Salicylanilides: Selective inhibitors of interleukin-12p40 production

Article abstract of DOI:10.1016/j.bmc.2008.07.024

Interleukin (IL)-12p40, a subunit component of both IL-12 and IL-23, is being widely studied for its role in inflammatory disease. As part of an effort to profile cellular signaling pathways across different cell types, we report salicylanilide inhibitors of IL-12p40 production in stimulated dendritic cells. Based on a hypothesis that a desirable therapeutic profile is one that could block IL-12p40 but not IL-6 production, we engaged in directed analoging. This resulted in salicylanilides with similar IL-12p40 related potency but enhanced selectivity relative to IL-6 production.

Full text of DOI:10.1016/j.bmc.2008.07.024

Bioorganic & Medicinal Chemistry 16 (2008) 8760–8764
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Salicylanilides: Selective inhibitors of interleukin-12p40 production
Michael E. Brown a, Jeffrey N. Fitzner a, Tracey Stevens b, Wilson Chin b, Clifford D. Wright b, Jim P. Boyce a,
*
a Medicinal Chemistry, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119, USA
b Inflammation, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Interleukin (IL)-12p40, a subunit component of both IL-12 and IL-23, is being widely studied for its role in
inflammatory disease. As part of an effort to profile cellular signaling pathways across different cell types,
we report salicylanilide inhibitors of IL-12p40 production in stimulated dendritic cells. Based on a
hypothesis that a desirable therapeutic profile is one that could block IL-12p40 but not IL-6 production,
we engaged in directed analoging. This resulted in salicylanilides with similar IL-12p40 related potency
but enhanced selectivity relative to IL-6 production.
Received 29 February 2008
Revised 30 June 2008
Accepted 14 July 2008
Available online 17 July 2008
Keywords:
Salicylanilide
Ó 2008 Published by Elsevier Ltd.
Interleukin-12p40 (IL-12p40)
Interleukin-12 (IL-12)
Interleukin-23 (IL-23)
Interleukin-6 (IL-6)
1. Introduction
Cl
NO2
O
OH
Br
O
OH
Cl
O
OH
O
1.1. Interleukin-12p40 biology
Cl
N
H
N
H
N
H
Cl
Interleukin (IL)-12p40 is a subunit component of both IL-12 and
IL-23. IL-12 is involved in type 1 helper T cell (Th1) mediated
inflammation in both normal immune defense as well as inflam-
matory diseases such as rheumatoid arthritis, asthma, psoriasis,
and Crohn’s disease.1–5 IL-23 has a divergent immunological role
in regulating T cells that produce other pro-inflammatory cyto-
kines. IL-12p40 may exist in monomeric or homodimeric (IL-
12p80) forms that have been recognized to possess their own
bio-activities, independent of IL-12 and IL-23, and which include
chemoattraction of macrophages and stimulated dendritic cells.6
1
2
3
Figure 1. Examples of bio-active salicylanilides.
2. Results and discussion
As a result of data analysis across an array of cell based as-
says, we found that salicylanilide 3 inhibited IL-12p40 and IL-6
production in stimulated primary human dendritic cells as well
as behaving similarly in a dendritic cell line assay (Table 1).13
Cytotoxicity profiling for compound 3 was assessed and did
not explain the IL-12p40 functional activity. With respect to
inhibition of IL-12p40 production, analog 4 indicated that the
amide carbonyl moiety of 3 is important for IL-12p40 (and IL-
6) activity.
Analog 5, with a nitro-group instead of the bromo-group fea-
tured in 3, resulted in a loss of activity toward IL-12p40 but lit-
tle change in IL-6 activity, relative to 3. A similar result was
observed with the isomeric nitro-salicylanilide 6. Compounds
7 and 8 demonstrate that other halogen substitution patterns,
on the anilide ring (i.e. ‘B’), are tolerated with respect to the
IL-12p40 assay but still little or no selectivity was observed rel-
ative to the IL-6 assay. In comparison with compound 3, analog
9, in which there is a hydrogen in place of the bromine on the
1.2. Bio-activity of salicylanilides
As a class, salicylanilides have been reported with a wide vari-
ety of interesting biological properties.7,8 For example, Efuamide,
1, has been reported to be immunosuppressive in experimental
allergic encephalomyelitis models in animals and to inhibit phos-
phorylation and signaling of erbB-2 resulting in cell cycle arrest.9,10
Niclosamide, 2, was originally discovered as a molluscicidal and
was subsequently developed as an orally administered, broad spec-
trum anthelmintic11,12 (see Fig. 1).
* Corresponding author. Tel.: +1 425 821 6971; fax: +1 206 217 5529.
E-mail addresses: boycej@amgen.com, jboyce2000@aol.com (J.P. Boyce).
0968-0896/$ - see front matter Ó 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.07.024

M. E. Brown et al. / Bioorg. Med. Chem. 16 (2008) 8760–8764
8761
Table 1
Table 3
Inhibition of IL-12p40 production in stimulated dendritic cells
Inhibition of IL-12p40 production in stimulated dendritic cells
Cl
OH
O
R7
R3
R4
Ar
HO
A
N
H
B
N
H
R6
R5
R2
Entry
9
Ar
IC50 (l
M)a
R1
IL-12p40
0.013
IL-6
>10
Entry
R1
R2
C(R3,R4)
R5
R6
R7
IC50
(l
M)a
IL-6
Cl
Cl
Cl
IL-12p40
3
4
5
6
7
8
Br
Br
NO2
H
Cl
Br
H
H
H
NO2
H
C@O
CH2
C@O
C@O
C@O
C@O
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
Cl
Cl
0.013
2.7
0.41
0.32
0.13
0.074
0.30
1.7
0.17
0.25
0.13
0.64
12
13
14
15
16
17
5.1
>10
>10
>10
>10
1.8
H
H
Cl
a
Data reported here are for a murine dendritic cell line stimulated with a mix-
0.045
>10
>10
0.20
>10
Cl
ture of CD40L, IFN
immunoassay.
c, and IL-18. IL-12p40 and IL-6 detection by fluorescence
Cl
OH
salicyl-ring (i.e. ring ‘A’), was found in our assays to behave
similarly to compound with respect to inhibition of IL-
N
3
12p40 production but to be much more selective against inhibi-
CF3
tion of IL-6 production (Table 2).14
We were interested in understanding the importance of the
phenol group and so analogs featuring other functional groups,
in place of the phenol, were prepared. We initially looked at O-al-
kyl ethers, such as the O-benzyl ether 10, but these generally
showed dramatic loss of inhibition of IL-12p40 production at
Cl
>10
CF3
CF3
our highest assay concentration (i.e. 10 lM). A promising excep-
tion to this, which is noted here and will be reported on more
fully elsewhere, is the related, non-salicylanilide compound 11
in which a sulfonamide group, with a pKa roughly close to that
of a phenol, was incorporated on the benzoyl ring.15 Halogen sub-
stitution on the anilide ring was generally favorable for IL-12p40
activity. The meta-chloro substituents of 9 are important for IL-
12p40 activity and appear to have an additive effect when com-
pared with compound 12 (Table 3). However, alternative halogen
substitution patterns, such as featured in analog 13 can result in
comparably active analogs. Hydrogen bond donor or acceptor
groups, such as appear in analogs 14 or 15, showed no inhibition
of IL-12p40 production.
18
0.12
0.74
a
See Table 1 comments for the IL-12p40 and IL-6 assays.
line ring of 9 are replaced with isosteric methyl groups, resulted in
complete loss of inhibitory activity in the IL-12p40 assay. The mid-
dling activity of analog 18 suggests an additional resonance (or
subtle steric) effect that favors chlorines over trifluoromethyl
groups at these meta-anilino positions (i.e. 9 vs 18).
Comparison of 9 with 17, in which the inductively electron-
withdrawing chlorine substituents at the meta position of the ani-
Table 4
Inhibition of IL-12p40 production in stimulated dendritic cells
CF3
O
Table 2
Inhibition of IL-12p40 production in stimulated dendritic cells
N
CF3
Cl
Cl
O
O
R1
O
21
Cl
Cl
N
H
Cl
OH
O
O
HO
N
Cl
R2
N
Cl
O
R1
Entry
R1
R2
IC50
IL-12p40
(l
M)a
22
IL-6
Entry
R1
IC50 (l
M)a
3
9
10
OH
OH
OBn
Br
H
H
0.013
0.013
>10
0.30
>10
>10
IL-12p40
IL-6
9
19
20
21
22
H
CH3
CH2CO2H
0.013
>10
>10
0.28
>10
>10
>10
>10
0.35
>10
O
S
O
NO2
Cl
11
H
0.24
0.22
N
H
a
a
See Table 1 comments for the IL-12p40 and IL-6 assays.
See Table 1 comments for the IL-12p40 and IL-6 assays.

8762
M. E. Brown et al. / Bioorg. Med. Chem. 16 (2008) 8760–8764
Lastly, simple substitution on the secondary amide nitrogen
O
of 9 is not tolerated as evidenced by analogs 19 and 20 (Table
4). Interestingly, the benzoxazinedione 21, which does not fea-
ture a secondary amide or an acidic phenol group but which
bares some intriguing resemblance to these salicylanilide inhibi-
tors, possessed some moderate IL-12p40 activity in our assay but
also inhibited IL-6 production. In contrast, the phthalimide 22,
which might also be considered to be a conformationally con-
strained analog of 9, lacked any inhibition of IL-12p40
production.
Broad counter-screening of 9 against a diverse collection of
recombinantly expressed proteins showed no significant inhibition
versus any of over 150 known and recombinantly expressed pro-
tein targets.16
a, b
OH
OH
6
O2N
2-hydroxy-4-nitro-benzoic acid
c, d, e
O
19
20
a, b
OH
10
f, g, h, e
OBn
2-benzyloxybenzoic acid
OH
O
i
O
22
O
3. Conclusions
3-hydroxyphthalic anhydride
We have reported that salicylanilides, such as 9, can potently
and selectively inhibit IL-12p40 production in dendritic cells. Fur-
ther elucidation of the mechanism of action, of the compounds de-
scribed here, will be reported.
Scheme 1. Example syntheses of salicylanilides. Reagents: (a) DCC, N-hydroxy-
succinimide, THF; (b) 3,5-dichloroaniline; (c) NaH, THF; (d) CH3I (e) Pd/C, H2; (f)
NaO-t-butyl, 1,2-dimethoxyethane; (g) tert-butyl bromoacetate; (h) TFA, CH2Cl2; (i)
3,5-dichloroaniline, AcOH, reflux.
4. Experimental methods
4.2. Experimental syntheses
4.1. General synthesis methods
4.2.1. N-(3,5-Dichlorophenyl)-2-hydroxy-4-nitrobenzamide (6)
A
solution of 2-hydroxy-4-nitro-benzoic acid (2.34 g,
12.8 mmol), N-hydroxysuccinimide (1.48 g, 12.8 mmol), and N,N0-
dicyclohexylcarbodiimide (2.68 g, 13.0 mmol) in THF (25 mL) was
stirred (rt, 3 h), and the resulting precipitate was filtered and the
filtrate concentrated in vacuo to afford a crude succinate ester
(2.97 g) which was used without further purification: MS (ꢁEI)
m/z 279 (Mꢁ, 100). A portion of this material (1.52 g, theor.
6.44 mmol) and 3,5-dichloroaniline (2.06 g, 12.7 mmol) was briefly
warmed (140 °C, internal, 10 min) as a melt, with stirring, then al-
lowed to cool (rt). After aqueous work-up (EtOAc/10% aq HCl), the
organic layer was dried (MgSO4), filtered, and concentrated in va-
cuo to afford a crude solid from which an analytical sample of 6
(130 mg, 0.397 mmol) was prepared by flash chromatography
(hexanes/ethyl acetate, 17:3). 1H NMR (500 MHz, DMSO-d6) d
11.71 (br s, 1H), 10.75 (br s, 1H), 7.89 (d, J = 10.0 Hz, 1H), 7.84 (s,
2H), 7.77 (d, J = 10.0 Hz, 2H), 7.37 (s, 1H); MS (ꢁEI) m/z 327
((Mꢁ, 70), 325 (Mꢁ, 100).
Unless noted otherwise, hydrogenations were performed
using a Parr 3911 hydrogenation apparatus and microwave irra-
diations were conducted with a CEM Explorer microwave appa-
ratus with auto-sampler. Chromatography-grade, anhydrous
solvents were used without further purification. Concentration
refers to distillation using a rotary evaporator (Buchi R-124).
Flash chromatography was performed on E. Merck #60, 230–
400 mesh silica gel. TLC analyses were performed using E.
Merck glass backed silica gel #60 plates, 0.25 mm thickness,
with 254 nm fluorescent indicator with visualization by UV
absorption, iodine chamber, or solutions of KMnO4 or other
common staining techniques. NMR spectra were collected with
a Varian Inova AS500 equipped with a Zymark auto-sampler
and spectral resonance shifts are reported after internal stan-
dardization to a characteristic resonance shift of the solvent.
Coupling constants refer to apparent peak multiplicities and
abbreviations used are s = singlet, d = doublet, t = triplet,
q = quartet, br = broad. HPLC analyses were performed on an
Agilent 1100 Series HPLC, equipped with a reverse phase col-
umn (Zorbax Rx-C8, 9.4 mm ID ꢀ 25 cm) and using an elution
gradient composed of increasing ratios of 0.1% v/v TFA in
CH3CN: 0.1% v/v TFA in H2O with monitoring of analytes by
UV absorption, evaporative light scattering (SEDEX ELSD) and
mass spec. detection (ESI/CI). Mass spectra were collected on
an Agilent 1100 MSD and are reported as percent intensity rel-
ative to the base peak. Except where noted otherwise, all re-
agents, including compound 14 [526-18-1], were purchased
from Sigma–Aldrich Chemical Co.
4.2.2. N-(3,5-Dichlorophenyl)-2-hydroxy-N-methylbenzamide
(19)
A solution of 2-benzyloxybenzoic acid (2.00 g, 8.76 mmol), N-
hydroxysuccinimide (1.21 g, 10.5 mmol) and N,N0-dicyclohexylcar-
bodiimide (1.99 g, 9.64 mmol) in anhydrous THF (30 mL) was stir-
red (rt, 4 h). The resulting precipitant was filtered and the filtrate
was concentrated in vacuo to afford N-hydroxysuccinyl 2-(benzyl-
oxy) benzoate as a crude intermediate that was used without fur-
ther purification (3.39 g): 1H NMR (500 MHz, DMSO-d6) d 7.93 (d,
J = 7.5, 1H), 7.73 (m, 1H), 7.50 (d, J = 7.5 Hz, 2H), 7.38 (m, 3H),
7.32 (d, J = 7.0 Hz, 1H), 7.16 (m, 1H), 5.30 (s, 2H), 2.89 (s, 4H);
13C NMR (125 MHz, DMSO-d6) d 170.4, 160.2, 158.8, 136.4, 136.4,
131.7, 128.4, 127.7, 127.0, 120.8, 114.5, 113.9, 69.8, 25.5. An ali-
quot of this crude succinate ester (1.00 g, 3.07 mmol) and 3,5-
dichloroaniline (1.50 g, 9.23 mmol) was briefly warmed as a melt
(10 min, 140 °C, internal). The resulting yellow solution was al-
lowed to cool (rt). The residue was diluted with EtOAc (100 mL)
and after aqueous washing (3 N HCl, brine), the organic layer was
dried (MgSO4) and concentrated in vacuo to afford a tan solid
which after crystallization (EtOAc) afforded 2-(benzyloxy)-N-(3,5-
dichlorophenyl)benzamide 10, CAS Registry [882158-06-7], as a
white crystalline solid (0.67 g, 1.80 mmol): 1H NMR (500 MHz,
Compounds 3 [439145-04-7], 4 [298215-62-0], 7 [642-84-2], 8
[254106-36-0], 9 [78154-58-2], 10 [882158-06-7 ], 13 [106480-
61-9], 15 [13563-04-7], 16 [5683-92-1], 17 [2819-59-2], and 21
[284664-73-9] were purchased from Maybridge-Thermo Fisher Sci-
entific Inc. Compound 11 [454691-62-4] was prepared according to
the general method of Thomas and Allanson and compound 12
[24448-71-3] was prepared according to the general method of
Liechti et al. and compounds 5 and 18 [744-58-1] were prepared
according to the general method of Muto et al.17–19 The following
are synthesis protocols for new compounds 6, 19, 20, and 22
(Scheme 1).

M. E. Brown et al. / Bioorg. Med. Chem. 16 (2008) 8760–8764
8763
CDCl3) d 10.09 (br s, 1H), 8.29 (d, J = 7.5 Hz, 1H), 7.54–7.52 (m, 6H),
7.15–7.12 (m, 2H), 7.08 (s, 2H), 7.97 (s, 1H), 5.19 (s, 2H); 13C NMR
(125 MHz, CDCl3) d 163.0, 156.7, 140.2, 134.9, 134.8, 133.8, 132.6,
129.8, 129.3, 129.0, 123.5, 122.0, 120.9, 117.6, 112.5, 72.0; MS (+EI)
m/z 374 (MH+, 70), 372 (MH+, 100). To a cold (0 °C bath) suspension
of 10 (100 mg, 0.269 mmol) and NaH (60% w/w oil disp., 16 mg,
theor. 0.4 mmol) in anhydrous THF (2 mL) was added neat methyl
iodide (0.2 mL, 3 mmol) and the resulting mixture was allowed to
warm (rt, 30 min) with stirring. The resulting mixture was care-
fully quenched with methanol (0.5 mL), filtered through a plug of
silica gel (hexanes/EtOAc, 5:1), and concentrated in vacuo to afford
4.2.4. 2-(3,5-Dichlorophenyl)-4-hydroxyisoindoline-1,3-dione (22)
solution of 3-hydroxyphthalic anhydride (701 mg,
A
4.27 mmol) and 3,5-dichloroaniline (692 mg, 4.27 mmol) in gla-
cial acetic acid (10 mL) was refluxed (2 h) and the resulting sus-
pension was allowed to cool (rt). The precipitant was collected,
washed (AcOH) and dried in vacuo to afford 22 as fine white
needles (1.17 g, 3.80 mmol): 1H NMR (500 MHz, DMSO-d6) d
11.20 (s, 1H), 7.70–7.59 (m, 2H), 7.58 (d, J = 4.0 Hz, 2H), 7.39
(t, J = 4.0 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) d 166.1, 164.9,
155.8, 136.4, 134.4, 133.8, 133.2, 127.4, 126.2, 123.5, 114.5;
MS (ꢁEI) m/z 308 (Mꢁ, 70), 306 (Mꢁ, 100).
2-(benzyloxy)-N-(3,5-dichlorophenyl)-N-methylbenzamide as
a
colorless oil (91 mg, 0.24 mmol) and which was used without fur-
4.3. General bio-assay methods
+
+
***
ther purification: MS (+EI)
m/z 388 (MH , 70), 386 (MH , 100). A
suspension of 2-(benzyloxy)-N-(3,5-dichlorophenyl)-N-methylb-
enzamide (72 mg, 0.19 mmol) and 5% Pd/C (wet Degussa-type
E101, 12 mg, theor. 0.0029 mmol Pd) in EtOAc (2 mL) was shaken
under a hydrogen atmosphere (60 psi, rt, 1 h). The resulting mix-
ture was filtered (diatomaceous earth) and the filtrate was concen-
trated in vacuo to afford a light orange oil which after flash
chromatography afforded 19 (44 mg, 0.15 mmol) as a colorless
oil: 1H NMR (500 MHz, CDCl3) d 7.25–7.24 (m, 2H), 7.03 (s, 2H),
6.97 (d, J = 8.5 Hz, 1H), 7.39 (d, J = 8.5 Hz, 1H), 6.54 (m, 1H), 3.46
(s, 3H); 13C NMR (125 MHz, CDCl3) d 171.5, 160.5, 147.0, 135.6,
133.5, 130.0, 127.2, 125.2, 125.2, 118.2, 115.2, 39.1; MS (+EI) m/z
298 (MH+, 70), 296 (MH+, 100).
All cell culture manipulations were performed in a biological
safety cabinet, unless otherwise indicated. Media and additives
were prepared and maintained under aseptic conditions with stor-
age in the refrigerator up to one month.
4.4. Fluorescence immunoassay protocol
The cytokine detection assays used a stimulatory mixture
consisting of muCD40L, muIL-18 and muIFNc to induce IL-
12p40 and IL-6 secretion by a murine dendritic cell line, ‘DC-
B’.20,21 DC-B cells were cultured in growth medium (i.e. recipe
follows below) with 60 ml total volume in T-175 tissue culture
flasks maintained in a 37 2 °C, 5 1% CO2 high humidity incu-
bator. Non-adherent cells were collected and diluted (1:3 v/v to
2.5 ꢀ 105/mL) every 48 h. DC-B cell growth medium was pre-
pared by combining the following: Hepes buffer (1 M, 10 mL,
pH 7.4); sodium pyruvate (10 mL, 100ꢀ, Gibco #11360-070);
MEM Vitamin Solution (4 mL, 100ꢀ, Gibco #1120-0511);
essential amino acids without glutamine (8 mL, 50ꢀ, Gibco
#11130-051); non-essential amino acids (4 mL, 100ꢀ, Gibco
#11140-050); PSG (10 mL); 2-ME (1 mL, 1000ꢀ, Gibco #21985-
023); FBS (10%, heat inactivated); GM-CSF (20 ng/mL), and di-
luted with bicarbonate (to 1 L). Assay medium for DC-B cells
was prepared by combining the following: in-house murine
GM-CSF (20 ng/mL, 10 mL), non-essential amino acids (1% aque-
ous, 10 mL); sodium pyruvate (1% aqueous, 10 mL), PSG (1%
aqueous, 10 mL), Hepes buffer (0.01 M, 10 mL), FBS (0.1% aque-
ous, heat inactivated), 50% aqueous Click’s Reagent. Immunoas-
say reagents include: in-house murine GM-CSF (500 g/mL),
murine IL-6 and IL-12p4. ELISA antibody pairs included purified
rat anti-mouse IL-6 monoclonal antibody (PharMingen #554400),
biotinylated rat anti-mouse IL-6 monoclonal antibody (PharMingen
#554402), purified rat anti-mouse IL-12p40/p70 monoclonal anti-
body (PharMingen #551219), biotinylated rat anti-mouse IL-
12p40/p70 monoclonal antibody (PharMingen #554476), DELFIA
Assay BufferTM (Wallac #1244-111), europium-labeled streptavidin,
(Wallac#1244-360, 0.1 mg/mL)and EnhancementSolutionTM (Wal-
lac #1244-105). Capture antibodies (i.e. IL12-p40 or IL-6) were
diluted (1:250 v/v) in PBS. The secondary capture antibody for IL-
12p40 and IL-6 were diluted in heat inactivated FBS (10% in PBS,
1:250 and 1:500, v/v dilution, respectively). Assay plates were
high-binding 384-well plates (Greiner #781077) and cell culture
plates were black wall/clear bottom 384-well plates (Costar #3712).
4.2.3. 2-(N-(3,5-Dichlorophenyl)-2-hydroxybenzamido)acetic
acid (20)
To a stirred solution of 10 (0.250 g, 0.672 mmol), prepared
according to the method described in the preceding section,
and sodium tert-butoxide (0.130 g, 1.34 mmol) in anhydrous
1,2-dimethoxyethane (7 mL) was added (rt, 5 min) neat tert-
butylbromoacetate (0.22 mL, 1.3 mmol) and the resulting solu-
tion was stirred (rt, 90 min). After aqueous work-up (EtOAc,
H2O), the organic layer was dried (MgSO4) and concentrated
in vacuo and filtered through a plug of silica gel (hexanes/
EtOAc, 5:1) to afford tert-butyl 2-(2-(benzyloxy)-N-(3,5-dichloro-
phenyl)benzamido)acetate as
a crude colorless oil (0.285 g)
which was used without further purification: MS (+EI) m/z
510 (MNa+, 20), 508 (MNa+, 30), 432 ((M-56)H+, 70), 430 ((M-
56)H+, 100). A suspension of this intermediate oil (0.285 g, the-
or. 0.586 mmol) and 5% Pd/C (wet Degussa-type E101, 22 mg,
theor. 0.0053 mmol Pd) in EtOAc (3 mL) was shaken under a
hydrogen atmosphere (60 psi, rt, 3 h). The resulting mixture
was filtered (diatomaceous earth) and the filtrate was concen-
trated in vacuo. The crystalline solid that was obtained was trit-
urated (hexanes) to afford tert-butyl 2-(N-(3,5-dichlorophenyl)-
2-hydroxybenzamido)acetate as
a
white crystalline solid
(0.148 g, 0.373 mmol): 1H NMR (500 MHz, CDCl3) d 10.2 (br s,
1H), 7.27–7.24 (m with CHCl3, ꢂ2H), 7.09 (s, 2H), 6.96 (d,
J = 8.0 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H), 6.55 (m, 1H), 4.41 (s,
2H), 1.50 (s, 9H); 13C NMR (125 MHz, CDCl3) d 171.3, 167.5,
160.1, 145.9, 135.5, 133.7, 129.9, 127.6, 125.6, 118.4, 118.2,
115.2, 82.9, 53.6, 28.0; MS (+EI) m/z 396 (MH+, 70), 394
(MH+, 100). A solution of tert-butyl 2-(N-(3,5-dichlorophenyl)-
2-hydroxybenzamido)acetate (109 mg, 0.275 mmol) in CH2Cl2:
TFA (1:1 v/v, 4 mL) was stirred (rt, 1 h) and then concentrated
in vacuo. The resulting solid was crystallized (hexanes/EtOAc)
to afford 20 as a white crystalline solid (50 mg, 0.15 mmol):
1H NMR (500 MHz, DMSO-d6) d 12.89 (br s, 1H), 9.87 (br s,
1H), 7.39 (m, 1H), 7.25 (s, 2H), 7.13 (m, 1H), 7.06 (d,
J = 7.5 Hz, 1H), 6.74–6.71 (m, 2H), 4.51 (s, 2H); 13C NMR
(125 MHz, DMSO-d6) d 170.4, 168.6, 153.5, 145.0, 133.1, 130.9,
128.4, 126.1, 125.9, 123.3, 118.8, 115.6, 50.5; MS (+EI) m/z
340 (MH+, 70), 338 (MH+, 100).
To solutions of the test compounds (40
lM in DMSO/assay
media, 0.4% v/v) in assay media (12.5 L per well in 384-well, cell
l
culture plates) were added DC-B cells in DELFIA Assay BufferTM
(27.5
l
L/well, cell concentration of 7.27 ꢀ 105/mL) followed by
the cytokine stimulation cocktail (10 lL/well, or assay media for
control wells) to achieve the desired final test compound concen-
tration and volume (i.e. 10 M, 50 L/well). Stimulation cocktail
l
l
was prepared so that the final concentration after the dilution
that was just described resulted in the desired concentration for

8764
M. E. Brown et al. / Bioorg. Med. Chem. 16 (2008) 8760–8764
each cytokine: murine CD40L (2 ng/mL), murine IL-18 (40 ng/mL)
and murine IFN (20 ng/mL). The resulting assay plates were
4.6. Calculated physical properties
c
incubated (18–24 h, 37 °C incubator with 5% CO2) after which
the supernatant (i.e. containing test compound or control) was
transferred to 384-well assay plates that were pre-coated, at
The calculated physical properties such as pKa were determined
using ACD/LogD SuiteTM 9.0 (ACD/Labs, Toronto, ON, Canada,
www.acdlabs.com).
4 °C, with capture antibody (i.e. 20 lL/well, either anti-IL-12p40
or anti-IL-6). These assay plates were then incubated (2 h, rt)
and then washed with PBST (i.e. 0.05% Tween 20 in PBS). A bio-
Supplementary data
tinylated secondary antibody (20
plates were incubated (1 h, rt). After washing with PBST, euro-
pium-labeled streptavidin (20 L/well, diluted 1:1670 v/v in Del-
lL/well) was added and the
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.07.024.
l
fia Assay BufferTM) was added and the plates were incubated
(30 min, rt). Microtiter plates were analyzed (europium counts
at 615 nm) using a fluorescence microplate reader (Victor2, Wal-
lac). Assay experimental data points were calculated as percent-
age of control (POC) and dose–response plots represent POC
versus concentration. Each concentration in the dose–response
plots was comprised of three assay experimental data points.
The concentrations tested were over a range of ten values (i.e.
0.0005, 0.0015, 0.0046, 0.014, 0.041, 0.12, 0.37, 1.11, 3.33, and
References and notes
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10.0, all
lM). IC50’s, defined as the inhibition at 50% of response,
were determined from these dose–response curves. Assay data
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(www.Tibco.com) and Activity Base 5.0 queried with SARgen
(www.idbs.com).
4.5. Cytotoxicity and recombinant protein profiling assays
Cytotoxicity was assessed in our dendritic cell line by using
the CellTiter-GloTM cell viability protocol (www.promega.com).
The dendritic cells, treated with test compound, were then stim-
ulated according to the same protocol described in the preceding
section for the IL-12p40 assay protocol (i.e. incubated 18 h with
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12. Compounds 1 and 2 were not included among the original compounds that we
evaluated for inhibition of IL-12p40 production.
13. Inhibition of IL-12p40 production was also assessed using primary human
dendritic cells however the human donor-to-donor cell variability of these
primary cells and the difficulty posed by their routine collection made their use
murine CD40L, murine IL-18, and murine IFNc). The CellTiter-
GloTM reagent was then added in equal volume to culture volume
and incubated (i.e. 2 min with orbital shaking to induce cell lysis
followed by 10 min of incubation without shaking). Luminescence
was then measured by fluorescence microplate reader. Cytotoxic-
ity was not observed, for any compound reported here, below
100 nM. Compounds demonstrating selective inhibition of IL-
12p40 production and including 3 and 9 were counter-screened
against a panel of 75ꢀ receptor binding and 71ꢀ enzyme inhibi-
tion assays as well as routine, in-house biochemical assays and in
no instance was significant activity observed that might indicate
difficult thus
development.
a murine dentritic cell line was used for routine SAR
14. A similar observation can be made by comparing compounds 13 and 8. One
hypothesis for this effect would invoke subtle differences in interactions with
different target proteins.
15. Calculated pKa’s for 11 (6.6) and 9 (8.2) were determined using ACD/LogD
SuiteTM 9.0, Advanced Chemistry Development, Inc., (ACD/Labs), Toronto, ON,
Canada, www.acdlabs.com.
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a target or class of targets (i.e. <30% inhibition at either 10
lM
or 25 M, depending upon the assay, as performed at Cerep,
l
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www.cerep.com). Experiments using the selective salicyanilide
inhibitors of IL-12p40 production, described here, as probes for
biochemical target identification are underway and will be re-
ported elsewhere.
21. Pasare, C.; Medzhitov, R. Science 2003, 299(5609), 1033–1036.