Translate this page into:
Solvent-free microwave-assisted synthesis of novel pyrazolo[4′,3′:5,6]pyrido[2,3-d]pyrimidines with potential antifungal activity
⁎Corresponding author. Fax: +57 2 3339240. jairo.quiroga@correounivalle.edu.co (Jairo Quiroga)
-
Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Novel fused pyrazolo[4′,3′:5,6]pyrido[2,3-d]pyrimidines 5 were prepared by a solvent-free microwave assisted reaction of heterocyclic o-aminonitriles 3 and cyanopyridines 4 in the presence of tBuOK as catalyst. This protocol provides a versatile procedure for the synthesis of the title compounds with the advantages of easy work-up, mild reaction conditions and good yields. All compounds were also tested for antifungal properties against two clinically important fungi; Candida albicans and Cryptococcus neoformans. Several compounds showed moderate activity against both fungi, being 5a the most active compound. Analysis of the antifungal behavior of properly grouped compounds allowed to determine that the position of the N in the pyrimidyl moiety per se does not play a role in the activity. In turn, the type of 4-R substituent appears to influence the activity. In addition to the above considerations, the lipophilicity of compounds measured as log P showed to be not related to the activity and regarding the dipole moment (D), no net correlation was observed, although it is the most active compounds (% inhibition >50%) that have a D ⩾ 7.5, mainly against C. albicans.
Keywords
Pyrazolopyridopyrimidines
o-Aminonitriles
Cyanopyridines
Microwave irradiation
Antifungal activity
1 Introduction
The pyrimidine core has been widely studied due to its presence in numerous natural products and structurally diverse synthetic derivatives (Lawen, 2003; Choudhury et al., 2008). Among pyrimidine-containing compounds, fused pyrimidines, particularly pyrido[2,3-d]pyrimidine derivatives (i.e. deazapteridines) have attracted much attention because they showed interesting bioactivities (Lunt et al., 1984; Bagley et al., 2001; Devi et al., 2003; Devi et al., 2004; Kanth et al., 2006; Bulicz et al., 2006; Tu et al., 2006; Tu et al., 2008) such as antipyretic, antibacterial, antitumor, antihistaminic (Piper et al., 1986; Kuyper et al., 1996; Quintela et al., 1997; Cordeu et al., 2007), diuretic, antifolate, calcium-channel-antagonist, anti-inflammatory (Parish et al., 1982; Pastor et al., 1994; Rosowsky et al., 1995).
The pyrimido[2,3-d]pyrimidine-7-ones I, piritrexim (II) and [1-(2-amino-6-aryl-pyrido[2,3-d]pyrimidin-7-yl)ureas] (III) showed to be inhibitors of cyclin-dependent kinases (Toogood, 2001), dihydrofolate reductase (Gangjee et al., 2003; Chan and Rosowsky, 2005; Chan et al., 2005) and receptor and non-receptor tyrosine kinases (Hamby et al., 1997; Dorsey et al., 2000; Wissing et al., 2004), respectively (Fig. 1).![Structures of some pyrido[2,3-d]pyrimidine derivatives with biological activity.](/content/184/2016/9/3/img/10.1016_j.arabjc.2015.03.002-fig1.png)
Structures of some pyrido[2,3-d]pyrimidine derivatives with biological activity.
The incidence of fungal infection has increased dramatically in recent years. The widespread use of antifungal drugs and their resistance against fungal infections has led to serious health hazards (Tandon et al., 2009). Although there are diverse available drugs for the treatment of systemic and superficial mycoses, they are not completely effective for their eradication (Brown and Wright, 2005). In addition, they all possess a certain degree of toxicity and quickly develop resistance due to the large-scale use. There is, therefore, an urgent need for new antifungal chemical structures alternatives to the existing ones (Mukherjee et al., 2003). In this sense, the pyrido[2,3-d]pyrimidine ring system is present in biologically active compounds which possess high antifungal properties. More specifically some of them show activity against dermatophytes, fungi causing the most important superficial mycoses in human beings (Quiroga et al., 2006).
Microwave irradiation (MWI) has emerged as a powerful tool for high-throughput organic synthesis. This source of energy can improve the yield and purity of the desired compounds in short reaction times through the precise control of parameters such as power irradiation, pressure and temperature (Martins et al., 2009; Kappe, 2004; Quiroga et al., 2010; Quiroga et al., 2012).
Due to our interest in the synthesis of potentially bioactive nitrogen-containing six-membered heterocyclic compounds (Insuasty et al., 2008; Insuasty et al., 2010; Quiroga et al., 1998), herein we report a versatile and efficient method for the preparation of pyrazolo[4′,3′:5,6]pyrido[2,3-d]pyrimidine derivatives, via cyclocondensation reaction between heterocyclic o-aminonitriles 3 and cyanopyridines 4. The starting o-aminonitriles 3 (6-aminopyrazolo[3,4-b]pyridine-5-carbonitriles) were obtained by a modified method described in the literature (Quiroga et al., 1999), through the interaction of 5-amino-3-methyl-1-phenylpyrazole 1 with different benzylidenemalononitriles 2, using ethanol as solvent and acetic acid as catalyst (Scheme 1).
Synthesis of heterocyclic ortho-aminonitrile derivatives 3.
2 Results and discussion
2.1 Chemistry
In our study, several conditions were tested at first including diverse solvents, temperatures and power of the microwave source in order to find the best reaction conditions for the synthesis of 5a. In all cases, reactions were carried out from o-aminonitrile 3a (R = Cl) and 4-cyanopyridine 4 as a model reaction (Scheme 2). When ethanol was used as the solvent and the mixture was subjected to reflux, the desired product 5a was obtained in low yield (30%, entry 1) after 9 h. When DMF was used as the refluxing solvent, almost the same yield was obtained (31%, entry 2) after 8 h. Significative improvements were obtained when the reaction was performed under MWI using DMF as the solvent (11 min, yield = 36%, entry 3) or under solvent-free MWI (10 min, yield = 41%, entry 4). The presence of tBuOK in the reaction media, which is a typical catalyst for such reactions (Olivieria et al., 2008), increased the efficiency of the MW as well as the reflux reactions. In MW under solvent-free conditions or with solvents or in reflux reactions, the improvement was evidenced for shorter times of reaction and higher yields (compares entries 4/5; 3/10; 1/8; 2/9). Another important finding was that when the MW potency is raised in the presence of tBuOK (entries 5–7), shorter times and higher yields were obtained (Table 1).![Synthesis of pyrazolo[4′,3′:5,6]pyrido[2,3-d]pyrimidine derivatives 5.](/content/184/2016/9/3/img/10.1016_j.arabjc.2015.03.002-fig3.png)
Synthesis of pyrazolo[4′,3′:5,6]pyrido[2,3-d]pyrimidine derivatives 5.
Entry
Solvent
Conditions
Time (min)
Yield (%)
1
Ethanol
Reflux
540
30
2
DMF
Reflux
480
31
3
DMF
MW (80 °C, 100 W)
11
36
4
Solvent-free
MW (100 °C, 150 W)
10
41
5
Solvent-free
tBuOK, MW (100 °C, 150 W)
8
52
6
Solvent-free
tBuOK, MW (100 °C, 200 W)
6
59
7
Solvent-free
tBuOK, MW (100 °C, 250 W)
5
61
8
Ethanol
tBuOK, Reflux
240
36
9
DMF
tBuOK, Reflux
210
40
10
DMF
tBuOK, MW (80 °C, 100 W)
9
48
It is worth mentioning that the lower yields were obtained when using 2-cyanopyridine in all cases (Table 2).
Compound
R
Product
Time (min)
Yield (%)
5a
Cl
Pyridin-4-yl
5
61
5b
Cl
Pyridin-3-yl
1
57
5c
Cl
Pyridin-2-yl
1
42
5d
OCH3
Pyridin-4-yl
2
60
5e
OCH3
Pyridin-3-yl
3
61
5f
OCH3
Pyridin-2-yl
2
42
5g
CH3
Pyridin-4-yl
5
62
5h
CH3
Pyridin-3-yl
6
56
5i
CH3
Pyridin-2-yl
3
48
5j
3,4-OCH2O
Pyridin-4-yl
5
59
5k
3,4-OCH2O
Pyridin-3-yl
5
50
Formation of the pyrazolo[4′,3′:5,6]pyridine[2,3-d]pyrimidine system was unequivocally established by NMR data of the products. The chemical shifts and multiplicities of the protons were in accordance with the expected values. For example signals for the protons of the phenyl and pyridine rings of compounds 5 were found between 7.05 and 9.60 ppm. The signal for NH2 appears as a broad singlet between 5.12 and 5.40 ppm, and the signals of the protons of CH3 appear as singlets between 1.70 and 1.90 ppm.
A possible mechanism of this cyclo-condensation reaction is outlined in Scheme 3. Presumably, the initial step is the addition of the amino group of the o-aminonitrile 3 to the nitrile group of the cyanopyridine 4 to amidine intermediate 6; the final step should be the amine–nitrile intramolecular condensation in 6 to afford 5a–k (Scheme 3).
Possible mechanistic route for the synthesis of compounds 5.
2.2 Antifungal activity
In order to have a look into the potential usefulness of these compounds as hits and heads of series for the development of antifungal drugs, we investigated the antifungal properties of compounds 5a–5k against two clinical important fungal species, Cryptococcus neoformans and Candida albicans. At first we used standardized strains of the American Type Culture Collection (ATCC) as the targets for testing antifungal activity and then, the most active compounds were tested against an expanded panel of clinical isolates in order to know the actual activity of the selected compounds against not only fungi from culture collections but from patients with fungal infections.
Results were expressed as the percentages of inhibition of each fungus in the range 250–0.98 μg/mL by using the standardized microbroth dilution method M-27A3 of Clinical and Laboratory Standards Institute (CLSI document, 2008), which assures confident and reproducible results.
The selection of C. neoformans was due to the fact that this opportunistic fungus is the main cause of cryptococcal meningoencephalitis, which has a high mortality rate among patients with profoundly impaired infections (Trpkovié et al., 2012). Even though new antifungal drugs have been developed in recent years, the availability of antifungal agents with anticryptococcal activity is still limited and sometimes the strains develop quickly resistance (Perkins et al., 2005). This scenario has motivated the search of new compounds that present antifungal properties against this fungus (Aguiar et al., 2012).
In turn, C. albicans is the fourth leading cause of nosocomial bloodstream infection (BSI) in intensive care units, causing fatal invasive candidiasis in a high percentage of patients (Pfaller and Diekema, 2007). As a consequence, new chemical structures with anticandidal activities are highly welcome.
For a more comprehensive analysis of the results, we grouped the compounds in two series: series (i) includes compounds with different pyridinyl moieties (pyridin-4-yl, 3-yl and 2-yl) and same R (Cl, OCH3 CH3 or 3,4-OCH2O) which allowed to have a look on the influence of the position of the N of the pyridinyl moiety on the antifungal activity; and series (ii) that includes compounds with same pyridinyl moiety (pyridin-4-yl, 3-yl or 2-yl) but different R (Cl, OCH3 CH3 and 3,4-OCH2O) which allowed to analyze the role played by the different R substituents in the antifungal activity. Compounds of series (i) were sub-divided in four sub-groups: (i.1) with R = Cl (5a, 5b, 5c); (i.2) with R = OCH3 (5d, 5e, 5f); (i.3) with R = CH3 (5g, 5h, 5i) and (i.4) with R = 3,4-OCH2O (5j, 5k). Comparative growth inhibition percentages of the compounds of each sub-group can be observed in Fig. 2.![Comparative antifungal activities of compounds of series (i) with different pyridin-yl moieties and similar R in position 4: (i.1) 5a, 5b and 5c; (i.2) 5d, 5e and 5f; (i.3) 5g, 5h and 5i; or in 3,4 (i.4): 5j and 5k against Cryptococcus neoformans [C.n.] or Candida albicans [C.a]. Amphotericin B (Amp) inhibits 100% growth at 1.0 μg/mL against C. albicans and 0.5 μg/mL against C. neoformans (curves of Amp are not included).](/content/184/2016/9/3/img/10.1016_j.arabjc.2015.03.002-fig5.png)
Comparative antifungal activities of compounds of series (i) with different pyridin-yl moieties and similar R in position 4: (i.1) 5a, 5b and 5c; (i.2) 5d, 5e and 5f; (i.3) 5g, 5h and 5i; or in 3,4 (i.4): 5j and 5k against Cryptococcus neoformans [C.n.] or Candida albicans [C.a]. Amphotericin B (Amp) inhibits 100% growth at 1.0 μg/mL against C. albicans and 0.5 μg/mL against C. neoformans (curves of Amp are not included).
In Fig. 2, sub-group i.1 (R = Cl) the highest activity was displayed by 5a which possesses a pyridin-4-yl moiety. Instead, in sub-groups i.2 and i.4 (R = OCH3 and —OCH2O— respectively) compounds with 4-pyridinyl moiety showed the lowest activity and compounds with 3-pyridinyl (5e, 5k) and 2-pyridinyl (5f) moieties were the most active ones. Compounds of group i.3 show dissimilar behavior against C. albicans and C. neoformans. From these results it is clear that the position of the N in pyridinyl moiety per se, does not play a role in the activity since i.e. compounds with pyridin-4-yl moieties are not the most active structures within each sub-series against C. albicans or C. neoformans. The same can be observed for compounds with pyridin-3-yl or 2-yl moieties (see Table 3 for values of the inhibition percentages).
Sub-series
Moiety
R
Cp
Concentrations in μg/mL
250
125
62.5
31.25
15.62
7.81
3.9
C. neoformans ATCC 32264
i.1
4-pyridinyl
Cl
5a
91.7 ± 2.8
57.3 ± 0.7
49.5 ± 1.6
13.75 ± 1.8
2.17 ± 0.4
0
0
3-pyridinyl
Cl
5b
17.5 ± 2.7
16.9 ± 1.8
14.0 ± 1.8
8.4 ± 1.3
3.4 ± 1.7
0
0
2-pyridinyl
Cl
5c
81.6 ± 1.9
48.6 ± 0.38
41.1 ± 0.5
18.8 ± 0.31
1.21 ± 0.79
0
0
i.2
4-pyridinyl
OCH3
5d
29.9 ± 1.9
19.7 ± 0.6
13.2 ± 1.08
9.04 ± 1.03
0
0
0
3-pyridinyl
OCH3
5e
72.5 ± 0.9
20.7 ± 0.4
19.9 ± 1.21
19.7 ± 1.2
7.9 ± 1.4
6.2 ± 1.3
3.6 ± 1.5
2-pyridinyl
OCH3
5f
79.7 ± 1.8
51.3 ± 1.5
33.2 ± 1.2
26.1 ± 1.9
19.3 ± 1.9
14.1 ± 1.1
6.9 ± 0.5
i.3
4-pyridinyl
CH3
5g
57.3 ± 1.1
33.09 ± 0.3
23.9 ± 0.3
0
0
0
0
3-pyridinyl
CH3
5h
24.7 ± 1.4
7.9 ± 2.1
7.2 ± 1.9
6.8 ± 1.9
0
0
0
2-pyridinyl
CH3
5i
64.5 ± 2.9
44.6 ± 1.6
35.6 ± 0.9
34.1 ± 1.9
22.9 ± 1.5
19.3 ± 1.1
9.4 ± 1.4
i.4
4-pyridinyl
—OCH2O—
5j
12.9 ± 1.1
10.5 ± 0.9
5.9 ± 0.5
4.7 ± 0.3
0
0
0
3-pyridinyl
—OCH2O—
5k
64.5 ± 1.5
62.5 ± 1.0
42.8 ± 0.6
40.7 ± 1.1
31.4 ± 1.4
19.5 ± 1.1
0
Amphotericin B at 2 μg/mL
100
100
100
100
100
100
100
C. albicans ATCC 10231
i.1
4-pyridinyl
Cl
5a
78.3 ± 0.3
31.0 ± 1.9
18.1 ± 1.7
12.9 ± 1.2
6.9 ± 1.6
5.7 ± 1.4
0
3-pyridinyl
Cl
5b
1.85 ± 0.1
0
0
0
0
0
0
2-pyridinyl
Cl
5c
8.12 ± 0.7
4.3 ± 0.4
2.3 ± 0.7
1.6 ± 0.4
1.1 ± 0.1
0
0
i.2
4-pyridinyl
OCH3
5d
7.3 ± 1.2
3.0 ± 0.6
2.1 ± 0.5
0
0
0
0
3-pyridinyl
OCH3
5e
62.2 ± 2.3
27.1 ± 1.1
16.5 ± 1.9
12.6 ± 0.1
0
0
0
2-pyridinyl
OCH3
5f
63.8 ± 2.0
23.7 ± 1.8
15.1 ± 0.4
9.4 ± 0.9
6.2 ± 0.7
1.4 ± 0.1
1.4 ± 0.2
i.3
4-pyridinyl
CH3
5g
76.9 ± 1.3
25.4 ± 1.5
24.1 ± 1.3
3.4 ± 0.5
0
0
0
3-pyridinyl
CH3
5h
58.5 ± 1.3
31.2 ± 1.8
18.4 ± 1.7
17.7 ± 2.8
11.1 ± 1.4
6.3 ± 1.1
5.3 ± 0.4
2-pyridinyl
CH3
5i
11.0 ± 0.6
3.2 ± 0.9
1.3 ± 0.1
0
0
0
0
i.4
4-pyridinyl
—OCH2O—
5j
22.1 ± 1.5
11.3 ± 0.6
2.9 ± 0.1
1.7 ± 0.1
0.3 ± 0.1
0
0
3-pyridinyl
—OCH2O—
5k
50.2 ± 2.1
34.9 ± 1.7
16.2 ± 0.3
7.0 ± 0.1
3.2 ± 0.2
0
0
Amphotericin B
100
100
100
100
100
100
100
Then, we tried to investigate the role (if any) played by the different R substituents in the activity. So, we compared compounds with same pyridinyl moiety (pyridin-4-yl, 3-yl or 2-yl) but different R (Cl, OCH3 CH3 and 3,4-OCH2O) [series (ii)] as follows: in Fig. 3, ii.1 we compared the compounds with pyridin-4-yl moiety but different R: 5a (Cl), 5d (OCH3), 5g (CH3) and 5j (OCH2O) against each fungi; in ii.2, compounds with pyridin-3-yl moiety 5b, 5e, 5h and 5k and in ii.3, compounds with pyridin-2-yl moiety 5c, 5f and 5i.![Comparative antifungal activities of compounds of series (ii) with different pyridin-yl moiety and similar 4-R: (ii.1) compounds with pyridine-4-yl and Cl (5a), OCH3 (5d), CH3 (5g) and 3,4-OCH2O (5j); (ii.2) compounds with pyridine-3-yl moiety 5b (Cl), 5e (OCH3), 5h (CH3) and 5k (3,4-OCH2O); (ii.3) compounds with pyridine-2-yl moiety 5c (Cl); 5f (OCH3); 5i (CH3) against Cryptococcus neoformans [C.n.] or Candida albicans [C.a.]. Amphotericin B inhibits 100% at 1.0 μg/mL against C. albicans and 0.5 μg/mL against C. neoformans.](/content/184/2016/9/3/img/10.1016_j.arabjc.2015.03.002-fig6.png)
Comparative antifungal activities of compounds of series (ii) with different pyridin-yl moiety and similar 4-R: (ii.1) compounds with pyridine-4-yl and Cl (5a), OCH3 (5d), CH3 (5g) and 3,4-OCH2O (5j); (ii.2) compounds with pyridine-3-yl moiety 5b (Cl), 5e (OCH3), 5h (CH3) and 5k (3,4-OCH2O); (ii.3) compounds with pyridine-2-yl moiety 5c (Cl); 5f (OCH3); 5i (CH3) against Cryptococcus neoformans [C.n.] or Candida albicans [C.a.]. Amphotericin B inhibits 100% at 1.0 μg/mL against C. albicans and 0.5 μg/mL against C. neoformans.
From Fig. 3 (whose percentages of inhibition can be queried in Table 4), we see that within compounds of group ii.1 5a, 5d, 5g and 5j those with R = Cl (5a) and R = CH3 (5g) (Fig. 3, ii.1) showed the best activity (91.7% and 57.3% inhibition growth against C. neoformans and 78.3% and 76.9% against C. albicans). Since chloro and methyl groups have nearly the same volume, the size of the substituent and not its electronic properties would seem to play a role in the antifungal activity in this ii.1 group. In contrast, within compounds of group ii.2 with pyridin-3-yl moiety, those with R = OCH3 (5e) and OCH2O (5k) showed the best activity against both fungi (72.5% and 64.5% against C. neoformans and 62.2% and 50.2% against C. albicans). The methylated derivative 5h also showed good activity but only against C. albicans. Within group ii.3, the substituents appear not to influence the antifungal activity since 5c, 5f and 5i show almost the same activity against C. neoformans and the behavior is dissimilar with C. albicans.
Compound
Log P
Dipole (D)
C.n.1 (% Inh)
C.a.1 (% Inh)
5a
7.27
7.6032
91.7
78.3
5b
7.27
6.9140
17.5
1.8
5c
7.69
7.1909
81.6
8.1
5d
6.58
8.4704
29.9
7.2
5e
6.58
9.2600
72.5
62.1
5f
7.01
9.2013
79.7
63.7
5g
7.20
8.5322
57.3
76.9
5h
7.20
7.9098
24.7
58.4
5i
7.62
8.6168
64.5
11.0
5j
6.49
7.4864
12.9
22.0
5k
6.46
8.9442
64.4
50.3
To deepen this analysis, the quantitative parameters log P and dipole moment (D) of each compound (5a–5k) were calculated and correlated with the activity. Both parameters were calculated using quantum mechanical at semiempirical level using Mopac, with the parametric method 3 (PM3). The molecular modeling was prepared using CS Chem-Office Software version 9.0 (Cambridge software) (C.S. Chem office, 2005). It is known that log P (logarithm of the partition coefficient in a biphasic system, e.g. n-octanol/water) describes the macroscopic hydrophobicity of a molecule which is a factor that many times determines its ability to penetrate the membranes of fungal cells and to reach the interacting sites, thus influencing the antifungal activity of compounds (Voda et al., 2004). D, that is the measure of net molecular polarity, tell us about the charge separation in a molecule. The larger the difference in electronegativities of bonded atoms, the larger the dipole moment.
Table 4 shows log P and D for all compounds tested along with the percentages of inhibition against both fungi at 250 μg/mL.
To determine if the log P has some influence in the activity, this parameter was plotted vs antifungal activity (against C. neoformans and C. albicans) in Fig. 4.
Log P vs inhibition percentage of Cryptococcus neoformans (left) and C. albicans (right) growth, by 5a–k at 250 μg/mL.
From Fig. 4, it is clear that log P, that is to say the lipophilicity of compounds, has no relationship with the activity, since compounds with the same log P such as 5a and 5b display completely different activities against both C. neoformans and C. albicans. Another clear example is the comparison of activities of 5e and 5d, which have almost the same log P and different activities.
In turn, to determine if D has some influence in the activity, D was plotted vs antifungal activity (against C. neoformans and C. albicans) in Fig. 5.
Dipole (D) vs inhibition percentage of the C. neoformans (left) and C. albicans (right) growth by 5a–k each at 250 μg/mL.
In Fig. 5, we can observe that there is not a net correlation between D and antifungal activity, but it is observed that most active compounds (% inhibition >50%) have a D ⩾ 7.5, mainly against C. albicans.
From Table 3 and both Figs. 4 and 5, it is clear that compound 5a showed the best activity against both fungi inhibiting more than 90% of the growth of C. neoformans and 78% of C. albicans and therefore, this compound deserves further attention.
2.2.1 Second order studies with clinical isolates
In order to gain insight into the potential of 5a not only against standardized strains but on clinical isolates of medical important fungi, 5a was tested at 250, 125, 62.5, 31.2 and 16.2 μg/mL against an extended panel of C. albicans and C. neoformans strains isolated from immunocompromised patients suffering fungal infections. Results are recorded in Table 5. For the sake of comparison, standardized strains of C. albicans ATCC 10231 and C. neoformans ATCC 32264 are included in the table. Ca: Candida albicans; Cn: Cryptococcus neoformans; IM: Instituto Malbrán, Buenos Aires; ATCC: American Type Culture Collection, Manassas, USA; CCC: Reference Center in Mycology, Rosario, Argentina. Amp = Amphotericin B.
Inhibition % of compound 5a
Amp
Strain
250 μg/mL
125 μg/mL
62.5 μg/mL
331.2 μg/mL
15.6 μg/mL
1
Ca
ATCC10231
78.3 ± 0.3
31.0 ± 1.9
18.1 ± 1.7
12.9 ± 1.2
6.9 ± 1.6
100
Ca
CCC 126
80.1 ± 0.4
58.2 ± 2.1
34.2 ± 1.5
14.3 ± 0.2
5.3 ± 0.6
100
Ca
CCC 127
70.8 ± 2.3
45.4 ± 1.0
32.7 ± 1.2
10.7 ± 0.8
4.2 ± 1.0
100
Ca
CCC 128
73.3 ± 0.8
55.0 ± 1.6
46.3 ± 1.4
17.4 ± 0.7
12.2 ± 1.3
100
Ca
CCC 129
84.3 ± 1.2
62.5 ± 1.1
50.7 ± 1.4
33.9 ± 0.2
10.9 ± 2.0
100
Ca
CCC 130
80.2 ± 1.2
53.2 ± 0.4
44.7 ± 1.3
22.5 ± 0.7
12.6 ± 1.3
100
Cn
ATCC 32264
91.7 ± 2.8
57.3 ± 0.7
49.5 ± 1.6
13.7 ± 1.8
2.1 ± 0.4
100
Cn
IM 983040
94.3 ± 1.4
68.3 ± 3.4
54.9 ± 2.3
23.4 ± 1.2
5.2 ± 0.7
100
Cn
IM 972724
97.8 ± 2.4
77.2 ± 2.3
35.5 ± 3.3
13.1 ± 1.3
0.3 ± 0.1
100
Cn
IM 042074
84.4 ± 1.7
76.8 ± 1.5
55.9 ± 2.6
33.6 ± 1.8
7.8 ± 1.2
100
Cn
IM 983036
92.3 ± 1.3
80.4 ± 1.0
52.4 ± 1.3
32.5 ± 2.4
12.3 ± 1.8
100
Cn
IM 00319
88.3 ± 1.2
68.5 ± 1.6
53.7 ± 1.4
39.3 ± 0.2
16.9 ± 2.5
100
Cn
IM 972751
83.4 ± 1.9
54.5 ± 1.5
22.7 ± 0.4
13.7 ± 1.3
6.7 ± 0.4
100
Cn
IM 031631
74.3 ± 1.5
55.8 ± 3.1
35.1 ± 1.9
15.2 ± 0.2
5.7 ± 0.2
100
Cn
IM 031706
87.5 ± 2.1
46.5 ± 1.2
37.1 ± 2.2
28.8 ± 1.2
12.3 ± 1.2
100
Cn
IM 961951
79.7 ± 2.8
69.5 ± 2.9
45.6 ± 1.2
20.9 ± 1.2
9.9 ± 0.3
100
Cn
IM 052470
88.2 ± 1.7
76.3 ± 1.1
45.3 ± 1.5
31.2 ± 0.3
14.7 ± 1.5
100
As it can be observed in Table 5, compound 5a exerts more than 80% of inhibition on 3 out the 5 C. albicans strains at 250 μg/mL, and more than 50% inhibition in 4 of the 5 strains at 125 μg/mL. Moreover, compound 5a produces more than 80% inhibition on 8/10 isolates of C. neoformans at 250 μg/mL, more than 50% inhibition in 9/10 isolates at 125 μg/mL and in 4/10 strains at 62.5 μg/mL, clearly indicating that 5a is a better inhibitor of C. neoformans than of C. albicans.
3 Conclusion
In this article we described the microwave-assisted synthesis of novel pyrazolo[4′,3′:5,6]pyrimido[2,3-d]pyrimidines 5 under solvent-free conditions. The described synthesis represents a versatile, practical and environmental friendly method for the preparation of compounds 5 with the advantages of easy work-up, mild reaction conditions and good yields. Regarding the antifungal activity, several compounds showed moderate activity against C. albicans and C. neoformans, being 5a the most active compound. Analysis of the antifungal activity of properly grouped compounds allowed to determine that the position of the N in the pyrimidyl moiety per se does not play a role in the activity. In turn, the type of 4-R substituent appears to play a role in the activity. Within compounds with pyridin-4-yl moiety (5a, 5d, 5g and 5j) those with R = Cl (5a) and R = CH3 (5g) showed the best activity. In contrast, within compounds with pyridin-3-yl moiety, those with R = OCH3 (5e) and OCH2O (5k) showed the highest inhibition percentage against both fungi. Within compounds with pyridin-2-yl, the substituents appear not to exert any influence in the antifungal activity. In addition to the above considerations, the lipophilicity of compounds measured as log P showed to be not related to the activity and there is not observed a net correlation between D and antifungal activity, although it is observed that the most active compounds (% inhibition >50%) have a D ⩾ 7.5, mainly against C. albicans.
4 Experimental
Commercially available starting materials, reagents and solvents were used as supplied. The TLC analysis was performed on Merck TLC-plates aluminum silica gel 60 F254. Melting point was measured using a Büchi melting point apparatus and was uncorrected. Microwave reactions were performed in glass vessels (10 mL) using a CEM Focused Microwave Synthesis System™ apparatus, Model Discover, with power output from 0 to 300 W. The IR analysis was performed on a Shimadzu FTIR 8400 spectrophotometer in KBr disks. 1H and 13C NMR spectra were run on a Bruker DPX 400 spectrometer operating at 400 MHz and 100 MHz respectively, using dimethyl sulfoxide-d6 as solvent and tetramethylsilane as internal standard. Mass spectra were obtained from Shimadzu GCMS-QP 2010 spectrometer (equipped with a direct inlet probe) operating at 70 eV. Elemental analysis was carried out using a Thermo Finnigan Flash EA1112 CHN (STIUJA) elemental analyzer.
4.1 General procedure for the preparation of pyrazolo[4′,3′:5,6]pyrido[2,3-d]pyrimidines 5
All experiments were carried out using a focused microwave reactor (CEM Discover TM). A mixture of ortho-aminonitrile 3 (0.3 mmol), cyanopyridine 4 (0.4 mmol) and tBuOK (10 mol%), was exposed to microwave irradiation from 1 to 6 min at 100 °C, a power of 250 W and 30 PSI of pressure. Then, the reaction mixture was treated with ethanol and the excess of solvent was removed under reduced pressure. Purification of products was performed using column chromatography in a mixture CHCl3/EtOH (20:1) as eluent.
4.1.1 4-(4-Chlorophenyl)-3-methyl-1-phenyl-7-(pyridin-4-yl)-1H-pyrazolo[4′,3′:5,6]pyrido[2,3-d]pyrimidin-5-amine 5a
Yellow solid, yield 61%, mp > 350. FTIR (KBr) υ(cm−1): 3496 (NH), 3038 (⚌C—H), 1600, 1569 (C⚌N and C⚌C). 1H NMR (400 MHz, DMSO-d6) δ: 1.78 (s, 3H, CH3), 5.13 (brs, 2H, NH2), 7.36 (t, 1H, J = 7.3 Hz, HAp), 7.61 (t, 2H, J = 7.7 Hz, HAo,), 7.76–7.86 (m, 4H, HAm, HBm), 8.29 (d, 2H, J = 4.6 Hz, H-2Py), 8.33(d, 2H, J = 8.1 Hz, HBo), 8.75 (d, 2H, J = 4.7 Hz, H-3Py). 13C NMR (100 MHz DMSO-d6) δ: 14.5 (CH3), 103.6 (C), 116.8 (C), 121.1 (CH), 122.6 (CH), 126.1 (CH), 129.6 (CH), 130.0 (CH), 131.0 (CH), 134.3 (C), 135.7 (C), 139.4 (C), 145.1 (C), 145.2 (C), 145.6 (C), 150.6 (CH), 152.6 (C), 159.6 (C), 162.4 (C), 164.9 (C). HR-MS calcd for C26H18ClN7 463.1312, found [M+ + K] 501.7842. [M++H] 463.8283. Anal. Calcd for C26H18ClN7·H2O: C, 64.90; H, 4.08; N, 20.34; found: C, 65.13; H, 3.82; N, 20.38.
4.1.2 4-(4-Chlorophenyl)-3-methyl-1-phenyl-7-(pyridin-3-yl)-1H-pyrazolo[4′,3′:5,6]pyrido[2,3-d]pyrimidin-5-amine 5b
Yellow solid, yield 57%, mp: 317–318. FTIR (KBr) υ(cm−1): 3470 (NH), 3058 (⚌C—H), 1550, 1510 (C⚌N and C⚌C). 1H NMR (400 MHz, DMSO-d6) δ: 1.74 (s, 3H, CH3), 5.10 (brs, 2H, NH2), 7.35 (t, 1H, J = 7.4 Hz HAp), 7.51–7.56 (m, 1H, H-2Py), 7.60 (t, 2H, J = 7.9 Hz, HAo), 7.79 (s, 4H, HAm, HBm), 8.33 (d, 2H, J = 7.9 Hz, HBo), 8.68–8.73 (m, 2H, H-3Py, H-4Py). 9.55 (s, 1H, H-5Py) 13C NMR (100 MHz DMSO-d6) δ: 14.5 (CH3), 103.4 (C), 116.6 (C), 121.2 (CH), 123.8 (CH), 126.2 (CH), 129.5 (CH), 130.0 (CH), 130.1 (CH), 133.8 (C), 134.8 (C), 135.7 (C), 136.0 (CH), 139.5(C), 145.0 (C), 145.2 (C), 150.2 (CH), 151.8 (CH), 152.3 (C), 159.7 (C), 162.7 (C), 164.7 (C). HR-MS calcd for C26H18ClN7 463.1312, found [M+ + K] 501.8709. [M++H] 463.9121.
4.1.3 4-(4-Chlorophenyl)-3-methyl-1-phenyl-7-(pyridin-2-yl)-1H-pyrazolo[4′,3′:5,6]pyrido[2,3-d]pyrimidin-5-amine 5c
Yellow solid, yield 42%, mp > 350. FTIR (KBr) υ(cm−1): 3485 (NH), 3040 (⚌C—H), 1574, 1547 (C⚌N and C⚌C). 1H NMR (400 MHz, DMSO-d6) δ: 1.70 (m, 3H, CH3), 7.36 (t, 1H, J = 7.3 Hz, HAp), 7.52–7.56 (m, 2H, HAo), 7.61–7.63 (m, 4H, HAm, HBm), 8.37 (d, 2H, J = 7.8 Hz, HBo), 8.58–8.63 (m, 2H, H-2Py, H-3Py), 8.83–8.70 (m, 2H, H-4Py, H-5Py). Not observed (brs, 2H, NH2). Compound 5c is barely soluble in dimethyl sulfoxide or any other solvent normally used for NMR spectroscopy; thus, made the registration of a high resolution 13C NMR spectrum impossible. HR-MS calcd for C26H18ClN7 463.1312, found [M+ + Na] 486.1490. [M+ + H] 463.3628.
4.1.4 4-(4-Methoxyphenyl)-3-methyl-1-phenyl-7-(pyridin-4-yl)-1H-pyrazolo[4′,3′:5,6]pyrido [2,3-d]pyrimidin-5-amine 5d
Yellow solid, yield 60%, mp: 332–333. υ(cm−1): 3494 (NH), 3044 (⚌C—H), 1620, 1574 (C⚌N and C⚌C). 1H NMR (400 MHz, DMSO-d6) δ: 1.90 (s, 3H, CH3), 3.94 (s, 3H, OCH3), 5.13 (brs, 2H, NH2), 7.28 (d, 2H, J = 8.6 Hz, HBo), 7.35 (t, 1H, J = 7.4 Hz, HAp), 7.55–7.63 (m, 4H, HAm, HBm), 8.32 (d, 2H, J = 5.9 Hz, H-2Py), 8.36 (d, 2H, J = 7.8 Hz, HAo), 8.75 (d, 2H, J = 5.9 Hz, H-3Py). 13C NMR (100 MHz DMSO-d6) δ: 14.4 (CH3), 56.1 (OCH3), 104.0 (C), 115.6 (CH), 117.6 (C), 121.1 (CH), 122.6 (CH), 126.1 (CH), 127.4 (C), 129.5 (CH), 130.0 (CH), 139.6 (C), 145.4 (C), 145.8 (C), 146.8 (C), 150.6 (CH), 152.4 (C), 159.7 (C), 161.3 (C), 162.4 (C), 165.1 (C). HR-MS calcd for C27H21N7O 459.1808, found [M+ + K] 498.9195. [M+ + H] 459.9754.
4.1.5 4-(4-Methoxyphenyl)-3-methyl-1-phenyl-7-(pyridin-3-yl)-1H-pyrazolo[4′,3′:5,6]pyrido [2,3-d]pyrimidin-5-amine 5e
Yellow solid, yield 61%, mp: 306–307. FTIR (KBr) υ(cm−1): 3468 (NH), 3056 (⚌C—H), 1575, 1508 (C⚌N and C⚌C). 1H NMR (400 MHz, DMSO-d6) δ: 1.81 (s, 3H, CH3), 3.92 (s, 3H, OCH3), 5.26 (brs, 2H, NH2), 7.28 (d, 2H, J = 8.4 Hz, HBo), 7.36 (t, 1H, J = 7.3 Hz, HAp), 7.51–7.58 (m, 1H, H-3Py) 7.58–7.67 (m, 4H, HAm, HBm), 8.35 (d, 2H, J = 8.0 Hz, HAo), 8.73 (d, 2H, J = 7.3 Hz, H-2Py, H-4Py), 9.57 (s, 1H, H-6Py). 13C NMR (100 MHz DMSO-d6) δ: 14.4 (CH3), 56.1 (OCH3), 103.8 (C), 115.6 (CH), 117.0 (C), 121.1 (CH), 123.7 (CH), 126.1 (CH), 127.5 (C), 129.5 (CH), 130.3 (CH), 133.9 (C), 136.0 (CH), 139.6 (C), 145.4 (C), 146.7 (C) 150.2 (CH), 151.7 (CH), 152.4 (C), 159.7 (C), 161.3 (C), 162.7 (C), 164.9 (C). HR-MS calcd for C27H21N7O 459.1808, found [M+ + K] 498.8764. [M+ + H] 459.9340.
4.1.6 4-(4-Methoxyphenyl)-3-methyl-1-phenyl-7-(pyridin-2-yl)-1H-pyrazolo[4′,3′:5,6]pyrido [2,3-d]pyrimidin-5-amine 5f
Yellow solid, yield 42%, mp > 350. FTIR (KBr) υ(cm−1): 3484 (NH), 3055 (⚌C—H), 1579, 1547 (C⚌N and C⚌C). 1H NMR (400 MHz, DMSO-d6) δ: 1.86 (s, 3H, CH3), 3.94 (s, 3H, OCH3), 7.27 (d, 2H, J = 8.1 Hz, HBo), 7.35 (t, 1H, J = 7.6 Hz, HAp), 7.53 (t, 1H, J = 6.2 Hz, H-4Py) 7.61 (d, 4H, J = 7.8 Hz, HAm, HBm), 7.97 (t, 1H, J = 7.8 Hz, H-3Py), 8.39 (d, 2H, J = 8.1 Hz, HAo), 8.48 (d, 1H, J = 7.9 Hz, H-2Py), 8.73 (d, 1H, 4.6 Hz, H-5Py). Not observed (brs, 2H, NH2). Compound 5f is barely soluble in dimethyl sulfoxide or any other solvent normally used for NMR spectroscopy; thus, made the registration of a high resolution 13C NMR spectrum impossible. HR-MS calcd for C27H21N7O 459.1808, found [M+ + K] 498.9088. [M+ + H] 459.9754.
4.1.7 3-Methyl-1-phenyl-7-(pyridin-4-yl)-4-(p-tolyl)-1H-pyrazolo[4′,3′:5,6]pyrido[2,3-d]pyrimidin-5-amine 5g
Yellow solid, yield 62%, mp: 339–340. FTIR (KBr) υ(cm−1): 3498 (NH), 3042 (⚌C—H), 1589, 1559 (C⚌N and C⚌C). 1H NMR (400 MHz, DMSO-d6) δ:1.82 (s, 3H, CH3), 5.22 (brs, 2H, NH2), 7.36 (t, 1H, J = 7.3 Hz, HAp), 7.49–7.72 (m, 6H, HBo, HAm, HBm), 8.31 (d, 2H, J = 5.2 Hz, H-3Py), 8.35 (d, 2H, J = 8.0 HAo), 8.76 (d, 2H, J = 5.1 Hz, H-2Py). 13C NMR (100 MHz DMSO-d6) δ: 14.3 (CH3), 21.4 (CH3), 103.8 (C), 116.9 (C), 121.1 (CH), 122.6 (CH), 126.1 (CH), 128.7 (CH), 129.5 (CH), 130.5 (CH), 132.7 (C), 139.6 (C), 140.4 (C), 145.4 (C), 145.8 (C), 146.8 (C), 150.6 (CH), 152.4 (C), 159.7 (C), 162.5 (C), 165.1 (C). HR-MS calcd for C27H22N7 443.1858, found [M+ + K] 482.8695. [M+ + H] 443.9281.
4.1.8 3-Methyl-1-phenyl-7-(pyridin-3-yl)-4-(p-tolyl)-1H-pyrazolo[4′,3′:5,6]pyrido[2,3-d]pyrimidin-5-amine 5h
Yellow solid, yield 56%, mp: 305–306. FTIR (KBr) υ(cm−1): 3468 (NH), 3054 (⚌C—H), 1564, 1547 (C⚌N and C⚌C). 1H NMR (400 MHz, DMSO-d6) δ: 1.81 (s, 3H, CH3), 5.21 (brs, 2H, NH2), 7.37 (t, 1H, J = 7.3 Hz, HAp), 7.58–7.66 (m, 7H, HBo, HAm, HBm, H-3Py), 8.35 (d, 2H, J = 8.0 Hz, HAo), 8.69–8.78 (m, 2H, H-2Py, H-4Py), 9.58 (s, 1H, H-6Py) 13C NMR (100 MHz DMSO-d6) δ: 14.5 (CH3), 21.5 (CH3), 103.4 (C), 116.6 (C), 120.9 (CH), 124.1 (CH), 126.1 (CH), 128.6 (CH), 129.7 (CH), 130.5 (CH), 132.6 (C), 133.5 (C), 136.1 (CH), 139.3 (C), 140 (C), 145.4 (C), 146.9 (C), 150.0 (CH), 151.9 (CH), 152.0 (C), 159.5 (C), 162.5 (C), 164.7 (C). EI MS (70 eV): m/z: 443(M+, 18), 354(16), 236 (17). Anal. Calcd for C27H21N7: C, 73.12; H, 4.77; N, 22.11; found: C, 73.07; H, 4.72; N, 22.06.
4.1.9 3-Methyl-1-phenyl-7-(pyridin-2-yl)-4-(p-tolyl)-1H-pyrazolo[4′,3′:5,6]pyrido[2,3-d]pyrimidin-5-amine 5i
Yellow solid, yield 48%, mp > 350. FTIR (KBr) υ(cm−1): 3481 (NH), 3057 (⚌C—H), 1569, 1544 (C⚌N and C⚌C). 1H NMR (400 MHz, DMSO-d6): 1.86 (s, 3H), 7.36 (t, 1H, J = 7.4 Hz, HAp), 7.52–7.57 (m, 4H, HBo, HBm), 7.95–8.01 (m, 2H, HAm), 8.03 (d, 2H, J = 7.7 Hz, H-3Py, H-4Py), 8.39 (d, 2H, J = 7.7 Hz, HAo), 8.63 (d, 2H, J = 4.6 Hz, H-2Py, H-5Py). Not observed (brs, 2H, NH2). Compound 5i is barely soluble in dimethyl sulfoxide or any other solvent normally used for NMR spectroscopy; thus, made the registration of a high resolution 13C NMR spectrum impossible. EI MS (70 eV): m/z: 443(M+, 83), 354(1), 236 (1). Anal. Calcd for C27H21N7: C, 73.12; H, 4.77; N, 22.11; found: C, 73.09; H, 4.74; N, 22.08.
4.1.10 4-(Benzo[d][1,3]dioxol-5-yl)-3-methyl-1-phenyl-7-(pyridin-4-yl)-1H-pyrazolo[4′,3′:5,6] pyrido[2,3-d]pyrimidin-5-amine 5j
Yellow solid, yield 59%, mp: 316–317. FTIR (KBr) υ(cm−1): 3488 (NH), 3045 (⚌C—H), 1570, 1559 (C⚌N and C⚌C). 1H NMR (400 MHz, DMSO-d6) δ: 1.82 (s, 3H, CH3), 5.36 (brs, 2H, NH2), 6.15 (s, 2H, OCH2O), 7.06 (d, 1H, J = 7.8 Hz, HAp), 7.16 (d, 1H, J = 7.9 Hz, HBo), 7.19–7.30 (m, 2H, HBo′, HBm), 7.51 (t, 2H, J = 7.7 Hz, HAm) 8.20–8.27 (m, 4H, H-2Py, HAo), 8.67 (d, 2H, J = 5.3 Hz, H-3Py). 13C NMR (100 MHz DMSO-d6) δ: 14.1 (CH3), 102.0 (CH2), 103.3 (C), 108.9 (CH), 109.1(CH), 116.6 (C), 120.2 (CH), 122.0 (CH), 125.6 (CH), 127.8 (C), 129.2 (CH), 138.8 (C), 144.8 (C), 145.0 (C), 145.9 (C), 148.1 (C), 148.6 (C), 150.1 (CH), 151.4 (C), 158.9 (C), 161.6 (C), 164.3 (C). EI MS (70 eV): m/z: 473(M+, 46), 369 (61), 313 (23), 236 (30). Anal. Calcd for C27H19N7O2: C, 68.49; H, 4.04; N, 20.71; found: C, 68.39; H, 4.01; N, 20.61.
4.1.11 4-(Benzo[d][1,3]dioxol-5-yl)-3-methyl-1-phenyl-7-(pyridin-3-yl)-1H-pyrazolo[4′,3′:5,6] pyrido[2,3-d]pyrimidin-5-amine 5k
Yellow solid, yield 50%, mp: 325–326. FTIR (KBr) υ(cm−1): 3478 (NH), 3058 (⚌C—H), 1571, 1518 (C⚌N and C⚌C). 1H NMR (400 MHz, DMSO-d6) δ: 1.88(s, 3H, CH3), 5.13 (brs, 2H, NH2), 6.24 (s, 2H, OCH2O), 7.16 (d, 1H, J = 9.0 Hz, HAp), 7.25 (d, 1H, J = 7.9 Hz, HBo), 7.32–7.38 (m, 2H, HBo′, HBm), 7.46–7.69 (m, 4H, H-2Py, H-3Py, HAm), 8.34 (d, 2H, J = 8.0 Hz, HAo), 8.72 (t, 2H, J = 6.1 Hz, H-4PY, H-6Py). 13C NMR (100 MHz DMSO-d6) δ: 14.0 (CH3), 101.9 (CH2), 103.1 (C), 108.9 (CH), 109.1 (CH), 116.3 (C), 120.2 (CH), 122.0 (CH), 123.5 (CH), 125.5 (CH), 127.9 (C), 129.1 (CH), 133.0 (C), 135.5 (CH), 138.8 (C), 144.8 (C), 145.9 (C), 148.1 (C), 148.6 (C), 149.5 (C), 151.4 (CH), 158.9 (C), 161.8 (C), 164.7 (C). EI MS (70 eV): EI MS: m/z: 473(M+, 100), 368 (11), 313 (12), 236 (19). Anal. Calcd for C27H19N7O2: C, 68.49; H, 4.04; N, 20.71; found: C, 68.41; H, 4.02; N, 20.62.
4.2 Biological evaluation
4.2.1 Antifungal activity
4.2.1.1 Microorganisms and media
For the antifungal evaluation, standardized strains from the American Type Culture Collection (ATCC), Rockville, MD, USA, Reference Center in Mycology (CEREMIC, CCC, Rosario, Argentina) and Instituto Malbrán (IM, Av. Velez Sarsfield 563, Buenos Aires) were used. Standardized strains: C. albicans ATCC 10231 and C. neoformans ATCC 32264; clinical isolates of C. albicans were provided by CCC and of C. neoformans were provided by IM. Voucher specimens of the isolated are presented in Table 6. Strains were grown on Sabouraud-chloramphenicol agar slants for 48 h at 30 °C, were maintained on slopes of Sabouraud-dextrose agar (SDA, Oxoid) and sub-cultured every 15 days to prevent pleomorphic transformations. Inocula were obtained according to reported procedures [29] and adjusted to 1–5 × 103 cells with colony forming units (CFU)/mL.
4.2.1.2 Fungal growth inhibition percentage determination
Broth microdilution techniques were performed in 96-well microplates according to the guidelines of the Clinical and Laboratory Standards Institute for yeasts (M27-A3) (Clinical and Laboratory Standards Institute). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts, Approved Standard [29]. For the assay, compound test wells (CTWs) were prepared with stock solutions of each compound in DMSO (maximum concentration ⩽ 1%), diluted with RPMI-1640, to final concentrations of 250–0.98 μg/mL. An inoculum suspension (100 μL) was added to each well (final volume in the well = 200 μL). A growth control well (GCW) (containing medium, inoculum, and the same amount of DMSO used in a CTW, but compound-free) and a sterility control well (SCW) (sample, medium, and sterile water instead of inoculum) were included for each fungus tested. Microtiter trays were incubated in a moist, dark chamber at 30 °C for 48 h for both yeasts. Microplates were read in a VERSA Max microplate reader (Molecular Devices, Sunnyvale, CA, USA). Amphotericin B was used as positive control. Tests were performed in triplicate. Reduction of growth for each compound concentration was calculated as follows: % of inhibition = 100 − (OD 405 CTW − OD 405 SCW)/(OD 405 GCW − OD 405 SCW). The means ± SEM were used for constructing the dose–response curves representing% inhibition vs concentration of each compound. Dose–response curves were constructed with SigmaPlot 11.0 software.
Acknowledgements
Authors wish to thank the COLCIENCIAS and Universidad del Valle for financial support.
References
- Farnesol inhibits in vitro growth of the Cryptococcus neoformans species complex with no significant changes in virulence-related exoenzymes. Vet. Microbiol.. 2012;159:375-380.
- [Google Scholar]
- A new and highly expedient synthesis of pyrido[2,3-d]pyrimidines. Tetrahedron Lett.. 2001;42:6585-6588.
- [Google Scholar]
- New targets and screening approaches in antimicrobial drug discovery. Chem. Rev.. 2005;105:759-774.
- [Google Scholar]
- Synthesis and pharmacology of pyrido[2,3-d]pyrimidinediones bearing polar substituents as adenosine receptor antagonists. Bioorg. Med. Chem. Lett.. 2006;14:2837-2849.
- [Google Scholar]
- C.S. Chem, Office, 2005. Version 9.0. Cambridge Soft Corporation, 100 Cambridge Park Drive, Cambridge, MA.
- Synthesis of the lipophilic antifolate piritrexim via a palladium(0)-catalyzed cross-coupling reaction. J. Org. Chem.. 2005;70:1364-1368.
- [Google Scholar]
- Design, synthesis, and antifolate activity of new analogues of piritrexim and other diaminopyrimidine dihydrofolate reductase inhibitors with ω-carboxyalkoxy or ω-carboxy-1-alkynyl substitution in the side chain. J. Med. Chem.. 2005;48:4420-4431.
- [Google Scholar]
- A chemoselective aniline–chloropyrimidine coupling in a competing electrophilic environment. Tetrahedron Lett.. 2008;49:102-105.
- [Google Scholar]
- CLSI, Clinical and Laboratory Standards Institute, 2008. Reference Method for Broth Dilution Antifungal Susceptibility Testing for Yeasts M27A3, 3rd ed. Approved Standard, vol. 28. Wayne Ed., Wayne (PA), No 14, pp 1–25.
- Biological profile of new apoptotic agents based on 2,4-pyrido[2,3-d]pyrimidine derivatives. Bioorg. Med. Chem.. 2007;15:1659-1669.
- [Google Scholar]
- A novel three-component one-pot synthesis of pyrano[2,3-d]pyrimidines and pyrido[2,3-d]pyrimidines using microwave heating in the solid state. Tetrahedron Lett.. 2003;44:8307-8310.
- [Google Scholar]
- Studies on uracils: a facile one-pot synthesis of oxazino[4,5-d]-, pyrano[2,3-d]-, pyrido[2,3-d]- and pyrimido[4,5-d]pyrimidines using microwave irradiation in the solid state. Tetrahedron Lett.. 2004;45:2405-2408.
- [Google Scholar]
- The Pyrido[2,3-d]pyrimidine Derivative PD180970 Inhibits p210Bcr-Abl Tyrosine Kinase and Induces Apoptosis of K562 Leukemic Cells. Cancer Res.. 2000;60:3127-3131.
- [Google Scholar]
- Synthesis and biological evaluation of 2,4-diamino-6-(arylaminomethyl)pyrido[2,3-d]pyrimidines as inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase and as antiopportunistic infection and antitumor agents. J. Med. Chem.. 2003;46:5074-5082.
- [Google Scholar]
- Structure−activity relationships for a novel series of pyrido[2,3-d]pyrimidine tyrosine kinase inhibitors. J. Med. Chem.. 1997;40:2296-2303.
- [Google Scholar]
- Synthesis of novel 6,6a,7,8-tetrahydro-5H-naphtho[1,2-e]pyrimido[4,5-b][1,4]diazepines under microwave irradiation as potential anti-tumor agents. Eur. J. Med. Chem.. 2008;43:1955-1962.
- [Google Scholar]
- Synthesis of novel 6,6a,7,8-tetrahydro-5H-naphtho[1,2-e]pyrimido[4,5-b][1,4]diazepines under microwave irradiation as potential anti-tumor agents. Eur. J. Med. Chem.. 2010;45:2841-2846.
- [Google Scholar]
- A new and highly expedient synthesis of pyrido[2,3-d]pyrimidines. Eur. J. Med. Chem.. 2006;41:1011-1016.
- [Google Scholar]
- Controlled microwave heating in modern organic synthesis. Angew. Chem. Int. Ed.. 2004;43:6250-6284.
- [Google Scholar]
- Pyrrolo[2,3-d]pyrimidines and pyrido[2,3-d]pyrimidines as conformationally restricted analogues of the antibacterial agent trimethoprim. Bioorg. Med. Chem.. 1996;4:593-602.
- [Google Scholar]
- Lunt, E., Newton, C.C., Katritzky, A.R., Rees, C.W., Boulton, A.J., Killop, A. Mc., 1984. Comprehensive Heterocyclic Chemistry, vol. 3. Pergamon Press, Oxford, 199–232 and 260–261.
- Clinical Trichophyton rubrum strain exhibiting primary resistance to terbinafine. Antimicrob. Agents Chemother.. 2003;47:82-86.
- [Google Scholar]
- Substituted pyrazolo[3,4-d]pyrimidines: microwave-assisted, solvent-free synthesis and biological evaluation. Helv. Chim. Acta. 2008;91:1336-1344.
- [Google Scholar]
- Syntheses and diuretic activity of 1,2-dihydro-2-(3-pyridyl)-3H-pyrido[2,3-d]pyrimidin-4-one and related compounds. J. Med. Chem.. 1982;25:98-102.
- [Google Scholar]
- Synthesis and structure of new pyrido[2,3-d]pyrimidine derivatives with calcium channel antagonist activity. Tetrahedron. 1994;50:8085-8098.
- [Google Scholar]
- Rates of antifungal resistance among Spanish clinical isolates of Cryptococcus neoformans var. neoformans. J. Antimicrob Chemother.. 2005;56:1144-1147.
- [Google Scholar]
- Epidemiology of invasive candidiasis: a persistent public health problem. Clin. Microbiol. Rev.. 2007;20:133-163.
- [Google Scholar]
- Syntheses and antifolate activity of 5-methyl-5-deaza analogs of aminopterin, methotrexate, folic acid, and N10-methylfolic acid. J. Med. Chem.. 1986;29:1080-1087.
- [Google Scholar]
- Synthesis and antihistaminic activity of 2-guanadino-3-cyanopyridines and pyrido[2,3-d]-pyrimidines. Bioorg. Med. Chem.. 1997;5:1543-1553.
- [Google Scholar]
- Synthesis of 6-cyanopyrido[2,3-d]pyrimidinones in the reaction of 6-amino-4-pyrimidinones with arylidene derivatives of malonodinitrile. J. Heterocyclic Chem.. 1998;35:1309-1311.
- [Google Scholar]
- Synthesis of 5-cyanopyrazolo[3,4-b]pyridines in the reaction of 5-amino-3-methyl-1-phenylpyrazole with arylidene derivatives of malonodinitrile and ethyl cyanoacetate. J. Heterocyclic Chem.. 1999;36:1311-1316.
- [Google Scholar]
- Microwave-assisted three-component synthesis and in vitro antifungal evaluation of 6-cyano-5,8-dihydropyrido[2,3-d]pyrimidin-4(3H)-ones. J. Heterocyclic Chem.. 2006;43:299-306.
- [Google Scholar]
- Microwave-assisted synthesis of pyrazolo[3,4-b]pyridine-spirocycloalkanediones by three-component reaction of 5-aminopyrazole derivatives, paraformaldehyde and cyclic β-diketones. Tetrahedron Lett.. 2010;51:4717-4719.
- [Google Scholar]
- Microwave-assisted synthesis of fused pyrazolo[3,4-b]pyrazines by the reaction of ortho-aminonitrosopyrazoles and cyclic β-diketones. Tetrahedron Lett.. 2012;53:3181-3187.
- [Google Scholar]
- Synthesis and antifolate activity of 2,4-diamino-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine analogues of trimetrexate and piritrexim. J. Heterocyclic Chem.. 1995;32:335-340.
- [Google Scholar]
- Synthesis of novel 6,6a,7,8-tetrahydro-5H-naphtho[1,2-e]pyrimido[4,5-b][1,4]diazepines under microwave irradiation as potential anti-tumor agents. Eur. J. Med. Chem.. 2009;44:3130-3137.
- [Google Scholar]
- Cyclin-dependent kinase inhibitors for treating cáncer. Med. Res. Rev.. 2001;21:487-498.
- [Google Scholar]
- In vitro antifungal activities of amphotericin B,5-fluorocytosine, fluconazole and itraconazole against Cryptococcus neoformans isolated from cerebrospinal fluid and blood from patients in Serbia. J. Med. Mycol.. 2012;22:243-248.
- [Google Scholar]
- New potential inhibitors of cyclin-dependent kinase 4: design and synthesis of pyrido[2,3-d]pyrimidine derivatives under microwave irradiation. Bioorg. Med. Chem. Lett.. 2006;16:3578-3581.
- [Google Scholar]
- An efficient chemoselective synthesis of pyrido[2,3-d]pyrimidine derivatives under microwave irradiation. Synthesis. 2008;3:369-376.
- [Google Scholar]
- A quantitative structure-antifungal activity relationship study of oxygenated aromatic essential oil compounds using data structuring and PLS regression analysis. J. Mol. Model.. 2004;10:76-80.
- [Google Scholar]
- Chemical proteomic analysis reveals alternative modes of action for pyrido[2,3-d]pyrimidine kinase inhibitors. Mol. Cell Proteomics. 2004;3:1181-1193.
- [Google Scholar]
