Pyrazolopyrimidines: Synthesis, effect on histamine release from rat peritoneal mast cells and cytotoxic activity

Article abstract of DOI:10.1016/S0223-5234(01)01225-9

A series of 1H-pyrazolo[3,4-d]pyrimidines (3-6) substituted at positions 1 (R₁ = Ph, H, tert-butyl and ribosetribenzoate), 4 (R₂ = chlorine, nitrogen and oxygen nucleophiles), and 6 (dimethylamino) have been synthesized and their effect on the release of histamine from rat peritoneal mast cells measured. After chemical stimulation, (polymer 48/80), several compounds (i.e. 3b, 4a, 4b, 4d, 4g, 5a), produce inhibition two to three times higher (40-60%) than DSCG but this action is lower after preincubation. 4b (R₁ = Ph, R₂ = NHCH₂Ph; 50-70% inhibition) and 5a (R₁ = H, R₂ = OMe; 50-55% inhibition) are the most active ones in both experiments. With ovoalbumin as stimulus, several pyrazolopyrimidines show inhibition similar to DSCG, the most active compounds being 6a-d (IC₅₀ = 12-16 μM; R₁ = ribosetribenzoate, R₂ = methoxy and amino). Compounds 4e (R₁ = t-butyl, R₂ = OMe) and 4g (R₁ = t-butyl, R₂ = piperidino) are inducers of the release of histamine (60 and 150% increase). Compounds 4b and 4c showed cytotoxic activity (IC₅₀ = 1 μg/mL) to HT-29 human colon cancer cells.

Full text of DOI:10.1016/S0223-5234(01)01225-9

Eur. J. Med. Chem. 36 (2001) 321–332
´
© 2001 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(01)01225-9/FLA
Original article
Pyrazolopyrimidines: synthesis, effect on histamine release from rat peritoneal
mast cells and cytotoxic activity
Jose´ M. Quintelaa, Carlos Peinadora, Mar´ıa J. Moreiraa, Amparo Alfonsob, Luis M. Botanab,
Ricardo Riguerac*
aDepartamento de Qu´ımica Fundamental e Industrial, Facultad de Ciencias, Universidad de La Corun˜a,
La Corun˜a E-15071, Spain
bDepartamento de Farmacologia, Facultad de Veterinaria, Universidad de Santiago, Lugo, Spain
cDepartamento de Qu´ımica Orga´nica, Facultad de Qu´ımica, Universidad de Santiago, Santiago de Compostela E-15706, Spain
Received 2 October 2000; revised 13 February 2001; accepted 13 February 2001
Abstract – A series of 1H-pyrazolo[3,4-d]pyrimidines (3–6) substituted at positions 1 (R1=Ph, H, tert-butyl and ribosetribenzoate),
4 (R2=chlorine, nitrogen and oxygen nucleophiles), and 6 (dimethylamino) have been synthesized and their effect on the release of
histamine from rat peritoneal mast cells measured. After chemical stimulation, (polymer 48/80), several compounds (i.e. 3b, 4a, 4b,
4d, 4g, 5a), produce inhibition two to three times higher (40–60%) than DSCG but this action is lower after preincubation. 4b
(R1=Ph, R2=NHCH2Ph; 50–70% inhibition) and 5a (R1=H, R2=OMe; 50–55% inhibition) are the most active ones in both
experiments. With ovoalbumin as stimulus, several pyrazolopyrimidines show inhibition similar to DSCG, the most active
compounds being 6a–d (IC50=12–16 mM; R1=ribosetribenzoate, R2=methoxy and amino). Compounds 4e (R1=t-butyl,
R2=OMe) and 4g (R1=t-butyl, R2=piperidino) are inducers of the release of histamine (60 and 150% increase). Compounds 4b
´
and 4c showed cytotoxic activity (IC50=1 mg/mL) to HT-29 human colon cancer cells. © 2001 Editions scientifiques et me´dicales
Elsevier SAS
Pyrazolopyrimidines / Histamine / Antihistaminic / Cytotoxicity
1. Introduction
We have recently described the action of pyridopy-
rimidines [14], pyridothienopyrimidines [15], and pyri-
dothienotriazines [16] as inhibitors and inducers of
the release of histamine from rat peritoneal mast cells.
A preliminary screening suggested us that some pyra-
The search for new drugs useful as inhibitors of the
release of histamine and other vasoactive mediators,
such as leukotrienes and prostaglandins, is an impor-
tant area of research in relation to the effective treat-
zol-fused heterocycles could be potentially interesting
ment of asthma. Disodium cromoglycate (DSCG®)
(figure 1), and nedocromil sodium [1–4] are two of
the most extensively used drugs for this purpose, but
a number of nitrogen and sulfur-containing hetero-
cyclic systems have also been explored and found to
produce effective oral antiallergics. Representative ex-
amples are found within the tetrazole [5–8], thienopy-
rimidine [9] (i.e. tiprinast®), pyrimidothienotriazine
[10, 11], thiophene (i.e. ketotifen®) and naphthyridine
[12, 13] systems.
* Correspondence and reprints
E-mail address: ricardo@usc.es (R. Riguera).
Figure 1.

322
J.M. Quintela et al. / European Journal of Medicinal Chemistry 36 (2001) 321–332
Figure 2.
cinoma) and MEL-28 (malignant melanoma) cell
lines.
in this process. In this paper, we describe our results
on the synthesis and action on the release of his-
tamine of a series of pyrazolopyrimidines (3–6)
(figure 2).
For the preparation of the title compounds we
devised a scheme that allowed for the introduction
along the process of diverse substituents at positions
1, 4 and 6 of the heterocycle. In this study we describe
the results on compounds having a dimethylamino
group fixed at C-6 and explore the effect of modifica-
tions on the other two positions (R1 =Ph, H, tert-bu-
tyl and ribosetribenzoate) and (R2 =nitrogen,
oxygen, chlorine).
It is known that the allergic response to a drug can
be caused not only by the onset immunological mech-
anisms (IgE-mediated) but by direct stimulation of
the mast cells with the chemical. Therefore, in this
study, we investigated the effect of the pyrazolopy-
rimidines under those two types of stimulus. The
amount of histamine released (inhibition and stimula-
tion) by rat peritoneal mast cells was measured after
immunological and chemical stimulus using ovoalbu-
min and polymer 48/80, respectively, as inducers. In
both experiments, the amount of histamine released
was evaluated with and without preincubation of the
pyrazolopyrimidine with the stimulus, and the data
compared with the amount released by DSCG taken
as reference.
2. Chemistry
The construction of fused pyrimidines from ortho-
aminonitriles and phosgene iminium salts has been
successfully applied to the preparation of many hete-
rocyclic fused systems [17]. For the synthesis of the
title pyrazolopyrimidines, we used that strategy start-
ing with N-substituted o-aminocyanopyrazoles (1a,b)
[18]. In this way, 1a,b were easily converted to the
corresponding chloroamidines (2a,b) by reaction with
In addition, the cytotoxicity of the pyrazolopyrim-
idines was evaluated in vitro for antiproliferative ac-
tivity against mouse P-388 (lymphoid neoplasm) and
human HT-29 (colon carcinoma), A-549 (lung car-

J.M. Quintela et al. / European Journal of Medicinal Chemistry 36 (2001) 321–332
323
phosgene iminium chloride. The pyrazolo[3,4-
d]pyrimidines (3a,b) were prepared by cyclization of
the chloroamidines (2a,b). Compounds 3a and 3b
were converted into a variety of pyrazolo[3,4-
d]pyrimidines (4a–h) by nucleophilic halide displace-
ment with nucleophiles such as primary and
secondary amines and the methoxide ion. In order to
prepare the pyrazolo[3,4-d]pyrimidine nucleosides, the
tert-Bu group of 4e–h was cleaved with formic acid at
90°C to give 5a–d, that were subsequently glycosi-
dated by reaction with 1-O-acetyl-3,4,5-tri-O-ben-
zoylribofuranose in the presence of SnCl4. All the
compounds gave satisfactory elemental analyses and
cal stimulus. In the experiments involving the
chemical stimulus, mast cells were obtained from
standard rats while immunological experiments were
carried out with ovoalbumin-sensitized rats [14]. The
inhibitory action was measured under two different
sets of experimental conditions; simultaneous addi-
tion of the stimulus and the drug to the cells, and with
preincubation (i.e. by addition of the chemical 10
min. before the stimulus). The results of these experi-
ments areshown graphically in figures 3–6. The his-
tamine released was calculated as a percentage of the
total histamine content of the cells. Figures 3–6 repre-
sent the variation of that percentage in comparison
with the histamine released by the stimuli controls
(48/80 or ovoalbumin). For comparative purposes,
the histamine released in the presence of the reference
compound, the mast cell stabilizer DSCG, has also
been included in the plots.
1
have spectral data (IR, MS, and H and 13C NMR)
that are consistent with the structures proposed.
3. Pharmacology
Experiments carried out in the absence of stimuli
showed that spontaneous histamine release was never
higher than 8%.
The pyrazolopyrimidines were used in concentra-
tions that cover the range from 15 to 40 mM, similar
to those of the reference compound DSCG (20 and 40
mM). In order to facilitate the comparison of our
The inhibition of the release of histamine by the
pyrazolopyrimidines (3–6) was measured as described
previously [16] using rat mast cells obtained from the
pleural and peritoneal cavities after lavages with sa-
line solution, and two different stimuli to provoke the
release of histamine; the polymer 48/80 as the chemi-
cal stimulus [19] and ovoalbumin as the immunologi-
Figure 3. Histamine release in rat peritoneal mast cells stimulated with 2 mg/mL of compound 48/80 in the presence of the
pyrazolopyrimidines. The compounds (100 mg/mL), were added to the cells simultaneously with the stimulus (no preincubation). The
activity data are normalized to the response of the control experiments (38.76 1.89% in the presence of 2 mg/mL of compound
48/80). The asterisks indicate the statistical significant results.

324
J.M. Quintela et al. / European Journal of Medicinal Chemistry 36 (2001) 321–332
Figure 4. Histamine release in rat peritoneal mast cells stimulated with 2 mg/mL of compound 48/80 in the presence of the
pyrazolopyrimidines. The compounds (100 mg/mL), were added to the cells 10 min. before the addition of the stimulus (10 min.
preincubation). The activity data are normalized to the response of the control experiments (39.89 9.35% in the presence of 2
mg/mL of compound 48/80). The asterisks indicate the statistical significant results.
Figure 5. Histamine release in rat peritoneal mast cells stimulated with 5 mg/mL of antigen (ovoalbumin) in the presence of the
pyrazolopyrimidines. The compounds (100 mg/mL), were added to the cells simultaneously with the stimulus (no preincubation). The
activity data are normalized to the response of the control experiments (15.05 3.06% in the presence of 5 mg/mL of antigen). The
asterisks indicate the statistical significant results.

J.M. Quintela et al. / European Journal of Medicinal Chemistry 36 (2001) 321–332
325
Figure 6. Histamine release in rat peritoneal mast cells stimulated with 5 mg/mL of antigen (ovoalbumin) in the presence of the
pyrazolopyrimidines. The compounds (100 mg/mL) were added to the cells 10 min. before the addition of the stimulus (10 min.
preincubation). The activity data are normalized to the response of the control experiments (22.53 3.04% in the presence of 5
mg/mL of antigen). The asterisks indicate the statistical significant results.
results with the literature, the data on figures 3–6 are
expressed in mg/mL. The IC50 values were also calcu-
lated (mM) and are shown in table 1. The cytotoxicity
of 3–6 was measured and the results are presented in
Section 4.4.
Overall, under chemical stimulation 4b, 4d and 5a are
the most active inhibitors independently of the mode of
addition of the stimulus and the heterocycle. Interest-
ingly, compound 4e, that is an inhibitor when added
simultaneously with the stimulus, is an inducer when it
is preincubated with the cells before addition of the
stimulus.
4. Results and discussion
4.1. Histamine release by chemical stimulus
Table I. Histamine release inhibition (IC50 mM) produced after
10 min. preincubation of the pyrazolopyrimidines with rat
peritoneal mast cells immunologically stimulated with ovoal-
bumin (5 mg/mL) a
All the pyrazolopyrimidines assayed were found to
present inhibitory action when the chemical stimulus
polymer 48/80 was added to the rat peritoneal mast cells
jointly with the compound (figure 3). The inhibition
values are between two and three times greater than
those obtained with the reference compound DSCG at
100–200 mg/mL. The most active are 3b, 4b, 4d, 4g and
5a, that produce about 50–60% less histamine than the
reference.
When the cells were preincubated with the com-
pounds before addition of the chemical inducer, (figure
4), compound 4e behaves as a stimulant of the release of
histamine while the rest of the compounds are inhibitors
although their activity is now reduced to a maximum
value of about twice that of DSCG (i.e. 4b and 6b).
Comp.
(preinc.)
IC50 (mM)
Comp.
(preinc.)
IC50 (mM)
DSCG
3a
20–40
36.3
36.9
29.5
30.8
21.5
30.8
5a
5b
5c
5d
6a
6b
6c
6d
51.2
37.2
40.6
26.2
15.8
14.0
14.4
12.1
4a
4b
4c
4d
4f
a Five concentrations of pyrazolopyrimidines (0.1, 1, 10, 100
and 500 mg/mL) have been used to calculate the data.

326
J.M. Quintela et al. / European Journal of Medicinal Chemistry 36 (2001) 321–332
4.2. Histamine release by immunological stimulus
compounds 6a–d with the larger substituent R1. In fact,
inhibitors similar or slightly better than DSCG are
found in all the other three series (R1=Ph, H, tert-bu-
tyl). As for the nature of substituent R2, it seems that
the activity is mainly associated to two types of sub-
stituents: amines (i.e. 4b and 4d), and ethers (i.e. 5a).
Preincubation produces a remarkable change in the
behaviour of 4e (R1=tert-butyl, R2=OMe) that is an
inhibitor in the simultaneous addition experiment but
becomes a stimulant when the experiment is carried out
under preincubation. Curiously, no such change of ac-
tivity is produced in the other pyrazolopyrimidines that
share with 4e the methoxy group at R2 (i.e. 4a, 5a).
For their part, the results obtained in the immunolog-
ical stimulated experiments, and the IC50 data indicate
that those pyrazolopyrimidines bearing the ribosetriben-
zoate substituent, are slightly more active as inhibitors
than the rest of the compounds assayed.
In addition to the experiments carried out with chem-
ical stimulation of the cells, the pyrazolopyrimidines
were tested for their action when the release of his-
tamine was induced by an effective immunological stim-
ulus such as the protein ovoalbumin.
Figure 5 shows the results of the tests carried out by
simultaneous addition of the immunological stimulus
and the pyrazol to the rat peritoneal mast cells. Again,
many compounds present an inhibitory activity similar
to that of DSCG, but the most interesting results are the
surge of 3b, 4e, 4h and principally 4g (150% more
histamine than the reference) as effective stimulants of
the release of histamine and the total lack of activity of
4b and 4c.
In the tests performed with the compound added 10
min. before the ovoalbumin, (preincubation; figure 6),
the general pattern of activity is similar to that of the
experiment without preincubation: Compounds 4e, 4g
and 4h are again inducers of the release of histamine
while the rest of the pyrazoles show about half or
similar (6b–d) activity to that of the reference.
In order to get a clearer comparative picture of the
potential interest of these compounds, experiments using
five concentrations of inhibitor have been carried and
the corresponding IC50 values were compared with those
of DSCG. The results, expressed in mM, are presented in
table 1. The values are in the range 12–50 mM, com-
pounds 6a–d being the most active of all. This confirms
that the pyrazolopyrimidines examined present an in-
hibitory action under immunological stimulus, similar to
that of the reference compound.
4.4. Cytotoxic activity
Pyrazolopyrimidines have been tested in vitro for
their cytotoxic activity against four standard tumoral
cell lines P-388 (lymphoid neoplasm) and human A-549
(lung carcinoma), HT-29 (colon carcinoma) and MEL-
28 (malignant melanoma) cell lines. Only two products,
4b and 4c, showed a sufficiently strong activity to de-
serve to be mentioned. (IC50=1 mg/mL towards HT-29
colon carcinoma cells). The other pyrazolopyrimidines,
were not cytotoxic at concentrations lower than 5–10
mg/mL.
5. Conclusions
Interestingly, preincubation shows a higher response
with immunological than with chemical stimulation.
This is probably related to the longer time the com-
pound has to modulate the signal used by the stimuli,
but in the case of chemical stimulation with compound
48/80 the inhibition is always higher when both drugs
are added together. We do not have an explanation to
this effect, but a competition to pass through the cellular
membrane might explain it, since compound 48/80 re-
quires interaction with G proteins to activate the re-
sponse [20].
In conclusion, several compounds containing the
pyrazolopyrimidine heterocyclic system are inhibitors
of the liberation of histamine, similar to DSCG, but
unfortunately no clear structure-activity pattern
emerges from that data apart from the higher activity
(50–70%) of those carrying the ribosetribenzoate sub-
stituent (6a–d) in the immunological experiments and
that of those with smaller ones (i.e. 4b, 4d and 5a)
when the cells are stimulated chemically. In addition,
some compounds are found to stimulate the release of
histamine in the range 50–150%. Particularly interest-
ing is 4e that stimulates the release of histamine
(35–60%), in both chemical and immunological ex-
periments. Compounds, 4g and 4h (30–150%) are
also stimulants but under immunological stimulation
only.
4.3. Structure–activity relationship
From a structural point of view, the results of the
chemical stimulated experiments, show no clear-cut pat-
tern of activity outside the lower relative activity of the

J.M. Quintela et al. / European Journal of Medicinal Chemistry 36 (2001) 321–332
327
The in vitro cytotoxicity of these heterocycles is low
in terms of activity (IC50 of 10 mg/mL or more) and
only 4b and 4c show some action with IC50 =1 mg/mL
towards human colon cancer cell line HT-29 only.
and for 10 more min. after addition of the stimulus.
Since we used compound 48/80 as a reference drug, each
experiment was carried out with parallel controls of
compound 48/80. Incubations were stopped by immers-
ing the tubes in a cold bath. After centrifugation at
1000×gmax for 5 min., the supernatants were collected
and decanted into other tubes for histamine determina-
tion. We used trichloroacetic acid (7%, final concentra-
tion) to precipitate the protein and avoid its interference
with histamine determination (the presence of 1 mg/mL
BSA interferes slightly with histamine determination).
Controls to determine spontaneous histamine release in
the absence of stimuli were executed in every experi-
ment. Spontaneous histamine release was never higher
than 8%.
6. Experimental
6.1. Biological methods
The histamine releaser compound 48/80, a condensa-
tion product of N-methyl-p-methoxy-phenethylamine,
was obtained from Sigma chemical Co. Orthophtalalde-
hyde for the spectrofluorimetric measurements was pur-
chased from Merck and the Bordetella pertussis used in
the sensitization of the rats was obtained from Wako.
Eagle’s minimum essential medium, Earle’s balanced
salts, nonessential aminoacids, (EMEM/neaa), and 1-
glutamine were purchased from Biosciences and fetal
calf serum (FCS) and trypsin from Seromed.
6.1.4. Histamine release assays
Histamine release was measured spectrofluorometri-
cally [22] both in the pellet and in the supernatants with
and without preincubation (10 min.) of each drug (100
mg/mL in the single dose experiments) with the cells
before addition of the stimulus. The fluorophore ob-
tained by reaction in alkaline medium of the 0.1%
solution of orthophtalaldehyde with the histamine re-
leased, is stabilized by acidification and remains useful
for measurement for about 2 h at room temperature. In
the experiments involving the chemical stimulus, the
mast cells were obtained from standard rats while the
experiments using immunological stimulus were carried
out with the ovoalbumin-sensitized rats. The results are
expressed as a percentage of histamine released with
respect to the total histamine content. Calculations were
done substracting spontaneous histamine values from
numerator and denominator. The trypan blue exclusion
test was used in order to ensure that histamine release
was not due to cytotoxicity. All the experiments were
repeated at least three times in duplicate and the data
was analyzed using the Student’s t-test for umpaired
data. A probability level of 0.05 or less was used for
statistical significance and the results are expressed as
mean S.E.M. Five concentrations of inhibitor (0.1, 1,
10, 100 and 500 mg/mL) were used in order to obtain the
IC50 values.
6.1.1. Mast cell preparation
Mast cells were obtained by lavage of pleural and
peritoneal cavities of Sprague–Dawley rats (200–400 g)
as described previously [21]. Physiological saline compo-
sition was (mM): Na+, 142.3; K+, 5.94; Ca2+, 1; Mg2+
,
1.2; Cl−, 126.1; CO32−, 22.85; PO4H2−, 1.2; SO24−, 1.2,
giving a final osmotic pressure of 300 5 mOms/kg H2O.
One mg/mL of sucrose and bovine serum albumin
(BSA) was also added to the solution, final pH being
7.0. The impurified cellular suspension contained 4–8%
mast cells, with an average of 1.5–2×106 mast cells per
rat.
6.1.2. Sensitization of rat mast cells
Sprague–Dawley rats weighting 200–300 g were sen-
sitized by intramuscular injection in the back extremities
of egg albumin (15 mg each rat) and adjuvant (9×109
killed Bordetella pertussis each rat) in saline solution.
Two weeks later, the rats were sacrificed and the mast
cells isolated as described.
6.1.3. Cell incubation
Twenty-five microliters of a freshly prepared concen-
trated solution of each drug in dimethylsulfoxide were
added to 0.9 ml of incubation medium. When the
medium reached 37°C, 25 ml of cell suspension, contain-
ing 1–1.5×105 mast cells, were added and the cells were
then incubated for 10 min. (in the experiments per-
formed without preincubation this step was omitted),
6.1.5. Antineoplasic assays
In vitro antitumor assays were carried out by an
adaptation of the method described by Bergeron et al.
[23] and the activity measured as IC50 against P-388
(ATCC CCL 46; suspension culture of a lymphoid
neoplasm from a DBA/2 mouse), A-549 (ATCC CCL

328
J.M. Quintela et al. / European Journal of Medicinal Chemistry 36 (2001) 321–332
185; monolayer culture of a human lung carcinoma),
HT-29 (ATCC HTB-38; monolayer culture of a human
colon carcinoma), and MEL-28 (ATCC HTB-72; mono-
layer culture of a human melanoma). The cells were
maintained in logarithmic growth in EMEM/neaa, sup-
reduced pressure and the residue was purified by flash
chromatography to yield the corresponding amide chlo-
ride.
6.2.2. 5-[Chloro(dimethylamino)-
plemented with 5% Fetal Calf Serum (FCS), 10−2
M
methyleneamino]-4-cyano-1-phenyl-1H-pyrazole (2a)
Using CH2Cl2/hexanes (2:1) as eluent. Yield 80%; m.p.
105–107°C. IR (cm−1): 2220 (CN); 1640. 1H NMR
(CDCl3): l=3.14 (s, 6H, NMe2); 7.32–7.59 (m, 5H,
C6H5); 7.75 (s, 1H, H-3). 13C NMR (CDCl3): l=40.2;
83.7; 114.2; 123.3; 127.3; 128.5; 138.0; 141.3; 143.0; 150.0.
MS (EI, m/z, %): 275 (M++2, 30); 273 (M+, 82). Anal.
Calc. C13H12N5Cl: C, 57.04; H, 4.42; N, 25.59. Found: C,
56.89; H, 4.59; N, 25.68%.
sodium bicarbonate and 0.1 g/L penicillin G+ 0.1 g/L
streptomycin sulfate and placed in 16 mm diameter wells
at 1×104 (P-388), 2×104 (A-549, MEL-28, and HT-29)
cells per well, respectively. The cytotoxicity was evalu-
ated by addition of 1 mL aliquots of a solution of the
compound in EMEM 5% FCS. A separate set of cultures
without test compounds to be added, was used as control
and to ensure that the cells remained in an exponential
phase of growth all along the test. After three days of
incubation at 37°C in a 10% CO2 and 98% H2O atmo-
sphere, the cells were trypsinized and counted in a
Coulter Counter ZM. All counts (net cells per well),
represent the average of duplicate wells. Comparison of
the counts with those of control cultures, allows the
calculation of the percentage of growth produced by each
compound. The results obtained from cultures treated
with different concentrations of each compound, are
used to generate dose–response curves from which IC50
values were derived.
6.2.3. 1-tert-butyl-5-[Chloro(dimethylamino)-
methyleneamino]-4-cyano-1H-pyrazole (2b)
Using hexanes/AcOEt (3:1) as eluent. Yield. 80%; m.p.
76–78°C. IR (cm−1): 2220 (CN); 1640. 1H NMR
(CDCl3): l=1.50 (s, 9H, C(CH3)3); 3.19 (s, 6H, NMe2);
7.48 (s, 1H, H-3). 13C NMR (CDCl3): l=28.8; 40.2;
60.4; 83.7; 114.6; 138.3; 142.1; 150.3. MS (EI, m/z, %):
255 (M++2, 13); 253 (M+, 51). Anal. Calc. C11H16N5Cl:
C, 52.07; H, 6.36; N, 27.60. Found: C, 52.30; H, 6.18; N,
27.81%.
6.2.4. Preparation of 4-chloropyrazolo[3,4-d]pyrimidines
(3a–b)
6.2. Chemistry
All reagents used were commercial grade chemicals
from freshly opened containers. Melting points were
determined on a Bu¨chi 510 apparatus and are reported
uncorrected. IR spectra were recorded on potassium
bromide disks on a Perkin–Elmer 783 spectrophotome-
ter. 1H and 13C NMR spectra were obtained on a Bruker
AC200F instrument at r.t. MS were obtained on a
VG-QUATTRO spectrometer. The Silica gel 60 HF254+
366 used for analytical thin layer chromatography and
the silica gel 60 (230–400 mesh) employed for flash
chromatography were purchased from Merck. Micro-
analyses for C, H, and N were performed by the Elemen-
tal Analyses General Service of the University of La
Corun˜a.
6.2.4.1. General procedure
A stream of dry hydrogen chloride was passed through
a solution of 1a or 1b in 1,2-dichlororethane for 1 h, the
solution was stirred at r.t. for 3 days. The solvent was
removed under reduced pressure and the residue was
purified by flash chromatography to yield 3a–b.
6.2.5. 4-Chloro-6-dimethylamino-1-phenyl-1H-
pyrazolo[3,4-d]pyrimidine (3a)
Using CH2Cl2/hexanes (1:1) as eluent. Yield 96%; m.p.
124–126°C. IR (cm−1): 2900; 1540. H NMR (CDCl3):
1
l=3.28 (s, 6H, NMe2); 7.25–8.27 (m, 5H, C6H5); 7.98
(s, 1H, H-3). 13C NMR (CDCl3): l=37.6; 107.4; 120.2;
125.7; 128.9; 133.8; 139.0; 154.3; 155.6; 160.5. MS (EI,
m/z, %): 275 (M++2, 36); 273 (M+, 100). Anal. Calc.
C13H12N5Cl: C, 57.04; H, 4.42; N, 25.59. Found: C,
57.30; H, 4.17; N, 25.70%.
6.2.1. Preparation of 5-[chloro(dimethylamino)-
methyleneamino]-4-cyanopyrazoles (2a–b)
6.2.1.1. General procedure
6.2.6. 1-tert-butyl-4-Chloro-6-dimethylamino-1H-
pyrazolo[3,4-d]pyrimidine (3b)
A solution of 1a or 1b [17] (8 mmol) and phosgeni-
minium chloride (12 mmol) in 1,2-dichloroethane (30
mL) was refluxed for 2 h. The solvent was removed under
Using CH2Cl2/hexanes (4:1) as eluent. Yield 74%; m.p.
1
110–111°C. IR (cm−1): 2970; 1610. H NMR (CDCl3):

J.M. Quintela et al. / European Journal of Medicinal Chemistry 36 (2001) 321–332
329
l=1.75 (s, 9H, C(CH3)3); 3.23 (s, 6H, NMe2); 7.75 (s,
6.2.11. 4-Benzylamino-6-dimethylamino-1-
phenylpyrazolo[3,4-d]pyrimidine (4b)
1H, H-3). 13C NMR (CDCl3): l=28.7; 37.5; 59.7;
107.4; 130.6; 153.9; 155.3, 159.4. MS (EI, m/z, %): 255
(M++2, 16); 253 (M+, 45). Anal. Calc. C11H16N5Cl: C,
52.07; H, 6.36; N, 27.60. Found: C, 51.89; H, 6.52; N,
27.78%.
Recrystallized from EtOH. Yield 60%; m.p. 118–
120°C. IR (cm−1): 3300 (NH); 2900; 1640. 1H NMR
(CDCl3): l=3.25 (s, 6H, NMe2); 4.80 (d, 2H, J=5.4
Hz, CH2); 5.5 (m, 1H, NH); 7.20–7.52, 8.29–8.35 (m, 10
H, C6H5); 7.78 (s, 1H, H-3). 13C NMR (CDCl3): l=
37.2; 45.0; 95.7; 120.2; 125.0; 128.7; 127.4; 127.7, 138.7;
132.1; 139.9; 156.6; 157.5; 161.1. MS (EI, m/z, %): 344
(M+, 52). Anal. Calc. C20H20N6: C, 69.75; H, 5.85; N,
24.40. Found: C, 69.87; H, 6.02; N, 24.11%.
6.2.7. Preparation of 6-dimethylamino-4-methoxy-
1H-pyrazolo[3,4-d]pyrimidines (4a–e)
6.2.7.1. General procedure
To a solution of sodium methoxide (0.05 g of Na, 2
mmol) in methanol (5 mL) was added 3a or 3b (0.5
mmol). The solution was refluxed until the starting
material had disappeared (TLC) (1–4 h). The solvent
was removed under reduced pressure and water (10 mL)
was added to the residue. The solution was neutralized
(2 N HCl) and the solid formed was filtered and recrys-
tallized to yield 4a,e.
6.2.12. 6-Dimethylamino-1-phenyl-4-piperidino-1H-
pyrazolo[3,4-d]pyrimidine (4c)
Recrystallized from EtOH. Yield 68%; m.p. 108–
1
110°C. IR (cm−1): 2900; 1560. H NMR (CDCl3): l=
1.72 (m, 6H, CH2); 3.23 (s, 6H, NMe2); 3.86–3.89 (m,
4H, NCH2); 7.24–7.52, 8.33–8.37 (m, 5H, C6H5); 7.91
(s, 1H, H-3). 13C NMR (CDCl3): l=24.5; 25.6; 37.1;
46.4; 95.6; 120.5; 124.9; 128.6; 134.5; 140; 156.8; 157.5;
161.1. MS (EI, m/z, %): 322 (M+, 100). Anal. Calc.
C18H22N6: C, 67.06; H, 6.88; N, 26.06. Found: C, 67.22;
H, 7.02; N, 25.76%.
6.2.8. 6-Dimethylamino-4-methoxy-1-phenyl-1H-
pyrazolo[3,4-d]pyrimidine (4a)
Recrystallized from EtOH. Yield 67%; m.p. 126–
1
128°C. IR (cm−1): 2900; 1640. H NMR (CDCl3): l=
3.27 (s, 6H, NMe2); 4.07 (s, 3H, CH3); 7.21–8.33 (m,
5H, C6H5); 7.93 (s, 1H, H-3). 13C NMR (CDCl3): l=
37.3; 53.3; 97.1; 120.2; 125.2; 133; 139.9; 157.4; 161.4;
163.6. MS (EI, m/z, %): 269 (M+, 19). Anal. Calc.
C14H15N5O: C, 62.44 H, 5.61;N, 26.01. Found: C, 62.67;
H, 5.80; N, 25.82%.
6.2.13. 6-Dimethylamino-1-phenyl-4-[4-(3,4-methylene-
dioxybenzyl)piperazino]-1H-pyrazolo[3,4-d]pyrimidine
(4d)
Recrystallized from EtOH. Yield 64%; m.p. 158–
1
160°C. IR (cm−1): w=2920; 1560. H NMR (CDCl3):
l=2.55–2.60 (m, 4H, NCH2); 3.22 (s, 6H, NMe2); 3.48
(s, 2H, CH2); 3.91–3.96 (m, 4H, NCH2); 5.97 (s, 2H,
OCH2O); 6.78–6.91 (m, 3H, C6H3CH2O2); 7.51–8.33
(m, 5H, C6H5); 7.88 (s, 1H, H-3). 13C NMR (CDCl3):
l=37.1; 45.2; 52.6; 62.7; 95.7; 100.9; 107.8; 109.4;
120.6; 122.2; 125.1; 128.6; 131.5; 134.3; 139.9; 146.7;
147.7; 157.1; 157.5; 161. MS (EI, m/z, %): 457 (M+, 12).
Anal. Calc. C25H27N7O2: C, 65.63; H, 5.95; N, 21.43.
Found: C, 65.58; H, 6.12; N, 21.18%.
6.2.9. 1-tert-butyl-6-Dimethylamino-4-methoxy-1H-
pyrazolo[3,4-d]pyrimidine (4e)
Recrystallized from EtOH. Yield 90%; m.p. 84–86°C.
1
IR (cm−1): 2980; 1610. H NMR (CDCl3): l=1.75 (s,
9H, C(CH3)3); 3.23 (s, 6H, NMe2); 4.04 (s, 3 H, CH3);
7.71 (s, 1H, H-3). 13C NMR (CDCl3): l=28.7; 37.2;
53.2; 59.1; 97.0; 129.5; 157.4; 160.5; 163.5. MS (EI, m/z,
%): 249 (M+, 50). Anal. Calc. C12H19N5O: C, 57.81; H,
7.68; N, 28.09. Found: C, 58.02; H, 7.80; N, 27.88%.
6.2.14. 4–Benzylamino–1–tert–butyl–6–
dimethylamino–1H-pyrazolo[3,4-d]pyrimidine (4f)
Using CH2Cl2 as eluent. Yield 84%; m.p. 190–192°C.
6.2.10. Preparation of 6-dimethylamino-1H-
pyrazolo[3,4-d]pyrimidines (4b–d and 4f–h)
1
IR (cm−1): 3300 (NH); 1640. H NMR (CDCl3): l=
1.76 (s, 9H, C(CH3)3); 3.20 (s, 6H, NMe2); 4.78 (d, 2H,
J= 5.86 Hz, CH2); 5.36 (s, 1H, NH); 7.32–7.41 (m, 5H,
C6H5); 7.56 (s, 1H, H-3). 13C NMR (CDCl3): l=28.7;
37.1; 44.9; 58.9; 95.9; 126.9; 127.3; 127.7; 128.6; 139.0;
128.6; 156.0; 160.6. MS (EI, m/z, %): 324 (M+, 91).
Anal. Calc. C18H24N6: C, 66.64; H, 7.46; N, 25.90.
Found: C, 66.89; H, 7.32; N, 25.79%.
6.2.10.1. General procedure
A solution of 3a or 3b (0.5 mmol) and the appropriate
amine (2 mmol) in THF (5 mL) was refluxed until the
starting material had disappeared (TLC) (1.5–8 h). The
solid was filtered and recrystallized or was purified by
flash chromatography.

330
J.M. Quintela et al. / European Journal of Medicinal Chemistry 36 (2001) 321–332
1
6.2.15. 1-tert-butyl-6-Dimethylamino-4-piperidino-1H-
pyrazolo[3,4-d]pyrimidine (4g)
1520. H NMR (CDCl3): l=3.22 (s, 6H, NMe2); 4.80
(d, 2H, J= 5.37 Hz, CH2); 5.40 (s, 1H, NH); 7.37–7.39
(m, 5H, C6H5); 7.68 (s, 1H, H-3). 13C NMR (CDCl3):
l=37.1; 44.9; 95.9; 126.9; 127.3; 127.7; 128.6; 139;
128.6; 156.0; 160.6. MS (EI, m/z, %): 268 (M+, 53).
Anal. Calc. C14H16N6: C, 62.67; H, 6.01; N, 31.32.
Found: C, 62.90; H, 5.92; N, 31.18%.
Recrystallized from EtOH. Yield 90%; m.p. 86–88°C.
IR (cm−1): w=2970; 1610. 1H NMR (CDCl3): l=1.62–
1.75 (m, 15H, C(CH3)3, CH2); 3.17 (s, 6H, NMe2);
3.82–3.85 (m, 4H, NCH2); 7.66 (s, 1H, H−3). 13C NMR
(CDCl3): l=24.6; 25.6; 28.5; 37.0; 46.4; 58.7; 95.9;
130.6; 156.9; 160.0. MS (EI, m/z, %): 302 (M+, 19).
Anal. Calc. C16H26N6: C, 63.54; H, 8.67; N, 27.79.
Found: C, 63.66; H, 8.84; N, 27.50%.
6.2.20. 6-Dimethylamino-4-piperidino-1H-
pyrazolo[3,4-d]pyrimidine (5c)
6.2.16. 1-tert-butyl-6-Dimethylamino-4-
[4-(3,4-methylenedioxybenzyl)piperazino]-1H-
pyrazolo[3,4-d]pyrimidine (4h)
Using AcOEt as eluent. Yield 94%; m.p. 225–227°C.
1
IR (cm−1): 3240 (NH); 2970; 1560. H NMR (CDCl3):
l=1.71 (br s, 6H, CH2); 3.28 (s, 6H, NMe2); 3.86–3.89
(m, 4H, NCH2); 7.79 (s, 1H, H-3). 13C NMR (CDCl3):
l=24.6; 25.6; 37.7; 46.4; 94.4; 133.9; 156.6; 158.8;
160.8. MS (EI, m/z, %): 246 (M+, 100). Anal. Calc.
C12H18N6: C, 58.51; H, 7.37; N, 34.12. Found: C, 58.34;
H, 7.28; N, 34.38%.
Using hexanes/AcOEt (4:1) as eluent. Yield 80%; m.p.
1
78–80°C. IR (cm−1): w=2980; 1600. H NMR (CDCl3):
l=1.72 (s, 9H, C(CH3)3); 2.04–2.83 (m, 4H, NCH2);
3.12 (s, 6H, NMe2); 3.32 (s, 2H, CH2); 3.46–4.07 (m,
4H, NCH2); 5.85 (s, 2H, OCH2O); 6.67–6.82 (m, 3H,
C6H3CH2O2); 7.61 (s, 1H, H-3). 13C NMR (CDCl3):
l=28.2; 36.7; 44.7; 52.2 (NCH2); 58.4; 62.3; 95.7; 100.5;
107.5; 109.0; 121.8; 130.3; 131.2; 146.7; 147.7; 156.7;
159.5. MS (EI, m/z, %): 437 (M+, 24). Anal. Calc.
C23H31N7O2: C, 63.14; H, 7.14; N, 22.41. Found: C,
62.88; H, 7.28; N, 22.64%.
6.2.21. 6-Dimethylamino-4-[4-(3,4-methylene-
dioxybenzyl)piperazino]-1H-pyrazolo[3,4-d]pyrimidine
(5d)
Using CH2Cl2/MeOH (26:1) as eluent. Yield 70%;
m.p. 168–170°C. IR (cm−1): 2980; 1600. 1H NMR
(CDCl3): l=2.47–2.87 (m, 4H, NCH2); 3.21 (s, 6H,
NMe2); 3.60 (s, 2H, CH2); 3.95–4.00 (m, 4H, NCH2);
5.95 (s, 2H, OCH2O); 6.76–6.87 (m, 3H, C6H3); 7.75 (s,
1H, H-3); 8.00 (br s, 1H, NH). 13C NMR (CDCl3):
l=37.8; 44.6; 51.9; 62.1; 94.4; 101.0; 108.0; 109.7;
122.8; 129.5; 133.7; 147.1; 147.7; 156.3; 156.6; 158.8. MS
(EI, m/z, %): 381 (M+, 12). Anal. Calc. C19H23N7O2: C,
59.83; H, 6.08; N, 25.71. Found: C, 60.05; H, 5.87; N,
25.90%.
6.2.17. Preparation of 6-dimethylamino-1H-
pyrazolo[3,4-d]pyrimidines (5a–d)
6.2.17.1. General procedure
A solution of 4e–h (6 mmol) in formic acid (2.5 mL)
was heated at 90°C until the starting material had
disappeared (TLC). The solvent was removed under
reduced pressure and the residue was recrystallized or
purified by flash chromatography to yield 5a–d.
6.2.22. Preparation of nucleosides (6a–d)
6.2.18. 6-Dimethylamino-4-methoxy-1H-
pyrazolo[3,4-d]pyrimidine (5a)
Recrystallized from EtOH. Yield 70%; m.p. 224–
1
226°C. IR (cm−1): 3100 (NH); 1610. H NMR (DMSO):
6.2.22.1. General procedure
l=3.14 (s, 6H, NMe2); 4.00 (s, 3H, CH3); 7.80 (s, 1H,
H-3); 12.9 (s, 1H, NH). 13C NMR (DMSO): l=36.9;
53.0; 94.7; 131.6; 159; 160.7; 162.8. MS (EI, m/z, %):
193 (M+, 100). Anal. Calc. C8H11N5O: C, 49.73 H,
5.74;N, 36.25. Found: C, 50.06; H, 5.96; N, 36.00%.
To a solution of 1-O-acetyl-3,4,5-tri-O-benzoylribo-
furanose (0.5 mmol) in dry acetonitrile (15 mL) SnCl4 (1
mmol) was added. The solution was stirred under inert
atmosphere at r.t. for 15 min. and then 5a–d (0.5 mmol)
was added and stirring was continued until starting
material had disappeared (TLC). The solvent was evap-
orated and a saturated solution of NaHCO3 was added.
The solution was extracted with AcOEt (3515 mL) and
dried (Na2SO4). After evaporation of the solvent, the
residue was purified by flash chromatography.
6.2.19. 4-Benzylamino-6-dimethylamino-1H-
pyrazolo[3,4-d]pyrimidine (5b)
Using hexanes/AcOEt (2:1) as eluent. Yield 40%; m.p.
248–250°C. IR (cm−1): 3360 (NH); 3160 (NH); 1600;

J.M. Quintela et al. / European Journal of Medicinal Chemistry 36 (2001) 321–332
331
6.2.23. 1-(2%,3%,5%-O-Tribenzoyl-d-ribofuranosyl)-
6-dimethylamino-4-methoxy-1H-pyrazolo[3,4-d]-
pyrimidine (6a)
100–102°C. [h]D= −50.87. IR (cm−1): 2960; 1730 (CO);
1600; 1530. H NMR (CDCl3): l=2.47–2.52 (m, 4H);
1
3.21 (s, 6H); 3.46 (s, 2H); 3.74–3.86 (m, 4H); 4.66–4.87
(m, 3H); 5.96 (s, 2H); 6.18 (d, J=1.96 Hz); 6.47 (m,
2H); 6.77–6.89 (m, 3H); 7.30–8.04 (m, 16H). 13C NMR
(CDCl3): l=37.4; 45.0; 52.5; 62.6; 64.3; 71.9; 75.9; 92.8;
92.8; 97.7; 100.9; 107.9; 109.5; 122.3; 124.2; 128.3; 128.4;
128.9; 129.4; 129.7; 129.8; 131.3; 133.1; 133.4; 133.6;
146.8; 147.7; 157.6; 161.3; 164.6; 165.0; 165.2; 166.0. MS
(FAB): 826 [(MH)+, 98]. Anal. Calc. C45H43N7O9: C,
65,44; H, 5.25; N, 11.87. Found: C, 65.19; H, 5.42; N,
11.99%.
Using CH2Cl2/AcOEt (10:1) as eluent. Yield 50%;
m.p. 152–154°C. [h]D= −77.1. IR (cm−1): 2960; 1760
1
(CO); 1640; 1560. H NMR (CDCl3): l=3.27 (s, 6H);
4.01 (s, 3 H); 4.66–4.88 (m, 3H), 6.21 (d, J= 0.88 Hz,
1H); 6.31 (m, 2H); 7.28–8.05 (m, 16H). 13C NMR
(CDCl3): l=37.5; 53.3; 64.1; 71.8; 75.9; 80.6; 93.0; 98.6;
123.7; 128.3; 128.5; 128.8; 129.4; 132.9; 133.3; 133.6;
129.7; 161.1; 164.5; 164.9; 165.0; 166.2. MS (FAB, m/z,
%): 638 [(MH)+, 19]. Anal. Calc. C34H31N5O8: C, 64.04;
H, 4.90; N, 10.98. Found: C, 63.88; H, 5.10; N, 11.17%.
Acknowledgements
6.2.24. 1-(2%,3%,5%-O-Tribenzoyl-d-ribofuranosyl)-
4-benzylamino-6-dimethylamino-1H-pyrazolo[3,4-d]-
pyrimidine (6b)
We acknowledge financial support from CICYT
(PM98-0227, FEDER-CICYT 1FD97-2157) and from
Xunta de Galicia (XUGA-10308-B95, XUGA-
20908B97, XUGA-PGIDT99PXI20906B and XUGA-
PGIDT99BIO20901). We are also grateful to Instituto
Biomar (Leo´n), for testing facilities.
Using CH2Cl2/AcOEt (4:1) as eluent. Yield 43%; m.p.
96–98°C. [h]D= −52.82. IR (cm−1): 3380 (NH); 1760
1
(CO); 1640; 1540. H NMR (CDCl3): l=3.20 (s, 6H);
4.58–4.86 (m, 5H); 5.80 (br s, 1H); 6.16 (s, 1H); 6.24–
6.31 (m, 2H); 7.29–8.05 (m, 21H). 13C NMR (CDCl3):
l=37.5; 44.7; 60.4; 64.3; 72.0; 75.9; 80.4; 92.8; 98.3;
122.9; 127.3; 128.6; 128.8; 129.1; 129.4; 129.5; 129.6;
129.7; 133.1; 133.2; 133.4; 133.6; 135.3; 138.7; 157.1;
161.8; 163.5; 165.0; 165.1; 165.5; 166.2; 169.8; 171.2. MS
(FAB): 713 [(MH)+, 40]. Anal. Calc. C40H36N6O7: C,
67.40; H, 5.09; N, 11.79. Found: C, 67.66; H, 4.86; N,
11.95%.
References
[1] Cox J., Beach J., Blair M., Clarke A., King J., Lee T., Loveday
D., Moss G., Orr T., Ritchie J., Sheard P., Adv. Drugs Res. 5
(1970) 115–196.
[2] Assem E., Ritcher A., Immunol. Lond. 21 (1971) 729–730.
[3] Foreman J., Garland L., Br. Med. J. 1 (1976) 820–821.
[4] Lal S., Malhotra S., Gribben D., Hoddre D., Thorax 39 (1984)
809.
6.2.25. 1-(2%,3%,5%-O-Tribenzoyl-d-ribofuranosyl)-
6-dimethylamino-4-piperidino-1H-pyrazolo[3,4-d]-
pyrimidine (6c)
[5] Unangst P.C., Brown R.E., Herzig D.J., J. Med. Chem. 23
(1980) 1251–1255.
Using CH2Cl2/AcOEt (100:1) as eluent. Yield 60%;
m.p. 96–98°C. [h]D= −62.3. IR (cm−1): 2940; 1750
(CO); 1580; 1520. 1H NMR (CDCl3): l=1.64– 2.00 (m,
6H); 3.22 (s, 6H); 3.70–3.75 (m, 4 H); 4.67–4.86 (m,
3H); 6.19 (s, 1H); 6.33 (d, 2H, J= 4.88 Hz); 7.31–8.03
(m, 16H). 13C NMR (CDCl3): l=24.4; 25.6; 37.4; 46.4;
64.4; 72.1; 76.0; 80.6; 92.8; 97.8; 129.7; 124.7; 127.9;
128.4; 128.5; 128.8; 128.9; 129.5; 133.1; 133.4; 133.5;
133.6; 147.5; 157.4; 161.5; 164.7; 164.9; 165.2; 166.2. MS
(FAB, m/z, %): 691 [(MH)+, 14]. Anal. Calc.
C38H38N6O7: C, 66.07; H, 5.54; N, 12.17. Found: C,
65.88; H, 5.70; N, 12.01%.
[6] Klaubert D.H., Sellstedt J.H., Ginosso C.J., Bell S.C., Capetola
S.J., J. Med. Chem. 24 (1981) 748–752.
[7] Phillip A., Jirkousky I., Martel R.R., J. Med. Chem. 23 (1980)
1372–1376.
[8] Schwender C.F., Sunday B.R., Kerbleski J.J., Herzig D.J., J.
Med. Chem. 23 (1980) 964–972.
[9] Madding G.D., Thompson M.D., J. Heterocyc. Chem. 24
(1987) 581–586.
[10] Youssefyeh R.D., Brown R.E., Uresh Shah J.W., Jones H.,
Loev B., Khandwala A., Liebowitz M.J., Jonnino-Goldman P.,
J. Med. Chem. 27 (1984) 1639–1643.
[11] Khandwala A., Van Inwegen R., Coutts S., Dally-Meade V.,
Youssefyeh R.D., Int. J. Immunopharmacol. 5 (1983) 491–502.
[12] Sherlock M.H., Kaminski J.J., Tom W.C., Lee J.F., Wong
S.C., Kretner W., Bryant R.W., McPhail A.T., J. Med. Chem.
31 (1988) 2108–2121.
6.2.26. 1-(2%,3%,5%-O-Tribenzoyl-d-ribofuranosyl)-
6-dimethylamino-4-[4-(3,4-methylenedioxybenzyl)-
piperazino]-1H-pyrazolo[3,4-d]pyrimidine (6d)
Using CH2Cl2/AcOEt (10:1) as eluent. Yield 5%; m.p.
[13] Sherwood J., Sehring S., Rizzo C., Chapman R.W., Siegel M.I.,
Egan R.W., J. Pharmacol. Exp. Ther. 247 (1988) 997–1003.
[14] Quintela J.M., Peinador C., Botana L., Estevez M., Riguera R.,
Biorganic Med. Chem. 5 (1997) 1543–1553.

332
J.M. Quintela et al. / European Journal of Medicinal Chemistry 36 (2001) 321–332
[15] Quintela J.M., Peinador C., Veiga C., Gonza´lez L., Botana L.M.,
Alfonso A., Riguera R., Biorg. Med. Chem. 6 (1998) 1911–1925.
[20] Mousli M., Bronner C., Bockaert J., Rouot B., Landry Y.,
Immunol. Lett. 25 (1990) 355–357.
[16] Quintela J.M., Peinador C., Veiga C., Botana L.M., Alfonso A.,
[21] Estevez M.D., Vieytes M.R., Louzao M.C., Botana L.M.,
Riguera R., Eur. J. Med. Chem. 33 (1998) 887–897.
Biochem. pharmacol. 47 (1994) 591–593.
[17] Quintela J.M., Peinador C., Moreira M.J., Alvarez-Sarande´s R.,
Recent Res. Dev. Org. Chem. 2 (1998) 409–418.
[22] Shore P.A., The chemical determination of histamine, in:
Glick D. (Ed.), Analysis of biogenic amines and their re-
lated enzymes, Wiley Interscience, Amsterdam, 1971, pp. 89–
92.
[18] Barald P.G., Cacciari B., Spalluto G., Pineda de las Infantas y
Villatoro M.J., Zocchi C., Dionisotti S., Ongini E., J. Med.
Chem. 39 (1996) 1164–1171.
[23] Bergeron R.J., Cavanaugh P.F., Kline S.J., Hughes R.G., Elliot
G.T., Porter C.W., Biochem. Biophys. Res. Comm. 121 (1984)
848–854.
[19] Scharemberg A.M., Kinet J.P., Chem. Immunol. 61 (1995)
72–87.
.