Discovery of a Potent and Specific M. tuberculosis Leucyl-tRNA Synthetase Inhibitor: (S)-3-(Aminomethyl)-4-chloro-7-(2-hydroxyethoxy)benzo[c][1,2]oxaborol-1(3H)-ol (GSK656)

Article abstract of DOI:10.1021/acs.jmedchem.7b00631

There is an urgent need to develop new and safer antitubercular agents that possess a novel mode of action. We synthesized and evaluated a novel series of 3-aminomethyl 4-halogen benzoxaboroles as Mycobacterium tuberculosis (Mtb) leucyl-tRNA synthetase (L

Full text of DOI:10.1021/acs.jmedchem.7b00631

Article
pubs.acs.org/jmc
Discovery of a Potent and Specific M. tuberculosis Leucyl-tRNA
Synthetase Inhibitor: (S)‑3-(Aminomethyl)-4-chloro-7-(2-
hydroxyethoxy)benzo[c][1,2]oxaborol-1(3H)‑ol (GSK656)
Xianfeng Li,† Vincent Hernandez,† Fernando L. Rock,† Wai Choi,† Yvonne S. L. Mak,† Manisha Mohan,†
Weimin Mao,† Yasheen Zhou,† Eric E. Easom,† Jacob J. Plattner,† Wuxin Zou,‡ Esther Perez-Herran,§
́
́
Ilaria Giordano,§ Alfonso Mendoza-Losana,§ Carlos Alemparte,§ Joaquín Rullas,§
Inigo Angulo-Barturen,∥,§ Sabrinia Crouch,§ Fatima Ortega,§ David Barros,*,§ and M. R. K. Alley†
́
̃
†Anacor Pharmaceuticals, Inc., 1020 E. Meadow Circle, Palo Alto, California 94303, United States
‡BioDuro LLC, Building E, No. 29, Life Science Park Road, Beijing 102206, P. R. China
§GlaxoSmithKline, Tres Cantos Medicines Development Campus, Severo Ochoa 2, Tres Cantos, Madrid 28760, Spain
ABSTRACT: There is an urgent need to develop new and safer antitubercular
agents that possess a novel mode of action. We synthesized and evaluated a novel
series of 3-aminomethyl 4-halogen benzoxaboroles as Mycobacterium tuberculosis
(Mtb) leucyl-tRNA synthetase (LeuRS) inhibitors. A number of Mtb LeuRS
inhibitors were identified that demonstrated good antitubercular activity with
high selectivity over human mitochondrial and cytoplasmic LeuRS. Further
evaluation of these Mtb LeuRS inhibitors by in vivo pharmacokinetics (PK) and
murine tuberculosis (TB) efficacy models led to the discovery of GSK3036656
(abbreviated as GSK656). This molecule shows potent inhibition of Mtb LeuRS
(IC50 = 0.20 μM) and in vitro antitubercular activity (Mtb H37Rv MIC = 0.08 μM). Additionally, it is highly selective for the Mtb
LeuRS enzyme with IC50 of >300 μM and 132 μM for human mitochondrial LeuRS and human cytoplasmic LeuRS, respectively.
In addition, it exhibits remarkable PK profiles and efficacy against Mtb in mouse TB infection models with superior tolerability
over initial leads. This compound has been progressed to clinical development for the treatment of tuberculosis.
methoxymycolic and ketomycolic acid, which are components
of the mycobacterial cell wall.12 However, bedaquiline has a
boxed warning related to possible cardiac toxicity13 and
delamanid (OPC-67683) induces QTc prolongation.14 In
addition there is cross-resistance between clofazimine and
bedaquiline15 as well as some of pre-existing resistance to
delamanid.16 There has been an increased activity toward the
discovery of new antitubercular agents.17−22 Other TB drug
candidates that are in the clinical development include
moxifloxacin, sutezolid, SQ109, and Q20321,22 (Figure 1).
We recently identified a series of 3-aminomethyl-
benzoxaboroles that target Mtb LeuRS (1−4, Figure 2).23
These Mtb LeuRS inhibitors require the boron atom for Mtb
LeuRS activity since it forms a bidentate covalent adduct with
the terminal nucleotide of tRNA, Ade76. The resulting covalent
adduct traps the 3′ end of tRNALeu in the editing site in a
nonproductive complex, inhibiting leucylation and thus protein
synthesis. The amino group of the (S)-aminomethyl side chain
at C-3 is critical for binding as it makes three hydrogen bonding
interactions. An important finding from this work is that 4-
halogen atom (especially Cl and Br) significantly improves Mtb
LeuRS activity, antitubercular activity against Mtb H37Rv, and
selectivity against other bacteria. This effort led to the discovery
INTRODUCTION
■
Tuberculosis (TB) is a life-threatening infectious disease caused
by the bacteria Mycobacterium tuberculosis (Mtb). The World
Health Organization (WHO) estimates that one-third of the
world’s population is infected with Mtb, resulting in 1.4 million
deaths in 2015.1 In the same year, there were additional 0.4
million people infected with HIV who also died of TB disease.1
The current treatment for TB infection requires a combination
therapy of four front-line drugs rifampin, isoniazid, pyrazina-
mide, and ethambutol for 6−9 months, often leading to
significant side effects and poor patient compliance. In addition,
emergence of multidrug-resistant TB (MDR-TB)2 and
extensively drug-resistant TB (XDR-TB)3 has rendered many
front-line and second-line drugs ineffective. Furthermore,
totally drug-resistant TB (TDR-TB) has recently emerged
that is resistant to all clinical drugs.4 Therefore, there is an
urgent need to develop new antitubercular agents that are
effective against TB, which possess a novel mode of action that
circumvents these resistances.5−8
Bedaquiline and delamanid have recently been approved by
the Food and Drug Administration (FDA) or the European
Medicines Agency (EMA) to treat MDR-TB, representing the
first new TB drugs in more than 40 years.9,10 Bedaquiline acts
by the novel mechanism of mycobacterial adenosine 5′-
triphosphate (ATP) synthase inhibition,11 while delamanid is
a nitroimidazole derivative that primarily inhibits synthesis of
Received: May 5, 2017
© XXXX American Chemical Society
A
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Figure 1. Bedaquiline, delamanid, and selected TB drug candidates in clinical trials.
described in Scheme 1, starting from 2-bromo-3-hydroxy-
benzaldehyde 37. Phenol 37 was converted to phenyl ether 38
by alkylation with alkyl bromide or alkyl iodide (when R is an
alkyl group) or through copper-mediated arylation with
arylboronic acid (when R is an aryl group). Incorporation of
boron was accomplished by reaction of 38 with pinacol
diborane in the presence of a palladium catalyst to give
boronate 39 or by reaction of 41 with n-butyllithium and
trimethyl borate followed by acidic workup to give boronic acid
42. The Henry reaction of 39 or 42 with nitromethane afforded
3-nitromethylbenzoxaborole 40.25 Chlorination or bromination
can be achieved through 3-nitromethylbenzoxaborole 40 or
through tert-butoxycarbonyl (Boc) protected 3-aminomethyl-
benzoxaborole 43. The removal of Boc group of compound 44
or the reduction of nitro group in compound 45 provided
corresponding C-4 chloro or bromo analogues (11−20, 23, 24)
as racemates. Compounds 23 and 24 were further resolved by
chiral supercritical fluid chromatography (SFC) separation to
provide active enantiomers 23a and 24a, respectively.
The chemistry used to synthesize enantiopure 7-substituted
compounds 5a−10a, 21a, and 22a (class A) is described in
Scheme 2. This modular approach would introduce C-7
substitution in appropriate enantiopure benzoxaborole inter-
mediates at a late stage of the synthesis and give active
enantiomers, thus avoiding a lengthy multistep route and chiral
separation. The enantiopure compound 46 with 3-hydrox-
ypropoxy substitution at C-7 position was made according to
literature procedure.26 Dealkylation of 46 with BBr3 followed
by Boc protection provided the key intermediate 51 with 7-
hydroxy substitution. Initial attempts to alkylate phenol 51 with
an alkyl bromide under standard conditions (K2CO3, DMSO,
room temperature) failed to give any desired 7-alkyoxy product,
presumably due to the presence of the neighboring boron atom
that forms an intramolecular complex and protects the phenol
from further reaction. Finally we found that the reaction of 51
with an alkyl bromide in the presence of potassium tert-
butoxide at 90 °C in DMSO afforded 7-substituted compound
52. Subsequent chlorination or bromination of compound 52,
followed by Boc removal, provided the C-4 chloro or bromo
analogs 7a−10a. Compounds 21a and 22a were made by
chlorination or bromination of Boc-protected intermediate 48,
followed by Boc removal. Compounds 5a and 6a were made
similarly from the enantiopure material 47.
Figure 2. 3-Aminomethyl 4-halogen benzoxaborole inhibitors.
of potent Mtb LeuRS inhibitors 3a and 4a that are orally
bioavailable and effective against Mtb in mouse TB infection
models but with potential toxicity issues from inhibition of
mammalian cytoplasmic LeuRS.23 Furthermore, a once daily
dose of 50 mg/kg 4a in a mouse acute TB infection model was
not tolerated and was a concern for an indication that involved
long-term therapy.
Therefore, we set out to expand structure−activity relation-
ship (SAR) of this 3-aminomethyl 4-halogen benzoxaborole
series with the goal to improve the selectivity of the Mtb LeuRS
inhibitors suitable for further development. From the binding
pharmacophore revealed by the crystal structure of 3a
complexed with Mtb LeuRS,23 the C-7 or C-6 position of the
benzoxaborole appears to be solvent-exposed and may be open
for structural modification. In the present work, we used 3a and
4a as lead compounds and focused on three modification sites
which are C-7, C-6, and C-6/C-7 positions. We investigated
various 7-substituted analogues represented by target com-
pounds 5−24 (class A) and a number of 6-substituted
compounds 25−32 (class B) as well as C-6/C-7 fused
compounds 33−36 (class C). In this study, we intentionally
made and evaluated both 4-Cl and 4-Br compounds in parallel,
providing useful SAR information on their relative magnitudes
in activity and selectivity. Further PK evaluation and efficacy
studies of these Mtb LeuRS inhibitors led to the identification
of a first-in-class boron-containing antitubercular agent 23a
(GSK656),24 which had a much better safety profile than 3a
and 4a.
RESULTS AND DISCUSSION
■
Chemistry. Throughout the article, compounds have been
numbered using the format 23, 23a, and 23b for the racemate,
the (S)-isomer, and the (R)-isomer of compound 23,
respectively. The chemistry used to synthesize racemic 7-
substituted compounds 11−20 and 23−24 (class A) is
B
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a
Scheme 1
a
Reagents and conditions: (a) when R = alkyl, alkyl bromide or alkyl iodide, K2CO3, DMF, heat; when R = aryl, arylboronic acid, Cu(OAc)2, TEA,
DCM; (b) (Pin)2B2, KOAc, PdCl2(dppf), THF, reflux; (c) CH3NO2, NaOH, water/THF; (d) ethylene glycol, TsOH, toluene, Dean−Stark
apparatus, reflux; (e) (1) BuLi, B(OMe)3, THF, −78 °C; (2) 2 N HCl, rt; (f) CH3NO2, NaOH, water/THF; (g) (1) Raney Ni, H2, NH3/EtOH,
then HCl/Et2O; (2) di-tert-butyl dicarbonate, TEA, DCM, 0 °C; (h) when X = Cl, NCS, DMF, heat or SO2Cl2, AcOH, rt; when X = Br, NBS,
CH3CN, heat; (i) TFA, DCM, then HCl; (j) when X = Cl, NCS, DMF, heat; when X = Br, NBS, CH3CN, heat; (k) Raney Ni, H2, NH3/EtOH, then
HCl/Et2O; (l) chiral SFC separation.
The chemistry used to make 6-substituted analogues (class
B) is described in Scheme 3 and Scheme 4. For this series of
compounds, 4-halogen substituent needs to be incorporated at
an early stage of the synthesis. As shown in Scheme 3, the 4-Cl
analogues were synthesized starting from 2-chloro-4,6-
dihydroxybenzaldehyde 53. After selected protection of 4-
hydroxy group, reaction of phenol 54 with triflic anhydride in
the presence of pyridine gave triflate 55. Borylation was
accomplished by reaction of triflate 55 with pinacol diborane in
the presence of a palladium catalyst. Subsequent multistep
manipulation of boronate 56 including Henry reaction, nitro
reduction, and alkylation provided the final 4-Cl compounds
(25, 27, 29, and 31). On the other hand, the 4-Br analogues
were synthesized starting from 2,6-dibromo-4-hydroxy-
benzaldehyde 60 as shown in Scheme 4. Alkylation of 60
followed by protection of aldehyde group provided symmetric
dibromide 62. Reaction of 62 with n-butyllithium and trimethyl
borate followed by acidic workup converted one bromide to
boronic acid 63. Subsequent multistep manipulation of boronic
acid 63 including Henry reaction and nitro reduction provided
the final 4-Br compounds (26, 28, 30, and 32).
The chemistry used to synthesize C-6/C-7 fused analogues
33−36 (class C) is described in Scheme 5. Reaction of 2-
bromo-3,4-dihydroxybenzaldehyde 65 with dibromomethane
provided five-membered dioxolane containing benzaldehyde 66
(n = 1). Similarly, reaction of 65 with 1,1,2,2-tetrabromoethane
provided six-membered dioxolane-containing benzaldehyde 66
(n = 2). Incorporation of boron in compound 66 was
accomplished by reaction with pinacol diborane in the presence
of a palladium catalyst to give boronate 67. The Henry reaction
of boronate 67 with nitromethane provided 3-nitromethyl-
benzoxaborole 68. Chlorination or bromination of 68 gave
corresponding C-4 chloro- or bromobenzoxaborole 69. The
reduction of nitro group in compound 69 gave the C-4 chloro
or bromo C-6/C-7 fused analogs 33−36. Compounds 35 and
36 were further separated into their active enantiomers 35a and
36a by chiral SFC separation.
Structure−Activity Relationships. The compounds were
evaluated in the Mtb LeuRS aminoacylation assay as well as in a
whole cell activity assay against Mtb H37Rv (Table 1). Since
cell penetration can vary between series, the MIC values of
these Mtb LeuRS inhibitors against Mtb H37Rv were used to
optimize the potency and drive SAR, while the biochemical
C
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a
Scheme 2
a
Reagents and conditions: (a) di-tert-butyl dicarbonate, TEA, DCM, 0 °C; (b) (1) when X = Cl, NCS, DMF, heat; when X = Br, NBS, CH3CN,
heat; (2) TFA, DCM, then HCl; (c) BBr3, DCM, −78 °C; (d) di-tert-butyl dicarbonate, TEA, DCM, 0 °C; (e) alkyl bromide, t-BuOK, DMSO, 90
°C; (f) (1) when X = Cl, NCS, DMF, heat or SO2Cl2, AcOH, rt; when X = Br, NBS, CH3CN, heat; (2) TFA, DCM, then HCl.
a
Scheme 3
a
Reagents and conditions: (a) dihydropyran, PPTS (cat.), DCM; (b) Tf2O, pyridine, DCM, 0 °C; (c) (Pin)2B2, PdCl2(dppf), KOAc, THF, reflux;
(d) CH3NO2, NaOH, THF; (e) conc HCl; (f) (1) Raney Ni, H2, NH3/EtOH, then HCl/EtOH; (2) di-tert-butyl dicarbonate, TEA, DCM, 0 °C; (g)
alkyl bromide or alkyl iodide, K2CO3, DMF, heat; (h) TFA, DCM, then HCl.
inhibition was used to gauge whether the inhibitors were on
target. Representative compounds with good antitubercular
activity (Mtb H37Rv MIC < 0.2 μM) were selected and tested
for selectivity in the human mitochondrial and cytoplasmic
LeuRS biochemical assays as well as activity against cytoplasmic
protein synthesis in intact human HepG2 cells (Table 2) to see
if we could improve on the safety profile of the initial leads.
Most of compounds were made and tested as racemates first
and separated into active (S)-isomers when racemates were
very active (Table 1). Generally, the (S)-enantiomer is 2-fold
more potent compared to the racemate and the (R)-enantiomer
is completely inactive as exemplified by compounds 23, 24, 35,
and 36. For these four compounds, the results from the
racemate and both enantiomers were given for comparison.
We first explored the size of 7-alkoxyl side chain of the Mtb
LeuRS inhibitors. The smaller 7-methoxy compounds 5a and
6a showed similar Mtb LeuRS potency and antitubercular
activity, compared to 7-ethoxy leads 3a and 4a. Increasing the
length of 7-alkoxy group provided 7-propoxy compounds 7a/
8a and butoxy compounds 9a/10a. These analogues with larger
7-alkoxy substitutions exhibited decreased Mtb LeuRS potency
and antitubercular activity by 2- to 5-fold, suggesting that
D
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a
Scheme 4
a
Reagents and conditions: (a) alkyl bromide, K2CO3, DMF, heat; (b) CH(OMe)3, H2SO4, MeOH, reflux; (c) (1) BuLi, B(OMe)3, THF, −78 °C;
(2) 2 N HCl, rt; (d) CH3NO2, NaOH, water/THF; (e) Raney Ni, H2, NH3/EtOH, then HCl/EtOH.
a
Scheme 5
a
Reagents and conditions: (a) CH2Br2 (n = 1) or BrCH2CH2Br (n = 2), K2CO3, DMF, heat; (b) (Pin)2B2, KOAc, PdCl2(dppf), dioxane, reflux; (c)
CH3NO2, NaOH, THF; (d) when X = Cl, NCS, DMF, heat or SO2Cl2, AcOH, rt; when X = Br, NBS, CH3CN; (e) Raney Ni, H2, NH3/EtOH, then
HCl/EtOH; (f) chiral SFC separation.
adding size and lipophilicity at 7-position is not beneficial for
biochemical and antitubercular activity. We also incorporated
aromatic moieties in 7-substitution and made six analogues with
one-, two, or three-atom linker between the two aromatics
(11−16). These large compounds showed decent Mtb LeuRS
activity; however, all of them lost antitubercular activity,
suggesting that adding hydrophobic aromatics in the molecule
was detrimental to their antitubercular activity. 5a and 6a were
selected for in vitro selectivity evaluation.
Since the addition of fluorine atoms to drugs is well-known
to have the potential to acquire additional interactions,
modulate potency and selectivity of bioactive compounds, we
did a fluorine scan of the 7-ethoxy side chain and explored the
effects of C-7 fluorine substitution on Mtb LeuRS and
antitubercular activity. Introduction of one fluorine atom in
the 7-ethoxy group (17 and 18) resulted in approximately
equivalent MIC values (considering compounds 17 and 18 are
racemates). Introduction of two fluorine atoms in the 7-ethoxy
group of compounds 19 and 20 resulted in slight decrease in
antitubercular activity.
and 22a with 3-hydroxypropoxy group at C-7 position
maintained Mtb LeuRS and antitubercular activity. To further
reduce the lipophilicity, the size of the three-carbon linker was
reduced to a two-carbon linker giving 23 and 24, both of which
were very active. After chiral separation, their corresponding S
enantiomers 23a and 24a were found to exhibit excellent
antitubercular activity (MIC = 0.08 μM and 0.08 μM,
respectively). Both 23a and 24a were selected for in vitro
selectivity determinations. It is interesting to note that 23a or
24a is present as a seven-membered tricyclic ring form based on
1H NMR in deuterated DMSO (Figure 3), suggesting that an
equilibrium exists between open and close forms depending on
solvent and environment. This dynamic equilibrium between
open and closed forms of the benzoxaborole pharmacophore
and its six- and seven-membered analogues has been studied by
Hall and his group.27
We also expanded chemical space and synthesized a number
of 6-substituted compounds 25−32 (class B), as described in
Schemes 3 and 4. These 6-substituted analogs showed decent
Mtb LeuRS activity in the range of 0.25−1.11 μM. However,
they exhibited poor antitubercular activity compared to the
corresponding 7-substituted analogs. Although 27 and 28
We also explored the impacts of a hydrophilic side chain at
C-7 position on the potency of the Mtb LeuRS inhibitors. 21a
E
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a
Table 1. In Vitro Activity of Compounds 1−36
F
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Table 1. continued
a
All compounds were prepared as HCl or TFA salts. Experimental procedures are described in the Experimental Section. Values are the mean of
b
duplicate or more experiments. Desired product with 7-phenoxy group (R = Ph) was not formed during the bromination step, and dibrominated
product 12 with 7-(4-bromophenoxy) group was isolated instead.
showed some MIC against Mtb H37Rv (0.6−1.3 μM), further
investigation of this subseries of Mtb LeuRS inhibitors was
discontinued due its poor antitubercular activity.
0.03 μM, respectively). 35a and 36a were selected for in vitro
selectivity evaluation.
In Vitro Selectivity. Although in vitro potency against Mtb
was not significantly improved over the initial analogs 3a and
4a, we evaluated the inhibitors against several different in vitro
selectivity assays in order to improve their safety profile
including a cell-based protein synthesis assay (Table 2).
Fortunately these series were effectively inactive against
human mitochondrial LeuRS with IC50 > 300 μM, which is
presumably due to this enzyme being editing defective.28
Compared to the leads 3a and 4a, the new inhibitors 5a, 6a,
23a, 24a, 35a all exhibited improved selectivity against human
cytoplasmic LeuRS and HepG2 protein synthesis assay by 2- to
9-fold, suggesting that decreasing lipophilicity or adding
polarity in the molecule could translate into improved
selectivity profiles. These results were consistent with and
further supported by our HepG2 cell toxicity EC50 data (48 h)
In another subseries to expand chemical space (class C), we
investigated a C-6/C-7 fused ring in the scaffold to determine
the structure−activity relationship by making compounds 33−
36, as described in Schemes 5. While 33 and 34 with a five-
membered dioxolane ring failed to demonstrate antitubercular
activity (MIC > 5 μM), compounds 35 and 36 with a six-
membered dioxane ring exhibited potent Mtb LeuRS and
antitubercular activity. Presumably the dioxolane-containing
compound 33 or 34 has an unfavorable conformation and/or
electronics that resulted in their poor cellular activity. The
dioxane-containing compounds 35 and 36 were separated into
their respective enantiomers 35a and 36a, both of which
exhibited potent antitubercular activity (MIC = 0.08 μM and
G
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7-hydroxyethoxy, and 6-/7-fused analog) were chosen for drug
metabolism and pharmacokinetics (DMPK) studies.
Table 2. In Vitro Selectivity Data for Representative
Compounds
a
DMPK. The PK of new compounds 6a, 23a, and 35a were
evaluated in mice (Table 3). All three new compounds
exhibited low clearance and excellent exposure, which are
consistent with previous PK results of 4a. The dose-normalized
(DN) AUC values for 6a, 23a, and 35a were 1.17, 2.03, and
2.54 h·μg/mL, respectively. After oral administration, the
DNAUC values of 6a, 23a, and 35a were 0.84, 2.96, and 1.26
h·μg/mL, corresponding to bioavailability of 72%, 100%, and
50%, respectively. These Mtb LeuRS inhibitors show low
molecular weight, low polar surface area (PSA), and clogD7.4
value similar to frontline TB drugs isoniazid, pyrazinamide, and
ethambutol. We believe that the physicochemical properties of
these compounds (such as low molecular weight, low PSA, and
favorable clogD7.4) contribute to their remarkable exposure and
bioavailability in the in vivo PK assays.
human
mito
human
cytoplasm
LeuRS
HepG2
protein
HepG2 cell
48 h
LeuRS
synthesis
selectivity
b
c
compd IC50 (μM) IC50 (μM) EC50 (μM)
index
EC50 (μM)
3a
>300
>300
>300
>300
>300
>300
38.8
66.9
260
19.6
30.5
105
97
196
65.8
47.1
327
74.5
381
292
69.5
43.2
4a
305
5a
1050
970
6a
>300
132
23a
24a
35a
36a
137
116
82.8
39.5
1713
1450
1035
1317
118
d
NT
96.6
118
d
NT
a
The human biological samples were sourced ethically, and their
research use was in accord with the terms of the informed consents.
Selectivity index is defined as a ratio of HepG2 protein synthesis EC50
over MIC against Mtb H37Rv. Values are the mean of duplicate or
more experiments. Readout performed with CellTiter Glo lumines-
cent viability assay kit (Promega). NT = not tested.
b
In Vivo Efficacy. To evaluate the ability of these Mtb LeuRS
inhibitors to treat tuberculosis, we examined 6a, 23a, and 35a
in an animal TB efficacy model. The results from a murine
acute TB infection model are shown in Table 4. In this TB
c
d
a
Table 4. Efficacy in a Mouse Model of Acute TB Infection
b
4a
6a
23a
35a
ED99 (mg/kg)
<1.0
3.8
0.4
70
a
Efficacy of compounds in a mouse model of acute TB infection under
QD dosing regimen (once a day). C57BL/6J mice were infected with
M. tuberculosis H37Rv intratracheally (∼105 CFU) and were dosed
starting on the following day after infection for 8 days. Mice were
sacrificed at least 24 h after the last drug administration. All animal
studies were ethically reviewed and carried out in accordance with
European Directive 2010/63/EU and the GSK Policy on the Care,
Figure 3. 3-Aminomethyl 4-halogen benzoxaborole inhibitors 23a and
24a.
b
Welfare and Treatment of Animals. Efficacy data for 4a was reported
obtained with these Mtb LeuRS inhibitors. The compounds 5a,
23a, and 24a exhibited the least activity against HepG2 cells
and thus were advanced further for in vivo testing. Also, it is
worthy to note that little difference exists in activity and
selectivity profiles between 4-Cl compound and 4-Br counter-
part, as exemplified by 3a vs 4a and 23a vs 24a. Compounds
6a, 23a, and 35a with different structural features (7-methoxy,
previously in ref 23.
infection model, 23a showed the best efficacy with ED99
(efficacious dose that gives 2 log colony-forming units (CFU)
reduction compared to the untreated control) of 0.4 mg/kg
among the four compounds evaluated. 6a is 10-fold less potent
a
Table 3. Physicochemical Properties and PK Parameters
b
c
b
c
c
parameter
4a
4a
6a
23a
35a
MW
285.9
0.95
271.9
0.60
64.71
5.0
239.5
−0.4
53.71
5.5
255.5
−0.10
73.94
30.0
1.5
clogD7.4
PSA
64.71
dose (mg/kg), iv
Vss (L/kg)
Cl (mL min−1 kg−1), iv
30
28.5
1.7
3.2
2.5
2.3
11.45
3.4
6.6
14.2
2.4
8.5
6.6
t1/2 (h)
3.1
3.6
2.9
AUC0−inf (h·μg/mL), iv
DNAUC0−inf (h·μg/mL per mg/kg), iv
dose (mg/kg), po
Cmax (μg/mL)
43.4
1.45
30
72.3
2.54
26.1
11.14
2.0
5.84
1.17
5.0
11.12
2.03
30.0
17.76
0.9
76.32
2.54
30.0
6.21
1.0
6.3
0.98
0.25
4.21
0.84
72
Tmax (h)
0.5
AUC0−24h (h·μg/mL), po
DNAUC0−24h (h·μg/mL), po
F (%)
57.6
1.92
∼100
77.86
2.98
∼100
88.24
2.94
∼100
37.67
1.26
50
a
All animal studies were ethically reviewed and carried out in accordance with European Directive 2010/63/EU and the GSK Policy on the Care,
b
Welfare and Treatment of Animals. Mouse PK studies were conducted by using CD-1 mice as described in ref 23. PK values for 4a by using CD-1
mice were from ref 23 and are presented for comparison. Mouse PK studies were conducted by using C57 mice as described in the Experimental
c
Section.
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a
Table 5. Efficacy in a Mouse Model of Chronic TB Infection
no treatment
linezolid
100
23a
dose (mg/kg)
log10 CFU
0.1
6.4 (0.6)
0.3
1
3
10
30
b
c
d
d
d
d
d
5.8 (0.3)
6.4 (0.3)
3.9 (0.2)
6.0 (0.1)
4.7 (0.2)
4.6 (0.2)
4.3 (0.1)
4.4 (0.2)
a
Efficacy of compounds in a mouse model of chronic TB infection under QD dosing regimen (once a day). C57BL/6J mice were infected with M.
tuberculosis H37Rv intratracheally (∼102 CFU) and were dosed once daily for 8 weeks starting 6 weeks after infection. Mice were sacrificed at least
24 h after the last drug administration. Every data point represents the mean values ( standard deviations) from 7 mice per group for untreated and
linezolid-treated groups and from 3 mice for 23a-treated groups. All animal studies were ethically reviewed and carried out in accordance with
European Directive 2010/63/EU and the GSK Policy on the Care, Welfare and Treatment of Animals. 6 weeks after infection. 14 weeks after
infection. Statistically significant difference compared to 14-week untreated mice (p < 0.05, ANOVA, Dunnett’s multiple comparison test).
b
c
d
formed in 96-well microtiter plates, using 80 μL of reaction mixtures
containing 50 mM HEPES−KOH (pH 8.0), 30 mM MgCl2 30 mM
KCl, 13 μM L-[14C]leucine (306 mCi/mmol, PerkinElmer), 15 μM
total E. coli tRNA (Roche, Switzerland), 0.02% (w/v) BSA, 1 mM
DTT, 0.2 pM LeuRS, and 4 mM ATP at 30 °C. Reactions were started
by the addition of 4 mM ATP. After 7 min, reactions were quenched
and tRNA was precipitated by the addition of 50 μL of 10% (w/v)
TCA and transferred to 96-well nitrocellulose membrane filter plates
(Millipore Multiscreen HTS, MSHAN4B50). Each well was then
washed three times with 100 μL of 5% TCA. Filter plates were then
dried under a heat lamp, and the precipitated L-[14C]leucine tRNALeu
was quantified by liquid scintillation counting using a Wallac
MicroBeta Trilux model 1450 liquid scintillation counter (PerkinElm-
er, Waltham, MA, USA). To determine the inhibitor concentration,
which reduces enzyme activity by 50% (IC50), increasing concen-
trations of compound inhibitors that covered the IC50 value were
incubated with LeuRS enzyme, tRNA, and L-leucine for 20 min.
Reactions were initiated by the addition of 4 mM ATP. Reactions were
stopped after 7 min and then precipitated and counted to quantify
radioactivity. IC50 values were determined using a four-parameter
logistic nonlinear regression model (GraphPad Software Inc. (La Jolla,
CA, USA). The only difference was with the human cytoplasmic
LeuRS aminoacylation assay when we used tRNA isolated from
Brewer’s yeast (Roche Diagnostics GmbH).
in terms of dose with ED99 of 3.8 mg/kg, compared to 23a. 35a
showed a significant decrease in efficacy with ED99 of 70 mg/kg
(>100-fold). The reason for an in vitro and in vivo disconnect
of 35a is not clear and is apparently not related to a lack of in
vitro potency or in vivo exposure. Unlike with 4a, which was
not tolerated at 50 mg/kg QD, 23a was well tolerated at the
maximum dose tested of 100 mg/kg. In a maximum single
tolerated dose study in healthy mice, 23a was well tolerated at
300 mg/kg and 4a was not tolerated at the lowest dose tested
of 150 mg/kg. Therefore, further evaluations of 23a in a murine
chronic TB infection model were performed in parallel with the
protein synthesis inhibitor linezolid (Table 5). 23a exhibited a
dose-dependent reduction in CFU from 0.1 mg/kg to 10 mg/
kg QD. Importantly, 10 mg/kg QD resulted in 2.1 log10
reduction in CFU compared with a 2.5 log10 reduction in
CFU for 100 mg/kg of linezolid QD. From these studies, 23a
was identified as having the best overall profiles, with
remarkable oral bioavailability and in vivo efficacy at low
doses in acute and chronic mouse TB infection models.
CONCLUSIONS
■
In this work, we designed and synthesized a new series of Mtb
LeuRS inhibitors by investigating various substituents in the
aromatic ring of the 3-aminomethyl 4-halogen benzoxaborole
scaffold. The SARs which include the influence of substitution
size, nature, and pattern at positions 6 and 7 were explored,
leading to the identification of a number of Mtb LeuRS
inhibitors with excellent antitubercular potency in the mid-
nanomolar range. These Mtb LeuRS inhibitors exhibited
improved selectivity over human cytoplasmic LeuRS as well
as protein synthesis inhibition in intact human cells over the
initial leads. Moreover, the superior tolerability of the new
compounds has enabled their further progression and
ultimately the selection of a clinical candidate. These Mtb
LeuRS inhibitors exhibited remarkable oral exposure, with small
molecular weight and low clogD similar to frontline TB drugs
isoniazid, pyrazinamide, and ethambutol. From these com-
pounds, 23a was identified as having the best overall profiles,
with excellent in vivo efficacy at low doses in acute and chronic
mouse TB infection models. As a result, 23a has been
progressed to clinical development for the treatment of
tuberculosis (ClinicalTrials.gov identifier NCT03075410).
Future publications will report other preclinical studies and
clinical trial results.
Determination of MIC for M. tuberculosis. Mycobacterium
tuberculosis H37Rv was grown at 37 °C in Middlebrook 7H9 broth
(Difco) supplemented with 0.025% Tween 80 and 10% albumin-
dextrose-catalase (ADC) or on Middlebrook 7H10 plates supple-
mented with 10% oleic acid-albumin-dextrose-catalase (OADC). The
antitubercular activity against extracellular of intracellular Mycobacte-
rium strains was performed as previously described.29
HepG2 Protein Synthesis EC50 Determination. Human liver
carcinoma HepG2 cells were grown in Dulbecco’s modified Eagle’s
medium containing 10% (v/v) fetal calf serum, 1 mM sodium
pyruvate, 0.1 mM nonessential amino acids, and 50 units/mL
penicillin−streptomycin at 37 °C with 5% CO2. HepG2 cells were
seeded in 96-well plates at 3000 cells/200 μL/well in cell culture
medium. Stock solutions of compounds were made in DMSO.
Increasing concentrations of compounds were added with the final
DMSO concentration being 0.5%. Vehicle-treated samples had DMSO
added at 0.5% with no compound. Cells were then grown in the
presence of the compounds at 37 °C in 10% CO2 for 2 days. After 48
h, the medium was removed and then replaced by fresh medium
containing 0.5 μCi of L-[14C]leucine and the same concentrations of
compounds. After incubation for 3 h, the medium was removed and 50
μL of trypsin−EDTA solution was added to each well. After 15 min,
the trypsinzed cells were transferred to a 96-well plate containing ice-
cold 20% (w/v) TCA. After 60 min on ice the samples were collected
over vacuum on 96-well glass fiber filter plate (Millipore, MSFBNB50)
and washed three times with 150 μL of ice-cold 10% (w/v) TCA. To
the dried filter plate 40 μL of scintillation cocktail was added, and
counts were obtained in Wallac MicroBeta Trilux model 1450 liquid
scintillation counter (PerkinElmer, Waltham, MA). IC50 values were
determined by curve-fitting using the Prism by GraphPad (San Diego,
CA, USA).
EXPERIMENTAL SECTION
■
LeuRS Aminoacylation Assay. An N-terminal six histidine-
tagged LeuRS from M. tuberculosis H37Rv and humans (cytoplasmic
and mitochondrial) were codon-optimized for E. coli (GenScript,
Piscataway, NJ, USA) and overexpressed and purified according to
Novagen (Madison, WI, USA) using an E. coli BL21(DE3) T7 RNA
polymerase overexpression strain. Aminoacylation assay was per-
HepG2 Cytotoxicity Assay. HepG2 (HB-8065) cells were
cultured with fresh medium (essential minimum Eagle medium,
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DOI: 10.1021/acs.jmedchem.7b00631
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EMEM, supplemented with 5% fetal calf serum and 2 mM L-
glutamine) the day before subculturing the plates. On the day of the
assay, cells (10 000 cells/well) were seeded in a black 96-well collagen
coated microplate with clear bottom, (Becton Dickinson) except in
column 11, which was dispensed only 100 mL of culture medium.
Stock solution from each test substances was prepared in 100%
DMSO. Ten serial 1:2 dilutions were prepared of each test compound,
and finally, a 1:200 dilution was made, in medium, to achieve a final
concentration of 0.5% of DMSO. Resazurin tablets (Merck) were
dissolved in phosphate buffer saline at a concentration of 0.0042%.
After 24 h of incubation of the cells (37 °C, 5% CO2, 95% relative
humidity), a volume of 150 μL of culture medium containing the
appropriate test concentrations of the compounds dilutions was added
to cells in two replicates. In column 12, only 150 μL of 0.5% DMSO
was added (blank control). Then, cells were exposed to test
compounds for 48 h. After that, medium was removed and resazurin
solution was added to each well and incubated for further 1.5 h.
Fluorescence was measured at an excitation wavelength of 515 nm and
an emission wavelength of 590 nm in a Microplate reader 1420
Multilabel HTS counter, Victor 2 (Wallac). Data were processed using
an Excel spreadsheet and GraphPad software analysis. Tox50 values
were calculated from the Sigmoidal dose−response (variable slope)
curves by nonlinear regression analysis.
Procedure for PK Studies. For pharmacokinetic studies, C57BL/
6 female mice (18−20 g) were used, and compound concentrations
were determined using peripheral whole blood. All animal studies were
ethically reviewed and carried out in accordance with European
Directive 2010/63/EU and the GSK Policy on the Care, Welfare and
Treatment of Animals.
4a was administered by intravenous route at 30 mg/kg single dose
in saline and by oral gavage at 30 mg/kg single dose in 1%
methylcellulose (1% MC). For iv route aliquots of 25 μL of blood
were taken from the lateral tail vein by puncture from each mouse (n =
5) at 5, 15, and 30 min and 1, 2, 4, and 8 h postdose; for oral route
aliquots of 25 μL of blood were taken by cardiac puncture for each
mouse (euthanized by CO2, n = 3 mice per time point) at 15, 30, and
45 min and 1, 1.5, 2, 3, 4, 8, and 24 h postdose.
23a was administered by intravenous route at 5 mg/kg single dose
in saline and by oral gavage at 30 mg/kg single dose in 1%
methylcellulose (1% MC). For iv route aliquots of 15 μL of blood
were taken from the lateral tail vein by puncture from each mouse (n =
3) at 5, 15, and 30 min and 1, 2, 4, 8, and 24 h postdose; for oral route
aliquots of 15 μL of blood were taken from the lateral tail vein by
puncture from each mouse (n = 3) at 15, 30, and 45 min and 1, 2, 4, 8,
and 24 h postdose.
35a was administered by intravenous route at 30 mg/kg single dose
in saline and by oral gavage at 30 mg/kg single dose in 1%
methylcellulose (1% MC). For iv route aliquots of 15 μL of blood
were taken from the lateral tail vein by puncture from each mouse (n =
3) at 5, 15, and 30 min and 1, 2, 4, 8, and 24 h postdose; for oral route
aliquots of 15 μL of blood were taken from the lateral tail vein by
puncture from each mouse (n = 3) at 15, 30, and 45 min and 1, 2, 4, 8,
and 24 h postdose.
aseptically removed, homogenized, and frozen. Homogenates were
plated onto 10% OADC−Middlebrook 7H11 medium and incubated
for 21 days at 37 °C. All animal studies were ethically reviewed and
carried out in accordance with European Directive 2010/63/EU and
the GSK Policy on the Care, Welfare and Treatment of Animals. The
viable CFU were converted to logarithms, which were then evaluated
by one-way analysis of variance, followed by multiple-comparison
analysis of variance by a one-way Tukey test (SigmaStat software
program). Differences were considered significant at the 95% level of
confidence.
Chemical Synthesis. Starting materials used were either
commercially available or prepared according to literature procedures
and had experimental data in accordance with those reported. 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 the exception of
compounds 8 and 30. 1H NMR spectra were recorded on a Varian or
Bruker 300 or 400 MHz spectrometer. Chemical shifts were reported
in ppm and were referenced to the appropriate residual solvent signals,
such as 2.49 ppm for DMSO-d6 and 7.26 ppm for CDCl3. LC−MS
data were obtained using an Agilent LC−MS 1200 with 6110 MS
detector. The mass spectrometer was equipped with an electrospray
ion source (ESI) operated in a positive or negative mode. Flash
column chromatography was typically preformed using silica gel 60
(230−400 mesh). Typical solvents used for flash chromatography or
thin layer chromatography (TLC) were mixtures of MeOH/DCM and
EtOAc/petroleum ether. HPLC analysis was performed on a Gilson
HPLC system using a Gemini 5 μm C18 column (150 mm × 4.6 mm
i.d.) or a Venusil MP C18 3 μm C18 column (100 mm × 4.6 mm i.d.).
Purification using prep-HPLC was accomplished using a Gilson HPLC
system with a Gemini 5 μm C18 column (150 mm × 21.2 mm i.d.).
Typically, the mobile phase used for HPLC was a linear gradient of
water (A) and acetonitrile (B). The water and acetonitrile were mixed
with 0.1% TFA or 0.1% HCOOH. The flow rate was maintained at
0.8−1.2 mL/min for analytic HPLC and 20 mL/min for prep-HPLC,
and the eluent was monitored with UV detector at 220 nm. Chiral
separation was performed on a Thar preparative SFC 80 system with a
Daicel Chiralpak AD-H 5 μm (250 mm × 20 mm i.d.), eluted with
carbon dioxide and methanol mobile phase. The flow rate was
maintained at 65−70 mL/min, and eluent was monitored with UV
detector at 220 nm. For enantiomeric excess determination, chiral
HPLC analysis was performed on a Thar analytical SFC system with a
Chiralpak AD-H 5 μm (150 mm × 4.6 mm i.d.) or a ChiralCel OJ-H 5
μm (150 mm × 4.6 mm i.d.) or a ChiralPak AY-H 5 μm (150 mm ×
4.6 mm i.d.), eluted with carbon dioxide and methanol mobile phase at
a flow rate of approximately 2.4−4 mL/min. The ee of all tested
enantiomers was >97%.
(S)-3-(Aminomethyl)-4-chloro-7-methoxybenzo[c][1,2]-
oxaborol-1(3H)-ol Hydrochloride (5a). This compound was
prepared by the similar method described below for the synthesis of
1
21a. H NMR (400 MHz, DMSO-d6) δ 9.22 (b, 1H), 8.20 (b, 3H),
7.54 (d, 1H, J = 9.0 Hz), 7.01 (d, 1H, J = 9.0 Hz), 5.33 (m, 1H), 3.82
(s, 3H), 3.56 (m, 1H), 2.92 (m, 1H). MS (ESI) m/z = 228 [M + H]+.
HPLC purity: 99.9% (220 nm). 5a had >99% ee based on starting
material 47.
LC−MS/MS was used as the analytical method for the establish-
ment of compound concentration in blood. Pharmacokinetic analysis
was performed by noncompartmental data analysis (NCA) with
Phoenix WinNonlin 6.3 (Pharsight, Certara L.P.), and supplementary
analysis was performed with GraphPad Prism 6 (GraphPad Software,
Inc.).
(S)-3-(Aminomethyl)-4-bromo-7-methoxybenzo[c][1,2]-
oxaborol-1(3H)-ol Hydrochloride (6a). This compound was
prepared by the similar method described below for the synthesis of
1
22a. H NMR (400 MHz, DMSO-d6) δ 9.21 (b, 1H), 8.41 (b, 3H),
Murine Model of Acute and Chronic TB Infections. Specific
pathogen-free, 8- to 10-week-old female C57BL/6 mice were
purchased from Harlan Laboratories and were allowed to acclimate
for 1 week. The experimental design for the acute assay has been
previously described.30 In brief, mice were intratracheally infected with
100 000 CFU/mouse of M. tuberculosis H37Rv. Compounds were
administered for 8 consecutive days starting 1 day after infection. For
the chronic assay, mice were intratracheally infected with 100 CFU/
mouse and the products administered daily (7 days a week) for 8
consecutive weeks starting 6 weeks after infection. Lungs were
harvested 24 h after the last administration, and all lung lobes were
7.66 (d, 1H, J = 8.8 Hz), 6.94 (d, 1H, J = 8.4 Hz), 5.34 (m, 1H), 3.81
(s, 3H), 3.62 (m, 1H), 2.85 (m, 1H). MS (ESI) m/z = 272 [M + H]+.
HPLC purity: 99.7% (220 nm). 6a had >99% ee based on starting
material 47.
(S)-3-(Aminomethyl)-4-chloro-7-propoxybenzo[c][1,2]-
oxaborol-1(3H)-ol Hydrochloride (7a). To a solution of (S)-3-
(aminomethyl)-7-(3-hydroxypropoxy)benzo[c][1,2]oxaborol-1(3H)-
ol hydrochloride 46 (30 g, 0.11 mol) and triethylamine (33.3 g, 0.33
mol) in DCM (300 mL) at 0 °C was added di-tert-butyl dicarbonate
(36 g, 0.165 mol). The mixture was stirred at rt for overnight. The
solvent was removed to afford (S)-tert-butyl (1-hydroxy-7-(3-hydroxy-
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propoxy)-1,3-dihydrobenzo[c][1,2]oxaborol-3-yl)methylcarbamate as
a white solid (38 g, yield 100%). To a stirred solution of the above
compound (10 g, 29.7 mmol) in CH2Cl2 (30 mL) at −78 °C was
added BBr3 (8.3 mL, 89.1 mmol) dropwise. The reaction was stirred at
−78 °C for 2 h and stirred at rt overnight. The mixture was poured
into ice−water and extracted with DCM. The aqueous layer was
adjusted to pH = 8 with sat. NaHCO3, and di-tert-butyl dicarbonate
(6.5 g, 29.7 mmol) was added. The reaction mixture was stirred at rt
overnight and then extracted with EtOAc (3 × 50 mL). The organic
layer was dried with anhydrous Na2SO4, filtered, and concentrated in
vacuo to give (S)-tert-butyl (1,7-dihydroxy-1,3-dihydrobenzo[c][1,2]-
oxaborol-3-yl)methylcarbamate (3 g, 36%). To a solution of the above
compound (600 mg, 2.15 mmol) in 10 mL of DMSO at 0 °C under
N2 was added t-BuOK (362 mg, 3.2 mmol). The reaction mixture was
stirred at rt for 30 min, followed by the addition of 1-bromopropane
(1.1 mL, 1.4 g, 10.7 mmol). The reaction mixture was stirred at rt for
12 h. After the reaction was quenched with ice−water, the mixture was
extracted with EtOAc (50 mL). The organic layer was washed with
brine (50 mL), dried over anhydrous Na2SO4, and concentrated in
vacuo. The residue was purified by silica gel chromatography, eluting
with EtOAc and petroleum ether (1:3) to give (S)-tert-butyl (1-
hydroxy-7-propoxy-1,3-dihydrobenzo[c][1,2]oxaborol-3-yl)-
methylcarbamate as colorless oil (110 mg, 16%). A solution of the
above compound (440 mg, 1.37 mmol) and N-chlorosuccinimide (183
mg, 1.37 mmol) in 20 mL of CH3CN was stirred at 90 °C for 1 h. The
mixture was concentrated in vacuo. The residue was purified by prep-
HPLC, eluting with water/acetonitrile gradient to give (S)-tert-butyl
(4-chloro-1-hydroxy-7-propoxy-1,3-dihydrobenzo[c][1,2]oxaborol-3-
yl)methylcarbamate as a white solid (89 mg, 18%). To a solution of
the above compound (89 mg, 0.25 mmol) in 10 mL of DCM was
added TFA (1 mL). The reaction mixture was stirred at rt for 1 h and
then concentrated in vacuo. After 1 M HCl in Et2O (10 mL) was
added, it was stirred for 1 h. The residue was filtered and dried in
3-(Aminomethyl)-4-bromo-7-(4-bromophenoxy)benzo[c]-
[1,2]oxaborol-1(3H)-ol Hydrochloride (12). This compound was
prepared by the similar method described below for the synthesis of
24. H NMR (300 MHz, CD3OD) δ 7.66−7.63 (d, 1H, J = 8.5 Hz),
1
7.51−7.46 (t, 2H, J = 7.9 Hz), 6.94−6.91 (d, 2H, J = 8.8 Hz), 6.85−
6.80 (t, 1H, J = 9.0 Hz), 5.44 (m, 1H), 3.94 (m, 1H), 3.09 (m, 1H).
MS (ESI) m/z = 414 [M + H]+. HPLC purity: 98.0% (220 nm).
3-(Aminomethyl)-7-(benzyloxy)-4-chlorobenzo[c][1,2]-
oxaborol-1(3H)-ol Hydrochloride (13). This compound was
prepared by the similar method described below for the synthesis of
1
23. H NMR (400 MHz, DMSO-d6) δ 9.20 (b, 1H), 8.29 (b, 3H),
7.49−7.31 (m, 6H), 7.04 (d, 1H, J = 8.8 Hz), 5.41 (m, 1H), 5.23 (s,
2H), 3.57 (m, 1H), 2.94 (m, 1H). MS (ESI) m/z = 304 [M + H]+.
HPLC purity: 96.8% (220 nm).
3-(Aminomethyl)-7-(benzyloxy)-4-bromobenzo[c][1,2]-
oxaborol-1(3H)-ol Hydrochloride (14). This compound was
prepared by the similar method described below for the synthesis of
1
24. H NMR (400 MHz, CD3OD) δ 7.62 (d, 1H, J = 8.0 Hz), 7.52−
7.32 (m, 5H), 7.00 (d, 1H, J = 8.0 Hz), 5.41 (m, 1H), 5.21 (s, 2H),
3.94 (m, 1H), 3.07 (m, 1H). MS (ESI) m/z = 348 [M + H]+. HPLC
purity: 99.6% (220 nm).
3-(Aminomethyl)-4-chloro-7-phenethoxybenzo[c][1,2]-
oxaborol-1(3H)-ol Hydrochloride (15). This compound was
prepared by the similar method described below for the synthesis of
23. 1H NMR (400 MHz, CD3OD) δ 7.45 (d, 1H, J = 8.0 Hz), 7.38 (d,
2H, J = 8.0 Hz), 7.31 (m, 2H), 7.22 (m, 1H), 6.97 (d, 1H, J = 8.0 Hz),
5.43 (m, 1H), 4.27 (t, 2H), 3.83 (m, 1H), 3.11 (t, 3H), 3.03 (m, 1H).
MS (ESI) m/z = 318 [M + H]+. HPLC purity: 99.6% (220 nm).
3-(Aminomethyl)-4-bromo-7-phenethoxybenzo[c][1,2]-
oxaborol-1(3H)-ol Hydrochloride (16). This compound was
prepared by the similar method described below for the synthesis of
24. 1H NMR (400 MHz, CD3OD) δ 7.60 (d, 1H, J = 8.0 Hz), 7.37 (d,
2H, J = 8.0 Hz), 7.30 (m, 2H), 7.22 (m, 1H), 6.91 (d, 1H, J = 8.0 Hz),
5.37 (m, 1H), 4.25 (t, 2H), 3.91 (m, 1H), 3.10 (t, 3H), 3.01 (m, 1H).
MS (ESI) m/z = 362 [M + H]+. HPLC purity: 100% (220 nm).
3-(Aminomethyl)-4-chloro-7-(2-fluoroethoxy)benzo[c][1,2]-
oxaborol-1(3H)-ol Hydrochloride (17). This compound was
prepared by the similar method described below for the synthesis of
23. 1H NMR (300 MHz, CD3OD) δ 7.46 (d, 1H, J = 9.0 Hz), 7.00 (d,
1H, J = 9.0 Hz), 5.43 (m, 1H), 4.81 (m, 1H), 4.65 (m, 1H), 4.35 (m,
1H), 4.28 (m, 1H), 3.81 (m, 1H), 3.04 (m, 1H). MS (ESI) m/z = 260
[M + H]+. HPLC purity: 100% (220 nm).
3-(Aminomethyl)-4-bromo-7-(2-fluoroethoxy)benzo[c][1,2]-
oxaborol-1(3H)-ol Hydrochloride (18). This compound was
prepared by the similar method described below for the synthesis of
24. 1H NMR (300 MHz, CD3OD) δ 7.61 (d, 1H, J = 9.0 Hz), 6.94 (d,
1H, J = 9.0 Hz), 5.36 (m, 1H), 4.81 (m, 1H), 4.65 (m, 1H), 4.37 (m,
1H), 4.27 (m, 1H), 3.89 (m, 1H), 3.04 (m, 1H). MS (ESI) m/z = 304
[M + H]+. HPLC purity: 97.8% (220 nm).
3-(Aminomethyl)-4-chloro-7-(2,2-difluoroethoxy)benzo[c]-
[1,2]oxaborol-1(3H)-ol Hydrochloride (19). This compound was
prepared by the similar method described below for the synthesis of
23. 1H NMR (300 MHz, CD3OD) δ 7.48 (d, 1H, J = 9.0 Hz), 7.02 (d,
1H, J = 9.0 Hz), 6.35 (m, 1H), 5.44 (m, 1H), 4.35 (m, 2H), 3.80 (m,
1H), 3.05 (m, 1H). MS (ESI) m/z = 278 [M + H]+. HPLC purity:
99.4% (220 nm).
1
vacuo to give (S)-7 as a white solid (71 mg, 97%). H NMR (400
MHz, DMSO-d6) δ 9.13 (b, 1H), 8.19 (b, 3H), 7.50 (d, 1H, J = 8.6
Hz), 6.99 (d, 1H, J = 8.7 Hz), 5.38 (m, 1H), 4.00 (t, 2H), 3.57 (m,
1H), 2.94 (m, 1H), 1.75 (m, 2H), 0.99 (t, 3H). MS (ESI) m/z = 256
[M + H]+. HPLC purity: 96.3% (220 nm). 7a had >99% ee based on
the use of starting material 46.
(S)-3-(Aminomethyl)-4-bromo-7-propoxybenzo[c][1,2]-
oxaborol-1(3H)-ol Hydrochloride (8a). This compound was
prepared by the similar method described above for the synthesis of
7a. 1H NMR (300 MHz, DMSO-d6) δ 9.09 (b, 1H), 8.03 (b, 3H), 7.63
(d, 1H, J = 8.6 Hz), 6.93 (d, 1H, J = 8.7 Hz), 5.26 (m, 1H), 3.98 (t,
2H), 3.63 (m, 1H), 3.00 (m, 1H), 1.75 (m, 2H), 0.98 (t, 3H). MS
(ESI) m/z = 300 [M + H]+. HPLC purity: 93.1% (220 nm). 8a had
>99% ee based on starting material 46.
(S)-3-(Aminomethyl)-7-butoxy-4-chlorobenzo[c][1,2]-
oxaborol-1(3H)-ol Hydrochloride (9a). This compound was
prepared by the similar method described above for the synthesis of
7a. 1H NMR (400 MHz, DMSO-d6) δ 9.11 (b, 1H), 8.11 (b, 3H), 7.50
(d, 1H, J = 8.4 Hz), 6.99 (d, 1H, J = 8.4 Hz), 5.36 (m, 1H), 4.04 (t,
2H), 3.57 (m, 1H), 2.96 (m, 1H), 1.72 (m, 2H), 1.47 (m, 2H), 0.94 (t,
3H). MS (ESI) m/z = 270 [M + H]+. HPLC purity: 97.2% (220 nm).
9a had >99% ee based on starting material 46.
(S)-3-(Aminomethyl)-7-butoxy-4-bromobenzo[c][1,2]-
oxaborol-1(3H)-ol Hydrochloride (10a). This compound was
prepared by the similar method described above for the synthesis of
7a. 1H NMR (400 MHz, DMSO-d6) δ 9.10 (b, 1H), 8.11 (b, 3H), 7.64
(d, 1H, J = 8.8 Hz), 6.96 (d, 1H, J = 8.4 Hz), 5.28 (m, 1H), 4.04 (t,
2H), 3.61 (m, 1H), 2.96 (m, 1H), 1.71 (m, 2H), 1.45 (m, 2H), 0.94 (t,
3H). MS (ESI) m/z = 314 [M + H]+. HPLC purity: 99.2% (220 nm).
10 had >99% ee based on starting material 46.
3-(Aminomethyl)-4-chloro-7-phenoxybenzo[c][1,2]-
oxaborol-1(3H)-ol Hydrochloride (11). This compound was
prepared by the similar method described below for the synthesis of
23. 1H NMR (300 MHz, CD3OD) δ 7.57−7.29 (m, 3H), 7.17 (t, 1H, J
= 7.4 Hz,), 7.06−6.97 (m, 2H), 6.79 (d, 1H, J = 8.6 Hz,), 5.51 (m,
1H), 3.88 (m, 1H), 3.08 (m, 1H). MS (ESI) m/z = 290 [M + H]+.
HPLC purity: 98.0% (220 nm).
3-(Aminomethyl)-4-bromo-7-(2,2-difluoroethoxy)benzo[c]-
[1,2]oxaborol-1(3H)-ol Hydrochloride (20). This compound was
prepared by the similar method described below for the synthesis of
1
24. H NMR (300 MHz, DMSO-d6) δ 9.27 (b, 1H), 8.32 (b, 3H),
7.65 (d, 1H, J = 9.0 Hz), 7.00 (d, 1H, J = 9.0 Hz), 6.40 (m, 1H), 5.33
(m, 1H), 4.40 (m, 2H), 3.58 (m, 1H), 2.92 (m, 1H). MS (ESI) m/z =
322 [M + H]+. HPLC purity: 98.3% (220 nm).
(S)-3-(Aminomethyl)-4-chloro-7-(3-hydroxypropoxy)benzo-
[c][1,2]oxaborol-1(3H)-ol Hydrochloride (21a). This compound
was prepared by the similar method described above for the synthesis
of 7a. 1H NMR (400 MHz, DMSO-d6) δ 8.00 (b, 3H), 7.43−7.41 (d,
1H, J = 8 Hz), 6.96−6.94 (d, 1H, J = 8 Hz), 5.45 (m, 1H), 4.39 (m,
2H), 4.16 (m, 2H), 3.60 (m, 1H), 3.10−2.80 (m, 1H), 2.00 (m, 2H).
MS (ESI) m/z = 272 [M + H]+. HPLC purity: 98.3% (220 nm). 21a
had >99% ee based on starting material 46.
K
DOI: 10.1021/acs.jmedchem.7b00631
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Journal of Medicinal Chemistry
Article
(S)-3-(Aminomethyl)-4-bromo-7-(3-hydroxypropoxy)benzo-
[c][1,2]oxaborol-1(3H)-ol Hydrochloride (22a). This compound
was prepared by the similar method described above for the synthesis
of 8a. 1H NMR (400 MHz, DMSO-d6) δ 8.31 (b, 3H), 7.65−7.63 (d,
1H, J = 8.0 Hz), 6.96−6.93 (d, 1H, J = 8.4 Hz), 5.29 (m, 1H), 4.11 (m,
2H), 3.70−3.50 (m, 3H), 2.91 (m, 1H), 1.89 (m, 2H). MS (ESI) m/z
= 316 [M + H]+. HPLC purity: 97.3% (220 nm). 22a had >99% ee
based on starting material 46.
mmol). The reaction mixture was stirred for 2 h at rt. After the
reaction was quenched with sat. NaHCO3 (150 mL), the resulting
mixture was extracted with EtOAc (2 × 200 mL). The combined
organic layers were dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by prep-HPLC,
eluting with water/acetonitrile gradient to give the Boc-protected 23
(4.6 g, yield 47%). The racemic Boc-protected 23 was separated on a
Thar preparative SFC 80 system with a Daicel Chiralpak AD-H 5 μm
(250 mm × 20 mm i.d.), eluting with carbon dioxide and methanol
mobile phase. The two isomers were obtained. Each compound (4.6 g,
12.9 mmol) was dissolved in 1 M HCl/Et2O (80 mL) and stirred at rt
for 1 h and concentrated in vacuo. The residue was washed with
acetonitrile (2 × 5 mL) and dried in vacuo to give the desired product
as a white solid. 23a (1.2 g, yield 32%). 1H NMR (300 MHz, DMSO-
d6) δ 8.31 (b, 3H), 7.51−7.48 (d, 1H, J = 9 Hz), 6.98−6.95 (d, 1H, J =
9 Hz), 5.59 (m, 1H), 4.70 (m, 1H), 4.36−4.23 (m, 3H), 3.57 (m, 1H),
3.02 (m, 1H). MS (ESI) m/z = 258 [M + H]+. HPLC purity: 99.7%
(220 nm). Analysis of the Boc-derivatized material using a ChiralCel
OJ-H analytical column showed the (S) enantiomer with a retention
time of 2.94 min and 99.9% ee. 23b (1.3 g, yield 34%). 1H NMR (300
MHz, DMSO-d6) δ 8.28 (b, 3H), 7.51−7.48 (d, 1H, J = 9 Hz), 6.98−
6.95 (d, 1H, J = 9 Hz), 5.58 (m, 1H), 4.69 (m, 1H), 4.36−4.23 (m,
3H), 3.59 (m, 1H), 3.02 (m, 1H). MS (ESI) m/z = 258 [M + H]+.
HPLC purity: 100% (220 nm). Analysis of the Boc-derivatized
material using a ChiralCel OJ-H analytical column showed the (R)
enantiomer with a retention time of 2.67 min and 97.8% ee.
3-(Aminomethyl)-4-bromo-7-(2-hydroxyethoxy)benzo[c]-
[1,2]oxaborol-1(3H)-ol Hydrochloride (24). A solution of 7-(2-
hydroxyethoxy)-3-(nitromethyl)benzo[c][1,2]oxaborol-1(3H)-ol (1.5
g, 5.9 mmol), Raney Ni (200 mg), and 2 M NH3 in EtOH (5 mL) in
ethanol (40 mL) was shaken under an atmosphere of hydrogen for 2 h
at rt. The mixture was filtered through a bed of Celite, and the filtrate
was concentrated in vacuo. The crude amine was dissolved in EtOH
(20 mL), and 1 M HCl in Et2O (30 mL) was added. After stirring for
1 h, the residue was filtered, washed with acetonitrile/hexane (2:1, 2 ×
20 mL), and dried in vacuo to give 3-(aminomethyl)-7-(2-
hydroxyethoxy)benzo[c][1,2]oxaborol-1(3H)-ol hydrochloride as a
white solid (700 mg, yield 46%). To a mixture of the above
compound (700 mg, 2.7 mmol) and triethylamine (879 mg, 8.7 mmol)
in dichloromethane (10 mL) at 0 °C was added di-tert-butyl
dicarbonate (948 mg, 4.4 mmol). The mixture was stirred at rt for 2
h. The reaction was quenched with sat. NaHCO3 (15 mL), and the
mixture was extracted with EtOAc (3 × 20 mL). The combined
organic layers were dried over anhydrous Na2SO4 and concentrated in
vacuo. The residue was purified by flash-column chromatography to
give tert-butyl (1-hydroxy-7-(2-hydroxyethoxy)-1,3-dihydrobenzo[c]-
[1,2]oxaborol-3-yl)methylcarbamate (500 mg, yield 57%). To a
solution of the above compound (0.5 mg, 1.5 mmol) and N-
bromosuccinimide (354 mg, 2.0 mmol) in CH3CN (15 mL) was
added 2,2′-azobis(2-methylpropionitrile) (27 mg). The mixture was
stirred for 1 h at 90 °C. The reaction mixture was concentrated in
vacuo, and the residue was purified by prep-HPLC, eluting with water/
acetonitrile gradient to give tert-butyl (4-bromo-1-hydroxy-7-(2-
hydroxyethoxy)-1,3-dihydrobenzo[c][1,2]oxaborol-3-yl)-
methylcarbamate (300 mg, yield 50%). A mixture of the above
compound (0.2 g, 0.5 mmol) in 1 M HCl/Et2O (10 mL) was stirred at
rt for 1 h and then concentrated in vacuo. The residue was washed
with acetonitrile (2 × 5 mL) and dried in vacuo to give 24 as a white
solid (140 mg, yield 83%). 1H NMR (300 MHz, DMSO-d6) δ 8.36 (b,
3H), 7.64−7.61 (d, 1H, J = 9 Hz), 6.93−6.60 (d, 1H, J = 9 Hz), 5.51
(m, 1H), 4.69 (m, 1H), 4.36−4.23 (m, 3H), 3.62 (m, 1H), 3.01 (m,
1H). MS (ESI) m/z = 302 [M + H]+. HPLC purity: 99.5% (220 nm).
(S)-3-(Aminomethyl)-4-bromo-7-(2-hydroxyethoxy)benzo-
[c][1,2]oxaborol-1(3H)-ol Hydrochloride (24a) and (R)-3-(Ami-
nomethyl)-4-bromo-7-(2-hydroxyethoxy)benzo[c][1,2]-
oxaborol-1(3H)-ol Hydrochloride (24b). The racemic tert-butyl (4-
bromo-1-hydroxy-7-(2-hydroxyethoxy)-1,3-dihydrobenzo[c][1,2]-
oxaborol-3-yl)methylcarbamate was separated on a Thar preparative
SFC 80 system with a Daicel Chiralpak AD-H 5 μm (250 mm × 20
mm i.d.), eluted with carbon dioxide and methanol mobile phase. Two
3-(Aminomethyl)-4-chloro-7-(2-hydroxyethoxy)benzo[c]-
[1,2]oxaborol-1(3H)-ol Hydrochloride (23). To 2-bromoethanol
(1.88 g, 15 mmol) was added dihydropyran (1.26 g, 15 mmol)
dropwise at 0 °C. The mixture was stirred for 30 min at 0 °C and then
2 h at rt. Subsequently, 2-bromo-3-hydroxybenzaldehyde (2.0 g, 10
mmol) was added, followed by potassium carbonate (1.52 g, 11
mmol), potassium iodide (332 mg, 2 mmol), and dry DMF (20 mL).
The reaction was stirred at 70 °C overnight. The mixture was cooled
to rt and diluted with diethyl ether (100 mL). After the inorganic salts
were removed by filtration, the filtrate was diluted with hexane (100
mL). The organic layer was washed with water (3 × 50 mL), dried
over Na2SO4, filtered, and concentrated in vacuo. The residue was
purified by silica gel chromatography, eluting with EtOAc and
petroleum ether (1:3) to give 2-bromo-3-(2-(tetrahydro-2H-pyran-2-
yloxy)ethoxy)benzaldehyde as yellow oil (3.0 g, yield 91%). A mixture
of the above compound (160 g, 0.49 mol), bis(pinacolato)diborane
(249 g, 0.98 mol), PdCl2(dppf) (20 g, 24.5 mmol), and KOAc (144 g,
1.47 mol) in DMF (2.0 L) was degassed and stirred at 90 °C
overnight. After the reaction was quenched with ice−water (4 L), the
reaction mixture was extracted with EtOAc (3 × 1.5 L). The combined
organic layers were washed with brine, dried over anhydrous Na2SO4,
and concentrated in vacuo. The residue was purified by silica gel
chromatography, eluting with EtOAc and petroleum ether (1:10 to
1:2) to give 3-(2-(tetrahydro-2H-pyran-2-yloxy)ethoxy)-2-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde as yellow oil (88 g,
yield 48%). To a solution of NaOH (4.8 g, 0.12 mol) in water (100
mL) was added nitromethane (18.3 g, 0.3 mol) at 5−10 °C. After
stirring for 15 min at 5−10 °C, cetyltrimethylammonium bromide (2.2
g, 6.0 mmol) was added. After the above compound (45 g, 0.12 mol)
was added slowly at 5−10 °C, the reaction mixture was stirred at rt for
5 h. Subsequently, the reaction mixture was acidified to pH = 1 using
diluted hydrochloric acid and stirred at rt overnight. The residue was
filtered and dried in vacuo to give 7-(2-hydroxyethoxy)-3-
(nitromethyl)benzo[c][1,2]oxaborol-1(3H)-ol as a white solid (14.5
g, yield 48%). To a solution of the above compound (29 g, 115 mmol)
in DMF (250 mL) at 80 °C was added a solution of N-
chlorosuccinimide (16.5 g, 123 mmol) in DMF (100 mL). The
mixture was stirred for 30 min at 80 °C. After cooling down to rt, the
reaction mixture was poured into ice−water and extracted with EtOAc
(3 × 200 mL). The combined organic layers were washed with brine,
dried over Na2SO4, filtered, and concentrated in vacuo. The residue
was purified by recrystallization from EtOAc and petroleum ether
(1:10) to give crude product 4-chloro-7-(2-hydroxyethoxy)-3-
(nitromethyl)benzo[c][1,2]oxaborol-1(3H)-ol (24 g). A solution of
the above compound (24 g, 83.5 mmol), Raney Ni (4.0 g), and 7 M
NH3 in MeOH (20 mL) in MeOH (300 mL) was shaken under an
atmosphere of hydrogen for 2 h at rt. The mixture was filtered through
a bed of Celite, and the filtrate was concentrated in vacuo. The crude
amine was dissolved in MeOH (20 mL), and 1 M HCl in Et2O (30
mL) was added. After stirring for 1 h, the residue was filtered, washed
with acetonitrile/hexane (2:1, 2 × 200 mL), and dried in vacuo to give
23 as a white solid (12 g, yield 49%). 1H NMR (300 MHz, DMSO-d6)
δ 8.19 (b, 3H), 7.51−7.48 (d, 1H, J = 9 Hz), 6.99−6.96 (d, 1H, J = 9
Hz), 5.54 (m, 1H), 4.69 (m, 1H), 4.36−4.23 (m, 3H), 3.58 (m, 1H),
3.03 (m, 1H). MS (ESI) m/z = 258 [M + H]+. HPLC purity: 99.7%
(220 nm).
(S)-3-(Aminomethyl)-4-chloro-7-(2-hydroxyethoxy)benzo[c]-
[1,2]oxaborol-1(3H)-ol Hydrochloride (23a) and (R)-3-(Amino-
methyl)-4-chloro-7-(2-hydroxyethoxy)benzo[c][1,2]oxaborol-
1(3H)-ol Hydrochloride (23b). To a mixture of 23 (8.0 g, 27.2
mmol) and triethylamine (10.2 g, 101 mmol) in dichloromethane
(250 mL) at 0 °C was added di-tert-butyl dicarbonate (11 g, 50.6
L
DOI: 10.1021/acs.jmedchem.7b00631
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Journal of Medicinal Chemistry
Article
isomers were obtained. Each compound (1.2 g, 3.1 mmol) in 1 M
HCl/Et2O (20 mL) was stirred at rt for 1 h and concentrated in vacuo.
The residue was washed with acetonitrile (2 × 5 mL) and dried in
vacuo to give the desired product as a white solid. 24a (900 mg, yield
90%). 1H NMR (300 MHz, DMSO-d6) δ 8.37 (b, 3H), 7.63−7.60 (d,
1H, J = 9 Hz), 6.92−6.89 (d, 1H, J = 9 Hz), 5.51 (m, 1H), 4.68 (m,
1H), 4.36−4.23 (m, 3H), 3.61 (m, 1H), 3.01 (m, 1H). MS (ESI) m/z
= 302 [M + H]+. HPLC purity: 98.4% (220 nm). Analysis of the Boc-
derivatized material using a ChiralCel AD-H analytical column showed
the (S) enantiomer with a retention time of 3.79 min and 98.0% ee.
24b (900 mg, yield 90%). 1H NMR (300 MHz, DMSO-d6) δ 8.38 (b,
3H), 7.63−7.60 (d, 1H, J = 9 Hz), 6.92−6.89 (d, 1H, J = 9 Hz), 5.50
(m, 1H), 4.68 (m, 1H), 4.35−4.23 (m, 3H), 3.61 (m, 1H), 3.00 (m,
1H). MS (ESI) m/z = 302 [M + H]+. HPLC purity: 99.5% (220 nm).
Analysis of the Boc-derivatized material using a ChiralCel AD-H
analytical column showed the (R) enantiomer with a retention time of
3.34 min and 98.1% ee.
3-yl)methylcarbamate (0.4 g, 25%) as oil. A mixture of the above
compound (0.4 g, 1.3 mmol), EtI (0.6 g, 3.8 mmol), and K2CO3 (0.5
g, 3.8 mmol) in 30 mL of acetone was stirred at reflux for 16 h. The
solvent was evaporated in vacuo. The residue was purified by silica gel
chromatography, eluting with MeOH and CH2Cl2 (1:20) to give tert-
butyl (4-chloro-6-ethoxy-1-hydroxy-1,3-dihydrobenzo[c][1,2]-
oxaborol-3-yl)methylcarbamate (0.43 g, 99%). To a stirred solution
of the above compound (0.43 g, 1.3 mmol) in 4 mL of CH2Cl2 was
added TFA (2 mL). The reaction was stirred at rt for 1 h. The solvent
was evaporated in vacuo. The residue was purified by prep-HPLC
followed by treating with conc HCl to give 25 as a white solid (20 mg,
6%). 1H NMR (400 MHz, CD3OD) δ 7.17 (s, 1H), 7.14 (s, 1H), 5.47
(m, 1H), 4.10 (q, 2H), 3.82 (m, 1H), 3.04 (m, 1H), 1.42 (t, 3H). MS
(ESI) m/z = 242 [M + H]+. HPLC purity: 97.0% (220 nm).
3-(Aminomethyl)-4-bromo-6-ethoxybenzo[c][1,2]oxaborol-
1(3H)-ol Hydrochloride (26). To a solution of 2,6-dibromo-4-
hydroxybenzaldehyde (2 g, 7.1 mmol) and potassium carbonate (2.96
g, 21.4 mmol) in DMF (50 mL) was added CH3CH2Br (2.3 g, 21.4
mmol). The mixture was stirred at rt for 3 h. After water was added,
the mixture was extracted with EtOAc (2 × 100 mL). The combined
organic layers were washed with brine (50 mL), dried over anhydrous
Na2SO4, and concentrated in vacuo to give 2,6-dibromo-4-ethoxy-
benzaldehyde as a white solid (2.1 g, 95%). To a stirred solution of the
above compound (2 g, 6.5 mmol) and HC(OMe)3 (1.38 g, 13 mmol)
in MeOH (30 mL) was added conc H2SO4 (1 drop) at rt. The
reaction was stirred at reflux for 1 h. MeONa was added until pH = 7.
After the solvent was removed, the residue was washed with petroleum
ether. The organic layer was concentrated in vacuo to afford 1,3-
dibromo-2-(dimethoxymethyl)-5-ethoxybenzene as oil (2.0 g, 87%).
To a solution of the above compound (500 mg, 1.4 mmol) in
anhydrous ether (20 mL) was added n-BuLi (2.5 M, 0.56 mL, 1.4
mmol) dropwise at −78 °C under N2. After the mixture was stirred at
−78 °C for 15 min, trimethyl borate (176 mg, 1.7 mmol) was added
dropwise. The resulting mixture was stirred at −78 °C for 30 min. The
reaction was quenched by adding 2 N HCl solution until pH = 1, and
the mixture was extracted with EtOAc (3 × 20 mL). The combined
organic layers were washed with water (10 mL) and brine (10 mL),
dried over anhydrous Na2SO4, and concentrated in vacuo to give 3-
bromo-5-ethoxy-2-formylphenylboronic acid (500 mg, crude). A
mixture of the above compound (500 mg, 1.8 mmol), NaOH (73
mg, 1.8 mmol) in H2O (10 mL) and THF (20 mL) was stirred for 30
min. Nitromethane (558 mg, 9.2 mmol) was added, and the reaction
was stirred at rt for 3 h. The mixture was adjusted to pH = 1 by adding
diluted HCl (2 N) and then extracted with EtOAc (3 × 30 mL). The
combined organic layers were washed with brine (50 mL), dried over
anhydrous Na2SO4, and concentrated in vacuo to give 4-bromo-6-
ethoxy-3-(nitromethyl)benzo[c][1,2]oxaborol-1(3H)-ol as oil (500
mg, crude). A mixture of the above compound (500 mg, 1.6 mmol),
NH3 in MeOH (7 N, 0.2 mL), and Raney Ni (100 mg) in MeOH (40
mL) was stirred under an atmosphere of hydrogen for 4 h at rt. The
mixture was filtered through a bed of Celite, and the filtrate was
concentrated in vacuo. The residue was purified by prep-HPLC
followed by treating with conc HCl to give 26 as a white solid (120
3-(Aminomethyl)-4-chloro-6-ethoxybenzo[c][1,2]oxaborol-
1(3H)-ol Hydrochloride (25). A solution of 2-chloro-4,6-dihydroxy-
benzaldehyde (6.2 g, 35.9 mmol), DHP (6.02 g, 71.7 mmol), and
PPTS (0.9 g) in 150 mL of CH2Cl2 was stirred at rt for 1.5 h. After the
reaction was quenched with sat. NaHCO3 solution (50 mL) at 0 °C,
the mixture was washed with brine (30 mL), dried over anhydrous
Na2SO4, and concentrated in vacuo. The residue was purified by silica
gel chromatography, eluting with EtOAc and petroleum ether (1:100)
to give 2-chloro-6-hydroxy-4-(tetrahydro-2H-pyran-2-yloxy)-
benzaldehyde as colorless oil (7.8 g, 85%). To a stirred solution of
the above compound (7.8 g, 30.4 mmol) and pyridine (10.9 mL) in
150 mL of CH2Cl2 at −10 °C was added Tf2O (7.66 mL). The
reaction was stirred at 0 °C for 2 h. After the reaction was quenched
with brine (35 mL), the mixture was extracted with EtOAc (100 mL).
The solvent was evaporated in vacuo. The residue was purified by silica
gel chromatography, eluting with EtOAc and petroleum ether (1:200)
to give 3-chloro-2-formyl-5-(tetrahydro-2H-pyran-2-yloxy)phenyl
trifluoromethanesulfonate as yellow oil (7.0 g, 59%). A mixture of
the above compound (3.4 g, 8.7 mmol), bis(pinacolato)diborane (4.0
g, 15.8 mmol), KOAc (2.3 g, 23.7 mmol), and PdCl2(dppf) (0.58 g) in
120 mL of 1,4-dioxane was degassed with N2 six times. The reaction
was stirred at 80 °C for 1 h. The solvent was evaporated in vacuo. The
residue was purified by silica gel chromatography, eluting with EtOAc
and petroleum ether (1:10) to give 2-chloro-4-(tetrahydro-2H-pyran-
2-yloxy)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde
as a white solid (3.1 g, 97%). To a stirred solution of the above
compound (2.2 g, 6.0 mmol) and CH3NO2 (1.1 g, 18.0 mmol) in 40
mL of THF was added a solution of NaOH (0.24 g, 6.0 mmol) in 40
mL of water. The reaction was stirred at rt for 3 h. The reaction
mixture was treated with citric acid to pH = 6 and was then extracted
with EtOAc (120 mL). The organic phase was washed with brine (15
mL) and dried over anhydrous Na2SO4. The solvent was evaporated in
vacuo to give crude 4-chloro-3-(nitromethyl)-6-(tetrahydro-2H-pyran-
2-yloxy)benzo[c][1,2]oxaborol-1(3H)-ol. A solution of the above
compound (3.3 g, 10.1 mmol) in 33 mL of conc HCl and 66 mL of
MeOH was stirred at reflux for 1 h. The solvent was evaporated in
vacuo to give 4-chloro-3-(nitromethyl)benzo[c][1,2]oxaborole-
1,6(3H)-diol (3 g, crude). It was used directly in the next step
without further purification. A mixture of the above compound (1.4 g,
5.8 mmol) and Raney Ni (0.5 g) in 5 mL of ammonium in ethanol (2
M, 5 mL) and 20 mL of ethanol was hydrogenated at an atmosphere
of hydrogen for 6 h. The mixture was filtered through a bed of Celite,
and the filtrate was concentrated in vacuo. The residue was dissolved
in 30 mL of ethanol and treated with 1 mL of conc HCl. The solvent
was evaporated in vacuo to give 3-(aminomethyl)-4-chlorobenzo[c]-
[1,2]oxaborole-1,6(3H)-diol hydrochloride as solid (1.3 g, 90%). To a
stirred solution of the above compound (1.3 g, 5.2 mmol) and Et3N
(1.16 g, 11.5 mmol) in 200 mL of CH2Cl2 at 0 °C was added Boc2O
(2.5 g, 11.5 mmol). The reaction was stirred at rt for 4 h. The mixture
was washed with water (20 mL), brine (20 mL), dried over anhydrous
Na2SO4, and concentrated in vacuo. The residue was purified by silica
gel chromatography, eluting with MeOH and CH2Cl2 (1:10) to give
tert-butyl (4-chloro-1,6-dihydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-
1
mg, 24%). H NMR (300 MHz, DMSO-d6) δ 9.77 (b, 1H), 8.19 (b,
3H), 7.41 (s, 1H), 7.32 (s, 1H), 5.31 (m, 1H), 4.10 (m, 2H), 3.64 (m,
1H), 2.87 (m, 1H), 1.36 (m, 3H). MS (ESI) m/z = 286 [M + H]+.
HPLC purity: 95.3% (220 nm).
3-(Aminomethyl)-4-chloro-6-propoxybenzo[c][1,2]-
oxaborol-1(3H)-ol Hydrochloride (27). This compound was
prepared by the similar method described above for the synthesis of
1
25. H NMR (400 MHz, CD3OD) δ 7.16 (s, 1H), 7.12 (s, 1H), 5.45
(m, 1H), 3.99 (t, 2H), 3.79 (m, 1H), 3.06 (m, 1H), 1.82 (m, 2H), 1.07
(t, 3H). MS (ESI) m/z = 256 [M + H]+. HPLC purity: 96.2% (220
nm).
3-(Aminomethyl)-4-bromo-6-propoxybenzo[c][1,2]-
oxaborol-1(3H)-ol Hydrochloride (28). This compound was
prepared by the similar method described above for the synthesis of
1
26. H NMR (300 MHz, DMSO-d6) δ 9.71 (b, 1H), 8.09 (b, 3H),
7.36 (s, 1H), 7.31 (s, 1H), 5.28 (m, 1H), 3.97 (m, 2H), 3.60 (m, 1H),
M
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2.86 (m, 1H), 1.74 (m, 2H), 0.98 (t, 3H). MS (ESI) m/z = 300 [M +
H]+. HPLC purity: 96.5% (220 nm).
[1,2]oxaborol-1(3H)-ol (1.3 g, yield 99%). To a mixture of the above
compound (200 mg, 0.8 mmol) in glacial acetic acid (15 mL) was
added sulfuryl dichloride (64 μL, 0.8 mmol) dropwise. The reaction
was stirred at rt for 1.5 h. After the reaction was quenched with water
(15 mL), the mixture was extracted with EtOAc (3 × 20 mL). The
combined organic layers were dried over anhydrous Na2SO4 and
concentrated in vacuo. The residue 6,7-dixoane-4-chloro-3-
(nitromethyl)benzo[c][1,2]oxaborol-1(3H)-ol was used directly for
the next step without purification. A mixture of the above compound,
Raney Ni (∼50 mg), and 2 M NH3 in EtOH (2 mL) in EtOH (10
mL) was shaken under an atmosphere of hydrogen for 3 h. The
mixture was filtered through a bed of Celite, and the filtrate was
concentrated in vacuo. The crude amine was dissolved in EtOAc (1
mL), and 1 M HCl in Et2O (8 mL) was added. After stirring for 1 h,
the residue was filtered, washed with hexane, and dried in vacuo to give
3-(Aminomethyl)-4-chloro-6-(3-hydroxypropoxy)benzo[c]-
[1,2]oxaborol-1(3H)-ol Hydrochloride (29). This compound was
prepared by the similar method described above for the synthesis of
1
25. H NMR (400 MHz, CD3OD) δ 7.36 (s, 1H), 7.18 (s, 1H), 5.48
(m, 1H), 4.13 (t, 2H), 3.80 (m, 1H), 3.75 (t, 2H), 3.05 (m, 1H), 2.02
(m, 2H). MS (ESI) m/z = 272 [M + H]+. HPLC purity: 95.3% (220
nm).
3-(Aminomethyl)-4-bromo-6-(3-hydroxypropoxy)benzo[c]-
[1,2]oxaborol-1(3H)-ol Hydrochloride (30). This compound was
prepared by the similar method described above for the synthesis of
1
26. H NMR (300 MHz, DMSO-d6) δ 9.72 (b, 1H), 8.13 (b, 3H),
7.39 (s, 1H), 7.33 (s, 1H), 5.29 (m, 1H), 4.08 (t, 2H), 3.63 (m, 1H),
3.57 (m, 2H), 2.86 (m, 1H), 1.87 (m, 2H). MS (ESI) m/z = 316 [M +
H]+. HPLC purity: 91.3% (220 nm).
1
35 as a white solid (90 mg, yield 38%, two steps). H NMR (400
3-(Aminomethyl)-4-chloro-6-(3-hydroxypropoxy)benzo[c]-
[1,2]oxaborol-1(3H)-ol Hydrochloride (31). This compound was
prepared by the similar method described above for the synthesis of
MHz, DMSO-d6) δ 9.29 (b, 1H), 8.25 (b, 3H), 7.08 (s, 1H), 5.30 (m,
1H), 4.27−4.23 (m, 4H), 3.48 (m, 1H), 2.84 (m, 1H). MS (ESI) m/z
= 256 [M + H]+. HPLC purity: 98.9% (220 nm).
1
25. H NMR (400 MHz, CD3OD) δ 7.22 (s, 1H), 7.20 (s, 1H), 5.48
(S)-3-(Aminomethyl)-4-chloro-7,8-dihydro[1,2]oxaborolo-
[3′,4′:3,4]benzo[1,2-b][1,4]dioxin-1(3H)-ol Hydrochloride (35a)
and (R)-3-(Aminomethyl)-4-chloro-7,8-dihydro[1,2]oxaborolo-
[3′,4′:3,4]benzo[1,2-b][1,4]dioxin-1(3H)-ol Hydrochloride
(35b). These compounds were prepared by the similar method
described above for the synthesis of 23a and 23b. 35a: 1H NMR (400
MHz, DMSO-d6) δ 9.34 (b, 1H), 8.26 (b, 3H), 7.12 (s, 1H), 5.33 (m,
1H), 4.30−4.26 (m, 4H), 3.52 (m, 1H), 2.86 (m, 1H). MS (ESI) m/z
= 256 [M + H]+. HPLC purity: 98.1% (220 nm). Analysis of the Boc-
derivatized material using a ChiralPak AY-H analytical column showed
the (S) enantiomer with a retention time of 1.64 min and >99% ee.
(m, 1H), 4.11 (t, 2H), 3.91 (t, 2H), 3.85 (m, 1H), 3.05 (m, 1H). MS
(ESI) m/z = 258 [M + H]+. HPLC purity: 98.4% (220 nm).
3-(Aminomethyl)-4-bromo-6-(3-hydroxypropoxy)benzo[c]-
[1,2]oxaborol-1(3H)-ol Hydrochloride (32). This compound was
prepared by the similar method described above for the synthesis of
1
26. H NMR (300 MHz, DMSO-d6) δ 9.72 (b, 1H), 8.11 (b, 3H),
7.39 (s, 1H), 7.32 (s, 1H), 5.28 (m, 1H), 4.91 (t, 1H), 4.04 (t, 2H),
3.75 (t, 2H), 3.70 (m, 1H), 2.86 (m, 1H). MS (ESI) m/z = 302 [M +
H]+. HPLC purity: 98.9% (220 nm).
3-(Aminomethyl)-4-chloro[1,3]dioxolo[4′,5′:5,6]benzo[1,2-
c][1,2]oxaborol-1(3H)-ol Hydrochloride (33). This compound was
prepared by the similar method described below for the synthesis of
1
35b: H NMR (400 MHz, DMSO-d6) δ 9.34 (b, 1H), 8.17 (b, 3H),
7.13 (s, 1H), 5.31 (m, 1H), 4.30−4.26 (m, 4H), 3.54 (m, 1H), 2.90
(m, 1H). MS (ESI) m/z = 256 [M + H]+. HPLC purity: 95.7% (220
nm). Analysis of the Boc-derivatized material using a ChiralPak AY-H
analytical column showed the (R) enantiomer with a retention time of
1.95 min and >99% ee.
1
35. H NMR (400 MHz, DMSO-d6) δ 9.65 (b, 1H), 7.98 (b, 3H),
7.24 (s, 1H), 6.17 (ds, 2H), 5.33 (m, 1H), 3.54 (m, 1H), 2.99 (m,
1H). MS (ESI) m/z = 242 [M + H]+. HPLC purity: 98.9% (220 nm).
3-(Aminomethyl)-4-bromo[1,3]dioxolo[4′,5′:5,6]benzo[1,2-
c][1,2]oxaborol-1(3H)-ol Trifluoroacetate (34). This compound
was prepared by the similar method described below for the synthesis
of 35 and obtained as TFA salt after prep-HPLC purification. 1H
NMR (400 MHz, DMSO-d6) δ 9.58 (b, 1H), 7.96 (b, 3H), 7.29 (s,
1H), 6.09 (ds, 2H), 5.18 (m, 1H), 3.52 (m, 1H), 2.90 (m, 1H). MS
(ESI) m/z = 286 [M + H]+. HPLC purity: 95.1% (220 nm).
3-(Aminomethyl)-4-bromo-7,8-dihydro[1,2]oxaborolo-
[3′,4′:3,4]benzo[1,2-b][1,4]dioxin-1(3H)-ol Trifluoroacetate
(36). This compound was prepared by the similar method described
above for the synthesis of 35 and obtained as TFA salt after prep-
1
HPLC purification. H NMR (400 MHz, DMSO-d6) δ 9.28 (b, 1H),
8.05 (b, 3H), 7.25 (s, 1H), 5.20 (m, 1H), 4.30−4.23 (m, 4H), 3.50 (m,
1H), 2.93 (m, 1H). MS (ESI) m/z = 300 [M + H]+. HPLC purity:
98.8% (220 nm).
3-(Aminomethyl)-4-chloro-7,8-dihydro[1,2]oxaborolo-
[3′,4′:3,4]benzo[1,2-b][1,4]dioxin-1(3H)-ol Hydrochloride (35).
A mixture of 2-bromo-3,4-dihydroxybenzaldehyde (4.5 g, 20.7 mmol),
1,2-dibromoethane (4.65 g, 24.8 mmol), and K2CO3 (8.9 g, 64.5
mmol) in DMF (40 mL) was stirred at 50 °C overnight. After H2O
(80 mL) was added, the mixture was extracted with EtOAc (2 × 80
mL). The combined organic layers were dried over anhydrous Na2SO4
and concentrated in vacuo. The residue was purified by silica gel
chromatography to give 5-bromo-2,3-dihydrobenzo[b][1,4]dioxine-6-
carbaldehyde (1.5 g, yield 30%). A mixture of the above compound
(2.68 g, 11.0 mmol), bis(pinacolato)diborane (4.0 g, 16.3 mmol),
KOAc (4.1 g, 41.8 mmol), and PdCl2(dppf) (310 mg, 4.2 mmol) in
dioxane (60 mL) was degassed for 15 min with N2. The reaction was
stirred at 100 °C for 3 h. After the reaction was quenched with ice−
water (25 mL), the mixture was extracted with EtOAc (3 × 70 mL).
The combined organic layers were washed with water (35 mL) and
brine (40 mL), dried over anhydrous Na2SO4, and concentrated in
vacuo. The residue was purified by silica gel chromatography to give 5-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzo[b]-
[1,4]dioxine-6-carbaldehyde (1.5 g, yield 47%). A mixture of the above
compound (1.52 g, 5.2 mmol), 10% NaOH solution (6 mL), and THF
(30 mL) was stirred for 0.5 h at rt. After nitromethane (980 mg 16.1
mmol) was added, the reaction was stirred at rt for 2 h. The mixture
was adjusted to pH = 2 by adding diluted HCl (2 N) and extracted
with EtOAc (3 × 60 mL). The combined organic layers were washed
with brine (50 mL), dried over anhydrous Na2SO4, and concentrated
in vacuo. The residue was purified by prep-HPLC, eluting with water/
acetonitrile gradient to give the 6,7-dixoane-3-(nitromethyl)benzo[c]-
(S)-3-(Aminomethyl)-4-bromo-7,8-dihydro[1,2]oxaborolo-
[3′,4′:3,4]benzo[1,2-b][1,4]dioxin-1(3H)-ol Hydrochloride (36a)
and (R)-3-(Aminomethyl)-4-bromo-7,8-dihydro[1,2]oxaborolo-
[3′,4′:3,4]benzo[1,2-b][1,4]dioxin-1(3H)-ol Hydrochloride
(36b). These compounds were resolved by the similar method
described above for the synthesis of 23a and 23b. 36a: 1H NMR (300
MHz, DMSO-d6) δ 9.28 (b, 1H), 8.19 (b, 3H), 7.23 (s, 1H), 5.22 (m,
1H), 4.29 (m, 4H), 3.56 (m, 1H), 2.88 (m, 1H). MS (ESI) m/z = 300
[M + H]+. HPLC purity: 97.9% (220 nm). Analysis of the Boc-
derivatized material using a ChiralCel AD-H analytical column showed
the (S) enantiomer with a retention time of 2.79 min and 97.6% ee.
1
36b: H NMR (300 MHz, DMSO-d6) δ 9.28 (b, 1H), 8.15 (b, 3H),
7.23 (s, 1H), 5.23 (m, 1H), 4.29 (m, 4H), 3.54 (m, 1H), 2.89 (m, 1H).
MS (ESI) m/z = 300 [M + H]+. HPLC purity: 96.5% (220 nm).
Analysis of the Boc-derivatized material using a ChiralCel AD-H
analytical column showed the (R) enantiomer with a retention time of
2.62 min and 99.6% ee.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (+34) 629170293. Fax: (+34) 918070550. E-mail:
david.a.barros@gsk.com.
ORCID
David Barros: 0000-0002-4099-0438
N
DOI: 10.1021/acs.jmedchem.7b00631
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Journal of Medicinal Chemistry
Article
(16) Stinson, K.; Kurepina, N.; Venter, A.; Fujiwara, M.; Kawasaki,
M.; Timm, J.; Shashkina, E.; Kreiswirth, B. N.; Liu, Y.; Matsumoto, M.;
Geiter, L. MIC of delamanid (OPC-67683) against Mycobacterium
tuberculosis clinical isolates and a proposed critical concentration.
Antimicrob. Agents Chemother. 2016, 60, 3316−3322.
Present Address
∥I.A.-B.: The Art of Discovery, SL, Biscay Science and
Technology Park, Astondo Bidea, BIC Bizkaia Building, No.
612, Derio 48160, Bizkaia, Basque Country, Spain.
Notes
(17) Martínez-Hoyos, M.; Perez-Herran, E.; Gulten, G.; Encinas, L.;
́
Alvarez-Gom
Ortega, F.; Angulo-Barturen, I.; Rullas-Trincado, J.; Blanco Ruano, D.;
Torres, P.; Castaneda, P.; Huss, S.; Fernandez Menendez, R.; Gonzalez
́ ́
ez, D.; Alvarez, E.; Ferrer-Bazaga, S.; García-Perez, A.;
The authors declare no competing financial interest.
́
́
́
̃
ABBREVIATIONS USED
■
Del Valle, S.; Ballell, L.; Barros, D.; Modha, S.; Dhar, N.; Signorino-
Gelo, F.; McKinney, J. D.; García-Bustos, J. F.; Lavandera, J. L.;
Sacchettini, J. C.; Jimenez, M. S.; Martín-Casabona, N.; Castro-Pichel,
J.; Mendoza-Losana, A. Antitubercular drugs for an old target: GSK693
as a promising InhA direct inhibitor. EBioMedicine. 2016, 8, 291−301.
CFU, colony-forming units; DNAUC, dose-normalized area
under the curve; dppf, 1,1′-bis(diphenylphosphino)ferrocene;
i.d., inside diameter; LeuRS, leucyl-tRNA synthetase; Mtb,
Mycobacterium tuberculosis; (pin)2, bis(pinacolato); TEA,
triethylamine; TDR-TB, totally drug-resistant tuberculosis;
XDR-TB, extensively drug-resistant tuberculosis
̌
̌
(18) Sink, R.; Sosic,
̌
I.; Zivec, M.; Fer
́
nandez-Menen
́
dez, R.; Turk, S.;
́
Pajk, S.; Alvarez-Gomez, D.; Lopez-Roman
́
́
, E. M.; Gonzales-Cortez,
C.; Rullas-Triconado, J.; Angulo-Barturen, I.; Barros, D.; Ballell-Pages,
L.; Young, R. J.; Encinas, L.; Gobec, S. Design, synthesis, and
evaluation of new thiadiazole-based direct inhibitors of enoyl acyl
carrier protein reductase (InhA) for the treatment of tuberculosis. J.
Med. Chem. 2015, 58, 613−624.
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