Boronic acid-containing aminopyridine- and aminopyrimidinecarboxamide CXCR1/2 antagonists: Optimization of aqueous solubility and oral bioavailability

Article abstract of DOI:10.1016/j.bmcl.2015.07.090

Abstract The chemokine receptors CXCR1 and CXCR2 are important pharmaceutical targets due to their key roles in inflammatory diseases and cancer progression. We have previously identified 2-[5-(4-fluoro-phenylcarbamoyl)-pyridin-2-ylsulfanylmethyl]-phenylboronic acid (SX-517) and 6-(2-boronic acid-5-trifluoromethoxy-benzylsulfanyl)-N-(4-fluoro-phenyl)-nicotinamide (SX-576) as potent non-competitive boronic acid-containing CXCR1/2 antagonists. Herein we report the synthesis and evaluation of aminopyridine and aminopyrimidine analogs of SX-517 and SX-576, identifying (2-{(benzyl)[(5-boronic acid-2-pyridyl)methyl]amino}-5-pyrimidinyl)(4-fluorophenylamino)formaldehyde as a potent chemokine antagonist with improved aqueous solubility and oral bioavailability.

Full text of DOI:10.1016/j.bmcl.2015.07.090

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Boronic acid-containing aminopyridine- and
aminopyrimidinecarboxamide CXCR1/2 antagonists:
Optimization of aqueous solubility and oral bioavailability
a,
Aaron D. Schuler a, , Courtney A. Engles , Dean Y. Maeda a, Mark T. Quinn b, Liliya N. Kirpotina b,
⇑
Winston N. Wicomb c, S. Nicholas Mason d, Richard L. Auten d,à, John A. Zebala a
a Syntrix Biosystems, 215 Clay Street NW, Suite B-5, Auburn, WA 98001, United States
b Department of Microbiology and Immunology, Montana State University, 960 Technology Boulevard, Bozeman, MT 59717, United States
c Infectious Disease Research Institute, 1616 Eastlake Avenue East, Seattle, WA 98102, United States
d Department of Pediatrics (Neonatal Medicine), DUMC Box 3373, Duke University, Durham, NC 27710, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The chemokine receptors CXCR1 and CXCR2 are important pharmaceutical targets due to their key roles
in inflammatory diseases and cancer progression. We have previously identified 2-[5-(4-fluoro-phenyl-
carbamoyl)-pyridin-2-ylsulfanylmethyl]-phenylboronic acid (SX-517) and 6-(2-boronic acid-5-trifluo-
romethoxy-benzylsulfanyl)-N-(4-fluoro-phenyl)-nicotinamide (SX-576) as potent non-competitive
boronic acid-containing CXCR1/2 antagonists. Herein we report the synthesis and evaluation of aminopy-
ridine and aminopyrimidine analogs of SX-517 and SX-576, identifying (2-{(benzyl)[(5-boronic acid-2-
Received 2 July 2015
Accepted 27 July 2015
Available online xxxx
Keywords:
CXCR1
CXCR2
Antagonist
COPD
pyridyl)methyl]amino}-5-pyrimidinyl)(4-fluorophenylamino)formaldehyde as
antagonist with improved aqueous solubility and oral bioavailability.
a potent chemokine
Ó 2015 Elsevier Ltd. All rights reserved.
Asthma
The chemokine receptors CXCR1 and CXCR2 are major targets of
drug development in the pharmaceutical industry1,2 due to their
key role in neutrophil chemotaxis and its association with inflam-
mation.3 The endogenous ligands for these G-protein coupled
receptors (GPCRs) include the CXCR2-specific growth-related
clinic a new structurally-similar inhibitor danirixin 1,18 with trials
currently recruiting patients for COPD and respiratory syncytial
virus infections. The CXCR2 antagonist navarixin (SCH527123,
2)19 from Merck utilized a cyclic urea bioisostere 3,4-diaminocy-
clobut-3-ene-1,2-dione instead of the diaryl urea to generate a
potent inhibitor that was evaluated in clinical trials for the treat-
ment of COPD,20,21 asthma,22 and psoriasis. Their Phase 2 trial
demonstrated safety and efficacy in moderate to severe COPD
patients.21 AstraZeneca’s pyrimidine-based CXCR1/2 inhibitors
AZD8309,23 AZD5069 3,24 and AZD4721 (structure undisclosed)
have been clinically evaluated to treat COPD25,26 and asthma.2
Dompé’s ketoprofen derivative reparixin 4, an inhibitor of CXCL8
receptor CXCR1 and CXCR2 activation,27,28 is being explored for
the reduction of post-surgical inflammation after transplantation
surgeries.2 Novartis29,30 and Pfizer23,31 also have active CXCR2
programs.
We have previously reported a novel class of dual allosteric
CXCR1/2 antagonists that utilize an aromatic boronic acid moiety
on a nicotinamide core.32,33 Our first-generation inhibitor 5 (SX-
517) exhibited anti-inflammatory activity in vivo, but its preclini-
cal development was halted due to its metabolic instability. A
focused SAR effort to increase metabolic stability led to the discov-
ery of our second-generation inhibitor 6 (SX-576), which was less
susceptible to oxidation of the boronic acid.32 Modeling of the
oncogene
a (GRO a, or CXCL1) and interleukin-8 (IL8, or CXCL8),
which binds to both receptors.4 Experiments with animals lacking
CXCR1 and/or CXCR2 activity have demonstrated that their inhibi-
tion could be beneficial in the treatment of several diseases, includ-
ing asthma,5 chronic obstructive pulmonary disease (COPD),6 and
inflammatory bowel disease7), cancer (melanoma,8 pancreatic,9,10
and colon11,12 cancer), Alzheimer’s disease,13 and traumatic brain
injury.14 Several previously reported CXCR1 and/or CXCR2 inhibi-
tors representing the key structural classes are summarized in
Figure 1. The diaryl urea CXCR2 antagonist SB656933 was evalu-
ated in clinical trials for the treatment of COPD15,16 and cystic
fibrosis,17 and GlaxoSmithKline has recently advanced into the
⇑
Corresponding author. Tel.: +1 253 833 8009; fax: +1 253 833 8127.
E-mail address: aschuler@syntrixbio.com (A.D. Schuler).
Present address: Paula’s Choice, 705 5th Avenue South, Suite 200, Seattle, WA
98104, United States.
à
Present address: Alamance Regional Medical Center, 1240 Huffman Mill Road,
Burlington, NC 27215, United States.
http://dx.doi.org/10.1016/j.bmcl.2015.07.090
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Schuler, A. D.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.07.090

2
A. D. Schuler et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
OH
OH
O
N
O
O
Cl
S
O
N
F
O
N
O
HN
N
O
F
S
N
N
F
N
N
N
H
S
O
O
H
H
H
H
OH
O
OH
3: AZD5069
2: Navarixin
1: Danirixin
F
F
O
O
H
N
N
N
O
N
N
S
B(OH)2
H
S
H
O
N
N
O
N
B(OH)2
R
5: R = H, SX-517
6: R = OCF , SX-576
4: Reparixin
7
3
Figure 1. Chemokine antagonists.
binding of 6 with CXCR2, constructed from the recently solved
structure of CXCR1,34 revealed that this class of antagonists has a
different binding model than previously described for other classes
of compounds. However, the poor aqueous solubility of 6 led us to
examine whether heteroatom replacement of the sulfur could
improve its oral bioavailability. Herein, we describe our focused
SAR studies that led to the identification of a third-generation com-
pound 7, which exhibited improved aqueous solubility and oral
bioavailability while maintaining activity in an animal model of
pulmonary inflammation.
Starting from our first- (5) and second- (6) generation inhibi-
tors, we synthesized compounds that maintained a boronic acid
in the 2-position of the phenyl ring, replacing the sulfur in 5 with
secondary (8) and tertiary (9) amines, utilizing either a bromoni-
cotinamide35 or triflatenicotinamide36 intermediate (10, 11),
depending on the reactivity of the amine. Generally, primary and
N-methyl secondary amines were coupled with the bromonicoti-
namide intermediate, and more sterically-hindered secondary
amines were coupled with the triflatenicotinamide intermediate.
The general synthesis of the inhibitors is shown in Scheme 1.
Shifting the position of the boronic acid to the 3- (12, 13) and 4-po-
sitions (14, 15) was also explored. The corresponding pyrimidine
compounds were also prepared from a chloropyrimidinamide37
intermediate (16), with the boronic acid in the 2- (17), 3- (18,
19), and 4-positions (20, 21). The corresponding pyrimidine analog
to 8 could not be prepared using our standard synthetic methods,
because the palladium-catalyzed conversion of the halogen to the
boronic acid pinacol ester instead led to reduction of the halogen
to an unsubstituted benzyl ring. Alternate routes to the desired
product also proved unsuccessful.
The compounds were screened for their ability to inhibit cal-
cium flux in a previously described cell-based assay.33,38 Briefly,
the synthesized compounds were incubated for 30 min with rat
basophilic leukemia (RBL) cells stably transfected with either
CXCR1 or CXCR2. After the addition of IL8, the release of intracellu-
lar calcium was measured via FLUO-4AM detection in a fluorescent
microplate reader. The activity of the compounds against CXCR1
and CXCR2 is summarized in Table 1. In contrast to what was
observed with our sulfur-containing compounds, the 2-position
for the boronic acid was not well tolerated. Instead, the 4-position
appeared to yield the most potent compounds, such as 21. The lack
of activity for the 2-position compounds was likely due to neigh-
boring group interactions between the boronic acid and the
amine.39 Due to these observations, the 4-boronic acid, 2-pyridinyl
analogs (22, 23) were prepared and found to maintain inhibitory
activity at CXCR2. These compounds also exhibited improved
H
H
N
H
N
O
N
O
O
ii, iii
i
F
F
F
N
N
N
N
Br
N
B(OH)2
X
R1 R2
R1 R2
8-9, 12-15, 22, 24,
25, 27, 30
10: X = Br
11: X = OTf
H
H
H
O
N
N
O
N
N
O
N
ii, iii
iv
F
F
F
N
N
N
N
N
Br
N
B(OH)2
Cl
16
R3 R4
R3 R4
7, 17-21, 23, 26,
28, 29, 31
Scheme 1. Reagents and conditions: (i) primary or secondary amine, DIPEA, anhyd
DMF or NMP, 120–160 °C, 16–72 h; (ii) PdCl2(dppf), bis(pinacolato)diboron, potas-
sium acetate, anhyd. DMF, 80 °C, 3–6 h; (iii) KHF2, then TMS–Cl/H2O or HCl/dioxane
or aq formic acid; (iv) primary or secondary amine, triethylamine, anhyd NMP, rt,
16–72 h.
aqueous solubility, making them good scaffolds for further
substitution.
The addition of a phenyl ring (24, 7) at the tertiary amine signif-
icantly improved potency of the inhibitors, in contrast to the addi-
tion of another pyridine ring (25, 26). Additions of
a
tetrahydropyran ring (27), carboxylic acid (28), or amine (29) in
efforts to further improve aqueous solubility, yielded inactive com-
pounds. However, the addition of a furan ring (30, 31) yielded com-
pounds with similar potency to the phenyl compounds. The third
generation inhibitors are summarized in Table 2. Interestingly,
aminopyrimidine-based 7 was significantly more potent than its
aminopyridine analog 24, while the aminopyridine-based 30 was
significantly more potent than its aminopyrimidine analog 31. It
was somewhat surprising that changing a single atom between
the paired compounds made such a difference in their activity.
Since they were the most potent in the cell-based assays, 740 and
3041 were selected for further evaluation.
Compared with 6, 7 and 30 were more potent inhibitors of
IL8-mediated calcium flux in RBL cells stably transfected with
CXCR1 or CXCR2, but they were not as potent at inhibiting calcium
flux in isolated neutrophils, and 7 (185 52 nM) was more potent
than 30 (321 96 nM). Due to its higher activity, 7 was selected for
further testing.
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A. D. Schuler et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Table 1
Inhibition of IL8-mediated intracellular calcium release in RBL cells stably transfected with CXCR1 or CXCR2
a
a
a
a
Compd
R1
R2
CXCR1 IC50 (nM)
CXCR2 IC50 (nM)
Compd
R3
R4
CXCR1 IC50 (nM)
CXCR2 IC50 (nM)
8, 9
H, Me
>5000
>5000
17
Me
>5000
>5000
12
13
H
611 62
733 85
410 40
18
19
H
1340 90
918 114
Me
4100 500
Me
2500 200
2500 400
14
15
22
H
4300 400
1400 200
1700 200
2200 400
1100 200
81 19
20
21
23
H
1700 300
681 80
2600 200
121 30
933 54
Me
Me
Me
Me
1700 100
a
Experiments performed in triplicate, reported as mean SE. Compound 6 had IC50 values of 31 4 nM and 21 6 nM versus CXCR1 and CXCR2, respectively.
Table 2
Inhibition of IL8-mediated intracellular calcium release in RBL cells stably transfected with CXCR1 or CXCR2
a
a
a
a
Compd R1
R2
CXCR1 IC50
(nM)
CXCR2 IC50
(nM)
Compd R3
R4
CXCR1 IC50
(nM)
CXCR2 IC50
(nM)
22
24
25
27
30
Me
1700 200
326 39
>5000
81 19
298 45
28
7
>5000
>5000
7
2
4
1
4850 250
2300 400
26
29
31
2050 430
>5000
4310 290
>5000
>5000
3.3 0.4
21
3
444 58
655 101
a
Experiments performed in triplicate, reported as mean SE.
To evaluate stability in vitro, 6 and 7 were incubated separately
in human plasma at a concentration of 1 M for 24 h at 37 °C.
would lead to improved systemic exposure in vivo, the pharma-
cokinetics of both 6 and 7 was evaluated in the rat. Compounds
6 and 7 were administered intravenously42 and orally43 at 1 mg/kg,
and the plasma concentrations of both compounds were deter-
mined by LC–MS/MS analysis, and calculated using a calibration
curve of the test compound spiked in rat plasma, which was run
concurrently with the samples. While orally-dosed 6 gave plasma
levels below the limit of quantification (LLOQ) for our system,44
7 was found to have a bioavailability of 24% from the aqueous oral
l
Samples were analyzed by LC–MS/MS at 0, 1, 4, and 24 h, and as
shown in Table 3 and 7 was found to be significantly more stable
than 6, suggesting it might have improved bioavailability.
However, there was no significant difference in their stability in
rat plasma (data not shown). Unlike 6, 7 was soluble in 0.1 N HCl
at 0.5 mg/mL, suggesting it would be soluble in stomach acid. In
order to evaluate whether the improved aqueous solubility of 7
Please cite this article in press as: Schuler, A. D.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.07.090

4
A. D. Schuler et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 3
Comparison of compounds 6 and 7
a
b
Compd
RBL cell line IC50
(nM)
Isolated human PMNs IC50 (nM)
Human plasma
stability (% remaining)
Solubility in 0.1 N HCl (mg/mL)
Rat PK AUC @
1 mg/kg
(l
mol-h/L)
CXCR1b
CXCR2b
CXCR1/2b
22
185 52
1 h
4 h
24 h
IV
Oral
6
7
31
7
4
2
21
4
6
1
3
62
90
34
71
2
62
Insoluble
0.5
5.40
4.37
<LLOQ
1.15
a
Experiments performed in triplicate, reported as mean SE.
IL8 used as agonist.
b
dose, despite having a slightly lower intravenous AUC (5.40 for 6 vs
4.37 for 7 mol-h/L).
l
To determine whether its improved aqueous solubility, plasma
stability, and PK parameters improved its in vivo characteristics, 7
was compared side-by-side with 6 in an ozone rat model of pul-
monary inflammation and found to be twice as effective at reduc-
ing neutrophil influx, when both compounds were given via
intravenous injection at a dose of 1 mg/kg (Fig. 2). In this model,
Sprague–Dawley rats (n = 4 per cohort) were given a single intra-
venous dose at t = 0 of either vehicle (negative and positive
groups), 6, or 7. The rats were then placed in either air (negative)
or 1 ppm ozone (positive, 6, and 7 groups) for 4 h. The rats were
sacrificed at t = 24 h, and the bronchoalveolar lavage fluid (BALF)
was collected. The cells were spun down, stained with Wright–
Giemsa and counted. In the negative group, no neutrophils were
observed when stained. Whereas 6 only produced a modest reduc-
tion in neutrophil influx, treatment with 7 led to a significant
reduction of neutrophil influx. This suggests that the improved
aqueous solubility of 7 may have led to increased systemic expo-
sure of the compound to circulating neutrophils.
Figure 2. Ozone rat model of pulmonary inflammation. ⁄⁄⁄p <0.001, ⁄⁄⁄⁄p <0.0001,
t-test of 6 or 7 versus positive control and 6 versus 7.
In conclusion, 7 is a potent CXCR1 and CXCR2 antagonist iden-
tified from a focused SAR effort to improve the aqueous solubility
and in vivo characteristics of our previous lead compounds.
Compound 7 is soluble in 0.1 N HCl, has improved plasma stability,
and is orally bioavailable in the rat. These improvements over our
prior lead compound 6 were further demonstrated in a head-to-
head comparison in a rat ozone model of pulmonary inflammation,
where 7 exhibited a more durable inhibitory effect than 6 after a
single intravenous dose. Compound 7 represents an improved lead
candidate for the treatment of inflammatory diseases, cancer, and
other diseases associated with CXCR1/2 activation. Further evalua-
tion of the biological activity and properties of 7 are currently
underway.
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This work was supported by National Institutes of Health grant
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pyridyl)methyl]amine. Without purification, the amine (1 equiv) was
combined with the chloropyrimidinamide intermediate (16, 1.5 equiv) in
anhydrous N-methyl-2-pyrrolidone (2 mL/mmol) in
a scintillation vial.
Triethylamine (2.0 equiv) was added and the reaction was left to stand at
room temperature for 38 h, before the solvent was removed via rotary
evaporation. The yellow oil was partitioned between ethyl acetate and water,
and the aqueous layer was re-extracted with ethyl acetate. The combined ethyl
acetate layers were dried over sodium sulfate, filtered, and concentrated under
vacuum. The crude product was purified via flash chromatography. The
purified product (1 equiv) was dissolved in anhydrous dimethylformamide
(6 mL/mmol) and degassed under vacuum. The solution was transferred to a
pressure bottle containing PdCl2(dppf) (0.08 equiv), bis(pinacolato)diboron
(3 equiv), and potassium acetate (3.2 equiv) and heated to 80 °C for 4.5 h, then
cooled to room temperature. The reaction was filtered through Celite, rinsing
with dimethylformamide, dried under vacuum, diluted with ethyl acetate,
dried over sodium sulfate, gravity filtered, and dried under vacuum. The crude
product was purified by flash chromatography to yield a white foam, which
was dissolved in methanol and diluted with water to form a thick white
suspension, which was concentrated on a rotary evaporator and filtered to
yield a white solid. The boronic acid pinacol ester (1 equiv) was dissolved in
methanol (18 mL/mmol) and formic acid (10 equiv) was added, followed by
dilution with water to yield a fine white precipitate. The water and methanol
were removed via lyophilization. The white powder obtained was suspended
in water with minimal methanol and cooled in the fridge. It was collected by
vacuum filtration and dried in a vacuum desiccator to yield compound 7. ESI-
MS m/z = 458.1 [M+H]+. Calcd for C24H21BFN5O3: C, 63.04; H, 4.63; N, 15.32.
Found: C, 63.30; H, 4.65; N, 15.22. 1H NMR (300 MHz, DMSO-d6) d 10.17 (s, 1H),
8.91–8.86 (m, 2H), 8.82 (s, 1H), 8.29 (s, 2H), 8.04 (d, 1H), 7.75–7.71 (m, 2H),
7.36–7.27 (m, 5H), 7.21–7.16 (m, 3H), 5.03 (s, 2H), 4.96 (s, 2H).
35. 6-Bromonicotinic
acid
(1 equiv)
was
dissolved
nitrogen atmosphere. N-
(1 equiv) was added,
in
anhydrous
dimethylformamide (1.25 mL/mmol) under
Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline
a
followed by 4-fluoroaniline (1 equiv), and the reaction was stirred at room
temperature for 18 h. The product was precipitated by dilution into water
(24:1 v/v), filtered, and washed to yield 10 as a white solid. ESI-MS m/z 294.9/
296.9 [M+H]+. Calcd for C12H8BrFN2O: C, 48.84; H, 2.73; N, 9.49. Found: C,
48.75; H, 2.57; N, 9.40. 1H NMR (500 MHz, DMSO-d6) d 10.54 (s, 1H), 8.92 (d,
1H), 8.25–8.23 (m, 1H), 7.86–7.85 (m, 1H), 7.79 (d, 2H), 7.24–7.23 (m, 2H).
36. 6-Hydroxynicotinic acid (1 equiv) was suspended in acetonitrile (0.6 mL/
mmol). Pyridine (0.005 equiv) was added, and the suspension was heated to
80 °C under nitrogen gas. Sulfonyl chloride (1.05 equiv) was added dropwise,
then stirred for 45 min before cooling to room temperature. A precipitate
formed that was collected by filtration and washed with cold acetonitrile
before drying under vacuum to yield the acid chloride as a tan solid, which was
41. 5-Bromo-2-formylpyridine (1 equiv) and 2-furfurylamine (1.5 equiv) were
dissolved in methanol (2 mL/mmol) in a round bottom flask and stirred at
room temperature for 3 h. Solid sodium borohydride (1.2 equiv) was to the
solution, and it was stirred for an additional 2 h. The methanol was removed
via rotary evaporation, and the residual yellow oil was partitioned between
ethyl acetate and saturated aqueous sodium bicarbonate, and the aqueous
layer was extracted twice with ethyl acetate. The combined ethyl acetate
extracts were dried over sodium sulfate, filtered, and concentrated under
vacuum to yield [(5-bromo-2-pyridyl)methyl](furfuryl)amine. Without
added under nitrogen gas to
pyridine (1.5 mL/mmol). The reaction was stirred overnight at room
temperature and precipitated from water (20:1 v/v). The
a solution of 4-fluoroaniline (1.1 equiv) in
hydroxynicotinamide was collected by filtration, washed with water, and
dried to constant weight at 90 °C overnight to yield an off-white solid. The
solid (1 equiv) was suspended in ethyl acetate (3 mL/mmol) with N-phenyl-
bis(trifluoromethyl sulfonimide) (1.5 equiv) and dimethylaminopyridine
(0.1 equiv). Diisopropylethylamine (3 equiv) was added, and the reaction was
heated to reflux for 1 h. It was cooled to room temperature, diluted with ethyl
acetate, and washed with water, saturated aqueous sodium bicarbonate, water,
and 1 N hydrochloric acid. The organic layer was concentrated and purified by
flash chromatography to yield 11 as a tan solid. ESI-MS m/z 365.3 [M+H]+. 1H
NMR (300 MHz, DMSO-d6) d 10.65 (s, 1H), 8.96 (d, 1H), 8.62–8.58 (m, 1H),
7.81–7.76 (m, 3H), 7.27 (t, 2H).
purification,
triflatenicotinamide intermediate (11, 1 equiv) in anhydrous N-methyl-2-
pyrrolidone (5 mL/mmol) in pressure bottle, layered with nitrogen.
the
amine
(1.5 equiv)
was
combined
with
the
a
Diisopropylethylamine (2.0 equiv) was added and the reaction was heated to
150 °C for 16 h, then cooled to room temperature before the solvent was
removed via rotary evaporation. The dark oil was diluted with ethyl acetate
and dried over sodium sulfate, filtered through a silica plug (ethyl acetate), and
concentrated under vacuum. The crude product was purified via flash
chromatography. The purified product (1 equiv) was dissolved in anhydrous
dimethylformamide (6 mL/mmol) and degassed under vacuum. The solution
37. 2-Chloropyrimidine-5-carboxylic acid (1 equiv) was dissolved in anhydrous
dimethylformamide (0.8 mL/mmol) under
Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline
a
nitrogen atmosphere. N-
(1 equiv) was added,
was transferred to
a pressure bottle containing PdCl2(dppf) (0.08 equiv),
followed by 4-fluoroaniline (1 equiv), and the reaction was stirred at room
temperature for 40 h. The product was partitioned between ethyl acetate and
water. The aqueous layer was washed with ethyl acetate, and combined
organic layers were dried over sodium sulfate, filtered, and dried under
vacuum. The crude product was purified by flash chromatography to yield 16
as a white solid. ESI-MS m/z 252.1 [M+H]+. 1H NMR (500 MHz, DMSO-d6) d
10.67 (s, 1H), 9.23 (s, 2H), 7.78–7.75 (m, 2H), 7.25 (t, 2H).
bis(pinacolato)diboron (3 equiv), and potassium acetate (3 equiv) and heated
to 80 °C for 4.5 h, then cooled to room temperature. The reaction was dried
under vacuum, diluted with ethyl acetate, dried over sodium sulfate, filtered
through a silica plug (ethyl acetate, then 10% methanol in ethyl acetate), and
dried under vacuum. The crude product was purified by C18 flash
chromatography and lyophilized to yield compound 30 as
a white fluffy
solid. ESI-MS m/z = 447.1 [M+H]+. Calcd for C23H20BFN4O4: C, 61.91; H, 4.52; N,
12.56. Found: C, 61.64; H, 4.65; N, 12.21. 1H NMR (500 MHz, DMSO-d6) d 10.05
(s, 1H), 8.83 (s, 1H), 8.70 (d, 1H), 8.29 (s, 2H), 8.03–7.99 (m, 2H), 7.76–7.74 (m,
2H), 7.59 (s, 1H), 7.19 (t, 2H), 7.08 (d, 1H), 6.86 (d, 1H), 6.40–6.38 (m, 2H), 4.96
(s, 2H), 4.91 (s, 2H).
38. Maeda, D. Y.; Quinn, M. T.; Schepetkin, I. A.; Kirpotina, L. N.; Zebala, J. A. J.
Pharmacol. Exp. Ther. 2010, 332, 145.
39. Hall, D. G. Boronic Acids: Preparation and Applications in Organic Synthesis and
Medicine; WILEY-VCH GmbH & Co. KGaA: Weinheim, 2005.
40. 5-Bromo-2-formylpyridine (1 equiv) and benzylamine (1 equiv) were
dissolved in methanol (2 mL/mmol) in a round bottom flask and stirred at
room temperature for 4 h. Solid sodium borohydride (1.2 equiv) was to the
solution, and it was stirred for an additional 2.5 h. The methanol was removed
via rotary evaporation, and the residual yellow oil was partitioned between
ethyl acetate and saturated aqueous sodium bicarbonate, and the aqueous
layer was extracted three additional times with ethyl acetate. The combined
ethyl acetate extracts were dried over sodium sulfate, filtered, and
42. Intravenous injections were performed using DPS (dimethylacetamide/
PEG400/0.9% saline 40:40:20) as the vehicle.
43. Oral gavage of 6 was performed using Labrasol as the vehicle as previously
described. Oral gavage of 7 was performed using 0.1 N HCl as the vehicle. The
use of this vehicle for oral gavage in rats has been previously described (Huang,
Y. T.; Liu, T. B.; Lin, H. C.; Yang, M. C.; Hong, C. Y. Pharmacology 1997, 54, 225).
44. The limit of detection on our LC–MS/MS was approximately 20 nM for
compound 6.
concentrated
under
vacuum
to
yield
(benzyl)[(5-bromo-2-
Please cite this article in press as: Schuler, A. D.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.07.090