Discovery of an orally bioavailable isoxazoline benzoxaborole (AN8030) as a long acting animal ectoparasiticide

Article abstract of DOI:10.1016/j.bmcl.2015.10.044

A novel series of isoxazoline benzoxaborole small molecules was designed and synthesized for a structure-activity relationship (SAR) investigation to assess the ectoparasiticide activity against ticks and fleas. The study identified an orally bioavailable molecule, (S)-3,3-dimethyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)benzo[c][1,2]oxaborol-1(3H)-ol (38, AN8030), which was long lasting in dogs (t_1/2 = 22 days). Compound 38 demonstrated 97.6% therapeutic effectiveness within 24 h of treatment, with residual efficacy of 95.3% against American dog ticks (Dermacentor variabilis) on day 30% and 100% against cat fleas (Ctenocephalides felis) on day 32 after a single oral dose at 50 mg/kg in dogs.

Full text of DOI:10.1016/j.bmcl.2015.10.044

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of an orally bioavailable isoxazoline benzoxaborole
(AN8030) as a long acting animal ectoparasiticide
a
a
a
a
a
Yong-Kang Zhang a, , Jacob J. Plattner , Eric E. Easom , Yasheen Zhou , Tsutomu Akama , Wei Bu ,
W. Hunter White b, Jean M. Defauw b, Joseph R. Winkle b, Terry W. Balko b, Shenghai Guo c, Jian Xue c,
Jianxin Cao d, Wuxin Zou e
⇑
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 Medicilon, Inc., 585 Chuanda Road, Zhangjiang High-tech Park, Pudong New Area, Shanghai 201200, China
d Shanghai ChemPartner, 998 Halei Road, Zhangjiang High-tech Park, Pudong New Area, Shanghai 201203, China
e BioDuro LLC, Building E, No. 29, Life Science Park Road, Beijing 102206, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of isoxazoline benzoxaborole small molecules was designed and synthesized for a struc-
ture–activity relationship (SAR) investigation to assess the ectoparasiticide activity against ticks and
fleas. The study identified an orally bioavailable molecule, (S)-3,3-dimethyl-5-(5-(3,4,5-trichlorophe-
nyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)benzo[c][1,2]oxaborol-1(3H)-ol (38, AN8030), which
was long lasting in dogs (t1/2 = 22 days). Compound 38 demonstrated 97.6% therapeutic effectiveness
within 24 h of treatment, with residual efficacy of 95.3% against American dog ticks (Dermacentor vari-
abilis) on day 30 and 100% against cat fleas (Ctenocephalides felis) on day 32 after a single oral dose at
50 mg/kg in dogs.
Received 26 September 2015
Revised 13 October 2015
Accepted 15 October 2015
Available online xxxx
Keywords:
Benzoxaborole
Isoxazoline
Structure–activity relationship
Ectoparasiticide
Tick and flea
Ó 2015 Elsevier Ltd. All rights reserved.
Isoxazoline compounds, as a new class of ectoparasiticide
agents inhibiting -aminobutyric acid (GABA)-gated and -gluta-
molecule 2, a series of its close analogs (3–38 in Figs. 3 and 4) was
designed and synthesized for a structure–activity relationship
(SAR) study to examine the effects from oxaborole 3-substituent
variation (2–6), fluoro position change on the benzoxaborole ben-
zene ring (7–9), substituent variation on the isoxazoline ring (10
vs 3, and 17–19), fluoro substitution on 3,3-dimethyls (20 vs 17),
di-substitution (11–16), tri-substitution (21–34) and tetra-substi-
tution on the left benzene ring (35 vs 36), and the chiral configura-
tion (37 vs 38). Herein, we report the chemistry, pharmacokinetic
results and ectoparasiticide activity against ticks and fleas.
The syntheses of compounds 2–36 were convenient and the
general route is shown in Scheme 1.4 Aldehyde 394 reacted with
hydroxylamine to give carbaldehyde oxime 40, which was con-
verted to carbimidoyl chloride 41 by reacting to N-chlorosuccin-
imide (NCS). The last step was the cycloaddition reaction of 41
with substituted styrene intermediates 42 to give the final com-
pounds 2–36. As an example, the experimental procedure for the
synthesis of 17, and its chiral separation method for obtaining 37
and 38 are described in the reference and note section.7a,b It has
been reported that the active enantiomer of Fluralaner has the
(S)-configuration of the carbon chiral center on the isoxazoline
ring.1a
c
L
mate-gated chloride channels, have recently been discovered and
drawn broad attention in the companion animal industry.1 Two
efficacious isoxazoline compounds1a–s (Fig. 1) have recently been
approved for the treatment of tick and flea infestations in dogs.1t,u
Previously, benzoxaboroles have been reported with selective
activity against parasites2 including the parasite, Plasmodium fal-
ciparum, the trypanosome, Trypanosoma brucei and the filarial
worm, Brugia malayi. As an extension of our research, we rational-
ized exploring the potential of benzoxaboroles as ectoparasiti-
cides for the treatment of ticks and fleas in animals including
cats and dogs.
It was disclosed that compound 1 (Fig. 2) has pesticide activities
against several pests tested including fall armyworm larvae (Spo-
doptera frugiperda) and mustard beetle larvae (Phaedon
cochleariae).3 The five-membered lactone scaffold in 1 has similar
pharmacophore feature to the oxaborole ring in 2. Both the lactone
carbonyl carbon in 1 and the boron in 2 are electron-deficient and
can interact with nucleophilic components. Therefore, starting from
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2015.10.044
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhang, Y.-K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.10.044

2
Y.-K. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
O
N
O
O
N
F3C
CF3
O
F3C
Cl
Cl
CF3
HN
HN
CF3
NH
NH
O
O
Cl
Fluralaner
Afoxolaner
Figure 1. Chemical structures of two typical isoxazoline compounds.
Activity of compounds 2–38 against larval-stage Lone star ticks
(Amblyomma americanum) was tested in an in vitro larval immer-
sion microassay (LIM assay).7c In this assay, tick larvae were sub-
merged in solutions containing compounds for 30 min, taken out
to air dry and then incubated for mortality-counting in 24 h. If
the compounds showed good activity in the LIM assay, they were
further tested for efficacy against nymphal-stage American dog
ticks (Dermacentor variabilis) on rats. In this in vivo rodent model,
tick nymphs were allowed to attach and begin feeding on rats for
O
N
O N
F3C
F3C
O
OH
Cl
Cl
B
O
O
Cl
Cl
1
2
Figure 2. Structural comparison of an initial isoxazoline benzoxaborole (2) with a
reported isoxazoline benzolactone pesticide (1).
X
Z
OH
7
6
5
Y
B 1
2
O
R1
N
3
4
R7
O
R6
R2
R5
R3
R4
3: R1 = R3 = Cl, R2 = R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
1
: R = R3 = Cl, R2 = R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Et;
4
5
6
7
1
: R = R3 = Cl, R2 = R4 = H, R5 = CF3, X = Y = Z = H, R6 & R7 = (CH2)4;
1
: R = R3 = Cl, R2 = R4 = H, R5 = CF3, X = Y = Z = H, R6 & R7 = (CH2)5;
1
: R = R3 = Cl, R2 = R4 = H, R5 = CF3, X = Y = H, Z = F, R6 = R7 = Me;
8: R1 = R3 = Cl, R2 = R4 = H, R5 = CF3, X = Z = H, Y = F, R6 = R7 = Me;
9: R1 = R3 = Cl, R2 = R4 = H, R5 = CF3, X = F, Y = Z = H, R6 = R7 = Me;
10: R1 = R3 = Cl, R2 = R4 = H, R5 = CF2CF3, X = Y = Z = H, R6 = R7 = Me;
11: R1 = R3 = Br, R2 = R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
: R1 = CF3, R3 = Cl, R2 = R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
: R1 = R3 = CF3, R2 = R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
: R1 = Cl, R2 = F, R3 = R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
12
13
14
15: R1 = R2 = Cl, R3 = R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
16: R1 = CF3, R2 = F, R3 = R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
17: R1 = R2 = R3 = Cl, R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
18: R1 = R2 = R3 = Cl, R4 = H, R5 = CH3, X = Y = Z = H, R6 = R7 = Me;
: R1 = R2 = R3 = Cl, R4 = H, R5 = CH2F, X = Y = Z = H, R6 = R7 = Me;
: R1 = R2 = R3 = Cl, R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = CH2F;
: R1 = R3 = Cl, R2 = F, R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
: R1 = R3 = Cl, R2 = Br, R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
19
20
21
22
23: R1 = R2 = R3 = F, R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
24: R1 = R3 = Cl, R2 = CF3, R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
25: R1 = R3 = Cl, R2 = CF2H, R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
26: R1 = R3 = Cl, R2 = Me, R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
: R1 = R2 = Cl, R3 = F, R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
: R1 = Cl, R2 = R3 = F, R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
: R1 = R3 = Br, R2 = Cl, R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
27
28
29
30: R1 = R3 = F, R2 = Cl, R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
31: R1 = R3 = Br, R2 = F, R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
32: R1 = R3 = Cl, R2 = OMe, R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
33: R1 = R3 = Cl, R2 = OCF3, R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
: R1 = R3 = Cl, R2 = OCH2CF3, R4 = H, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
: R1 = R2 = R3 = R4 = Cl, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
: R1 = R3 = Cl, R2 = R4 = F, R5 = CF3, X = Y = Z = H, R6 = R7 = Me;
34
35
36
Figure 3. Chemical structures of the isoxazoline benzoxaboroles of compound 2 analogs investigated in a SAR study to identify novel ectoparasiticides.
Please cite this article in press as: Zhang, Y.-K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.10.044

Y.-K. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Table 1
N
N
O
O
OH
OH
Activity of compounds 2–38 in in vitro tick LIM assay and in vivo tick rat model
F3C
F3C
B
O
B
O
Compound
In vitro activity in LIM assaya
Activity in rat modelb
Activityc
EC50
(lM)
Cl
Cl
2
3
100% at 100
100% at 100
l
l
M
M
20.6
27.7
21.8% at 12 mpk
100% at 10 mpk
53.1% at 5 mpk
13.7% at 2.5 mpk
0% at 25 mpk
0% at 25 mpk
0% at 25 mpk
0% at 25 mpk
0% at 25 mpk
0% at 25 mpk
7% at 25 mpk
100% at 10 mpk
77.7% at 5 mpk
14.7% at 2.5 mpk
100% at 25 mpk
83.5% at 25 mpk
100% at 25 mpk
1.9% at 5 mpk
100% at 25 mpk
6.1% at 5 mpk
15.6% at 25 mpk
1.8% at 5 mpk
100% at 5 mpk
12.1% at 1 mpk
19.1% at 25 mpk
0% at 5 mpk
Cl
Cl
Cl
Cl
37
(R)-isomer
38 (AN8030)
(S)-isomer
4
5
6
7
8
9
10
11
0% at 100
0% at 100
0% at 100
0% at 300
l
l
l
l
M
ntd
nt
nt
nt
288.8
nt
Figure 4. Two enantiomers of racemic compound 17.
M
M
M
80% at 300
l
M
X
X
0% at 100
0% at 100
l
M
OH
OH
lM
nt
37.6
Y
Y
B
B
a
100% at 100
lM
O
O
N
OHC
HO
R1
R7
R7
R6
R6
Z
Z
12
13
14
98% at 100
26% at 100
l
l
M
M
40
117.2
13.9
39
40
100% at 300
lM
15
16
17
18
19
89% at 300
97% at 300
100% at 100
l
M
57.1
49.8
28.6
nt
X
R2
OH
R5
Y
B
l
M
R3 R4
O
b
42
c
Cl
lM
2 - 36
R7
R6
N
OH
Z
0% at 100
0% at 100
0% at 100
l
l
l
M
41
M
nt
28% at 25 mpk
0% at 5 mpk
20
21
M
nt
21.1
0% at 25 mpk
100% at 5 mpk
16.3% at 2.5 mpk
100% at 25 mpk
0% at 5 mpk
88% at 25 mpk
8% at 5 mpk
100% at 25 mpk
0% at 5 mpk
Scheme 1. General route for syntheses of 2–36. Reagents and conditions: (a) NH2-
OHꢀHCl, NaOAc, THF, H2O, rt, 16 h; (b) NCS, DMF, 45 °C, 3 h; (c) 42, TEA, DMF, rt,
16 h.
100% at 300
l
M
M
22
23
24
25
100% at 300
l
64.2
76% at 300
100% at 300
100% at 300
100% at 300
l
M
118.7
134.6
76.4
24 h. Rats in treated groups were orally administered compounds,
and 48 h after treatment, live and dead ticks were removed from
animals and counted.7d
It was very encouraging that the first molecule synthesized for
this program, compound 2, showed excellent activity in the LIM
l
l
l
M
M
100% at 25 mpk
0% at 5 mpk
26
27
M
31.9
58.6
0% at 25 mpk
100% at 25 mpk
3.9% at 5 mpk
100% at 25 mpk
0% at 5 mpk
100% at 25 mpk
97.3% at 5 mpk
0% at 2.5 mpk
41.3% at 25 mpk
0% at 5 mpk
100% at 25 mpk
96.9% at 5 mpk
0% at 2.5 mpk
0% at 25 mpk
25% at 25 mpk
0% at 25 mpk
25.5% at 25 mpk
0% at 5 mpk
assay (100% at 100 lM and EC50 = 20.6 lM; see Table 1) and
90% at 300
100% at 300
100% at 300
lM
moderate in vivo efficacy (21.8% at 12 mg/kg) in the rodent model.
Subsequent design of a molecule for improving metabolic stability
and pharmacokinetic profile by installing 3,3-dimethyl groups on
the oxaborole ring lead to 3, which had better in vivo efficacy
(100% at 10 mg/kg and 53.1% at 5 mg/kg). Further increase of the
3,3-substituent sizes using diethyl, cyclopentyl and cyclohexyl
groups for more lipophilicity diminished the activity (4–6).
Introducing a fluorine atom to the 4-, 6- or 7-position of the ben-
zoxaborole benzene ring also resulted in loss of activity (7–9).
Replacement of trifluoromethyl in 3 with a larger pentafluoroethyl
group substituted on the isoxazoline ring (10) depleted the
activity. Structural modification was subsequently directed to the
substituents on the left-side benzene ring. Among variations of
molecules 11–16 with di-substituents at R1 and R3, dibromo com-
pound 11 had similar activity to dichloro molecule 3. An additional
group was then introduced at the R2 position. With R2 = Cl, the
efficacy of 17 was improved by 2-fold as compared to 3 (100% vs
53.1% at 5 mg/kg). The R5 group effect was further examined by
using CF3 in 17, CH3 in 18 or CH2F in 19. Molecules 18 and 19 lost
activity. Changing 3,3-dimethyl in 17 to 3,3-di(fluoromethyl) in 20
dramatically reduced the activity. Molecules 21–34 were gener-
ated to investigate the effect of the tri-substituents (R1, R2 and
R3) variation. This resulted in three compounds (21, 29 and 31)
with excellent in vivo efficacies close to the lead compound (17).
It was noticed that even slightly polar groups such as methoxy,
trifluoromethoxy and trifluoroethoxy, decreased activity (32–34).
Compounds 35 and 36 with tetra-substituents at R1–R4 positions
had good activity but were less active than 17.
28
29
lM
34.0
23.0
lM
30
31
82% at 300
l
M
nt
100% at 300
lM
44.2
32
33
34
35
100% at 300
85% at 300 lM
1% at 300 lM
100% at 300
lM
67.1
257.7
nt
l
M
M
99
36
100% at 300
l
32.2
100% at 25 mpk
0% at 5 mpk
0% at 5 mpk
100% at 5 mpk
87.8% at 2.5 mpk
0% at 1 mpk
37
38
0% at 100
lM
nt
15.3
100% at 300
lM
a
Experimental procedure for the in vitro LIM assay is described in reference and
note section.7c
b
Experimental procedure for the in vivo rat model is described in reference and
note section;7d ‘mpk’ stands for ‘mg/kg’.
c
Percentage of larval mortality at 100 lM or 300 lM of the tested compound.
Not tested.
d
The SAR study identified four top tier molecules (17, 21, 29 and
31) having excellent activity both in the in vitro LIM assay and in
the in vivo rat model. Molecule 17 was very stable when tested
Please cite this article in press as: Zhang, Y.-K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.10.044

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Y.-K. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 2
Pharmacokinetic parameters of molecule 17 in male ratsa
Intravenous dose (2 mpk)
PK parameter
Oral dose (10 mpk)
Cmax g/mL)
tmax (h)
PK parameter
Cmax
(
lg/mL)
1.82
80
1031
3005
25.4
27.3
(
l
2.25
4.67
66.0
77.5
24.7
52
CL (mL/h/kg)
Vc (mL/kg)
Vss (mL/kg)
AUC0–1 (h ꢁ
t1/2 (h)
AUC72h (h ꢁ
l
g/mL)
AUC0–1 (h ꢁ
lg/mL)
lg/mL)
t1/2 (h)
Bioavailability (%)
a
Abbreviations: Cmax, maximum concentration of drug in plasma; CL, clearance; Vc, apparent volume of central compartment of pharmacokinetic model; Vss, volume of
distribution at steady state; tmax, time to maximum concentration of drug in plasma; AUC0–1, area under the curve extrapolated to infinity; t1/2, half-life.
In order to determine the molecule’s pharmacokinetic parame-
ters in dogs, blood samples were taken during the dog efficacy
Table 3
Efficacy of molecule 38 against the tick and the flea in dogsa
study and the plasma concentrations of 38 were analyzed. It
reached high maximum plasma concentration (8.4 lg/mL) in 26 h
after oral dosing, and had long t1/2 (22 day) and high exposure
(Table 4). The plasma concentration during 48 h of day 28 to day
Geometric mean percentage efficacy against the
Efficacy against
the cat fleab
American dog tick (Dermacentor variabilis)b
Day 1
97.6
Day 2
100
Day 7
100
Day 14
96.7
Day 21
100
Day 30
95.3
Day 32
100
30 was estimated to be in the range of 1.7–1.8 lg/mL, which corre-
a
sponded to 95.3% efficacy against ticks on day 30.
Dogs in the treated group were administered with
a
single oral dose at
In summary, a novel series of isoxazoline benzoxaborole small
molecules was discovered to be active against ticks and fleas. Four
close analogs (17, 21, 29 and 31) were identified to have 95–100%
efficacy at 5 mg/kg against the American dog tick in a rat model.
Molecule 38 (AN8030) was long lasting in the dog (t1/2 = 22 day)
and demonstrated 95.3% efficacy on day 30 against the tick and
100% efficacy against the cat flea on day 32 in dogs with
50 mg/kg single oral dose. Its minimum effective plasma
50 mg/kg. Experimental procedure for the in vivo dog model is described in
7e
reference and note section.
b
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.
Table 4
Pharmacokinetic parameters of molecule 38 in dogsa
concentration in dogs was estimated to be 1.7–1.8
achieve >95% efficacy against ticks.
lg/mL to
Oral dose (50 mpk in capsules)
Cmax g/mL)
C28day g/mL)
tmax (h)
PK parameter
(
l
8.4
1.8
26.0
(l
References and notes
AUC0–672h (h ꢁ
l
g/mL)
2220
3794
22
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2. (a) Zhang, Y.-K.; Plattner, J. J.; Freund, Y.; Easom, E. E.; Zhou, Y.; Gut, J.;
Rosenthal, P. J.; Waterson, D.; Gamo, F.-J.; Angulo-Barturen, I.; Ge, M.; Li, Z.; Li,
L.; Jian, Y.; Cui, H.; Wang, H.; Yang, J. Chem. Lett. 2011, 21, 644; (b) Zhang, Y.-K.;
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Gamo, F.-J.; Sanz, L. M.; Ge, M.; Li, Z.; Li, L.; Wang, H.; Cui, H. Chem. Lett. 2012, 22,
1299; (c) Zhang, Y.-K.; Plattner, J. J.; Easom, E. E.; Waterson, D.; Ge, M.; Li, Z.; Li,
L.; Jian, Y. Tetrahedron Lett. 2011, 52, 3909; (d) Zhang, Y.-K.; Plattner, J. J.; Easom,
E. E.; Liu, L.; Retz, D. M.; Ge, M.; Zhou, H.-H. J. Label. Compd. Radiopharm. 2012,
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Yarlett, N.; Zhang, Y.-K.; Hernandez, V.; Xia, Y.; Freund, Y.; Abdulla, M.; Ang, K.
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AUC0–1 (h ꢁ
t1/2 (day)
lg/mL)
a
Plasma concentration at each time point was averaged (n = 4, four treated dogs
in the efficacy study). For abbreviations, see footnote under Table 2.
in rat and dog liver microsome stability assays (92–100% remain-
ing in 2 h with or without NADPH). The result of a pharmacokinetic
study indicated it had low clearance, high volume distribution,
long t1/2 and high exposure in the rat (Table 2), which is ideal for
eliminating ectoparasites such as ticks and fleas that take blood
meals through host skin.
Compound 17 was separated by chiral chromatography for con-
tinued evaluation. (R)-Enantiomer 37 had no activity against the
tick. (S)-Enantiomer 38 (AN8030 in Fig. 4) had 87.8% efficacy at
2.5 mg/kg in the in vivo rodent model (Table 1). Since this research
program was intended to identify a molecule to treat dogs with tick
and flea infestation, molecule 38 was tested in dogs.7e 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 38 at a dose of 50 mg/kg. Tick counts and classification
were conducted on Days 1, 2, 7, 14, 21 and 30 (48 h after infestation).
Following removal of ticks on Day 30, all dogs were infested with
adult cat fleas. Cat flea counts were conducted on Day 32 (48 h after
infestation). The efficacy results are shown in Table 3. Against the
American dog tick, molecule 38 demonstrated 98% efficacy within
24 h oftreatmentand100%within48 htreatment, excellentresidual
efficacy of 97–100% through Day 21 and 95% on Day 30. On Day 32, it
yielded 100% control of fleas. Oral treatment with this molecule was
well tolerated by all dogs and there were no abnormal clinical signs
or adverse effects to 38.
3. Murata, T.; Yoneta, Y.; Mihara, J.; Domon, K.; Hatazawa, M.; Araki, K.; Shimojo,
E.; Shibuya, K.; Ichihara, T.; Goergens, U.; Voerste, A.; Becker, A.; Franken, E.-M.;
Mueller, K.-H., WO2009112275.
Please cite this article in press as: Zhang, Y.-K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.10.044

Y.-K. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
4. Akama, T.; Balko, T. W.; Defauw, J. M.; Plattner, J. J.; White, W. H.; Winkle, J. R.;
Zhang, Y.-K.; Zhou, Y., US20130131016.
5. White, W. H.; Plummer, P. R.; Kemper, C. J.; Miller, R. J.; Davey, R. B.; Kemp, D. H.;
Hughes, S.; Smith, C. K., II; Gutiérrez, J. A. J. Med. Entomol. 2004, 1034, 41.
6. Gutiérrez, J. A.; Zhao, X.; Kemper, C. J.; Plummer, P. R.; Bauer, S. M.; Hutchens, D.
E., ; Smith, C. K., II; White, W. H. J. Med. Entomol. 2006, 43, 526.
stock solution at a concentration of at least 10 mM. Using 96-well microtiter
plates, an aliquot of the 10 mM sample was subsequently diluted in a water-
based solution containing 1% ethanol and 0.2% Triton X-100, to obtain the
desired concentration (typically 0.3 mM or lower) of compound in a volume of
0.1 ml (minimum n = 3 replicates per compound or concentration). Approxi-
mately 30–50 Lone star tick larvae (Amblyomma americanum) were submerged
into each well containing compounds. After a 30 min immersion period, larvae
were removed with a wide-bore pipette tip in 0.05 ml of fluid, dispensed into a
commercial paper tissue biopsy bag which was sealed at the top with a plastic
dialysis clip, inverted and allowed to air dry for 60 min. Bags containing larvae
were then incubated at approximately 27°C and >90% relative humidity. After
24 h, bags were opened, live and dead larvae were counted and percent larval
mortality was calculated.
7. Experimental procedures for the syntheses of 17, 37 and 38 (AN8030) and for
the biological evaluation of the compounds are described below.
(a) Synthesis of 3,3-dimethyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-
4,5-dihydroisoxazol-3-yl)benzo[c][1,2]oxaborol-1(3H)-ol (17): To
a mixture of
1-hydroxy-3,3-dimethyl-1,3-dihydrobenzo[c][1,2]oxaborole-5-carbaldehyde
(560 mg, 2.95 mmol) and NH2OHꢀHCl (246 mg, 3.54 mmol) in THF (6 mL) and
H2O (1.5 mL) at room temperature (rt) was added NaOAc (339 mg, 4.13 mmol).
The reaction mixture was stirred at rt for 16 h, diluted with H2O and extracted
with ethyl acetate (2 ꢁ 10 mL). The combined organic layers were washed with
brine, dried over Na2SO4, filtered and concentrated under reduced pressure to
give the desired oxime (620 mg, 100% yield) as a white solid. To a solution of
1-hydroxy-3,3-dimethyl-1,3-dihydrobenzo[c][1,2]oxaborole-5-carbaldehyde
oxime (1.38 g, 6.73 mmol) in DMF (15 mL) at rt was added N-chlorosuccinimide
(1.07 g, 8.03 mmol). The reaction mixture was warmed to 45 °C, stirred for 3 h
and cooled to rt. The mixture was poured into ice-water (20 mL) and extracted
with ethyl acetate (2 ꢁ 20 mL). The combined organic layers were washed with
brine, dried over Na2SO4, filtered and concentrated under reduced pressure to
give the desired carbimidoyl chloride (1.7 g; 100% yield) as a white solid. To a
solution of N,1-dihydroxy-3,3-dimethyl-1,3-dihydrobenzo[c][1,2]oxaborole-
5-carbimidoyl chloride (850 mg, 3.55 mmol) and 1,2,3-trichloro-5-(3,3,3-
trifluoroprop-1-en-2-yl)benzene (1.47 g, 5.32 mmol) in DMF (10 mL) at rt was
added TEA (0.5 mL, 3.55 mmol). The reaction mixture was stirred at rt for 16 h,
poured into ice-water and extracted with ethyl acetate (20 mL). The organic
layer was washed with brine, dried over Na2SO4, filtered and concentrated
under reduced pressure. The residue was purified by column chromatography to
give the desired final compound 3,3-dimethyl-5-(5-(3,4,5-trichlorophenyl)-
5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl) benzo[c][1,2]oxaborol-1 (3H)-ol
(510 mg; 30% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): d 9.25 (s,
1H), 7.86 (s, 2H), 7.80-7.75 (m, 2H), 7.72 (d, J = 7.2 Hz, 1H), 4.46 (d, J = 19.2 Hz,
1H), 4.35 (d, J = 18.4 Hz, 1H), 1.503 (s, 3H), 1.496 (s, 3H) ppm; HPLC purity:
95.3% at 220 nm and 96.8% at 254 nm; MS: m/z = 478.4 (M+, ESI+).
(b) Synthesis of (R)-3,3-dimethyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-
4,5-dihydroisoxazol-3-yl)benzo[c][1,2]oxaborol-1(3H)-ol (37) and (S)-3,3-
dimethyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-
yl)benzo[c][1,2]oxaborol-1(3H)-ol (38, AN8030): Compounds 37 and 38 were
obtained as peak 2 and peak 1, respectively, by separation of the racemic
mixture of 3,3-dimethyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-
dihydroiso-xazol-3-yl)benzo[c][1,2]oxaborol-1(3H)-ol 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. ꢁ 15 cm length), mobile phase CO2/MeOH = 70/30 (w/w), flow rate
2.0 mL/min, detector wave length UV 220 nm, and temperature 35 °C. Analytical
data for 37: 1H NMR (500 MHz, DMSO-d6): d 9.22 (s, 1H), 7.84 (s, 2H), 7.77-7.75
(m, 2H), 7.70 (d, J = 7.2 Hz, 1H), 4.45 (d, J = 18.0 Hz, 1H), 4.34 (d, J = 18.5 Hz, 1H),
1.486 (s, 3H), 1.479 (s, 3H) ppm; HPLC purity: 99.0% at 220 nm and 98.7% at
254 nm; Chiral HPLC purity: 100% at 254 nm; MS: m/z = 478.0 (M+, ESI+).
Analytical data for 38 (AN8030): 1H NMR (500 MHz, DMSO-d6): d 9.22 (s, 1H),
7.84 (s, 2H), 7.77-7.75 (m, 2H), 7.70 (d, J = 7.2 Hz, 1H), 4.45 (d, J = 18.5 Hz, 1H),
4.34 (d, J = 18.5 Hz, 1H), 1.486 (s, 3H), 1.478 (s, 3H) ppm; HPLC purity: 100%
at 220 nm and 100% at 254 nm; Chiral HPLC purity: 100% at 254 nm; MS:
m/z = 478.2 (M+, ESI+).
(d) Method for testing efficacy of compounds against nymphal-stage American dog
ticks (Dermacentor variabilis) on rats: Evaluations were conducted using
a
modified version of the assay as described in a literature Ref. 6. This assay may
be modified by simply using different tick species (the reference describes
Amblyomma americanum ticks), such as Dermacentor variabilis or Rhipicephalus
sanguineus ticks, as well as different life-stages (larval, nymphal or adult).
Further, the reference describes using topical application methods, but oral,
transdermal and subcutaneous injection routes of administration may be used.
In these studies, adult male or female rats, approximately 300 g in size, were
randomly assigned to a treatment group or a control (untreated negative control
or fipronil positive control) group. Each group consisted of three (3) to five (5)
rats. One day before treatment (Day-1), rats were infested with approximately
ten (10) D. variabilis tick nymphs, which were allowed to attach and begin
feeding for 24 h. On Day 0, rats in treated groups were orally administered
compounds dissolved in polyethylene glycol-300, propylene glycol and water, at
point dosages of 5–25 mg/kg bodyweight. Fipronil was prepared in similar
fashion and administered orally at 10 mg/kg bodyweight. On Day 2, approxi-
mately forty-eight (48) hours after treatment, live and dead ticks were removed
from animals and counted. Live tick counts were transformed using the natural
logarithm transformation plus one (Ln count + 1); addition of one to each count
served to adjust for counts that were zero. Geometric mean (GM) group tick
counts were obtained via back-transformation of group mean transformed
counts and subtracting one. The contemporaneous negative control group was
used for comparison to the compound treatment groups for the calculation of
percent efficacy (% reduction in live tick counts). GM percent efficacy of
treatments was calculated using the following formula: % efficacy = [(A ꢂ B)/
A] ꢁ 100%, where A = GM number of live ticks in control group and B = GM
number of live ticks in treated group.
(e) Efficacy of 38, administered orally at 50 mg/kg, against adult American dog tick
(Dermacentor variabilis) and cat flea (Ctenocephalides felis) infestations on dogs.
The therapeutic (knockdown) and residual efficacy of 38, administered orally at
a
point dose of 50 mg/kg bodyweight, was evaluated against adult tick
(Dermacentor variabilis) and adult cat flea (Ctenocephalides felis) infestations on
dogs. Eight (8) male and female beagle dogs were allocated to either an
untreated, negative control group or 38 group (n = 4 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
38 (50% technical active, 47% microcrystalline cellulose and 3% croscarmellose
sodium, w/w) at
a point dose of 50 mg/kg. D. variabilis tick counts and
classification were conducted on Day 1 (thumb count, approximately 24 h after
treatment) and Days 2, 7, 14, 21 and 30 (approximately 48 h 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 h after infestation). Geometric mean
(GM) percent efficacy of treatments against both tick species and fleas was
calculated using the following formula: % efficacy = [(A ꢂ B)/A] ꢁ 100%, where
A = GM number of live ticks or fleas control; B = GM number of live ticks or fleas
treated.
(c) Method for testing activity of compounds against larval-stage Lone star ticks
(Amblyomma americanum) in
a larval immersion microassay: The larval
immersion microassay was conducted as described in detail in a published
article.5 Compounds were formulated in dimethylsulfoxide (DMSO) to prepare a
Please cite this article in press as: Zhang, Y.-K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.10.044