinc(II) Complex with [12]aneN₄(= 1,4,7,10-Tetraazacyclodode-

cane) and AZT (= 3 ’-Azido-3 ’-deoxythymine). Highly Selective

Recognition of Thymidine and Its Related Nucleosides by a Zinc(II)

Macrocyclic Tetraamine Complex with Novel Complementary

Associations. J. Am. Chem. Soc. 1993, 115, 6730−6737. (b) Kikuta,

E.; Murata, M.; Katsube, N.; Koike, T.; Kimura, E. Novel Recognition

of Thymine Base in Double-Stranded DNA by Zinc(II)−Macrocyclic

Tetraamine Complexes Appended with Aromatic Groups. J. Am.

Chem. Soc. 1999, 121, 5426−5436. (c) Kimura, E.; Kikuta, E. Why

Zinc in Zinc Enzymes? From Biological Roles to DNA Based-

Selective Recognition. J. Biol. Inorg Chem. 2000, 5, 139−155.

(d) Kimura, E.; Kikuta, E. Macrocyclic Zinc(II) Complexes for

Selective Recognition of Nucleobases in Single- and Double-Stranded

Polynucleotides. Prog. React. Kinet. Mech. 2000, 25, 1−64. (e) Aoki,

S.; Kimura, E. Zinc −Nucleic Acid Interaction. Chem. Rev. 2004, 104,

769−788. (f) del Mundo, I. M.; Siters, K. E.; Fountain, M. A.;

Morrow, J. R. Structural Basis for Bifunctional Zinc(II) Macrocyclic

Complex Recognition of Thymine Bulges in DNA. Inorg. Chem. 2012,

51, 5444−5457.

(25) (a) Kodama, M.; Kimura, E. Thermodynamic and Kinetic

Effects of 12-Membered Macrocycles Polarographic Studies of

1,4,7,10-Tetra-azacyclododecanecopper(II). J. Chem. Soc., Dalton

Trans. 1976, 116−120. (b) Ohshima, R.; Kitamura, M.; Morita, A.;

Shiro, M.; Yamada, Y.; Ikekita, M.; Kimura, E.; Aoki, S. Design and

Synthesis of a Fluorescent Probe for Zn²⁺, 5,7-Bis(N,N -dimethylami-

nosulfonyl)-8-hydroxyquinoline-Pendant 1,4,7,10-Tetraazacyclodode-

cane and Zn²⁺-Dependent Hydrolytic and Zn²⁺-Independent Photo-

chemical Reactivation of Its Benzenesulfonyl-Caged Derivative. Inorg.

Chem. 2010, 49, 888−899.

(32) (a) Richman, J. E.; Atkins, T. J. Nitrogen Analogs of Crown

Ethers. J. Am. Chem. Soc. 1974, 96, 2268−2270. (b) Atkins, T. J.;

Richman, J. E.; Oettle, W. F. Macrocyclic Polyamines: 1,4,7,10,13,16-

Hexaazacyclooctadecane. Org. Synth. 2003, 6, 652. (c) Cao, R.;

8543

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Mu

̈

ller, P.; Lippard, S. J. Tripodal Tris-tacn and Tris-dpa Platforms for

(44) (a) Hartman, T.; Carlsson, J. Radiation Dose Heterogeneity in

Receptor and Antigen Mediated Boron Neutron Capture Therapy.

Radiother. Oncol. 1994, 31, 61−75. (b) Hosmane, N. S.; Maguire, J.

A.; Zhu, Y.; Takagaki, M. Boron and Gadolinium Neutron Capture

Therapy for Cancer Treatment; World Scientific Publishing: Singapore,

2012. (c) Ono, K.; Tanaka, H.; Tamari, Y.; Watanabe, T.; Suzuki, M.;

Masunaga, S.-i. Proposal for Determining Absolute Biological

Effectiveness of Boron Neutron Capture Therapy ―the Effect of

10B(n,α )⁷Li Dose can be Predicted from the Nucleocytoplasmic

Ratio or the Cell Size. J. Radiat. Res. 2019, 60, 29−36.

(45) (a) Kimura, E.; Ikeda, T.; Aoki, S.; Shionoya, M. Macrocylic

Zinc(II) Complexes for Selective Recognition of Nucleobases in

Single- and Double-Stranded Polynucleotides. J. Biol. Inorg Chem.

1998, 3, 259−267. (b) Zulkefeli, M.; Sogon, T.; Takeda, K.; Kimura,

E.; Aoki, S. Design and Synthesis of a Stable Supramolecular Trigonal

Prism Formed by the Self-Assembly of a Linear Tetrakis(Zn²⁺

cyclen) Complex and Trianionic Trithiocyanuric Acid in Aqueous

Solution and Its Complexation with DNA (Cyclen = 1,4,7,10-

Tetraazacyclododecane). Inorg. Chem. 2009, 48, 9567−9578.

(46) Aoki, S.; Kikuchi, C.; Kitagawa, Y.; Hasegawa, Y.; Sonoike, S.;

Saga, Y.; Hatanaka, M. Evaluation of Zn²⁺Coordination Structures in

Chiral Zn²⁺Complexes Based on Shape Measurement Factors:

Relationships between Activity and the Coordination Structure. Eur. J.

Inorg. Chem. 2019, 4740−4751.

(47) The complexation properties of metal-free [15]aneN₅and

larger macrocyclic polyamines with organic anions were reported in

refs.^24b,29,48On the other hand, the complexation of their Zn²⁺

complexes with dT and other imide-type guest molecules is yet to

be studied. To the best of our knowledge, this is the ﬁrst observation

of the electrostatic interaction of Zn²⁺−[15]aneN₅complexes with the

double-stranded DNA and the details will be reported elsewhere (we

consider that it is not easy to directly and quantitatively compare the

aﬃnity of ctDNA with these B-carriers, which exhibit diﬀerent

interaction modes with DNA).

(48) (a) Lehn, J. M. Supramolecular Chemistry: Concepts and

Perspectives; VCH: Weinheim, New York, 1995. (b) Kimura, E.

Macrocyclic Polyamines as Biological Cation and Anion Complex-

ones: An Application to Calculi Dissolution. Biomimetic and

Bioorganic Chemistry; Topics in Current Chemistry; Springer-Verlag

Berlin: Heidelberg, 1985; Vol. 128, pp 113−141. (c) Kimura, E.

Macrocyclic Polyamines with Intelligent Functions. Tetrahedron 1992,

48, 6175−6217.

Assembling Phosphate-Templated Trimetallic Centers. J. Am. Chem.

Soc. 2010, 132, 17366−17369. (d) Brown, A.; Bunchuay, T.; Crane,

C. G.; White, N. G.; Thompson, A. L.; Beer, P. D. A Bis-

Triazacyclononane Tris-Pyridyl N₉-Azacryptand “Beer Can ” Receptor

for Completion of Alkali Metal and Lead(II) Cations. Chem.Eur. J.

2018, 24, 10434−10442.

(33) Pieters, G.; Cazzolaro, A.; Bonomi, R.; Prins, L. J. Self-assembly

and Selective Exchange of Oligoanions on the Surface of Monolayer

Protected Au Nanoparticles in Water. Chem. Commun. 2012, 48,

1916−1918.

(34) (a) Qu, D.-H.; Wang, Q.-C.; Ren, J.; Tian, H. A Light-Driven

Rotaxane Molecular Shuttle with Dual Fluorescence Addresses. Org.

Lett. 2004, 6, 2085−2088. (b) Liu, Y.; Zhang, S.; Miao, Q.; Zheng, L.;

Zong, L.; Cheng, Y. Fluorescent Chemosensory Conjugated Polymers

Based on Optically Active Polybinaphthyls and 2,2 ’-Bipyridyl Units.

Macromolecules 2007, 40, 4839−4847.

(35) (a) Corey, E. J.; Danheiser, R. L.; Chandrasekaran, S. New

Reagents for the Intermolecular Pinacolic Coupling of Ketones and

Aldehydes. J. Org. Chem. 1976, 41, 260−265. (b) Chen, C.-Y.; Chen,

C.-T. Reaction-Based and Single Fluorescent Emitter Decorated

Ratiometric Nanoprobe to Detect Hydrogen Peroxide. Chem.Eur. J.

2013, 19, 16050−16057.

(36) Cappuccio, F. E.; Suri, J. T.; Cordes, D. B.; Wessling, R. A.;

Singaram, B. Evaluation of Pyranine Derivatives in Boronic Acid

Based Saccharide Sensing: Significance of Charge Interaction Between

Dye and Quencher in Solution and Hydrogel. J. Fluoresc. 2004, 14,

521−533.

(37) (a) Takeuchi, M.; Mizuno, T.; Shinmori, H.; Nakashima, M.;

Shinkai, S. Fluorescence and CD Spectroscopic Sugar Sensing by a

Cyanine-appended Diboronic Acid Probe. Tetrahedron 1996, 52,

1195−1204. (b) Pathak, R.; Nhlapo, J. M.; Govender, S.; Michael, J.

P.; van Otterlo, W. A. L.; de Koning, C. B. A Concise Synthesis of

Novel Naphtho[α ]carbazoles and Benzo[c ]carbazoles. Tetrahedron

2006, 62, 2820−2830.

(38) Kimura, E.; Aoki, S.; Koike, T.; Shiro, M. A Tris(Zn^II−1,4,7,10-

tetraazacyclododecane) Complex as a New Receptor for Phosphate

Dianions in Aqueous Solution. J. Am. Chem. Soc. 1997, 119, 3068−

3076.

(39) (a) Itoh, S.; Kitamura, M.; Yamada, Y.; Aoki, S. Chiral Catalysts

Dually Functionalized with Amino Acid an Zn²⁺Complex

Components for Enantioselective Direct Aldol Reactions Inspired

by Natural Aldolases: Design, Synthesis, Complexation Properties,

Catalytic Activities, and Mechanistic Study. Chem.Eur. J. 2009, 15,

10570−10584. (b) Itoh, S.; Sonoike, S.; Kitamura, M.; Aoki, S.

Design and Synthesis of Chiral Zn²⁺Complexes Mimicking Natural

Aldolases for Catalytic C −C Bond Forming Reactions in Aqueous

Solution. Int. J. Mol. Sci. 2014, 15, 2087−2118.

(40) Rodal, S. K.; Skretting, G.; Garred, Ø.; Vilhardt, F.; van Deurs,

B.; Sandvig, K. Extraction of Cholesterol with Methyl-β -Cyclodextrin

Perturbs Formation of Clathrin-Coated Endocytic Vesicles. Mol. Biol.

Cell 1999, 10, 961−974.

(41) (a) Macia, E.; Ehrlich, M.; Massol, R.; Boucrot, E.; Brunner, C.;

Kirchhausen, T. Dynasore, a Cell-Permeable Inhibitor of Dynamin.

Dev. Cell 2006, 10, 839−850. (b) Dutta, D.; Donaldson, J. G. Search

for Inhibitors of Endocytosis. Cell. Logist. 2012, 2, 203−208.

(42) (a) Lagana, A.; Vadnais, J.; Le, P. U.; Nguyen, T. N.; Laprade,

R.; Nabi, I. R.; Noël, J. Regulation of the Formation of Tumor Cell

Pseudopodia by the Na⁺/H⁺Exchanger NHE1. J. Cell Sci. 2000, 113,

3649−3662. (b) Koivusalo, M.; Welch, C.; Hayashi, H.; Scott, C. C.;

Kim, M.; Alexander, T.; Touret, N.; Hahn, K. M.; Grinstein, S.

Amiloride Inhibits Micropinocytosis by Lowering Submembranous

pH and Preventing Rac1 and Cdc42 Signaling. J. Cell Biol. 2010, 188,

547−563.

(43) (a) Schneider, C. A.; Rasband, W. S.; Eliceiri, K. W. NIH Image

to ImageJ: 25 Years of Image Analysis. Nat. Methods 2012, 9, 671−

675. (b) Guzmán, C.; Bagga, M.; Kaur, A.; Westermarck, J.; Abankwa,

D. ColonyArea: An ImageJ Plugin to Automatically Quantify Colony

Formation in Clonogenic Assays. PloS One 2014, 9, No. e92444.

8544

https://doi.org/10.1021/acs.jmedchem.1c00445

J. Med. Chem. 2021, 64, 8523−8544

Article Doi

Design, Synthesis, and Biological Evaluation of Boron-Containing Macrocyclic Polyamines and Their Zinc(II) Complexes for Boron Neutron Capture Therapy

DOI: 10.1021/acs.jmedchem.1c00445

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.1c00445

Full text of DOI:10.1021/acs.jmedchem.1c00445

Products guided by the article

R&D Labs maybe for 1198150-45-6

Relevant to this article

Hot Product