Design, synthesis, and evaluation of quinazoline T cell proliferation inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.07.004

We report here on a class of quinazoline molecules that inhibit T cell proliferation. The most potent compound N-p-tolyl-2-(3,4,5-trimethoxyphenyl) quinazolin-4-amine (S101) and its close analogs were found to inhibit the proliferation of T cells from human peripheral blood mononuclear cells (PBMC) and Jurkat cells, with IC₅₀ in the sub-micromolar range. The inhibitor induced G2 cell cycle arrest but did not inhibit IL-2 secretion. The anti-proliferative effect correlated with inhibition of the tyrosine phosphorylation of SLP-76, a molecular element in the signaling pathway of the T cell receptor (TCR). The inhibitor restrained proliferation of lymphocytes with much higher potency than non-hematopoietic cells. This new class of specific T cell proliferation inhibitors may serve as lead molecules for the development of agents aimed at diseases in which T cell signaling plays a role and agents to induce tolerance to grafted tissues or organs.

Full text of DOI:10.1016/j.bmc.2010.07.004

Bioorganic & Medicinal Chemistry 18 (2010) 6404–6413
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and evaluation of quinazoline T cell proliferation inhibitors
*
Idit Sagiv-Barfi, Ester Weiss, Alexander Levitzki
Unit of Cellular Signaling, Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Givat Ram,
Jerusalem 91904, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
We report here on a class of quinazoline molecules that inhibit T cell proliferation. The most potent com-
pound N-p-tolyl-2-(3,4,5-trimethoxyphenyl)quinazolin-4-amine (S101) and its close analogs were found
to inhibit the proliferation of T cells from human peripheral blood mononuclear cells (PBMC) and Jurkat
cells, with IC50 in the sub-micromolar range. The inhibitor induced G2 cell cycle arrest but did not inhibit
IL-2 secretion. The anti-proliferative effect correlated with inhibition of the tyrosine phosphorylation of
SLP-76, a molecular element in the signaling pathway of the T cell receptor (TCR). The inhibitor restrained
proliferation of lymphocytes with much higher potency than non-hematopoietic cells. This new class of
specific T cell proliferation inhibitors may serve as lead molecules for the development of agents aimed at
diseases in which T cell signaling plays a role and agents to induce tolerance to grafted tissues or organs.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 1 June 2010
Revised 1 July 2010
Accepted 2 July 2010
Available online 8 July 2010
Keywords:
T cells
Proliferation
Quinazoline
1. Introduction
phosphodiesterase, which is expressed in lymphocytes and mono-
cytes.18–20
Organ transplantation elicits a complex series of immunologic
processes that are generally categorized as inflammation, immu-
nity, tissue repair, and structural reinforcement of damaged tis-
sues. Macrophages and T cells mediate inflammation by the
secretion of proinflammatory cytokines, for example, interleukin-
2 (IL-2), and by the activation of biochemical cascades such as
the classic complement cascade. The medical community is in need
of novel immunosuppressive agents.1 Immunosuppressive drugs
fall into five groups: (i) regulators of gene expression; (ii) alkylat-
ing agents; (iii) inhibitors of de novo purine synthesis; (iv) inhibi-
tors of de novo pyrimidine synthesis and (v) inhibitors of kinases
and phosphatases.2 Glucocorticoids exert immunosuppressive
and anti-inflammatory activity mainly by inhibiting the expression
of the genes for interleukin-2 and other mediators.3 Cyclophospha-
mide suppresses immune responses mediated by B-lympho-
cytes.4,5 Methotrexate and its polyglutamate derivatives suppress
inflammatory responses through release of adenosine.5 Mycophen-
olic acid and mizoribine inhibit inosine monophosphate dehydro-
genase.6,7 Mycophenolic acid induces apoptosis of activated
T-lymphocytes.8,9 A leflunomide metabolite and brequinar inhibit
dihydroorotate dehydrogenase.10,11 Cyclosporine and FK-506/
tacrolimus inhibit the phosphatase activity of calcineurin.12–16
Rapamycin inhibits signal transduction from the IL-2, epidermal
growth factor and other cytokine receptors.17 Immunosuppressive
and anti-inflammatory compounds in development include
inhibitors of p38 kinase and of the type IV isoform of cyclic AMP
Immunosuppressive medications are associated with toxicity
due to their non-specific immunosuppressive effects. Reducing
immunosuppression can prevent side effects related to over-
immunosuppression. However, since the intrinsic immunosup-
pressive requirements for each donor recipient pair are unknown,
immunosuppressive minimization carries a potential risk of under-
immunosuppression and consequent acute rejection, premature
graft loss and death. A promising future application of immuno-
suppressive drugs is their use in regimens designed to induce tol-
erance to allografts. The first step in finding such drugs is a search
for agents that inhibit T cell proliferation by novel mechanisms, as
the currently used agents, which all possess non-specific broad
immunosuppressive effects. In our search for compounds that inhi-
bit T cell proliferation we chose a known chemical scaffold, the
quinazoline moiety, which has been utilized to develop tyrosine
phosphorylation inhibitors. Quinazolines serve as the backbone
for a range of inhibitors. Quinazoline-based tyrosine kinase inhib-
itors include: the reversible EGFR inhibitors, Gefitinib/Iressa/
ZD1839 and Erlotinib/Tarceva; the dual EGFR-HER2 inhibitor,
Lapatinib/Tykerb/GW572016; and the irreversible EGFR inhibitor,
CI-1033 (reviewed in21). Quinazolines also form the basis for inhib-
itors of serine/threonine kinases, such as CDK,22 and for a thymi-
dylate synthase inhibitor.23–26
In this study, we report on the identification of a new quinazo-
line family of T cell proliferation inhibitors that operate by a novel
mechanism, not yet fully defined. We examined the potency of
these compounds on T cell proliferation of peripheral blood mono-
nuclear cells (PBMC). We identified N-p-tolyl-2-(3,4,5-trimethoxy-
phenyl)quinazolin-4-amine (S101- dubbed 1) as the most potent
* Corresponding author. Tel.: +972 2 6585404; fax: +972 2 6512958.
E-mail address: alex.levitzki@mail.huji.ac.il (A. Levitzki).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.004

I. Sagiv-Barfi et al. / Bioorg. Med. Chem. 18 (2010) 6404–6413
6405
inhibitor. Here we explore its activity and the structure–activity
relationship of a family of compounds related to 1.
was formed. The precipitate was washed with water, filtered, and
dried under high vacuum to afford (2-chloro-quinazolin-4-yl)-p-
tolyl-amine (5.65 g, 0.021 mol, 70%). Mp 198–199 °C; 1H NMR
(300 MHz, DMSO-d6) d 2.27 (s, 3H), 7.21 (d, J = 7.8 Hz, 2H), 7.50
(d, J = 7.2 Hz, 3H), 7.83 (t, J = 8.5 Hz, 2H), 8.50 (t, J = 8.0 Hz, 1H).
2. Materials and methods
2.1. Chemistry
2.2.4. 1 p-Tolyl-[2-(3,4,5-trimethoxy-phenyl)-quinazolin-4-yl]-
amine27
Thin-layer chromatography was carried out on Merck alumi-
num sheets, silica gel 60 F254 and visualized with UV light at
254 nm. Preparative column chromatography was performed on
Merck silica gel 60 (70–230 mesh). High-pressure liquid chroma-
tography was performed on a Merck Hitachi HPLC, which included
an L 6200 pump, a D 6000A interphase, L-4250 UV detector and AS-
4000 autosampler. Integration employed the HSM HPLC System
Manager, Merck KgaA, Darmstadt and Hitachi Instruments, Inc.,
San Jose. Reversed-phase preparative HPLC was performed with a
C-18 column (218TPL022 Vydac).
Mass spectrometry was performed using an LCQDUO, from
ThermoQuest of Finnigan, and NMR was on a Bruker AMX 300.
Chemical shifts d are given in ppm referring to the signal center
using the solvent peaks for reference: CDCl3 7.26/77.0 ppm and
DMSO-d6 2.49/39.7 ppm.
Amixtureof(2-chloro-quinazolin-4-yl)-p-tolyl-amine((2-chloro-
quinazolin-4-yl)-p-tolyl-amine, 3.76 g, 0.014 mol), ethylene-glycol-
dimethyl-ether/water (1 L/120 mL), 3,4,5-trimethoxyphenylboron-
ic acid (2.714 g, 0.014 mol), and sodium bicarbonate (3.6 g) was
degassed with argon for 15 min. Pd(dppf)Cl2 (0.84 g) was added,
and the mixture was heated to reflux overnight. After cooling to
room temperature CH2Cl2 (1 L) and H2O (500 mL) were added. The
organic and aqueous layers were separated, the aqueous layer was
extracted with CH2Cl2 (2ꢀ 500 mL), and the combined organic layers
were dried over anhydrous sodium sulfate. The organic solvent was
removed under reduced pressure. The crude product was purified by
MeOH. Filtration in a Büchner apparatus afforded p-tolyl-[2-(3,4,5-
trimethoxy-phenyl)-quinazolin-4-yl]-amine (3.15 g, 0.0078 mol
56%). Mp >250 °C; 1H NMR (300 MHz, DMSO-d6) d 2.33 (s, 3H),
3.74 (s, 3H), 3.91 (s, 6H), 6.81(s, 2H), 7.24 (d, J = 8.4 Hz, 2H), 7.33
(d, J = 7.3 Hz, 2H), 7.58 (t, J = 8.2 Hz, 1H), 7.83 (t, J = 7.2 Hz, 2H),
8.54 (d, J = 7.5 Hz, 1H). Anal. (C24H23N3O3) C, H, N.
Combustion elemental analysis was performed using a Perkin-
Elmer 2400 Elemental Analyzer.
All solvents for HPLC analysis and purification were from J.T. Ba-
ker, BDH or Bio-Lab Ltd (Israel). Reagents for chemical synthesis
were from Frutarom Acros (Geel, Belgium), Fluka (Taufkirchen,
Germany), or Sigma–Aldrich (Steinheim, Germany).
2.2.5. 1 mesylate:28 m-tolyl-[2-(3,4,5-trimethoxy-phenyl)-
quinazolin-4-yl]-amine methanesulfonic salt
m-Tolyl-[2-(3,4,5-trimethoxy-phenyl)-quinazolin-4-yl]-amine
(1, 100 mg 0.25 mmol) was dissolved in THF at room temperature.
Melting point was determined using a Fisher–John melting
point apparatus (Fisher Scientific).
Methanesulfonic acid (1.2 equiv 20 lL, 0.3 mmol) was added to the
mixture. The mixture was mixed at room temperature until a pre-
cipitate was observed. The THF was decanted and the precipitate
was washed three times with 3 mL of diethyl ether, the ether
was removed and the precipitate was dried under nitrogen stream
to afford yellow crystals of m-tolyl-[2-(3,4,5-trimethoxy-phenyl)-
quinazolin-4-yl]-amine methanesulfonic salt (119 mg, 0.24 mmol,
96%). Mp >250 °C; 1H NMR (300 MHz, DMSO-d6) d 2.33 (s, 3H),
3.74 (s, 3H), 3.91 (s, 6H), 6.81(s, 2H), 7.24 (d, J = 7.7 Hz, 2H), 7.33
(d, J = 6.7 Hz, 2H), 7.58 (t, J = 7.1 Hz, 1H), 7.83 (t, J = 7.8 Hz, 2H),
8.54 (d, J = 8.0 Hz, 1H).
2.2. Synthesis
Synthetic procedures and analytical data for compounds 2–19,
21–38 are listed in Supporting Information. All tested compounds
possessed a purity of not less than 95%.
2.2.1. p-Tolyamine
4-Nitrotoluene (7.95 g, 5.8 mmol) in a solution of EtOH/H2O 9:1
(8 mL) was added dropwise to a mixture of hydrazine hydrate and
Raney nickel (7 mL) (14.4 mol) in aq EtOH (10 mL) at 60 °C. After
reflux was attained, an additional quantity of hydrazine hydrate
(2 mL) was added. The mixture was left to reflux for 25 min, cooled
to room temperature, filtered and evaporated to afford p-toly-
amine (5.2 g, 0.048 mol, 84%). Mp 52–53 °C; 1H NMR (300 MHz,
DMSO-d6) d 6.27 (s, 2H), 6.63 (d, J = 5.9 Hz, 2H), 6.81 (t, J = 7 Hz,
1H), 7.20 (t, J = 7 Hz, 2H).
2.2.6. Demethylation of 1: 5-(4-p-tolylamino-quinazolin-2-yl)-
benzene-1,2,3-triol (20)
An aryl methoxy derivative p-tolyl-[2-(3,4,5-trimethoxy-phe-
nyl)-quinazolin-4-yl]-amine (1, 30 mg, 7.5 eꢁ5 mol) was dissolved
in anhydrous DCM (minimal volume, not fully dissolved); the
round flask was then sealed. BBr3 (76 lL, 0.111 g, 205 g/mol,
d = 1.45, 2 equiv/per methoxy group, here 6 equiv were used
5.5 eꢁ4 mol), was added drop wise (from a syringe) and the reac-
tion mixture was stirred at room temperature for 1 h. The degree of
conversion was monitored by HPLC until full conversion was
achieved, then the reaction was quenched with methanol and
evaporated to low volume. Another wash was performed using
methanol, followed by a wash with acetonitrile and evaporation,
to obtain 5-(4-p-tolylamino-quinazolin-2-yl)-benzene-1,2,3-triol
(24 mg, 6.6 eꢁ5 mol, 90%). Mp >250 °C; 1H NMR (300 MHz,
DMSO-d6) d 2.35 (s, 3H), 6.75 (s, 2H), 7.22 (d, J = 8.0 Hz, 2H), 7.35
(d, J = 7.5 Hz, 2H), 7.56 (t, J = 8.0 Hz, H), 7.82 (t, J = 8.2 Hz, 2H),
8.24 (d, J = 7.5 Hz, 1H). Anal. (C21H17N3O3) C, H, N.
2.2.2. 2,4-Dichloroquinazoline27
P(O)Cl3 (40 mL) was stirred at room temperature for 20 min and
then was added to a flask containing 2,4-quinolinedione (10 g,
0.06 mol). The mixture was heated to reflux for 48 h. The brown
solution was cooled to 50 °C, poured into cold water (0 °C,
40 mL) while stirring vigorously. The aqueous mixture was main-
tained at a temperature below 30 °C during the quench. The cold
precipitate was filtered, washed with cold water (3ꢀ 60 mL) and
dried under high vacuum to afford 8 g (0.04 mol) of 2,4-dichloro-
quinazoline (71%). Mp 118 °C; 1H NMR (300 MHz, DMSO-d6) d
7.58 (t, J = 7.6 Hz, 1H), 7.84 (t, J = 6.5 Hz, 2H), 8.16 (d, J = 6.9 Hz, H).
2.2.3. (2-Chloro-quinazolin-4-yl)-p-tolyl-amine27
2.3. Biology
A mixture of 2,4-dichloroquinazoline (5.8 g, 0.029 mol), p-toly-
amine (3.5 g, 0.032 mol), and potassium acetate (3.72 g, 0.038 mol)
in THF/water (66 mL/30 mL) was stirred at room temperature for
16 h. Water (66 mL) was added to the mixture, and a precipitate
2.3.1. Culture conditions
Jurkat cells (human, acute T cell leukemia line) and human
peripheral blood mononuclear cells (PBMC) were grown in RPMI

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I. Sagiv-Barfi et al. / Bioorg. Med. Chem. 18 (2010) 6404–6413
medium supplemented with 100 U/mL penicillin, 100
tomycin. All cultures were incubated in a humidified incubator at
37 °C in 5% CO2.
l
g/mL strep-
from 2c mice were stimulated by irradiated splenocytes from
BALB/c mice in the presence of the evaluated compound at the con-
centrations specified. Cultures were incubated for 72 h in 96-well
plates.
NIH 3T3 (mouse embryo fibroblasts), A-431 (human epithelial-
like skin carcinoma cells), MDA-MB-468 (human epithelial-like
breast carcinoma), PANC-1 (human epithelial-like pancreatic carci-
noma), PC-3 (human prostate cancer cells), HEK293 (human embry-
onic kidney cells), and A375 (human malignant melanoma), were
grown in DMEM medium (Biological Industries, Beit Haemek), sup-
plemented with 100 U/mL penicillin, 100 mg/mL streptomycin.
MDA-MB-231 (human breast adenocarcinoma) were grown with
Leibovitz’s L-15 Medium (Biological Industries, Beit Haemek), sup-
plemented with 100 U/mL penicillin, 100 mg/mL streptomycin.
Primary human keratinocytes were maintained in keratinocyte
growth medium (KGM): DMEM, 25% Nutrient mixture F-12 (HAM),
2.3.6. Methylene blue assay
The inhibitor-treated cultures and controls were fixed in glutar-
aldehyde, 0.05% final concentration, for 10 min at room tempera-
ture. After washing, the plates were stained with 0.1% methylene
blue in 0.1 M borate buffer, for 60 min at room temperature. The
plates were rigorously washed to remove excess dye and dried.
The dye taken up by cells was eluted in 0.1 N HCl for 60 min at
37 °C, and the OD of the solution was read at 620 nm. In prelimin-
ary titration experiments, linear readings were obtained for
1 ꢀ 103 to 4 ꢀ 104 cells/well.
10% fetal bovine serum, 5 lg/mL insulin, 0.4 lg/mL hydrocortisone,
0.1 nM cholera toxin, 10 ng/mL epidermal growth factor,
2.3.7. IL-2 secretion
1.8 ꢀ 10ꢁ4 M adenine, 5
lg/mL transferrin, 2 nM T3, 100,000 U/L
Jurkat or PBMC cells were treated for 12 h with 1 lM, 0.5 lM or
penicillin, 100
lg/L streptomycin, and 0.1 mg/mL amphotericin.
no inhibitor. Cells (1 ꢀ 106) were plated in 24-well plates (Nunc)
The primary keratinocytes were cultured from small biopsy
and stimulated by C305 (dilution 1:500), anti-CD28 (1:100), PMA
specimens.
(50 ng/ml) or ionomycin (1 lM) for 12 h. Alternately, cells were
stimulated for 24, 48, and 72 h in MLR. After stimulation cells were
2.3.2. T cell isolation and stimulation
centrifuged at 130g for 5 min at room temperature.
Blood samples were taken from healthy donors and loaded on a
Ficoll density gradient to purify the PBMC population. T cells were
purified by human anti-CD3 using an autoMACS instrument (Milt-
enyi Biotec, Bergisch-Gladbach, Germany), according to the manu-
facturer’s instructions. The identity of T cells was confirmed by
flow cytometry, as they stained positive for CD3. Cells were stimu-
Supernatants were collected and IL-2 was assayed using the IL-
2 ELISA kit (BioSource International USA, KH0022) according to the
manufacturer’s instructions. Absorbance was read at 450 nm in an
ELISA plate reader (ELx800 BIO-TEK Instruments Inc.).
2.3.8. Cell cycle analysis
lated with ConA (10
ences), or LPS (50
for 72 h in the presence of the inhibitor, and their proliferation
was measured in the BrdU incorporation assay as will be later
described.
l
g/mL), ODN2006 (0.5
l
g/mL; Enzo Life Sci-
The effects of the inhibition of the cell cycle were analyzed with
a fluorescence-activated cell sorter (FACS). Cells were grown in
60 mm diameter plates (Nunc) and were treated with inhibitor
for 12 and 24 h. After the indicated time, cells were pooled,
l
g/mL; Sigma–Aldrich, Steinheim, Germany),
2.3.3. Antibodies
Table 1
Antibodies used in this study were: Hybridoma C305, specific
for the Jurkat Ti beta chain of TCR (from Dr. D. Yablonski, Technion,
Israel); anti-ZAP-70 (05-253) from Upstate Biotechnology (Lake
Placid, NY); anti-SLP-76 (sc-9062) from Santa Cruz Biotech (Santa
Cruz, CA); Hybridoma 4G-10 specific for phosphorylated tyrosine
(from our collection); anti-CD28 (MCA709XZ) from Serotec (Cergy
Saint-Christophe, France); anti-PGSK3 (9331) and anti-glyceralde-
hyde-3-phosphate dehydrogenase (GAPDH) (2118) from Cell Sig-
naling Technology (Beverly, MA).
(3,4,5-Trimethoxyphenyl)quinazoline 1–19
R
NH
N
O
N
O
O
2.3.4. DNA 5-bromo 20-deoxyuridine (BrdU) incorporation assay
Cells were depleted of fetal calf serum (FCS) for 12 h. They were
then stimulated to proliferate by addition of ConA (10 lg/mL) for
A
R
IC50 in PBMC IC50 in Jurkat
(nM) SD
(nM) SD
PBMC from healthy individuals and for Jurkat cells, FCS (10%) for
HEK293 and A375 cells, PDGF (30 ng/mL) for NIH 3T3 cells, and
EGF (30 ng/mL) for MDA-MB-231, cells (1 ꢀ 105) were cultured in
96-well plates for 3 days in the presence or absence of inhibitors
at the concentrations specified in the text. Each condition was
tested in duplicate wells.
Proliferation was assessed by BrdU incorporation using a color-
imetric ELISA kit (Roche, Germany). Subsequent to labeling with
10 lM BrdU, DNA was denatured and the cells were incubated
with anti-BrdU-POD, prior to the addition of colorimetric substrate
(TMB). The reaction product was quantified by measuring absor-
bance at 450 nm in an ELISA plate reader (ELx800 BIO-TEK Instru-
ments Inc.).
1
4-Methyl phenyl
Phenyl
4-Trifluoromethoxy phenyl
4-Aminobenzyl
4-Hydroxy phenyl
4-Chloro phenyl
4-Methoxy phenyl
3,4-Difluoro-benzyl
1H-Indazol-6-yl
70 0.05
280 0.04
400 0.00
380 0.04
300 0.14
300 0.05
500 0.14
200 0.00
450 0.07
600 0.28
750 0.21
380 0.04
850 0.07
750 0.05
500 0.14
300 0.14
430 0.11
3250 2.47
2400 2.26
29 0.23
380 0.18
180 0.11
480 0.18
230 0.04
530 0.32
1100 0.14
730 0.39
600 0.14
700 0.35
700 0.35
600 0.00
580 0.18
2600 3.39
1100 0.14
730 0.39
1500 0.71
600 0.14
850 0.07
2
3
4
5
6
7
8
9
10 1H-Indazol-5-yl
11 5-{4-[2-(1H-Indol-3-yl)-ethylamino]}
12 Benzo[1,3]dioxol-5-yl
13 5-[4-(2-Hydroxymethyl-phenylamino)]
14 2,4-Dimethy phenyl
15 4-Methoxy benzyl
16 3,4-Dichloro-benzyl
17 3H-Benzoimidazol-5-yl
18 Benzyl
2.3.5. Mixed lymphocyte reaction (MLR)
In the mixed lymphocyte reaction, stimulator cells cause re-
19 4-Octyloxy-phenyl
sponder cells to proliferate, owing to alloreactivity.29 Splenocytes

I. Sagiv-Barfi et al. / Bioorg. Med. Chem. 18 (2010) 6404–6413
6407
Table 2
pelleted by centrifugation, washed in phosphate-buffered saline
(PBS), and fixed with cold 70% ethanol for a minimum of 12 h. Cells
were re-pelleted and resuspended in PBS. RNase A (50 g/mL) was
added in the dark for 30 min followed by 5 g/mL of propidium io-
(3,4,5-Trihydroxyphenyl)quinazoline 20–38
R
l
NH
l
dide for FACS analysis (Becton–Dickinson FACScan).
N
2.3.9. Cell stimulation and lysis
OH
OH
Jurkatcells werewashedin PBScontainingCa2+ andMg2+ (Biolog-
ical Industries, Beit Haemek), preheated to 37 °C for 15 min, and
were either mock stimulated with PBS, or were stimulated for
1 min with C305 (1:500). Cells were then collected and lysed at a
concentration of 2 ꢀ 107vcells/mL in cold lysis buffer containing 1%
NP-40, 10 mM Tris (pH 7.8), 150 mM NaCl, 1 mM phenylmethylsul-
N
OH
B
R
IC50 in PBMC IC50 in Jurkat
(nM) SD
(nM) SD
fonyl fluoride (PMSF), 2 lg/mL pepstatin A, 1 lg/mL leupeptin,
10 mM sodium pyrophosphate, 0.4 mM EDTA, 0.4 mM sodium
orthovanadate, and 50 mM NaF, 50 mM beta-glycerol phosphate
pH 7.5 and 20 mM sodium pyrophosphate pH 7.5. After 20 min at
4 °C, the lysates were cleared by centrifugation in a microfuge
(Eppendorf, centrifuge 5427R) at 16,000g for 10 min at 4 °C. The
supernatants were used directly for immunoprecipitation.
20 4-Methyl phenyl
21 Phenyl
22 4-Trifluoromethoxy phenyl
23 4-Aminobenzyl
34 4-Hydroxy phenyl
25 4-Chloro phenyl
26 4-Methoxy phenyl
27 3,4-Difluoro-benzyl
28 1H-Indazol-6-yl
850 0.21
650 0.49
2780 0.26
570 0.24
970 0.84
550 0.21
500 0.14
280 0.04
500 0.14
780 0.67
1240 0.74
5500 6.36
900 0.14
730 0.04
3260 0.49
2600 0.24
4340 0.74
7000 3.50
4530 0.31
850 0.21
5500 3.50
3240 0.31
670 0.22
1120 0.28
4250 5.30
550 0.21
600 0.21
690 0.30
710 0.87
1970 0.74
800 0.28
650 0.21
3700 4.67
5430 0.76
3640 0.54
2570 0.82
950 0.07
4250 1.30
2.3.10. Western blot
29 1H-Indazol-5-yl
Western blots were prepared on nitrocellulose using standard
techniques. Membranes were blocked in TBST buffer (10 mM
Tris–HCl, pH 7.4 (TBS), 0.2% Tween 20, and 170 mM NaCl) contain-
ing 5% low-fat milk, then probed overnight with the appropriate
primary antibody. After incubation with horseradish peroxidase-
conjugated secondary antibody, immunoreactive proteins were
visualized using an enhanced chemiluminescence (ECL) detection
reagent.
30 5-{4-[2-(1H-Indol-3-yl)-ethylamino]}
31 Benzo[1,3]dioxol-5-yl
32 5-[4-(2-Hydroxymethyl-phenylamino)]
33 2,4-Dimethy phenyl
34 4-Methoxy benzyl
35 3,4-Dichloro benzyl
36 3H-Benzoimidazol-5-yl
37 Benzyl
38 4-Octyloxy-phenyl
2.3.11. Immunoprecipitation
Bead preparation: 500 lL lysis buffer were mixed with 30 lL
protein A (Roche, Germany) and the desired antibody, stirred at
room temperature for 1 h, spun down and dried by vacuum. Cell ly-
sates were obtained as described above and incubated with the
Scheme 1. Synthetic approaches to compounds 1–19. Reagents and conditions: (i) POCl3,
D; (ii) hydrazine hydrate, Raney nickel, D; (iii) potassium acetate, THF/DDW; (iv)
ethylene glycol dimethyl-ether/DDW, Pd(dppf)Cl2.

6408
I. Sagiv-Barfi et al. / Bioorg. Med. Chem. 18 (2010) 6404–6413
beads for 1.5 h at 4 °C in a tumbling apparatus. The cells were cen-
trifuged and washed three times with lysis buffer (the same lysis
buffer used for cell lysis). Sample buffer was added and the sam-
ples were boiled for 3 min at 100 °C. The supernatants were sub-
jected to SDS–PAGE and Western blotting.
3. Results and discussion
3.1. Chemical synthesis
We hypothesized that we might be able to find T cell prolifera-
tion inhibitors among compounds possessing chemical scaffolds
used to generate protein kinase inhibitors, since protein kinases
play crucial roles in signaling processes and cellular communica-
tion. We therefore designed compounds based on the quinazoline
moiety, an element that is present in many tyrosine phosphoryla-
tion inhibitors and a few serine/threonine kinase inhibitors. In our
search for new specific inhibitors of T cell proliferation, we gener-
ated two libraries of quinazoline-based compounds. The first library
(Table 1, compounds 1–19, Scheme 1) possessed a 3,4,5-trime-
thoxyphenyl quinazoline skeleton. The trimethoxy-phenyl moiety
was tested because it is a common pharmacophore for a variety
2.3.12. SLP-76 kinase assay
ZAP-70 was immunoprecipitated as described above. The im-
mune complexes were washed two times with lysis buffer and
two times with kinase buffer (50 mM Tris, pH 7.5, 100 mM NaCl,
and 10 mM MgCl2). The beads were resuspended in 30
buffer containing 3 g of His-tagged, full-length human SLP-76
protein (kindly provided by Dr. Yablonski, Technion, Israel), and
the kinase reactions were initiated by the addition of 10 l kinase
buffer containing 5 M ATP. The reactions proceeded for 15 min at
30 °C and were stopped by the addition of 6 L EDTA (100 mM)
and sample buffer. The results were analyzed by Western blot.30
ll of kinase
l
l
l
l
of inhibitors, including inhibitors of tubulin polymerization31
;
Scheme 2. Synthetic approaches to compounds 20–38. Reagents and condition: BBr3, methylene chloride, rt.
Figure 1. The effect of 1 on the growth of various cell lines. (A) BrdU uptake of the cells without stimulation. (B) BrdU uptake of the stimulated cells: ConA (10
lg/mL)
activated PBMC, serum activated HEK293 and A375, PDGF (30 ng/mL) activated NIH 3T3, EGF (30 ng/mL) activated MDA-MB-231 with increasing inhibitor concentrations. (C)
Growth curves of the selected cells without inhibitor and after treatment with 1
values of the non-stimulated and stimulated cells as detailed in (B).
lM inhibitor, relevant quantification (RQ) to growth without inhibitor’s treatment. (D) IC50

I. Sagiv-Barfi et al. / Bioorg. Med. Chem. 18 (2010) 6404–6413
6409
dihydrofolate reductase inhibitor,32 tyrosine and phosphoinositide
kinases33; and an HSP-90 inhibitor.34
of 70 nM for the inhibition of PBMC proliferation and 30 nM for the
inhibition of Jurkat cell proliferation (Table 1).
The second library (Table 2, compounds 20–38, Scheme 2) pos-
sessed a 3,4,5-trihydroxyphenyl quinazoline skeleton, which was
introduced to reduce hydrophobicity. These compounds were ob-
tained by a multistep synthesis, starting from the chlorination of
2,4-quinolinedione by P(O)Cl3 to give 2,4-dichloroquinazoline, fol-
lowed by the addition of the corresponding aromatic amines. The
reaction products were coupled with 3,4,5-trimethoxyphenylbo-
ronic acid catalyzed by Pd(II). Compounds 20–38 were prepared
by the demethylation of compounds 1–19.
3.3. Structure–activity relationships
We evaluated the role of the aromatic amine ring on the activity
(Table 1). Elimination of the methyl group (compound 2) caused a
threefold increase in the IC50 of PBMC proliferation and a 10-fold
increase in the IC50 of Jurkat cell proliferation. The addition of an-
other methyl at position 2 (compound 14) reduced the potency of
the compound 10-fold in both cell lines. A bulky OCF3 group (com-
pound 3) increased the IC50 by fivefold for PBMC and sixfold for
Jurkat. OH or Cl groups at position 4 were similar in their potencies
(compounds 5 and 6). To assess the affect of flexibility and bond
expansion, we replaced the aromatic amine ring with a benzyl
amine (compounds 2, 18 and 7, 15). This substitution increased
the IC50 approximately fivefold. 4-Aminobenzyl was found to be
more active than benzyl alone (compounds 4 and 18). Dichloro-
and difluoro-benzyls were similar in their abilities to inhibit prolif-
eration. Replacing the ring with indazole, indole, benzodioxol or
imidazole (compounds 10, 11, 12, 17, respectively) failed to im-
prove activity.
3.2. Inhibition of T cell proliferation
Since we were not prejudiced towards any specific protein ki-
nase, we used a biological assay for T cell proliferation. We tested
our libraries for inhibition of proliferation of PBMC and Jurkat cells
(Tables 1 and 2). Cell proliferation was measured by BrdU uptake.
Jurkat cells do not need to be stimulated to proliferate. PBMC cells
were stimulated by a non-specific T cell mitogen, Concanavalin A
(ConA). All of the compounds that were tested inhibited prolifera-
tion at micromolar or sub-micromolar concentrations. The chemi-
cal entity 1 possessed the highest inhibition potency, with an IC50
Figure 2. Activation and inhibition of PBMC, CD3+ cells and PBMC depleted of CD3+ cells. Flow cytometry. (A) PBMC; (B) CD3+ cells; (C) PBMC depleted of CD3+ cells. PBMC
were stimulated with (10 g/mL) ConA, (0.5 g/mL) ODN2006 (Enzo Life Sciences), or (50 g/mL) LPS (D), CD3+ cells were stimulated with ConA (E), and PBMC depleted of
l
l
l
CD3+ cells were stimulated with both ODN2006 and LPS, in the presence and absence of inhibitor (F). Their proliferation was measured by uptake of BrdU and normalized to
the maximal proliferation in the absence of inhibitor.

6410
I. Sagiv-Barfi et al. / Bioorg. Med. Chem. 18 (2010) 6404–6413
Trihydroxy compounds were less active than their trimethoxy
PBMC consist of a mixture of mononucleated cells, mostly T
cells. To identify the cell type that was inhibited by 1, we first puri-
fied T cells from PBMC with an autoMACS apparatus (Miltenyi Bio-
tec), using anti-CD3 antibodies. We were now able to compare
three cell populations: total PBMC, CD3+ cells purified from PBMC,
and PBMC that had been depleted of CD3+ cells. In order to test the
effect of 1 on the various cell populations, we made use of three
modes of stimulation: (1) ConA, which stimulates T cells exclu-
sively; (2) ODN2006 (Oligodeoxynucleotide 50-tcgtcgttttgtcgtttt
gtcgtt-30), which stimulates mainly non-T cells, via Toll-like Recep-
tor-9 (TLR9), which is highly expressed on dendritic cells, B-lym-
phocytes and natural killer (NK) cells and (3) lipopolysaccharide
(LPS), which stimulates mostly B cells, macrophages and some T
cells. To test the inhibition by 1 of PBMC proliferation, we used
all three stimuli, namely ConA, ODN2006, and LPS. ConA-stimu-
lated proliferation was strongly inhibited by 1, with an IC50 of
analogs (Table 2), with the exception of compound 27, in which
the methyl group of the aromatic amine in compound 8 was
substituted by difluoro-benzyl.
3.4. Compound 1 specifically inhibits the CD3+ cell population
To characterize the mode of action of 1 in more detail, we first
examined whether 1 inhibits non-hematopoietic cell lines. We
studied the effect of 1 on the growth of four cell lines: NIH 3T3
(mouse embryonic fibroblasts), A-431 (human vulval carcinoma
cells), MDA-MB-468 (human breast carcinoma cells), and PANC-
1
(human pancreatic carcinoma cells) (Supplementary data,
Fig. 1). We also tested the inhibitors on human keratinocytes.
The inhibitor slightly affected these cell types at high concentra-
tions, at which the growth of Jurkat and PBMC was strongly
inhibited (Fig. 1C). We also examined the effect of 1 on BrdU up-
take by stimulated and non-stimulated PC-3 (human prostate
cancer), HEK293 (human embryonic kidney), NIH 3T3, A375 (hu-
man malignant melanoma), and MDA-MB-231 (human breast
adenocarcinoma). The effect of the inhibitor on the proliferation
of the various cells had IC50 values higher by two orders of mag-
nitude in comparison to the inhibitory IC50 for PBMC and Jurkat
cells (Fig. 1D).
ꢂ0.1
lM. ODN2006 and LPS-stimulated proliferation of PBMCs
was inhibited to
a
lesser extent, with an IC50 of ꢂ0.5
lM
(Fig. 2D), which was similar to the IC50 of 1 for PBMCs that had
been depleted of CD3+ cells, whether stimulated by ODN2006 or
LPS (Fig. 2F). The proliferation of isolated CD3+ cells was very
strongly inhibited by 1, with an IC50 that was slightly higher than
0.1
lM (Fig. 2E). These data show that 1 specifically inhibits the
proliferation of CD3+ T cells, and has a much lesser effect on the
Figure 3. The effect of 1 on the cell cycle of PBMC and Jurkat cells. (A) Cellular G2 arrest and apoptosis as a result of 1 treatment. Distribution in sub-G1 (represented by M1),
G1 (M2), S (M3), or G2/M (M4) phase was determined by FACS analysis. Jurkat (1 and 2) 1 without inhibitor, and 2 in the presence of 0.5 M 1. PBMC (3 and 4) 3 without
inhibitor, and 4 in the presence of 0.5 M 1. The x-axis shows DNA content; and the y-axis shows the number of cells. (B) Graphic quantification of the cell cycle.
l
l

I. Sagiv-Barfi et al. / Bioorg. Med. Chem. 18 (2010) 6404–6413
6411
non-T cell fraction of the PBMC. These results suggest that 1 affects
cell growth factors that are unique to CD3+ T cells.
Cyclosporine A and rapamycin act synergistically to inhibit im-
mune responses by preventing the IL-2-driven clonal expansion
of T cells.36,37
3.5. Compound 1 specifically arrests T cells in G2
Jurkat cells, which originate from leukemia, divide with no need
of exogenous IL-2 stimulation since they produce their own,
whereas PBMC proliferation requires exogenous IL-2 for growth.
As expected, IL-2 secretion levels were lower in cells isolated from
the blood (PBMC) than in Jurkat cells (Fig. 4A and B). We analyzed
the effect of 1 on IL-2 secretion in both Jurkat (Fig. 4A) and PBMC
(Fig. 4B) cells, using a variety of T cell stimuli. Cells were stimu-
lated with an antibody to the TCR alone or in combination with
anti-CD28 or phorbol 12-myristate 13-acetate (PMA). In order to
bypass the TCR, we also stimulated the cells with a combination
of ionomycin, PMA and anti-CD28 (Fig. 4A). We were surprised
to discover that the inhibitor did not act by repressing IL-2 secre-
tion. Irrespective of the mode of stimulation, compound 1 had no
effect on IL-2 levels in the medium, up to a concentration of
FACS analysis revealed that treatment of PBMC and Jurkat cells
with 1 led to arrest at the G2 phase of the cell cycle. The fraction of
cells in G2 increased from 30% in the untreated cells to 50% in the
treated cells. The sub-G1 population increased from 2% to 9.5%,
indicating that some cells underwent apoptosis (Fig. 3).
3.6. Compound 1 inhibited T cell proliferation without affecting
IL-2 secretion
Compound 1 showed a remarkable ability to inhibit T cell pro-
liferation in a selective manner, as observed in CD3+ cells, PBMC
and Jurkat cells. IL-2 is a cytokine secreted in an autocrine manner
by T cells to induce their own proliferation. The production of IL-2
determines whether a T cell will proliferate and become an armed
effector cell, where the most important function of the co-stimula-
tory signal is to promote the synthesis of IL-2.35 The central impor-
tance of IL-2 in initiating adaptive immune responses is well
illustrated by the drugs that are commonly used to suppress unde-
sirable immune responses. The immunosuppressive drugs cyclo-
sporine A and FK506 (tacrolimus) inhibit IL-2 production by
disrupting the signaling through the T cell receptor, whereas rapa-
mycin (sirolimus) inhibits signaling through the IL-2 receptor.
5
lM inhibitor.
In vivo, T cells are stimulated by neighboring cells. In order to
test the effect of 1 on cell-stimulated IL-2 secretion, the superna-
tants of splenocytes after 24, 48, and 72 h MLR were collected
and analyzed for their IL-2 levels (Fig. 4C). Without inhibitor, or
in the presence of S145 (compound 20) levels of IL-2 decreased
after 72 h, probably due to the consumption of IL-2 by proliferating
cells. This effect was not seen with compound 1, because the cells
did not proliferate. Treatment with cyclosporine A, which is known
to repress IL-2 transcription, led to a marked decrease in IL-2 levels
Figure 4. IL-2 secretion after stimulation. IL-2 levels in the growth medium were measured by ELISA after centrifuging out the cells. (A) Jurkat cells. (B) PBMC. Four modes of
stimulation were used in order to examine the inhibitor’s ability to affect diverse events in the T cell. First, cells were stimulated using anti-TCR antibody, attached to the
plate. The coupling of the antibody to TCR initiates a signal transduction cascade, leading to cell proliferation and secretion of IL-2. Second, anti-TCR antibody was used in
combination with an antibody to the CD28 molecule. This combination of signaling via the TCR as well as the co-stimulatory effect induced higher levels of IL-2. Third,
ionomycin (a calcium ionophore), phorbol 12-myristate 13-acetate (PMA; a PKC activator) and the antibody for CD28, were used in combination. This stimulation activated
parallel pathways and gave rise to the highest IL-2 level. Fourth, ConA stimulation served as control to proliferation assays. The experiment was executed with both Jurkat and
PBMC. 1 did not affect IL-2 secretion in either cell type, irrespective of the mode of stimulation. PBMC had lower IL-2 levels. (C) MLR; splenocytes from 2c mice were
stimulated by irradiated splenocytes from BALB/c mice in the presence of the evaluated compound at the concentrations specified. Cultures were incubated for 72 h in 96-
well plates. Supernatants were collected and IL-2 was assayed for 24, 48, and 72 h in the presence of either compound 1 or compound 20, and Cyclosporine A L.

6412
I. Sagiv-Barfi et al. / Bioorg. Med. Chem. 18 (2010) 6404–6413
in the medium, as early as 24 h after initiation of treatment. In the
presence of 1, no visible decrease in IL-2 secretion was detectable
even at 1 lM inhibitor (Fig. 4C). At higher concentrations, no viable
cells remained. These findings lead us to conclude that the dra-
matic inhibition by 1 of proliferation is caused by a distinct, novel
mechanism that does not involve IL-2 secretion.
tated the ZAP-70 kinase from the treated/untreated cells, and
tested its ability to phosphorylate recombinant SLP-76 protein as
substrate (Fig. 5B). Compound 1 inhibited the activity of ZAP-70,
as well as tyrosine phosphorylation of SLP-76, in a dose-dependent
manner, indicating that 1 acts upstream of SLP-76. Although in
SLP-76 deficient Jurkat cells, no IL-2 is produced40 at the concen-
trations we tested (up to 1 lM), SLP-76 phosphorylation was re-
3.7. Compound 1 inhibited tyrosine phosphorylation of SLP-76
duced but not abolished. Presumably the remaining SLP-76
activity was sufficient to allow IL-2 secretion.
We were intrigued by the properties of 1 and its analogs, espe-
cially by the finding that IL-2 secretion was not inhibited, in con-
trast to known T cell anti-proliferative agents, and by the effect
of 1 on the cell cycle. We therefore began to search for cellular tar-
gets of 1.
The T cell receptor and co-receptor are associated with the Src-
family protein kinases, Fyn and Lck. It is thought that binding of a
peptide:MHC ligand to the T cell receptor and co-receptor brings
together CD4, the T cell receptor complex, and CD45.38 This allows
the CD45 tyrosine phosphatase to remove inhibitory phosphate
groups on Fyn and Lck. Phosphorylation of the f chains on the
receptor enables them to bind and activate the cytosolic tyrosine
kinase ZAP-70.39 ZAP-70 phosphorylates the adaptor protein SLP-
76 (Src homology 2 (SH2) domain-containing leukocyte protein
4. Conclusion
Clinical transplantation has become a routine procedure, and its
successful utilization depends on MHC matching, immunosuppres-
sive drugs, and technical skill. Today, most transplants require gen-
eralized immunosuppression of the recipient. This can be toxic and
increases the risk of cancer and infection. Induction of specific tol-
erance, which would suppress the response to the graft without
compromising host defense, is now considered the ‘holy grail’ of
immunology.
Compound 1, described here, shows a remarkable ability to in-
hibit T cell proliferation in a selective manner. Compound 1 in-
duces G2 arrest, as well as an increase in the sub-G1 population
of cells. Our results suggest that 1 affects cell growth factors that
are unique to CD3+ T cells. We believe that the reduction in SLP-
76 phosphorylation is not the only mechanism by which our inhib-
itor exhibits proliferation arrest. This information is currently guid-
ing us in the search for the 1 target, directing our search to distinct
signaling features of these cells (e.g., specific cytokines, unique sig-
naling molecules, unique kinases). The apparent selective effect of
compound 1 on T cells and its lower activity against other cell
types indicate that compound 1 may be a potential lead for an
inhibitor of graft rejection. In the future we intend to examine if
the systemic administration of 1 or one of its derivatives during
graft transplantation, can lead to unresponsiveness towards anti-
gens presented by the graft, without harming the body’s ability
to react toward a pathogen.
of 76 kDa), which in turn leads to the activation of PLC-c by Tec ki-
nases and the activation of Ras by guanine-nucleotide exchange
factors. Thus, SLP-76 is one of the key elements in T cell signaling
following T cell receptor activation.
Jurkat cells were treated with 1 for 16 h, and SLP-76 phosphor-
ylation was measured by immunoprecipitation of SLP-76 protein
followed by Western blotting with an antibody to the phosphory-
lated tyrosines of the protein (Fig. 5A). We also immunoprecipi-
97 kDa
Anti P-Y (4G-10)
66 kDa
T cell proliferation is crucial for development of psoriasis, a
common T cell-mediated autoimmune disorder where primary on-
set of skin lesions is followed by chronic relapses. In an animal
model, the blocking of T cell proliferation led to the inhibition of
psoriasis development.41 Thus, 1 can also be considered a potential
lead for the development of agents against graft rejection as well as
psoriasis and other inflammatory diseases.
97 kDa
Anti Slp-76
66 kDa
45 kDa
GAPDH
30 kDa
Stimulation
Acknowledgments
Inhibitor
No Inhibitor
0.5µM
1µM
The authors thank Professor Yair Reisner and Dr. Esther Bachar-
Lustig from the Weizmann Institute of Science, Rehovot, Israel. This
study was partially supported by Algen Biopharmaceticals, Ltd,
Jerusalem, Israel.
97 kDa
Anti P-Y (4G-10)
GAPDH
66 kDa
45 kDa
Supplementary data
30 kDa
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.07.004.
Stimulation
Inhibitor
No inhibitor
0.5µM
1µM
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