Inhibition of Fc epsilon RI-mediated activation of mast cells by 2,3,4-trihydropyrimidino[2,1-a]isoquinolines.

Article abstract of DOI:10.1021/jm9706628

Assays based on reporter gene technology represent today an important tool in the pharmaceutical industry for discovering novel compound classes interfering with the activation and signaling of target cells after stimulation. Here we describe a reporter g

Full text of DOI:10.1021/jm9706628

1050
J . Med. Chem. 1998, 41, 1050-1059
In h ibition of F cERI-Med ia ted Activa tion of Ma st Cells by
2,3,4-Tr ih yd r op yr im id in o[2,1-a ]isoqu in olin es
Dieter Scholz,* Hannelore Schmidt, Eva E. Prieschl, Robert Csonga, Winfried Scheirer, Virginia Weber,
Anna Lembachner, Gunther Seidl, Gudrun Werner, Peter Mayer, and Thomas Baumruker
Department of Immunology, Novartis Forschungsinstitut GmbH, Brunnerstrasse 59, A-1235 Vienna, Austria
Received October 1, 1997
Assays based on reporter gene technology represent today an important tool in the pharma-
ceutical industry for discovering novel compound classes interfering with the activation and
signaling of target cells after stimulation. Here we describe a reporter gene assay targeting
mast cell activation by IgE plus antigen, established in an attempt to identify substances
preventing type I allergy (allergic rhinitis, allergic conjunctivitis, allergic asthma, and acute
and chronic urticaria). The assay is based on a murine mast cell line designated CPII,
stimulation by IgE plus antigen, and a reporter gene construct with the TNFR promoter linked
to luciferase as a read-out system. Via screening about 50 000 substances, compound 2 was
found to inhibit the reporter gene induction in the submicromolar range in this assay.
Analogues of compound 2 of the 2,3,4-trihydropyrimidino[2,1-a]isoquinoline type were synthe-
sized starting from 2-alkyl-substituted benzonitriles via aminolysis with 1,3-diaminopropane,
dimetalation of 2-substituted 2-phenyl-1,4,5,6-tetrahydropyrimidines with n- and sec-butyl-
lithium, reaction with carboxylic acid methyl esters, and finally acidic dehydration. From about
50 derivatives, compound 41 was selected as a lead structure with an IC50 of 0.2 µM and a
TC50 of 2.7 µM. In a first profiling in secondary assays, it effectively interfered with the
production of mediators such as TNFR, IL-4, IL-6, IL-13, and leukotriene synthesis as measured
by the corresponding ELISAs. In addition, a passive cutaneous anaphylaxis in mice (a typical
type I reaction) is inhibited to more than 90% by compound 41, when administered
intradermally 90 min before challenge.
In tr od u ction
relates to the success of cyclic undecapeptides and
macrolides as specific inhibitors of the TCR/CD28
signaling in T-cells and their widespread use in the
clinical practice of transplantation surgery.6,7 To screen
for compounds interfering with the signaling in mast
cells, a reporter gene assay (RGA) in the murine mast
cell line CPII was established.4 It uses a stably inte-
grated DNA construct of the human TNFR promoter
linked to luciferase as a reporter gene and TNFR 3′-
nontranslated sequences.8,9 The validation of this read-
out system in CPII cells in transient transfections was
demonstrated recently.9 Upon triggering this recombi-
nant cell line with IgE plus antigen/allergen, a strong
induction (10-30-fold) of luciferase activity can be
detected which parallels the induction of the endogenous
proinflammatory mediators (histamine, leukotrienes,
and cytokines). In particular, it is an ideal surrogate
parameter for measuring the endogenous TNFR produc-
tion, a cytokine shown to contribute to the allergic late-
phase inflammation in a murine in vivo model by
applying neutralizing antibodies.10
Triggering of mast cells via the FcꢀRI is the final step
in a complex chain of events in atopic individuals,
resulting in diseases such as allergic rhinitis, conjunc-
tivitis, acute and chronic urticaria, and acute allergic
asthma.1 While antihistaminika of the first and second
generation has proven to be effective to relieve most of
the characteristic symptoms, the bronchoconstriction in
asthma, the nasal blockage in rhinitis, and the axon
reflex in the skin are unaffected by these drugs.1 In
addition, late-phase reactions resulting in an enhanced
and sustained inflammation are not a direct conse-
quence of histamine, strongly suggesting that other
mediators are involved in the pathogenic picture ob-
served in these patients. After FcꢀRI stimulation mast
cells release a vast amount of leukotrienes and cytok-
ines/chemokines into the tissue in a tightly and timely
controlled fashion.2-4 The cytokines and chemokines in
particular serve as a clear link to the inflammatory
process that is characteristic for these diseases and
could also explain how chronic manifestations develop.
In an attempt to prevent mediator release from mast
cells in general, two intervention strategies can be fol-
lowed: First, inhibition of receptor cross-linking by
preventing the binding of IgE and polyvalent allergen
(or in the case of certain types of chronic urticaria of
IgG autoantibodies) to the FcꢀRIsanti-IgE antibodies,
currently in clinical trials, are an example of this type
of approach;5 second, inhibition of the signaling cascades
initiating at the FcꢀRI after such bindingsthis approach
More than 25 000 pure compounds and 22 000 broths
were screened in this assay for their inhibitory potential.
Here, we show that 2,3,4-trihydropyrimidino[2,1-a]-
isoquinolines efficiently inhibit not only the RGA but
also endogenous TNFR, IL-4, IL-6, and IL-13 production
as well as the degranulation reaction (hexosaminidase
release) and arachidonic acid metabolism (leukotrienes).
The in vivo effect of this class of compounds is demon-
strated in a passive cutaneous anaphylaxis in mice,
S0022-2623(97)00662-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 02/27/1998

Inhibition of FcꢀRI-Mediated Activation of Mast Cells
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1051
Sch em e 1
Sch em e 3
Sch em e 2
clone 12, was generated which harbors a TNFR pro-
moter-luciferase-TNFR 3′ construct stably integrated
into its genome. Upon triggering with IgE plus antigen
(Ag), clone 12 cells do not only induce the pathophysi-
ologically relevant mediators histamine, hexosamini-
dase, leukotriene C4 (LTC4), and a variety of cytokines/
chemokines but also upregulate luciferase activity. The
latter generates a cost-effective, nonradioactive, robust
primary read-out system for activation of those cells by
IgE plus Ag (Figure 1). FK506, a macrolide immuno-
suppressant, is able to prevent this activation ef-
fectively9 and served as a positive standard in the initial
screening. Organic solvents were tolerated without any
inhibitory effect up to 1.0% for both DMSO and EtOH.
High-throughput screening was done at a single dose
of 10 µM for pure compounds and with 50 µg/well for
broths and plant extracts. Compounds and broths were
considered to be a “hit” if a more than 50% inhibition
of induction was found and confirmed at this concentra-
where the parental compound (2) inhibits this experi-
mental allergic reaction by 54% while the lead com-
pound (41) prevents it by 92%.
Ch em istr y
2,3,4-Trihydropyrimidino[2,1-a]isoquinolines substi-
tuted in position 6 were synthesized as published11
(Scheme 1), with the modification of using sec-butyl-
lithium instead of n-butyllithium as the second basic
equivalent. This improved the overall yield and gave
consistent results. 10-Anilino derivatives were synthe-
sized according to Scheme 2, starting from commercially
available 4-amino-2-methylbenzonitrile. Compounds
substituted in positions 6 and 7 were synthesized
according to Scheme 3. All amidines and 2,3,4-trihy-
dropyrimidino[2,1-a]isoquinolines were purified on silica
gel as hydrochlorides due to superiority in terms of yield
and purity compared to the free bases.
tion where less than 20% toxicity was observed.
A
chemical derivatization program was started if in ad-
dition the initial “hits” showed some specificity by failing
to inhibit two further RGA at a concentration of 10 µM
(the human T-cell line J urkat after anti-CD3/CD28
stimulation, read-out IL-2, the murine DC18 antigen-
presenting cell line after IgG complex stimulation, read-
Biology
The recently established mouse mast cell line CPII
conveyed the starting point for the search of signaling
inhibitors.4 A genetically modified subline, designated

1052 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Scholz et al.
F igu r e 1. Comparison of the reporter gene read-out to pathophysiological parameters of CPII activation. Panels from left to
right represent hexosaminidase release (% release in comparison to total hexosaminidase content), leukotriene release (% release
in comparison to total leukotriene content), murine TNFR production (pg/mL), and TNFR reporter gene assay (luciferase values).
All experiments were done in triplicate, and SD is indicated. Values for the nonstimulated, IgE-plus-antigen-stimulated (IgE/
Ag), and solvent (DMSO control; at the highest concentration later used in the compound screening) treated CPII clone 12 cells
are shown for all parameters.
Ta ble 1. Influence of Substituents in Position 6 on the
out TNFR).12,13 In total 25 368 pure compounds and
Inhibition Potency in the TNFR Reporter Gene, Cell Toxicity,
and Hexosaminidase Release Assays
22 603 broths were tested with a hit rate of 0.08% and
0.04%, respectively. Pure compounds fall into eight
chemical classes, broths belong to four natural product
classes.
For guiding the chemical derivatization program, a
hexosaminidase release assay and an extended cellular
toxicity assay (over 3 days) were performed for all the
derivatives in addition to the RGA. The best derivative
(“lead”) was evaluated further by measuring the inhibi-
tion of the production of various mast cell cytokines and
leukotrienes in ELISAs. The final proof of efficacy for
this compound class was done by a passive cutaneous
anaphylaxis in mice.
Resu lts
Compound 2 is one of the initial hits in this mast cell
reporter gene assay that fulfilled the outlined criteria
of inhibition, toxicity, and selectivity at the test con-
centration of 10 µM. Its IC50 in the RGA assay was
determined to be 0.8 µM, the IC50 in the hexosaminidase
release assay to be 0.5 µM, and the toxicity (TC50) as
measured by an XTT test over 3 days on clone 12 cells
at 3.2 µM. On the basis of these data, a chemical
derivatization program was started to improve the
biological activity of this compound and in parallel
reduce its toxic potential. As shown in Table 1 aliphatic
chains (3, 4) or phenyl rings substituted with polar
groups instead of the trimethylsilyl group (6, 7) abol-
ished the inhibitory effect. Also, inserting a C,C double
bond (9) or CH2O (10) that enlarges the distance of the
aromatic substituent to ring B resulted in inactive
compounds. Substitution of the trimethylsilyl group by
a tert-butyl group led to a slightly less active and less
toxic derivative (8). As a consequence we investigated
the influence of lipophilic substituted aromatic rings in
position 6 (Table 2). None of the three compounds in
this series resulted in a significant improvement; how-
ever, activity was not lost by the modification. Com-
pound 14 bearing a 2-phenylethyl substitution in the
para position was active in the same range as compound
2. This determined the importance of the substituent
for the inhibitory effect with respect to distance, ring
structure, and lipophilicity.
a
CPII mouse mast cells stable transfected by a TNFR promoter-
driven luciferase reporter construct were incubated for 1 h with
the substance and then triggered by a (dinitrophenyl)albumin/
anti-dinitrophenyl-IgE complex; incubation time: 6 h. CPII cells
were incubated for 3 days with substance in growth medium
(RPMI 1640 + 10% FCS). Cell growth is measured by formazane
formation (XTT staining). c CPII mouse mast cells were treated
overnight with substance and anti-dinitrophenyl-IgE. Degranu-
lation was then induced by the addition of (dinitrophenyl)albumin
for 2 h and quantified by colorimetric measurement of released
hexosaminidase.
b
No improvement was seen within a series of six deriva-
tives, with compound 18 (the best out of this series)
being in the same range as compound 2. This indicates
the importance of a certain overall lipophilicity of the
molecule, a fact often observed in a cell-based assay (see
Discussion). We therefore decided to study the influence
of simultaneous substitutions in positions 6 and 7 (Table
4). Compound 33, which is unsubstituted in position 6
and has a benzyl substitution in position 7 was inactive.
If compound 2 is benzyl-substituted in position 7 (34),
reduced inhibition of hexosaminidase release is ob-
served. Exchange of the trimethylsilyl group to a tert-
We next investigated the contribution of substitutions
of ring A (Table 3) to the activity of this compound class.

Inhibition of FcꢀRI-Mediated Activation of Mast Cells
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1053
Ta ble 2. Influence of Substituted Aromatic Rings in Position 6
on the Inhibition Potency in the TNFR Reporter Gene, Cell
Toxicity, and Hexosaminidase Release Assays
Ta ble 4. Influence of Substituents in Positions 5 and 6 on the
Inhibition Potency in the TNFR Reporter Gene, Cell Toxicity,
and Hexosaminidase Release Assays
a-c See corresponding footnotes in Table 1.
Ta ble 3. Influence of Anilides in Position 2 on the Inhibition
Potency in the TNFR Reporter Gene, Cell Toxicity, and
Hexosaminidase Release Assays
a-c See corresponding footnotes in Table 1.
recent findings of the effect of FK506 on these mediators
in mast cells. Here proinflammatory cytokines (like
TNFR) are inhibited at 10 times lower concentrations
than growth factors (like IL-13).14 Also the production
of leukotrienes was inhibited in the same range, while
the phosphorylation (activation) of the MAP kinase erk
1/2, a key kinase in this pathway, was unaffected
(Figure 3) (see Discussion). All these findings charac-
terize compound 41 as being of interest for interfering
with these important mediators in allergic diseases.
To finally prove the efficacy of this compound class
in type I allergic reactions, a passive cutaneous ana-
phylaxis in mice was performed. This (exclusively) IgE-
driven in vivo skin reaction was done to exclude the
possibility that substituted isoquinolines are specific
inhibitors of our selected screening system (CPII and
clone 12 mast cells). Both the parental compound (2)
as well as the selected lead compound (41) were inhibit-
ing the extravasation, if applied intradermally 90 min
before an allergic challenge (Figure 4). The observed
inhibition ranged from 54% (for 2) to 92% (for 41),
further demonstrating the improved activity of our lead
compound. The direct effect of the compounds on mast
cell activation is indicated by their inability to inhibit
an extravasation provoked by the administration of
histamine. It shows that substituted isoquinolines
comprise novel and suitable potential drugs for the
treatment of type I allergic diseases.
a-c See corresponding footnotes in Table 1.
butyl group (35) led to reduced activity, although this
exchange from C to Si had nearly no influence on the
activity in earlier cases (e.g., 2, 8). Polar substitution
in position 7 (36) again abolished the activity. 4-Ethox-
ybenzyl substitution in position 7 reduced the inhibition
of hexosaminidase release to some extent (37, 38),
whereas a 4-(2-phenylethyl)phenyl substituent in posi-
tion 6 and in addition a benzyl group in position 7 (39)
was again as active as 34 but showed enhanced cellular
toxicity. An n-butyl group in position 7 in combination
with the 4-tert-butylphenyl group in position 6 (40)
showed clearly improved activity compared to compound
34. Finally, switching to the 4-(2-phenylethyl)phenyl
side chain in position 6 in addition to the n-butyl group
in position 7 (41) enhanced the activity compared to
compound 2 by a factor of 4, with a cell toxicity in the
same range as that of compound 2.
We therefore studied the biological activity of com-
pound 41 in more detail. The compound did not inhibit
the IgE interaction with the FcꢀRI, as measured in a
cell-free ELISA system (data not shown). A dose-
dependent inhibition, however, at slightly higher con-
centrations as in the RGA was observed for the cytok-
ines TNFR, IL-4, IL-6, and IL-13 (Figure 2). This
difference in effective concentration can be explained
in part by a certain amount of preformed mediators in
mast cells. The differing IC50 values for inhibition of
the various cytokines (from 0.25 to 0.45 µM) parallel
Discu ssion
Signaling/activation inhibitors at various levels have
provided valuable pharmaca for the treatment of im-
munological disorders and include such diverse drugs
as FK506 and glucocorticoids.1 Depending on their
target specificity, they are either broadly antiinflam-
matory (glucocorticoids) or more or less restricted to
T-cell-mediated phenomena (FK506). Mast cell-specific
signaling inhibitors which would prevent all symptoms
related to type I allergy in the indications allergic

1054 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Scholz et al.
F igu r e 3. Leukotriene release, but not erk 1/2 activation,
affected by compound 41. (A) Dose titration of compound 41
on leukotriene synthesis. Release is shown on the y-axis in %
endogenous LT; concentration of compound and stimulation
conditions are given on the x-axis. All experiments were done
in triplicate, and SD is indicated. (B) Western blot detecting
erk 1/2 and phospho-erk 1/2 after 15 min of activation of CPII
clone 12 cells in untreated, stimulated, and stimulated and
compound 41-treated cells. Stimulations, inhibitor (compound
41), and solvent are indicated on the x-axis.
F igu r e 2. Inhibition of mast cell cytokine release by com-
pound 41. Dose titration of the solvent (DMSO) and compound
41 on the reporter gene assay is shown in the top left and right
panels. Middle and bottom panels show dose titration of
compound 41 on the inhibition of TNFR (IC50 ) 0.3 µM), IL-4
(IC50 ) 0.25 µM), IL-6 (IC50 ) 0.25 µM), and IL-13 (IC50 ) 0.45
µM) in CPII clone 12 cells. Release is shown on the y-axis in
pg; concentration of compound and stimulation conditions are
given on the x-axis. All experiments were done in triplicate,
and SD is indicated.
high-throughput screening) were linked to the tran-
scriptional activation of cytokines/chemokines.16,17 All
these late-signaling molecules are triggered by common,
mostly tyrosine-based phosphorylation events initiated
at the â and γ chain of the tetrameric FcꢀRI after cross-
linking.18-20 This primarily includes the lyn and syk
kinases, a number of adaptor proteins such as Shc, SOS,
and Grb2, and Ca2+ influx.21-26 The inhibitory effect
of 2,3,4-trihydropyrimidino[2,1-a]isoquinolines on all
pathophysiological mediators would strongly argue for
an interference with one of these central steps relatively
proximal to the receptor, thereby inhibiting all further
downstream pathways. From the structure it is not
unlikely that the strongly basic 2,3,4-trihydropyrimi-
dino[2,1-a]isoquinolines interfere by forming salt bridges
to the phosphorus acids, thereby preventing the binding
of the important SH2 domains in the tyrosine kinases
and adaptor proteins. Interference with the inositol
triphosphate-mediated Ca2+ release from internal stores
mechanistically based on the same salt bridge-building
ability is also plausible.
rhinitis, allergic conjunctivitis, allergic asthma, and
acute and chronic urticaria still need to be developed.
Such potential drugs require a relatively high specificity
due to the fact that allergic diseases are highly uncom-
fortable and disturbing but usually not life-threatening
(fatal asthma and anaphylactic reactions excluded).
Therefore, besides toxicity, effects of compounds espe-
cially on T-cells and other antigen-presenting cells must
be excluded to prevent general immunosuppression in
those patients. This was the basis for choosing an IL-2
RGA in the J urkat T-cell line and a TNFR RGA in the
antigen-presenting DC18 cell line as initial counter-
screening assays for specificity.12,13
Several signaling pathways in mast cells were re-
cently revealed; however, their interplay and cross-talk
still need to be elucidated. PI-3 kinase catalytic activity
was shown to be important for the degranulation
reaction (hexosaminidase release) and LT synthesis but
is dispensable for cytokine/chemokine induction.15 On
the contrary, the MAP kinase pathway (erk 1/2 serine/
threonine kinase) and the phosphatase calcineurin (the
target for FK506, the positive standard control in the
The finding that erk 1/2 phosphorylation (activation)
is not inhibited by this compound is interesting and also
allows a different interpretation. The erk 1/2 MAP
kinase directly activates PLCγ1, the enzyme releasing

Inhibition of FcꢀRI-Mediated Activation of Mast Cells
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1055
dried over P2O5 (this is essential to obtain good yields), was
dissolved in 30 mL of absolute tetrahydrofuran (THF), and 1.31
mL (8.8 mmol) of N,N,N′,N′-tetramethylethylenediamine,
cooled to -10 °C and lithiated29,30 with 6.6 mL (8.6 mmol) of
a 1.3 M solution of n-butyllithium in hexane, was added
followed by addition of 3.38 mL (4.4 mmol) of a 1.3 M solution
of sec-butyllithium in hexane. The reaction mixture was kept
for 6 h at -50 °C; then 4 mmol of the chosen methyl ester
dissolved in 3 mL of absolute THF was added. The whole
procedure was run under the protecting atmosphere of argon.
The reaction mixture was allowed to warm to room temper-
ature overnight and then was poured into 100 mL of saturated
ammonium chloride solution, and the aqueous solution was
extracted with methylene chloride. The organic phase was
dried and evaporated, and the residue was dissolved in 30 mL
of toluene; 270 mg of p-toluenesulfonic acid was added, and
the solution was refluxed for 3 h. Then toluene was evapo-
rated in vacuo, the residue dissolved in methylene chloride,
and the resulting solution extracted with a mixture of NaOH
(20%) and saturated NaCl solution. The organic phase was
dried and the solvent evaporated in vacuo. Adding methanolic
HCl to the residue, evaporation of excess solvent, and chro-
matography of the resulting hydrochloride with silica gel
(methylene chloride/methanol) gave the product.
B. Syn th esis of Sta r tin g Ma ter ia ls for Com p ou n d s
Ca r r yin g a Su bstitu en t in P osition 7 (Wittig Rea ction
a n d Hyd r ogen a tion ). 2-Cyanobenzaldehyde 24 (3.57 g, 27.2
mmol) was added at 0 °C in 10 mL of absolute THF to a
solution of the Wittig ylid, prepared from 42 mmol of a
triphenylphosphonium bromide, predried over P2O5, and 40
mmol of potassium tert-butylate in THF. The solution was
stirred for 30 min at room temperature, then diluted with
methylene chloride, and washed with 1 M HCl. The organic
layer was separated, dried, and evaporated to dryness. Chro-
matography of the residue over silica gel (toluene/ethyl acetate)
gave the product in varying amounts of cis/trans isomers. This
mixture was dissolved in 250 mL of ethanol (in case of poor
solubility methylene chloride was added) and, after addition
of 1.4 g of Pd (5%) on charcoal, was hydrogenated at room
temperature and atmospheric pressure. After filtration and
evaporation of the solvent, the residue was chromatographed
on silica gel or used crude for the synthesis of the 2-(2-
substituted phenyl)-4,5-dihydro-6H-pyrimidines as described
in ref 11. Ring closures to 2,3,4-trihydropyrimidino[2,1-a]-
isoquinolines were done as described above.
6-[4-(Tr im eth ylsilyl)p h en yl]-3,4-d ih yd r o-2H-p yr im id o-
[2,1-a ]isoqu in olin e Hyd r och lor id e (2‚HCl). Procedure A,
1, and 4-(trimethylsilyl)benzoic acid methyl ester were used;
the product was chromatographed on silica gel (CH2Cl2/CH3-
OH: 8/1), yield 50% of 2‚HCl (amorphous): 1H NMR (CDCl3)
δ 0.60 (s, 9H, (CH3)3), 2.17 (quin, 2H, CH2), 4.01 (t, 2H, NCH2),
4.18 (t, 2H, NCH2), 6.79 (s, 1H, dCH), 7.43-7.51 (m, 3H, ArH),
7.60-7.75 (m, 4H, ArH), 9.38 (dd, 1H, ArH), 11.78 (bs, 1H,
H+); MS 333 (MH+). Anal. (C21H24N2Si‚HCl) C, H, N.
6-Me t h yl-3,4-d ih yd r o-2H -p yr im id o[2,1-a ]isoq u in o-
lin e Hyd r och lor id e (3‚HCl). Procedure A, 1, and acetic acid
methyl ester were used; the product was chromatographed on
silica gel (CH2Cl2/CH3OH: 8/1), yield 41% of 3‚HCl: mp 210
°C subl; 1H NMR (CDCl3/CD3OD) δ 2.24 (quin, 2H, CH2), 2.60
(s, 3H, CH3), 3.74 (t, 2H, NCH2), 4.39 (t, 2H, NCH2), 7.00 (s,
1H, dCH), 7.63-7.72 (m, 2H, ArH), 7.82-7.90 (m, 1H, ArH),
8.40 (d, 1H, ArH); MS 199.2 (MH+). Anal. (C13H14N2‚HCl) C,
H, N.
F igu r e 4. Passive cutaneous anaphylaxis (PCA) is inhibited
by 2,3,4-trihydropyrimidino[2,1-a]isoquinolines: (A) dose-de-
pendent inhibition of an IgE-plus-antigen-provoked PCA by
compound 2, (B) dose-dependent inhibition of an IgE-plus-
antigen-provoked PCA by compound 41, (C) control reaction
that substituted isoquinolines do not inhibit a PCA provoked
by histamine. The score of the PCA in mm2 of dye extravasa-
tion is given on the y-axis; stimulations, compounds, and
concentrations are given on the x-axis, and SD is indicated.
arachidonic acid from the membrane as the first step
in the leukotriene pathway. That leukotriene produc-
tion is inhibited but not this kinase suggests that
further signaling pathways are required for the overall
induction/activation. We have recently described such
an additional signaling pathway in CPII cells which is
blocked by inhibitors of atypical PKCs, primarily PKCµ.17
This pathway is required in addition to the MAPK
pathway and Ca2+ influx for the activation of all so far
tested mast cell functions and seems to be equally
important as the other two for the overall activation.
In this respect it is interesting that PKCµ is membrane-
bound and dependent on lipids for its activity.27 This
could be an explanation for the requirement of a certain
amount of lipophilicity of the inhibitors. Currently in
vitro kinase assays after immunoprecipitation of PKCµ
are being performed to address this question.
Exp er im en ta l Section
Ch em istr y: Gen er a l. 1H NMR spectra were recorded with
a Bruker WC-250 or AMX-500 spectrometer; chemical shifts
are reported in ppm (δ) relative to internal Me4Si. Elemental
analyses were performed by the Analytical Department, No-
vartis, Basel, Switzerland, and are within (0.4% of the
theoretical values. Analytical thin-layer chromatography was
performed on silica gel 60 F254 glass plates (HPTLC, Merck).
Preparative column chromatography was performed on silica
gel (40-63 µm) under pressure (≈0.2 mPa). Solvents were
AR grade and were used without further purification. All
reagents were obtained from commercial suppliers and were
used without further purification. Evaporations were carried
out in vacuo with a rotary evaporator. Melting points were
determined with a thermovar apparatus (Reichert-J ung) and
are not corrected.
Gen er a l P r oced u r es: A. Rin g Closu r e of 2-(2-Meth -
ylp h en yl)-4,5-d ih yd r o-6H-p yr im id in e to 2,3,4-Tr ih yd r o-
p yr im id in o[2,1-a ]isoqu in olin es. (Slightly modified proce-
dure of ref 11; 2,3-dihydroimidazo[2,1-a]isoquinolines have
been synthesized analogously28.) All yields relate to purified
products (via chromatography), in general they are between
22 and 50%. The exceptions, 12 (9%) and 40 (7%), are due to
purification problems. 1 (840 mg, 4 mmol) (2-(2-methylphe-
nyl)-4,5-dihydro-6H-pyrimidine hydrochloride),11 carefully pre-
6-Hexyl-3,4-dih ydr o-2H-pyr im ido[2,1-a ]isoqu in olin e Hy-
d r och lor id e (4‚HCl). Procedure A, 1, and hexoic acid methyl
ester were used; product crystallized from toluene, yield 35%
of 4‚HCl (amorphous, hygroscopic): 1H NMR (CDCl3) δ 0.96
(t, 3H, CH3), 1.20-1.58 (m, 6H, (CH2)3), 1.62-1.77 (m, 2H,
CH2), 2.25 (quin, 2H, CH2), 2.76 (t, 2H, CH2), 3.85 (t, 2H,
NCH2), 4.22 (t, 2H, NCH2), 6.65 (s, 1H, dCH), 7.45 (d, 1H,
ArH), 7.60-7.79 (m, 2H, ArH), 9.25 (d, 1H, ArH), 11.42 (bs,
1H, H+); MS 269.0 (MH+). Anal. (C18H24N2‚HCl) C, H, N.
6-P h e n yl-3,4-d ih yd r o-2H -p yr im id o[2,1-a ]isoq u in o-
lin e Hyd r och lor id e (5‚HCl). Procedure A, 1, and benzoic

1056 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Scholz et al.
acid methyl ester were used; the product was chromatographed
2H, NCH2), 6.82 (s, 1H, dCH), 7.42-7.95 (m, 12H, ArH), 9.38
(dd, 1H, ArH), 11.69 (bs, 1H, H+); MS 337.2 (MH+). Anal.
(C24H20N2‚HCl) C, H, N.
on silica gel (CH2Cl2/CH3OH: 8/1), yield 40% of 5‚HCl: mp
1
200 °C subl; H NMR (CDCl3) δ 2.10 (quin, 2H, CH2), 3.96 (t,
2H, NCH2), 4.05 (t, 2H, NCH2), 6.77 (s, 1H, dCH2), 7.10-7.18
(m, 2H, ArH), 27.22 (t, 1H, ArH), 7.42-7.44 (m, 2H, ArH),
7.50-7.55 (m, 1H, ArH), 7.60 (dt, 1H, ArH), 7.73 (dt, 1H, ArH),
8.95 (d, 1H, ArH), 11.42 (bs, 1H, H+); MS 261.2 (MH+). Anal.
(C18H16N2‚HCl‚0.6H2O) C, H, N.
6-[4-(Ben zyloxy)p h en yl]-3,4-d ih yd r o-2H-p yr im id o[2,1-
a ]isoqu in olin e Hyd r och lor id e (13‚HCl). Procedure A, 1,
and 4-(benzyloxy)benzoic acid methyl ester were used; the
product was chromatographed on silica gel (CH2Cl2/CH3OH:
1
9/1), yield 29% of 13‚HCl: mp 252-260 °C (amorphous); H
6-(3,4,5-Tr im eth oxyp h en yl)-3,4-d ih yd r o-2H-p yr im id o-
[2,1-a ]isoqu in olin e Hyd r och lor id e (6‚HCl). Procedure A,
1, and 3,4,5-trimethoxybenzoic acid methyl ester were used;
the product was chromatographed on silica gel (CH2Cl2/CH3-
OH: 8/1), yield 35% of 6‚HCl: mp 225-230 °C; 1H NMR
(CDCl3) δ 2.12 (quin, 2H, CH2), 3.86-4.07 (m, 11H, 3 OCH3,
NCH2), 4.21 (t, 2H, NCH2), 6.74 (s, 1H, dCH2), 6.85 (s, 2H,
ArH), 7.42-7.55 (m, 2H, ArH), 7.63 (t, 1H, ArH), 9.16 (d, 1H,
ArH), 11,43 (bs, 1H, H+); MS 351 (MH+). Anal. (C21H22N2O3‚-
HCl) C, H, N.
6-(4-Ch lor op h en yl)-3,4-d ih yd r o-2H-p yr im id o[2,1-a ]iso-
qu in olin e Hyd r och lor id e (7‚HCl). Procedure A, 1, and
4-chlorobenzoic acid methyl ester were used; the product was
chromatographed on silica gel (CH2Cl2/CH3OH: 7/1), yield 26%
of 7‚HCl (amorphous): 1H NMR (CDCl3) δ 2.20 (quin, 2H,
CH2), 3.90 (t, 2H, NCH2), 4.03 (t, 2H, NCH2), 6.82 (s, 1H, dCH),
7.40-7.51 (ABX, 4H, ArH), 7.58 (dd, 1H, ArH), 7.78-7.82 (m,
2H, ArH), 9.00 (dd, 1H, ArH), 11,40 (bs, 1H, H+); MS 294
(MH+). Anal. (C18H15N2Cl‚HCl) C, H, N.
6-(4-ter t-Bu tylp h en yl)-3,4-d ih yd r o-2H-p yr im id o[2,1-a ]-
isoqu in olin e Hyd r och lor id e (8‚HCl). Procedure A, 1, and
4-tert-butylbenzoic acid methyl ester were used; the product
was chromatographed on silica gel (CH2Cl2/CH3OH: 8/1), yield
45% of 8‚HCl: mp 242-250 °C (amorphous); 1H NMR (CDCl3)
δ 1.41 (s, 9H, (CH3)3), 2.17 (quin, 2H, CH2), 4.01 (t, 2H, NCH2),
4.18 (t, 2H, NCH2), 6.78 (s, 1H, dCH), 7.42-7.79 (m, 7H, ArH),
9.38 (dd, 1H, ArH), 11.73 (bs, 1H, H+); MS 317.2 (MH+). Anal.
(C22H24N2‚HCl‚0.8H2O) C, H, N.
6-St yr yl-3,4-d ih yd r o-2H -p yr im id o[2,1-a ]isoq u in olin e
Hyd r och lor id e (9‚HCl). Procedure A, 1, and cinnamic acid
methyl ester were used; the product was chromatographed on
silica gel (CH2Cl2/CH3OH: 8/1), yield 36% of 9‚HCl: mp 231-
240 °C (amorphous); 1H NMR (CDCl3/CD3OD) δ 2.33 (quin,
2H, CH2), 3.78 (t, 2H, NCH2), 4.44 (t, 2H, NCH2), 7.23 (dd,
1H, dCH), 7.28 (d, 1H, dCH), 7.32 (s, 1H, dCH), 7.38-7.47
(m, 4H, ArH), 7.63 (td, 2H, ArH), 7.74 (dt, 1H, ArH), 7.84 (d,
1H, ArH), 7.91 (dt, 1H, ArH), 8.41 (td, 1H, ArH); MS 287.2
(MH+). Anal. (C20H18N2‚HCl) C, H, N.
NMR (CDCl3) δ 2.10 (quin, 2H, CH2), 3.96 (t, 2H, NCH2), 4.12
(t, 2H, NCH2), 5.18 (s, 2H, OCH2), 6.76 (s, 1H, dCH), 7.18 (td,
2H, ArH), 7.38-7.76 (m, 10H, ArH), 9.32 (dd, 1H, ArH), 11.55
(bs, 1H, H+); MS 366.0 (MH+). Anal. (C25H22N2O‚HCl) C, H,
N.
6-(4-P h en eth ylph en yl)-3,4-dih ydr o-2H-pyr im ido[2,1-a ]-
isoqu in olin e Hyd r och lor id e (14‚HCl). Procedure A, 1, and
4-phenethylbenzoic acid methyl ester were used; the product
was chromatographed on silica gel (CH2Cl2/CH3OH: 8/1), yield
49% of 14‚HCl: mp 200 °C subl (amorphous); 1H NMR (CDCl3)
δ 2.17 (quin, 2H, CH2), 3.01 (s, 4H, CH2CH2), 3.96 (t, 2H,
NCH2), 4.08 (t, 2H, NCH2), 6.78 (s, 1H, dCH), 7.20-7.59 (m,
10H, ArH), 7.62-7.79 (m, 2H, ArH), 9.38 (dd, 1H, ArH), 11.55
(bs, 1H, H+); MS 364.0 (M+). Anal. (C26H24N2‚HCl) C, H, N.
5-(Diben zyla m in o)-2-m eth ylben zon itr ile (16). 15 (396
mg, 3 mmol) (5-amino-2-methylbenzonitrile) was dissolved in
3 mL of dimethylformamide, and 1.07 mL (9 mmol) of benzyl
bromide and 1.243 g (9 mmol) of K2CO3 were added at room
temperature. After the suspension stirred for 5 h at room
temperature, 552 mg (4 mmol) of K2CO3 and 357 µL (3 mmol)
of benzyl bromide were added in addition. After 24 h at room
temperature, the mixture was poured into water and extracted
with methylene chloride, the organic phase was washed twice
with water and dried, the solvent was evaporated under
reduced pressure, and the residue was chromatographed over
silica gel (toluene/ethyl acetate: 98/2), yield 899 mg (96%) of
16, clear syrup: 1H NMR (CDCl3) δ 2.40 (s, 3H, CH3), 4.61 (s,
6H, N(CH2)2), 6.81 (dd, 1H, ArH), 6.90-7.08 (ABX, 2H, ArH),
7.12-7.40 (m, 10H, ArH); MS 313.2 (MH+). Anal. (C22H20N2)
C, H, N.
Dib en zyl[4-m et h yl-3-(1,4,5,6-t et r a h yd r op yr im id in -2-
yl)p h en yl]a m in e Hyd r och lor id e (17‚HCl). 16 (876 mg, 2.8
mmol) was dissolved in 5 mL of ethylene glycol and 280 µL
(3.36 mmol) of 1,3-diaminopropane; 319 mg (1.68 mmol) of
p-toluenesulfonic acid was added, the solution was kept for 4
days at 200 °C, and then after cooling the reaction mixture
was diluted with CH2Cl2 and extracted with a solution of
NaOH (20%) and saturated sodium chloride. The organic
phase was separated, dried, evaporated, and transformed to
the hydrochloride with methanolic HCl. The residue was
chromatographed on silica gel (CH2Cl2/CH3OH: 7/1), yield 363
mg (35%) of 17‚HCl: mp 215-222 °C (acetic acid isopropyl
6-[(4-Ch lor oph en oxy)m eth yl]-3,4-dih ydr o-2H-pyr im ido-
[2,1-a ]isoqu in olin e Hyd r och lor id e (10‚HCl). Procedure A,
1, and (4-chlorophenoxy)acetic acid methyl ester were used;
the product was chromatographed on silica gel (CH2Cl2/CH3-
OH: 8/1), yield 23% of 10‚HCl: mp 182-190 °C; 1H NMR
(CDCl3/CD3OD) δ 2.23 (quin, 2H, CH2), 3.80 (t, 2H, NCH2),
4.40 (t, 2H, NCH2), 5.18 (s, 2H, OCH2), 6.92 (td, 2H, ArH),
7.08 (s, 1H, dCH), 7.26 (td, 2H, ArH), 7.58-7.82 (m, 3H, ArH),
8.87 (d, 1H, ArH); MS 325.1 (MH+). Anal. (C19H17N2OCl‚-
HCl) C, H, N.
1-(3,4-Dih yd r o-2H-p yr im id o[2,1-a ]isoqu in olin -6-yl)-2-
p h en yleth yla m in e Dih yd r och lor id e (11‚2HCl). Procedure
A, 1, and N-(tert-butoxycarbonyl)-L-phenylalanine methyl ester
were used (reaction leads to cleavage of the BOC protecting
group); the product was chromatographed on silica gel (CH2-
1
ester); H NMR (CDCl3) δ 1.70 (quin, 2H, CH2), 2.22 (s, 3H,
CH3), 3.34 (t, 4H, (NCH2)2), 4.59 (s, 4H, (CH2)2), 6.63 (dd, 1H,
ArH), 6.82-6.90 (m, 2H, ArH), 7.16-7.38 (m, 10H, ArH), 11.55
(bs, 1H, H+); MS 370.0 (M+). Anal. (C25H27N2‚HCl) C, H, N.
Diben zyl[6-(4-ter t-bu tylp h en yl)-3,4-d ih yd r o-2H-p yr im -
ido[2,1-a ]isoqu in olin -10-yl]am in e Hydr och lor ide (18‚HCl).
Procedure A, 17‚HCl, and 4-tert-butylbenzoic acid methyl ester
were used; the product was chromatographed on silica gel
(CH2Cl2/CH3OH: 9/1), yield 56% of 18‚HCl, amorphous: 1H
NMR (CDCl3) δ 1.38 (s, 9H, C(CH3)3), 2.04 (quin, 2H, CH2),
3.81 (t, 2H, NCH2), 3.89 (t, 2H, NCH2), 5.00 (s, 4H, N(CH2)2),
6.64 (s, 1H, dCH), 7.08-7.76 (m, 16H, ArH), 8.37 (d, 1H), 11.24
(bs, 1H, H+); MS 512 (MH+). Anal. (C36H37N3‚HCl) C, H, N.
[6-(4-ter t-Bu tylph en yl)-3,4-dih ydr o-2H-pyr im ido[2,1-a ]-
isoqu in olin -10-yl]a m in e Hyd r och lor id e (19‚HCl). 18‚HCl
(110 mg, 0.2 mmol) was dissolved in 10 mL of ethanol and
acidified with ethanol/HCl to pH 3; 50 mg of Pd (5%) on
charcoal was added and the reaction mixture hydrogenated
at room temperature and atmospheric pressure for 3 h. After
filtration the organic solvent was evaporated under reduced
pressure, and the residue was chromatographed on silica gel
(CH2Cl2/CH3OH: 9/1), yield 61 mg (92%) of 19‚HCl: mp 250
°C dec (methanol/diethyl ether); 1H NMR (CDCl3) δ 1.35 (s,
9H, C(CH3)3), 2.04 (quin, 2H, CH2), 3.70 (t, 2H, NCH2), 3.92
Cl2/CH3OH: 4/1), yield 22% of 11‚2HCl: mp 138-145 °C; [R]25
D
-0.7° (c ) 1, C2H5OH); 1H NMR (CDCl3/CD3OD) δ 2.01-2.25
(m, 2H, CH2), 3.06-3.21 (m, 2H, CH2), 3.49-3.78 (m, 2H,
NCH2), 4.01-4.18 (m, 1H, CHNH2), 4.46-4.62 (m, 2H, NCH2),
7.20-7.28 (m, 5H, ArH), 7.40 (s, 1H, dCH), 7.74 (dt, 1H, ArH),
7.84-7.95 (m, 2H, ArH), 8.32 (dd, 1H, ArH); MS 304 (MH+).
Anal. (C20H21N3‚1.4HCl‚2H2O) C, H, N.
6-Bip h en ylyl-3,4-d ih yd r o-2H-p yr im id o[2,1-a ]isoqu in o-
lin e Hyd r och lor id e (12‚HCl). Procedure A, 1, and 4-phe-
nylbenzoic acid methyl ester were used; the product was
chromatographed on silica gel (CH2Cl2/CH3OH: 7/1), yield 9%
1
of 12‚HCl: mp 272-280 °C (CH2Cl2/ethyl acetate); H NMR
(CDCl3) δ 2.19 (quin, 2H, CH2), 4.01 (t, 2H, NCH2), 4.22 (t,

Inhibition of FcꢀRI-Mediated Activation of Mast Cells
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1057
(t, 2H, NCH2), 6.62 (s, 1H, dCH), 7.09 (dd, 1H, ArH), 7.24 (td,
2H, ArH), 7.33 (d, 1H, ArH), 7.46 (td, 2H, ArH), 7.78 (d, 1H,
ArH), 10.04 (bs, 1H, H+); MS 332 (MH+). Anal. (C22H25N3‚-
HCl) C, H, N.
3.07-3.18 (m, 2H, CH2), 7.18-7.36 (m, 7H, ArH), 7.49 (dt, 1H,
ArH), 7.60 (dd, 1H, ArH); MS 208.2 (MH+). Anal. (C15H13N)
C, H, N.
2-[3-(Dim eth yla m in o)p r op yl]ben zon itr ile (26). Proce-
dure B, 24, and [2-(dimethylamino)ethylidene]triphenyl-
phosphorane were used; the product was chromatographed on
silica gel (CH2Cl2/CH3OH: 6/1), yield 52% of 26, syrup: 1H
NMR (CD3OD) δ 1.86 (quin, 2H, CH2), 2.27 (s, 6H, N(CH3)2),
2.40 (dd, 2H, CH2), 2.88 (dd, 2H, CH2), 7.25-7.38 (m, 2H, ArH),
7.51 (dt, 1H, ArH), 7.62 (dd, 1H, ArH); MS 189.1 (MH+). Anal.
(C12H16N2‚H2O) C, H, N.
1-[6-(4-ter t-Bu tylp h en yl)-3,4-d ih yd r o-2H-p yr im id o[2,1-
a ]isoq u in olin -10-yl]-3-cycloh exylu r ea H yd r och lor id e
(20‚HCl). 19‚HCl (100 mg, 0.27 mmol) was dissolved in 5 mL
of dry THF, and 38 µL (0.3 mmol) of cyclohexylisocyanate was
added. The reaction mixture was kept at 70 °C for 24 h, the
solvent evaporated, and the residue chromatographed on silica
gel (CH2Cl2/CH3OH: 96/4), yield 37 mg (30%) of 20‚HCl: mp
235-240 °C; 1H NMR (CDCl3/CD3OD) δ 1.18-1.61 (m, 5H,
(CH2)3), 1.42 (s, 9H, C(CH3)3), 1.63-1.75 (m, 1H, CH), 1.80-
2.02 (m, 4H, 2 CH2), 2.24 (quin, 2H, CH2), 3.64-3.78 (m, 1H,
NCH), 3.80 (t, 2H, NCH2), 4.18 (t, 2H, NCH2), 7.08 (s, 1H,
dCH), 7.48 (dt, 2H, ArH), 7.62 (td, 2H, ArH), 7.75-7.78 (m,
2H, ArH), 8.43 (d, 1H, ArH); MS 457.6 (MH+). Anal.
(C29H36N4O‚HCl) C, H, N.
2-[2-(4-Eth oxyp h en yl)eth yl]ben zon itr ile (27). Proce-
dure B, 24, and (4-ethoxybenzylidene)triphenylphosphorane
were used; the product was chromatographed on silica gel
(toluene/ethyl acetate: 9/1), yield 94% of 27, syrup: 1H NMR
(CDCl3) δ 1.18 (t, 3H, CH3), 2.80-2.96 (m, 2H, CH2), 3.08-
3.20 (m, 2H, CH2), 3.98 (q, 2H, OCH2), 6.80 (td, 2H, ArH), 7.10
(td, 2H, ArH), 7.22 (m, 2H, ArH), 7.47 (dt, 1H, ArH), 7.62 (dd,
1H, ArH); MS 252.1 (MH+). Anal. (C17H17NO) C, H, N.
2-P en tylben zon itr ile (28). Procedure B, 24, and butyle-
netriphenylphosphorane were used; the product was chro-
matographed on silica gel (toluene/ethyl acetate: 9/1), yield
76% of 28, syrup: 1H NMR (CDCl3) δ 0.82-0.94 (m, 3H, CH3),
1.29-1.41 (m, 4H, CH2CH2), 1.62-1.68 (m, 2H, CH2), 2.83 (AB,
2H, CH2), 7.22-7.34 (m, 2H, ArH), 7.49 (dt, 1H, ArH), 7.60
(dd, 1H, ArH); MS 173 (M+). Anal. (C12H15N) C, H, N.
2-(2-P h en et h ylp h en yl)-1,4,5,6-t et r a h yd r op yr im id in e
(29). 25 reacted as described in ref 11. The product was
chromatographed on silica gel (CH2Cl2/CH3OH: 6/1), yield 70%
1-[6-(4-ter t-Bu tylp h en yl)-3,4-d ih yd r o-2H-p yr im id o[2,1-
a ]isoq u in olin -10-yl]-3-p h en ylt h iou r ea H yd r och lor id e
(21‚HCl). 19‚HCl (100 mg, 0.27 mmol) was dissolved in 5 mL
of THF and 0.5 mL of DMF, 72 µL (0.6 mmol) of phenyl
isothiocyanate was added ,and the reaction mixture was kept
at 60 °C for 20 h. After evaporation of the solvent, the residue
was chromatographed on silica gel (CH2Cl2/CH3OH: 96/4), yield
1
99 mg (79%) of 21‚HCl: mp 200 °C dec; H NMR (CDCl3) δ
1.47 (s, 9H, C(CH3)3), 2.17 (quin, 2H, CH2), 3.84 (t, 2H, NCH2),
4.10 (t, 2H, NCH2), 6.84 (s, 1H, dCH), 7.17 (t, 1H, ArH), 7.25-
7.36 (m, 3H, ArH), 7.43 (td, 2H, ArH), 7.75 (d, 1H, ArH), 7.80
(td, 2H, ArH), 7.76-7.80 (m, 2H, ArH), 8.52 (s, 1H, NH), 8.97
(d, 1H, ArH), 10.08 (s, 1H, NH), 10.85 (bs, 1H, H+); MS 467
(MH+). Anal. (C29H30N4S‚HCl) C, H, N.
1
of 29: mp 208-212 °C; H NMR (CD3OD) δ 2.09 (quin, 2H,
CH2), 2.81-3.06 (m, 4H, CH2CH2), 3.40-3.51 (m 4H, (NCH2)2),
7.03-7.59 (m, 9H, ArH); MS 265.2 (MH+). Anal. (C18H20N2‚-
HCl) C, H, N.
1-[6-(4-ter t-Bu tylp h en yl)-3,4-d ih yd r o-2H-p yr im id o[2,1-
a ]isoqu in olin -10-yl]-3-(2,4-d ich lor op h en yl)u r ea Hyd r o-
ch lor id e (22‚HCl). 19‚HCl (100 mg, 0.27 mmol) was dis-
solved in 5 mL of THF and cooled to -15 °C. Then 56 mg (0.3
mmol) of 2,4-dichlorophenyl isocyanate was added, and the
reaction mixture was allowed to come to room temperature
slowly overnight. The solvent was evaporated under reduced
pressure, and the residue was chromatographed on silica gel
(CH2Cl2/CH3OH: 96/4), yield 106.5 mg (76%) of 22‚HCl,
amorphous powder: 1H NMR (CDCl3/CD3OD/DMSO-d6) δ 1.37
(s, 9H, C(CH3)3), 2.20 (quin, 2H, CH2), 3.82 (t, 2H, NCH2), 4.10
(t, 2H, NCH2), 6.85 (s, 1H, dCH), 7.18 (dd, 1H, ArH), 7.234-
7.36 (m, 1H, ArH), 7.38 (td, 2H, ArH), 7.50 (td, 2H, ArH), 7.61
(d, 1H, ArH), 8.11 (d, 1H, ArH), 8.25 (d, 1H, ArH), 8.42 (dd,
1H, ArH); MS 519 (MH+). Anal. (C29H28Cl2N4O‚HCl) C, H,
N.
2-[2-[3-(Dim eth ylam in o)pr opyl]ph en yl]-1,4,5,6-tetr ah y-
d r op yr im id in e (30). 26 was transformed as described in ref
11. The product was chromatographed on silica gel (CH2Cl2/
CH3OH: 9/1 f 2/1), yield 73% of 30, very hygroscopic: 1H NMR
(CDCl3) δ 1.89-2.20 (m, 4H, 2 CH2), 2.64-2.81 (m, 2H, CH2),
2.75 (s, 6H, N(CH3)2), 2.08 (t, 2H, NCH3), 3.64 (t, 2H, NCH2),
7.38-7.61 (m, 3H, ArH); MS 246 (MH+). Due to hygroscopic
problems, no further characterization; 30 was reacted im-
mediately after drying to 36.
2-[2-[2-(4-Eth oxyp h en yl)eth yl]p h en yl]-1,4,5,6-tetr a h y-
d r op yr im id in e Hyd r och lor id e (31‚HCl). 27 was trans-
formed as described in ref 11. The product was chromato-
graphed on silica gel (CH2Cl2/CH3OH: 9/1 f 2/1), yield 79% of
31‚HCl, precipitated from CH3OH with diethyl ether: mp
189-196 °C; 1H NMR (CDCl3) δ 1.36 (t, 3H, CH3), 1.90 (quin,
2H, CH2), 2.80-2.87 (m, 2H, CH2), 2.88-2.97 (m, 2H, CH2),
3.28 (t, 4H, 2 NCH2), 3.98 (q, 2H, OCH2), 6.78 (td, 2H, ArH),
6.90 (td, 2H, ArH), 7.11-7.28 (m, 3H, ArH), 7.34 (dt, 1H, ArH),
9.42 (bs, 1H, H+); MS 309.2 (MH+). Anal. (C20H24N2O‚HCl)
C, H, N.
N-[6-(4-ter t-Bu tylp h en yl)-3,4-dih yd r o-2H-p yr im ido[2,1-
a ]isoq u in olin -10-yl]-2-p yr id in -2-yla cet a m id e Dih yd r o-
ch lor id e (23‚2HCl). 2-Pyridylacetic acid (45 mg, 0.27 mmol)
was dissolved in 5 mL of DMF, and the reaction mixture was
cooled to -10 °C. Then 259 µL (1.5 mmol) of diisopropylamine,
211 mg (0.405 mmol) of (benzotriazol-1-yloxy)tripyrrolidino-
phosphonium hexafluorophosphate, and 100 mg (0.27 mmol)
of 19‚HCl were added, and the solution was allowed to come
to room temperature overnight. After diluting with CH2Cl2
the reaction mixture was extracted with 0.1 N NaOH and H2O
and dried, and the organic solvent was evaporated under
reduced pressure. The residue was dissolved in methanol and
acidified to pH 5 with methanolic HCl and the solvent
evaporated. The resulting dihydrochloride was chromato-
graphed on silica gel (CH2Cl2/CH3OH: 96/4), yield 59.6 mg
2-(2-P en tylp h en yl)-1,4,5,6-tetr a h yd r op yr im id in e (32).
28 was transformed as described in ref 11. The product was
chromatographed on silica gel (CH2Cl2/CH3OH: 9/1), yield 82%
of 32, free base recrystallized from toluene/n-hexane: mp 98-
1
105 °C; H NMR (CDCl3) δ 0.87 (t, 3H, CH3), 1.22-1.40 (m,
4H, 2 CH2), 1.60 (quin, 2H, CH2), 1.83 (quin, 2H, CH2), 2.78
(AB, 2H, CH2), 3.63 (t, 4H, 2 NCH2), 7.18-7.25 (m, 4H, ArH);
MS 231.2 (MH+). Anal. (C15H22N2) C, H, N.
7-Ben zyl-3,4-dih yr o-2H-pyr im ido[2,1-a ]isoqu in olin e Hy-
d r och lor id e (33‚HCl). Procedure A, 29, and formic acid
methyl ester were used; the product was chromatographed on
silica gel (CH2Cl2/CH3OH: 4/1), yield 48% of 33‚HCl: mp 228-
1
(49%) of 23‚2HCl: mp 212-220 °C; H NMR (CDCl3) δ 1.38
1
(s, 9H, C(CH3)3), 2.08 (quin, 2H, CH2), 3.81-4.01 (m, 4H, CH2-
CO, NCH2), 4.17 (t, 2H, NCH2), 6.78 (s, 1H, dCH), 7.14-7.76
(m, 8H, ArH), 8.62-8.90 (m, 3H, ArH), 11.04 (bs, 1H, H+),
11.43 (s, 1H, NH); MS 450 (M+). Anal. (C29H30N4O‚2HCl) C,
H, N.
240 °C; H NMR (CDCl3) δ 2.20 (quin, 2H, CH2), 3.82 (t, 2H,
NCH2), 4.11 (s, 2H, CH2), 4.23 (t, 2H, NCH2), 6.80 (s, 1H,
dCH), 7.16-7.38 (m, 5H, ArH), 7.62-7.78 (m, 3H, ArH), 9.17
(d, 1H, ArH), 11.40 (bs, 1H, H+); MS 274.1 (MH+). Anal.
(C19H18N2‚HCl) C, H, N.
2-P h en eth ylben zon itr ile (25). Procedure B, 24, and
benzylidene triphenylphosphorane were used; the product was
chromatographed on silica gel (toluene/ethyl acetate: 9/1), yield
65% of 25, syrup: 1H NMR (CDCl3) δ 2.96-3.04 (m, 2H, CH2),
7-Ben zyl-6-[4-(tr im eth ylsila n yl)p h en yl]-3,4-d ih yr o-2H-
pyr im ido[2,1-a ]isoqu in olin e Hydr och lor ide (34‚HCl). Pro-
cedure A, 29, and 4-(trimethylsilyl)benzoic acid methyl ester
were used; the product was chromatographed on silica gel

1058 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Scholz et al.
(CH2Cl2/CH3OH: 9/1), yield 23% of 34‚HCl: mp 232-240 °C
(CH3OH, (C2H5)2O); 1H NMR (CDCl3) δ 0.53 (s, 9H, Si(CH3)3),
2.18 (quin, 2H, CH2), 3.82-3.98 (m, 4H, 2 NCH2), 3.99 (s, 2H,
CH2), 7.00 (d, 2H, ArH), 7.18-7.39 (m, 5H, ArH), 7.58-7.63
(m, 5H, ArH), 9.22 (d, 1H, ArH), 11.60 (bs, 1H, H+); MS 422
(M+). Anal. (C28H30N2Si‚HCl) C, H, N.
7-Ben zyl-6-(4-ter t-bu tylp h en yl)-3,4-d ih yd r o-2H-p yr im -
id o[2,1-a ]isoqu in olin e Hyd r och lor id e (35‚HCl). Proce-
dure A, 29, and 4-tert-butylbenzoic acid methyl ester were
used; the product was chromatographed on silica gel (CH2Cl2/
CH3OH: 9/1), yield 31% of 35‚HCl, amorphous: 1H NMR
(CDCl3) δ 1.38 (s, 9H, C(CH3)3), 2.15 (quin, 2H, CH2), 3.78-
3.84 (m, 4H, 2 NCH2), 3.92 (s, 2H, CH2), 6.97-7.03 (m, 2H,
ArH), 7.14-7.30 (m, 5H, ArH), 7.50 (td, 2H, ArH), 7.63-7.74
(m, 3H, ArH), 8.97-9.01 (m, 1H, ArH); MS 407.2 (MH+). Anal.
(C29H30N2‚HCl) C, H, N.
7-Bu tyl-6-(4-p h en eth ylp h en yl)-3,4-d ih yd r o-2H-[2,1-a ]-
isoqu in olin e Hyd r och lor id e (41). Procedure A, 32, and
4-phenethylbenzoic acid methyl ester were used; the product
was chromatographed on silica gel (CH2Cl2/CH3OH: 9/1), yield
1
38% of 41: mp 194-202 °C (amorphous); H NMR (CDCl3) δ
0.77 (t, 3H, CH3), 1.22 (quin, 2H, CH2), 1.41 (quin, 2H, CH2),
2.06 (quin, 2H, CH2), 2.43 (AB, 2H, CH2), 2.95-3.08 (m, 4H,
CH2CH2), 3.71 (t, 2H, NCH2), 3.83 (t, 2H, NCH2), 7.16-7.38
(m, 9H, ArH), 7.71-7.86 (m, 3H, ArH), 9.32 (d, 1H, ArH), 11.42
(bs, 1H, H+). Anal. (C30H32N2‚HCl) C, H, N.
Biology: Sta ble Rep or ter Gen e Cell Lin e (Clon e 12).
CPII cells (5 × 107) were transfected by electroporation using
a TNFR promoter-luciferase-TNFR 3′ reporter gene plasmid
as recently described.8,9 After 24 h in culture, cells were
selected for viability via lymphoprep (Nycomed Pharma AS,
Oslo, Norway) and seeded at a density of 1 × 104 cells/well in
100 µL of medium in a 96-well plate. Selection pressure for
G418 (Gibco BRL Life Technologies Ltd., Paisley, U.K.)
resistance encoded in cis on the reporter gene plasmid was
applied 24 h later (500 µg/mL G418). Eighteen G418-resistant
cell lines were obtained out of 550 wells, from which eight
expressed luciferase upon IgE plus Ag stimulation. Cell
culturing, stimulation, and luciferase read-outs were per-
formed as described.4,8
ELISA for Cytok in es. ELISAs for TNFR (Genzyme,
Cambridge, MA), IL-4 (Endogen, Woburn, MA), IL-6 (R&D,
Minneapolis, MN), and IL-13 (R&D) were performed according
to the protocol provided by the manufacturer; 1 × 105 cells
were pretreated with the indicated compounds 1 h prior to
stimulation. Supernatants were analyzed for cytokine produc-
tion 4 h later.
[2-[6-(4-ter t-Bu t ylp h en yl)-3,4-d ih yd r o-2H -p yr im id o-
[2,1-a ]isoqu in olin -7-yl]eth yl]d im eth yla m in e Hyd r och lo-
r id e (36‚HCl). Procedure A, 29, and 4-tert-butylbenzoic acid
methyl ester were used; the product was chromatographed on
silica gel (CH2Cl2/CH3OH: 7/1 f 4/1), yield 17% of 36‚HCl,
precipitated from CH3OH with diethyl ether: mp 175-190 °C
1
(hygroscopic); H NMR (CDCl3) δ 1.41 (s, 9H, C(CH3)3), 2.18
(quin, 2H, CH2), 2.42 (s, 6H, N(CH3)2), 2.87-2.96 (m, 4H, 2
CH2), 2.67 (t, 2H, NCH2), 3.87 (t, 2H, NHC2), 7.48 (td, 2H,
ArH), 7.75 (td, 2H, ArH), 7.86 (dt, 1H, ArH), 8.09 (dt, 1H, ArH),
8.18 (d, 1H, ArH), 8.48 (d, 1H, ArH); MS 388 (MH+). Due to
hygroscopy, no microanalysis was possible.
7-(4-Eth oxyben zyl)-6-(3-n a p h th a len -1-ylp r op yl)-3,4-d i-
h yd r o-2H -p yr im id o[2,1-a ]isoq u in olin e H yd r och lor id e
(37‚HCl). Procedure A, 31‚HCl, and 4-(1-naphthyl)butyric
acid methyl ester were used; the product was chromatographed
on silica gel (CH2Cl2/CH3OH: 95/5), yield 48% of 37‚HCl,
precipitated from CH3OH with diethyl ether: mp 192-200 °C;
1H NMR (CDCl3) δ 1.38 (t, 3H, CH3), 1.82-2.04 (m, 2H, CH2),
2.06-2.20 (m, 2H, CH2), 2.73-2.86 (m, 2H, CH2), 2.90 (t, 2H,
CH2), 3.81 (t, 2H, CH2), 3.93 (q, 2H, CH2), 3.93 (s, 2H, CH2),
4.10 (t, 2H, CH2), 6.61-6.70 (m, 4H, ArH), 7.25 (dd, 1H, ArH),
7.41-7.58 (m, 2H, ArH), 7.58-7.71 (m, 4H, ArH), 7.71-7.90
(m, 3H, ArH), 9.01 (dd, 1H, ArH), 11.15 (bs, 1H, H+); MS 487.3
(MH+). Anal. (C34H34N2O‚HCl) C, H, N.
7-(4-Eth oxyben zyl)-6-(4-p h en eth ylp h en yl)-3,4-d ih ydr o-
2H-p yr im id o[2,1-a ]isoqu in olin e Hyd r och lor id e (38‚HCl).
Procedure A, 31‚HCl, and 4-phenethylbenzoic acid methyl
ester were used; the product was chromatographed on silica
gel (CH2Cl2/CH3OH: 95/5), yield 38% of 38‚HCl, precipitated
from CH3OH with diethyl ether: mp 235-240 °C; 1H NMR
(CDCl3) δ 1.37 (t, 3H, CH3), 2.12 (quin, 2H, CH2), 2.92-3.04
(m, 4H, CH2CH2), 3.81-8.84 (m, 4H, NCH2, CH2), 3.89 (t, 2H,
NCH2), 3.96 (q, 2H, OCH2), 6.74 (td, 2H, ArH), 6.87 (td, 2H,
ArH), 7.13-7.29 (m, 9H, ArH), 7.56-7.61 (m, 3H, ArH), 9.27
(d, 1H, ArH), 11.57 (bs, 1H, H+); MS 498.1 (MH+). Anal.
(C35H34N2O‚HCl) C, H, N.
ELISA for Leu k otr ien es. The ELISA for leukotriene
synthesis (Amersham, Little Chalfont, U.K.) was performed
according to the protocol provided by the manufacturer; 1 ×
104 cells were pretreated with the indicated compounds 1 h
prior to stimulation. Supernatants were analyzed for leukot-
riene production 4 h later.
Wester n Blot. Cells were lysed in sample buffer (Novex,
San Diego, CA) containing 2% â-mercaptoethanol, and 20 µL
was loaded on a precast Tris-glycine 4-20% gradient gel
(Novex). Proteins were transferred to PVDF membranes
(Novex) by electroblotting. Membranes were blocked with 5%
skim milk in PBS containing 0.1% Tween-20 (Biorad Labora-
tories, Hercules, CA) and incubated with the primary antibody
overnight. The antibodies for erk 1/2 and p-erk 1/2 were
purchased from New England Biolabs (New England Biolabs,
Beverly, MA). After being washed three times with PBS/
Tween, membranes were incubated at room temperature for
1 h with the secondary antibody (anti-rabbit IgG/alkaline
phosphatase; New England Biolabs) and washed four times
with PBS/Tween. Detection by chemiluminescence (CDP Star,
New England Biolabs) was perfomed as described by the
manufacturer.
Hexosa m in id a se Assa y. CPII cells (5 × 104) were incu-
bated in 200 µL of Tyrode/BSA buffer (1.5 mM CaCl2, 5 mM
KCl, 1 mM MgCl2, 130 mM NaCl, 10 mM Hepes, 0.1% (w/v)
D-glucose, 0.1% (w/v) BSA) with or without compounds and
75 ng of murine IgE (BioMakor, Rehovot, Israel) for 18 h. Cells
were stimulated by adding 20 ng of DNP-BSA (Calbiochem
Corp., La J olla, CA) for 100 min. After centrifugation 100 µL
of supernatants was mixed with 100 µL of substrate solution
(3.8 mM p-nitrophenyl-m-acetylglucosamine; Sigma, St. Louis,
MO) and 0.1 M NaOAc, pH 4.5 (Sigma), and incubated for 90
min at 37 °C. The reaction was terminated by the addition of
100 µL of 2 M NaOH and measured using a microplate reader
at a wavelength of 405 nm.
XTT Test. Clone 12 cells (1 × 104) were subjected to an
XTT Test (Boehringer Mannheim, Mannheim, Germany) ac-
cording to the protocol provided by the manufacturer.
P a ssive Cu ta n eou s An a p h yla xis (P CA). Female mice
(Charles River) were sensitized after anesthesia (ip) with 225
µg of Ketalar (Parke Davis, Berlin, Germany) and 30 µg of
Rompun (Bayer, Leverkusen, Germany) by intracutaneous
injection of 5 µg in 10 µL of mouse IgE anti-DNP (BioMakor)
7-Ben zyl-6-(4-ph en eth ylph en yl)-3,4-d ih yd r o-2H-p yr im -
id o[2,1-a ]isoqu in olin e Hyd r och lor id e (39‚HCl). Proce-
dure A, 29, and 4-phenethylbenzoic acid methyl ester were
used; the product was chromatographed on silica gel (CH2Cl2/
CH3OH: 98/2 f 9/1); yield 52% of 39‚HCl, precipitated from
CH3OH with diethyl ether: mp 182-190 °C; 1H NMR (CDCl3)
δ 2.12 (quin, 2H, CH2), 2.97 (s, 4H, CH2CH2), 3.79 (t, 2H,
NCH2), 3.90-4.01 (m, 4H, CH2, NCH2), 6.97 (dd, 2H, ArH),
7.11-7.38 (m, 12H, ArH), 7.61-7.70 (m, 3H, ArH), 9.24-9.32
(m, 1H, ArH), 11.50 (bs, 1H, H+); MS 454.2 (MH+). Anal.
(C33H30N2‚HCl) C, H, N.
7-Bu tyl-6-(4-ter t-bu tylp h en yl)-3,4-d ih yd r o-2H-[2,1-a ]i-
soqu in olin e Hyd r och lor id e (40‚HCl). Procedure A, 32, and
4-tert-butylbenzoic acid methyl ester were used; the product
was chromatographed on silica gel (CH2Cl2/CH3OH: 9/1), yield
7% of 40‚HCl, amorphous: 1H NMR (CDCl3) δ 0.77 (t, 3H,
CH3), 1.22 (quin, 2H, CH2), 1.38-1.45 (m, 11H, CH2, C(CH3)3),
2.14 (quin, 2H, CH2), 2.43 (AB, 2H, CH2), 3.78 (t, 2H, NCH2),
3.92 (t, 2H, NCH2), 7.21 (td, 2H, ArH), 7.58 (td, 2H, ArH),
7.74-7.83 (m, 3H, ArH), 9.37 (d, 1H, ArH), 11.42 (bs, 1H, H+);
MS 372.2 (MH+). Anal. (C26H32N2‚HCl) C, H, N.

Inhibition of FcꢀRI-Mediated Activation of Mast Cells
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1059
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Ack n ow led gm en t. We thank D. J aksche for excel-
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