Hybrid Molecules Containing a 7-Chloro-4-aminoquinoline Nucleus and a Substituted 2-Pyrazoline with Antiproliferative and Antifungal Activity

Twenty-four new hybrid analogues (15–38) containing 7-chloro-4-aminoquinoline and 2-pyrazoline N-heterocyclic fragments were synthesized. Twelve of the new compounds were evaluated against 58 human cancer cell lines by the U.S. National Cancer Institute (NCI). Compounds 25, 30, 31, 36, and 37 showed significant cytostatic activity, with the most outstanding GI50 values ranging from 0.05 to 0.95 µM. The hybrid compounds (15–38) were also evaluated for antifungal activity against Candida albicans and Cryptococcus neoformans. From the obtained results some structure–activity relationships were outlined.


Introduction
The substituted 2-pyrazoline moiety (II, Scheme 1) represents a structural component of significant interest in the field of Medicinal Chemistry, due to their prominent pharmacological effects, such as antimicrobial, antimycobacterial, antifungal, antiamoebic, anti-inflammatory, analgesic, antidepressant, and anticancer activities [1][2][3]. Compounds with this moiety also possess other biological properties like Nitric Oxide Synthase (NOS) inhibition and Cannabinoid CB1 receptor antagonism, among others [4].
On the other hand, structure-activity relationship studies on 4-aminoquinolines showed that the 7-chloro-4-aminoquinoline nucleus (I, Scheme 1) that is present in pharmacologically active substances displays a broad range of biological activities [5][6][7][8]. Therefore, the incorporation of this active pharmacophore into the structure of new heterocyclic compounds might improve their biological activity.
Based on the above considerations and as a part of our current project devoted to the synthesis of novel nitrogen-containing heterocyclic compounds with biological activity [9][10][11][12][13], we decided to attempt the synthesis of hybrid compounds composed of both the 7-chloro-4-aminoquinoline nucleus The synthesized compound were tested against an ample panel of cancer cell lines within the Developmental Therapeutics Program (DTP) at the National Cancer Institute (NCI) due to the fact that the moiety 2-pyrazoline [11,12] has demonstrated antitumor activities. Furthermore, considering that several antineoplastic compounds have proven valuable antifungal activities [14,15], we tested them as antifungals against two clinically important fungi, Candida albicans and Cryptococcus neoformans. It is important to take into account the limited number of efficacious antifungal drugs, which are not completely effective for the eradication of mycoses. There is, therefore, an urgent need for new antifungal chemical structures as alternatives to the existing ones [16]. Even though the precursors 1-12 for the synthesis of these pyrazolines are very similar, different reaction conditions had to be used for the synthesis of each particular series, as shown in Scheme 2. It was observed that the electron density of the substituents on the aryl ring next to the α,β-unsaturated ketone highly influenced the rates of the cyclocondensation reactions. Electron-donating groups (EDG) increased the reaction time, while electron-withdrawing groups (EWG) favored the reaction; this may be due to the fact that EWG increased the electrophilic character of the carbonyl carbon atom, while the EDG decreased it.
The effective cyclocondensation of the 1,3-dielectrophilic system (C=C- C=O) in compounds 1-12 was confirmed by means of NMR spectroscopy (see Experimental section). For the discussion, we will take N-phenyl pyrazoline 27 as an example ( Figure 1). Even though the precursors 1-12 for the synthesis of these pyrazolines are very similar, different reaction conditions had to be used for the synthesis of each particular series, as shown in Scheme 2. It was observed that the electron density of the substituents on the aryl ring next to the α,β-unsaturated ketone highly influenced the rates of the cyclocondensation reactions. Electron-donating groups (EDG) increased the reaction time, while electron-withdrawing groups (EWG) favored the reaction; this may be due to the fact that EWG increased the electrophilic character of the carbonyl carbon atom, while the EDG decreased it.
The effective cyclocondensation of the 1,3-dielectrophilic system (C=C- C=O) in compounds 1-12 was confirmed by means of NMR spectroscopy (see Experimental section). For the discussion, we will take N-phenyl pyrazoline 27 as an example ( Figure 1). In the 1 H-NMR spectrum (DMSO-d6) of the compound 27, the signals corresponding to the three chemically and magnetically non-equivalent protons of the pyrazoline ring B appeared as a spin coupling system AMX. Three double-doublets are observed at 3.13, 3.93, and 5.52 ppm. They belong to methylenic protons on the diastereotopic center C-4′ (HA and HM) and to the methine proton H-5′ (HX) of the pyrazoline ring, with coupling constants of 2 JAM = 17.4, 3 JAX = 6.1 and 3 JMX = 12.1 Hz. Downfield, 13 signals with multiplicity according to their substitution pattern were assigned; eight of them correspond to the aromatic protons of the three aryl rings A, C, and D and the H-3 quinoline proton between 6.73 and 7.76 ppm; four signals between 7.59 and 8.49 ppm belong to the quinoline protons H-2, H-5, H-6, and H-8, and the N-H proton was observed at 9.19 ppm. Additionally, the 26 magnetically different carbons were found at their respective chemical shifts in the 13 C-NMR spectrum of compound 27.
In addition, the structures of compounds 15-38 were also confirmed through electron impact mass spectrometry (EIMS). The molecular ion peaks (M +· ) of all compounds were observed at their respective molecular mass and the fragmentation pattern was in good agreement with the already reported for 2-pyrazoline derivatives [17]. Furthermore, the molecular ion peak of each compound was found to be the most stable fragment or base peak in 75% of the synthesized compounds. Mass spectrometry data on all the synthesized pyrazoline derivatives 15-38 are provided in the Experimental Section.

Antiproliferative Activity
As a preliminary screening, compounds 15-38 were submitted to the Developmental Therapeutics Program (DTP) at the National Cancer Institute (NCI) for evaluation of their antiproliferative activity against different human tumor cell lines. Twelve (16,18,19,22,24,25,28, 30, 31, 34, 36, and 37) of the submitted compounds were selected and subjected to a preliminary evaluation against 58 tumor cell lines at a single dose of 10 µM and 48 h of incubation. The output from the single dose screening was reported as a mean graph available for analysis by the COMPARE program (data not shown). The results of this first assay showed that only compounds 25, 30, 31, 36, and 37 were active. It was observed that none of the pyrazolines containing EWG or weakly activating groups like methyl at R and R′ position (see Scheme 2) were active in the cancer cell lines.
The active compounds 25, 30, 31, 36, and 37 passed to a second evaluation step in order to determine their cytostatic activity against 58 tumor cell lines of leukemia, melanoma, lung, colon, brain, breast, ovary, kidney, and prostate. The results were expressed in the following parameters according to previously published protocols [18][19][20][21]: GI50, which is the molar concentration of the In the 1 H-NMR spectrum (DMSO-d 6 ) of the compound 27, the signals corresponding to the three chemically and magnetically non-equivalent protons of the pyrazoline ring B appeared as a spin coupling system AMX. Three double-doublets are observed at 3.13, 3.93, and 5.52 ppm. They belong to methylenic protons on the diastereotopic center C-4 1 (H A and H M ) and to the methine proton H-5 1 (H X ) of the pyrazoline ring, with coupling constants of 2 J AM = 17.4, 3 J AX = 6.1 and 3 J MX = 12.1 Hz. Downfield, 13 signals with multiplicity according to their substitution pattern were assigned; eight of them correspond to the aromatic protons of the three aryl rings A, C, and D and the H-3 quinoline proton between 6.73 and 7.76 ppm; four signals between 7.59 and 8.49 ppm belong to the quinoline protons H-2, H-5, H-6, and H-8, and the N-H proton was observed at 9.19 ppm. Additionally, the 26 magnetically different carbons were found at their respective chemical shifts in the 13 C-NMR spectrum of compound 27.
In addition, the structures of compounds 15-38 were also confirmed through electron impact mass spectrometry (EIMS). The molecular ion peaks (M +¨) of all compounds were observed at their respective molecular mass and the fragmentation pattern was in good agreement with the already reported for 2-pyrazoline derivatives [17]. Furthermore, the molecular ion peak of each compound was found to be the most stable fragment or base peak in 75% of the synthesized compounds. Mass spectrometry data on all the synthesized pyrazoline derivatives 15-38 are provided in the Experimental Section.

Antiproliferative Activity
As a preliminary screening, compounds 15-38 were submitted to the Developmental Therapeutics Program (DTP) at the National Cancer Institute (NCI) for evaluation of their antiproliferative activity against different human tumor cell lines. Twelve (16,18,19,22,24,25,28, 30, 31, 34, 36, and 37) of the submitted compounds were selected and subjected to a preliminary evaluation against 58 tumor cell lines at a single dose of 10 µM and 48 h of incubation. The output from the single dose screening was reported as a mean graph available for analysis by the COMPARE program (data not shown). The results of this first assay showed that only compounds 25, 30, 31, 36, and 37 were active. It was observed that none of the pyrazolines containing EWG or weakly activating groups like methyl at R and R 1 position (see Scheme 2) were active in the cancer cell lines.
The active compounds 25, 30, 31, 36, and 37 passed to a second evaluation step in order to determine their cytostatic activity against 58 tumor cell lines of leukemia, melanoma, lung, colon, brain, breast, ovary, kidney, and prostate. The results were expressed in the following parameters according to previously published protocols [18][19][20][21]: GI 50 , which is the molar concentration of the compounds required to inhibit 50% of the growth of cell lines (relative to untreated cells), and LC 50 , which is a parameter of cytotoxicity that reflects the molar concentration needed to kill 50% of the cells [22]. The active compounds were evaluated at five concentration levels (100, 10, 1.0, 0.1, and 0.01 µM) and the test consisted of a 48-h continuous drug exposure protocol using sulforhodamine B (SRB) protein assay to estimate cell growth [18][19][20][21]. As an interesting result, compounds 25, 30, 31, 36, and 37 exhibited significant cytostatic activity, with GI 50 values lower than 1.0ˆ10´6 M against several human cancer cell lines. The biological response elicited by these compounds could be attributed, in part, to the EDG on ring C (4-OCH 3 and 3,4,5-triOCH 3 ). With the exception of compound 31, compounds 30, 36, and 37 were more active (in terms of the average number of cell lines) than compound 25, which is presumably due to the meta substitution on ring A.
A comparison between the values of GI 50 of compounds 25, 30, 31, 36, 37, and the standard drug (adriamycin) against several tumor cell lines showed that all of these compounds have similar or even better cytostatic activity than the reference drug, as observed in Table 2. In comparison to adriamycin (GI 50 = 0.12 µM), compound 30 showed better activity against the UACC-62 cell line with a GI 50 = 0.05 µM. Compound 30 was also potent against KAKI-1 (GI 50 = 0.68 µM) as compared to adriamycin (GI 50 = 0.95 µM). Interestingly, all of the compounds previously mentioned (25, 30, 31, 36, 37) showed better cytostatic activity than adriamycin (GI 50 = 6.46 µM) by a remarkable difference in GI 50 values, going from 0.34 to 2.30 µM against HCT-15 tumor cell line. In Table 2, the compounds that exhibited promising cytostatic activity against different cell lines are highlighted in gray.

Antifungal Activity
In order to have a look into the potential usefulness of these compounds as candidates for the developments of new antifungal drugs, we investigated the antifungal properties of compounds 15-38 against two clinically important fungal species, C. neoformans and C. albicans. 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 incidence among HIV patients with impaired defenses [23]. High rates of fungal persistence and frequent disease relapse have motivated the search for new compounds that display antifungal properties against this fungus [24].
Moreover, 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 [25]. For this reason, the development of new potential anticandidal drugs is crucial.
To assess antifungal activities, the standardized microbroth dilution method M-27A3 for yeasts of the Clinical and Laboratory Standards Institute was used [26]. Percentages of growth inhibition of each fungus were found using all compounds 15-38 with a concentration range within 250-3.9 µg/mL, which allowed for the determination of MIC 100 , MIC 80 , and MIC 50 .
For a more comprehensive analysis of the results, we grouped all compounds (15-38) into two series (i, ii) that differ only in the position of the diaryl-azole substituent on the A ring (Table 3): series (i) includes compounds 15-26 with the diaryl-azole moiety in the p-position of ring A; series (ii) includes compounds 27-38 with the same moiety in the m-position. Each series was sub-divided into two sub-groups, (i.1) and (ii. 1), which comprise compounds with an un-substituted ring D (15-20 and 27-32, respectively), and (i. 2) and (ii. 2), which include compounds with a p-Cl substituted ring D (21-26 and 33-38, respectively). Table 3 shows the MIC 100 , MIC 80 , and MIC 50 of each compound. It can be observed that MIC 50 values displayed by several compounds (i.e., 25, 32, 36) were highly promising.
To gain insight into the structure-activity relationships of the whole series, we compared first the antifungal behavior of all compounds of series (i) with those of series (ii) against both fungi. We focused on the last column (MIC 50 ) of Table 3. This column contains 24 MIC 50 values (against both fungi) for compounds 15-26 of group (i), and 24 MIC 50 values of compounds 27-38 of group (ii). Then, the concentration values of MIC 50 within group (i) (>250, 250, 125, 62.5, 31.2, 15.6. 7.8, 3.9, and <3.9 µg/mL) were analyzed to determine the number of times they were repeated within this series against both fungi and a percentage of occurrence of each MIC 50 value was calculated (respective to the 24 total MIC 50 values obtained in this group). The same analysis was applied to the compounds in group (ii). With these data, a comparative graph was produced ( Figure 2A). Lastly, the antifungal properties of all compounds in series (i) (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) and those in series (ii) (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38) were analyzed against each fungus separately; these data are presented in Figure 2B,C. all compounds in series (i) (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) and those in series (ii) (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38) were analyzed against each fungus separately; these data are presented in Figure 2B,C. As can be seen in Figure 2A, within group (i) 40% of all MIC50 values fall into the value 250 µg/mL, while no compounds produced MIC50 values of 7.8 µg/mL or lower. Instead, 21% of the compounds within group (ii) fall into the 250 µg/mL concentration, while 4% fall into 7.8 and <3.9 µg/mL. From these results, it is clear that compounds of series (ii) exhibit better antifungal properties than those of series (i) against both fungi. Regarding the behavior against each fungus separately, series (ii) showed a higher percentage of lower MIC50 values over (i) against C. neoformans ( Figure 2C). Also, the same trend can be observed against C. albicans, where compounds of series (i) showed a much higher percentage of MIC50 values at 250 µg/mL than compounds in series (ii). These results indicate that the position of the azole moiety does play a role in the antifungal activity and that compounds with this moiety in  series (i) includes compounds 15-26 with the diaryl-azole moiety in the p-position of ring A; series (ii) includes compounds 27-38 with the same moiety in the m-position. Each series was sub-divided into two sub-groups, (i.1) and (ii. 1), which comprise compounds with an un-substituted ring D (15-20 and 27-32, respectively), and (i. 2) and (ii. 2), which include compounds with a p-Cl substituted ring D (21-26 and 33-38, respectively). Table 3 shows the MIC100, MIC80, and MIC50 of each compound. It can be observed that MIC50 values displayed by several compounds (i.e., 25, 32, 36) were highly promising. series (i) includes compounds 15-26 with the diaryl-azole moiety in the p-position of ring A; series (ii) includes compounds 27-38 with the same moiety in the m-position. Each series was sub-divided into two sub-groups, (i.1) and (ii. 1), which comprise compounds with an un-substituted ring D (15-20 and 27-32, respectively), and (i. 2) and (ii. 2), which include compounds with a p-Cl substituted ring D (21-26 and 33-38, respectively). Table 3 shows the MIC100, MIC80, and MIC50 of each compound. It can be observed that MIC50 values displayed by several compounds (i.e., 25, 32, 36) were highly promising. series (i, ii) that differ only in the position of the diaryl-azole substituent on the A ring (Table 3): series (i) includes compounds 15-26 with the diaryl-azole moiety in the p-position of ring A; series (ii) includes compounds 27-38 with the same moiety in the m-position. Each series was sub-divided into two sub-groups, (i.1) and (ii. 1), which comprise compounds with an un-substituted ring D (15-20 and 27-32, respectively), and (i. 2) and (ii. 2), which include compounds with a p-Cl substituted ring D (21-26 and 33-38, respectively). Table 3 shows the MIC100, MIC80, and MIC50 of each compound. It can be observed that MIC50 values displayed by several compounds (i.e., 25, 32, 36) were highly promising. series (i, ii) that differ only in the position of the diaryl-azole substituent on the A ring (Table 3): series (i) includes compounds 15-26 with the diaryl-azole moiety in the p-position of ring A; series (ii) includes compounds 27-38 with the same moiety in the m-position. Each series was sub-divided into two sub-groups, (i.1) and (ii. 1), which comprise compounds with an un-substituted ring D (15-20 and 27-32, respectively), and (i. 2) and (ii. 2), which include compounds with a p-Cl substituted ring D (21-26 and 33-38, respectively). Table 3 shows the MIC100, MIC80, and MIC50 of each compound. It can be observed that MIC50 values displayed by several compounds (i.e., 25, 32, 36) were highly promising. series (i, ii) that differ only in the position of the diaryl-azole substituent on the A ring (Table 3): series (i) includes compounds 15-26 with the diaryl-azole moiety in the p-position of ring A; series (ii) includes compounds 27-38 with the same moiety in the m-position. Each series was sub-divided into two sub-groups, (i.1) and (ii. 1), which comprise compounds with an un-substituted ring D (15-20 and 27-32, respectively), and (i. 2) and (ii. 2), which include compounds with a p-Cl substituted ring D (21-26 and 33-38, respectively). Table 3 shows the MIC100, MIC80, and MIC50 of each compound. It can be observed that MIC50 values displayed by several compounds (i.e., 25, 32, 36) were highly promising. series (i, ii) that differ only in the position of the diaryl-azole substituent on the A ring (Table 3): series (i) includes compounds 15-26 with the diaryl-azole moiety in the p-position of ring A; series (ii) includes compounds 27-38 with the same moiety in the m-position. Each series was sub-divided into two sub-groups, (i.1) and (ii. 1), which comprise compounds with an un-substituted ring D (15-20 and 27-32, respectively), and (i. 2) and (ii. 2), which include compounds with a p-Cl substituted ring D (21-26 and 33-38, respectively). Table 3 shows the MIC100, MIC80, and MIC50 of each compound. It can be observed that MIC50 values displayed by several compounds (i.e., 25, 32, 36) were highly promising. series (i, ii) that differ only in the position of the diaryl-azole substituent on the A ring (Table 3): series (i) includes compounds 15-26 with the diaryl-azole moiety in the p-position of ring A; series (ii) includes compounds 27-38 with the same moiety in the m-position. Each series was sub-divided into two sub-groups, (i.1) and (ii. 1), which comprise compounds with an un-substituted ring D (15-20 and 27-32, respectively), and (i. 2) and (ii. 2), which include compounds with a p-Cl substituted ring D (21-26 and 33-38, respectively). Table 3 shows the MIC100, MIC80, and MIC50 of each compound. It can be observed that MIC50 values displayed by several compounds (i.e., 25, 32, 36) were highly promising. For a more comprehensive analysis of the results, we grouped all compounds (15-38) into two series (i, ii) that differ only in the position of the diaryl-azole substituent on the A ring (Table 3): series (i) includes compounds 15-26 with the diaryl-azole moiety in the p-position of ring A; series (ii) includes compounds 27-38 with the same moiety in the m-position. Each series was sub-divided into two sub-groups, (i.1) and (ii. 1), which comprise compounds with an un-substituted ring D (15-20 and 27-32, respectively), and (i. 2) and (ii. 2), which include compounds with a p-Cl substituted ring D (21-26 and 33-38, respectively). Table 3 shows the MIC100, MIC80, and MIC50 of each compound. It can be observed that MIC50 values displayed by several compounds (i.e., 25, 32, 36) were highly promising. For a more comprehensive analysis of the results, we grouped all compounds (15-38) into two series (i, ii) that differ only in the position of the diaryl-azole substituent on the A ring (Table 3): series (i) includes compounds 15-26 with the diaryl-azole moiety in the p-position of ring A; series (ii) includes compounds 27-38 with the same moiety in the m-position. Each series was sub-divided into two sub-groups, (i.1) and (ii. 1), which comprise compounds with an un-substituted ring D (15-20 and 27-32, respectively), and (i. 2) and (ii. 2), which include compounds with a p-Cl substituted ring D (21-26 and 33-38, respectively). Table 3 shows the MIC100, MIC80, and MIC50 of each compound. It can be observed that MIC50 values displayed by several compounds (i.e., 25, 32, 36) were highly promising. For a more comprehensive analysis of the results, we grouped all compounds (15-38) into two series (i, ii) that differ only in the position of the diaryl-azole substituent on the A ring (Table 3): series (i) includes compounds 15-26 with the diaryl-azole moiety in the p-position of ring A; series (ii) includes compounds 27-38 with the same moiety in the m-position. Each series was sub-divided into two sub-groups, (i.1) and (ii. 1), which comprise compounds with an un-substituted ring D (15-20 and 27-32, respectively), and (i. 2) and (ii. 2), which include compounds with a p-Cl substituted ring D (21-26 and 33-38, respectively). Table 3 shows the MIC100, MIC80, and MIC50 of each compound. It can be observed that MIC50 values displayed by several compounds (i.e., 25, 32, 36) were highly promising. which allowed for the determination of MIC100, MIC80, and MIC50. For a more comprehensive analysis of the results, we grouped all compounds (15-38) into two series (i, ii) that differ only in the position of the diaryl-azole substituent on the A ring (Table 3): series (i) includes compounds 15-26 with the diaryl-azole moiety in the p-position of ring A; series (ii) includes compounds 27-38 with the same moiety in the m-position. Each series was sub-divided into two sub-groups, (i.1) and (ii. 1), which comprise compounds with an un-substituted ring D (15-20 and 27-32, respectively), and (i. 2) and (ii. 2), which include compounds with a p-Cl substituted ring D (21-26 and 33-38, respectively). Table 3 shows the MIC100, MIC80, and MIC50 of each compound. It can be observed that MIC50 values displayed by several compounds (i.e., 25, 32, 36) were highly promising. which allowed for the determination of MIC100, MIC80, and MIC50. For a more comprehensive analysis of the results, we grouped all compounds (15-38) into two series (i, ii) that differ only in the position of the diaryl-azole substituent on the A ring (Table 3): series (i) includes compounds 15-26 with the diaryl-azole moiety in the p-position of ring A; series (ii) includes compounds 27-38 with the same moiety in the m-position. Each series was sub-divided into two sub-groups, (i.1) and (ii. 1), which comprise compounds with an un-substituted ring D (15-20 and 27-32, respectively), and (i. 2) and (ii. 2), which include compounds with a p-Cl substituted ring D (21-26 and 33-38, respectively). Table 3 shows the MIC100, MIC80, and MIC50 of each compound. It can be observed that MIC50 values displayed by several compounds (i.e., 25, 32, 36) were highly promising.  To gain insight into the structure-activity relationships of the whole series, we compared first the antifungal behavior of all compounds of series (i) with those of series (ii) against both fungi. We focused on the last column (MIC50) of Table 3. This column contains 24 MIC50 values (against both fungi) for compounds 15-26 of group (i), and 24 MIC50 values of compounds 27-38 of group (ii). Then, the concentration values of MIC50 within group (i) (>250, 250, 125, 62.5, 31.2, 15.6. 7.8, 3.9, and <3.9 µg/mL) were analyzed to determine the number of times they were repeated within this series against both fungi and a percentage of occurrence of each MIC50 value was calculated (respective to the 24 total MIC50 values obtained in this group). The same analysis was applied to the compounds in group (ii). With these data, a comparative graph was produced (Figure 2A). Lastly, the antifungal properties of To gain insight into the structure-activity relationships of the whole series, we compared first the antifungal behavior of all compounds of series (i) with those of series (ii) against both fungi. We focused on the last column (MIC50) of Table 3. This column contains 24 MIC50 values (against both fungi) for compounds 15-26 of group (i), and 24 MIC50 values of compounds 27-38 of group (ii). Then, the concentration values of MIC50 within group (i) (>250, 250, 125, 62.5, 31.2, 15.6. 7.8, 3.9, and <3.9 µg/mL) were analyzed to determine the number of times they were repeated within this series against both fungi and a percentage of occurrence of each MIC50 value was calculated (respective to the 24 total MIC50 values obtained in this group). The same analysis was applied to the compounds in group (ii). With these data, a comparative graph was produced (Figure 2A). Lastly, the antifungal properties of To gain insight into the structure-activity relationships of the whole series, we compared first the antifungal behavior of all compounds of series (i) with those of series (ii) against both fungi. We focused on the last column (MIC50) of Table 3. This column contains 24 MIC50 values (against both fungi) for compounds 15-26 of group (i), and 24 MIC50 values of compounds 27-38 of group (ii). Then, the concentration values of MIC50 within group (i) (>250, 250, 125, 62.5, 31.2, 15.6. 7.8, 3.9, and <3.9 µg/mL) were analyzed to determine the number of times they were repeated within this series against both fungi and a percentage of occurrence of each MIC50 value was calculated (respective to the 24 total MIC50 values obtained in this group). The same analysis was applied to the compounds in group (ii). With these data, a comparative graph was produced (Figure 2A). Lastly, the antifungal properties of To gain insight into the structure-activity relationships of the whole series, we compared first the antifungal behavior of all compounds of series (i) with those of series (ii) against both fungi. We focused on the last column (MIC50) of Table 3. This column contains 24 MIC50 values (against both fungi) for compounds 15-26 of group (i), and 24 MIC50 values of compounds 27-38 of group (ii). Then, the concentration values of MIC50 within group (i) (>250, 250, 125, 62.5, 31.2, 15.6. 7.8, 3.9, and <3.9 µg/mL) were analyzed to determine the number of times they were repeated within this series against both fungi and a percentage of occurrence of each MIC50 value was calculated (respective to the 24 total MIC50 values obtained in this group). The same analysis was applied to the compounds in group (ii). To gain insight into the structure-activity relationships of the whole series, we compared first the antifungal behavior of all compounds of series (i) with those of series (ii) against both fungi. We focused on the last column (MIC50) of Table 3. This column contains 24 MIC50 values (against both fungi) for compounds 15-26 of group (i), and 24 MIC50 values of compounds 27-38 of group (ii). Then, the concentration values of MIC50 within group (i) (>250, 250, 125, 62.5, 31.2, 15.6. 7.8, 3.9, and <3.9 µg/mL) were analyzed to determine the number of times they were repeated within this series against both fungi and a percentage of occurrence of each MIC50 value was calculated (respective to the 24 total MIC50 values obtained in this group). The same analysis was applied to the compounds in group (ii). To gain insight into the structure-activity relationships of the whole series, we compared first the antifungal behavior of all compounds of series (i) with those of series (ii) against both fungi. We focused on the last column (MIC50) of Table 3. This column contains 24 MIC50 values (against both fungi) for compounds 15-26 of group (i), and 24 MIC50 values of compounds 27-38 of group (ii). Then, the concentration values of MIC50 within group (i) (>250, 250, 125, 62.5, 31.2, 15.6. 7.8, 3.9, and <3.9 µg/mL) were analyzed to determine the number of times they were repeated within this series against both fungi and a percentage of occurrence of each MIC50 value was calculated (respective to the 24 total MIC50 values obtained in this group). The same analysis was applied to the compounds in group (ii). To gain insight into the structure-activity relationships of the whole series, we compared first the antifungal behavior of all compounds of series (i) with those of series (ii) against both fungi. We focused on the last column (MIC50) of Table 3. This column contains 24 MIC50 values (against both fungi) for compounds 15-26 of group (i), and 24 MIC50 values of compounds 27-38 of group (ii). Then, the concentration values of MIC50 within group (i) (>250, 250, 125, 62.5, 31.2, 15.6. 7.8, 3.9, and <3.9 µg/mL) were analyzed to determine the number of times they were repeated within this series against both fungi and a percentage of occurrence of each MIC50 value was calculated (respective to the 24 total To gain insight into the structure-activity relationships of the whole series, we compared first the antifungal behavior of all compounds of series (i) with those of series (ii) against both fungi. We focused on the last column (MIC50) of Table 3 To gain insight into the structure-activity relationships of the whole series, we compared first the antifungal behavior of all compounds of series (i) with those of series (ii) against both fungi. We focused on the last column (MIC50) of Table 3 To gain insight into the structure-activity relationships of the whole series, we compared first the antifungal behavior of all compounds of series (i) with those of series (ii) against both fungi. We focused on the last column (MIC50) of Table 3 To gain insight into the structure-activity relationships of the whole series, we compared first the antifungal behavior of all compounds of series (i) with those of series (ii) against both fungi. We focused on the last column (MIC50) of Table 3 To gain insight into the structure-activity relationships of the whole series, we compared first the antifungal behavior of all compounds of series (i) with those of series (ii) against both fungi. We focused on the last column (MIC50) of Table 3 As can be seen in Figure 2A, within group (i) 40% of all MIC 50 values fall into the value 250 µg/mL, while no compounds produced MIC 50 values of 7.8 µg/mL or lower. Instead, 21% of the compounds within group (ii) fall into the 250 µg/mL concentration, while 4% fall into 7.8 and <3.9 µg/mL. From these results, it is clear that compounds of series (ii) exhibit better antifungal properties than those of series (i) against both fungi. Regarding the behavior against each fungus separately, series (ii) showed a higher percentage of lower MIC 50 values over (i) against C. neoformans ( Figure 2C). Also, the same trend can be observed against C. albicans, where compounds of series (i) showed a much higher percentage of MIC 50 values at 250 µg/mL than compounds in series (ii). These results indicate that the position of the azole moiety does play a role in the antifungal activity and that compounds with this moiety in the m-position are better antifungal candidates than those with the azole moiety in the p-position.
From the previous analyses, it was determined that within the whole series of hybrids of N-aryl-substituted pyrazolines with 7-chloro-4-aminoquinoline nucleus tested , compounds of the sub-series (ii. 2) with the aryl-azole moiety in m-position of ring A and a p-Cl substituted ring D were the most active, mainly against C. neoformans. To corroborate the higher antifungal activity of these (ii-2) compounds against C. neoformans over C. albicans, a MIC 50 values' comparison of 33-38 against both fungi is shown in Figure 3.

General Information
Commercially available starting materials, reagents, and solvents were used as supplied. Microwave irradiation reactions were performed in glass vessels (10 mL) using a CEM Discover Focused Microwave Synthesis System™ apparatus (Matthews, NC, USA), with power output from 0 to 300 W. TLC analyses were performed on Merck (EMD Millipore, Billerica, MA, USA) silica gel 60 F254 aluminum plates. Melting points were determined in a Büchi (Instrumart, South Burlington, VT, USA) melting point apparatus and are uncorrected. IR spectra were performed on a Shimadzu (Scientific Instruments, Seattle, WA, USA) FTIR 8400 spectrophotometer in KBr disks. The 1 H-and 13 C-NMR spectra were run on a Bruker (Karlsruhe, Germany) DPX 400 spectrophotometer operating at 400 MHz and 100 MHz respectively, using dimethylsulfoxide-d6 as solvents and tetramethylsilane as internal reference. The mass spectra were obtained on a Hewlett Packard (Scientific Instrument Services, Ringoes, NJ, USA) HP Engine-5989 spectrometer (equipped with a direct inlet probe) operating at 70 eV. The elemental analyses were obtained using a Thermo-Finnigan Flash EA1112 CHN (Elemental Microanalysis Ltd., (Okehampton, Devon, UK) elemental analyzer.

General Procedure for the Synthesis of the Precursors 1-12
Using the same previously reported method [15], precursors 1-12 were obtained.

General Procedure for the Preparation of Compounds 15-20
A mixture of 4-(7-chloroquinolin-4-yl) amino chalcone 1-6 (0.11 mmol), phenylhydrazine 13 (0.33 mmol) in glacial acetic acid (10 mL) was submitted to microwave irradiation for 12 min at 250 W As can be observed in Figure 3, the MIC 50 values of compounds 33-38 in C. neoformans are much lower than those exhibited by C. albicans, thus suggesting a higher sensitivity of C. neoformans against this group of compounds. In this figure, it is evidenced that among the compounds of (ii.2) sub-group, compound 36 was by far the most active one, mainly against C. neoformans, and thus it is the best anti-cryptococcal candidate of the compounds in this work.

General Information
Commercially available starting materials, reagents, and solvents were used as supplied. Microwave irradiation reactions were performed in glass vessels (10 mL) using a CEM Discover Focused Microwave Synthesis System™ apparatus (Matthews, NC, USA), with power output from 0 to 300 W. TLC analyses were performed on Merck (EMD Millipore, Billerica, MA, USA) silica gel 60 F254 aluminum plates. Melting points were determined in a Büchi (Instrumart, South Burlington, VT, USA) melting point apparatus and are uncorrected. IR spectra were performed on a Shimadzu (Scientific Instruments, Seattle, WA, USA) FTIR 8400 spectrophotometer in KBr disks. The 1 H-and 13 C-NMR spectra were run on a Bruker (Karlsruhe, Germany) DPX 400 spectrophotometer operating at 400 MHz and 100 MHz respectively, using dimethylsulfoxide-d 6 as solvents and tetramethylsilane as internal reference. The mass spectra were obtained on a Hewlett Packard (Scientific Instrument Services, Ringoes, NJ, USA) HP Engine-5989 spectrometer (equipped with a direct inlet probe) operating at 70 eV. The elemental analyses were obtained using a Thermo-Finnigan Flash EA1112 CHN (Elemental Microanalysis Ltd., (Okehampton, Devon, UK) elemental analyzer.

General Procedure for the Synthesis of the Precursors 1-12
Using the same previously reported method [15], precursors 1-12 were obtained.

General Procedure for the Preparation of Compounds 15-20
A mixture of 4-(7-chloroquinolin-4-yl) amino chalcone 1-6 (0.11 mmol), phenylhydrazine 13 (0.33 mmol) in glacial acetic acid (10 mL) was submitted to microwave irradiation for 12 min at 250 W and 120˝C. Once the reaction mixture was cooled to room temperature, the resulting solution was neutralized with concentrated ammonium hydroxide. Then, crushed ice was added to the solution and a solid was precipitated, collected by vacuum filtration, washed thoroughly with water, dried, and recrystallized from ethanol.  (Compounds 25, 30, 31, 36, and 37). Furthermore, these compounds displayed higher cytostatic activity against several cell lines compared to the standard drug, adriamycin. Regarding the antifungal activity, compounds 15-38 showed activity against C. albicans and C. neoformans with varied MIC 50 values between <3.9 and 250 µg/mL. However, among the different sub-groups, the compounds in series (ii) showed better activity than those in group (i) against both fungi, These results indicate that the position of the azole moiety does play a role in the antifungal activity and that compounds with this moiety in the m-position are better antifungal candidates than those with the azole moiety in the p-position. Within the two sub-groups in series (ii), the best activity was displayed by compounds with a p-Cl moiety in the aryl ring D. It could be showed that C. neoformans had higher sensitivity to these compounds than the other fungus tested (C. albicans).
Compound 36 exhibited the highest antifungal properties, becoming an interesting candidate for new antifungal studies.