Synthesis and biological evaluation of 1,2,4- triazinylphenylalkylthiazolecarboxylic acid esters as cytokine-inhibiting antedrugs with strong bronchodilating effects in an animal model of asthma

Article abstract of DOI:10.1021/jm049479m

The influx of leukocytes (eosinophils, lymphocytes, and monocytes) into the airways and their production of proinflammatory cytokines contribute to the severity of allergic asthma. We describe here the synthesis and pharmacological evaluation of a series

Full text of DOI:10.1021/jm049479m

J. Med. Chem. 2005, 48, 2167-2175
2167
Synthesis and Biological Evaluation of
1,2,4-Triazinylphenylalkylthiazolecarboxylic Acid Esters as Cytokine-Inhibiting
Antedrugs with Strong Bronchodilating Effects in an Animal Model of Asthma
Eddy J. Freyne,† Jean F. Lacrampe,‡ Frederik Deroose,† Gustaaf M. Boeckx,† Marc Willems,†
Werner Embrechts,† Erwin Coesemans,† Johan J. Willems,† Jerome M. Fortin,‡ Yannick Ligney,‡
Lieve L. Dillen,§ Willy F. Cools,§ Jan Goossens,§ David Corens,† Alex De Groot,† and Jean P. Van Wauwe*,§
Medicinal Chemistry Department and Department of Inflammation, Johnson & Johnson Pharmaceutical Research and
Development, a Division of Janssen Pharmaceutica N.V., Turnhoutseweg 30, B-2340 Beerse, Belgium, and Campus de
Maigremont BP 615, 27106 Val de Reuil, France
Received June 30, 2004
The influx of leukocytes (eosinophils, lymphocytes, and monocytes) into the airways and their
production of proinflammatory cytokines contribute to the severity of allergic asthma. We
describe here the synthesis and pharmacological evaluation of a series of triazinylphenylalkyl-
thiazolecarboxylic acid esters that were designed to act as lung-specific antedrugs and inhibitors
of the production of interleukin (IL)-5, a primary eosinophil-activating and proinflammatory
cytokine. Closer examination of the hydroxypropyl ester, 15, indicated its high metabolic
stability (t1/2 > 240 min) in human lung S9 fraction but rapid conversion (t1/2 ) 15 min) into
the pharmacologically inactive carboxylic acid by human liver preparations. In stimulated
human whole blood cultures, 15 reduced not only the production of IL-5 (IC50 ) 78 nM) but
also the biosynthesis of the monocyte chemotactic proteins MCP-1 (IC50 ) 220 nM), MCP-2
(IC50 ) 580 nM), and MCP-3 (IC50 ) 80 nM). In vivo, intratracheal administration of 15 (6
mg/animal) to allergic sheep, either before (-4 h) or after (+1.5 h) the pulmonary allergen
challenge, completely abrogated the late-phase airway response and reduced the bronchial
hyperreactivity to inhaled carbachol.
Introduction
Allergic asthma is a chronic lung disease character-
ized by airflow obstruction, symptoms of cough, breath-
lessness and chest tightness, increased airway respon-
siveness together with evidence of airway inflammation.1
Inflammatory phenomena include denudation of the
airway epithelium, edema of the submucosa, smooth
muscle hypertrophy, and the infiltration or activation
of inflammatory cells, in particular eosinophils and T
lymphocytes.2 The activated T lymphocytes belong to
the Th-2 cell subpopulation and display a cytokine
production profile that includes interleukin (IL)-4, IL-
5, monocyte chemotactic protein (MCP)-1, MCP-2, and
MCP-3. IL-4 induces the synthesis of IgE antibodies,3
whereas IL-5 is responsible for growth, differentiation,
activation, and survival of eosinophils.4 MCP-1, MCP-
2, and MCP-3 attract or activate basophils, monocytes,
or eosinophils in the airways.5 Thus, suppression or
antagonism of proallergic cytokines may represent a
viable approach for the treatment of asthma6,7 Through
compound library screening using the phytohemagglu-
tinin (PHA)-activated human whole blood assay,8 we
identified a series of 1-phenyl-substituted 6-azauracil
derivatives with micromolar IL-5 inhibiting efficacy.9
Figure 1. R146225 (1).
nanomolar potent, orally active IL-5 inhibitor capable
of reducing the pulmonary infiltration of eosinophils in
Cryptococcus-challenged mice.10
However, chronic dosing of 1 to pregnant rats and
rabbits induced signs of teratogenesis and efforts to
develop this compound were halted. However, convinced
of the intrinsic antiasthmatic properties of compound
1, we sought to eliminate the teratogenic side effect by
designing 1-substituted 6-azauracil-containing ante-
drugs, i.e., compounds that exert their desired topical
effect in the lung (target tissue) but are rapidly con-
verted into inactive/nontoxic metabolites after entry into
the systemic circulation.11-14
Subsequent targeted chemical synthesis resulted in
the characterization of R146225 (Figure 1, 1) as a
Synthetic efforts aimed at combining the 1-substitut-
ed 6-azauracil pharmacophore with “labile” carboxylic
ester functionalities14-16 led to the synthesis of a series
of triazinylphenylalkylcarboxylic acid esters with lung-
specific antedrug characteristics.
* Author to whom correspondence should be addressed. Phone:
+ 32-14 602464. Fax: + 32-14 605403. E-mail: jvwauwe@prdbe.jnj.com.
† Medicinal Chemistry Department, Belgium.
‡ Medicinal Chemistry Department, France.
§ Department of Inflammation, Belgium.
10.1021/jm049479m CCC: $30.25 © 2005 American Chemical Society
Published on Web 12/29/2004

2168 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Freyne et al.
Table 1. Metabolic Stability of Compounds in Human Lung and Liver S9 Fractions and Their IL-5 Inhibitory Effects
metabolic stability
(% conversion at 60 min)
IL-5 inhibition
(% inhibition)
compound
R
synthesis method
human lung
human liver
10-6
M
10-7
M
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
ethyl
A
A
A
A
B
B
B
B
B
B
B
B
B
B
B
B
B
C
C
C
C
17
1
29
10
76
7
94
67
85
58
79
0
isopropyl
94
83
89
0
CH2-CH2-CH2-OH
t-butyl
8
0
0
H
0
methyl
47
23
18
32
27
27
23
6
20
24
34
36
3
49
68
86
72
87
80
68
65
55
94
61
38
1
93
96
88
91
92
90
96
73
90
88
91
92
100
93
83
85
84
69
51
54
56
56
69
55
87
62
74
75
96
66
70
57
n-propyl
n-butyl
n-pentyl
CH2-cprop
isobutyl
CH2-CHdCH2
CH2-CHdC(Me)2
CH2-C≡CH
CH2-CH2-Ph
CH2CN
CH2-CF3
CH2-C≡C-Me
CH2-CH2-OH
CH2-CH2-OPh
CH2(CO)Me
0
21
19
1
73
64
Chemistry
at 120 °C and without isolation was further hydrolyzed
with concentrated HCl at reflux to the corresponding
acid 10. Decarboxylation to give the 6-azauracil deriva-
tive 11, was done in thioglycolic acid at 100 °C. Finally,
conversion of the nitrile 11 to the pivotal intermediate
thioamide 12 was completed in pyridine/diisopropyl-
ethylamine (DIPEA), ensuring a constant flow of H2S
through the solution at 80 °C.
For the synthesis of the target compounds 13-33
(Scheme 2), three methods were elaborated. Following
method A, the ester derivatives 13-15 were formed via
direct cyclization of compound 12 with the appropriate
R-bromo-â-oxo-benzenepropionate esters17,20 34-36 in
DMF. For compound 15 (R ) t-Bu), 12 was first
condensed with tert-butyl R-bromo-â-oxo-benzenepropi-
onate 37 in CH3CN in the presence of K2CO3. The actual
cyclization to the ester derivative 16 was carried out in
refluxing t-BuOH. Conversion to the acid 17 was proven
to go smoothly in CF3COOH at room temperature.
Following method B, compound 17 was alkylated di-
rectly with the appropriate alkyl halides in DMF using
NaHCO3 as a base at a temperature of 70 °C, giving
the corresponding ester variations 18-28. Finally, in
method C, the acid 17 was activated using CDI in DMF
at 40 °C. Then, the reactive imidazolylcarbonyl inter-
mediate initially formed was treated further with the
appropriate alcohol and DBU affording compounds 29-
33.
Further extension of our chemical program around 1
resulted in the discovery of triazinylphenyl-alkylthiazole
derivatives as a novel family of nonchiral IL-5 inhibitors
that served as a template for the synthesis of potential
antedrugs.17 The title compounds are compiled in Table
1, and the general procedures for their total syntheses
are outlined in Schemes 1 and 2. The pivotal intermedi-
ate, thioamide 12 (Scheme 1), was prepared from the
commercially available 2,6-di-chloro-4-nitroanisole in 10
steps. From 2,6-dichloro-4-nitroanisole 2, selective dis-
placement of the methoxy group was achieved with the
sodium salt of ethyl cyanoacetate in dimethylformamide
(DMF) at 5 °C giving 3. The ethoxycarbonyl group was
next removed in a mixture of dimethyl sulfoxide (DMSO)/
H2O using LiCl at 165 °C to produce compound 4.
Double methylation of 4 was obtained using MeI in
tetrahydrofuran (THF) in the presence of benzyltriethyl-
ammonium chloride (BTEAC) and 50% NaOH at 50 °C
affording 5. Conversion to the corresponding aniline 6
was carried out by catalytic hydrogenation with Raney
Ni in MeOH at room temperature. This aniline deriva-
tive was used immediately for the construction of the
triazine-3,5(2H,4H)-dione ring. The synthesis of such a
6-azauracil ring had been reported.18 We used an
adapted procedure of 19 and our anticoccidial product
Clinacox.19 In this method, 4-amino-2,6-dichloro-R,R-
dimethylbenzeneacetonitrile (6) was converted to the
diazonium salt 7 using an aqueous NaNO2 solution in
a mixture of HOAc and concentrated HCl at 10 °C.
Compound 7 was not isolated, but after NaOAc was
added, subsequently coupled further with the sym-
metrical malonyldiurethane to the intermediate 8. Ring
cyclization to 9 was carried out using KOAc in HOAc
Pharmacology
Compounds were tested in vitro for their metabolic
stability and inhibitory activity on IL-5 production.
Metabolic stability was assessed in human lung and
liver S9 fractions and expressed as percent of compound

Cytokine-Inhibiting Antedrugs for Asthma Treatment
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2169
Scheme 1. Synthesis of the Key Thioamide Intermediate 12a
a Reagents and conditions: (a) NaH, NCCH2COOEt, N2 atm, DMF, 5 °C to room temperature; (b) LiCl, DMSO/H2O (5:2), 165 °C; (c)
50% NaOH, MeI, BTEAC, THF, 50 °C, 6 h; (d) H2 (3 bar), Raney Ni (5%), MeOH, room temperature; (e) NaNO2, H2O, HOAc, conc HCl,
10 °C; (f) NaOAc, CH2(CONHCOOEt)2, 10 °C; (g) KOAc, HOAc, 120 °C, 3 h; (h) conc HCl, reflux, 4 h; (i) HSCH2COOH, 100 °C, 4 h; (j)
H2S, DIPEA, pyridine, 80 °C.
converted after a 60 min incubation at 37 °C, whereas
IL-5 production was determined by ELISA in the
supernatant of human whole blood cultures stimulated
by PHA for 48 h.
16, 18, 26, 28, 29, 30, and 32 invariably displayed
unfavorable antedrug characteristics. Taken together,
these data led us to select 15 for additional experiments
to document its potential as a lung-directed antedrug.
First, time course studies using human samples to
determine the metabolic stability of 15 indicated that
its metabolic half-life (t1/2) exceeded 240 min in plasma
and lung S9 fraction but was as short as 15 min in liver
S9 preparation. Additional stability experiments that
were done using tissues from sheep (the species to be
used for in vivo evaluation) indicated that 15 is meta-
bolically stable in sheep plasma and lung (t1/2 > 240
min) but rather labile (t1/2 ) 60 min) in liver prepara-
tions, comparable to the human data.
Additionally, a 60 min incubation of 15 with human
liver S9 fraction and subsequent LC/MSMS analysis of
the reaction mixture identified the carboxylic acid 17
as the sole metabolic product. This compound had no
effect on the whole blood production of IL-5 (Table 1),
indicating that 15 was metabolized to a pharmacologi-
cally inactive product.
As presented in Table 1, most esters were rather
resistant to metabolic conversion when incubated with
lung S9 fraction. Conversion rates below 10% were
obtained for compounds 14, 15, 16, 25, 30, and 31. By
contrast, when incubated with liver S9 fraction, several
compounds, such as 15, 20, 21, 22, 23, 25, 27, and 32
were readily (>70% conversion) metabolized. Taken
together, only compounds 15 and 25 could be identified
as compounds having a metabolic stability that was high
in lung and low in liver S9 fractions. Because none of
the tested esters was converted for more than 10% when
incubated with fresh human plasma (data not shown),
we used PHA-stimulated human whole blood cultures
to assess their effect on the production of IL-5. As listed
in Table 1, when tested at 10-7 M, all esters suppressed
by more than 50% the production of this cytokine.
However, the strongest IL-5 production inhibitors 14,

2170 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Scheme 2. Synthesis of Target Compounds 13-33a
Freyne et al.
a Reagents and conditions: (a) method A, PhCOCH(Br)COOR 34-36, HO(CH2)3OH/DMF or EtOH/DMF or K2CO3, CH3CN; (b)
PhCOCH(Br)COOt-Bu 37, K2CO3, CH3CN, room temperature; (c) t-BuOH, reflux; (d) CF3COOH, room temperature, 4 h; (e) method B,
acid 17, appropriate RX (X ) Br, I), NaHCO3, DMF, 70 °C; (f) method C: acid 17, appropriate ROH, CDI, DBU, DMF, 40 °C.
Figure 2. Effect of 15 (10-6 M) on cytokine protein production in PHA-stimulated human whole blood.
The breadth of cytokine inhibitory activity of 15 was
then examined by determining the compound’s effect on
the PHA-induced whole blood production of other T-cell-
derived cytokines besides IL-5. As shown in Figure 2,
when tested at a concentration of 10-6 M, 15 not only
reduced the production of IL-5 (83 ( 7% inhibition) but
also suppressed the synthesis of IL-4 (49 ( 11% inhibi-
tion), IL-8 (46 ( 11% inhibition), MCP-1 (69 ( 8%
inhibition), MCP-2 (53 ( 5% inhibition), and MCP-3 (83
( 8% inhibition).
respectively. However, the compound barely affected the
generation of IL-1 (24 ( 4% inhibition), IL-2 (23 ( 7%
inhibition), IL-6 (8 ( 17% inhibition), IL-10 (3 ( 8%
inhibition), IFN-γ (2 ( 2% inhibition), or TNF (18 ( 6%).
Finally, we examined the antiasthmatic effects of 15
in a model of Ascaris suum antigen-induced airway
responses in conscious allergic sheep. In the vehicle
trial, the antigen challenge enhanced the specific lung
resistance (SRL expressed in liters × cm H2O/(L/s)) from
0.98 ( 0.02 to peak and late phase responses of 4.04 (
0.81 and 2.09 ( 0.09, respectively. Pretreatment (4 h
before the Ascaris challenge) with 6 mg of aerosolized
15 significantly reduced the late-phase bronchoconstric-
Concentration-response experiments indicated that
15 inhibited the production of IL-5, MCP-1, MCP-2, and
MCP-3 with IC50 values of 78, 220, 580, and 80 nM,

Cytokine-Inhibiting Antedrugs for Asthma Treatment
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2171
Figure 4. Effect of aerosolized 15 (6 mg, administered 1.5 h
after allergen exposure) on (A) early- and late-phase bron-
choconstriction and (B) airway responsiveness in allergen-
challenged Ascaris-sensitive sheep (n ) 5). Bronchoconstriction
was measured as the percentage of increase of specific lung
resistance (SRL) over a 8 h period after antigen challenge
(mean ( SE, n ) 5; * p < 0.01 vs vehicle), and airway
responsiveness (expressed in cumulative breath units of
inhaled carbachol to induce a 400% increase in specific lung
resistance) was measured before and 24 h postantigen chal-
lenge.
Figure 3. Effect of aerosolized 15 (6 mg, administered at 4 h
before allergen exposure) on airway responses in allergen-
challenged Ascaris-sensitive sheep. (A) Early- and late-phase
bronchoconstriction was assessed as the percentage of increase
of specific lung resistance (SRL) over a 8 h period after antigen
challenge (mean ( SE, n ) 5; * p < 0.01 vs vehicle). (B) Airway
hyperreactivity was determined by the change in the cumula-
tive carbachol dose, expressed as breath units needed to induce
a 400% increase in SRL measured 24 h after the antigen
challenge.
and became virtually undetectable (<5 ng/mL) at the
120 min time point.
tion, although it had no effect on the early-phase
reaction (Figure 3A). Also, as shown in Figure 3B, 15
completely blocked the development of airway hyper-
reactivity, as evidenced by its ability to prevent the fall
in the PC400 values, i.e., the provocative dose of inhaled
carbachol that caused a 400% increase of SRL.
Of importance, delaying the administration of 15 to
1.5 h after the antigen challenge still produced marked
suppressive effects on the late-phase response and
airway responsiveness (Figure 4A and B).
To assess the extent of systemic exposure, sheep (n
) 5) received an intratracheal administration of aero-
solized 15 (6 mg) and blood was drawn at 5, 10, 20, 60,
120, and 240 min later. Plasma concentrations of 15
reached a maximal value of 28.4 ( 4.5 ng/mL (5 × 10-8
M) at 5 min after treatment decreasing rapidly to less
than 2 ng/mL at 60 min. Levels of the carboxylic acid
metabolite 17 peaked to 17.2 ( 3.1 ng/mL at 10 min
Conclusions
We have synthesized a series of 1,2,4-triazinylphen-
ylalkylcarboxylic acid esters that inhibit the production
of IL-5 and have the potential to act as lung-specific
antedrugs. On the basis of its favorable metabolic and
pharmacological properties, the hydroxypropyl ester 15
was selected for more detailed evaluation. This com-
pound showed high metabolic stability (t1/2 > 240 min)
in human lung S9 samples but was rapidly (t1/2 ) 15
min) converted by human liver S9 fraction to the
pharmacologically inactive carboxylic acid 17. Further-
more, when profiled for its cytokine specificity, 15 was
found to suppress at submicromolar potency the human
whole blood production of IL-5 and MCP-1, -2, and -3
and, to a lesser extent, the biosynthesis of IL-4 and IL-
8, while the generation of several other cytokines (IL-

2172 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Freyne et al.
g, 6.0 mmol), DMSO (5 mL) and water (2 mL) were added at
room temperature. The reaction flask was placed in an oil bath
that was preheated at 165 °C. The reaction mixture was stirred
for 30 min at 165 °C (CO2 gas evolution). The dark red-brown
reaction solution was poured out into ice-water, and this
mixture was extracted DIPE (3×). The combined organic
extracts were dried (MgSO4) and filtered, and the filtrate was
concentrated in vacuo on a water bath at 40 °C, yielding an
off-white solid. The compound was crystallized from DIPE (25
mL), filtered off, washed with DIPE and n-hexane, and dried
in vacuo (50 °C) to give the pure title compound 4: 1.04 g
(68%), mp 106 °C. Anal. (C8H4Cl2N2O2) C,H,N.
2,6-Dichloro-r,r-dimethyl-4-nitrobenzeneacetoni-
trile (5). To a solution of compound 4 (577.6 g, 2.51 mol),
methyl iodide (1,069 g, 7.33 mol), and BTEAC (57 g, 0.251 mol)
in THF (5 L), 50% NaOH (960 mL) was added in a dropwise
manner. The mixture was stirred vigorously at 50 °C for 6 h.
The reaction mixture was poured out on ice-water and
extracted with DIPE. The combined organic extracts were
dried (MgSO4) and filtered, and the filtrate was concentrated
under reduced pressure. The residual solid was purified by
column chromatography on silica gel (30%EtOAc/70% hexane).
The fractions containing pure product were combined and
concentrated in vacuo to dryness. The resulting solid was
stirred up in n-hexane, filtered off, and dried in vacuo to give
5: 457 g (70%).
1, IL-2, IL-6, IL-10, IFN-γ, and TNF) was barely
affected.
As each of these cytokines can trigger proallergic
activities,3-5,21 inhibition of their production may ex-
plain the antiallergic effect of 15 in the Ascaris sheep
model of asthma. Indeed, intratracheal administration
of aerosolized 15 reduced the antigen-induced late-phase
bronchoconstriction and development of airway respon-
siveness. The compound abrogated these late phase
reactions without affecting the immediate bronchial
response, in line with the noninvolvement of cytokines
during the early stage of the bronchoconstriction reac-
tion. An intriguing feature is that compound 15 gener-
ated its effect when administered both before (prophyl-
actic mode) and after (curative mode) the antigen
challenge. This curative activity appears to be excep-
tional in this sheep model and has not been reported
for a number of antiallergic drugs, including the corti-
costeroid budenoside22 and the mast cell tryptase in-
hibitor APC 366.23
Additonally, the intratracheal administration of 15
resulted in short-lived and minimal plasma exposure,
supporting its characterization as a compound with
potential use in the topical treatment of allergic disor-
ders.
4-Amino-2,6-dichloro-r,r-dimethylbenzeneacetoni-
trile (6). A mixture of 2,6-dichloro-R,R-dimethyl-4-nitroben-
zeneacetonitrile (5; 17.1 g, 66 mmol) in MeOH (200 mL) was
hydrogenated under pressure (3 bar) for 1 h, using Raney Ni
(5%) at room temperature. The catalyst was filtered through
Celite and washed with MeOH, and the filtrate was concen-
trated in vacuo to afford 5: 15.0 g (>95%). The compound was
immediately used further in the preparation of 10.
Experimental Section
All of the air and moisture sensitive reactions were con-
ducted under a nitrogen atmosphere. All of the reagents and
starting materials were obtained from commercial sources and
used without further purification. THF, DMF, CH2Cl2, CHCl3,
toluene, and pyridine were dried over 3 Å molecular sieves.
DIPE refers to diisopropyl ether, DIPA refers to N,N-diiso-
propylethylamine, DBU refers to 1,8-diazabicyclo[5.4.0]undec-
7-ene, CDI refers to 1,1′-carbonyldiimidazole, NBS refers to
N-bromosuccinimide, and BTEAC refers to N,N,N′-benzyltri-
ethylammonium chloride. 1H and 13C NMR spectra were
recorded on Bruker AMX 400, DPX 400, and DPX 360
spectrometers. DMSO-d6 was used as solvent, unless otherwise
mentioned. The chemical shifts are expressed in δ, parts per
million (ppm), downfield from internal tetramethylsilane
(TMS). The peak patterns are shown as the following ab-
breviations: s ) singlet, d ) doublet, t ) triplet, q ) quartet,
m ) multiplet, br ) broad. Melting points were determined
on a Kofler or Buchi B545 instrument and are uncorrected.
Elemental analyses were performed on a Carlo-Erba EA1110
analyzer. Analytical results were within (0.4% of the theoreti-
cal values unless otherwise noted.
2-[3,5-Dichloro-4-(1-cyano-1-methylethyl)phenyl]-2,3,4,5-
tetrahydro-3,5-dioxo-1,2,4-triazine-6-carboxylic Acid (10).
A solution of 4-amino-2,6-dichloro-R, R-dimethylbenzeneac-
etonitrile (6; 77.6 g, 0.34 mol) in HOAc (700 mL) and
concentrated HCl (102 mL) was stirred under a nitrogen
atmosphere at 10 °C for 30 min. A solution of NaNO2 (24.8 g,
0.36 mol) in H2O (50 mL) was added dropwise over 30 min
maintaining the temperature around 10 °C forming the
corresponding diazonium salt 7. After an additional 80 min
at 10 °C, a mixture of NaOAc (83.64 g, 1.02 mol) and
malonyldiurethane (92.1 g, 0.374 mol) was added in one
portion. After 40 min, the reaction mixture was poured out
on ice-water (2 L). The precipitate was filtered off, washed
with water, and dissolved in CH2Cl2. The organic layer was
dried (MgSO4), filtered, and evaporated under reduced pres-
sure affording diethyl N,N′-[2-[[3,5-dichloro-4-(1-cyano-1-me-
thylethyl)phenyl]hydrazono]-1,3-dioxo-1,3-propanediyl]dicar-
bamate (8): 135 g (84%). The compound was dissolved in
HOAc (1 L) and treated with KOAc (27.25 g, 0.28 mol), and
the reaction mixture was refluxed for 3 h at 120 °C forming
ethyl [[2-[3,5-dichloro-4-(1-cyano-1-methylethyl)phenyl]-2,3,4,5-
tetrahydro-3,5-dioxo-1,2,4-triazin-6-yl]carbonyl]carbamate (9).
Subsequently, concentrated HCl (70 mL, 0.84 mol) was added,
and after an additional stirring for 4 h at reflux, the mixture
was allowed to warm to room temperature. After being stirred
at ambient temperature over the weekend, the reaction
mixture was poured out on ice and extracted with CH2Cl2. The
organic extracts were dried (MgSO4), filtered, and concentrated
in vacuo to give the title compound 10: 100,6 g (98%, over
two steps from 8).
Silica gel thin-layer chromatography was performed on
precoated plates Kieselgel 60F254 (E. Merck, AG, Darmstadt,
Germany). Silicagel chromatography was performed with
Kieselgel 60 (0.063-0.200 mm) (E. Merck, AG, Darmstadt,
Germany).
Ethyl 1,3-Dichloro-r-cyano-4-nitrobenzeneacetate (3).
To a cooled (5 °C), stirred solution of ethyl cyanoacetate (122.4
g, 1.08 mol) in DMF (1 L), NaH dispersion (60% mineral oil)
(40.5 g, 1.08 mol) was added in portions under a N2 atmo-
sphere. After the addition was completed, the mixture was
stirred further at 5 °C for 1 h until evolution of hydrogen had
stopped. Nitro derivative 2 (200 g, 0.9 mol, commercially
available) was added, and the mixture was allowed to warm
to room temperature overnight under stirring. The reaction
mixture was poured onto ice and adjusted to pH ) 3 with
concentrated HCl. The resulting precipitate was filtered off,
washed with water, and taken up in CH2Cl2. The organic layer
was dried (MgSO4), filtered, and concentrated in vacuo. The
resulting solid was stirred up in n-hexane and dried in vacuo
overnight (60 °C) to give 3: 261.3 g (96%).
2,6-Dichloro-4-(4,5-dihydro-3,5-dioxo-1,2,4-triazin-2(3H)-
yl)-r,r-dimethylbenzeneacetonitrile (11). A stirred sus-
pension of 10 (111.6 g, 0.28 mol) in thioglycolic acid (250 mL)
was heated for 4 h at 100 °C, then allowed to cool to room
temperature, and stirred further overnight. The reaction
mixture was poured out on ice and extracted three times with
CH2Cl2. The combined organic extracts were dried (MgSO4)
and filtered, and the solvent was removed in vacuo. Toluene
was added and concentrated under reduced pressure (3×). The
residual material was purified by short column chromatogra-
2,6-Dichloro-4-nitrobenzeneacetonitrile (4). To a stirred
mixture of nitro derivative 3 (2 g, 6.6 mmol) and LiCl (0.254

Cytokine-Inhibiting Antedrugs for Asthma Treatment
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2173
phy on silica gel using solvent (98% CH2Cl2/2% CH3OH). The
pure fractions were collected, and the solvent was removed in
vacuo. The residual product was stirred up in DIPE, filtered
off, and washed with DIPE in vacuo at 50 °C for 16 h yielding
11 (39.3 g, 44%).
2,6-Dichloro-4-(4,5-dihydro-3,5-dioxo-1,2,4-triazin-2(3H)-
yl]-r,r-dimethylbenzeneethane-thioamide (12). A stirred
solution of 11 (56 g, 0.172 mol) and DIPEA (67 g, 0.662 mol)
in pyridine (500 mL) was heated at 80 °C. At this temperature,
H2S gas was bubbled through the reaction mixture for 24 h.
After an additional stirring at room temperature over the
weekend, the solvent was removed in vacuo. The crude
material was purified by HPLC chromatography on silicagel
(Novasep column, diameter 80 mm; silicagel 20-45 µm) using
93%CH2Cl2/7% MeOH as solvent system affording thioamide
12: 24 g (40%).
1-Methylethyl r-Bromo-â-oxo-benzenepropionate (35).
To a solution of 1-methylethyl â-oxo-benzenepropionate24(3 g,
1.5 mmol) in CH2Cl2 (75 mL) was added a solution of Br2 (0.67
mL, 1.3 mmol) in CH2Cl2 (5 mL) in a dropwise manner at 10
°C. The reaction mixture was stirred further for an additional
1 h and then treated with 10% K2CO3 solution. The organic
layer was separated, dried (MgSO4), filtered, and concentrated
in vacuo giving the crude bromide 35 (3.6 g, 87%), which was
used subsequently in the synthesis of compound 14.
Ethyl r-Bromo-â-oxo-benzenepropionate (34) and 1,1-
Dimethylethyl R-Bromo-â-oxo-benzenepropionate (37).
Bromide 3420 and 37 were synthesized from the commercially
available ethyl â-oxo-benzenepropionate and 1,1-dimethylethyl
â-oxo-benzenepropionate,25 respectively, according to the method
for the preparation of 35. Compound 34 and 37 were im-
mediately used further in the synthesis of 13 and 16, respec-
tively.
3-Hydroxypropyl r-Bromo-â-oxo-benzenepropionate
(36). To a vigorously stirred solution of 3-hydroxypropyl-â-oxo-
benzenepropionate26 (26.0 g, 0.097 mol) in CHCl3 (250 mL),
NBS (20.3 g, 0.114 mol) was added in portions over 2 h at room
temperature under a nitrogen atmosphere. After an additional
stirring for 2 h, the reaction mixture was treated with an
aqueous NaHCO3 solution. The organic layer was separated,
dried (MgSO4), filtered, and concentrated in vacuo. The
residual bromide 36 (30.1 g, 85%) was immediately used
further in the preparation of compound 15.
Representative Procedure for the Synthesis of the
Target Compounds (13-15, 16). Method A. Compounds
13-15 and 16 were synthesized via direct ring closing reaction
of the thioamide 12 with the appropriate R-bromo-â-oxo-
benzenepropionate ester derivatives 34-37.
3-Hydroxypropyl 2-[1-[2,6-Dichloro-4-(4,5-dihydro-3,5-
dioxo-1,2,4-triazin-2(3H)-yl)phenyl]-1-methylethyl]-4-
phenyl-5-thiazolecarboxylate (15). To a stirred solution of
bromide 36 (30.1 g, 0.1 mol) in DMF (300 mL) under a N2
atmosphere, 1,3-propane-diol (30 mL) and subsequently thio-
amide 12 (35.9 g, 0.1 mol) were added. The mixture was stirred
at ambient temperature overnight and then for an additional
3.5 h at 70 °C. The reaction mixture was partially concentrated
in vacuo, poured out in ice-water, and extracted with CH2-
Cl2. The combined organic extracts were dried (MgSO4),
filtered, and concentrated in vacuo giving a residual solid that
was purified by chromatography on a short column of silica
gel (99% CH2Cl2/1% MeOH to 98% CH2Cl2/2% MeOH). After
an additional purification via HPLC on silica gel (100% CH2-
Cl2 to 97.5% CH2Cl2/2.5% MeOH), the compound was obtained
as a white solid 15: 20.9 g (37%), mp 137 °C. Anal. (C25H22-
Cl2N4O5S) C, H, N.
sized from 10 according to method A using CH3CN as solvent
and K2CO3 as base (30%), mp 215 °C. Anal. (C25H22Cl2N4O4S)
C, H, N.
1,1-Dimethylethyl 2-[1-[2,6-Dichloro-4-(4,5-dihydro-3,5-
dioxo-1,2,4-triazin-2(3H)-yl)phenyl]-1-methylethyl]-4-
phenyl-5-thiazolecarboxylate (16). Compound 16 was syn-
thesized from 12 according to method A, in which the initial
reaction with tert-butyl R-bromo-â-oxo-benzenepropionate 37
was performed in CH3CN using K2CO3 as base, and the
subsequent ring closing reaction was carried out in refluxing
t-BuOH to afford 16 (50%), mp 130 °C. Anal. (C26H24Cl2N4O4S)
C, H, N.
Representative Procedure for the Synthesis of the
Target Compounds (18-28). Method B: Propyl 2-[1-[2,6-
Dichloro-4-(4,5-dihydro-3,5-dioxo-1,2,4-triazin-2(3H)-yl)-
phenyl]-1-methylethyl]-4-phenyl-5-thiazolecarboxylate
(19). A mixture of compound 16 (29 g, 0.0518 mol) in CF3-
COOH (200 mL) was stirred at room temperature for 4 h. The
reaction mixture was poured out on ice, and the precipitate
was washed with water and then taken up in CH2Cl2. The
organic layer was washed with water, dried (MgSO4), filtered,
and concentrated in vacuo. The residual solid was purified by
column chromatography on silica gel (20-45 µm; 97% CH2-
Cl2/3% MeOH/0.1% AcOH). The pure fractions were combined
and concentrated in vacuo, and the residual solid was recrys-
tallized (CH3CN) to afford 2-[1-[2,6-dichloro-4-(4,5-dihydro-3,5-
dioxo-1,2,4-triazin-2(3H)-yl)phenyl]-1-methylethyl]-4-phenyl-
5-thiazolecarboxylic acid (17): 25.0 g (95%), mp >250 °C.
To a mixture of the acid 17 (2 g, 4 mmol) and NaHCO3 (0.33
g, 4 mmol) in DMF (8 mL), a mixture of n-propyl bromide (0.48
g, 4 mmol) in DMF (2 mL) was added at room temperature
and heated for 6 h at 70 °C. The reaction mixture was poured
out on ice, acidified with HCl (3 N), and extracted with EtOAc.
The organic extracts were washed with water, dried (MgSO4),
filtered, and concentrated in vacuo. The residual product was
purified by column chromatography on silica gel (15-40 µm;
99.25% CH2Cl2/0.75% MeOH). The pure fractions were com-
bined and concentrated in vacuo, and the residue was crystal-
lized (diethyl ether/DIPE) to yield compound 19: 0.56 g (54%),
mp 172 °C. Anal. (C25H22Cl2N4O4S) C, H, N.
Methyl 2-[1-[2,6-Dichloro-4-(4,5-dihydro-3,5-dioxo-1,2,4-
triazin-2(3H)-yl)phenyl]-1-methylethyl]-4-phenyl-5-thia-
zolecarboxylate (18). Compound 18 was synthesized from
17 and methyl iodine according to method B (8%), mp 175 °C.
Anal. Calcd for (C23H18Cl2N4O4S): C, 53.39; H, 3.51; N, 10.83.
Found: C, 52.39; H, 3.38 N, 10.38.
Butyl 2-[1-[2,6-Dichloro-4-(4,5-dihydro-3,5-dioxo-1,2,4-
triazin-2(3H)-yl)phenyl]-1-methylethyl]-4-phenyl-5-thia-
zolecarboxylate (20). Compound 20 was synthesized from
17 and n-butyl bromide according to method B (11%), mp 130
°C. Anal. (C26H24Cl2N4O4S) C, H, N.
Pentyl 2-[1-[2,6-Dichloro-4-(4,5-dihydro-3,5-dioxo-1,2,4-
triazin-2(3H)-yl)phenyl]-1-methylethyl]-4-phenyl-5-thia-
zolecarboxylate (21). Compound 21 was synthesized from
17 and n-pentyl bromide according to method B (23%), mp 134
°C. Anal. (C27H26Cl2N4O4S) C, H, N.
Cyclopropylmethyl 2-[1-[2,6-Dichloro-4-(4,5-dihydro-
3,5-dioxo-1,2,4-triazin-2(3H)-yl)phenyl]-1-methylethyl]-
4-phenyl-5-thiazolecarboxylate (22). Compound 22 was
synthesized from 17 and cyclopropylmethyl bromide according
to method B (15%), mp 100 °C. Anal. (C26H22Cl2N4O4S) C, H,
N.
2-Methylpropyl 2-[1-[2,6-Dichloro-4-(4,5-dihydro-3,5-
dioxo-1,2,4-triazin-2(3H)-yl)phenyl]-1-methylethyl]-4-
phenyl-5-thiazolecarboxylate (23). Compound 23 was syn-
thesized from 17 and isobutyl bromide according to method B
(17%), mp 149 °C. Anal. Calcd for (C26H20Cl2N4O4S): C, 55.82;
H, 4.32; N, 10.01. Found: C, 55.63; H, 4.09; N, 9.93.
Ethyl 2-[1-[2,6-Dichloro-4-(4,5-dihydro-3,5-dioxo-1,2,4-
triazin-2(3H)-yl)phenyl]-1-methylethyl]-4-phenyl-5-thia-
zolecarboxylate (13). Compound 13 was synthesized from
12 according to method A using an EtOH/DMF mixture (2/
1,v/v) (27%), mp 80 °C. Anal. (C24H20Cl2N4O4S) C, H, N.
1-Methylethyl 2-[1-[2,6-Dichloro-4-(4,5-dihydro-3,5-di-
oxo-1,2,4-triazin-2(3H)-yl)phenyl]-1-methylethyl]-4-phen-
yl-5-thiazolecarboxylate (14). Compound 14 was synthe-
2-Propenyl 2-[1-[2,6-Dichloro-4-(4,5-dihydro-3,5-dioxo-
1,2,4-triazin-2(3H)-yl)phenyl]-1-methylethyl]-4-phenyl-5-
thiazolecarboxylate (24). Compound 24 was synthesized
from 17 and allyl bromide according to method B (15%), mp
172 °C. Anal. (C25H20Cl2N4O4S) C, H, N.

2174 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Freyne et al.
3-Methyl-2-butenyl 2-[1-[2,6-dichloro-4-(4,5-dihydro-
3,5-dioxo-1,2,4-triazin-2(3H)-yl)phenyl]-1-methylethyl]-
4-phenyl-5-thiazolecarboxylate (25). Compound 25 was
synthesized from 17 and 3,3-dimethylallyl bromide according
to method B (11%), mp 90 °C. Anal. Calcd for (C27H24-
Cl2N4O4S): C, 56.75; H, 4.23; N, 9.80. Found: C, 56.56; H, 4.33;
N, 9.36.
2-Propynyl 2-[1-[2,6-Dichloro-4-(4,5-dihydro-3,5-dioxo-
1,2,4-triazin-2(3H)-yl)phenyl]-1-methylethyl]-4-phenyl-5-
thiazolecarboxylate (26). Compound 26 was synthesized
from 17 and propargyl bromide according to method B (19%),
mp 180 °C. Anal. (C25H18Cl2N4O4S) C, H, N.
2-Phenylethyl 2-[1-[2,6-Dichloro-4-(4,5-dihydro-3,5-di-
oxo-1,2,4-triazin-2(3H)-yl)phenyl]-1-methylethyl]-4-phen-
yl-5-thiazolecarboxylate (27). Compound 27 was synthe-
sized from 17 and phenethyl bromide according to method B
(17%), mp 146 °C. Anal. Calcd for (C30H24Cl2N4O4S): C, 59.31;
H, 3.98; N, 9.22. Found: C, 58.86; H, 3.88; N, 9.19.
Cyanomethyl 2-[1-[2,6-Dichloro-4-(4,5-dihydro-3,5-di-
oxo-1,2,4-triazin-2(3H)-yl)phenyl]-1-methylethyl]-4-phen-
yl-5-thiazolecarboxylate (28). Compound 28 was synthe-
sized from 17 and bromoacetonitrile according to method B
(12%). Anal. (C24H17Cl2N5O4S) C, H, N.
Representative Procedure for the Synthesis of the
Target Compounds (29-33). Method C: Phenoxyethyl
2-[1-[2,6-Dichloro-4-(4,5-dihydro-3,5-dioxo-1,2,4-triazin-
2(3H)-yl)phenyl]-1-methylethyl]-4-phenyl-5-thiazolecar-
boxylate (32). A mixture of the acid 17 (1.35 g, 2.7 mmol)
and CDI (0.57 g, 3.5 mmol) in DMF (10 mL) was stirred for 1
h at 40 °C. Phenoxyethanol (0.74 g, 5.4 mmol) in DBU (0.4 g,
2.7 mmol) was added, and the reaction mixture was stirred
further for 3 h at 40 °C. The mixture was poured out on ice
and acidified with HCl (3 N). The precipitate was filtered off,
washed with water, and taken up in CH2Cl2. The organic
solution was washed with water, dried (MgSO4), and concen-
trated in vacuo. The residual solid was purified by chroma-
tography on silica gel (99%CH2Cl2/1%MeOH). Compound 32
was obtained from n-pentane: 1.2 g (72%), mp 80 °C. Anal.
(C30H24Cl2N4O5S) C, H, N.
2,2,2-Trifluoroethyl 2-[1-[2,6-Dichloro-4-(4,5-dihydro-
3,5-dioxo-1,2,4-triazin-2(3H)-yl)phenyl]-1-methylethyl]-
4-phenyl-5-thiazolecarboxylate (29). Compound 29 was
synthesized from 17 and 2,2,2-trifluoroethanol according to
method C (36%), mp 180 °C. Anal. Calcd for (C24H17Cl2-
F3N4O4S): C, 49.24; H, 2.93; N, 9.57. Found: C, 48.62; H, 2.97;
N, 9.37.
2-Butynyl 2-[1-[2,6-Dichloro-4-(4,5-dihydro-3,5-dioxo-
1,2,4-triazin-2(3H)-yl)phenyl]-1-methylethyl]-4-phenyl-5-
thiazolecarboxylate (30). Compound 30 was synthesized
from 17 and 2-butyn-1-ol according to method C (49%), mp
184 °C. Anal. (C26H20Cl2N4O4S) C, H, N.
2-Hydroxyethyl 2-[1-[2,6-Dichloro-4-(4,5-dihydro-3,5-
dioxo-1,2,4-triazin-2(3H)-yl)phenyl]-1-methylethyl]-4-
phenyl-5-thiazolecarboxylate (31). Compound 31 was syn-
thesized from 17 and ethylene glycol according to method C
(17%), mp 174 °C. Anal. (C24H20Cl2N4O5S) C, H, N.
acetonitrile were added, and the samples were vortexed. After
centrifugation, supernatants were evaporated, and the resi-
dues were dissolved in 100 µL of 50% acetonitrile containing
0.05% formic acid for analysis by LC-MSMS. Briefly, 10 µL
samples were injected on a Symmetry C18
column (50 mm ×
2.1 mm, 3.5 µm; Milford, MA. The mobile phase was 0.05%
formic acid (solvent A). Solvent B consisted of acetonitrile with
0.05% formic acid. A shallow gradient at a flow rate of 0.5 mL/
min was performed to ensure an elution time of 3-10 min. A
1
split ratio of
/ is applied after the photodiode array (PDA)
5
detector allowing 100 µL/min to enter the mass spectrometer.
Quantitative analysis was performed using the MRM transi-
tion of the parent ion to the daughter ion.
Cytokine Production. Blood from adult healthy volun-
teers was anticoagulated in heparin (12.5 U/mL) and cultured
as described.8 Briefly, blood samples were diluted 3-fold in
culture medium, and 300 µL fractions were preincubated for
15 min in the presence or absence of test compound (10-6
-
10-9 M). Then cultures were stimulated by the addition of 100
µL of PHA (final concentration of 5 µg/mL) and incubated at
37 °C for 24 or 48 h (in the case of IL-5) in a humidified 6%
CO2 atmosphere. Cell supernatants were then removed and
stored at -20 °C. Cytokine levels were determined by ELISA
using the Duoset antibodies (R&D Systems, Abingdon, U.K.)
according to the manufacturer’s instructions. Concentrations
were calculated from standard curves using recombinant
cytokines supplied by R&D Systems.
Antigen-Induced Airway Responses in Sheep. Airway
responsiveness was assessed in a sheep model of atopic asthma
as described.22,23 Adult sheep (mean body weight 30 kg) that
had shown positive allergic skin and airway responses to
Ascaris suum antigen were used. Mean pulmonary flow
resistance (RL) was calculated from the analysis of 5-10
breaths by using the esophageal balloon catheter technique.
Immediately after the determination of RL, thoracic gas volume
(Vtg) was measured in a constant-volume body plethysmograph
to obtain the specific lung resistance (SRL ) RL × Vtg). Aerosols
of Ascaris suum extract (82 000 protein nitrogen units/mL in
phosphate buffered saline) were generated using a disposable
medical nebulizer and directed into the animals via a plastic
T-piece, which had one end connected to the inspiratory port
of a Harvard respirator and the other end to the nasotracheal
tube. Ascaris aerosols were delivered at a tidal volume of 500
mL and a rate of 20 breaths/min for 20 min. Aerosols of 15
(dissolved at 2 mg/mL in a solution containing 2 mg/mL
tromethamine, 100 mg/mL hydroxypropyl-â-cyclodextrin, 30
mg/mL mannitol, and 3 mg/mL N-methyl-glucamine (final pH
8.5)) and of carbachol (0.25, 0.5, 1, 2, and 4% w/v in phosphate
buffered saline) were aerosolized and delivered by the same
nebulizer system.
Airway responsiveness was assessed from cumulative dose-
response curves to nebulized carbachol by measuring SRL
immediately after inhalation of buffer (phosphate buffered
saline) and after each consecutive administration of 10 breaths
of increasing concentrations of carbachol (0.25, 0.5, 1, 2, and
4% w/v in buffer). The cumulative carbachol dose (in breath
units), that increases SRL by 400% over the postbuffer value
(i.e., PC400) was calculated by interpolation from the dose-
response curve. One breath unit (BU) was defined as one
breath of an aerosol solution containing 1% w/v carbachol.
2-Oxo-propyl 2-[1-[2,6-Dichloro-4-(4,5-dihydro-3,5-di-
oxo-1,2,4-triazin-2(3H)-yl)phenyl]-1-methylethyl]-4-phen-
yl-5-thiazolecarboxylate (33). Compound 33 was synthe-
sized from 17 and 1-hydroxy-2-propanone according to method
C (30%), mp 226 °C. Anal. (C25H20Cl2N4O5S) C, H, N.
Pharmacology. Materials. For in vitro experiments,
compounds were dissolved at 5 mM in DMSO and ap-
propriately diluted with culture medium (RPMI 1640 supple-
mented with 2 mM glutamine, 100 U/mL penicillin and 10 µg/
mL streptomycin). PHA was obtained from Murex (Dartford,
U.K.). S9 preparations of human liver and lung were obtained
from Gentest and Analytical Biological Services, respectively.
Human Lung and Liver S9 Incubations. Compounds
(10-6 M) were incubated at 37 °C in 100 µL of phosphate
buffered saline with human liver S9 fraction (10 µg protein/
ml; obtained from BD Biosciences) or human lung S9 prepara-
tion (50 µg protein/ml; obtained from Analytical Biological
Services). After 0, 15, and 60 min of incubation, 3 volumes of
Baseline airway responsiveness was determined in five
sheep 2-4 days before start of vehicle or compound treatment.
On the challenge day, baseline SRL measurements were
repeated, and the animals were treated with aerolized com-
pound or vehicle either 0.5, 2, 4, or 16 h before or 1.5 h after
the challenge with Ascaris suum antigen. Every sheep served
as its own control and was treated with vehicle as well as with
test compound in experiments that were separated by at least
14 days. Changes in SRL were monitored hourly from 1 to 6 h
1
after challenge and on the half-hour from 6
/ to 8 h after
2
challenge. The compound’s effect on airway responsiveness was
measured 24 h after Ascaris challenge.

Cytokine-Inhibiting Antedrugs for Asthma Treatment
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2175
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Acknowledgment. We thank Mireille Vanderh-
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