De novo design of nonpeptidic compounds targeting the interactions between interferon-α and its cognate cell surface receptor

Article abstract of DOI:10.1021/jm701182y

Type 1 interferons (IFN) bind specifically to the corresponding receptor, IFNAR. Agonists and antagonists for IFNAR have potential therapeutic value in the treatment of viral infections and systemic lupus erythematosus, respectively. Specific sequences on

Full text of DOI:10.1021/jm701182y

2734
J. Med. Chem. 2008, 51, 2734–2743
De Novo Design of Nonpeptidic Compounds Targeting the Interactions between Interferon-r
and its Cognate Cell Surface Receptor
Angelica M. Bello,† Tanushree Bende,† Lianhu Wei,† Xiaoyang Wang,† Beata Majchrzak-Kita,† Eleanor N. Fish,†,‡,4 and
Lakshmi P. Kotra*,†,§,4,
Center for Molecular Design and Preformulations, Toronto General Research Institute, Toronto General Hospital, Toronto, ON M5G 2C4 Canada,
Department of Immunology, Departments of Pharmaceutical Sciences and Chemistry, and McLaughlin Center for Molecular Medicine,
UniVersity of Toronto, Toronto, Ontario, Canada, and Department of Chemistry and Biochemistry, The UniVersity of North Carolina at Greensboro,
Greensboro, North Carolina 27412
ReceiVed September 19, 2007
Type 1 interferons (IFN) bind specifically to the corresponding receptor, IFNAR. Agonists and antagonists
for IFNAR have potential therapeutic value in the treatment of viral infections and systemic lupus
erythematosus, respectively. Specific sequences on the surface of IFN, IFN receptor recognition peptides
(IRRPs) mediate the binding and signal transduction when IFN interacts with IFNAR. Structural features of
two such IRRPs, IRRP-1 and IRRP-3, were used as templates to design small molecule mimetics. In silico
screening was used to identify the molecular structural features mimicking their surface characteristics. A
set of 26 compounds were synthesized and their ability to interfere with IFN-IFNAR interactions was
investigated. Two compounds exhibited antagonist activity, specifically, blocking IFN-inducible Stat
phosphorylation Stat complex-DNA binding. Design principles revealed here pave the way toward a novel
series of small molecules as antagonists for IFN-IFNAR interactions.
Introduction
responsible for this differential recognition.12,13 In recent years,
the signaling events that follow IFN-R/ꢀ engagement of IFNAR
have been extensively characterized.14,15 Accumulating evidence
suggests that distinct differences in critical amino acid residues
among the different type-1 IFNs determine the nature of the
ligand–receptor interaction and the subsequent responses.10,14,16,17
At the outset, these interactions between IFN (the ligand) and
IFNAR-1 and IFNAR2 (the receptor) domains may be charac-
terized as protein–protein interactions. Due to the important roles
that IFNs play (i) in protection from viral infections, in cancer,
(ii) for their role in other autoimmune diseases, and (iii)
cognizant that the potency of a particular IFN resides in the
nature of the ligand–receptor interaction for individual IFNs,11
we undertook a program of research to develop small molecule
IFN mimetics, focusing on receptor interactive domains.
Interferons (IFNsa) are a family of biologically active proteins
classified as type 1, type 2 and type 3.1 Type 1 IFNs mediate
diverse biological effects, including antiviral and antiproliferative
responses, and several cell type-restricted responses of im-
munological relevance, such as maturation and differentiation
of dendritic cells and modulation of B, T, and NK cell
responses.2 IFNs have widespread clinical application as
therapeutic agents for the treatment of viral infections, especially
in the context of hepatitis B and C (HBV and HCV) infections.3
IFNs are clinically useful against a variety of solid tumors and
hematological malignancies, including chronic myelogenous
leukemia (CML), multiple myeloma, and hairy cell leukemia,
and for the treatment of multiple sclerosis.3–5 Deregulation of
IFNs is implicated in certain autoimmune diseases such as
systemic lupus erythematosus (SLE).6–8
Type 1 IFNs comprise a family of homologous helical bundle
cytokines that in humans include 14 IFN-R subtypes and single
IFN-ꢀ, IFN- κ, and IFN-ω subtypes. There is >50% seque-
nce homology among all type-1 IFNs. IFNs R, ꢀ, ω, and κ
activate the IFN receptor, IFNAR, engaging the two subunits
of this receptor, IFNAR-1 and IFNAR-2.9 Each of these subunits
possesses a separate ligand-binding domain required for a
functional ligand–receptor complex.10,11 Mutational studies
indicated that the binding of IFN-R and IFN-ꢀ elicits subtly
different cellular responses, and the subunit IFNAR-2 is
In the past several years, mimicking protein–protein interac-
tions has attracted attention for therapeutics design.18,19 Inter-
faces between protein–protein complexes are typically large and
often flat surfaces, thus providing both opportunities and
challenges for nonpeptidic molecular engineering.20 Recently,
we disclosed a de novo approach using in silico screening and
designing novel scaffolds to target protein–protein interactions.21
Here we outline a molecular approach involving in silico
screening and a selection process to design a series of nonpep-
tidic pyrimidine based molecules that mimic targeted regions
on the surface of the IFN molecule. It is anticipated that the
compounds designed to bind to specific interactive subdomains
in either IFNAR-1 or IFNAR-2 will compete and block the
binding of IFN to the complete extracellular pocket described
by IFNAR. Such molecules could be characterized as antagonists
for IFN-IFNAR interactions. An ideal agonist will bind to and
activate IFNAR. IFNAR antagonists will have potential utility
in the treatment of autoimmune diseases such as systemic lupus
erythematosus (SLE) and other diseases where IFN is implicated
in pathogenesis. Design, synthesis, and functional assay results
of this novel class of compounds are presented here, paving
the way for the design of antagonists to IFNAR.
* Corresponding Author: Mailing address: #5-356, Toronto Medical
Discoveries Tower/MaRS Center, 101 College Street, Toronto, Ontario,
Canada M5G 1L7. Tel. (416) 581-7601, Fax . (416) 581-7621, E-mail:
lkotra@uhnres.utoronto.ca.
†
Toronto General Research Institute.
Department of Immunology, University of Toronto.
Departments of Pharmaceutical Sciences and Chemistry, University
‡
§
of Toronto.
4
McLaughlin Center for Molecular Medicine.
The University of North Carolina at Greensboro.
a Abbreviations: IFN, interferon; IFNAR, interferon-R/ꢀ receptor; Stat,
signal transducers and activator of transcription.
10.1021/jm701182y CCC: $40.75
2008 American Chemical Society
Published on Web 04/05/2008

De NoVo Design of Nonpeptidic Compounds
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2735
Scheme 1a
a Reagents and conditions: (a) furan or thiophene, n-BuLi, ether, -40 °C; (b) DDQ, NaOH; (c) NaOEt, reflux; (d) n-BuLi, ether, N,N-dimethyl-2[(4′-
bromophenyl)thio]ethylamine, -40 °C; (e) DDQ, NaOH.
85%. Hydrochloride salt was prepared using the method described
for compound 1.
Experimental Section
Computer Modeling. An Octane2 workstation or a SGI
Onyx3800 supercomputer was used to conduct in silico screening,
visualization, and other computational tasks. Sybyl suite of software
and Unity suite were used for database searches, comparisons, and
other related tasks. Except when specified, default parameters and
constraints were used in database searches.
2-({4-[2-{[2-(Dimethylamino)ethyl]thio}-6-(2-furyl)pyrimidin-
4-yl]phenyl}thio)-N,N-dimethyl Ethanamine (3). Compound 3
was synthesized from compound 1 (500 mg, 2 mmol) and 2-(4-
bromophenylthio)-N,N-dimethylethanamine (624 mg, 2.4 mmol)
using the method described for compound 28a, and the product
was obtained in 72% yield (617 mg). Compound 3 (215 mg, 0.5
mmol) was dissolved in a small amount of methanol (1 mL). Then
a solution of HCl in diethyl ether (3 mL, 1M) was added dropwise
at 0 °C and the mixture was stirred for 30 min. Diethyl ether (20
mL) was added, and the precipitated solid was filtered and dried
under vacuum to obtain the corresponding hydrochloride salt for
compound 3.
2-{[4-(4-{[2-(Dimethylamino)ethyl]thio}phenyl)-6-(2-thienyl)py-
rimidin-2-yl]thio}-N,N-dimethyl Ethanamine (4). Starting from
compound 2 (500 mg, 1.88 mmol) and N,N-dimethyl-2[(4′-
bromophenyl)thio]ethylamine (588 mg, 2.26 mmol), compound 4
was synthesized using the method described for compound 28a,
with the final yield of 668 mg (76%).
Synthesis. General. All reactions involving air- or moisture-
sensitive reagents were performed under nitrogen atmosphere. All
solvents and reagents were obtained from commercial sources and
dried using common procedures. Column chromatography was
performed using 60 Å (70–230 mesh) silica gel. NMR spectra were
recorded on a Varian spectrometer (300 or 400 MHz for 1H; 75 or
100 MHz for 13C). Chemical shifts are reported in δ ppm using
tetramethylsilane as the reference. Mass spectra were determined
in a Q-Star mass spectrometer using either ESI or EI technique.
Purity of the compounds were established using Waters Delta 600
HPLC (symmetry C18, 3.5 µm, 4.6 × 75 mm column), equipped
with a 600 controller, and a 996 photodiode array detector using
isocratic conditions (15 min; 0.5 mL/min flow rate; 60 to 100%
0.1% acetic acid in water/0–40% methanol). All solvents for HPLC
were obtained from commercial sources and filtered through Waters
membranes, 47 mm GHP 0.45 µm (Pall Corporation), and degassed
with helium. Samples for HPLC were filtered through Waters
Acrodisc syringe filters.
2-Chloro-4-(2-furyl) Pyrimidine (28a). Furan (15 mmol) was
treated with n-butyl lithium (5 mmol) in anhyd diethyl ether at -40
°C for 30 min. Then a solution of 2-chloropyrimidine (4.5 mmol) in
diethyl ether (10 mL) was added dropwise at -40 °C, and the resultant
mixture was stirred at the same temperature for 1 h. The temperature
of the reaction mixture was slowly allowed to rise to 0 °C and the
stirring was continued for an additional hour. The reaction was
quenched with water (0.5 mL) in THF (3 mL) and stirred for an
additional 15 min. The reaction mixture was treated with DDQ (5
mmol) in THF (6 mL), stirred at room temperature for 25 min, and
cooled in an ice bath. The reaction mixture was treated with hexanes
(6 mL) and then with a cold solution of NaOH (3 M, 5 mL, 15 mmol)
and stirred for 5 min. The organic layer was separated, washed with
brine, and dried with anhyd Na2SO4. Solvent was evaporated to dryness
and the residue was purified by column chromatography (hexanes/
CH2Cl2) to yield 28a (92% yield; Scheme 1).
2-Chloro-4-(2-thienyl) Pyrimidine (28b). This compound was
synthesized from 2-chloropyrimidine (515 mg, 4.5 mmol) and
thiophene (1.26 g, 15 mmol) using the procedure described for
compound 28a, and the product 28b was isolated as a solid (832
mg, 94% yield; Scheme 1).
2-{[4-(2-Furyl)pyrimidin-2-yl]thio}-N,N-dimethyletha-
namine (1). Metallic sodium (120 mg, 5.2 mmol) was added to
anhyd ethanol (10 mL), and the resulting solution of sodium
ethoxide was treated with 2-(dimethylamino)ethanethiol hydro-
chloride (370 mg, 2.6 mmol). The mixture was stirred for 15 min
at rt, then treated with compound 28a (2 mmol), and stirred for an
additional 12 h at 70 °C . The solvent was evaporated under
vacuum, and the crude product was purified by column chroma-
tography (hexanes/NEt3/EtOH, 80:15:5) to obtain compound 1 as
an oil (404 mg, 81%; Scheme 1).
General Method for the Synthesis of 31, 32, and 33 (Scheme
2). 2,4-Dichloro-6-ethoxypyrimidine (31a), 4,6-Dichloro-2-
ethoxypyrimidine (32a), and 4-Chloro-2,6-diethoxypyrimidine
(33a). Sodium (125 mg, 5.54 mmol) was dissolved in absolute
ethanol (25 mL) at 0 °C and stirred for 15 min under argon. 2,4,6-
Trichloropyrimidine 29 (1 g, 5.54 mmol) was added to the above
reaction mixture and was stirred at the same temperature for an
additional 24 h. Evaporation of the solvent and separation via
column chromatography of the crude product produced compounds
31a (821 mg, 78%), 32a (160 mg, 15%), and minor 33a (34 mg,
3%).
2,4-Dichloro-6-isopropoxypyrimidine (31b), 4,6-Dichloro-2-
isopropoxyrimidine (32b), and 4-Chloro-2,6-diisopropoxypyri-
midine (33b). Sodium (6.27 mg, 2.73 mmol) was dissolved in
anhyd isopropanol (25 mL) at 0 °C and stirred for 15 min under
argon. 2,4,6-Trichloropyrimidine 29 (500 mg, 2.73 mmol) was
added to the above reaction mixture and was stirred at the same
temperature for 24 h. Evaporation of the solvent and separation
via column chromatography of the crude product produced com-
pounds 31b (428 mg, 76%), 32b (108 mg, 19%), and 33b (40 mg,
3%).
2,2′-[(6-Ethoxypyrimidine-2,4-diyl)bis(oxy)]bis(N,N-dimeth-
ylethanamine) (5). To a mixture of sodium (58 mg, 2.5 mmol) in
anhyd THF (25 mL) was added an equivalent amount of N,N-
dimethylethanolamine (223 mg, 2.5 mmol), and the solution was
stirred at 0 °C. Then compound 31a (193 mg, 1 mmol) dissolved
in anhyd THF (5 mL) was added and continued stirring at 0 °C.
After completion of the reaction, the solvent was evaporated under
reduced pressure, and the residue was purified by column chro-
matography (CHCl3/MeOH/Et3N, 92:5:3) to afford compound 5
(275 mg, 92% yield).
2,2′-[(6-Isopropoxypyrimidine-2,4-diyl)bis(oxy)]bis(N,N-di-
methylethanamine) (6). To a mixture of sodium (2.5 equiv) in
anhyd THF (25 mL) was added an equivalent amount of N,N-
dimethylethanolamine (2.5 equiv), and the solution was stirred at
0 °C. Then compound 31b (1 equiv), dissolved in 5 mL of anhyd
THF, was added and the stirring was continued at 0 °C. After the
completion of the reaction, the solvent was evaporated and the
N,N-Dimethyl-2-{[4-(2-thienyl)pyrimidin-2-yl]thio}ethanamine
(2). This compound was synthesized from 28b (393 mg, 2 mmol)
using the same method described for compound 1. Yield: 451 mg,

2736 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Bello et al.
Scheme 2a
a Reagents and conditions: (a) sodium, 0 °C, 24 h; (b) N,N-dimethylethanolamine sodium, THF, 0 °C, 24 h; (c) 2-methoxyethylamine, THF, reflux; (d)
isopropoxy propylamine, ethanol, Et3N, 45 °C, 24 h; (e) Et3N, DMF, 150 °C, 8 h; (f) NaH, Et3N, ROH, THF, reflux, 14–24 h.
residue was purified by column chromatography (CHCl3/MeOH/
Et3N ) 92:5:3) to afford compound 6 (98%).
dispersion in mineral oil, 200 mg, 5 mmol) in anhyd THF (5 mL)
at 0 °C for 15 min. A solution of compound 34a (274 mg, 1 mmol)
and triethylamine (1 mL) in anhyd THF (15 mL) at 0 °C was slowly
added, and the reaction mixture was refluxed overnight. After
cooling to room temperature, the solution was quenched with cold
water (10 mL) and extracted with ethyl acetate (3 × 20 mL). The
combined organic layers were washed with brine (2 × 15 mL),
dried with sodium sulfate, concentrated, and purified by column
chromatography (EtOAc/MeOH/Et2O/TEA ) 10:1:3:1) to yield
compound 9 as a pale yellow syrup (283 mg, 89%).
4-(3-Aminopropoxy)-6-ethoxy-N-(3-isopropoxypropyl)pyrimi-
din-2-amine (10). This compound was synthesized from 31a (274
mg, 1 mmol) and 3-aminopropanol (375 mg, 5 mmol) using the
procedure described for compound 9. The crude product was
purified by chromatography (CHCl3/MeOH/TEA ) 9:1:0.5) to
obtain 283 mg (90% yield) product as pale yellow liquid.
4-(4-Aminobutoxy)-6-ethoxy-N-(3-isopropoxypropyl)pyrimi-
din-2-amine (11). This compound was synthesized from 31a (274
mg, 1 mmol) and 4-amino-1-butanol (446 mg, 5 mmol) using the
procedure described for compound 9, and the reaction was refluxed
for 20 h under nitrogen atmosphere. The crude product was purified
by column chromatography (CHCl3/MeOH/Et3N ) 9:1:0.5) to
obtain compound 11 as a pale yellow syrup (150 mg, 46%).
4-(2-(Dimethylamino)ethoxy)-6-isopropoxy-N-(3-isopropoxypro-
pyl)pyrimidin-2-amine (12). This compound was synthesized from
31b (288 mg, 1 mmol) and N,N-dimethylethanolamine (445 mg, 5
mmol) using the procedure described for compound 9, and the
reaction was refluxed for 24 h under nitrogen atmosphere. The crude
product was purified by column chromatography (CHCl3/MeOH/
TEA ) 9:1:0.5) to obtain compound 12 as a pale yellow liquid
(302 mg, 89%).
2,6-Diisopropoxy-N-(2-methoxyethyl)pyrimidin-4-amine (7).
Compound 33b (300 mg, 1.3 mmol) was dissolved in anhyd THF
(5 mL) and 2-methoxyethylamine (293 mg, 3.9 mmol) was added.
The reaction mixture was refluxed overnight. Evaporation of the
solvent and the purification of the crude by column chromatography
yielded compound 7 (339 mg, 97% yield).
4-Chloro-6-ethoxy-N-(3-isopropoxypropyl)pyrimidin-2-
amine (34a). Compound 31a (1 g, 5.18 mmol) and TEA (1 mL)
were dissolved in anhyd ethanol (70 mL) and then 3-isopro-
poxypropylamine (1.75 g, 15 mmol) was added. The reaction
mixture was stirred at 45 °C overnight. The solvent was evaporated
under reduced pressure, and the residue was purified by chroma-
tography (hexanes/CHCl3/Et2O ) 5:5:1) to obtain compound 34a
as a white solid (1.23 g, 87%).
4-Chloro-6-isopropoxy-N-(3-isopropoxypropyl)pyrimidin-2-
amine (34b). Compound 34b was synthesized from compound 31a
(500 mg, 2.59 mmol) and isopropanol (50 mL) using the procedure
followed for 34a. Yield ) 626 mg (84%).
6-Ethoxy-N2-(3-isopropoxypropyl)-N4,N4-dimethylpyrimidine-
2,4-diamine (8). A solution of compound 34a (273 mg, 1 mmol)
and triethylamine (1 mL) in DMF (20 mL) was stirred at 150 °C
for 8 h. The reaction mixture was cooled to room temperature, and
the solvent was evaporated under vacuum. The residue was taken
in water (20 mL) and was extracted with ethyl acetate (3 × 20
mL). The combined organic layers were washed with brine (2 ×
15 mL), dried (sodium sulfate), and concentrated under vacuum.
The crude was purified by column chromatography (hexane/
chloroform/diethyl ether ) 2:2:1) to obtain compound 34a as a
syrup (180 mg, 64%).
4-(2-(Dimethylamino)ethoxy)-6-ethoxy-N-(3-isopropoxypro-
pyl)pyrimidin-2-amine (9). N,N-Dimethylethanolamine (445 mg,
5 mmol) was added to a suspension of sodium hydride (60%
4-(3-Aminopropoxy)-6-isopropoxy-N-(3-isopropoxypropyl)py-
rimidin-2-amine (13). This compound was synthesized from 31b
(288 mg, 1 mmol) and 3-aminopropanol (375 mg, 5 mmol) using

De NoVo Design of Nonpeptidic Compounds
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2737
Scheme 3a
a Reagents and conditions: (a) EtOH, 40 °C, 24 h; (b) NaH, THF, DIEA, 40 °C, 20 h; (c) NaH, THF, N,N-dimethylethanolamine, 0.3 equiv of DIEA, 60
°C, 24 h.
the procedure described for compound 9. The product was isolated
as a pale yellow liquid (280 mg, 86%).
THF (2 mL), sodium hydride (166 mg, 4.15 mmol; 60% dispersion
in mineral oil) was added, and the mixture was stirred for 15 min
at 0 °C. Then 4,6-dichloro-N-(2-methoxyethyl)pyrimidin-2-amine
36a (840 mg, 3.78 mmol) and a catalytic amount of DIEA (0.13
mL, 0.756 mmol) were added, and the mixture was stirred at 40
°C for 20 h under a nitrogen atmosphere. The reaction mixture
was quenched with saturated aqueous ammonium chloride solution,
extracted with ethyl acetate (3 × 15 mL) and the combined organic
layers were washed with brine. The organic phase was dried over
anhyd sodium sulfate and concentrated under vacuum. The residue
was purified by column chromatography (silica gel) using a gradient
elution (CHCl3/MeOH/TEA 99:0:1f97:2:1) to afford compound
40a as a pale yellow liquid (727 mg, 70%).
4-(2-Aminoethoxy)-6-chloro-N-(3-isopropoxypropyl)pyrimi-
din-2-amine (40b). Compound 40b was synthesized from 4,6-
dichloro-N-(3-isopropoxypropyl)pyrimidin-2-amine 36b (1 g, 3.78
mmol) and 2-aminoethanol 38 (y ) 2; 231 mg, 3.78 mmol) using
the procedure described for compound 40a. Yield: 655 mg, 69%.
4-(4-Aminobutoxy)-6-chloro-N-(3-isopropoxypropyl)pyrimi-
din-4-amine (40c). Compound 40c was synthesized from 4,6-
dichloro-N-(3-isopropoxypropyl)pyrimidin-2-amine 36b (1 g, 3.78
mmol) and 4-aminobutan-1-ol 38 (y ) 4; 337 mg, 3.78 mmol) using
the procedure described for compound 40a. Yield: 982 mg, 82%.
General Method for the Synthesis of 36 and 37 (Scheme 3).
4,6-Dichloro-N-(2-methoxyethyl)pyrimidin-2-amine (36a) and
2,6-Dichloro-N-(2-methoxyethyl)pyrimidin-4-amine (37a). To a
solution of 2,4,6-trichloropyrimidine 29 (7 g, 38.1 mmol) in 15
mL of anhyd ethanol, 2-methoxyethanamine 35a (2.861 g, 38.1
mmol) was added. The reaction mixture was stirred at 40 °C for
24 h, and then the solvent was removed under reduced pressure.
The crude product was purified by column chromatography (hexane/
EtOAc, 9:1) to obtain compound 36a (4.89 g, 58%) and compound
37a (2.54 g, 30%) as a white solid.
4,6-Dichloro-N-(3-isopropoxypropyl)pyrimidin-2-amine (36b)
and 2,6-Dichloro-N-(3-isopropoxypropyl)pyrimidin-4-amine
(37b). This compound was synthesized from 2,4,6-trichloropyri-
midine 29 (7 g, 38.1 mmol) and 3-isopropoxypropan-1-amine 35b
(4.462 g, 38.1 mmol) by the method described for compound 36a
and 37a. Compound 36b: white solid (5.91 g, 59%); compound
37b: white solid (3.13 g, 31%).
General Procedure for the Synthesis of Compounds 40a-40c
and 41a-41e (Scheme 3). 4-(4-Aminobutoxy)-6-chloro-N-(2-
methoxyethyl)pyrimidin-2-amine (40a). To an ice-cooled solution
of 4-amino-1-butanol 38 (y ) 4; 337 mg, 3.78 mmol) in anhyd

2738 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Bello et al.
2-(2-Aminoethoxy)-6-chloro-N-(2-methoxyethyl)pyrimidin-4-
amine (41a). Compound 41a was synthesized from 2,6-dichloro-
N-(2-methoxyethyl)pyrimidin-4-amine 37a (1 g, 4.5 mmol) and
2-aminoethanol 39a (275 mg, 4.5 mmol) using the procedure
described for compound 40a. Yield: 720 mg, 65%.
Scheme 4a
2-(3-Aminopropoxy)-6-chloro-N-(2-methoxyethyl)pyrimidin-
4-amine (41b). Compound 41b was synthesized from 2,6-dichloro-
N-(2-methoxyethyl)pyrimidin-4-amine 37a (1 g, 4.5 mmol) and
3-amino-1-propanol 39b (338 mg, 4.5 mmol) using the procedure
described for compound 40a. Yield: 850 mg, 72%.
2-(3-Aminopropoxy)-6-chloro-N-(3-isopropoxypropyl)pyrimi-
din-4-amine (41c). Compound 41c was synthesized from 2,6-
dichloro-N-(3-isopropoxypropyl)pyrimidin-4-amine 37b (1 g, 3.78
mmol) and 3-amino-1-propanol 39b (284 mg, 3.78 mmol) using
the procedure described for compound 40a. Yield: 810 mg, 71%.
2-[4-(2-Aminoethyl)phenoxy]-6-chloro-N-(3-isopropoxypropyl)
pyrimidin-4-amine (41d). Compound 41d was synthesized from 2,6-
dichloro-N-(3-isopropoxypropyl)pyrimidin-4-amine 37b (280 mg, 1.06
mmol) and 4-(2-aminoethyl)phenol 39c (145 mg, 1.06 mmol) using
the procedure described for compound 40a. Yield: 240 mg, 62%.
2-[4-(2-Aminoethyl)phenoxy]-6-chloro-N-(2-methoxyethyl)py-
rimidin-4-amine (41e). Compound 41d was synthesized from 2,6-
dichloro-N-(2-methoxyethyl)pyrimidin-4-amine 37a (378 mg, 1.70
mmol) and 4-(2-aminoethyl)phenol 39c (233 mg, 1.70 mmol) using
the procedure described for compound 40a. Yield: 380 mg, 69%.
General Procedure for the Synthesis of Compounds 14–21
(Scheme 3). 4-(4-Aminobutoxy)-6-(2-(dimethylamino)ethoxy)-
N-(2-methoxyethyl)pyrimidin-2-amine (14). Sodium hydride (360
mg, 9 mmol, 60% dispersion in mineral oil) was added to an ice-
cooled solution of N,N-dimethylethanolamine (802 mg, 9 mmol)
in anhyd THF (15 mL), and the mixture was stirred for 30 min at
0 °C. Then 4-(4-aminobutoxy)-6-chloro-N-(2-methoxyethyl)pyri-
midin-2-amine 40a (495 mg, 1.8 mmol) and a catalytic amount of
DIPEA (0.1 mL, 0.5 mmol) were added, and the mixture was stirred
at 60 °C for 24 h under a nitrogen atmosphere. The reaction mixture
was quenched with saturated aqueous NH4Cl solution, and the crude
was extracted with ethyl acetate (3 × 20 mL). The combined ethyl
acetate layers were washed with brine (15 mL), dried over anhyd
sodium sulfate, and concentrated under vacuum. The residue was
purified on a silica gel column chromatography using gradient
elution with CHCl3/MeOH/TEA (97:0:3 to 92:4:4) to yield com-
pound 14 as a light yellow liquid (420 mg, 71%). Compound 14
was converted into its hydrochloride salt following the method
described for the hydrochloride salt of compound 3.
4-(2-Aminoethoxy)-6-(2-(dimethylamino)ethoxy)-N-(3-isopro-
poxypropyl)pyrimidin-2-amine (15). Compound 15 was synthe-
sized from 4-(2-aminoethoxy)-6-chloro-N-(3-isopropoxypropyl)py-
rimidin-2-amine 40b (491 mg, 1.70 mmol) and N,N-dimethylethanol
amine (758 mg, 8.5 mmol) using the procedure described for
compound 14. Yield: 380 mg, 65%.
4-(4-Aminobutoxy)-6-[2-(dimethylamino)ethoxy]-N-(3-isopro-
poxypropyl)pyrimidin-4-amine (16). Compound 15 was synthe-
sized from 4-(4-aminobutoxy)-6-chloro-N-(3-isopropoxypropyl)py-
rimidin-2-amine 40c (500 mg, 1.6 mmol) and N,N-dimethylethanol
amine (713 mg, 8 mmol) using the procedure described for
compound 14. Yield: 0.384 g, 66%.
2-(2-Aminoethoxy)-6-(2-(dimethylamino)ethoxy)-N-(2-meth-
oxyethyl)pyrimidin-4-amine (17). Compound 17 was synthesized
from 2-(2-aminoethoxy)-6-chloro-N-(2-methoxyethyl)pyrimidin-4-
amine 41a (493 mg, 2 mmol) and N,N-dimethylethanol amine (890
mg, 10 mmol) using the procedure described for compound 14.
Yield: 380 mg, 64%.
2-(3-Aminopropoxy)-6-(2-(dimethylamino)ethoxy)-N-(2-meth-
oxyethyl)pyrimidin-4-amine (18). Compound 18 was synthesized
from 2-(3-aminopropoxy)-6-chloro-N-(2-methoxyethyl)pyrimidin-
4-amine 41b (495 mg, 1.9 mmol) and N,N-dimethylethanol amine
(847 mg, 9.5 mmol) using the procedure described for compound
14. Yield: 470 g, 79%.
a Reagents and conditions: (a) 1H-pyrazole-1-carboxamidine ·HCl/DIEA,
rt, 40 h.
pyl)pyrimidin-4-amine 41c (430 mg, 1.42 mmol) and N,N-
dimethylethanol amine (633 mg, 7.1 mmol) using the procedure
described for compound 14. Yield: 480 mg, 82%.
2-(4-(2-Aminoethyl) phenoxy)- 6-(2-(dimethylamino)ethoxy)-
N-(3-isopropoxypropyl)pyrimidin-4-amine (20). Compound 20
was synthesized from 2-(4-(2-aminoethyl)phenoxy)-6-chloro-N-(3-
isopropoxypropyl)pyrimidin-4-amine 41d (183 mg, 0.5 mmol) and
N,N-dimethylethanol amine (223 mg, 2.5 mmol) using the procedure
described for compound 14. Yield: 120 mg, 56%.
2-(4-(2-Aminoethyl)phenoxy)-6-(2-(dimethylamino)ethoxy)-N-
(2-methoxyethyl)pyrimidin-4-amine (21). Compound 21 was
synthesized from 2-(4-(2-aminoethyl)phenoxy)-6-chloro-N-(2-meth-
oxyethyl)pyrimidin-4-amine 41e (130 mg, 0.4 mmol) and N,N-
dimethylethanolamine (178 mg, 2 mmol) using the procedure
described for compound 14. Yield: 90 mg, 61%.
General Procedure for the Synthesis of Compounds 22–26
(Scheme 4). 1-(4-(6-(2-(Dimethylamino)ethoxy)-2-(2-methoxy-
ethylamino)pyrimidin-4-yloxy)butyl)guanidine Hydrochloride
(22). 4-(4-Aminobutoxy)-6-(2-(dimethylamino)ethoxy)-N-(2-meth-
oxyethyl)pyrimidin-2-amine 16 (50 mg, 0.15 mmol) was dissolved
in DMF (2 mL) and was treated with 1H-pyrazole-1-carbox-
amidine·HCl (23 mg, 0.16 mmol) and DIEA (27 µL, 0.16 mmol).
The reaction mixture was stirred at room temperature under nitrogen
for 40 h. Solvent was evaporated under reduced pressure, and the
crude product was washed with several portions of ether and dried.
The crude product was recrystallized from MeOH/ether to obtain
compound 22 as a white solid (44 mg, 72%).
1-(4-(6-(2-(Dimethylamino)ethoxy)-2-(3-isopropoxypropylami-
no)pyrimidin-4-yloxy)butyl)guanidine Hydrochloride (23). Com-
pound 23 was synthesized from 4-(4-aminobutoxy)-6-(2-(dimeth-
ylamino)ethoxy)-N-(3-isopropoxypropyl)pyrimidin-2-amine 14 (50
mg, 0.13 mmol) and 1H-pyrazole-1-carboxamidine·HCl (23 mg,
0.16 mmol) using the procedure described for compound 22. Yield:
46 mg, 82%.
1-(2-(4-(2-(Dimethylamino)ethoxy)-6-(2-methoxyethylami-
no)pyrimidin-2-yloxy)ethyl)guanidine Hydrochloride (24). Com-
pound 24 was synthesized from 2-(2-aminoethoxy)-6-(2-(dimeth-
ylamino)ethoxy)-N-(2-methoxyethyl)pyrimidin-4-amine 17 (45 mg,
0.15 mmol) and 1H-pyrazole-1-carboxamidine·HCl (23 mg, 0.16
mmol) using the procedure described for compound 22. Yield: 45
mg, 80%.
2-(3-Aminopropoxy)-6-(2-(dimethylamino)ethoxy)-N-(3-iso-
propoxypropyl)pyrimidin-4-amine (19). Compound 19 was syn-
thesized from 2-(3-aminopropoxy)-6-chloro-N-(3-isopropoxypro-
1-(3-(4-(2-(Dimethylamino)ethoxy)-6-(2-methoxyethylami-
no)pyrimidin-2-yloxy)propyl)guanidine Hydrochloride (25). Com-
pound 25 was synthesized from 2-(3-aminopropoxy)-6-(2-(di-

De NoVo Design of Nonpeptidic Compounds
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2739
Figure 1. Ortho view of the structure of IFN-R2a (pdb: 1ITF). The
IRRP regions are illustrated as a Connolly surface: IRRP-1 (residues
29–35), IRRP-2 (residues 78–95), and IRRP-3 (residues 123–140) are
depicted in red, green, and blue-green, respectively.
methylamino)ethoxy)-N-(2-methoxyethyl)pyrimidin-4-amine 18 (45
mg, 0.14 mmol) and 1H-pyrazole-1-carboxamidine·HCl (23 mg,
0.16 mmol) using the procedure described for compound 22. Yield:
48 mg, 85%.
1-(3-(4-(2-(Dimethylamino)ethoxy)-6-(3-isopropoxypropyl)ami-
no)pyrimidin-2-yloxy)propyl)guanidine Hydrochloride (26). Com-
pound 26 was synthesized from 2-(3-aminopropoxy)-6-(2-(di-
methylamino)ethoxy)-N-(2-methoxyethyl)pyrimidin-4-amine 19 (45
mg, 0.13 mmol) and 1H-pyrazole-1-carboxamidine·HCl (23 mg,
0.16 mmol) using the procedure described for compound 22. Yield:
48 mg, 87%.
Figure 2. Stereoview of the structural features on surface of human
IFN-R2a used in the in silico screens targeting IRRP-1 and IRRP-3
regions (panels A and B, respectively). Amino acids are shown in
capped-stick models. Grey spheres represent the centers of each selected
feature. Connolly surface is depicted as a mesh in green and the
backbone of the interferon is shown as a ribbon.
Design Strategy. The strategy for the design of IFN mimics to
create small molecule antagonists considered (i) the availability of
hot spot regions on IFN-R, (ii) the availability of the three-
dimensional structures of IFNs-R/ꢀ, and (iii) the possibility to map
small molecules onto the hot spot regions on the IFN.12,13,22,23 Site-
directed mutagenesis studies further identified three regions of
conserved residues among IFN-Rs, the AB-loop spanning Cys29-
Phe36 (AB domain), the helix C spanning Glu78-Asp95 (C
domain), and the helix D along with the adjacent DE loop spanning
Tyr122-Ala139 (D domain), as functionally important regions
interacting with the IFNAR.14,24 These three regions are labeled
as IRRP-1, IRRP-2, and IRRP-3, respectively (IRRP ) interferon
receptor recognition peptide). When IRRPs are mapped onto the
X-ray structure of IFN-R2a, these regions concentrate on two sides
of the protein (Figure 1).16,23,25 The solvent accessible surface areas
of IRRP-1, IRRP-2, and IRRP-3 are 457, 839, and 865 Å2,
respectively. Each face of the IFN binds to one subdomain of
IFNAR: IRRP-1 and IRRP-3 bind to an IFNAR2 subdomain and
the IRRP-2 region binds to an IFNAR-1 subdomain.11 Productive
interactions with both IFNAR1 and IFNAR2 are essential for a
molecule to function as an agonist. However, for an antagonist, it
would suffice to bind to either IFNAR1 or IFNAR2 such that an
IFN molecule cannot productively bind to both receptor subdomains
to activate the receptor and elicit a biological response.11 We
initially targeted the regions of IRRP-1 and IRRP-3 as the templates
to mimic on the surface of IFN. Because these two IRRPs are
contiguous and bind to the IFNAR2 portion of the receptor,
molecules mimicking either of these IRRPs are anticipated to
interfere with IFN-inducible receptor-mediated activities. In other
words, such small molecules would be antagonists at the IFNAR.
hydrophobic centers (Hp1 and Hp2, respectively); Nꢁ of the residue
Lys31 and the midpoint between NH1 and NH2 atoms in the residue
Arg33 were defined as the positively charged centers (Pos1 and
Pos2, respectively; Figure 2A). An in silico database search using
UNITY suite of software was performed using the above definition
for the molecular search against three databases, with a total of
over 300000 molecules: the NCI database, the Maybridge database,
and the LeadQuest database.26 The search criteria included the
identification of the hit molecules matching at least three of the
four features (Hp1, Hp2, Pos1, and Pos2). A distance tolerance of
1 Å was set between any two features and a tolerance of 1 Å was
set as a volume constraint. In the first filtration process, 1010
compounds were selected that satisfy the above criteria. These
molecules were further screened for their molecular complexity and
synthetic feasibility. Finally, three compounds 42 (NCI-119030),
43 (1502–09435), and 44 (CD11398) were identified from the above
in silico filtration process (Figure 3A,C).27 These molecules were
further refined and arrived at the focused library featuring com-
pounds 5-26 represented by species II in Figure 4C.
Another in silico search targeting IRRP-3 was initiated to design
a second small molecule mimicking this region. IRRP-3 on human
IFN-R2a spans the residues 122–139. Side chains from six residues,
namely, Arg125, Leu128, Lys131, Glu132, Lys133, and Lys134,
were used as the hot spot regions for the mimicry of IRRP-3. Based
on the structural features of these six residues on the surface of
IFN-R2a, the following structural features were selected for the in
silico search: the midpoint between the atoms NH1 and NH2 of
the residue Arg125, Nꢀ atoms in Lys131, Lys133, and Lys134
(designated as Pos1, Pos2, Pos3, and Pos4, respectively); addition-
ally, the midpoint between the atoms CD1 and CD2 on the residue
Leu128 (designated as Hp) and the midpoint between OE1 and
OE2 on the residue Glu132 (designated as Neg) were assigned as
hydrophobic and negatively charged centers, respectively (Figure
2B). These six regions were selected on the basis of their position
The three-dimensional structure of the IRRP-1 region in IFN-
R2a was used as a template to perform the molecular design. Based
on the structural analyses of the IRRP-1 segment, four residues
were chosen as the key mimicry residues. These are the hydrophobic
side chain of Leu30, the hydrophobic aromatic side chain on His34,
and the positively charged groups on Lys31 and Arg33. The
midpoint between the atoms CD1 and CD2 on the residue Leu30
and the center of the imidazole ring of His34 were defined as

2740 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Bello et al.
structure targeting IRRP-3 (Figure 3B,C).28 Thus, a small set of
compounds 1–4 were designed to evaluate the potential of this
designed core mimicking IRRP-3. Below, chemical synthesis for
all the designed compounds, 1–26 is outlined.
In Vitro Activity Evaluation. Two series of experiments were
conducted to evaluate the antagonistic effects of the designed
compounds 1–26 on IFN-inducible events. First, all compounds
(100 µm) were screened for their inhibitory effects on IFN-induced
Stat-1 tyrosine phosphorylation. Next, we examined their ability
to interfere with IFN-inducible Stat complex formation and DNA
binding. Daudi cells were either left untreated or treated with IFN
(100 pg) for 15 min in the presence or absence of the target
compounds, as indicated. Cell lysates were then prepared and
resolved by SDS-PAGE, and the proteins were transferred to
nitrocellulose and immunoblotted (WB) for phospho-Stat1, using
standard protocols.29 The membranes were stripped and were
reprobed for Stat1. Alternatively, ꢀ-actin was used as loading
control.
For the evaluation of Stat1 complex formation, Daudi cells were
either left untreated or treated with IFN in the presence or absence
of compounds for 15 min. Nuclear extracts from these cells were
prepared, mixed with a 32P-labeled palindromic IFN-responsive
element (32P-pIRE) probe to assess IFN-inducible Stat1:Stat1 and
Stat1:Stat3 dimer formation and DNA binding, and resolved by
native agarose gel electrophoresis.30 Only those compounds that
showed potential inhibition of IFN activity in the Stat1 phospho-
rylation assay were subjected to this radiolabeled electrophoretic
mobility shift assay (EMSA) to further confirm their inhibitory
activities.
Results and Discussion
Chemistry. Compounds 1-4 were synthesized according to
Scheme 1. Either furan (for compounds 1 and 3) or thiophene
(for 2 and 4) were added to 2-chloropyrimidine after activation
with n-butyl lithium. The crude products were subjected to
oxidation by the treatment with DDQ to yield 28a and 28b.31
Compounds 1 and 2 were obtained by nucleophilic substitution
of chlorine on 28a and 28b by N,N-(dimethylamino)ethanethiol
in the presence of metallic sodium, respectively.32 Compounds
1 and 2 were further substituted at position 6 by the addition-
dehydrogenation using lithiated N,N-dimethyl-2[(4′-bromophe-
nyl)thio]ethylamine followed by the treatment with DDQ to
yield compounds 3 and 4.33
The substituted pyrimidines 5-13 were synthesized starting
from 2,4,6-trichloropyrimidines (Scheme 2). 2,4,6-Trichloro-
pyrimidine was first treated with an appropriate alkoxide to
obtain 2- and 4-monosubstituted derivatives, as well as a small
amount of 2,4-disubstituted derivative. These regioisomers were
separated by silica gel column chromatography. Subsequent
treatment of 4-alkoxy pyrimidine derivative (31a and 31b) with
isopropoxy propylamine in the presence of ethanol and triethyl-
amine led to the regioselective substitution at position 2 yielding
34a and 34b. Treatment of compounds 31a and 31b with N,N-
dimethylethanolamine yielded compound 5 and 6, respectively.
Treatment of 33b with 2-methoxyethylamine in THF under
refluxing conditions provided access to compound 7. Substitu-
tion with various alkoxides in the presence of triethyl amine
afforded the substitution at 6-position to obtain the final
pyrimidine derivatives 8-13.
Figure 3. (A) Stereoview of the overlap of IRRP-1 region and the
two hits 42 (in red) and 43 (in yellow) identified from the chemical
databases search. Orange spheres represent the search criteria based
on the structure of IRRP-1: sphere at 12 o’clock represents a cationic
moiety (Pos1), at 3 o’clock represents a hydrophobic moiety (Hp1), at
4 o’clock represents the second cationic moiety (Pos2), and at 6 o’clock
represents the second hydrophobic moiety (Hp2). (B) Stereoview of
the overlap of 45 onto the IRRP-3 segment of IFN-R2a. The backbone
of the protein is shown as a ribbon in magenta, the residues of IRRP-3
are shown in green in capped stick representation, and 45 is shown in
ball-and-stick representation color-coded according to atom type. White
spheres represent the six functional regions that were used in in silico
database search. (C) Chemical structures of 42, 44, and 45.
Compounds 14-21 were synthesized using the protocols in
Scheme 3 starting from 2,4,6-trichloropyrimidine. When com-
pound 29 was treated with an alkoxyamine (methoxy and
isopropyloxy derivatives), regioisomers 36 and 37 were isolated
in 2:1 ratio. These two regioisomers were separated and were
further treated with various aminoalkoxides in presence of
diisopropylethylamine at 40 °C to afford regioselective substitu-
tion at 4- and 2-positions on 36 and 37 to yield compounds
exposed to the solvent and their hydrophobic/electronic charge
characteristics. A distance tolerance of 1 Å was set between any
two features and a tolerance of 1 Å was set as a volume constraint.
A total of 192 compounds were identified from the three databases
matching at least three of the six features used from the IRRP-3
region. These compounds were further screened manually for their
chemical nature, feasibility of synthesis, and/or druggability features,
ultimately selecting one molecule 45 (NCI-619009) as a “hit”

De NoVo Design of Nonpeptidic Compounds
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2741
Figure 4. (A) Schematic of compound 43 and its morphosis into species I mimicking IRRP-1 interactions. (B) Stereoview of the overlap of a
model compound onto the IRRP-1 segment of IFN-R2a. IRRP-1 residues are in cyan and species II is shown as a ball-and-stick model. (C) Chemical
library based on general scaffold I (also see Scheme 1).
40a-c and 41a-e, respectively. Nucleophilic substitution at
the 6-position on compounds 40a-c and 41a-e was carried
out with the appropriate aminoalkoxide at 60 °C in the presence
of sodium hydride and DIEA to afford the target compounds
14-21. A remarkable increase in the yields was observed when
proton scavengers such as diisopropyl ethylamine or triethyl
amine were used in THF as a solvent rather than DMF, which
produced lower yields and side products. Guanidinylation of
the primary amine derivatives 14, 16–19 was achieved by using
pyrazole-1-carboxamidine under basic conditions to yield
compounds 22-26.34 All designed compounds 1–26 were
confirmed for their purity for subsequent biological activity
evaluations.
This effort started with the hypothesis that one could mimic
the “functional hot spots” on the surface of the protein–ligand
IFN-R2a using small molecules. While the design principles
for agonist activity due to such small molecules could be quite
complicated, it was envisioned that (i) mimics of smaller regions
of the surface of IFN-R2a could be feasible and (ii) such small
molecules would interfere with the binding of this ligand at its
receptor. Such molecules would most likely exhibit the antago-
nist activity. The above synthesized two series of compounds,
namely, 1–26 were evaluated for their ability to competitively
inhibit IFN in the cell-based assays.
Activation of the IFNAR that results in comprehensive signal
transduction requires that both IFNAR1 and IFNAR2 be
activated, for example, the ligand has to interact with both
subdomains.11 This activation involves the phosphorylation-
activation of IFNAR2- and IFNAR1-associated kinases, Jak1
and Tyk2, respectively, then the phosphorylation of the intra-
cellular domains of the receptor subunits on specific tyrosine
residues by these kinases. This results in the recruitment of Stat
proteins to the receptor and their phosphorylation-activation,
specifically the Stat proteins Stat1, Stat2, Stat3, and Stat5. Once
phosphorylated, Stats will form complexes: Stat1:Stat1, Stat1:
Stat3, Stat3:Stat3, Stat2:Stat1/IRF-9, and Stat5:CrkL. These
activated Stat complexes translocate to the nucleus where they
bind to specific target elements in the promoters of IFN sensitive
genes, thereby activating their transcription. IFN-sensitive genes
mediate the different IFN-inducible biological responses.35

2742 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Bello et al.
at IFNAR (Figure 5C). Viewed altogether, compounds 3 and 4
function as antagonists of IFN-IFNAR interactions.
We disclose here the design of small molecule mimics based
on the features of specific surface regions on the IFN, as
antagonists for IFNAR. Two regions IRRP-1 and IRRP-3 on
the surface of IFN-R2a were used to design the molecular
templates de novo. An iterative in silico screening was
conducted to design a small focused library of compounds and
yielded two moderately potent antagonist molecules. Identifica-
tion of agonists using structure-based design is the next step in
this challenge, and most likely, such agonist molecules may
have to consider additional structural features in the design
process. Principles disclosed here pave the way to a novel series
of small molecules that could potentially be used to design
mimics of various functional hotspots on the surface of
interferon.
Acknowledgment. This work was supported by a grant from
the Canadian Institutes of Health Research (E.N.F. and L.P.K.).
L.P.K. is a recipient of an Rx&D HRF-CIHR research career
award. An infrastructure grant from the Ontario Innovation Trust
provides support for the Molecular Design and Information
Technology Centre (MDIT).
Supporting Information Available: 1H NMR, 13C NMR, UV,
and HPLC purity data for the synthesized compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
References
(1) Platanias, L. C. Mechanisms of type-I- and type-II-interferon-mediated
signalling. Nat. ReV. Immunol. 2005, 5, 375–386.
(2) Guilhot, F.; Chastang, C.; Michallet, M.; Guerci, A.; Harousseau, J. L.;
Maloisel, F.; et al. Interferon R-2b combined with cytarabine versus
interferon alone in chronic myelogenous leukemia: French myeloid
leukemia study group. New Engl. J. Med. 1997, 337, 270–271.
(3) Ahmed, A.; Keefe, E. B. Overview of interferon therapy for chronic
hepatitis C. Clin. LiVer Dis. 1999, 3, 757–773.
(4) Vedantham, S.; Gamliel, H.; Golomb, H. M. Mechanism of interferon
action in hairy cell leukemia: A model of effective cancer biotherapy.
Cancer Res. 1992, 52, 1056.
(5) Billiau, A.; Kieseier, B. C.; Hartung, H. P. Biologic role of interferon
beta in multiple sclerosis. J. Neurol. 2004, 251, 1110–1114.
(6) Lee, P. Y.; Reeves, W. H. Type I interferons as a target of treatment
in SLE. Endocr. Metab. Immune Disord. Drug Targets 2006, 6, 323–
330.
(7) Kyogoku, C.; Tsuchiya, N. A compass that points to lupus: Genetic
studies on type I interferon pathway. Genes Immunol. 2007, 8, 445–
455.
Figure 5. Antagonistic effects of IFN mimetics on IFN-inducible Stat1
phosphorylation. (A) Daudi cells were either left untreated or treated
with IFN alfacon-1 in the absence or in the presence of compounds 3,
4, 1, and 2 for 15 min. (B) In dose–response assays, cells were either
left untreated (lane 1), treated with IFN alfacon-1(lane 2) or IFN
alfacon-1 plus varying doses of 3 (IFN+20, IFN+50 and IFN+80 µM
in lanes 3–5, respectively) and 4 (IFN+20, IFN+50, IFN+80 and
IFN+100 µM in lanes 3–6, respectively) and the extent of Stat1
phosphorylation determined as per panel A. ꢀ-Actin served as the
loading control. (C) Daudi cells were either left untreated (-) (lane 1)
or treated with IFN alfacon-1 (+) (lane 2) in the presence of compound
3 (lane 3) or 4 (lane 4) for 15 min, as indicated. Nuclear extracts were
prepared, mixed with radiolabeled pIRE, resolved by native agarose
gel electrophoresis, then the pIRE-bound Stat complexes visualized by
autoradiography, as shown.
(8) Koutouzov, S.; Mathian, A.; Dalloul, A. Type I interferons and
systemic lupus erythematosus. Autoimmun. ReV. 2006, 5, 554–562.
(9) Cohen, B.; Novick, D.; Barak, S.; Rubinstein, M. Mol. Cell. Biol. 1995,
15, 4208–4214.
(10) Mogensen, K. E.; Lewerenz, M.; Reboul, J.; Lutfalla, G.; Uze, G.
The type I interferon receptor: Structure, function and evolution of a
family business. J. Interferon Cytokine Res. 1999, 19, 1069–1098.
(11) Kumaran, J.; Wei, L.; Kotra, L. P., Fish, E. N. A structural basis for
interferon-R-receptor interactions. FASEB J. 2007, 21, 3288–3296.
(12) Chill, J. H.; Nivasch, R.; Levy, R.; Albeck, S.; Schreiber, G.; Anglister,
J. The human interferon receptor: NMR-based modeling, mapping of
the IFN-R2 binding site, and observed ligand-induced tightening.
Biochemistry 2002, 41, 3575–3585.
(13) Chill, J. H.; Quadt, S. R.; Levy, R.; Schreiber, G.; Anglister, J. The
human type I interferon receptor: NMR Structure reveals the molecular
basis of ligand binding. Structure 2003, 791, 802.
(14) Platanias, L. C.; Fish, E. N. Signaling pathways activated by
interferons. Exp. Hematol. 1999, 27, 1583–1592.
In a first series of experiments, we examined the effects of
all synthesized compounds on the phosphorylation activation
of Stat1 in Daudi lymphoblastoid cells. In time course studies
(5–30 min), our data revealed that none of the compounds
induced the phosphorylation of Stat1 on tyrosine residues (data
not shown). Next, because we predicted that these compounds
may interfere with IFN alfacon-1 binding to IFNAR, we
examined the influence of these compounds on IFN-inducible
Stat1 phosphorylation. The data in Figure 5A,B demonstrate
that compounds 3 and 4 inhibit the IFN-inducible phosphory-
lation of Stat1, in a dose-dependent manner. In subsequent
studies, focusing on compounds 3 and 4, we examined whether
they would exhibit a similar inhibitory effect on the DNA-
binding activity of IFN-inducible Stat1:Stat1 and Stat1:Stat3
complexes. In the electrophoretic mobility shift assays (EMSA)
using the pIRE as a probe for Stat complex binding, compounds
3 and 4 reduced IFN-inducible Stat1:Stat1-pIRE and Stat1:Stat3-
pIRE complex formation thus antagonizing the effects of IFN
(15) Kalvakolanu, D. V. Alternate interferon signaling pathways. Phar-
macol. Ther. 2003, 100, 1–29.
(16) Fish, E. N. Definition of receptor binding domains in interferon-R.
J. Interferon Res. 1992, 12, 257–266.
(17) Muller, U.; Steinhoff, U.; Ries, L. F. L.; Hemmi, S.; Pavlovic, J.;
Zinkernagel, R. M.; Aguet, M. Functional role of type I and II
interferons in antiviral defense. Science 1994, 264, 1918–1921.
(18) Yohannes, D. Disruption of protein-protein interactions. Annu. Rep.
Med. Chem. 2003, 38, 295–304.

De NoVo Design of Nonpeptidic Compounds
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2743
(19) Cochran, A. G. Antagonists of protein-protein interactions. Chem.
Biol. 2000, 7, R85–R94.
were obtained as part of the UNITY software version 4.0, Tripos Inc.,
St. Louis, MO.
(20) Boger, D. L.; Deshrnais, J.; Capps, K. Solution-phase combinatorial
libraries: Modulating cellular signalling by targeting protein-protein
or protein-DNA interactions. Angew. Chem., Int. Ed. 2003, 4138,
4176.
(21) Tan, C.; Wei, L.; Ottensmeyer, F. P.; Goldfine, I.; Yip, C. C.; Batey,
R. A.; Kotra, L. P. Structure-based de novo design of ligands using a
three-dimensional model of the insulin receptor. Bioorg. Med. Chem.
Lett. 2004, 14, 1407–1410.
(22) Roisman, L. C.; Piehler, J.; Trosset, J.-Y.; Scheraga, H. A.; Schreiber,
G. Structure of the interferon-receptor complex determined by
distance constraints from double-mutant cycles and flexible docking.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 13231–13236.
(23) Klaus, W.; Gsell, B.; Labhardt, A. M.; Wipf, B. The three-dimensional
high resolution structure of human interferon R-2a determined by
heteronuclear NMR spectroscopy in solution. J. Mol. Biol. 1997, 274,
661–675.
(24) Waine, G. J.; Thymms, M. J.; Brandt, E. R.; Cheetham, B. F.; Linnane,
A. W. Structure-function study of the region encompassing residues
26–40 of human interferon-R4: Identification of residues important
for antiviral and antiproliferative activities. J. Interferon Res. 1992,
12, 43–48.
(25) Korn, A. P.; Rose, D. R.; Fish, E. N. Three-dimensional model of a
human interferon-R consensus sequence. J. Interferon Res. 1994, 14,
1–9.
(26) Unity software program from Tripos Inc. (St. Louis, MO) was used
to conduct database searches, in conjunction with Sybyl molecular
modeling program. Searches were conducted on an Octane2 dual
processor computer or a 44-processor Onyx3800 supercomputer.
(27) CAS Registry No. for compound 42, 903633-37-4, and for compound
44, 671188-22-0. All the hit compounds were extracted from either
NCI Database, LeadQuest Database or Maybridge database, and these
(28) Schinazi, R. F.; Strekowski, L. Unfused heteropolycyclic compounds
as visucides, especially against human immunodeficiency virus. PCT/
WO 8911279, 1989.
(29) Masters, J.; Hinek, A. A.; Uddin, S.; Platanias, L. C.; Zeng, W. Q.;
McFadden, G.; Fish, E. N. Poxvirus infection rapidly activates tyrosine
kinase signal transduction. J. Biol. Chem. 2001, 276, 48371–48375.
(30) Ghislain, J. J.; Fish, E. N. Application of genomic DNA affinity
chromatography identifies multiple interferon-alpha-regulated Stat2
complexes. J. Biol. Chem. 1996, 271, 12408–12413.
(31) Strekowski, L.; Harden, D. B.; Grubb, W. B.; Patterson, S. E.; Czarny,
A.; Mokrosz, M. J.; Cegla, M. T.; Wydra, R. L. Synthesis of 2-chloro-
4,6-di(heteroaryl)pyrimidines. J. Heterocycl. Chem. 1990, 27, 1393–
1400.
(32) Strekowski, L.; Wilson, W. D.; Mokrosz, J. L.; Mokrosz, M. J.; Harden,
B. D.; Tanious, F. A.; Wydra, R. L.; Crow, S. A. Quantitative
structure-activity relationship analysis of cation-substituted polyaro-
matic compounds as potentiators (amplifiers) of bleomycin-mediated
degradation of DNA. J. Med. Chem. 1991, 34, 580–588.
(33) Wilson, W. D.; Strekowski, L.; Tanious, F. A.; Watson, R. A.;
Mokrosz, J. L.; Strekowska, A.; Webster, G. D.; Neidle, S. Binding
of unfused aromatic cations to DNAsThe influence of molecular twist
on intercalation. J. Am. Chem. Soc. 1988, 110, 8292–8299.
(34) Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. 1H-Pyrazole-1-
carboxamidine hydrochloride: An attractive reagent for guanylation
of amines and its application too peptide synthesis. J. Org. Chem.
1992, 57, 2497–2502.
(35) Brierley, M. M.; Fish, E. N. Stats: Multi-faceted regulators of
transcription. J. Interferon Cytokine Res. 2005, 25, 733–744.
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