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Article

New Trifluoromethyl Triazolopyrimidines as Anti-Plasmodium falciparum Agents

by
Núbia Boechat
1,*,
Luiz C. S. Pinheiro
1,
Thiago S. Silva
1,2,
Anna C. C. Aguiar
3,4,
Alcione S. Carvalho
1,
Monica M. Bastos
1,
Carolina C. P. Costa
1,
Sergio Pinheiro
5,
Angelo C. Pinto
2,
Jorge S. Mendonça
1,
Karen D. B. Dutra
1,5,
Alessandra L. Valverde
5,
Osvaldo A. Santos-Filho
1,
Isabela P. Ceravolo
3 and
Antoniana U. Krettli
3,4
1
Departamento de Síntese Orgânica, Instituto de Tecnologia em Fármacos-Farmanguinhos, Fundação Oswaldo Cruz, Manguinhos, Rio de Janeiro, RJ 21041-250, Brazil
2
Departamento de Química Orgânica, Instituto de Química, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, RJ 21941-590, Brazil
3
Laboratório de Malária, Instituto de Pesquisas René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, MG 30190-002, Brazil
4
Programa de Pós Graduação em Medicina Molecular, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, MG 30000-000, Brazil
5
Departamento de Química Orgânica, Instituto de Química, Universidade Federal Fluminense, Campus do Valonguinho, Niterói, RJ 24020-141, Brazil
*
Author to whom correspondence should be addressed.
Molecules 2012, 17(7), 8285-8302; https://doi.org/10.3390/molecules17078285
Submission received: 31 May 2012 / Revised: 27 June 2012 / Accepted: 29 June 2012 / Published: 10 July 2012
(This article belongs to the Section Medicinal Chemistry)
Browse Figures
Versions Notes

Abstract

:
According to the World Health Organization, half of the World’s population, approximately 3.3 billion people, is at risk for developing malaria. Nearly 700,000 deaths each year are associated with the disease. Control of the disease in humans still relies on chemotherapy. Drug resistance is a limiting factor, and the search for new drugs is important. We have designed and synthesized new 2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine derivatives based on bioisosteric replacement of functional groups on the anti-malarial compounds mefloquine and amodiaquine. This approach enabled us to investigate the impact of: (i) ring bioisosteric replacement; (ii) a CF3 group substituted at the 2-position of the [1,2,4]triazolo[1,5-a]pyrimidine scaffold and (iii) a range of amines as substituents at the 7-position of the of heterocyclic ring; on in vitro activity against Plasmodium falciparum. According to docking simulations, the synthesized compounds are able to interact with P. falciparum dihydroorotate dehydrogenase (PfDHODH) through strong hydrogen bonds. The presence of a trifluoromethyl group at the 2-position of the [1,2,4]triazolo[1,5-a]pyrimidine ring led to increased drug activity. Thirteen compounds were found to be active, with IC50 values ranging from 0.023 to 20 µM in the anti-HRP2 and hypoxanthine assays. The selectivity index (SI) of the most active derivatives 5, 8, 11 and 16 was found to vary from 1,003 to 18,478.

Graphical Abstract

1. Introduction

According to the World Health Organization (WHO), half of the World’s population, approximately 3.3 billion people, is at risk of contracting malaria. Nearly 700,000 deaths are associated with this disease annually. One in five childhood deaths in Africa are believed to be due to malaria [1,2].
In Brazil, a slight reduction in malaria cases was reported in 2009; however, a yearly total of over 306,000 cases was recently reported, most of which occurred in the Amazonia region [3]. Among the five known species of malaria that affect humans, three are found in Brazil: P. falciparum, P. malariae and P. vivax, with the latter causing 80% of the malaria cases diagnosed [3].
No effective vaccine is available yet for human use, although several promising antigens are undergoing clinical trials among endemic populations [4]. Control of malaria in Latin America relies on a specific therapeutic drug, chloroquine, used in association with other blood schizonticidal antimalarial drugs. Primaquine is also used in the treatment of P. vivax to prevent late malaria relapses caused by remaining liver forms [2]. In light of the rapid growth and spread of chloroquine-resistant P. falciparum and P.vivax strains, the development of new and more effective blood schizonticidal drugs is required. Several models are available to evaluate such new therapeutic agents [5].
The medicinal chemistry of fluorine-containing molecules has contributed greatly to the development of new drugs used in a wide range of diseases. A fluorine atom is often introduced to modify both the chemical reactivity and the physical and biological properties of organic compounds. One of the most widespread fluorine-containing functional groups in bioactive molecules is the trifluoromethyl moiety. It is a highly electronegative substituent that can exert significant electronic influence on neighboring groups. The trifluoromethyl substituent is also one of the most lipophilic groups known, making it useful for improving the targeting of molecules to enzyme active sites [6,7,8,9,10,11,12].
Many heterocyclic compounds have been developed in an attempt to find new drugs to treat malaria [13,14,15,16,17,18,19,20,21,22]. In 2004, our research group described the synthesis of 5-methyl-7-N'-(N,N-diethylpentane-1,4-diamine)-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine derivatives [23,24]. These compounds contained a trifluoromethyl group on the [1,2,4]triazolo[1,5-a]pyrimidine as a ring bioisostere of mefloquine and N1,N1-diethylpentane-1,4-diamine to mimic the chloroquine pharmacophore (Figure 1). However, these compounds showed poor antimalarial activity. Phillips and co-workers recently confirmed our proposal, demonstrating that [1,2,4]triazolo[1,5-a]pyrimidines inhibit P.falciparum dihydroorotate dehydrogenase (PfDHODH) and kill the parasite [25,26,27,28,29].
Molecules 17 08285 g001 550
Figure 1. Strategy used in 2004 to obtain 5-methyl-7-N'-(N,N-diethylpentane-1,4-diamine)-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (29) as an antimalarial bioisostere of quinoline.
Figure 1. Strategy used in 2004 to obtain 5-methyl-7-N'-(N,N-diethylpentane-1,4-diamine)-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (29) as an antimalarial bioisostere of quinoline.
Molecules 17 08285 g001
Continuing our research by using standard medicinal chemistry and modeling principles, such as isosteric replacement, we have designed new antimalarial agents 4–29 that are derivatives of the [1,2,4]triazolo[1,5-a]pyrimidine compounds (Figure 2). These derivatives were based on ring bioisosterism with mefloquine and amodiaquine. Different arylamines 4–17, 22–27 and aliphatic amines 18–21, 28, 29 were incorporated into the structure to investigate the importance of the substituent at the 7-position. Investigation of the impact of CF3 in the 2-position of the [1,2,4]triazolo[1,5-a]pyrimidine scaffold was prioritized. Twenty-six derivatives 4–29 from this series were designed, synthesized and evaluated in vitro against a Plasmodium falciparum chloroquine-resistant W2 clone strain.
Molecules 17 08285 g002 550
Figure 2. Rational approach to the design of [1,2,4]triazolo[1,5-a]pyrimidine derivatives 4–29.
Figure 2. Rational approach to the design of [1,2,4]triazolo[1,5-a]pyrimidine derivatives 4–29.
Molecules 17 08285 g002

2. Results and Discussion

2.1. Synthesis of Compounds

The synthetic route to [1,2,4]triazolo[1,5-a]pyrimidine derivatives 4–29 is shown in Figure 3. Condensation of 3-amino-1,2,4-triazoles 1a–c with ethyl acetoacetate or ethyl 4,4,4-trifluoroacetoacetate in refluxing toluene, in the presence of catalytic p-toluenesulfonic acid, gave [1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-ones 2a–d in 50–90% yields after 24 h, as previously described in the literature [23,24,30,31]. Spectroscopic data of compounds 2a–d were in agreement with the literature, and X-ray crystallography showed 2c as their keto-tautomers [24]. Compounds 2a–d were easily chlorinated with phosphorus oxychloride under reflux for 6 h, affording the respective 7-chloro[1,2,4]triazolo[1,5-a]pyrimidines 3a–d in 58–90% yields. Compounds 3a and 3b showed identical spectroscopic data to those reported in the literature [23,25,26,27,28,29,30,31]. Spectroscopic data of compounds 3c and 3d were in agreement with the proposed structures. Reaction of compounds 3a–d with several amines produced the target compounds 4–29 in 30–90% yields.
Molecules 17 08285 g003 550
Figure 3. Synthesis of [1,2,4]triazolo[1,5-a]pyrimidines 4–29.
Figure 3. Synthesis of [1,2,4]triazolo[1,5-a]pyrimidines 4–29.
Molecules 17 08285 g003

2.2. Molecular Modeling

It is known that [1,2,4]triazolo[1,5-a]pyrimidine derivatives interact with the enzyme dihydroorotate dehydrogenase (DHODH) [25,32]. Thus, docking calculations were performed for the newly synthesized compounds to verify their binding modes with this enzyme fromP. falciparum (PfDHODH) available in the Protein Data Bank as PDB ID code 3I65. Each compound was modeled, and 1,000 steps of energy minimization were performed by the steepest descent method using Gasteiger-Hückel charges and a dielectric constant of 80 in the Tripos force field [33]. The structures were further optimized using the conjugated gradient method.
Ligand-enzyme docking simulations were performed with the molecular docking algorithm MolDock [34] using the Molegro Virtual Docker 4.3.0. MolDock uses a heuristic search algorithm (i.e., termed guided differential evolution), which combines differential evolution and a cavity-prediction algorithm. The docking scoring function is an extension of the piecewise linear potential (PLP) [34]. After the ligands and protein coordinates were imported, all structural parameters, including bond type, hybridization, explicit hydrogen, charges, and flexible torsions, were assigned using the automatic preparation function in the Molegro Virtual Docker software. For each compound, 100 docking runs were performed with the initial population of 150 individuals. After each compound was docked, it was energy-minimized into the active site of the enzyme.
All synthesized compounds were docked into PfDHODH. The known PfDHODH inhibitor DSM1 that was co-crystallized with the enzyme was used as the reference molecule during the docking simulations [32].
Enzyme residues H185 and R265 and the water molecule W15 found in the crystal structure of PfDHODH act as “molecular anchors” for binding molecules 4–29 at the active site. Such “anchors” are actually hydrogen bonds formed between 4–29 and the enzyme residues or the water molecule. Each [1,2,4]triazolo[1,5-a]pyrimidine 4–29 interacts with R265 by forming a hydrogen bond through N-4. An additional hydrogen bond can be present between N-1 of the pyrimidine ring and H185. The frequency with which hydrogen bonds are formed between the W15 molecule and CF3 groups at the 2-position of [1,2,4]triazolo[1,5-a]pyrimidine rings of the majority of compounds was remarkable. Twenty-two of the 26 synthesized compounds (compounds 4–13, 17–27, 29) showed the CF3–W15 interaction. Consequently, the CF3 group must be carefully considered for the development of potential new lead inhibitors of PfDHODH. Figure 4 shows the R265–N-4 and the CF3–W15 interactions.
Molecules 17 08285 g004 550
Figure 4. Compound 5 docked into the active site of PfDHODH.
Figure 4. Compound 5 docked into the active site of PfDHODH.
Molecules 17 08285 g004

2.3. Continuous Cultures and in Vitro Assays with P. falciparum-Infected Erythrocytes

The P. falciparum W2 clone, which is chloroquine-resistant and mefloquine-sensitive [35], was maintained in continuous culture. Briefly, the parasites were kept as described [36] at 37 °C in human erythrocytes (A+) in complete medium (RPMI 1640 supplemented with 10% human sera blood group A+, 2% glutamine, and 7.5% NaHCO3) either in Petri dishes in a candle jar or in 25-cm culture flasks in an environment containing a gas mixture atmosphere (3% O2, 5% CO2 and 91% N2). Before testing, the ring-stage parasites were synchronized by sorbitol [37]; the suspension was adjusted for parasitemia and hematocrit as described below for each test used. The infected red blood cells were distributed in a 96-well microtiter plate (Corning, Santa Clara, CA, USA), 180 μL/well, to which 20 μL of different concentrations of test drugs and controls had previously been added. The maximum concentration 50 µg/mL (~157 μM) was tested two or three times; compounds are considered inactive at equal or higher doses.
The effects of compounds against the W2 P. falciparum blood cultures were evaluated through incorporation of 3H hypoxanthine (Perkin Elmer, Waltham, MA, USA) by the parasites [38]. Alternatively, compound effects were also examined using monoclonal antibodies to a commercially available parasite histidine and alanine-rich protein (HRP2) (MPFM ICLLAB-55A®, MPFG55P ICLLAB®, Immunology Consultants Laboratory Inc. (ICL), Portland, OR, USA), as described previously [39]. The [3H]-hypoxanthine assay was performed with 1% of parasitemia and 1% of hematocrit, and the level of isotope incorporation was read in a beta-counter (Perkin Elmer). The anti-HRP2 test was performed with 0.05% parasitemia and 1.5% hematocrit, and the quantification of protein was determined using a specific read at 450 nm in a spectrophotometer (SpectraMax340PC384, Molecular Devices, Sunnyvale, CA, USA). Drug activities were expressed by the half-maximal inhibitory dose (IC50) compared to the drug-free controls and estimated using the curve-fitting software Origin 8.0 (OriginLab Corporation, Northampton, MA, USA) [40].

2.4. Cell Cultures and Cytotoxicity Tests

The human hepatoma cell line (HepG2) was cultured in 75-cm2 sterile flasks with RPMI 1640 medium supplemented with 10% heat-inactivated fetal and 40 mg/L gentamicin in a 5% CO2 atmosphere at 37 °C. For in vitro cytotoxicity experiments, the cell monolayer was trypsinized, washed with culture medium, distributed in a flat-bottomed 96-well plate (5 × 103 cells/well), and finally incubated for 18 h at 37 °C for cell adherence.
For cytotoxicity testing, we used the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, performed as described in the literature [41]. The HepG2 cell line was incubated with 20 µL of the compounds in different concentrations (200–25 µg/mL) for 24 h in an atmosphere of 5% CO2 at 37 °C.
For the MTT assay, which evaluates mitochondrial viability, 20 µL of MTT solution (5 mg/mL) was added, and the plates were incubated for an additional 3 h. After incubation, the supernatant was carefully removed from the wells, followed by addition of 100 µL DMSO with thorough mixing. Optical densities at 570 and 630 nm (background) were determined by an ELISA reader.
Cell viability was expressed as the percentage of control absorbance obtained in untreated cells after subtracting the absorbance from appropriate background. Lastly, the minimum lethal dose for 50% of the cells (MLD50) was determined as previously described in the literature [42]. The ratio between MLD50 and drug activity (IC50) in vitro was used to determine the selectivity index (SI).
The synthesized compounds with different substituents in the 2-, 5-, and 7-positions of the [1,2,4]triazolo[1,5-a]pyrimidine scaffold were tested against the W2-chloroquine-resistant P. falciparum clone. The anti-P. falciparum activity and cytotoxicity results of the 26 new [1,2,4]triazolo[1,5-a]pyrimidine derivatives are summarized in Table 1. Among them, 13 showed low IC50 values (ranging from 0.023 to 20 µM) in the anti-HPR2 and [3H]-hypoxanthine incorporation tests. The other compounds showed high IC50 values (>20 µM) or were inactive. Values of SI greater than 10 are considered non-toxic, which was the case for all new compound tested herein. Derivatives 5, 8, 11 and 16 were the most active, least toxic, and had the highest SI values (from 1,003 to 18,478).
Table
Table 1. Evaluation of anti-plasmodial activity against a chloroquine-resistant W2 clone of P. falciparum, cytotoxicity against a human hepatoma cell line (HepG2) and drug selectivity index (SI) of compounds 4–29 and chloroquine.
Table 1. Evaluation of anti-plasmodial activity against a chloroquine-resistant W2 clone of P. falciparum, cytotoxicity against a human hepatoma cell line (HepG2) and drug selectivity index (SI) of compounds 4–29 and chloroquine.
CompoundsIC50 (µM) * Plasmodium FalciparumMDL50 HepG2 (µM)SI MDL50/IC50
Anti-HRP2Hypoxanthine
42.2Nd326148
50.023 ± 0.002Nd42518,478
63 ± 210.2 ± 5>682>227
71.83 ± 1.101.22373203
80.55 ± 0.660.11 ± 0.05>5521,003
92.7 ± 2.213.32 ± 1.10320118
10>69.9>148.9337Inactive
110.4 ± 0.091.5 ± 1.2>619>1,547
122.5 ± 0.050.36 ± 0.1498>199
131.47 ± 0.110.83 ± 0.58320218
143.84 ± 0.4015.05 ± 10.03515134
15>162.3149.3415Inactive
160.30.4 ± 0.064461486
1712.3123.42269Inactive
188.69 ± 0.4610.0351564
19>174.0>174.0>697Inactive
2039 ± 1757 ± 13>666Inactive
21>184.5>184.5>738Inactive
22>156.4>170.0394Inactive
2336.93 ± 11.3631.25 ± 17.04<71Inactive
242.1Nd>746>355
2521 ± 215 ± 0.7<93Inactive
2629.7333.45 ± 14.86<93Inactive
2797.37 ± 22.4778.65 ± 7.49>749Inactive
28>172.4>172.4>689Inactive
2973 ± 1739.1>558Inactive
Chloroquine0.220.234904,200
* IC50 < 10 µM active; >10 and <20 µM partially active; >20 µM inactive; Nd: not determined.
Taken together, the in vitro data of anti-P. falciparum and its toxicological activities show that these four compounds are promising candidates for the development of a novel anti-malarial therapy. Decreased biological activity resulted from substitution with cyclic secondary amines 19–21 and alkylamines 28, 29. Moreover, except in the case of pyrazolyl derivative 24, compounds containing azoles or heteroaromatics as substituents were inactive (compounds 22, 23, 25–27).

3. Experimental

1H-, 13C- and 19F-Nuclear Magnetic Resonance (NMR) spectra were obtained at 400.00 MHz, 100.00 MHz and 376.00 MHz, respectively, on a Bruker Avance instrument equipped with a 5-mm probe, using tetramethylsilane as the internal standard. Chemical shifts (δ) reported in ppm and coupling constants (J) in Hertz. Fourier transform infrared (FT-IR) absorption spectra were recorded on a Shimadzu mode IR Prestige-21 spectrophotometer by reflectance in KBr. GC/MS experiments were conducted using a model 6,890 N gas chromatograph (Agilent, Palo Alto, CA, USA) equipped with a 7,683 B auto sampler coupled with a model MS 5,973 N single quadrupole mass spectrometer (Agilent). The GC was equipped with a HP-5MS capillary column 30 m in length, 0.25 mm in diameter, with a 0.25-μm film thickness. The temperature program began at 50 °C, then increased to 300 °C at a rate of 10 °C/min and held for 10 minutes. The helium flow rate was 0.5 mL/min. Melting points (m.p.) were determined with a Büchi model B-545 apparatus. TLC was carried out using silica gel F-254 glass plates (20 × 20 cm). All other reagents and solvents used were analytical grade.

3.1. General Procedure for Preparing [1,2,4]Triazolo[1,5-a]pyrimidin-7(4H)-ones (2a–d)

The [1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-ones 2a,b were prepared according to methodology described in the literature [23,24,30,31]. A mixture of a 3-amino-1,2,4-triazole derivative (1c) (20 mmol) and ethyl acetoacetate or ethyl 4,4,4-trifluoroacetoacetate (15 mL) was stirred at room temperature for 30 minutes. To the mixture was added toluene (30 mL) and catalytic p-toluenesulfonic acid. The reaction was heated under reflux for 24 h. The resulting solid was then cooled to RT, filtered, washed with toluene, and dried. Compounds 2c,d were used without purification.
5-Methyl-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one (2c) Yield: 90%; m.p. 266–267 °C; IR (KBr, cm−1): 3,445 (NH); 1702 (C=O); 1,659 (C=N); 761 (CF3). 1H-NMR (CD3OD, δ): 2.34 (s, 3H, CH3); 5.98 (s, 1H, H-6); 13.59 (s, 1H, NH). 13C-NMR (CD3OD, δ): 18.75; 99.26; 119.20 (q, 1J = 268.8 Hz, C-9); 151.56; 151.99 (q, J = 39 Hz, C-2); 152.74; 155.27. 19F-NMR (CD3OD, δ): −64.97 (s, 3F). GC-MS (70 eV) m/z (%): 218; 199; 190; 69 (100); 68; 53.
2,5-bis(Trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one (2d) Yield: 50%; m.p. 176–178 °C; IR (KBr, cm−1): 3,489 (NH); 1,708 (C=O); 1,608; 761 (CF3). 1H-NMR (CD3OD, δ): 6.61 (s, 1H, H-6). 13C-NMR (CD3OD, δ): 101.33; 120.50 (q, 1J = 268.0 Hz); 121.27 (q, 1J = 272.0 Hz); 142.92 (q, J = 37.5 Hz); 153.53. 19F-NMR (CD3OD, δ): −67.73 (s, 3F); −67.73 (s, 3F). GC-MS (70 eV) m/z (%): 272; 152 (100); 69; 53; 43.

3.2. General Procedure for Preparing 7-Chloro[1,2,4]triazolo[1,5-a]pyrimidines 3a–d

The 7-chloro[1,2,4]triazolo[1,5-a]pyrimidine 3a,b were prepared according to methodology described in the literature [23,30,31]. To a [1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one derivative 2c,d (4.5 mmol) was added phosphorus oxychloride (10 mL). The mixture was stirred under reflux for 4 h. Excess solvent was removed under reduced pressure and the resulting material was carefully added to 50 mL of crushed ice, which was then basified to pH 12 with NaOH (6 M aq.) and stirred for 40. The mixture was diluted with water (30 mL) and extracted with chloroform (3 × 30 mL). The combined organic solution was washed with water (3 × 50 mL), dried (magnesium sulfate), filtered, and concentrated under vacuum.
7-Chloro-5-methyl-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (3c). Yield: 90%; m.p. 98–100 °C. IR (KBr, cm−1): 3,062 (CH); 1,614 (C=N); 1,523; 1,487; 1,062; 1,215; 1,190; 752 (CF3). 1H-NMR (CD3OD, δ): 2.74 (s, 3H, CH3); 7.65 (s, 1H, H-6). 13C-NMR (CD3OD, δ): 25.31; 115.18; 120.74 (q, J = 268.6 Hz, C-9); 157.52 (q, J = 41.6 Hz, C-5); 141.32; 170.14. 19F-NMR (CD3OD, δ): −67.27 (s, 3F). GC-MS (70 eV) m/z (%): 238; 236; 216; 201; 76; 69 (100); 51.
7-Chloro-2,5-bis(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (3d). Yield: 80%; m.p. 107–108 °C. IR (KBr, cm−1): 3,130 (CH); 1,639 (C=N); 823; 625; 684 (CF3). 1H-NMR (CD3OD, δ): 7.68 (s, 1H, H-6). 13C-NMR (CD3OD, δ): 112.39; 117.90 (q, J = 274.0 Hz); 118.60 (q, J = 270.0 Hz); 137.30 (q, J = 40.4 Hz); 155.29; 158.37; 158.90 (q, J = 40.7 Hz). 19F-NMR (CD3OD, δ): −68.22 (s, 3F); −65.83 (s, 3F). GC-MS (70 eV) m/z (%): 292; 290; 271; 69 (100).

3.3. General Procedure for Preparing 5-Methyl-7-aryl/cycloalkylamine[1,2,4]triazolo[1,5-a]pyrimidines 4–21

A mixture of a 7-chloro[1,2,4]triazolo[1,5-a]pyrimidine derivative 3b–d and the appropriate amine (1 equivalent) in ethanol (10 mL) was stirred at room temperature for 16–18 h. The reaction mixture was concentrated and poured into 50 mL of ice-cold water. The precipitate was collected by filtration and washed with water (10 mL) to give 4–21 in yields of 50–90%.
5-Methyl-7-(naphthalen-1-ylamine)-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (4). Yield: 85%; m.p. 186–188 °C. IR (KBr, cm−1): 3,516 (N-H); 3,304; 1,627 (C=N); 1,575; 1,514; 1,448; 1,396; 1,369; 1,301; 1,282; 1,211, 1,195; 1,139; 806; 781 (CF3). 1H-NMR (CD3OD, δ): 2.36 (s, 3H, CH3); 5.90 (s, 1H, H-6); 7.65–7.55 (m, 4H, H-5'-H-8'); 8.01–7.95 (m, 3H, H-2'-H-4'). 13C-NMR (CD3OD, δ): 25.03; 92.49; 121.13 (q, J = 268.6 Hz, C-9); 123.54; 126.64; 127.09; 128.14; 128.51; 129.83; 130.25; 131.43; 133.17; 136.40; 150.15; 156.98 (d; J = 39.3 Hz; C-2);157.42; 168.41. 19F-NMR (CD3OD, δ): −67.21 (s, 3F). GC/MS m/z (%): 343 (100); 324; 274; 190; 177; 127.
5-Methyl-7-(naphthalen-2-ylamine)-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (5). Yield: 90%; m.p. 163–164 °C. IR (KBr, cm−1): 3,062 (NH); 1,662 (C=N); 1,589; 1,506; 1,467; 1,369; 1,309; 1,288; 1,192; 1,184; 1,153; 848; 829; 779 (CF3). 1H-NMR (CD3OD, δ): 2.60 (s, 3H, CH3); 6.67 (s, 1H, H-6); 7.63–7.58 (m, 3H, H-1'-H-4'); 7.97–8.11 (m, 4H, H-5'-H-8'). 13C-NMR (CD3OD, δ): 25.19; 92.31; 121.07 (q, J = 214.8 Hz, C-9); 123.94; 124.41; 127.63; 128.14; 128.85; 128.95; 131.02; 133.62; 134.91; 135.31; 148.63; 156.71 (q, J = 31.3 Hz, C-2); 157.32; 168.57. 19F-NMR (CD3OD, δ): −67.47 (s, 3F). GC/MS m/z (%): 343 (100); 328; 247; 190; 177; 127.
5-Methyl-7-phenylamine-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (6). Yield: 85%; m.p. 151–152 °C. IR (KBr, cm−1): 3,653 (N-H); 3,062 (CH-aromatic); 2,999; 1,631 (C=N); 1,589; 1,512; 1,429; 1,369; 1,305; 1,286; 1,197, 1,170; 1,139; 777; 721 (CF3). 1H-NMR (CD3OD, δ): 2.48 (s, 3H, CH3); 6.46 (s, 1H, H-6); 7.50–7.36 (m, 5H, H-2'-H-6'). 13C-NMR (CD3OD, δ): 25.18; 92.13; 121.05 (q, J = 268.5 Hz, C-9); 126.28; 128.49; 131.08; 137.56; 148.83; 156.79 (q, J = 39 Hz, C-2); 157.39; 168.55. 19F-NMR (CD3OD, δ): −67.22 (s, 3F). GC/MS m/z (%): 293 (100); 294; 278; 224; 190; 177; 77.
7-(3-Chlorophenylamine)-5-methyl-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (7). Yield: 50%; m.p. 136–138 °C. IR (KBr, cm−1): 3,603 (NH); 3,062 (CH-aromatic); 2,893; 1,627 (C=N); 1,589; 1,504; 1,431; 1,323; 1,192; 1,141; 1,072; 848; 779 (CF3). 1H-NMR (CD3OD; δ): 2.52 (s, 3H, CH3); 6.55 (s, 1H, H-6); 7.38 (d, J = 7.5 Hz, H-6'); 7.42 (d, J = 8 Hz, 1H, H-4'); 7.50 (t, J = 8.0 Hz, 1H, H-'); 7.51 (d, J = 2.0 Hz, 1H, H-2'). 13C-NMR (CD3OD, δ): 25.23; 92.46; 121.02 (q, J = 214.9 Hz, C-9); 124.42; 126.17; 128.34; 132.31; 136.49; 139.15; 148.43; 156.77 (q, J = 31.3 Hz, C-2); 157.34; 168.85. 19F-NMR (CD3OD, δ): −67.20 (s, 3F). GC/MS m/z (%): 327 (100); 328; 329; 312; 258; 190; 177; 111.
7-(3,4-Dichlorophenylamine)-5-methyl-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (8). Yield: 90%; m.p. 136–138 °C. IR (KBr, cm−1): 3,606 (NH); 3,062; 3,005 (CH-aromatic); 2,360; 1,672 (C=N); 1,585; 1,512; 1,469; 1,388; 1,315; 1,288; 1,195; 1,149; 833; 779 (CF3).1H-NMR (CD3OD, δ): 2.53 (s, 3H, CH3); 6.57 (s, 1H, H-6); 7.43 (dd, J = 2.0 Hz; J = 8.75 Hz, 1H, H-6'); 7.65 (d, J = 9.0 Hz, 1H, H-5'); 7.66 (d, J = 2.5 Hz, 1H, H-2'). 13C-NMR (CD3OD, δ): 25.23; 92.66; 121.03 (q, J = 268.5 Hz, C-9); 125.85; 128.05; 131.77; 132.79; 134.61; 137.78; 148.31; 156.86 (q, J = 39.2 Hz, C-2); 157.38; 168.97. 19F-NMR (CD3OD, δ): −67.27 (s; 3F). GC/MS m/z (%): 361 (100); 362; 363; 346; 292; 190; 177; 145.
7-(2,4-Dichlorophenylamine)-5-methyl-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (9). Yield: 80%; m.p. 217–218 °C. IR (KBr, cm−1): 3,128 (NH); 3,066 (CH-aromatic); 1,624 (C=N); 1,577; 1,508; 1,469; 1,307; 1,288; 1,188; 1,168; 1,141; 1,099; 1,056; 844; 779 (CF3). 1H-NMR (CD3OD, δ): 2.58 (s, 3H, CH3); 6.20 (s, 1H, H-6); 7.60 (dd, J = 2.0 Hz, J = 6.8 Hz, 1H, H-5'); 7.65 (d, J = 6.8 Hz, 1H, H-6'); 7.82 (d, J = 2.0 Hz, 1H, H-3'). 13C-NMR (CD3OD, δ): 25.13; 92.78; 121.02 (q, J = 215 Hz, C-9); 130.08; 131.74; 131.81; 133.52; 134.42; 135.94; 148.69; 156.96 (q, J = 31 Hz, C-2); 157.29; 168.85. 19F-NMR (CD3OD, δ): −67.17 (s, 3F). GC/MS m/z (%): 361; 346; 328; 326 (100); 190; 177; 145.
7-(2,6-Difluorophenylamine)-5-methyl-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (10). Yield: 80%; m.p. 129–130 °C. IR (KBr, cm−1): 3,383 (NH); 3,082; 2,357; 1,631 (C=N); 1,577; 1,516; 1,477; 1,373; 1,296; 1,242; 1,195; 1,161; 1,006; 794 (CF3). 1H-NMR (CD3OD, δ): 2.51 (s, 3H, CH3); 6.14 (s, 1H, H-6); 7.22 (m, 2H, H-3', H-5'); 7.55–7.49 (m, 1H, H-4'). 13C-NMR (CD3OD, δ): 25.12; 92.81; 113.82 (dd; J = 15.45 Hz, J = 3.2 Hz, 2C, C-3', C-5'); 121.01 (q, J = 214.9 Hz, C-9); 131.49 (t;J = 7.15 Hz; 2C; C-1', C-4'); 148.69; 157.02 (q, J = 31.5 Hz; C-2); 157.25; 160.36 (dd; J = 200.2 Hz, J = 2.2 Hz, 2C, C-2', C-6'); 169.01. 19F-NMR (CD3OD, δ): −67.29 (s; 3F); −119.77 (s; 2F). GC/MS m/z (%): 329 (100); 330; 310; 260; 190; 177.
7-(4-Methoxyphenylamine)-5-methyl-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (11). Yield: 60%; m.p. 163–164 °C. IR (KBr, cm−1): 3,606 (NH); 3,007 (CH-aromatic); 2,050; 1,629 (C=N); 1,606; 1,579; 1,510; 1,433; 1,377; 1,288; 1,269; 1,242; 1,031; 1,193, 1,168; 1,136, 823; 779 (CF3). 1H-NMR (CD3OD, δ): 2.46 (s, 3H, CH3); 3.84 (s, 3H, OCH3); 6.29 (s. 1H, H-6); 7.03 (d; J = 9.2 Hz, 2H, H-2'; H-6'); 7.34 (d, J = 9.2 Hz, 2H, H-3', H-5'). 13C-NMR (CD3OD, δ): 25.11; 56.22; 91.88; 116.28; 121.04 (q, J = 268.9 Hz; C-9); 128.27; 129.88; 149.45; 156.79 (d, J = 38.7 Hz, C-2); 157.38; 160.62; 168.36. 19F-NMR (CD3OD, δ): −67.24 (s, 3F). GC/MS m/z (%): 323 (100); 324; 308; 254; 190; 177.
7-(3,5-Dimethoxyphenylamine)-5-methyl-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (12). Yield: 90%; m.p. 154–156 °C. IR (KBr, cm−1): 3,383 (NH); 3,007 (CH-aromatic); 2,968; 2,843; 1,629 (C=N); 1,604; 1,581; 1,502; 1,431; 1,346; 1,309; 1,284; 1,251; 1,205; 1,155; 1,053; 1,155; 979; 839; 777 (CF3). 1H-NMR (CD3OD, δ in ppm): 2.50 (s, 3H, CH3); 3.79 (s, 6H, OCH3); 6.55 (s, 1H, H-6); 6.44 (t, J = 2.4 Hz, 1H, H-4'); 6.58 (d, J = 2.4 Hz; 2H; H-2', H-6’). 13C-NMR (CD3OD, δ in ppm): 25.08; 56.20; 92.68; 100.08; 104.29; 121.01 (q, J = 269 Hz, C-9); 139.06; 148.67; 156.75 (q, J = 39 Hz, C-2); 157.19; 163.38; 168.36. 19F-NMR (CD3OD, δ in ppm): −67.19 (s, 3F). GC/MS m/z (%): 353 (100); 354; 352; 338; 282; 190; 177; 137.
7-(3,5-Dichlorophenylamine)-5-methyl-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (13). Yield: 85%; m.p. 207–208 °C. IR (KBr, cm−1): 3,319 (NH); 3,084; 3,062; 1,624 (C=N); 1,560; 1,510; 1,433; 1,371; 1,319; 1,286 (C=N); 1,193; 1,174; 1,143; 806; 779 (CF3). 1H-NMR (CD3OD, δ): 2.54 (s; 3H; CH3); 6.61 (s; 1H; H-6); 7.41 (t; J = 0.9 Hz; 1H; H-4'); 7.48 (d; J = 1.2 Hz; 2H; H-2', H-6'). 13C-NMR (CD3OD, δ): 25.38; 93.03; 121.11 (q; J = 215 Hz; C-9); 124.54; 128.02; 137.37; 140.50; 148.11; 156.94 (q; J = 31.3 Hz; C-2); 157.41; 169.11. 19F-NMR (CD3OD, δ): −67.23 (s; 3F). GC/MS m/z (%): 361; 362; 363; 346; 292; 190 (100); 177; 145.
7-(3,5-Dimethoxyphenylamine)-2,5-dimethyl[1,2,4]triazolo[1,5-a]pyrimidine (14). Yield: 90%; m.p. 112–114 °C. IR (KBr, cm−1): 3,518 (NH); 1,629 (C=N); 1,585; 1,519; 1,483; 1,388; 1,357; 1,311; 1,284; 1,203; 1,155; 931; 817. 1H-NMR (CD3OD, δ in ppm): 2.45 (s, 3H, CH3); 2.52 (s, 3H, CH3); 3.80 (s, 6H, OCH3); 6.42 (s, 1H, H-6); 6.44 (dd, J = 1.6 Hz, 1H, H-4'); 6.56 (d, J = 2.0 Hz; 2H; H-2', H-6'). 13C-NMR (CD3OD, δ in ppm): 14.78; 24.96; 56.17; 91.03; 99.49; 103.81; 139.51; 147.41; 157.23; 163.28; 165.29; 166.38. GC/MS m/z (%): 299 (100); 298; 284; 269; 136; 123.
7-(3,5-Dichlorophenylamine)-2,5-dimethyl[1,2,4]triazolo[1,5-a]pyrimidine (15). Yield: 85%; m.p. 128–130 °C. IR (KBr, cm−1): 3,560 (NH); 3,138 (CH-aromatic); 1,612 (C=N); 1,591; 1,564; 1,525; 1,454; 1,371; 1,361; 1,321; 1,114; 935; 835; 804. 1H-NMR (CD3OD, δ): 2.49 (s, 3H, CH3); 2.52 (s, 3H, CH3); 6.48 (s, 1H, H-6); 7.39 (dd, J = 1.2 Hz, 1H, H-4'); 7.45 (d, J = 1.2 Hz; 2H; H-2', H-6'). 13C-NMR (CD3OD, δ): 14.80; 25.01; 91.24; 123.86; 127.37; 123.18; 137.37; 140.77; 146.75; 157.22; 165.48; 166.87. GC/MS m/z (%): 307; 309; 272; 145; 136 (100); 123; 109.
2,5-Dimethyl-7-(4-(trifluoromethyl)phenylamine)-[1,2,4]triazolo[1,5-a]pyrimidine (16). Yield: 80%; m.p. 191–193 °C. IR (KBr, cm−1): 3,211 (NH); 1,328; 1,604 (C=N); 1,564; 844. 1H-NMR (CD3OD, δ): 2.47 (s, 3H, CH3); 2.52 (s, 3H, CH3); 6.54 (s, 1H, H-6); 7.77 (d, J = 8.0 Hz, 2H, H-2', H-6'); 7.63 (d, J = 8.0 Hz; 2H; H-3', H-5'). 13C-NMR (CD3OD, δ): 14.76; 24.98; 91.19; 125.12; 125.63 (q; J = 215 Hz; CF3); 128.09; 129.05 (q; J = 215 Hz; C4'); 141.96; 146.70; 157.30; 165.51; 166.85. 19F-NMR (CD3OD, δ): −63.79 (s, 3F). GC/MS m/z (%): 307 (100); 145; 136; 123.
7-(3,5-Dichlorophenylamine)-2,5-bis(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (17). Yield: 50%; m.p 248–249 °C. IR (KBr, cm−1): 3,624 (NH); 3,313; 3,120; 1,658 (C=N); 1,622; 1,591; 1,510; 1,454; 1,394; 1,323; 1,301; 1,217; 1,163; 1,139; 1,051; 968; 858; 773 (CF3). 1H-NMR (CD3OD, δ): 7.17 (t; J = 1.2 Hz; 1H; H-4’); 7.22 (s; 1H; H-6); 7.90 (d; J = 1.2 Hz; 2H; H-2', H-6'). 13C-NMR (CD3OD, δ): 106.17; 119.68; 120.07 (q; J = 217.5 Hz; C-10); 120.68 (q; J = 214.7 Hz; C-9); 125.10; 136.39 (q; J = 31 Hz; C-5); 136.49; 142.02; 157.13 (q; J = 31 Hz; C-2); 157.91; 158.37. 19F-NMR (CD3OD, δ): −67.82 (s; 3F); −70.08 (s; 3F); GC/MS m/z (%): 414 (100); 415; 417; 396; 380; 345; 145.
7-Cyclohexylamine-5-methyl-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (18). Yield: 75%; m.p 128–130 °C. IR (KBr, cm−1): 3,344 (NH); 3,012; 2,941; 2,860; 2,798 (C–H); 1,624 (C=N); 1,589; 1,516; 1,452; 1,427; 1,367; 1,321; 1,303; 1,286; 1,203; 1,172, 1,155; 796 (CF3). 1H-NMR (CD3OD, δ): 1.24–3.67 (m; 11H, CH2 and CH); 6.47 (s; 1H; H-6). 13C-NMR (CD3OD, δ): 25.0; 26.14; 26.39; 33.22; 53.37; 90.83; 120.92 (q; J = 214.9 Hz; C-9); 148.68; 156.34 (q; J = 31.1 Hz; C-2); 157.17; 167.93. 19F-NMR (CD3OD, δ): −67.28 (s; 3F; F-9); GC/MS m/z (%) 299; 280; 270; 230; 217 (100); 190; 177.
4-(5-Methyl-7-(4-morpholinyl)-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (19). Yield: 70%; m.p. 215–216 °C. IR (KBr, cm−1): 3,051; 2,972; 2,912; 2,960; 2,870 (C–H); 1618 (C=N); 1,564; 1,442; 1,371; 1,323; 1,296; 1,274; 1,213; 1,197; 1,180; 1,120; 773; (CF3). 1H-NMR (CD3OD, δ): 3.91 (m, 8H, CH2); 2.58 (s, 3H, CH3); 6.63 (s, 1H, H-6). 13C-NMR (CD3OD, δ): 25.04; 49.94; 67.35; 97.35; 120.95 (q, J = 268.8 Hz; C-9); 156.04 (q, J = 39.1 Hz; C-2); 152.01; 158.75; 168.57. 19F-NMR (CD3OD, δ): −67.46 (s, 3F). GC/MS m/z (%): 287 (100); 288; 268; 256; 244; 230; 218; 202; 177.
5-Methyl-7-(4-methylpiperazin-1-yl)-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (20). Yield: 90%; m.p. 158–160 °C. IR (KBr, cm−1): 3,047; 2,976; 2,947; 2,910; 2,850; 2,800 (C-N); 1,618 (C=N); 1,554; 1,512; 1,450; 1,431; 1,371; 1,323; 1,294; 1,213; 1,190; 1,141; 777 (CF3). 1H-NMR (CD3OD, δ): 2.38 (s, 3H, CH3); 2.56 (s, 3H, N-CH3); 2.68 (m, 4H); 3.94 (m, 4H); 6.63 (s, 1H, H-6). 13C-NMR (CD3OD, δ): 25.03; 46.13; 49.19; 55.34; 97.56; 120.95 (q, J = 268.5 Hz, C-9); 151.86; 156.00 (q, J = 39.1 Hz, C-2); 158.72; 168.44. 19F-NMR (CD3OD, δ): −67.39 (s, 3F). GC/MS m/z (%): 300; 285; 231; 70 (100).
5-Methyl-7-(pyrrolidin-1-yl)-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (21). Yield: 90%; m.p. 234–236 °C. IR (KBr, cm−1): 2,987; 2,949; 2,877 (C-N); 1,629 (C = N); 1,571; 1,508; 1,444; 1,371; 1,355; 1,309; 1,261; 1,211; 1,192; 1,138; 771 (CF3). 1H-NMR (CD3OD, δ): 2.07–2.10 (m, 8H); 2.55 (s, 3H, CH3); 6.19 (s, 1H, H-6). 13C-NMR (CD3OD, δ): 24.71; 26.47; 52.36; 121.04 (q, J = 268.4 Hz, C-9); 149.40; 155.84 (q, J = 38.6 Hz, C-2); 159.00; 167.12. 19F-NMR (CD3OD, δ): −67.79 (s, 3F). GC/MS m/z (%): 271; 252; 202 (100).

3.4. General Procedure for Preparing 5-Methyl-7-substituted[1,2,4]triazolo[1,5-a]pyrimidines 22–27

A mixture of 7-chloro-5-methyl-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidine (3c) and the appropriate azole or amine (1 equivalent) in DMF (3 mL) was stirred at 120 °C for 12 h. The organic solvent was then removed under reduced pressure. The solid was collected and recrystallized from EtOH/H2O to give 22–27 a yield of 50–72%.
5-Methyl-7-(pyridin-2-ylamine)-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (22). Yield: 50%; m.p 174–177 °C. IR (KBr, cm−1): 3,643 (NH); 3,161 (CH-aromatic); 1,631 (C=N); 1,136; 775 (CF3). 1H-NMR (CD3OD, δ): 2.64 (s; 3H; CH3); 7.81 (s; 1H; H-6); 8.43–7.16 (m; 4H; H-3'-H-6'); 8.22 (s; NH). 13C-NMR (CD3OD, δ): 25.64; 97.34; 115.97; 120.91; 121.01 (q; J = 215.0 Hz; C-9); 139.86; 145.21; 148.87; 153.40; 156.39 (q; J = 31.0 Hz; C-2); 156.81; 169.33. 19F-NMR (CD3OD, δ): −67.47 (s; 3F; F-9).
5-Methyl-2-(trifluoromethyl)-7-(5-(trifluoromethyl)-3-amine-4H-1,2,4-triazolyl)[1,2,4]triazolo[1,5-a]pyrimidine (23). Yield: 60%; m.p. 217–219 °C. IR (KBr, cm−1): 3,398; 3,334 (NH); 3,111 (CH-aromatic); 1,643; 1,633 (C=N); 1,190; 758; 750 (CF3). 1H-NMR (CD3OD, δ): 2.83 (s, 3H, CH3); 7.73 (s, 1H, H-6). 13C-NMR (CD3OD, δ): 25.6; 93.7; 121.01 (q, J = 268.5 Hz, C-9); 125.59 (q, J = 269 Hz, C-18); 125.75; 128.14; 129.61 (q, J = 33 Hz, C-15); 140.1; 147.97; 156.85 (q, J = 39 Hz, C-2); 158.0; 159.7. 19F-NMR (CD3OD, δ): −67.20 (s, 3F); −68.53 (s, 3F). GC/MS m/z (%): 352 (100); 333; 310; 283.
5-Methyl-7-(1H-pyrazol-1-yl)-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (24). Yield: 70%; m.p. 221–223 °C. IV (KBr, cm−1): 3,128 (NH); 1,631; 1,566; 1,195; 1,153; 783 (CF3). 1H-NMR (CD3OD): 2.79 (s, 3H, CH3), 7.93 (s, 1H, H-6); 9.38 (d, J = 2.8 Hz, H-3'); 8.03 (d, J = 1.6 Hz, H-5'); 6.78 (dd, J = 2.8 Hz, 1.6 Hz, H-4'). 13C-NMR (CD3OD, δ): 25.58; 30.81; 102.79; 111.56; 120.78 (q, J = 270 Hz, C-9); 134.82; 143.49; 146.32; 157.70 (q, J = 40 Hz, C-2); 170.64. 19F-NMR (CD3OD, δ): −67.38 (s, 3F). GC/MS m/z (%): 268 (100); 119.
7-(1H-Imidazol-1-yl)-5-methyl-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (25). Yield: 72%; m.p. 157–159 °C. IV (KBr, cm−1): 3,159 (NH); 3,047; 1,631(C=N); 1,192; 783 (CF3). 1H-NMR (CD3OD, δ): 2.81 (s, 3H, CH3); 7.74 (s, 1H, H-6); 7.33 (s, 1H, H-5'); 8.14 (s, 1H, H-4'); 8.87 (s, 1H, H-2'). 13C-NMR (CD3OD, δ): 25.58; 104.92;. 120.73 (q, J = 270 Hz, C-9); 120.91; 131.16; 139.70; 141.39; 157.71 (q, J = 40 Hz, C-2); 171.21. 19F-NMR (CD3OD, δ): −67.37 (s, 3F); GC/MS m/z (%):268 (100); 241; 214; 146.
5-Methyl-7-(1H-1,2,4-triazol-1-yl)-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (26). Yield: 65%; m.p. 173–175 °C. IV (KBr, cm−1): 3,076; 1,639 (C=N); 1,566; 1,193; 777 (CF3). 1H-NMR (CD3OD, δ): 2.84 (s, 3H, CH3), 7.99 (s, 1H, H-6); 8.43 (s, 1H, H-5'); 9.98 (s, 1H, H-2'). 13C-NMR (CD3OD, δ): 25.75; 103.95; 120.69 (q, J = 214 Hz, C-9); 141.01; 148.31; 154.58; 157.85 (q, J = 32 Hz, C-2); 171.41. 19F-NMR (CD3OD, δ): −67.59 (s, 3F); GC/MS m/z (%): 269 (100); 250; 242; 92.
5-Methyl-7-(1H-pyrrol-1-yl)-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (27). Yield: 60%; m.p. 198–200 °C. IV (KBr, cm−1): 3,375; 1,620 (C=N); 1,192; 744 (CF3). 1H-NMR (CD3OD, δ): 2.69 (s, 3H, CH3), 7.57 (s, 1H, H-6); 7.67–6.46 (m, 4H). 13C-NMR (CD3OD, δ): 25.12; 106.29; 112.82; 119.50; 120.91 (q, J = 269 Hz, C-9); 127.98; 141.05; 157.29 (q, J = 50 Hz, C-2); 168.30; 19F-NMR (CD3OD, δ): −67.20 (s, 3F). GC/MS m/z (%): 267 (100).

3.5. General Procedure for Preparing 5-Methyl-7-N'-(N,N-diethylpentane-1,4-diamine)[1,2,4]triazolo [1,5-a]pyrimidines 28, 29

A mixture of a 7-chloro[1,2,4]triazolo[1,5-a]pyrimidine derivative 3a,c (1 mmol) and the appropriate amine (1.5 mmol) in ethanol (10 mL) was stirred at room temperature for 43 h. Excess solvent was removed under reduced pressure, giving the respective derivative 28 and 29 a yield of 80–83%, as pale yellow oils.
5-Methyl-7-N'-(N,N-diethylpentane-1,4-diamine)[1,2,4]triazolo[1,5-a]pyrimidine (28). Yield: 80%. IV (neat, cm−1): 3,420 (NH); 1,651; 1,601 (C=N); 1,219; 1,149; 992. 1H-NMR (acetone-d6, δ): 1.03 (t; 6H; J = 7 Hz, CH2-CH3); 1.23 (d; 3H; J = 7.5 Hz, CH3); 1.35 (m; 4H, CH2-CH2); 2.01 (s; 3H, CH3); 2.56–2.45 (m; 7H, CH2-CH3; CH2 and CH); 4,12 (sl; 1H, NH); 6,38 (s; 1H, H-6); 8,26 (s; 1H, H-2). 13C-NMR (acetone-d6, δ): 13.3; 16.20; 19.9; 22.9; 24.0; 30.6; 46.7; 53.8; 101.1; 140.9; 154.2; 158.5; 162.7. GC/MS m/z (%): 290; 275; 230; 218; 86 (100).
5-Methyl-7-N'-(N,N-diethylpentane-1,4-diamine)-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine (29). Yield: 83%. IV (neat, cm−1): 3,434 (NH); 1,650; 1,604 (C=N); 1,218; 1,151; 995; 772 e 747 (CF3). 1H-NMR (400 MHz, CD3OD, δ): 1.06 (t; 6H; J = 8 Hz, CH2-CH3); 1.18 (d; 3H; J = 6 Hz, CH3); 1.70–1.45 (m; 4H, CH2-CH2); 2.56 (s; 3H, CH3); 2.80–2.45 (m; 7H, CH2-CH3; CH2 and CH); 4.15 (sl; 1H, NH); 6.12 (s; 1H, H-6). 13C-NMR (100 MHz, CD3OD, δ): 9.7; 16.0; 19.3; 21.8; 24.2; 33.5; 45.9; 51.9; 101.1; 118.9 (q; J = 269 Hz; C-9); 144.9; 152.2 (q; J = 14.5 Hz; C-2); 158.5; 163.7. 19F-NMR (376 MHz, CD3OD, δ): −66.43 (s, 3F); GC/MS m/z (%):358; 86 (100); 69; 58.

4. Conclusions

One important strategy in drug design is the chemical modification of available drugs to develop novel, biologically active compounds. This approach seeks to improve the “druggability” of analogues, thus reducing the chance of causing parasite resistance [43]. We have synthesized 26 new derivatives of the [1,2,4]triazolo[1,5-a]pyrimidine system, with different substituents at the 2-, 5- and 7-positions of that ring system; these compounds exhibited a range of anti-P. falciparum activities. The data suggest that these compounds can be used as potential agents against malaria.
The results show that compounds containing an arylamine substituent in the 7-position of [1,2,4]triazolo[1,5-a]pyrimidine exhibit anti-plasmodial activity against the W2 chloroquine-resistant P. falciparum clone, with IC50 values of 0.023 to 20 µM. This trend is exemplified by compounds 5 (2-naphthyl), 8 (3,4-diCl), 11 (4-OCH3), and 16 (4-CF3). In compound 5 the naphythylamine substituent at the 7-position has an important contribution to anti-Plasmodium falciparum activity, when compared with compounds 8 and 11, which contain arylamine groups. However, compound 16 having 4-CF3-phenylamine as a substituent at 7-position was more important than CF3 group at 2-position.
None of these compounds were toxic to HepG2 cells. The substituent groups at the 7-position of the [1,2,4]triazolo[1,5-a]pyrimidine ring were found to play an important role in the anti-Plasmodium activity. The trifluoromethyl group as a substituent at the 2-position of the [1,2,4]triazolo[1,5-a]pyrimidine ring contributed to increased anti-plasmodial activity in several compounds (5, 8, 11).
Docking simulations of the synthesized compounds with PfDHODH are in accordance with the crystallographic investigation published elsewhere [32], which suggests that the presence of “molecular anchors” formed by specific hydrogen bonds between the ligands and the enzyme should be considered carefully for the design of potential new lead compounds. The residues involved in these hydrogen bonds are H185 and R265. Moreover, additional hydrogen bonds between nearly all of the compounds and a water molecule (W15) should also be considered for the stabilization of the ligand-enzyme interaction. The presence of a CF3 group can facilitate such hydrogen bonds.

Acknowledgments

The authors thank the Coordination of Improvement of Higher Education (CAPES) and the National Council of R&D of Brazil (CNPq) for the fellowships granted. We also thank the Foundation for Research of the State of Rio de Janeiro (FAPERJ), the Foundation for Research of the State of Minas Gerais (FAPEMIG), the Technological Development Program on Products for Health (PDTIS) and CNPq-MCT/MS (PRONEX Rede Malaria) for financial support.

References

  1. 10 Facts on Malaria. Available online: http://www.who.int/features/factfiles/malaria/en/index.html (accessed on 22 March 2011).
  2. World Health Organization (WHO). Global report on antimalarial drug efficacy and drug resistance: 2000–2010. 2010. Available online: http://www.who.int/malaria/en (accessed on 22 March 2011).
  3. Oliveira-Ferreira, J.; Lacerda, M.V.G.; Brasil, P.; Ladislau, J.L.B.; Tauil, P.L.; Daniel-Ribeiro, C.T. Malaria in Brazil: An overview. Malaria J. 2010, 9, 115–130. [Google Scholar] [CrossRef]
  4. Agnandji, S.T.; Lell, B.; Soulanoudjingar, S.S.; Fernandes, J.F.; Abossolo, B.P.; Conzelmann, C.; Methogo, B.G.; Doucka, Y.; Flamen, A.; Mordmüller, B.; et al. First results of phase 3 trial of RTS,S/AS01 malaria vaccine in African children. N. Engl. J. Med. 2011, 365, 1863–1875. [Google Scholar]
  5. Krettli, A.U.; Adebayo, J.O.; Krettli, L.G. Testing of natural products and synthetic molecules aiming at new antimalarials. Curr. Drug Targets 2009, 10, 261–270. [Google Scholar] [CrossRef]
  6. Purser, S.; Moore, P.R.; Swallow, S.; Gouverneur, V. Fluorine in medicinal chemistry. Chem. Soc. Rev. 2008, 37, 320–330. [Google Scholar] [CrossRef]
  7. Welch, J.T.; Eswarakrishnan, S. Fluorine in Bioorganic Chemistry; John Wiley & Sons: New York, NY, USA, 1991. [Google Scholar]
  8. Ojima, I. Fluorine in Medicinal Chemistry and Chemical Biology; John Wiley & Sons: Chichester, UK, 2009. [Google Scholar]
  9. Kirk, K.L. Fluorination in medicinal chemistry: Methods, strategies, and recent developments. Org. Process. Res. Dev. 2008, 12, 305–321. [Google Scholar] [CrossRef]
  10. Park, B.K.; Kitteringham, N.R.; O’Neill, P.M. Metabolism of fluorine containing drugs. Ann. Rev. Pharmacol. Toxicol. 2001, 41, 443–470. [Google Scholar] [CrossRef]
  11. Müller, K.; Faeh, C.; Diederich, F. Fluorine in pharmaceuticals: Looking beyond intuition. Science 2007, 317, 1881–1886. [Google Scholar] [CrossRef]
  12. Boechat, N.; Bastos, M.M. Trifluoromethylation of carbonyl compounds. Curr. Org. Synth. 2010, 7, 403–413. [Google Scholar] [CrossRef]
  13. Milner, E.; McCalmont, W.; Bhonsle, J.; Caridha, D.; Cobar, J.; Gardner, S.; Gerena, L.; Goodine, D.; Lanteri, C.; Melendez, V.; et al. Anti-malarial activity of a non-piperidine library of next-generation quinoline Methanols. Malaria J. 2010, 9, 51–61. [Google Scholar] [CrossRef]
  14. Biot, C.; Chibale, K. Novel approaches to antimalarial drug discovery. Infect. Dis. Drug Targets 2006, 6, 173–204. [Google Scholar] [CrossRef]
  15. Blackie, M.A.L.; Yardley, V.; Chibale, K. Synthesis and evaluation of phenylequine for antimalarial activity in vitro and in vivo. Bioorg. Med. Chem. Lett. 2010, 20, 1078–1080. [Google Scholar] [CrossRef]
  16. Iwaniuk, D.P.; Whetmore, E.D.; Rosa, N.; Ekoue-Kovi, K.; Alumasa, J.; Dios, A.C.; Roepe, P.D.; Wolf, C. Synthesis and antimalarial activity of new chloroquine analogues carrying a multifunctional linear side chain. Bioorg. Med. Chem. 2009, 17, 6560–6566. [Google Scholar] [CrossRef]
  17. Delarue-Cochin, S.; Paunescu, E.; Mães, L.; Mouray, E.; Sergheraert, C.; Grellier, P.; Melnyk, P. Synthesis and antimalarial activity of new analogues of amodiaquine. Eur. J. Med. Chem. 2008, 43, 252–260. [Google Scholar] [CrossRef]
  18. Casagrande, M.; Basilico, N.; Parapini, S.; Romeo, S.; Taramelli, D.; Sparatore, A. Novel amodiaquine congeners as potent antimalarial agents. Bioorg. Med. Chem. 2008, 16, 6813–6823. [Google Scholar] [CrossRef]
  19. Guglielmo, S.; Bertinaria, M.; Rolando, B.; Crosetti, M.; Fruttero, R.; Yardley, V.; Croft, S.L.; Gasco, A. A new series of amodiaquine analogues modified in the basic side chain with in vitro antileishmanial and antiplasmodial activity. Eur. J. Med. Chem. 2009, 44, 5071–5079. [Google Scholar]
  20. Jonet, A.; Dassonville-Klimpt, A.; da Nascimento, S.; Leger, J.; Guillon, J.; Sonnet, P. First enantioselective synthesis of 4-aminoalcohol quinoline derivatives through a regioselective SN2 epoxide opening mechanism. Tetrahedron Asymmetry 2011, 22, 138–148. [Google Scholar] [CrossRef]
  21. Zhu, S.; Zhang, Q.; Gudise, C.; Meng, L.; Wei, L.; Smitha, E.; Kong, Y. Synthesis and evaluation of naphthyridine compounds as antimalarial agents. Bioorg. Med. Chem. Lett. 2007, 17, 6101–6106. [Google Scholar] [CrossRef]
  22. Mello, H.; Echevarria, A.; Bernardino, A.M.; Canto-Cavalheiro, M.; Leon, L.L. Antileishmanial pyrazolopyridine derivatives: Synthesis and structure-activity relationship analysis. J. Med. Chem. 2004, 47, 5427–5432. [Google Scholar] [CrossRef]
  23. Dutra, K.D.B. Síntese e Avaliação Antimalárica de Novos Análogos Triazolopirimidínicos da Cloroquina. MSc. Thesis, Departamento de Química Orgânica, Universidade Federal Fluminense, Niterói, RJ, Brazil, 2004. [Google Scholar]
  24. Boechat, N.; Dutra, K.D.B.; Valverde, A.L.; Wardell, S.; Low, J.N.; Glidewell, C. Hydrogen-bonded chains in 5-methyl-2-trifluoromethyl-1,2,4-triazolo-[1,5-a]pyrimidin-7(4H)-one and hydrogen- bonded chains of rings in 5-amino-3-trifluoromethyl-1H-1,2,4-triazolo-5-methyl-2-trifluromethyl-1,2, 4-triazolo[1,5-a]pyrimidin-7(4H)-one (1/1) the co-crystal of a reaction product and one of its precursors. Acta Crystallogr. Sect. C-Cryst. Struct. Commun. 2004, 60, 733–736. [Google Scholar] [CrossRef]
  25. Gujjar, R.; Marwaha, A.; El Mazouni, F.; White, J.; White, K.L.; Creason, S.; Shackleford, D.M.; Baldwin, J.; Charman, W.N.; Buckner, F.S.; et al. Identification of a metabolically stable triazolopyrimidine-based dihydroorotate dehydrogenase inhibitor with antimalarial activity in mice. J. Med. Chem. 2009, 52, 1864–1872. [Google Scholar] [CrossRef]
  26. Phillips, M.A.; Gujjar, R.; Malmquist, N.A.; White, J.; El Mazouni, F.; Baldwin, J.; Rathod, P.K. Triazolopyrimidine-based dihydroorotate dehydrogenase inhibitors with potent and selective activity against the malaria parasite Plasmodium Falciparum. J. Med. Chem. 2008, 51, 3649–3653. [Google Scholar] [CrossRef]
  27. Phillips, M.A.; Rathod, P.K. Plasmodium dihydroorotate dehydrogenase: A promising target for novel anti-malarial chemotherapy. Infect. Dis. Drug Targets 2010, 10, 226–239. [Google Scholar]
  28. Gujjar, R.; El Mazouni, F.; White, K.L.; White, J.; Creason, S.; Shackleford, D.M.; Deng, X.; Charman, W.N.; Bathurst, I.; Burrows, J.; et al. Lead optimization of aryl and aralkyl amine-based triazolopyrimidine inhibitors of plasmodium falciparum dihydroorotate dehydrogenase with antimalarial activity in mice. J. Med. Chem. 2011, 54, 3935–3949. [Google Scholar]
  29. Coteron, J.M.; Marco, M.; Esquivias, J.; Deng, X.; White, K.L.; White, J.; Koltun, M.; El Mazouni, F.; Kokkonda, S.; Katneni, K.; et al. Structure-guided lead optimization of triazolopyrimidine-ring substituents identifies potent Plasmodium falciparum dihydroorotate dehydrogenase inhibitors with clinical candidate potential. J. Med. Chem. 2011, 54, 5540–5561. [Google Scholar]
  30. Zohdi, H.F. Reactions with 3-amino-5-(trifluoromethyl)-1,2,4-triazole: A simple route to fluorinated polysubstituted triazolo[1,5-a]pyrimidine and triazolo[5,1-c]triazine derivatives. J. Chem. Res. 1997, 392–393. [Google Scholar] [CrossRef]
  31. Erkin, A.V.; Krutikov, V.I. Formation, structure and heterocyclization of aminoguanidine and ethyl acetoacetate condensation products. Russ. J. Gen. Chem. 2009, 79, 1204–1209. [Google Scholar] [CrossRef]
  32. Deng, X.; Gujjar, R.; Mazouni, F.E.; Kaminsky, W.; Malmquist, N.A.; Goldsmith, E.J.; Rathod, P.K.; Phillips, M.A. Structural platicity of malaria dihydroorotate dehydrogenase allows selective binding of diverse chemical scaffolds. J. Biol. Chem. 2009, 284, 26999–27009. [Google Scholar]
  33. Tripos 1699 South Hanley Road, St. Louis, MO 63144–2319, USA.
  34. Molegro ApS. C. F. Moellers Alle 8, Building 1110, DK-8000 Aarhus C, Denmark.
  35. Oduola, A.M.; Weatherly, N.F.; Bowdre, J.H.; Desjardins, R.E. Plasmodium falciparum: Cloning by single-erythrocyte micromanipulation and heterogeneity in vitro. Exp. Parasitol. 1988, 66, 86–95. [Google Scholar] [CrossRef]
  36. Trager, W.; Jensen, J. Human malaria parasites in continuous culture. Science 1976, 193, 673–675. [Google Scholar]
  37. Lambros, C.; Vanderberg, J. Synchronization of plasmodium falciparum erythrocytic stages in culture. J. Parasitol. 1979, 65, 418–420. [Google Scholar] [CrossRef]
  38. Desjardins, R.; Canfield, C.; Haynes, J.; Chulay, J. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob. Agents Chemother. 1979, 16, 710–718. [Google Scholar] [CrossRef]
  39. Noedl, H.; Wongsrichanalai, C.; Miller, R.; Myint, K.; Looareesuwan, S.; Sukthana, Y.; Wongchotigul, V.; Kollaritsch, H.; Wiedermann, G.; Wernsdorfer, W. Plasmodium falciparum: Effect of anti-malarial drugs on the production and secretion characteristics of histidine-rich protein II. Exp. Parasitol. 2002, 102, 157–163. [Google Scholar] [CrossRef]
  40. Carvalho, L.; Brandão, M.; Santos-Filho, D.; Lopes, J.; Krettli, A. Antimalarial activity of crude extracts from brazilian plants studied in vivo in plasmodium berghei-infected mice and in vitro against plasmodium falciparum in culture. Braz. J. Med. Biol. Res. 1991, 24, 1113–1123. [Google Scholar]
  41. Denizot, F.; Lang, R. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods 1986, 89, 271–277. [Google Scholar] [CrossRef]
  42. Madureira, A.M.; Martins, A.P.; Gomes, M.; Paiva, J.; Cunha, A.P.; Rosário, V. Antimalarial activity of medicinal plants used in traditional medicine in S. Tome and Principe Island. J. Ethnopharmacol. 2002, 81, 23–29. [Google Scholar] [CrossRef]
  43. Na-Bangchang, K.; Karbwang, J. Current status of malaria chemotherapy and the role of pharmacology in antimalarial drug research and development. Fundam. Clin. Pharmacol. 2009, 23, 387–409. [Google Scholar] [CrossRef]
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Boechat, N.; Pinheiro, L.C.S.; Silva, T.S.; Aguiar, A.C.C.; Carvalho, A.S.; Bastos, M.M.; Costa, C.C.P.; Pinheiro, S.; Pinto, A.C.; Mendonça, J.S.; et al. New Trifluoromethyl Triazolopyrimidines as Anti-Plasmodium falciparum Agents. Molecules 2012, 17, 8285-8302. https://doi.org/10.3390/molecules17078285

AMA Style

Boechat N, Pinheiro LCS, Silva TS, Aguiar ACC, Carvalho AS, Bastos MM, Costa CCP, Pinheiro S, Pinto AC, Mendonça JS, et al. New Trifluoromethyl Triazolopyrimidines as Anti-Plasmodium falciparum Agents. Molecules. 2012; 17(7):8285-8302. https://doi.org/10.3390/molecules17078285

Chicago/Turabian Style

Boechat, Núbia, Luiz C. S. Pinheiro, Thiago S. Silva, Anna C. C. Aguiar, Alcione S. Carvalho, Monica M. Bastos, Carolina C. P. Costa, Sergio Pinheiro, Angelo C. Pinto, Jorge S. Mendonça, and et al. 2012. "New Trifluoromethyl Triazolopyrimidines as Anti-Plasmodium falciparum Agents" Molecules 17, no. 7: 8285-8302. https://doi.org/10.3390/molecules17078285

APA Style

Boechat, N., Pinheiro, L. C. S., Silva, T. S., Aguiar, A. C. C., Carvalho, A. S., Bastos, M. M., Costa, C. C. P., Pinheiro, S., Pinto, A. C., Mendonça, J. S., Dutra, K. D. B., Valverde, A. L., Santos-Filho, O. A., Ceravolo, I. P., & Krettli, A. U. (2012). New Trifluoromethyl Triazolopyrimidines as Anti-Plasmodium falciparum Agents. Molecules, 17(7), 8285-8302. https://doi.org/10.3390/molecules17078285

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