Benzoxaborole Antimalarial Agents. Part 4. Discovery of Potent 6-(2-(Alkoxycarbonyl)pyrazinyl-5-oxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaboroles

Article abstract of DOI:10.1021/acs.jmedchem.5b00678

A series of 6-hetaryloxy benzoxaborole compounds was designed and synthesized for a structure-activity relationship (SAR) investigation to assess the changes in antimalarial activity which result from 6-aryloxy structural variation, substituent modification on the pyrazine ring, and optimization of the side chain ester group. This SAR study discovered highly potent 6-(2-(alkoxycarbonyl)pyrazinyl-5-oxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaboroles (9, 27-34) with IC₅₀s = 0.2-22 nM against cultured Plasmodium falciparum W2 and 3D7 strains. Compound 9 also demonstrated excellent in vivo efficacy against P. berghei in infected mice (ED₉₀ = 7.0 mg/kg).

Full text of DOI:10.1021/acs.jmedchem.5b00678

Article
pubs.acs.org/jmc
Benzoxaborole Antimalarial Agents. Part 4. Discovery of Potent 6‑(2-
(Alkoxycarbonyl)pyrazinyl-5-oxy)-1,3-dihydro-1-hydroxy-2,1-
benzoxaboroles
Yong-Kang Zhang,*,† Jacob J. Plattner,† Eric E. Easom,† Robert T. Jacobs,† Denghui Guo,§
Virginia Sanders,† Yvonne R. Freund,† Brice Campo,‡ Philip J. Rosenthal,§ Wei Bu,†
Francisco-Javier Gamo,∥ Laura M. Sanz,∥ Min Ge,⊥ Liang Li,⊥ Jie Ding,⊥ and Yin Yang⊥
†Anacor Pharmaceuticals, Inc., 1020 East Meadow Circle, Palo Alto, California 94303, United States
‡International Center Cointrin, Medicines for Malaria Venture, Block G, 20 route de Pre-
́
Bois, POB 1826, CH-1215 Geneva,
Switzerland
§Department of Medicine, University of California, San Francisco, Box 0811, San Francisco, California 94143, United States
∥Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, 28760 Tres Cantos, Spain
⊥Acesys Pharmatech, Inc., Science and Technology Building 24B, 5 Xing Mo Fan Road, Nanjing 21009, China
S
* Supporting Information
ABSTRACT: A series of 6-hetaryloxy benzoxaborole com-
pounds was designed and synthesized for a structure−activity
relationship (SAR) investigation to assess the changes in
antimalarial activity which result from 6-aryloxy structural
variation, substituent modification on the pyrazine ring, and
optimization of the side chain ester group. This SAR study discovered highly potent 6-(2-(alkoxycarbonyl)pyrazinyl-5-oxy)-1,3-
dihydro-1-hydroxy-2,1-benzoxaboroles (9, 27−34) with IC50s = 0.2−22 nM against cultured Plasmodium falciparum W2 and 3D7
strains. Compound 9 also demonstrated excellent in vivo efficacy against P. berghei in infected mice (ED90 = 7.0 mg/kg).
changing substituents on the pyrazine ring (10−26) and
exploring the pyrazinyl side chain ester group (27−35). These
compounds have been synthesized and evaluated against P.
falciparum. Representative compounds were further tested in a
mouse model. Herein we report the synthesis and in vitro SAR
of compounds 1−40 and safety, in vitro stability, pharmaco-
kinetic, and in vivo efficacy results for selected compounds.
INTRODUCTION
■
Malaria represents a continuing public health problem for close
to half of the world’s population. It is a parasitic infection that
was responsible for an estimated 198 million clinical cases and
584000 deaths worldwide in 2013, mostly among children
under the age of five.1 The most important causative parasite,
Plasmodium falciparum, is transmitted to humans by infected
mosquitoes and is responsible for most malarial severe
morbidity and mortality. Current therapies to treat malaria
caused by P. falciparum are heavily reliant on artemisinin-based
combination therapies. However, emergence of resistance to
the endoperoxide component of the combination has recently
been identified, and resistance to older antimalarial drugs is
already widespread.2−4 This situation has brought renewed
urgency to discovering new medications that counter resistance
and that are safe and easy for use in the most vulnerable
populations.5−10
Boron-containing benzoxaboroles have shown potent activity
against P. falciparum, with the best reported IC50 at 26 nM.11−14
A new scaffold with a 6-aryloxy benzoxaborole structural feature
caught our attention from early library screening against P.
falciparum. The screening identified a 6-(4-carboxyphenoxy)-
1,3-dihydro-1-hydroxy-2,1-benzoxaborole (1 in Figure 1) with
an IC50 of 120 nM. In a continuing effort to identify
benzoxaboroles with better antimalarial in vitro and in vivo
activities, we further explored structure−activity relationships
(SARs) by varying the 6-aryloxy group (2−9 in Figure 1),
CHEMISTRY
■
Methods for the preparation of a diverse number of substituted
benzoxaboroles have recently been published.15−22 The
chemistry for the syntheses of compounds 1, 2, 6, 8, and 9
was described previously.23,24 Scheme 1 illustrates the simple
displacement methods for the syntheses of compounds 3, 7,
and 10. The synthesis of benzoxaborole 36 was reported
previously,24 and intermediate esters 37 and 38 were easily
prepared from the corresponding commercial carboxylic acids.
The methods for the syntheses of compounds 4 and 5 are
shown in Scheme 2. Nucleophilic substitution of a fluorine
atom in 41 by the hydroxyl group in 40 generated 42, followed
by esterification, giving the methyl ester 43. Catalytic
boronylation converted the bromo compound 43 to boron
compound 44. Reduction of the aldehyde of 44 followed by
Received: May 1, 2015
© XXXX American Chemical Society
A
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tetrazole side chain, respectively. Reduction of the ester in 9
afforded the hydroxymethyl analogue 12.
The chemistry for the syntheses of compounds 14 and 22 is
shown in Scheme 4. The carboxylic acid of 45 was converted to
the ester 48, which was reduced to give the aldehyde
compound 49. A coupling reaction of intermediate 49 with
36 generated 50, which led to the amine compound 14 by
reductive amination and the oxime analogue 22 by reacting
with hydroxylamine.
Scheme 5 illustrates the syntheses for compounds 15−21.
Amide intermediates 46 and 51−56 were obtained by
converting the acid 45 to its isobutylcarbonyl anhydride or its
carbonyl chloride followed by reacting with the corresponding
amines or sulfonamide. Finally, compounds 15−21 were
generated by coupling the amides 46 and 51−56 with 6-
hydroxy benzoxaborole 36.
The chemistry for the preparation of compounds 24 and 35
is shown in Scheme 6. The carboxylic acid group in 45 was
converted to the amide of 57, which was then cyclized to 58.
The chlorine atom in 58 was replaced by the hydroxyl
benzoxaborole 36 to give 24. Ring-opened ester product 35 was
also obtained because of the hydrolysis of the oxazoline ring
under higher temperature.
For the synthesis of compound 25, the chemistry is
illustrated in Scheme 7. From a common intermediate 48,
compound 59 was obtained by hydrazine exchange with the
ester methoxy group. Further acetylation of 59 gave 60, which
was cyclized to generate 61. The substitution reaction between
61 and 36 provided the final product 25.
Figure 1. Chemical structures of benzoxaboroles with 6-aryloxy
variation (1−9), substituent changes on the pyrazinyl ring (10−26),
and alterations in the side chain ester group (27−35).
Syntheses of ester compounds 27−34 are shown in Scheme
8. The carboxylic acid in 45 was transformed to the
corresponding ester intermediates 62−69, which underwent a
substitution reaction with the common intermediate 6-hydroxy
benzoxaborole 36 to provide the final ester compounds 27−34.
acidification provided the ester benzoxaborole 5, which was
hydrolyzed to the acid compound 4.
As shown in Scheme 3, acid compound 45 was converted to
the amide 46 followed by dehydration, resulting in cyano
compound 47. Displacement of a chlorine atom in 47 by the
phenoxy moiety in compound 36 gave the desired compound
11, which was reduced to generate compound 13, containing
the aminomethyl side chain. The nitrile compound 11 was also
converted to 23 and 26, with the carboximidamide and
RESULTS AND DISCUSSION
■
Structure−Activity Relationships. The in vitro inhibitory
activities of compounds 1−35 against the 3D7 (chloroquine
sensitive) and W2 (chloroquine resistant) strains of P.
a
Scheme 1. Synthesis of Compounds 3, 7, and 10
a
Reagents and conditions: (a) KHMDS, 1,4-dioxane, 0 °C to rt, 15 min, then 80 °C, 22 h, and HCl acidification; (b) Cs2CO3, DMF, 50 °C, 1.5 h;
(c) Cs2CO3, DMF, 75 °C, 0.5 h.
B
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a
Scheme 2. Synthesis of Compounds 4 and 5
a
Reagents and conditions: (a) Cs2CO3, DMF, 80 °C, 16 h; (b) SOCl2, MeOH, 0−60 °C, 16 h; (c) Pin2B2, Pd(PPh3)2Cl2, KOAc, 1,4-dioxane, N2,
100 °C, 0.5 h; (d) NaBH4, MeOH, 0 °C to rt, 1 h, then 1 N HCl; (e) LiOH·H2O, IPA: H2O (4:1), 40 °C, 1 h, then 1 N HCl.
a
a
Scheme 3. Synthesis of Compounds 11−13, 23, and 26
Scheme 4. Synthesis of Compounds 14 and 22
a
Reagents and conditions: (a) SOCl2, MeOH, 0−60 °C, 16 h; (b)
DIBAL-H, THF, −78 °C, 2 h; (c) intermediate 36, Cs2CO3, DMF, 50
°C, 2 h; (d) Me2NH·HCl, NaBH(OAc)3, DCM, rt, 16 h; (e) NH2OH·
HCl, NaOH, EtOH, rt, 3 h.
position ortho to the carboxyl functional group, the acid
compound 4 had submicromolar activity against W2 and 3D7
strain parasites, and its ester analogue 5 was about 5-fold more
active. With two nitrogens, both meta to the carboxyl functional
group, compound 6, containing a pyrimidine ring, had
decreased activity. When one of the two nitrogens was
moved to the position ortho to the carboxyl functional group,
compound 7, with a pyridazine ring, showed excellent activity.
When the left-side aromatic ring was changed to a pyrazine ring
with one nitrogen ortho to the carboxyl functional group and
the other ortho to the oxygen bridge, the acid compound 8 was
somewhat less active, whereas its ester compound 9 was
extremely potent, with low nanomolar IC50 values. With the
identification of the pyrazine ring, compounds 10−26 were
designed and synthesized to investigate the effects of left-side
substituent variation on antimalarial activity. As shown in
a
Reagents and conditions: (a) (1) SOCl2, DCM, 50 °C, 16 h, (2)
NH4OH; (b) (CNCl)3 DMF, rt, 1 h; (c) intermediate 36, Cs2CO3,
DMF, 50 °C, 2 h; (d) LiAlH4, THF, 0 °C, 50 min; (e) NH2OH·HCl,
Na2CO3, H2O, 40 °C, 2 h; (f) NaN3, NH4Cl, DMF, rt, 1 h; (g)
LiAlH4, THF, 0 °C, 50 min.
falciparum were determined (Table 1). Compounds 1−9 (see
Figure 1) were designed to examine the effects of the left-side
aromatic moiety, carboxylic acid (COOH) and ester (COOR)
groups on antimalarial activity. Compound 1, containing a
COOH group, showed submicromolar (0.12 μM) activity
against the W2 strain, whereas its ester analogue 2 was without
apparent antimalarial activity. Addition of a nitrogen atom
ortho-to-oxygen bridge in compound 3 did not improve
activity. However, when the nitrogen was moved to the
C
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a
a
Scheme 5. Synthesis of Compounds 15−21
Scheme 8. Syntheses of Ester Compounds 27−34
a
Reagents and conditions: (a) (1) isobutyl chloroformate or SOCl2,
(2) NH4OH for 46, MeNH2 for 51, EtNH2 for 52, Me2NH for 53,
NH2CH2CH2OH for 54, NH2CH2CH2NMe2 for 55, or i-PrSO2NH2
for 56; (b) for 15−20, intermediate 36, Cs2CO3, DMF, 50 °C, 2 h,; for
21, intermediate 36, NaOMe, DMF, 60 °C, 16 h.
a
Reagents and conditions: (a) (1) SOCl2, 80 °C, 2 h, (2)
corresponding alcohol for specific ester intermediates 62−69; 50 °C,
1 h; (b) intermediate 36, Cs2CO3, DMF, 50 °C, 0.5−2 h.
group is critical for potent activity. Therefore, several other
ester compounds (27−35) were designed and synthesized to
further explore the effect of ester side chain variation on
antimalarial activity. Linear and primary esters such as n-butyl
ester 30 had outstanding activity, which was better than that of
tertiary and more hindered analogues such as t-butyl ester (31).
Ester compound 35, containing a terminal amine group on the
ester side chain, had decreased potency, presumably because
the charged amino group decreased permeability into parasites.
Cytotoxicity Results. Representative compounds were
selected for in vitro cytotoxicity tests using human Jurkat
cells (Table 2). In general, the tested compounds showed low
cytotoxicities against this cell line.
a
Scheme 6. Synthesis of Compounds 24 and 35
In Vitro Stability and Pharmacokinetic Profiles of 8, 9,
and 15. Stability testing showed that the amide 15 was stable
in both mouse and human liver microsomes, but the ester 9 had
poor stability (Table 3). However, because of the excellent in
vitro potency of the ester 9, we planned to investigate its in vivo
efficacy. Therefore, 9 and two closely related compounds, 8 (an
acid) and 15 (an amide), were selected for pharmacokinetic
profiling. The acid 8 had high plasma concentration, good
exposure, and good bioavailability after oral administration in
mice (Table 3). The ester 9 was bioavailable after oral dosing
and was extensively metabolized to the acid 8 in vivo, resulting
in higher plasma concentration and exposure of the metabolite
acid 8 compared to the parent ester 9. An amide functional
group is more stable than an ester upon hydrolysis in vivo.
Indeed, the amide 15 showed improved plasma concentration,
exposure, half-life, and bioavailability compared to 9.
Production of the in vivo metabolite 8 from the amide 15
was minor, with <1% conversion.
In Vivo Efficacy in a P. berghei Mouse Model. Four
compounds (4, 8, 15, and 9) were tested in a P. berghei mouse
model. These compounds were selected for testing based on
their chemical structural features, in vitro activities, phys-
icochemical properties, and pharmacokinetic results. Com-
pound 4 has a carboxylic acid group linked to a pyridine ring.
Compound 8 has a pyrazine ring replacing the pyridine ring in
4. These compounds showed similar in vitro activities.
Compounds 15 and 9 are a carboxamide and a carboxylic
methyl ester of 8, respectively. Compounds were dosed by oral
administration once a day for 4 consecutive days, beginning 1 h
after infection by intraperitoneal administration of P. berghei-
infected erythrocytes. The acids 4 and 8 and the amide 15
offered good in vivo efficacy. The ester 9 was the most
a
Reagents and conditions: (a) HOBT, EDC, 2-amino-2-methyl-1-
propanol, TEA, DCM, rt, 30 min; (b) SOCl2, rt, 2 h; (c) intermediate
36, Cs2CO3, DMF, 60 °C, 2 h; (d) intermediate 36, Cs2CO3, DMF, 80
°C, 1 h.
a
Scheme 7. Synthesis of Compound 25
a
Reagents and conditions: (a) NH2NH2, H2O, MeOH, 0 °C, 16 h; (b)
AcCl, NMM, DCM, rt, 1 h; (c) POCl3, 110 °C, 30 min; (d)
intermediate 36, Cs2CO3, DMF, 80 °C, 45 min.
Figure 1, a broad range of structural variation was studied for
the left-side group, including hydrogen (10), cyano (11),
hydroxymethyl (12), aminomethyl (13), dimethylaminomethyl
(14), carboxamides (15−20), N-sulfonyl carboxamide (21),
oxime (22), carboximidamide (23), oxazoline ring (24),
oxadiazole ring (25), and tetrazole (26) groups. Activities of
these compounds varied widely. Oxazoline and oxadiazole
compounds (24 and 25) were designed to be bioisosteres of
the carboxylic ester group in 9. Their relatively poor
antimalarial activity might indicate that the carboxylic ester
D
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a
Table 1. In Vitro Activity Results for Compounds 1−35 against Cultured P. falciparum
b
a
a
entry
cLogD7.4
IC50 (nM) (W2)
IC50 (nM) (3D7)
entry
cLogD7.4
IC50 (nM) (W2)
IC50 (nM) (3D7)
1
−0.37
3.60
120 10
1090 170 (72 h)
20
−0.53
−0.25
1.33
6660 1350
190 10
>5000
0
c
d
2
0%
c
nt
d
21
130 10
3
2.98
0%
nt
22
1770 470
3000 720
4
−1.64
1.74
170 10
540 60
23
0.01
>10000
800 70
>10000
>10000
0
>5000
0
5
23
80%
2
c
120
0
24
1.76
1460 210
d
6
1.80
nt
25
−0.21
1.31
0
0
>5000
>5000
4.4
0
0
7
1.11
82 16
150 10
1.4 0.3
9380 270
470 80
440 20
100 40
26
8
−2.35
1.13
300 40 (72 h)
27
1.47
0.5 0.008
1.2 0.1
1
0
0
0
2
9
1.9
0
28
1.88
1.4
10
11
12
13
14
15
16
17
18
19
1.18
>5000
0
29
2.34
0.5 0.007
0.2 0.04
2.5
1.39
3250 490
2240 240
470 30
30
2.33
0.7
0.50
31
32
1.96
19
1
22
−0.27
1.12
110
6
2.67
0.4 0.07
2
0
>10000
0
>5000
0
33
2.71
0.7 0.001
2.9
0
2
0.23
210 50
240 30
350 130
320 50
34
2.85
6.6
2
8.9
0.48
35
−0.24
190 50
250 40
e
0.82
120
8
870 220
960 50
artemisinin
chloroquine
atovaquone
6
3
na
e
0.72
150 10
>10000
21
6
na
e
−0.27
0
5900 3200
na
1
0.1
a
Experimental procedures for the in vitro assays for the W2 and 3D7 strains are described in Supporting Information. IC50 values were determined
from two independent replicates for W2 strain and three replicates for 3D7 strain, and standard deviations were calculated. Artemisinin and
chloroquine were used as reference controls for W2 strain assay, and atovaquone was used for 3D7 assay. Compounds were incubated with parasites
b
c
for 48 h unless otherwise noted. cLogD7.4 was calculated using ChemAxon software under the condition of pH = 7.4. The value shown is
d
e
percentage inhibition at 5 μM. Not tested. Not applicable.
from compound 4, additional benefit with compounds 8 and
15, and the greatest efficacy with the ester 9 (Table 4).
Physicochemical properties such as lipophilicity, in vitro
potency, and pharmacokinetics were assessed. The acids, 4 and
8, have −1.64 and −2.35 of cLogD7.4 values, respectively,
indicating that the carboxylic group is negatively charged
(COO−), likely resulting in inefficient penetration of parasite-
infected erythrocytes. The amide 15 has improved lipophilicity
and PK profile, but its in vitro activity was not high. The ester 9
Table 2. In Vitro Cytotoxicity Results for Representative
Compounds
a
compd
IC50 (μM, Jurkat cell)
compd
IC50 (μM, Jurkat cell)
5
>100
>100
7.59
27
28
29
30
31
33
16.8
>100
17.6
8
9
15
17
18
48.2
>100
>100
52.4
79.9
>100
a
The experimental procedure for the in vitro cytotoxicity assay was
described previously.11
was extremely active in vitro and its lipophilicity (cLogD7.4
=
1.13) suggests good cell penetration,25 which may contribute
importantly to its low in vivo ED90. However, the metabolic
instability and less favored PK parameters of 9 need to be
further addressed. Our goal is to improve PK parameters while
maintaining desired physicochemical properties of 9.
efficacious among the four compounds tested with ED90 = 7.0
mg/kg (Table 4). Considering survival of experimental animals,
control mice reached lethal parasitemias within 6−8 days. All
four tested compounds extended survival, with modest benefits
a
Table 3. In Vitro Stability in Liver Microsomes and Pharmacokinetic Parameters for 8, 9, and 15 in Mice
stability in liver microsome
oral PK parameters
intravenous PK parameters
% remain
at 1 h,
mouse +
NADPH
% remain
at 1 h,
% remain
at 1 h,
human +
NADPH
% remain
at 1 h,
mouse −
human −
Cmax
Tmax
(h)
AUC0‑inf
(h·μg/mL)
T1/2
(h)
F
CL
Vss
AUC0−inf
T1/2
(h)
compd
NADPH
NADPH (μg/mL)
(%) (mL/h/kg) (mL/kg) (h·μg/mL)
b
e
f
8 (an acid)
nd
nd
0
nd
0
nd
0
19.5
0.16
0.25
0.25
8.62
0.23
1.0
67
23
2326
848
926
4.3
0.4
1.1
0.3
9 (an
ester)
0
0.10
12463
c
d
8 from 9
0.92
1.38
0.25
0.25
0.61
2.29
1.5
2.1
29
99
0.99
2.2
0.13
0.4
15 (an
amide)
97
100
87
100
2261
973
c
a
Abbreviations: Cmax, maximum concentration of drug in plasma; Tmax, time to maximum concentration of drug in plasma; AUC0−inf, area under the
b
curve extrapolated to infinity; T1/2 terminal half-life; F, oral bioavailability; CL, clearance; Vss, volume of distribution at steady state. Oral dosing at
30 mg/kg and intravenous dosing at 10 mg/kg. Oral dosing at 5 mg/kg and intravenous dosing at 5 mg/kg. The acid 8 detected from studies of
the ester 9 because there was conversion of 9 to 8 in vivo. Not determined. This is AUC0−2h
c
d
e
f
.
E
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a
Table 4. Percent Survival of Mice and Efficacies of 4, 8, 9, and 15 in a P. berghei Mouse Malaria Model
percent survival of mice on specific days (%)
d
ED90
b
compd
oral dosage
d4
d5
d6
d7
d8
d9
d10
d11
d12
d13
d14
d15
d16-d19 d20 (mg/kg)
4
100 mg/kg
33 mg/kg
11 mg/kg
3 mg/kg
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
60
80
60
40
20
0
0
21.1
0
0
0
1 mg/kg
60
8
9
100 mg/kg
33 mg/kg
11 mg/kg
3 mg/kg
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
60
100
80
60
40
20
0
60
40
20
60
40
20
0
0
0
30.3
60
20
1 mg/kg
80
40
0
100 mg/kg
33 mg/kg
11 mg/kg
3 mg/kg
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
80
100
100
80
100
100
80
100
100
80
100
20
0
60
0
40
40
20
0
7.0
40
40
40
0
1 mg/kg
40
0
c
15
200 mg/kg
100 mg/kg
33 mg/kg
11 mg/kg
3 mg/kg
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
80
100
100
100
100
60
40
60
60
60
0
0
0
0
0
38.7
1 mg/kg
60
40
0
e
CQ 30 mg/kg
vehicle
100
100
100
100
100
40
100
20
100
0
100
100
100
100
100
100
100
100
20
NA
NA
a
Compounds were formulated in a vehicle composed of polyethylene glycol 300, propylene glycol, and water (weight ratio, 55/25/20), and dosed by
b
c
oral administration once a day for 4 consecutive days. D4 to d20 represent the days of experiment. Compound 15 was tested in a separate
experiment, which was stopped on day 18. Mice in the chloroquine (CQ) control group had 100% survival on day 18 and those in the vehicle
d
control group had 100% survival on day 4 and 0% on day 5. ED90s were based on comparisons of parasitemias with those of mice treated only with
e
vehicle on day 4 after the onset of therapy; Not applicable.
composed of buffer A (H2O containing 0.1% phosphoric acid) and
buffer B (CH3CN). The column was eluted with a gradient of 95%
In summary, we identified and characterized a benzoxaborole
buffer A and 5% buffer B to 40% buffer A and 60% buffer B over 10
CONCLUSION
■
chemical series that was highly potent against P. falciparum.
Carboxylic ester 6-(2-(alkoxycarbonyl)pyrazinyl-5-oxy)-1,3-di-
min, followed by 40% buffer A and 60% buffer B for 1 min, followed
by a gradient over 1 min to 95% buffer A and 5% buffer B that was
hydro-1-hydroxy-2,1-benzoxaboroles (9, 27−34) demonstrated
potent in vitro activity, with IC50 values of 0.2 to 22 nM against
cultured parasites. Compound 9 also showed excellent in vivo
efficacy (ED90 = 7.0 mg/kg).
maintained for three more minutes at a flow rate of 1.0 mL/min with a
column temperature of 30 °C and UV detection at 220 and 254 nm.
Silica gel 60 nm (200−425 mesh, Thermo Fisher Scientific, Waltham,
Massachusetts, USA) was used for flash column chromatography.
Pharmacokinetic study with experimental animals was designed and
performed with reference to the guidelines of the Institutional Animal
Care and Use Committee (Anacor Pharmaceuticals, Inc., AN-7−2007)
and reviewed by members of the committee. The in vivo efficacy work
with experimental animals was performed in accordance with the
institutional guidelines as defined by the Institutional Animal Care and
Use Committee (UCSF, AN109984).
Methods for Testing in Vitro Antimalarial Activity. W2 strain
P. falciparum parasites were cultured in human erythrocytes and
RPMI-1640 culture media with either 10% human serum or 0.5%
Albumax serum substitute under 3% O2, 5% CO2, and 92% N2.
Parasites synchronized to ring stage by treatments with 5% D-sorbitol
were cultured with serial dilutions of benzoxaboroles from 5 to 10 mM
stocks in 96-well microplate cultures including 200 μL of media/well,
2% hematocrit, and 1% parasitemia. At the completion of 48 h
incubations, when untreated cultures contained new rings, parasites
were fixed with 2% formaldehyde for 48 h, and 5 μL aliquots were
transferred to another 96-well plate containing 150 μL/well of staining
solution (100 mM NH4Cl, 0.1% Triton X-100, and 1 nM YOYO-1 in
EXPERIMENTAL SECTION
■
General Methods. Starting materials and solvents purchased from
chemical companies were used without further purification except
where noted. High performance liquid chromatography (HPLC) was
used to determine the purity of the compounds synthesized. The data
confirmed that the target compounds generally had ≥95% of purity
with exceptions where noted. Proton (1H) NMR spectra were
recorded at room temperature on Bruker 300 or 400 instruments
(Bruker Corporation, Billerica,Massachusetts, USA) using DMSO-d6
as solvent. Chemical shifts are given in parts per million (ppm).
Electrospray ionization−mass spectrometry (ESI−MS) and LC−MS
were carried out on an API2000 (AB Sciex, Framingham,
Massachusetts, USA) or a Finnigan LCQ mass spectrometer (Thermo
Fisher Scientific, Waltham, Massachusetts, USA). HPLC was
conducted on an Agilent 1200 system (Agilent Technologies, Santa
Clara, California, USA) using a BDS Hypersil C-18 column (150 mm
× 4.6 mm, 5 μm, 120 nm pore size, Thermo Fisher Scientific,
Waltham, Massachusetts, USA). The mobile phase used was
F
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PBS). Parasites per erythrocyte were then determined by flow
cytometry from plots of forward scatter against fluorescence
(excitation 488 nm, emission 520 nm) using a FacSort flow cytometer
(Beckton Dickinson) equipped with an AMS Loader (Cytek
Development). All values were normalized to percent control activity,
and IC50s were calculated using the Prism 3.0 program (GRAPHPAD
Software). Goodness of fit was assessed by R2 values, and meaningful
dose−response curves yielded R2 values >0.95.
weight) were infected by the intraperitoneal injection of 6 × 106
Plasmodium berghei-infected erythrocytes collected from a previously
infected animal. Mice were treated once daily by oral gavage with
various concentrations of test compounds, beginning 1 h after initial
infection, for 4 consecutive days. All compounds were formulated in a
vehicle composed of polyethylene glycol 300, propylene glycol and
water (weight ratio, 55/25/20), and administered in a total volume of
100 μL. Beginning on day 4 after infection, parasitemias were counted
daily by examination of Giemsa stained smears. All counts within a
single experiment were performed by the same investigator. The ED90
of the compounds was defined as the mean dose (mg/kg; 5 mice per
dose tested) that reduced parasitemia by 90% compared to that in
vehicle treated controls 4 days after the initiation of therapy. The ED90
was estimated using nonlinear regression analysis with Graphpad
Prism 5 software. When parasitemias exceeded 50%, animals were
euthanized. Chloroquine (30 mg/kg) was used as a positive control.
Animal survival and morbidity was monitored for 21 days post-
infection.
Ethyl 6-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-
yloxy)nicotinate (3). To a solution of 36 (1.2 g, 8.0 mmol) in 1,4-
dioxane (100 mL) was added KHMDS (48 mL, 0.5 M in toluene, 24
mmol, 3 equiv) at 0 °C. The reaction was stirred at room temperature
(rt) for 15 min, and then intermediate 37 (2.97g, 16 mmol) was added
at 0 °C. The reaction mixture was stirred at 80 °C for 22 h and
quenched by adding cold brine at 0 °C. The solution pH was adjusted
to 3 with 1 N HCl, and the mixture was extracted with ethyl acetate
(EA). The organic phase was separated, combined, washed with brine,
dried over anhydrous Na2SO4, and filtered. The residue after rotary
evaporation was purified by column chromatography (silica gel,
DCM:MeOH = 40:1, v/v) to the final desired product 3 (0.109 g,
yield 4.6%) as a light-yellow solid. 1H NMR (300 MHz, DMSO-d6): δ
9.22 (s, 1H), 8.68 (s, 1H), 8.32 (d, 1H), 7.50−7.45 (m, 2H), 7.32 (d,
1H), 7.17 (d, 1H), 5.01 (s, 2H), 4.35 (q, 2H), 1.35 (t, 3H) ppm.
HPLC area: 98.2% at 220 nm and 99.3% at 254 nm. MS: m/z = 300
(M + 1, ESI+).
Methyl 5-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-
yloxy)picolinate (5) and 5-(1-Hydroxy-1,3-dihydrobenzo[c]-
[1,2]oxaborol-6-yloxy)picolinic Acid (4). To a solution of 40
(3.0 g, 21.56 mmol, 1.1 equiv) in DMSO (33 mL) were added 41 (4.0
g, 19.61 mmol) and Cs2CO3 (8.1 g, 58.83 mmol, 3 equiv) in one
portion at rt. The reaction mixture was stirred at 80 °C overnight.
After completion, the reaction mixture was poured into 2 N HCl (300
mL) and extracted with EA (3 × 100 mL). The organic phases were
washed with brine, dried over MgSO4, filtered, and evaporated to a
solid (6 g). The residue was purified by column chromatography to
the desired product 42 as a white solid (4.0 g, 63% yield). To a
solution of 42 (6.3 g, 19.56 mmol) in MeOH (12 mL) was added
SOCl2 (3.6 mL, 48.90 mmol, 2.5 equiv) under N2. Then the reaction
was heated to 60 °C and stirred overnight. The mixture was
evaporated to a residue, which was poured into NaHCO3 (50 mL) and
extracted with EA (3 × 20 mL). The combined organic phases was
washed with brine, dried over Na2SO4, filtered, and evaporated to 43
as solid (3.8 g, 93% yield). To a solution of 43 (0.8 g, 2.38 mmol),
Pin2B2 (0.9 g, 3.57 mmol, 1.5 equiv), and KOAc (0.7 g, 7.14 mmol, 3
equiv) in 1,4-dioxane (12 mL) was added Pd(PPh3)2Cl2 (170 mg, 0.24
mmol, 0.1 equiv) under N2. The reaction was heated at 100 °C for 0.5
h. Then the reaction was cooled to rt, poured into water (100 mL),
and extracted with EA (3 × 3 mL). The combined organic phases were
washed with brine, dried over Na2SO4, filtered, and evaporated to a
residue. The residue was purified by silica gel column (PE:EA = 5:1)
to the desired product 44 (0.5 g, 62% yield) as an oil. To a solution of
44 (0.4 g, 1.04 mmol) in MeOH (5.2 mL) was added NaBH4 (40 mg,
1.04 mmol, 1 equiv) in portions at 0 °C. The reaction was stirred at rt
for 1 h. The reaction was quenched with 1 N HCl (4 mL), stirred for
another 0.5 h, and extracted with EA (3 × 20 mL). The combined
organic phase was washed with brine, dried over Na2SO4, filtered, and
evaporated. The residue was purified by column chromatography to
the desired product 5 as white solid (0.2 g, 71% yield). 1H NMR (300
MHz, DMSO-d6): δ 9.26 (s, 1H), 8.50 (d, 1H), 8.07 (d, 1H), 7.54−
7.32 (m, 4H), 5.02 (s, 2H), 3.86 (s, 3H) ppm. HPLC purity: 99.6% at
Inhibition of 3D7 strain parasites was assessed using the [3H]-
hypoxanthine incorporation assay.26 Briefly, parasites were cultured in
human erythrocytes (from the Spanish Red Cross blood bank) using
RPMI-1640 culture media (Gibco) supplemented with 0.5% Albumax
II (Invitrogen), 2% D-sucrose (Sigma-Aldrich), 0.3% glutamine
(Sigma-Aldrich), and 5 mM hypoxanthine (Sigma-Aldrich). Cultures
were maintained at 37 °C at an atmosphere of 5% O2, 5% CO2, and
95% N2. To assess inhibition, asynchronous parasite cultures with 0.5%
parasitemia and 2% hematocrit were exposed to 3-fold serial dilutions
of test compounds for 24 h in 96-well plate cultures (Costar no. 3894).
After 24 h, [3H]-hypoxanthine was added, plates were incubated for an
additional 24 h, and parasites were harvested on glass fiber filters
(Wallac no. 1450-421) using a Cell Harvester 96 (TOMTEC,
PerkinElmer). Filters were dried on scintillator sheets (MeltiLex A,
PerkinElmer no. 1450-441) to determine incorporation of [3H]-
hypoxanthine. Radioactivity was measured using a microbeta counter
(PerkinElmer). Data are normalized using the incorporation of the
positive control (infected erythrocytes without drug). IC50 values were
determined using Excel and Grafit 5 software. Values were determined
from at least three independent experiments, and standard deviations
were calculated.
Method for Testing in Vitro Stability in Liver Microsomes.
The aliquot of benzoxaborole sample stock solution (1 mg/mL in
DMSO) was added to a 96-well plate containing mouse or human liver
microsomes in the presence or absence of NADPH. The final
concentrations of benzoxaborole, NADPH, and liver microsomes were
1 μM benzoxaborole, 1 mM NADPH, and 0.5 mg/mL microsomal
protein in 100 mM potassium phosphate buffer (pH 7.4), and the total
volume was 200 μL per well. All samples were tested in duplicate. One
set of preincubation samples (absolute zero) of benzoxaborole was
initially stored at −80 °C during the incubation period to rule out
thermal instability and served as a control for the initial concentration.
Incubations of benzoxaborole in liver microsomes without NADPH
served as negative controls, while incubations of liver microsomes
only, with or without NADPH, served as background controls for
metabolite identification experiments. The samples were incubated at
37 °C in a shaking water-bath, and samples were obtained at 0, 15, 30,
60, and 120 min. As a positive control, dextromethorphan prepared by
spiking hepatocytes with 10 mM DMSO stock solution was incubated
in the presence of NADPH. The final concentration of dextro-
methorphan was 5 μM, and the total incubation volume was 200 μL
per well. The samples were incubated at 37 °C in a shaking water-bath,
and the sampling times were 0, 15, 30, 60, and 120 min. All
incubations were terminated by addition of 600 μL of cold acetonitrile.
An additional 80 μL of cold acetonitrile containing the internal
standard at 1 μg/mL was then added. The samples were vortexed for
10 min and centrifuged for 20 min at approximately 4000 rpm. The
supernatants were analyzed by LC/MS/MS.
Method for Pharmacokinetics Measurement. Adult female
CD-1 mice received the benzoxaborole test material either by
intravenous injection or by oral gavage in formulated solutions. For
benzoxaborole 8, the doses were 10 mg/kg for iv and 30 mg/kg for
oral, and the formulation used 100% saline. For benzoxaborole 9, the
dose was 5 mg/kg for both iv and oral, and the formulation used
polyethylene glycol 400, dimethylacetamide, and water (ratio 45/10/
45, pH = 5.8). For benzoxaborole 15, the dose was also 5 mg/kg for
both iv and oral, and the formulation used polyethylene glycol 300,
propylene glycol, and water (ratio 55/25/20, pH = 5.9). Blood
samples were collected and analyzed for drug content using HPLC
coupled to tandem mass spectrometry.
Method for Testing in Vivo Efficacy Using a P. berghei
Mouse Model. Female Swiss Webster Mice (average of 20 g body
G
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220 nm and 99.3% at 254 nm. Mass: m/z = 286.1 (M + 1, ESI+). To a
solution of 5 (0.3 g, 1.05 mmol) in 2-propanol (8.9 mL) was added
LiOH·H2O (88 mg, 2.10 mmol, 2 equiv) in H2O (2.6 mL) in one
portion at 0 °C. The reaction mixture was stirred at 40 °C for 2 h. The
reaction was quenched with 1 N HCl (2 mL) and extracted with EA (3
× 5 mL). The combined organic phase was washed with brine, dried
over Na2SO4, filtered, and evaporated. The residue was purified by
column chromatography to the desired product 4 as white solid (0.2 g,
74% yield). 1H NMR (300 MHz, DMSO-d6): δ 13.00 (br s, 1H), 9.37
(s, 1H), 8.44 (s, 1H), 8.02 (d, 1H), 7.52−7.28 (m, 4H), 4.99 (s, 2H)
ppm. HPLC area: 100% at 220 nm and 100% at 254 nm. Mass: m/z =
272 (M + 1, ESI+).
with 1 N HCl, and extracted with EA (2 × 50 mL). The organic phases
were combined, washed with brine, dried over anhydrous Na2SO4, and
filtered. The residue after rotary evaporation was purified by column
1
chromatography to the product 12 (210 mg, 27% yield). H NMR
(300 MHz, DMSO-d6): δ 9.17 (s, 1H), 8.43 (s, 1H), 8.17 (s, 1H),
7.46−7.43 (m, 2H), 7.26 (d, 1H), 5.46 (t, 1H), 4.98 (s, 2H), 4.55 (d,
2H) ppm. HPLC area: 94% at 254 nm. Mass: m/z = 259 (M + H, ESI
+).
6-(5-(Aminomethyl)pyrazin-2-yloxy)benzo[c][1,2]oxaborol-
1(3H)-ol Hydrochloride (13). To a solution of LiAlH4 (285 mg, 7.5
mmol, 3 equiv) in THF (8 mL) was added a solution of 11 (630 mg,
2.5 mmol, 1 equiv) in THF (4.5 mL). The reaction mixture was stirred
at 0 °C for 50 min, diluted with H2O (5 mL), dissolved with MeOH
(50 mL), and filtered. The residue after rotary evaporation was
purified by column chromatography to a solid. The solid was stirred
with 4 N HCl/MeOH and then concentrated to the desired solid
Methyl 6-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-
yloxy)pyridazine-3-carboxylate (7). To a solution of 38 (172
mg, 1 mmol, 1 equiv) in DMF (4 mL) were added 36 (150 mg, 1
mmol, 1 equiv) and Cs2CO3 (813 mg, 2.5 mmol, 2.5 equiv) at rt. The
reaction was stirred at 50 °C for 2 h, poured into 2 N HCl (100 mL),
and extracted with EA (3 × 50 mL). The combined organic phase was
washed with brine, dried over MgSO4, filtered, and concentrated in
vacuo. The residue was purified by column chromatography to the
desired product 7 (40 mg, 14% yield) as a white solid. 1H NMR (300
MHz, DMSO-d6): δ 9.30 (s, 1H), 8.25 (d, 1H), 7.51−7.61 (m, 3H),
7.39 (d, 1H), 5.04 (s, 2H), 3.93 (s, 3H) ppm. HPLC area: 96.1% at
220 nm and 96.5% at 254 nm. Mass: m/z = 287 (M + 1, ESI+).
6-(Pyrazin-2-yloxy)benzo[c][1,2]oxaborol-1(3H)-ol (10). To a
solution of 39 (0.3 g, 2.8 mmol, 1.5 equiv) in DMF (10 mL) were
added Cs2CO3 (1.5 g, 4.75 mmol, 2.5 equiv) and 36 (0.28 g, 1.9
mmol). The reaction mixture was stirred at 75 °C for 0.5 h. After
completion, the reaction was diluted with H2O (20 mL) and extracted
with EA (2 × 10 mL). The combined organic phase was washed with
brine (20 mL), dried over anhydrous Na2SO4, and filtered. The
residue after rotary evaporation was purified by column chromatog-
1
product 13 (22 mg, 27% yield). H NMR (300 MHz, DMSO-d6): δ
9.28 (br s, 1H), 8.63 (s, 1H), 8.42 (br s, 3H), 8.30 (s, 1H), 7.51−7.49
(m, 2H), 7.30 (d, 1H), 5.02 (s, 2H), 4.19 (q, 2H) ppm. HPLC area:
97.5% at 220 nm. Mass: m/z = 258 (M + H, ESI+).
6-(5-((Dimethylamino)methyl)pyrazin-2-yloxy)benzo[c][1,2]-
oxaborol-1(3H)-ol (14). A solution of 45 (1.58g, 10 mmol) in SOCl2
(33.3 mL) was stirred at 80 °C for 2 h and then concentrated under
reduced pressure. MeOH (20 mL) was added and stirred at 50 °C for
2 h. The mixture was concentrated in vacuo. The residue was mixed
with H2O (30 mL) and extracted with EA (2 × 10 mL). The organic
phases were combined, washed with brine, dried over MgSO4, filtered,
and concentrated. The residue was purified by column chromatog-
raphy on silica gel (EA:PE = 10%) to the methyl ester 48 (1.4 g, 81%
yield). To a solution of 48 (500 mg, 2.9 mmol) in THF (26 mL) was
added DIBAL-H (9 mL, 9 mmol, 3.1 equiv) dropwise at −78 °C with
good stirring. The reaction was stirred at −78 °C for 2 h, poured into
water (300 mL), and acidified with 1 N HCl to pH = 3. The resulted
solution was extracted with EA (2 × 100 mL). The organic phases
were combined, washed with brine, dried over MgSO4, filtered, and
evaporated. It was purified by column chromatography on silica gel
(EA:PE = 20%) to 49 (205 mg, 49% yield). To a solution of 49 (105
mg, 0.733 mmol, 1.1 equiv) and 36 (100 mg, 0.667 mmol) in DMF
(2.2 mL) was added Cs2CO3 (434 mg, 1.33 mmol, 2 equiv). The
reaction mixture was stirred at 50 °C for 1 h, poured into water, and
extracted with EA (2 × 20 mL). The organic phases were washed with
brine, dried over MgSO4, filtered, and evaporated. Then the residue
was washed with n-hexane and dried in vacuo to the product 50 (110
mg, 64% yield). A solution of 50 (60 mg, 0.234 mmol) and
dimethylamine hydrochloride (23 mg, 0.281 mmol, 1.2 equiv) in
DCM (1.2 mL) was stirred at rt for 1.5 h. Then NaBH(OAc)3 (198
mg, 0.94 mmol, 4 equiv) was added, and the reaction mixture was
stirred at rt overnight. It was triturated with DCM (20 mL), filtered,
and the filtrate concentrated. The residue was purified by column
chromatography on silica gel (MeOH:DCM = 10%) to the product 14
(38 mg, 56% yield). 1H NMR (300 MHz, DMSO-d6): δ 9.21 (s, 1H),
8.51 (s, 1H), 8.19 (s, 1H), 7.49−7.47 (m, 2H), 7.31 (d, 1H), 5.01 (s,
2H), 3.72 (s, 2H), 2.31 (s, 6H) ppm. HPLC area: 98.1% at 220 nm
and 97% at 254 nm. Mass: m/z = 286 (M + H, ESI+).
1
raphy to the desired product 10 (50 mg, 8% yield). H NMR (300
MHz, DMSO-d6): δ 9.24 (s, 1H), 8.57 (d, 1H), 8.37 (d, 1H), 8.19 (dd,
1H), 7.49−7.47 (m, 2H), 7.31 (dd, 1H), 5.01 (s, 2H) ppm. HPLC
area: 96.5% at 220 nm and 95.9% at 254 nm. Mass: m/z = 229 (M +
H, ESI+).
5-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-
pyrazine-2-carbonitrile (11). To a solution of 45 (1.0 g, 6.3 mmol,
1 equiv) in DCM (31 mL) was added SOCl2 (31 mL) at rt. The
reaction was heated to 50 °C overnight and then concentrated. The
residue after rotary evaporation was mixed with NH3·H2O and then
extracted with DCM (20 mL). The organic phase was washed with
saturated Na2CO3 (50 mL) and H2O (50 mL), dried over anhydrous
MgSO4, and filtered. The residue after rotary evaporation was purified
by column chromatography to the amide compound 46 (0.6 g, 67%
yield). To a solution of 46 (0.6 g, 3.80 mmol) in DMF (10 mL) was
added (CNCl)3 (0.84g, 4.56 mmoL, 1.2 equiv) at rt. The mixture was
stirred at rt for 1 h before H2O (100 mL) was added. The mixture was
extracted with EA (3 × 40 mL). The organic phases were combined,
washed with 0.5 N HCl (2 × 50 mL), saturated Na2CO3 (2 × 50 mL),
and brine (300 mL), dried over anhydrous MgSO4, and filtered. The
residue after rotary evaporation was purified by column chromatog-
raphy to 47 (0.3 g, 60% yield). To a solution of 47 (110 mg, 0.80
mmol, 1.2 equiv) in DMF (2.2 mL) were added 36 (100 mg, 0.67
mmol) and Cs2CO3 (0.54g, 1.68 mmol, 2.5 equiv) in one portion at rt.
The reaction mixture was stirred at 50 °C for 2 h, poured into 2 N
HCl (100 mL), and then extracted with EA (3 × 50 mL). The organic
phases were combined, washed with brine, dried over MgSO4, filtered,
and evaporated to a solid (150 mg). The residue was purified by
column chromatography to the desired product 11 as a white solid (40
mg, 25% yield). 1HNMR (300 MHz, DMSO-d6): δ 9.26 (s, 1H), 8.76
(s, 1H), 8.74 (s, 1H), 7.51−7.48 (m, 2H), 7.36 (dd, 1H), 5.01 (s, 2H)
ppm. HPLC area: 99.2% at 220 nm and 98.6% at 254 nm. Mass: m/z =
505 (2M − 1, ESI−).
5-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-
pyrazine-2-carboxamide (15). A solution of 46 (516 mg, 3 mmol),
36 (450 mg, 3 mmol, 1 equiv), and Cs2CO3 (2.46g, 7.5 mmol, 2.5
equiv) in DMF (6 mL) was stirred at 50 °C for 2 h. It was poured into
1 N HCl (100 mL) and extracted with EA (4 × 100 mL). The
combined organic phase was washed with brine, dried over MgSO4,
filtered, and concentrated in vacuo. The residue was purified by
column chromatography to the desired product 15 (460 mg, 56%
yield) as a solid. 1H NMR (300 MHz, DMSO-d6): δ 9.24 (s, 1H), 8.70
(s, 1H), 8.57 (s, 1H), 8.08 (br s, 1H), 7.71 (br s, 1H), 7.52−7.49 (m,
2H), 7.35 (dd, 1H), 5.02 (s, 2H) ppm. HPLC area: 98.1% at 220 nm
and 98.8% at 254 nm. Mass: m/z = 272 (M + 1, ESI+).
6-(5-(Hydroxymethyl)pyrazin-2-yloxy)benzo[c][1,2]-
oxaborol-1(3H)-ol (12). To a suspension of LiAlH4 (342 mg, 9
mmol, 3 equiv) in THF (10 mL) was added dropwise a solution of 9
(860 mg, 3.0 mmol) in THF (5 mL). The reaction mixture was stirred
at 0 °C for 50 min, diluted with H2O (40 mL), adjusted to pH = 3
5-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-N-
methylpyrazine-2-carboxamide (16). This compound was pre-
pared by the similar method described above for the synthesis of 15.
1H NMR (300 MHz, DMSO-d6): δ 9.24 (s, 1H), 8.72−8.70 (m, 1H),
H
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8.68 (s, 1H), 8.58 (s, 1H), 7.52−7.49 (m, 2H), 7.36 (dd, 1H), 5.03 (s,
2H), 2.82 (d, 3H) ppm. HPLC area: 100.0% at 220 nm and 96.5% at
254 nm. Mass: m/z = 286 (M + H, ESI+) and 308 (M + Na, ESI+).
N-Ethyl-5-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-
yloxy)pyrazine-2-carboxamide (17). This compound was prepared
4 mmol), 2-amino-2-methylpropan-1-ol (430 mg, 4.8 mmol, 1.2
equiv), HOBT (702 mg, 5.2 mmol, 1.3 equiv), and EDC (1g, 5.2
mmol, 1.3 equiv) in DCM (40 mL, c = 0.1) was added TEA (1.2 g, 12
mmol, 3 equiv). The reaction was stirred at rt for 30 min, and a normal
workup resulted in the desired product 57 (670 mg, 80% yield) as
yellow oil. To SOCl2 (6 mL) was added 57 (400 mg, 1.65 mmol), and
the solution was stirred at rt for 2 h. The reaction solution was
concentrated to dryness, and the residue was dissolved in DCM (30
mL), adjusted to pH 8−9 with saturated NaHCO3, and then
separated. The organic phase was washed with brine, dried over
MgSO4, filtered, and concentrated in vacuo. The residue was purified
by column chromatography to the desired product 58 (180 mg, 50%
yield) as a white solid. To a solution of 58 (85 mg, 0.4 mmol) in DMF
(4 mL) were added 36 (60 mg, 0.4 mmol, 1 equiv) and Cs2CO3 (325
mg, 1 mmol, 2.5 equiv) at rt. The reaction mixture was stirred at 60 °C
for 2 h, poured into 2 N HCl (100 mL), and extracted with EA (3 ×
50 mL). The combined organic phase was washed with brine, dried
over MgSO4, filtered, and concentrated in vacuo. The residue was
purified by column chromatography to the desired product 24 (12 mg,
1
by the similar method described above for the synthesis of 15. H
NMR (500 MHz, DMSO-d6): δ 9.25 (s, 1H), 8.77 (t, 1H), 8.68 (s,
1H), 8.58 (s, 1H), 7.52−7.49 (m, 2H), 7.35 (dd, 1H), 5.03 (s, 2H),
3.37−3.27 (m, 2H), 1.15 (t, 3H) ppm. HPLC area: 99% at 220 nm
and 99.3% at 254 nm. Mass: m/z = 300 (M + H, ESI+).
5-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-
N,N-dimethylpyrazine-2-carboxamide (18). This compound was
prepared by the similar method described above for the synthesis of
1
15. H NMR (300 MHz, DMSO-d6): δ 9.23 (s, 1H), 8.54 (s, 1H),
8.38 (s, 1H), 7.51−7.49 (m, 2H), 7.35 (dd, 1H), 5.02 (s, 2H), 3.02 (s,
6H) ppm. HPLC area: 99.6% at 220 nm and 99.2% at 254 nm. Mass:
m/z = 300 (M + 1).
5-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-N-
(2-hydroxyethyl)pyrazine-2-carboxamide (19). This compound
was prepared by the similar method described above for the synthesis
1
12% yield) as a white solid. H NMR (300 MHz, DMSO-d6): δ 9.25
1
of 15. H NMR (300 MHz, DMSO-d6): δ 9.29 (s, 1H), 8.69 (s, 1H),
(s, 1H), 8.65 (s, 1H), 8.62 (s, 1H), 7.48−7.51 (m, 2H), 7.35 (dd, 1H),
5.02 (s, 2H), 4.14 (s, 2H), 1.30 (s, 6H) ppm. HPLC area: 95.3% at
220 nm and 94.9% at 254 nm. Mass: m/z = 326 (M + H, ESI+).
6-(5-(5-Methyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yloxy)benzo-
[c][1,2]oxaborol-1(3H)-ol (25). To a solution of 48 (172 mg, 1
mmol) in MeOH (7 mL) was added a solution of hydrazine hydrate
(0.11 mL, 2 mmol, 2 equiv) in MeOH dropwise at 0 °C. The reaction
mixture was stirred for 16 h, filtered, and concentrated to the desired
product 59 (100 mg, 58% yield). To a solution of 59 (100 mg, 0.58
mmol) and NMM (203 mg, 1.74 mmol, 3 equiv) in DCM (5 mL) was
added AcCl (46 mg, 0.58 mmol, 1 equiv) dropwise. The mixture was
stirred at rt for 1 h, diluted with H2O (10 mL), and extracted with
DCM (20 mL). The organic phase was washed with 1 N HCl (10
mL), 1N NaHCO3 (15 mL), and brine (15 mL), dried over anhydrous
Na2SO4, and filtered. The residue after rotary evaporation was purified
by column chromatography to the desired product 60 (95 mg, 76%
yield). The solution of 60 (90 mg, 0.42 mmol, 1 equiv) in POCl3 (1.8
mL) was stirred at 110 °C for 0.5 h. After completion, the reaction was
diluted with ice water (10 mL), washed with 1N NaHCO3 (10 mL),
and extracted with EA (15 mL). The organic phase was washed with
brine (10 mL), dried over anhydrous Na2SO4, and filtered. The
residue after rotary evaporation was purified by column chromatog-
raphy to the desired product 61 (62 mg, 76% yield). To a solution of
61 (60 mg, 0.306 mmol) in DMF (1 mL) were added Cs2CO3 (251
mg, 0.765 mmol, 2.5 equiv) and 36 (46 mg, 0.306 mmol, 1 equiv).
The reaction mixture was stirred at 80 °C for 45 min, diluted with
H2O (10 mL), and extracted with EA (10 mL). The organic phase was
washed with brine (10 mL), dried over anhydrous Na2SO4, and
filtered. The residue after rotary evaporation was purified by column
8.66−8.61 (m, 2H), 7.51−7.49 (m, 2H), 7.36 (dd, 1H), 5.03 (s, 2H),
4.82 (t, 1H), 3.55−3.49 (m, 2H), 3.40−3.35 (m, 2H) ppm. HPLC
area: 97.8% at 220 nm and 99.0% at 254 nm. Mass: 316 (M + 1, ESI
+).
N-(2-(Dimethylamino)ethyl)-5-(1-hydroxy-1, 3-
dihydrobenzo[c][1,2]oxaborol-6-yloxy)pyrazine-2-carboxa-
mide hydrochloride (20). This compound was prepared by the
similar method described above for the synthesis of 15 and was
converted to HCl salt. 1H NMR (300 MHz, DMSO-d6): δ 9.63 (br s,
1H), 9.27 (s, 1H), 9.05 (t, 1H), 8.73 (s, 1H), 8.65 (s, 1H), 7.53−7.50
(m, 2H), 7.35 (d, 1H), 5.03 (s, 2H), 3.62−3.68 (m, 2H), 3.28−3.32
(m, 2H), 2.82 (s, 6H) ppm. HPLC area: 99.5% at 220 nm and 99.6%
at 254 nm. Mass: m/z = 343 (M + 1, ESI+).
5-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)-N-
(isopropylsulfonyl)pyrazine-2-carboxamide (21). This com-
pound was prepared by the similar method described above for the
1
synthesis of 15. H NMR (300 MHz, DMSO-d6): δ 11.87 (br s, 1H),
9.25 (s, 1H), 8.76 (s, 1H), 8.65 (s, 1H), 7.54−7.50 (m, 2H), 7.37 (dd,
1H), 5.03 (s, 2H), 3.78 (septet, 1H), 1.33 (d, 6H) ppm. HPLC area:
96.7% at 220 nm and 97.9% at 254 nm. Mass: m/z = 378 (M + 1, ESI
+).
(E)-5-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-
yloxy)pyrazine-2-carbaldehyde oxime (22). To a solution of 50
(100 mg, 0.39 mmol) and hydroxylamine hydrochloride (31 mg, 0.45
mmol, 1.14 equiv) in EtOH (1.36 mL) was added sodium hydroxide
solution (3N, 0.26 mL). The reaction mixture was stirred at rt for 2 h,
poured into water (5 mL), and extracted with EA (2 × 10 mL). The
organic phases were combined, washed with brine, dried over MgSO4,
filtered, and concentrated. The residue was purified by column
chromatography on silica gel (MeOH:DCM = 1%) to 22 (38 mg, 36%
yield). 1H NMR (300 MHz, DMSO-d6): δ 11.77 (s, 1H), 9.25 (s, 1H),
8.56 (s, 1H), 8.52 (s, 1H), 8.14 (s, 1H), 7.50−7.47(m, 2H), 7.35 (d,
1H), 5.02 (s, 2H) ppm. HPLC area: 94.6% at 220 nm and 98.8% at
254 nm.
1
chromatography to the desired product 25 (35 mg, 50% yield). H
NMR (300 MHz, DMSO-d6): δ 9.25 (s, 1H), 8.84 (s, 1H), 8.73 (s,
1H), 7.54−7.49 (m, 2H), 7.36 (dd, 1H), 5.01 (s, 2H), 2.60 (s, 3H)
ppm. HPLC area: 96.8% at 220 nm and 95.5% at 254 nm. Mass: m/z =
311 (M + H, ESI+) and 333 (M + Na).
(Z)-N′-Hydroxy-5-(1-hydroxy-1,3-dihydrobenzo[c][1,2]-
oxaborole-6-yloxy)pyrazine-2-carboximidamide (23). To a
solution of 11 (250 mg, 0.99 mmol) and hydroxylamine hydrochloride
(137 mg, 1.98 mmol, 2.0 equiv) in H2O (3 mL) was added a solution
of Na2CO3 (209 mg, 19.8 mmol, 2.0 equiv) in H2O (0.9 mL). The
reaction mixture was stirred at 40 °C for 3 h, poured into water (20
mL), and extracted with EA (4 × 15 mL). The organic phases were
combined, washed with brine, dried over MgSO4, filtered, and
concentrated. The residue was purified by column chromatography on
silica gel (MeOH:DCM = 1%) to the product 23 (34 mg, 12% yield).
1H NMR (300 MHz, DMSO-d6): δ 9.99 (s, 1H), 9.23 (s, 1H), 8.59 (s,
6-(5-(1H-Tetrazol-5-yl)pyrazin-2-yloxy)benzo[c][1,2]-
oxaborol-1(3H)-ol (26). To a solution of 11 (200 mg, 0.75 mmol) in
DMF (2 mL) were added NH4Cl (68 mg, 1.27 mmol, 1.7 equiv) and
NaN3 (82 mg, 1.27 mmol, 1.7 equiv). The reaction mixture was stirred
at 95 °C overnight, diluted with H2O (40 mL), and extracted with EA
(2 × 20 mL). The aqueous phase was adjusted to pH < 7 with 0.5 N
HCl and extracted with EA (2 × 30 mL). The combined organic
phases were washed with brine (2 × 30 mL), dried over anhydrous
Na2SO4, and filtered. The residue after rotary evaporation was purified
by column chromatography to the desired product 26 (40 mg, 18%
yield). 1H NMR (300 MHz, DMSO-d6): δ 9.32 (s, 1H), 9.06 (s, 1H),
7.18 (d, 1H), 7.09 (s, 1H), 6.87 (dd, 1H), 4.86 (s, 2H) ppm. HPLC
area: 99.8% at 220 nm and 93.9% at 254 nm. Mass: m/z = 319 (M +
Na, ESI+).
1H), 8.52 (s, 1H), 7.52−7.43 (m, 2H), 7.34 (dd, 1H), 5.88 (s, 2H),
5.02 (s, 2H) ppm. HPLC area: 100% at 220 nm and 98.2% at 254 nm.
Mass: m/z = 287 (M + H, ESI+).
6-(5-(4,4-Dimethyl-4,5-dihydrooxazol-2-yl)pyrazin-2-yloxy)-
benzo[c][1,2]oxaborol-1(3H)-ol (24). To a solution of 45 (632 mg,
Ethyl 5-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-
yloxy)pyrazine-2-carboxylate (27). A solution of 45 (2g, 12.62
I
DOI: 10.1021/acs.jmedchem.5b00678
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
mmol) in SOCl2 (42 mL) was stirred at 80 °C for 2 h and then
concentrated directly. The residue was dissolved with EtOH (40 mL)
and then stirred at 50 °C for 2 h. The reaction was evaporated to
dryness, poured into H2O (40 mL), and extracted with EA (3 × 50
mL). The combined organic phase was washed with brine, dried over
MgSO4, filtered, and concentrated in vacuo to give the ethyl ester 62
(2.0 g, 85% yield). The solution of 62 (205 mg, 1.1 mmol), 36 (150
mg, 1 mmol, 1 equiv), and Cs2CO3 (815 mg, 2.5 mmol, 2.5 equiv) in
DMF (3.3 mL) was stirred at 50 °C for 1 h. After completion, the
reaction was poured into water (30 mL), adjusted to pH = 3 with 2 N
HCl, and extracted with EA (3 × 20 mL). The combined organic
phase was washed with brine, dried over MgSO4, filtered, and
concentrated in vacuum. The residue was purified by column
2-Amino-2-methylpropyl 5-(1-Hydroxy-1,3-dihydrobenzo-
[c][1,2]oxaborol-6-yloxy)pyrazine-2-carboxylate Hydrochlor-
ide (35). To a solution of 58 (85 mg, 0.4 mmol, 1 equiv) in DMF
(2 mL) was added 36 (60 mg, 0.4 mmol) and Cs2CO3 (325 mg, 1
mmol, 2.5 equiv) at rt. The mixture was stirred at 80 °C for 1 h,
poured into 1 N HCl (20 mL) and concentrated directly to a residue.
The residue was purified by column chromatography to give 35 (17
1
mg, 17% yield) as a light-yellow solid. H NMR (300 MHz, DMSO-
d6): δ 9.27 (s, 1H), 9.04 (s, 1H), 8.70 (s, 1H), 8.23 (br s, 3H), 7.54−
7.48 (m, 2H), 7.35 (d, 1H), 5.01 (s, 2H), 4.32 (s, 2H), 1.33 (s, 6H)
ppm. HPLC area: 97.6% at 220 nm and 97.8% at 254 nm. Mass: m/z =
344 (M + H, ESI+).
ASSOCIATED CONTENT
* Supporting Information
1
chromatography to the desired product 27 (55 mg, 25% yield). H
■
S
NMR (500 MHz, DMSO-d6): δ 9.24 (s, 1H), 8.76 (s, 1H), 8.67 (s,
1H), 7.54−7.50 (m, 2H), 7.37 (dd, 1H), 5.03 (s, 2H), 4.36 (q, 2H),
1.33 (t, 3H) ppm. HPLC area: 95.8% at 220 nm and 95.6% at 254 nm.
Mass: m/z = 301 (M + H, ESI+).
Figures illustrating mice survival and efficacy ED90 data. The
Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jmedchem.5b00678.
Isopropyl 5-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-
6-yloxy)pyrazine-2-carboxylate (28). This compound was pre-
pared by following the methodology similar to that described above for
AUTHOR INFORMATION
Corresponding Author
*Phone: 650-543-7513. Fax: 650-543-7660. E-mail: yzhang@
anacor.com, yongkangzhang@yahoo.com.
■
1
27. H NMR (500 MHz, DMSO-d6): δ 9.23 (s, 1H), 8.74 (s, 1H),
8.65 (s, 1H), 7.53−7.50 (m, 2H), 7.36 (dd, 1H), 5.21−5.16 (septet,
1H), 5.03 (s, 2H), 1.33 (d, 6H) ppm. HPLC area: 97.4% at 220 nm
and 98.7% at 254 nm. Mass: m/z = 315 (M + H, ESI+).
Isobutyl 5-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-
yloxy)pyrazine-2-carboxylate (29). This compound was prepared
by following the methodology similar to that described above for 27.
1H NMR (500 MHz, DMSO-d6): δ 9.24 (s, 1H), 8.77 (s, 1H), 8.68 (s,
1H), 7.54−7.50 (m, 2H), 7.36 (dd, 1H), 5.03 (s, 2H), 4.11 (d, 2H),
2.06−2.00 (m, 1H), 0.97 (d, 6H) ppm. HPLC area: 98.3% at 220 nm
and 97.4% at 254 nm. Mass: m/z = 351 (M + Na, ESI+).
n-Butyl 5-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-
yloxy)pyrazine-2-carboxylate (30). This compound was prepared
by following the methodology similar to that described above for 27.
1H NMR (300 MHz, DMSO-d6): δ 9.24 (s, 1H), 8.74 (s, 1H), 8.65 (s,
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Medicines for Malaria Venture (MMV) and the
National Institutes of Health (AI095324) for financial support.
We also thank Dr. David Sullivan for calculating cLogD7.4
values of the compounds.
ABBREVIATIONS USED
■
NMR, nuclear magnetic resonance; HPLC, high performance
liquid chromatography; ESI, electrospray ionization; DMSO,
dimethyl sulfoxide; KHMDS, potassium hexamethyldisilazide;
EA, ethyl acetate; DCM, dichloromethane; TLC, thin layer
chromatography; PE, petroleum ether; DMF, N,N-dimethyl-
formamide; THF, tetrahydrofuran; m-CPBA, meta-chloroper-
benzoic acid; HOBT, hydroxybenzotriazole; EDC, 1-ethyl-3-(3-
(dimethylamino)propyl)carbodiimide; NMM, N-methylmor-
pholine
1H), 7.51−7.47 (m, 2H), 7.34 (dd, 1H), 5.01 (s, 2H), 4.30 (t, 2H),
1.72−1.63 (m, 2H), 1.45−1.33 (m, 2H), 0.90 (t, 3H) ppm. HPLC
area: 95.6% at 220 nm and 93.2% at 254 nm. Mass: m/z = 351 (M +
Na, ESI+).
tert-Butyl 5-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-
6-yloxy)pyrazine-2-carboxylate (31). This compound was pre-
pared by following the methodology similar to that described above for
1
27. H NMR (300 MHz, DMSO-d6): δ 9.26 (s, 1H), 8.71 (s, 1H),
8.64 (s, 1H), 7.51−7.49 (m, 2H), 7.35 (dd, 1H), 5.02 (s, 2H), 1.55 (s,
9H) ppm. HPLC area: 98.8% at 220 nm and 99.0% at 254 nm. Mass:
m/z = 351 (M + Na, ESI+).
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■
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27. H NMR (500 MHz, DMSO-d6): δ 9.29 (s, 1H), 8.84 (s, 1H),
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