Design and synthesis of boron-containing PDE4 inhibitors using soft-drug strategy for potential dermatologic anti-inflammatory application

Article abstract of DOI:10.1016/j.bmcl.2010.02.010

PDE4 inhibitors are a validated approach as anti-inflammatory agents but are limited by systemic side effects including emesis. We report a soft-drug strategy incorporating a carboxylic ester group into boron-containing PDE4 inhibitors leading to the discovery of a series of benzoxaborole compounds with good potency (for example IC₅₀ = 47 nM of compound 2) and low emetic activity. These compounds are intended for dermatological use further limiting possible systemic side effects.

Full text of DOI:10.1016/j.bmcl.2010.02.010

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2270–2274
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of boron-containing PDE4 inhibitors using soft-drug
strategy for potential dermatologic anti-inflammatory application
a
a
a
a
Yong-Kang Zhang a, , Jacob J. Plattner , Tsutomu Akama , Stephen J. Baker , Vincent S. Hernandez ,
*
Virginia Sanders a, Yvonne Freund a, Richard Kimura a, Wei Bu a, Karin M. Hold a, Xiao-Song Lu b
a Anacor Pharmaceuticals, Inc., 1020 E. Meadow Circle, Palo Alto, CA 94303, USA
b NAEJA Pharmaceutical Inc., 4290-91A Street, 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:
PDE4 inhibitors are a validated approach as anti-inflammatory agents but are limited by systemic side
effects including emesis. We report a soft-drug strategy incorporating a carboxylic ester group into
boron-containing PDE4 inhibitors leading to the discovery of a series of benzoxaborole compounds with
good potency (for example IC50 = 47 nM of compound 2) and low emetic activity. These compounds are
intended for dermatological use further limiting possible systemic side effects.
Received 21 December 2009
Revised 30 January 2010
Accepted 2 February 2010
Available online 6 February 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Benzoxaborole
Phosphodiesterase
Soft-drug
Dermatology
Anti-inflammatory
Cyclic nucleotide phosphodiesterase (PDE) enzymes have been
drug targets in many diseases and several non-specific PDE inhib-
itors, such as theophylline and doxofylline (see Fig. 1), have been
approved to treat patients.1
O
O
O
H
N
O
H3C
O
N
H3C
O
N
N
N
N
N
CH3
N
CH3
Doxofylline
The phosphodiesterase 4 (PDE4) is one of the eleven families of
PDEs and, during the last 30 years, PDE4 inhibitors have been a sig-
nificant focus of research and development by numerous organiza-
tions in the quest to discover therapeutic agents for the treatment
of inflammation-associated diseases such as asthma and chronic
obstructive pulmonary disease (COPD).2 More than 30 new chem-
ical entities (NCEs) of PDE4 inhibitors have progressed to various
clinical stages, but only a small portion of these NCEs are currently
in active development at advanced clinical phases.2a–c The chal-
lenges for developing PDE4 inhibitors are multi-factorial including
lack of efficacy in many cases and, more often, safety-related is-
sues. Vomiting, nausea, arteritis and immunosuppression are
among the serious adverse events of PDE4 inhibitors.2d–f The re-
ported side effects take place in the brain, gastrointestinal tract,
arteries or whole body due to systemic exposure of PDE4
inhibitors.
Theophylline
Figure 1. Theophylline has been launched as oral sustained release formulation for
treating asthma and respiratory disease (once daily or twice daily). Doxofylline has
been launched in Italy for treating asthma in expectation of having fewer side
effects than its close analog, theophylline.
ment for potential topical treatment of psoriasis and atopic
dermatitis. For a topically used drug, the possible systemic side ef-
fects are expected to be relatively low as compared to for the sys-
temic usage. In an ideal case, a topically applied drug exerts its
therapeutic action in the target area of the skin and then converts
into inactive and non-toxic metabolites if any drug penetrates the
skin and reaches systemic circulation. This so-called soft-drug
strategy4 may further improve the therapeutic index of boron-con-
Topical therapeutic application of boron-containing NCEs has
been one of our focuses3 and recently two boron-containing
PDE4 inhibitors (AN2728 & AN2898; see Fig. 2)3d–f have been iden-
tified as anti-inflammatory agents undergoing clinical develop-
OH
NC
X
B
AN2728 (X = H)
AN2898 (X = CN)
O
O
* Corresponding author. Tel.: +1 650 543 7513; fax: +1 650 543 7660.
E-mail address: yzhang@anacor.com (Y.-K. Zhang).
Figure 2. Chemical structures of AN2728 and AN2898 that are in clinical
development.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.010

Y.-K. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2270–2274
2271
Table 1
O
OH
OH
PDE4 IC50 results for benzoxaborole compounds 1, 2, 7–16 and AN2728
NC
B
B
EtO
O
O
OH
1
7
N
O
N
O
Y
B
6
O
2
1
2
X
O
5
3
4
Figure 3. A boron-containing PDE4 inhibitor 1 was used as a starting point of a soft-
drug approach for the discovery of more potent carboxylic ester compound 2 with
fewer side effects.
Compds
X
Y
PDE4 IC50 (lM)
1
N
CN
0.18
2
7
8
N
N
N
COOEt
COOH
COOMe
0.047
5% inhib@10uM
0.084
X
Br
Br
X
a
9
10
11
AN2728
12
13
14
15
16
N
N
N
CH
CH
CH
CH
CH
CH
COOPr-n
COOPr-i
COOBu-n
CN
COOH
COOMe
COOEt
0.13
0.113
0.0945
0.49
6.42
0.287
0.219
0.763
0.13
+
N
O
CHO
HO
CHO
N
Cl
5a: X=CN; 5b: X=COOEt
3a: X=CN
4
3b: X=COOEt
b
O
OH
c
X
B
X
B
O
O
COOPr-n
COOPr-i
N
O
CHO
N
O
1: X=CN; 2: X=COOEt
6a: X=CN; 6b: X=COOEt
d
OH
OH
ROOC
e
B
HOOC
The acid compound 12 was conveniently converted to the corre-
sponding esters under normal esterification condition.5
B
O
O
N
O
N
O
7
Since the goal of the soft-drug approach is to identify a potent
boron-containing PDE4 inhibitor with an ester group that may con-
vert to the corresponding carboxylic acid with much less activity
against PDE4, screening against PDE4 of compounds 1, 2, 7–16
and AN2728 have been performed using the previously described
procedure3f and the results are summarized in Table 1.
8: R=Me; 9: R= n-Pr;
10: 11:
R= i-Pr;
R= n-Bu
Scheme 1. Reagents and conditions: (a) K2CO3, DMF, 65–80 °C overnight; (b)
bis(pinacolato)diboron, PdCl2(dppf)2, 1,4-dioxane, N2, 80 °C 14 h; (c) NaBH4, EtOH,
and then 6 N HCl, H2O, EtOH (overall yield 60% for 2 from 1st step; (d) NaOH, H2O,
MeOH, rt overnight, and then HCl, H2O, yield 95%; (e) for 8 (R = Me), H2SO4, MeOH,
reflux overnight, N2, yield 15%; for 9–11 (R = n-Pr, i-Pr and n-Bu), CDI, DCM/THF/
DMF (1:1:1, v/v/v), N2, rt overnight, and then ROH and catalytic amount NaH, reflux,
2–4 h, yield 61%, 72% and 69%, respectively.
As a starting point, 5-(5-cyano-2-pyridyloxy) benzoxaborole (1)
has a reasonable potency of IC50 = 0.18 lM against PDE4. After the
cyano group is replaced with carboxylic esters, improvement of
potencies was observed with IC50 in the range of 47–130 nM. The
ester alkyl group has an impact on the potency and the sequence
of the potency is Et (2) > Me (8) ꢀ n-Bu (11) > i-Pr (10) ꢀ n-Pr (9),
which is not correlated to the lipophilicity. Further modification
of the functional group to COOH (7) resulted in the loss of activity
taining PDE4 inhibitors. This article describes our soft-drug ap-
proach starting from compound 1 (see Fig. 3) to the discovery of
a series of benzoxaborole carboxylic ester compounds, such as
compound 2, with good potency against PDE4.
(5% inhibition at 10 lM) against PDE4. For the 5-phenoxybenzox-
The synthetic methodologies for the preparation of
cyanopyridyloxybenzoxaborole (1), the ester compounds (2, 8–11)
and their corresponding carboxylic acid (7) are shown in
Scheme 1.5 Nucleophilic substitution of the chloro atom in 3a and
3b by the phenolic group in 2-bromo-5-hydroxybenzaldehyde 4 in
the presence of a base provided the coupling products 5a and 5b
which were catalytically boronylated to replace the bromo with
bis(pinacolato)diboron generating intermediates 6a and 6b. Reduc-
tion of compounds 6a and 6b with sodium borohydride converted
the aldehyde moiety into hydroxymethyl group that was simulta-
neously cyclized to the adjacent borate and subsequently hydro-
aborole series, very similar activity pattern was observed, and
the ester compounds (13–16) are generally more potent than the
cyano (AN2728) and the acid (12). Again, the acid (12) is the least
potent among this series. For cross comparison between these two
pyridyloxy and phenoxy series, the pyridyloxybenzoxaboroles are
more potent except the carboxylic compound (7). Since the most
potent ester (2) and least potent acid (7) fit the soft-drug strategy
well, more anti-inflammatory screenings for selected cytokine re-
lease inhibitions from THP-1 cells3f were conducted and the data
are shown in Table 2.
IC50s of the acid 7 against release of TNF-
IL-10 are greater than 10 M indicating this acid compound is inac-
tive of inhibiting cytokine release whereas the ester 2 has good
potencies of IC50s in the range between 0.10 and 0.46 M. These
a, IL-2, IFN-c, IL-5 and
lyzed to benzoxaborole
1 and 2 upon addition of aqueous
l
hydrochloride. Hydrolysis of compound 2 afforded acid compound
7 thatwas used for the preparation of other esteranalogues 8–11 un-
der normal esterification conditions.5
For the SAR study, non-pyridyl phenoxybenzoxaborole acid (12)
and esters (13–16) were also synthesized as shown in Scheme 2.
l
activity differences, in addition to the two compound’s PDE4 po-
tency difference, further support the soft-drug approach of using
active ester compound that may hydrolyze in the systemic circula-
tion into the acid. Therefore, plasma stabilities of ester compounds
(2, 8–11 and 13–16) and their conversion into the corresponding
OH
OH
HOOC
ROOC
B
a
B
Table 2
O
O
Cytokine inhibition results for compounds 2 (ester) and 7 (acid)
O
O
a
a
a
a
a
Compds TNF-
a
IC50
IL-2 IC50
M)
IFN-
c
IC50
IL-5 IC50
M)
IL-10 IC50
M)
13:
14:
R = Me;
R = Et;
12
(
lM)
(
l
(lM)
(
l
(l
15: R = n-Pr;16: R = i-Pr.
Ester 2
Acid 7
0.20
>10
0.10
>10
0.10
>10
0.20
>10
0.46
>10
Scheme 2. Reagents and conditions: (a) for 13 and 14 (R = Me and Et), H2SO4,
MeOH or EtOH, reflux overnight, N2; for 15 and 16 (R = n-Pr and i-Pr), H2SO4, n-
PrOH or i-PrOH, 100 °C two days, N2.
Values are means of triplicate.3f
a

2272
Y.-K. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2270–2274
Table 3
100
Stability and conversion results of compounds 2, 8–11 and 13–16 in mouse plasma
Ester 2
Acid 7
and PBS buffera
10
1
Compds
Ester group
% Change
of ester in
PBS
% Formed
of acid in
PBS
% Change
of ester in
plasma
% Formed
of acid in
plasma
2
8
9
10
11
13
14
15
16
COOEt
À2.4
À9.8
À13.6
2.4
À8.9
À8.9
À3.2
À8.8
À10.6
ND (7)
0.4 (7)
0.5 (7)
1.4 (7)
0.4 (7)
ND (12)
ND (12)
ND (12)
ND (12)
À72.5
À42.4
À92.5
À60.5
À98.0
À43.6
À76.2
À94.9
À58.9
ND (7)
0.1
0.01
COOMe
COOPrn
COOPri
COOBun
COOMe
COOEt
31.6 (7)
79.8 (7)
36.4 (7)
76.6 (7)
29.4 (12)
53.6 (12)
58.8 (12)
27.6 (12)
0
1
2
3
4
5
6
Time (h)
COOPrn
COOPri
Figure 5. Ester 2 and acid 7 plasma concentrations after oral dose of ester 2 in mice
(CD-1, n = 3) at 100 mg/kg single dosage (1%CMC).
a
Values are means of triplicate after 1 h incubation in mouse plasma (CD-1,
female, K2EDTA) or PBS buffer (pH 7.4) at 37 °C. ND = not determined.
100
Ester 2
Acid 7
10
acid compounds (7 and 12) are very critical. LC–MS analysis of es-
ter samples in plasma and PBS buffer gave the results6 as shown in
Table 3.
1
0.1
In general, the percentage loss of the esters and their conver-
sions into the corresponding acids in PBS buffer are very minor
as indicated by the data in Table 3. In plasma, high percentages
of the esters (42–98%) disappeared were mainly converted to their
corresponding acids. Consistently in both of pyridyl and phenyl
series, the stability order in mouse plasma is COOMe > COOPri >
COOEt > COOPrn ꢀ COOBun, which might be the combining effects
of various esterase activities and susceptibility for hydrolysis, and
steric hindrance of ester compounds. It can be concluded that the
esters studied were hydrolyzed to the corresponding acid as a ma-
jor product in mouse plasma at the physiological condition.
Furthermore, pharmacokinetic properties of ester compound 2
were investigated and the acid compound 7 was monitored simul-
taneously.7 The results are summarized in Figs. 4–6 and Table 4.
Following subcutaneous dose of ester compound 2 in mice at
100 mg/kg single dosage, analysis of the plasma samples indicates
that the ester rapidly converts to the corresponding acid which in-
creases to its maximum concentration at 2 h (Fig. 4).
After oral administration, the ester converts rapidly to the acid
with maximum concentration at 15 min ( Fig. 5) indicating an
instantaneous hydrolysis during the absorption and distribution
of the ester to the blood. A very similar result was observed when
the ester compound was dosed intravenously as shown in Figure 6.
The similar declined rates of ester and acid indicates the conver-
sion rate of ester to acid is the rate limiting step. As presented in
Table 4, the ester has a very short t1/2’s and tmax’s (0.25–1.03 h) that
fit the criteria for soft-drug profile. The subcutaneous bioavailabil-
ity is high (F% = 97.8%) in comparison to the oral bioavailability
(F% = 10.8%).
0.01
0
1
2
3
4
5
6
Time (h)
Figure 6. Ester 2 and acid 7 plasma concentrations after intravenous (iv) dose of
ester 2 in mice (CD-1, n = 3) at 25 mg/kg single dosage (MO-1).
Table 4
Pharmacokinetic parameters of ester 2 in mice plasma
b
Dosing
t1/2 (h)
tmax (h)
Cmax
g/mL)
F%
AUClast
(h g/mL)
AUCinf pred
(h g/mL)
a
method
(
l
l
l
sc
po
iv
NA
NA
1.03
0.50
0.25
NA
12.7
4.39
12.5
97.8
10.8
100
14.5
1.66
NA
15.2
1.68
3.88
a
Mice (CD-1, not fasted) were dosed at 100 mg/kg single dosage for sc and po,
and 25 mg/kg single dosage for iv (n = 3 per group).
b
Calculated with non-compartment model and iv bioavailability (F%) is consid-
ered to be 100%. NA = not available.
Since esterase is present in the skin, the ester compound 2 was
also tested in phorbol ester-induced mouse ear edema model3f to
investigate its in vivo efficacy by skin penetration under dermato-
logic treatment. As summarized in Table 5, compound 2 showed
significant inhibition against the ear edema caused by phorbol es-
ter after dosing at 1 mg/ear  2 suggesting that this compound has
good skin bioavailability, skin stability and in vivo anti-inflamma-
tory activity. Rolipram exhibited similar activity only at a higher
dosage in this model.
The ester compound 2 in vitro plasma stability and in vivo phar-
macokinetic results above indicate its rapid conversion to the
corresponding PDE4-inactive acid, and presumably this might lead
to less systemic toxicity of the compound. Because there is a
100
Ester 2
Acid 7
10
1
Table 5
Inhibitory activity of 2 against phorbol ester-induced mouse ear edema
Compound
Dose (mg/ear)
Inhibition (%)
0.1
0
1
2
3
4
5
6
2
1
1
1
3
59
68
6
Time (h)
Dexamethasone
Rolipram
Figure 4. Ester 2 and acid 7 plasma concentrations after subcutaneous (sc) dose of
ester 2 in mice (CD-1, n = 3) at 100 mg/kg dose (MO-1).
53

Y.-K. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2270–2274
2273
Table 6
A.; Benkovic, S. J.; Cusack, S.; Alley, M. R. K. Science 2007, 316, 1759; (c) Baker, S.
J.; Akama, T.; Zhang, Y.-K.; Sauro, V.; Pandit, C.; Singh, R.; Kully, M.; Khan, J.;
Plattner, J. J.; Benkovic, S. J.; Lee, V.; Maples, K. R. Bioorg. Med. Chem. Lett. 2006,
16, 5963; (d) Akama, T., Baker, S. J., Zhang, Y.-K., Zhou, H., Hernandez, V.,
Sanders, V., Plattner, J. J. Abstracts, 65th Annual Meeting of the American
Academy of Dermatology, Feb 2–6, 2007; Washington, DC, USA; (e) Akama, T.,
Freund, Y., Kimura, R., Baker, S. J., Zhang, Y.-K., Hernandez, V., Zhou, H., Sanders,
V., Maples, K. R., Plattner, J. J. Abstracts, International Investigative Dermatology
2008 Conference, May 14–17, 2008, Kyoto, Japan.; (f) 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.
Emesis test results of ester 2 in Suncus murinus8
Compd
po dose
(mg/kg)
N a Number of Onset of
Last of
Vomitingb
vomiting
episodes
vomiting vomitingb ( )
at (min)b (min)
Rolipram 10
1
2
3
1
2
3
4
1
2
3
4
8
11
19
0
0
0
0
1
1
0
15
3
2
NV
NV
NV
NV
37
4
11
39
7
+
+
+
2
2
10 or 30
NV
NV
NV
NV
NV
NV
NV
NV
NV
NV
NV
NV
+
+
NV
NV
4. (a) Bodor, N. Trends Pharmacol. Sci. 1982, 3, 53; (b) Bodor, N. Adv. Drug Res. 1984,
13, 255.
5. Synthesis of 6-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yloxy)nicotinonitrile
(1): This compound was prepared with 3a as starting material using the same
procedures described below for compound 2.
100
Synthesis of ethyl 6-(1-hydroxy-1,3-dihydrobenzo[c][1,2] oxaborol-5-yloxy)nico-
tinate (2): To a mixture of ethyl 6-chloronicotinate (3b, 18.6 g, 0.1 mol) and 2-
bromo-5-hydroxy benzaldehyde (4, 20.1 g, 0.1 mol) in dry DMF (200 mL) was
added K2CO3 (20.8 g, 1.5 equiv) under nitrogen atmosphere and the mixture was
stirred at 65–80 °C for 30.5 h. After being cooled to room temperature, the
mixture was filtered, evaporated and pumped overnight to give brown oil
(38.26 g) with 81.5% coupling conversion to ethyl 6-(4-bromo-3-formyl-
phenoxy)nicotinate (5b) as indicated by 1H NMR. 1H NMR (300 MHz, DMSO-
d6): d 10.17 (s, 1H), 8.66–8.65 (m, 1H), 8.33 (dd, J = 8.7&2.4 Hz, 1H), 7.86 (d,
J = 8.7 Hz, 1H), 7.60 (d, J = 2.7 Hz, 1H), 7.50 (dd, J = 8.4&2.7 Hz, 1H), 7.23 (dd,
J = 8.7& 0.6 Hz, 1H), 4.30 (q, J = 7.2 Hz, 2H) and 1.29 (t, J = 7.2 Hz, 3H) ppm. To
the solution of the resulting oil intermediate (5b) in 1,4-dioxane (450 mL) was
added bis(pinacolato)diboron (30.5 g, 0.12 mol), KOAc (29.5 g, 0.3 mol) and
PdCl2(dppf)2 (1.95 g, 2.5 mol %). The mixture was degassed with N2 and heated
at 80 °C for 14 h with stirring. The resulting dark mixture was filtered and
evaporated. The residue was dissolved in minimum volume of EtOAc, passed
through a short silica gel column eluted with hexane/EtOAc (2:1) to remove the
dark color giving brown oil (46.4 g) mainly containing ethyl 6-(3-formyl-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)nicotinate (6b). 1H NMR
(300 MHz, DMSO-d6): d 10.39 (s, 1H), 8.67–8.65 (m, 1H), 8.35–8.31 (m, 1H), 7.83
(d, J = 8.1 Hz, 1H), 7.64 (d, J = 2.7 Hz, 1H), 7.50 (dd, J = 7.8 & 2.7 Hz, 1H), 7.24 (d,
J = 8.1 Hz, 1H), 4.30 (q, J = 7.2 Hz, 2H), 1.34 (s, 12H) and 1.29 (t, J = 7.2 Hz, 3H)
ppm. To the solution of the pinacolboron aldehyde (6b, 46.4 g) in EtOH (450 mL,
200 proof) at 0 °C was added NaBH4 (5 g) in portions and the mixture was
stirred overnight with slow increasing to room temperature. The mixture was
cooled with ice bath again and water (50 mL) was added and followed with slow
addition of 6 N HCl (50 mL). After being stirred for 30 min, the mixture was
evaporated to remove EtOH and then water (200 mL) was added, neutralized
with NaHCO3-saturated water. The mixture was extracted with EtOAc,
concentrated and loaded to a short and big silica gel column eluted with
hexane/EtOAc (2:1, v/v) to remove dark impurity. The oil obtained contained
pinacol impurity that also complicates 1H NMR spectrum. The oil was dissolved
in minimum acetone, and then water was added slowly with sonication at same
time to participate the solid product. The solid was collected by filtration and
washed with pentane and hexane, dried overnight under high vacuum to give
ethyl 6-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yloxy)nicotinate (2) as
a cream solid (17.84 g) in 60% overall yield (three steps). Mp 110–113 °C. 1H
NMR (300 MHz, DMSO-d6): d 9.21 (s, 1H), 8.68 (d, J = 2.4 Hz, 1H), 8.30 (dd, J = 8.4
& 2.1 Hz, 1H), 7.76 (d, J = 8.1 Hz, 1H), 7.21 (d, J = 1.5 Hz, 1H), 7.15 (d, J = 8.7 Hz,
1H), 7.12 (dd, J = 7.8 & 2.1 Hz, 1H), 4.97 (s, 2H), 4.30 (q, J = 7.5 Hz, 2H) and 1.29
(t, J = 7.5 Hz, 3H) ppm. Purity (HPLC): 95.3% at 220 nm and 95.4% at 254 nm. MS:
m/z = 300 (M+1, ESI+) and m/z = 298 (MÀ1, ESIÀ).
NV
NV
0
a
Body weights of Suncus murinus are between 50 and 70 g. Experimental pro-
cedure is given in the reference section.8
b
The symbol of ‘+’ stands for positive result with vomiting and ‘NV’ for no
vomiting.
possibility that variable amount of a topically applied PDE4 inhib-
itor, depending on the dosage, could enter the systemic circulation,
the ester compound 2 was tested in an emesis model and com-
pared to rolipram8 to verify that this soft-drug approach could re-
sult in a potent PDE4 compound with less systemic side effect. As
shown in Table 6, all of the animals in the group dosed with roli-
pram at 10 mg/kg (po, 3 animals) had vomiting episodes (38-time
episodes with 57 min in total). In comparison, no vomiting was ob-
served for the two groups dosed with ester compound 2 at 10 mg/
kg (po, 4 animals) and at 30 mg/kg (po, 4 animals). Half of the ani-
mals (2/4 animals) in the group with 100 mg/kg oral dosage had a
single vomiting episode less than a minute during the 90 min
observation time. The results demonstrated that ester compound
2 has less emetic activity than rolipram, and the low emesis is pos-
sibly due to ester’s conversion to PDE4-inactive acid, which is con-
sistent with the soft-drug approach.
In summary, the soft-drug strategy has been used for designing
boron-containing chemical entities for topical anti-inflammatory
application. Two structural series, 5-(substituted-pyridyloxy)ben-
zoxaborole and 5-(substituted-phenoxy)benzoxaborole, have been
synthesized. In general, esters show good potency against PDE4
while the acid is inactive. One of the ester compounds, 5-(5-eth-
oxycarbonyl-2-pyridyloxy) benzoxaborole (2), has broad anti-
inflammatory activities and converts rapidly to the corresponding
inactive acid (7) in vitro in plasma and in vivo in mice. This ester
compound has demonstrated in vivo anti-inflammatory activity
and improved safety properties with less emetic activity as com-
pared to rolipram. More studies on the benzoxaborole compounds
are in progress for their potential topical application.
Synthesis of 6-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yloxy)nicotinic acid
(7): Ethyl 6-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yloxy)nicotinate
(2, 2.99 g, 10 mmol) was dissolved in freshly opened THF (100 mL), and 1 N
NaOH (38 mL) was added. The mixture was stirred at room temperature under
N2 overnight. Then 6 N HCl (6.5 mL) was added, rotary evaporated to remove
THF, filtered and washed with water and hexane. The solid was dried overnight
under high vacuum to afford the bis-acid compound 7 (2.57 g, 9.48 mmol, yield
94.8%) as a slightly brown solid. Mp >200 °C. 1H NMR (300 MHz, DMSO-d6): d
13.21 (s, 1H), 9.21 (s, 1H), 8.65 (d, J = 2.1 Hz, 1H), 8.28 (dd, J = 8.4 & 2.4 Hz, 1H),
7.76 (d, J = 7.8 Hz, 1H), 7.21 (d, J = 1.5 Hz, 1H), 7.14–7.11 (m, 2H) and 4.97 (s, 2H)
ppm. Purity (HPLC): 97.2% at 220 nm and 97.8% at 254 nm. MS: m/z = 272 (M+1,
ESI+) and m/z = 270 (MÀ1, ESIÀ).
References and notes
1. (a) Currie, G. P.; Butler, C. A.; Anderson, W. J.; Skinner, C. Br. J. Clin. Pharmacol.
2008, 65, 803; (b) Marx, D.; Tassabehji, M.; Heer, S.; Huettenbrink, K.-B.;
Szelenyi, I. Pulm. Pharmacol. Ther. 2002, 15, 7; (c) Boot, J. D.; De Haas, S. L.; Van
Gerven, J. M. A.; De Smet, M.; Leathem, T.; Wagner, J.; Denker, A.; Miller, D.; Van
Doorn, M. B. A.; Schoemaker, R. C.; Cohen, A. F.; Diamant, Z. Pulm. Pharmacol.
Ther. 2008, 21, 573; (d) Dini, F. L.; Cogo, R. Curr. Med. Res. Opin. 2001, 16, 258; (e)
Rxlist.com.
2. (a) www.iddb3.com.; (b) Fitzgerald, M. F.; Fox, J. C. Drug Discovery Today 2007,
12, 479; (c) McKenna, J. M.; Muller, G. M. In Cyclic Nucleotide Phosphodiesterases
in Health and Disease; Beavo, J., Francis, S. H., Houslay, M. D., Eds.; CRC/Taylor &
Francis: Boca Raton, 2007; pp 667–699; (d) Giembycz, M. A. In Cyclic Nucleotide
Phosphodiesterases in Health and Disease; Beavo, J., Francis, S. H., Houslay, M. D.,
Eds.; CRC/Taylor & Francis: Boca Raton, 2007; pp 649–665; (e) Giembycz, M. A.
Br. J. Pharmacol. 2008, 155, 288; (f) Spina, D. Br. J. Pharmacol. 2008, 155, 308.
3. (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)
Fernando, R.; Mao, W.; Yaremchuk, A.; Tukalo, M.; Crépin, T.; Zhou, H.; Zhang,
Y.-K.; Hernandez, V.; Akama, T.; Baker, S. J.; Plattner, J. J.; Shapiro, L.; Martinis, S.
Synthesis of methyl 6-(1-hydroxy-1,3-dihydrobenzo[c][1,2] oxaborol-5-yloxy)nicotinate
(8): The mixture of acid 7 (0.8 g, 2.95 mmol) and 96%H2SO4 (1 g) in MeOH (130 mL)
was refluxed overnight under N2. Normal work-up and flash column
chromatography over silica gel eluted with hexane/EtOAc (1:1, v/v) provided the
title methyl ester compound 8 as a white solid (0.127 g, yield 15%). Mp 156–158 °C.
1H NMR (300 MHz, DMSO-d6): d 9.22 (s, 1H), 8.68 (dd, J = 2.4 & 0.6 Hz, 1H), 8.31 (dd,
J = 8.7 & 2.4 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.22 (d, J = 1.2 Hz, 1H), 7.17–7.11 (m, 2H),
4.97(s,2H)and3.84(s,3H)ppm. Purity(HPLC):98.0%at220 nmand100%at254 nm.
MS: m/z = 286 (M+1, ESI+) and m/z = 284 (MÀ1, ESIÀ).
Synthesis of n-propyl 6-(1-hydroxy-1,3-dihydrobenzo[c][1,2] oxaborol-5-yloxy)nicotinate
(9): The mixture of acid 7 (0.5 g, 1.84 mmol) and a coupling agent CDI (0.66 g,
4.06 mmol, 2.2 equiv) in a mixed solvent of CH2Cl2 (50 mL), THF (30 mL) and DMF
(40 mL) was stirred at rt overnight under N2. Then anhydrous n-PrOH (30 mL) was
injected into the mixture, and catalytic amount of NaH (60%, 10 mg) was added. The
mixture was refluxed under N2 for 2 h and then evaporated. Theresidue was dissolved
in EtOAc, washed with 0.5 N HCl, then with NaHCO3 solution (pH 8), dried and

2274
Y.-K. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2270–2274
evaporated. The sticky solid was dissolved in minimum acetone followed by addition
6. Benzoxaborole stabilities in mouse plasma and buffer were tested by using the
following procedure. Four 96-well plates were divided into two groups for
handling compound solutions in mouse plasma (CD-1, Female, K2EDTA,
purchased from Bioreclamation) and in phosphate buffer saline (PBS, pH 7.4,
purchased from Sigma). To two plates (one for time 0 and the other for time
of hexane with sonication and cooling to generate the title n-propyl ester product 9 as
an off-white solid (0.354 g, 1.13 mmol, yield 61.3%). Mp 89–94 °C. 1H NMR (300 MHz,
DMSO-d6): d 9.22 (s, 1H), 8.69 (d, J = 2.4 Hz, 1H), 8.31 (dd, J = 8.7 & 2.7 Hz, 1H), 7.76 (d,
J = 7.8 Hz, 1H), 7.22 (s, 1H), 7.17–7.12 (m, 2H), 4.97 (s, 2H), 4.22 (t, J = 6.3 Hz, 2H), 1.70
(sextet, J = 6.9 Hz, 2H) and 0.94 (t, J = 7.2 Hz, 3H) ppm. Purity (HPLC): 98.3% at 220 nm
and 98.3% at 254 nm. MS: m/z = 314 (M+1, ESI+) and m/z = 312 (MÀ1, ESIÀ).
Synthesis of isopropyl 6-(1-hydroxy-1,3-dihydrobenzo[c][1,2] oxaborol-5-yloxy)nico-
tinate (10): This compound was prepared by adapting the procedure described above
for 9 with increase of refluxing time to 4 h. Yield 71.8%. Mp 95–101 °C. 1H NMR
(300 MHz, DMSO-d6): d 9.21 (s, 1H), 8.66 (d, J = 2.4 Hz, 1H), 8.29 (dd, J = 8.7 & 2.1 Hz,
1H), 7.76 (d, J = 7.8 Hz, 1H), 7.21 (d, J = 1.8 Hz, 1H), 7.16–7.11 (m, 2H), 5.12 (septet,
J = 6.0 Hz, 1H), 4.97 (s, 2H) and 1.30 (d, J = 6.3 Hz, 6H) ppm. Purity (HPLC): 98.2% at
220 nm and 96.6% at 254 nm. MS: m/z = 314 (M+1, ESI+) and m/z = 312 (MÀ1, ESIÀ).
Synthesis of n-butyl 6-(1-hydroxy-1,3-dihydrobenzo[c][1,2] oxaborol-5-yloxy)nico-
tinate (11): This compound was prepared by using the procedure described above
for 10. Yield 68.5%. Mp 75–80 °C. 1H NMR (300 MHz, DMSO-d6): d 9.22 (s, 1H), 8.68–
8.67 (m, 1H), 8.32–8.29 (dm, Jd = 8.7 Hz, 1H), 7.76 (d, J = 7.5 Hz, 1H), 7.22 (s, 1H), 7.17–
7.12 (m, 2H), 4.97 (s, 2H), 4.26 (t, J = 6.3 Hz, 2H), 1.66 (pentatet, J = 7.5 Hz, 2H), 1.39
(sextet, J = 7.5 Hz, 2H) and 0.90 (t, J = 7.5 Hz, 3H) ppm. Purity (HPLC): 100% at 220 nm
and 100% at 254 nm. MS: m/z = 328 (M+1, ESI+).
60 min), 90
PBS was added. Ten microliters of each compound PBS solution (50
added to the well resulting in 5 M final sample concentration. Time 60 min
plate was placed in water bath set at 37 °C with mild shaking and time 0 min
plate was left on ice and was quenched with 300 L of internal standard
working solutions (ISWS, 0.1 g/mL in acetonitrile or methanol depending on
the structures of compounds tested) on ice after the preparation. After
incubation for 1 h, the time 60 min plate was placed on ice and then 300 L of
l
L of plasma was added to each well. To other two plates, 90
lL of
l
M) was
l
l
l
l
ISWS was added to each well. Both plasma plates were vortexed and then
centrifuged at approximate 1800 rpm for 10 min. To a 96-well autosampler
plate, 200 lL of supernatant of all samples were transferred and proceed for LC/
MS/MS analysis (MS instrument API3000 assembled with Shimadzu LC-10AD
system). Each compound was incubated and processed in triplicate.
7. Pharmacokinetic parameters were calculated using a non-compartment model
for iv, po and sc in WinNonlin Pro, ver. 5.2. Following single iv dosing at 25 mg/
kg of the ester dissolved in MO-1 (1-methyl-2-pyrrolidone/propylene glycol/
Synthesis of 4-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yloxy)benzoic acid (12):
This compound was prepared by hydrolysis from AN2728 and the procedure was
reported previously.3f
PEG-400, 10:40:50, v/v/v), ester reached maximum plasma concentration (Cmax
of 12.5 g/mL at 5 min. The mean half-time of ester was 1.03 h and mean
clearance was 6.44 L/h/mL. After po and sc dosing of the ester suspended in 1%
carboxymethylcellulose (CMC) at 100 mg/kg/route, the mean Cmax of 4.39 g/mL
and 12.7 g/mL were achieved at 0.25 h for po and 0.25 h for sc, respectively.
The mean bioavailability was approximately 10.8% for po dosing and 97.8% for
sc dosing. The measurable acid was observed and its profiles were similar to its
corresponding ester in all dosing groups. The acid was not quantified and no
pharmacokinetics parameters were generated.
)
l
Synthesis of methyl 4-(1-hydroxy-1,3-dihydro-benzo[c][1,2]oxaborol-5-yloxy)ben-
zoate (13): To a solution of acid 12 (800 mg, 2.96 mmol) in methanol (50 mL) was
added 4 drops of conc. H2SO4. The resulting solution was refluxed overnight under N2.
Solvent was evaporated under vacuum. The residue was dissolved in EtOAc (30 mL)
and washed with saturated NaHCO3 (30 mL). Then the mixture was acidified to pH 3
with 1 N HCl. The organic layer was dried over MgSO4, filtered, and evaporated. The
crude product was purified by silica gel column chromatography eluted with 10%
EtOAc/hexane providing 630 mg of the methyl ester compound 13 in 75% yield. 1H
NMR 400 MHz (400 MHz, DMSO-d6): d 9.22 (s, 1H), 7.98 (d, J = 9.0 Hz, 2H), 7.78 (d,
J = 7.8 Hz, 1H), 7.30–7.10 (m, 4H), 4.97 (s, 2H) and 3.83 (s, 3H) ppm. Purity (HPLC):
96.2% at 220 nm and 96.2 % at 254 nm. MS: m/z = 285 (M+1, ESI+).
l
l
8. (a) Emetic activities of rolipram and ester 2 were tested with the procedure as
described below. Male animals (Suncus murinus, body weights 50–70 g,
purchased from CLEA Japan Inc.) were administered orally with rolipram at
10 mg/kg or ester compound
carboxymethylcellulose (1% CMC) at
2
at 10, 30 and 100 mg/kg in 1%
dosing volume of 10 mL/kg. Then
a
Synthesis of ethyl 4-(1-hydroxy-1,3-dihydro-benzo[c][1,2]oxaborol-5-yloxy)ben-
zoate (14): This compound was prepared in 39% yield by using the procedure above
for 13 with EtOH as the solvent. 1H NMR (400 MHz, DMSO-d6): d 9.21 (s, 1H), 7.98 (d,
J = 9.0 Hz, 2H), 7.78 (d, J = 8.2 Hz, 1H), 7.11–7.03 (m, 4H), 4.96 (s, 2H), 4.30 (q,
J = 7.4 Hz, 2H) and 1.31 (t, J = 7.0 Hz, 3H) ppm. Purity (HPLC): 97.7% at 220 nm and
99.5% at 254 nm. MS: m/z = 299 (M+1, ESI+).
Synthesis of n-propyl 4-(1-hydroxy-1,3-dihydro-benzo[c][1,2]oxaborol-5-yloxy)ben-
zoate (15): This compound was prepared using the procedure above for 13 with n-
PrOH as the solvent. 1H NMR (400 MHz, DMSO-d6): d 9.21 (s, 1H), 7.99 (d, J = 9.0 Hz,
2H), 7.78 (d, J = 7.8 Hz, 1H), 7.16–7.05 (m, 4H), 4.96 (s, 2H), 4.22 (t, J = 6.7 Hz, 2H),
1.76–1.65 (m, 2H) and 0.96 (t, J = 7.41 Hz, 3H) ppm. Purity (HPLC): 96.5% at 220 nm
and 97.6% at 254 nm. MS: m/z = 313 (M+1, ESI+).
Synthesis of isopropyl 4-(1-hydroxy-1,3-dihydro-benzo[c][1,2]oxaborol-5-yloxy)ben-
zoate (16): This compound was prepared using the procedure described above for 13
with i-PrOH as the solvent in 60% yield. 1H NMR (400 MHz, DMSO-d6): d 9.22 (s, 1H),
7.98 (d, 2H), 7.78 (d, 1H), 7.13–7.05 (m, 4H), 5.16–5.08 (m, 1H), 4.96 (s, 2H) and 1.33 (d,
6H). HPLC purity: 97.4% at 220 nm and 98.2% at 254 nm. MS: m/z = 313 (M+1, ESI+).
animals were observed for retching and emetic responses for 90 min post-
dosing. A positive emetic episode was defined as disgorgement of the stomach
contents generally preceded by retching and its occurrence in one or more of 3
or 4 test animals was considered significant. Additionally, the number, onset
and duration of emetic episodes were recorded; (b) Structure of rolipram [4-(3-
(cyclopentyloxy)-4-methoxyphenyl)pyrrolidin-2-one, C Log P = 1.715, molecu-
lar weight = 275.34]:
O
HN
O
O
Rolipram