Synthesis of 3-(1,2,3-triazol-1-yl)- and 3-(1,2,3-triazol-4-yl)-substituted pyrazolo[3,4-d]pyrimidin-4-amines via click chemistry: Potential inhibitors of the Plasmodium falciparum PfPK7 protein kinase

Article abstract of DOI:10.1039/b906482f

Efficient routes to 3-(1,2,3-triazol-1-yl)- and 3-(1,2,3-triazol-4-yl) pyrazolo[3,4-d]pyrimidin-4-amines using a one-pot two-step reaction are presented. The two routes give easy access to two different isomers of 1,4-disubstituted triazoles and the targe

Full text of DOI:10.1039/b906482f

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of 3-(1,2,3-triazol-1-yl)- and 3-(1,2,3-triazol-4-yl)-substituted
pyrazolo[3,4-d]pyrimidin-4-amines via click chemistry: potential inhibitors
of the Plasmodium falciparum PfPK7 protein kinase
Michael Klein,a Peter Dine´r,a Dominique Dorin-Semblat,b Christian Doerigb and Morten Grøtli*a
Received 1st April 2009, Accepted 27th May 2009
First published as an Advance Article on the web 2nd July 2009
DOI: 10.1039/b906482f
Efficient routes to 3-(1,2,3-triazol-1-yl)- and 3-(1,2,3-triazol-4-yl)pyrazolo[3,4-d]pyrimidin-4-amines
using a one-pot two-step reaction are presented. The two routes give easy access to two different
isomers of 1,4-disubstituted triazoles and the target compounds are obtained from a variety of readily
available aromatic and aliphatic halides without isolation of potentially unstable organic azide
intermediates. Two compounds show activity towards the PfPK7 kinase (IC50 10–20 mM) of P.
falciparum, the organism responsible for the most virulent form of malaria, and can be regarded as hits
useful for further development into lead compounds.
Introduction
The purine ring is the most ubiquitous nitrogen-containing
heterocycle in nature, and it is the core structure of adenine
and guanine in nucleic acids (RNA and DNA).1 In addition,
purines are involved in many metabolic processes as the co-factors
associated with a large number of enzymes and receptors, e.g.
ATP and GTP, which play key roles in cell signalling and other
fundamental biological processes.2
The ATP binding site in protein kinases is highly conserved,
and therefore, the development of highly ATP competitive
kinase inhibitors is a difficult task. However, there are regions
within the binding cleft that are not occupied by ATP, and these
regions (hydrophobic pockets) show a higher degree of structural
Fig. 1 Schematic picture of the binding of inhibitors 1 and 2 to the Btk
diversity between members of the kinase family than the ATP
binding regions. This provides opportunities for the discovery
or design of selective and small molecule ATP-competitive
inhibitors.3 For example, Shokat et al. have shown that the Abl
kinase was effectively inhibited by the substituted 4-amino-
pyrazolo[3,4-d]pyrimidine II, largely through interaction with
such “non-ATP binding” pockets.4 Further, Pan et al. showed that
compound I inhibits the Btk kinase in nanomolar concentration.5
The common motif for these 4-amino-pyrazolo[3,4-d]-
pyrimidine scaffolds is the “bent” geometry of the substituent in
the 3-position of the pyrazole ring (Fig. 1). The bent structure
allows the inhibitor to interact with the additional pocket
of the active site, thereby increasing the inhibition and the
specificity.
kinase and the Abl kinase, respectively.
of the pyrazole ring in the 3-position with differently substituted
triazole rings using a 1,4-regioselective copper-catalysed azide–
alkyne cycloaddition (CuAAC).6,7
The 1,3-dipolar cycloaddition, often referred to as click chem-
istry, has been used in various applications, e.g. organic synthesis,
drug discovery, and chemical biology due to the high reaction
yield and simple reaction and purification conditions of the “click
chemistry”.7–11
The use of click chemistry allows the 4-substituent of the
1,2,3-triazole ring to be easily varied through the use of different
azides or alkynes. Furthermore, the 1,4-disubstituted triazoles
will have the “bent” geometry that should be useful for targeting
kinases with extended lipophilic pockets with a bent shape.
Here we wish to present two efficient routes to 3-(1,2,3-triazol-1-
yl)- and 3-(1,2,3-triazol-4-yl)-pyrazolo[3,4-d]pyrimidin-4-amines
using a one-pot two-step reaction. The two routes developed give
easy access to both 1- and 4-substituted triazole rings in the
3-position of the pyrazole ring. Two compounds show activity
in micromolar concentration (IC50 10–20 mM) towards the PfPK7
kinase in P. falciparum, the organism responsible for the most
virulent form of malaria, and can be regarded as hits useful for
further developments into lead compounds.
In our search for new scaffolds that could be used in
the development of kinase inhibitors, we became interested
in the 4-aminopyrazolo[3,4-d]pyrimidines. Our approach to 4-
aminopyrazolo[3,4-d]pyrimidines involves the functionalisation
aDepartment of Chemistry, University of Gothenburg, Kemiva¨gen 10, 41296
Gothenburg, Sweden. E-mail: grotli@chem.gu.se; Fax: (+)46 31 7723840;
Tel: (+)46 31 7722905
bINSERM Unit 609, Wellcome Centre for Molecular Parasitology, 120
University Place, Glasgow, UK G12 8TA and INSERM U609, Global Health
Institute, Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), Station 19,
CH-1015 Lausanne, Switzerland
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Results and discussion
Starting from the common precursor 1, the target compounds 6
and 7 were synthesised according to two complementary strategies
(Scheme 1). In Route 1, compound 2 is synthesised via the
palladium-catalysed Sonogashira coupling followed by a copper-
catalysed [3 + 2]-cycloaddition with an azide, resulting in the
formation of target compound 3-(1,2,3-triazol-4-yl)-substituted
pyrazolo[3,4-d]pyrimidin-4-amines 6. In Route 2, compound 1 is
converted into the azide 3, which is reacted in situ via the copper-
catalysed [3 + 2]-cycloaddition with different alkynes resulting
in formation of 3-(1,2,3-triazol-1-yl)-substituted pyrazolo[3,4-
d]pyrimidin-4-amines 7.
Scheme 2 (i) and (ii) Compound 1 (1–3 mmol), acetylene (4 eq.)
Pd(PPh3)4 (2 mol%), CuI (20 mol%) and Amberlite IRA-67 (5 eq.), THF,
MW (120 ◦C, 15 min). (iii) Compound 8, NaOH (2 N), THF/MeOH (1:1),
r.t., 22 h. (iv) Compound 9, NaOH, toluene, reflux, 2h.
In some cases, the purification of compound 8 becomes difficult
due to the formation of triphenylphosphine oxide in the reaction.
Therefore, an alternative approach to the Sonogashira coupling
was developed involving a different alkyne, 2-methyl-3-yn-2-ol.
Under the same conditions as for the synthesis of compound 8,
compound 9 was prepared in 74% yield. Due to the additional hy-
droxyl group, the resulting product is more polar than compound 8
and triphenylphosphine oxide could be removed completely with
flash chromatography using chloroform/methanol as an eluent.
Furthermore, the propargyl alcohol is much cheaper than TMS
acetylene. Compound 9 was deprotected using catalytic amounts
of sodium hydroxide in dry refluxing toluene yielding compound
2 in 91%.
Since the synthesis of different organic azides is time consuming
and in some cases very risky, several one-pot procedures have
previously been developed.12,13 In these procedures, the organic
azides are generated in situ either via the nucleophilic substitution
of the alkyl halides by sodium azide or by the copper-catalysed
reaction between an aromatic iodide and sodium azide, followed
by the copper-catalysed [3 + 2]-cycloaddition reaction.
Scheme 1 Strategies for the synthesis of the target compounds 6 and 7.
From the starting material 1, the terminal alkyne 2 was
synthesised according to two different procedures involving a
Sonogashira coupling with a protected acetylene, followed by a
deprotection of the alkyne (Scheme 2). Initially, 1 was reacted
with TMS-acetylene in the presence of Pd(PPh3)4 (2 mol%) and
CuI (20 mol%) in THF using microwave-assisted heating at 120 ◦C
to yield the protected alkyne 8 in excellent yield (91%). In order to
facilitate the work-up the weak ion exchange resin (IRA-67) was
used as base, which after the reaction could simply be filtered off.
The TMS-group was easily deprotected with sodium hydroxide in
THF/MeOH in good yield (87%).
In order to find the optimal conditions for our reaction, a
small screening of copper catalysts, solvents, reaction time and
temperature was performed for the pyrazolo[3,4-d]pyrimidine
system (Table 1). Starting with the commonly used solvent system
tert-butanol/water and copper sulfate with copper wire under
microwave irradiation (125 ◦C, 15 min), formation of the product
6a was observed but the conversion was not complete according
to 1H NMR (66%, entry 1).
The solvents were changed to THF and DMF:water (4:1), but
no reaction was observed and the starting material was fully
recovered (entries 2 and 3). The influence of the copper catalyst was
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Table 1 Screening of different solvents, catalysts, and reaction conditions
for the Cu(I)-catalysed [3 + 2] cycloadditiona
Entry Solvent
Cu-cat.
T/◦C Time/min Conv.b
1
2
3
4
5
6
7
tBuOH:H2O (1:1) Cu0, CuSO4
125
125
125
15
15
15
15
15
15
30
66:33
n.r.
n.r.
88:12
THF
Cu0, CuSO4
DMF:H2O (4:1) Cu0, CuSO4
DMF:H2O (4:1) CuSO4/Naasc 125
DMF:H2O (4:1) CuSO4/Naasc 140
DMF:H2O (4:1) CuSO4/Naasc 140
DMF:H2O (4:1) CuSO4/Naasc 125
Full conv.
26:1c
Full conv.
a Compound 2 (0.25 mmol), benzylbromide (1.2 eq.), NaN3 (1.2 eq.),
solvents (2 ml), CuSO4 (20 mol%, 1 M solution in H2O), MW-heating (fixed
hold-time). b Determined by 1H NMR (2:6a). c 2.4 eq. benzylbromide and
azide.
Scheme 3 (i) Compound 2 (0.25 mmol), alkyl bromide or aromatic iodide
(1.2 eq.), NaN3 (1.2 eq.), DMF:H2O (4:1) (2.5 ml), CuSO4 (20 mol%, 1 M
solution in H2O), sodium ascorbate (40 mol%), MW-heating (130 ◦C,
30 min, fixed hold-time).
investigated by exchanging the copper wire for sodium ascorbate
(Naasc) (entries 3 and 4). Switching to copper sulfate/sodium
ascorbate gave a high conversion (88:12) of the starting material
(entry 4). The conversion was further improved by increasing
the amount of alkylbromide (2.4 eq.) a◦nd sodium azide (2.4 eq.)
and increasing the temperature to 140 C (entries 5 and 6). Full
conversion was also observed at 125 ◦C after prolonged reaction
time (entry 7).
With the optimised results in hand, compounds 6a–b were
synthesised in good to moderate yield (66–86%, Scheme 3) using
CuSO4/sodium ascorbate (20 mol%/40 mol%) as the copper
catalyst in a DMF:H2O (4:1) solvent system and microwave
irradiation for the heating (130 ◦C, 30 min).
Using the same conditions, the aromatic iodides 4c–j were
reacted with alkyne 2 to give products 6c–j in moderate to excellent
yield (45–95%) considering that the reaction is a two-step one-pot
reaction.
The same one-pot procedure that was used in the synthesis
of compounds 6c–j was also used for the synthesis of compounds
7a–h in which the azide 3 is generated in situ from compound 1 and
then further reacts with the alkyne in the copper-catalysed [3 + 2]-
cycloaddition reaction to yield products 7a–h (Scheme 4). The
reactions proceeded in good to excellent yield for both aliphatic
(5a–b) and aromatic alkynes (5c–h) (78–91% yield), with the
exception of the aromatic compounds (5f–g) having an amine
group and a nitro group substituent on the aromatic ring (58%
and 67%, respectively).
Scheme 4 (i) Alkyne 5 (0.25 mmol), compound 1, (1.2 eq.), NaN3 (1.2 eq.),
DMF:H2O (4:1) (2.5 ml), CuSO4 (20 mol%, 1 M solution in H2O), sodium
ascorbate (40 mol%), MW-heating (130 ◦C, 30 min, fixed hold-time).
structural studies included compounds based on the same pyra-
zolopyrimidine scaffold as presented in this work.14,15 Therefore,
we were interested in investigating the potential use of our
compounds as leads towards PfPK7 inhibitors.
P. falciparum is the most virulent species of human malaria
parasites and is responsible for the most severe forms of the disease.
Biological evaluation of inhibitors towards the
Plasmodium falciparum protein kinase PfPK7
Recently, several X-ray structures of Plasmodium falciparum
(P. falciparum) protein kinase 7 (PfPK7) in complex with different
inhibitors have been disclosed. The molecules used in these
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Malaria causes over two million deaths annually, predominantly
among children from sub-Saharan Africa. P. falciparum is becom-
ing resistant to currently available antimalarial treatments, which
has led to an urgent and continuing search for new methods of
control.16,17 Within malaria parasites, inhibition of protein kinases
can modulate intracellular protein phosphorylation events, just as
in other eukaryotes. Some P. falciparum kinases are not related to
any eukaryote protein kinase family, and these may be validated as
potential P. falciparum-specific drug targets.18,19 Onesuchemerging
target is PfPK7, an ‘orphan’ plasmodial kinase which is distantly
related to the MAPKK family of kinases (mitogen-activated
protein kinase kinase).20,21 Disruption of the pfpk7 gene suggests
that PfPK7 is involved in a pathway regulating cell proliferation
and development: P. falciparum clones in which the PfPK7 locus
has been inactivated are viable, but their asexual growth rate is
about half that observed in wild-type parasites.22 This phenotype
suggests that PFPK7 inactivation through chemical inhibition
would be expected to slow parasite growth and, hence, decrease
the virulence of infection.
The effect of molecules 6a–i and 7a–h on the activity of
recombinant PfPK7 was tested in in vitro inhibition assays
and an initial test was performed at 10 mM for compounds
6a–i and 7a–h. The assay measured the efficacy of compounds
6a–i and 7a–h at decreasing myelin basic protein (MBP, the
exogenous substrate used in the assay) phosphorylation, as semi-
quantitatively determined by autoradiography (Fig. 2).
Fig. 3 Inhibition of recombinant PfPK7 by compounds 6c (top panel)
and 6g (bottom panel). The autoradiographic data (MBP phosphoryla-
tion) used to construct the curves are shown as insets. See Experimental
procedures for details.
The pyrazolopyrimidine ring is buried within the active site and
makes one hydrogen bond with PfPK7 to the backbone amide
group of Met120 within the hinge region and another hydrogen
bond to the backbone carbonyl moiety of Glu118, which is a
similar binding to that seen for the AMPPNP ligand in the X-ray
structure (Fig. 4). In PfPK7, a hydrophobic pocket is accessible
to ATP-competitive inhibitors, which has previously been shown
in X-ray structures of PfPK7.15 The docking suggests that the
Fig. 2 Inhibition of recombinant PfPK7 by compounds 6a–i and 7a–h.
The efficacies are the averages from three measurements at 10 mM
compound concentration. See Experimental procedures for details.
Two of the compounds, 6c and 6g, showed a significant decrease
in MBP phosphorylation at 10 mM concentration and IC50 values
for these compounds were determined to be ~20 and ~10 mM, re-
spectively (Fig. 3). Interestingly, although activity was significantly
decreased in the presence of micromolar range concentrations of
the inhibitors, we were unable to completely inhibit the kinase
with the compounds, even at 1 mM. Nevertheless, these results
demonstrate that the molecules with the pyrazolopyrimidine
scaffold developed in this study do indeed affect PfPK7 activity.
To understand the binding mode of the active compounds, we
docked 6c (Glide, XP mode) into the X-ray structure of PfPK7
(2PML).23,24 The pyrazolopyrimidine scaffold was superimposed
with the adenylyl imidodiphosphate (AMP-PNP) ligand in the
X-ray structure and the protein–ligand complex was optimised
using the OPLS force field.24
Fig. 4 Docking of compound 6c into the ATP-pocket of PfPK7 (purple:
AMP-PNP). Compound 6c is suggested to have the same binding mode
as ATP and to also bind to the additional hydrophobic part of the ATP
pocket.
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4-phenyl-(1,2,3-triazol-1-yl)-moiety of compound 6c binds to the
hydrophobic pocket within the ATP cleft and could explain the
inhibitory effect of compound 6c.
C8H10IN5 (302.9981): C, 31.70; H, 3.33; N, 23.11. Found C, 31.84;
H, 3.40; N, 23.19.
General procedure A for preparation of 3-ethynyl substituted
4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidines (8 and 9)
Conclusions
4-Amino-3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidine 1 (1–
3 mmol), Pd(PPh3)4 (2 mol%), CuI (20 mol%) and Amberlite IRA-
67 (5 eq.) were suspended in of THF (12 ml) in a 20 ml microwave
vessel. To the stirring mixture the appropriate acetylene (4 eq.) was
added, the vessel was sealed and heated to 120 ◦C for 15 min (ramp
time: 240 s, pre-stirring: 20 s, high, fixed hold time: on). After
cooling to room temperature, the reaction mixture was filtered
through Celite and washed with CHCl3. The solvent was removed
in vacuo at 30 ◦C. The crude product was purified by flash column
chromatography on silica gel (MeOH : CHCl3 = 1 : 20).
An efficient strategy for the preparation of of 3-(1,2,3-triazol-
1-yl)- and 3-(1,2,3-triazol-4-yl)pyrazolo[3,4-d]pyrimidin-4-amines
has been developed. The target compounds are obtained by a
convenient one-pot procedure from a variety of readily available
aromatic and aliphatic halides without isolation of potentially
unstable organic azide intermediates. Two compounds show ac-
tivity towards the PfPK7 kinase (IC50 10–20 mM) in P. falciparum,
the organism responsible for the most virulent form of malaria.
These are some of the very few inhibitors identified targeting the
PfPK7 kinase and should be useful starting points for further
development into lead compounds.
4-Amino-1-isopropyl-3-trimethylsilanylethynyl-1H-pyrazolo[3,4-
d]pyrimidine (8)
Experimental part
Compound 1 (981 mg, 3.237 mmol) was converted to compound
8 (800 mg, 91% yield) by general procedure A. Rf (MeOH:CHCl3
General
1
1H (400 MHz) and 13C (100 MHz) NMR spectra were obtained
from a JEOL JNM-EX 400 spectrometer. The symbol “+”
indicates CH3 and CH groups identified from distortionless
enhancement of polarisation transfer (DEPT) spectra. Column
chromatography was performed by manual flash chromatography
(wet packed silica, 0.04–0.063 mm) or by automated column
chromatography on Biotage SP-4 using pre-packed columns.
Microwave reactions were performed in a Biotage Initiator reactor
with fixed hold time. IR spectra were recorded on a Perkin-
Elmer 16 PC spectrometer. Elemental analyses were performed at
Kolbe Mikroanalytisches Laboratorium, Mu¨lheim and der Ruhr,
Germany. X-ray structures with inhibitors were used as starting
point for all dockings. The PfPK7 kinase was prepared according
to the standard procedure in the Schro¨dinger package. Docking
was performed by using Glide (Schro¨dinger) with extraprecision
(XP) settings and standard parameters for ligand docking.
1:40) = 0.21. H NMR (400 MHz, CDCl3) d 0.28 ppm (s, 9H,
3
-Si-(CH3)3), 1.54 (d, JHH = 6.6 Hz, 6H, CH3-CH), 5.10 (sept,
3JHH = 6.6 Hz, 1H, CH3-CH-N), 8.31 (s, 1H, N-CH-N). 13C NMR
(100 MHz, CDCl3) d -0.15 ppm (+, -Si-(CH3)3), 22.14 (+, CH3-
CH), 49.61 (+, CH3-CH-N), 96.69 (Cquart), 100.94 (Cquart), 102.34
(Cquart), 125.97 (Cquart), 152.45 (Cquart), 156.28 (+), 158.10 (Cquart). IR
(KBr) n = 3453 cm - 1, 3326, 3113, 2975, 2163, 1657, 1595, 1574,
1252, 862, 846. Anal. Calcd for C13H19N5Si: C, 57.11; H, 7.00; N,
25.61. Found C, 57.24; H, 7.10; N, 25.48.
4-(4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-
methyl-but-3-yn-2-ol (9)
The general procedure A using 1 (303 mg, 1.000 mmol) afforded
192 mg (74%) of 9 as colorless solid. Rf (MeOH:CHCl3=
1:40) = 0.21. 1H NMR (400 MHz, CDCl3) d 1.46 ppm (d,
3JHH = 6.6 Hz, 6H, CHN-(CH3)2), 1.62 (s, 6H, CCO-(CH3)2), 5.02
(sept, 3JHH = 6.6 Hz, 1H, (CH3)2-CH-N), 8.21 (s, 1H, N-CH-N).
13C NMR (100 MHz, CDCl3) d = 22.01 ppm (+, CHN-(CH3)2),
31.33 (+, -CCO-(CH3)2), 49.51 (+, CH3-CH-CH3), 65.33 (Cquart),
74.12 (Cquart), 99.68 (Cquart), 101.81 (Cquart), 125.69 (Cquart), 152.04
(Cquart), 155.84 (+), 157.95 (Cquart). IR (film) n 3459 cm-1, 3329,
2981, 2934, 1638, 1573. Anal. Calcd for C13H17N5O: C, 60.21; H,
6.61; N, 27.01. Found C, 60.23; H,6.64; N, 27.04.
4-Amino-3-iodo-1-(isopropyl)pyrazolo[3,4-d]pyrimidine (1)
4-Amino-3-iodo-pyrazolo[3,4-d]pyrimidine (2000 mg, 7.66 mmol)
and K2CO3 (2115 mg, 15.32 mmol) were suspended in 12 ml
of dry DMF in a 20 ml microwave vessel. To this mixture,
2-chloropropane (0.77 ml, 662 mg, 8.426 mmol) were added and
the sealed tube was heated to 200 ◦C for 5 min. (20 s of pre-stirring
and fixed hold time: on). After cooling of the reaction mixture to
room temperature, additional 2-chloropropane (0.35 ml, 301 mg,
3.83 mmol) was added and the microwave vessel was heated
again to 200 ◦C for 5 min. After cooling to ambient temperature,
the reaction mixture was diluted with DMF and filtered. The
solvent was removed in vacuo at 80 ◦C and the residue was
co-distilled with toluene three times. The crude product was
purified by flash column chromatography on silica gel (MeOH :
CHCl3 = 1 : 20) to give 2205 mg (95%) of 2 as fine yellow needles.
Rf (MeOH : CHCl3 = 1 : 40) = 0.29. Mp 203–204 ◦C. 1H NMR
(400 MHz, CDCl3) d 1.55 ppm (d, 3JHH = 6.6 Hz, 6H), 5.09 (sept,
3JHH = 6.6 Hz, 1H), 6.17 (bs, 2H, -NH2), 8.32 (s, 1H). 13C NMR
(100 MHz, CDCl3) d 22.27 ppm (+), 49.89 (+), 85.62 (Cquart), 104.25
(Cquart), 153.17 (Cquart), 155.88 (+), 157.63 (Cquart). Anal. Calcd for
4-Amino-1-isopropyl-3-ethynyl-1H-pyrazolo[3,4-d]pyrimidine (2)
Method 1. Alcohol 9 (151 mg, 0.582 mmol) was suspended
in 5 ml of dry toluene (dried over Na/benzophenone) and
crushed NaOH (84 mg, 2.10 mmol) was added. The reaction
mixture was stirred at reflux for 2h. The solvent was removed
in vacuo at 30 ◦C and the residue was purified by flash column
(MeOH:CHCl3 = 1:80) to give 106 mg (91%) of 2 as a pale
brownish solid.
Method 2. Compound 8 (713 mg, 2.61 mmol) was dissolved
in THF (20 ml) and MeOH (15 ml) and 2 N NaOH was added
and the reaction mixture was stirred at room temperature. After
22 h, the reaction was quenched by the addition of 30 ml of
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brine. The mixture was extracted with EtOAc (3 ¥ 60 ml). The
combined, organic layers were dried over MgSO4 and the solvent
was removed in vacuo. The crude product was purified with flash
column chromatography on silica gel (MeOH:CHCl3 = 1:40) to
give 454 mg (87%) of 2 as a pale colored solid. Rf (MeOH :
22.16 ppm (+), 36.79 (-), 48.91 (+), 52.16 (-), 98.76 (Cquart), 121.41
(+), 127.44 (+), 128.79 (+), 129.11 (+), 135.43 (Cquart), 136.76
(Cquart), 142.68 (Cquart), 153.63 (Cquart), 156.28 (+), 158.77 (Cquart).
IR (KBr) n 3439 cm-1, 3282, 3117, 2978, 2932, 1652, 1601, 1561.
Anal. Calcd for C18H20N8: C, 62.05; H, 5.79; N, 32.16. Found C,
62.18; H, 5.71; N, 32.04.
◦
1
CHCl3 = 1 : 40) = 0.18. Mp 188–189 C. H NMR (400 MHz,
3
CDCl3) d = 1.54 ppm (d, JHH = 6.6 Hz, 6H, CH3-CH), 3.49
3
(s, 1H, Csp-H), 5.11 (sept, JHH = 6.6 Hz, 1H, CH3-CH-N), 8.33
General procedure C for preparation of triazoles from aromatic
iodides
(s, 1H, N-CH-N). 13C NMR (100 MHz, CDCl3) d = 22.12 ppm
(+, CH3-CH), 49.67 (+, CH3-CH-N), 76.19 (+), 82.51 (Cquart),
124.93 (Cquart), 152.45 (Cquart), 156.08 (+), 158.02 (Cquart). IR (film)
n 3447 cm-1, 3307, 3221, 3069, 2984, 2109, 1651, 1595, 1567. Anal.
calcd for C10H11N5: C, 59.69; H, 5.51; N, 34.80. Found C, 59.67;
H, 5.46; N, 34.71.
3-Ethynyl-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine
(0.25 mmol), aromatic iodide (1.2 eq.), NaN3 (1.44 eq.), L-proline
(20 mol%), Na2CO3 (20 mol%), sodium ascorbate (40 mol%) were
suspended in DMSO : H2O (2.5 ml, 9 : 1) mixture in a 2–5 ml
microwave vessel. To the stirring mixture CuSO4 (20 mol%, as a
1 M solution i◦n H2O) were added and the vessel was sealed and
heated to 130 C for 30 min (ramp time: 35 s, pre-stirring: 20 s,
high, fixed hold time: on). After cooling to room temperature,
the reaction mixture was poured into aqueous ammonia solution
(80 ml of H2O, 20 ml of conc. NH4OH). The layers were separated
and the water layer was extracted with EtOAc three times. The
combined organic layers were washed with H2O and brine and
dried over MgSO4. The solvent was removed in vacuo at 30 ◦C and
the residue was further purified with column chromatography.
General procedure B for preparation of triazoles from
non-aromatic/benzylic bromides
3-Ethynyl-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine
(0.25 mmol), benzylbromide (1.2 eq.), NaN3 (1.2 eq.), sodium
ascorbate (40 mol%) were suspended in of a DMF : H2O (2.5 ml,
4 : 1) mixture in a 2–5 ml microwave vessel. To the stirring mixture
CuSO4 (20 mol%, as a 1 M solution in H2O) was added and the
vessel was sealed and heated to 130 ◦C for 30 min (ramp time:
40 s, pre-stirring: 20 s, high, fixed hold time: on). After cooling to
room temperature, the reaction mixture was poured into aqueous
ammonia solution (80 ml of H2O, 20 ml of conc. NH4OH). The
layers were separated and the water layer was extracted with
EtOAc three times. The combined organic layers were washed with
H2O and brine and dried over MgSO4. The solvent was removed
in vacuo at 30 ◦C and the residue was further purified with column
chromatography.
4-Amino-1-isopropyl-3-(1-phenyl-1H-[1,2,3]triazol-4-yl)-1H-
pyrazolo[3,4-d]pyrimidine (6c)
The general procedure C using 2 (50 mg, 0.248 mmol) afforded
75 mg (95%) of 6c as colorless solid. Mp 251–252 ◦C. Rf
(MeOH:CHCl3= 1:20) = 0.30. 1H NMR (400 MHz, CDCl3)
3
d 1.58 ppm (d, JHH = 6.6 Hz, 6H, NCH-(CH3)2), 5.18 (sept,
3JHH = 6.6 Hz, 1H, (CH3)2-CH-N), 6.12 (bs, 1H, NH-), 7.48–
7.62 (m, 3H), 7.82–7.86 (m, 2H), 8.35 (s, 1H, N-CH-N), 8.61 (s,
1H, N-CH-C), 9.44 (bs, 1H, NH-). 13C NMR (100 MHz, CDCl3)
d 22.24 ppm (+), 49.06 (+), 98.92 (Cquart), 119.32 (+), 120.83 (+),
129.54 (+), 130.16 (+), 135.11 (Cquart), 136.90 (Cquart), 143.46 (Cquart),
153.78 (Cquart), 156.41 (+), 158.70 (Cquart). IR (KBr) n 3432 cm-1,
3295, 3099, 2983, 2934, 1727, 1663, 1564, 1505. Anal. Calcd for
C16H16N8: C, 59.99; H, 5.03; N, 34.98. Found C, 60.02; H, 5.05; N,
35.01.
4-Amino-3-(1-benzyl-1H-[1,2,3]triazol-4-yl)-1-isopropyl-1H-
pyrazolo[3,4-d]pyrimidine (6a)
The general procedure B using 2 (50 mg, 0.248 mmol) afforded
71 mg (86%) of 6a as colorless sticky oil. Rf (MeOH:CHCl3=
1:80) = 0.04. 1H NMR (400 MHz, CDCl3) d 1.51 ppm (d,
3
3JHH = 6.6 Hz, 6H, CH3-CH), 5.09 (sept, JHH = 6.6 Hz, 1H,
CH3-CH-N), 5.58 (s, 2H, N-CH2-Ph), 7.30–7.42 (m, 5H, Ph), 8.06
(s, 1H, C-CH-N), 8.33 (s, 1H, N-CH-N). 13C NMR (100 MHz,
CDCl3) d 22.03 ppm (+, CH3-CH), 48.87 (+, CH3-CH-N), 54.68
(-,N-CH2-Ph), 98.77 (Cquart), 121.17 (+), 128.39 (+), 129.18 (+),
129.41 (+), 134.19 (Cquart), 135.50 (Cquart), 143.09 (Cquart), 153.39
(Cquart), 156.32 (+), 158.60 (Cquart). IR (film) n 3439 cm-1, 3296,
3105, 2981, 2934, 1661, 1560.). Anal. Calcd for C17H18N8: C, 61.06;
H, 5.43; N, 33.51. Found C, 60.94; H, 5.40; N, 33.36.
4-Amino-3-[1-(4-fluoro-phenyl)-1H-[1,2,3]triazol-4-yl]-1-
isopropyl-1H-pyrazolo[3,4-d]pyrimidine (6d)
The general procedure C using 2 (50 mg, 0.248 mmol) afforded
53 mg (65%) of 6d as pale yellow solid. Mp 265–266 ◦C. Rf
(MeOH:CHCl3= 1:20) = 0.28. 1H NMR (400 MHz, CDCl3)
3
d 1.58 ppm (d, JHH = 6.6 Hz, 6H, NCH-(CH3)2), 5.17 (sept,
3JHH = 6.6 Hz, 1H, (CH3)2-CH-N), 6.09 (bs, 1H, NH-), 7.25–7.32
(m, 2H), 7.79–7.85 (m, 2H), 8.35 (s, 1H, N-CH-N), 8.56 (s, 1H, N-
CH-C), 9.38 (bs, 1H). 13C NMR (100 MHz, CDCl3) d 22.24 ppm
(+, -CH3, isopropyl), 49.08 (+, -CH, isopropyl), 98.90 (Cquart),
4-Amino-1-isopropyl-3-(1-phenethyl-1H-[1,2,3]triazol-4-yl)-1H-
pyrazolo[3,4-d]pyrimidine (6b)
The general procedure B using 2 (50 mg, 0.248 mmol) afforded
57 mg (66%) of 6b as colorless solid. Mp 197–198 ◦C. Rf
(MeOH:CHCl3= 1:20) = 0.30. 1H NMR (400 MHz, CDCl3)
2
117.19 (d, JCF = 23.1 Hz, “ortho-C”, +), 119.48 (+), 122.87 (d,
4
3JCF = 9.2 Hz, “meta-C”, +), 133.18 (d, JCF = 2.3 Hz, “para-
3
d 1.54 ppm (d, JHH = 7.0 Hz, 6H, NCH-(CH3)2), 3.29 (t,
C”, Cquart), 134.96 (Cquart), 143.62 (Cquart), 153.80 (Cquart), 156.42
3
1
3JHH = 7.7 Hz, 2H), 4.66 (t, JHH = 7.7 Hz, 2H), 5.13 (sept,
(+), 158.66 (Cquart), 162.97 (d, JCF = 250.6 Hz, “ipso-C”, Cquart).
3JHH = 7.0 Hz, 1H, (CH3)2-CH-N), 6.36 (bs, 1H, NH-), 7.13–
7.19 (m, 2H), 7.23–7.35 (m, 3H), 8.01 (s, 1H, N-CH-C), 8.32 (s,
1H, N-CH-N), 9.48 (bs, 1H). 13C NMR (100 MHz, CDCl3) d
IR (KBr) n 3432 cm-1, 3296, 3149, 2982, 2934, 1659, 1565, 1516,
1235, 622. Anal. Calcd for C16H15FN8: C, 56.80; H, 4.47; N, 33.12.
Found C, 56.88; H, 4.42; N, 33.19.
3426 | Org. Biomol. Chem., 2009, 7, 3421–3429
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4-Amino-3-[1-(4-chloro-phenyl)-1H-[1,2,3]triazol-4-yl]-1-
isopropyl-1H-pyrazolo[3,4-d]pyrimidine (6e)
153.19 (Cquart), 156.15 (+), 158.25 (Cquart). IR (KBr) n 3439 cm-1,
3370, 2980, 1635, 1563, 1500. Anal. Calcd for C16H17N9: C, 57.30;
H, 5.11; N, 37.59. Found C, 57.34; H, 5.13; N, 38.02.
The general procedure C using 2 (50 mg, 0.248 mmol) afforded
1
53 mg (60%) of 6e as pale yellow solid. H NMR (400 MHz,
4-Amino-3-[1-(2-nitrophenyl)-1H-[1,2,3]triazol-4-yl]-1-isopropyl-
1H-pyrazolo[3,4-d]-pyrimidine (6i)
3
DMSO) d 1.51 ppm (d, JHH = 6.6 Hz, 6H), 5.08 (sept,
3JHH = 6.6 Hz, 1H), 7.69–7.75 (m, 2H, AA¢BB¢) 8.09 (bs, 1H),
8.11–8.18 (m, 2H, AA¢BB¢) 8.24 (s, 1H), 8.96 (bs, 1H), 9.42 (s,
1H). 13C NMR (100 MHz, DMSO) d 21.77 ppm (+), 48.47 (+),
97.83 (Cquart), 120.75 (+), 122.34 (+), 129.92 (+), 133.64 (Cquart),
134.25 (Cquart), 135.15 (Cquart), 142.47 (Cquart), 153.24 (Cquart), 156.21
(+), 158.24 (Cquart). IR (KBr) n 3453 cm-1(sh), 3331, 3307, 3116,
2977, 2931, 1654, 1565, 1499, 1284, 1257, 587, 509. Anal. Calcd
for C16H15ClN8: C, 54.16; H, 4.26; N, 31.58. Found C, 54.20; H,
4.28; N, 31.61.
The general procedure C using 2 (49 mg, 0.244 mmol) af-
forded 40 mg (45%) of 6i as yellow solid. Mp 256–257 ◦C. Rf
1
(MeOH:CHCl3= 1:20) = 0.35. H NMR (400 MHz, CDCl3) d
3
3
1.57 ppm (d, JHH = 6.6 Hz, 6H), 5.17 (sept, JHH = 6.6 Hz,
1H), 6.07 (bs, 1H, -NH), 7.67–7.72 (m, 1H), 7.75–7.81 (m, 1H),
7.83–7.89 (m, 1H), 8.12–8.19 (m, 1H), 8.36 (s, 1H), 8.48 (s, 1H),
9.26 (bs, 1H). 13C NMR (100 MHz, CDCl3) d 22.23 ppm (+), 49.10
(+), 98.92 (Cquart), 122.67 (+), 126.07 (+), 127.92 (+), 130.11 (Cquart),
131.51 (+), 134.18 (+), 134.60 (Cquart), 143.56 (Cquart), 144.75 (Cquart),
153.80 (Cquart), 156.41 (+), 158.59 (Cquart). IR (KBr) n 3432 cm-1
(sh), 3299, 3104, 2980, 2935, 1651, 1609, 1564, 1538, 1352, 1256,
641. Anal. Calcd for C16H15N9O2: C, 52.60; H, 4.14; N, 34.50.
Found C, 52.63; H, 4.16; N, 34.53.
4-Amino-3-[1-(4-methoxy-phenyl)-1H-[1,2,3]triazol-4-yl]-1-
isopropyl-1H-pyrazolo[3,4-d]pyrimidine (6f)
The general procedure C using 2 (50 mg, 0.248 mmol) afforded
53 mg (81%) of 6f as pale yellow solid. Mp 261–262 ◦C. Rf
(MeOH:CHCl3= 1:20) = 0.30. 1H NMR (400 MHz, CDCl3)
4-Amino-3-[1-p-tolyl-1H-[1,2,3]triazol-4-yl]-1-isopropyl-1H-
pyrazolo[3,4-d]pyrimidine (6j)
3
d 1.59 ppm (d, JHH = 6.6 Hz, 6H), 3.91 (s, 3H), 5.17 (sept,
3JHH = 6.6 Hz, 1H), 7.06–7.11 (m, 2H, AA¢BB¢), 7.71–7.77 (m, 2H,
AA¢BB¢), 8.35 (s, 1H), 8.52 (s, 1H). 13C NMR (100 MHz, CDCl3) d
22.23 ppm (+, CHN-(CH3)2), 49.15 (+, CH3-CH-CH3), 55.91 (+),
115.17 (+), 119.49 (+), 122.47 (+), 130.27 (Cquart), 135.45 (Cquart),
143.14 (Cquart), 153.58 (Cquart), 155.73 (+), 158.36 (Cquart), 160.47
(Cquart). IR (KBr) n 3432 cm-1, 3302, 3114, 2988, 2935, 1656, 1606,
1563, 1517, 1255, 1039, 621. Anal. Calcd for C17H18N8O: C 58.27;
H 5.18; N 31.98. Found C, 58.30; H, 5.20; N, 32.01.
The general procedure C using 2 (50 mg, 0.248 mmol) afforded
62 mg (76%) of 6j as pale yellow solid. Mp 284–285 ◦C. Rf
(MeOH:CHCl3= 1:20) = 0.30. 1H NMR (400 MHz, CDCl3)
3
d 1.59 ppm (d, JHH = 6.6 Hz, 6H, CHN-(CH3)2), 5.17 (sept,
3JHH = 6.6 Hz, 1H, (CH3)2-CH-N), 6.01 (bs, 1H, -NH), 7.35–7.41
(m, 2H, AA¢BB¢), 7.69–7.74 (m, 2H, AA¢BB¢), 8.35 (s, 1H, N-CH-
N), 8.56 (s, 1H, -CHtriazol), 9.48 (bs, 1H, -NH).13C NMR (100 MHz,
CDCl3) d 21.37 ppm (+), 22.23 (+), 49.05 (+), 98.90 (Cquart), 119.30
(+), 120.73 (+), 130.65 (+), 134.61 (Cquart), 135.22 (Cquart), 139.77
(Cquart), 143.30 (Cquart), 153.77 (Cquart), 156.40 (+), 158.69 (Cquart). IR
(film) n 3439 (sh) cm-1, 3298, 3098, 2980, 1663, 1560, 1520. Anal.
Calcd for C17H18N8: C, 61.06; H, 5.43; N, 33.51. Found C, 61.14;
H, 5.37; N, 33.40.
4-Amino-3-[1-(2-pyridyl)-1H-[1,2,3]triazol-4-yl]-1-isopropyl-1H-
pyrazolo[3,4-d]-pyrimidine (6g)
The general procedure C using 2 (49 mg, 0.244 mmol) af-
forded 37 mg (48%) of 6g as yellow solid. Rf (MeOH:CHCl3=
1:20) = 0.30. 1H NMR (400 MHz, CDCl3) d 1.58 ppm (d,
3
3JHH = 6.6 Hz, 6H), 5.17 (sept, JHH = 6.6 Hz, 1H), 6.17 (bs,
General procedure D for preparation of triazoles from compound 1
1H, -NH), 7.39–7.44 (m, 1H), 7.94–8.01 (m, 1H), 8.21–8.25 (m,
1H), 8.35 (s, 1H), 8.54–8.59 (m, 1H), 9.18 (s, 1H), 9.37 (bs, 1H).
13C NMR (100 MHz, CDCl3) d 22.23 ppm (+), 49.04 (+), 98.94
(Cquart), 114.08 (+), 118.75 (+), 124.32 (+), 134.95 (Cquart), 139.47
(+), 143.20 (Cquart), 149.03 (+), 153.73 (Cquart), 156.27 (+), 156.79
(Cquart), 158.65 (Cquart). IR (KBr) n 3439 cm-1 (sh), 3295, 3111, 2977,
2936, 2874, 1663, 1563, 1471, 1031, 775. Anal. Calcd for C15H15N9
(321.1450): C 56.07; H 4.71; N 39.23. Found C,56,10; H, 4.73; N,
39.26.
3 - Iodo - 1 - isopropyl - 1H - pyrazolo[3,4 - d]pyrimidin - 4 - amine
(0.50 mmol), alkyne (1.2 eq.), NaN3 (1.44 eq.), L-proline
(20 mol%), Na2CO3 (20 mol%), sodium ascorbate (40 mol%) were
suspended in DMSO : H2O (2.5 ml, 9 : 1) mixture in a 2–5 ml
microwave vessel. To the stirring mixture CuSO4 (20 mol%, as a
1 M solution i◦n H2O) were added and the vessel was sealed and
heated to 130 C for 30 min (ramp time: 35 s, pre-stirring: 20 s,
high, fixed hold time: on). After cooling to room temperature,
the reaction mixture was poured into aqueous ammonia solution
(80 ml of H2O, 20 ml of conc. NH4OH). The layers were separated
and the water layer was extracted with EtOAc three times. The
combined organic layers were washed with H2O and brine and
dried over MgSO4. The solvent was removed in vacuo at 30 ◦C and
the residue was further purified with column chromatography.
4-Amino-3-[1-(3-aminophenyl)-1H-[1,2,3]triazol-4-yl]-1-isopropyl-
1H-pyrazolo[3,4-d]-pyrimidine (6h)
The general procedure C using 2 (49 mg, 0.244 mmol) afforded
◦
1
42 mg (52%) of 6h as pale brownish solid. Mp 262–263 C. H
NMR (400 MHz, DMSO) d 1.51 ppm (d, 3JHH = 6.6 Hz, 6H), 5.07
(sept, 3JHH = 6.6 Hz, 1H), 5.57 (bs, 2H), 6.67–6.73 (m, 1H), 7.11–
7.16 (m, 1H), 7.19–7.24 (m, 2H), 8.07 (bs, 1H), 8.24 (s, 1H), 9.02
(bs, 1H), 9.18 (s, 1H). 13C NMR (100 MHz, DMSO) d 21.72 ppm
(+), 48.44 (+), 105.20 (+), 107.31 (+), 114.48 (+), 120.18 (+), 130.22
(+), 134.41 (Cquart), 137.10 (Cquart), 142.09 (Cquart), 150.10 (Cquart),
4-Amino-3-(4-benzyl-1H-1,2,3-triazol-1-yl)-1-isopropyl-1H-
pyrazolo[3,4-d]pyrimidine (7a)
The general procedure D using 1 (150 mg, 0.49 mmol) afforded
142 mg (87%) of 7a as yellow solid. Mp 247–248 ◦C. Rf
This journal is
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Org. Biomol. Chem., 2009, 7, 3421–3429 | 3427
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(MeOH:CHCl3= 1:20) = 0.30. 1H NMR (400 MHz, CDCl3)
4-Amino-1-isopropyl-3-[4-(4-methylphenyl)-1H-1,2,3-triazol-1-yl]-
1H-pyrazolo[3,4-d]pyrimidine (7e)
3
d 1.53 ppm (d, JHH = 6.6 Hz, 6H), 4.20 (s, 2H), 5.16 (sept,
3JHH = 6.6 Hz, 1H), 6.47 (bs, 1H), 7.22–7.39 (m, 5H), 8.19 (s,
1H), 8.35 (s, 1H), 8.77 (s, 1H). 13C NMR (100 MHz, CDCl3)
d = 22.04 ppm (+), 32.35 (-), 49.12 (+), 92.32 (Cquart), 119.68 (+),
127.02 (+), 128.99 (+), 129.02 (+), 137.28 (Cquart), 138.46 (Cquart),
148.18 (Cquart), 153.58 (Cquart), 156.90 (+), 157.59 (Cquart). IR (KBr)
n = 3453 cm-1 (sh), 3349, 3098, 2978, 2928, 1654, 1568, 617. Anal.
Calcd for C17H18N8: C 61.06; H 5.43; N 33.51. Found C 61.20; H
5.49; N 33.39.
The general procedure D using 1 (150 mg, 0.49 mmol) afforded
148 mg (90%) of 7e as colorless solid. Mp 293–294 ◦C. Rf
1
(MeOH:CHCl3= 1:20) = 0.30. H NMR (400 MHz, CDCl3) d
1.58 ppm (d, 3JHH = 6.6 Hz, 6H), 5.21 (sept, 3JHH = 6.6 Hz, 1H),
2.43 (s, 3H), 5.94 (bs, 1H), 7.29–7.34 (m, 2H, AA¢BB¢), 7.83–7.88
(m, 2H, AA¢BB¢), 8.38 (s, 1H), 8.71 (s, 1H), 8.76 (bs, 1H). 13C
NMR (100 MHz, CDCl3) d 21.60 ppm (+), 22.13 (+), 49.21 (+),
91.83 (Cquart), 117.12 (+), 126.14 (+), 126.87 (Cquart), 129.96 (+),
137.18 (Cquart), 139.17 (Cquart), 148.28 (Cquart), 153.77 (Cquart), 157.11
(+), 157.53 (Cquart). IR (KBr) n 3439 cm-1, 3342, 3155, 3100, 2984,
2932, 1658, 1579 (sh), 1567, 612. Anal. Calcd for C17H18N8: C
61.06; H 5.43; N 33.51. Found C, 61.06; H, 5.42; N, 33.54.
4-Amino-1-isopropyl-3-(4-phenethyl-1H-1,2,3-triazol-1-yl)-1H-
pyrazolo[3,4-d]pyrimidine (7b)
The general procedure D using 1 (150 mg, 0.49 mmol) afforded
143 mg (84%) of 7b as colorless solid. Mp 187–188 ◦C. Rf
1
4-Amino-3-[4-(3-aminophenyl)-1H-1,2,3-triazol-1-yl]-1-isopropyl-
1H-pyrazolo[3,4-d]pyrimidine (7f)
(MeOH:CHCl3= 1:10) = 0.36. H NMR (400 MHz, CDCl3) d
3
1.54 ppm (d, JHH = 6.6 Hz, 6H), 3.05–3.20 (m, 4H), 5.17 (sept,
3JHH = 6.6 Hz, 1H), 6.87 (bs, 1H), 7.17–7.35 (m, 5H), 8.21 (s,
1H), 8.36 (s, 1H), 8.77 (bs, 1H). 13C NMR (100 MHz, CDCl3)
d 22.03 ppm (+), 27.56 (-), 35.42 (-), 49.04 (+), 92.30 (Cquart),
119.21 (+), 126.46 (+), 128.58 (+), 128.70 (+), 137.30 (Cquart),
140.92 (Cquart), 147.85 (Cquart), 153.54 (Cquart), 156.82 (+), 157.69
(Cquart). IR (KBr) n 3432 cm-1 (sh), 3332, 3138, 2980, 2929, 1653,
1569, 608. Anal. Calcd for C18H20N8: C 62.05; H 5.79; N 32.16.
Found C, 62.09; H, 5.81; N, 32.17.
The general procedure D using 1 (150 mg, 0.49 m◦mol) afforded
1
95 mg (58%) of 7f as greyish solid. Mp 287–288 C. H NMR
(400 MHz, CDCl3) d 1.58 ppm (d, 3JHH = 6.6 Hz, 6H), 5.21 (sept,
3JHH = 6.6 Hz, 1H), 3.83 (bs, 2H), 5.99 (bs, 1H), 6.69–6.79 (m,
1H), 7.20–7.38 (m, 3H), 8.38 (s, 1H), 8.70 (s, 1H), 8.76 (bs, 1H).
13C NMR (100 MHz, CDCl3) d 22.13 ppm (+), 49.22 (+), 92.44
(Cquart), 112.57 (+), 115.83 (+), 116.54 (+), 117.51 (+), 130.25 (+),
130.62 (Cquart), 137.16 (Cquart), 147.25 (Cquart), 148.31 (Cquart), 153.76
(Cquart), 157.06 (+), 157.50 (Cquart). IR (KBr) n 3451 cm-1, 3370,
3128, 2985, 1655, 1616, 1567. Anal. Calcd for C16H17N9: C 57.30;
H 5.11; N 37.59. Found C,57.32; H, 5.13; N, 38.02.
4-Amino-1-isopropyl-3-(4-phenyl[1,2,3]triazol-1-yl)-1H-
pyrazolo[3,4-d]pyrimidine (7c)
The general procedure D using 1 (90 mg, 0.297 mmol) af-
forded 86 mg (91%) of 7c as colorless solid. Mp >300 ◦C. Rf
(MeOH:CHCl3= 1:20) = 0.30. 1H NMR (400 MHz, CDCl3)
4-Amino-1-isopropyl-3-[4-(2-nitrophenyl)-1H-1,2,3-triazol-1-yl]-
1H-pyrazolo[3,4-d]pyrimidine (7g)
3
d 1.59 ppm (d, JHH = 6.6 Hz, 6H, CHN-(CH3)2), 5.22 (sept,
The general procedure D using 1 (150 mg, 0.49 mmol) afforded
120 mg (67%) of 7g as colorless solid. Mp 245–246 ◦C. Rf
3JHH = 6.6 Hz, 1H, (CH3)2-CH-N), 6.03 (bs, 1H, -NH), 7.39–
7.54 (m, 3H), 7.95–7.99 (m, 2H), 8.38 (s, 1H, N-CH-N), 8.75 (bs,
1H, -NH), 8.75 (s, 1H, CHtriazol). 13C NMR (100 MHz, CDCl3)
d = 22.13 ppm (+), 49.24 (+), 92.45 (Cquart), 117.49 (+), 126.23 (+),
129.16 (+), 129.28 (+), 129.70 (Cquart), 137.14 (Cquart), 148.19 (Cquart),
153.77 (Cquart), 157.09 (+), 157.54 (Cquart). IR (KBr) n 3448 cm-1,
3339, 3101, 1658, 1567, 1584. Anal. Calcd for C16H16N8: C, 59.99;
H, 5.03; N, 34.98. Found C, 60.02; H, 5.05; N, 35.01.
1
(MeOH:CHCl3= 1:40) = 0.29. H NMR (400 MHz, CDCl3) d
3
3
1.58 ppm (d, JHH = 6.6 Hz, 6H), 5.21 (sept, JHH = 6.6 Hz,
1H), 6.37 (bs, 1H), 7.57–7.63 (m, 1H), 7.71–7.76 (m, 1H), 7.90–
8.03 (m, 2H), 8.38 (s, 1H), 8.61 (bs, 1H), 8.75 (s, 1H). 13C NMR
(100 MHz, CDCl3) d 22.09 ppm (+), 49.32 (+), 92.44 (Cquart), 120.59
(+), 124.00 (Cquart), 124.65 (+), 129.98 (+), 131.43 (+), 132.93 (+),
136.77 (Cquart), 143.03 (Cquart), 148.68 (Cquart), 153.73 (Cquart), 157.04
(+), 157.48 (Cquart). IR (KBr) n 3381 cm-1, 3189, 3151, 2987, 2934,
1653, 1582, 1563, 1527. Anal. Calcd for C16H15N9O2: C 52.60; H
4.14; N 34.50. Found Found C 52.45; H 4.20; N 34.42.
4-Amino-1-isopropyl-3-[4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-
yl]-1H-pyrazolo[3,4-d]pyrimidine (7d)
The general procedure D using 1 (150 mg, 0.49 mmol) afforded
134 mg (78%) of 7d as colorless solid. Mp 282–283 ◦C. Rf
(MeOH:CHCl3= 1:20) = 0.30. 1H NMR (400 MHz, CDCl3)
4-Amino-1-isopropyl-3-[4-(2-pyridyl)-1H-1,2,3-triazol-1-yl]-1H-
pyrazolo[3,4-d]pyrimidine (7h)
3
d 1.59 ppm (d, JHH = 6.6 Hz, 6H), 3.89 (s, 3H), 5.21 (sept,
The general procedure D using 1 (150 mg, 0.49 mmol) afforded
122 mg (78%) of 7h as colorless solid. Mp 247–248 ◦C. Rf
3JHH = 6.6 Hz, 1H), 5.93 (bs, 1H), 7.01–7.06 (m, 2H, AA¢BB¢),
7.86–7.92 (m, 2H, AA¢BB¢), 8.38 (s, 1H), 8.66 (s, 1H), 8.77 (bs,
1H). 13C NMR (100 MHz, CDCl3) d 22.13 ppm (+), 49.20 (+),
55.62 (+), 92.44 (Cquart), 114.69 (+), 116.63 (+), 122.35 (Cquart),
127.59 (+), 137.19 (Cquart), 148.07 (Cquart), 153.78 (Cquart), 157.13
(+), 157.53 (Cquart), 160.42 (Cquart). IR (KBr) n 3424 cm-1 (sh),
3340, 3103, 2989, 2936, 1650, 1569, 1492, 1251. Anal. Calcd for
C17H18N8O: C 58.27; H 5.18; N 31.98. Found C, 58.29; H, 5.20; N,
32.01.
1
(MeOH:CHCl3= 1:40) = 0.29. H NMR (400 MHz, CDCl3) d
1.57 ppm (d, 3JHH = 6.6 Hz, 6H), 5.20 (sept, 3JHH = 6.6 Hz, 1H),
6.32 (bs, 1H), 7.29–7.36 (m, 1H), 7.81–7.89 (m, 1H), 8.20–8.26
(m, 1H), 8.38 (s, 1H), 8.67 (s, 1H,), 8.68 (s, 1H), 9.14 (s, 1H).
13C NMR (100 MHz, CDCl3) d 22.12 ppm (+), 49.17 (+), 92.46
(Cquart), 120.09 (+), 120.72 (+), 123.74 (+), 136.93 (Cquart), 137.29
(+), 148.68 (Cquart), 149.49 (Cquart), 149.99 (+), 153.73 (Cquart), 157.02
(+), 157.54 (Cquart). IR (KBr) n 3340 cm-1, 3190, 3097, 2976, 1658,
3428 | Org. Biomol. Chem., 2009, 7, 3421–3429
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1579, 1565, 1528. Anal. Calcd C15H15N9: C 56.07; H 4.71; N 39.23.
Found C 56.15; H 4.12; N 39.16.
3 J. J.-L. Liao, J. Med. Chem., 2007, 50, 409–424.
4 A. C. Dar, M. S. Lopez and K. M. Shokat, Chem. Biol., 2008, 15,
1015–1022.
5 Z. Pan, H. Scheerens, S.-J. Li, B. E. Schultz, P. A. Sprengeler, L. C.
Burrill, R. V. Mendonca, M. D. Sweeney, K. C. K. Scott, P. G. Grothaus,
D. A. Jeffery, J. M. Spoerke, L. A. Honigberg, P. R. Young, S. A.
Dalrymple and J. T. Palmer, Chem. Med. Chem, 2007, 2, 58–61.
6 V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless, Angew.
Chem., Int. Ed., 2002, 41, 2596–2599.
PfPK7 inhibition assays
Recombinant PfPK7 was expressed in E. coli as a glutathione-S-
transferase fusion protein and purified as described previously.20
Kinases assays were performed in a standard reaction (30 ml)
containing 0.5 mg GST-PfPK7, 20mM Tris-HCl pH 7,5, 20mM
MgCl2, 2mM MnCl2, 15 mM cold ATP, 2,5 mCi and 5 mg of
MBP (Myelin Basic Protein) as a substrate. The compounds were
tested at 10 mM in the first instance. To determine IC50 values
for compounds 6c and 6g, kinase reactions were performed with
a range of concentrations of the inhibitors; the concentration
of DMSO vehicle was identical in all reactions (10% final). All
reactions were performed in triplicate and the data indicate the
ave◦rage values ( SD). The reaction proceeded for 30 min at
30 C and was stopped by addition of Laemmli buffer. Samples
were boiled for 3 min and analysed by electrophoresis on 12%
SDS-polyacrylamide gels. The gels were dried and subjected to
autoradiography and quantification in a phosphorimager.
7 M. Meldal, C. W. Tornoe and C. Wenzel, Chem. Rev., 2008, 108, 2952–
3015.
8 L. V. Lee, M. L. Mitchell, S. J. Huang, V. V. Fokin, K. B. Sharpless and
C. H. Wong, J. Am. Chem. Soc., 2003, 125, 9588–9589.
9 Q. Wang, T. R. Chan, R. Hilgraf, V. V. Fokin, K. B. Sharpless and
M. G. Finn, J. Am. Chem. Soc., 2003, 125, 3192–3193.
10 A. Brik, J. Muldoon, Y. C. Lin, J. H. Elder, D. S. Goodsell, A. J. Olson,
V. V. Fokin, K. B. Sharpless and C. H. Wong, ChemBioChem, 2003, 4,
1246–1248.
11 P. Wu and V. V. Fokin, Aldrichimica Acta, 2007, 40, 7–17.
12 A. K. Feldman, B. Colasson and V. V. Fokin, Org. Lett., 2004, 6, 3897–
3899.
13 P. Appukkuttan, W. Dehaen, V. V. Fokin and E. Van der Eycken, Org.
Lett., 2004, 6, 4223–4225.
14 N. Bouloc, J. M. Large, E. Smiljanic, D. Whalley, K. H. Ansell, C. D.
Edlin and J. S. Bryans, Bioorg. Med. Chem. Lett., 2008, 18, 5294–5298.
15 A. Merckx, A. Echalier, K. Langford, A. Sicard, G. Langsley, J. Joore,
C. Doerig, M. Noble and J. Endicott, Structure, 2008, 16, 228–238.
16 A. R. Renslo and J. H. McKerrow, Nat. Chem. Biol., 2006, 2, 701–710.
17 R. G. Ridley, Microbes Infect., 2002, 4, 155–156.
Acknowledgements
18 C. Doerig, O. Billker, D. Pratt and J. Endicott, Biochimica et Biophysica
Acta (BBA) - Proteins & Proteomics, 2005, 1754, 132–150.
19 P. Pattanaik, J. Raman and H. Balaram, Curr. Top. Med. Chem., 2002,
2, 483–505.
20 D. Dorin, J.-P. Semblat, P. Poullet, P. Alano, J. P. D. Goldring,
C. Whittle, S. Patterson, D. Chakrabarti and C. Doerig, Molecular
Microbiology, 2005, 55, 184–186.
21 J. C. Kissinger, B. P. Brunk, J. Crabtree, M. J. Fraunholz, B. Gajria,
A. J. Milgram, D. S. Pearson, J. Schug, A. Bahl, S. J. Diskin, H.
Ginsburg, G. R. Grant, D. Gupta, P. Labo, L. Li, M. D. Mailman,
S. K. McWeeney, P. Whetzel, C. J. Stoeckert and D. S. Roos, Nature,
2002, 419, 490–492.
22 D. Dorin-Semblat, A. Sicard, C. Doerig, L. Ranford-Cartwright and
C. Doerig, Eukaryot. Cell, 2008, 7, 279–285.
This work was financed by the European Commission (the
CELLCOMPUT project, Contract No 043310). Work in the CD
laboratory is supported by INSERM, and by the FP6 (SIGMAL
and ANTIMAL projects, BioMalPar Network of Excellence)
and FP7 (MALSIG project) programmes of the European
Commission.
References
1 R. Helmut, Chem. Biodivers., 2004, 1, 361–401.
2 A. P. Schmidt, D. R. Lara and D. O. Souza, Pharmacol. Ther., 2007,
116, 401–416.
23 Glide [4.0], Schro¨dinger, LLC., New York, 2005.
24 Macromodel [9.1], Schro¨dinger, LLC., New York, 2007.
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