Multiple biological active 4-aminopyrazoles containing trifluoromethyl and their 4-nitroso-precursors: Synthesis and evaluation
Abstract
4-Nitroso-3-trifluoromethyl-5-alkyl[(het)aryl]pyrazoles were synthesized via one-pot nitrosation of 1,3- diketones or their lithium salts followed by treatment of hydrazines. Reduction of nitroso-derivatives made it possible to obtain 4-amino-3-trifluoromethylpyrazoles chlorides. According to computer-aided calculations, all synthesized compounds are expected to have acceptable ADME profile for drug design. Tuberculostatic, antibacterial, antimycotic, antioxidant and cytotoxic activities of the compounds were evaluated in vitro, while their analgesic and anti-inflammatory action was tested in vivo along with acute toxicity studies.
N-Unsubstituted 4-nitrosopyrazoles were the most effective tuberculostatics (MIC to 0.36 mg/ml) and antibacterial agents against Streptococcus pyogenes (MIC to 7.8 mg/ml), Staphylococcus aureus, S. aureus MRSA and Neisseria gonorrhoeae (MIC to 15.6 mg/ml). 4-Nitroso-1-methyl-5- phenylpyrazole had the pronounced antimycotic action against a wide range of fungi (Trichophyton rubrum, T. tonsurans, T. violaceum, T. interdigitale, Epidermophyton floccosum, Microsporum canis with MIC 0.38e12.5 mg/ml). N-Unsubstituted 4-aminopyrazoles shown high radical-scavenging activity in ABTS test, ORAC/AAPH and oxidative erythrocyte hemolysis assays. 1-Methyl-5-phenyl-3- trifluoromethylpyrazol-4-aminium chloride revealed potential anticancer activity against HeLa cells (SI > 1351). The pronounced analgesic activity was found for 4-nitroso- and 4-aminopyrazoles having phenyl fragment at the position 5 in “hot plate” test. The most of the obtained pyrazoles had a moderate acute toxicity.
Introduction
Although a pyrazole moiety is rarely found in natural products, it appears to be a perspective scaffold for designing biologically active molecules. The functionalization of the pyrazole backbone allowed multifunctional compounds to be obtained that are able to bind to biological targets in different ways, leading to various pharmaceutical activities [1e5]. There is an increasing number of drug discovery programs that are using aminopyrazoles as a ver- satile framework [6,7]. Many of bioactive pyrazoles are 4-amino- derivatives, e.g., pyrazolone analgesic-antipyretics (amino- phenazone, metamizole, etc.), which are known to interact with several biological targets (Fig. 1). Recently, N-myristoyltransferase inhibitor was found to be useful for treatment of the deadly African trypanosomiasis (sleeping sickness) caused by Trypanosoma brucei [8]. 4-Aminopyrazole chemotype is present in compounds that are currently in clinical trial, for example, AT7519 as a potent inhibitor of several CDK [9] and AT9283 as a multitargeted kinase inhibitor with potent aurora kinase activity [10].
Moreover, 1,2,4,5-functionalized 4-aminopyrazoles possess antibacterial [11e13], antifungal [11,13], cytotoxic [14] actions and show inhibitory activity against urease [15]. 4-Aminopyrazoles were used as initial synthons for the synthesis of Sildenafil (Viagra®) (Fig. 1) and its derivatives [16], antineoplastic and antibiotic drug Formycin A [17], and bioactive 4-hetarylated derivatives [18], acyla- mides [19], pyrazolo[4,3-d]pyrimidines [20,21], pyrazoloxadiazoles [22], pyrazolo[4,3-c][1,2,6]benzothiadiazocine [23], pyrazolo[3,4-f] pyrrolo[1,2-a][1,4][]diazepines [24] and 4-(het)arylazopyrazoles [25] as disperse dyes. Therefore, 4-aminopyrazole core may serve as an advantageous starting point for different drug discovery programs.
The literature data show a variety of synthetic routes to obtain 4-aminopyrazoles. The convenient precursors to their synthesis are 4-nitrosopyrazoles, because the reduction of the nitroso group can be carried out under mild conditions, without affecting the other functional groups in the molecule [18,19,21,26e28]. There is another approach, which is based on the catalytic reduction of 4- nitropyrazoles [16,18,19,23,27], but it requires preliminary nitra- tion of pyrazoles to be carried out under rather severe conditions [19,27,29]. An example of 4-arylazopyrazoles reduction into amino derivatives is known [25]. By the ThorpeeZiegler reaction, it is possible to obtain 4-aminopyrazoles having (het)aroyl [11e14,22,30e34] or cyano [13,15,21,35] substituent at the position 5.
However, this method requires using hardly accessible initial reagents. The original process is described for 4-aminopyrazole synthesis via the reaction of enaminones with diazonium tetra- fluoroborate [36].
Although fluorinated pyrazoles are, undoubtedly, promising for being used in the pharmaceutical and agrochemical industries [37,38], there is no or very few information on fluorine-containing analogues in literature. The present studies are encouraged by the discovery of selective COX-2 inhibitors among (trifluoromethyl) pyrazole derivatives, e.g., drug celecoxib [39e41].
4-Amino-3-trifluoromethylpyrazoles having alkyl, aryl or acyl substituents were obtained through the catalytic reduction of 4-nitropyrazoles with hydrogen (at p = 810 kPa) in the presence of Pd/C [42,43], zinc in acetic acid [44], iron in hydrochloric acid or potassium borohydride under the action of CuCl or SnCl2 [45]. This approach is made complicated by the need for the preliminary synthesis of 4-nitropyrazoles via nitration with HNO3/H2SO4 at high temperature; and the process may occur non-selectively [42,43].
There are no data available on the application of 4- nitrosopyrazoles for polyfluoroalkyl-containing 4-aminopyrazoles synthesis. However, this approach may be regarded as very promising so long as both introduction and reduction of the nitroso-group are carried out under mild conditions ensuring effi- ciency and selectivity of the processes. The feasibility of this approach can be evidenced by the synthesis of 4-amino-5-phenyl- 4-trifluoromethylpyrazole via one-pot nitrosation of 4,4,4- trifluoro-1-phenylbutane-1,3-dione [46] or its lithium salt [47] followed by simultaneous cyclization and reduction with hydrazine hydrate.
Herein, we have developed the effective approaches to the synthesis of trifluoromethyl-containing 4-aminopyrazoles using 4- nitrosopyrazoles as precursors (Fig. 2). In view of the multi- targeted nature on many pyrazole-containing drugs, we have assessed new trifluoromethylated pyrazoles as causing some of the most expected types of biological activity, such as antibacterial (including antituberculosis), antimycotic, antitumor, analgesic and anti-inflammatory ones; and we have determined their radical- scavenging activity as well.
Although 4-nitrosopyrazoles obtained in our study are viewed mainly as intermediates for synthesis of amino-derivatives, we paid special attention to the assessment of their biological activity. To the best of our knowledge, the release of nitric oxide (NO) using nitrosopyrazoles has thus far been unreported. However, it can be expected that these compounds are capable of performing such a function.
Nitric oxide is known to be an important regulator and mediator involved in many physiological processes, e.g., vasodila- tion, neurotransmission, decreasing platelet aggregation, immune system reactions, regulation of smooth muscle tone, memory state, etc. [48,49]. Nitric oxide metabolism disorders play a crucial role in the pathogenesis of a variety of serious conditions, such as septic shock, arterial hypertension, coronary heart disease, stroke, heart failure, diabetes mellitus, neurodegenerative diseases [50,51]. Multiple research have been done [52,53] on designing combinational NO-NSAIDs having an anti-inflammatory moiety and the source of the NO molecule.
We also recognize a rather useful aspect of studying the bio- logical activity of nitroso- and aminopyrazoles, because nitroso- and amino-compounds are the metabolites of many medicines [54e57]. In this regard, the study of biological activity of this class of compounds is of great importance.
Results and discussion
Chemistry
4-Nitrosopyrazoles bearing alkyl, (het)aryl, carboxyl sub- stituents at the positions 3 and 5 are known to be obtained by cyclization of 2-hydroxyimino-1,3-diketones with hydrazines [58]. The reduction of 4-nitrosopyrazoles into 4-amino derivatives was realized with hydrogen in the presence of Pd/C catalyst [59] or zinc in acetic acid [60]. The photosensitized reduction of 4- nitrosopyrazoles was described, with titanium dioxide being used as a photocatalyst in the presence of trimethylamine [61]. Occa- sionally, heating of 2-hydroxyimino-1,3-diketones with excess of hydrazine hydrate immediately resulted in 4-aminopyrazole, because cyclization and reduction occurred simultaneously [62].
To synthesize 4-nitroso-3-polyfluoroalkylpyrazoles 5, we applied an earlier introduced one-pot method based on the nitro- sation of 1,3-diketones 1 and the subsequent condensation of in- termediates 2-hydroxyimino-1,3-diketones 3 with hydrazines [63,64]. In this work, in addition to 1,3-diketones 1, we used their lithium salts 2, which are intermediates in Claisen synthesis of polyfluoroalkyl-1,3-diketones 1 from esters of polyfluorocarboxylic acids and methyl ketones under the action of lithium hydride [65].
It was found that lithium phenyl- and thienyl-1,3-diketonates 2a,b and diketone 1b in the reactions of successive nitrosation and cyclization with hydrazine hydrate firstly result in 3-hydroxy- 4-nitrosopyrazolines 4a,b, which then produce 4-nitrosopyrazoles 5a,b after dehydration (Scheme 1). Moreover, dehydration of compound 4a was carried out under heating, while product 4b was dehydrated on silica gel during flash-chromatography. Unlike the previous transformations, furanyl- and tolyl-1,3-diketones 1c,d and their lithium salts 2c,d yielded 4-nitrosopyrazoles 5c,d immedi- ately. The using of methyl hydrazine in one-pot nitrosation and cyclization of 1,3-diketones 1a-c and their lithium salts 2a-c resulted in 4-nitrosopyrazoles 5e-g.
It should be noted that, in earlier studies [64], the products 4a and 5a,d would be obtained from 4-aryl-1,1,1-trifluorobutane-2,4- dione 1a following an analogous three-component method. In the present research, however, these products were synthesized based on lithium diketonate 2a. Comparing the products yields, a conclusion can be made that using lithium salt 2a is more efficient than using diketone 1a.
In contrast to 1,1,1-trifluoropentane-2,4-dione 1e [64], its lithium salt 2e reacted with hydrazine hydrate to yield 4- nitrosopyrazole 5h immediately, while with methyl hydrazine resulted in two isomeric 3-CF3- and 5-CF3-4-nitrosopyrazoles 5i and 6 at a ratio of 2 to 1 (Scheme 2), which were separated by a column chromatography. The same isomeric mixture has been obtained by D.A. Fletcher et al. [66] based on 1,1,1-trifluoropentane- 2,4-dione, but this process required more time.
The synthesis of 4-nitrosopyrazoles bearing N-aryl substituent proceeded ambiguously. Earlier, we have isolated 5-hydroxy-4- hydroxyimino-5-trifluoromethyl-1-phenylpyrazoline by one-pot reaction of 1,1,1-trifluoropentane-2,4-dione 1e with nitrosating reagent (sodium nitrite in acetic acid) and phenyl hydrazine [64]. Nitrosation of lithium 1,2-diketonates 2a,d and cyclization with aryl hydrazines carried out as a one-pot reaction also resulted in the formation of 5-hydroxypyrazolines 7a,b (Scheme 3).
However, cyclocondensation of preliminary obtained 2-hydroxyimino-1,3- diketones 3a,d with aryl hydrazines allowed us to synthesize 4- nitrosopyrazoles 5j,k. 2-Hydroxyimino-1,3-diketones 3a,d reacted with aryl hydrazine to form 5-CF3-pyrazoline 7a,c similarly to three-component synthesis. Another cyclization direction can be explained by changing of medium acidity due to the presence of lithium cations obviously affecting as a buffer. We failed to dehy- drate 5-hydroxypyrazolines 7a-c owing to they seemed to exist as stable hydroxyimine tautomers.
All pyrazoles 5a-k, 6 have a characteristic color from bright blue to dark green for nitroso compounds. However, it may be typical for N-unsubstituted pyrazoles 5a-d to have nitroso-oxime tautom- erism. The structure of 4-nitrosopyrazole 5c was confirmed by X- Ray diffraction analysis (XRD) (Fig. 3, CCDC 2006858). Albeit there are no substituents at the nitrogen atom position in this compound, the position of hydrogen atom allowed us to attribute the com- pound to 3-CF3-isomer. Analysis of XRD data showed that the oxyygen atom O2 of nitroso group turns toward trifluoromethyl substituent and binds with proton H7A0 of the furan ring of another pyrazole molecule (the distance is 2.669 Å, not shown in Fig. 3).
The structure of compound 5c forms dimeric chains, which are bound by the intermolecular hydrogen bond (IHB) between the nitrogen atom N2 of one pyrazole molecule and the proton H1’ of another molecule (the bond length is 2.191 Å).
The 3-CF3-regioisomeric structure of N-substituted 4- nitrosopyrazoles 5g,k was proved by XRD data (Fig. 4, CCDC 2007108 and Fig. 5, CCDC 2006860, respectively). The crystalline structure of compound 5g comprises two crystallographically in- dependent molecules. Binding of these molecules was found to occur through CeHep-stacking between the proton H1Ab of one molecule and the furan ring of another molecule, with the bond lengths of 2.803 Å. Molecules of pyrazole 5k having a phenyl- sulfamide fragment form dimers in the crystals due to the IHB formation between amino- and sulfo-groups (the bond length is 2.071 Å).
Biology
Considering the multi-targeted nature of many pyrazolo- containing drugs, we have evaluated new polyfluoroalkyl-4- nitroso- and -4-amino-derivatives for a number of more expected types of biological activity applying mainly a phenotypic screening approach, which allows most of the possible biological targets and metabolizing enzymes to be involved in a single screen.
However, it is obvious that for the 4-nitrosopyrazoles 5, not only the nitroso group itself is responsible for the antimicrobial action, but also its combination with other structural fragments, since high antibacterial activity is shown by pyrazoles 5a-c,h, which have a combination of NO and NH fragments in the cycle, and pyrazoles 5e-g, which combine NO and NMe fragments, have a more pro- nounced antifungal effect compared to N-unsubstituted analogues of 5a-c.
Radical-scavenging activity
Among pyrazole derivatives, high effective antioxidants have been found [85,86]. The most known of them is Edaravone (3- methyl-1-phenyl-2-pyrazolin-5-one; Radicut, Mitsubishi Tanabe Pharma Corporation) used for the treatment of the acute stage of cerebral infarction [87,88] and for amyotrophic lateral sclerosis treatment [89]. In both indications, it is believed that the effect of drug is developed due to its ability to scavenge free radicals and thus prevent oxidative stress related damage to neurons.
Antioxidant activity of the synthesized pyrazoles 5e9 was evaluated using two radical-scavenging assays: the ABTS and ORAC-FL tests.
The ABTS assay is based on spectrophotometric determination of a decrease in absorbance of a stable dark green ABTS cation- radical (ABTS·+) solution after its interaction with an antioxidant compound [90]. The measurements were performed as previously described in detail [91]. Trolox was used as a reference antioxidant, and ascorbic acid as a positive control. The results were expressed as TEAC values (Trolox equivalent antioxidant capacity) and IC50 values and are presented in Table 4.
Additionally, a set of compounds was evaluated in the oxygen radical absorbance capacity (ORAC-FL) assay with 2,20-azobis-(2- methylpropionamidine) dihydrochloride (AAPH) as a free peroxyl radical generator [92]. In this method, fluorescein (FL) is used as a fluorescent probe. The method is based on measuring the decrease in the intensity of fluorescence with time, which characterizes the degree of decay of the fluorescent probe under the influence of peroxyl radicals. In the presence of antioxidants, the degree of decay of the fluorescent probe decreases and, accordingly, the fluorescence intensity increases. In these tests, Quercetin was used as a reference antioxidant. The results also are shown in Table 4.
In the ABTS test, all 4-nitrosopyrazoles 5 showed very weak radical-scavenging activity or did not show it at all. This result is quite unexpected because nitroso compounds are characterized with the radical binding activity, and some of them are used as spin traps [93] or as inhibitors of radical polymerization [94]. The ability of 4-hydroxyimino-5-hydroxy-1-phenylpyrazoline 7a to bind the ABTS radical was significantly higher (TEAC = 0.44). The intro- duction of a methanesulfonyl group into the 1-phenyl substituent (Ar = C6H4SO2Me-4) reduced the antiradical activity for pyrazoline 7b; whereas, the presence of a p-tolyl moiety instead of the phenyl in N-phenylsulfamide-substituted pyrazoline 7c led to an increase in TEAC value to 0.48.
The maximum antiradical activity was shown by N-unsub- stituted 4-aminopyrazolones 9a,b,d and their hydrochlorides 8a,b (Scheme 4, R1 = H), which, in contrast to nitroso-containing pre- cursors 5, showed high TEAC values exceeding that of Trolox (with the exception of salt 8a, for which TEAC = 0.58). Moreover, free amines 9a,b,d proved to be rather fast antioxidants, the maximum binding of the ABTS radical was observed after 10 min. Introducing methyl (8e,f,j, 9e) or methanesulfophenyl (9g) group to the nitro- gen atom at position 2 of the pyrazole ring led to an almost com- plete absence of antiradical activity for the corresponding 4- aminopyrazoles 8e,f,j and 9e,g.
According to ORAC assay, all the tested compounds did not exceed the antioxidant capacity of Quercetin at both tested con- centrations. Interestingly, the inverse concentration e activity relationship was reproducibly observed for salt 8b and its base 9b: the higher concentration (50 mM) provided less antiradical pro- tection than the lower one (10 mМ), which is, possibly, a sign of nonlinear relationship of concentration and radical-trapping ac- tivity in this assay.
Overall, the results on antiradical activity for the most active hydroxyimino- and aminoderivatives 7b,c, 8a,b and 9a,b obtained in both tests are in a fairly good correspondence (TEAC or IC50 values in ABTS test and % of fluorescence restored at 10 mM in ORAC test). However, compound 8e, one of the best in ORAC assay, does not scavenge ABTS radicals.
Acute toxicity
Prior to the in vivo assessment, the compounds were evaluated for acute toxicity in mice, and some structural features associated with toxicity of the compounds were analyzed.
According to OECD Guideline for testing of chemicals, minimal amount of animals, i.e. three mice, was involved in a single dose evaluation. When administered intraperitoneally (i/p), the tested nitroso- and amino-pyrazoles show different levels of toxicity depending on the substituents around the main pyrazole core (Table 6). The expected LD50 values for most of the tested com- pounds are equal or greater than 150 mg/kg or, for some of them, greater than 300 mg/kg. The representatives of 4-aminoderivatives subset appear to be slightly more toxic than the ones containing a 4-nitroso-group: 5a vs 8a and 5k vs 8k. For a subset of 4-nitroso- derivatives, a non-substituted nitrogen atom in the pyrazole core is associated with higher toxicity values when they are compared with ones for the corresponding N-methyl-substituted analogues (5a vs 5e; 5b vs 5f and 5c vs 5g).
A similar trend has been described for 3-trifluoromethyl-1H-pyrazol-3-oles containing H or a methyl group at N1 [102]. Substitution of phenyl group at C5 for heteryl (2- furanyl and 2-thienyl) leads to increasing the toxicity for N1eH derivatives in both, nitroso- and amino-pyrazoles, sets (5a vs 5b and 5c; 8a vs 8b; 9a vs 9b), while the data is not consistent and sufficient for drawing an analogous suggestion in case of N1eMe derivatives (5e vs 5f or 5g; 8a vs 8e). Expected LD50 values exceed 300 mg/kg for 4-hydroxyiminopyrazolines 7b and 7c, as well as for compound 5k. Overall acute toxicity data for tested compounds does not correlate with cytotoxicity data against HDF.
Due to the high acute toxicity, the compounds 5b, 5c, 5h, 8b and 9b were excluded from further in vivo evaluation.
Conclusions
In summary, we developed simple and efficient methods for synthesis of 3-trifluoromethyl-containing 4-nitroso- and 4- aminopyrazoles bearing the various substituents at the position 1 and 5 as well as for synthesis of 4-hydroxyiminopyrazolines.
According to calculated ADME parameters, all obtained pyrazole derivatives could be considered as potentially possessing favorable pharmacokinetic profile. The synthesized compounds revealed multiple bioactivity depending on the nature of peripheral sub- stituents at the pyrazole core.
The 4-nitrosopyrazoles with unsubstituted NH group had the high tuberculostatic and antibacterial activity, while the pro- nounced antimycotic action was typical for methyl-substituted analogues. 4-Aminopyrazoles did not reveal the essential antibac- terial and antimycotic activity.
The N-unsubstituted 4-aminopyrazoles and their hydrochlo- rides were found to have a high ability to scavenge the free radicals, while N-substituted analogues were inactive or significantly less active. Regardless of N-substituent, 4-nitrosopyrazoles were prac- tically inactive in the ABTS, ORAC tests and erythrocyte membrane stability assay. Some of 4-hydroxyiminopyrazolines possessed a moderate antiradical action.
The cytotoxicity investigation of synthesized pyrazoles showed that their action on cancer HeLa cells and on human dermal fibroblasts depended on substituents combination. We found among 4-nitrosopyrazoles the substances that showed cytotoxicity to the both cell lines and compounds with more selective anti- cancer action. 4-Aminopyrazoles were found to be cytotoxic against cancer cells.
The acute toxicity evaluation of the pyrazoles showed that most of them had a moderate toxicity. However, the introduction of thienyl moiety at the position 5 significantly increased the acute toxicity both for 4-nitroso- and 4-amino-derivatives.
The pronounced analgesic activity was revealed for 4-nitroso- and 4-aminopyrazoles having phenyl fragment at the position 5; and the amino-derivatives were found to be more effective than nitroso-analogues. 4-Hydroxyimino-5-hydroxypyrazolines also had a considerable analgesic action. It was revealed that only 4- amino-5-phenylpyrazoles had the appreciable anti-inflammatory activity among all the series of the tested compounds.
The analysis of structure-activity-toxicity relationships showed that there is a research potential for studying the 4-amino-3- trifluoromethyl-5-phenylpyrazoles and 4-hydroxyimino-5- hydroxy-1-arylpyrazolines, Lithium Chloride aimed at further chemical modifica- tions and biological action optimization.