Design, synthesis, and structure-activity relationship of 7-propanamide benzoxaboroles as potent anticancer agents

Article abstract of DOI:10.1021/acs.jmedchem.9b00736

Benzoxaboroles, as a novel class of bioactive molecules with unique physicochemical properties, have been shown to possess excellent antimicrobial activities with tavaborole approved in 2014 as an antifungal drug. Although urgently needed, the investigation of benzoxaboroles as anticancer agents has been lacking so far. In this study, we report the design, synthesis, and anticancer structure-activity relationship of a series of 7-propanamide benzoxaboroles. Compounds 103 and 115 showed potent activity against ovarian cancer cells with IC₅₀ values of 33 and 21 nM, respectively. Apoptosis was induced by these compounds and colony formation was effectively inhibited. Furthermore, they also showed excellent efficacy in ovarian tumor xenograft mouse model.

Full text of DOI:10.1021/acs.jmedchem.9b00736

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Article
Design, Synthesis, and Structure-Activity Relationship of 7-
Propanamide Benzoxaboroles as Potent Anticancer Agents
jiong zhang, Jinyi Zhang, Guiyun Hao, Weixiang Xin, Fei Yang, Mingyan Zhu, and Huchen Zhou
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00736 • Publication Date (Web): 20 Jun 2019
Downloaded from http://pubs.acs.org on June 21, 2019
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Design, Synthesis, and Structure-Activity Relationship of
-Propanamide Benzoxaboroles as Potent Anticancer Agents
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Jiong Zhang, Jinyi Zhang, Guiyun Hao, Weixiang Xin, Fei Yang, Mingyan Zhu,
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Huchen Zhou*
State Key Laboratory of Microbial Metabolism, School of Pharmacy, Shanghai Jiao
Tong University, 200240 Shanghai, China
ABSTRACT: Benzoxaboroles, as a novel class of bioactive molecules with unique
physicochemical properties, have been shown to possess excellent antimicrobial
activities with tavaborole approved in 2014 as an antifungal drug. Although urgently
needed, the investigation of benzoxaboroles as anticancer agents has been lacking so
far. In this study, we report the design, synthesis, and anticancer structure-activity
relationship of a series of 7-propanamide benzoxaboroles. Compounds 103 and 115
showed potent activity against ovarian cancer cells with IC50 values of 33 and 21 nM.
Apoptosis was induced by these compounds and colony formation was effectively
inhibited. Furthermore, they also showed excellent efficacy in ovarian tumor
xenograft mouse model.
INTRODUCTION
During the past fifteen years, boron-containing compounds have gradually
emerged as a novel class of useful structures that possess fascinating chemical
1
-3
properties and unique biological activitites.
Due to the rare existence of
boron-containing organic compounds in nature and the historical lack of experience in
the development of boron-containing drugs, they are largely ignored in drug discovery
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with assumed concerns on their toxicity and lack of druglikeness. It was till 2003
when the world saw the first approved boron-containing drug, i.e. bortezomib
(Velcade) (Figure 1), a boronic acid-based proteasome inhibitor for the treatment of
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multiple myeloma. Interestingly, although benzoxaboroles, a class of cyclic boronic
5,6
acids, were first synthesized and studied in 1950s its medical usage had remained
literally unknown for fifty years until tavaborole (Kerydin) (Figure 1) was approved
7
,8
in 2014 as a topical treatment of fungal infection of the toenails. As the second
benzoxaborole drug, crisaborole (Eucrisa) was approved in 2016 as a nonsteroidal
9
topical treatment of atopic dermatitis. There are also a number of compounds in the
pipelines as well, including acoziborole for trypanosomiasis, GSK656 for tuberculosis,
10-12
and AN13762 for malaria.
We have reported a number of potent anti-parasitic and
1
3-17
antibacterial benzoxaboroles such as compounds 1a and 1b in the past.
At the
same time, the antiviral and anti-inflammatory properties have also been reported by
1
8,19
researchers.
OH
B
O
OH
B
O
O
OH
B
H
N
NC
N
N
N
H
OH
F
O
O
Tavaborole
Crisaborole
Bortezomib
O
OH
OH
OH
O
B
B
O
O
H2N
1a
1b
Figure 1. Chemical structures of bortezomib, tavaborole, crisaborole, and compounds
a and 1b.
Today, cancer still remains one of the major causes of human death despite of
efforts and progress in the past decades. About 2.8 and 0.6 million cancer deaths
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21
occurred in 2015 alone in China and United States, respectively. While
chemotherapy remains the main treatment, clinically used anticancer drugs are
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2,23
generally limited by toxicity, side effects, and resistance.
Thus, it remains urgent
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to develop novel anti-cancer agents.
Benzoxaboroles have unique physicochemical properties that are distinct from
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any previously known drug classes. In light of their recently proven druglikeness
and success in anti-infective application, it is imperative and urgent to explore the
potential of benzoxaboroles as anticancer agents. However, an investigation of their
anticancer activity has been lacking to date. Although several preliminary efforts
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toward this goal have been reported, the anticancer activity of benzoxaboroles has not
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been convincingly defined in those studies.
We have previously reported a small
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group of chalcone-benzoxaborole hybrids as preliminary anticancer agents. Here we
report the design, synthesis, and structure-activity relationship of a series of
7
-propanamide benzoxaboroles with potent anticancer activity. Compounds 103 and
115 showed inhibitory activity against ovarian cancer cells with IC50 values of 33 and
21 nM and excellent selectivity between cancerous and normal cells. They effectively
induced cancer cell apoptosis, and inhibited colony formation. Furthermore, they
demonstrated satisfactory efficacy and safety in ovarian tumor xenograft mouse
model.
RESULTS AND DISCUSSION
Chemistry. Various amines were synthesized following the procedure shown in
Scheme 1. Analines 4 and 5 were prepared by Boc protection of the corresponding
aminoalkylated analines 2 and 3. The acetylenylanalines 12-14 were prepared from
iodoanilines 6-8 by installation of the trimethylsilylacetylenyl group under the
conditions of Pd(PPh
3
)
2
Cl , CuI, and triethylamine followed by removal of the the
2
trimethylsilyl group by KOH. Phenylanalines 36-55 were prepared by
Suzuki-Miyaura coupling reaction of arylboronic acids 15-32 and bromoanilines
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3-35.
a
Scheme 1. Synthesis of various amine intermediates
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a
2 3 2 2
Reagents and conditions: (a) Boc O, THF; (b) trimethylsilylacetylene, Pd(PPh ) Cl , CuI, THF,
TEA; (c) KOH, MeOH; (d) Pd(PPh
3
)
2
Cl
2
, sat. aq. Na
2
CO
3
, EtOH, toluene.
a
Scheme 2. Synthesis of 7-propanamide benzoxaboroles
a
Reagents and conditions: (a) KMnO
O, DMF; (f) n-BuLi, B(i-PrO)
PCC, Celite, DCM; (i) ethyl (triphenylphosphoranylidene)acetate, toluene; (j) Pd/C, H
NaOH, MeOH, H O; (l) RNH , EDCI, DCM.
4
, t-BuOH, H
2
O; (b) SOCl
2
; (c) MeOH, Et
, THF; (g) 6 M HCl, MeOH; (h)
, EtOH; (k)
3 4
N; (d) LiBH ,
MeOH, THF; (e) DHP, p-TsOH-H
2
3
2
2
2
The 7-propanamide benzoxaborole derivatives 66-115 were prepared following
the procedures shown in Scheme 2. The key aldehyde intermediate 62 was prepared
2
7
as we previously reported. Aldehyde 62 was converted to ester 63 by Wittig reaction.
Compound 63 underwent reduction by Pd/C and H and hydrolysis under alkaline
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condition, to give carboxylic acid 65. Subsequent condensation with various amines
under the conditions of EDCI gave the 7-propanamide benzoxaboroles 66-115.
a
Scheme 3. Synthesis of analog compounds
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H
O
O
HO
O
N
O
OH
B
O
OH
B
O
OH
B
O
(A)
a
b
6
3
126
127
O
O
R
(
B)
HO
HO
b
N
H
1
28
129-132
O
O
(C)
HO
CHO
c
d
e
Br
Br
Br
Br
OH
O
OMOM
OMOM
OMOM
5
9
133
134
135
O
O
O
HO
O
f
g
a
Br
OMOM
OMOM
OMOM
1
36
137
138
RHN
O
RHN
O
RHN
O
b
h
i
OH
O
CHO
OMOM
OMOM
139-141
142-144
145-147
a
Reagents and conditions: (a) NaOH, MeOH, H
2 2
O; (b) RNH , EDCI, DCM; (c) MOMCl, DIPEA,
THF; (d) PCC, Celite, DCM; (e) ethyl (triphenylphosphoranylidene)acetate, toluene; (f) NaBH
NiCl ·6H O; (g) tributyl(vinyl)tin, Pd(PPh , DMF; (h) NaIO , K OsO , THF, H O; (i) 4 M
aqueous HCl, THF.
4
,
2
2
3
)
4
4
2
4
2
As shown in Scheme 3, acrylate 63 was hydrolyzed to acrylic acid 126 which
was subsequently coupled with analine to give acrylamide 127. Compounds 129-132
were prepared by the condensation of hydrocinnamic acid and corresponding amines.
Meanwhile, three non-boron analogs 145-147 were synthesized as shown in Scheme
3C. First, the diol 59 was converted to mono-MOM protected compound 133 which
was oxidized by PCC to give aldehyde 134. Ester 135 was obtained by Wittig reacion
and then reduced to 136. Next, a vinyl group was introduced by Stille reaction to give
compound 137 which was subsequently hydrolyzed under alkaline condition. Next,
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condensation with various amines gave compounds 139-141, which was followed by
oxidation of the vinyl group to give aldehydes 142-144. Finally, compounds 142-144
were hydrolyzed which was followed by cyclization under the acidic condition to
provide lactols 145-147.
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Discovery of Initial Hit Compound 69. In order to explore the
therapeutic value of benzoxaborole derivatives with high efficiency, we have
previously developed a type of versatile formyl benzoxaborole intermediates that
could be obtained in large scale and rapidly derivatized into benzoxaboroles bearing a
27
variety of functional groups at the 4-, 5-, 6-, or 7-position of the phenyl ring. The
7
-propionic acid derivative 65 was consequently obtained and found to possess potent
2
8
antimalarial activity. However, it did not show any antiproliferation activity on
cancer cells. In order to explore the anticancer potential of benzoxaboroles, the amide
analogs 66-72 were synthesized. As shown in Table 1, compounds 66-68 with
aliphatic chains showed no inhibitory activity on cancer cell proliferation.
Interestingly, the phenyl compound 69 gave submicromolar inhibitory potency on the
three cancer cell lines tested (SKOV3: ovarian carcinoma, MDA-MB231: breast
cancer, HCT116: colon colorectal carcinoma), while toxicity was low on normal cells
(
WI-38: human lung fibroblast). However, when the phenyl group was spaced by one
or two carbons from the amide bond (compounds 70 and 71), the anticancer potency
was abolished. The thiazolyl substitution in compound 72 also gave abolished activity.
Table 1. The inhibitory effect of compounds 65-72 on cancerous and normal cell
proliferation
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H
N
O
R
OH
B
O
a
IC50 (μM)
Structure
MDA-
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SKOV3
HCT116 WI-38
MB231
6
5
>100
>100
>100
>100
66
>100
>100
>100
0.20
>100
>100
>100
0.23
>100
>100
>100
0.35
>100
>100
>100
30
6
6
7
8
69
7
7
0
1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.59
72
Doxorubicin 0.032 0.0069 0.023
a
IC50 indicates compound concentration required to inhibit cell viability by 50%. Values are
expressed as the mean of triplicate experiments.
Modification on Phenyl Group of Compound 69 did not Improve
Antiproliferation Potency. We explored the effect of various substitutions of the
phenyl ring of compound 69 on its anticancer activity as shown in Table 2. First, the
para-substituted phenyl groups (compounds 73-84) were explored. The fluoro
compound 73 gave submicromolar activity that was slightly weaker than the
unsubstituted compound 69. When the larger halogen atoms were introduced, the
inhibitory activity gradually decreased from chloro compound 74 to iodo compound
76 that showed near 100-fold decreased activity as compared to the fluoro compound
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3. Analogs 77-84 also showed a similar tendency of lower activity associated with
bulkier substituents. The methyl and methoxy compounds 77 and 80 gave
submicromolar activity, while larger substituents in compounds 78, 79, 81, 83, and 84
all gave significantly diminished activity. However, an exception is compound 82
with a bulky Boc group that showed significantly higher activity than its shorter
analog 81, which may be due to the flexibility of alkyl chain by addition of an extra
carbon atom. Next, we investigated the effect of moving the substituents from para-
to meta- and ortho- positions. The m-bromo analog 85 showed improved potency as
compared to the p-bromo compound 75. The p-iodo compound 86 also showed
similar tendency. This observation implied that the meta-position may be a favorable
spot for further optimization. Therefore, larger groups such as isopropyl and
acetylenyl were introduced into the meta-position in compounds 87 and 88, which
both showed submicromolar activity. Interestingly, the ortho-substituted analogs 89
and 90 also showed good activity. The disubstituted and trisubstituted analogs showed
diminished activity as shown by compounds 92 and 93. However, the
m,p-disubstituted analog 91 gave good inhibitory activity. Thus, the simple halogen or
alkyl modifications on the phenyl ring only achieved inhibitory activity comparable to
the unsubstituted compound 69. This series of compounds implied that bulky groups
may be tolerated as demonstrated by compound 82, and substitutions at the ortho- and
meta-position of the phenyl ring are more favorable than the para-substitution. At the
same time, it was demonstrated that selectivity between cancerous and normal cells is
tunable depending on the substitution patterns. For example, compound 82 showed
nearly no selectivity while compound 77 showed good selectivity, although these two
compounds possess similar antiproliferative potency. Thus, it proved feasible to
fine-tune the structure-activity relationship to obtain good selectivity between
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cancerous and normal cells while retaining the antiproliferative potency. These
observations led to the design and synthesis of the naphthyl and biphenyl compounds
in Table 3 in order to improve the potency.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
4
4
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4
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5
5
5
5
5
5
5
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0
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9
0
Table 2. The inhibitory effect of compounds 73-93 on cancerous and normal cell
proliferation
H
N
O
R
OH
B
O
a
IC50 (μM)
Structure
MDA-
SKOV3
HCT116 WI-38
MB231
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7
7
7
7
7
7
8
8
8
8
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8
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0.72
5.0
0.44
3.6
5.3
33
0.65
12
12
>100
42
9.2
46
51
50
49
0.86
6.3
1.1
6.6
33
4.5
45
>100
>100
55
4.2
19
0.90
>100
2.0
0.85
29
1.7
24
>100
>100
4.8
1.3
>100
36
2.3
>100
>100
1.4
2.3
>100
83
>100
>100
20
0.54
0.63
0.72
5.3
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87
0.51
0.98
0.20
0.82
0.60
>100
19
1.6
1.6
1.6
22
>100
1.6
8
8
8
9
3.6
0.43
1.4
0.93
1.3
0
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9
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6
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2
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5
6
7
8
9
0
90
18
9
9
1
2
0.87
>100
71
1.2
15
>100
64
>100
74
93
a
IC50 indicates compound concentration required to inhibit cell viability by 50%. Values are
expressed as the mean of triplicate experiments.
Discovery of Biphenyl Compounds with Significantly Improved
Antiproliferation Potency. As shown in Table 3, the 1-naphthyl compound 94
gave antiproliferation activity comparable to the phenyl compound 69, but its
selectivity between cancer cells and normal cells was low. The 2-naphthyl analog 95
showed a 10-fold decrease of potency. Next, biphenyl compounds 96-98 were
explored. The biphenyl-2-yl and 4-yl compounds 96 and 98 only showed moderate
activity, however the 3-yl compound 97 showed submicromolar potency and good
selectivity between cancerous and normal cells. Based on this observation and the
chemical accessibility of various biphenyls, we took compound 97 for further
structural modification. Introduction of a fluoro atom at the 4’-position gave
compound 100 which is the first compound with IC50 value below 100 nM in this
work. It also showed good selectivity (44 fold) between SKOV3 and normal cells.
However, the 3’-fluoro compound 99 and the chloro compounds 101 and 102 only
gave slightly diminished potency. When the hydrogen bond acceptor group, methoxy,
was installed in compounds 103, 104, and 105, the potency was further improved for
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all three compounds. The most potent compound 103 gave an IC50 value as low as 33
nM which is the most active compound so far in this study. At the same time, it also
showed good selectivity between cancerous and normal cells (100 fold selectivity:
SKOV3 vs. WI-38 cells). When the methoxy group was replaced by the
electronegative trifluoromethyl group (compounds 106 and 107), they showed more
than 10-fold decrease of activity. The thioether, trifluoromethyl, and cyano derivatives
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
08-113 all showed submicromolar activity that is comparable to the unsubstituted
compound 97, but no improvement of potency. When acetyl group, which is a
hydrogen bond acceptor, was introduced in compounds 114 and 115, the potency was
significantly improved. Especially in compound 115, an IC50 value of 21 nM on
SKOV3 cells was achieved, which represents the highest potency in this study. It also
showed good selectivity between cancerous and normal cells (180 fold selectivity:
SKOV3 vs. WI-38 cells), which is better than the low potency compounds such as the
phenyl compound 69 (150 fold selectivity: SKOV3 vs. WI-38 cells). We believe with
a further elucidation of the intracellular targets, there is sufficient space to further
improve the selectivity by rational design.
Table 3. The inhibitory effect of compounds 94-115 on cancerous and normal cell
proliferation
H
N
O
R
OH
B
O
a
IC50 (μM)
Structure
MDA- HCT
MB231 116
SKOV3
WI-38
94
0.17
1.4
0.15
1.8
0.20
1.7
1.2
9
5
>100
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2
2
2
2
2
2
3
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3
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5
5
5
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9
6
26
18
23
77
9
9
7
8
0.25
48
0.22
8.8
0.89
13
12
32
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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8
9
0
1
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8
9
0
1
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7
8
9
0
1
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7
8
9
0
9
9
0.48
0.57
0.25
0.46
0.55
37
100
101
0.093
4.1
0.30
0.22
0.25
0.26
0.35
0.38
58
9.8
3.2
Cl
1
1
02
03
0.033
0.037 0.26
0.088 0.16
0.040 0.053
1
1
04
05
0.048
0.044
1.4
1.7
1
1
1
06
07
08
0.46
0.56
0.15
0.46
0.64
0.15
0.31
0.73
0.11
9.8
25
2.0
1
1
09
10
0.14
0.39
0.14
0.41
0.18
0.58
3.8
23
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F3C
1
1
1
1
1
11
12
13
14
15
0.50
0.12
0.51
0.11
0.38
0.55
0.14
0.41
26
7.3
12
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.34
0.068
0.021
0.052 0.086
0.056 0.11
2.2
3.7
O
a
IC50 indicates compound concentration required to inhibit cell viability by 50%. Values are
expressed as the mean of triplicate experiments.
Table 4. The inhibitory effect of compounds 116-147 on cancer cell proliferation
a
IC50 (μM)
Structure
MDA-
HCT
116
WI-38
>100
>100
>100
>100
>100
SKOV3
MB231
116
117
6-
6-
6-
5-
5-
62
49
21
59
90
77
21
3.7
19
81
56
29
>100
>100
93
O
1
1
1
18
19
20
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5
5
5
5
5
6
O
1
21
5-
4-
4-
4-
79
78
66
76
63
46
>100
>100
>100
>100
58
122
123
0
1
2
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8
9
0
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9
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9
0
88
93
50
O
1
1
24
25
57
41
48
>100
>100
>100
>100
>100
>100
>100
>100
>100
65
tb
32
1
27
>100
>100
>100
>100
129
1
1
30
31
24
25
87
40
>100
>100
O
H
N
O
O
>100
>100
132
3.2
78
2.7
12
4.0
22
13
42
1
45
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61
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52
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49
18
93
1
1
1
1
1
1
1
1
1
1
2
2
2
2
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3
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5
6
0
1
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
>100
a
IC50 indicates compound concentration required to inhibit cell viability by 50%. Values are
expressed as the mean of triplicate experiments.
Both Oxaborole Ring and 7-Propanamide Substitution are
Essential for Antiproliferation Potency. In order to evaluate the essentiality
of the oxaborole moiety and the 7-propanamide substitution, a number of control
compounds were synthesized and tested as shown in Table 4. In compounds 103, 105,
and 115, when the substituting position on the phenyl was changed from 7- to 4-, 5-,
and 6- as in compounds 116-124, the antiproliferation activity was significantly
diminished. Furthermore, the parent benzoxaborole 125 and the antifungal drug
tavaborole (tb) did not show any antiproliferation activity at all, which suggests that
the 7-propanamide substitution is essential for the antiproliferation activity.
Compound 127 with a double bond showed abolished activity as compared to its
saturated counterpart 69, which suggests that the saturated chain is favorable owing to
its conformational flexibility. When the oxaborole ring was removed as in compounds
1
29-132 or when the boron was replaced by carbon as in lactols 145-147, they
showed either completely abolished activity or significantly diminished activity as
compared to their corresponding benzoxaboroles 69, 103, 105, and 115. This
observation suggests that the oxaborole moiety is essential for the antiproliferation
activity. Thus, both the oxaborole ring and the 7-propanamide substitution are
essential for the potent antiproliferation activity of the compounds in this study.
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6
Membrane Permeability and Pharmacokinetic Profile of
Compounds 103 and 115. Compounds 103 and 115 were evaluated for their
membrane permeability and pharmacokinetic properties. Compound 103 showed high
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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8
9
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9
0
1
2
3
4
5
6
7
8
9
0
-6
permeability in the caco-2 assay with a high Papp (AB 19.5 and BA 26.510
cm/s) and a low efflux ratio of 1.36. Compound 115 showed a medium permeability
-6
with a moderate Papp (AB 3.95 and BA 2.2310 cm/s) and a low efflux ratio of
0
.56. In the PK study as shown in Table 5, when dosed orally at 10 mg/kg compound
1
03 showed an oral bioavailability of 19.5% and a half-life (T1/2) of 2.2 h. While
compound 115 has a very low bioavailability of 4.6% which may ascribe to its
relatively low permeability, and a half-life (T1/2) of 2.0 h. Both compounds showed
reasonable peak plasma concentration (Cmax) (434 and 373 ng/mL) and AUC values of
5
51 and 1920 h·ng/mL.
a
Table 5. Oral (po) and intravenous (iv) pharmacokinetic (PK) parameters of
compounds 103 and 115 in SD Rats
Dose
T
max
C
max
AUCinf
CL
V
ss
T
1/2
F
(mg/kg) (h)
(ng/mL) (h*ng/mL) (mL/h/kg)
(mL/kg) (h)
(%)
iv
2
NA
0.2
NA
434
NA
373
551
547
1920
459
3750
NA
7630
NA
1.4
2.2
0.6
2.0
NA
19.5
NA
4.6
103
po
iv
10
2
NA
0.25
1050
NA
882
NA
115
po
10
a
PK parameters were based on mean plasma concentration time profiles of three
−
animals per time points. PK parameters were calculated by noncompartmental
analysis using DAS 2.0.
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Compound 103 (M)
0
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
10
Annexin V
Figure 2. Induction of apoptosis on SKOV3 cells by compound 103: Flow cytometry
analysis of apoptotic SKOV3 cells induced by compound 103 at different
concentrations. Cells were treated with vehicle or compound 103 at 1.0, 3.0, and 10
μM, respectively, for 48 h.
Apoptosis was Induced by Compound 103. To determine whether the
proliferative inhibition induced by compound 103 was attributed to apoptosis,
SKOV3 cells were treated with vehicle and compound 103 at different concentrations
(
(
1, 3, or 10 M) for 48 h and then stained with annexin V-FITC and propidium iodide
PI). As shown in Figure 2, the percentages of apoptotic SKOV3 cells were
determined by flow cytometry. Compound 103 induced apoptosis in a
concentration-dependent manner, resulting in 28.2%, 31.6%, and 51.3% of apoptotic
cells (early and late apoptosis) at 1, 3, and 10 M, respectively.
Compound103 (M)
0
0.01
0.1
1
10
c-Caspase 9
c-Caspase 3

-actin
0
0.25
0.5
1
2
c-PARP
-actin
Figure 3. Western blot analysis of biological markers for apoptosis induction by
compound 103 on H460 cells for 48 h.
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2
2
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4
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5
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5
5
5
5
6
Cleavage of PARP and Caspase 3/9 was Induced by Compound
1
03. To further elucidate the potential mechanisms of apoptosis induced by
compound 103, cleaved PARP and caspase 3/9 were determined by Western blotting
as the markers of apoptosis. As shown in Figure 3, treatment of H460 cells with
compound 103 triggered cleavage of PARP and caspase 3/9 at low concentrations,
which indicated that compound 103 induced the apoptosis at least partially through
the cleavage of caspase 3/9 and PARP.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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9
0
1
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9
0
1
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9
0
1
2
3
4
5
6
7
8
9
0
Compound103
(M)
A
0
0.0625
0.125
0
.25
0.5
1
B
Figure 4. Inhibitory effects of compound 103 on colony formation of SKOV3 cells.
A) Cells were treated with compound 103 at various concentrations. Digital image
was taken and all visible colonies were counted. (B) Data were normalized to
untreated control. Error bars represent standard deviations from triplicates. The
statistical significance was obtained with one-way ANOVA (p < 0.0001).
(
Colony Formation was Inhibited by Compound 103. The ability of
compound 103 to inhibit the colony formation of SKOV3 cells was tested. As shown
in Figure 4, the exposure of SKOV3 cells to low concentrations of compound 103
resulted in a significant inhibition of colony formation. The cancer cell growth was
completely abolished at the concentration of 500 nM and above.
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A
C
E
B
1200
1
200
900
600
Vehicle
Vehicle
103 (2 mg)
900
115 (2 mg)
115 (10 mg)
1
03 (10 mg)
ns
*
*
**
600
300
0
*
3
00
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
3
6
9
12 15 18 21 24 27 30 33 36
Days after treatment
0
3
6
9
12 15 18 21 24 27 30 33 36
Days after treatment
25
D
25
20
15
10
5
2
1
1
0
5
0
5
0
ns
ns
ns
ns
Vehicle
Vehicle
1
1
03 (2 mg)
115 (2 mg)
115 (10 mg)
03 (10 mg)
0
0
3
6
9
12 15 18 21 24 27 30 33 36
Days after treatment
0
3
6
9 12 15 18 21 24 27 30 33 36
Days after treatment
F
1.5
1.5
1.0
0.5
0.0
1
0
.0
.5
ns
ns
*
**
0.0
Vehicle
103 (2 mg)
103 (10 mg)
Vehicle
115 (2 mg)
115 (10 mg)
Figure 5. The in vivo anticancer activity of compounds 103 and 115 on SKOV3
xenograft mouse model. Female nude BALB/c mice were administered by
intraperitoneal injection with vehicle, compounds 103, and 115 at a dosage of 2 and
1
0 mg/kg, respectively (n = 6 each group), once a day for 33 days. Average tumor
volume (A, B), average body weight (C, D), and average tumor weight (E, F) were
measured every three days. Data are expressed as the mean ± SE (n = 6). * indicates P
<
0.05 and ** indicates P < 0.01 vs. control.
Tumor Growth in Xenograft Mouse Model was Reduced. Finally,
we evaluated the in vivo efficacy of compounds 103 and 115 in SKOV3 xenograft
mouse model. Six BALB/c mice per group were administered via intraperitoneal
injection with vehicle, compounds 103, and 115 at a dosage of 2 or 10 mg/kg once a
day for 33 days. As shown in Figure 5, compounds 103 and 115 both significantly
inhibited tumor growth in a dose-dependent manner as measured by tumor volume
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5
5
6
and weight. At the same time, the body weight of the treated mice which did not show
any significant weight loss, which implied that the two compounds may have desired
toxicity properties, but further comprehensive toxicity evaluation is needed.
0
1
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5
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8
9
0
1
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0
1
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5
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9
0
CONCLUSION
Benzoxaboroles, as a novel class of bioactive molecules with unique chemical
properties, have been shown to possess excellent antimicrobial activities with
tavaborole approved in 2014 as an antifungal drug. Although urgently needed, the
investigation of benzoxaboroles as anticancer agents has been lacking to this date. In
this work, based on an antimalarial benzoxaborole 65 which does not show any
anticancer activity, a series of 7-propanamide derivatives were designed and
synthesized to give an initial hit compound 69 which showed submicromolar
antiproliferation activity on three cancer cell lines. Further structure-activity
relationship studies found that replacement of the phenyl by biphenyl and installation
of hydrogen bond acceptor group were critical for the significant improvement of
antiproliferation potency. Both the benzoxaborole moiety and the 7-propanamide
modifications are essential for the anticancer activity. Thus, it led to the identification
of a series of potent anticancer compounds. Among them, the most potent compound
115 showed an IC50 value of 21 nM on ovarian cancer cells and near 200-fold
selectivity versus normal cells. More importantly, it also demonstrated in vivo efficacy
and low toxicity on tumor xenograft mouse model at a dosage of 5 mg/kg.
This work is the first to show that benzoxaboroles may serve as potent and
efficacious anticancer agents. Although there are existing reports commenting on their
anticancer activity, the reported potency at cellular level was not ideal and in vivo
3
0
efficacy was not reported. However, the intracellular target of our benzxoaboroles
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remains to be elucidated. They were demonstrated to effectively induce cancer cell
apoptosis through the caspase 3/9 and PARP pathways and inhibited colony formation.
In previous reports, the biological activities of benzoxaboroles were mainly assessed
by phenotypic screening while the cellular targets largely remained unknown.
Although leucyl-tRNA synthetases (LeuRS) has been identified as the cellular target
1
1
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of the antifungal benzoxaborole tavaborole, compounds 103 and 115 did not inhibit
human LeuRS, which is consistent with our previous reported observation that the
16
eukaryotic helix blocked the 7-position of the benzoxaborole. Tavaborole did not
show any anticancer activity, either. At the same time, benzoxaborole has been used as
a novel chemotype in the development of enzyme inhibitors. An interesting example
31,32
was the carbonic anhydrase inhibitors reported by Winum and Simone et al.
As a
continuous development, we are currently making efforts to decipher the cellular
targets of the anticancer benzoxaboroles reported herein.
EXPERIMENTAL SECTION
Chemistry. General Procedures. All solvents and reagents were purchased
from commercial suppliers and used without further purification if not indicated.
Column chromatography was performed using Huanghai silica gel (48-75 m). NMR
spectra were recorded on AVANCE 400 (Bruker). Chemical shifts () are expressed in
parts per million (ppm) relative to residual solvent as an internal reference. High
resolution mass spectra were obtained on an Agilent 6530 Accurate-Mass Quadrupole
Time-of-Flight (Q-TOF) LC/MS. High performance liquid chromatography (HPLC)
analysis was performed on an Agilent 1200 with a flow rate of 1 mL/min and a
gradient of 10% MeCN/90% H O to 100% MeCN in 20 min using a DAD detector.
2
An Agilent Eclipse XDB-C18 column (4.6 mm × 150 mm, 5 μm) was used. Purity
was based on the integrated UV chromatogram (220 nm). All compounds used for
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2
2
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5
6
biological evaluation have purity of ≥ 95%. Melting points were measured on a
SGWX-4 melting point apparatus.
t-Butyl (4-aminophenyl)carbamate (4). To a solution of p-aminoanaline
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
3.24 g, 30 mmol) and K
(2.18 g, 10 mmol) dropwise over 0.5 h. After stirred overnight at rt, the mixture was
diluted with H O (70 mL) and extracted with DCM (50 mL × 3). The organic phase
was combined, washed with brine, dried over anhydrous Na SO , and evaporated in
2
CO
3
(1.52 g, 11 mmol) in THF (30 mL) was added Boc
2
O
2
2
4
vacuum followed by purification on silica gel column chromatography (petroleum
1
ether/EtOAc = 2:1) to give compound 4 (2.02 g, 100%) as a yellow solid. H NMR
(
400 MHz, DMSO-d
H), 4.72 (s, 2H), 1.44 (s, 9H) ppm; C NMR (100 MHz, DMSO-d
128.4, 120.1, 113.8, 78.0, 28.1 ppm.
6
): δ 8.77 (s, 1H), 7.06 (d, J = 8.2 Hz, 2H), 6.46 (d, J = 8.2 Hz,
1
3
2
6
): δ 153.0, 143.8,
t-Butyl (4-aminobenzyl)carbamate (5). Compound 5 (0.97 g, 53.4%)
1
was prepared following a similar procedure to compound 4 as a white solid. H NMR
(
400 MHz, CDCl
.18 (d, J = 5.2 Hz, 2H), 3.64 (s, 2H), 1.45 (s, 9H) ppm; C NMR (100 MHz, CDCl
δ 155.8, 145.6, 128.8, 128.7, 115.1, 79.2, 44.3, 28.4 ppm.
3
): δ 7.07 (d, J = 8.2 Hz, 2H), 6.64 (d, J = 8.2 Hz, 2H), 4.70 (s, 1H),
1
3
4
3
):
2
-((Trimethylsilyl)ethynyl)aniline (9). To a solution of 2-iodoaniline (1.1
g, 5.0 mmol), Pd(PPh
3
)
2
Cl (105.3 mg, 0.15 mmol), and CuI (19.0 mg, 0.1 mmol) in a
2
mixture of dry THF (10 mL) and TEA (3 mL) was added trimethylsilylacetylene (915
μL, 7.0 mmol) dropwise over 1 h. After stirred overnight at rt, the mixture was
evaporated in vacuum and the residue was purified by silica gel column
chromatography (petroleum ether/EtOAc = 50:1) to obtain compound 9 as a yellow
1
oil (797 mg, 83.9%). H NMR (400 MHz, DMSO-d
6
): δ 7.13 (d, J = 7.6 Hz, 1H), 7.07
(
dd, J = 8.0, 7.6 Hz, 1H), 6.71 (d, J = 8.0 Hz, 1H), 6.49 (t, J = 7.6 Hz, 1H), 5.28 (s,
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13
2
1
H), 0.24 (s, 9H) ppm; C NMR (100 MHz, DMSO-d
6
): δ 149.7, 131.6, 129.9, 115.7,
13.7, 105.4, 102.6, 98.6, 0.0 ppm.
3-((Trimethylsilyl)ethynyl)aniline (10). Compound 10 (888 mg, 93.4%)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
was prepared following a similar procedure to compound 9 as a yellow oil. H NMR
(400 MHz, DMSO-d ): δ 6.99 (t, J = 7.8 Hz, 1H), 6.63 (s, 1H), 6.57 (t, J = 7.8 Hz,
6
1
3
2
1
H), 5.20 (s, 2H), 0.20 (s, 9H) ppm; C NMR (100 MHz, DMSO-d
6
): δ 149.2, 129.6,
22.9, 119.4, 117.0, 115.3, 106.8, 92.7, 0.5 ppm.
4-((Trimethylsilyl)ethynyl)aniline (11). Compound 11 (565 mg, 59.4%)
1
was prepared following a similar procedure to compound 9 as a yellow solid. H
NMR (400 MHz, DMSO-d
5.53 (s, 2H), 0.18 (s, 9H) ppm; C NMR (100 MHz, DMSO-d
33.3, 117.0, 113.9, 108.6, 107.9, 90.5, 0.7 ppm.
2-Ethynylaniline (12). To a solution of KOH (4.46 g, 32.3 mmol) in MeOH
20 mL) was added a solution of 9 (612 mg, 3.23 mmol) in DCM (20 mL). After
6
): δ 7.09 (d, J = 8.4 Hz, 2H), 6.49 (d, J = 8.4 Hz, 2H),
1
3
6
): δ 150.1, 137.5,
1
(
stirred at rt for 3 h, the reaction mixture was filtered. The filtrate was concentrated
and purified by silica gel column chromatography (petroleum ether/EtOAc = 10:1) to
1
obtain compound 12 as a yellow oil (210 mg, 55.5%). H NMR (400 MHz, CDCl
3
): δ
7
.33 (d, J = 7.6 Hz, 1H), 7.15 (dd, J = 8.0, 7.6 Hz, 1H), 6.71-6.66 (m, 2H), 4.24 (s,
1
3
2H), 3.38 (s, 1H) ppm; C NMR (100 MHz, CDCl
3
): δ 148.5, 132.6, 130.1, 117.8,
1
14.3, 106.6, 82.4, 80.6 ppm.
3
-Ethynylaniline (13). Compound 13 (208 mg, 43.1%) was prepared
1
following a similar procedure to compound 12 as a yellow oil. H NMR (400 MHz,
DMSO-d
.96 (s, 1H) ppm; C NMR (100 MHz, DMSO-d
117.0, 115.2, 84.9, 79.5 ppm.
6
): δ 7.00 (t, J = 7.8 Hz, 1H), 6.64 (s, 1H), 6.59-6.57 (m, 2H), 5.20 (s, 2H),
1
3
3
6
): δ 149.2, 129.6, 122.4, 119.5,
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1
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
5
5
5
5
5
6
4
-Ethynylaniline (14). Compound 14 (267 mg, 64.4%) was prepared
1
following a similar procedure to compound 12 as a brown solid. H NMR (400 MHz,
DMSO-d ): δ 7.11 (d, J = 8.6 Hz, 2H), 6.50 (d, J = 8.6 Hz, 2H), 5.49 (s, 2H), 3.76 (s,
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
1
H) ppm; C NMR (100 MHz, DMSO-d
6
): δ 149.4, 132.7, 113.4, 107.6, 85.0, 77.1
ppm.
1
,1’-Biphenyl-2-amine (36). To a solution of 2-bromoaniline (1.0 g, 5.8
mmol) in a mixture of toluene (25 mL) and EtOH (9 mL) was added Pd(PPh Cl
CO
3 mL). The reaction mixture was refluxed overnight under nitrogen atmosphere. The
O (30 mL) and extracted with DCM (30 mL × 3).
The organic phase was combined, dried over anhydrous Na SO , and evaporated in
3
)
2
2
(
(
203.5 mg, 0.29 mmol), phenylboronic acid (708 mg, 5.8 mmol), and sat aq Na
2
3
reaction mixture was diluted with H
2
2
4
vacuum. The residue was purified by silica gel column chromatography (petroleum
1
ether/EtOAc = 25:1) to obtain compound 36 as a white solid (837 mg, 85.1%). H
NMR (400 MHz, DMSO-d
6
): δ 7.46-7.40 (m, 4H), 7.33 (t, J = 7.0 Hz, 1H), 7.04 (t, J
=
7.6 Hz, 1H), 6.97 (d, J = 7.6 Hz, 1H), 6.76 (d, J = 8.0 Hz, 1H), 6.63 (t, J = 7.4 Hz,
1
3
1
H), 4.75 (s, 2H) ppm; C NMR (100 MHz, DMSO-d
6
): δ 144.9, 139.6, 129.9, 128.7,
128.5, 128.1, 126.7, 125.7, 116.7, 115.2 ppm.
1
,1’-Biphenyl-3-amine (37). Compound 37 (338.7 mg, 68.9%) was
1
prepared following a similar procedure to compound 36 as a yellow oil. H NMR (400
MHz, CDCl
3
): δ 7.58-7.56 (m, 2H), 7.44-7.40 (m, 2H), 7.33 (t, J = 7.4 Hz, 1H), 7.23
(
t, J = 7.8 Hz, 1H), 7.00 (d, J = 7.6 Hz, 1H), 6.91 (t, J = 2.0 Hz, 1H), 6.68 (dd, J = 8.0,
1
3
2
.0 Hz, 1H), 4.24 (s, 2H), 3.71 (s, 2H) ppm; C NMR (100 MHz, CDCl
3
): δ 146.7,
142.4, 141.3, 129.6, 128.6, 127.2, 127.1, 117.6, 114.0, 113.8 ppm.
1
,1’-Biphenyl-4-amine (38). Compound 38 (195.2 mg, 39.7%) was
1
prepared following a similar procedure to compound 36 as a brown solid. H NMR
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(
400 MHz, CDCl
3
): δ 7.55-7.53 (m, 2H), 7.43-7.38 (m, 4H), 7.27 (t, J = 7.4 Hz, 1H),
1
3
6
.76 (d, J = 8.4 Hz, 2H), 3.74 (s, 2H) ppm; C NMR (100 MHz, CDCl
3
): δ 145.8,
141.1, 131.6, 128.6, 128.0, 126.4, 126.2, 115.4 ppm.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
’-Fluoro-1,1’-biphenyl-3-amine (39). Compound 39 (493.4 mg, 90.7%)
1
was prepared following a similar procedure to compound 36 as a yellow oil. H NMR
(
400 MHz, DMSO-d
6
): δ 7.46 (dd, J = 10.8, 7.8 Hz, 1H), 7.40 (d, J = 7.8 Hz, 1H),
7
6
.35 (d, J = 10.8 Hz, 1H), 7.17-7.12 (m, 1H), 7.11 (t, J = 7.8 Hz, 1H), 6.87 (s, 1H),
1
3
.80 (d, J = 7.8 Hz, 1H), 6.61 (d, J = 7.8 Hz, 1H), 5.19 (s, 2H) ppm; C NMR (100
): δ 162.5 (d, J = 241.8 Hz), 149.1, 143.5 (d, J = 7.6 Hz), 139.4 (d, J
2.1 Hz), 130.6 (d, J = 8.6 Hz), 129.5, 122.4, 114.4, 113.7, 113.6 (d, J = 20.8 Hz),
13.9 (d, J = 21.6 Hz), 112.1 ppm.
MHz, DMSO-d
6
=
1
4
’-Fluoro-1,1’-biphenyl-3-amine (40). Compound 40 (407.8 mg, 75.1%)
1
was prepared following a similar procedure to compound 36 as a yellow oil. H NMR
(400 MHz, CDCl
3
): δ 7.51(dd, J = 8.8, 5.4 Hz, 2H), 7.22 (t, J = 7.8 Hz, 1H), 7.10 (t, J
=
8.8 Hz, 2H), 6.93 (d, J = 7.8 Hz, 1H), 6.85 (t, J = 2.0 Hz, 1H), 6.67 (dd, J = 7.8, 2.0
1
3
Hz, 1H), 3.71 (s, 2H) ppm; C NMR (100 MHz, CDCl
146.7, 141.4, 137.4 (d, J = 3.3 Hz), 129.7, 128.5 (d, J = 7.9 Hz), 117.4, 115.4 (d, J =
1.3 Hz), 114.0, 113.6 ppm.
3
): δ 162.3 (d, J = 244.4 Hz),
2
3
’-Chloro-1,1’-biphenyl-3-amine (41). Compound 41 (481.1 mg, 81.3%)
1
was prepared following a similar procedure to compound 36 as a yellow solid. H
NMR (400 MHz, DMSO-d
6
): δ 7.57 (s, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.45 (t, J = 7.8
Hz, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.11 (t, J = 7.8 Hz, 1H), 6.85 (s, 1H), 6.79 (d, J =
1
3
7.8 Hz, 1H), 6.60 (d, J = 7.8 Hz, 1H), 5.19 (s, 2H) ppm; C NMR (100 MHz,
DMSO-d ): δ 149.1, 143.2, 139.2, 133.4, 130.5, 129.5, 126.8, 126.0, 125.0, 114.2,
13.7, 112.0 ppm.
6
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2
2
2
2
2
2
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’-Chloro-1,1’-biphenyl-3-amine (42). Compound 42 (564.0 mg, 95.3%)
1
was prepared following a similar procedure to compound 36 as a yellow solid. H
NMR (400 MHz, DMSO-d
6
): δ 7.56 (d, J = 8.6 Hz, 2H), 7.47 (d, J = 8.6 Hz, 2H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
.10 (t, J = 7.8 Hz, 1H), 6.82 (s, 1H), 6.76 (d, J = 7.8 Hz, 1H), 6.58 (d, J = 7.8 Hz,
1
3
1H), 5.18 (s, 2H) ppm; C NMR (100 MHz, DMSO-d ): δ 149.2, 139.8, 139.5, 131.9,
6
1
29.5, 128.7, 128.2, 114.3, 113.5, 112.0 ppm.
3
’-Methoxy-1,1’-biphenyl-3-amine (43). Compound 43 (465.0 mg,
7
0.9%) was prepared following a similar procedure to compound 36 as a yellow oil.
1
H NMR (400 MHz, DMSO-d
6
): δ 7.33 (t, J = 8.0 Hz, 1H), 7.12-7.05 (m, 3H), 6.89
(
dd, J = 8.0, 2.0 Hz, 1H), 6.83 (t, J = 2.0 Hz, 1H), 6.77 (d, J = 7.6 Hz, 1H), 6.56 (dd, J
1
3
= 8.0, 1.5 Hz, 1H), 5.13 (s, 2H), 3.80 (s, 3H) ppm; C NMR (100 MHz, DMSO-d
6
): δ
59.5, 149.0, 142.6, 140.7, 129.7, 129.3, 118.8, 114.4, 113.2, 112.6, 112.1, 111.9,
5.0 ppm.
1
5
4
’-Methoxy-1,1’-biphenyl-3-amine (44). Compound 44 (347.5 mg,
6
0.1%) was prepared following a similar procedure to compound 36 as a yellow solid.
1
H NMR (400 MHz, CDCl
3
): δ 7.49 (d, J = 8.8 Hz, 2H), 7.20 (t, J = 8.0 Hz, 1H),
6
3
.97-6.94 (m, 3H), 6.87 (t, J = 2.0 Hz, 1H), 6.64 (dd, J = 8.0, 2.0 Hz, 1H), 3.85 (s,
13
H), 3.72 (s, 2H) ppm; C NMR (100 MHz, CDCl ): δ 159.0, 146.7, 142.0, 133.9,
3
129.6, 128.1, 117.3, 114.0, 113.5, 113.4, 55.3 ppm.
3
’,5’-Methoxy-1,1’-biphenyl-3-amine (45). Compound 45 (425.0 mg,
7
9.7%) was prepared following a similar procedure to compound 36 as a yellow oil.
1
H NMR (400 MHz, DMSO-d
6
): δ 7.08 (t, J = 7.8 Hz, 1H), 6.84 (s, 1H), 6.77 (d, J =
7
.8 Hz, 1H), 6.67 (d, J = 2.2 Hz, 2H), 6.57 (dd, J = 7.8, 1.2 Hz, 1H), 6.47 (t, J = 2.2
1
3
Hz, 1H), 5.14 (s, 2H), 3.85 (s, 3H), 3.79 (s, 6H) ppm; C NMR (100 MHz,
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DMSO-d
6
): δ 160.6, 148.9, 143.3, 140.8, 129.2, 114.4, 113.3, 112.2, 104.6, 98.9, 55.1
ppm.
3’-Trifluoromethoxy-1,1’-biphenyl-3-amine (46). Compound 46
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
674.9 mg, 91.7%) was prepared following a similar procedure to compound 36 as a
1
yellow oil. H NMR (400 MHz, DMSO-d
6
): δ 7.60 (dt, J = 8.0, 1.4 Hz, 1H), 7.54 (t, J
=
7.8 Hz, 1H), 7.48 (s, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.13 (t, J = 7.8 Hz, 1H), 6.89 (s,
13
1
H), 6.81 (d, J = 7.8 Hz, 1H), 6.64 (d, J = 8.0 Hz, 1H), 5.23 (s, 2H) ppm; C NMR
100 MHz, DMSO-d ): δ 149.2, 148.8, 143.4, 139.1, 130.6, 129.5, 125.5, 120.1 (q, J
= 254.7 Hz), 119.3, 118.7, 114.3, 113.8, 112.0, ppm.
(
6
4
’-Trifluoromethoxy-1,1’-biphenyl-3-amine (47). Compound 47
(553.0 mg, 75.1%) was prepared following a similar procedure to compound 36 as a
1
yellow solid. H NMR (400 MHz, DMSO-d
6
): δ 7.66 (d, J = 8.4 Hz, 2H), 7.39 (d, J =
8
.4 Hz, 2H), 7.11 (t, J = 7.8 Hz, 1H), 6.86 (s, 1H), 6.77 (d, J = 7.8 Hz, 1H), 6.62 (d, J
1
3
= 7.8 Hz, 1H), 5.21 (s, 2H) ppm; C NMR (100 MHz, DMSO-d
6
): δ 149.2, 147.5,
1
40.3, 139.4, 129.5, 128.1, 121.2, 120.1 (q, J = 254.5 Hz), 114.3, 113.5, 112.1 ppm.
3
’-Methylthio-1,1’-biphenyl-3-amine (48). Compound 48 (355.2 mg,
5
6.9%) was prepared following a similar procedure to compound 36 as a yellow oil.
1
H NMR (400 MHz, DMSO-d ): δ 7.38-7.35 (m, 2H), 7.31 (dt, J = 7.8, 1.4 Hz, 1H),
6
7.22 (dt, J = 7.6, 1.4 Hz, 1H), 7.09 (t, J = 7.8 Hz, 1H), 6.84 (t, J = 1.8 Hz, 1H), 6.77 (d,
1
3
J = 7.6 Hz, 1H), 6.56 (dd, J = 7.8, 1.4 Hz, 1H), 5.16 (s, 2H), 2.52 (s, 3H) ppm; C
NMR (100 MHz, DMSO-d ): δ 149.0, 141.7, 140.3, 138.5, 129.3, 129.2, 124.5, 123.6,
23.0, 114.3, 113.3, 112.0, 14.6 ppm.
6
1
4
’-Methylthio-1,1’-biphenyl-3-amine (49). Compound 49 (456.0 mg,
7
3.1%) was prepared following a similar procedure to compound 36 as a yellow solid.
1
H NMR (400 MHz, DMSO-d ): δ 7.50 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H),
6
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.09 (t, J = 7.8 Hz, 1H), 6.83 (s, 1H), 6.76 (d, J = 7.8 Hz, 1H), 6.55 (d, J = 7.8 Hz,
1
3
H), 5.14 (s, 2H), 3.35 (s, 3H) ppm; C NMR (100 MHz, DMSO-d ): δ 149.0, 140.1,
6
137.5, 136.8, 129.3, 126.8, 126.2, 113.9, 113.0, 111.6, 14.6 ppm.
0
1
2
3
4
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7
8
9
0
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2
3
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5
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7
8
9
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0
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’-Trifluoromethyl-1,1’-biphenyl-3-amine (50). Compound 50 (619.3
mg, 89.8%) was prepared following a similar procedure to compound 36 as a yellow
1
oil. H NMR (400 MHz, DMSO-d
6
): δ 7.85-7.84 (m, 2H), 7.65-7.59 (m, 2H), 7.16 (t,
J = 7.8 Hz, 1H), 6.99 (s, 1H), 6.84 (d, J = 7.8 Hz, 1H), 6.71 (d, J = 7.8 Hz, 1H), 5.27
1
3
(
s, 2H) ppm; C NMR (100 MHz, DMSO-d
6
): δ 149.3, 142.1, 139.3, 130.4, 129.9,
129.7, 129.6, 129.5, 125.6, 123.5 (t, J = 3.8 Hz), 122.9, 122.7 (t, J = 3.9 Hz), 114.5,
1
14.0, 112.3 ppm.
4’-Trifluoromethyl-1,1’-biphenyl-3-amine (51). Compound 51 (561.0
mg, 81.4%) was prepared following a similar procedure to compound 36 as a yellow
1
solid. H NMR (400 MHz, DMSO-d
6
): δ 7.76 (s, 4H), 7.14 (dd, J = 8.0, 7.6 Hz, 1H),
1
3
6.90 (s, 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.64 (d, J = 8.0 Hz, 1H), 5.25 (s, 2H) ppm; C
NMR (100 MHz, DMSO-d ): δ 149.2, 145.0, 139.2, 129.6, 127.1, 125.6 (t, J = 3.8
Hz), 114.4, 114.0, 112.1 ppm.
6
3’-Cyano-1,1’-biphenyl-3-amine (52). Compound 52 (507.1 mg, 89.8%)
1
was prepared following a similar procedure to compound 36 as a yellow oil. H NMR
(400 MHz, DMSO-d
6
): δ 7.98 (t, J = 1.6 Hz, 1H), 7.89 (dt, J = 7.8, 1.6 Hz, 1H), 7.78
(
dt, J = 7.8, 1.2 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.13 (t, J = 7.8 Hz, 1H), 6.87 (t, J =
1
.6 Hz, 1H), 6.83 (d, J = 7.8 Hz, 1H), 6.62 (dd, J = 7.8, 1.6 Hz, 1H), 5.22 (s, 2H) ppm;
1
3
C NMR (100 MHz, DMSO-d
6
): δ 149.2, 142.1, 138.6, 131.2, 130.6, 129.9, 129.8,
129.6, 118.8, 114.3, 113.9, 112.0, 111.8 ppm.
4
’-Cyano-1,1’-biphenyl-3-amine (53). Compound 53 (438.8 mg, 77.7%)
1
was prepared following a similar procedure to compound 36 as a yellow solid. H
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NMR (400 MHz, DMSO-d
6
): δ 7.88 (d, J = 8.2 Hz, 2H), 7.74 (d, J = 8.2 Hz, 2H),
7
.14 (t, J = 7.8 Hz, 1H), 6.89 (s, 1H), 6.83 (d, J = 7.8 Hz, 1H), 6.64 (dd, J =7.8, 1.6
1
3
Hz, 1H), 5.26 (s, 2H) ppm; C NMR (100 MHz, DMSO-d
6
): δ 149.3, 145.5, 138.9,
1
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2
2
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3
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3
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32.6, 129.6, 127.2, 118.9, 114.4, 114.3, 112.1, 109.5 ppm.
3
’-Acetyl-1,1’-biphenyl-3-amine (54). Compound 54 (544.8 mg, 88.7%)
1
was prepared following a similar procedure to compound 36 as a yellow oil. H NMR
(
400 MHz, DMSO-d
6
): δ 8.11 (t, J = 1.6 Hz, 1H), 7.92 (d, J = 7.8 Hz, 1H), 7.81 (d, J
=
7.8 Hz, 1H), 7. 57 (t, J = 7.8 Hz, 1H), 7.14 (t, J = 7.8 Hz, 1H), 6.93 (t, J = 1.6 Hz,
1H), 6.84 (d, J = 7.8 Hz, 1H), 6.63 (dd, J = 7.8, 1.6 Hz, 1H), 5.22 (s, 2H), 2.63 (s, 3H)
13
ppm; C NMR (100 MHz, DMSO-d
6
): δ 197.9, 149.2, 141.4, 139.9, 137.2, 131.0,
1
29.5, 129.1, 126.9, 125.8, 114.4, 113.5, 112.1, 26.8 ppm.
4
’-Acetyl-1,1’-biphenyl-3-amine (55). Compound 55 (523.3 mg, 85.4%)
1
was prepared following a similar procedure to compound 36 as a yellow solid. H
NMR (400 MHz, CDCl
3
): δ 8.01 (d, J = 8.6 Hz, 2H), 7.92 (d, J = 8.6 Hz, 2H), 7.25 (t,
J = 7.8 Hz, 1H), 7. 02 (d, J = 7.8 Hz, 1H), 6.93 (t, J = 1.8 Hz, 1H), 6.73 (dd, J = 7.8,
1
3
1
.8 Hz, 1H), 3.79 (s, 2H), 2.63 (s, 3H) ppm; C NMR (100 MHz, CDCl
3
): δ 197.8,
146.9, 146.0, 141.0, 135.7, 129.8, 128.8, 127.1, 117.6, 114.9, 113.7, 26.6 ppm.
Ethyl
(E)-3-(1,3-dihydro-1-hydroxy-2,1-benzoxaborol-7-yl)
acrylate (63). To a solution of aldehyde 62 (1.68 g, 10.4 mmol) in toluene (150 mL)
was added ethyl (triphenylphosphoranylidene)acetate (4.34 g, 12.4 mmol). After
stirred overnight at rt, the reaction mixture was evaporated in vacuum followed by
purification on silica gel column chromatography (petroleum ether/EtOAc = 2:1) to
1
obtain compound 63 as a white solid (1.6 g, 66.7%). H NMR (400 MHz, DMSO-d
6
):
δ 9.33 (s, 1H), 8.10 (d, J = 16.2 Hz, 1H), 7.81 (d, J = 7.6 Hz, 1H), 7.52 (t, J = 7.6 Hz,
1H), 7.44 (d, J = 7.6 Hz, 1H), 6.80 (d, J = 16.2 Hz, 1H), 5.02 (s, 2H), 4.19 (q, J = 7.0
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