Synthesis and activities of tricyclic pyrido[3,4-g ]quinazolines, pyrazolo[3,4-g ]quinoxalines and pyrroloindazole regioisomers

Heteroaromatics constitute an important class of organic compounds with wide-ranging applications. This account, including patent literature, describes reported synthetic methods leading to tricyclic nitrogen-containing heterocyclic systems (pyrido[3,4-g ]quinazolines, pyrazolo[3,4-g ]quinoxalines and pyrroloindazoles) which are found in many biologically active compounds.


Introduction
Heteroaromatics containing systems such as indole, indazole or pyrazole constitute a large family of organic compounds with a wide range of applications in biology [1][2][3][4] as active ingredients or molecular probes due to their biological and physicochemical properties.Some heteroaromatic derivatives are also of high interest due to their application in organic optoelectronic materials. 5Therefore, the development of new synthetic methodologies allowing access to these classes of compounds has given rise to numerous studies. 6As part of our work aiming at identifying new biologically active skeletons, we are particularly interested in the design and synthesis of novel structural series with protein kinase inhibitory potencies.The scaffolds presented Figure 1 are found in potent protein kinase inhibitors (targeting Pim kinases, except for the pyrido [3,4-g]quinazoline series which exhibited activities toward DYRK1A/CLK1).Synthesis and properties of various series such as dihydropyrrolocarbazoles 4 and indolocarbazoles 7 have already been reviewed while very few examples of pyrazolo [4,3-a]phenanthridines can be found in the literature.Therefore, due to our interest in these tricyclic systems we decided to focus this account on pyrido [3,4g]quinazolines, pyrazolo [3,4-g]quinoxalines and pyrroloindazole regioisomers (Figure 2).To the best of our knowledge, none of these fully aromatic systems has been reported so far, the majority of syntheses concerning dihydropyrroloindazole derivatives.Moreover, the synthesis of some series was never or rarely reported, such as pyrrolo [3,4-

Synthesis and biological activities of pyrido[3,4-g]quinazolines
The pyrido [3,4-g]quinazoline scaffold was designed in our group on the basis of a structure-activity relationship study performed around meridianins, marine alkaloids isolated from the ascidian Aplidium meridianum, possessing an indole moiety substituted in the 3-position by a 2-aminopyrimidine ring.The best activities were found for CLK1 and DYRK1A kinases where nanomolar potencies were encountered.After molecular modelling studies, the design of a second generation of inhibitors ended up with this new pyridoquinazoline series (Scheme 1). 8

AUTHOR(S)
Scheme 1. Design and synthesis of pyrido [3,4-g]quinazolines. [8][9][10] The synthesis of this pyridoquinazoline scaffold was achieved via intermediate 2, a tetrasubstituted benzene derivative obtained in seven steps from benzoic acid derivative 1 using trivial aromatic chemistry (Scheme 1).The upper substituents are necessary to establish the aminopyrimidine moiety and the lowers to form the pyridine part.The isoquinoline ring system was first formed under microwave irradiation in the presence of ammonia in methanol.Next, oxidation of 3 using MnO 2 led to the isoquinoline 4.Then, the regioselective introduction of a nitro group at the ortho position to the chlorine atom was necessary to ensure a suitable reactivity of the system, with respect to amidine or guanidinium salts, in order to obtain the tricyclic system 6.Catalytic hydrogenation of 6 led to the corresponding amino derivatives.][10] The reaction of compound 6 (R 1 = H) with organolithium or Grignard reagents allowed the introduction of alkyl/aryl substituents at the 5-position (R 3 ) while electrophilic aromatic substitution led to the functionalization of the 9-position (R 4 = Br). 11All compounds were evaluated towards CDK5/p25, CK1/ε, GSK-3/, DYRK1A and CLK1.Nanomolar activities were observed toward DYRK1A/CLK1.Nitro derivatives were generally more active towards CLK1 in comparison to DYRK1A whereas amino analogues could exert potent activities towards both CLK1 and DYRK1A or, in some cases were more active towards DYRK1A.Therefore, this series affords interesting research tools to study DYRK1A/CLK1 protein kinases.
An example of a related compound (14, Scheme 2) is reported in a patent dealing with the identification of factor IXa inhibitors. 12In this case, the formation of the pyridoquinazoline derivative scaffold (compound 13) was achieved by cyclization under basic conditions of the corresponding 6-acylamino-7carboxamidoquinazoline 12 prepared in 3 steps from 6-amino-7-cyano-2H-isoquinoline-1-one 9. Scheme 2. Structure and synthesis of factor IXa inhibitors containing a pyridoquinazoline moiety. 12
Finally, a recent patent application showed that the pyrazoloindazolone scaffold can be obtained via Bartoli indole synthesis from 4-nitroindazolone 49.Compound 50 is expected to be developed as a drug to treat atherosclerosis. 26heme 7. Synthesis of pyrrolo[2,3-e]indazole-3-one 50. 26

Pyrrolo[3,2-e]indazoles
The regioisomeric 1,6-dihydropyrrolo[3,2-e]indazole synthesis has been reported by S. M. Bronner starting from the indolynes generated from indole derivatives 51 and 52 (Scheme 8). 27These reactive intermediates were trapped with ethyl diazoacetate to give the [3,2-e]-and the [2,3-g]-regioisomers in varying proportion depending on the substitution at the 6-position of the starting indole derivative.The 1,6-dihydropyrrolo[3,2e]indazole was favored in the case of a bromine substitution, and this preference was reversed in the case of R = H.Scheme 8. Indolyne trapping with ethyldiazoacetate leading to 53 or 54. 27other strategy was reported in 2014 by M. Chakrabarty and collaborators, 28 starting from 5aminoindazole 55, which was iodinated/acylated to give 57.Using a Sonogashira/Cacchi procedure from involving consecutive palladium-catalyzed Sonogashira coupling, aminopalladation and reductive elimination, the 1,6-dihydropyrrolo[3,2-e]indazoles 58 were obtained in good yields (Scheme 9).Contrarily to the preceding example, the regioselectivity of the reaction was controlled by the appropriate functionalization at the 4-and 5-positions of compound 57.This strategy appeared recently in a PCT patent application of Radius Pharmaceuticals, focused on the preparation of substituted indoles as estrogen receptor-modulating compounds. 29Compounds 59 were reported (Scheme 9), showing high activity towards the MCF7 cell line, with IC 50 values lower than 1 nM in proliferation inhibition assays in the presence of estradiol at a concentration of 10 pM.Scheme 9. Preparation of 1,6-dihydropyrrolo[3,2-e]indazoles 58 via a Sonogashira/Cacchi procedure. 29tructure of compounds 59.

Pyrrolo[2,3-g]indazoles
The first references of pyrrolo[2,4-g]indazole preparation dates from the 1960's in patent applications by American Cyanamide 36,37 , reporting compounds with analgesic and anti-inflammatory activity. 36Reaction of 76 with hydrazines and subsequent aromatization using Pd/C in refluxing cumene led to a series of differently substituted compounds 78 (Scheme 14).This strategy has also been used for the synthesis of compounds 80 in a work dedicated to preparation of potential inhibitors of soluble guanylate cyclase. 38In this work, DDQ was used instead of Pd/C for the dehydrogenation, and the protecting group of the pyrrole moiety was changed to ethoxymethyl or the SEM group.The same approach was used for the preparation of pyrroloindazoles in the [3,4-g] (see below) or [3,2-g] 39 series.Scheme 14.General structure of compounds 78 36,37 or 80 38 .Preparation of compounds 79 from 76. 36 As presented above for the preparation of pyrrolo[2,3-e]indazoles (Scheme 10), the work by Borza et al. was also carried out from 6-aminoindazole to get the [2,3-g]-regioisomers. 30 Previously, Fischer indolization was also reported in the [2,3-g]-series, from 6-hydrazinoindazoles 81 (Scheme 15). 40Formation of the pyrrole nucleus could also be achieved by reaction of 6-aminoindazoles 83 with arylglyoxal hydrates and cyclic 1,3dicarbonyl compounds to give 86 41 , or by condensation of 83 or 87 with benzoin, to give 7,8-diphenyl derivatives 85 and 86 42 , (Scheme 15).The pyrrolo [3,2-f]indazole scaffold could also be formed using this reaction, starting from 6-amino-7-chloroindazole. 42 Scheme 15.Fischer indolization from 6-hydrazinoindazole 81. 40Three-component reaction using 6aminoindazole 83, arylglyoxal hydrates and cyclic 1,3-dicarbonyl compounds. 41Condensation of indazoles 83 and 87 with benzoin. 42 2011, we reported the preparation of diversely substituted pyrrolo[2,3-g]indazoles. 43,44 The method involved a palladium-catalyzed annulation from 6-amino-7-iodoindazole 90 and terminal or internal alkynes using Pd(PPh 3 ) 4 as a palladium source and XPhos as a ligand (Scheme 16).High yields were attained, and a single regioisomer could be obtained as in the cases of examples presented Scheme 16.This synthetic method was used to prepare a series of potential Pim kinase inhibitors of general structure 92.In particular, high inhibitory potency was observed towards Pim-1 and Pim-3 protein kinases, with IC 50 values in the submicromolar range (92, R 1 = R 2 = H, R 3 = NH 2 , IC 50 Pim-3 = 33 nM).Scheme 16.Palladium-catalyzed synthesis of pyrrolo[2,3-g]indazole derivatives 91. 43General structure 92 of potential Pim kinase inhibitors. 44milarly to the [3,2-e]-series (see Scheme 11), fused pyrazole derivatives of isatin have been prepared from 6-aminoindazole 45 or 6-amino-5-methylindazole. 46The synthetic method is similar, with the use of chloral hydrate and hydroxylamine hydrochloride to give a 2-(hydroxyimino)acetamide intermediate which was cyclized under acidic conditions.

Pyrrolo[3,2-g]indazoles
This last example has been reported in a patent application deposited by the Memory Pharmaceuticals Corporation, in a work related to the preparation of ligands for the α7 nicotinic acetylcholine receptor. 49The synthesis from the adequately functionalized indazole 97 (Scheme 19).A Negishi cross-coupling was first carried out to introduce the 2-carbon atom chain necessary for pyrrole formation.Reduction of the nitro group and acetal hydrolysis/cyclization led to compound 100 in 72% yield.Scheme 19.Synthesis of 1,8-dihydropyrrolo[3,2-g]indazole 100. 49

Conclusions
Nitrogen-containing heteroaromatic are particularly interesting due to their various areas of application, especially in biology.The development of efficient methods allowing access to these scaffolds has sparked many efforts, and synthetic approaches using either multi-step synthesis, multi-component or metalcatalyzed reactions enabled the preparation of heterocyclic rings and the expansion of the chemical space in this domain.Nevertheless, the synthesis of many heterocyclic systems remains challenging in order to enlarge the scope of synthetic methodologies, control the regioselectivity or overcome reactivity and stability issues.This account, focused on pyrido [3,4-g]quinazolines, pyrazolo [3,4-g]quinoxalines and pyrroloindazoles, explored the existing synthetic methods to obtain these structures, some of them demonstrating potential biological activities, particularly protein kinase inhibitory potencies.The literature analysis showed that some series have not or sparsely been reported (e.g.pyrrolo[2,3-f]indazoles, pyrrolo [3,4-e]indazoles, pyrrolo [3,4f]indazoles), or that the existing synthetic methods can limit the choice of substitution and functionalization, highlighting that the development of new methodologies for the preparation of simple but challenging heteroaromatic moieties is still needed.

Figure 1 .
Figure 1.Nitrogen-containing heteroaromatic scaffolds found in protein kinase inhibitors developed by our group.