Amines derived from 5- Nitroindazole with antiprotozoal properties (Machine-translation by Google Tr

专利摘要:
Amines derived from 5-nitroindazole with antiprotozoal properties. The present invention relates to three families of amines derived from 5-nitroindazole [1- (aminoalkyl) indazolinones, 3- (aminoalkoxy) indazoles and 3- (alkylamino) indazoles] which possess antiprotozoal properties and their use for the manufacture of medicaments, preferably for the treatment of infections caused by pathogenic protozoa of the families Trypanosomatidae and Trichomonadidae, such as Chagas disease, leishmaniasis and trichomonosis. (Machine-translation by Google Translate, not legally binding)
公开号:ES2653674A1
申请号:ES201700741
申请日:2017-10-20
公开日:2018-02-08
发明作者:José Antonio ESCARIO GARCIA-TREVIJANO；Alicia GÓMEZ BARRIO；Juan José NOGAL RUIZ；Cristina Rosa FONSECA BERZAL；Alexandra IBÁÑEZ ESCRIBANO；Vicente Jesús ARÁN REDÓ；Christophe DARDONVILLE；Nerea VELA ORTEGA；Sergio Sifontes Rodriguez；Alfredo Irenaldo MENESES MARCEL
申请人:Centro De Bioactivos Qumicos Facultad De Ciencias de la Universidad Central Marta Abreu De Las Villas；Centro De Bioactivos Qumicos Facultad De Ciencias de la Univ Central Marta Abreu De Las Villas；Consejo Superior de Investigaciones Cientificas CSIC；Universidad Complutense de Madrid；
IPC主号:

专利说明:


Amines derived from 5-nitroindazole with antiprotozoal properties Technical sector
The present invention relates to three families of amines derived from 5-nitroindazole [formulas (1), (11) and (111)], their preparation and their use in the manufacture of medicaments for the treatment of parasitosis, particularly for caused by protozoa of the Trypanosomatidae (Trypanosoma, Leishmania) and Trichomonadidae (Trichomonas) families. The invention is therefore encompassed within the pharmaceutical sector. State of the art
Several of the most important parasitosis are caused by pathogenic protozoa; Many of these diseases have a special prevalence in underdeveloped areas of the Earth, barely arouse the interest of large pharmaceutical companies, and are considered "neglected tropical diseases." Most existing medications for treatment have a number of drawbacks such as low efficacy, high toxicity, high prices, complex administration protocols, etc. , so that the development of new chemotherapeutic agents for Most of these diseases is an urgent need.
The pathogenic protozoan Trypanosoma cruzi is the etiologic agent of Chagas disease or American trypanosomiasis; This ailment is endemic to 21 Latin American countries, where it causes more than 7,000 deaths annually and maintains around 25 million people at risk of contracting the infection. In the area originally affected by the disease, it is transmitted mainly through the contaminated feces of hematophagous bedbugs, although there are alternative routes of infection such as the digestive route, the transfusion of contaminated blood or the congenital route. As a consequence of international migrations produced in recent decades, Chagas disease is an emerging pathology in countries such as the United States or Spain, considering that it currently affects about 7 million people worldwide (WHO, Investing to overcome the global impact of Neglected Tropical Oiseases, Third WHO report on Neglected Tropical Oiseases,
2015).
After infection, the initial acute phase of Chagas disease is usually mild or asymptomatic and lasts 1-2 months. The infection persists throughout life, although 60-70% of those infected enter what is called the indeterminate phase of the disease, and never develop clinical manifestations. However, the remaining 30-40%, 10-30 years after the initial infection develop the chronic phase of the disease, characterized by a very serious cardiomyopathy and / or severe digestive problems (megacolon and megaesophagus).
There is no vaccine available for Chagas disease, and the only two drugs available, nitroheterocycles nifurtimox and benznidazole, are quite effective in the acute phase, but show limited efficacy in the chronic phase. On the other hand, both compounds can cause severe side effects, although benznidazole is generally better tolerated by patients and is considered the treatment of choice for this disease.
Although compounds with antihagasic activity and different potential molecular targets of the parasite have been described in the literature, there are currently no therapeutic alternatives that can replace nifurtimox and benznidazole. Some antifungal azoles, inhibitors of the synthesis of sterols, have been especially promising for their high efficacy in animal models; however, a recent clinical study conducted with posaconazole has shown that this compound is much less effective than benznidazole, at least as a single therapy (Molina, 1. et al., N. Engl. J. Med. 2014, 370 , 1899-1908). Very recently, the description of some very effective imidazo-and triazolopyrimidines, which act through the proteasome inhibition of several pathogenic trypanosomatids (Trypanosoma, Leishmania) (Khare, S. et al., Nature 2016, 537, has also been of interest. 229-233).
In relation to this topic, the inventors' work group has described in recent years the synthesis and anti-gasic properties of many 5-nitroindazole derivatives; Due to its direct relationship with the present application, we can highlight the activity against T. cruzi of various 1,2-disubstituted 5-nitroindazolin-3-ones [Vega, M.
C. et al., Eur. J. Med. Chem. 2012, 58, 214-227; Escario, J. A. et al., P 201500769 (2710-2015), PCT / ES 2016/000119 (); Fonseca-Berzal, C. et al., Eur. J. Med. Chem. 2016, 115, 295-310). The trypanocidal properties of some 3-alkoxy-1-alkylindazoles have also been described (Arán, VJ et al., Bioorg. Med. Chem. 2005, 13, 31973207; Boiani, L. et al., Eur. J. Med. Chem. 2009, 44, 1034-1040; Rodríguez, J. et al., Eur. J. Med. Chem. 2009, 44, 1545-1553; Rodríguez, J. et al., Bioorg. Med. Chem. 2009, 17, 8186-8196; Muro, B. et al., Eur. J. Med. Chem. 2014, 74, 124-134); these last
5 compounds, however, have the substituents at positions 1 and 3 of the indazole ring and, therefore, are not directly related to those included in this invention.
On the other hand, leishmaniasis is a parasitic disease caused by different
10 species of protozoa of the genus Leishmania, which is transmitted through the bite of hematophagous dipterans of the group of sandflies. This disease is distributed throughout most of the world, especially in tropical and subtropical areas. It appears in 98 countries, many of them underdeveloped. It is estimated that 1.3 million new cases, 1 million cutaneous leishmaniasis and 300,000 cases appear every year.
15 visceral leishmaniasis, estimating that the latter form is responsible for 20,000 to
50,000 annual deaths (WHO, Investing to overcome the global impact of Neglected Tropical Oiseases. Third WHO report on Neglected Tropical Oiseases, 2015). In humans, depending on the specific Leishmania species involved, the disease has three main clinical forms: cutaneous, mucocutaneous and leishmaniasis.
20 visceral, the latter usually deadly without treatment.
Nor in this case there are effective vaccines, so chemotherapy is the only existing treatment. For many years, and still today, derivatives of pentavalent antimony (sodium stibogluconate and meglumine antimony) have been the first treatment alternative in many countries. As a second line of treatment we can mention pentamidine, amphotericin B, miltefosine and paromomycin, and more recently, sitamaquine (Santos, DO et al., Parasitol. Res. 2008, 103, 1-10; Singh, N. et al., Asian Paco J. Trop. Med. 2012, 5, 485-497). Proteasome-inhibiting imidazo-and triazolopyrimidines also seem very promising
30 mentioned when talking about Chagas disease (Khare, S. et al., Nature 2016, 537, 229-233).
Also in this case it is necessary to obtain new antileishmanial agents of lower cost, greater effectiveness and with less adverse side effects. Although nitroheterocycles are not currently used for the treatment of leishmaniasis, the leishmanicidal properties of some compounds of this group such as trypanocidal fexinidazole have recently been described (Wyllie, S. et al., Sci. Transl. Med. 2012, 4, 119re1) or the broad spectrum antiparasitic nitazoxanide and some analogs (Chan-Bacab, MJ et al., J. Antimicrob. Chemother. 2009, 63, 1292-1293). In this context, the leishmanicidal properties of some 3-alkoxy-1-alkyl-5-nitroindazoles have been studied in recent years (Boiani, L. et al., Eur. J. Med. Chem. 2009, 44, 10341040; Marín, C. et al., Acta Trop. 2015, 148, 170-178). As mentioned above, these compounds have the substituents at positions 1 and 3 of the indazole ring and are not directly related to those included in this invention.
Another disease caused by a pathogenic protozoan is trichonosis, whose etiologic agent is Trichomonas vaginalis. This sexually transmitted infection (STI) accounts for more than 50% of curable cases of this type of ailments in the world. According to WHO, the number of cases in adults was estimated at more than 276.4 million in 2008 (Harp, DF & Chowdhuri, l., Eur. J. Obstet. Gynecol. Reprod. Biol. 2011, 157, 39 ; WHO, Global incidence and prevalence of selected curable sexually transmitted diseases -2008, 2012).
This disease shows a wide range of clinical manifestations; The man is frequently an asymptomatic carrier and potential transmitter of the infection, while in women it usually produces more or less severe inflammation of the genitourinary ducts accompanied by a characteristic leucorrhoea, erythema, pruritus, dysuria, infertility, etc. It has been associated with problems in pregnancy and premature births, increases the risk of development of cervical and prostate neoplasia and also of coinfection with other STIs of bacterial, viral or fungal origin.
The reference drug for the treatment of trichonosis is nitroheterocycle metronidazole. This product and its analogue tinidazole, are the only two drugs accepted by the Food and Drug Administration (FDA) to treat this infection. These drugs are, in general, effective, but we must look for alternatives for those patients who are hypersensitive or to treat infections caused by isolates.
resistant to nitroimidazoles (ca. 10% today, but probably increasing).
In this regard, the tricomonicidal activity of various 5-nitroindazole derivatives has recently been described, standing out for their relationship with the present invention, 3-alkoxy-2-alkylindazoles (Escario, JA et al., P 201500769 (), PCT / ES 2016/000119 (); Ibáñez-Escribano, A. et al., Parasitology 2016, 143, 34-40; Fonseca-Berzal, C. et al., Eur. J. Med. Chem. 2016, 115, 295 -310). On the other hand, the tricomonicidal activity of other indazole derivatives has been described which, having the substituents in other ring positions, are not directly related to those included in this invention: 3-alkoxy-1-alkylindazoles (Arán, VJ et al., Bioorg. Med. Chem. 2005, 13, 3197-3207; Ibáñez-Escribano, A. et al., Parasitology 2016, 143, 3440), 3-alkoxyindazoles, indazol-3-0Is 1-substituted, indazolin- 2-substituted 3-ones and condensed indazolin-3-ones (Marrero-Ponce, Y. et al., Curro Drug Discov. Technol. 2005, 2,245-265; Marrero-Ponce, Y. et al., Bioorg. Med. Chem. 2006, 14, 6502-6524).
Many of the 5-nitroindazole derivatives discussed when treating Chagas disease, leishmaniasis and trichomonosis are, in general, lipophilic compounds with poor water solubility and poor pharmacokinetic properties. This problem has been tried to solve previously through some derivatives that contain carboxyl groups in their structure and form water-soluble salts, but unfortunately, they lack significant activity [Escario, JA et al., P 201500769 (), PCT / ES 2016/000119 (); Fonseca-Berzal, C. et al. , Eur. J. Med. Chem. 2016, 115, 295-310].
Description of the invention The present invention relates to three groups of compounds derived from 5-nitroindazole that possess biological activity against pathogenic protozoa of various genera such as Trypanosoma, Leishmania and Trichomonas. The compounds of general formulas (1) and (11) are structurally very different from the drugs currently used for the treatment of these infections and are significantly less cytotoxic; Although they are based on other compounds previously prepared by the team, they have been designed to be soluble in water and present better pharmacokinetic properties, by introducing primary, secondary or secondary amino groups.
tertiary, capable of forming water-soluble salts with appropriate inorganic or organic acids.
On the other hand, we have no evidence that antiparasitic properties have been described for the compounds of the general formula (111), derived from the transposition of certain derivatives of the general formula (11).
Thus, a first aspect of the invention relates to three different types of compounds, of general formulas (1), (11) Y (111).
Formula (1) Formula (11) Formula (111)
In the compounds of formulas (1) and (11), NR1R2 may be an amino, alkylamino group
or dialkylamino, a cyclic secondary amine moiety such as the pyrrolidino group, or groups
Phthalimide (which are prepared as synthetic intermediates of the compounds with final NH2 group), and n may be 2-3; In the compounds of formula (111), NR1R2 may be an alkylamino, (w-hydroxyalkyl) amino, or alkyl (w-hydroxyalkyl) amino group. In compounds of formula (1), when n = 2, the compound in which NR1R2 is a piperidine group is specifically excluded, since its activity has been previously described
20 vs. epimastigotes of T. cruzi (Mura, F. et al., J. Spectrosc. Dyn. 2013, 3, article 8).
In a more preferred embodiment, the present invention relates to a compound [formula (1)] that is selected from the following list:
2-Benzyl-1- [2- (dimethylamino) ethyl] -5-nitro-1,2-dihydro-3H-indazol-3-one (hydrochloride) 2-Benzyl-5-nitro-1- (2-pyrrolidinoethyl) ) -1, 2-dihydro-3H-indazol-3-one (oxalate) 2-Benzyl-1- (3-phthalimidopropyl) -5-nitro-1, 2-dihydro-3H-indazol-3-one 2-Benzyl -1- (2-phthalimidoethyl) -5-nitro-1, 2-dihydro-3H-indazol-3-one 1- (2-Aminoethyl) -2-benzyl-5-nitro-1, 2-dihydro-3H- indazol-3-one (hydrochloride)
1- (3-Aminopropyl) -2-benzyl-5-nitro-1, 2-dihydro-3H-indazol-3-one (hydrochloride) 2-Benzyl-1- [3- (methylamino) propyl] -5- nitro-1, 2-dihydro-3H-indazol-3-one (hydrochloride)
2-Benzyl-1- [3- (dimethylamino) propyl] -5-nitro-1, 2-dihydro-3H-indazol-3-one (hydrochloride) 2-Benzyl-5-nitro-1- (3-piperidinopropyl ) -1, 2-dihydro-3H-indazol-3-one (oxalate)
or its solvates or prodrugs, or other salts.
In another more preferred embodiment, the present invention relates to a compound[formula (11)] that is selected from the following list:2-Benzyl-3- (2-phthalimidoethoxy) -5-nitro-2H-indazole2-Benzyl-3- (3-phthalimidopropoxy) -5-nitro-2H-indazole2-Benzyl-3- [2- (dimethylamino) ethoxy] -5-nitro-2H-indazole (hydrochloride)3- (3-Am inop ropoxy) -2-ben ci1-5-nitro-2H-indazol (hydrochloride)
or its solvates or prodrugs, or other salts.
In another more preferred embodiment, the present invention relates to a compound [formula (111)] that is selected from the following list: 2-Benzyl-3-methylamino-5-nitro-2H-indazol 2 -Benci 1-3- [(2 -h idroxieti I) ami no] -5-nitro-2H-indazol 2 -Benci 1-3 - [(3-hydroxy prop il) ami no] -5-n itro-2H-ind azol 2-Benzyl -3- [(2-hydroxyethyl) methylamino] -5-nitro-2H-indazol 2 -Ben ci1-3 - [(3-h id roxi propi 1) methyl ami no] -5-n itro-2H-i ndazol
or its solvates or prodrugs.
In a third aspect, the present invention relates to the process of obtaining some of the compounds of general formula (1) or (11) by alkylation of 5-nitroindazolin-3-one 1; Due to the tautomeric nature of the latter, some of the aforementioned compounds are obtained at the same time by reacting the starting product under various conditions with alkylating agents, as shown in the Scheme
1. As detailed in the examples, alkylation of the represented tautomeric form, "indazolin-3-one", leads to compounds 2-5 [general formula (1)], while alkylation of the tautomeric form quot. ; 3-hydroxy-2H-indazolquot; leads to compounds 6 and 7 [general formula (11)].
or N '(Jd
___ or 02 I N-Bn ::.-.... N /
,
2,3,4,5 R 6, 7 R
2 [CH2hNMe2 3 [CH2hPrd 4.6 [CH2l2Ft
5, 7 [CH2hFt
(Ft: phthalimido; Prd: pyrrolidino)
Scheme 1. Synthetic route for the preparation of 2-benzylindazolin-3-one 2- substituted 2-5 [formula (1)] and 3-alkoxy-2-benzylindazoles 6 and 7 [formula (11)].
Other compounds of the general formula (1) are obtained by chemical transformation from precursors containing conveniently w-functionalized chains in position 1 of the indazole ring, as detailed in the examples. Thus, the removal of the phthaloyl protecting group from precursors 4 and 5 causes primary amines 8 and 9 (Scheme 2), and the nucleophilic replacement of bromine of compound 10 with amines.
10 primary or secondary gives rise to amines 11-13 (Scheme 3).
02N ~ J_Bn ___ .02N ~ N-Bn VN '__ VN'
n [CH2JnPht n [CH2JnNH2 4 2 8 2 5 3 9 3
Scheme 2, Synthetic route for the preparation of 1- (w-aminoalkyl) -2-benzylindazolin-3-ones 8 and 9 [formula (1)].
10 R
eleven NHMe
12 NMe2
13 Pip (piperidino)
Scheme 3. Synthetic route for the preparation of 1- (3-aminopropyl) -2-benzylindazolin-3-ones 11-13 [formula (1)].
In a fourth aspect, the present invention relates to the method of obtaining
5 some of the compounds of general formula 11 (16 and 19) and 111 (21-25) from the corresponding 3- (w-bromoalkoxy) indazoles (14, 15) and amines, or from the 3- (w-phthalimidoalkoxy) derivatives (6, 7) by elimination of the phthaloyl group followed by intramolecular transposition of the 3- (w-aminoalkoxy) indazoles (17-20) initially obtained (Scheme 4).
A fifth aspect of this invention relates to the use of a compound of general formula (1), (11) or (111) for the preparation of a medicament for the treatment of diseases caused by protozoa, preferably American trypanosomiasis (Chagas disease ), leishmaniasis and trichomonosis, caused by parasites of the
15 genera Trypanosoma, Leishmania and Trichomonas, respectively.
A sixth aspect of the present invention relates to a pharmaceutical composition comprising a compound of general formula (1), (11) or (111) together with acceptable adjuvants or pharmaceutical carriers; optionally said composition may contain
20 also other active ingredients. 02N ~~ c ~~: Br
V N 'n
14 2
15 3
__n _ = _2 _.02N ~~ c ~~: NMe2
Me2NH V N '16
n
6 2
7 3
--one --
O R or R
[17J
[18]
or, N
22 23
2 H 19 3 H 2 Me [20J 3 Me
R,
~ [CH '[quot; OH
Y' :;-
N-Bn
~
N /
n R n R 2 H 24 3 H 2 Me 25 3 Me
Scheme 4, Synthetic route for the preparation of 3- (w-aminoalkoxy) -2-benzylindazoles 16 and 19 [formula (11)] and 3-alkylamino-2-benzylindazoles 21-25 [formula (111)]. Compounds included in square brackets [17, 18 and 20] are reaction intermediates that were not isolated.
EMBODIMENT OF THE INVENTION The present invention is further illustrated by the following examples, which are not intended to limit its scope.
EXAMPLE 1, Preparation of compounds 2-5 [formula (1)] and 6, 7 [formula (11)] from 2-benzyl-5-nitroindazolinone 1, Final amines 2 and 3, as well as Phthalimide intermediate derivatives 4 and 5 were obtained by alkylation of 2-benzyl-5-nitroindazolin-3-one 1 (Aran, VJ et al., Liebigs Ann. 1996, 683-691) with the corresponding functionalized alkyl halides (Scheme
15 1); this type of reaction usually leads to mixtures of 1,2-disubstituted indazolinones (2-5) and of the isomeric 3-alkoxy-2-alkylindazoles; The latter compounds are in many cases minority reaction products (Vega, MC et al., Eur. J. Med. Chem. 2012, 58,214-227; Fonseca-Berzal, C. et al., Eur. J. Med. Chem 2016, 115, 295-310) and, in this case, only 3- (w-phthalimidoalkoxy) derivatives 6 and 7 were isolated.
Example 1a. Preparation of 1- [2- (dialkylamino) eti / jindazolinones 2 and 3. In the case of compound 2, a stirred mixture of 2-benzylindazolinone 1 (1.35 g, 5.01 mmol), 2- chloride (dimethylamino) ethyl (hydrochloride) (1.44 g, 10.00 mmol) and K2C03 (2.76 g, 19.97 mmol) in dimethylformamide (DMF; 50 mL) is heated at 100 oC for 24 h; 5 subsequently, heating is continued for three days, adding additional portions of the chloride (1.44 g) Y of K2C03 (2.76 g) every 24 h. To obtain compound 3, a similar procedure is followed from N- (2-chloroethyl) pyrrolidine (hydrochloride) (1.44 g, 10.00 mmol); in this case the reaction is carried out in butanone (100 mL) and refluxed for 3 days. In both cases, the reaction mixture is then evaporated to dryness and, after the addition of H20 (100 mL), extracted with CHCb (4 x 50 mL). The organic phase is dried (MgSO4), concentrated, and applied to a silica gel flash chromatography column that is eluted with CHCb / MeOH mixtures (50/1 to 25/1). The fractions containing the main reaction product evaporate to dryness; in the case of compound 2, the residue is treated with a conc. HCI solution. 15 (1.0 mL) in EtOH (50 mL) and then evaporated to deliver the desired product in the form of the corresponding hydrochloride; in the case of compound 3, the product obtained after column chromatography is treated with a saturated solution of oxalic acid in EtOH (10 mL) and the mixture obtained is dripped onto diethyl ether (100 mL). The precipitated oxalate is collected by filtration, washed with diethyl ether (2 x 20 mL)
20 and air dried.
2-Benzyl-1- [2- (dimeti / amino) ethyl} -5-nitro-1, 2-dihydro-3H-indazol-3-one (hydrochloride) (2).Yield: 0.74 g (39%). P. f. 286-288 oC (EtOH / H20). 1 H NMR [300 MHz, (CD3) zSO):OR 11, 34 (sa, 1 H, NW), 8.55 (d, J = 2.1 Hz, 1 H, 4-H), 8.45 (dd, J = 9.3, 2.1 Hz , 1 H, 6-H),25 7.81 (d, J = 9.3 Hz, 1 H, 7-H), 7.29 (m, 5H, aromatic H Bn), 5.29 (s, 2H, CH2 Bn), 4, 54 (t,J = 7.5 Hz, 2H, 1'-H), 2.99 (t, J = 7.5 Hz, 2H, 2'-H), 2.71 (s, 6H, Me); 13C NMR [75 MHz,(CD3) 2S0): OR 161.09 (C-3), 149.10 (C-7a), 142.18 (C-5), 135.91 (C-1 Bn), 128.75 (C- 3.5 Bn), 127.95 (C-4 Bn), 127.58 (C-6), 127.50 (C-2, -6 Bn), 120.37 (C-4), 116.73 (C-3a),112.53 (C-7), 50.54 (C-2 '), 44.65 (CH2 Bn), 42.20 (Me), 41.45 (C-1'); MS (ES +): miz (%)
30 341 (100) ([M + Hn, 703 (26) ([2M + Nan. Cal. Anal. For C18H21 CIN4 0 3 (376.84): C 57.37; H 5.62; N 14.87 Found: C 57.59; H 5.87; N 14.59.
2-Benzyl-5-nitro-1- (2-pyrrolidinoethyl) -1, 2-dihydro-3H-indazol-3-one (oxalate) (3). Yield: 1.10 g (48%). P. f. 216-218 oC (MeOH). 1 H NMR [300 MHz, (CD3) zSO]: Ó
8.53 (d, J = 2.2 Hz, 1 H, 4-H), 8.40 (dd, J = 9.0, 2.2 Hz, 1 H, 6-H), 7.73 ( d, J = 9.0 Hz, 1 H, 7-H), 7.35-7.20 (m, 5H, aromatic H Bn), 5.18 (s, 2H, CH2 Bn), 4.27 (sa, 2H, 1'-H), 2.79 (sa, 6H, 1'-HY 2-, 5-H pyrrolidino), 1.69 (sa, 4H, 3-, 4-H pyrrolidino); 13C NMR [75 MHz, (CD3h SO]: OR 165.01 (CO oxalate), 161, 21 (C-3), 149.34 (C-7a), 141, 82 (C-5), 135.93 (C-1 Bn), 128.75 (C-3, -5 Bn), 127.90 (C-4 Bn), 127.39 (C-2, -6 Bn), 127.33 (C-6 ), 120.35 (C-4), 116.23 (C-3a), 112.45 (C-7), 53.17 (C-2, -5 pyrrolidino), 49.39 (C-2 ') , 44.84 (CH2 Bn), 43.55 (C-1 '), 22.83 (C-3, -4 pyrrolidino); MS (ES +): miz (%) 367 (100) ([M + Hn , 755
(10) ([2M + Nan. Anal calc. For C22H24N40 7 (456.45): C 65.78; H 4.42; N 12.27. Found: C 65.99; H 4.21; N 12.42.
Example 1b Preparation of 1- (w-phthalimidoalkyl) indazolinones 4, 5 and 3- (w-phthalimidoalkoxy) indazoles 6, 7. In order to obtain the 2-phthalimidoethyl derivatives 4 and 6, a stirred mixture of 2-benzylindazolinone 1 ( 1.00 g, 3.72 mmol), N- (2-bromoethyl) phthalimide (1.27 g, 5.00 mmol) and K2C03 (0.55 g, 3.98 mmol) in DMF (15 mL) is heated to 100 oC for 5 h; Next, an additional amount of N- (2-bromoethyl) phthalimide (1.00 g) is added and heating is continued for an additional 12 h. The solvent is evaporated to dryness and, after the addition of H20 (50 mL), the precipitate is collected by filtration, washed with water and air dried. The solid obtained is suspended in CHCb (10 mL) and the insoluble product 6 (0.32 g) is collected by filtration, washed with CHCb (2 x 5 mL) and air dried. The filtrate is concentrated and applied to a silica gel flash chromatography column, which is eluted with CHCb / acetone mixtures (50/1 to 20/1) to deliver, in this order of elution, an additional amount of compound 6 and then compound 4.
The 3-phthalimidopropyl derivatives 5 and 7 are prepared following a similar procedure, using the corresponding amount of N- (3-bromopropyl) phthalimide (1.34 g, 5.00 mmol). The reaction ends in this case in 3 h. After evaporating the DMF and adding water, the mixture is extracted with chloroform (3 x 50 mL). The organic solution is dried (MgSO4), concentrated and chromatographed as described above, providing, in this order of elution, compounds 7 and 5.
2-Benzyl-1- (2-phthalimidoefil) -5-nifro-1, 2-dihydro-3H-indazol-3-one (4). Yield: 1.07 g (65%). P. f. 190-192 oC (1-PrOH). 1H NMR [300 MHz, (CD3) 2S0]: OR 8.33 (d, J = 2.4 Hz, 1 H, 4-H), 7.87 (dd, J = 9.0, 2.4 Hz , 1 H, 6-H), 7.66 (m, 4H, phthalimido H), 7.36 (d, J = 9.0
12
Hz, 1 H, 7-H), 7.25 (m, 5H, aromatic H Bn), 5.20 (s, 2H, CH2 Bn), 4.36 (t, J = 5.1 HZ, 2H , 1'-H), 3.63 (t, J = 5.1 Hz, 2H, 2'-H); 13C NMR [75 MHz, (CD3) 2S0]: OR 166.99 (phthalimido CO), 161.11 (C-3), 149.40 (C-7a), 140.93 (C-5), 136, 00 (C-1 Bn), 134.32 (C-4, -5 phthalimido), 130.98 (C-1, -2 phthalimido), 128.68 (C-3, -5 Bn), 127.83 (C-4 Bn), 127.37 (C-2, -6 Bn), 126.39 (C-6), 122.84 (C-3, -6 phthalimide), 120.12 (C-4) , 116.31 (C-3a), 111.48 (C7), 45.30 (C-1 '), 44.72 (CH2 Bn), 34.29 (C-2'); MS (ES +): miz (%) 443 (100) ([M + Ht), 465 (11) ([M + Nat), 885 (20) ([2M + Ht), 907 (6) ([2M + Nan. Anal heating for C24H1sN40 s (442.42): C 65.15; H 4.10; N 12.66. Found: C 65.40; H 3.97; N 12.57.
2-Benzyl-1- (3-phthalimidopropyl) -5-nitro-1, 2-dihydro-3H-indazol-3-one (5). Yield: 0.93 9 (55%). P. f. 202-204 oC (1-PrOH). 1H NMR [300 MHz, (CD3) 2S0]: OR 8.48 (d, J = 2.0 Hz, 1 H, 4-H), 8.34 (dd, J = 9.0, 2.0 Hz , 1 H, 6-H), 7.81 (m, 4H, phthalimido H), 7.71 (d, J = 9.0 Hz, 1H, 7-H), 7.22 (m, 5H, H aromatics Bn), 5.22 (s, 2H, CH2 Bn), 4.13 (t, J = 7.2 Hz, 2H, 1'-H), 3.49 (t, J = 6.9 Hz , 2H, 3'-H), 1.60 (m, 2H, 2'-H); 13C NMR [75 MHz, (CD3) 2S0]: OR 167.82 (Phthalimide CO), 161.14 (C-3), 148.93 (C-7a), 141.45 (C-5), 136, 08 (C-1 Bn), 134.22 (C-4, -5 phthalimido), 131.72 (C-1, -2 phthalimido), 128.63 (C-3, -5 Bn), 127.75 (C-4 Bn), 127.22 (C-6 and C-2, -6 Bn), 122.91 (C-3, -6 phthalimido), 120.40 (C-4), 115.92 ( C-3a), 112.21 (C-7), 45.02 (C-1 '), 44.96 (CH2 Bn), 34.86 (C-3'), 24.82 (C-2 ' ); MS (lE): miz (%) 456 (100) (M +), 426 (18), 218 (15), 188 (67), 160 (83), 130 (29), 104 (18). Anal. cale. for C2sH20N40 S (456.45): C 65.78; H 4.42; N 12.27. Found: C 65.50; H, 4.67; N 12.51.
2-Benzyl-3- (2-phthalimidoethoxy) -5-nitro-2H-indazole (6). Yield: 0.49 9 (30%). P. f. 249251 ° C (MeN02). 1H NMR [300 MHz, (CD3hSO, 80 OC]: OR 8.81 (d, J = 2.1 Hz, 1 H, 4-H), 7.86 (dd, J = 9.6, 2.1 Hz, 1 H, 6-H), 7.82 (m, 4H, phthalimido H), 7.48 (d, J = 9.6 Hz, 1 H, 7-H), 7.18 (m, 5H , Aromatic H Bn), 5.36 (s, 2H, CH2 Bn), 4.97 (t, J = 5.4 Hz, 2H, 1'-H), 4.14 (t, J = 5, 4 Hz, 2H, 2'-H); 13C NMR [75 MHz, (CD3hSO, 80 OC]: OR 167.19 (phthalimido CO), 148.72 (C-3), 146.46 (C-7a) , 140.25 (C-5), 135.11 (C-1 Bn), 133.88 (C-4, -5 phthalimido), 131.19 (C-1, -2 phthalimido), 127.90 ( C-3, -5 Bn), 127.19 (C-2, -4, -6 Bn), 122.55 (C-3, -6 phthalimido), 119.73 (C-6), 119.32 (C-4), 117.64 (C-7), 104.47 (C-3a), 70.49 (C-1 '), 51, 24 (CH2 Bn), 37.05 (C-2' ); MS (ES +): miz (%) 174 (90), 443 (100) ([M + Hn, 465 (14) ([M + Nan. Anal. Heating for C24H1SN40S (442.42): C 65 , 15; H 4.10; N 12.66. Found: C 65.03; H 4.27; N 12.47.
2-Benzyl-3- (3-phthalimidopropoxy) -5-nitro-2H-indazole (7). Yield: 0.68 g (40%). P. f. 183-185 oC (1-PrOH). 1H NMR [300 MHz, (CD3) 2S0, 80 oC): OR 8.84 (d, J = 1.9 Hz, 1H, 4-H), 7.88 (dd, J = 9.6, 1, 9 Hz, 1 H, 6-H), 7.82 (m, 4H, phthalimido H), 7.51 (d, J = 9.6 Hz, 1H, 7-H), 7.30 (m, 5H , Aromatic H Bn), 5.44 (s, 2H, CH2 Bn), 4.79 (t, J = 5.4 Hz, 2H, 1'-H), 3.83 (t, J = 6, 3 Hz, 2H, 3'-H), 2.17 (m, 2H, 2'-H); 13C NMR [75 MHz, (CD3hSO]: OR 168.07 (phthalimido CO), 149.62 (C-3), 146.81 (C-7a), 140.11 (C-5), 135.73 ( C-1 Bn), 134.26 (C-4, -5 phthalimido), 131.76 (C-1, -2 phthalimido), 128.63 (C-3, -5 Bn), 127.95 (C -2.6 Bn), 127.90 (C-4 Bn), 122.96 (C-3, -6 phthalimido), 121.08 (C-4), 119.88 (C-6), 117, 99 (C-7), 104.95 (C-3a), 71.40 (C-1 '), 51.67 (CH2 Bn), 34.11 (C-3'), 28.21 (C- 2 '); MS (lE): mIz (%) 456 (1) (M +), 368 (2), 188 (100), 160 (64), 130 (16). Calc. Analysis for C2sH20N40 S (456 , 45): C 65.78; H 4.42; N 12.27. Found: C 65.86; H 4.67; N 12.09.
EXAMPLE 2. Preparation of 1- (w-aminoalkyl) indazolinones 8 and 9 [formula (1)] by deprotection of the corresponding w-phthalimidoalkyl derivatives 4 and 5. Removal of the phthaloyl protecting group of compounds 4 and 5 (described in Example 1) with MeNH2 / H20 led to the final 1- (w-aminoalkyl) indazolinones 8 and 9 (Scheme 2).
For this, a suspension of the corresponding 1- (w-phthalimidoalkyl) indazolinone (4 or 5; 2.50 mmol) in MeNH2 / H20 (40% w / w; 20 mL) was heated at 80 ° C for 4 h. The reaction was allowed to cool to room temperature, the formed amine was extracted with chloroform (4 x 50 mL) and the organic solution was evaporated to dryness. In each case, the product obtained was dissolved in HCI N (50 mL) and extracted with ether (4 x 50 mL); The aqueous phase was evaporated to dryness and the residue was triturated with ether (20 mL), providing the desired amines as the corresponding hydrochlorides, which were collected by filtration and dried.
1- (2-Aminoethyl) -2-benzyl-5-nitro-1, 2-dihydro-3H-indazol-3-one (hydrochloride) (8). Yield: 0.84 g (96%). P. f. 261-263 oC (EtOH / H20). 1H NMR [300 MHz, (CD3hSO]: OR 8.53 (d, J = 2.4 Hz, 1 H, 4-H), 8.41 (dd, J = 9.3, 2.4 Hz, 1 H, 6-H), 8.33 (sa, 3H, NH3 +), 7.68 (d, J = 9.3 Hz, 1 H, 7-H), 7.27 (m, 5H, aromatic H. Bn), 5.29 (s, 2H, CH2 Bn), 4.39 (t, J = 6.9 Hz, 2H, 1'-H), 2.80 (t, J = 6.9 Hz, 2H , 2'-H); 13C NMR [75 MHz, (CD3hSO]: OR 160.79 (C-3), 148.23 (C-7a), 141.64 (C-5), 135.93 (C -1 Bn), 128.77 (C-3, -5 Bn), 127.91 (C-4 Bn), 127.38 (C-6), 127.26 (C-2, -6 Bn), 120.45 (CA), 116.06 (C-3a), 111.88 (C-7), 44.63 (CH2 Bn), 43.56 (C-1 '), 35.41 (C-2 '); MS (ES +): miz (%) 313 (100) ([M + Hn 335
(4) ([M + Nan 625 (57) ([2M + Hn 647 (26) ([2M + Nan. Anal calc. For C16H17CIN40 3 (348.78): C 55.10; H 4.91; N 16.06 Found: C 55.37; H 4.76; N 15.85.
1- 1- (3-Aminopropyl) -2-benzyl-5-nitro-1, 2-dihydro-3H-indazol-3-one (hydrochloride) (9).
Yield: 0.86 g (95%). P. f. 202-204 oC (1-PrOH). 1H NMR [300 MHz, (CD3) 2S0j: OR 8.53 (d, J = 2.4 Hz, 1 H, 4-H), 8.40 (dd, J = 9.0, 2.4 Hz, 1 H, 6-H), 8.03 (sa, 3H, NH3 +), 7.75 (d, J = 9.0 Hz, 1 H, 7-H), 7.27 (m, 5H, aromatic H .Bn), 5.20 (s, 2H, CH2 Bn), 4.20 (t, J = 7.2 Hz, 2H, 1'-H), 2.71 (sa, 2H, 3'-H) , 1.61 (m, 2H, 2'-H); 13C NMR [75 MHz, 10 (CD3) 2S0j: OR 161.22 (C-3), 149.07 (C-7a), 141.61 (C-5), 136.10 (C-1 Bn), 128.69 (C-3.5 Bn), 127.85 (C-4 Bn), 127.42 (C-2, -6 Bn), 127.31 (C-6), 120.51 (C- 4), 116.10 (C-3a), 112.36 (C-7), 44.65 (CH2 Bn), 44.30 (C-1 '), 36.07 (C-3'), 23 , 92 (C-2 '); MS (ES +): miz (%) 327 (100) ([M + Hn 349 (10) ([M + Nan 653 (25) ([2M + Hn 675 (18) ([2M + Nan. Anal calc. for C17H19CIN403 (362.81): C 56.28; H 5.28; N 15.44. Found: C 56.12; H
15 4.99; N 15.71.
EXAMPLE 3. Preparation of 1- (3-aminopropyl) indazolinones 11-13 [formula (1)]from 1- (3-bromopropyl) indazolinone 10.The treatment of 1- (3-bromopropyl) indazolinone 10 (Fonseca-Berzal, C. et al., Eur. J.
20 Med. Chem. 2016,115,295-310) with primary (MeNH2) or secondary amines (Me2NH, piperidine) gave rise to the final products 11-13 (Scheme 3). Analogous reactions starting from analogue 2-bromoethyl did not lead to the expected products, since in these cases the halogenated derivative preferably underwent dehydrohalogenation to the corresponding 1-vinylindazolinone (Fonseca-Berzal, C. et
25 al., Eur. J. Med. Chem. 2016, 115, 295-310).
In the case of 3-methylamino (11) and 3-dimethylamino (12) derivatives, a mixture of 1 (3-bromopropyl) indazolinone 10 (0.98 g, 2.51 mmol) and the corresponding amine (MeNH2 / EtOH 33% w / w or Me2NH / EtOH 33% w / w; 30 mL) was stirred at room temperature 30 for 24 h. The mixture was evaporated to dryness, the formed amine was extracted with chloroform (4 x 50 mL) and the organic solution was evaporated again. The product obtained was dissolved in HCI N (50 mL) and extracted with ether (4 x 50 mL); the aqueous phase was evaporated to dryness and the residue was triturated with ether (20 mL), providing the
Desired amines such as the corresponding hydrochlorides, which were collected by filtration and dried.
In the case of the 3-piperidino derivative (13), a mixture of the 3-bromopropyl derivative 10 (0.98 g, 2.51 mmol) and piperidine (0.45 g, 5.28 mmol) in EtOH (50 mL) it was stirred at room temperature for 48 h and then refluxed for 3 h. The reaction was evaporated to dryness and the residue was dissolved in HCI N (50 mL) and extracted with ether (4 x 50 mL). The aqueous phase was made alkaline with KZC03, and the free amine was extracted with chloroform (4 x 50 mL). The residue obtained by evaporation of the solvent was treated with oxalic acid (salt.) In ethanol (5 mL) and then the amine oxalate was precipitated by the addition of ether (100 mL). The salt obtained was collected by filtration and dried.
2-Benzyl-1- [3- (methylamino) propylj-5-nitro-1, 2-dihydro-3H-indazol-3-one (hydrochloride)
(eleven). Yield: 0.90 g (95%). P. f. 186-188 oC (1-PrOH). 1 H NMR [300 MHz, (CD3) zSO]: OR 8.61 (sa, 2H, NHz +), 8.52 (d, J = 2.1 Hz, 1 H, 4-H), 8.40 (dd , J = 9.0, 2.1 Hz, 1 H, 6-H), 7.74 (d, J = 9.0 Hz, 1 H, 7-H), 7.27 (m, 5H, H aromal. Bn), 5.20 (s, 2H, CHz Bn), 4.19 (t, J = 7.5 Hz, 2H, 1'-H), 2.81 (t, J = 7.5 Hz , 2H, 3'-H), 2.44 (s, 3H, Me), 1.64 (m, 2H, 2'-H); 13C NMR [75 MHz, (CD3) zSO]: OR 161, 19 (C-3), 148.96 (C-7a), 141, 63 (C5), 136.07 (C-1 Bn), 128, 72 (C-3, -5 Bn), 127.86 (C-4 Bn), 127.38 (C-2, -6 Bn), 127.34 (C-6), 120.51 (C-4 ), 116.13 (C-3a), 112.28 (C-7), 45.30 (C-3 '), 44.71 (CHz Bn), 44.20 (C1'), 32.25 ( Me), 22.44 (C-2 '); MS (ES +): miz (%) 341 (100) ([M + Hn, 681 (18) ([2M + Hn Cal. Anal. For C18HzlCIN403 (376.84): C 57.37; H 5.62; N 14.87 Found: C 57.20, H 5.87, N 14.77.
2-Benzyl-1- [3- (dimethylamino) propyl] -5-nitro-1, 2-dihydro-3H-indazol-3-one (hydrochloride)
(12). Yield: 0.87 g (89%). P. f. 176-178 oC (2-PrOH). 1 H NMR [300 MHz, (CD3) zSO]: OR 10.90 (sa, 1 H, NW), 8.53 (d, J = 2.1 Hz, 1 H, 4-H), 8.39 ( dd, J = 9.3, 2.1 Hz, 1 H, 6-H), 7.75 (d, J = 9.3 Hz, 1 H, 7-H), 7.28 (m, 5H, H aromal. Bn), 5.22 (s, 2H, CHz Bn), 4.19 (t, J = 7.5 Hz, 2H, 1'-H), 2.96 (t, J = 7.5 Hz, 2H, 3'-H), 2.59 (s, 6H, Me), 1.69 (m, 2H, 2'-H); 13C NMR [75 MHz, (CD3) zSO]: OR 161, 10 (C-3), 148.78 (C-7a), 141.59 (C5), 136.14 (C-1 Bn), 128, 74 (C-3, -5 Bn), 127.85 (C-4 Bn), 127.38 (C-2, -6 Bn), 127.31 (C-6), 120.47 (C-4 ), 116.09 (C-3a), 112.22 (C-7), 53.44 (C-3 '), 44.76 (CHz Bn), 44.30 (C1'), 41, 85 ( Me), 20.80 (C-2 '); MS (ES +): miz (%) 355 (100) ([M + Hn, 377 (7) ([M + Nan, 731 (7) ([2M + Nan Anal. Heating for C19H23CIN403 (390.86): C 58.38; H 5.93; N 14.33 Found: C 58.09; H 5.98; N 14.57.
2-Benzyl-5-nitro-1- (3-piperidinopropyl) -1, 2-dihydro-3H-indazol-3-one (oxalate) (13).
5 Yield: 1.03 g (85%). P. f. 178-180 oC (MeOH). 1H NMR [300 MHz, (CD3hSO): OR 8.52 (d, J = 2.1 Hz, 1 H, 4-H), 8.45 (sa, 1 H, NW), 8.38 (dd, J = 9.3, 2.1 Hz, 1 H, 6-H), 7.68 (d, J = 9.3 Hz, 1 H, 7-H), 7.28 (m, 5H, aromatic H .Bn), 5.18 (s, 2H, CH2 Bn), 4.09 (t, J = 7.2 Hz, 2H, 1'-H), 2.86 (ma, 6H, 3'-H, 2-, 6-H piperidino), 1.63 (sa, 6H, 2'-H, 3-, 5-H piperidino), 1.44 (sa, 2H, 4-H piperidino); 13C NMR [75 MHz, (CD3hSO): OR 164.79 (CO
10 oxalate), 161.04 (C-3), 148.85 (C-7a), 141.58 (C-5), 136.11 (C-1 Bn), 128.76 (C-3, - 5 Bn), 127.85 (C-4 Bn), 127.33 (C-2, -6 Bn), 127.28 (C-6), 120.44 (C-4), 116.11 (C -3a), 112.23 (C-7), 52.82 (C-3 '), 51.93 (C-2, -6 piperidino), 44.75 (CH2 Bn), 44.37 (C- 1 '), 22.58 (C-3, -5 piperidino), 21.42 (C-4 piperidino), 20.66 (C-2'); MS (ES +): miz (%) 395 (100) ([M + Hn, 417 (8) ([M + Nan, 811 (4) ([2M + Nan Anal. Heating for C24H2SN407 (484.50): C
15 59.50; H 5.83; N 11, 56. Found: C 59.75; H 5.57; N 11.57.
EXAMPLE 4. Preparation of 3- [2- (dimethylamino) ethoxy] indazole 16 [formula (11)) afrom 3- (2-bromoethoxy) indazole 14.The treatment of 3- (2-bromoethoxy) indazol 14 (Fonseca-Berzal, C. et al., Eur. J. Med.
20 Chem. 2016, 115, 295-310) with a secondary amine such as Me2NH resulted in the expected 2 (dimethylamino) ethoxy derivative 16 (Scheme 4).
To this end, a mixture of 3- (2-bromoethoxy) indazole 14 (0.94 g, 2.50 mmol) and Me2NH / EtOH (33% w / w; 20 mL) was stirred at room temperature for 24 h. Be
25 evaporated the mixture to dryness and the residue was extracted with chloroform (4 x 50 mL); The organic phase was evaporated, the residue was dissolved in HCI N (50 mL) and the solution was extracted with ether (4 x 50 mL). The aqueous phase was evaporated to dryness and the residue was triturated with ether (20 mL) to provide the desired amine hydrochloride, which was collected by filtration and dried.
30 2-Benzyl-3- [2- (dimethylamino) ethoxy} -5-nitro-2H-indazole (hydrochloride) (16). Yield: 0.87 g (92%). P. f. 177-179 oC (2-PrOH). 1H NMR [300 MHz, (CD3hSO): OR 11.39 (sa, 1 H, NW), 8.94 (d, J = 2.1 Hz, 1 H, 4-H), 7.91 (dd, J = 9.6, 2.1 Hz, 1 H, 6-H), 7.55 (d, J = 9.6 Hz, 1 H, 7-H), 7.30 (m, 5H, aromatic H .Bn), 5.65 (s, 2H, CH2 Bn), 5.19 (t, J = 4.2 Hz, 2H, 1'-H), 3.63 (t, J = 4.2 Hz, 2H, 2'-H), 2.84 (s, 6H, Me); 13C NMR [75 MHz, (CD3hSOj: OR 148.81 (C-3), 146.82 (C-7a), 140.27 (C-5), 136.00 (C-1 Bn), 128.53 (C-3, -5 Bn), 128.03 (C-2, -6 Bn), 127.79 (C-4 Bn), 121.14 (C-4), 119.88 (C-6) , 118.07 (C-7), 104.84 (C-3a), 67.60 (C-1 '), 55.32 (C-2'), 51.68 (CH2 Bn), 42.48 (Me); MS (ES ·): miz (%) 341
(100) ([M + Hn Anal. Cale for C18H21CIN40 3 (376.84): C 57.37; H 5.62; N 14.87. Found: C 57.10; H 5.43; N 14 , 99.
EXAMPLE 5. Preparation of 3- (3-aminopropoxy) indazol 19 [(formula (11)], 3 (methylamino) indazol 21 [formula (111)] and 3 - [(w-hydroxyalkyl) amino] indazoles 22 And 24. [formula (111)] from 3- (w-phthalimidoalkoxy) derivatives 6 and 7. the removal of the phthaloyl moiety of compounds 6 and 7 (Example 1) with MeNH2 / H20 resulted in amines 17 and 19; these compounds were easily detected by thin layer chromatography (TLC) in the reaction mixtures and even 19 could be isolated with low yield; these products, however, were not stable, undergoing intramolecular transposition to the corresponding 3- [(whidroxyalkyl) aminojindazoles 22 and 24. In both cases, 3- (methylamino) indazole 21 was also isolated from these reactions, derived from the intermolecular nucleophilic substitution of the 3-alkoxy group of 6/17 and 7/19 by the rest of MeNH2 present in the reaction medium (Scheme 4).
With this objective, a suspension of the corresponding 3- (phthalimidoalkoxy) indazole (6 or 7) (2.50 mmol) in MeNH2 / H20 (40% w / w; 20 ml) was heated at 80 oC for 4 h (for 22 )
or 20 h (for 24). The reaction was allowed to cool to room temperature, extracted with chloroform (4 x 50 ml) and the organic solution evaporated to dryness. Starting from 6, the residue was chromatographed on silica gel flash column eluting with chloroform / acetone mixtures (30/1 to 5/1) to lead, in this order of elution, to compounds 21 (49 mg, 7%) and 22. Starting from 7, the column was eluted with chloroform / ethanol mixtures (50/1 to 10/1) to obtain compounds 21 (0.26 g, 37%) and 24 and then with chloroform / methanol (20/1 to 5/1) to obtain 3- (3-aminopropoxy) indazoI19; The latter was isolated as the corresponding hydrochloride by dissolution in HCI N (15 ml), followed by evaporation to dryness.
3- (3-Aminopropoxy) -2-benzyl-5-nitro-2H-indazole (hydrochloride) (19). Yield: 36 mg (4%). P. f. 194-196 oC (previous softening) (2-PrOH). 1 H NMR [500 MHz,
(CD3) 2S0): OR 8.91 (d, J = 2.1 Hz, 1 H, 4-H), 8.03 (sa, 3H, NH3 +), 7.91 (dd, J = 9.6 , 2.1 Hz, 1 H, 6-H), 7.55 (d, J = 9.6 Hz, 1 H, 7-H), 7.33 (m, 5H, Bn aromat. H), 5 , 47 (s, 2H, Bn CH2), 4.88 (t, J = 5.9 Hz, 2H, 1'-H), 3.01 (t, J = 6.0 Hz, 2H, 3'- H), 2.16 (m, 2H, 2'-H); 13C NMR [125 MHz, (CD3) 2S0): OR 149.54 (C-3), 146.81 (C-7a), 140.17 (C-5), 135.79 (Bn C1), 128, 68 (Bn C-3, -5), 127.92 (Bn C-2, -4, -6), 121, 15 (C-4), 119.94 (C-6), 118.07 (C7 ), 104.90 (C-3a), 70.59 (C-1 '), 51.72 (Bn CH2), 35.78 (C-3'), 27.24 (C-2 '); MS (ES +): miz (%) 327 (100) ([M + Hn, 349 (13) ([M + Nat). Cal. Anal for C17H19CIN40 3 (362.81): C 56.28; H 5 , 28; N 15.44. Found: C 56.01; H 5.57; N 15.17.
2-Benzyl-3-methylamino-5-nitro-2H-indazole (21). P. f. 185-187 oC (2-PrOH). 1 H NMR [300 MHz, (CD3) 2S0]: OR 8.79 (d, J = 2.1 Hz, 1 H, 4-H), 7.79 (dd, J = 9.6, 2.1 Hz, 1 H, 6-H), 7,407.14 (m, 7H, 7-H, NH, aromatic H Bn), 5.38 (s, 2H, CH2 Bn), 3.23 (d, J = 4.8 Hz, 3H, Me); 13C NMR [75 MHz, (CD3) 2S0): OR 147.72 (C-7a), 146.51 (C-3), 137.18 (C-5), 136.17 (C-1 Bn), 128.50 (C-3, -5 Bn), 127.51 (C-4 Bn), 127.25 (C-2, -6 Bn), 123.65 (C-4), 120.32 (C6 ), 115.78 (C-7), 107.40 (C-3a), 51.04 (CH2 Bn), 31.13 (Me); MS (lE): miz (%) 282 (100) (M +), 236 (5), 191 (21), 163 (3), 145 (6), 117 (11), 102 (11). Anal. calc. for C1sH14N40 2 (282.30): C 63.82; H 5.00; N 19.85. Found: C 63.57; H 5.27; N 19.67.
2-Benzyl-3 - [(2-hydroxyethyl) aminoj-5-nitro-2H-indazole (22). Yield: 0.70 9 (90%). P.
F. 171-173 oC (MeCN). 1H NMR [300 MHz, (CD3) 2S0): OR 8.80 (d, J = 2.1 Hz, 1H, 4-H), 7.81 (dd, J = 9.6, 2.1 Hz, 1H, 6-H), 7.38-7.10 (m, 7H, 7-H, NH, aromatic H Bn), 5.44 (s, 2H, CH2 Bn), 4.94 (sa, 1 H, OH), 3.66 (sa, 4H, 1'-, 2'-H); 13C NMR [75 MHz, (CD3) 2S0]: OR 147.79 (C-7a), 145.90 (C-3), 137.36 (C-5), 136.30 (C-1 Bn), 128.50 (C-3, -5 Bn), 127.50 (C-4 Bn), 127.34 (C-2, -6 Bn), 123.31 (C-4), 120.25 (C -6), 116.00 (C-7), 107.17 (C-3a), 59.97 (C-2 '), 51.18 (CH2 Bn), 46.97 (C-1'); MS (lE): miz (%) 312 (63) (M +), 281 (37), 221 (5), 191 (7), 102 (6), 91 (100). Anal. calc. for C16H16N403 (312.32): C 61.53; H 5.16; N, 17.94. Found: C 61, 70; H 4.97; N 17.57.
2-Benzyl-3 - [(3-hydroxypropyl) aminoj-5-nitro-2H-indazole (24). Yield: 0.44 9 (54%).
P. f. 154-156 oC (2-PrOH). 1H NMR [300 MHz, (CD3) 2S0]: OR 8.81 (d, J = 2.1 Hz, 1H, 4-H), 7.80 (dd, J = 9.6.2.1 Hz, 1H, 6-H), 7.38-7.10 (m, 7H, 7-H, NH, aromatic H Bn), 5.42 (s, 2H, CH2 Bn), 4.63 (t, J = 5.1 Hz, 1 H, OH), 3.66 (m, 2H, 1'-H), 3.52 (m, 2H, 3'-H), 1.80 (m, 2H, 2 ' -H); 13C NMR [75 MHz, (CD3) 2S0): OR 147.77 (C-7a), 145.71 (C-3), 137.42 (C5), 136.23 (C-1 Bn), 128, 51 (C-3, -5 Bn), 127.51 (C-4 Bn), 127.28 (C-2, -6 Bn), 123.46
(C-4), 120.28 (C-6), 115.98 (C-7), 107.05 (C-3a), 58.02 (C-3 '), 51.17 (CH2 Bn) , 41.46 (C1 '), 32.37 (C-2'); MS (lE): miz (%) 326 (100) (M +), 281 (24), 235 (18), 205 (8), 191 (16), 102 (10). Anal. calc. for C17H18N403 (326.35): C 62.57; H 5.56; N 17.17. Found: C 62.83; H 5.77; N 16.93.
5 EXAMPLE 6. Preparation of 3- (methylamino) indazol 21 and 3 - [(whidroxyalkyl) methylamino] indazoles 23 and 25 [formula (111)] from 3- (wbromoalkoxy) indazoles 14 and 15. Both the compound 14 as its analog 3-bromopropoxy 15 (Fonseca-Berzal, C. et al.,
10 Eur. J. Med. Chem. 2016, 115, 295-310), treated with a primary amine such as MeNH2 initially led to the corresponding 3- (w-aminoalkoxy) derivatives 18 and 20 which, as described for analogues 17 and 19 (Example 5), were quickly transposed to 3 - [(w-hydroxyalkyl) methylamino) indazoles 23 and 25, respectively; 3- (methylamino) indazole 21 could also be isolated from these processes (Scheme 4).
For this, a suspension of the corresponding 3- (w-bromoalkoxy) indazole (14 or 15) (2.50 mmol) in MeNH2 / EtOH (33% w / w; 30 mL) was stirred at room temperature for 20 h (for 23) or 4 days (for 24). The mixture was evaporated to dryness and the residue was chromatographed on silica gel flash column using chloroform / acetone mixtures.
20 (30/1 to 10/1). Following this elution order, the derived methylamino 21 [77 mg (11%) from 14 was first obtained; 0.13 g (18%) from 15) and then the corresponding transposed products 23 and 25.
2-Benzyl-3 - [(2-hydroxyethyl) methylaminoj-5-nitro-2H-indazole (23). Yield: 0.70 9
25 (86%). P. f. 111-113 oC (2-PrOH). 1H NMR [300 MHz, (CD3hSO]: or 8.86 (d, J = 2.4 Hz, 1 H, 4-H), 7.93 (dd, J = 9.6, 2.4 Hz, 1 H , 6-H), 7.60 (d, J = 9.6 Hz, 1 H, 7-H), 7.38-7.20 (m, 5H, aromatic H Bn), 5.61 (s , 2H, CH2 Bn), 4.78 (t, J = 5.4 Hz, 1 H, OH), 3.54 (m, 2H, 2'H), 3.32 (t, J = 4.5 Hz, 2H, 1'-H), 3.02 (s, 3H, Me); 13C NMR [75 MHz, (CD3hSO]: or 148.11 (C-3), 147.63 (C-7a), 140.33 (C-5), 136.44 (C-1 Bn), 128.51 (C-3, -5Bn), 127.69
30 (C-2, -6 Bn), 127.60 (C-4 Bn), 120.73 (C-4), 119.64 (C-6), 118.28 (C-7), 112, 61 (C-3a), 58.47 (C-2 '), 58.29 (C-1'), 52.24 (CH2 Bn), 41.26 (Me); MS (lE): miz (%) 326 (42) (M +), 295 (100), 249 (13), 235 (21), 205 (11), 192 (7), 161 (7), 116 (11 ), 91 (49). Anal. calc. for C17H18N403 (326.35): C 62.57; H 5.56; N 17.17. Found: C 62.30; H 5.77; N 17.01.
2-Benzyl-3 - {(3-hydroxypropyl) methylamino] -5-nitro-2H-indazole (25). Yield: 0.65 9(76%). Oil that solidifies over time; p. F. 67-69 oC. 1H NMR [300 MHz, (CD3hSO):OR 8.83 (d, J = 2.1 Hz, 1 H, 4-H), 7.93 (dd, J = 9.3, 2.1 Hz, 1H, 6-H), 7.62 ( d, J = 9.3 Hz,1H, 7-H), 7.38-7.12 (m, 5H, aromatic H Bn), 5.55 (s, 2H, CH2 Bn), 4.45 (t, J = 5.1 Hz, 1 HOUR,OH), 3.37 (m, 2H, 3'-H), 3.32 (t, J = 6.9 Hz, 2H, 1'-H), 2.94 (s, 3H, Me), 1 , 62 (m, 2H, 2 'H); 13C NMR [75 MHz, (CD3) 2S0): OR 147.94 (C-7a), 147.62 (C-3), 140.51 (C-5), 136.31(C-1 Bn), 128.55 (C-3, -5 Bn), 127.66 (C-4 Bn), 127.52 (C-2, -6 Bn), 120.53 (C-4 ), 119.68(C-6), 118.43 (C-7), 112.58 (C-3a), 58.04 (C-3 '), 52.83 (C-1'), 52.36 (CH2 Bn ), 41.46(Me), 30.58 (C-2 '); MS (lE): miz (%) 340 (81) (M +), 295 (100), 249 (48), 205 (51), 174(10), 159 (7), 130 (7), 116 (21), 102 (6). Anal. calc. for C18H2oN4 0 3 (340.38): C 63.52;H 5.92; N, 16.46. Found: C 63.24; H 5.77; N 16.71.
EXAMPLE 7. In vitro study of the activity and selectivity index ofderivatives of indazole on Trypanosoma cruzi.These studies are performed following a sequential in vitro screening protocolpublished in the literature and followed previously by the inventors (Fonseca-Berzal,
C. et al., Bioorg. Med. Chem. Lett. 2013, 23, 4851-4856; Fonseca-Berzal, C. et al., Eur.
J. Med. Chem. 2016, 115, 295-310). First, the activity of the products against epimastigotes of T. cruzi (extracellular form present in the vector insect) and their nonspecific toxicity against 1929 fibroblasts (host cells of amastigotes) are simultaneously evaluated. Compounds that have a selectivity in epimastigotes equal to or greater than that of the reference drug, benznidazole, are selected for further study in amastigotes, more significant from the point of view of human disease as the intracellular forms of the parasite present in mammals
Example 7a. Activity against epimastigotes of T. cruzi.To determine in vitro activity on epimastigotes, axenic cultures were used.of T. cruzi, strain Cl clone B5 stably transfected with the p geneE. coli galactosidase (lacZ). the activity is obtained from a methodcolorimetric that determines the red chlorophenol released from its p-Dgalactopyranoside (CPRG) by parasites that remain alive after treatment.the compounds, dissolved in dimethyl sulfoxide (DMSO), were tested at
final concentrations in the culture medium of 256-0,125 J.lM following a procedure described (Fonseca-Berzal, C. et al., Bioorg. Med. Chem. Lett. 2013, 23, 4851-4856). From the corresponding dose-response curves, the concentration necessary to inhibit 50% of the growth of the compounds was estimated for each compound.
5 epimastigotes (Clso epimastigotes). The activity results, shown in Table 1, are expressed as the mean of the Clso in epimastigotes ± standard deviation (OS), from the values obtained in three independent experiments. It can be seen that seven 1,2-disubstituted indazolinones [formula (1); 2, 3, 8, 9 AND 11
10 13], a 3-aminoalkoxy-2-benzylindazole [formula (11); 19] And three 3-alkylamino-2-benzylindazoles [formula (111); 21, 22 and 24] show activity values of the same order
or higher than benznidazole. Special attention is paid to primary 8 and 9 and secondary 11, much more active (Cl so ::; 0.5 iJM) against T. cruzi epimastigotes than the benznidazole reference drug (Clso = 26.6 iJM) And that a tertiary amine
15 related, derived from piperidine, previously described (Cl so = 9.0 iJM) (Mura, F. et al., J. Spectrosc. Dyn. 2013, 3, article 8).
Table 1. In vitro activity against extracellular (epimastigote) and intracellular (amastigote) forms of T. cruzi Cl-B5, nonspecific cytotoxicity in murine fibroblasts 1929, 20 expressed as Clso and Clso, respectively, and selectivity indices (18).
Comp. Clim epimastigotes (J.lM)CLso L929 (J.lM)Isa epimastigotesCl so amastigotes (J.lM)ISbamastigotes
2 10.63 ± 0.18gt; 256gt; 24.083.93 ± 1.11gt; 65.14
3 23.36 ± 5.89gt; 256gt; 10.96
4 89.73 ± 6.07gt; 256gt; 2.85
5 gt; 256gt; 256NO
6 gt; 256gt; 256NO
7 gt; 256gt; 256NO
8 0.35 ± 0.00237.21 ± 27.93677.740.29 ± 0.03817, 96
9 0.24 ± 0.12161, 36 ± 22.55672.330.25 ± 0.13645.44
eleven 0.50 ± 0.19242.91 ± 6.23485.820.71 ± 0.02342.13
12 0.94 ± 0.05103.86 ± 31, 97110.491.24 ± 0.1783.76
13 16.95 ± 0.79gt; 256gt; 15.1015.25 ± 2.70gt; 16.79
16 41, 76 ± 6.59126.59 ± 17.323.03
19 14.07 ± 2.67102.32 ± 8.127.27
twenty-one 17.55 ± 6.90113.00 ± 18.476.44
22 22.46 ± 1.91gt; 256gt; 11.401.37 ± 0.26> 186.86
2. 3 58.47 ± 2.12159.29 ± 2.612.72
24 13.43 ± 8.24200.65 ± 27.7014.945.52 ± 0.0436.35
25 31, 76 ± 1, 32100.58 ± 13.703.17
Benznidaz01 26.55 ± 4.53gt; 256gt; 9.640.50 ± 0.03gt; 512
selectivity indices for epimastigotes (IS = LC50 L929 / C15o epimastigotes).Selectivity indexes for amastigotes (IS = LC50 L929 / C15 or amastigotes).NO: not determined.-: Not evaluated in amastigotes for not reaching epimastigotes the minimum of
5 selectivity established by the reference drug (IScomposite <; ISBenznidazole).
Example 7b. I cite non-specific toxicity on murine fibroblasts L929 and determination of selectivity indices (IS). In order to determine whether anti-T activity. cruzi of the studied indazoles is
10 specific and that are not toxic to mammalian cells, their unspecific cytotoxicity in fibroblasts was evaluated by fluorimetry using resazurine, a redox indicator that undergoes a color change and emits fluorescence in the presence of metabolically active cells (Fonseca-Berzal, C. et al ., Bioorg. Med. Chem. Lett. 2013, 23, 4851-4856).
The compounds studied were added to the culture medium at final concentrations 256-0,125 µm, and for each of them the concentration necessary to inhibit 50% of cell growth (LC50 L929) was calculated from the corresponding dose curve -reply. The activity results, shown in Table 1, are expressed as the mean of the LC50 ± OS, from the values obtained in three experiments
20 independent. From these data, the corresponding selectivity indices were calculated (IS = LC50 L929 / C15 or epimastigotes). It can be seen that the most active compounds against epimastigotes (8, 9, 11 and 12) have IS (110.5-677.7) much higher than the reference drug (> 9.6). Other compounds with interesting anti-epimastigote activity (2, 3, 13, 19, 21, 22 and 24)
they were, however, quite toxic to fibroblasts, so their IS (6.4 to> 24.1) were not as satisfactory.
Example 7e Activity against amastigotes of T. cruzi.
These tests were performed on the same strain used when studying the activity against epimastigotes, also using the CPRG chromogenic substrate, following previously described protocols (Fonseca-Berzal, C. et al., Parasitol. Res. 2014, 113, 1049- 1056). For this, L929 cells were infected with trypomastigotes derived from cell culture (TDC), which quickly transformed intracellularly into amastigotes. This study was carried out with the compounds that showed an IS similar or superior to that of the reference drug (benznidazole) against epimastigotes, determining, as described above (Example 7a), the concentration necessary to inhibit 50% of the growth of amastigotes (Clso amastigotes) from the corresponding dose-response curves. Activity results are expressed as the mean of Clso ± OS, based on the values found in three independent experiments. The activities obtained, as well as the corresponding IS for amastigotes are shown in Table 1.
It can be seen that most of the effective compounds against epimastigotes also show a similar activity (8, 9, 11-13) or, as with benznidazole, superior (2, 22 and 24) against amastigotes. Compounds 8 and 9 stand out (Clso = 0.29 and 0.25 ~ M, respectively), with activity higher than that of benznidazole (Clso = 0.50 ~ M) and excellent IS values (> 645). These results are especially interesting from the point of view of human disease, taking into account the relevant role played by amastigotes, intracellular forms present in mammalian cells.
In this sense, many compounds meet the requirements of activity and IS against amastigotes of a strain of T. cruzi of the TcVI type necessary to be considered good starting points (hits) for the development of new antihagasic drugs (Clso: S; 10 ~ M or <5 ~ M and IS> 10; Don, R. & loset, J.-R., Parasitology 2014, 141, 140146; Chatelain, E., J. Biomol. Screen. 2015, 20, 22 -35).
The results described here confirm our hypothesis that the introduction of w-aminoalkyl groups in position 1 of the 2-benzylindazolinones [formula (1)] leads to compounds that maintain high trypanocidal activity, while conferring, through the corresponding ammonium salts, a high water solubility. On the other hand, the activity shown by compound 19 [formula (11)] is very interesting. given that other similar 3-alkoxy-2-alkylindazoles described previously, with substituents of another nature, show little anti-gasic activity. Finally, the remarkable trypanocidal activity of some 3- (alkylamino) indazoles, e. g., 22 and 24 [formula (111)], especially against amastigotes, has not been described before, so this type of structure constitutes a promising new skeleton for the further development of new inhazole-derived antihagasic drugs.
EXAMPLE 8. Activity; n v; lro of indazole derivatives against promastigotes of Le; shman; a amazonens; s and determination of selectivity indices (15). Given the close relationship between the genera Trypanosoma and Leishmania, both belonging to the Trypanosomatidae family, some of the 1,2-substituted indazolinones [formula (1): 2, 8, 9, 11 and 12] and 3- (alkylamino) indazoles [formula (111): 21, 22 and 24] active against T. cruzi were also tested against L. amazonensis following a previously published procedure (Sifontes-Rodríguez, S. et al., Mem. Inst. Oswaldo Cruz 2015, 110 , 166-173). For this, the promastigotes of L. amazonesis (MHOM / BR / 77 / L TB0016) were grown at 26 oC in the middle of Schneider supplemented with 10% fetal bovine serum, sodium penicillin (200 IU / mL) and streptomycin (200 IJg / mL). The compounds to be tested were previously dissolved in dimethyl sulfoxide and then serial dilutions were carried out with 96-well plate culture medium to which fresh promastigote cultures were added to achieve a final density of 5 x 105 promastigotes / mL. Eight concentrations of each product were tested and each concentration was tested in quadruplicate. After 72 h of incubation at 26 oC, 20 IJL of 3 mM resazurine was added to each well and incubated for another 3 h under the same conditions. The plates were subsequently read in a fluorescence reader, the growth inhibition values associated with each concentration were calculated and the mean inhibitory concentrations (C150) were estimated by non-linear adjustment to sigmoid curves. Each product was tested four times. Simultaneously the activity of amphotericin B (deoxycholate), used as a reference drug, was tested. On the other hand, in order to determine the corresponding selectivity indexes (IS) against mammalian cells, the unspecific cytotoxicity values previously obtained for murine fibroblasts L929 (Example 7b, Table 1) were used. Activity values
5 obtained, as well as the corresponding IS, are listed in Table 2.
The high activity (IC5o <4 iJM) of 1,2-substituted indazolinones 8, 9 and 11 [formula (1)] and of 3- (alkylamino) indazoles 21, 22 and 24 [formula (111)] versus to promastigotes of L. amazonensis. These values are much higher than the 10 found for amphotericin B, a very effective leishmanicide drug but also very toxic and of high cost in its liposomal formulations. The IC50 values obtained for our compounds are, on the other hand, in the same order as those described for two other reference drugs (miltefosine and pentamidine) against promastigotes of this species. For miltefosine IC50 values of 15 16.8 iJM (Trinconi, CT et al., J. Antimicrob. Chemother. 2016,71, 1314-1322) and 3.4 iJM (Santa-Rita, RM et al. ., J. Antimicrob. Chemother. 2004, 54, 704-710), while for pentamidine they have been 29.9 iJM (Dutra, LA et al., Antimicrob. Agents Chemother. 2014, 58, 4837-4847) And 4.8 iJM (de Melas, JLR et al., Eur. J. Med. Chem. 2015, 103,409-417). This remarkable leishmanicidal activity of some indazolinones 1,2
20 disubstituted, e. g., 8, 9 and 11 [formula (1)] and 3- (alkylamino) indazoles, e. g., 21, 22 and 24 [formula (111)], has not been described before, so both types of indazole derivatives are promising for the development of new leishmanicidal drugs.
Table 2. In vitro activity against extracellular forms (promastigotes) of L. amazonesis (MHOM / BR / 77 / L TB0016), nonspecific cytotoxicity in murine fibroblasts L929, expressed as IC50 and LC50, respectively, and selectivity indices (IS ).
Comp. Clso promastigotes (IlM)CLso L929 (IlM)Isa promastigotes
2 35.82 ± 1.91gt; 256gt; 7.15
8 1.04 ± O, 18237.21 ± 27.93228.09
9 3.42 ± 1.52161, 36 ± 22.5547.18
eleven 2.34 ± 0.39242.91 ± 6.23103.81
12 12.83 ± 0.72103.86 ± 31, 978.10
twenty-one 1.82 ± 1.33113.00 ± 18.4762.09
22 1.61 ± 1.12gt; 256gt; 159.01
24 1.34 ± 0.71200.65 ± 27.70149.74
Amphotericin B 0.044 ± 0.014
selectivity indices for promastigotes (IS = LC50 L929 / C15o promastigotes).
EXAMPLE 9. In vitro study of the activity and selectivity index of
5 derivatives of indazole on Trichomonas vaginalis. These studies aimed at the search for new tricomonicidal agents are based on a sequential screening model composed of several phases that, acting as a filter, allow to move to the next level of study only those products that show significant activity values (Ibáñez-Escribano, A. et al., J. Microbe.
10 Methods 2014, 105, 162-167). First, indazole derivatives are evaluated against trophozoites of a T. vagina / is isolate sensitive to metronidazole (reference drug) and, simultaneously, against Vero cells to detect their possible nonspecific toxicity. Compounds that show relevant activity against the isolate sensitive to the reference drug are then subjected to evaluation against a
15 isolated from T. vagina / is metronidazole-resistant.
Example 9a. Activity against T. vaginalis trophozoites.First, indazole derivatives were evaluated against trophozoites of theJH31A4 isolated from T. vagina / is (ATCC), sensitive to the reference drug metronidazole.
20 In vitro screening is carried out by evaluating the growth percentage of a controlled culture after 24 hours in contact with different concentrations of the compound to be evaluated, following a previously described procedure (Ibáñez Escribano, A. et al., Mem. Insto Oswa / do Cruz 2012, 107, 637-643). To determine the corresponding C1 5o, a stock solution of the compounds to be evaluated in dimethyl sulfoxide is prepared
25 (DMSO) and are tested in a range of six different final concentrations in successive serial double dilutions, starting from a maximum concentration of 300 IJM. Viable cells after treatment are determined by taking advantage of their ability to reduce the resazurine redox dye to resorufin, as discussed in Example 7b (Ibáñez Escribano, A. et al., Mem. Insto Oswa / do Cruz 2012, 107, 637-643).
30 IC50 values are calculated from the average obtained from at least two independent experiments. All compounds are evaluated in triplicate in each test, obtaining an OS below 10%.
The IC50 values obtained for the isolate of T. vaginalis JH31A4 sensitive to metronidazole, as well as the 95% confidence intervals, are shown in Table 3. The compounds that passed the first evaluation filter in T. vaginalis, presenting values of IC50 lt; 50 iJM, were 1,2-disubstituted indazolinones 8 and 11-13 [formula (1)], 3-aminoalkoxy-2-benzylindazoles 16 and 19 [formula (11)], and 3-alkylamino-2-benzylindazoles 21- 23 and 25 [formula (111)]. Compounds 19 [formula (11)], Y 23 and 25 [formula (111)] should be noted, which, although less active than metronidazole (C15o = 1.4 iJM), presented relevant activities against the parasite with IC50 values of 5.6, 8.5 and 10.0 iJM, respectively.
Some of the compounds that showed a relevant activity against the JH31A4 isolate (19, 22, 23 and 25) were also subjected to evaluation against trophozoites of the metronidazole-resistant IR 78 isolate, following an in vitro screening procedure identical to that described above.
The results obtained against the IR 78 isolate are shown in Table 4. The IC50 values corresponding to compounds 19 and 25 (8.5 and 11.0 iJM, respectively) are similar to those obtained in the metronidazole sensitive isolate, which highlights the lack of cross resistance between these compounds and the reference drug. Thus, the skeletons of 3- (w-aminoalkoxy) -2-benzylindazole [formula (11)] and 3 - [(w-hydroxyalkyl) amino] -2-benzylindazole [formula (111)] of compounds 19 and 25, respectively, are of interest for the development of drugs for the treatment of infections caused by T. vaginalis isolates resistant to 5-nitroimidazoles.
Table 3. In vitro activity against trophozortos of T. vaginalis JH31A4, nonspecific cytotoxicity against Vero cells, expressed as IC50 and CC50, respectively, and selectivity indices (IS).
CC50
Comp.
(fAM)
2 276.2 [200.2-540.7] 3 NO
4 61.9 [49.4-78.5]5 NO6 NO7 93.1 [71.4-126.5]8 49.4 [29.8-101, 3] gt; 300 gt; 6.19 170.9 [146.0-205.7]
11 48.9 [38.8-61.4] gt; 300 gt; 6.112 40.8 [30.2-55.6] gt; 300 gt; 7.413 41.3 [26.5-71.6] gt; 300 gt; 7.316 41, 3 [26, 5-71, 6] gt; 300 gt; 7.319 5.6 [4.7-6.4] 104.1 C 18.721 45.4 [38.6-53.3] gt; 300 gt; 6.622 15.7 [11, 7-20.3] gt; 300 gt; 19.123 8.5 [6.4-10.8] gt; 300 gt; 35.224 52.7 [46.8-59.2]10.0 [9.1-11.0] gt; 300 gt; 16.2
Metronidazole 1.4 [1, 1-1.8] gt; 600 gt; 419.6
NO: Not determined by absence of antiparasitic activity.-: Not evaluated against Vero cells due to low Clso values.a Between brackets, 95% confidence intervals.Bindices of selectivity (IS = Clso trophozoites / CCso Vero cells).95% confidence interval: 78.5-138.5.
Table 4. In vitro activity against T. vaginalis IR 78 and non-specific cytotoxicity against Vera cells, expressed as Clso and CCso, respectively.
Comp. Cl so (¡.tM) to CCso (¡.tM) IS
10 19 8.5 [7.6-9.6] 22 34.8 [30.7-39.3] 23 49.3 [34.3-71, 0] 25 11, 0 [6.5-14, 4] Metronidazole 2.6 [2.2-3.2]104.1 b gt; 300 gt; 300 gt; 300 gt; 600gt; 12.2 gt; 8.6 gt; 6.1 gt; 27.4 gt; 143.0
a Between brackets, 95% confidence intervals.
95% confidence interval: 78.5-138.5.
Example 9b. Non-specific cytotoxicity on Vera cells and index determination
of selectivity (IS).
This assay was carried out using the Vero CCL-81 cell line (ATCC) and
only for those molecules that had shown significant activity in
5 vitra versus T. vaginalis trophozoites (Clso <50 IJM). Its purpose is to determine if
studied indazoles have a specific activity on the protozoan, lacking
of nonspecific toxicity to mammalian cells. The method used to
determine the concentrations of products capable of producing 50% of
Cytotoxicity (CCso) is based on the reduction of resazurin, as described
l O in Examples 7b, 8 and 9a. The CCso values are calculated from the average obtained
after performing at least two independent experiments. Each concentration
it is evaluated in triplicate obtaining an OS below 10%, also existing
a growth control that assumes 0% non-specific cytotoxic activity.
fifteen The CCso values obtained in this study, as well as the indexes of selectivity (IS)
are collected in Tables 3 and 4. Most of the compounds tested (8, 11-13,
16, 21 -23, and 25) showed low unspecific cytotoxic activity (CCso> 300 IJM) and,
in the case of sensitive isolate JH31A4, reasonable dellS values (> 6.1 agt; 35.2).
Compound 19 was somewhat more toxic, but due to its high activity still
twenty reached a remarkable IS (18.7). The dellS values obtained for the metronidazole isolate
IR resistant 78 are of the same order (> 6.1 agt; 27.4).
The results described in this invention confirm our hypothesis that the 2
alkyl-3- (w-aminoalkoxy) indazoles, e. g., 19 [formula (11)), maintain the activity
25 tricomonicide described for some analogs containing in position 3 alkoxy groups
singles (Ibáñez-Escribano, A. et al., Parasitology 2016, 143, 34-40; Fonseca-Berzal,
C. et al. , Eur. J. Med. Chem. 2016, 115, 295-310), but they have the advantage of presenting
better pharmacokinetic properties due to the basic amino groups that allow
the preparation of water soluble salts. Moreover, the remarkable activity of some 3
30 (alkylamino) indazoles, e. g., 22, 23 and 25 [formula (111)], had not been described with
before, so this type of structure constitutes a new skeleton
promising for the development of new tricomonicides derived from indazole.

权利要求:
Claims (12)
[1]
1. Compounds derived from 5-nitroindazole, of general formulas (1), (11) Y (111).

[CH21nNR1R2
5 Formula (1) Formula (11) Formula (111)
where:
-in the compounds of formulas (1) and (11), NR1R2 may be an amino group,
alkylamino or dialkylamino, a cyclic secondary amine moiety such as
10 pyrrolidino group, or phthalimido groups, and n can be 2-3;
-in the compounds of formula (111), NR1R2 may be an alkylamino group,
(w-hydroxyalkyl) amino, or alkyl (w-hydroxyalkyl) amino;
-in compounds of formula (1), when n = 2, the
derivative in which NR1R2 is piperidino.
fifteen
or its possible salts, solvates or prodrugs.
[2]
2. A compound of general formula (1) according to claim 1, selected from the
following list:
twenty 2-Benzyl-1- [2- (dimethylamino) ethyl] -5-nitro-1, 2-dihydro-3H-indazol-3-one
(hydrochloride)
2-Benzyl-5-nitro-1- (2-pyrrolidinoethyl) -1, 2-dihydro-3H-indazol-3-one (oxalate)
2-Benzyl-1- (3-phthalimidopropyl) -5-nitro-1, 2-d ihydro-3H-indazol-3-one
2-Benzyl-1- (2-phthalimidoethyl) -5-nitro-1, 2-dihydro-3H-indazol-3-one
25 1- (2-Aminoethyl) -2-benzyl-5-nitro-1, 2-dihydro-3H-indazol-3-one (hydrochloride)
1- (3-Aminopropyl) -2-benzyl-5-nitro-1, 2-dihydro-3H-indazol-3-one (hydrochloride)
2-Benzyl-1- [3- (methylamino) propyl] -5-nitro-1, 2-dihydro-3H-indazol-3-one
(hydrochloride)
2-Benzyl-1- [3- (dimethylamine ino) propyl] -5-nitro-1, 2-d ihydro-3H-indazol-3-one
30 (hydrochloride)
2-Benzyl-5-nitro-1- (3-piperidinopropyl) -1, 2-dihydro-3H-indazol-3-one (oxalate)
or its solvates or prodrugs, or other salts.
[3]
3. A compound of the general formula (11) according to claim 1, selected from the
following list:2-Benzyl-3- (2-phthalimidoethoxy) -5-nitro-2H-indazole2-Benzyl-3- (3-phthalimidopropoxy) -5-nitro-2H-indazole2-Benzyl-3- [2- (d-ethylamino) ethoxy] -5-nitro-2H-indazole (hydrochloride)3- (3-Am i non-propoxy) -2-benzyl-5-n itro-2H-ind azol (hid roelo ru ro)
or its solvates or prodrugs, or other salts.
[4]
4. A compound of the general formula (111) according to claim 1, selected from the
following list:2-Benzyl-3-methylamino-5-nitro-2H-indazole2 -Benci 1-3 - [(2-hydroxyeti I) a m i n o] -5-n itro-2H-i ndazol2-Benzyl-3 - [(3-hydroxypropyl) amino] -5-nitro-2H-indazole2 -Benci 1-3 - [(3-h idroxi p rop i 1) meti lam i no] -5-n itro-2H-i ndazol2 -Benci 1-3 - [(2-hydroxyeti1) methylamine and non] -5-n itro-2H-indazol
or its solvates or prodrugs.
[5]
5. Process for the preparation of compounds of general formula (1) and (11) by treating 5-nitroindazolinone with w- (dialkylamino) alkyl halides or w-phthalimidoalkyl halides, as set forth in Scheme 1.
[6]
6. Procedure for the preparation of 1- (w-aminoalkyl) indazolinones of the general formula (1) from the corresponding w-phthalimidoalkyl derivatives, by elimination of the phthaloyl protecting group, as set forth in Scheme 2.
[7]
7. Procedure for the preparation of 1- (w-aminoalkyl) indazolinones of general formula (1) from the corresponding halides and the necessary secondary or tertiary amines, as set out in Scheme 3.
[8]
8. Process for the preparation of 3- (w-aminoalkoxy) indazoles of general formula (11) from a) of the corresponding halides and tertiary amines
necessary, or b) of the corresponding w-phthalimidoalkoxy derivatives, by elimination of the phthaloyl protecting group, as set out in Scheme 3.
[9]
9. Procedure for the preparation of 3- (alkylamino) indazoles of the general formula
5 (111) from a) of the corresponding halides and the necessary primary amines, or b) of the corresponding w-phthalimidoalkoxy derivatives, by elimination of the phthaloyl protecting group, as set out in Scheme 3.
[10]
10. Use of the compounds of claims 1-4 for the preparation of a
10 medicine intended for the treatment of diseases caused by pathogenic protozoa of the Trypanosomatidae (Trypanosoma, Leishmania) and Trichomonadidae (Trichomonas) families.
[11 ]
eleven . A pharmaceutical composition that includes any of the compounds defined in claims 1-4 and at least one pharmaceutically acceptable excipient.
[12]
12. A pharmaceutical composition according to claim 11, which may optionally also contain other active ingredients.

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公开号 | 公开日

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WO2019077174A1|2019-04-25|

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ES2653674B2|2018-09-18|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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WO2017072374A1|2015-10-27|2017-05-04|Universidad Complutense De Madrid|5-nitroindazole derivatives and use thereof as antiprotozoal agents|

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ES201700741A|ES2653674B2|2017-10-20|2017-10-20|Amines derived from 5- Nitroindazole with antiprotozoal properties|ES201700741A| ES2653674B2|2017-10-20|2017-10-20|Amines derived from 5- Nitroindazole with antiprotozoal properties|

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PCT/ES2018/000075| WO2019077174A1|2017-10-20|2018-10-19|Amines derived from 2-benzyl-5-nitroindazole with antiprotozoal properties against trypanosoma, leishmania and trichomonas|