Structure-activity relationships of 6-(aminomethylphenoxy)-benzoxaborole derivatives as anti-inflammatory agent

Article abstract of DOI:10.1016/j.bmcl.2013.01.072

A series of novel 6-(aminomethylphenoxy)benzoxaborole analogs was synthesized for the investigation of the structure-activity relationship of the inhibition of TNF-alpha, IL-1beta, and IL-6, from lipopolysaccharide stimulated peripheral blood mononuclear cells. Compounds 9d and 9e showed potent activity against all three cytokines with IC₅₀ values between 33 and 83 nM. Chloro substituted analog 9e (AN3485) is considered to be a promising lead for novel anti-inflammatory agent with a favorable pharmacokinetic profile.

Full text of DOI:10.1016/j.bmcl.2013.01.072

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1680–1683
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationships of 6-(aminomethylphenoxy)-
benzoxaborole derivatives as anti-inflammatory agent
a
a
a
a
Tsutomu Akama a, , Charlotte Virtucio , Chen Dong , Richard Kimura , Yong-Kang Zhang ,
James A. Nieman b, Rashmi Sharma b, Xiaosong Lu b, Marcelo Sales b, Rajeshwar Singh b, Anne Wu a,
Xiao-Qing Fan a, Liang Liu a, Jacob J. Plattner a, Kurt Jarnagin a, Yvonne R. Freund a
⇑
a Anacor Pharmaceuticals Inc., 1020 E. Meadow Circle, Palo Alto, CA 94303, USA
b NAEJA Pharmaceuticals Inc., 4290-91A St. Edmonton, Alberta, Canada T6E 5V2
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 6-(aminomethylphenoxy)benzoxaborole analogs was synthesized for the investigation
of the structure–activity relationship of the inhibition of TNF-alpha, IL-1beta, and IL-6, from lipopolysac-
charide stimulated peripheral blood mononuclear cells. Compounds 9d and 9e showed potent activity
against all three cytokines with IC50 values between 33 and 83 nM. Chloro substituted analog 9e
(AN3485) is considered to be a promising lead for novel anti-inflammatory agent with a favorable phar-
macokinetic profile.
Received 11 December 2012
Revised 12 January 2013
Accepted 16 January 2013
Available online 29 January 2013
Keywords:
Benzoxaborole
Ó 2013 Elsevier Ltd. All rights reserved.
Structure–activity relationships
Anti-inflammatory
Cytokine
Tumor necrosis factor-alpha
Interleukin-1beta
Interleukin-6
Pharmacokinetics
The clinical success of various biologic therapeutics has demon-
strated that several cytokines are important therapeutic targets for
chronic inflammatory diseases. Therapeutics targeting tumor
pharmacophore, we have discovered a number of novel anti-infec-
tive and anti-inflammatory agents.4 As part of this research, we
have created a library of boron-containing compounds which has
proven to be a useful source for both biochemical and phenotypic
screening. In the current project, we screened our library for the
necrosis factor-
a (TNF-a) are used for rheumatoid arthritis (RA),
psoriasis, inflammatory bowel disease (IBD), and more.1 The biolo-
gics targeting interleukin-1b (IL-1b) are approved for either RA or
cryopyrin-associated periodic syndrome and the interleukin-6
(IL-6) receptor monoclonal antibody is approved for RA, Crohn’s
disease, and Castman’s disease.1 Although biologics have been
highly effective and represent an important therapeutic advance,
there remain a number of limitations for this class of agents, such
as administration by injection, high cost, and poor or partial effi-
cacy in considerable numbers of individuals. This leaves an unmet
medical need for alternative therapies.1–3 An orally or topically
administered small molecule which blocks multiple inflammatory
cytokines would meet the needs for ease of administration, lower
cost and potentially increased efficacy.
inhibitory activity against the release of three cytokines, TNF-a,
IL-1b, and IL-6, from lipopolysaccharide (LPS) stimulated peripheral
blood mononuclear cells (PBMCs) in order to identify compounds
that inhibit secretion of all three cytokines. Compounds 1a and 1b
(Fig. 1) were found to show IC50 values ranging from 0.28 to
1.6
l
M against all three cytokines. Preliminary structure–activity
OH
O
B
3'
O
R
4'
Anacor Pharmaceuticals is focused on discovering new thera-
peutic agents by inclusion of boron atom into small molecule scaf-
folds. Using the benzoxaborole and related scaffolds as a novel drug
R
R
3'-CH2NH2 HCl
1b 4'-CH2NH2 HCl
3'-CH2NMe2 HCl
3'-NH2
1f 4'-NH2
3'-CO2H
1a
1e
1c
1g
1d 4'-CH2NMe2 HCl 1h 4'-CO2H
⇑
Corresponding author. Tel.: +1 650 543 7527; fax: +1 650 543 7660.
E-mail address: takama@anacor.com (T. Akama).
Figure 1. Chemical structures of compounds 1a–h.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.072

T. Akama et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1680–1683
1681
2
5
X
OH
F
Br
OH
O
Br
3
a
F
HO
B
R
R
+
O
+
CHO
CHO
4
NC
7a
4a-d
3
R
F
R
8
2a
2b
2c
b
2-CN
X
OH
7b Cl
4-CN-2-OMe
O
B
O
B
a
5-CN-2-Me
O
O
O
O
R
2d 3-
R
O
CHO
X
F
R
CN
X
R
b
b
5a-d
9d
9e
9f
9a
F
CH2NH2 HCl
9b Cl CN
9c
Cl CH2NH2 HCl
Cl NHCOCH3
c
c
d
Cl CONH2
OH
2'
5'
Scheme 2. Synthesis of compounds 9a–e. Reagents and conditions: (a) K2CO3,
DMSO, 80–90 °C, overnight (33–61%); (b) LAH, THF, 0 °C to rt, 1 h, then 4 M HCl in
1,4-dioxane (43–68%); (c) aq NaOH, MeOH, 50 °C, 2 h (61%), (d) Ac2O, pyridine, rt
(79%).
O
B
3'
O
R
4'
R
R
d
d
d
e
6a 2'-CN
6b
6e 2'-CH2NH2 HCl
6f
4'-CN-2'-OMe
6c 5'-CN-2'-Me
6d
4'-CH2NH2-2'-OMe HCl
OH
O
6g 5'-CH2NH2-2'-Me HCl
6h 3'-CH2-morpholino HCl
6i 3'-CH2-(4-Me-piperazino) 2HCl
B
O
3'-CHO
H2N
f
OH
a
O
10
O
B
Scheme 1. Synthesis of compounds 6a–g. Reagents and conditions: (a) K2CO3, DMF,
80 °C, overnight (70–91%); (b) bis(pinacolato)diboron, PdCl2(dppf) CH2Cl2, KOAc,
1,4-dioxane, 90 °C, 2–16 h (46–88%); (c) NaBH4, MeOH, 0 °C to rt, 1 h, then HCl (42–
80%); (d) LAH, THF, 0 °C to rt, 1 h, then 4 M HCl in 1,4-dioxane (11–43%); (e)
morpholine, CH(OEt)3, NaBH(OAc)3, EtOH, CH2Cl2, rt, 1 h (34%); (f) 1-methylpiper-
azine, CH(OEt)3, NaBH(OAc)3, EtOH, CH2Cl2, rt, 1 h (27%).
O
b
OH
HO
O
B
O
O
1f
HO
11
Scheme 3. Synthesis of compounds 10 and 11. Reagents and conditions: (a)
NH4OH, EDC, HOBT, DMF, rt, 24 h (44%); (b) BH3, THF, 0 °C to rt (quant).
relationships (SAR) observed from the screening indicated that the
primary aminomethyl group would be important for the activity.
Based on this finding, further chemical modification was initiated.
Herein we describe the in vitro SAR of novel 6-(aminomethyl-
phenoxy)benzoxaborole derivatives as anti-inflammatory agents
F
Br
SH
S
Br
a,b
+
OH
CHO
NC
c,d
NC
with inhibitory activity against TNF-
a, IL-1b, and IL-6 production
12
13
2
in PBMCs and the mouse pharmacokinetics of the most potent
compounds 9d and 9e.
OH
S
B
HCl
H2N
O
There are a number of ways to prepare substituted benzoxabo-
role derivatives.4 Syntheses of compounds 1a–h were reported
previously.5 Synthesis of 6-(aminomethylphenoxy)benzoxaborole
analogs (6e–i) are shown in Scheme 1. SNAr reactions between
substituted phenols (2a–d) and 2-bromo-4-fluorophenol (3) gave
diphenylethers (4a–d). Miyaura-borylation6 converted the bro-
mide into pinacol borates (5a–d). The formyl group of compounds
5a–d was reduced by NaBH4 followed by acidic aqueous work-up
to afforded benzoxaboroles 6a–d. The cyano group of 6a–c was re-
duced to aminomethyl group by LAH to give aminomethyl deriva-
tives 6e–g. Compound 6d was converted into morpholinomethyl
(6h) and 4-methylpiperazinomethyl (6i) derivatives by reductive
amination.
14
Scheme 4. Synthesis of compound 14. Reagents and conditions: (a) K2CO3, DMF,
80 °C (44%); (b) NaBH4, MeOH, 0 °C to rt (100%); (c) (i-PrO)3B, n-BuLi, THF, ꢀ78 °C to
rt (62%); (d) LAH, THF, 0 °C to rt, then 4 M HCl in 1,4-dioxane, THF, Et2O (26%).
to form the benzoxaborole scaffold. Then the cyano group was re-
duced to the aminomethyl group with LAH to give 14.
The compounds were tested for the inhibitory activity against
the release of the three cytokines, TNF-a, IL-1b, and IL-6 from
PBMCs stimulated by LPS.8 The results are summarized in Table 1.
Two aminomethylphenoxy analogs (1a,b) showed IC50 values
The synthesis of fluoro- and chloro-substituted analogs (9d and
9e7) and some related analogs are shown in Scheme 2. SNAr reac-
tions between 7a,b and 85 successfully gave the desired products
9a,b. The cyano group of 9a,b was reduced to aminomethyl group
by LAH. Compound 9b was partially hydrolyzed with sodium
hydroxide in methanol to give the carboxamide (9c). Compound
9e was treated with acetic anhydride to give the acetamide (9f).
Compounds 10 and 11 were prepared from compound 1f5 by
the amide coupling using EDC and borane reduction, respectively,
as shown in Scheme 3.
The thioether analog 14 was synthesized as shown in Scheme 4.
Compounds 12 and 2 were coupled under basic condition followed
by NaBH4 reduction to give 13. Compound 13 was mixed with tri-
isopropyl borate followed by the slow addition of n-BuLi at ꢀ78 °C
ranging from 0.28 to 1.6 lM against the release of the three cyto-
kines. N,N-Dimethylaminomethyl (1c,d) and 3-amino (1e) analogs
lost the activity. The 4-amino analog (1f) showed moderate activity
against TNF-a (9.1 lM) and IL-1b (4.8 lM), but not against IL-6.
The 3-carboxy analog (1g) was slightly less potent than the 3-ami-
nomethyl analog (1a) against all three cytokines. The 4-carboxy
analog (1h) showed twofold to sixfold less potent IC50 values than
the 4-aminomethyl analog (1b). Relocation of the aminomethyl
group to 20-position (6e) resulted in loss of activity. Installation
of methyl (6f) or methoxy (6g) to the same phenyl ring resulted
in decrease in activity. Both morpholinomethyl (6h) and N-methyl-
piperazinomethyl (6i) derivatives also showed decreased activity.
When a fluoro (9d) or a chloro (9e) atom was introduced to
the 20-position of the 4-aminomethyl derivative (1b), activity was

1682
T. Akama et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1680–1683
Table 1
was not tolerated even with the smallest methyl groups, suggest-
ing that the polarity would be important for the activity. The most
potent compounds (9d,e) showed broader spectrum of activity
than several standard anti-inflammatory agents. A corticosteroid,
In vitro IC50 results of compounds against three cytokines released from PBMCsa,b
Compound
IC50
(l
M) TNF-
a
IC50
(
lM) IL1-b
IC50 (lM) IL-6
1a
1b
1c
1d
1e
1f
1g
1h
6a
6b
6c
6e
6f
6g
6h
6i
9a
9b
9c
9d
9e
9f
10
11
0.42
1.6
>10
>10
>10
9.1
0.32
0.28
>10
>10
>10
4.8
0.34
1.4
Clobetasol, showed very potent activity against TNF-
but moderate activity against IL-1b (4.4 M) and IL-6 (4.2
other steroid, dexamethasone, showed IC50 values of 17 and 9.5 nM
against TNF- and IL-1b, respectively, while it showed only 43%
inhibition against IL-6 at 10 M. A p38 MAP kinase inhibitor, SB-
203580, was active against TNF- and IL-1b with IC50 values of
1.0 M and 0.34 M, respectively, but not active against IL-6
(>10 M).
a
(7.0 nM),
>10
>10
>10
>10
0.57
4.0
l
lM). An-
a
l
0.75
4.4
0.40
1.9
a
>10
>10
>10
>10
>10
>10
5.9
>10
>10
>10
>10
>10
>10
>10
>10
1.5
>10
4.4
7.1
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
l
l
l
Mouse pharmacokinetics was examined for the most potent
compounds 9d and 9e.9 The results are summarized in Table 2.
Both compounds showed similar clearance, volume of distribution,
and AUC after intravenous administration. Compound 9e; how-
ever, showed higher Cmax (10-fold), AUC (fivefold), and bioavail-
ability (4.5-fold) after oral dosing. Although 9d showed slightly
more potent IC50 values against cytokine production assay, 9e
showed fivefold higher oral exposure in vivo PK study, suggesting
that 9e would show better oral efficacy in vivo.
Oral administration of 9e actually showed dose-dependent sup-
pression of LPS-induced TNF-a and IL-6 production in mice with an
ED90 of 30 mg/kg, and suppressed collagen-induced arthritis in
mice over a 20-day period (35 mg/kg, BID).10 Topically applied
compound 9e also showed significant inhibition of mouse ear
swelling induced by either phorbol ester or oxazolone.10
Compounds 9d and 9e showed almost equal activity against the
release of all three cytokines tested, which was distinct from the
reference compounds, clobetasol, dexamethasone, and SB203580.
Compound 9e does not inhibit typical drug targets of anti-inflam-
matory agents, such as cyclooxygenases, phosphodiesterase-4, p38
MAP kinase, or JAK kinases.10 Although the mechanism of action of
the benzoxaborole analogs described in this Letter is unknown, the
inhibition of cytokine release from PBMCs by compound 9e is not
due to cell general cytotoxicity, and appears to be down stream of
TLRs and up stream of the transcriptional machinery.10 Multiple
benzoxaborole analogs, such as AN2690 (Tevaborole) for onycho-
mycosis,11 AN2728 for atopic dermatitis and psoriasis,4b and
SCYX-7158 for human African trypanosomiasis,12 are being clini-
cally developed and showing good tolerability in human. Metabo-
lism studies of several of these compounds show a general pattern
of oxidative deboronation13 or typical transformations of groups,
such as primary alcohol moieties in parts of the molecule distant
from the boron atom.14 The compounds in development are free
of gentotoxicy and long term carcinogenicity signals.15 Compound
9e (AN3485) is considered to be a promising lead for novel anti-
inflammatory agent with potent inhibitory activity against the re-
lease of three cytokines and favorable PK profile in mice. Further
structural optimization, in vivo efficacy studies in both oral and
topical inflammatory disease models, and the studies on its mech-
anism of action are in progress.
>10
>10
>10
0.041
0.083
>10
0.043
0.052
>10
0.033
0.067
>10
>10
>10
>10
>10
>10
>10
14
10
>10
>10
Clobetasol
Dexamethasone
SB-203580
0.0070
0.017
1.0
4.4
0.0095
0.34
4.2
43%c
>10
a
IC50 values are means of P2 experiments for all; except 1c and Clobetasol for
TNF- ; and 1e, 1f, 6i, and 14 for IL-1b which are n = 1. IC50 for compounds with
<40% inhibition at 100
all resulted in between 80% and 100% inhibition at 100
a
l
M are reported as >10
lM. Compounds with reported IC50
s
lM; except that 40–79%
inhibition was observed for 1h, 6f, 6h, and 6i for IL1-b; and 1b for IL-6.
b
All curves had Hill slopes between 0.5 and 1.8; except, 1g and 14 for TNF-
a; 1h,
1g, and 6f for IL1-b which displayed Hill slopes >2. Compounds 1f for TNF-
a; and 1b
for IL1-b and IL-6 had a Hill slope of <0.5.
c
% inhibition at 10 lM.
Table 2
In vivo mouse PK parameters of 9d and 9e
Compound
9d
9e
IV (5 mg/kg)
(n = 3)
CL (mL/h/kg)
Vss (mL/kg)
2280
2140
1.74
0.65
2210
2870
2.26
1.39
AUC (h
lg/mL)
Terminal t1/2 (h)
PO (10 mg/kg)
(n = 3)
Cmax g/mL)
Tmax (h)
AUC (h
Terminal t1/2 (h)
Bioavailability (%)
(
l
0.142
0.25
0.533
2.43
15
1.45
0.25
2.66
2.82
67
l
g/mL)
increased 18- to 39-fold against TNF-a, fivefold to sixfold against
IL-1b, and 20- to 42-fold against IL-6 compared to 1b, respectively.
IC50 values of these two compounds against three cytokines were
ranging from 33 to 83 nM. Although the fluoro analog (9d) seemed
to be slightly more potent than the chloro analog (9e), the differ-
ence might be within experimental error. The carboxamide analogs
(9c and 10), the acetamide (9f), and the hydroxymethyl analog (11)
were all inactive. Replacement of the ether linker of 1b with sulfur
linker (14) resulted in losing activity. The cyano derivatives (6a–c,
and 9a,b) did not suppress cytokine secretion. Interestingly, both
aminomethyl (1a,b) and carboxy analogs (1g,h) showed similar
activity, while aniline analogs (1e,f) did not. These results indicate
that a chargeable functional group is required at either 30- or 40-po-
sition and the charged atoms have to be at least one atom distance
from the phenyl ring. N,N-Dialkylation of the aminomethyl group
References and notes
1. Kopf, M.; Bachmann, M. F.; Marsland, B. J. Nat. Rev. Drug Disc. 2010, 9, 703.
2. Le, G. T.; Abbenante, G. Curr. Med. Chem. Lett. 2005, 12, 2963.
3. Peifer, C.; Wagner, G.; Laufer, S. Curr. Top. Phram. Med. Chem. 2006, 6, 113.
4. (a) Baker, S. J.; Zhang, Y.-K.; Akama, T.; Lau, A.; Zhou, H.; Hernandez, V.; Mao,
W.; Alley, M. R. K.; Sanders, V.; Plattner, J. J. J. Med. Chem. 2006, 49, 4447; (b)
Akama, T.; Baker, S. J.; Zhang, Y.-K.; Hernandez, V.; Zhou, H.; Sanders, V.;
Freund, Y.; Kimura, R.; Maples, K. R.; Plattner, J. J. Bioorg. Med. Chem. Lett. 2009,
19, 2129; (c) Zhang, Y.-K.; Plattner, J. J.; Akama, T.; Baker, S. J.; Hernandez, V.;
Sanders, V.; Freund, Y.; Kimura, R.; Bu, W.; Hold, K. M.; Lu, X. S. Bioorg. Med.
Chem. Lett. 2010, 20, 2270; (d) Ding, D.; Zhao, Y.; Meng, Q.; Xie, D.; Nare, B.;
Chen, D.; Bacchi, C. J.; Yarlett, N.; Zhang, Y.-K.; Hernandez, V.; Xia, Y.; Freund,
Y.; Abdulla, M.; Ang, K. H.; Ratnam, J.; McKerrow, J. H.; Jacobs, R. T.; Zhou, H.;
Plattner, J. J. ACS Med. Chem. Lett. 2010, 1, 165; (e) Ding, C. Z.; Zhang, Y.-K.; Li, X.;
Liu, Y.; Zhang, S.; Zhou, Y.; Plattner, J. J.; Baker, S. J.; Liu, L.; Duan, M.; Jarvest, R.

T. Akama et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1680–1683
1683
L.; Ji, J.; Kazmierski, W. M.; Tallant, M. D.; Wright, L. L.; Smith, G. K.; Crosby, R.
M.; Wang, A. A.; Ni, Z.-J.; Zou, W.; Wright, J. Bioorg. Med. Chem. Lett. 2010, 20,
7317; (f) Zhang, Y.-K.; Plattner, J. J.; Freund, Y. R.; Easom, E. E.; Zhou, Y.; Gut, J.;
Rosenthal, P. J.; Waterson, D.; Gamo, F.-J.; Angulo-Barturen, I.; Ge, M.; Li, Z.;
Jian, Y.; Cui, H.; Wang, H.; Yang, J. Bioorg. Med. Chem. Lett. 2011, 21, 644; (g)
Zhang, Y.-K.; Plattner, J. J.; Freund, Y. R.; Easom, E. E.; Zhou; Ye, L.; Zhou, H.;
Waterson, D.; Gamo, F.-J.; Sanz, L. M.; Ge, M.; Li, M.; Li, L.; Wang, H.; Cui, H.
Bioorg. Med. Chem. Lett. 2012, 22, 1299; (h) Freund, Y. R.; Akama, T.; Alley, M. R.
K.; Antunes, J.; Dong, C.; Jarnagin, K.; Kimura, R.; Nieman, J. A.; Maples, K. R.;
Plattner, J. J.; Rock, F.; Sharma, R.; Singh, R.; Sanders, V.; Zhou, Y. FEBS Lett.
2012, 586, 3410.
supplemented with 10% FBS, 2 mM glutamine and 10 mM HEPES, in a 95% air/
lL were seeded in each well of a 96-
well culture plate for 3 h, then pre-incubated with test articles for 15 min,
before stimulation with 1 lg/mL of LPS in a final volume of 200 lL. After 24 h,
the cell culture supernatants were collected for cytokine determinations using
Cisbio HTRF cytokine determination kits. Compounds were prepared using an
eight point 1:10 dilution series starting at 100 lM; the resulting dose–response
curves were fit to the four parameter sigmoid equation.
5% CO2 incubator. 5 ꢁ 105 PBMCs in 100
9. CD-1 mice were divided into 16 groups with three mice/group, and receive 9d
or 9e by either intravenous (IV) injection with 5 mg/kg in a solution comprising
50% saline, 40% PGE400 and 10% DMSO (50/40/10 Saline/PGE400/DMSO) (pH
5.7), or by a gavage (PO) with 10 mg/kg in 70/25/5 PGE400/PG/DMSO solution.
Blood samples were collected at eight time points during a 24 h period with
three mice/time point. Drug concentrations in plasma were quantitated by LC/
MS/MS16 with the sensitivity of P16 ng/mL for 9d and P8 ng/mL for 9e. The
mean plasma concentration-time profiles were analyzed using WinNonlin Pro
version 5.2 with a two compartmental model for the IV data and a non-
compartmental model for PO data.
10. Dong, C.; Sexton, H.; Gertrudes, A.; Akama, T.; Virtucio, C.; Chen, C.-W.; Fan, X.;
We, A.; Bu, W.; Liu, L.; Feng, L.; Jarnagin, K.; Freund, Y. J. Pharmacol. Exp. Ther
2013, 344, 436.
11. Alley, M. R. K.; Baker, S. J.; Beutner, K. R.; Plattner, J. J. Expert Opin. Investig.
Drugs 2007, 16, 157.
5. Xia, Y.; Cao, K.; Zhou, Y.; Alley, M. R. K.; Rock, F.; Mohan, M.; Meewan, M.;
Baker, S. J.; Lux, S.; Ding, C. Z.; Jia, G.; Kully, M.; Plattner, J. J. Bioorg. Med. Chem.
Lett. 2011, 21, 2533.
6. Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem. 1995, 60, 7508.
7. Synthesis of 6-(4-(aminomethyl)-2-chlorophenoxy)benzo[c][1,2]oxaborol-1(3H)-ol
(9e): To
a solution of 3H-benzo[c][1,2]oxaborole-1,6-diol (8) (300 mg,
2.00 mmol) in DMSO (30 mL) were added K2CO3 (828 mg, 6.00 mmol) and 3-
chloro-4-fluoro-benzonitrile (7b) (933 mg, 6.00 mmol). The reaction was
heated at 90 °C for 7 h. After cooling the reaction mixture to room
temperature, EtOAc (50 mL) was added. The organic layer was washed with
water (5 ꢁ 50 mL). The organic layer was evaporated under vacuum. The
residue was purified by reverse phase chromatography to afford 3-chloro-4-(1-
hydroxy-1,3-dihydro-benzo[c][1,2]oxaborol-6-yloxy)-benzonitrile
(9b)
12. Jacobs, R. T.; Plattner, J. J.; Don, R. Curr. Med. Chem. 2011, 11, 01.
13. Bu, W.; Akama, T.; Chanda, S.; Sullivan, D.; Ciaravino, V.; Jarnagin, K.; Freunf, Y.;
Sanders, V.; Chen, C.-W.; Fan, X.; Heyman, I.; Liu, L. J. Pharm. Biomed. Anal. 2012,
70, 344.
14. Liu, L.; Baker, S. J.; Fitzsimmons, M.; Watney, A.; Bu, W.; Potchoiba, M.; Qi, M.;
Hernandez, V.; Alley, M. R. K.; Feng, L.; Heyman, I.; Chanda, S. 17th North
American Regioanl International Society for the Study of Xenobiotics, Atlanta,
GA, 2011, Poster 84 (http://www.anacor.com/pdf/ISSX2011_GSK2251052_
DME.pdf).
15. Chen, C.-W.; Sharper, V.; Chanda, S. Abstract of Papers, 51st Society od
Toxicology Annual Meeting, San Francisco, CA, 2012; Abstract 2252, http://
www.anacor.com/pdf/SOT_poster.pdf.; Ciaravino, V.; Heyman, I.; Plattner, J.;
Chanda, S. Abstract of Papers, 51st Society od Toxicology Annual Meeting, San
Francisco, CA, 2012; Abstract 2786, http://www.anacor.com/pdf/SOT2012%20
Poster%20VC0306.pdf.; Zane, L.; Heerincks, F.; Hughes, M. Abstract of Papers,
41st European Society for Dermatological Research Annual Meeting, Barcelona,
Spain, 2011; Abstract 242, http://www.anacor.com/pdf/ESDR_AN2728_242.
pdf.; Heyman, I.; Liu. L.; Chanda, S. Abstract of Papers, 41st European Society for
Dermatological Research Annual Meeting, Barcelona, Spain, 2011; Abstract
131, http://www.anacor.com/pdf/ESDR_AN2690_131.pdf.
(190 mg, 33%). 1H NMR (400 MHz, DMSO-d6) d ppm 9.24 (s, 1H), 8.22 (s, 1H),
7.77 (d, J = 7.8 Hz, 1H), 7.50 (d, J = 8.2 Hz, 1H), 7.34 (s, 1H), 7.28 (d, J = 8.2 Hz,
1H), 7.01 (d, J = 8.6 Hz, 1H), 4.99 (s, 2H); ESIMS (m/z): 284 (MꢀH)ꢀ; HPLC:
96.4% (220 nm), 96.0% (maxplot). To a solution of compound 9b (136 mg,
0.480 mmol) in anhydrous THF (60 mL) was added lithium aluminum hydride
(1 M/ether, 1.19 mL, 1.19 mmol) at 0 °C. The reaction was stirred for 2 h. Then
the reaction was quenched with 1 M HCl (30 mL). MeOH (50 mL) was added
and the solution was filtered. The filtrate was evaporated under vacuum. The
residue was purified by reverse phase chromatography (biotage, gradient
MeOH/H2O from 10% to 100%). To a suspension of 9e free base in MeOH (5 mL)
was added 4 M HCl in 1,4-dioxane (0.2 mL). The mixture became a clear
solution then precipitates formed, which were collected by filtration to afford
9e (106 mg, 68%) as a white solid. 1H NMR (400 MHz, DMSO-d6) d ppm 9.19 (s,
1H), 8.18 (br s, 3H), 7.75 (s, 1H), 7.44–7.39 (m, 2H), 7.19–7.10 (m, 3H), 4.98 (s,
2H), 4.03 (q, J = 5.5 Hz, 2H); ESIMS (m/z): 290 (M+H)+; HPLC: 95.9% (220 nm),
96.9% (maxplot).
8. Cell isolation, culture and stimulation: human buffy coats from healthy donors
were obtained from Stanford Blood Center (Palo Alto, CA). PBMCs were isolated
from pools of human buffy coats from healthy donors. Pools of buffy coats from
eight donors by using Histopaque-1077, aliquoted and frozen in liquid
nitrogen. Human PBMCs, monocytes and T cells were cultured in RPMI 1640
16. Jia, L.; Tomaszewski, J. E.; Hanrahan, C.; Coward, L.; Noker, P.; Gorman, G.;
Nikonenko, B.; Protopopova, M. Br. J. Pharm. 2005, 144, 80.