Optimization of isoxazoline amide benzoxaboroles for identification of a development candidate as an oral long acting animal ectoparasiticide

Article abstract of DOI:10.1016/j.bmcl.2016.04.093

Novel isoxazoline amide benzoxaboroles were designed and synthesized to optimize the ectoparasiticide activity of this chemistry series against ticks and fleas. The study identified an orally bioavailable molecule, (S)-N-((1-hydroxy-3,3-dimethyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)methyl)-2-methyl-4-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)benzamide (23), with a favorable pharmacodynamics profile in dogs (C_max = 7.42 ng/mL; T_max = 26.0 h; terminal half-life t_1/2 = 127 h). Compound 23, a development candidate, demonstrated 100% therapeutic effectiveness within 24 h of treatment, with residual efficacy of 97% against American dog ticks (Dermacentor variabilis) on day 30 and 98% against cat fleas (Ctenocephalides felis) on day 32 after a single oral dose at 25 mg/kg in dogs.

Full text of DOI:10.1016/j.bmcl.2016.04.093

Accepted Manuscript
Optimization of Isoxazoline Amide Benzoxaboroles for Identification of a De-
velopment Candidate as an Oral Long Acting Animal Ectoparasiticide
Yong-Kang Zhang, Jacob J. Plattner, Eric E. Easom, Tsutomu Akama, Yasheen
Zhou, W. Hunter White, Jean M. Defauw, Joseph R. Winkle, Terry W. Balko,
Jianxin Cao, Zhixin Ge, Jianzhang Yang
PII:
S0960-894X(16)30491-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.04.093
BMCL 23865
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 April 2016
26 April 2016
27 April 2016
Please cite this article as: Zhang, Y-K., Plattner, J.J., Easom, E.E., Akama, T., Zhou, Y., Hunter White, W., Defauw,
J.M., Winkle, J.R., Balko, T.W., Cao, J., Ge, Z., Yang, J., Optimization of Isoxazoline Amide Benzoxaboroles for
Identification of a Development Candidate as an Oral Long Acting Animal Ectoparasiticide, Bioorganic &
Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.04.093
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Optimization of Isoxazoline Amide Benzoxaboroles for
Identification of a Development Candidate as an Oral Long
Acting Animal Ectoparasiticide
Yong-Kang Zhang a,*, Jacob J. Plattner a, Eric E. Easom a, Tsutomu Akama a, Yasheen Zhou a,
W. Hunter White b, Jean M. Defauw b, Joseph R. Winkle b, Terry W. Balko b, Jianxin Cao c,
Zhixin Ge c, Jianzhang Yang d
a Anacor Pharmaceuticals, Inc., 1020 E. Meadow Circle, Palo Alto, CA 94303, USA;
b Elanco Animal Health Research and Development, a Division of Eli Lilly & Company, 2500 Innovation Way, Greenfield, IN 46140,
USA;
c Shanghai ChemPartner, 998 Halei Road, Zhangjiang High-tech Park, Pudong New Area, Shanghai 201203, China;
d Sundia MediTech Company, Ltd., Building 8, 388 Jialilue Road, Zhangjiang High-Tech Park, Shanghai 201203, China.
This is where the receipt/accepted dates will go; Received Month XX, 2000; Accepted Month XX, 2000 [BMCL RECEIPT]
Abstract— Novel isoxazoline amide benzoxaboroles were designed and synthesized to optimize the ectoparasiticide activity of this
chemistry series against ticks and fleas. The study identified an orally bioavailable molecule, (S)-N-((1-hydroxy-3,3-dimethyl-1,3-
dihydrobenzo[c][1,2]oxaborol-6-yl)methyl)-2-methyl-4-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-
yl)benzamide (23), with a favorable pharmacodynamics profile in dogs (Cmax = 7.42 ng/ml; Tmax = 26.0 h; terminal half-life t1/2 = 127 h).
Compound 23, a development candidate, demonstrated 100% therapeutic effectiveness within 24 hours of treatment, with residual
efficacy of 97% against American dog ticks (Dermacentor variabilis) on day 30 and 98% against cat fleas (Ctenocephalides felis) on day
32 after a single oral dose at 25 mg/kg in dogs.
Isoxazolines are
ectoparasiticide agents inhibiting γ-aminobutyric acid
(GABA)-gated and L-glutamate-gated chloride
a
relatively new class of
We previously reported a novel series of isoxazoline
benzoxaborole small molecules exhibiting excellent
oral-systemic activity against ticks and fleas; the lead
compound (AN8030, Fig. 2) was shown to provide 95%
efficacy against American dog ticks (D. variabilis) on
day 30, following a single oral dose at 50 mg/kg in
dogs.2
channels, with several examples approved for use in the
companion animal industry.1 Three efficacious
1a-t
isoxazoline compounds
(Fig. 1) have been approved
for the treatment of tick and flea infestations in dogs.1u-w
Figure 2. Structure of an isoxazoline benzoxaborole lead providing at least 1 month
of ectoparasitic activity against experimental tick and flea infestations on dogs 2
As a continuation of the research program with a goal of
lowering the dosage while maintaining acceptable
Figure 1. Chemical structures of three typical isoxazoline compounds

clinical effectiveness (i.e., ≥ 95%) for at least one
month, we designed and synthesized a series of novel
isoxazoline amide benzoxaboroles (1-23, Figs. 3 and 4)
for a structure-activity relationship (SAR) study to
optimize the ectoparasitic activity. Specifically, these
molecules were designed to examine the effects of
oxaborole 3-substituent variation (1 vs 2, 9 and 11; 7 vs
10, 13 and 14), amide N-substituent change (2 vs 3),
halogen addition on the C(5)-aryl (4 vs 5, and 2 vs 6
and 7), linking position variation to the benzoxaborole
(2 vs 4, 5 vs 6, and 7 vs 8), linkage length (2 vs 15, 6 vs
16, and 7 vs 17), substituent changes on the benzylic
carbon between the amide and benzoxaborole (7 vs 18
and 19), and the chiral configuration (20 vs 21, and 22
vs 23). Herein, we report the synthesis, pharmacokinetic
profile and ectoparasiticide activity against ticks and
fleas of this “extended” series.
The synthetic routes used for the preparation of
compounds 1-19 are outlined in Schemes 1-3.3a In
3b
Scheme 1, aldehyde 24 reacted with hydroxylamine
to give the resulting oxime 25, which was reduced to the
aminomethyl intermediate 26. Amidation reaction
between amine 26 and acid 27 gave the corresponding
amides (1, 2, 4-14). Reductive amination of 24 with
methylamine provided the amine 28, which was
followed by reaction with acid 27 to generate 3.
Aldehyde 24 reacted with nitromethane providing 29,
which was reduced to aminoethyl molecule 30 followed
by an amidation reaction with 27, giving final
compounds 15-17. In Scheme 2, Grignard reaction of 31
with MeMgBr generated the ketone alcohol 32, which
was protected to give 33 followed by catalytic
boronylation to afford 34. Deprotection of 34 and
subsequent simultaneous cyclization provided 35 which
was converted to oxime 36. Reduction of the oxime
group in 36 yielded amine 37, which reacted with acid
27 to produce the final amide 18. In Scheme 3, the
diester 38 reacted with MeMgBr to give the dialcohol
39 followed by protection to provide 40, which was
catalytically boronylated to afford 41. Hydrolysis of 41
generated the alcohol 42 that was converted to the azide
43. The azide group in 43 was reduced to the amine of
44, which reacted with the acid 27 to produce the final
amide 19. The racemic compounds 6 and 7 were
separated by chiral SFC to obtain each enantiomer (Fig.
4). As an example, the experimental procedure for the
synthesis of 7, and its chiral separation method for
obtaining 22 and 23 are described in the reference and
note section.4a, 4b
Figure 3. Chemical structures of the isoxazoline amide benzoxaboroles investigated
in a SAR study to identify novel ectoparasiticides.
Scheme 1. Routes for syntheses of 1-17. Reagents and conditions: (a) NH2OH·HCl,
NaOAc, THF, H2O, rt, 16 h; (b) Zn dust, AcOH, 40 oC, 4 h; (c) HATU, DIPEA,
DMF, rt, 16 h; (d) MeNH2, NaBH(OAc)3, AcOH, THF, rt, 16 h; (e) BEP, DIPEA, rt,
16 h; (f) MeNO2, AcOH, NH4OAc, 100 oC, 3 h; (g) H2, Pd(OH)2, MeOH, 3N
HCl/H2O, rt, 8 h; (h) HATU, DIPEA, DMF, rt, 16 h.
Figure 4. Enantiomers of racemic compounds 6 and 7.

treatment, live and dead ticks were removed from
animals and counted.2
Table 1. Activity of compounds 1-23 in in vitro tick LIM assay and in
vivo tick rat model a
In vitro activity in LIM assay
Activity in
rat model
Compound
Activity b EC50 (uM)
1
2
100% at 300 uM
100% at 300 uM
61.3
27.6
93.0% at 25 mpk
100% at 25 mpk
100% at 10 mpk
0% at 5 mpk
3
4
5
40.3% at 300 uM
100% at 300 uM
100% at 300 uM
314.5
57.0
36.7
100% at 25 mpk
13.9% at 10 mpk
96.0% at 25 mpk
16.3% at 10 mpk
100% at 25 mpk
100% at 10 mpk
39.2% at 5 mpk
100% at 25 mpk
100% at 10 mpk
10.0% at 5 mpk
100% at 25 mpk
100% at 10 mpk
100% at 5 mpk
27.3% at 2.5 mpk
100% at 25 mpk
95.6% at 10 mpk
11.2% at 5 mpk
nt c
6
7
100% at 300 uM
100% at 300 uM
52.4
89.8
Scheme 2. Synthetic route for preparation of 18. Reagents and conditions: (a)
MeMgBr, THF, 0 oC to rt, 16 h, Argon; (b) EtOCH2Cl, DIPEA, DCM, 40 oC, 16 h;
(c) Pin2B2, Pd(dppf)Cl2, KOAc, 1,4-dioxane, 80 oC, 16 h, Argon; (d) 6N HCl, THF,
rt, 16 h; (e) NH2OH·HCl, NaOAc, THF, H2O, rt, 16 h; (f) Zn dust, AcOH, 40 oC, 0.5
h; then (Boc)2O, Et3N, DCM, rt, 3 h; then 6N HCl, MeOH, rt, 16 h; (g) X = Cl for
27, HATU, Et3N, DMF, rt, 16 h.
8
100% at 300 uM
57.1
9
10
100% at 300 uM
100% at 300 uM
62.1
44.6
100% at 25 mpk
100% at 10 mpk
100% at 5 mpk
97.3% at 25 mpk
0% at 10 mpk
74.3% at 25 mpk
0% at 10 mpk
11
12
13
94.6% at 300 uM
93.1% at 300 uM
34.8% at 300 uM
149.5
149.6
nt c
89.4% at 25 mpk
0% at 10 mpk
14
15
16
98.0% at 300 uM
45.8% at 300 uM
92.9% at 300 uM
118.9
326.3
189.1
32.7% at 25 mpk
0% at 25 mpk
100% at 25 mpk
59% at 10 mpk
4.3% at 5 mpk
97.3% at 25 mpk
32.4% at 10 mpk
0% at 5 mpk
100% at 25 mpk
96.0% at 10 mpk
90.4% at 5 mpk
12.4% at 2.5 mpk
22.9% at 25 mpk
0% at 10 mpk
5.6% at 25 mpk
100% at 10 mpk
93.8% at 5 mpk
80.7% at 2.5 mpk
11.5% at 1 mpk
0% at 25 mpk
100% at 10 mpk
100% at 5 mpk
100% at 2.5 mpk
0% at 1 mpk
17
18
79.8% at 300 uM
100% at 300 uM
200.4
66.3
19
56.6% at 300 uM
187.7
Scheme 3. Synthetic route for preparation of 19. Reagents and conditions: (a) 6 eq
MeMgBr, THF, 0 oC to rt, 16 h, Argon; (b) EtOCH2Cl, DIPEA, DCM, 40 oC, 16 h;
(c) Pin2B2, Pd(dppf)Cl2, KOAc, 1,4-dioxane, 80 oC, 16 h, Argon; (d) 6N HCl, THF,
rt, 5 h; (e) NaN3, TFA, CHCl3, -5 oC to 0 oC for 15 min and rt for 16 h; (f) H2, Pd/C
(10%), rt, 5 h; (g) X = Cl for 27, HATU, DIPEA, THF, rt, 16 h.
20
21
0% at 300 uM
100% at 300 uM
nt c
23.9
22
23
0% at 300 uM
100% at 300 uM
nt c
96.6
Activity of compounds 1-23 against larval-stage Lone
Star ticks (Amblyomma americanum) was tested in an in
vitro larval immersion microassay (LIM assay).2 In this
assay, tick larvae were submerged in solutions
containing compounds for 30 min, taken out to air dry,
and incubated for 24 h, at which time mortality was
assessed. If the compounds showed good activity in the
LIM assay, they were advanced for in vivo testing
against nymphal-stage American dog tick (D. variabilis)
infestations on rats. In this in vivo rodent model, tick
nymphs were allowed to attach and begin feeding on
a Experimental procedures for the in vitro LIM assay and the in vivo rat model was
previously described in a reference;2 “mpk” stands for “mg/kg”. Percentage of
b
larval mortality at 300 µM of the tested compound. c Not tested.
Starting with compound 1 that had good activity in the
LIM assay (100% at 300 M and EC50 = 61.3 µM; see
Table 1) and encouraging in vivo efficacy (93.0% at 25
mg/kg) in the rodent model, compound 2 was designed
to improve metabolic stability and its pharmacokinetic
profile by installing 3,3-dimethyl groups on the
oxaborole ring. This structural modification resulted in
rats for 24 h.
Rats in treated groups received
compounds administered orally, and 48h after

better in vivo efficacy (100% at 25 or 10 mg/kg). N-
methylation on the amide decreased the activity (3,
13.9% at 10 mg/kg). Switching the linking position on
the benzoxaborole from 6- to 5-position also decreased
the activity (4, 16.3% at 10 mg/kg). Replacement of the
hydrogen on the left phenyl with a halogen, such as F or
Cl, generally improved the activity in the examples of 5
(100% at 10 mg/kg) vs 4, 6 (10% at 5 mg/kg) vs 2 (0%
at 5 mg/kg), and 7 (100% at 5 mg/kg) vs 2. Decreased
activity of 8 (11.2% at 5 mg/kg, vs 7) reconfirmed the
previous observation that linking 6-position to the
benzoxaborole provided better activity, which led us to
focus on this chemistry. Replacement of 3,3-dimethyl
tolerated by all dogs and there were no abnormal
clinical signs or adverse effects to 23.
Table 2. Efficacy of Molecule 23 against the Tick and the Flea in Dogs.a
Efficacy
Geometric mean percentage efficacy against the American
against
dog tick (Dermacentor variabillis) b
the cat flea b
Day 1
100
Day 7
100
Day 14
100
Day 21
100
Day 30
97
Day 32
98
a Dogs in the treated group (n = 6) were administered with a single oral dose at 25
mg/kg. Experimental procedure for the in vivo dog model is described in reference
b
and note section.4c
The same group of treated dogs was used for the cat flea
(Ctenocephalides felis) efficacy test on day 32 after a month long tick efficacy
study.
groups
with
3,3-di(fluoromethyl)
substituents
maintained the activity (9 and 10 vs 2 and 7,
respectively). Increasing the 3,3-substituent size using
diethyl and spirocyclopentyl groups for more
lipophilicity decreased the activity (11-14). Increasing
the linkage length between the amide and the
benzoxaborole from a carbon to two carbons also
resulted in the decreased activity (15-17). With regard
to the substitution on the methylene moiety between the
In order to determine the compound’s pharmacokinetic
parameters in dogs, blood samples were taken during
the dog efficacy study and the plasma concentrations of
23 were analyzed. It reached high maximum plasma
concentration (7.4 g/mL) in 26 h after oral dosing, and
had long t1/2 (127 h) and high exposure (Table 3). The
plasma concentrations on day 21 and day 30 were 0.491
µg/mL and 0.134 µg/mL, respectively. Therefore, the
97% efficacy against ticks on dogs on day 30 needed
plasma concentration of ≥ 0.134 µg/mL. The minimum
effective plasma concentration of a previous lead
AN8030 in dogs was estimated to be 1.7-1.8 µg/mL to
achieve >95% efficacy against ticks.2 Molecule 23 was
approximately 12-fold more efficacious than AN8030 to
achieve >95% efficacy on day 30.
amide and the benzoxaborole,
maintained the activity (18) while dimethyl substituents
significantly lowered the activity (19).
a
methyl group
The SAR study identified three top tier molecules (7,
10 and 18) that exhibited 90 – 100% efficacy at 5 mg/kg
in the in vivo rat model, and two second tier compounds
(5 and 6) that exhibited 100% efficacy at a slightly
higher dose of 10 mg/kg. Two compounds (6 and 7)
from each of the tiers were selected for chiral
chromatography separation for continued evaluation.
Two of the enantiomers (20 and 22, Fig. 4) had no
activity against the tick whereas the other enantiomers
(21 and 23) had 80.7% and 100% efficacy at 2.5 mg/kg
in the in vivo rodent model, respectively (Table 1). It
has been reported that the active enantiomer of
Fluralaner has the (S)-configuration of the carbon chiral
center on the isoxazoline ring.1a Thus, we reasoned that
the active enantiomers 21 and 23 have (S)-
configuration. Since this research program was intended
to identify a molecule to treat dogs with tick and flea
infestation, molecule 23 was advanced for testing in
dogs.4c In this study, dogs were infested with adult
American dog ticks on Days -1, 5, 12, 19, and 28. On
Day 0, dogs were orally administered gelatin capsules
containing 23 at a dose of 25 mg/kg. Therapeutic
efficacy was evaluated 24 h after treatment (Day 1),
with residual efficacy being evaluated 48 h after each
subsequent infestation (corresponding to Days 7, 14, 21
and 30). Following removal of ticks on Day 30, all
dogs were infested with adult cat fleas (Ctenocephalides
felis). Flea counts were conducted on Day 32 (48 h
after infestation). The efficacy results are shown in
Table 2. Against the American dog tick, molecule 23
demonstrated 100% efficacy within 24 h of treatment,
excellent residual efficacy of 100% through Day 21 and
97% on Day 30. On Day 32, it yielded 98% control of
fleas. Oral treatment with this molecule was well
Table 3. Pharmacokinetic Parameters of Molecule 23 in Dogs.a
Oral dose (25 mpk in capsules)
Cmax (µg/mL)
PK parameter
7.42
C21day (µg/mL)
0.491
C30day (µg/mL)
tmax (h)
AUC0-888 h (h×µg/mL)
AUC0-inf (h×µg/mL)
t1/2 (h)
0.134
26.0
1344
1357
127
a Plasma concentration at each time point was averaged (n = 6, six treated dogs in
the efficacy study). Abbreviations: Cmax, maximum concentration of drug in plasma;
tmax, time to maximum concentration of drug in plasma; AUC0-inf, area under the
curve extrapolated to infinity; t1/2, half-life.
In summary, a novel series of isoxazoline amide
benzoxaboroles was discovered to be active against
ticks and fleas. Two close analogs (7 and 10) were
identified to have 100% efficacy at 5 mg/kg against the
American dog tick in a rat model. Enantiomer 23
demonstrated 97% efficacy on day 30 against American
dog ticks (Dermacentor variabilis) and 98% efficacy
against the cat flea on day 32 in dogs with 25 mg/kg
single oral dose. Its minimum effective plasma
concentration in dogs was estimated to be ≥ 0.134
µg/mL to achieve 97% efficacy against ticks. This new
compound was selected as a development candidate for
potential treatment of ectoparasitic infestations to
companion animals.

methanol (100 mL) was added HCl (20 mL, 65.75 mmol). The reaction
mixture was stirred at rt for 16 h. The solvent was removed to give the
purified 6-(aminomethyl)-3,3-dimethylbenzo[c][1,2]oxaborol-1(3H)-ol
hydrochloride (1.5g; yield 50% over 4 steps). MS: m/z=192.1 (M+1,
ESI+). To a solution of 4-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-
4-5-dihydroisoxazol-3-yl)-2-methyl-benzoic acid (3.39g, 7.51mmol) and
HATU (4.18g, 11 mmol) in DMF (50mL) was added DIPEA (2.84 g, 22
mmol). The reaction mixture was stirred at rt for 10 min and then 6-
(aminomethyl)-3,3-dimethylbenzo[c][1,2]oxaborol-1(3H)-ol
References and Notes
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G. P.; Shoop, W. L.; Xu, M., WO2007079162; (d) Zhao, C.; Casida, J. E.
J. Agric. Food Chem. 2014, 62, 1019; (e) Garcia-Reynaga, P.; Zhao, C.;
Sarpong, R.; Casida, J. E. Chem. Res. Toxicol. 2013, 26, 514; (f) Gassel,
M.; Wolf , C.; Noack, S.; Williams, H.; Ilg, T. Insect Biochem. Mol. Bio.
2014, 45, 111; (g) Rohdich, N.; Roepke, R. K. A.; Zschiesche, E.
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K. A.; Nuernberger, M. C. Parasit. Vectors 2014, 7, 84; (i) Kilp, S.;
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R. K. A.; Nuernberger, M. C. Parasit. Vectors 2014, 7, 86; (k) Walther, F.
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hydrochloride (1.4g, 6.15mmol) was added. The reaction mixture was
stirred overnight. Water was added and the mixture was extracted three
times with EA. The combined organic layers were washed with brine,
dried over Na2SO4, filtered and concentrated under reduced pressure. The
residue was purified by column chromatography over silica gel eluted
with PE:EA (2:1 to 1:1) to give the final compound 7 (2.02 g; yield
1
56.9%) as a white solid. H NMR (500 MHz, DMSO-d6): δ 9.06 (s, 1H),
8.94 (t, J = 6.0 Hz, 1H), 7.85 (s, 2H), 7.65-7.60 (m, 3H), 7.50-7.40 (m,
3H), 4.49 (d, J = 6.0 Hz, 2H), 4.38 (d, J = 18.5 Hz, 1H), 4.35 (d, J = 18.5
Hz, 1H), 2.39 (s, 3H), 1.44 (s, 6H) ppm; HPLC purity: 99.09% at 220 nm
and 99.01% at 254 nm; MS: m/z = 624.8 (M+1, ESI+).
(b)
Synthesis
of
(R)-N-((1-hydroxy-3,3-dimethyl-1,3-
dihydrobenzo[c][1,2]oxaborol-6-yl)methyl)-2-methyl-4-(5-(3,4,5-
trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)benzamide
(22)
and
(S)-N-((1-hydroxy-3,3-dimethyl-1,3-
dihydrobenzo[c][1,2]oxaborol-6-yl)methyl)-2-methyl-4-(5-(3,4,5-
trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)benzamide
(23): Compounds 22 and 23 were obtained as peak 2 and peak 1,
respectively, by separation of the racemic mixture of N-((1-hydroxy-3,3-
dimethyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)methyl)-2-methyl-4-(5-
(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)
benzamide with chiral column chromatography. The racemic mixture
was dissolved in the solvent of mobile phase and separated by
supercritical fluid (SFC) chiral chromatography. The chromatography
conditions used were: column CHIRALCEL OJ-H (column size: 0.46 cm
I.D. × 15cm length), mobile phase CO2/MeOH = 65/35 (v/v), flow rate
2.0 mL/min, detector wave length UV 220nm, and temperature 35oC.
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www.ema.europa.eu; (v) www.fda.gov and www.merial.com; (w)
www.zoetis.com and www.ema.europa.eu.
2. Zhang, Y.-K.; Plattner, J. J.; Easom, E. E.; Zhou, Y.; Akama, T.; Bu,
W.; White, W. H.; Defauw, J. M.; Winkle, J. R.; Balko, T. W.; Guo, S.;
Xue, J.; Cao, J.; Zou, W. Bioorg. & Med. Chem. Lett. 2015, 25, 5589.
1
Analytical data for 22: H NMR (400 MHz, DMSO-d6): δ 9.04 (s, 1H),
8.91 (t, J = 6.0 Hz, 1H), 7.84 (s, 2H), 7.64-7.60 (m, 3H), 7.50-7.38 (m,
3H), 4.48 (d, J = 6.0 Hz, 2H), 4.42 (d, J = 18.4 Hz, 1H), 4.36 (d, J = 18.4
Hz, 1H), 2.39 (s, 3H), 1.44 (s, 6H) ppm; HPLC purity: 99.46% at 220 nm
and 98.05% at 254 nm; Chiral HPLC purity: 97.05% at 220 nm; MS: m/z
= 625.0 (M+1, ESI+). Analytical data for 23: 1H NMR (400 MHz,
DMSO-d6): δ 9.04 (s, 1H), 8.91 (t, J = 6.0 Hz, 1H), 7.84 (s, 2H), 7.64-
7.60 (m, 3H), 7.50-7.38 (m, 3H), 4.48 (d, J = 6.0 Hz, 2H), 4.38 (d, J =
18.2 Hz, 1H), 4.35 (d, J = 18.2 Hz, 1H), 2.39 (s, 3H), 1.44 (s, 6H) ppm;
HPLC purity: 97.78% at 220 nm and 97.97% at 254 nm; Chiral HPLC
purity: 97.64% at 220 nm; MS: m/z = 625.0 (M+1, ESI+).
3. (a) Akama, T.; Balko, T. W.; Defauw, J. M.; Plattner, J. J.; White, W.
H.; Winkle, J. R.; Zhang, Y.-K.; Zhou, Y., US20130131017; (b) Akama,
T.; Balko, T. W.; Defauw, J. M.; Plattner, J. J.; White, W. H.; Winkle, J.
R.; Zhang, Y.-K.; Zhou, Y., US20130131016.
4. Experimental procedures for the syntheses of 7, 22 and 23 and for the
biological evaluation of the compounds are described below.
(a)
Synthesis
of
N-((1-hydroxy-3,3-dimethyl-1,3-
(c) Efficacy of 23, administered orally at 25 mg/kg, against adult
American dog tick (Dermacentor variabilis) and cat flea
(Ctenocephalides felis) infestations on dogs. The therapeutic
(knockdown) and residual efficacy of 23, administered orally at a point
dose of 25 mg/kg bodyweight, was evaluated against adult tick
(Dermacentor variabilis) and adult cat flea (Ctenocephalides felis)
infestations on dogs. Twelve (12) male and female beagle dogs were
allocated to either an untreated, negative control group or 23 group (n=6
dogs per group). Dogs were infested with approximately 50 unfed, adult
D. variabilis ticks on Days -1, 5, 12, 19, and 28. On Day 0, dogs were
orally administered gelatin capsules containing 23 (50% technical active,
47% microcrystalline cellulose and 3% croscarmellose sodium, w/w) at a
point dose of 25 mg/kg. D. variabilis tick counts and classifications were
conducted on Day 1 (thumb count, approximately 24 hours after
treatment) and Days 7, 14, 21 and 30 (approximately 48 hours after
infestation). Following removal of D. variabilis ticks on Day 30, all dogs
were infested with approximately 100 unfed, adult cat fleas (C. felis). Cat
flea counts were conducted on Day 32 (approximately 48 hours after
infestation). Geometric mean (GM) percent efficacy of treatments against
both tick species and fleas was calculated using the following formula: %
Efficacy = [(A – B)/A] × 100%, where A = GM number of live ticks or
dihydrobenzo[c][1,2]oxaborol-6-yl)methyl)-2-methyl-4-(5-(3,4,5-
trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)benzamide
(7): To
a
solution of 1-hydroxy-3,3-dimethyl-1,3-dihydrobenzo
[c][1,2]oxaborole-6-carbaldehyde (5.0 g, 26.3 mmol) and NH2OH·HCl
(2.2 g, 31.6 mmol) in THF (50 mL) and H2O (10 mL) at rt was added
NaOAc (3.2 g, 39.5 mmol). The reaction mixture was stirred for 2 h and
diluted with H2O. The mixture was extracted with ethyl acetate (EA) and
the organic layer was separated. The organic solution was washed with
brine, dried over Na2SO4, filtered and concentrated under reduced
pressure
to
give
crude
1-hydroxy-3,3-dimethyl-1,3-
dihydrobenzo[c][1,2]oxaborole-6-carbaldehyde oxime as light yellow
solid. To a solution of the oxime (2.7g, 13.15mmol) in AcOH (30 mL) at
rt was added zinc dust (2.56 g, 39.45 mmol). The reaction mixture was
stirred at 40 oC for 4 h under argon. Methanol was added and the mixture
was filtered over Celite. The filtrate was concentrated and dissolved in
ethyl acetate. The organic layer was washed with water. The water layer
was lyophilized
to
give the
crude 6-(aminomethyl)-3,3-
dimethylbenzo[c][1,2]oxaborol-1(3H)-ol as light yellow solid. To a
solution of the crude amine compound (13.15mmol) and (Boc)2O (5.26g,
26.3mmol) in DCM (50mL) was added Et3N (5.6mL, 39.45mmol) at rt.
The reaction mixture was stirred at rt for 3 h. The mixture was poured
into water and extracted with DCM. The organic layer was washed with
water, dried over Na2SO4, filtered and concentrated under reduced
fleas control;
B = GM number of live ticks or fleas treated.
pressure
to
give
tert-butyl
((1-hydroxy-3,3-dimethyl-1,3-
dihydrobenzo[c][1,2]oxaborol-6-yl)methyl)carbamate. MS: m/z=314.0
(M+23, ESI+). To a solution of the Boc-protected amine (13.15 mmol) in

Graphical Abstract
Optimization of Isoxazoline Amide Benzoxaboroles for Identification of a
Development Candidate as an Oral Long Acting Animal Ectoparasiticide
Yong-Kang Zhang*, Jacob J. Plattner, Eric E. Easom, Tsutomu Akama, Yasheen Zhou,
W. Hunter White, Jean M. Defauw, Joseph R. Winkle, Terry W. Balko, Jianxin Cao,
Zhixin Ge, Jianzhang Yang
Novel isoxazoline amide benzoxaboroles were designed and synthesized to optimize the ectoparasiticide
activity of this chemistry series against ticks and fleas. The study identified an orally bioavailable molecule,
(S)-N-((1-hydroxy-3,3-dimethyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)methyl)-2-methyl-4-(5-(3,4,5-
trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)benzamide (23), with
a
favorable
pharmacodynamics profile in dogs (Cmax = 7.42 ng/ml; Tmax = 26.0 h; terminal half-life t1/2 = 127 h).
Compound 23, a development candidate, demonstrated 100% therapeutic effectiveness within 24 hours of
treatment, with residual efficacy of 97% against American dog ticks (Dermacentor variabilis) on day 30
and 98% against cat fleas (Ctenocephalides felis) on day 32 after a single oral dose at 25 mg/kg in dogs.