Novel pyrrolobenzoxaboroles: Design, synthesis, and biological evaluation against Trypanosoma brucei

Article abstract of DOI:10.1016/j.ejmech.2014.04.079

Human African trypanosomiasis is a fatal parasitic infection caused by the protozoan Trypanosoma brucei. The development of novel antitrypanosomal agents is urgently needed. Here we report the synthesis and structure-activity relationship of a new class of benzoxaboroles as antitrypanosomal agents. These compounds showed antiparasitic IC₅₀ values ranging from 4.02 to 0.03 μg/mL and satisfactory cytotoxicity profile. Three of the lead compounds were demonstrated to cure the parasitic infection in a murine acute infection model. The structure-activity relationship of the pyrrolobenzoxaboroles are also discussed.

Full text of DOI:10.1016/j.ejmech.2014.04.079

European Journal of Medicinal Chemistry 81 (2014) 59e75
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel pyrrolobenzoxaboroles: Design, synthesis, and biological
evaluation against Trypanosoma brucei
,
,
Puhua Wu a 1, Jiong Zhang a 1, Qingqing Meng a, Bakela Nare b, Robert T. Jacobs b,
Huchen Zhou a
,
*
a State Key Laboratory of Microbial Metabolism, and School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China
b Scynexis, Inc., P.O. Box 12878, Research Triangle Park, NC 27709-2878, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Human African trypanosomiasis is a fatal parasitic infection caused by the protozoan Trypanosoma brucei.
The development of novel antitrypanosomal agents is urgently needed. Here we report the synthesis and
structureeactivity relationship of a new class of benzoxaboroles as antitrypanosomal agents. These
Received 31 January 2014
Received in revised form
25 April 2014
Accepted 26 April 2014
Available online 29 April 2014
compounds showed antiparasitic IC50 values ranging from 4.02 to 0.03 mg/mL and satisfactory cytotox-
icity profile. Three of the lead compounds were demonstrated to cure the parasitic infection in a murine
acute infection model. The structureeactivity relationship of the pyrrolobenzoxaboroles are also
discussed.
Keywords:
Ó 2014 Elsevier Masson SAS. All rights reserved.
Human African trypanosomiasis
Antitrypanosomal agent
Indolobenzoxaboroles
Pyrrolobenzoxaboroles
Structureeactivity relationship
1. Introduction
increasing drug resistance [4,5]. Recently, various combinations of
eflornithine, melarsoprol, and nifurtimox have shown improved
Human African trypanosomiasis (HAT or sleeping sickness), a
fatal parasitic disease caused by the protozoan Trypanosoma brucei,
is transmitted to human hosts by the bite of tsetse flies. The disease
threatens thirty-seven sub-Saharan countries which corresponds
approximately to one-third of Africa’s total land area and a popu-
lation of about 60 million. HAT has become the second greatest
cause of death, ahead of HIV/AIDS, in those areas [1,2]. Currently,
there are five drugs available for the treatment, namely, pentami-
dine, suramin, melarsoprol, eflornithine, and nifurtimox. Although
having been used for decades to a century, the exact intracellular
targets of these drugs remain elusive with the exception of eflor-
nithine which irreversibly inhibits ornithine decarboxylase. Pent-
amidine, suramin, melarsoprol, and nifurtimox have been found to
disrupt mitochondrial processes, glycolysis, redox metabolism, and
induce oxidative attack, respectively [3]. These drugs suffer from
serious drawbacks: low efficacy, severe toxic side effects, and
efficacy compared with monotherapy [6]. But combinations con-
taining melarsoprol resulted in very high rates of severe adverse
effects such as loss of consciousness and even death. At the same
time, the development of new medicines against HAT underwent a
serious setback when the development of pafuramidine maleate
(DB289) failed due to nephrotoxicity [7]. Of the 336 new chemical
entities proved during 2000e2011, only 1% contributed to neglec-
ted diseases while none of them was for the treatment of HAT [8].
Therefore, discovery and development of novel antitrypanosomal
drugs are still in great need and present challenges for medicinal
chemists.
Benzoxaboroles, which were reported as early as 1957 by Tors-
sell [9], are cyclic hemiesters of phenylboronic acids. Since AN2690
(5-fluorobenzoxaborole) succeeded in clinical trials for the treat-
ment of fungal infection [10,11], much attention has been paid to
the application of this unique scaffold in medicinal chemistry. It has
been utilized in the development of compounds for the treatment
of periodontal disease [12], psoriasis, and atopic dermatitis [13].
We have previously reported C(6)-substituted benzoxaboroles
as antitrypanosomal agents [14e18]. In this work, we report the
development of a new series of 6-pyrrolobenzoxaboroles (Fig. 1)
derived from the initial 6-indolylbenzoxaboroles. First, exploration
of indolyl derivatives (I) gave only one active compound.
Abbreviations: HAT, human African trypanosomiasis; HIV, human immunode-
ficiency virus; AIDS, acquired immune deficiency syndrome; T. brucei, Trypanosoma
brucei; b.i.d., twice a day; i.p., intraperitoneal.
* Corresponding author.
E-mail addresses: hczhou@sjtu.edu.cn, huchen.zhou@gmail.com (H. Zhou).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2014.04.079
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

60
P. Wu et al. / European Journal of Medicinal Chemistry 81 (2014) 59e75
Fig. 1. Discovery of a new 6-pyrrolobenzoxaborole scaffold.
Subsequent replacement of the indolyl group with a pyrrolyl group
(II) significantly improved the overall antitrypanosomal activity.
Further structureeactivity relationship studies revealed that mov-
ing the substituents from the pyrrolyl C(20) to the C(30) (III) gave
significantly
more
potent
antitrypanosomal
6-
pyrrolobenzoxaboroles with IC50 values as low as 0.03
mg/mL and
satisfactory cytotoxicity profile. Three of the lead compounds, 50,
68, and 78, were demonstrated to effectively clear the parasitic
infection in a murine acute infection model.
2. Chemistry
The 6-indolobenzoxaboroles 6e17 were prepared from 2-
bromo-4-fluoro-benzaldehyde (1) (Scheme 1). After protection of
the aldehyde in the form of acetal 2, a nucleophilic replacement
reaction by indole gave the indolyl intermediate 3. After depro-
tection and reduction, compound 5 was converted to the 6-
indolobenzoxaborole 6 by a one-pot treatment: first with n-BuLi
and triisopropyl borate to install boron; and subsequently with
hydrochloric acid to induce the spontaneous cyclization. Com-
pound 6 was further reacted with various arylthiols in the presence
of iodine to give 30-thioethers 7e15 [19]. Thioether 7 was oxidized
to sulfoxide 16 or sulfone 17 by addition of H2O2 at ambient or
elevated temperature, respectively.
Scheme 1. Synthesis of 6-indolobenzoxaboroles. Reagents and conditions: (a)
ethylene glycol, p-TsOH, toluene; (b) indole, Cs2CO3, DMF; (c) 1 M HCl, THF; (d) NaBH4,
MeOH; (e) B(i-PrO)3, n-BuLi, THF, ꢀ78 ꢁC to RT; (f) aq. HCl; (g) ArSH, I2, EtOH, H2O; (h)
H2O2, AcOH, RT; (i) H2O2, AcOH, 75 ꢁC.
The 6-pyrrolobenzoxaborole scaffold 24 was prepared from 2-
bromo-4-nitrotoluene (18) in six steps (Scheme 2). The aniline in-
termediate 21 was condensed with 2,5-dimethoxytetrahydrofuran
to give the pyrrolyl intermediate 22 which was reduced and sub-
jected to the one-pot boronylation to yield the parent 6-
pyrrolobenzoxaborole 24 that would serve as the common inter-
mediate for the synthesis of various derivatives as described below.
First, the parent 6-pyrrolobenzoxaborole 24 was readily con-
verted to 20-thioethers 31e33 after the treatment with various
phenylthiophthalimides [20] (28e30) in the presence of
MgBr2(Et2O)2 (Scheme 3A) [21]. Second, formylation of the 6-
pyrrolobenzoxaborole 24 with DMF/POCl3 gave 20-formyl com-
pound 34 which was subsequently oxidized to carboxylic acid 35
(Scheme 3B). The carboxylic acid 35 would serve as a common
intermediate in the synthesis of amides 36e40 under standard
EDC/HOBt-assisted coupling condition. However, the methyl- and
ethyl- amides (42 and 43) eluded this approach and were prepared
by nucleophilic reaction with trichloroacetyl derivative 41 (Scheme
3C) [22].
chloride gave solely 30-acetyl compound 48, VilsmeiereHaack re-
action was employed to obtain its 20-analog 80 (Scheme 4B) [24].
To demonstrate the essentiality of the 30-carbonyl and the
oxaborole moiety for good antitrypanosomal potency, compound
81 wherein the carbonyl was removed and compounds 84 and 92
wherein the oxaborole ring was either removed or replaced were
synthesized as described in Scheme 5. Compound 81 was obtained
by reduction of the compound 50 with sodium borohydride and
boron trifluoride diethyl etherate (Scheme 5A). Compound 84 was
prepared via FriedeleCrafts reaction of benzoyl chloride and com-
pound 83 which was synthesized from phenylamine and 2,5-
dimethoxytetrahydrofuran (Scheme 5B). Compound 92 was syn-
thesized from alcohol 23 in eight steps (Scheme 5C). The 30-
FriedeleCrafts reactions catalyzed by anhydrous stannic chlo-
ride was used to introduce arylacyl or alkylacyl substituents mainly
on the pyrrolyl C(30) position (48e64, 69e75, 77e79), while the
C(20)-acylated regio-isomers (44e47) were obtained at the same
time in separable yields (Scheme 4A) [23]. In these cases, the 20-acyl
to 30-acyl ratio ranges from 1:5 to 1:2. Removal of acetyl from
compound 62 gave compound 65. Treatment of the methyl ether 59
with boron tribromide afforded its demethylated analog 66.
Removal of Fmoc group from compounds 63 and 64 gave com-
pounds 67 and 68, respectively. Removal of the phthalimide pro-
tection of compound 75 by basic and subsequent acidic hydrolysis
produced compound 76. As the acylation reaction with acetyl
Scheme 2. Synthesis of the 6-pyrrolobenzoxaborole core structure. Reagents and
conditions: (a) KMnO4, pyridine, H2O; (b) SOCl2, reflux; (c) MeOH, Et3N; (d)
SnCl2$2H2O, ethyl acetate, reflux; (e) 2,5-dimethoxytetrahydrofuran, AcOH, reflux; (f)
LiBH4, THF, 0 ꢁC; (g) B(i-PrO)3, n-BuLi, ꢀ78 ꢁC to RT; (h) 2 M HCl.

P. Wu et al. / European Journal of Medicinal Chemistry 81 (2014) 59e75
61
Scheme 3. Synthesis of 6-pyrrolobenzoxaboroles bearing arylsulfenyl or amide sub-
stituents. Reagents and conditions: (a) chlorine, n-pentane, 0 ꢁC; (b) phthalimide, Et3N,
DMF; (c) compound 24, MgBr2(Et2O)2, N,N-dimethylacetamide; (d) DMF, POCl3, DCM,
0
ꢁC to RT; (e) AgNO3, 6 M NaOH, H2O, 0 ꢁC; (f) the corresponding amine, EDC, HOBt,
DCM, RT; (g) trichloroacetyl chloride, 75 ꢁC; (h) the corresponding amine, DCM, 0 ꢁC to
RT.
phenylacylpyrrolyl intermediate 89 was obtained from 23 after
sequential methylation, formylation, reduction, acetylation, and
FriedeleCrafts reaction. Deacetylation of compound 89 followed by
oxidation gave aldehyde 91 which was subsequently demethylated
to yield the desired hemiacetal 92.
Scheme 5. Synthesis of compounds without 30-carbonyl or oxaborole functionalities.
Reagents and conditions: (a) NaBH4, BF3$Et2O, THF; (b) 2,5-dimethoxytetrahydrofuran,
AcOH, reflux; (c) benzoyl chloride, SnCl4, DCM, RT; (d) CH3I, NaH, THF; (e) n-BuLi, DMF,
THF, ꢀ78 ꢁC; (f) NaBH4, MeOH, RT; (g) Ac2O, Et3N, DCM; (h) benzoyl chloride, SnCl4,
3. Results and discussion
DCM, ꢀ40 ꢁC; (i)
1 M NaOH, 1 M HCl, RT; (j) PCC, celite, DCM, RT; (k) BBr3,
3.1. Inhibition of T. brucei parasite growth
DCM, ꢀ78 ꢁCe0 ꢁC; (l) MeOH, RT.
Indole is a privileged scaffold that is widely found in clinically
used drugs and natural products [25,26]. We initiated our explo-
ration of new antitrypanosomal benzoxaboroles by incorporating
this privileged structure. A whole cell-based screening approach
was taken with the aim to identify new compounds that can
effectively inhibit the growth of T. brucei parasites.
However, the indolobenzoxaborole 6 and its thioether de-
rivatives showed disappointing activity with only one active com-
pound, namely the sulfoxide 16 (Table 1). Assuming that the
rigidity of the indolobenzoxaborole structure may have led to the
low activity via reduced solubility and permeability, we decided to
reduce the indolyl ring size by replacing it with a pyrrolyl group.
Indeed, the parent 6-pyrrolobenzoxaborole 24 showed an inhibi-
tory IC50 of 1.98 m
g/mL (Table 2). The 20-thioether derivatives 31e33
were synthesized to compare with the indolylthioethers. The
monochloro- compounds 31 and 32 showed activity similar to
compound 16 while the dichloro- compound 33 was inactive.
In order to explore the C(20) position in a more efficient manner,
we decided to introduce a carboxylate group which can be easily
converted to amides for rapid divergent derivatization. The car-
boxylic acid 35 (Table 2) showed a 15-fold increase in potency
Scheme 4. Synthesis of 20- and 30-acylpyrrolylbenzoxaboroles. Reagents and condi-
tions: (a) the corresponding acyl chloride, SnCl4, DCM, RT; (b) 1 M NaOH, RT, then 1 M
HCl; (c) BBr3, DCM, ꢀ80 ꢁC to RT; (d) piperidine, acetone or trichloromethane, 0 ꢁC to
RT; (e) 6 M HCl, MeOH, reflux; (f) N,N-dimethylacetamide, POCl3, DCM, 0 ꢁC to RT.
(IC50 ¼ 0.13
mg/mL) as compared to the parent pyrrolobenzox-
aborole 24, which suggests the importance of introducing a
hydrogen bond acceptor and donor on the pyrrole ring. Carboxylic

62
P. Wu et al. / European Journal of Medicinal Chemistry 81 (2014) 59e75
Table 1
demonstrated by compounds 50e52 which showed significantly
improved activity as compared to their 20-acyl analogs 45e47. This
observation thus prompted us to focus our following exploration on
the 30-acyl derivatives.
Effect of substituted 6-indolobenzoxaboroles on T. brucei growth inhibition and
cytotoxicity.a
As shown in Tables 3e4, a series of 30-acylpyrrolobenzoxabor-
oles were explored. The chlorophenyl compounds 51, 53, and 54
gave reduced activity as compared to the parent phenyl compound
50. The methylphenyl and ethylphenyl compounds 55e58, with the
exception of analog 57 (IC50 ¼ 0.56
the ortho- position, gave comparable inhibitory activity (IC50 ¼ 0.08
and 0.09 g/mL) to the parent phenyl compound 50. The methox-
yphenyl compounds 59e61 all showed good inhibitory activity, but
the trimethoxyphenyl compound 61 exhibited the highest cyto-
mg/mL) that has the methyl at
Compd
R
T. brucei IC50
(m
g/mL)
L929 IC50 (mg/mL)
6
7
>5
>5
>10
>10
m
toxicity (L929 IC50 ¼ 1.15
mg/mL) among all compounds discussed in
8
9
>5
>5
>10
>10
this work. Polar groups such as acetoxyl, hydroxyl and amino
groups, were subsequently introduced onto the phenyl ring as
represented by compounds 62 and 65e68 which showed excellent
inhibitory activity. Among them, compounds 62, 65, and 68
10
>5
>10
Table 2
Effect of 20-substituted 6-pyrrolobenzoxaboroles on T. brucei growth inhibition and
cytotoxicity.a
11
12
>5
>5
>10
>10
13
14
15
>5
>5
>5
>10
>10
>10
Compd
R
T. brucei IC50
(
mg/mL)
L929 IC50 (mg/mL)
24
31
1.98
1.16
>10
>10
32
33
2.89
>10
>10
16
1.56
>10
>10
17
>5
>5
a
IC50: growth inhibition of T. b. brucei 427 strain (
g/mL). References: pentamidine (IC50: 0.009
g/mL).
m
m
g/mL). L929: IC50 against
g/mL) and suramin (IC50:
L929 cells (
0.007
m
35
36
0.13
3.21
>10
>10
m
37
2.85
>10
acid 35 was subsequently modified to give amides 36e40, while
amides 42 and 43 were obtained via an alternative tri-
chloromethylketone intermediate. The bulky or small alkyl groups
were explored as in compounds 36e37 and 42e43, which showed
much reduced activity as compared to the carboxylic acid 35. The
phenyl-containing derivatives 38e40 gave relatively better activity
42
43
38
0.86
1.85
0.80
>10
>10
>10
than the alkyl amides with IC50 values below 1
pounds showed satisfactory cytotoxicity profile with IC50 values
above 10 g/mL against mouse lung fibroblast L929 cells.
mg/mL. All com-
m
Encouraged by the above preliminary result, we decided to
explore the effect of introducing a carbonyl group as a hydrogen
bond acceptor to either the C(20) or the C(30) of the pyrrole ring.
FriedeleCrafts reaction gave 30-acyl products as the major products
with separable yields of 20-acyl products, thus providing us the
opportunity to directly compare the antitrypanosomal effect of the
two regio-isomers as shown in Table 3. Although the acetyl com-
pounds 80/48 and dichloro-compounds 44/49 showed comparable
activity for the regioeisomer pairs, the 30-acyl compounds gener-
ally showed apparently favorable antitrypanosomal activity as
39
0.70
0.68
>10
>10
40
a
IC50: growth inhibition of T. b. brucei 427 strain (
g/mL). References: pentamidine (IC50: 0.009
g/mL).
m
m
g/mL). L929: IC50 against
g/mL) and suramin (IC50:
L929 cells (
0.007
m
m

P. Wu et al. / European Journal of Medicinal Chemistry 81 (2014) 59e75
63
Table 3
Table 4
Effect of 20-acyl versus 30-acyl substitutions on T. brucei growth inhibition and
Effect of different 30-acyl substitutions on T. brucei growth inhibition and cytotox-
cytotoxicity.a
icity.a
Compd
R
Position T. brucei IC50
(
m
g/mL) L929 IC50 (mg/mL)
Compd
R
T. brucei IC50
(m
g/mL)
L929 IC50 (mg/mL)
80
20
0.69
>10
53
0.21
>10
44
45
46
20
1.43
>10
>10
>10
54
55
56
57
58
59
0.35
0.08
0.09
0.56
0.07
0.11
>10
>10
>10
>10
>10
>10
20
20
0.34
0.70
47
20
0.40
>10
48
49
30
30
0.56
1.12
>10
>10
50
51
30
0.09
>10
60
61
0.17
0.16
>10
30
30
0.28
0.16
>10
>10
1.15
52
a
IC50: growth inhibition of T. b. brucei 427 strain (
g/mL). References: pentamidine (IC50: 0.009
g/mL).
m
m
g/mL). L929: IC50 against
g/mL) and suramin (IC50:
L929 cells (
0.007
m
62
0.03
10
m
65
66
0.04
0.06
9.31
exhibited the highest inhibitory activity (IC50 ¼ 0.03e0.04
mg/mL)
among all compounds discussed in this work. Expansion of the size
of the phenyl group or extension of the linker to the phenyl group
(compounds 69e71) did not enhance the activity as compared to
the phenyl parent compound 50. Next, alkyl ketones 72e76 were
investigated. When compared to their methyl analog 48, the larger
propyl, pentyl, and cyclohexyl analogs 72e74 gave increased ac-
>10
67
68
0.18
0.03
>10
3.9
tivity (IC50 ¼ 0.09e0.13
mg/mL) which is comparable to the phenyl
derivative 50. However, addition of a terminal amino group to the
alkyl chain significantly reduced the activity of compound 76.
Finally, thiophenyl ketones 77e79 were shown to have inhibitory
activity similar to their phenyl analogs. With the exception of
compounds 61 and 69, the 30-acyl compounds all showed satis-
factory cytotoxicity profile. Although compounds 62, 65, and 68
have low L929 IC50 values, their selectivity indices are still well
above 100.
69
0.40
5.59
70
71
0.29
0.19
>10
>10
In order to demonstrate the importance of the 30-carbonyl ox-
ygen and the oxaborole moiety in maintaining the anti-
trypanosomal activity of the 30-acylpyrrolylbenzoxaboroles, we
obtained the deoxy analog 81, oxaborole-deleted analog 84, and
compound 92 whose boron atom was replaced by a carbon atom.
They all showed significantly diminished activity as described in
Table 5, thus demonstrating that the carbonyl oxygen and the
oxaborole ring are indispensable for the antiparasitic activity.
72
73
74
0.11
0.09
0.13
>10
>10
>10
(continued on next page)

64
P. Wu et al. / European Journal of Medicinal Chemistry 81 (2014) 59e75
Table 4 (continued )
Table 5
The effect of deletion of 30-carbonyl or the oxaborole functionalities on T. brucei
Compd
R
T. brucei IC50
(m
g/mL)
L929 IC50 (mg/mL)
growth inhibition and cytotoxicity.a
Compd
Structure
T. brucei IC50
(m
g/mL)
L929 IC50 (mg/mL)
75
0.72
>10
81
2.17
>10
76
77
4.02
0.08
>10
>10
78
0.07
0.11
>10
>10
84
>5
>10
79
a
IC50: growth inhibition of T. b. brucei 427 strain (
g/mL). References: pentamidine (IC50: 0.009
g/mL).
m
m
g/mL). L929: IC50 against
g/mL) and suramin (IC50:
L929 cells (
0.007
m
m
92
>5
8.18
3.2. Antitrypanosomal efficacy in a murine model
a
IC50: growth inhibition of T. b. brucei 427 strain (
g/mL). References: pentamidine (IC50: 0.009
g/mL).
m
m
g/mL). L929: IC50 against
g/mL) and suramin (IC50:
L929 cells (
0.007
m
In order to evaluate the in vivo efficacy of the pyrrolobenzox-
aboroles discussed above, five of the lead compounds, namely
compounds 50, 58, 66, 68, and 78, were tested in a murine model of
acute blood stage T. brucei infection (Fig. 2). Treatment with each
compound (50 mg/kg b.i.d., i.p.) was initiated 24 h post-infection,
and was continued for 5 days. The ethylphenyl and hydrox-
yphenyl compounds 58 and 66 showed moderate extension of the
survival time of the infected mice but failed to eradicate the
infection. The phenyl, aminophenyl, and methylthiophenyl com-
pounds 50, 68, and 78 gave 100% survival rate and clearance of
blood parasites on day 30.
m
in 10 min) or method B (a flow rate of 1 mL/min and a gradient of
10% MeOH/90% H2O containing 0.1% TFA to 100% MeOH in 20 min)
using
(4.6 ꢂ 150 mm, 5
UV chromatogram (254 nm). Column chromatography was per-
formed using Huanghai silica gel (38e54 m). Melting points were
a
VWD detector.
A Waters Symmetry C18 column
mm) was used. Purity was based on the integrated
m
measured on a SGWX-4 melting point apparatus.
5.1.2. 2-(2-Bromo-4-fluorophenyl)-1,3-dioxolane (2)
To
a solution of 2-bromo-4-fluoro-benzaldehyde (2.43 g,
12 mmol) in toluene (50 mL) were added ethylene glycol (7.44 g,
120 mmol) and p-toluenesulfonic acid monohydrate (0.23 g,
1.2 mmol). After heated to reflux and stirred overnight, the mixture
was washed with saturated NaHCO3, water, and brine, and dried
over anhydrous Na2SO4. The residue after rotary evaporation was
purified by column chromatography over silica gel to give com-
pound 2 as an oil (2.07 g, 70.0% yield). 1H NMR (400 MHz, CDCl3):
4. Conclusion
We have discovered 6-pyrrolobenzoxaboroles as a new class of
potent antitrypanosomal agents. Three of the lead compounds
were demonstrated to cure the parasitic infection in a murine acute
infection model with complete clearance of the parasites in the
blood. However, the mechanism of action of these 6-
pyrrolobenzoxaboroles is unclear and we are currently undertak-
ing the task to elucidate it. Whole-cell phenotypic screening has
been a very useful approach in antiparasitic drug discovery and the
subsequent identification of the cellular targets would further
provide much needed novel therapeutic targets [27]. We believe
these novel 6-pyrrolobenzoxaboroles will serve both as potential
therapeutic agents and as molecular tools to explore new antipar-
asitic targets.
d
7.58 (dd, J ¼ 8.6, 6.1 Hz,1H), 7.30 (dd, J ¼ 8.2, 2.5 Hz,1H), 7.04 (ddd,
J ¼ 8.6, 8.2, 2.5 Hz, 1H), 6.03 (s, 1H), 4.16e4.09 (m, 2H) and 4.08e
4.00 (m, 2H) ppm.
5.1.3. 2-(2-Bromo-4-(indol-1-yl)-phenyl)-1,3-dioxolane (3)
To a solution of compound 2 (200 mg, 0.81 mmol) in DMF (5 mL)
were added Cs2CO3 (395 mg, 1.21 mmol) and indole (98.4 mg,
0.81 mmol). The mixture was heated to reflux and stirred for 2 h.
The mixture was poured into water (20 mL), extracted with ether,
and dried over anhydrous Na2SO4. The residue after rotary evapo-
ration was purified by column chromatography over silica gel to
give compound 3 as an oil (120 mg, 43.3% yield). 1H NMR (400 MHz,
5. Experimental section
5.1. Chemistry
CDCl3):
d
7.76 (d, J ¼ 8.3 Hz, 1H), 7.75 (d, J ¼ 2.1 Hz, 1H), 7.68 (dd,
J ¼ 7.9,1.2 Hz,1H), 7.56 (ddd, J ¼ 8.2,1.0, 0.8 Hz,1H), 7.50 (dd, J ¼ 8.3,
2.1 Hz, 1H), 7.30 (d, J ¼ 3.3 Hz, 1H), 7.25 (ddd, J ¼ 8.2, 7.1, 1.2 Hz, 1H),
7.2 (ddd, J ¼ 7.9, 7.1, 1.0 Hz, 1H), 6.7 (dd, J ¼ 3.3, 0.8 Hz, 1H), 6.14 (s,
1H), 4.23e4.17 (m, 2H) and 4.16e4.10 (m, 2H) ppm.
5.1.1. General
NMR spectra were recorded on a MercuryPlus 400 (Varian) or
Mercury 300 (Varian). Chemical shifts (d) are expressed in parts per
million (ppm) relative to residual solvent as an internal reference.
High resolution mass spectra were obtained on a Micromass GCT.
High performance liquid chromatography analysis was performed
on a Varian ProStar 230 with method A (a flow rate of 1 mL/min and
a gradient of 10% MeCN/90% H2O containing 0.1% TFA to 100% MeCN
5.1.4. (2-Bromo-4-(indol-1-yl))-benzyl alcohol (5)
To a solution of compound 3 (2.47 g, 7.2 mmol) in THF (20 mL)
was added 1 M HCl (10 mL) dropwise and stirred for 3 h. After it was

P. Wu et al. / European Journal of Medicinal Chemistry 81 (2014) 59e75
65
100
80
60
40
20
0
100
80
Untreated
Untreated
Compound 50
50 mg/kg, b.i.d
Compound 68
50 mg/kg, b.i.d
60
40
20
0
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Day Post-Infection
Day Post-Infection
100
80
60
40
20
0
100
80
60
40
20
0
Untreated
Compound 58
50 mg/kg, b.i.d
Compound 66
50 mg/kg, b.i.d
Untreated
Compound 78
50 mg/kg, b.i.d
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Day Post-Infection
Day Post-Infection
Fig. 2. Female BALB/c mice were inoculated with 600 T. b. brucei 221 parasites. Compounds 50, 68 and 78 gave complete eradication of T. b. brucei parasites. Treatment with
compounds 58 and 66 extended survival time of the infected mice but failed to cure them. Compounds were administered at a dosage of 50 mg/kg, b. i. d. Suramin was the reference
compound.
neutralized with saturated NaHCO3, the mixture was extracted
with EtOAc, and dried over anhydrous Na2SO4. After evaporation
the residue was redissolved in CH3OH (30 mL). To this solution
NaBH4 (0.4 g,10.8 mmol) was added and stirred for 1 h. The mixture
was quenched with water (10 mL), evaporated, extracted with
EtOAc, and dried over anhydrous Na2SO4. The residue after rotary
evaporation was purified by column chromatography over silica gel
to give compound 5 as an oil (2.01 g, 92.6% yield). 1H NMR
(0.5 mL) was added dropwise a solution of I2 (56 mg, 0.22 mmol) in
EtOH (0.5 mL). After it was refluxed for 2 h, the mixture was poured
into ice water, extracted with EtOAc, washed with water, and dried
over anhydrous Na2SO4. The residue after rotary evaporation was
purified by column chromatography over silica gel to give com-
pound 7 as a solid (60 mg, 76.2% yield). 1H NMR (400 MHz, DMSO-
d6):
d
9.36 (s, 1H), 8.08 (s, 1H), 7.95 (d, J ¼ 2.1 Hz, 1H), 7.75 (dd,
J ¼ 8.1, 2.1 Hz, 1H), 7.65 (d, J ¼ 8.1 Hz, 1H), 7.57 (d, J ¼ 8.3 Hz, 1H),
7.53 (d, J ¼ 7.8 Hz, 1H), 7.27 (dd, J ¼ 8.3, 7.1 Hz, 1H), 7.22 (dd, J ¼ 8.9,
2.2 Hz, 2H), 7.20 (dd, J ¼ 7.8, 7.1 Hz,1H), 6.86 (dd, J ¼ 8.9, 2.2 Hz, 2H),
5.10 (s, 2H) and 3.69 (s, 3H) ppm; 13C NMR (100 MHz, DMSO-d6):
(400 MHz, CDCl3):
d
7.73 (d, J ¼ 2.1 Hz, 1H), 7.70 (d, J ¼ 7.8 Hz, 1H),
7.63 (d, J ¼ 8.2 Hz, 1H), 7.56 (d, J ¼ 8.2 Hz, 1H), 7.49 (dd, J ¼ 8.2,
2.1 Hz, 1H), 7.30 (d, J ¼ 3.3 Hz, 1H), 7.25 (dd, J ¼ 8.2, 7.1 Hz, 1H), 7.21
(dd, J ¼ 7.8, 7.1 Hz, 1H), 6.70 (d, J ¼ 3.3 Hz, 1H) and 4.8 (s, 2H) ppm.
d
157.8, 152.5, 137.2, 136.1, 134.0, 129.5, 129.1, 128.0, 127.0, 125.8,
123.3, 123.0, 121.2, 119.2, 114.7, 110.9, 104.6, 69.9, 55.1 ppm; HRMS
(ESI): [M þ H]þ C22H19BNO3S calcd 388.1179, found 388.1167; mp:
99e100 ꢁC; HPLC: purity 98.3%, retention time 16.1 min with
method A.
5.1.5. 1,3-Dihydro-1-hydroxy-6-(indol-1-yl)-2,1-benzoxaborole (6)
To a solution of compound 5 (400 mg, 1.32 mmol) in anhydrous
THF (80 mL) at ꢀ78 ꢁC under nitrogen atmosphere was added 1.6 M
n-BuLi dropwise in THF (1.82 mL, 2.90 mmol). After 15 min, B(i-
PrO)3 (0.76 mL, 2.90 mmol) was added dropwise at ꢀ78 ꢁC. The
mixture was allowed to warm to r.t. gradually and stirred for
overnight. After addition of 2 M HCl (10 mL), the mixture was
stirred for 2 h and evaporated. The residue was dissolved in EtOAc,
washed with water, and dried over anhydrous Na2SO4. The residue
after rotary evaporation was purified by column chromatography
over silica gel to give compound 6 as a solid (249 mg, 75.7% yield).
5.1.7. 6-(3-(4-Hydroxyphenylsulfenyl)-indol-1-yl)-1,3-dihydro-1-
hydroxy-2,1-benzoxaborole (8)
Compound 8 (235 mg, 68.4% yield) as a solid was prepared
following a similar procedure to compound 7. 1H NMR (400 MHz,
DMSO-d6):
d
9.44 (s, 1H), 9.36 (s, 1H), 8.03 (s, 1H), 7.94 (d, J ¼ 2.1 Hz,
1H), 7.74 (dd, J ¼ 7.7, 2.1 Hz, 1H), 7.64 (d, J ¼ 7.7 Hz, 1H), 7.57e7.54
(m, 2H), 7.28e7.19 (m, 2H), 7.15 (d, J ¼ 8.2 Hz, 2H), 6.68 (d,
J ¼ 8.2 Hz, 2H) and 5.10 (s, 2H) ppm; 13C NMR (100 MHz, DMSO-d6):
1H NMR (400 MHz, DMSO-d6):
d
9.34 (s, 1H), 7.90 (d, J ¼ 2.1 Hz, 1H),
7.69e7.65 (m, 3H), 7.60 (d, J ¼ 8.1 Hz, 1H), 7.52 (d, J ¼ 8.2 Hz, 1H),
7.20 (dd, J ¼ 8.2, 7.1 Hz, 1H), 7.12 (dd, J ¼ 7.8, 7.1 Hz, 1H), 6.69 (d,
J ¼ 3.3 Hz,1H) and 5.06 (s, 2H) ppm; 13C NMR (100 MHz, DMSO-d6):
d 155.9, 152.2, 137.0, 135.9, 133.5, 129.7, 129.3, 126.7, 125.6, 125.4,
123.0, 122.8, 120.9, 119.0, 115.8, 110.7, 105.2, 69.7 ppm; HRMS (ESI):
[M þ H]þ C21H17BNO3S calcd 374.1022, found 374.1001; mp: 221e
224 ꢁC; HPLC: purity 98.9%, retention time 14.8 min with method A.
d
151.8, 138.0, 135.2, 128.9, 128.6, 126.6, 125.4, 122.8, 122.2, 120.9,
120.1, 110.1, 103.3, 69.8 ppm; HRMS (ESI): [M þ Na]þ C15H12BNO2Na
calcd 272.0859, found 272.0799; mp: 145e147 ꢁC; HPLC: purity
95.6%, retention time 19.1 min with method A.
5.1.8. 6-(3-(3,4-Dichlorophenylsulfenyl)-indol-1-yl)-1,3-dihydro-1-
hydroxy-2,1-benzoxaborole (9)
Compound 9 (283 mg, 82.7% yield) as a solid was prepared
5.1.6. 6-(3-(4-Methoxyphenylsulfenyl)-indol-1-yl)-1,3-dihydro-1-
hydroxy-2,1-benzoxaborole (7)
To a mixture of compound 6 (50 mg, 0.20 mmol) and 4-
methoxyphenylthiol (0.22 mmol) in EtOH (4 mL) and water
following a similar procedure to compound 7. 1H NMR (400 MHz,
DMSO-d6):
d
9.36 (s, 1H), 8.21 (s, 1H), 7.98 (d, J ¼ 2.1 Hz, 1H), 7.78
(dd, J ¼ 8.1, 2.1 Hz, 1H), 7.67 (d, J ¼ 8.1 Hz, 1H), 7.60 (d, J ¼ 8.4 Hz,
1H), 7.51 (m, 2H), 7.38 (d, J ¼ 2.2 Hz, 1H), 7.32 (ddd, J ¼ 8.3, 7.1,

66
P. Wu et al. / European Journal of Medicinal Chemistry 81 (2014) 59e75
1.2 Hz, 1H), 7.25 (ddd, J ¼ 7.9, 7.1, 1.0 Hz, 1H), 7.07 (dd, J ¼ 8.4, 2.2 Hz,
1H) and 5.11 (s, 2H) ppm; 13C NMR (100 MHz, CDCl3):
153.0, 139.6,
5.1.13. 6-(3-(4-Nitrophenylsulfenyl)-indol-1-yl)-1,3-dihydro-1-
hydroxy-2,1-benzoxaborole (14)
d
138.3, 137.2, 134.4, 133.0, 130.6, 129.9, 129.0, 127.8, 127.6, 126.6,
125.5, 123.9, 122.7, 121.9, 119.9, 111.2, 102.8, 71.3 ppm; HRMS (ESI):
[M þ H]þ C21H15BCl2NO2S calcd 426.0294, found 426.0272; mp:
104e105 ꢁC; HPLC: purity 99.3%, retention time 17.8 min with
method A.
Compound 14 (75 mg, 23.3% yield) as a solid was prepared
following a similar procedure to compound 7. 1H NMR (300 MHz,
DMSO-d6):
d
9.38 (s,1H), 8.26 (s, 1H), 8.10 (d, J ¼ 8.8 Hz, 2H), 8.00 (d,
J ¼ 1.9 Hz, 1H), 7.80 (dd, J ¼ 8.1, 1.9 Hz, 1H), 7.68 (d, J ¼ 8.2 Hz, 1H),
7.63 (d, J ¼ 8.1 Hz, 1H), 7.49 (d, J ¼ 7.8 Hz, 1H), 7.36 (dd, J ¼ 8.2,
7.1 Hz, 1H), 7.32 (d, J ¼ 8.8 Hz, 2H), 7.24 (dd, J ¼ 7.8, 7.1 Hz, 1H) and
5.12 (s, 2H) ppm; 13C NMR (100 MHz, DMSO-d6):
d 152.9, 148.7,
5.1.9. 6-(3-(3-Chlorophenylsulfenyl)-indol-1-yl)-1,3-dihydro-1-
hydroxy-2,1-benzoxaborole (10)
144.7, 137.0, 136.5, 135.7, 129.0, 127.2, 126.1, 125.4, 124.1, 123.7, 123.1,
121.8, 118.8, 111.4, 99.6, 69.9 ppm; HRMS (ESI): [M
þ
H]þ
Compound 10 (86 mg, 59.2% yield) as a solid was prepared
C21H16BN2O4S calcd 403.0924, found 403.0907; mp: 115e118 ꢁC;
following a similar procedure to compound 7. 1H NMR (400 MHz,
HPLC: purity 99.1%, retention time 20.1 min with method A.
DMSO-d6):
d
9.36 (s, 1H), 8.19 (s, 1H), 7.98 (d, J ¼ 2.0 Hz, 1H), 7.78
(dd, J ¼ 8.1, 2.0 Hz, 1H), 7.66 (d, J ¼ 8.1 Hz, 1H), 7.59 (d, J ¼ 8.2 Hz,
1H), 7.52 (d, J ¼ 7.8 Hz, 1H), 7.32e7.09 (m, 6H) and 5.11 (s, 2H) ppm;
5.1.14. 6-(3-(Naphthalen-2-ylsulfenyl)-indol-1-yl)-1,3-dihydro-1-
hydroxy-2,1-benzoxaborole (15)
13C NMR (100 MHz, CDCl3):
d 152.8, 141.4, 138.4, 137.2, 134.9, 134.4,
130.1, 129.9, 127.8, 126.5, 125.9, 125.3, 124.3, 123.8, 122.7, 121.8,
120.0, 111.1, 103.2, 71.3 ppm; HRMS (ESI): [M þ H]þ C21H16BClNO2S
calcd 392.0683, found 392.0658; mp: 104e105 ꢁC; HPLC: purity
99.2%, retention time 18.3 min with method A.
Compound 15 (77 mg, 25.6% yield) as a solid was prepared
following a similar procedure to compound 7. 1H NMR (400 MHz,
DMSO-d6):
d
9.34 (s, 1H), 8.21 (s, 1H), 8.01 (d, J ¼ 2.1 Hz, 1H), 7.84e
7.80 (m, 3H), 7.74e7.66 (m, 3H), 7.59 (d, J ¼ 8.3 Hz, 1H), 7.52 (d,
J ¼ 7.8 Hz, 1H), 7.47e7.40 (m, 2H), 7.35e7.33 (m, 1H), 7.30 (dd,
J ¼ 8.3, 7.1 Hz,1H), 7.20 (dd, J ¼ 7.8, 7.1 Hz,1H), and 5.10 (s, 2H) ppm;
5.1.10. 6-(3-(4-Chlorophenylsulfenyl)-indol-1-yl)-1,3-dihydro-1-
hydroxy-2,1-benzoxaborole (11)
13C NMR (100 MHz, DMSO-d6):
d 152.6, 137.2, 136.3, 135.6, 134.9,
133.2, 131.0, 129.5, 128.5, 127.6, 127.1, 126.8, 126.7, 126.0, 125.4,
124.8, 123.7, 123.4, 123.0, 121.4, 119.1, 111.1, 102.3, 69.9 ppm; HRMS
(ESI): [M þ H]þ C25H19BNO2S calcd 408.123, found 408.1207; mp:
139e142 ꢁC; HPLC: purity 96.7%, retention time 21.2 min with
method A.
Compound 11 (162 mg, 51.6% yield) as a solid was prepared
following a similar procedure to compound 7. 1H NMR (300 MHz,
DMSO-d6):
d
9.34 (s, 1H), 8.17 (s, 1H), 7.97 (d, J ¼ 2.1 Hz, 1H), 7.76
(dd, J ¼ 8.1, 2.1 Hz, 1H), 7.65 (d, J ¼ 8.1 Hz, 1H), 7.58 (d, J ¼ 8.2 Hz,
1H), 7.49 (d, J ¼ 7.9 Hz, 1H), 7.32 (dd, J ¼ 8.2, 7.1 Hz, 1H), 7.31 (d,
J ¼ 8.8 Hz, 2H), 7.22 (dd, J ¼ 7.9, 7.1 Hz, 1H), 7.15 (d, J ¼ 8.8 Hz, 2H)
and 5.09 (s, 2H) ppm; 13C NMR (100 MHz, CDCl3):
d 152.4, 138.1,
5.1.15. 6-(3-(4-Methoxyphenylsulfinyl)-indol-1-yl)-1,3-dihydro-1-
hydroxy-2,1-benzoxaborole (16)
137.5, 137.0, 134.0, 130.8, 129.9, 128.8, 127.6, 127.4, 126.3, 123.5,
122.5, 121.5, 119.9, 111.0, 103.5, 71.2 ppm; HRMS (ESI): [M]þ
C21H15BClNO2S calcd 391.0605, found 391.0602; mp: 100e102 ꢁC;
HPLC: purity 99.0%, retention time 18.3 min with method A.
To a solution of compound 7 (100 mg, 0.26 mmol) in AcOH
(10 mL) at 10 ꢁC was added 4 M H2O2 in AcOH (0.13 mL) dropwise.
The mixture was stirred for 2 h at r.t. After evaporation the mixture
was dissolved in EtOAc, washed with saturated NaHCO3, water, and
brine, and dried over anhydrous Na2SO4. The residue after rotary
evaporation was purified by column chromatography over silica gel
to give compound 16 as a solid (65 mg, 62.4% yield). 1H NMR
5.1.11. 6-(3-(2-Hydroxyphenylsulfenyl)-indol-1-yl)-1,3-dihydro-1-
hydroxy-2,1-benzoxaborole (12)
Compound 12 (160 mg, 53.4% yield) as a solid was prepared
following a similar procedure to compound 7. 1H NMR (300 MHz,
(400 MHz, DMSO-d6):
d
9.39 (s, 1H), 8.38 (s, 1H), 7.94 (d, J ¼ 2.1 Hz,
1H), 7.75 (dd, J ¼ 7.9, 2.1 Hz, 1H), 7.67 (d, J ¼ 7.9 Hz, 3H), 7.51 (d,
J ¼ 8.3 Hz, 1H), 7.44 (d, J ¼ 7.6 Hz, 1H), 7.28 (dd, J ¼ 8.3, 7.1 Hz, 1H),
7.13 (dd, J ¼ 7.6, 7.1 Hz, 1H), 7.12 (d, J ¼ 7.9 Hz, 2H), 5.11 (s, 2H) and
DMSO-d6):
d
9.97 (s, 1H), 9.36 (s, 1H), 8.05 (s, 1H), 7.95 (d, J ¼ 2.0 Hz,
1H), 7.75 (dd, J ¼ 8.1, 2.0 Hz, 1H), 7.64 (d, J ¼ 8.1 Hz, 1H), 7.58 (d,
J ¼ 8.2 Hz, 1H), 7.49 (d, J ¼ 7.6 Hz, 1H), 7.30 (dd, J ¼ 8.2, 7.2 Hz, 1H),
7.19 (dd, J ¼ 7.6, 7.2 Hz, 1H), 6.95e6.89 (m, 1H), 6.82 (d, J ¼ 7.9 Hz,
1H), 6.60 (d, J ¼ 7.9 Hz, 2H) and 5.10 (s, 2H) ppm; 13C NMR
3.79 (s, 3H) ppm; 13C NMR (100 MHz, DMSO-d6):
d 160.8, 153.1,
136.7,136.6,135.5,132.3,127.3,126.2,124.0,123.8,123.1,121.8,119.7,
118.8, 114.6, 111.4, 69.9, 55.4 ppm; HRMS (ESI): [M
þ
H]þ
(100 MHz, DMSO-d6): d 153.3, 152.5, 137.2, 136.3, 134.8, 129.9, 127.0,
C22H19BNO4S calcd 404.1128, found 404.1125; mp: 119e120 ꢁC;
126.2,125.9,125.7, 124.4, 123.3,123.0,121.2, 119.5, 119.3,114.6,111.0,
102.4, 69.9 ppm; HRMS (ESI): [M þ H]þ C21H17BNO3S calcd
374.1022, found 374.1021; mp: 133e136 ꢁC; HPLC: purity 98.4%,
retention time 14.4 min with method A.
HPLC: purity 99.7%, retention time 15.1 min with method A.
5.1.16. 6-(3-(4-Methoxyphenylsulfonyl)-indol-1-yl)-1,3-dihydro-1-
hydroxy-2,1-benzoxaborole (17)
5.1.12. 6-(3-(4-Fluorophenylsulfenyl)-indol-1-yl)-1,3-dihydro-1-
hydroxy-2,1-benzoxaborole (13)
To a solution of compound 7 (80 mg, 0.13 mmol) in AcOH (5 mL)
at 10 ꢁC was added 4 M H2O2 in AcOH (0.2 mL) dropwise. The
mixture was stirred for 2 h at 75 ꢁC. After evaporation, the mixture
was dissolved in EtOAc, washed with saturated NaHCO3, water, and
brine, and dried over anhydrous Na2SO4. The residue after rotary
evaporation was purified by column chromatography over silica gel
to give compound 17 as a solid (61 mg, 56.3% yield). 1H NMR
Compound 13 (80 mg, 26.5% yield) as a solid was prepared
following a similar procedure to compound 7. 1H NMR (400 MHz,
DMSO-d6):
d
9.37 (s, 1H), 8.15 (s, 1H), 7.97 (d, J ¼ 2.1 Hz, 1H), 7.77
(dd, J ¼ 8.1, 2.1 Hz, 1H), 7.65 (d, J ¼ 8.1 Hz, 1H), 7.59 (d, J ¼ 8.2 Hz,
1H), 7.51 (dd, J ¼ 7.8, 1.1 Hz, 1H), 7.30 (ddd, J ¼ 8.2, 7.1, 1.1 Hz, 1H),
7.22 (dd, J ¼ 8.9, 5.1 Hz, 2H), 7.20 (dd, J ¼ 7.8, 7.1 Hz, 1H), 7.12 (t,
J ¼ 8.9 Hz, 2H) and 5.11 (s, 2H) ppm; 13C NMR (100 MHz, DMSO-d6):
(300 MHz, DMSO-d6):
d
10.31 (s, 1H), 8.38 (s, 1H), 7.99 (d, J ¼ 9.0 Hz,
2H), 7.89e7.86 (m, 1H), 7.58 (m, 1H), 7.45 (d, J ¼ 7.9 Hz, 1H), 7.37e
d
160.8 (d, J ¼ 240.0 Hz), 152.6,137.1,136.3,134.8,133.5,129.3,128.3,
7.29 (m, 2H), 7.11 (d, J ¼ 9.0 Hz, 2H), 7.11e7.06 (m, 2H), 5.10 (s, 2H)
128.2, 127.1, 126.0, 123.4, 123.0, 121.4, 119.0, 116.1, 115.9, 111.1, 102.8,
69.8 ppm; HRMS (ESI): [M þ H]þ C21H16BFNO2S calcd 376.0979,
found 376.0965; mp: 91e95 ꢁC; HPLC: purity 98.7%, retention time
19.5 min with method A.
and 3.80 (s, 3H) ppm; 13C NMR (100 MHz, DMSO-d6):
d 170.3, 162.6,
156.4, 137.8, 135.9, 134.4, 132.8, 131.0, 128.8, 124.2, 123.6, 122.8,
122.4, 119.3, 117.5, 115.0, 114.6, 111.8, 111.3, 60.7, 55.7 ppm; HRMS
(ESI): [M þ H þ CH3OH]þ C23H13NO6S calcd 452.1339, found

P. Wu et al. / European Journal of Medicinal Chemistry 81 (2014) 59e75
67
452.1150; mp: 177e179 ꢁC; HPLC: purity 98.3%, retention time
17.3 min with method A.
5.1.22. 1,3-Dihydro-1-hydroxy-6-(pyrrol-1-yl)-2,1-benzoxaborole
(24)
To a solution of compound 23 (500 mg,1.98 mmol) in anhydrous
THF (80 mL) was added dropwise 1.6 M n-BuLi in THF (2.72 mL,
4.36 mmol) at ꢀ78 ꢁC under nitrogen. After stirred for 15 min
at ꢀ78 ꢁC, B(i-PrO)3 (1 mL, 4.36 mmol) was added dropwise. The
reaction mixture was allowed to warm to r.t. gradually, and stirred
for overnight at r.t. After addition of 2 M HCl (10 mL), the mixture
was evaporated, extracted with EtOAc, and dried over anhydrous
Na2SO4. The residue after rotary evaporation was purified by col-
umn chromatography over silica gel to give compound 24 as a solid
5.1.17. 2-Bromo-4-nitrobenzoic acid (19)
To a mixture of 2-bromo-4-nitrotoluene (25 g, 116 mmol), pyr-
idine (100 mL) and water (200 mL) was added KMnO4 (102 g,
642 mmol) in portions. After the mixture was refluxed for 5 h, an
additional amount of KMnO4 (27.5 g, 174 mmol) was added. The
mixture was refluxed and stirred for overnight, then filtered over
celite. The filtrate was acidified with concentrated HCl (110 mL),
extracted with EtOAc (600 mL), dried over anhydrous Na2SO4, and
evaporated under vacuum to give compound 19 as a solid (19.4 g,
(224 mg, 57% yield). 1H NMR (400 MHz, DMSO-d6):
d 9.27 (s, 1H),
68.1% yield). 1H NMR (400 MHz, CD3OD):
d
8.43 (d, J ¼ 2.1 Hz, 1H),
7.79 (d, J ¼ 2.2 Hz, 1H), 7.65 (dd, J ¼ 8.2, 2.2 Hz, 1H), 7.47 (d,
J ¼ 8.2 Hz, 1H), 7.29 (t, J ¼ 2.1 Hz, 2H), 6.25 (t, J ¼ 2.1 Hz, 2H) and
8.18 (dd, J ¼ 8.5, 2.1 Hz, 1H) and 7.86 (d, J ¼ 8.5 Hz, 1H) ppm; mp:
5.03 (s, 2H) ppm; 13C NMR (100 MHz, DMSO-d6):
d 140.4, 124.4,
153e155 ꢁC.
124.1, 122.2, 120.4, 111.9, 70.9 ppm; HRMS (ESI): [M þ H]þ
C11H11BNO2 calcd 200.0883, found 200.0884; mp: 120e122 ꢁC;
HPLC: purity 98.5%, retention time 14.9 min with method A.
5.1.18. 2-Bromo-4-nitrobenzoic acid methyl ester (20)
Compound 19 (5.7 g, 23.0 mmol) was dissolved in SOCl2 (50 mL).
This mixture was heated to reflux and stirred for 4 h. After evap-
oration, to the residue was added CH3OH (50 mL) and TEA (4.8 mL)
dropwise at 0 ꢁC. The mixture was stirred for 1 h at r.t. After
evaporation the residue was dissolved in EtOAc, washed with wa-
ter, and dried over anhydrous Na2SO4. The residue after rotary
evaporation was purified by column chromatography over silica gel
to give compound 20 as a solid (5.66 g, 94.6% yield). 1H NMR
5.1.23. N-(4-chlorophenylthio)-phthalimide (28)
To a stirred solution of 4-chlorobenzenethiol (6.9 mmol) in n-
pentane (15 mL), chlorine gas was added at 0 ꢁC until the solution
turned red orange to obtain the sulfenyl chloride. The sulfenyl
chloride solution was added dropwise to a stirred solution of
phthalimide (6.9 mmol) and TEA (9.0 mmol) in DMF (15 mL). The
mixture was stirred for 30 min, transferred to a large beaker, and
then cold water was added. The suspension was filtered to give
compound 28 as a solid (967 mg, 48.4% yield). 1H NMR (400 MHz,
(400 MHz, CDCl3):
d
8.55 (d, J ¼ 2.1 Hz, 1H), 8.24 (dd, J ¼ 8.5, 2.1 Hz,
1H), 7.96 (d, J ¼ 8.5 Hz, 1H) and 4.02 (s, 3H) ppm; mp: 83e86 ꢁC.
5.1.19. 2-Bromo-4-aminobenzoic acid methyl ester (21)
CDCl3):
d
7.93 (dd, J ¼ 8.6, 2.4 Hz, 2H), 7.79 (dd, J ¼ 8.6, 2.4 Hz, 2H),
To a solution of compound 20 (4.5 g, 17.0 mmol) in EtOAc
(100 mL) was added SnCl2$2H2O (38.3 g, 0.17 mol). The mixture was
heated to reflux and stirred for 4 h. The mixture was poured into
saturated NaHCO3 (500 mL) and EtOAc (370 mL). The organic layer
was washed with brine (300 mL), and dried over anhydrous MgSO4.
The residue after rotary evaporation was purified by column
chromatography over silica gel to give compound 21 as a solid
7.57 (d, J ¼ 8.5 Hz, 2H) and 7.29 (d, J ¼ 8.5 Hz, 2H) ppm; mp: 179e
182 ꢁC.
5.1.24. N-(2-chlorophenylthio)-phthalimide (29)
Compound 29 (972 mg, 48.7% yield) as a solid was prepared
following a similar procedure to compound 28. 1H NMR (400 MHz,
(3.68 g, 94.2% yield). 1H NMR (400 MHz, CDCl3):
d
7.75 (d, J ¼ 8.5 Hz,
CDCl3):
d
8.00 (dd, J ¼ 8.6, 2.4 Hz, 2H), 7.85 (dd, J ¼ 8.6, 2.4 Hz, 2H),
7.37e7.34 (m, 1H), 7.16e7.14 (m, 2H) and 6.92e6.90 (m, 1H) ppm;
mp: 217e219 ꢁC.
1H), 6.92 (d, J ¼ 2.2 Hz, 1H), 6.57 (dd, J ¼ 8.5, 2.2 Hz, 1H), 4.04 (brs,
2H) and 3.86 (s, 3H) ppm; mp: 96e98 ꢁC.
5.1.20. 2-Bromo-4-(pyrrol-1-yl)-benzoic acid methyl ester (22)
To a solution of compound 21 (3 g, 13.0 mmol) in AcOH (50 mL)
was added 2,5-dimethoxytetrahydrofuran (2.15 g, 16.3 mmol). The
mixture was heated to reflux and stirred for 4 h. After evaporation,
the mixture was dissolved in EtOAc, washed with saturated
NaHCO3, and dried over anhydrous Na2SO4. The residue after rotary
evaporation was purified by column chromatography over silica gel
to give compound 22 as a solid (3.06 g, 84.3% yield). 1H NMR
5.1.25. N-(3,4-dichlorophenylthio)-phthalimide (30)
Compound 30 (670 mg, 37.0% yield) as a solid was prepared
following a similar procedure to compound 28. 1H NMR (400 MHz,
CDCl3):
d
7.95 (dd, J ¼ 8.6, 2.4 Hz, 2H), 7.81 (dd, J ¼ 8.6, 2.4 Hz, 2H),
7.68 (d, J ¼ 2.0 Hz, 1H), 7.43 (dd, J ¼ 8.4, 2.0 Hz, 1H) and 7.39 (d,
J ¼ 8.4 Hz, 1H) ppm; mp: 171e173 ꢁC.
(400 MHz, CDCl3):
d
7.92 (d, J ¼ 8.5 Hz, 1H), 7.70 (d, J ¼ 2.3 Hz, 1H),
5.1.26. 6-(2-(4-Chlorophenylsulfenyl)-pyrrol-1-yl)-1,3-dihydro-1-
hydroxy-2,1-benzoxaborole (31)
7.35 (dd, J ¼ 8.5, 2.3 Hz,1H), 7.10 (t, J ¼ 2.2 Hz, 2H), 6.38 (t, J ¼ 2.2 Hz,
2H) and 3.93 (s, 3H) ppm; mp: 50e53 ꢁC.
A solution of compound 24 (200 mg, 1.0 mmol), MgBr2(Et2O)2
(16.6 mg, 0.05 mmol) and compound 28 (1.1 mmol) in DMF (10 mL)
was heated to reflux and stirred for overnight. The mixture was
poured into ice water (20 mL), extracted with EtOAc, and dried over
anhydrous Na2SO4. The residue after rotary evaporation was puri-
fied by column chromatography over silica gel to give compounds
5.1.21. 2-Bromo-4-(pyrrol-1-yl)-benzyl alcohol (23)
To a solution of compound 22 (1.1 g, 3.92 mmol) in anhydrous
THF (20 mL) was added dropwise 2 M LiBH4 in THF (2.95 mL,
5.89 mmol) at 0 ꢁC under nitrogen. The mixture was stirred for
overnight at r.t. then quenched with water. After evaporation, the
mixture was extracted with EtOAc, and dried over anhydrous
Na2SO4. The residue after rotary evaporation was purified by col-
umn chromatography over silica gel to give compound 23 as a solid
31 as a solid (20 mg, 5.8% yield). 1H NMR (400 MHz, CD3OD):
d 7.44
(s, 1H), 7.36e7.29 (m, 2H), 7.16 (dd, J ¼ 3.0, 1.8 Hz, 1H), 7.12 (dd,
J ¼ 8.7 Hz, 2H), 6.79 (dd, J ¼ 8.7 Hz, 2H), 6.69 (dd, J ¼ 3.6, 1.8 Hz, 1H),
6.38 (dd, J ¼ 3.6, 3.0 Hz, 1H) and 5.05 (s, 2H) ppm; 13C NMR
(0.92 g, 93.8% yield). 1H NMR (400 MHz, CDCl3):
1H), 7.52 (d, J ¼ 8.3 Hz, 1H), 7.36 (dd, J ¼ 8.3, 2.2 Hz, 1H), 7.06 (t,
J ¼ 2.1 Hz, 2H), 6.36 (t, J ¼ 2.1 Hz, 2H) and 4.76 (s, 2H) ppm; mp: 92e
94 ꢁC.
d
7.59 (d, J ¼ 2.2 Hz,
(100 MHz, CD3OD): d 154.5, 139.9, 139.5, 132.2, 130.3, 129.8, 129.1,
128.4, 122.6, 122.5, 119.0, 110.7, 72.0 ppm; [M þ H]þ C17H14BClNO2S
calcd 342.0527, found 342.0524; mp: 105e107 ꢁC; HPLC: purity
98.1%, retention time 25.4 min with method A.

68
P. Wu et al. / European Journal of Medicinal Chemistry 81 (2014) 59e75
5.1.27. 6-(2-(2-Chlorophenylsulfenyl)-pyrrol-1-yl)-1,3-dihydro-1-
hydroxy-2,1-benzoxaborole (32)
found 266.0595; mp: 204e205 ꢁC; HPLC: purity 96.2%, retention
time 11.8 min with method A.
Compound 32 (40 mg, 11.7% yield) as a solid was prepared
following a similar procedure to compound 31. 1H NMR (400 MHz,
5.1.31. 1-(1,3-Dihydro-1-hydroxy-2,1-benzoxaborol-6-yl)-pyrrole-
2-carboxylic acid propylamide (36)
CD3OD):
d
7.48 (s, 1H), 7.34e7.33 (m, 2H), 7.23 (dd, J ¼ 3.0, 1.8 Hz,
1H), 7.20 (dd, J ¼ 7.8, 1.4 Hz, 1H), 7.07 (ddd, J ¼ 7.8, 7.5, 1.4 Hz, 1H),
7.00 (ddd, J ¼ 7.8, 7.5, 1.7 Hz, 1H), 6.72 (dd, J ¼ 3.7, 1.8 Hz, 1H), 6.54
(dd, J ¼ 7.8, 1.7 Hz, 1H), 6.43 (dd, J ¼ 3.7, 3.0 Hz, 1H) and 5.05 (s, 2H)
To a solution of compound 35 (20 mg, 0.083 mmol) in CH2Cl2
(3 mL) was added EDC (18.8 mg, 0.098 mmol) and HOBt (13.3 mg,
0.098 mmol) at 0 ꢁC. After the mixture was stirred at r.t. for 30 min,
n-propylamine (5.8 mg, 0.098 mmol) in CH2Cl2 (0.2 mL) was added
dropwise. The mixture was stirred for overnight, washed with
water, and dried over anhydrous Na2SO4. The residue after rotary
evaporation was purified by column chromatography over silica gel
to give compound 36 as a solid (10 mg, 42.9% yield). 1H NMR
ppm; 13C NMR (100 MHz, CDCl3):
d 153.2, 139.1, 138.6, 130.0, 129.4,
129.3, 127.9, 127.4, 127.2, 126.8, 126.1, 122.3, 121.6, 117.0, 110.3,
71.2 ppm; HRMS (ESI): [M þ H]þ C17H14BClNO2S calcd 342.0527,
found 342.0526; mp: 106e109 ꢁC; HPLC: purity 97.7%, retention
time 20.6 min with method B.
(300 MHz, CDCl3):
d
7.64 (s, 1H), 7.40 (s, 2H), 6.88 (dd, J ¼ 2.7, 1.6 Hz,
5.1.28. 6-(2-(3,4-Dichlorophenylsulfenyl)-pyrrol-1-yl)-1,3-dihydro-
1-hydroxy-2,1-benzoxaborole (33)
1H), 6.74 (dd, J ¼ 3.6, 1.6 Hz, 1H), 6.29 (dd, J ¼ 3.6, 2.7 Hz, 1H), 5.08
(s, 2H), 3.24 (q, J ¼ 6.6 Hz, 2H), 1.53e1.43 (m, 2H) and 0.87 (t,
J ¼ 7.2 Hz, 3H) ppm; 13C NMR (100 MHz, DMSO-d6):
d 161.0, 152.7,
Compound 33 (50 mg, 13.3% yield) as a solid was prepared
following a similar procedure to compound 31. 1H NMR (400 MHz,
140.1, 128.6, 128.1, 127.7, 127.4, 121.9, 113.8, 108.7, 70.3, 40.8, 23.0,
11.9 ppm; HRMS (ESI): [M þ Na]þ C15H17BN2O3Na calcd 307.123,
found 307.1222; mp: 148e149 ꢁC; HPLC: purity 99.4%, retention
time 12.9 min with method A.
CDCl3):
d
7.60 (s, 1H), 7.31e7.30 (m, 2H), 7.18 (d, J ¼ 8.4 Hz, 1H), 7.12
(dd, J ¼ 3.0, 1.7 Hz, 1H), 6.93 (d, J ¼ 2.2 Hz, 1H), 6.75 (dd, J ¼ 3.6,
1.7 Hz,1H), 6.70 (dd, J ¼ 8.4, 2.2 Hz, 1H), 6.39 (dd, J ¼ 3.6, 3.0 Hz,1H)
and 5.04 (s, 2H) ppm; 13C NMR (100 MHz, CDCl3):
d 153.3, 139.7,
138.6, 133.0, 130.6, 129.5, 129.4, 128.3, 127.6, 127.4, 125.4, 122.0,
121.6, 117.3, 110.2, 71.2 ppm; HRMS [M þ H]þ C17H13BCl2NO2S calcd
376.0137, found 376.0133; mp: 92e94 ꢁC; HPLC: purity 98.3%,
retention time 21.3 min with method A.
5.1.32. 1-(1,3-Dihydro-1-hydroxy-2,1-benzoxaborol-6-yl)-pyrrole-
2-carboxylic acid cyclohexylamide (37)
Compound 37 (67 mg, 50.4% yield) as a solid was prepared
following a similar procedure to compound 36. 1H NMR (300 MHz,
DMSO-d6):
d
9.25 (s, 1H), 7.83 (d, J ¼ 8.1 Hz, 1H), 7.55 (d, J ¼ 2.1 Hz,
5.1.29. 1,3-Dihydro-6-(2-formyl-pyrrol-1-yl)-1-hydroxy-2,1-
benzoxaborole (34)
1H), 7.43 (d, J ¼ 8.1 Hz, 1H), 7.31 (dd, J ¼ 8.1, 2.1 Hz, 1H), 7.01 (dd,
J ¼ 2.7, 1.7 Hz, 1H), 6.83 (dd, J ¼ 3.7, 1.7 Hz, 1H), 6.21 (dd, J ¼ 3.7,
2.7 Hz, 1H), 5.03 (s, 2H), 3.54e3.51 (m, 1H), 1.72e1.54 (m, 4H) and
To DMF (0.92 mL, 12.0 mmol) at 0 ꢁC under nitrogen was added
POCl3 (1.1 mL, 12.0 mmol) dropwise. The mixture was stirred for
15 min at r.t. then cooled to 0 ꢁC. A solution of compound 24 (2.1 g,
11.0 mmol) in CH2Cl2 (20 mL) was added dropwise. The mixture
was stirred for 1 h at r.t., and refluxed for 15 min. After the mixture
was cooled to r.t., aq. NaOAc (5.3 g, 66.0 mmol) was added. The
mixture was extracted with CH2Cl2, and dried over anhydrous
Na2SO4. The residue after rotary evaporation was purified by col-
umn chromatography over silica gel, and recrystallization with
acetone/petroleum ether to give compound 34 as a solid (1.1 g,
1.30e1.07 (m, 6H) ppm; 13C NMR (100 MHz, DMSO-d6):
d 160.2,
152.7, 140.1, 128.5, 128.2, 127.6, 127.3, 121.9, 113.8, 108.6, 70.3, 48.1,
32.9, 25.7, 25.3 ppm; HRMS (ESI): [M þ Na]þ C18H21BN2O3Na calcd
347.1543, found 347.1531; mp: 158e160.5 ꢁC; HPLC: purity 95.0%,
retention time 16.1 min with method A.
5.1.33. 1-(1,3-Dihydro-1-hydroxy-2,1-benzoxaborol-6-yl)-pyrrole-
2-carboxylic acid benzylamide (38)
43.6% yield). 1H NMR (300 MHz, DMSO-d6):
d 9.49 (s, 1H), 9.29 (s,
Compound 38 (50 mg, 36.6% yield) as a solid was prepared
following a similar procedure to compound 36. 1H NMR (400 MHz,
1H), 7.67 (t, J ¼ 1.4 Hz, 1H), 7.51 (d, J ¼ 1.4 Hz, 2H), 7.43 (dd, J ¼ 2.6,
DMSO-d6):
d
9.25 (s, 1H), 8.66 (t, J ¼ 5.9 Hz, 1H), 7.58 (d, J ¼ 2.0 Hz,
1.7 Hz, 1H), 7.20 (dd, J ¼ 4.0, 1.7 Hz, 1H), 6.44 (dd, J ¼ 4.0, 2.6 Hz, 1H)
and 5.05 (s, 2H) ppm; 13C NMR (100 MHz, DMSO-d6):
d 178.7, 153.3,
1H), 7.42 (d, J ¼ 8.1 Hz, 1H), 7.34 (dd, J ¼ 8.1, 2.0 Hz, 1H), 7.32e7.21
(m, 5H), 7.06 (dd, J ¼ 2.6, 1.7 Hz, 1H), 6.93 (dd, J ¼ 3.7, 1.7 Hz, 1H),
6.25 (dd, J ¼ 3.7, 2.6 Hz, 1H), 5.03 (s, 2H) and 4.32 (d, J ¼ 5.9 Hz, 2H)
137.5, 132.1, 132.0, 128.5, 127.4, 122.7, 122.1, 110.8, 69.8 ppm; HRMS
(ESI): [M þ Na]þ C12H10BNO3Na calcd 250.0651, found 250.0646;
mp: 140e142 ꢁC; HPLC: purity 97.1%, retention time 13.2 min with
method B.
ppm; 13C NMR (100 MHz, DMSO-d6):
d 160.4, 152.3, 139.9, 139.6,
128.2, 128.1, 127.7, 127.2, 127.0, 126.9, 126.6, 121.4, 113.7, 108.3, 69.8,
41.9 ppm; HRMS (ESI): [M þ Na]þ C19H17BN2O3Na calcd 355.1230,
found 355.1218; mp: 178e180 ꢁC; HPLC: purity 98.1%, retention
time 15.1 min with method A.
5.1.30. 1-(1,3-Dihydro-1-hydroxy-2,1-benzoxaborol-6-yl)-pyrrole-
2-carboxylic acid (35)
To 6 M NaOH (10 mL) at 0 ꢁC under nitrogen was added a so-
lution of AgNO3 (1.5 g, 8.8 mmol) in water (2 mL) dropwise. The
mixture was stirred for 15 min at 0 ꢁC, then compound 34 (500 mg,
2.2 mmol) was added in small portions. After warming to r.t.
gradually the mixture was stirred for overnight. After filtration, the
filtrate was acidified with concentrated HCl (10 mL), extracted with
EtOAc (600 mL), and dried over anhydrous Na2SO4. The residue
after rotary evaporation was purified by column chromatography
over silica gel to give compound 35 as a solid (150 mg, 26.6% yield).
5.1.34. 1-(1,3-Dihydro-1-hydroxy-2,1-benzoxaborol-6-yl)-pyrrole-
2-carboxylic acid 4-methoxy-benzylamide (39)
Compound 39 (66 mg, 44.4% yield) as a solid was prepared
following a similar procedure to compound 36. 1H NMR (300 MHz,
DMSO-d6):
d
9.26 (s, 1H), 8.59 (t, J ¼ 6.0 Hz, 1H), 7.56 (d, J ¼ 2.1 Hz,
1H), 7.43 (d, J ¼ 8.1 Hz, 1H), 7.33 (dd, J ¼ 8.1, 2.1 Hz, 1H), 7.16 (d,
J ¼ 8.7 Hz, 2H), 7.05 (dd, J ¼ 2.7, 1.7 Hz, 1H), 6.89 (dd, J ¼ 3.8, 1.7 Hz,
1H), 6.85 (d, J ¼ 8.7 Hz, 2H), 6.23 (dd, J ¼ 3.8, 2.7 Hz, 1H), 5.03 (s,
2H), 4.23 (d, J ¼ 6.0 Hz, 2H) and 3.71 (s, 3H) ppm; 13C NMR
1H NMR (400 MHz, DMSO-d6):
d 12.10 (s, 1H), 9.25 (s, 1H), 7.60 (d,
J ¼ 2.1 Hz, 1H), 7.46 (d, J ¼ 8.1 Hz, 1H), 7.42 (dd, J ¼ 8.1, 2.1 Hz, 1H),
7.16 (dd, J ¼ 2.6, 1.8 Hz, 1H), 6.99 (dd, J ¼ 3.8, 1.8 Hz, 1H), 6.29 (dd,
J ¼ 3.8, 2.6 Hz,1H) and 5.05 (s, 2H) ppm; 13C NMR (100 MHz, DMSO-
(100 MHz, DMSO-d6): d 160.4, 158.1, 152.3, 139.6, 131.8, 128.3, 128.2,
127.6, 127.3, 127.0, 121.4, 113.6, 108.3, 69.8, 55.0, 41.3 ppm; HRMS
(ESI): [M þ Na]þ C20H19BN2O4Na calcd 385.1336, found 385.1318;
mp: 138e140 ꢁC; HPLC: purity 97.4%, retention time 15.1 min with
method A.
d6): d 161.5, 153.3, 139.7, 130.6, 129.1, 128.1, 124.2, 122.0, 119.1, 109.5,
70.3 ppm; HRMS (ESI): [M þ Na]þ C12H10BNO4Na calcd 266.0601,

P. Wu et al. / European Journal of Medicinal Chemistry 81 (2014) 59e75
69
5.1.35. 1-(1,3-Dihydro-1-hydroxy-2,1-benzoxaborol-6-yl)-pyrrole-
2-carboxylic acid 2-phenyl-ethylamide (40)
C14H15BN2O3Na calcd 293.1073, found 293.1086; mp: 174e175 ꢁC;
HPLC: purity 95.6%, retention time 12.8 min with method B.
Compound 40 (72 mg, 50.7% yield) as a solid was prepared
following a similar procedure to compound 36. 1H NMR (300 MHz,
5.1.39. (2,4-Dichlorophenyl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-2-yl)-methanone (44)
Acetone-d6):
d
8.18 (s, 1H), 7.66 (d, J ¼ 2.0 Hz, 1H), 7.47 (d, J ¼ 8.1 Hz,
1H), 7.37 (dd, J ¼ 8.1, 2.0 Hz, 1H), 7.32e7.18 (m, 5H), 7.01 (dd, J ¼ 2.7,
To a solution of 2,4-dichlorobenzoyl chloride (3.0 mmol) in
CH2Cl2 (10 mL) was added dropwise anhydrous SnCl4 (0.36 mL,
3.0 mmol) at r.t. This mixture was stirred for 15 min at r.t. before a
solution of compound 24 (300 mg, 1.5 mmol) in CH2Cl2 (10 mL) was
added slowly. After stirred for 1 d, the mixture was quenched with
saturated NaHCO3, washed with water, and brine, and dried over
anhydrous Na2SO4. The residue after rotary evaporation was puri-
fied by column chromatography over silica gel to give compound 44
1.7 Hz,1H), 6.78 (dd, J ¼ 3.8,1.7 Hz,1H), 6.24 (dd, J ¼ 3.8, 2.7 Hz,1H),
5.11 (s, 2H), 3.52e3.45 (m, 2H) and 2.85 (t, J ¼ 7.4 Hz, 2H) ppm; 13
C
NMR (100 MHz, DMSO-d6): d 160.4, 152.2, 139.5, 139.4, 128.6, 128.3,
128.1, 127.4, 127.3, 126.8, 126.0, 121.4, 113.4, 108.2, 69.8, 35.2 ppm;
HRMS (ESI): [M þ Na]þ C20H19BN2O3Na calcd 369.1387, found
369.1378; mp: 173.5e175 ꢁC; HPLC: purity 99.0%, retention time
15.7 min with method A.
as a solid (30 mg, 5.4% yield). 1H NMR (400 MHz, CDCl3):
d 7.69 (d,
5.1.36. Trichloromethyl-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-2-yl)-methanone (41)
J ¼ 1.8 Hz, 1H), 7.49 (dd, J ¼ 8.1, 1.8 Hz, 1H), 7.43 (d, J ¼ 1.7 Hz, 1H),
7.42 (d, J ¼ 8.1 Hz, 1H), 7.38 (J ¼ 8.2 Hz, 1H), 7.29 (dd, J ¼ 8.2, 1.7 Hz,
1H), 7.11 (dd, J ¼ 2.6, 1.5 Hz, 1H), 6.67 (dd, J ¼ 3.9, 1.5 Hz, 1H), 6.32
(dd, J ¼ 3.9, 2.6 Hz, 1H), 5.15 (s, 2H) and 4.97 (s, 1H) ppm; 13C NMR
Compound 24 (50 mg, 0.25 mmol) was dissolved in tri-
chloroacetyl chloride (3 mL). The mixture was heated at 75 ꢁC for
0.5 h. The reaction was quenched with saturated NaHCO3, extracted
with EtOAc, and dried over anhydrous Na2SO4. The residue after
rotary evaporation was purified by column chromatography over
silica gel to give compound 41 as a solid (20 mg, 23.0% yield). 1H
(100 MHz, CDCl3):
d 182.3, 153.5, 139.5, 137.8, 136.3, 133.0, 132.8,
131.2, 130.4, 130.1, 129.1, 127.7, 126.9, 124.8, 121.7, 110.3, 71.1 ppm;
HRMS (ESI): [M þ H]þ C18H14BCl2NO3 calcd 372.0366, found
372.0363; mp: 146e149 ꢁC; HPLC: purity 98.7%, retention time
19.14 min with method B.
NMR (400 MHz, CD3OD):
d
7.67 (dd, J ¼ 4.3, 1.6 Hz, 1H), 7.51 (d,
J ¼ 2.0 Hz, 1H), 7.49 (d, J ¼ 8.1 Hz, 1H), 7.34 (dd, J ¼ 8.1, 2.0 Hz, 1H),
7.28 (dd, J ¼ 2.6, 1.6 Hz, 1H), 6.45 (dd, J ¼ 4.3, 2.6 Hz, 1H) and 5.13 (s,
2H) ppm; 13C NMR (100 MHz, DMSO-d6):
d 171.8, 154.1, 139.4, 135.9,
5.1.40. Phenyl-(1-(1,3-dihydro-1-hydroxy-2,1-benzoxaborol-6-yl)-
pyrrol-2-yl)-methanone (45)
128.9, 128.0, 125.0, 122.6, 122.1, 111.1, 96.2, 70.4 ppm; HRMS (ESI):
[M þ Na]þ C13H9BCl3NO3Na calcd 365.9639, found 365.9642; mp:
132e134 ꢁC; HPLC: purity 99.1%, retention time 10.7 min with
method A.
Compound 45 (55 mg, 12.1% yield) as a solid was prepared
following a similar procedure to compound 44. 1H NMR (400 MHz,
CDCl3):
d
7.87 (d, J ¼ 7.4 Hz, 2H), 7.58 (s, 1H), 7.57e7.53 (t, J ¼ 7.4 Hz,
1H), 7.46e7.37 (m, 4H), 7.08 (dd, J ¼ 2.8,1.4 Hz,1H), 6.90 (dd, J ¼ 3.8,
1.4 Hz, 1H), 6.33 (dd, J ¼ 3.8, 2.8 Hz, 1H), 5.77 (s, 1H) and 5.03 (s, 2H)
ppm; 13C NMR (100 MHz, CDCl3):
d 185.1, 153.1, 140.0, 139.1, 132.2,
5.1.37. 1-(1,3-Dihydro-1-hydroxy-2,1-benzoxaborol-6-yl)-pyrrole-
2-carboxylic acid methylamide (42)
131.6, 131.3, 129.7, 128.8, 128.3, 127.4, 123.8, 121.8, 109.7, 71.2 ppm;
HRMS (ESI): [M þ H]þ C18H15BNO3 calcd 304.1145, found 304.1152;
mp: 120e124 ꢁC; HPLC: purity 97.0%, retention time 17.8 min with
method B.
To a solution of compound 41 (180 mg, 0.52 mmol) in CH2Cl2
(10 mL) was added dropwise 30% methylamine in EtOH (0.15 mL,
1.04 mmol) at 0 ꢁC. This mixture was stirred for 2 d at r.t. The
mixture was quenched with 2 M HCl, extracted with EtOAc, washed
with water, and brine, and dried over anhydrous Na2SO4. The res-
idue after rotary evaporation was purified by column chromatog-
raphy over silica gel to give compound 42 as a solid (56 mg, 42.0%
5.1.41. (2-Chlorophenyl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-2-yl)-methanone (46)
yield). 1H NMR (400 MHz, CD3OD):
d
7.51 (d, J ¼ 2.0 Hz, 1H), 7.43 (d,
Compound 46 (60 mg, 8.9% yield) as a solid was prepared
following a similar procedure to compound 44. 1H NMR (400 MHz,
J ¼ 8.1 Hz, 1H), 7.37 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.97 (dd, J ¼ 2.7, 1.7 Hz,
1H), 6.80 (dd, J ¼ 3.8, 1.7 Hz, 1H), 6.25 (dd, J ¼ 3.8, 2.7 Hz, 1H), 5.00
CDCl3):
d
7.69 (d, J ¼ 1.9 Hz, 1H), 7.50 (dd, J ¼ 8.1, 1.9 Hz, 1H), 7.44e
(s, 2H) and 2.76 (s, 3H) ppm; 13C NMR (100 MHz, DMSO-d6):
d 161.5,
7.28 (m, 5H), 7.10 (dd, J ¼ 2.7, 1.4 Hz, 1H), 6.66 (dd, J ¼ 3.9, 1.4 Hz,
1H), 6.35 (dd, J ¼ 3.9, 2.7 Hz, 1H), 5.45 (brs, 1H) and 5.14 (s, 2H)
152.7, 140.1, 128.6, 127.9, 127.8, 127.5, 121.9, 113.8, 108.7, 70.3,
26.0 ppm; HRMS (ESI): [M þ Na]þ C13H13BN2O3Na calcd 279.0917,
found 279.0911; mp: 195e197 ꢁC; HPLC: purity 97.3%, retention
time 12.0 min with method B.
ppm; 13C NMR (100 MHz, DMSO-d6):
d 182.1, 153.0, 138.9, 138.8,
133.3, 131.1, 130.6,130.0,129.6,129.0, 128.4,127.2, 126.8, 124.1, 121.7,
110.1, 69.8 ppm; HRMS (ESI): [M þ H]þ C18H14BClNO3 calcd
338.0755, found 338.0757; mp: 131e135 ꢁC; HPLC: purity 98.1%,
retention time 18.9 min with method A.
5.1.38. 1-(1,3-Dihydro-1-hydroxy-2,1-benzoxaborol-6-yl)-pyrrole-
2-carboxylic acid ethylamide (43)
To a solution of compound 41 (180 mg, 0.52 mmol) in CH2Cl2
(10 mL) was added dropwise 70% ethylamine in water (0.25 mL,
3.13 mmol) at 0 ꢁC. This mixture was stirred for overnight at r.t. The
mixture was quenched with 2 M HCl, extracted with EtOAc, washed
with water, and brine, and dried over anhydrous Na2SO4. The res-
idue after rotary evaporation was purified by column chromatog-
raphy over silica gel to give compound 43 as a solid (60 mg, 42.5%
5.1.42. (4-Fluorophenyl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-2-yl)-methanone (47)
Compound 47 (30 mg, 4.7% yield) as a solid was prepared
following a similar procedure to compound 44. 1H NMR (400 MHz,
CDCl3):
d
7.90 (dd, J ¼ 8.6, 5.5 Hz, 2H), 7.62 (s, 1H), 7.42 (dd, J ¼ 8.1,
1.8 Hz, 1H), 7.39 (d, J ¼ 8.1 Hz, 1H), 7.13 (t, J ¼ 8.6 Hz, 2H), 7.10 (dd,
J ¼ 2.9, 1.2 Hz, 1H), 6.87 (dd, J ¼ 3.8, 1.2 Hz, 1H), 6.35 (dd, J ¼ 3.8,
2.9 Hz, 1H), 5.14 (brs, 1H) and 5.16 (s, 2H) ppm; 13C NMR (100 MHz,
yield). 1H NMR (400 MHz, CD3OD):
d
7.52 (s, 1H), 7.43 (d, J ¼ 8.1 Hz,
1H), 7.38 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.98 (dd, J ¼ 2.7, 1.7 Hz, 1H), 6.81
(dd, J ¼ 3.9, 1.7 Hz, 1H), 6.25 (dd, J ¼ 3.9, 2.7 Hz, 1H), 5.00 (s, 2H),
3.24 (q, J ¼ 7.2 Hz, 2H) and 1.12 (t, J ¼ 7.2 Hz, 3H) ppm; 13C NMR
CDCl3):
d
183.6, 165.3 (d, J ¼ 250.0 Hz), 153.2, 140.0, 135.3 (d,
J ¼ 3.0 Hz), 132.1 (d, J ¼ 9.0 Hz),131.6, 131.1, 128.8,127.3, 123.5,121.9,
115.4 (d, J ¼ 22.0 Hz), 109.7, 71.2 ppm; HRMS (ESI): [M þ H]þ
C18H14BFNO3 calcd 322.1051, found 322.1051; mp: 114e117 ꢁC;
HPLC: purity 95.2%, retention time 13.5 min with method A.
(100 MHz, DMSO-d6): d 160.3, 152.2, 139.6, 128.1, 127.5, 127.2, 126.9,
121.4, 113.2, 108.2, 69.8, 33.3, 14.8 ppm; HRMS (ESI): [M þ Na]þ

70
P. Wu et al. / European Journal of Medicinal Chemistry 81 (2014) 59e75
5.1.43. Methyl-(1-(1,3-dihydro-1-hydroxy-2,1-benzoxaborol-6-yl)-
pyrrol-3-yl)-methanone (48)
5.1.48. (4-Chlorophenyl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (53)
Compound 48 (40 mg, 16.6% yield) as a solid was prepared
Compound 53 (40 mg, 11.8% yield) as a solid was prepared
following a similar procedure to compound 44. 1H NMR (400 MHz,
following a similar procedure to compound 44. 1H NMR (400 MHz,
CD3OD):
d
8.00 (dd, J ¼ 2.1, 1.7 Hz, 1H), 7.79 (d, J ¼ 2.1 Hz, 1H), 7.67
Acetone-d6): d 8.29 (s, 1H), 7.94e7.92 (m, 3H), 7.88e7.87 (m, 1H),
(dd, J ¼ 8.2, 2.1 Hz, 1H), 7.53 (d, J ¼ 8.2 Hz, 1H), 7.26 (dd, J ¼ 3.0,
7.79 (dd, J ¼ 8.4, 2.0 Hz, 1H), 7.60e7.55 (m, 3H), 7.44e7.42 (m, 1H),
2.1 Hz, 1H), 6.75 (dd, J ¼ 3.0, 1.7 Hz, 1H), 5.14 (s, 2H) and 2.47 (s, 3H)
6.85e6.84 (m, 1H) and 5.09 (s, 2H) ppm; 13C NMR (100 MHz,
ppm; 13C NMR (100 MHz, DMSO-d6):
d
192.3, 152.1, 138.2, 127.2,
DMSO-d6): d 187.9, 152.3, 138.1, 137.8, 136.5, 130.5, 128.6, 126.4,
125.1, 123.3, 122.7, 121.9, 121.5, 109.8, 69.7, 27.0 ppm; HRMS (ESI):
[M þ H]þ C13H13BNO3 calcd 242.0988, found 242.0990; mp: 189e
191 ꢁC; HPLC: purity 97.6%, retention time 14.0 min with method B.
125.0, 123.6, 122.8, 122.2, 122.0, 111.6, 69.8 ppm; HRMS (ESI):
[M þ Na]þ C18H13BClNO3Na calcd 360.0575, found 360.0503; mp:
149e151 ꢁC; HPLC: purity 96.3%, retention time 18.8 min with
method B.
5.1.44. (2,4-Dichlorophenyl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (49)
5.1.49. (3-Chlorophenyl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (54)
Compound 49 (95 mg, 17.0% yield) as a solid was prepared
following a similar procedure to compound 44. 1H NMR (400 MHz,
Compound 54 (40 mg, 7.9% yield) as a solid was prepared
following a similar procedure to compound 44. 1H NMR (400 MHz,
CDCl3):
d
7.74 (d, J ¼ 1.9 Hz, 1H), 7.51 (dd, J ¼ 8.2, 1.9 Hz, 1H), 7.49
CDCl3):
d
7.84 (dd, J ¼ 2.2, 1.7 Hz, 1H), 7.78 (d, J ¼ 2.1 Hz, 1H), 7.74
(dd, J ¼ 2.4, 1.6 Hz, 1H), 7.44e7.40 (m, 3H), 7.32 (dd, J ¼ 8.2, 1.8 Hz,
1H), 7.10 (dd, J ¼ 2.8, 2.4 Hz, 1H), 6.80 (dd, J ¼ 2.8, 1.6 Hz, 1H), 5.13 (s,
(ddd, J ¼ 8.2, 1.6, 1.2 Hz, 1H), 7.60 (dd, J ¼ 2.1, 1.6 Hz, 1H), 7.53 (dd,
J ¼ 8.2, 2.1 Hz, 1H), 7.50 (dd, J ¼ 7.9, 2.1 Hz, 1H), 7.44 (d, J ¼ 8.2 Hz,
1H), 7.41 (dd, J ¼ 8.2, 7.9 Hz, 1H), 7.11 (dd, J ¼ 3.1, 2.2 Hz, 1H), 6.85
(dd, J ¼ 3.1, 1.7 Hz, 1H), 5.30 (brs, 1H) and 5.13 (s, 2H) ppm; 13C NMR
2H) and 5.03 (brs, 1H) ppm; 13C NMR (100 MHz, DMSO-d6):
d 187.6,
153.0, 138.9, 138.4, 135.2, 131.4, 130.5, 130.0, 127.9, 127.8, 126.4,
124.1, 123.24, 123.21, 122.7, 111.2, 70.3 ppm; HRMS (ESI): [M þ H]þ
C18H13BCl2NO3 calcd 372.0366, found 372.0367; mp: 168e170 ꢁC;
HPLC: purity 95.4%, retention time 19.3 min with method B.
(100 MHz, DMSO-d6):
d 187.7, 152.4, 141.2, 138.1, 133.4, 131.4, 130.5,
128.0, 126.5, 124.8, 123.7, 122.8, 122.3, 122.2, 111.6, 69.8 ppm; HRMS
(ESI): [M þ Na]þ C18H13BClNO3Na calcd 360.0575, found 360.0577;
mp: 161e163 ꢁC; HPLC: purity 97.2%, retention time 19.1 min with
method B.
5.1.45. Phenyl-(1-(1,3-dihydro-1-hydroxy-2,1-benzoxaborol-6-yl)-
pyrrol-3-yl)-methanone (50)
Compound 50 (105 mg, 23.1% yield) as a solid was prepared
5.1.50. (4-Methylphenyl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (55)
following a similar procedure to compound 44. 1H NMR (400 MHz,
CDCl3):
d
7.89 (d, J ¼ 7.1 Hz, 2H), 7.80 (d, J ¼ 1.6 Hz, 1H), 7.63 (dd,
Compound 55 (40 mg, 12.8% yield) as a solid was prepared
J ¼ 2.4, 1.6 Hz, 1H), 7.58e7.44 (m, 5H), 7.11 (dd, J ¼ 2.7, 2.4 Hz, 1H),
following a similar procedure to compound 44. 1H NMR (400 MHz,
6.88 (dd, J ¼ 2.7, 1.6 Hz, 1H), 5.53 (s, 1H) and 5.15 (s, 2H) ppm; 13C
CD3OD):
d
7.76 (d, J ¼ 8.2 Hz, 2H), 7.73 (d, J ¼ 2.1 Hz, 1H), 7.70 (dd,
NMR (100 MHz, CDCl3):
d 191.1, 152.7, 139.8, 139.3, 131.8, 129.1,
J ¼ 2.2, 1.7 Hz, 1H), 7.63 (dd, J ¼ 8.2, 2.1 Hz, 1H), 7.47 (d, J ¼ 8.2 Hz,
1H), 7.33 (d, J ¼ 8.2 Hz, 2H), 7.28 (dd, J ¼ 3.1, 2.2 Hz, 1H), 6.79 (dd,
J ¼ 3.1, 1.7 Hz, 1H), 5.09 (s, 2H) and 2.41 (s, 3H) ppm; 13C NMR
128.5, 126.6, 126.4, 124.4, 123.2, 122.6, 121.6, 112.7, 71.1 ppm; HRMS
(ESI): [M þ H]þ C18H15BNO3 calcd 304.1145, found 304.1143; mp:
148e151 ꢁC; HPLC: purity 99.4%, retention time 17.4 min with
method B.
(100 MHz, CDCl3):
d 190.7, 152.7, 142.3, 139.4, 137.2, 129.3, 129.1,
126.6, 126.3, 124.5, 123.1, 122.6, 121.5, 112.7, 71.1, 21.7 ppm; HRMS
(ESI): [M þ H]þ C19H17BNO3 calcd 318.1301, found 318.1305; mp:
161e163 ꢁC; HPLC: purity 96.1%, retention time 18.3 min with
method B.
5.1.46. (2-Chlorophenyl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (51)
Compound 51 (120 mg, 17.8% yield) as a solid was prepared
following a similar procedure to compound 44. 1H NMR (400 MHz,
5.1.51. (3-Methylphenyl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (56)
CDCl3):
d
7.76 (d, J ¼ 1.8 Hz, 1H), 7.51 (dd, J ¼ 8.2, 1.8 Hz, 1H), 7.48e
7.33 (m, 6H), 7.11 (dd, J ¼ 2.8, 2.3 Hz, 1H), 6.83 (dd, J ¼ 2.8, 1.5 Hz,
Compound 56 (50 mg, 12.6% yield) as a solid was prepared
1H), 5.44 (brs, 1H) and 5.14 (s, 2H) ppm; 13C NMR (100 MHz, DMSO-
following a similar procedure to compound 44. 1H NMR (400 MHz,
d6): d 188.1, 152.5, 139.5, 137.9, 131.0, 129.9, 129.6, 128.7, 127.1, 126.9,
CDCl3):
d
7.81 (d, J ¼ 1.8 Hz, 1H), 7.68e7.62 (m, 3H), 7.53 (dd, J ¼ 8.2,
126.2, 123.7, 122.8, 122.6, 122.2, 110.8, 69.8 ppm; HRMS (ESI):
[M þ H]þ C18H14BClNO3 calcd 338.0755, found 338.0757; mp: 165e
168 ꢁC; HPLC: purity 97.3%, retention time 17.6 min with method B.
1.8 Hz, 1H), 7.44 (d, J ¼ 8.2 Hz, 1H), 7.36e7.34 (m, 2H), 7.10e7.09 (m,
1H), 6.87e6.86 (m, 1H), 6.08 (brs, 1H), 5.14 (s, 2H) and 2.42 (s, 3H)
ppm; 13C NMR (100 MHz, DMSO-d6):
d 189.2, 152.2, 139.3, 138.2,
137.8, 132.2, 128.9, 128.3, 125.9, 125.7, 125.3, 123.6, 122.7, 122.1,
121.7,111.6, 69.7, 20.9 ppm; HRMS (ESI): [M þ H]þ C19H17BNO3 calcd
318.1301, found 318.1308; mp: 180e182 ꢁC; HPLC: purity 99.5%,
retention time 18.7 min with method A.
5.1.47. (4-Fluorophenyl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (52)
Compound 52 (140 mg, 21.8% yield) as a solid was prepared
following a similar procedure to compound 44. 1H NMR (400 MHz,
CDCl3):
d
7.93 (dd, J ¼ 8.4, 5.5 Hz, 2H), 7.81 (d, J ¼ 1.9 Hz, 1H), 7.64
5.1.52. (2-Methylphenyl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (57)
(dd, J ¼ 2.2, 1.7 Hz, 1H), 7.54 (dd, J ¼ 8.2, 1.9 Hz, 1H), 7.45 (d,
J ¼ 8.2 Hz, 1H), 7.16 (t, J ¼ 8.4 Hz, 2H), 7.11 (dd, J ¼ 3.0, 2.2 Hz, 1H),
Compound 57 (140 mg, 29.4% yield) as a solid was prepared
6.87 (dd, J ¼ 3.0, 1.7 Hz, 1H), 5.70 (brs, 1H) and 5.16 (s, 2H) ppm; 13
C
following a similar procedure to compound 44. 1H NMR (400 MHz,
NMR (100 MHz, DMSO-d6):
d
187.6, 164.0 (d, J ¼ 250.0 Hz), 152.3,
CDCl3):
d
7.75 (d, J ¼ 2.0 Hz, 1H), 7.50 (dd, J ¼ 8.2, 2.0 Hz, 1H), 7.46e
138.1, 135.7 (d, J ¼ 3.0 Hz), 131.3 (d, J ¼ 9.0 Hz), 126.1, 125.0, 123.6,
122.7, 122.1, 121.8, 115.3 (d, J ¼ 22.0 Hz), 111.6, 69.7 ppm; HRMS
(ESI): [M þ H]þ C18H14BFNO3 calcd 322.1051, found 322.1059; mp:
160e162 ꢁC; HPLC: purity 95.8%, retention time 17.7 min with
method B.
7.41 (m, 3H), 7.37e7.34 (m, 1H), 7.28e7.22 (m, 2H), 7.09 (dd, J ¼ 3.0,
2.2 Hz, 1H), 6.82 (dd, J ¼ 3.0, 1.7 Hz, 1H), 5.53 (brs, 1H), 5.13 (s, 2H)
and 2.41 (s, 3H) ppm; 13C NMR (100 MHz, DMSO-d6):
d 191.6, 152.3,
140.0, 138.1, 135.2, 130.7, 129.6, 127.4, 126.9, 126.3, 125.3, 123.6,
122.9, 122.3, 122.1, 111.1, 69.8, 19.3 ppm; HRMS (ESI): [M þ Na]þ

P. Wu et al. / European Journal of Medicinal Chemistry 81 (2014) 59e75
71
C19H16BNO3Na calcd 340.1121, found 340.1052; mp: 134e135 ꢁC;
2H), 5.14 (s, 2H) and 2.14 (s, 3H) ppm; 13C NMR (100 MHz, CDCl3):
HPLC: purity 99.4%, retention time 18.1 min with method B.
d 190.4, 171.0, 152.7, 139.7, 139.3, 129.4, 128.0, 126.5, 126.4, 124.5,
123.0, 122.7, 121.7, 112.6, 71.2, 65.9, 21.1 ppm; HRMS (ESI): [M þ H]þ
C21H19BNO5 calcd 376.1356, found 376.1359; mp: 160e162 ꢁC;
HPLC: purity 95.3%, retention time 16.5 min with method B.
5.1.53. (4-Ethylphenyl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (58)
Compound 58 (160 mg, 32.1% yield) as a solid was prepared
following a similar procedure to compound 44. 1H NMR (400 MHz,
5.1.58. (4-((((9H-fluoren-9-yl)-methoxycarbonyl)-amino)-methyl)-
phenyl)-(1-(1,3-dihydro-1-hydroxy-2,1-benzoxaborol-6-yl)-pyrrol-
3-yl)-methanone (63)
CD3OD):
d
7.81 (d, J ¼ 8.0 Hz, 2H), 7.77 s (s, 1H), 7.75 (s, 1H), 7.66 (d,
J ¼ 8.2 Hz, 1H), 7.51 (d, J ¼ 8.2 Hz, 1H), 7.37 (d, J ¼ 8.0 Hz, 2H), 7.31e
7.30 (m, 1H), 6.82e6.81 (m, 1H), 5.12 (s, 2H), 2.72 (q, J ¼ 7.6 Hz, 2H)
and 1.28 (t, J ¼ 7.6 Hz, 3H) ppm; 13C NMR (100 MHz, DMSO-d6):
Compound 63 (160 mg, 4.8% yield) as a solid was prepared
following a similar procedure to compound 44. 1H NMR (400 MHz,
d
188.7, 152.2, 147.8, 138.1, 136.7, 128.8, 127.8, 125.7, 125.3, 123.5,
CDCl3):
d
7.83 (d, J ¼ 8.1 Hz, 2H), 7.78e7.75 (m, 3H), 7.62e7.59 (m,
122.7, 122.0, 121.6, 111.6, 69.7, 28.0, 15.1 ppm; HRMS (ESI): [M þ H]þ
C20H19BNO3 calcd 332.1458, found 332.1457; mp: 161e163 ꢁC;
HPLC: purity 98.6%, retention time 14.4 min with method A.
2H), 7.53 (d, J ¼ 8.2 Hz, 1H), 7.44e7.28 (m, 8H), 7.13e7.12 (m, 1H),
6.88e6.87 (m, 1H), 5.78 (brs, 1H), 5.26e5.22 (m, 1H), 5.13 (s, 2H),
4.50 (d, J ¼ 6.5 Hz, 2H), 4.45e4.43 (m, 2H) and 4.23 (t, J ¼ 6.5 Hz,1H)
ppm; mp: 120e124 ꢁC.
5.1.54. (4-Methoxyphenyl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (59)
5.1.59. (4-(((9H-fluoren-9-yl)-methoxycarbonyl)-amino)-phenyl)-
(1-(1,3-dihydro-1-hydroxy-2,1-benzoxaborol-6-yl)-pyrrol-3-yl)-
methanone (64)
Compound 59 (50 mg, 15.0% yield) as a solid was prepared
following a similar procedure to compound 44. 1H NMR (400 MHz,
CD3OD):
d
7.92 (d, J ¼ 8.9 Hz, 2H), 7.79 (d, J ¼ 2.1 Hz, 1H), 7.76 (dd,
Compound 64 (50 mg, 3.7% yield) as a solid was prepared
J ¼ 2.2, 1.7 Hz, 1H), 7.67 (dd, J ¼ 8.2, 2.1 Hz, 1H), 7.52 (d, J ¼ 8.2 Hz,
1H), 7.32 (dd, J ¼ 3.1, 2.2 Hz, 1H), 7.06 (d, J ¼ 8.9 Hz, 2H), 6.81 (dd,
J ¼ 3.1, 1.7 Hz, 1H), 5.12 (s, 2H) and 3.89 (s, 3H) ppm; 13C NMR
following a similar procedure to compound 44. 1H NMR (300 MHz,
CD3OD): d 7.84e7.79 (m, 3H), 7.75 (s, 1H), 7.73 (s, 1H), 7.71e7.69 (m,
2H), 7.63 (dd, J ¼ 8.4, 1.5 Hz, 1H), 7.56e7.48 (m, 3H), 7.41e7.29 (m,
6H), 6.80e6.81 (m, 1H), 5.09 (s, 2H), 4.53 (d, J ¼ 6.2 Hz, 2H) and 4.27
(t, J ¼ 6.2 Hz, 1H) ppm; mp: 142e147 ꢁC.
(100 MHz, DMSO-d6):
d 187.7, 162.1, 152.1, 138.2, 131.6, 130.8, 125.4,
125.3, 123.5, 122.7, 122.0, 121.4, 113.7, 111.7, 69.7, 55.3 ppm; HRMS
(ESI): [M þ Na]þ C19H16BNO4Na calcd 356.1070, found 356.0992;
mp: 155e159 ꢁC; HPLC: purity 95.1%, retention time 18.0 min with
method B.
5.1.60. (4-Hydroxymethyl-phenyl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (65)
A solution of compound 62 (45 mg, 0.12 mmol) in 1 M aq. NaOH
(10 mL) was stirred at r.t. for 3 h. The mixture was acidified with
1 M aq. HCl (15 mL) to afford crude product. The crude product was
washed with EtOAc to give compound 65 as a solid (29 mg, 72.5%
5.1.55. (2-Methoxyphenyl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (60)
Compound 60 (130 mg, 25.9% yield) as a solid was prepared
following a similar procedure to compound 44. 1H NMR (400 MHz,
yield). 1H NMR (400 MHz, CD3OD):
d
7.87 (d, J ¼ 8.0 Hz, 2H), 7.79e
CDCl3):
d
7.75 (d, J ¼ 2.1 Hz, 1H), 7.50 (dd, J ¼ 8.2, 2.1 Hz, 1H), 7.46
7.77 (m, 2H), 7.68 (d, J ¼ 8.0 Hz, 1H), 7.53 (d, J ¼ 8.0 Hz, 3H), 7.34e
(dd, J ¼ 2.2, 1.7 Hz, 1H), 7.44e7.39 (m, 3H), 7.05 (dd, J ¼ 3.1, 2.2 Hz,
1H), 7.02e6.98 (m, 2H), 6.80 (dd, J ¼ 3.1, 1.7 Hz, 1H), 5.44 (brs, 1H),
5.13 (s, 2H) and 3.81 (s, 3H) ppm; 13C NMR (100 MHz, DMSO-d6):
7.33 (m, 1H), 6.84e6.83 (m, 1H), 5.13 (s, 2H) and 4.72 (s, 2H) ppm;
13C NMR (100 MHz, DMSO-d6):
d 189.7, 152.8, 146.9, 138.6, 138.0,
129.1, 126.7, 126.4, 125.8, 124.1, 123.4, 122.6, 122.3, 112.2, 70.2,
63.0 ppm; HRMS (ESI): [M þ H]þ C19H17BNO4 calcd 334.1251, found
334.1253; mp: 227e230 ꢁC; HPLC: purity 95.1%, retention time
16.5 min with method B.
d
189.7, 156.7, 152.7, 138.6, 131.5, 130.7, 128.6, 127.7, 126.4, 123.9,
123.3, 122.4, 122.3, 120.6, 112.4, 111.4, 70.3, 55.9 ppm; HRMS (ESI):
[M þ Na]þ C19H16BNO4Na calcd 356.1070, found 356.0991; mp:
177e179 ꢁC; HPLC: purity 96.5%, retention time 16.5 min with
method B.
5.1.61. (4-Hydroxyphenyl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (66)
5.1.56. (3,4,5-Trimethoxyphenyl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (61)
To a solution of compound 59 (300 mg, 0.9 mmol) in CH2Cl2
(30 mL) was added BBr3 (0.36 mL) at ꢀ80 ꢁC under nitrogen at-
mosphere. After stirred for overnight at r.t. the mixture was
quenched with water, extracted with EtOAc, and dried over anhy-
drous Na2SO4. The residue after rotary evaporation was purified by
column chromatography over silica gel to give compound 66 as a
Compound 61 (54 mg, 9.1% yield) as a solid was prepared
following a similar procedure to compound 44. 1H NMR (400 MHz,
CD3OD):
d
7.83 (dd, J ¼ 2.2, 1.7 Hz, 1H), 7.78 (d, J ¼ 2.1 Hz, 1H), 7.67
(dd, J ¼ 8.1, 2.1 Hz, 1H), 7.53 (d, J ¼ 8.1 Hz, 1H), 7.34 (dd, J ¼ 3.0,
2.2 Hz, 1H), 7.20 (s, 2H), 6.84 (dd, J ¼ 3.0, 1.7 Hz, 1H), 5.13 (s, 2H),
3.90 (s, 6H) and 3.86 (s, 3H) ppm; 13C NMR (100 MHz, DMSO-d6):
solid (40 mg, 13.9% yield). 1H NMR (400 MHz, DMSO-d6):
d 10.20 (s,
1H), 9.32 (s, 1H), 7.92 (d, J ¼ 1.5 Hz, 1H), 7.84e7.82 (m, 2H), 7.79 (d,
J ¼ 8.4 Hz, 2H), 7.55 (d, J ¼ 8.2 Hz, 1H), 7.47 (dd, J ¼ 2.9, 2.2 Hz, 1H),
6.88 (d, J ¼ 8.4 Hz, 2H), 6.74 (dd, J ¼ 2.9, 1.6 Hz, 1H) and 5.04 (s, 2H)
d
188.7, 153.1, 152.7, 141.1, 138.7, 134.9, 126.4, 126.3, 125.7, 124.1,
123.3, 122.6, 122.5, 112.3, 106.8, 106.7, 106.6, 70.3, 60.5, 56.6,
56.5 ppm; HRMS (ESI): [M þ Na]þ C21H20BNO6Na calcd 416.1281,
found 416.1297; mp: 147e149 ꢁC; HPLC: purity 95.8%, retention
time 17.0 min with method B.
ppm; 13C NMR (100 MHz, DMSO-d6):
d 187.7, 161.0, 152.1, 138.3,
131.2, 130.2, 125.5, 125.1, 123.5, 122.8, 122.0, 121.3, 115.1, 111.8,
69.8 ppm; HRMS (ESI): [M þ H]þ C18H15BNO4 calcd 320.1094, found
320.1097; mp: 192e194 ꢁC; HPLC: purity 96.4%, retention time
16.7 min with method A.
5.1.57. (4-Benzyl acetate)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol -6-yl)- pyrrol-3-yl)-methanone (62)
Compound 62 (88 mg, 11.7% yield) as a solid was prepared
5.1.62. (4-(Aminomethyl)phenyl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (67)
following a similar procedure to compound 44. 1H NMR (400 MHz,
CDCl3 þ D2O):
d
7.87 (d, J ¼ 8.0 Hz, 2H), 7.80 (d, J ¼ 1.7 Hz,1H), 7.62e
A solution of compound 63 (187 mg, 0.34 mmol) and piperidine
(2 mL) in CHCl3 (20 mL) was stirred at 20 ꢁC for overnight. The
residue after rotary evaporation was purified by recrystallization
7.61 (m, 1H), 7.53 (dd, J ¼ 8.2, 1.7 Hz, 1H), 7.46 (d, J ¼ 8.0 Hz, 2H),
7.44 (d, J ¼ 8.2 Hz, 1H), 7.12e7.11 (m, 1H), 6.88e6.87 (m, 1H), 5.18 (s,

72
P. Wu et al. / European Journal of Medicinal Chemistry 81 (2014) 59e75
with EtOAc and CH3OH to give compound 67 as a solid (50 mg,
44.3% yield). 1H NMR (400 MHz, CD3OD):
d 193.9, 152.0, 141.5, 138.2, 128.3, 128.1, 126.7, 125.7, 124.8, 123.2,
d
7.87 (d, J ¼ 8.0 Hz, 2H),
122.7, 121.8, 121.4, 109.8, 69.7, 40.1, 29.8 ppm; HRMS (ESI): [M þ H]þ
C20H19BNO3 calcd 332.1458, found 332.1442; mp: 122e124 ꢁC;
HPLC: purity 96.1%, retention time 18.7 min with method B.
7.61e7.60 (m, 1H), 7.57 (d, J ¼ 8.0 Hz, 2H), 7.47e7.46 (m, 1H), 7.28e
7.25 (m, 2H), 7.19 (d, J ¼ 7.9 Hz, 1H), 6.79e6.78 (m, 1H), 4.89 (s, 2H)
and 4.15 (s, 2H) ppm; 13C NMR (100 MHz, DMSO-d6):
d 188.8, 152.1,
147.0, 138.1, 137.4, 128.6, 127.1, 125.7, 125.4, 123.5, 122.7, 122.1, 121.7,
111.6, 69.7, 45.0 ppm; HRMS (ESI): [M-H þ Na]þ C19H16BN2O3Na
calcd 354.1152, found 354.1307; mp: 300 ꢁC decomposed; HPLC:
purity 95.2%, retention time 12.7 min with method B.
5.1.67. (n-Propyl)-(1-(1,3-dihydro-1-hydroxy-2,1-benzoxaborol-6-
yl)-pyrrol-3-yl)-methanone (72)
Compound 72 (104 mg, 38.6% yield) as a solid was prepared
following a similar procedure to compound 44. 1H NMR (300 MHz,
DMSO-d6):
d
9.30 (s, 1H), 8.16 (dd, J ¼ 2.2, 1.7 Hz, 1H), 7.91 (d,
5.1.63. (4-Aminophenyl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (68)
J ¼ 2.1 Hz, 1H), 7.78 (dd, J ¼ 8.2, 2.1 Hz, 1H), 7.53 (d, J ¼ 8.2 Hz, 1H),
7.40 (dd, J ¼ 3.0, 2.2 Hz, 1H), 6.66 (dd, J ¼ 3.0, 1.7 Hz, 1H), 5.05 (s,
2H), 2.78 (t, J ¼ 7.3 Hz, 2H), 1.63 (h, J ¼ 7.3 Hz, 2H) and 0.93 (t,
A solution of compound 64 (200 mg, 0.37 mmol) and piperidine
(2 mL) in acetone (20 mL) was stirred at 20 ꢁC for overnight. The
residue after rotary evaporation was washed with 1% citric acid,
extracted with EtOAc, and dried over anhydrous Na2SO4. The res-
idue after rotary evaporation was purified by column chromatog-
raphy over silica gel and washed with ether to give compound 68 as
J ¼ 7.3 Hz, 3H) ppm; 13C NMR (100 MHz, DMSO-d6):
d 192.3, 152.1,
138.2, 127.2, 125.1, 123.3, 122.7, 121.9, 121.5, 109.8, 69.7, 27.0 ppm;
HRMS (ESI): [M þ H]þ C15H17BNO3 calcd 270.1301, found 270.1303;
mp: 122e124 ꢁC; HPLC: purity 95.6%, retention time 16.8 min with
method B.
a solid (30 mg, 25.5% yield). 1H NMR (400 MHz, DMSO-d6):
d 9.3 (s,
1H), 7.92 (s, 1H), 7.81 (dd, J ¼ 8.3 Hz, 1H), 7.79 (dd, J ¼ 2.3, 1.7 Hz,
1H), 7.69 (d, J ¼ 8.6 Hz, 2H), 7.54 (d, J ¼ 8.3 Hz, 1H), 7.43 (dd, J ¼ 3.0,
2.3 Hz, 1H), 6.69 (dd, J ¼ 3.0, 1.7 Hz, 1H), 6.61 (d, J ¼ 8.6 Hz, 2H), 5.93
(brs, 2H) and 5.04 (s, 2H) ppm; 13C NMR (100 MHz, DMSO-d6):
5.1.68. (n-Pentyl)-(1-(1,3-dihydro-1-hydroxy-2,1-benzoxaborol-6-
yl)-pyrrol-3-yl)-methanone (73)
Compound 73 (80 mg, 18.8% yield) as a solid was prepared
following a similar procedure to compound 44. 1H NMR (400 MHz,
d
187.1, 152.0, 138.4, 131.2, 126.8, 125.9, 124.3, 123.4, 122.8, 121.9,
CDCl3):
d
7.80 (d, J ¼ 2.0 Hz, 1H), 7.70 (dd, J ¼ 2.2, 1.6 Hz, 1H), 7.53
120.9, 113.2, 111.9, 69.8 ppm; HRMS (ESI): [M þ H]þ C18H16BN2O3
calcd 319.1254, found 319.1252; mp: 167e169 ꢁC; HPLC: purity
97.0%, retention time 16.1 min with method A.
(dd, J ¼ 8.2, 2.0 Hz, 1H), 7.44 (d, J ¼ 8.2 Hz, 1H), 7.04 (dd, J ¼ 2.9,
2.2 Hz, 1H), 6.77 (dd, J ¼ 2.9, 1.6 Hz, 1H), 5.15 (s, 2H), 2.78 (t,
J ¼ 7.4 Hz, 2H), 1.71e1.78 (m, 2H), 1.65e1.63 (m, 2H), 1.36e1.34 (m,
2H) and 0.90 (t, J ¼ 6.9 Hz, 3H) ppm; 13C NMR (100 MHz, DMSO-d6):
5.1.64. Naphthalene-1-yl-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (69)
d 195.0, 152.0, 138.3, 126.9, 124.6, 123.3, 122.7, 121.8, 121.4, 109.8,
69.7, 38.5, 31.0, 24.1, 21.9, 13.8 ppm; HRMS (ESI): [M þ Na]þ
C17H20BNO3Na calcd 320.1434, found 320.1368; mp: 124e126 ꢁC;
HPLC: purity 95.4%, retention time 19.2 min with method B.
Compound 69 (25 mg, 7.0% yield) as a solid was prepared
following a similar procedure to compound 44. 1H NMR (400 MHz,
DMSO-d6):
d
9.25 (s, 1H), 8.10 (d, J ¼ 8.3 Hz, 1H), 8.06e8.02 (m, 2H),
7.84 (d, J ¼ 2.1 Hz, 1H), 7.77 (dd, J ¼ 7.2, 1.2 Hz, 1H), 7.76 (dd, J ¼ 8.3,
2.3 Hz, 1H) 7.69 (dd, J ¼ 2.0, 1.7 Hz, 1H), 7.63e7.50 (m, 5H), 6.79 (dd,
J ¼ 3.1, 1.7 Hz, 1H) and 5.02 (s, 2H) ppm; 13C NMR (100 MHz, DMSO-
5.1.69. Cyclohexyl-(1-(1,3-dihydro-1-hydroxy-2,1-benzoxaborol-6-
yl)-pyrrol-3-yl)-methanone (74)
Compound 74 (90 mg, 23.1% yield) as a solid was prepared
d6):
d 190.7, 152.3, 138.0, 137.5, 133.3, 130.2, 129.9, 128.3, 127.3,
following a similar procedure to compound 44. 1H NMR (400 MHz,
126.8, 126.6, 126.2, 126.1, 125.1, 124.8, 123.5, 122.7, 122.2, 122.0,
111.2, 69.7, ppm; HRMS (ESI): [M þ H]þ C22H17BNO3 calcd 354.1301,
found 354.1300; mp: 166e168 ꢁC; HPLC: purity 96.2%, retention
time 18.7 min with method B.
DMSO-d6): d 9.34 (s, 1H), 8.20e8.19 (m, 1H), 7.92 (s, 1H), 7.78 (d,
J ¼ 8.2 Hz, 1H), 7.54 (d, J ¼ 8.2 Hz, 1H), 7.41e7.40 (m, 1H), 6.65e6.64
(m,1H), 5.03 (s, 2H), 3.13e3.14 (m,1H),1.77e1.65 (m, 5H),1.40e1.33
(m, 4H) and 1.19e1.18 (m, 1H) ppm; 13C NMR (100 MHz, DMSO-d6):
d
198.2, 152.0, 138.3, 125.8, 124.5, 123.3, 122.7, 121.9, 121.5, 110.1,
5.1.65. Benzyl-(1-(1,3-dihydro-1-hydroxy-2,1-benzoxaborol-6-yl)-
pyrrol-3-yl)-methanone (70)
69.7, 45.9, 29.3, 25.6, 25.2 ppm; HRMS (ESI): [M
þ
Na]þ
C18H20BNO3Na calcd 332.1434, found 332.1357; mp: 161e163 ꢁC;
Compound 70 (40 mg, 12.6% yield) as a solid was prepared
HPLC: purity 98.9%, retention time 19.1 min with method A.
following a similar procedure to compound 44. 1H NMR (400 MHz,
CDCl3):
d
7.76 (d, J ¼ 2.0 Hz, 1H), 7.71 (dd, J ¼ 2.1, 1.8 Hz, 1H), 7.50
5.1.70. 2-(4-(1-(1,3-Dihydro-1-hydroxy-2,1-benzoxaborol-6-yl)-
pyrrol-3-yl)-4-oxo-butyl)-isoindole-1,3-dione (75)
(dd, J ¼ 8.1, 2.0 Hz, 1H), 7.43 (d, J ¼ 8.1 Hz, 1H), 7.33e7.29 (m, 4H),
7.24e7.21 (m, 1H), 7.02 (dd, J ¼ 2.9, 2.1 Hz, 1H), 6.80 (dd, J ¼ 2.9,
1.8 Hz, 1H), 5.56 (brs, 1H), 5.14 (s, 2H) and 4.09 (s, 2H) ppm; 13C
Compound 75 (40 mg, 19.3% yield) as a solid was prepared
following a similar procedure to compound 44. 1H NMR (400 MHz,
NMR (100 MHz, DMSO-d6):
d
192.8, 152.7, 138.7, 136.4, 130.0, 128.7,
CDCl3):
d
7.83 (dd, J ¼ 8.5, 2.3 Hz, 2H), 7.78 (d, J ¼ 2.0 Hz, 1H), 7.68
127.0, 126.8, 126.0, 123.9, 123.3, 122.4, 122.3, 110.8, 70.3, 46.0 ppm;
HRMS (ESI): [M þ H]þ C19H17BNO3 calcd 318.1301, found 318.1303;
mp: 159e161 ꢁC; HPLC: purity 97.6%, retention time 17.4 min with
method B.
(dd, J ¼ 8.5, 2.3 Hz, 2H), 7.66e7.65 (dd, J ¼ 2.3, 1.7 Hz, 1H), 7.52 (dd,
J ¼ 8.2, 2.0 Hz, 1H), 7.43 (d, J ¼ 8.2 Hz, 1H), 7.02 (dd, J ¼ 3.0, 2.3 Hz,
1H), 6.73 (dd, J ¼ 3.0, 1.7 Hz, 1H), 5.38 (brs, 1H), 5.14 (s, 2H), 3.81 (t,
J ¼ 6.9 Hz, 2H), 2.87 (t, J ¼ 7.3 Hz, 2H) and 2.14 (dd, J ¼ 7.3, 6.9 Hz,
2H) ppm; 13C NMR (100 MHz, DMSO-d6):
d 194.1, 168.1, 152.2, 138.4,
5.1.66. (2-Phenylethyl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (71)
134.4, 131.8, 126.8, 124.9,123.4, 123.0, 122.9,122.0, 121.6, 110.0, 69.9,
37.3, 35.9, 23.1 ppm; HRMS (ESI): [M þ Na]þ C23H19BN2O5Na calcd
437.1285, found 437.1195; mp: 158e160.5 ꢁC; HPLC: purity 97.4%,
retention time 18.0 min with method A.
Compound 71 (100 mg, 30.2% yield) as a solid was prepared
following a similar procedure to compound 44. 1H NMR (400 MHz,
CDCl3):
d
7.77 (d, J ¼ 1.8 Hz, 1H), 7.67 (dd, J ¼ 2.2, 1.7 Hz, 1H), 7.50
(dd, J ¼ 8.2, 1.8 Hz, 1H), 7.43 (d, J ¼ 8.2 Hz, 1H), 7.29 (dd, J ¼ 7.8,
6.9 Hz, 2H), 7.25 (d, J ¼ 7.8 Hz, 2H), 7.19 (t, J ¼ 6.9 Hz, 1H), 7.03 (dd,
J ¼ 2.5, 2.2 Hz, 1H), 6.77 (dd, J ¼ 2.5, 1.7 Hz, 1H), 5.75 (brs, 1H), 5.14
(s, 2H) and 3.14e3.03 (m, 4H) ppm; 13C NMR (100 MHz, DMSO-d6):
5.1.71. 4-Amino-1-(1-(1,3-dihydro-1-hydroxy-2,1-benzoxaborol-6-
yl)-pyrrol-3-yl)-butan-1-one (76)
A solution of compound 75 (100 mg, 0.24 mmol) in 1 M aq.
NaOH (5 mL) was stirred at r.t. for 30 min before 1 M aq. HCl (8 mL)

P. Wu et al. / European Journal of Medicinal Chemistry 81 (2014) 59e75
73
was added and the mixture was extracted with EtOAc. After
evaporation the residue was dissolved in CH3OH and 6 M aq. HCl
(5 mL), and was refluxed for 2 d. After evaporation of organic phase,
the residue was filtered and recrystallized with EtOAC and CH3OH
to give compound 76 as a solid (33 mg, 48.4% yield). 1H NMR
Na2SO4. The residue after rotary evaporation was purified by col-
umn chromatography over silica gel to give compound 80 as a solid
(50 mg, 10.0% yield). 1H NMR (300 MHz, CD3OD):
d 7.50 (s, 1H),
7.47e7.44 (m, 1H), 7.39e7.35 (m, 1H), 7.30e7.28 (m, 1H), 7.12e7.11
(m, 1H), 6.38e6.35 (m, 1H), 5.15 (s, 2H) and 2.43 (s, 3H) ppm; 13C
(400 MHz, CD3OD):
d
8.38 (dd, J ¼ 2.2, 1.8 Hz, 1H), 7.85 (d, J ¼ 2.0 Hz,
NMR (100 MHz, DMSO-d6): d 171.8, 154.1, 139.4, 135.9, 128.9, 128.0,
1H), 7.73 (dd, J ¼ 8.2, 2.0 Hz, 1H), 7.59 (d, J ¼ 8.2 Hz, 1H), 7.54 (dd,
J ¼ 3.1, 2.2 Hz, 1H), 6.99 (dd, J ¼ 3.1, 1.8 Hz, 1H), 5.16 (s, 2H), 4.09 (t,
J ¼ 7.5 Hz, 2H), 3.52 (t, J ¼ 8.0 Hz, 2H) and 2.36e2.44 (m, 2H) ppm;
125.0, 122.6, 122.1, 111.1, 96.2, 70.4 ppm; HRMS (ESI): [M þ Na]þ
C13H12BNO3Na calcd 264.0808, found 264.0803; mp: 100e104 ꢁC;
HPLC: purity 99.0%, retention time 16.4 min with method A.
13C NMR (100 MHz, DMSO-d6):
d 178.3, 153.6, 138.1, 130.4, 124.8,
123.9, 122.7, 122.3, 113.9, 110.0, 70.9, 51.7, 33.8, 20.2 ppm; HRMS
(ESI): [M ꢀ OH]þ C15H16BN2O2 calcd 267.1299, found 267.1275; mp:
250 ꢁC decomp.; HPLC: purity 96.5%, retention time 14.8 min with
method A.
5.1.76. 6-(3-Benzyl-pyrrol-1-yl)-1,3-dihydro-1-hydroxy-2,1-
benzoxaborole (81)
To a solution of compound 50 (150 mg, 0.48 mmol) in THF
(5 mL) was added NaBH4 (181.4 mg, 4.8 mmol) portionwise slowly.
After 10 min BF3(Et2O) (1.08 mL) was added dropwise slowly. The
mixture was stirred for 2 h, and quenched with saturated NaHCO3.
After evaporation the residue was extracted with EtOAc, washed
with water, and brine, and dried over anhydrous Na2SO4. The res-
idue after rotary evaporation was purified by column chromatog-
raphy over silica gel to give compound 81 as a solid (40 mg, 28.8%
5.1.72. (Thiophen-2-yl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (77)
Compound 77 (24 mg, 7.7% yield) as a solid was prepared
following a similar procedure to compound 44. 1H NMR (400 MHz,
CDCl3):
d
7.86 (d, J ¼ 3.8 Hz, 1H), 7.83 (dd, J ¼ 2.1 Hz, 1H), 7.81 (dd,
J ¼ 2.4, 1.6 Hz, 1H), 7.63 (d, J ¼ 4.9 Hz, 1H), 7.55 (dd, J ¼ 8.1, 2.1 Hz,
1H), 7.45 (d, J ¼ 8.1 Hz, 1H), 7.16 (dd, J ¼ 4.9, 3.8 Hz, 1H), 7.10 (dd,
J ¼ 2.7, 2.4 Hz, 1H), 6.94 (dd, J ¼ 2.7, 1.6 Hz, 1H), 5.76 (brs, 1H) and
yield). 1H NMR (400 MHz, CDCl3):
d
7.70 (d, J ¼ 2.1 Hz, 1H), 7.48 (dd,
J ¼ 8.2, 2.1 Hz, 1H), 7.37 (d, J ¼ 8.2 Hz, 1H), 7.34e7.30 (m, 4H), 7.22e
7.21 (m, 1H), 7.05 (dd, J ¼ 2.6, 2.5 Hz, 1H), 6.87e6.86 (m, 1H), 6.21
(dd, J ¼ 2.6, 1.9 Hz, 1H), 5.13 (s, 2H), 5.12 (brs, 1H) and 3.90 (s, 2H)
5.15 (s, 2H) ppm; 13C NMR (100 MHz, DMSO-d6):
d 180.1, 152.3,
144.3, 138.2, 133.4, 132.5, 128.5, 125.0, 124.9, 123.7, 122.8, 122.2,
121.8, 111.4, 69.8 ppm; HRMS (ESI): [M þ H]þ C16H13BNO3S calcd
310.0709, found 310.0711; mp: 145e150 ꢁC; HPLC: purity 97.3%,
retention time 16.8 min with method B.
ppm; 13C NMR (100 MHz, CDCl3):
d 150.8, 141.9, 140.3, 128.9, 128.5,
126.0, 125.9, 123.6, 122.3, 121.7, 119.6, 117.6, 111.5, 71.2, 33.7 ppm;
HRMS (ESI): [M þ Na]þ C18H16BNO2Na calcd 312.1172, found
312.1163; mp: 112e114 ꢁC; HPLC: purity 98.2%, retention time
19.9 min with method A.
5.1.73. (5-Methylthiophen-2-yl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (78)
5.1.77. 1-Phenyl-pyrrole (83)
Compound 78 (40 mg, 12.4% yield) as a solid was prepared
To a solution of phenylamine (1.5 g, 15.6 mmol) in AcOH (20 mL)
was added 2,5-dimethoxytetrahydrofuran (2.56 g, 19.2 mmol). Af-
ter refluxed for 4 h, it was evaporated, re-dissolved in EtOAc,
washed with saturated NaHCO3, and dried over anhydrous Na2SO4.
The residue after rotary evaporation was purified by column
chromatography over silica gel to give compound 83 as an oil (1.3 g,
following a similar procedure to compound 44. 1H NMR (400 MHz,
CDCl3):
d
7.82 (d, J ¼ 2.1 Hz, 1H), 7.78 (dd, J ¼ 2.4, 1.7 Hz, 1H), 7.68 (d,
J ¼ 3.7 Hz, 1H), 7.54 (dd, J ¼ 8.1, 2.1 Hz, 1H), 7.44 (d, J ¼ 8.1 Hz, 1H),
7.09 (dd, J ¼ 3.0, 2.4 Hz, 1H), 6.91 (dd, J ¼ 3.0, 1.7 Hz, 1H), 6.83 (dd,
J ¼ 3.7, 0.9 Hz, 1H), 5.70 (brs, 1H), 5.15 (s, 2H) and 2.56 (s, 3H) ppm;
13C NMR (100 MHz, DMSO-d6):
d
179.7, 152.2, 147.8, 142.1, 138.2,
58.2% yield). 1H NMR (400 MHz, CDCl3):
d 7.45e7.39 (m, 4H), 7.27e
133.0, 127.2, 124.9, 124.5, 123.6, 122.7, 122.1, 121.5, 111.3, 69.7,
15.4 ppm; HRMS (ESI): [M þ H]þ C17H15BNO3S calcd 324.0866,
found 324.0870; mp: 148e150 ꢁC; HPLC: purity 95.3%, retention
time 18.0 min with method B.
7.23 (m, 1H), 7.10 (t, J ¼ 2.1 Hz, 2H) and 6.36 (t, J ¼ 2.1 Hz, 2H) ppm.
5.1.78. Phenyl-(1-phenyl-pyrrol-3-yl)-methanone (84)
To a solution of compound 83 (300 mg, 2.1 mmol) and benzoyl
chloride (353.4 mg, 2.52 mmol) in CH2Cl2 (10 mL) was added
anhydrous SnCl4 (0.36 mL, 3.15 mmol) dropwise slowly. After stir-
red for 2 h, the mixture was quenched with saturated NaHCO3,
washed with water, and brine, and dried over anhydrous Na2SO4.
The residue after rotary evaporation was purified by column
chromatography over silica gel to give compound 84 as an oil
5.1.74. (3-Methylthiophen-2-yl)-(1-(1,3-dihydro-1-hydroxy-2,1-
benzoxaborol-6-yl)-pyrrol-3-yl)-methanone (79)
Compound 79 (60 mg, 18.6% yield) as a solid was prepared
following a similar procedure to compound 44. 1H NMR (400 MHz,
CDCl3):
d
7.80e7.79 (m, 2H), 7.55 (dd, J ¼ 8.2, 1.8 Hz, 1H), 7.44 (d,
J ¼ 8.2 Hz, 1H), 7.41 (d, J ¼ 4.9 Hz, 1H), 7.08e7.07 (m, 1H), 7.00 (d,
(230 mg, 44.2% yield). 1H NMR (400 MHz, CDCl3):
d 7.91e7.89 (m,
J ¼ 4.9 Hz, 1H), 6.95e6.94 (m, 1H), 5.58 (brs, 1H), 5.15 (s, 2H) and
2H), 7.61 (dd, J ¼ 2.4, 1.7 Hz, 1H), 7.55e7.50 (m, 1H), 7.48e7.31 (m,
2.58 (s, 3H) ppm; 13C NMR (100 MHz, DMSO-d6):
d 181.6, 152.2,
6H), 7.30e7.34 (m, 1H), 7.10 (dd, J ¼ 2.9, 2.4 Hz, 1H) and 6.89 (dd,
143.3,138.1,134.5,131.9,129.6,127.1,124.9,123.4,122.8,122.0,121.6,
111.4, 69.7, 15.9 ppm; HRMS (ESI): [M þ H]þ C17H15BNO3S calcd
324.0866, found 324.0867; mp: 150e152 ꢁC; HPLC: purity 97.7%,
retention time 18.1 min with method B.
J ¼ 2.9, 1.7 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3):
d 190.7, 139.8,
131.5, 129.8, 128.9, 128.2, 127.1, 126.2, 126.1, 121.2, 121.1, 112.4 ppm;
HRMS (ESI): [M þ H]þ C17H14NO calcd 248.1057, found 248.1073;
HPLC: purity 95.3%, retention time 19.9 min with method A.
5.1.75. Methyl-(1-(1,3-dihydro-1-hydroxy-2,1-benzoxaborol-6-yl)-
pyrrol-2-yl)-methanone (80)
5.1.79. 1-(3-Bromo-4-(methoxymethyl)-phenyl)-pyrrole (85)
To a solution of compound 23 (3 g, 12.0 mmol) in anhydrous THF
(50 mL) was added NaH (60% in mineral oil 0.72 g, 0.018 mol). The
mixture was stirred for 10 min at r.t. before CH3I (0.7 mL,
14.0 mmol) was added dropwise slowly. After stirred for 6 h, the
mixture was quenched with water and 2 M aq. HCl. After evapo-
ration the residue was extracted with EtOAc, washed with water,
and brine, and dried over anhydrous Na2SO4. The residue after
rotary evaporation was purified by column chromatography over
To DMF (0.17 mL, 2.4 mmol) in anhydrous CH2Cl2 (5 mL) at 0 ꢁC
under nitrogen was added POCl3 (0.22 mL, 2.4 mmol) dropwise. The
mixture was stirred for 1 h at r.t. then cooled to 0 ꢁC. After a solution
of compound 24 (0.4 g, 2 mmol) in CH2Cl2 (5 mL) was added
dropwise, the mixture was stirred for 1 d at r.t., and refluxed for
15 min. After cooled to r.t., aq. NaOAc (0.88 g, 11 mmol) was added
before it was extracted with CH2Cl2, and dried over anhydrous

74
P. Wu et al. / European Journal of Medicinal Chemistry 81 (2014) 59e75
silica gel to give compound 85 as a solid (3 g, 93.9% yield). 1H NMR
(400 MHz, CDCl3):
5.1.85. 2-(Methoxymethyl)-5-(3-benzoyl-pyrrol-1-yl)-
benzaldehyde (91)
d
7.61 (d, J ¼ 2.2 Hz, 1H), 7.51 (d, J ¼ 8.3 Hz, 1H),
7.36 (dd, J ¼ 8.3, 2.2 Hz, 1H), 7.06 (t, J ¼ 2.2 Hz, 2H), 6.36 (t,
To a solution of compound 90 (88 mg, 0.27 mmol) in CH2Cl2
(10 mL) were added PCC (88.3 mg, 0.41 mmol) and Celite (132 mg).
After stirred for 2 h at r.t., the mixture was purified by column
chromatography over silica gel to give compound 91 as a solid
J ¼ 2.2 Hz, 2H), 4.54 (s, 2H) and 3.49 (s, 3H) ppm; mp: 59e62 ꢁC.
5.1.80. 2-(Methoxymethyl)-5-(pyrrol-1-yl)-benzaldehyde (86)
To a solution of compound 85 (200 mg, 0.75 mmol) in anhy-
drous THF (10 mL) was added dropwise 1.6 M n-BuLi in THF
(2.72 mL, 4.36 mmol) at ꢀ78 ꢁC under nitrogen. After stirred for
5 min at ꢀ78 ꢁC, DMF (0.34 mL, 4.5 mmol) was added dropwise. The
reaction was quenched with 0.5 M HCl immediately. After evapo-
ration, the residue was extracted with EtOAc, washed with water,
and brine, and dried over anhydrous Na2SO4. The residue after
rotary evaporation was purified by column chromatography over
silica gel to give compound 86 as a solid (122 mg, 75.6% yield). 1H
(78 mg, 90.0% yield). 1H NMR (400 MHz, CDCl3):
d 10.28 (s, 1H), 7.93
(d, J ¼ 2.4 Hz,1H), 7.88 (d, J ¼ 7.6 Hz, 2H), 7.72 (d, J ¼ 8.3 Hz,1H), 7.67
(dd, J ¼ 2.4, 1.7 Hz, 1H), 7.63 (dd, J ¼ 8.3, 2.4 Hz, 1H), 7.57 (t,
J ¼ 7.2 Hz, 1H), 7.49 (dd, J ¼ 7.6, 7.2 Hz, 2H), 7.17 (dd, J ¼ 2.9, 2.4 Hz,
1H), 6.91 (dd, J ¼ 2.9, 1.7 Hz, 1H), 4.87 (s, 2H) and 3.50 (s, 3H) ppm;
mp: 79e81 ꢁC.
5.1.86. 5-(3-Benzoyl-pyrrol-1-yl)-2-(hydroxymethyl)benzaldehyde
and its hemiacetal isomer (92)
NMR (400 MHz, CDCl3):
d
10.28 (s, 1H), 7.90 (d, J ¼ 2.3 Hz, 1H), 7.65
To a solution of compound 91 (75 mg, 0.24 mmol) in anhydrous
CH2Cl2 (10 mL) was added BBr3 (0.07 mL, 0.7 mmol) dropwise
slowly at ꢀ78 ꢁC. The reaction mixture was allowed to warm to 0 ꢁC
gradually before quenched with water, extracted with CH2Cl2,
washed with water, and brine, and dried over anhydrous Na2SO4.
The residue after rotary evaporation was purified by column
chromatography over silica gel to give 92 as a solid (60 mg, 83.6%
yield). The compound exists as two tautomers in solution:
aldehyde:hemiacetal ¼ 4:5. 1H NMR (400 MHz, CD3OD): aldehyde:
(d, J ¼ 8.3 Hz, 1H), 7.62 (dd, J ¼ 8.3, 2.3 Hz, 1H), 7.15 (t, J ¼ 2.2 Hz,
2H), 6.39 (t, J ¼ 2.2 Hz, 2H), 4.86 (s, 2H) and 3.50 (s, 3H) ppm; mp:
49e53 ꢁC.
5.1.81. 2-(Methoxymethyl)-5-(pyrrol-1-yl)-benzyl alcohol (87)
To a solution of compound 86 (1.2 g, 5.6 mmol) in CH3OH
(20 mL) was added NaBH4 (0.21 g, 5.6 mmol) portionwise slowly.
After stirred for 30 min at r.t., the mixture was quenched with
water. After evaporation, the residue was extracted with EtOAc,
washed with water, and brine, and dried over anhydrous Na2SO4.
The residue after rotary evaporation gave crude compound 87
which was used without further purification.
d
10.31 (s,1H), 8.12 (s,1H), 7.89e7.85 (m, 3H), 7.67 (d, J ¼ 8.2 Hz,1H),
7.62e7.52 (m, 4H), 7.46e7.45 (m,1H), 6.87e6.86 (m,1H) and 5.06 (s,
2H) ppm; hemiacetal: 7.89e7.85 (m, 3H), 7.79e7.77 (m, 2H), 7.62e
d
7.52 (m, 4H), 7.35e7.34 (m, 1H), 6.84e6.83 (m,1H), 5.90 (s, 1H), 4.78
(d, J ¼ 10.5 Hz, 1H) and 4.74 (d, J ¼ 10.5 Hz, 1H) ppm; 13C NMR
5.1.82. 2-(Methoxymethyl)-5-(pyrrol-1-yl)-benzyl acetate (88)
To a solution of compound 87 (1.0 g, 4.6 mmol) in CH2Cl2
(20 mL), TEA (2.05 mL, 13.8 mmol) and Ac2O (0.65 mL, 7 mmol)
were added successively. After stirred for overnight at r.t., the
mixture was quenched with 1 M aq. HCl. After evaporation, the
residue was extracted with EtOAc, and dried over anhydrous
Na2SO4, evaporated, and purified by column chromatography over
silica gel to give compound 88 as an oil (1.13 g, 95% yield). 1H NMR
(100 MHz, CD3OD): d 193.1, 192.8, 142.9, 141.5, 140.8, 140.7, 139.8,
136.5, 134.6, 134.0, 133.6, 133.2, 133.1, 130.0, 129.6, 127.7, 127.3,
126.6, 125.4, 122.8, 122.7, 122.2, 121.7, 120.7, 119.6, 113.7, 113.4, 101.8,
75.9 and 71.4 ppm; HRMS (ESI): [M þ H]þ C19H16NO3 calcd
306.1130, found 306.1104; HPLC: purity 98.2%, retention time
19.9 min with method A.
5.2. Biology
(400 MHz, CDCl3):
d
7.47 (d, J ¼ 2.2 Hz, 1H), 7.44 (d, J ¼ 8.2 Hz, 1H),
7.34 (dd, J ¼ 8.2, 2.2 Hz,1H), 7.12 (t, J ¼ 2.1 Hz, 2H), 6.37 (t, J ¼ 2.1 Hz,
5.2.1. In vitro T. brucei assay
2H), 5.25 (s, 2H), 4.54 (s, 2H), 3.43 (s, 3H) and 2.14 (s, 3H) ppm.
All in vitro antiparasitic assays were conducted with the
bloodstream-form T. brucei brucei 427 strain. Parasites were
cultured in T-25 vented cap flasks and kept in humidified in-
cubators at 37 ꢁC and 5% CO2. The parasite culture medium was
complete HMI-9 medium (Hirumi, H.; Hirumi, K. Continuous
cultivation of T. brucei bloodstream forms in a medium containing a
low concentration of serum protein without feeder cell layers.
J. Parasitol. 1989, 75, 985e989) containing 10% FBS, 10% Serum Plus
medium, and penicillin/streptomycin. To ensure log growth phase,
trypanosomes were subcultured at appropriate dilutions every 2e3
5.1.83. 2-(Methoxymethyl)-5-(3-benzoyl-pyrrol-1-yl)-benzyl
acetate (89)
To a solution of compound 88 (760 mg, 2.93 mmol) and benzoyl
chloride (453.2 mg, 3.22 mmol) in CH2Cl2 (40 mL) was added
anhydrous SnCl4 (0.41 mL, 3.5 mmol) dropwise slowly at ꢀ40 ꢁC.
The reaction mixture was allowed to warm to 0 ꢁC gradually before
it was quenched with saturated NaHCO3, washed with water, and
brine, and dried over anhydrous Na2SO4. The residue after rotary
evaporation was purified by column chromatography over silica gel
to give compound 89 as an oil (160 mg, 15.0% yield). 1H NMR
days. The log phase cultures were diluted 1:10 in HMI-9, and 10 mL
was counted using hemocytometer to determine parasite concen-
tration. Parasites were diluted to 2 ꢂ 105/mL in HMI-9 to generate a
2-fold working concentration for assay. Compounds to be tested
(400 MHz, CDCl3):
d
7.88 (d, J ¼ 7.7 Hz, 2H), 7.62 (dd, J ¼ 2.4, 1.6 Hz,
1H), 7.55 (d, J ¼ 7.3 Hz, 1H), 7.50e7.46 (m, 4H), 7.36 (dd, J ¼ 8.1,
2.4 Hz,1H), 7.10 (dd, J ¼ 2.9, 2.4 Hz, 1H), 6.87 (dd, J ¼ 2.9, 1.6 Hz, 2H),
5.23 (s, 2H), 4.53(s, 2H), 3.42 (s, 3H) and 2.13 (s, 3H) ppm.
were serially diluted in DMSO, and 0.5
HMI-9 in triplicate 96-well plates using a Biomek NX liquid handler.
Parasites from the diluted stock were added to each well (50 L)
mL was added to 49.5 mL
m
5.1.84. 2-(Methoxymethyl)-5-(3-benzoyl-pyrrol-1-yl)-benzyl
alcohol (90)
using a Multidrop 384 dispenser to give a final concentration of
1.0 ꢂ 105/mL parasites in 0.4% for DMSO. Trypanosomes were
incubated with compounds for 72 h at 37 ꢁC with 5% CO2. Resazurin
A solution of compound 89 (100 mg, 0.27 mmol) in 1 M aq.
NaOH (1 mL) and CH3OH (30 mL) was stirred at r.t. for 15 min. The
mixture was quenched with 1 M aq. HCl. After evaporation, the
residue was extracted with EtOAc, washed with water, and brine,
and dried over anhydrous Na2SO4. The residue after rotary evapo-
ration gave crude compound 90 which was used without further
purification.
(20 mL of 12.5 mg/mL stock) from SigmaeAldrich was added to each
well, and plates were incubated for an additional 2e4 h. Assay
plates were read using an EnVision plate reader at an excitation
wavelength of 544 nm and emission of 590 nm. Triplicate data
points were averaged to generate sigmoidal dose response curve
and to determine IC50 values using XLfit curve fitting software from

P. Wu et al. / European Journal of Medicinal Chemistry 81 (2014) 59e75
75
IDBS (Guildford, U.K.). IC50 values were measured in triplicate with
an error range of ꢃ0.2 M. Suramin and pentamidine are used as
positive control, and typical average IC50 values are 0.007 g/mL
(0.005 M) and 0.009 g/mL (0.026 M), respectively.
Appendix A. Supplementary data
m
m
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.079.
m
m
m
5.2.2. In vitro mammalian cell cytotoxicity assay
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supplemented with 10% fetal bovine serum and 1% penicillin/
streptomycin. L929 cells were maintained below confluent levels
by sub-culturing at 1:10 dilution twice weekly using 0.05% trypsin
for detachment. Sub-confluent L929 cells were trypsinized, resus-
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Acknowledgments
We thank National Natural Science Foundation of China
(81222042 and 81102313), the Ministry of Science and Technology
of China (2009CB918404 and 2012CB518001), and the E-Institutes
of Shanghai Universities (EISU) Chemical Biology Division for
financial support of this work.