Identification of [1,2,3]triazolo[4,5-d ]pyrimidin-7(6H)-ones as novel inhibitors of chikungunya virus replication

Article abstract of DOI:10.1021/jm401844c

Chikungunya virus (CHIKV) is a re-emerging Alphavirus that is transmitted to humans by Aedes mosquitoes. Currently, there are still no drugs or vaccines available for the treatment or prevention of this disease. Although traditionally restricted to Africa and Asia, the adaptation of the virus to Aedes albopictus, a mosquito species with an almost worldwide distribution, has contributed to the geographical spread of this virus in the past decade. Here, we report on a new family of compounds named [1,2,3]triazolo[4,5-d]pyrimidin- 7(6H)-ones that inhibit CHIKV replication in the low micromolar range with no toxicity to the host (Vero) cells. The most potent compound in this series has an EC₅₀ value below 1 μM with no cytotoxicity detected up to 668 μM, therefore affording a selectivity index greater than 600. Interestingly, the compounds have little or no antiviral activity on the replication of other members of the Togaviridae family. The exploration and study of this class of selective inhibitors of CHIKV replication will contribute to deeper insights into the CHIKV life cycle and may be a first step toward the development of a clinical drug candidate.

Full text of DOI:10.1021/jm401844c

Article
pubs.acs.org/jmc
Identification of [1,2,3]Triazolo[4,5‑d]pyrimidin-7(6H)‑ones as Novel
Inhibitors of Chikungunya Virus Replication
Alba Gigante,† María-Dolores Canela,† Leen Delang,‡ Eva-María Priego,† María-Jose Camarasa,†
́
Gilles Querat,§ Johan Neyts,‡ Pieter Leyssen,‡ and María-Jesus Perez-Perez*,†
́
́
́
†Instituto de Química Med
́
ica (CSIC), Juan de la Cierva 3, Madrid E-28006, Spain
‡Rega Institute for Medical Research, KU Leuven, Leuven B-3000, Belgium
§UMR190, Emergence des Pathologies Virales, Aix-Marseille Univ. IRD French Institute of Research for Development, EHESP
French School of Public Health, 27 Bd Jean Moulin, Marseille 13005, France
S
* Supporting Information
ABSTRACT: Chikungunya virus (CHIKV) is a re-emerging Alphavirus
that is transmitted to humans by Aedes mosquitoes. Currently, there are still
no drugs or vaccines available for the treatment or prevention of this disease.
Although traditionally restricted to Africa and Asia, the adaptation of the
virus to Aedes albopictus, a mosquito species with an almost worldwide
distribution, has contributed to the geographical spread of this virus in the
past decade. Here, we report on a new family of compounds named
[1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones that inhibit CHIKV replication
in the low micromolar range with no toxicity to the host (Vero) cells. The
most potent compound in this series has an EC50 value below 1 μM with no
cytotoxicity detected up to 668 μM, therefore affording a selectivity index greater than 600. Interestingly, the compounds have
little or no antiviral activity on the replication of other members of the Togaviridae family. The exploration and study of this class
of selective inhibitors of CHIKV replication will contribute to deeper insights into the CHIKV life cycle and may be a first step
toward the development of a clinical drug candidate.
to a new vector, Aedes albopictus.8,9 As this mosquito species has
a wider distribution than the traditional vector Aedes aegypti,
this could contribute to a broader spread of the virus. In
December 2013, the first autochthonous CHIKV cases were
INTRODUCTION
■
Chikungunya virus (CHIKV) is an insect-borne viral disease
that was first described during an outbreak in southern
Tanzania in 1952.1 Taxonomically, CHIKV is a RNA virus of
confirmed on the Caribbean island of St. Martin. Since then,
the Alphavirus genus of the Togaviridae family and belongs
more specifically to the Semliki Forest virus (SFV) complex.
CHIKV is transmitted to humans by virus-carrying Aedes
mosquitoes. CHIKV infection causes an illness with symptoms
that are similar to those of dengue fever: first, there is an acute
febrile phase that lasts for two to five days, followed by a
CHIKV has been and is currently still spreading to neighboring
countries,10 and it is also possible that CHIKV will be able to
spread to the American mainland in the near future.
There are no specific drugs to prevent or cure the
chikungunya infection. Treatment is primarily directed at
relieving the symptoms, more in particular the joint pain by
prolonged arthralgic disease that affects the joints of the
extremities.2 Most patients fully recover, but in 10% of the
administration of corticosteroids or NSAIDs. Although
researchers have recently reported on the development of a
cases, joint pain may persist for several months or even years.
new candidate vaccine to protect against CHIKV infection,11
Occasional cases of eye, neurological, and heart complications
have been reported, as well as gastrointestinal complaints.3,4
there is still no approved commercial CHIKV vaccine available.
Up to now, only a few examples of selective inhibitors of
During the past 50 years, several re-emergences of CHIKV
CHIKV replication have been described.12−18 Therefore, there
have occurred in Africa and Asia. Starting in February 2005, a
is still an unmet need for new classes of potent and selective
major outbreak of CHIKV infections occurred in the islands of
inhibitors of CHIKV replication.19 To this end, a joint
the Indian Ocean, more in particular in the French territory La
screening program was set up between our laboratories to
́
Reunion. There were 270 000 estimated cases, nearly half of the
island population, and 237 reported deaths.5 Subsequently, this
epidemic spread to other regions of India and Southeast Asia,
and transmission was reported for the first time in Europe, in a
localized outbreak in northeastern Italy.6 Cases have now been
reported in more than 40 countries.7 Interestingly, these
outbreaks have been related to the ability of the virus to adapt
identify such novel compounds. The selective antiviral activity
of a structurally diverse collection of chemical compounds from
CSIC (Madrid) was evaluated for selective antiviral activity in a
Received: November 28, 2013
© XXXX American Chemical Society
A
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a
virus-cell-based assay for CHIKV replication at KU Leuven.
One of the chemical samples, coded TP274, portrayed an
antiviral activity/cytotoxicity profile that matched our hits
selection criteria, which are protection of the host cells from
any signs of virus-induced effects without eliciting a significant
adverse effect on cell or monolayer morphology and cell
metabolism (Figure 1). Chemical validation of this hit sample
Scheme 1. Synthesis of Triazolopyrimidines and Analogues
Figure 1. Dose−response curve (in μM) obtained for the chemical
sample coded TP274 as identified in a general screening. The anti-
CHIKV activity is represented in white circles while the cytotoxic/
cytostatic effect is represented in black squares.
by high-performance liquid chromatography−mass spectrome-
try (HPLC-MS) analysis revealed a 1:1 mixture of two
compounds. Both entities were separated by chromatography,
structurally characterized, and individually evaluated for anti-
CHIKV activity. This revealed that the chloro compound 120
was inactive while the 7-oxo derivative 2 (Figure 2) showed the
antiviral activity.
a
Reagents and conditions: (a) NH3/MeOH, MW, 70 °C, 30 min (3a,
66% yield); (b) MeNH2/MeOH, rt, 2 h (3b, 67% yield); (c) MeONa/
MeOH, MW, 100 °C, 20 min (3c, 61% yield); (d) MeI, DBU, DMA,
rt, 16 h (4, 86% yield); (e) CH3COONa, DMF, MW, 120 °C, 1 h (6a,
62% yield); (f) HCl/dioxane, MW, 100 °C, 2 h (6b, 61% yield).
and MeI in N,N-dimethylacetamide afforded exclusively the 6-
methyl derivative 4 (86% yield). Finally, reaction of the 6-
chloropurine 5a21 with sodium acetate in DMF afforded the
hypoxanthine derivative 6a (62% yield). Reaction of the 6-
chloro-8-methyl purine (5b)21 with 1 N HCl in dioxane yielded
the purinone derivative 6b (61% yield).
Figure 2. Compounds present in the sample coded TP274.
On the basis of the good activity/toxicity profile of
compound 2, its low molecular weight, and the clear need for
new compounds that selectively inhibit CHIKV replication, a
synthetic program was initiated to prepare structural analogues
and to explore the structure−activity relationship of this
compound class in more detail. The synthesis and exploration
of the relationship between the compound structure and anti-
CHIKV activity is reported here in detail.
The evaluation of this first series of compounds in the virus-
cell-based assay (see the Antiviral Activity section) revealed that
all these modifications have a deleterious effect on the antiviral
activity. Therefore, the next series of compounds kept the
[1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-one as the heterocyclic
base and focused on exploring different substituents at the aryl
ring. To this end, the approach employed for the synthesis of
compound 1 was used, incorporating a final hydrolysis step
from the 7-chloro to the keto form, as shown in Scheme 2.
Reaction of an equimolar amount of differently substituted
anilines (7) with 4,6-dichloro-5-aminopyrimidines (8) in
isobutanol in the presence of HCl at 150 °C for 10 min
under microwave irradiation afforded the 4-chloro-5,6-diami-
nopyrimidines 9a−m in good to excellent yields, that in many
cases were isolated by filtration.20−22 Then, reaction of 9a−m
with NaNO2 in CH2Cl2 in the presence of HCl at room
temperature for 30 min afforded the triazolo derivatives 10a−
m. Finally, treatment of 10a−m with sodium acetate in DMF
afforded the [1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones 11a−
m. Saponification of the ethyl ester 11m afforded the carboxylic
acid 11n.
RESULTS AND DISCUSSION
■
Chemistry. On the basis of the structure of compound 2,
our first series of modifications was meant to evaluate the
impact on the antiviral activity of small substituents at positions
6 and/or 7 of the [1,2,3]triazolo[4,5-d]pyrimidine or
replacement of this heterocyclic base by a purine analogue
(Scheme 1) while keeping a 3-acetyl substituent at the aryl ring.
Thus, reaction of the 7-chloro derivative 120 with ammonia or
methylamine in MeOH afforded the 7-amino and 7-
methylamino derivatives (3a and 3b, respectively) in good
yields. Similarly, treatment of 1 with MeONa in MeOH led to
the 7-OMe derivative 3c in 61% yield. Reaction of 2 with DBU
B
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a
Scheme 2. Triazolopyrimidin-7-ones Differently Substituted at the Aryl Ring
a
Reagents and conditions: (a) isobutyl alcohol, HCl (cat), MW, 150 °C, 10 min to 1 h (71−98% yield); (b) NaNO2, HCl 1 N/DCM (1:1), rt, 30
min (56−75% yield); (c) CH3COONa, DMF, MW, 120 °C, 1 h (50−83% yield); (d) KOH 20%, DMF, MW, 100 °C, 30 min (92% yield).
a
Scheme 3. Synthesis of Triazolopyrimidones from Aminocarboxamides
a
Reagents and conditions: (a) cyanoacetamide, EtONa, EtOH, reflux, 2 h (13, 15% yield and 14, 68% yield); (b) cyanoacetamide, NaH, DMF, 0 °C,
30 min, then addition of 12 in DMF, rt, 1 h (13, 70% yield); (c) R1COOEt, EtONa, refluxing EtOH, 1−8 h (8−79% yield); (d) N,N′-
diisopropylcarbodiimide, 90 °C, 16 h (56% yield).
The biological evaluation of compounds 11a−n showed a
number of compounds with antiviral activity, the best of which
was the 3-isopropoxyderivative 11e with an EC50 value 1.4-fold
better than that of the acetyl derivative 2. Therefore, the next
series of modifications were based on compound 11e.
The construction of the [1,2,3]triazolo[4,5-d]pyrimidine
starting from 4,6-dichloro-5-aminopyrimidines, as shown in
Scheme 2, has been very convenient for exploring substituents
at the aryl ring or even at positions 6 and/or 7 of the
heterocyclic base but has serious limitations to incorporate
different substituents at position 5 of the heterocycle, since the
nature of this substituent is determined by the substituent at
position 2 in the parent pyrimidine. A more reasonable pathway
to introduce diversity at position 5 of the triazolo[4,5-
d]pyrimidine could be devised based on a ring-closing reaction
performed on 5-amino-1,2,3-triazolo-4-carboxamides with
different esters (Scheme 3). The required 5-amino-1,2,3-
triazolo-4-carboxamides could be obtained by reaction of
arylazides with cyanoacetamide in basic media.23 This approach
has been extensively explored in the literature for 1-benzyl
derivatives, but the described examples of 1-aryl derivatives are
much scarcer.24−27 Thus, reaction of 1-azido-3-isopropoxyben-
zene (12, Scheme 3)28 with cyanoacetamide in the presence of
EtONa in refluxing EtOH afforded a mixture of two isomeric
compounds, whose 1H NMR spectra showed significant
differences affecting the NH protons. In particular, the minor
C
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product showed a unique signal at 6.37 ppm corresponding to
two protons, while the major isomer showed two well-defined
signals at 14.67 and 6.55 ppm, both of them corresponding to a
single proton. These compounds were assigned as the expected
5-aminotriazol derivative 13 (15% yield) and the corresponding
Dimroth isomer 14 (68%) arising by Dimroth rearrangement of
the internal N-aryl group to the more thermodynamically stable
exocyclic position.24,29
Treatment of 18 with cyanoacetamide in DMF in the presence
of NaH afforded the 5-amino-1H-1,2,3-triazole-4-carboxamide
19 as the unique compound in 77% yield. Finally, reaction of
19 with ethyl acetate in the presence of sodium ethoxide led to
the benzylic derivative 20 in 75% yield.
Antiviral Activity. The compounds were evaluated for their
potential inhibitory effect on the in vitro replication of CHIKV
in Vero cells (Table 1). Chloroquine was included as a
Mechanistic considerations on similar analogues suggest the
Dimroth rearrangement is favored by the use of protic
solvents.30 Thus, reaction of 12 with a preformed solution of
cyanoacetamide and NaH in DMF afforded the 5-aminotriazol
13 in 70% yield with no detection of the Dimroth isomer.
Finally, reaction of 13 with different esters in the presence of
EtOH/EtONa afforded a variety of 5-substituted triazolo[4,5-
d]pyrimidines (15a−g) (Scheme 3). It should be mentioned
that, under these cyclization conditions, the Dimroth rearrange-
ment was a competing reaction, so the quicker the cyclization,
the lower the formation of the Dimroth isomer. In this way,
different linear or branched alkyl or aromatic substituents were
incorporated at position 5 of the heterocycle. In addition,
reaction of the 5-aminotriazol 13 with 1,1′-carbonyldiimidazole
in DMF at 90 °C afforded the triazolopyrimidin-5,7-dione 16 in
56% yield.
A similar synthetic scheme was used to prepare a benzylic
analogue of compound 11e incorporating a methylene unit
between the aryl ring and the [1,2,3]triazolo[4,5-d]-
pyrimidinone as shown in Scheme 4. The starting azide 18
was obtained in 89% yield in two steps by reaction of the
benzylic alcohol 17 with mesyl chloride and further substitution
reaction of the mesyl derivative thus formed with NaN3.
Table 1. Antiviral Evaluation of the Triazolopyrimidines and
Analogues against CHIKV in Vero Cells
a
b
c
d
compd
EC50 (μM)
EC90 (μM)
CC50 (μM)
1
>174
>174
>174
2
19
2
38 16
309 48
>443
>743
3a
3b
3c
4
225 33
>443
>746
514 55
495 34
>706
>441
>441
>441
>441
6a
6b
127 10
>440
161 27
>440
491
>703
11a
11b
11c
11d
11e
11f
348 36
460 13
179 44
>777
28
6
>777
32 11
235
47
7
>764
23
6
4
>604
12
156 43
425
>704
318
>370
206 78
538
11g
11h
11i
>370
131 11
>490
187 21
>490
>793
>784
11j
326 53
169
>743
>743
11k
11l
>701
594 100
>737
>461
>461
a
11m
11n
15a
15b
15c
15d
15e
15f
202 53
>399
331 64
>399
322
Scheme 4. Synthesis of the Benzylic Derivative 20
>638
68
3
4
>147
104 32
>668
1
18 18
137
115 16
>399
215 63
>638
>399
280
>638
>638
204 72
75 19
>435
>360
277 128
82 22
>696
15g
16
>144
>435
20
167 10
232 62
21 18
>872
chloroquine
11
7
89 28
a
All data are mean values
standard deviation for at least three
b
independent experiments. 50% effective concentration or calculated
concentration of compound that is required to protect 50% of the cells
c
against cytopathic effects caused by the viral infection. 90% effective
concentration or calculated concentration of compound that is
required to protect 90% of the cells against cytopathic effects caused
d
by the viral infection. 50% cytotoxic concentration or calculated
concentration of compound that reduces the overall cell metabolic
activity (by a combined cytotoxic, cytostatic, and antimetabolic effect)
to 50%.
reference compound.13 The antiviral activity is expressed as the
50% effective concentration (EC50) and the 90% effective
concentration (EC90), which is indicative of the concentration
of compound that is required to inhibit the virus-induced
cytopathic effect on the host cell by 50% and 90%, respectively.
The overall antimetabolic effect of the compounds, indicative
for the adverse effect on noninfected host cells (combined
cytotoxic, cytostatic, and antimetabolic effect), is expressed as
a
Reagents and conditions: (a) MsCl, DMAP, DMF, rt, 5 h, then
addition of NaN3, rt, 4 h (89% yield); (b) cyanoacetamide, NaH,
DMF, 0 °C, 1 h, then addition of 18, DMF, rt, 1 h (77% yield); (c)
CH3COOEt, EtONa, refluxing EtOH, 8 h (75% yield).
D
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CC50 and is the calculated concentration of compound that
reduces the measured metabolic activity of compound-treated
cells by 50%. The evaluation of the first series of compounds
(3a−c, 4, 6a, 6b) revealed that a carbonyl was required at
position 7 of the heterocyclic base to elicit antiviral activity, as
shown for compound 2. Those compounds with a NH2,
NHMe, or OMe at position 7 (compounds 3a−c, respectively)
were significantly less active or inactive. Also, the NH at
position 6 should be unsubstituted according to the lack of
activity of the N-Me derivative 4. When the triazolopyrimidine
in compound 2 is replaced by the analogous imidazopyrimidine
(compound 6a), some antiviral activity could be observed but
at EC50 values 7-fold higher than that of compound 2. If, in
addition, a methyl group is incorporated at position 8 of the
purine (compound 6b), the antiviral activity is lost.
Table 2. Antiviral Evaluation of the Triazolopyrimidine 2
and 15b against Different Laboratory Strains and Clinical
Isolates of CHIKV, SFV, and SINV in Vero Cells
compd
2
15b
a
a
species
virus (strain)
899 (lab)
EC50 (μM) EC50 (μM)
Chikungunya
19
25
26
2
2
2.6
1
LR2006-OPY1 (lab)
Venturini (Italy 2008)
Congo 95 (2011)
St. Martin (2013)
Vietnam (lab)
2.6 0.5
1.4 0.01
0.75 0.4
2.9 0.05
219
6.4 0.05
24 0.5
>464
Semliki forest virus
Sindbis virus
HRsp (lab)
>464
69
8
a
50% effective concentration or calculated concentration of compound
Regarding the substituents at the aryl ring (compounds 11a−
n), the requirements for antiviral activity are also quite strict. In
particular, those compounds with a methoxy (11b), a Cl (11c),
a benzoyl (11d), or an isopropoxy (11e) at position 3, together
with the acetyl of the hit compound 2, produced the best
inhibitory activity. However, when the methoxy substituent is at
position 2, the EC50 is more than 10-fold higher than when the
methoxy is at position 3 (compound 11a vs 11b). Additionally,
moving the acetyl group from position 3 to position 4
(compound 2 vs 11j) results in a 17-fold decrease in antiviral
efficacy. Therefore, these data stress the importance of the
substituent on the aryl ring at position 3. The best EC50 values
are obtained when this substituent is an isopropoxy (compound
11e). Interestingly, compound 11i, with an acetyl at position 3
at the aryl ring but a hydrogen at position 5 of the base, is
inactive, indicating the importance of this position of the
heterocyclic base in the antiviral activity. This became more
obvious with the evaluation of compounds 15a−g and 16. For
this series, some compounds showed antiviral activity (15a−c,
f, or g), although in several cases, this was accompanied by
some cytotoxicity according to the CC50 values. Interestingly,
compound 15b with an ethyl substituent at position 5 showed
very interesting EC50 and EC90 values, with no cytotoxicity up
to 668 μM. Thus, compound 15b with a selectivity index of
222, is the most potent and selective compound among these
triazolopyrimidines and has a better profile than the reference
compound chloroquine. Finally, the benzyl derivative 20 was at
least 9-fold less potent than its corresponding aryl analogue 2.
The hit compound 2 and the most potent compound of this
series 15b were evaluated for selective antiviral activity against
several CHIKV clinical isolates (Table 2), including the new
strain from St. Martin that is currently responsible for an
epidemy in the West Indies. On average, compound 15b was
10 times more active than the original hit compound 2,
regardless of the clinical strain tested, with EC50 values in the
low micromolar range and a best value of 0.75 μM for CHIK
Congo. Interestingly, both compounds were significantly more
active by a factor of 4 against the African Congo strain than
against the four other strains. Interestingly, antiviral activity was
also found for compounds 2 and particularly 15b against the
recently emerged St. Martin strain (Caribbean 2013). No
cytotoxicity up to 300 μM was observed in Vero E6 cells using
the same experimental settings as the antiviral assay.
that is required to protect 50% of the cells against cytopathic effects
caused by the viral infection.
a selection of other viruses (i.e., Coxsackievirus B3 e.a.; data not
shown).
The triazolopyrimidines that were the subject of this study
are confirmed to be highly selective inhibitors of CHIKV
replication and not of related and unrelated viruses, which
points toward a mechanism of action that involves a highly
specific target in the CHIKV life cycle.
CONCLUSIONS
■
Here, we report that several 3-aryl-[1,2,3]triazolo[4,5-d]-
pyrimidin-7(6H)-ones are potent and selective inhibitors of
CHIKV replication. These compounds are easily accessible in
two to three synthetic steps using two complementary
approaches: (1) starting from 6-chloropyrimidine-4,5-diamines,
treatment with sodium nitrite and hydrolysis of the 7-chloro to
the keto form; or (2) starting from arylazides that react with
cyanoacetamide followed by a ring-closing reaction with
different esters. The structural requirements for anti-CHIKV
activity have been clearly established both at the level of the
heterocyclic base and the aryl substitution. On the basis of
these modifications, compound 15b was identified as a potent
and selective inhibitor of CHIKV replication with a better
profile than the reference compound chloroquine and a
selectivity index greater than 200. Interestingly, this class of
compounds specifically targets CHIKV among the members of
viruses of the Togaviridae family. Further optimization of the
antiviral activity is ongoing, and mechanism of action studies
are being carried out to identity the molecular target of this
novel family of anti-CHIKV compounds.
EXPERIMENTAL SECTION
■
Chemistry Procedures. Melting points were obtained on a
Reichert-Jung Kofler apparatus and are uncorrected. The elemental
analysis was performed with a Heraeus CHN-O-RAPID instrument.
The elemental compositions of the compounds agreed to within
0.4% of the calculated values. For all the tested compounds,
satisfactory elemental analysis was obtained supporting greater than
95% purity. Electrospray mass spectra were measured on a quadrupole
mass spectrometer equipped with an electrospray source (Hewlett-
Packard, LC/MS HP 1100). Compounds containing a Cl showed the
typical Cl isotopic pattern in the MS spectra. 1H and 13C NMR spectra
were recorded on a Varian INNOVA 300 operating at 299 MHz (1H)
and 75 MHz (13C), respectively, and a Varian INNOVA-400 operating
at 399 MHz (1H) and 99 MHz (13C), respectively.
In the virus test panel, also other Togaviridae family
members such as the Sindbis virus and Semliki Forest virus
were included, but no or only marginal antiviral activity was
detected. Similarly, a broad-spectrum evaluation of the
compound did not reveal any selective antiviral activity against
Analytical TLC was performed on silica gel 60 F254 (Merck)
precoated plates (0.2 mm). Spots were detected under UV light (254
E
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nm) and/or charring with ninhydrin. Separations on silica gel were
performed by preparative centrifugal circular thin-layer chromatog-
raphy (CCTLC) on a ChromatotronR (Kiesegel 60 PF254 gipshaltig
(Merck)), with layer thicknesses of 1 and 2 mm and flow rates of 4 or
8 mL/min, respectively. Flash column chromatography was performed
with silica gel 60 (230−400 mesh) (Merck).
Microwave reactions were performed using the Biotage Initiator 2.0
single-mode cavity instrument from Biotage (Uppsala). Experiments
were carried out in sealed microwave process vials utilizing the
standard absorbance level (400 W maximum power). The temperature
was measured with an IR sensor on the outside of the reaction vessel.
HPLC-MS Analysis of the Sample TP274. The sample coded
TP274 was analyzed through a HPLC Waters 2695 instrument
connected to a Waters Micromass ZQ spectrometer and a photodiode
array detector. The column used was a Sunfire C-18 (150 mm × 19
mm × 5 μm), and the flow rate was 1 mL/min. Solvents used were
CH3CN for bottle A and H2O (0.1% HCOOH) for bottle B. The
gradient used was from 10% A to 100% A in 10 min. HPLC-MS
analysis revealed a 1:1 mixture of two compounds: a peak at 3.52 min
with m/z 287.6 and Cl isotopic pattern (compound 1), and a second
peak at 5.04 min with m/z 269.1 (compound 2).
4H, Ar). 13C NMR (DMSO-d6, 100 MHz): δ 26.8 (CH3), 27.6 (CH3),
55.6 (OCH3), 125.2, 121.4, 126.6 (Ar), 129.3 (C-7a), 131.1, 136.5,
138.7 (Ar), 151.6 (C-3a), 161.6 (C-7), 168.0 (C-5), 197.8 (CO). Anal.
(C14H13N5O2): C, H, N.
3-(3′-Acetylphenyl)-5,6-dimethyl-3H-[1,2,3]triazolo[4,5-d]-
pyrimidin-7(6H)-one (4). To a flask charged with 2 (90 mg, 0.33
mmol) in N,N-dimethylacetamide (2 mL), iodomethane (27 μL, 0.43
mmol) and DBU (66 μL, 0.44 mmol) were added. The reaction was
stirred at room temperature overnight under argon atmosphere. Then,
a hexane/diethyl ether (1:1) mixture (10 mL) was added, and the
resultant suspension was cooled at −20 °C. The precipitate was
dissolved in dichloromethane (20 mL) and washed with saturated
aqueous NaHCO3 (15 mL). The organic layer was dried over Na2SO4,
filtered, and evaporated to dryness. The residue was purified by flash
chromatography (dichloromethane/methanol) to yield 80 mg (86%)
of 4 as a yellow solid. mp: 166−168 °C. EM (ES, positive mode): m/z
284 (M + H)+. 1H NMR (DMSO-d6, 300 MHz): δ 2.66 (s, 3H, CH3),
2.68 (s, 3H, CH3), 3.57 (s, 3H, CH3), 7.80−8.57 (m, 4H, Ar). 13C
NMR (DMSO-d6, 100 MHz): δ 24.2 (CH3), 26.9 (CH3), 30.8 (CH3),
121.0, 126.1, 128.1 (Ar), 128.6 (C-7a), 130.4, 135.6, 138.0 (Ar), 146.8
(C-3a), 155.5 (C-5), 162.1 (C-7), 197.1 (CO). Anal. (C14H13N5O2):
C, H, N.
9-(3′-Acetylphenyl)-2-methyl-1H-purin-6(9H)-one (6a). To a
solution of 5a21 (154 mg, 0.54 mmol) in methanol (10.0 mL),
sodium methoxide (3.0 mmol) and 2-mercaptoethanol were added.
The reaction was heated in a microwave reactor at 100 °C for 90 min.
After cooling, the product was isolated by filtration and dried to obtain
89 mg (62%) of 6a as a yellowish solid. mp: 286−287 °C. MS (ES,
positive mode): m/z 269 (M + H)+. 1H NMR (300 MHz, DMSO-d6):
δ 2.22 (s, 3H, CH3), 2.64 (s, 3H, CH3), 7.67 (pt, J = 7.8 Hz, 1H, H-
5′), 7.91 (d, J = 7.8 Hz, 1H, H-6′), 8.18 (m, 2H, H-2′, H-4′), 8.46 (s,
1H, H-8). Anal. (C14H12N4O2): C, H, N.
3-(3′-Acetylphenyl)-5-methyl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-
7(6H)-one (2). A microwave vial was charged with 120 (80 mg, 0.29
mmol) and sodium acetate (70 mg, 0.88 mmol) in anhydrous DMF
(1.3 mL) and was irradiated at 120 °C for 1 h. After work-up, the
residue was purified by CCTLC in the Chromatothron (dichloro-
methane/methanol, 30:1) to yield 65 mg (83%) of 2 as a yellow solid.
1
mp 257−259 °C. MS (ES, positive mode): m/z 270 (M + H)+. H
NMR (DMSO-d6, 300 MHz) δ: 2.45 (s, 3H, CH3), 2.67 (s, 3H, CH3),
7.83−8.56 (m, 4H, Ar), 12.79 (br s, 1H, NH). 13C NMR (DMSO-d6,
100 MHz) δ: 22.1 (CH3), 27.4 (CH3), 121.7, 126.9, 129.2 (Ar), 129.3
(C-7a), 136.1, 138.4, 130.8 (Ar), 149.3 (C-3a), 156.2 (C-5), 161.3 (C-
7), 197.5 (CO). Anal. (C13H11N5O2): C, H, N.
9-(3-Acetylphenyl)-2,8-dimethyl-1H-purin-6(9H)-one (6b). To a
solution of 6b21 (63 mg, 0.20 mmol) in dioxane (5.0 mL), a 1 N HCl
aqueous solution was added. The reaction was heated in a microwave
reactor at 100 °C for 2 h. The mixture was extracted with ethyl acetate
(15 mL), and the organic layer was washed with a saturated aqueous
Na2CO3 solution (10 mL) and brine (10 mL), dried over Na2SO4,
filtered, and evaporated to dryness in vacuo. The residue was purified
by flash column chromatography (dichloromethane/methanol) to
yield 35 mg (61%) of 6b as a solid. mp: 282−283 °C. MS (ES, positive
3-(3′-Acetylphenyl)-7-amino-5-methyl-3H-[1,2,3]-triazolo[4,5-d]-
pyrimidin-7(6H)-one (3a). A microwave vial containing 120 (100 mg,
0.34 mmol) in a solution of NH3 in methanol (2.0 M, 4 mL) was
sealed and heated in a microwave reactor at 70 °C for 30 min. After
cooling, the resulting precipitate was filtered and purified by flash
column chromatography (dichloromethane/methanol) to yield 60 mg
(66%) of 3a as a solid. mp: 286−287 °C. MS (ES, positive mode): m/
1
z 269 (M + H)+. H NMR (DMSO-d6, 300 MHz): δ 2.50 (s, 3H,
1
mode): m/z 283 (M + H)+. H NMR (400 MHz, DMSO-d6): δ 2.26
CH3), 2.69 (s, 3H, COCH3), 7.82 (pt, J = 7.9 Hz, 1H, H-5′), 8.10 (d, J
= 7.8 Hz, 1H, H-6′), 8.17 (s, 1H, NH), 8.45 (d, J = 8.1 Hz, 1H, H-4′),
8.50 (s, 1H, NH), 8.70 (s, 1H, H-2′). 13C NMR (DMSO-d6, 100
MHz): δ 26.7 (CH3), 27.6 (COCH3), 120.9, 123.8, 126.1 (Ar), 128.6
(C-7a), 130.9, 137.1,138.6 (Ar), 150.3 (C-3a), 156.7 (C-7), 168.0 (C-
5), 197.9 (CO). Anal. (C13H12N6O): C, H, N.
(s, 3H, CH3), 2.30 (s, 3H, CH3), 2.62 (s, 3H, CH3), 7.74 (m, 2H, H-
5′, H-6′), 8.01 (dd, J = 2.4, 1.1 Hz, 1H, H-2′), 8.10 (ddd, J = 5.0, 3.5,
1.7 Hz, 1H, H-4′). Anal. (C15H14N4O2): C, H, N.
General Procedure for the Reaction of 4,6-Dichloropyrimi-
dines with Substituted Anilines (9a−m). A microwave vial was
charged with the corresponding aniline (7, 1.0 mmol), the 4,6-
dichloropyrimidine (8, 1.0 mmol), isobutanol (2.5 mL), and 37%
aqueous HCl (0.07 mL/mmol). The reaction vessel was sealed and
heated in a microwave reactor at 150 °C for 10−30 min. After cooling,
the reaction mixture was worked up as indicated in each case.
6-Chloro-N4-(2′-methoxyphenyl)-2-methylpyrimidine-4,5-dia-
mine (9a). Following the general procedure, a microwave vial was
charged with 5-amino-4,6-dichloro-2-methylpyrimidine (200 mg, 1.12
mmol), 2-methoxyaniline (126 μL, 1.12 mmol), isobutanol (2.8 mL),
and 37% aqueous HCl (84 μL). After cooling, the product was isolated
by filtration and dried to obtain 291 mg (98%) of 9a as a beige solid.
3-(3′-Acetylphenyl)-5-methyl-7-methylamino-3H-[1,2,3]-triazolo-
[4,5-d]pyrimidin-7(6H)-one (3b). A solution of 120 (100 mg, 0.34
mmol) in methylamine in methanol (2.0 M, 10 mL) was stirred at
room temperature for 2 h. After volatiles removal, the residue was
purified by flash column chromatography (dichloromethane/meth-
anol) to yield 64 mg (67%) of 3b as a solid. mp: 220−221 °C. MS
1
(ES, positive mode): m/z 283 (M + H)+. H NMR (DMSO-d6, 300
MHz) δ: 2.55 (s, 3H, CH3), 2.68 (s, 3H, COCH3), 3.05 (d, J = 4.6 Hz,
3H, NHCH3), 7.82 (pt, J = 7.9 Hz, 1H, H-5′), 8.10 (d, J = 7.8 Hz, 1H,
H-6′), 8.45 (d, J = 8.0 Hz, 1H, H-4′), 8.70 (s, 1H, H-2′), 8.92 (s, 1H,
NH). 13C NMR (DMSO-d6, 100 MHz): δ 27.2 (CH3), 27.6
(COCH3), 27.8 (NHCH3), 120.9, 124.4. 126.1 (Ar), 128.6 (C-7a),
130.9, 137.1, 138.6 (Ar), 150.3 (C-3a), 156.7 (C-7), 168.0 (C-5),
197.9 (CO). Anal. (C14H14N6O): C, H, N.
1
mp: 241−243 °C. MS (ES, positive mode): m/z 265 (M + H)+. H
NMR (DMSO-d6, 300 MHz) δ: 2.27 (s, 3H, CH3), 3.81 (s, 3H,
OCH3), 6.58 (s, 2H, NH2), 6.96 (m, H-6′), 7.14 (m, 2H, H-4′, H-5′),
7.75 (m, 1H, H-3′), 8.68 (s, 1H, NH).
3-(3′-Acetylphenyl)-7-methoxy-5-methyl-3H-[1,2,3]-triazolo[4,5-
d]pyrimidin-7(6H)-one (3c). A microwave vial was charged with 120
(100 mg, 0.32 mmol) and sodium methoxide (86 mg, 1.59 mmol) in
methanol (4 mL) and was irradiated at 100 °C for 20 min. After
volatiles removal, the residue was purified by CCTLC in the
Chromatothron (dichloromethane/methanol, 30:1) to yield 55 mg
(61%) of 3c as a white solid. mp: 158−160 °C. EM (ES, positive
The synthesis of compounds 9b−m was performed following a
similar procedure, and all details and analytical and spectroscopic data
are included in the Supporting Information.
General Procedure for the Synthesis of 7-Chloro-3H-
[1,2,3]triazolo[4,5-d]pyrimidines (10a−m) Starting from 4,5-
Diaminopyrimidines. To a suspension of the corresponding 4,5-
diaminopyrimidine (9a−m, 1.00 mmol) in dichloromethane (3.5 mL/
mmol), NaNO2 (1.05 mmol) and 1 N HCl (3.5 mL/mmol) were
1
mode): m/z 284 (M + H)+. H NMR (DMSO-d6, 300 MHz): δ 2.69
(s, 3H, CH3), 2.72 (s, 3H, CH3), 4.23 (s, 3H, OCH3), 7.86−8.67 (m,
F
dx.doi.org/10.1021/jm401844c | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
added. The mixture was stirred at room temperature for 30 min. The
reaction was diluted with dichloromethane. The organic layer was
washed with saturated aqueous NaHCO3, dried over Na2SO4, filtered,
and evaporated to dryness.
cyanoacetamide (326 mg, 3.88 mmol) in anhydrous DMF (5.9 mL)
was added at 0 °C. After 1 h, a solution of 1228 (624 mg, 3.52 mmol)
in anhydrous DMF (5.9 mL) was slowly added and stirring was
continued at room temperature for 1 h. Volatiles were removed. The
residue obtained was purified by flash column chromatography
(dichloromethane/methanol, 20:1) to yield 641 mg (70%) of 13 as
a yellowish solid.
General Procedure for the Synthesis of [1,2,3]Triazolo[4,5-
d]pyrimidin-7(6H)-ones (15a−g) from the 5-Amino-1H-1,2,3-
triazole-4-carboxamide 13. To a solution of Na (5.00 mmol) in
ethanol (10 mL/mmol), the carboxamide 13 (1.00 mmol) and the
appropriate ester (4.00 mmol) were added. The reaction mixture was
heated at reflux for 1−8 h. After cooling, the reaction was concentrated
to dryness. The residue was dissolved in water and acidified by
addition of acetic acid, and the precipitate thus formed was isolated by
filtration and purified as specified.
5-Ethyl-3-(3′-isopropoxyphenyl)-3H-[1,2,3]triazolo[4,5-d]-
pyrimidin-7(6H)-one (15b). Following the general procedure, to a
solution of Na (44 mg, 1.90 mmol) in ethanol (3.8 mL), 13 (100 mg,
0.38 mmol) and ethyl propionate (176 μL, 1.52 mmol) were added.
The reaction mixture was refluxed for 8 h. After work-up, a precipitate
was obtained that was purified by CCTLC (dichloromethane/
methanol, 20:1) to yield 15b (60 mg, 53%) as a white solid. mp:
242−244 °C. MS (ES, positive mode): m/z 300 (M + H)+. 1H NMR
(400 MHz, DMSO-d6): δ 1.23 (t, J = 7.5 Hz, 3H, CH3), 1.31 (d, J =
6.0 Hz, 6H, CH(CH3)2), 2.71 (q, J = 7.5 Hz, 2H, CH2), 4.69 (hept, J =
6.0 Hz, 1H, CH), 7.06 (d, J = 8.0, 1H, H-4′), 7.51 (pt, J = 8.1 Hz, 1H,
H-5′), 7.58 (d, J = 8.0 Hz, 1H, H-6′), 7.63 (s, 1H, H-2′), 12.70 (s 1H,
NH). 13C NMR (100 MHz, DMSO-d6): δ 11.5 (CH3), 22.1
(CH(CH3)2), 28.1 (CH2), 70.3 (CH), 109.2, 114.0, 116.6 (Ar),
129.4 (C-7a), 131.1, 136.9 (Ar), 149.1 (C-3a), 156.3 (C-7), 158.5
(Ar), 165.0 (C-5). Anal. (C15H17N5O2): C, H, N.
7-Chloro-3-(2′-methoxyphenyl)-5-methyl-3H-[1,2,3]triazolo[4,5-
d]pyrimidine (10a). Following the general procedure, to a suspension
of 9a (296 mg, 1.12 mmol) in dichloromethane (3.9 mL), NaNO2 (81
mg, 1.17 mmol) and 1 N HCl (3.9 mL) were added. After work-up,
233 mg (75%) of 10a were obtained as a pale brown solid. mp: 156−
1
158 °C. MS (ES, positive mode): m/z 276 (M + H)+ ; H NMR
(DMSO-d6, 300 MHz): δ 2.34 (s, 3H, CH3), 3.74 (s, 3H, OCH3), 7.15
(ddd, J = 7.6, 1.7, 1.1 Hz, 1H, H-5′), 7.32 (dd, J = 8.5, 1.1 Hz, 1H, H-
3′), 7.47 (dd, J = 7.8, 1.7 Hz, 1H, H-6′), 7.62 (ddd, J = 8.4, 7.5, 1.7 Hz,
1H, H-4′).
The synthesis of compounds 10b−m was performed following a
similar procedure, and all details and analytical and spectroscopic data
are included in the Supporting Information.
General Procedure for the Synthesis of [1,2,3]Triazolo[4,5-
d]pyrimidin-7(6H)-ones (11a−m). To a solution of the correspond-
ing 7-chloro-3H-[1,2,3]triazolo[4,5-d]pyrimidine (10) (1.00 mmol) in
anhydrous DMF (4.5 mL/mmol), sodium acetate (3.00 mmol) was
added. The reaction was microwave-irradiated at 120 °C for 1 h. The
mixture was dissolved in dichloromethane and washed with brine. The
organic layer was dried over Na2SO4, filtered, and evaporated to
dryness. The residue was purified as indicated in each case.
3-(2′-Methoxyphenyl)-5-methyl-3H-[1,2,3]triazolo[4,5-d]-
pyrimidin-7(6H)-one (11a). Following the general procedure, to a
solution of 10a (170 mg, 0.62 mmol) in anhydrous DMF (2.8 mL),
sodium acetate (154 mg, 1.85 mmol) was added. After work-up, the
residue was purified by precipitation with dichloromethane/hexane to
yield 79 mg (50%) of 11a as a beige solid. mp: 235−237 °C. MS (ES,
positive mode): m/z 258 (M + H)+. 1H NMR (DMSO-d6, 400 MHz):
δ 2.34 (s, 3H, CH3), 3.75 (s, 3H, OCH3), 7.16 (m, 1H, H-5′), 7.33 (d,
J = 8.5, 1H, H-3′), 7.48 (dd, J = 7.8, 1.3 Hz, 1H, H-4′), 7.63 (m, 1H,
H-6′), 12.63 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz): δ 21.8
(CH3), 56.5 (OCH3), 113.5, 121.1, 123.3, 127.9 (Ar), 129.2 (C-7a),
132.7 (Ar), 150.8 (C-3a), 154.9 (Ar), 156.3 (C-5), 160.7 (C-7). Anal.
(C12H11N5O2): C, H, N.
The synthesis of compounds 15a, c−g was performed following a
similar procedure, and all details and analytical and spectroscopic data
are included in the Supporting Information.
3-(3′-Isopropoxyphenyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine-5,7-
(6H,4H)-dione (16). To a solution of 13 (120 mg, 0.50 mmol) in DMF
(2.5 mL), 1,1′-carbonyldiimidazole (89 mg, 0.55 mmol) was added.
The mixture was stirred at 90 °C overnight. Then, it was concentrated
to dryness, and the residue was suspended in acetone. The precipitate
thus formed was filtered to yield 81 mg (56%) of 16 as a white solid.
The synthesis of compounds 11b−m was performed following a
similar procedure, and all details and analytical and spectroscopic data
are included in the Supporting Information.
1
mp: 286−288 °C. MS (ES, positive mode): m/z 288 (M + H)+. H
5-Amino-1-(3-isopropoxyphenyl)-1H-1,2,3-triazole-4-carboxa-
mide (13). Procedure A. To a solution of Na (71 mg, 3.08 mmol) in
ethanol (3 mL), cyanoacetamide (130 mg, 1.54 mmol) was slowly
added for 30 min. This was followed by the addition of the azide 1228
(273 mg, 1.54 mmol) and reflux for 2 h. The mixture was concentrated
to dryness. The crude obtained was dissolved in water (10 mL),
acidified by addition of acetic acid, and extracted with ethyl acetate (2
× 10 mL). The combined organic phases were washed with a solution
of NaHCO3 and brine, dried on Na2SO4, filtered, and evaporated. The
residue was purified by flash chromatography (hexane/ethyl acetate,
1:1). The fastest moving fractions afforded 274 mg (68%) of 14 as a
yellow solid while the slowest fractions afforded 60 mg (15%) of 13.
Data for ((3′-isopropoxyphenyl)amino)-1H-1,2,3-triazole-4-carboxa-
mide (14): mp: 212−213 °C. MS (ES, positive mode): m/z 262 (M +
NMR (400 MHz, DMSO-d6): δ 1.25 (d, J = 6.0 Hz, 6H, CH(CH3)2),
4.63 (hept, J = 6.1 Hz, 1H, CH), 6.97 (m, 1H, H-4′), 7.31 (s, 1H, H-
2′), 7.40 (m, 2H, H-5′, H-6′), 8.34 (s, 1H, N4H),10.63 (s, 1H, N6H).
13C NMR (100 MHz, DMSO-d6): δ 22.2 (CH3), 70.1 (CH), 110.0,
115.3, 116.2 (Ar), 121.0 (C-7a), 130.8 (Ar), 135.2 (C-3a), 136.5 (Ar),
154.3 (C-5), 157.5 (C-7), 158.3 (Ar). Anal. (C13H13N5O3): C, H, N.
1-(Azidomethyl)-3-isopropoxybenzene (18). To a solution of 17
(185 mg, 1.11 mmol) in anhydrous DMF (1.4 mL), DMAP (163 mg,
1.34 mmol) and CH3SO2Cl (104 μL, 1.34 mmol) were added. The
reaction mixture was stirred at room temperature for 5 h. Then, NaN3
was added and stirring was continued at room temperature for 4 h. It
was diluted with water (20 mL) and extracted with diethyl ether (4 ×
10 mL). The organic layer was dried over Na2SO4, filtered, and
evaporated to dryness to yield 226 mg (89%) of 18 as an oil. 1H NMR
(300 MHz, DMSO-d6): δ 1.25 (d, J = 6.1 Hz, 6H, CH(CH3)2), 4.38
(s, 2H, CH2), 4.61 (hept, J = 6.0 Hz, 1H, CH), 6.89 (m, 3H, H-2′, H-
4′, H-6′), 7.28 (pt, J = 7.7 Hz, 1H, H-5′).
1
H)+. H NMR (400 MHz, DMSO-d6): δ 1.25 (d, J = 6.0 Hz, 6H,
CH(CH3)2), 4.56 (sept, J = 6.0 Hz, 1H, CH), 6.42 (d, J = 7.7 Hz, 1H,
H-4′), 6.96 (d, J = 7.7 Hz, 1H, H-6′), 7.13 (t, J = 8.1 Hz, 1H, H-5′),
7.21 (s 1H, H-2′), 7.53 (s, 1H, NH-amide), 7.86 (s, 1H, NH-amide),
8.53 (s, 1H, NH-amine), 14.67 (s, 1H, NH-triazol). Anal. Calcd. for
(C12H15N5O2): C, H, N. Data for 13: mp: 123−125 °C. MS (ES,
positive mode): m/z 262 (M + H)+. 1H NMR (400 MHz, DMSO-d6):
δ 1.29 (d, J = 6.0 Hz, 6H, CH(CH3)2), 4.70 (hept, J = 5.7 Hz, 1H,
CH), 6.37 (s, 2H, NH2), 7.08 (m, 3H, H-2′, H-4′, H-6′), 7.26 (s, 1H,
5-Amino-1-(3′-isopropoxybenzyl)-1H-1,2,3-triazole-4-carboxa-
mide (19). To a suspension of NaH (60% in mineral oil, 37 mg, 1.55
mmol) in anhydrous DMF (1.4 mL) at 0 °C, a solution of
cyanoacetamide (79 mg, 0.94 mmol) in anhydrous DMF (1.4 mL)
was added. After 1 h, a solution of 18 (196 mg, 0.86 mmol) in
anhydrous DMF (1.4 mL) was slowly added and stirring was
continued at room temperature for 1 h. Volatiles were removed. The
residue obtained was purified by flash column chromatography
(dichloromethane/methanol, 20:1) to yield 183 mg (77%) of 19 as
a white solid. mp: 171−173 °C. MS (ES, positive mode): m/z 276 (M
CONH2), 7.47 (pt, J = 7.9 Hz, 1H, H-5′), 7.62 (s, 1H, CONH2). 13
C
NMR (100 MHz, DMSO-d6): δ 22.4 (CH3), 70.4 (CH), 111.6, 116.5,
116.9 (Ar), 122.3 (C-4), 131.3, 136.6 (Ar), 145.3 (C-5), 158.9 (Ar),
165.0 (CO). Anal. (C12H15N5O2): C, H, N.
Procedure B. To a suspension of NaH (60% in mineral oil, 152 mg,
6.24 mmol) in anhydrous DMF (5.9 mL), a solution of
1
+ H)+. H NMR (300 MHz, DMSO-d6): δ 1.23 (d, J = 6.0 Hz, 6H,
G
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Journal of Medicinal Chemistry
Article
CH(CH3)2), 4.54 (hept, J = 6.0 Hz, 1H, CH), 5.35 (s, 2H, CH2), 6.38
(s, 2H, NH2), 6.71 (m, 2H, H-2′, H-6′), 6.83 (d, J = 7.9 Hz, 1H, H-
4′), 7.08 (s, 1H, CONH2), 7.23 (pt, J = 8.1 Hz, 1H, H-5′), 7.45 (s, 1H,
CONH2).
supplemented with 25 μL of medium containing 0.1% DMSO, and
four cell control wells were supplemented with 50 μL of medium.
After 15 min, 25 μL of a virus mix containing the appropriate amount
of viral stock diluted in medium was added to the 96-well plates. Cells
were cultivated for 2 days after which 100 μL of the supernatant was
collected and transferred to 96 square well plates preloaded with 400
μL of RAV-1 lysis buffer from Macherey Nagel “NucleoSpin 96 virus
kit”. Viral RNA was extracted (NucleoSpin 96 virus kit running on an
Eppendorf epMotion 5075 liquid handler automat) and quantified by
real time RT-PCR to quantify the amount of viral RNA, which is
representative for the amount of progeny virions that are produced
(SuperScript III Platinium one-step RT-PCR with Rox from
Invitrogen). The four control wells were replaced by four 2-log
dilutions of an appropriate T7-generated RNA standards. IC50 values
were determined by fitting a sigmoidal curve on values of virus yield
inhibition (in %) versus the log values of drug concentration
(Kaleidagraph software).
3-(3-Isopropoxybenzyl)-5-methyl-3H-[1,2,3]triazolo[4,5-d]-
pyrimidin-7(6H)-one (20). To a solution of Na (41 mg, 1.80 mmol) in
ethanol (3.6 mL), 19 (100 mg, 0.36 mmol) and ethyl acetate (230 μL,
1.45 mmol) were added. The reaction mixture was heated at reflux for
8 h. After cooling, the reaction was concentrated to dryness. The
residue was dissolved in water, acetic acid was added, and the
precipitate was isolated by filtration to yield 81 mg (75%) of 20 as a
white solid. mp: 186−187 °C. MS (ES, positive mode): m/z 300 (M +
1
H)+. H NMR (400 MHz, DMSO-d6): δ 1.22 (d, J = 6.0 Hz, 6H,
CH(CH3)2), 2.41 (s, 3H, CH3), 4.56 (hept, J = 6.1 Hz, 1H, CH), 5.65
(s, 2H, CH2), 6.72 (m, 3H, H-2′, H-4′, H-6′), 7.22 (pt, J = 8.1 Hz, 1H,
H-5′). 13C NMR (100 MHz, DMSO-d6): δ 22.1 (CH3), 22.4 (CH3),
50.0 (CH2), 69.8 (CH), 115.6, 115.6, 120.1 (Ar), 128.7 (C-7a), 130.6,
137.7 (Ar), 149.7 (C-3a), 156.6 (C-5), 158.3 (C-7), 160.7 (Ar). Anal.
(C15H17N5O2): C, H, N.
ASSOCIATED CONTENT
* Supporting Information
■
Cells and Virus Strains. CHIKV Indian Ocean strain 899 (Genbank
S
FJ959103.1) was generously provided by Prof. S. Gunther (Bernhard
̈
Synthesis and spectroscopic data of compounds 9b−m, 10b−
m, 11b−n, and 15a, 15c−g; elemental analysis of compounds
2, 3a−c, 4, 6a, 6b, 11a−n, 13, 15a−g, 16, and 20. This material
is available free of charge via the Internet at http://pubs.acs.org.
Nocht Institute for Tropical Medicine, Hamburg, Germany). CHIKV
strain LR2006_OPY1 (GenBank DQ443544.2) and the clinical
isolates Venturini (Italy 2008), Congo 95 (2011), and St. Martin
(2013) belong to the collection of viruses at the UMR 190, Marseille,
France. Sindbis virus (SINV, strain HRsp, GenBank J02363.1) and the
Semliki Forest virus (SFV, Vietnam strain, GenBank EU350586.1)
belong to the collection of viruses at the Rega Institute of Medical
Research, Belgium. All viruses were propagated in African green
monkey kidney cells (Vero cells (ATCC CCL-81)).
AUTHOR INFORMATION
Corresponding Author
*Phone: 34 91 2587579; fax: 34 91 5644853; e-mail: mjperez@
iqm.csic.es.
■
Vero cells were maintained in cell growth medium composed of
minimum essential medium (MEM Rega-3, Gibco, Belgium)
supplemented with 10% fetal bovine serum (FBS, Integro, The
Netherlands), 1% L-glutamine (Gibco), and 1% sodium bicarbonate
(Gibco). The antiviral assays were performed in virus growth medium
that is the respective cell growth medium supplemented with 2%
(instead of 10%) FBS. All cell cultures were maintained at 37 °C in an
atmosphere of 5% CO2 and 95−99% humidity.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
A.G. and M.-D.C have both of them a JAE predoctoral
fellowship financed by the CSIC and the FSE (Fondo Social
Europeo). L.D. has a postdoctoral fellowship of the FWO
Antiviral and Antimetabolic Assays. Vero cells were seeded in 96-
well tissue culture plates (Becton Dickinson, Aalst, Belgium) at a
density of 2.5 × 104 cells/well in 100 μL of assay medium and were
allowed to adhere overnight. Next, a compound dilution series was
prepared in the medium on top of the cells after which the cultures
were infected with 0.01 MOI of CHIKV 899 inoculum in 100 μL of
assay medium. A similar setup was used for SINV and SFV. Each assay
was performed in multiplicate (at least in 3-fold) in the same test, and
assays were repeated independently to assess for interexperiment
variability. On day 7 postinfection (p.i.), the plates were processed
using the MTS/PMS method as described by the manufacturer
(Promega, The Netherlands). The 50% effective concentration (EC50),
which is defined as the compound concentration that is required to
inhibit virus-induced cytopathic effect by 50%, was determined using
logarithmic interpolation. The overall antimetabolic effect of the
compounds, which is representative for the cytostatic, cytotoxic, and
antimetabolic effect, was evaluated in uninfected cells, also by means of
the MTS/PMS method. The 50% cytostatic/cytotoxic concentration
(CC50; i.e., the concentration of compound that reduces the overall
metabolic activity of the cells by 50%) was calculated using logarithmic
interpolation. All assay wells were checked microscopically for minor
signs of virus-induced CPE or alterations of the cell or monolayer
morphology and were used to distinguish between true hit compounds
and mere cytoprotective compounds.
́
Belgium. We thank Maria Nares, Stijn Delmotte, and Tom
Bellon for excellent technical assistance. This work has been
supported by grants of the Spanish CICYT (SAF2009-13914-
C02-01 and SAF2012-39760-C02-01), BIPEDD-2-CM
(S2010/BMD-2457), and by EU F7 project SILVER.
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