S(+)-4-(1-phenylethylamino)quinazolines as inhibitors of human immunoglobuline E synthesis: Potency is dictated by stereochemistry and atomic point charges at N-1

Article abstract of DOI:10.1021/jm010888h

Since the pathogenesis of allergic diseases is associated with elevated levels of immunoglobulin E (IgE), we developed a high throughput reporter gene assay in a human B-cell line to screen for low molecular weight IgE inhibitory compounds. Monitoring the IL-4 driven IgE-germline promoter activity (IgE-GLP), we discovered 4-(1-phenylethylamino)qinazolines as potent inhibitors of IgE-germline gene expression. Testing of the individual enantiomers (1, 2) revealed that only the S(+) enantiomer 1 was active. A cell viability assay done in the same cell line in parallel discriminated the dose-dependent inhibition from a general antiproliferative effect. The observed correlation of the inhibitory potencies found in the reporter gene assay with those measured by IgE-ELISA in primary human splenocytes provided evidence that the blockade of IgE synthesis is the direct consequence of IgE-germline gene inhibition, thereby validating the reporter gene assay. Parallel synthesis in solution rapidly provided a series of analogues of compound I with modifications in the phenethylamine side chain and the quinazoline core for SAR studies. Increasing the lipophilicity of the arylalkylamine moiety yielded S(+)-4-(1-(2-naphthyl)ethylamino)quinazoline (6) as the most potent inhibitor (IC₅₀ of 14 nM) while the R(-) enantiomer was again found to be inactive. Within the set of S enantiomers, quantum mechanical calculations revealed that the IgE inhibitory activity can be quantitatively described by the charge at N-1 of the heterocyclic core and to a lesser extent by the molar refractivity. These results demonstrate the importance of electron-deficient fused 4-aminopyrimidines and lipophilic side chains for biological activity. The strong preference for the S configuration of the phenethylamine side chain is remarkable insofar as biological activity for fused 4-(1-phenylethylamino)pyrimidines has been published for the R enantiomers only (EGFR tyrosine kinase inhibition).

Full text of DOI:10.1021/jm010888h

J . Med. Chem. 2001, 44, 3031-3038
3031
S(+)-4-(1-P h en yleth yla m in o)qu in a zolin es a s In h ibitor s of Hu m a n
Im m u n oglobu lin e E Syn th esis: P oten cy Is Dicta ted by Ster eoch em istr y a n d
Atom ic P oin t Ch a r ges a t N-1
Michael Berger,† Bettina Albrecht, Attila Berces, Peter Ettmayer,* Wolfgang Neruda, and
Maximilian Woisetschla¨ger
Novartis Forschungsinstitut, Brunnerstrasse 59, Vienna A-1235, Austria
Received March 30, 2001
Since the pathogenesis of allergic diseases is associated with elevated levels of immunoglobulin
E (IgE), we developed a high throughput reporter gene assay in a human B-cell line to screen
for low molecular weight IgE inhibitory compounds. Monitoring the IL-4 driven IgE-germline
promoter activity (IgE-GLP), we discovered 4-(1-phenylethylamino)qinazolines as potent
inhibitors of IgE-germline gene expression. Testing of the individual enantiomers (1, 2) revealed
that only the S(+) enantiomer 1 was active. A cell viability assay done in the same cell line in
parallel discriminated the dose-dependent inhibition from a general antiproliferative effect.
The observed correlation of the inhibitory potencies found in the reporter gene assay with those
measured by IgE-ELISA in primary human splenocytes provided evidence that the blockade
of IgE synthesis is the direct consequence of IgE-germline gene inhibition, thereby validating
the reporter gene assay. Parallel synthesis in solution rapidly provided a series of analogues
of compound 1 with modifications in the phenethylamine side chain and the quinazoline core
for SAR studies. Increasing the lipophilicity of the arylalkylamine moiety yielded S(+)-4-(1-
(2-naphthyl)ethylamino)quinazoline (6) as the most potent inhibitor (IC50 of 14 nM) while the
R(-) enantiomer was again found to be inactive. Within the set of S enantiomers, quantum
mechanical calculations revealed that the IgE inhibitory activity can be quantitatively described
by the charge at N-1 of the heterocyclic core and to a lesser extent by the molar refractivity.
These results demonstrate the importance of electron-deficient fused 4-aminopyrimidines and
lipophilic side chains for biological activity. The strong preference for the S configuration of
the phenethylamine side chain is remarkable insofar as biological activity for fused 4-(1-
phenylethylamino)pyrimidines has been published for the R enantiomers only (EGFR tyrosine
kinase inhibition).
In tr od u ction
to interfere with its production or to block its function.
Both approaches provided proof of concept that inhibi-
tion of IgE has therapeutic effects in animal models and
in allergic patients. The small molecular weight com-
pound suplatast tosylate has been described as an
inhibitor of IL-4 and IL-6 production in T-cells, thereby
leading to IgE inhibition in B-lymphocytes. In allergic
patients, the drug improved clinical symptoms, which
correlated with a decrease in serum IgE antibody
levels.6 Soluble IL-4 receptor7 and mutant IL-4 proteins8
were shown to block IgE production, resulting in the
inhibition of IgE-mediated allergic reactions.9 To inter-
fere with the function of IgE, humanized non-anaphy-
lactogenic anti-IgE antibodies such as rhuMAb-E25 are
currently in clinical development. These antibodies
interfered with virtually all aspects of IgE function in
vitro and in vivo and had therapeutic effects in patients
with allergic asthma and allergic rhinitis due to their
selective neutralization and inhibition of IgE.10
The pathogenesis of allergic diseases such as atopic
dermatitis (AD), allergic asthma, and allergic rhinitis
is associated with elevated levels of immunoglobulin E
(IgE). IgE binds to its high-affinity receptor FcꢀRI,
which is expressed on mast cells, basophils, monocytes,1
and Langerhans cells,2 or to FcꢀRII (CD23) found on
B-cells, monocytes, and dendritic cells.3 Interaction of
allergens with IgE/FcꢀRI complexes on mast cells and
basophils leads to receptor cross-linking and degranu-
lation of these cells as measured by the release of
pharmacological mediators (e.g., histamine, cytokines,
and chemokines) responsible for the pathogenesis of
allergic diseases.4 Recent studies established the in-
volvement of IgE/allergen immune complexes in the
enhancement and redirection of IgE-mediated allergen
presentation,5 leading to the perpetuation and worsen-
ing of allergic conditions. Therefore, IgE appears to be
critically involved in both immediate early and later
chronic manifestations of the allergic immune response.
On the basis of the central role of IgE in allergic
diseases, a number of strategies were developed either
On the basis of this evidence, it can be anticipated
that a specific low molecular weight inhibitor of IgE
synthesis in B-lymphocytes will have similar therapeu-
tic effects in allergic patients with the advantages of
an orally available drug. So far, there is no such drug
on the market or in clinical development.
* To whom correspondence should be addressed. Phone: +43-1-
86634-9038. Fax: +43-1-86634-582. E-mail: peter.ettmayer@
pharma.novartis.com.
Searching for such low molecular weight inhibitors,
we developed a high-throughput screening reporter gene
† Current address: Intervet, Althanstrasse 14, Vienna A-1090,
Austria.
10.1021/jm010888h CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/03/2001

3032 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Berger et al.
assay in a human B-cell line monitoring early steps of
IgE isotype switching. The screening for compounds that
interfered with the IL-4 and anti-CD40 mAb induced
IgE-germline promoter activity but not with cell vi-
ability led to identification of (S)-4-(1-phenylethylami-
no)quinazoline as a potent and selective inhibitor. The
inhibitory potencies measured in the reporter gene
assay for both enantiomers correlated with the inhibi-
tion of IgE synthesis in primary human splenocytes,
thereby validating the HTS assay. Parallel synthesis in
solution utilizing polymer-supported quenching re-
agents provided a series of analogues of compound 1 for
SAR studies. Here, we report the effect of the stereo-
chemistry in the phenethylamine side chain and the
charge properties of the heterocyclic core of fused 4-(1-
phenylethylamino)pyrimidines on the inhibition of the
IgE-germline promoter activity.
Biology
IgE is produced by a subset of B-lymphocytes that are
generated during an allergic immune response as the
result of IgE isotype switching. This differentiation
process is driven by direct contact of the B-cells with
activated T-helper (TH) cells and by TH-cell-derived
interleukin-4 (IL-4) or IL-13. IL-4 induces transcription
from the IgE-germline promoter (IgE-GLP) through the
ꢀ-switch region and the Cꢀ gene locus. This activation
is essential and primes the B-cells for the subsequent
DNA switch recombination step, which is mainly driven
by signals originating from the T/B-cell contact, such
as the interaction of the TH-cell-expressed CD40 ligand
with its CD40 receptor on B-cells. The recombined
genome allows for the generation of productive IgE
transcripts and the synthesis and secretion of IgE
protein.11 In cell culture systems, T/B cell contact can
be mimicked by cultivation of primary B-cells with
agonistic anti-CD40 monoclonal antibodies. Thus, a
blockade of the IL-4 induced activity of the IgE-GLP
should result in diminished IgE isotype switching and
IgE synthesis. To be able to screen for inhibitors of IgE-
GLP activation, a reporter gene assay was developed.
The human B-cell line BL2 was stably transfected with
a plasmid in which the luciferase reporter gene is under
the control of a 630 bp fragment encompassing all
regulatory DNA elements known to be involved in the
IL-4 response.12 The cytokine inducibility of the lu-
ciferase gene was compared with the one of the endog-
enous IgE-germline gene. Figure 1A demonstrates that
IL-4 led to a ca. 7-fold induction of luciferase expression,
while stimulation with anti-CD40 mAb had no effect.
The combination of the two reagents acted synergisti-
cally to achieve a 35-fold up-regulation of reporter gene
expression. Synergy of the IL-4 and CD40 mediated
induction of IgE-GLP activity has been shown before
in primary human B-cells.13 Essentially the same
results were obtained when steady-state levels of IgE-
germline transcripts were quantitated in the same cells
(Figure 1B). From that we conclude that this reporter
gene assay closely mimics the situation seen during the
early steps in IgE isotype switching and thus is useful
for screening for inhibitors. B-cell viability, as deter-
mined by mitochondrial activity, was assessed in paral-
lel to discriminate from general antiproliferative effects.
F igu r e 1. Comparison of the cytokine inducibility of the
luciferase gene and the endogenous IgE-germline gene. Trans-
fectant cells were stimulated with IL-4, anti-CD40 monoclonal
antibodies (mAb), or both for 24 h. The antibodies were cross-
linked with sheep antimouse IgG antibodies (SAM) to mimic
the trimeric conformation of the natural ligand for CD40.
Induced IgE-GLP activity was assessed either by luciferase
assays for the transgene promoter or by detection of IgE-
germline transcripts (IgE glRNA) for the endogenous promoter.
Ch em istr y
The 4-aminopyrimidines were prepared by parallel
synthesis in solution, utilizing polymer-supported quench-
ing reagents.14 The substituted 4-chloropyrimidines
were reacted with 2 equiv of amines in the presence of
polymer-supported triethylamine (P-TEA, Scheme 1) to
achieve complete turnover. Because of the different
reactivity of the educts, the reactions had to be divided
into three sets: (A) reaction in THF at 65 °C for 16 h
(1-15); (B) DMF/n-butanol, 100 °C, 16 h (16-18, 20,
23-30); and (C) neat, 160 °C, 1 h (19, 21, 22) (Tables 1
and 2). After filtration the reaction mixtures were
incubated with Wang aldehyde resin15 (P-CHO) in
tetrahydrofuran containing 1% of acetic acid for 16 h
at room temperature to remove excess of amine by Schiff
base formation. The catalytic amount of acetic acid was
found to be essential for complete removal of the
primary amine in the presence of the secondary amine
in the product. Acidic ion-exchange resin also tended
to bind the product. Removal of the solvents provided
the final product in near-quantitative yield and greater
than 90% purity. Further purification was done by
crystallization (1-5, 19, 22) or by automated prepara-
tive HPLC on reversed phase using water/acetonitrile
gradients containing 0.1% TFA (6-13, 16-18, 20, 21,
24-25). The latter compounds were tested as their TFA
salts. Purity and homogenicity of the final compounds
1
were assessed by RP-HPLC, TLC, and H NMR spec-
troscopy.
Resu lts a n d Discu ssion
Screening for inhibitors of IL-4/anti-CD40 mAb in-
duced IgE-germline promoter activation revealed race-

S(+)-4-(1-Phenylethylamino)quinazolines
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 3033
Sch em e 1. Representative Example of the Parallel Synthesis Scheme in Solution Utilizing Polymer-Supported
Quenching Reagents
Ta ble 1. Variation in the Side Chain of 4-(1-Phenylethylamino)quinazolines
20
c
yielda purityb
mp
(°C)
[R]D
(deg)
MH+
IgE-GLP-RGA,
CellProl_XTT,c
R
method
(%)
(%)
MW
(ESI, m/z) IC50 ( SEM (n)d IC50 ( SEM (n)d
1
2
3
4
5
6
7
8
9
(S)-1-phenylethylamine
(R)-1-phenylethylamine
benzylamine
(S)-1-cyclohexylethylamine
(R)-1-cyclohexylethylamine
(S)-1-(2-naphthyl)ethylamine
(R)-1-(2-naphthyl)ethylamine
(S)-1-(4-bromophenyl)ethylamine
(S)-1-(4-methylphenyl)ethylamine
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B
55
67
66
76
71
80
60
68.7
74.6
75
81
58
72.9
64
80
99
61
99.6
100
123-124 +169.1 249.3
123-124 -168.5 249.3
250.1
250.1
236.0
256.1
256.0
300.1
300.0
328.0
264.1
280.1
280.1
264.1
266.0
262.0
262.0
264.0
222.0
127 ( 14 (19)
>2000 (4)
>2000 (4)
81 ( 9 (5)
>2000 (3)
14 ( 3 (5)
>2000 (2)
72 ( 14 (4)
46 ( 7 (4)
45 ( 8 (4)
23 ( 3 (4)
147 ( 25 (5)
149 ( 88 (5)
>2000 (4)
1015 ( 212 (4)
>2000 (2)
>2000 (2)
>2000 (4)
>2000 (3)
>2000 (3)
>2000 (3)
>2000 (3)
1633 ( 300 (3)
>2000 (2)
>2000 (2)
>2000
100
100
100
100
100
100
171
235.3
167-168 +124.5 255.4
167-168 -128.7 255.4
+199.6 299.4 + 114
-199.4 299.4 + 114
328.2 + 114
263.3 + 114
279.3 + 114
279.3 + 114
263.3 + 114
265.3 + 114
+ 4.0 261.3
-2.1 261.3
263.3 + 114
221.3 + 114
95.8
10 (S)-1-(4-methoxyphenyl)ethylamine
11 (S)-1-(3-methoxyphenyl)ethylamine
12 (S)-1-phenylpropylamine
13 (S)--2-amino-2-phenylethanol
14 (S)-1-aminoindane
15 (R)-1-aminoindane
16 N-methyl-1-phenylethylamine
17 anilin
95.8
94.8
99.6
98.7
94.7
94.7
99.4
97.9
>2000 (2)
>2000
>2000 (3)
>2000 (3)
>2000 (3)
>2000 (3)
>2000 (2)
>2000 (2)
a
b
Isolated yields after purification (recrystallization or preparative HPLC). Purity of the final products by HPLC, 254 nm. c Determined
d
in ethanol, c ) 1 g/100 mL. Mean values from n independent IC50 determinations (in nM) are given ( the standard error.
Ta ble 2. Variation in the Heterocyclic Core of (Fused) 4-(1-Phenylethylamino)pyrimidines
yielda purityb
mp
(°C)
MH+
(ESI, m/z) IC50 ( SEM (n)c IC50 ( SEM (n)c
IgE-GLP-RGA,
CellProl_XTT,
R
method
(%)
(%)
MW
1
quinazolin-4-yl
A
B
C
B
C
C
B
B
B
B
B
B
B
B
55
78
60
41
71
33
31
27
48
65
53
37
37
21
99.6
99.3
100
99.0
99.2
99.3
99.4
95.2
92.7
99.6
123-124 249.3
309.4 + 114
248.3
127 ( 14 (19)
>2000 (2)
701 ( 87 (5)
670 ( 123 (5)
>2000 (2)
>2000 (3)
931 ( 204 (3)
>2000 (2)
>2000 (2)
>2000 (2)
>2000 (4)
>2000 (2)
>2000 (4)
>2000 (4)
>2000 (2)
>2000 (4)
>2000 (3)
>2000 (2)
>2000 (2)
>2000 (2)
>2000 (3)
1476( 317 (4)
>2000 (2)
>2000 (2)
18 6,7-dimethoxyquinazolin-4-yl
19 quinolin-4-yl
20 7-chlorquinolin-4-yl
21 2-methylquinolin-4-yl
22 isoquinolin-1-yl
23 2-methylthiopyrimidin-4-yl
24 2-amino-6-methylpyrimidin-4-yl
25 6-methylpyrrolo[2,3-d]pyrimidin-4-yl
26 purin-6-yl
27 thieno[2,3-d]pyrimidin-4-yl
28 5-methylthieno[2,3-d]pyrimidin-4-yl
29 thieno[3,2-d]pyrimidin-4-yl
30 7-methylthieno[3,2-d]pyrimidin-4-yl
310.1
249.1
283.0
263.1
249.1
246.0
229.1
253.1
240.0
256.0
270.0
256.0
270.0
87-89
80-81
282.8 + 114
262.4 + 114
248.3
245.3
228.3 + 114
252.3 + 114
239.3
255.3
269.4
100
370 ( 70 (4)
130 ( 12 (5)
>2000 (2)
91.5
99.6
92.9
255.3
269.4
>2000 (2)
a
b
Isolated yields after purification (recrystallization or preparative HPLC). Purity of the final products by HPLC, 254 nm. c Mean
values from n independent IC50 determinations (in nM) are given ( the standard error.
mic 4-(1-phenylethylamino)quinazoline as hit. To de-
termine the role of the stereochemistry on the biological
activity, the individual enantiomers 1 and 2 were
synthesized and tested in the reporter gene assay (Table
1). Luciferase expression was blocked by the S(+)
enantiomer 1 in a dose-dependent manner with an IC50
of 125 nM (Figure 2), while the corresponding R(-)
enantiomer 2 did not inhibit the IL4-driven germline
promotor activity up to the highest concentration tested
(2 µM). The viability of the B-cells, assessed in parallel,
was only effected at 20 times higher concentrations of
compound 1 (IC50 of 2.5 µM, Figure 2).
On the basis of the observed enantioselective inhibi-
tion in the reporter gene assay, compounds 1 and 2
qualified as tools to validate the concept that the
blockade of IL-4-dependent IgE-germline gene tran-
scription should lead to inhibition of IgE switch recom-
bination and IgE secretion from B-cells. Human sple-
nocytes were cultivated with IL-4 and agonistic anti-
CD40 mAb in the presence of increasing concentrations

3034 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Berger et al.
and 12 might indicate a preference for the meta
substitution.
Replacing the (S)-1-phenylethylamino group in 1 with
a (S)-1-phenylpropylamino moiety (12) or (S)-phenyl-
alaninol (13) resulted in IC50 values comparable to that
of 1. Substantially decreased potency was observed
when the 1-phenylpropylamino group in 12 was re-
placed with the 1-aminoindane moiety (14, 15). Inter-
estingly this time the R enantiomer 15 was found to
have a weak biological activity (IC50 of 1015 nM) while
the S enantiomer 14 was inactive up to 2 µM. When
the 4-amino group in quinazoline 1 was methylated (16),
no inhibitory activity was observed up to the highest
tested concentration. A similar loss of potency was
observed when the 1-phenylethylamino moiety was
replaced by aniline (17). Both findings highlight the
importance of an R-branched, S-configurated, liophilic
secondary amine in position 4 of the quinazoline for IgE
inhibitory activity.
F igu r e 2. Inhibition of IgE-germline promoter activity in
BL-2 cells by 1 (O, duplicates). Parallel cultures were quan-
titated for cell viability (×, duplicates) in the presence of 1.
To establish the role of the quinazoline core of 1 in
the IL-4 driven IgE-germline promoter inhibition, we
studied a diverse set of substituted heterocycles, keeping
the S-phenylethylamine side chain constant (Table 2,
Figure 4). 6,7-Dimethoxy substitution on the quinazo-
line ring resulted in complete loss of potency (18). We
also found that the nitrogen in position 3 of the
quinazoline can be replaced by carbon but the resulting
quinoline 19 is 5 times less active than the lead
compound 1. Substitution on the quinoline ring is
tolerated in position 7 (Cl, 20) and not allowed in
position 2 (CH3, 21). Isoquinoline 22 (replacement of the
nitrogen in position 1 by carbon) was found to be
inactive. A similar observation as with the quinolines
20 and 21 was made for substituted pyrimidines 23 and
24. While weak activity was measured for the 2-meth-
ylthiopyrimidine (23, IC50 of 931 nM), no activity was
obtained for the 2-amino-6-methylpyrimidine derivative
24. Replacing the benzene ring in the quinazoline core
with pyrrole and imidiazole yielded the pyrrolo[2,3-d]-
pyrimidine 25 and the purine derivative 26, both being
inactive up to 2 µM. When the benzene ring was
replaced by thiophene, only the thieno[2.3d]pyrimidines
27 and 28 inhibited the IgE promoter activity with
similar potencies as the lead compound 1. The thieno-
[3.2d] pyrimidines (29, 30) were inactive up to the
highest tested concentration (2 µM).
To better understand the structural properties that
are beneficial for the inhibition of IgE synthesis within
this compound class, besides the pronounced preference
for the S enantiomers, we calculated several physico-
chemical properties for the S enantiomers (1, 4, 6, 8-14,
18-30) and compared them with the biological activity
measured in the reporter gene assay. Interestingly, we
discovered that the activity as plotted by log(IC50)
correlated well with the charge at the N atom (QN) as
shown in Figure 5. The atomic point charges were
calculated by density functional quantum chemistry
calculations as described in the Experimental Section
under “Computational Details”.
F igu r e 3. Dose-response curves of quinazolins 1 (O) and 2
(4) on IgE synthesis in human splenocytes. Data are presented
as mean ( SEM (n ) 4-12).
of 1 or 2. After 9 days, IgE was quantitated in the cell
supernatants by ELISA. Figure 3 demonstrates that 1
but not 2 blocked IgE production in a dose-dependent
manner. The IC50 value of 110 nM measured for 1 is
very similar to the one calculated in the IgE-GLP
reporter gene assay. From these data we conclude that
the observed block in IgE synthesis is a direct conse-
quence of inhibition of IgE-germline gene inhibition.
Therefore, the basic concept could be validated and
verified in relevant target cells.
To further substantiate the observed enantioselectiv-
ity for biological activity, the corresponding nonchiral
4-benzylquinazoline (3) and both enantiomers of the
4-(1-cyclohexylethyl)amino (4/5) and 4-(1-naphthyethyl)-
aminoquianzoline (6/7) were tested in the reporter gene
assay (Table 1). Nonchiral 3 did not effect the IL-4
driven induction of the germline transcript up to 2000
nM, highlighting the importance of the R branching of
the 4-amino substituent. When the 1-phenylethylamino
group in 1 and 2 was replaced by the 1-cyclohexylethyl-
amino group, the S(+) enantiomer (4) was found to 1.5
times more potent than 1 and more than 25 times more
active than R(-)-5. Further enhancing the lipophilicity
of the side chain by incorporation of (S)-1-(2-naphthyl)-
ethylamine yielded 6, the most potent derivative within
this series (IC50 of 14 nM). The R(-) enantiomer 7 was
again found to be inactive at the highest tested concen-
tration (2 µM). Bromo (8), methyl (9), and methoxy (11,
12) substitutions on the phenylethylamine residue in 1
were also well tolerated, leading to 3-5 times increased
potencies compared to 1. Comparing the potencies of 11
Classifying compounds 21 and 23 as outliers, the
inverse correlation can be described by a linear correla-
tion coefficient R of -0.94. The pyrimidine 21 and the
quinoline 22 are both less active than the point charge

S(+)-4-(1-Phenylethylamino)quinazolines
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 3035
F igu r e 4. Modifications of the heterocyclic core of 4-(1-phenylethylamino)pyrimidines.
electrons to N1, whereas the S atom on C6 of the
pyrimidines 27 and 28 lowers the negative charge on
N1.
To evaluate the PK properties, compounds 4 and 5
were administered to mice orally (4, 30 mg/kg) and by
intravenous injection (4 and 5, 10 mg/kg) for plasma
level determination by LC-MS. After oral administra-
tion, plasma levels of 4 were below the detection limit.
After intravenous administration, the R(-) enantiomer
5 was found to be rapidly cleared from the system (10
mg/kg, AUC0-5h 0.92 µM h, Cl 43.6 [kg-1 h-1]. Because
of acute toxicity, no PK data could be obtained for the
biologically active S(+) enantiomer 4.
Con clu sion s
S(+)-4-(1-Arylethylamino)qinazolines were discovered
as potent and enantioselective inhibitors of the IL4-
driven IgE-germline promoter activity in human B-
lymphocytes. A cell viability assay proved that the
inhibition is not due to a general antiproliferative effect.
The potencies found in the reporter gene assay could
be correlated with those measured by IgE-ELISA in
primary human splenocytes, thereby providing evidence
that the block of IgE synthesis is the direct consequence
of IgE-germline inhibition. Within the set of S enanti-
omers, quantum mechanical calculations revealed that
the IgE inhibitory activity can be quantitatively de-
scribed by the charge at N-1 of the heterocyclic core and
to a lesser extent by the molar refractivity. These results
show the importance of electron-deficient fused 4-ami-
nopyrimidines and of lipophilic side chains for biological
activity in addition to the strong preference for the S
configuration of the phenethylamine side chain. The
S-enantioselective inhibition is remarkable insofar as
biological activity for fused 4-(1-phenylethylamino)-
pyrimidines has been published for the R enantiomers
only. 2 was reported as an ATP site directed reversible
inhibitor of the epidermal growth factor receptor (EGFR)
tyrosine kinase (IC50 of 86nM), whereas the S(+) enan-
tiomer 1 shows no activity up to 10 µM in the EGFR
assay.17 For the corresponding pyrimido[5,4-b]indoles,18
benzothieno[2,3-d]pyrimidines18 and pyrrolo[2,3-d]py-
rimidines,19 where data on EGFR tyrosine kinase
inhibition is published, a similar preference for the R
enantiomers is observed (Table 3). While the molecular
target for the IgE-germline promoter inhibitors remains
unknown, the potential correlation of the IgE inhibitory
activity with the acute toxicity in mice could point
F igu r e 5. Correlation between the calculated point charge
QN and log(IC50) for a subset of 4-aminopyrimidines. Filled
circles were used in the R calculation and the straight line fit
(-). Circles are labeled with the compound number.
would predict. It is interesting to note that both outliers
are substituted at the 2 position, and the lack of potency
could be attributed to unfavorable interaction of this
position with the unknown receptor. Molar refractivity16
(polarizability) correlated to a lesser extent with log-
(IC50) (CMR, R ) -0.55; the size of the circles plotted
in Figure 5 corresponds to the CMR values), and there
was a cross-correlation between CMR and QN with a
correlation coefficient (R) of 0.60. No correlation of log-
(IC50) and CLOGP was observed (R ) -0.32). On this
basis, the activity can be well represented by only one
parameter, the QN point charge, in the following equa-
tion:
log(IC50) ) -5.33((0.70) - [16.75((1.50)]QN (1)
It is remarkable that within a range of 3 orders of
magnitude the IC50 values of 19 out of 21 compounds
fit the equation well (Figure 5). With the above density
functional quantum chemistry calculations the lack of
activity of the dimethoxy-substituted quinazoline 18 can
be rationalized by the +M effect of the methoxy sub-
stitution increasing the negative charge QN at the N1
atom of the pyrimidine ring. The same argument
provides a rationale for why the thieno[2.3d]pyrimidines
27 and 28 are more active than the corresponding
thieno[3.2d]pyrimidines 29 and 30. The +M effect of the
S atom on C5 of the pyrimidine (29, 30) donates

3036 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Berger et al.
Ta ble 3: Blockage of EGF-Stimulated Tyrosinephosphorylation
in EGFR-Positive A 431 Cells
resin, Novabiochem, 2.66 mmol/g, 0.5 mmol) together with
acetic acid (70 µL). The mixture was agitated on an orbital
shaker overnight and filtered, and the resin was washed with
THF (2 × 2 mL). After removal of the solvent in vacuo the
product was obtained in typically around 90% purity and near-
quantitative yield and could be further purified by preparative
HPLC.
Gen er a l Meth od B. A reaction vial (10 mL) was charged
with polymer-supported TEA (140 mg, diethylaminometh-
ylpolystyrene, Fluka, 3.2 mmol/g, 0.45 mmol) and purged with
argon for 10 min. 4-Chloropyrimidine or a 4-chloroquinazoline
derivative (0.15 mmol) dissolved in DMF (0.6 mL) was added
followed by a solution of the corresponding amine in 1-butanol
(0.5 M, 0.6 mL). Subsequently 1-butanol (1.8 mL) was added
and the suspension was agitated at 100 °C for 16 h. After
cooling to room temperature, the mixture was filtered, the
resin was washed with DMF (2 × 2 mL), and the filtrates were
combined in scintillation vials. The solvent was removed in
vacuo and the residue dissolved in THF containing 0.1% acetic
acid (4 mL) and treated with P-CHO as described in general
method A.
EGFR,
IC50 (nM)
IgE-GLP-RGA,
IC50 (nM)
R
/
quinazolin-4-yl
quinazolin-4-yl
1
2
S
R
S
R
R
>104 a
86a
127
>2000
2,4,9-triazafluoren-1-yl
2,4,9-triazafluoren-1-yl
benzothieno[3,2-d]-
pyrimidin-4-yl
6-phenylpyrrolo[2,3-d]-
pyrimidin-4-yl
>105 b
419b
538b
R
10-50c
a
b
IC50 values as published in ref 17. IC50 value as published
in ref 18. c IC50 value as published in ref 19.
toward a common target. N-Alkyl-4-aminoquinazolines
are reported to be the most potent synthetic inhibitors
of the NADH-ubiquinone reductase activity of complex
I (the function of the energy-conserving enzyme complex
is particularly crucial in tissues such as neurons that
heavily rely on mitochondrial ATP- and NAD-linked
pathways).20 Unfortunately, nothing is published about
the enantioselectivity of complex I inhibition. The
potential correlation between IgE inhibition in human
splenocytes and complex I inhibition is the topic of
ongoing investigations.
Gen er a l Meth od C. A V-bottom-shaped reaction vial (1
mL) was charged with 4-chlorochinolin (0.3 mmol) or
a
1-chloroisoquinolin derivative (0.3 mmol). The corresponding
amine (0.6 mmol) was added, and the mixture was heated to
160 °C for 60 min. After cooling to room temperature, the
resulting mixture was dissolved in DMF and purified by
preparative HPLC.
Com p u ta tion a l Deta ils. The lowest energy conformation
was searched by systematic bond rotation and the geometry
optimized corresponding to the potential described in Merk
Molecular Force Field (1994).23 Subsequently, the electronic
structure was calculated at the level of gradient-corrected
density functional theory as implemented in the J aguar24
quantum chemistry program. The exchange-correlation func-
tional included the XR exchange potential by Slater25 and the
local correlation of Vosko, Wilk, and Nusair (VWN)26 as well
as self-consistent gradient corrections by Becke (1988)27 and
Perdew (1986)28 for exchange and correlation, respectively. The
basis set was 6-31G**. Atomic point charges were calculated
by the Mulliken population analysis.29 This fairly high level
of theory may not be necessary for QSAR calculations.
However, we found that for systems where electronic structure
plays an important role in the activity relationship, the higher
level of theory pays off by providing a better correlation. In
addition, even these high-level calculations require only 20
min/molecule on a Silicon Graphics Origin 2000 workstation.
Biology. 1. Cell Cu ltu r e a n d Rea gen ts. The Epstein-
Barr virus negative Burkitt’s lymphoma B-cell line BL2 was
carried in Yssel’s medium (25) supplemented with 10% heat-
inactivated fetal calf serum (GIBCO Laboratories, Grand
Island, NY), 100 U/mL penicillin, and 100 µg/mL streptomycin.
Cells were kept at 37 °C with 5% CO2. Purified human
recombinant IL4 was obtained from Novartis AG (Basle, CH)
with a specific activity of 0.5 U/ng. The hybridoma cell line
producing mouse monoclonal antihuman CD40 antibodies was
obtained from Dr. Shu man Fu, University of Virginia,
Charlotteville, VA.
2. RNA isola tion a n d Nor th er n blot a n a lysis. Total RNA
was isolated from cells by the RNAzol method (Biotecx
Laboratories, Inc., Houston, TX) according to the instructions
of the manufacturer. Aliquots (15 µg) were subjected to
electrophoresis in a 1.2% denaturing agarose gel and trans-
ferred to a nylon membrane (GeneScreen, DuPont, Boston,
MA). The membranes were stained with 0.04% methylene blue
in 0.5 M sodium acetate (pH ) 5.2) for 10 min at room
temperature followed by several washes in water to localize
and quantify rRNA before hybridization. The RNA was
hybridized at 65 °C to a 32P-labeled oligonucleotide probe
derived from the Cꢀ1 exon (5′-gccacacggcaggtgaacatctgcttg-3′),
which was labeled at the 3′ end using the terminal transferase
kit (Roche). The blots were washed for 30 min at 62 °C in wash
mix 1 (3 × SSC, 5% SDS, 100 mM Na‚PO4, pH ) 7.0, 10 ×
Denhardt solution) followed by a short wash in 1 × SSC, 1%
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Neolab
automated melting apparatus and are uncorrected. The ranges
1
given reflect the results from two independent melts. H and
13C NMR spectra were recorded on Bruker AC 250 or Avance
400 systems. Optical rotation data were obtained using a
Perkin-Elmer model 241 polarimeter. Analytical HPLC analy-
ses were performed on a HP 1100 system with a multiwave-
length detector using a Waters Xterra column (4.6 mm × 50
mm C18, 3.5 µm) and a flow rate of 2 mL/min. The column was
eluted with a linear gradient of 10-95% acetonitrile in water
containing 0.1% TFA in 6 min. Preparative HPLC separations
were performed on a Gilson system (model 322 pump, model
215 liquid handler, model 155 dual-wavelength detector) using
a Waters Prep Nova-Pak cartridge (25 mm × 100 mm C18, 6
µm) and a flow rate of 25 mL/min. The column was eluted with
a linear gradient of 10-90% acetonitrile in water containing
0.1% TFA in 13 min. MS analyses were obtained on a HP
model 1100 system with direct flow injection into a Thermo-
quest (Finnigan) MS detector with electrospray ionization.
Parallel syntheses were performed on a Quest 210 (Argonaut)
in 5 mL reaction vessels or on a Tecan Combitec reaction block.
Vacuum removal of solvents was achieved using an IR dancer
(Hettlab) or a Genevac centrifuge. 4-Chloro-6,7-dimethoxy-
quinazoline, 4-chlorothieno[2,3-d]pyrimidine, 4-chloro-5-me-
thylthieno[2,3-d]pyrimidine, 4-chlorothieno[3,2-d]pyrimidine,
and 4-chloro-7-methylthieno[3,2-d]pyrimidine were purchased
from Maybridge. 4-Chloroquinazolin21 and 4-chloro-6-meth-
ylpyrrolo[2,3-d]pyrimidine22 were prepared from their corre-
sponding pyrimidinols.
Gen er a l Meth od A. A reaction vessel for a Quest parallel
synthesizer (5 mL) was charged with polymer-supported TEA
(140 mg; diethylaminomethylpolystyrene, Fluka, 3.2 mmol/g,
0.45 mmol) and purged with argon for 10 min. 4-Chloro-
quinazoline (24.7 mg, 0.15 mmol) dissolved in THF (1 mL, 0.15
mM) was added followed by the corresponding amine (0.3
mmol) dissolved in THF (1 mL, 0.3 mM). The suspension was
then agitated at 65 °C for 16 h. After cooling to room
temperature, the mixture was filtered, the resin was washed
with THF (2 × 2 mL), and the filtrates were combined in
scintillation vials. P-CHO was added (180 mg, Aldehyde Wang

S(+)-4-(1-Phenylethylamino)quinazolines
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 3037
(3) Kikutani, H.; Suemura, M.; Owaki, H.; Nakamura, H.; Sato, R.;
Yamasaki, K.; Barsumian, E. L.; Hardy, R. R.; Kishimoto, T. Fc
epsilon receptor, a specific differentiation marker transiently
expressed on mature B cells prior to isotype switching. J . Exp.
Med. 1986, 164, 1455-1469. Vercelli, D.; J abara, H. H.; Lee, B.
W.; Woodland, N.; Geha, R. Human recombinant interleukin-4
induces Fc epsilon R2/CD23 on normal human monocytes. J .
Exp. Med. 1988, 167, 1406-1416. Bieber, T.; Rieger, A.; Neu-
christ, C.; Prinz, J . C.; Rieber, E. P.; Boltz-Nitulescu, G.;
Scheiner, O.; Kraft, D.; Ring, J .; Stingl, G. Induction of Fc epsilon
R2/CD23 on human epidermal Langerhan’s cells by recombinant
interleukin-4 and gamma-interferon. J . Exp. Med. 1989, 170,
309-314.
SDS at 62 °C. Specific signals were visualized by exposure to
X-ray films (Kodak BioMax MR) for 3 days. Then the blot was
stripped and reprobed with a 32P-labeled oligonucleotide probe
specific for the glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) gene as control.
3. In d u ction of IgE Syn th esis in Hu m a n Sp len ocytes.
Normal human splenocytes were seeded at 1 × 105 cells/mL
in 96-well round-bottom plates and cultivated with 50 ng/mL
IL-4 and 1 µg/mL anti-CD40mAb for 9 days. Compounds were
dissolved in DMSO, diluted to suitable concentrations with
culture medium, and added to the cells 2 h before the inducers.
Supernatants from nine identical culture wells were pooled
into three replicates, and IgE was quantitated by isotype-
specific ELISA. Half-maximal inhibition was calculated using
Origin 6.0 logistic fit.
4. Gen er a tion of Sta ble Rep or ter Gen e Tr a n sfecta n ts
a n d Lu cifer a se Assa ys. A 630 bp DNA fragment encompass-
ing all relevant regulatory elements12 of the human IgE-
germline promoter was cloned into the vector pGL2-basic
(Promega, Wisconsin, WI) in which a neomycin gene resistance
cassette had been incorporated. BL2 cells were transfected by
electroporation, and stable clones were selected in 1.8 mg/mL
G418 (Gibco). Functional clones were further selected by
induction with IL-4 and anti-CD40mAb for 48 h. The best
responder clone was seeded at 1 × 106 cells/mL in a 96-well
plate and induced for 48 h with 10 ng/mL IL-4, 1 µg/mL anti-
CD40mAb, and 1 µg/mL sheep antimouse IgG (Grub, Vienna)
to cross-link the CD40 antibodies. Then the cells were lysed
and luciferase activity was determined, according to the
instructions of the manufacturer, using the dual-luciferase
reporter assay system (Promega). In parallel cultures, cell
viability was quantitated as assessed by intact mitochondria
(EZ4U, Biomedica, Vienna, Austria). In both assays, duplicates
were analyzed. Half-maximal inhibition was calculated using
Origin 6.0 logistic fit.
5. P h a r m a cology. For po administration the compounds
were dissolved in ethanol/Labrafil M2125/corn oil, 150:350:
500 w/w/w. The solutions were diluted with an equal part of
isotonic glucose solution immediately before use. For intrave-
nous administration the compounds were dissolved in Cremo-
phor EL/ethanol, 3:1 w/w, and diluted with nine parts of
isotonic glucose solution before use. Nonfasted female mice
(strain Balb/c) were dosed with the compounds either perorally
(po) per gavage or intravenously (iv) by injection into the tail
vein. Blood samples were collected from three animals at six
time points. Plasma was prepared from heparin-stabilized
blood by centrifugation and stored at -20 °C until analysis.
Analysis was done after protein precipitation using methanol
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followed by centrifugation on
a HP1090 chromatograph
equipped with a Merck Superspher RP 8 column.
Ack n ow led gm en t. The authors are grateful to
Peter Nussbaumer for helpful discussions and a critical
review of the manuscript. We thank Heinrich Aschauer
for the pharmacokinetic studies and Elena Macoratti
for technical assistance.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR and 13C
NMR data for all new compounds (4-6, 14-16, 19-21, 23-
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Internet at http://pubs.acs.org.
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