T ; Zhang

Z Wang

kinases dédiés au traitement de maladies néoplasiques et des complications diabétiques

Keywords: inhibiteurs de tyrosine kinase, aldose réductase, VEGFR2 TK, ALR2, aminooxazoles tyrosine kinase inhibitors, aldose reductase, VEGFR2 TK, ALR2, aminooxazoles, oxotriazinoindoles

triazine-3-(2H)-thione catalysed by base .................

Introduction 1. Introduction

Le cancer est sans aucun doute l'une des maladies les plus graves et potentiellement plus mortelles au monde. Le développement d'un cancer est un processus en plusieurs étapes impliquant une initiation et un état de progression. Lors de l'initiation de la tumeur, une seule cellule est mutée par des modifications de l'ADN entraînant ensuite une prolifération anormale.

Cette prolifération donne lieu à un grand nombre de cellules filles contenant la mutation créée par l'initiateur. La progression tumorale se poursuit alors que des mutations supplémentaires se produisent dans les cellules de la population tumorale, suivies d'une sélection pour des cellules à croissance plus rapide. Cette sélection contribue au développement de la tumeur, de sorte que la tumeur devient à croissance plus rapide et maligne.

L'angiogenèse est un processus complexe contrôlé par des facteurs de croissance produits dans le corps. Au cours de l'angiogenèse, de nouveaux vaisseaux sanguins sont formés à partir de vaisseaux préexistants et ce processus est physiologiquement crucial pendant le développement embryonnaire et la guérison des plaies. D'autre part, l'angiogenèse est également impliquée dans la croissance des tumeurs. La compréhension des processus de signalisation cellulaire au cours des dernières décennies a conduit à l'identification du VEGFR2 TK comme cible intéressante pour une thérapie anticancéreuse. Il a été démontré que de petites molécules inhibitrices de VEGFR2 TK suppriment la croissance du cancer et les métastases, conduisant à un traitement efficace du cancer, principalement en combinaison avec d 'autres agents.

Le Diabète sucré (DM) est également une maladie bien connue induite par un groupe de troubles métaboliques caractérisés par un taux anormalement élevé de sucre dans le sang. Les personnes atteintes de diabète sucré (DM) ont soit un manque de sécrétion d'insuline (DM type I), soit l'insuline ne peut pas être utilisée correctement (DM type II), ce qui conduit à diverses pathologies. Il peut endommager les minuscules vaisseaux des reins, du foie, des yeux ou du système nerveux entraînant une cataracte diabétique, une rétinopathie, une neuropathie ou encore une néphropathie. Toutes ces complications pathologiques diabétiques sont associées à une voie polyol, qui dans un premier temps convertit le glucose en sorbitol par une enzyme, l'aldose réductase. Outre une concentration élevée de sorbitol, il en résulte une accumulation excessive d'espèces intracellulaires réactives de l'oxygène (ROS) dans divers tissus. Des études récentes ont montré que les inhibiteurs de l'ALR2 pouvaient prévenir ou retarder l'apparition des complications diabétiques chroniques, par conséquent, l'inhibition de l'activité de l'ALR2 a ouvert une voie prometteuse pour le traitement des complications liées au diabète.

La première partie de la thèse est consacrée au développement d'aminooxazoles inhibiteurs de VEGFR2 TK prédits in silico. La deuxième partie de la thèse est axée sur le développement d'oxotriazinoindoles comme nouveaux inhibiteurs d'ALR2, y compris la conception, la synthèse, l'activité biologique et l'évaluation des résultats obtenus. Ces études et les résultats obtenus peuvent servir d'outil précieux pour une meilleure compréhension de la conception des médicaments.

Dans le cadre du premier projet, nous visons à synthétiser une série d'inhibiteurs prédits de VEGFR2 TK comportant le noyau N,4-diaryloxazole-2-amine NBM(1-5,7) représentant un ensemble de régioisomères comportant le noyau N,5-diaryloxazole-2-amine BM(1-6) préparés antérieurement à ce travail. Etant donné qu'un oxygène est un isostère d'un azote dans un cycle oxazole et vice versa, des interactions de liaison similaires des inhibiteurs avec Cys917 à partir du site de liaison ATP sont attendues pour les deux régioisomères. Un composé manquant pour cette étude BM7 a également été synthétisé. Enfin nous avons également déterminé les activités biologiques des nouveaux composés préparés afin d'évaluer notre hypothèse de bioisostérie régioisomérique (RegBio). (Figure 1) Finalement, les activités et les sélectivités de tous les composés préparés ont été déterminées.

(Figure 3) Dans le dernier projet, nous nous sommes concentrés sur la synthèse et l'étude SAR de OTI-(6-9) en tant que nouveaux analogues N-benzyliques de OTI-3 afin d'améliorer l'activité inhibitrice d'ALR2 en exploitant davantage la poche interactive avec le benzyle. (Figure 4) Le VEGFR2 TK est activé lorsqu'un ligand (VEGF) se lie au domaine extracellulaire d'un récepteur, ce qui conduit à sa dimérisation avec son voisin et à la stabilisation de ce complexe.

Après cela, plusieurs résidus tyrosine intracellulaires de chaque monomère de récepteur sont transphosphorylés par son récepteur partenaire. La phosphorylation et l'activation de ces deux protéines réceptrices conduisent à une activation et à l'initiation de diverses réponses et voies cellulaires. (Figure 7) L'expression de VEGFR2 dans les cellules cancéreuses est régulée à la hausse pendant la croissance tumorale par des stimuli environnementaux tels que l'hypoxie ou certaines mutations génétiques. L'état d'hypoxie favorise la sécrétion de VEGF-A, qui se lie au VEGFR2 TK exprimé sur les cellules endothéliales vasculaires (Figure 8). L'expression du VEGF-A est un marqueur pronostique chez les patients atteints de diverses tumeurs malignes. Par conséquent, l'inhibition de l'angiogenèse avec des médicaments anti-angiogéniques efficaces peut entraîner la suppression de la croissance tumorale. Ces agents sont divisés en plusieurs classes selon leur mécanisme d'action. (Figure 8)

Inhibiteurs du VEGF

Le bevacizumab (nom commercial : Avastin®) est devenu le premier inhibiteur de l'angiogenèse en tant qu'anticorps monoclonal recombinant humanisé en se liant au VEGF-A.

Le bevacizumab a été approuvé pour la première fois par la FDA USA en 2004 pour un traitement du cancer colorectal et plus tard pour d'autres types graves de cancers tels que les tumeurs du poumon, du sein ou encore du cerveau. Les principaux effets indésirables du bevacizumab sont l'hypertension, les saignements de nez, les maux de tête et les éruptions

Résultats et discussions

En (Figure 13) Cependant, si de grandes quantités de glucose sont présentes dans des conditions d'hyperglycémie, l'hexokinase est saturée et environ 33% du glucose global est métabolisé par une voie polyol. Alors que la plupart des cellules nécessitent l'action de l'insuline pour que le glucose pénètre dans la cellule, les cellules de la rétine, des reins et des tissus nerveux sont indépendantes de l'insuline, de sorte que le glucose se déplace à travers la membrane cellulaire, quelle que soit l'action de l'insuline. Les cellules utiliseront le glucose comme énergie comme d'habitude, et l'excès de glucose entrera dans la voie des polyols.

La voie des polyols

La voie des polyols est un processus en deux étapes qui convertit le glucose en fructose. Il

Diabète sucré (DM) et complications du diabète

Le diabète sucré de type I est un trouble métabolique du pancréas, un organe qui produit l'hormone insuline. Cette dernière est une hormone peptidique, qui joue un rôle clé dans la régulation de diverses bio-macromolécules, en particulier la régulation de la glycémie.

Normalement, lorsque le taux de glucose est élevé, les cellules bêta du pancréas sécrètent (Figure 16)

Propriétés et fonction d'ALR2

Figure 16. La structure du site actif de l'enzyme ALR2.

Aldéhyde réductase

Inhibiteurs d'ALR2

Au cours des 40 dernières années, de nombreux inhibiteurs d'ALR2 ont été développés, mais seulement un candidat (epalrestat) a été approuvé comme médicament au Japon, en Chine et en Inde. La plupart des autres inhibiteurs mis au point n'ont pas été approuvés pour les essais cliniques, principalement à cause de leurs faibles efficacités in vivo, leurs inconvénients pharmacocinétiques et leurs effets secondaires liés à l'inhibition non sélective de l'aldéhyde réductase (ALR1).

Dérivés d'acide carboxylique

Les acides carboxyliques représentent la classe la plus importante et la plus grande des ARI, car le groupe carboxylate de l'inhibiteur occupe une poche de liaison anionique du site actif d'ALR2. Par contre, de faibles valeurs de pKa entravent la perméabilité de la membrane dans des conditions de pH physiologiques et conduisent à un mauvais profil pharmacocinétique de ces ARI. À l'instar de ces inhibiteurs, l'epalrestat, un inhibiteur non compétitif et réversible de l'ALR2 (contenant un anneau de rhodanine) utilisé pour le traitement de la neuropathie diabétique, a été développé en 1983 et approuvé au Japon, en Chine et en Inde pour l'amélioration des symptômes de neuropathie subjective et des modifications anormales du rythme cardiaque associées à une neuropathie diabétique périphérique. (Figure 17) L'imirestat, contenant un anneau fluorène planaire, a également atteint la phase 3 mais a été retiré par la suite en raison de sa toxicité. (Figure 19) En conclusion, ces acides carboxyliques ainsi que les dérivés de spirohydantoïne n'ont pas été approuvés par la FDA en tant qu'inhibiteurs d'ALR2. Par conséquent, dans la recherche récente d'inhibiteurs ARI efficaces, il est nécessaire de trouver de nouveaux squelettes actifs.

Résultats et discussions

Le point de départ du deuxième projet est l'acide 2- (3-thioxo-2H-[1,2,4]triazino [5,6-b] Sur la base de ces résultats, nous pouvons conclure que le remplacement isostérique du soufre par de l'oxygène joue un rôle important à la fois pour l'inhibition de l'ALR2 et sa sélectivité.

Conclusion générale 4. Conclusion générale

La thèse a été divisée en deux parties : Recent studies have shown that ALR2 inhibitors could prevent or delay the onset chronic diabetic complications, therefore inhibition of ALR2 activity has opened a promising way for a treatment of complications connected with DM.

Manuscript en Anglais

Resume of the thesis

The first part of the dissertation thesis is devoted to the development of in silico predicted aminooxazole VEGFR2 TK inhibitors. The second part of the thesis is focused on the development of novel oxotriazinoindole ALR2 inhibitors including design, synthesis, biological assay and evaluation of obtained results. These studies and achieved results may provide a great insight into a medicinal chemistry and could serve as a valuable tool for better understanding of drug design.

Within the first project we aimed to synthetize a series of predicted N,4-diaryloxazole-2-amine VEGFR2 TK inhibitors NBM(1-5,7) as a set of regioisomers to the previously prepared, highly active N,5-diaryloxazole-2-amine analogues BM(1-6). Since an oxygen is an isostere to a nitrogen in an oxazole ring and vice versa, similar binding interactions with Cys917 from ATP-binding site is expected for both regioisomers. A missing compound for this study BM7 was synthetized as well. After successful preparation we also determined biological activities for novel compounds in order to evaluate our hypothesis of Regioisomeric Bioisostery (RegBio). (Figure 22) In the third project we aimed to develop a synthetic methodology for recently discovered and highly active aldose reductase (ALR2) inhibitor CMTI. After that, we were focus on a development of CMTI sulfur/oxygen isosteric analogue OTI and its derivatives OTI-(1-5)

possessing oxotriazinoindole scaffold. At the end activities and selectivities of all prepared compounds were determined. (Figure 24) Within the last project we were focus on a synthesis and a SAR study of novel N-benzyl OTI-3 analogues OTI-(6-9) in order to improve ALR2 inhibitory activity by further exploitation of a benzyl interactive pocket. Chapter 3. Vascular endothelial growth factor receptor (VEGFR)

Vascular endothelial growth factor receptor (VEGFR)

Very important signal proteins that stimulate the formation of new blood vessels are vascular endothelial growth factors (VEGFs). This sub-family of growth factors are produced by cells and are involved in vasculogenesis (de novo production of endothelial cells) and angiogenesis (production of new vessels from pre-existing ones). 6 The normal function of VEGFs is to create new blood vessels after injury and during an embryonic development. On the other hand, VEGFs are overexpressed in many tumor cells leading to tumor angiogenesis, proliferation and metastasis. Each from VEGF ligands binds in a specific manner to three VEGF receptors of tyrosin kinase (VEGFR TKs). 7 Tyrosin kinases are enzymes that specifically phorphorylate tyrosine amino acid residues of peptide targets by high-energy containing molecules ATP.

There are three main subtypes of VEGFR TKs, numbered 1, 2 and 3. VEGFR1 (Flt-1) is expressed on haematopoietic stem cells, monocytes and macrophages, VEGFR2 (Flk-1, KDR) mainly regulates vascular endothelial cell fuction and promotes their proliferation, migration, permeability and survival. Therefore, VEGFR2 is crucial for a tumor development.

VEGFR3 (Flt-4) regulates lymphatic endothelial cell functions. (Figure 26)

VEGFR2 tyrosin kinase

VEGFR2 TK (kinase insert domain receptor (KDR) or foetal liver kinase (Flk-1)) is a transmembrane tyrosine kinase receptor consisted of an extracellular region (composed of seven immunoglobuline (Ig)-like domains), a short transmembrane domain and an intracellular region (containing tyrosine kinase domain). 8 (Figure 27) VEGFR2 TK is activated when a ligand (VEGF) binds to the extracellular domain of a receptor which leads to dimerization of a receptor with a neighboring receptor and stabilization of this complex. After that, multiple intracellular tyrosine residues of each receptor monomer are trans phosphorylated by its partner receptor. Phosphorylation and activation of these two receptor proteins lead to an activation and initiation of various cellular responses and pathways. 9,10 (Figure 28) VEGFR2 expression in cancer cells is upregulated during tumor growth by environmental stimuli such as hypoxia or genetic mutations. 11 The hypoxia state promotes secretion of VEGF-A, which bind to VEGFR2 TK expressed on vascular endotielial cells. (Figure 29) Expression of VEGF-A is a prognostic marker in patients with variety of malignancies. Therefore, inhibition of angiogenesis with effective anti-angiogenic drugs may results in suppression of tumor growth. 12 These agents are divided into several classes according to mechanism of action. 13 (Figure 29)

Biological assay activity (VEGFR2 TK)

For investigation of the IC50 activity of inhibitors, a radioactive in vitro kinase assay can be used. This method utilizes substrate, kinase and 32 P-or 33 P-labeled ATP (on gamma phosphate) to transfer a radioactive phosphate group to a tyrosine residues containing substrate. The product of the reaction is spotted on a phosphocellulose (P81) paper and washed, removing excess of radiolabeled ATP. The transfered phosphate group can be visualized and quantified using a phosphorimager. 25 Chapter 4. Synthesis of aminooxazole VEGFR2 TK inhibitors

Synthesis of aminooxazole VEGFR2 TK inhibitors

Diaryloxazole-2-amine moiety represents an interesting pharmacophoric fragment found in many biological active compounds such as antitumor, immunosuppressive and antiinflammatory compounds. Among large number of heterocyclic compounds, an oxazole ring is characterized by various biological properties, such as antibacterial, hypoglycemic and antiinflammatory activities. 27 There are two regioisomeric forms of aminooxazoles: 2,5disubstituted aminooxazole and 2,4-disubstituted aminooxazole. (Figure 32)

Synthesis of N,5-diaryloxazole-2-amines

The most common and efficient synthesis of the N,5-diaryloxazole-2-amine derivatives T47 was described by Harris in 2005 by the reaction of various α-azidoketones T45 and isocyanates or isothiocyanates T46 in the presence of PPh3 (or pPPh3). 28 The yields of desired oxazoles T47 were recorded in very broad range (from 1 to 95 %) and depended on the structure of substrates and reaction conditions. The best yields of oxazoles T47

were obtained with electron-donating substituents on α-azidoketones T45, whereas structure of isothiocyanates T46 had weaker impact on the yield of products. Reactions in dioxane at 90 °C within 30 min also provided higher yields. A proposed mechanism of the oxazole-2-amine core formation is described below. (Scheme 6) In the first step, an azaylide T48 is prepared by a reaction between α-azidoketone T45 and PPh3 analogically to the Staudinger reaction. A similar study was described on various alkyl and aryl substituted hydroxyketones T83 and cyanamides T84. 38 The yields of oxazoles T85 were from 40 to 90 %. (Scheme 15)

Scheme 15. Synthesis of various aminooxazoles T85 carried out from hydroxyketones T83 and cyanamides

T84.

Another usefull method for a preparation of

Palladium-catalyzed cross-coupling reactions

In organic chemistry, a palladium-catalyzed cross-coupling reaction is a process, where two fragments are joined together with catalysis of a trasition metal-palladium. 40 This versatile reaction has become very popular in modern organic and medicinal chemistry owing to effective formation of carboncarbon or carbonheteroatom bonds. There are several types of cross-coupling reaction according to type of coupling reagents used in the reaction. Within this dissertation thesis, we used Suzuki-Miyaura and Stille coupling reactions for the synthesis of majority of designed VEGFR2 TK inhibitors. In general, all palladium-catalyzed reactions involves: 1) oxidative addition, 2) transmetallation and 3) reductive elimination.

Suzuki-Miyaura (SM) cross-coupling reaction

The Suzuki-Miyaura (SM) cross-coupling reaction was first described in 1979 by Prof. Akira Suzuki. 41 It is widespread transition metal-catalyzed reaction, which utilizes organoboron compounds as a coupling partner. Popularity and broad application for this coupling arise from relatively stable, readily available and less toxic organoboron reagents relative to organotin or organozinc reagents. Mechanism of SM coupling reaction is described below. (Scheme 17)

40 King, A.O.; Yasuda, N. Organomet. Chem., 2004, 6, 205 -245. 41 Miyuara, N.; Suzuki, A. J. Chem. Soc., Chem. Commun. 1979, 19, 866 -867. Scheme 17. Mechanism of SM cross-coupling reaction.

Oxidative addition

In the first step, the palladium (0) catalyst C1 is oxidized to palladium (II) and coupled with an aryl halide to form organopalladium complex C2. A wide-variety of aryl halides in SM coupling are used, but bromides, iodides and "pseudohalids" triflates are most useful. Since oxidative addition is the rate determining step in the cycle, the reactivity decreases in the order of I > OTf > Br > Cl. 42 Aryl halides are also activated by electron-withdrawing groups (EWD) in orto and para position that increase their reactivity in oxidative addition. 43

Transmetalation

In transmetalation step, an organic group Ar 2 attached on boron spieces is transferred to the Pd II) complex C2 to give a new Pd (II) complex C3. The exact mechanism of transmetalation is still not clear and remains to be discovered. This step requires a presence of the base in order to increase electron density of boron atom. Lima et al. proposed two main pathways that explaining a role of the base in SM cross-coupling reaction. 44 The first pathway A: halogen or triflate is exchanged after oxidative addition with a nucleophilic species, which can be an anion of the used base or anion present in the solution. Resulting Pd (II) -OR (alkoxy, hydroxy or acetoxy) complex is considered to be more reactive in transmetalation step relative toward Pd (II) -X complex . The second pathway B explains a role of the base differently. The base attacks a boron atom to get more electronic rich borate and increases its reactivity toward palladium in the C2 complex. (Scheme 18) Commonly employed bases for this process are K3PO4, K2CO3, KOH or even fluoride anions could be used (KF). (Scheme 18)

Scheme 18. Two proposed pathways A and B of the base in SM cross-coupling reaction.

Reductive elimination

The final step is reductive elimination where a required product is eliminated from C3 and palladium (II) catalyst is regenerated to Pd (0) C1.

Types of boronic derivatives

Over the last decade, a wide range of boronic derivatives for SM cross-coupling reaction have been developed. Most common are arylboronic acids ArB(OH)2, aryl boronic esters ArB(OR)2, aryl trifluoroborate salts [ArBF3] (-) or popular aryl boronic acid pinacol esters ArBpin. (Figure 33) The reactivity of borane in transmetalation depends on its Lewis acidity. Therefore, electron withdrawing substituents increase the reactivity of the borane, which suggests higher reactivity of boronic acids than esters. 45 In boronic esters, σ-donating ability of carbon leads to a stronger conjugation of oxygen lone pairs with a neighboring boron atom, which results to reduced Lewis acidity. 46 On the other hand, boronic esters exhibit stability toward column chromatography and liquid esters could be distilled.

45 ChemLer, S. R.; Trauner, D.; Danishefsky, S. J. Angew. Chem. Int. Ed., 2001, 40, 4544 -4568. 46 Lennox, A.J.J.; Lloyd-Jones, G.C.; Chem. Soc. Rev., 2014, 43, 412 -443.

Ligands in SM-cross coupling reactions

Ligands play important role during the cross-coupling reactions due to their electron donating and bulky features, which can stabilize Pd (0) catalyst and promote oxidative addition and reductive elimnation. 49 Very effective and common are monodentate phoshpine ligands, widely used in both laboratory and industry. Among them, triphenylphospine (PPh3) is characterised by high ability to donate eletrons to the palladium (0) and accelerate the reaction. In order to facilite reactivity of the various coupling reactions, Netherton et al. proposed and replaced aromatic rings of PPh3 47 E. P. Gillis, M. D. Burke, J. Am. Chem. Soc., 2007, 129, 6716 -6717. 48 E. P. Gillis, M. D. Burke, J. Am. Chem. Soc., 2008, 130, 14084 -14085. 49 Miyaura, N.; Suzuki, A. Chem. Rev., 1995, 95, 2457 -2483. with bulkier and more electron-rich alkyl groups, which result to a higher catalytic reactivity. 50 (Figure 35) (to substrate or temperature) or must be reduced in situ from Pd (II) to Pd (0) (e.g. Pd(OAc)2 and PdCl2). Developed electron-rich and bulky ligands mitigate these limitations and overally improved properties of ligand. 52 In the figure below, functions of all particular R-substituents at various position of a ligand are explained. 53 (Figure 36)

50 Netherton, M. R. Pyridyl ligand in the structure provides additional stability and bulky -R substituents improve reductive elimination. Phenylimidazolium moiety binds the palladium more tightly than traditional phosphines and prevents metal dissociation. 55 PEPPSI-i Pr is very efficient in Negishi, Suzuki, Sonogashira or Kumada coupling as well as in Buchwald-Hartwig amination and Heck coupling.

Stille cross-coupling reaction

Another versatile cross-coupling reaction, widely used in organic synthesis, is Stille reaction.

This type of coupling includes the same steps as SM cross-coupling, but organotin compounds (or organostannanes) instead of organoborones are used without basic activation. 56 Chem. Int. Ed. Engl., 1986, 25, 508 -524 the synthesis of natural products. Panek et al. described a total synthesis of antibiotic (+)mycotrienol using Stille coupling in macrocyclization step. 57 Also, in a total synthesis of antitumor alkaloid Ircinal A includes a Stille reaction. 58 (Scheme 20) Scheme 20. Application of Stille coupling reaction in total syntheses of natural products.

Additives in Stille coupling reactions

The most frequent additive to the Stille coupling reaction is copper (I) which may enhance the rate of reaction by 10 3 -fold more. 59 In polar solvents stannane is exchange with copper, which results to organocuprate reagent readily transmetalated with palladium catalyst. (Scheme 21) Lithium chloride is another effective rate accelerant of Stille coupling reaction. It is believed that chloride anion either replace another halogen atom X on palladium leading to higher reactivity for transmetalation or coordinate Pd (0) centre resulting to enhanced rate of oxidative addition. In addition, LiCl salts increase the polarity of solvents leading to easier leaving of anionic ligand (-Cl, -Br, -OTf etc.). 60 The same favorable effect is also attributed to fluoride 57 Masse, C. E.; Yang, M.; Solomon, J.; Panek, J. S. J. Am. Chem. Soc., 1998, 120, 4123 -4134. 58 Martin, S. F.; Humphrey, J. M.; Ali, A.; Hillier, M. C. J. Am. Chem. Soc., 1999, 121, 866 -867. 59 Farina, V. Pure Appl. Chem., 1996, 68, 73 -78. 60 Farina, V.; Kapadia, S.; Brishnan, B.; Wang, C.; Liebeskind, L. S. J, Org. Chem., 1994, 59, 5905 -5911. anion (CsF), that could increase the rate of reaction. Moreover, fluoride anions could act as scanvenger for stannane side products, making them easily to remove. 61 (Scheme 21) Scheme 21. An effect of copper (I) and fluoride anion in Stille coupling reaction.

Other type is Negishi coupling that utilizes reaction of organic halides or triflates with organozinc compounds forming new C-C bonds. Palladium is largely used as a catalyst, but in some cases also nickel could replace it. 62 Negishi coupling was used in 2003 by Novartis in a synthesis of a phosphodiesterase inhibitor PDE472, considered as a leading structure for treatment of asthma. 63 The Heck coupling is another palladium-catalyzed reaction of aromatic / vinyl halide or triflate with an alkene in the presence of a base (e.g. synthesis of naproxen). 64,65 Other useful reaction is Sonogashira cross-coupling (organohalide with alkyne) used in synthesis of an alkaloid (+)-(S)-laudanosine. 66 (Scheme 22) 61 Mee, S. P. H.; Lee, V.; Baldwin, J. E. Angew. Chem. Int. Ed., 2004, 43, 1132 -1136. 62 King, A.O.; Okukado, N.; Negishi, E. J. Chem. Soc., Chem. Commun., 1977, 19, 683 -684. 63 Manley, P.W.; Acemoglu, M.; Marterer, W.; Pachinger, W. Org. Proc. Res. Dev., 2003, 7, 436 -445. 64 Heck, R. F. Org. React., 1982, 27, 345 -390. 65 De Vries, J.G. Can. J. Chem., 2001, 79, 5 -6. 66 Mujahidin, D.; Doye, S. Eur. J. Org. Chem., 2005, 2005, (10) Based on available data from literature the most common procedure for preparation of N,4diaryloxazole-2-amines is heterocyclization between α-bromoacetophenones and arylureas under various conditions (see in the chapter 4 of this dissertation thesis). Following the most promising described procedures, we were not able to prepare the desired N,4-diaryloxazole-2amine 10. Instead of that, our colleagues confirmed 1,5-diarylimidazoline-2,4-dione 10a and so our group accidentaly developed a novel synthesis of a hydatoin compounds. The structure of 10a has been confirmed also by an X-ray analysis. 70 Synthesis of the pyrimidine inhibitor NBM2 with the same reaction conditions as in the synthesis of its regioisomeric compound BM2 was the most problematic. Accordingly, for all inhibitors a conformation penalty is expected. In case of N-regioisomers the lower activity can be explained also by weaker H-bond between an oxygen from oxazole and Cys917 compare to H-bond of O-regioisomers between a nitrogen from an oxazole and the same backbone amino acids. Within this part the Biomagi hypothesis was confirmed and both regioisomers are VEGFR2 TK inhibitors. .

Synthesis of N,4-diaryloxazole-2-amine precursor

Determination of biological activity and evaluation of the project

Synthesis of N-[5-(ethylsulfonyl)-2-hydroxyphenyl]acetamide (2)

To a solution of 3.00 g (14.9 mmol, 1.00 mol eq) 2-amino-4-(ethylsulfonyl)phenol ( 1) in 50 mL of EA (abs), acetic anhydride 1.69 mL (17.9 mmol, 1.20 mol eq) was added within 10 min under Ar. Then the mixture was stirred 1.5 h at 50 °C. Within this time grey solid material was precipitated. The mixture was cooled to rt and stirred in an ice bath for 20 min. Obtained solid material was filtered off, washed with H2O and dried under reduced pressure to yield 3.05 g (12.5 mmol, 84 %) of desired aniline 2.

Novelty: N-[5-(ethylsulfonyl) 4-(Ethylsulfonyl)-2-isothiocyanato-1-methoxybenzene (5) To a solution of 1.00 g (4.65 mmol, 1.00 mol eq) aniline 4 in 20 mL dioxane (abs), 428 μL (5.58 mmol, 1.20 mol eq) of thiophosgene (CSCl2) was added dropwise within 30 min. Then the reaction mixture was stirred 1 h at 70 °C. After complete comsuption of aniline 4, the reaction mixture was cooled down, 20 mL of NaHCO3 (saturated aq solution) was added and stirred for 5 min. Subsequently, dioxane and volatile parts were evaporated and 40 mL of EA added. The obtained solution was extracted with 3 x 15 mL of NaHCO3 (saturated aq solution)

and brine (3 x 15 mL). The organic layer was separated, dried over Na2SO4, filtered and concentrated under reduced pressure. Resulted oily crude product was stored in a refrigerator to provide 980 mg (3.81 mmol, 82 %) of 4-(ethylsulfonyl)-2-isothiocyanato-1methoxybenzene (5) in the form of a grey solid compound.

Novelty: 4-(ethylsulfonyl)-2-isothiocyanato-1-methoxybenzene ( 5) is described in the literature by its M.p., 1 H-NMR, 13 Hz, H-C( 3)), 6.39 (br s, 2H, -NHCONH2), 3.96 (s, 3H, -OCH3), 3.15 (q, 2H, J(CH2,CH3) = 7.4

Hz, -COOCH2CH3), 1.10 (t, 3H, J(CH2,CH3) = 7.4 Hz, -COOCH2CH3). (10) Scheme 34. Synthesis of N,4-diaryloxazole-2-amine general intermediate 10.

Synthesis of N,4-diaryloxazole-2-amine general intermediate

Synthesis of 1-(3-bromophenyl)-2-hydroxyethan-1-one (8)

To a solution of 4.64 g (82. 5)), 7.96 (dd, 1H, J(C2,C4) = J(C2,C6) = 1.9 Hz, H-CC( 2)), 7.76 (ddd, 1H, J(C5,C6) = 7.9 Hz, J(C2,C6) = 1.9 Hz, J(C4,C6) = 1.1 Hz, H-CC(6)), 7.52 (dd, 1H, J(A3,A4) = 8.6 Hz, J(A4,A6) = 2.3 Hz, H-CA(4)), 7.51 (ddd, 1H, J(C4,C5) = 7.9 Hz, J(C2,C4) = 1.9 Hz, J(C4,C6) = 1.1 Hz, H-CC(4)), 7.41 (dd, 1H, J(C4,C5) = J(C5,C6) = 7.9 Hz, H-CC( 5)), 7.28 (d, 1H, J(A3,A4) = 8.6 Hz, H-CA( 3)), 3.97 (s, 3H, CH3O-), 3.24 (q, 2H, J(CH2,CH3) = 7.3 Hz, CH3CH2SO2-), 1.18 (t, 3H, J(CH2,CH3) = 7.3 Hz, CH3CH2SO2-).

13 C-NMR (100 MHz, DMSO-d6): δ 157. 2, 152.2, 137.8, 134.1, 131.4, 130.8, 130.6, 130.2, 129.2, 127.9, 124.1, 123.0, 122.7, 117.2, 111.5, 56.8 Synthesis of 2-chlorotetrahydro-2H-pyran [START_REF]Preparation of the 1,2,4-triazine-3-(2H)thione catalysed by HOAc or NaOAc / HOAc An alternative way for the synthesis of 1,2,4-triazine-3-thione in acidic conditions was described in 2000 by J. Hlaváč et al. 124 The anion of the acid T102 obtained from isatine T101 was treated with thiosemicarbazide to give aniline T103 in 70 % overall yield. The final reaction step from T103 to T104 was performed at refluxing AcOH. Based on 1 H-NMR spectrum the ratio of obtained isomers T104 to T105 was 10 : 3. (Scheme 56) Scheme 56. Preparation of isomers T104 and T105 from isatine T101. Another preparation of 1,2,4-triazine-3(2H)-thiones T109 and T111 were described on aceanthrylene-1,2-dione T108 in acid-catalysed reaction 127 as well as on substituted thiosemicarbazone T110 catalysed by AcONa in AcOH at reflux[END_REF] In the first step gaseous HCl was generated in situ by adding conc. H2SO4 dropwise to sodium chloride in a two-neck round-bottom flask. Generated gas was bubbled through a stirred solution of 7.00 g (83. the reaction mixture at 0 °C and stirred overnight at rt. The reaction was quenched with ice at 0 °C, extracted with Et2O (3 x 15 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. An obtained red oily product was purified by vaccum distillation (130 °C at 1 mbar) to provide 4.27 g (2.81 mmol, 82 %) of protected imidazole 14 as a white waxen compound.

Novelty: 1-(tetrahydro-2H-pyran-2-yl)-1H-imidazole ( 14) is described in the literature by its 1 H-NMR, 13 C-NMR and IR. 79 A solution of imidazole 14 (900 mg, 5.92 mmol, 1.00 mol eq) in 10 mL of THF (abs) was added slowly at -30 °C to a mixture of 9.25 mL (14.8 mmol, 2.50 mol eq) n-BuLi (1.6 M in hexanes) and 2.21 mL (14.8 mmol, 2.50 mol eq) of TMEDA (abs) in 15 mL of THF (abs). The reaction mixture was stirred for 30 min at the same temperature and 1 h at rt. Subsequently, reaction mixture was cooled down to -30 °C and 4.00 mL (14.8 mmol, 2.50 mol eq) of Bu3SnCl in 10 mL of THF (abs) was added dropwise and stirred for 20 min at the same temperature and overnight at rt. Then, the mixture was quenched with H2O (30 mL) and extracted with EA (3 x 20 mL). Combined organic layer was washed with brine (3 x 15 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. A crude product was purified by FLC (EA / cHex: gradient from 1 / 1 to 4 / 1) to afford 850 mg (1.93 mmol, 33 %) of imidazole 15 as a red oily material.

Novelty: 1-(tetrahydro-2H-pyran-2-yl)-5-(tributylstannyl)-1H-imidazole (15) is not described in the literature. 13 C-NMR (100 MHz, CDCl3): 138. 7, 137.5, 128.4, 85.3, 67.5, 31.4, 29.0, 27.3, 24.8, 22.6, 13.7 (-CH2CH3), 10.3 (-CH2CH3).

FT-IR (solid, cm -

Synthesis of 3-bromo-1-(triisopropylsilyl)-1H-pyrrole (17)

A solution of 1.00 g (4.48 mmol, 1.00 mol eq) of pyrrole 16 in 15 mL of THF (abs) was cooled to -78 °C. Subsequently 797 mg (4.48 mmol, 1.00 mol eq) of freshly crystallized NBS was added portionwise to the reaction mixture. Resulting solution was stirred for 1.5 h at the same temperature and another 1.5 h at room temperature. Afterwards, the reaction was quenched with NaHCO3 (sat aq solution), extracted with Et2O (3 x 15 mL), combined organic layer washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. Obtained oily product was filtered throught a thin layer of silica gel (cHex / EA = 30 / 1) yielding 1075 mg (3.56 mmol, 79 %) of pyrrole 17 with traces of its α-isomer as colorless oil.

Novelty: 3-bromo-1-(triisopropylsilyl)-1H-pyrrole ( 17) is described in the literature by its 1 H-NMR, 13 C-NMR and IR. 80 A solution of 550 mg (1.82 mmol, 1.00 mol eq) pyrrole 17 in 15 mL of THF (abs) was cooled to -78 °C and 1.37 mL (2.18 mmol, 1.20 mol eq) of n-BuLi (1.6 M in pentane) was added dropwise. Resuting mixture was stirred for 30 min at the same temperature. Subsequently 677 mg (3.64 mmol, 2.00 mol eq) of pinacol boronate 18 was added to the mixture and stirred 1 h x 1H, H-CC(4 and 6)), 7.30 (dd, 1H, J(C4,C5 and C5,C6) = 8.1 and 7.7 Hz, H-CC( 5)), 7.20 -7.17 13 C-NMR (100 MHz, CDCl3): δ 155.6, 150. 5, 140.5, 136.3, 131.2, 130.9, 129.1, 128.9, 127.5, 124.8, 124.6, 122.4, 2 x 122.3, 119.0, 115.7, 155.1, 109.5, 106.5, 56.

Synthesis of N-[5-(ethylsulfonyl)-2-methoxyphenyl]-4-[3-(pyrimidin-2-yl)phenyl]oxazol-

2-amine (NBM2)

A mixture of 150 mg (0.34 mmol, 1.00 mol eq) oxazole 10, 39.6 mg (0.03 mmol, 0.10 mol eq) of Pd(PPh3)4, 15.7 mg (0.37 mmol, 1.10 mol eq) of LiCl and 37.0 mg (0.37, 1.10 mol eq) of CuCl in 10 mL of THF (abs) was purged by Ar for 10 min. Subsequently 252 mg (0.68 mmol, 2.00 mol eq) of tributylstannane 20 was added and the mixture was sealed and stirred at 70 °C overnight. Then the reaction was cooled down and filtered througt a thin layer of silicagel.

Afterwards, the solution was diluted with 10 mL of EA, quenched with 10 mL of KF (sat aq solution) and stirred for 4 h. The organic layer was separated, washed with 10 mL of H2O, dried over Na2SO4, filtered and evaporated under reduced pressure. The crude product was purified by FLC (Hex / EA = 1 / 2) and crystallized with Hex / EA yielding 10.0 mg (0.03 mmol, 10 %) of desired oxazole NBM2. N, 13.57.

Novelty: N-[5-(ethylsulfonyl)-2-methoxyphenyl]-4-[3-(pyrimidin-2-yl)phenyl]oxazol-2- amine (

Synthesis of N-[5-(ethylsulfonyl)-2-methoxyphenyl]-4-[3-(thiophen-3-yl)phenyl]oxazol-2amine (NBM4)

A suspension of 50.0 mg (0.11 mmol, 1.00 mol eq) of oxazole 10, 41.0 mg (0.17 mmol, 1.50 mol eq) of MIDA-boronate 22, 2.60 mg (0.01 mmol, 0.11 mol eq) of Pd(OAc)2 and 9.40 mg (0.02 mmol, 0.22 mol eq) of SPhos in 5 mL of THF (abs) was bubbled by Ar for 10 min. Then the solution of 175 mg (0.82 mmol, 7.50 mol eq) K3PO4 in 1 mL of H2O was also bubbled by Ar for 10 min and added to the reaction mixure. Resulting mixture was sealed and stirred overnight at 90 °C. The reaction was cooled down, diluted with 20 mL of EA and washed with brine (3 x 15 mL). The organic layer was dried over Na2SO4, filtered and a solution concentrated under reduced pressure. The crude product was purified by FLC (EA / cHex = 1 / 1) to yield 25.3 mg (0.06 mmol, 50 %) of desired oxazole NBM4 as a red solid compound.

Novelty: N-[5-(ethylsulfonyl)-2-methoxyphenyl]-4- [3-(thiophen-3-yl) ), 3.26 (q, 2H, J(CH2,CH3) = 7.4 Hz, CH3CH2SO2-), 1.17 (t, 3H, J(CH2,CH3) = 7.4 Hz,

CH3CH2SO2-).

13 C-NMR (100 MHz, DMSO-d6): δ 157. [START_REF] Hz | A3 and A6))[END_REF]152.0,141.6,139.1,135.9,132.2,130.6,129.8,2 x 129.4,127.7,126.6,125.8,123.9,123.0,122.7,121.5,116.9,111.4,, 50.4 (CH3CH2SO2-), 8.0 (CH3CH2SO2-).

FT-IR (solid, cm

Synthesis of N-[5-(ethylsulfonyl)-2-methoxyphenyl]-5-{3-[1-(tetrahydro-2H-pyran-2-yl)-

1H-imidazol-5-yl]phenyl}oxazol-2-amine (29)

A solution of 150 mg (0.35 mmol, 1.00 mol eq) oxazole 28 and 24.0 mg (34.2 µmol, 0.10 mol eq) of Pd(PPh3)2Cl2 in 4 mL of DMF (abs) was bubbled by Ar for 10 min. Afterwards 378 mg (0.86 mmol, 2.50 mol eq) of tributylstannne 15 was added and the reaction stirred at 100 °C and MW irradiated for 1 h. After that TLC analysis confirmed a presence of a new product without 28 and side products. The reaction mixture was cooled down, diluted with 50 mL of brine and extracted with EA (3 x 15 mL). Combined organic layer was washed with brine (4 x 15 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. Obtained oily product was diluted in CH3CN, extracted with n-hexane (3 x 15 mL) and separated organic layer evaporated with RVE. The crude product was purified by FLC (SiO2, from 100 % of EA to EA / MeOH : 20 / 1) yielding 125 mg (0.25 mmol, 72 %) of oxazole 29 as an orange solid compound.

Novelty: 3)), 3.99 (s, 3H, CH3O-), 3.21 (q, 2H, J(CH2,CH3) = 7.3

N-[5-(ethylsulfonyl)-2-methoxyphenyl]-5-{3-[1-(tetrahydro-2H-pyran-2-yl)-1H- imidazol-5-yl]phenyl}oxazol-2-
Hz, CH3CH2SO2-), 1.12 (t, 3H, J(CH2,CH3) = 7.3 Hz, CH3CH2SO2-).

13 C-NMR (125 MHz, DMSO-d6): 156.4, 152.0, 145. 0, 140.1, 136.4, 136.1, 130.6, 129.6, 129.3, 128.5, 123.8, 122.8, 122.7, 120.9, 119.0, 116.1, 113.4, 111.3, 56.8 According the above results we decided to perform hydrolysis of a cyano group first and then carry out a coupling reaction. Unfortunately, Suzuki coupling reaction with the amide derivative 36b provided the dehalogenation product 36d instead of desired product BM9. (Scheme 41)

Scheme 41. Unexpected preparation of 36d by dehalogenation of starting compound 36b.

We proposed a mechanism of dehalogenation, which was initiated by hydrolysis of dimethylformamide (DMF) in a basic solution at 100 °C. Formed sodium formate attacks palladium (II) in D1 intermediate and releases a bromide anion to form intermediate D2.

Subsequently, a hydride anion from formate moiety is transferred to Pd (II) with simultaneous releasing of CO2. Subsequent reductive elimination gives a product D4. (Scheme 42) Scheme 42. A proposed mechanism of debromination of 36b by decomposition products of DMF.

Therefore we decided to use dimethylacetamide (DMA) as a solvent, which does not contain hydrogen as in DMF. But in this case after 16 h at 100 °C we observed only a starting compound 36b. The obvious reason of non-reactivity of 36b is a presence of an amide group. In basic conditions of Suzuki coupling reaction an internal amine group of 36d is deprotonated, which leads to delocalization of an anion and form more nucleophilic amide group, which could either disrupt Pd (0) oxidative addition or favour Pd (II) According to unsatisfactory results we decided not to continue with preparation of three-point interacting pyrrole derivatives BM9 and BM10. We used the same synthetic pathway for 69 as was successfully applied for the intermediate 10.

(Scheme 34) Synthesis started from benzonitrile 32, which underwent α-oxidation with PhI(OAc)2 and subsequent hydrolysis yielding 66 in 75 % yield. Reaction of benzonitrile 66 with triflic anhydride in a presence of 2,6-lutidine provided a mixture of two major products 67

and 68 (based on TLC analysis), which was used in the next step without further purification.

According to analogues of N-oxazole intermediate 10 (67 %), the mixture (67 + 68) had to be added portionwise to the urea 6 for 2 days at 45 °C. In this case no product 69 was obtained, According to described 1 H-NMR chemical shifts for α-trifluoromethanesulfonyl acetophenone from the literature, we assigned the products 9 and 67 to desired triflate reagents. 84 Then we suggested that side products 9a and 68 could be dimers, formed from triflates 9 and 67 and their Our hypothesis was also supported by fact, that proposed dimer 68 was easily separated by FLC on SiO2 compare to relative low stability of triflates. On the other hand, 19 F-NMR and IR spectra 84 Vedejs, E.; Engler, D.A.; Mullins, M.J. J. Org. Chem., 1977, 42, 3109 -3113. confirmed a presence of unexpected -SO2CF3 group and obtained MS spectrum did not corespond directly with a structure of dimer 68. Based on a proved presence of -SO2CF3 group in the structure 68 and markedly lower chemical shift of its α-methylene group in 1 H-and mainly 13 C-NMR in comparison to starting compound 66 (65.9 vs 45.2 ppm), we also proposed benzonitrile 70. (Figure 52) Comparable spectral analyses and reaction of similar trifluoromethanesulfonyl compounds in the literatures also supported our hypothesis.

Synthesis of 4-acetyl-2-bromobenzonitrile (32)

A solution of 3.50 g (16.4 mmol, 1.00 mol eq) acetofenone 31 in 30 mL of DCM (abs) was cooled down to -10 °C under Ar. Then, 2.33 mL (19.6 mmol, 1.20 mol eq) of t BuONO and 3.03 mL (24.5 mmol, 1.50 mol eq) of Et2O . BF3 were added dropwise and the resulting mixture stirred at rt for 4 h. After precipitation of diazonium salt, the mixture was diluted with 30 mL of Et2O, the precipitate filtered off, washed with Et2O and dried by HV. Obtained salt was suspended in 40 mL of toluene and cooled to -5 °C. After that, the solution of 1.90 g (21.3 mmol, 1.30 mol eq) CuCN and 3.60 g (73.6 mmol, 4.50 mol eq) NaCN in 60 mL of H2O was added dropwise while the temperature was kept below 5 °C. When addition was completed, the mixture was stirred at 0 °C for 30 min, warmed to 60 °C and stirred another 2 h. After cooling down to rt, 40 mL of EA was added, extracted with H2O (3 x 20 mL) and brine (3 x 20).

Synthesis of 2-bromo-4-(2-bromoacetyl)benzonitrile (34)

A solution of trimethylphenylammonium tribromide 33 (460 mg, 1.23 mmol, 1.10 mol eq) in 15 mL of THF (abs) was added dropwise to the solution of 250 mg (1.12 mmol, 1.00 mol eq) benzonitrile 32 in 15 mL of THF (abs) at rt within 30 min. After that, the reaction mixture was stirred another 1 h at the same temperature. Then the reaction mixture was evaporated, an obtained solid material dissolved in 30 mL of EA, extracted with H2O (3 x 20 mL) and brine ( 3x 10). The organic layer was dried over Na2SO4, filtered and evaporated under reduced pressure. 5)), 7.80 (d, 1H, J(5,6) = 8.1 Hz, H-C( 6)), 4.39 (s, 2H, -COCH2Br).

13 C-NMR (150 MHz,CDCl3,: δ 189.1 138.0,134.9,133.4,127.8,126.3,120.5,116.4,. Anal. calcd for C9H5Br2NO (302.95): C, 35.68; H, 1.66; N, 4.62; found: 35.77; H: 1.90; N, 4.88.

Synthesis of 4-(2-azidoacetyl)-2-bromobenzonitrile (35)

A solution of 500 mg (1.65 mmol, 1.00 mol eq) benzonitrile 34 in 15 mL of acetone was cooled down to 0 °C. Then, 127 mg (1.98 mmol, 1.20 mol eq) of NaN3 dissolved in 2 mL of H2O was added dropwise at 0 °C and resulting mixture stirred for 15 min at rt. Subsequently, acetone was evaporated with RVE, remaining material was dissolved in 15 mL of EA, washed with H2O (2 x 20 mL) and brine (2 x 15 mL). Separated organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The obtained oily product was purified by trituration with Hex / EA yielding 415 mg (1.57 mmol, 95 %) 4)), 135. [START_REF] Hz | A3 and A6))[END_REF]132.5,126.8,126.5,120.7,116.3,. A solution of 100 mg (0.39 mmol, 1.00 mol eq) isothiocyanate 5 and 122 mg (1.17 mmol, 1.20 mol eq) of PPh3 in 10 mL of dioxane (abs) was placed into a 100 °C preheated bath. Then, 185 mg (0.70 mmol, 1.80 mol eq) of azide 35 dissolved in 20 mL of dioxane (abs) was dropwise added to the mixture within 30 min. Then, the reaction mixture was stirred another 1 h at the same temperature. After that, the solvent was evaporated and the crude product purified by FLC (EA / cHex: gradient from 50 % to 100 % of EA). Subsequent trituration of the obtained product with benzene (to remove traces of PPh3=S) provided 63.0 mg (0.14 mmol, 35 %) 1 H-NMR (400 MHz, DMSO-d6): δ 10.02 (br s, 1H, -NH-), 8.73 (d, 1H, J(4,6) = 2.2 Hz, H-CA( 6)), 8.01 (d, 1H, J(3,5) = 1.6 Hz, H-CC( 3)), 7.99 (d, 1H, J(5,6) = 8.2 Hz, H-CC( 6)), 7.93 (s, 1H, H-CB(4)), 7.73 (dd,1H,J(5,6) = 8.2 Hz,J(3,5) = 1.6 Hz, H-CC( 5)), 7.54 (dd, 1H, J(3,4) = 8.6 Hz, J(4,6) = 2.2 Hz, H-CA(4)), 7.29 (d, 1H, J(3,4) = 8.6 Hz, H-CA( 3)), 3.98 (s, 3H, -OCH3), 3.21 (q, 2H, J(CH2,CH3) = 7.3 Hz, -SO2CH2CH3), 1.12 (t, 3H, J(CH2,CH3) = 7.3 Hz, -SO2CH2CH3).

FT IR (solid, cm

13 C-NMR (150 MHz, DMSO-d6): δ 157.4 (CB( 2)), 151.8 (CA( 2)), 141.4,135.5,133.5,130.2,128.2,128.0,126.0,125.2,123.0,121.6,117.4,116.5,111.4,111.1,, 49.7 (-SO2CH2CH3), 7.4 (-SO2CH2CH3). Anal. calcd for C19H16BrN3O4S (462.32): C, 49.36; H, 3.49; N, 9.09; found: C, 49.46; H, 3.58; N, 9.25.

Synthesis of 4-{2-{[5-(ethylsulfonyl)-2-methoxyphenyl]amino}oxazol-4-yl}-2-(1H-pyrrol-3-yl)benzonitrile (BM8)

A mixture of 25.0 mg (0.05 mmol, 1.00 mol eq) oxazole 36, 28.3 mg (0.08 mmol, 1.60 mol eq) of pinacol boronate 19 and 6.26 mg (5.42 µmol, 0.11 mol eq) of Pd(PPh3)4 in 4 mL of DMF placed in a microwave vial was purged by Ar for 10 min. Then the solution of 11.0 mg (0.10 mmol, 2.00 mol eq) Na2CO3 in 1 mL of H2O was also purged by Ar for 10 min and added to the reaction. Resulting mixture was stirred and heated in a microwave reactor at 100 °C for 1 h. After that TLC analysis confirmed a presence of only one new compound with traces of one side product. The reaction was cooled down, diluted with 10 mL of EA and washed with brine

(5 x 15 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by FLC (EA / Hex = 1 / 1) and crystallized from EA yielding 14.0 mg (0.03 mmol, 58 %) of desired oxazole BM8 as a red solid material.

Novelty:

4-{2-{ [5-(ethylsulfonyl)

CH3CH2SO2-).

13 C-NMR (150 MHz, DMSO-d6): δ 157. 3,152.2,143.4,140.6,135.2,132.4,130.6,128.9,126.5,123.2,121.5,120.4,120.1,119.8,119.7,118.3,116.6,111.5,107.6,105.3,, 50.1 (CH3CH2SO2-), 7.8 (CH3CH2SO2-). N,12.49;Found: C,61.95;H,4.90;N,12.82.

FT-IR (solid, cm

Synthesis

CH3CH2SO2-).

13 C-NMR (100 MHz, DMSO-d6): δ 157.6, 152. 2,143.1,137.8,135.5,132.4,131.9,130.6,129.3,128.9,127.0,123.3,122.1,122.0,119.6,116.8,111.5,106.4,104.5,, 50.2 (CH3CH2SO2-), 7.8 (CH3CH2SO2-). Anal. calcd for C22H19N5O4S (449.49): C,58.79;H,4.26;N,15.58;Found: C,58.95;H,4.44;N,15.48. 2)), 4.12 and 3.52 (2 x m, 2 x 1H, 2 x H-CE( 6)), 4.01 (s, 3H, CH3O-), 3.18 (q, 2H,

FT-IR

Synthesis

J(CH2,CH3) = 7.4 Hz, CH3CH2SO2-), 2.13 -1.63 (m, 6H, H-CE(3-5)), 1.31 (t, 3H, J(CH2,CH3) = 7.4 Hz, CH3CH2SO2-).
13 C-NMR (100 MHz, CDCl3, MH-179C-19): δ 156. 2, 150.6, 143.5, 136.7, 134.3, 133.9, 131.9, 131.1, 130.8, 128.3, 128.2, 125.9, 124.9, 123.1, 122.3, 117.8, 115.8, 110.8, 109.7, 82.8, 68.2, 56.4 (CH3O-), 50.7 (CH3CH2SO2-), 30. 8,24.7,23.1,. To the solution of 70.0 mg (0.13 mmol, 1.00 mol eq) oxazol 37 dissolved in 6 mL of DCM, 1.70 mL (2.60 mmol, 20.0 mol eq) of CF3COOH (conc) was added and the mixture stirred at rt overnight. Then, solvent and CF3COOH volatile parts were evaporated with RVE and resulting oil dissolved in DCM. Obtained solution was extracted with NaHCO3 (saturated aq solution) ( 2x 15 mL) while a formation of precipitate was observed. Resulting suspension was evaporated to a half of its volume and put to a refrigerator overnight. After 16 h an obtained solid product was filtered off, washed with small amount of cold water and dried by HV to provide 30.0 mg (0.07 mmol, 50 %) of oxazole BM14 as a white solid compound. 3)), 4.01 (s, 3H, CH3O-), 3.21 (q, 2H, J(CH2,CH3) = 7.4 Hz, CH3CH2SO2-), 1.12 (t, 3H, J(CH2,CH3) = 7.4 Hz, CH3CH2SO2-).

FT-IR

Novelty

13 C-NMR (150 MHz, DMSO-d6, MH-185E-19): δ 157. 5, 152.2, 143.4, 138.7, 136.9, 136.7, 135.1, 132.4, 130.6, 128.9, 126.6, 123.3, 121.0, 120.9, 120.2, 116.7, 116.4, 111.5, 104.6, 56.8 (CH3O-), 50.2 (CH3CH2SO2-), 7.8 (CH3CH2SO2-).

FT-IR (solid, cm To a solution of 687 mg (12.3 mmol, 5.50 mol eq) KOH in 30 mL of MeOH (abs), 500 mg (2.23 mmol, 1.00 mol eq) of benzonitrile 32, suspended in ca 40 mL of MeOH (abs), was added and cooled to 0 °C. Afterwards, 791 mg (2.46 mmol, 1.10 mol eq) of PhI(OAc)2 was added portionwise within 10 min and stirred for another 10 min at the same temperature. After 2.5 h of stirring at rt, reaction mixture was evaporated by RVE, resulting solid material diluted in 50 mL of EA and extracted with H2O (3 x 30 mL) and brine (3 x 30 mL). Obtained oil was dissolved in 20 mL of MeOH and 15 mL of 2 M HCl (aq) was added. After 2 h of stirring at 60 °C, TLC analysis confirmed a presence of one product with traces of two side products.

Afterwards, reaction mixture was evaporated by RVE, diluted in 50 mL of EA and extracted with H2O (3 x 30 mL) and brine (3 x 30 mL). Combined organic layer was dried over Na2SO4, filtered and evaporated under reduced pressure. Crude product was triturated with a mixture of cHex / EA to yield 360 mg (1.50 mmol, 67 %) of benzonitrile 66 as a brown solid compound.

Novelty: 2-bromo-4-(2-hydroxyacetyl)benzonitrile (66) is not described in the literature. 3,134.9,132.1,126.3,120.7,116.3,. Benzonitrile 66 120 mg (0.50 mmol, 1.00 mol eq) was dissolved in 30 mL of DCM (abs) and cooled to -78 °C. At this temperature 120 µl (108 mg, 1.00 mmol, 2.00 mol eq) of 2,6-lutidine was added with subsequent dropwise addition of 168 µl (284 mg, 1.00 mmol, 2.00 mol eq) of Tf2O. Reaction mixture was stirred for 2 h while its temperature went to rt. Afterwards, volatile parts were evaporated by RVE, obtained crude product dissolved in 30 mL of EA and extracted with H2O (1 x 30 mL) and brine (1 x 30 mL). Combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to yield 150 mg of a mixture of two products.

FT-IR

Due to expected low stability of triflates, desired product 67 was not separated from side product 68 and was used as a mixture (67 / 68 : 61 / 39 % according to 1 H-NMR) for a next reaction.

Novelty: 2-(3-bromo-4-cyanophenyl)-2-oxoethyl trifluoromethanesulfonate (67) is not described in the literature.

1 H-NMR analysis (determined from a mixed 1 H-NMR spectrum):

8.2 Hz, J(3,5) = 1.6 Hz, H-C( 5)), 7.86 (d, 1H, J(5,6) = 8.2 Hz, H-C(6)), 5.61 (s,2H,.

Due to instability, other characteristicts for 67 were not determined. Side product 68 revealed high stability on SiO2, therefore we separated it from a mixture by FLC (SiO2, eluent: cHex / EA : 3 / 1) and performed spectral analysis. Unfortunately, its structure has not been exactly determined yet.

M.p.: 100.0 -126.0 [cHex / EA] (dec). 13 C-NMR (100 MHz, CDCl3): δ 189.0 (-COCH2R), 138. 1,134.8,133.0,127.3,126.2,120.4,116.2,. Chapter 8. Aldose reductase (ALR2) in diabetes mellitus

Aldose reductase (ALR2) in diabetes mellitus

The second part of this dissertation thesis is dedicated to another serious and well-know disease diabetes mellitus (DM). The most important factor implicated in DM and its related diseases is blood sugar. Glucose is a hexose sugar, which circulates in the blood of human. It is important source of energy for cells and it is also stored as polymer glycogene in a liver. 91 In the normal aerobic circumstances (non-diabetic conditions), D-glucose is phosphorylated by hexokinase and subsequently metabolized through several metabolic steps to CO2 and water. This cascade produces energy mostly in the form of energetically rich molecules like NADH and ATP (adenosine triphosphate). 92

Glycolysis is a metabolic pathway that converts glucose into pyruvate and the free energy is released in a form of ATP and NADH. 93 Produced pyruvate molecules are then transported into the mitochondrial matrix, where they are oxidized by coenzyme A to form CO2, acetyl-CoA and NADH. 94 Resulted acetyl-CoA molecules enter to citric acid cycle (or Krebs cycle) and by the series of reactions are oxidized to CO2 and produce energy-rich ATP. 95 The cycle also reduces NAD + However, if large amounts of glucose are present under hyperglycemic conditions, hexokinase becomes saturated and around 33 % of overall glucose is metabolized through a polyol pathway.

While most of the cells require the action of insulin for glucose to gain entry into the cell, the cells of retina, kidney and nervous tissues are insulin-independent, so glucose moves across the cell membrane, regardless of the action of insulin. The cells will use glucose for energy as usually, and excess of glucose will enter the polyol pathway. 97

The polyol pathway

The polyol pathway is a two-step process which converts the glucose to the fructose. It uses an aldose reductase (ALR2) and a sorbitol dehydrogenase as catalysts. 98 (Figure 54) The polyol pathway reduces D-glucose and oxidizes NADPH to NADP + . Subsequently sorbitol dehydrogenase oxidizes sorbitol to fructose, which produces NADH from NAD + . 97 NADPH promotes glutathione production via the polyol pathway and deficiency of NADPH (e.g. its consumption via the polyol pathway) causes oxidative stress and hemolysis. NADPH is important to prevent accumulation of reactive oxygen species (ROS) and damaging of cells. Furthermore, produced fructose causes glycation of proteins and fats and enhances formation of advanced glycation end products (AGEs), which production causes oxidative stress and 97 Brownlee, M. Nature, 2001, 414, 813 -820. 98 Bonnefont-Rousselot, D. Curr . Opin .Clin. Nutr. 2002, 5, 561 -568. inflammation. 99 What more, the sorbitol is highly polar compound and hardly penetreates through the cell membranes. Its accumulation causes cell swelling leading to osmotic imbalanceas and causes various diabetic complications such as cataract, retinopathy, neuropathy and nephropathy. 100

Diabetes mellitus (DM) and diabetic complications

Diabetes mellitus type I is a metabolic disorder of a pancreas, an organ that produces the hormone insulin. Insuline is peptide hormone, which plays a key role in the regulation of variety of biomacromolecules, especially a blood glucose level. Normally, when the glucose level is high, the beta cells of the pancreas secrete the insulin and release it into the blood stream. Then, the glucose is absorbed into the cell thanks to glucose transporter activated by a complex of insuline with its receptor and glucose can be converted directly to energy or to energy storages: either glycogen via glycogenesis or fats via lipogenesis. 101 (Figure 55) 99 Vistoli, G.; De Maddis, D.; Cipak, A.; Zarkovic, N.; Carini, M.; Aldini, G. Free Radic. Res. 2013, 47, 3 -27. 100 Kinoshita, J.H. Invest. Ophth. Vis. Sci. 1974, 10, 713 -724. 101 Stryer, L. Biochemistry (4 th ed.), W.H. Freeman andCompany, New York, 1995, 773-774, ISNB: 0-7167-2009-4.

Properties and function of ALR2

Aldose reductase (ALR2) is a first enzyme of a polyol pathway and belongs to the aldo-keto reductase enzyme superfamily. It catalyses NADPH-dependent reduction of a wide variety of carbonyl compounds, e.g. glucose. 103 The enzyme is a single polypeptide domain composed of 315 amino acid residues folded into a β/α-barrel structural motif containing eight parallel β strands. 104 The active site is located in a large cavity in the C-terminal end of the β barrel and the NADPH cofactor is situated at the top of the β/α-barrel with the nicotinamide ring stands out in the center of the barrel.

An active site of ALR2

According to analysis of crystal structures of ALR2, the structure of its active site has been proposed. The first binding pocket of the ALR2 active site consists of a rigid anion binding pocket, composed of Trp20, Trp111, Tyr48, His110 amino acid side chains and amide group of NADP + . The second is a specificity pocket, consisted of Leu 300, Cys298, Cys303, Trp111 and Phe122. 105 This pocket is characteristic for its high degree of flexibility and the amino acid residues lining this pocket are not present in other aldo-keto reductases such as aldehyde reductase e.g. ALR1. The third is a hydrophobic pocket formed from Phe122, Trp219, Trp20 and Trp111 residues. 106 (Figure 56)

Aldehyde reductase

Aldehyde reductase (ALR1) is another oxidoreductase closely related to the aldose reductase (ALR2). Both enzymes share 65 % of structural homology and these similarities play important role in a common inhibitor non-selectivity. The co-inhibition of related oxidoreductases may create undesirable side effects. 107 A main role of the ALR1 is a detoxification, because it specifically metabolises toxic aldehydes such as 3-deoxyglucosone (3DG), hydroxynonenal (HNE), which may arise from consequences connected with oxidative stress in 105 Oates, P.J. Curr. Drug. Targets,2008, 9, 14-36. 106 El-Kabbani, O.; Darmanin, C.; Schneider, T.R.; Hazemann, I.; Ruiz, F.; Oka, M.; Joachimiak, A.; Schulze-Briese, C.; Tomizaki, T.; Mitschler, A.; Podjarny, A. Proteins, 2004, 55, 805 -813. 107 El-Kabbani, O.; Wilson, D. K.; Petrash, J. M.; Quiocho, F. A. Mol. Vis. 1998, 4, 19 -25. hyperglycemia. 108 In addition, it is responsible also for ascorbic acid synthesis in animals by reduction of D-glucoronate to D-gulonate. 109 Therefore ALR1 is an antitarget compare to an important antidiabetic target ALR2.

Inhibitors of ALR2

Over the last 40 years, numerous of ALR2 inhibitors have been developed, but only an epalrestat is available as a drug in Japan, China and India. 110 Most of structurally different ARIs have been refused in the clinical trials mainly for their low in vivo efficacies, pharmacokinetic drawbacks and side effects affected by non-selective inhibition of aldehyde reductase (ALR1).

Carboxylic acid derivatives

Carboxylic acids are the most important and the largest class of ARIs, because carboxylic anion group of the inhibitor occupies an anion binding pocket of the active site of ALR2. On the other hand, low pKA values hinder membrane permeability in physiological pH conditions and lead to a poor pharmacokinetic profile of such ARIs. Epalrestat is a noncompetitive and reversibile ALR2 inhibitor (containing rhodanine ring) used for treatment of diabetic neuropathy. 111 It was developed in 1983 and approved in Japan, China and India for the improvement of subjective neuropathy symptoms and abnormal changes in heart beat associated with a diabetic peripheral neuropathy. (Figure 57) Second very important ALR2 inhibitor is tolrestat, a potential drug for a treatment of diabetic complications, which was approved for several markets, but in 1997 withdrawn due to its liver toxicity in 3 rd phase of a clinical trial and finally never received FDA approval. 112 (Figure 58) Zenarestat is a potential drug for a treatment of cataract, diabetic retinopathy and neuropathy.

Zenarestat together with zopolrestat proceeded into clinical trials. In the 2 nd phase of clinical study they showed beneficial effect on nerve conduction velocity, but the 3 rd phase of trials showed that zenarestat therapy was linked with renal toxicity in a small number of patients. Its structurally similar inhibitor ponalrestat also failed in clinical trials due to the lack of efficacy.

(Figure 58)

Spirohydantoin and spirosuccinimide derivatives

The second important class of ALR2 inhibitors includes spirohydantoin derivatives, where the hydantoin group occupies the anion binding pocket similarly as occupies anion group of carboxylic derivatives. 113 Sorbinil, a strong ALR2 inhibitor with chromane substructure, has been shown to be an excelent inhibitor of ALR2 for both in vivo and in vitro assays.

Unfortunately, in clinical trials sorbinil was found, that increase a risk for developing of hypersensitivity such as skin rash, fever and myalgia (muscle pain). 114 These adverse reactions may be attributed to the poor selectivity of sorbinil with aldehyde reductase (ALR1). 115 Based on the structure of the sorbinil, several spirohydantoin inhibitors have been developed. Addition of a carbamoyl or methyl group at the sorbinil skeleton resulted in fidarestat and M79175, resp. Replacement of chromane ring with isoquinoline-1,3(2H,4H)-dione and introduction the spirosuccinimide ring produced minalrestat. Another similar inhibitor is ranirestat, which was developed for treatment of diabetic neuropathy 116 and now it is in the 3 rd phase of a clinical trial in Japan. Imirestat, containing planar fluorene ring, was also in the 3 rd phase of clinical trial and later withdrawn due to its toxicity. (Figure 59) 113 Fukushi, S.; Merola, L.O.; Kinoshita, J.H. Invest. Ophth. Vis. Sci., 1980, 3, 313 -315. 114 Jaspan, J.B.; Herold, K.; Bartkus, C. Am. J. Med. 1985, 5, 24 -37. 115 Barski, O.A.; Gabbay, K.H.; Grimshaw, C.E.; Bohren, K.M. Biochemistry, 1995, 35, 11264 -11275. 116 Negoro, T.; Murata, M.; Ueda, S.; Fujitani, B.; Ono, Y.; Kuromiya, A.; Komiya, M.; Suzuki, K. J. Med. Chem., 1998, 21, 4118 -4129. the D-glucoronate by ALR1 (IC50 = 41 µM) and selectivity factor SF(IC50 ALR1 / IC50 ALR2) = 400. (Figure 61) For the better understanding of interactions of CMTI with an active site of ALR2, a crystal structure of ALR2 in complex with CMTI was investigated. Two molecules of the CMTI inhibitor are present in a complex. First one as expected in the binding pocket and the second one in a neighbourhood of the entrance of the ALR2 active site. (Figure 62) A carboxylate group well occupies the anion binding pocket with interactions to His110, Tyr48 and Trp111.

A sulfur atom of CMTI forms a crucial H-bond with Leu300 and the remaining interactions have hydrophobic character. (Figure 62) Thanks to the high activity and selectivity of CMTI, excellent drug-like properties (Lipinski rules of five) 120 and water solubility, CMTI appears to be a promising lead compound suitable for further drug development.

Biological assay activity (ALR2)

Ability of ALR2 inhibitor to inhibit reduction of D,L-glyceraldehyde is a significant marker for an investigation of a remaning ALR2 activity. A partially purified ALR2 enzyme from rat eye lenses is used for an in vitro test.

Synthesis of ALR2 inhibitors possessing triazinoindole scaffold

In the previous chapter we described development of the effective ALR2 inhibitor CMTI, which possesses triazinoindole scaffold and its structure has become promising and suitable for further development. In the second part of this dissertation thesis we proceeded with optimization of CMTI skeleton and its analogues and therefore this chapter is devoted to the preparation of triazinoindole compounds. In 2000, the preparation of the 1,2,4-triazine-3-(2H)-thione T96 was also described 122 Gladych, J.M.Z.; Hornby, R.; Hunt, J.H.; Jack, D. J. Med. Chem. 1972, 15, 277 -281. 123 Kgokong, J.L.; Smith, P.P.; Matsabisa, G.M. Bioorg. Med. Chem. 2005, 13, 2935-2942. using basic conditions. 124 Synthesis started from 7-(6-azauracil-5-yl)isatine thiosemicarbazone T95, which was refluxed in 5 % aqueous solution of K2CO3 yielding 5H-triazino [5,6-b] Scheme 54. The preparation of the 4,5-dihydro-3H- [1,2,4]triazino [5,6-b]indole-3-thione T98 from thiosemicarbazone T97.

Similar results were obtained by refluxing of 7-trifluoromethylisatine T99 in aquenous solution of K2CO3 within 3 h. 126 (Scheme 55) 124 Hlaváč, J.; Slouka, J.; Hradil, P.; Lemr, K. J. Heterocyclic Chem., 2000, 37, 115 -118. 125 For determination of an antioxidant activity, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test was employed to evaluate a radical scavenging efficacy of CMTI, OTI and OTI-2.

Considerably decreased antioxidant activity of OTI (SF > 2300) and OTI-2 (SF > 1177) was recorded in comparison to CMTI (SF > 303). Nevertheless, higher scavenging activity of both inhibitors OTI and OTI-2 was observed relative to a standard melatonin.

In addition, we also determined an activity of CMTI and OTI on human recombinant aldose reductase AKR1B1. Human small intestine aldose reductase AKR1B10 was used as an antitarget, characterized by 71 % structural homology to AKR1B1. Also in this assay OTI (IC50 = 66 nM and SF = 1000) exhibited higher inhibitory activity and selectivity than CMTI (IC50 = 102 nM and SF = 200). (Figure 68) To conclude, OTI revealed 3-fold higher inhibitory activity on a rat ALR2 and 1.5-fold higher activity on human AKR1B1 compare to CMTI. What more, OTI exhibited 8-fold higher ALR1 / ALR2 (rat) and 5-fold higher AKR1B10 / AKR1B1 (human) selectivity compare to CMTI.

These results have been recently published.

Synthesis of ethyl 2-(2,3-dioxoindolin-1-yl)acetate (39)

A mixture of 2.00 g (13.6 mmol, 1.00 mol eq) isatine ( 38) and 630 mg (15.0 mmol, 1.10 mol eq) of CaH2 in 15 mL of DMF (abs) was stirred at 100 °C for 1 h under Ar and then cooled to 40 °C. At this temperature 4.37 mL (40.8 mmol, 3.00 mol eq) of ethyl chloroacetate was added within 10 min into the reaction mixture. The resulting mixture was heated to 100 °C for 5 h and then allowed to cool to rt. The reaction was poured into a vigorously stirred aqueous 0.5 M HCl solution (50 mL), which resulted in a red precipitate. The obtained solid material was filtered off and washed with H2O. The crude product was further purified by trituration in a mixture of (dd, 1H, J(8,9) = 7.6 Hz, J(7,9) = 1.4 Hz, H-C( 9)), 7.68 -7.65 (m, 2H, H-C(6 and7)), 7.41 (ddd, 1H, J(7,8) = 8.2 Hz, J(8,9) = 7.6 Hz, J(6,8) = 1.4 Hz, H-C( 8)), 5.00 (s, 2H, NCH2COOH).

Synthesis of 2-(2-oxoindolin-3-ylidene)hydrazinecarboxamide (41)

To a solution of 3.00 g (20.4 mmol, 1.00 mol eq) isatine 38 in 40 mL of EtOH, 2.73 g (24.5 mmol, 1.20 mol eq) of semicarbazide hydrochloride was added and the mixture refluxed for 30 min. Then, 40 mL of water was added to the reaction and the mixture cooled to rt and stirred ), 7.23 (dd, 1H, J(A5,A6) = 7.7 Hz, J(A4,A6) = 1.4 Hz, H-CA(6)), 7.07 (ddd, 1H, J(A4,A5) = 8.3 Hz, J(A3,A4) = 7.9 Hz, J(A4,A6) = 1.4 Hz, H-CA(4)), 6.68 (dd, 1H, J(A3,A4) = 7.9 Hz, J(A3,A5) = 1.4 Hz, H-CA( 3)), 6.53 (ddd, 1H, J(A4,A5) = 8.3 Hz, J(A5,A6) = 7.7 Hz, J(A3,A5) = 1.4 Hz, H-CA( 5)), 5.35 (br s, 2H, -NH2).

13 C-NMR (150 MHz, DMSO-d6): δ 157. 4, 149.8, 147.6, 143.4; 131.0, 130.2; 116.3, 115.8, 115.4. A suspension of 2.77 g (13.5 mmol) 42 in 50 mL of conc. HOAc was refluxed for 3 h. After cooling to 0 °C 20 mL of water was added and resulting suspension stirred for another 30 min in an ice bath. An obtained solid material was filtered off, washed with water, dried under IR lamp and reduce pressure to yield 2.23 g (12.0 mmol, 88 %) of 2H- [1,2,4]triazino [5,6-b] A mixture of 1.40 g (7.52 mmol, 1.00 mol eq) 43 and 474 mg (11.30 mmol, 1.50 mol eq) of CaH2 in 15 mL of DMF (abs) was stirred at 60 °C for 40 min under Ar and then cooled to rt. Then 1.61 mL (15.04 mmol, 2.00 mol eq) of ethyl chloroacetate was added dropwise to the reaction mixture within 10 min. Afterwards the mixture was heated to 50 °C for 3 h. After 3 h a TLC analysis confirmed formation of products 44, 45 and the starting material 43 in a ratio: 44/ 45 / 43 = 2.0 / 2.5 / 1.0. The mixture was cooled to rt and poured into a stirred aqueous 0.5 M HCl solution (50 mL), which resulted an orange precipitate. A solution obtained after filtration was extracted with EA (5 x 10 mL), washed with brine and water. A combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. A crude product was purified by FLC (SiO2, DCM / MeOH = 14 / 1) to give 450 mg (1.65 mmol, 22 %) of monoester 44 as a white solid compound and 539 mg (1.51 mmol, 20 %) 9)), 7.58 (ddd, 1H, J(6,7) = 8.0 Hz, J(7,8) = 7.3 Hz, J(7,9) = 1.0 Hz, H-C( 7)), 7.56 (dd, 1H, J(6,7) = 8.0 Hz, J(6,8) = 1.0 Hz, H-C( 6)), 7.34 (ddd, 1H, J(8,9) = 8.0 Hz, J (7,8) = 7.3 Hz, J(6,8) = 1.0 Hz, H-C( 8)), 5.03 (s, 2H, NCH2COOEt), 4.18 (q, 2H, J(CH2,CH3) = 7.1 Hz, -OCH2CH3), 1.22 (t, 3H, J(CH2,CH3) = 7.1 Hz, -OCH2CH3).

13 C-NMR (150 MHz, DMSO-d6): δ 167.9 (-CH2COO-); 154. 8, 154.5, 144.2, 132.6; 131.2, 123.6, 121.4, 118.0, 111.6; 62.0 (-OCH2CH3), 42.3 (-CH2COOEt), 14.4 (-OCH2CH3). To a solution of 200 mg (0.74 mmol, 1.00 mol eq) ester 44 in 20 mL of MeOH, 59.0 mg (1.48 mmol, 2.00 mol eq) of NaOH in 5 mL of water was added and the resulted mixture refluxed for 2 h. After consumption of starting material 44 (confirmed by TLC analysis) the mixture was cooled down in an ice bath and acidified with 1 M aq HCl to adjust pH = 4. After 10 min of stirring formation of precipitate was observed. The mixture was stirred for another 20 min in an ice bath. Then the precipitate was filtered off and dried under reduced pressure to yield 113 mg (0.46 mmol, 63 %) of OTI as a pale orange solid product.

FT-IR

Experiment 2

Compound CMTI 100 mg (0.38 mmol, 1.00 mol eq) was added to a solution of 30.0 mg (0.75 mmol, 2.00 mol eq) NaOH in 5 mL of water. An another solution consisting of 122 mg (0.77 mmol, 2.00 mol eq) KMnO4 and 84.0 mg (2.10 mmol, 5.50 mol eq) of NaOH in 5 mL of water was added dropwise to the above mixture and a resulting suspension was stirred at rt for 30 min. Then, the solid material was filtered off, a filtrate acidified with 1 M aq HCl to pH = 1 and refluxed for 5 min to perform a clear solution, cooled down and concentrated to a half of its volume by RVE. The additionally obtained OTI crude product was filtered off and washed with water. Collected product was purified by trituration in a mixture of hexole / ethyl acetate to afford 84.0 mg (0.34 mmol, 89 %) of OTI as a pale orange compound.

Novelty : 2-(3-oxo-2H-[1,2,4]triazino [5,6-b]indol-5(3H)-yl)acetic acid (OTI) is not described in the literature. To a solution of 70.0 mg (0.22 mmol, 1.00 mol eq) ester 46 in 15 mL of MeOH, 18.0 mg (0.45 mmol, 2.00 mol eq) of NaOH in 5 mL water was added and the mixture refluxed for 1 h. After consumption of starting material 46 (confirmed by TLC analysis) the reaction was cooled down in an ice bath and acidified with 1 M aq HCl to pH = 4. After 20 min of stirring, formation of precipitate was observed and the mixture was stirred for another 20 min. Then, the precipitate was filtered off, washed with ice water and dried under reduce pressure to yield 28.0 mg (0.10 mmol, 44 %) of OTI-2 as a white solid product. To a solution of 60.0 mg (0.22 mmol, 1.00 mol eq) ester 44 in 5 mL of DMF (abs), 19.0 mg (0.44 mmol, 2.00 mol eq) of CaH2 was added and the reaction was stirred at 50 °C under Ar for 30 min. Then, the mixture was cooled to rt, 102 µl (0.88 mmol, 4.00 mol eq) of benzyl chloride was added dropwise. The resulted solution was stirred at 80 °C within 4 h under Ar. After consumption of ester 44 (confirmed by TLC analysis) the reaction was cooled down, 10 mL of water added and stirred for 20 min in an ice bath which led to formation of a precipitate.

Obtained solid material was filtered off, washed with water and dried under reduced pressure to yield 20.0 mg (0.06 mmol) of product 47. The filtrate was extracted with EA (3 x 10 mL). A combined organic layer was washed with brine and water, dried by Na2SO4, filtered off and concentrated under reduced pressure to yield another 44.0 mg (0.12 mmol) of product 47.

Overall yield of ester 47 was 64.0 mg, (0.18 mmol, 80 %).

Novelty: ethyl 2- (2-benzyl-3-oxo-2H-[1,2,4]triazino [5,6-b]indol-5(3H)-yl)acetate ( 47 To a solution of 64.0 mg (0.18 mmol, 1.00 mol eq) 47 in 15 mL of MeOH, 14.0 mg (0.35 mmol, 2.00 mol eq) of NaOH in 5 mL of water was added and the mixture refluxed for 2 h. After consumption of 47 (confirmed by TLC analysis) the reaction mixture was cooled down in an ice bath and acidified with 1 M aq HCl to pH = 4. After 10 min formation of precipitate was observed and the mixture was stirred for 20 min. Then, the precipitate was filtered off, washed with ice water and dried under reduce pressure to yield 25.0 mg (82.8 µmol, 47 %) of acid OTI-3 as an orange solid product. To a suspension of 25.0 mg (0.90 mmol, 1.00 mol eq) ester 44 in 5 mL of DCM (abs), 24 µl (0.14 mmol, 1.60 mol eq) of DIPEA (abs), 13 µl (0.18 mmol, 2.00 mol eq) of acetyl chloride and 11.0 mg (0.09 mmol, 1.00 mol eq) of DMAP were added stepwise and resulting mixture stirred at 40 °C under Ar. After 1 h of stirring TLC analysis confirmed presence of product 48 without any side products. Then, the reaction mixture was extracted with water (3 x 10 mL), combined organic layer dried over Na2SO4, filtered and concentrated under reduced pressure to yield 26.0 mg (0.80 mmol, 79 %) of the crude product 48 as an orange solid compound.

Novelty: ethyl 2-(2-acetyl-3-oxo-2,3-dihydro-5H- [1,2,4]triazino [5,6-b]indol-5-yl)acetate ( 48) is not described in the literature.

1 H-NMR (300 MHz, DMSO-d6): δ 7.99 (dd, 1H, J(8,9) = 7.6 Hz, J(7,9) = 1.2 Hz, H-C( 9)), 7.67 (ddd, 1H, J(7,8) = 8.2 Hz, J(6,7) = 7.6 Hz, J(7,9) = 1.2 Hz, H-C( 7)), 7.57 (dd, 1H, J(6,7) = 7.6 Hz, J(6,8) = 1.2 Hz, H-C(6)), 7.37 (ddd, 1H, J(7,8) = 8.2 Hz, J(8,9) = 7.6 Hz, J(6,8) = 1. 2Hz, H-C( 8)), 5.03 (s, 2H, -CH2COOEt), 4.19 (q, 2H, J(CH2,CH3) = 7.1 Hz, -OCH2CH3), 2.68 (s, 3H, -COCH3), 1.23 (t, 3H, J(CH2,CH3) = 7.1 Hz, -OCH2CH3).

Due to instability of the compound 48 its M.p., 13 C-NMR, IR, MS and el. anal. were not determined. To a suspension of 25.0 mg (0.10 mmol, 1.00 mol eq) ester 44a (prepared by reaction of OTI and CH2N2, not shown) in 5 mL of DCM (abs), 25 µl (0.15 mmol, 1.50 mol eq) of DIPEA (abs), 24 µl (0.20 mmol, 2.00 mol eq) of pivaloyl chloride and 12.0 mg (0.10 mmol, 1.00 mol eq) of DMAP were added stepwise and resulting mixture was stirred at 30 °C under Ar. After 15 min of stirring TLC analysis confirmed presence of product 49 without any side products. Then, the reaction mixture was extracted with water (3 x 10 mL), combined organic layer dried by Na2SO4, filtered and concentrated under reduced pressure to yield 26.0 mg (0.80 mmol, 79 %) of crude product 49 as an orange solid compound.

Synthesis

Novelty: methyl 2-(3-oxo-2-pivaloyl-2,3-dihydro-5H- [1,2,4]triazino [5,6-b]indol-5-yl)acetate (49) is not described in the literature.

Project 4 -Synthesis and SAR study of novel OTI derivatives

OTI-(6-9)

The last project was dedicated to a development of ALR2 inhibitors with inhibitory activity than leading inhibitor OTI-3, developed in the previous project. 131 According to intermolecular interaction analysis we discovered an unoccupied pocket located in a close proximity to N (2) benzyl moiety of OTI-3 consisting of Ala299, Leu301 and Trp219. In order to utilize these residues for additional interactions, we proposed novel N-benzyloxotriazinoindole derivatives OTI-(6-9). (Scheme 67 and Figure 69)

Evaluation of OTI-(6-9) ALR2 inhibitory activity

Inhibitors OTI-(7-10) were tested for their ability to inhibit the reduction of D,Lglyceraldehyde using ALR2 isolated from the rat eye lenses. Unsubstituted benzyl analogue OTI-6 was used as a reference inhibitor. To assess selectivity we used a structurally related detoxification enzyme (an antitarget), aldehyde reductase (ALR1) isolated from the rat kidneys.

(Table 1) 1) According to predicted binding position of OTI-7 in an active site of ALR2 (PDB: 4QX4) a present cyano group formed an additional H-bond with Ala299

(2.4 Å) and a benzyl moiety showed two good π-π stacking interctions with Trp219 (3.9 and 4.6 Å) from an investigated interactive pocket of ALR2. (Figure 70)

Synthesis of 1,2-phenylenedimethanol (51)

A solution of phtalic anhydride 50 (440 mg, 2.97 mmol, 1.00 mol eq) in 15 mL of THF (abs)

was added dropwise at 0 °C to the stirred suspension of LiAlH4 (226 mg, 5.96 mmol, 2.00 mol eq) in 20 mL of THF (abs) within 30 min. Then, the mixture was stirred overnight at room temperature. Afterwards, the reaction was cooled to 0 °C and 10 mL of isopropanol added slowly, followed by methanol (10 mL) and water (10 mL). Obtained wax-like precipitate was filtered through Celite and obtained filtrate washed with EA and DCM. Organic solution was evaporated and aqueous mixture extracted with EA (3x 20 mL) and DCM (3x 10 mL).

Combined organic layer was dried over Na2SO4, filtered and concentrated over reduced pressure totally yielding 385 mg (2.79 mmol, 94 %) of diol 51 as a colourless oil, which solidified by storing in a refrigerator.

Novelty: 1,2-phenylenedimethanol ( 51) is described in the literature by its M.P., 1 H-NMR, 13 7,136.2,130.6,128.7,128.6,128.3,66.4,62.3,31.0,30.5,25.4,19.4. To a solution of 100 mg (0.41 mmol, 1.00 mol eq) of OTI dissolved in 30 mL of EtOH (abs),

1.00 mL of H2SO4 (conc) was added and the reaction mixture stirred at reflux under Ar. After 6 h TLC analysis confirmed a presence of a new product without starting compound. The mixture was cooled down and excess of EtOH was evaporated by RVE. Crude product was dissolved in 30 mL of DCM, washed with water (5 x 30 mL) and saturated aq solution of NaHCO3 (3 x 30 mL). Separated organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure yielding 85.0 mg (0.31 mmol, 76 %) of ester 44 as an orange solid product.

Novelty: ethyl 2- (3-oxo-2,3-dihydro-5H-[1,2,4]triazino [5,6-b]indol-5-yl)acetate ( 44) is described in the literature by its M.P., 1 H-NMR, 13 C-NMR, IR , MS and elemental analysis. 5H- [1,2,4]triazino [5,6-b]indol-5-yl}acetate (54) is not described in the literature.

Anal. calcd for C19H16N4O4 (364.36): C,62.63;H,4.43;N,15.38 Found: C,62.75;H,4.49;N,15.55. Reaction of benzonitrile 66 with Tf2O provided a mixture of probably containing desired triflate 67 and the stable side product 68, which structure has not been defined yet.

The third project was focused on a development of inhibitors of aldose reductase (ALR2), implicated in various chronic diabetic complications. Herein, we developed a synthesis of very efficient ALR2 inhibitor cemtirestat (CMTI), which synthesis had not been described before. We also developed CMTI bioisosteric analogue OTI, containing an oxygen instead of a sulphur, and its derivatives OTI-(1-3). Synthesis of the acyl and pivaloyl analogues OTI-4 and OTI-5 was not feasible due to their chemical instability. The most active inhibitor OTI exhibited 3-fold higher inhibitory activity than CMTI and remarkable 8-fold higher selectivity (SF > 2380, ALR2 & ALR1). Similar results we observed in assay on humam enzymes with 1.5-fold higher activity of OTI and 5-fold higher selectivity (AKR1B1 & AKR1B10). On the other hand, antioxidant activity assay revealed lower activity for OTI in comparison to sulphur containing CMTI. Based on all these results, OTI inhibitor has become a promissing lead compound suitable for further development towards inhibition of ALR2.

In the last project we performed a SAR study on the N-benzylated ALR2 inhibitor OTI-3, described in the previous project. According to molecular modeling we detected an unoccupied pocket in the active site of ALR2. Therefore four N-benzyl substituted derivatives OTI-(6-9) were proposed, possessing various functional groups attached on a benzyl moiety.

After synthesis and screening we have seen that all predicted compounds OTI-(6-9) exhibited higher inhibitory activity than their unsubstituted analogue OTI-3, which proved the proposed additional interactions in the ALR2 binding pocket. We also proposed hypothesis of lower inhibitory activity of derivatives OTI-(6,8,9) compare to OTI-7 and proposed further analogues OTI-(10-17) with expected better properties.

To sum up the dissertation thesis, we have prepared 78 compounds, from which 48 were new. From 30 predicted final compounds, we synthetized 22 while 16 of them were biologically evaluated (IC50, VEGFR2 TK, ALR2 and ALR1). a -Due to instability of product other analyses were not determined b -Synthesis and full experimental analysis is decribed by Peter Šramel in his dissertation thesis c -Synthesis of these four intermediate was not an aim of this thesis, therefore other experimental analyses and characterization were not determined and described in the experimental part of the thesis. d -Synthesis of this product was not an aim of this thesis, therefore other characterizations are not described in the experimental part of the thesis. e -Due to instability of the product, the structure is determined from a mixed spectrum with 68 f -19 F NMR g -NOE NMR h -HMBC NMR i -HSQC NMR Chapter 14. Scientific contribution

Figure 1 2 )Figure 2 .

 122 Figure 1 Bioisostérie régioisomérique (RegBio) -série des aminooxazoles N,5-disubstitués BM(1-7) les plus actifs et leurs paires régioisomères NBM (1-7) possédant un noyau aminooxazole N,4-disubstitué.

Figure 3 .

 3 Figure 3. Remplacement bioisostérique du soufre par de l'oxygène dans le CMTI et la structure de l'OTI et des anlogues proposés OTI-(1-5).

Figure 4 .Chapitre 1 . 2 .Figure 5 .

 4125 Figure 4. Les structures des dérivés de N-benzyl(oxotriazinoindole) proposés OTI-(6-9).

2. 1 .Figure 6 .

 16 Figure 6. La structure de la tyrosine kinase VEGFR2 humaine.

Figure 7 .

 7 Figure 7. a) Etat inactif du récepteur tyrosine kinase b) Un récepteur tyrosine kinase homodimère activé.

Figure 8 .

 8 Figure 8. Inhibition de VEGFR2 TK par diverses voies.

9 )Figure 9 .

 99 Figure 9. Structures et activités des inhibiteurs de tyrosine kinases VEGFR2.

Figure 10 .

 10 Figure 10. Structures des inhibiteurs les plus actifs de VEGFR2 TK BM(1-7) s'appuyant sur le motif oxazole-2amine N,5-disubstituée.

Figure 11 .Schéma 1 .Schéma 2 .Figure 12 .

 111212 Figure 11. Une structure générale d'oxazole-2-amines N,4-disubstituées NBM(1-7) proposée basée sur des oxazole-2-amines N,5-disubstituées BM(1-7) possédant divers substituants hétérocycliques (Ar) dans une position méta.

Figure 13 .Chapitre 2 . 3 .Schéme 3 .

 13233 Figure 13. Structures des inhibiteurs de 2e génération conçus BM8 et BM(11-15) possédant la capacité d'effectuer des interactions en trois points avec le SBCP.

  utilise une aldose réductase (ALR2) et une sorbitol déshydrogénase comme catalyseurs. (Figure 14) La voie polyol réduit le D-glucose et oxyde le NADPH en NADP +. Par la suite, la sorbitol déshydrogénase oxyde le sorbitol en fructose, qui produit du NADH à partir de NAD + . Le NADPH favorise la production de glutathion via la voie des polyols et conduit à une carence en NADPH provoquant un stress oxydatif et une hémolyse. Le NADPH est important pour empêcher l'accumulation d'espèces réactives de l'oxygène (ROS) et l'endommagement des cellules. En outre, le fructose produit provoque la glycation des protéines et des graisses et améliore la formation de produits finaux de glycation avancée (AGE), dont la production provoque un stress oxydatif ainsi qu'une inflammation. De plus, le sorbitol est un composé hautement polaire et pénètre à peine à travers les membranes cellulaires. Son accumulation provoque un gonflement des cellules conduisant à des déséquilibres osmotiques et entraîne diverses complications diabétiques telles que la cataracte, la rétinopathie, la neuropathie et la néphropathie.

Figure 14 .

 14 Figure 14. Une illustration de la voie des polyols et de son implication dans les complications diabétiques.

Figure 15 .

 15 Figure 15. Transport du glucose à travers la membrane cellulaire, médié par l'insuline.

3. 4 .

 4 Un site actif d'ALR2 D'après l'analyse des structures cristallines d'ALR2, une structure de son site actif a été proposée. La première poche de liaison du site actif d'ALR2 est constituée d'une poche de liaison anionique rigide, composée de chaînes latérales d'acides aminés Trp20, Trplll, Tyr48, His110 et d'un groupe amide de NADP+. La seconde, est une poche de spécificité et est constituée de Leu 300, Cys298, Cys303, Trp111 et Phe122. Cette poche est caractéristique par son degré élevé de flexibilité et les résidus d'acides aminés tapissant cette poche ne sont pas présents dans d'autres aldo-céto réductases telles que l'aldéhyde réductase, par ex. ALR1. La troisième est une poche hydrophobe formée de résidus Phe122, Trp219, Trp20 et Trp111.

Figure 17 .Figure 18 .

 1718 Figure 17. Structure et activité de l'epalrestat.

Figure 19 .

 19 Figure 19. Structures et activités des plus importants inhibiteurs de la spirohydantoïne ALR2.

Figure 20 .Figure 21 .

 2021 Figure 20. Les structures du CMTI, ses dérivés analogues de l'oxygène OTI et OTI-(1-3) avec des activités IC50.

Matúš Hlaváč :

 Hlaváč Rational development of kinase inhibitors dedicated to treatment of neoplastic diseases and diabetic complications Université de Strasbourg, Ecole européene de Chimie, Polymères et Matériaux (ECPM), UMR CNRS 7042 LIMA, Strasbourg, France. Comenius University in Bratislava, Faculty of Natural Sciences, Bratislava, Slovakia. VEGFR2 tyrosine kinase receptors are transmembrane proteins that are crucial for tissue regeneration, regulation of important cell cycles and vascular endothelial cell function. On the other hand, enhanced activity of VEGFR2 TK causes uncontrolled division of cancer cells and formation of tumor. Accordingly, a development of small molecule VEGFR2 TK inhibitors has become a promising and effective way for cancer treatment. Another equally serious disease is diabetes mellitus, characterized by absence of insulin or its resintance. Aldose reductase (ALR2) plays an important role in a formation of various heart and diabetic complications and has become an important therapeutic target in treatment of illnesses related to diabetes mellitus. Within the first part of this dissertation thesis we described a development of 14 predicted aminooxazole VEGFR2 TK inhibitors. We successfully synthetized novel N,4disubstituted aminooxazole inhibitors NBM(1-7), which are regioisomeric pairs to the previously developed N,5-disubstituted inhibitors BM(1-7). Afterwards, we proposed more effective derivatives BM(8-16) and six from them prepared. Evaluation of their activity is currently in progress. Second part of the thesis was dedicated to the development of ALR2 inhibitors. Based on the structure of the previously developed lead compound CMTI we Introduction 5. IntroductionCancer is undoubtedly one of the most serious and potentially life-threatening illnesses in the World.1 The development of a cancer is a multistep process involving an initiative and a progressive state. In the tumor initiation, a one single cell is mutated by changes in DNA, which results to abnormal proliferation. This proliferation giving rise to large number of daughter cells containing the mutation created by the initiator.2 Tumor progression continues as additional mutations occur within cells of tumor population followed by selection for more rapidly growing cells. This selection continues to tumor development, so tumor become more rapidgrowing and malignant.3 Angiogenesis is a complex process controlled by growth factors produced in the body. During the angiogenesis new blood vessels are formed from pre-existing ones and it´s physiologically crucial during an embryonic development and healing of wounds.4 On the other hand, angiogenesis is also involved in growth of tumors. The understanding of cell signaling processes in the last decades led to the identification of VEGFR2 TK as an interesting target for an anticancer therapy. It has been demostrated that small-molecule VEGFR2 TK inhibitors suppress cancer growth and metastasis leading to an effective cancer treatment mainly in combination with other agents.Diabetes mellitus (DM) is also a well-know disease characterised by abnormal high level of blood sugar. People with diabetes mellitus (DM) have either a lack of secretion of insulin (DM type I) or insulin can´t be used properly (DM type II), which lead to variety of pathology conditions. It can damage the tiny vessels in kidneys, hearth, eyes or nervous system leading to diabetic cataract, retinopathy, neuropathy and nephropathy.5 All these diabetic pathological complications are associated with a polyol pathway, which in the first step converts glucose to sorbitol by an enzyme aldose reductase. Beside of high concentration of sorbitol it results to an excessive accumulation of intracellular reactive oxygen species (ROS) in various tissues.

Figure 22 . 1 - 7 )Figure 23 .

 221723 Figure 22. Regioisomeric bioisostery (RegBio) -series of the most active N,5-disubstituted aminooxazoles BM(1-7) and their regioisomeric pairs NBM(1-7) possessing N,4-disubstituted aminooxazole core.

Figure 24 .

 24 Figure 24. Bioisosteric replacement of sulphur with oxygen in CMTI and the structure of OTI and its proposed derivatives OTI-(1-5).

Figure 25 .

 25 Figure 25. The structures of proposed N-benzyl(oxotriazinoindole) derivatives OTI-(6-9).

Figure 26 .

 26 Figure 26. A schematic illustration of each vascular endothelial growth factor receptors (VEGFRs) with their growth factors (VEGF(A-E), PLGF) and their cellular and physiological effects.

Figure 27 .

 27 Figure 27. The structure of human VEGFR2 tyrosin kinase.

Figure 28 .

 28 Figure 28. a) an inactive state of tyrosin kinase receptor b) an activated homodimeric tyrosin kinase receptor.

Figure 29 .

 29 Figure 29. Inhibition of VEGFR2 TK by various pathways.

  (Figure 31) Developed VEGFR2 TK inhibitors were assayed and their IC50 values were determined by testing at 10 concentrations (1 x 10 -4 M to 3 x 10 -9 M) for each compound. All active compounds bound to VEGFR2 TK by concentration dependent manner. Biological assays were performed by ProQinase GmbG, Freiburg, Germany. 26

Figure 31 .

 31 Figure 31. Schematic view of the radiometric kinase assay.

Figure 32 .

 32 Figure 32. The structures of N,5-diaryloxazole-2-amine and N,4-diaryloxazole-2-amine scaffolds.

Figure 33 .Figure 34 .

 3334 Figure 33.The structures of the most common aryl boronic derivatives used in SM cross-coupling reaction.

Figure 35 .

 35 Figure 35. The most common phosphine ligands used in cross-coupling reactions.

Figure 36 .Figure 37 .

 3637 Figure 36. Structural and functional features of particular substituents attached on dialkylbiarylphospine. The structures of the most utilized ligands are depicted as well.

Figure 38 .

 38 Figure 38. General structure of PEPPSI catalysts.

Figure 39 . 7 )Figure 40 . 1 - 7 )Figure 41 . 9 . 1 .Scheme 24 . 7 .

 39740174191247 Scheme 22. Examples of Negishi, Heck and Sonogashira coupling reactions.

  Scheme 27. Unexpected formation of hydantoin 10a instead of desired N,4-diaryloxazole-2-amine precursor 10.

Figure 42 .

 42 Figure 42. X-ray analysis of compound 10.

  Within this project I have successfuly prepared inhibitor BM7 and six N-regioisomeric pairs NBM(1-5,7) to the N,5-diaryloxazole-2-amine (O-regioisomeric) inhibitors BM(1-7). We also determined the most of their in vitro IC50 (VEGFR2 TK) activities. 26 Inhibitory activity of imidazole derivatives BM7 and NBM7 remain to be determined. All N,4-diaryloxazole-2amine inhibitors NBM(1-6) exhibited lower inhibitory IC50 activity (micromolar range) in comparison to their nM regioisomeric pairs BM(1-7). (Figure 43) All VEGFR2 TK inhibitors BM(1-7) and NBM(1-7) are predicted to bind a kinase active pocket in their "U" shaped conformation compare to the expected more relaxed "L" shaped conformation in solution.

Figure 43 . 1 .

 431 Figure 43. Comparison of structures and activities of O-regioisomers BM(1-7) with their regioisomeric partners NBM(1-7).

1 H

 1 -NMR (400 MHz, CDCl3): δ 7.67 (s, 1H, H-C(2)), 7.09 -7.07 (m, 2H, H-C(4 and 5)), 5.23 (dd, 1H, J(A2,A3) = 9.5 Hz, J(A2,A3) = 2.4 Hz, H-CA(2)), 4.07 and 3.68 (2 x m, 2 x 1H, 2 x H-CA(6)), 2.08 -1.58 (m, 6H, 3 x -CH2-from CA(3)-CA(5)).79 Primas, N.; Mahatsekake, C.; Bouillon, A.; Lancelot, J-Ch.; de Oliviera Santos, J.S.; Lohier, J-F.; Rault, S.Tetrahedron, 2008, 64, 4596 -4601.

1 H

 1 -NMR (400 MHz, CDCl3): δ 7.84 (s, 1H, H-C(2)), 7.03 (s, 1H, H-C(4)), 5.06 (dd, 1H, J(A2,A3) = 9.5 Hz, J(A2,A3) = 2.4 Hz, H-CA(2)), 4.02 and 3.61 (2 x m, 2 x 1H, 2 x H-CA(6)), 2.10 -1.40 (m, 6H, 3 x -CH2-from H-CA(3)-CA(5)), 1.51 (m, 2H, -SnCH2CH2-), 1.33 (m, 2H, -SnCH2CH2-), 1.06 (m, 2H, -CH2CH3), 0.89 (t, 3H, J(CH2,CH3) = 7.3 Hz, -CH2CH3).

  1 ): 2954 (m), 2923 (s), 2852 (m), 1506 (w), 1464 (m), 1376 (w), 1357 (w), 1259 (w), 1240 (w), 1204 (w), 1177 (w), 1111 (w), 1083 (s), 1044 (s), 997 (m), 911 (m), 876 (m), 825 (m), 769 (w), 661 (s), 595 (m), 550 (w), 506 (w), 458 (w).

1 H

 1 -NMR (400 MHz, CDCl3): 6.74 (dd, 1H, J(2,5) = 2.3 Hz, J(2,4) = 1.5 Hz, H-C(2)), 6.63 (dd, 1H, J(4,5) = 2.8 Hz, J(2,5) = 2.3 Hz, H-C(5)), 6.30 (dd, 1H, J(4,5) = 2.8 Hz, J(2,4) = 1.5 Hz, H-C(4)), 1.43 (sep, 3H, J(CH,(CH3)2) = 7.4 Hz, 3 x (CH3)2CHSi-)), 1.10 (d, 18H, J(CH,(CH3)2) = 7.4 Hz, 3 x (CH3)2CHSi-)).

1 HFigure 44 .

 144 Figure 44. Synthesis of N,4-diaryloxazole-2-amine VEGFR2 TK inhibitors NBM(1-7).

  (m, 1H, H-CD(2)), 6.92 (d, 1H, J(A3,A4) = 8.6 Hz, H-CA(3)), 6.79 (dd, 1H, J(D4,D5) = 4.9 Hz, J(D2,D5) = 2.4 Hz, H-CD(5)), 6.56 (dd, 1H, J(D4,D5) = 4.9 Hz, J(D2,D4) = 2.4 Hz, H-CD(4)), 3.94 (s, 3H, CH3O-), 3.10 (q, 2H, J(CH2,CH3) = 7.4 Hz, CH3CH2SO2-), 1.25 (t, 3H, J(CH2,CH3) = 7.4 Hz, CH3CH2SO2-).

  NBM2) is not described in the literature. 60.0 mg (0.14 mmol, 1.00 mol eq) oxazole 10, 23.0 mg (0.21 mmol, 1.50 mol eq) of boronic acid 21 and 15.9 mg (0.01 mmol, 0.10 mol eq) of Pd(PPh3)4 in 3 mL of DMF (abs) was bubbled by Ar for 10 min. A solution of 29.5 mg (0.28 mmol, 2.00 mol eq) Na2CO3 in 1.0 mL of H2O was bubbled by Ar for 10 min and added to the reaction. Resulting mixture was sealed and stirred overnight at 100 °C. Then TLC analysis confirmed a presence only a new product with traces of a side product. The reaction was cooled down, diluted with 10 mL of EA and washed with brine (3 x 15 mL). The separated organic layer was dried over Na2SO4, filtered and evaporated under reduced pressure. The crude product was purified by FLC (DCM / MeOH = 50 / 1) and resulting material triturated with EA / cHex yielding 23.0 mg (0.05 mmol, 39 %) of desired oxazole NBM3 as a white solid compound.Novelty:4-[3-(1H-pyrazol-3-yl)phenyl]-N-[5-(ethylsulfonyl)-2-methoxyphenyl]oxazol-2amine (NBM3) is not described in the literature. M.p.: 88.0 -114.0 °C (dec) [DCM / MeOH].

  50.0 mg (0.11 mmol, 1.00 mol eq) of oxazole 10, 32.0 mg (0.16 mmol, 1.45 mol eq) of pinacol boronate 23 and 12.7 mg (0.01 mmol, 0.10 mol eq) of Pd(PPh3)4 in 2 mL of DMF (abs) was bubbled by Ar for 10 min. A solution of 23.3 mg (0.22 mmol, 2.00 mol eq) Na2CO3 in 1 mL of H2O was bubbled by Ar for 10 min and added to the reaction mixture. Resulting mixture was sealed and stirred overnight at 100 °C. After that TLC analysis confirmed a presence of a new product with traces of the starting compound 10. The reaction was cooled down, diluted with 10 mL of EA and washed with brine (3 x 15 mL). The separated organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by FLC (DCM / MeOH = 30 / 1), resulting material triturated with Et2O / cHex and dried under reduced pressure yielding 15.0 mg (0.04 mmol, 31 %) of oxazole NBM5 as a white solid compound.Novelty:4-[3-(1H-pyrazol-4-yl)phenyl]-N-[5-(ethylsulfonyl)-2-methoxyphenyl]oxazol-2amine(NBM5) is not described in the literature. M.p.: 209 -222 °C (dec) [DCM / MeOH]. 1H, J(A4,A6) = 2.1 Hz, H-CA(6)), 8.27 (s, 1H, H-CB(5)), 8.22 and 7.99 (2 x br, 2 x 1H, H-CD(3 and 5)), 8.07 (dd, 1H, J(C2,C4) = J(C2,C6) = 1.4 Hz, H-CC(2)), 2 x 7.57 (2 x dd, 2 x 1H, J(C4,C5) = J(C5,C6) = 7.7 Hz, J(C2,C4) = (C2,C6) = 1.4 Hz, H-CC(4 and 6)), 7.52 (dd, 1H, J(A3,A4) = 8.5 Hz, J(A4,A6) = 2.1 Hz, H-CA(4)), 7.41 (dd, 1H, J(C4,C5) = J(C5,C6) = 7.7 Hz, H-CC(5)), 7.28 (d, 1H, J(A3,A4) = 8.5 Hz, H-CA(3)), 3.98 (s, 3H, CH3O-), 3.25 (q, 2H, J(CH2,CH3) = 7.4 Hz, CH3CH2SO2-), 1.17 (t, 3H, J(CH2,CH3) = 7.4 Hz, CH3CH2SO2-).

  (solid, cm -1 ) 3350 (m), 2962 (w), 2112 (w), 1629 (s), 1556 (m), 1488 (w), 1418 (m), 1348 (w), 1304 (m), 1264 (s), 1230 (m), 1179 (w), 1143 (m), 1072 (s), 1039 (m), 1019 (m), 949 (m), 926 (w), 892 (w), 865 (w), 796 (s), 637 (m), 611 (s), 595 (s), 524 (s).MS (ESIm/z): 425.1 (80 %) [M+H]+ , 447.1 (100 %) [M+Na]+ , 463.1 (20 %) [M+K] 100 mg (0.23 mmol, 1.00 mol eq) oxazole 10 and 16.0 mg (22.8 µmol, 0.10 mol eq) of Pd(PPh3)2Cl2 in 4 mL of DMF (abs) was bubbled in by Ar for 10 min. Afterwards 252 mg (0.57 mmol, 2.50 mol eq) of tributylstannne 15 was added to the reaction mixture and stirred at 100 °C in a microwave reactor for 1 h. After that TLC analysis confirmed a presence of a new product without 10 and side products. The reaction was cooled down, diluted with 50 mL of brine and resulting mixture was extracted with EA (3 x 15 mL). Combined organic layer was washed with brine (4 x 15 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. Obtained oily product was diluted in CH3CN, extracted with n-hexane (3 x 15 mL) and concentrated with RVE. The crude product was purified by FLC (SiO2, from 100 % of EA to EA / MeOH : 20 / 1) yielding 80.2 mg (0.16 mmol, 69 %) of desired oxazole 25 as an orange solid compound.Novelty: N-[5-(ethylsulfonyl)-2-methoxyphenyl]-4-{3-[1-(tetrahydro-2H-pyran-2-yl)-1H-imidazol-5-yl]phenyl}oxazol-2-amine (25) is not described in the literature. of 60.0 mg (0.20 mmol, 1.00 mol eq) oxazole 25 in 20 mL of CHCl3, 2.00 mL (4.00 mmol, 20 mol eq) of HCl (2M in Et2O) was added and the mixture stirred at 50 °C overnight. Resulting imidazolium salt (white precipitate) was filtered off, obtained solid triturated with a mixture of cHex / EA and subsequently dissolved in 50 mL of H2O. After addition of 50 mL of NaHCO3 (sat aq solution) the mixture was extracted with EA (3 x 20 mL).Combined organic layer was extracted with saturated aq solution of NaHCO3 (3 x 15 mL) and brine (3 x 15 mL), dried over Na2SO4, filtered and concentrated under reduced pressure yielding 32.0 mg (0.08 mmol, 64 %) of oxazole NBM7 as a yellow-white powder.Novelty:4-[3-(1H-imidazol-5-yl)phenyl]-N-[5-(ethylsulfonyl)-2-methoxyphenyl]oxazol-2amine (NBM7) is not described in the literature.

Chapter 7 .Figure 45 .Figure 46 .

 74546 Figure 45. The structures of the most active p-substituted VEGFR2 TK inhibitors 60-62 with their position and proposed interactions within the SBCP pocket of VEGFR2 TK (PDB: 1Y6A). IC50 activities in upper row are from lit. and in the last row are measured by us in ProQinase.

Figure 47 . 1 )Figure 48 .

 47148 Figure 47. A synergic effect of disubstituted aminooxazole 63 on VEGFR2 TK inhibitory activity.

Figure 49 .

 49 Figure 49. Predicted interacting poses of some three-point interacting VEGFR2 TK inhibitors BM(8 and 11-14).

Figure 50 .

 50 Figure 50. Structures of N,5-disubstituted oxazole-2-amines BM(8 and 11-16) and their regioisomers NBM(8 and 11-16).

10 .

 10 Scheme 47. Unsuccessful preparation of N-regioisomeric intermediate 69 in comparison to the synthesis of less substituted compound 10.

hydroxyethanone compounds 8

 8 Scheme 48. A proposed mechanism of a dimer 68 formation.

10. 5 .

 5 Evaluation of biological activityIn this project we have prepared proposed SBCP interacting inhibitors BM(8 and 11-15), containing pyrrole-3-yl, pyrazole-3-yl and imidazole-4-yl moieties. (Figure53) Synthesis of inhibitor BM(16) is still in progress. Pyrrole inhibitor BM8 with p-cyano group exhibited nanomolar IC50 activity. Meanwhile we also determined an inhibitory activity of N-regioisomer NBM1, containing pyrrole-3-yl heterocycle in meta position (described in the Project 1). It exhibited > 100 µM IC50 activity in comparison to its regioisomeric pair BM1 possessing nanomolar IC50 activity. This unexpected results did not correspond with our aim to find the most appropriate heterocycle for both N-and O-regioisomers and planned synthesis of two pyrrole derivatives BM(9-10) was discontinued. Inhibitory activities of derivatives BM(11-15) will be determined.

Figure 53 .

 53 Figure 53. Structures of prepared three-point SBCP interacting inhibitors BM(8 and 11-14) and other planned BM(15-16) derivatives.

8. 4

 4 Hz, J(2,6) = 2.0 Hz, H-C(6)), 6.75 (d, 1H, J(5,6) = 8.4 Hz, H-C(5)), 4.59 (br s, 2H, -NH2), 2.50 (s, 3H, -COCH3).

  The crude product was purified by trituration with Hex / EA yielding 236 mg (0.78 mmol, 70 %) of benzonitrile 34 as a red solid material. Novelty: 2-bromo-4-(2-bromoacetyl)benzonitrile (34) is not described in the literature. M.p.: 93.0 -95.0 °C [Hex / EA].

1 H

 1 -NMR (600 MHz, CDCl3): δ 8.27 (d, 1H, J(3,5) = 1.5 Hz, H-C(3)), 8.00 (dd, 1H, J(5,6) = 8.1 Hz, J(3,5) = 1.5 Hz, H-C(

FT

  IR (solid, cm -1 ): 3085 (w), 2943 (w), 2230 (w), 2083 (w), 1806 (w), 1709 (s), 1590 (w), 1546 (w), 1388 (s), 1274 (m), 1185 (s), 1141 (m), 1043 (m), 1015 (m), 927 (w), 887 (w), 832 (s), 750 (w), 687 (m), 668 (s), 600 (m), 549 (m), 460 (m).

  of benzonitrile 35 as a red solid compound. Novelty: 4-(2-azidoacetyl)-2-bromobenzonitrile (35) is not described in the literature. M.p.: 89.0 -101.0 °C [Hex / EA] (dec).

1 H

 1 -NMR (600 MHz, CDCl3): δ 8.20 (d, 1H, J(3,5) = 1.5 Hz, H-C(3)), 7.92 (dd, 1H, J(5,6) = 8.1 Hz, J(3,5) = 1.5 Hz, H-C(5)), 7.81 (d, 1H, J(5,6) = 8.1 Hz, H-C(6)), 4.55 (s, 2H, -COCH2N3).

  13 C-NMR (150 MHz, CDCl3): δ 191.3 (-COCH2N3), 138.3 (C(

  -1 ): 3378 (w), 2962 (w), 2906 (w), 2231 (w), 2203 (w), 2101 (s), 2007 (w), 1793 (w), 1702 (s), 1595 (w), 1548 (w), 1477 (w), 1388 (m), 1344 (w), 1267 (m), 1231 (m), 1199 (s), 1042 (m), 1023 (m), 982 (w), 919 (m), 828 (m), 771 (w), 674 (m), 655 (m), 603 (m), 555 (m), 524 (w), 453 (w). MS (ESI m/z): 234.9 (60 % for Br 79 ) [M-N2-e -] -, 236.9 (60 % for Br 81 ) [M-N2-e -] -(unstable compound; negatie mode). Anal. calcd for C9H5BrN4O (265.07): C, 40.78; H, 1.90; N, 21.14; found: 40.65; H, 1

  of aminooxazole 36 as a red solid material. Novelty: 2-bromo-4-{2-[5-(ethylsulfonyl)-2-methoxyphenylamino]oxazol-5-yl}benzonitrile (36) is not described in the literature. M.p.: 253.0 -256.0 °C (dec.) [Hex / EA].

FT

  IR (solid, cm -1 ): 2227 (w), 1602 (m), 1572 (s), 1520 (w), 1482 (w), 1424 (w), 1306 (m), 1264 (m), 1141 (m), 1122 (s), 1084 (m), 1016 (m), 887 (w), 825 (m), 795 (m), 719 (s), 640 (w), 575 (w), 552 (w), 525 (m), 478 (m), 422 (m), 411 (m). MS (ESI m/z): 462.1 (100 % for Br 79 ) [M+H] + , 464.1 (98 % for Br 81 ) [M+H] + (positive mode).

  120 mg (0.26 mmol, 1.00 mol eq) oxazole 36, 44.0 mg (0.39 mmol, 1.50 mol eq) of boronic acid 21 and 30.0 mg (0.03 mmol, 0.10 mol eq) of Pd(PPh3)4 in 4 mL of DMF (abs) was purged by Ar for 10 min. A solution of 55.0 mg (0.52 mmol, 2.00 mol eq) Na2CO3 in 1 mL of H2O was purged by Ar for 10 min, subsequently added to the reaction mixture and stirred and irradiated at 100 °C in a microwave reactor for 1 h. Then TLC analysis confirmed a presence of a new product without 36 and some traces of two side products. The reaction was cooled down, diluted with 20 mL of H2O and 5 mL of 2 M HCl (aq) was added. Resulting mixture was extracted with EA (3 x 15 mL) and organic layer washed with brine (3 x 15 mL).The separated organic layer dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by FLC (DCM / MeOH = 50 / 1) yielding 63.0 mg (0.14 mmol, 52 %) of desired oxazole BM11 as a gray solid compound.Novelty: 4-{2-{[5-(ethylsulfonyl)-2-methoxyphenyl]amino}oxazol-4-yl}-2-(1H-pyrazol-3yl)benzonitrile (BM11) is not described in the literature. M.p.: 238.5 -242.5 °C (dec) [DCM / MeOH].

  (solid, cm -1 ): 3415 (w), 3285 (w), 2921 (w), 2215.1 (m), 1735 (w), 1601 (m), 1574 (s), 1503 (m), 1432 (m), 1299 (m), 1263 (s), 1185 (w), 1143 (s), 1123 (s), 1025 (m), 922 (m), 877 (m), 826 (m), 776 (s), 716 (s), 598 (m), 525 (m), 491 (s), 450 (m). MS (ESI m/z): 448.1 (100 %) [M-H] + (negative mode).

  50.0 mg (0.11 mmol, 1.00 mol eq) oxazole BM9 in 2 mL of CF3COOH (TFA) (conc) and 0.5 mL of H2SO4 (conc) was stirred at 70 °C for 3 h. Then TLC analysis confirmed a presence of a new product without BM9 and some traces of side products. The reaction was cooled down, 100 mL of H2O added and extracted with DCM (3 x 15 mL). Combined organic layer was extracted with saturated solution of NaHCO3 (3 x 15 mL) and brine (2 x 15 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was triturated with cHex / EA to provide 32.0 mg (0.07 mmol, 62 %) of desired oxazole BM12 as a white solid compound.Novelty:4-(2-((5-(ethylsulfonyl)-2-methoxyphenyl)amino]oxazol-4-yl}-2-(1H-pyrazol-3yl)benzamide (BM12) is not described in the literature. 50.0 mg (0.11 mmol, 1.00 mol eq) oxazole BM9 in 2 mL of H2O and 1 mL of H2SO4 (conc) was stirred at 100 °C overnight. Then TLC analysis confirmed a presence of a new product with traces of the amidic intermediate BM12. (also present after prolonged reaction time) The reaction was cooled down, 100 mL of H2O was added, extracted with DCM (3 x 15 mL) and a combined organic layer washed with brine (3 x 15 mL). The crude product was purified by FLC (MeOH / EA = 1 / 10) yielding 30.0 mg (0.06 mmol, 58 %) of carboxylic acid BM13 as a white solid compound. Novelty: 4-{2-[(5-(ethylsulfonyl)-2-methoxyphenyl)amino]oxazol-4-yl}-2-(1H-pyrazol-3yl)benzoic acid (BM13) is not described in the literature. M.p.: 200 -270 °C (dec) [MeOH / EA]. 1 H-NMR (600 MHz, DMSO-d6): δ 2 x 15.10 (2 x br s, 2 x 1H, -COOH and H-ND(1)), 9.78 (br s, 1H, -NH-), 8.79 (d, 1H, J(A4,A6) = 2.2 Hz, H-CA(6)), 7.88 (d, 1H, J(C3,C5) = 1.4 Hz, H-70.0 mg (0.15 mmol, 1.00 mol eq) oxazole 36 and 10.5 mg (14.9 µmol, 0.10 mol eq) of Pd(PPh3)2Cl2 in 3 mL of DMF (abs) was purged by Ar for 10 min. Afterwards 165 mg (0.37 mmol, 2.50 mol eq) of imidazole 15 was added to the reaction and the mixture stirred and irradiated at 100 °C in a microwave reactor for 1 h. Then TLC analysis confirmed a presence of a new product without 36 together with some traces of side products. The reaction was cooled down, diluted with 20 mL of EA and resulting mixture extracted with EA (3 x 15 mL). Separated organic layer was washed with brine (4 x 15 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. Obtained oily product was diluted in CH3CN, extracted with n-hexane (3 x 15 mL) and separated CH3CN layer evaporated with RVE. The crude product 37 was purified by FLC (EA / cHex: gradient from 2 / 1 to 100 % of EA) to yield 65.0 mg (0.12 mmol, 80 %) of desired oxazole 37 as an orange solid compound. Novelty: 4-{2-{[5-(ethylsulfonyl)-2-methoxyphenyl]amino}oxazol-5-yl}-2-[1-(tetrahydro-2H-pyran-2-yl)-1H-imidazol-5-yl]benzonitrile (37) is not described in the literature. M.p.: 144.0 -148.0 [EA].

  (solid, cm -1 ): 2942 (m), 2221 (w), 1603 (-CN, m), 1571 (s), 1487 (w), 1422 (w), 1344 (w), 1307 (w), 1263 (m), 1229 (w), 1207 (w), 1142 (m), 1122 (s), 1082 (m), 1042 (m), 1018 (w), 910 (m), 823 (m), 794 (m), 720 (s), 657 (m), 626 (w), 598 (w), 553 (w), 526 (m), 491 (m). MS (ESI m/z): 450.11 (100 %) [M-THP+2H] + , 534.13 (10 %) [M+H] + (positive mode).

  : 4-{2-{[5-(ethylsulfonyl)-2-methoxyphenyl]amino}oxazol-5-yl}-2-(1H-imidazol-5yl)benzonitrile (BM14) is not described in the literature. M.p.: 255.0 -257.5 [DCM].

.

  MS(ESI m/z): 450.07 (100 %) [M+H] + (positive mode).Anal. calcd for C22H19N5O4S (449.49):C, 58.79; H, 4.26; N, 15.58; Found: C, 58.84; H, 440.0 mg (0.08 mmol, 1.00 mol eq) oxazole 37 in 1 mL of CF3COOH (TFA) (conc) and 0.25 mL of H2SO4 (conc) was stirred at 70 °C for 4 h. Then TLC analysis confirmed a presence of a new product without 37 and some traces of side products. The reaction was cooled down, 100 mL of H2O added and extracted with DCM (3 x 15 mL). Combined organic layer was extracted with saturated solution of NaHCO3 (3 x 15 mL) and brine (2x 15 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was triturated with MeOH / DMSO to provide 19.0 mg (0.07 mmol, 55 %) of desired oxazole BM15 as a white solid compound. ethylsulfonyl)-2-methoxyphenyl)amino)oxazol-5-yl)-2-(1Himidazol-5-yl)benzamide (BM15) is not described in the literature. M.p.: 227 -245 °C (dec) [DCM].

1 H

 1 -NMR (400 MHz, CDCl3): δ 8.22 (d, 1H, J(3,5) = 1.6 Hz, H-C(3)), 7.94 (dd, 1H, J(5,6) = 8.1 Hz, J(3,5) = 1.6 Hz, H-C(5)), 7.82 (d, 1H, J(5,6) = 8.1 Hz, H-C(6)), 4.89 (s, 2H, -COCH2OH), 3.29 (br s, 1H, -OH).

13 C

 13 -NMR (100 MHz, CDCl3): δ 196.4 (-COCH2OH), 137.

  (solid, cm -1 ): 3256 (m), 2988 (w), 2230 (w, -CN), 1702 (s), 1550 (m), 1373 (m), 1271 (m), 1224 (s), 1099 (m), 1042 (m), 985 (s), 892 (w), 844 (s), 761 (m), 675 (m), 608 (m). MS (ESI m/z): 238.1 (100 % for Br 79 ) [M-H] -, 240.1 (100 % for Br 81 ) [M-H] -(negative mode).

1 H

 1 -NMR (400 MHz, CDCl3): δ 8.26 (d, 1H, J(3,5) = 1.6 Hz, H-C(3)), 7.99 (dd, 1H, J(5,6) = 8.2 Hz, J(3,5) = 1.6 Hz, H-C(5)), 7.82 (d, 1H, J(5,6) = 8.2 Hz, H-C(6)), 4.68 (s, 2H, -COCH2Z).

  (solid, cm -1 ): 3086 (w), 2230 (m), 2230 (w, -CN), 1713 (s), 1547(m), 1393 (m), 1311 (w), 1279 (w), 1269 (w), 1195 (s, -S=O), 1142 (m), 1043 (w), 1020 (w), 904 (w), 838 (s), 794 (s), 722 (s), 672 (s), 604 (w), 582 (w), 554 (m), 460 (m).

Scheme 51 .

 51 Scheme 51. An overview of metabolism of the D-glucose.

Figure 54 .

 54 Figure 54. An illustration of the polyol pathway and its implication in the diabetic complications.

Figure 55 .

 55 Figure 55. Transport du glucose à travers une membrane cellulaire médiée par l'insuline.

Figure 56 .

 56 Figure 56. The structure of the active site of ALR2 enzyme.

Figure 57 .

 57 Figure 57. Structure and activity of the epalrestat.

Figure 58 .

 58 Figure 58. Structures of the most developed ARIs possessing carboxylic functionality.

Figure 59 .

 59 Figure 59. Structures and activities of the most important ALR2 spirohydantoin inhibitors.

Figure 61 .

 61 Figure 61. Structure, activity and selectivity of cemtirestat (CMTI).

Figure 62 .Figure 63 .

 6263 Figure 62. The crystal structure of CMTI in a complex with ALR2. a) Two molecules of CMTI are located in the complex. b) Binding mode of the deep buried molecule of CMTI in the ALR2 complex.

12. 1 .

 1 Scheme 52. The synthesis of 5H-triazino[5,6-b]indole-3-thiones T94 from thiosemicarbazone T93.

  Scheme 53. Synthesis of the 5H-triazino[5,6-b]indole-3-thione T96 from thiosemicarbazone T95.

Scheme 57 .Figure 64 .Figure 65 . 1 . 2 . 1 . 13 . 3 .Figure 67 .

 57646512113367 Scheme 57. Formation of 1,2,4-triazine-3(2H)-thione T109 and T111 from aceanthrylene-1,2-dione T108 or substituted thiosemicarbazone T110.

Figure 68 .

 68 Figure 68. Comparison of determined inhibitory activity and selectivity of CMTI and OTI.

  131

  Scheme 64. Synthesis of triazinoindol ALR2 inhibitor cemtirestat (CMTI).

FT

  -IR (solid, cm -1 ): 3491 (w), 3377 (w, NH), 3018 (m, NH2), 1699 (s, C=O), 1609 (s, C=O), 1582 (m), 1542 (m), 1465 (m), 1311 (m), 1246 (m), 1154 (m), 1040 (w), 848 (m), 751 (s), 639 (m), 552 (m), 431 (s). MS (ESI m/z): 202.9 (100 %) [M-H] -(negative mode).

  indol-3(5H)-one (43) as a yellow solid product.

  of diester 45 as an orange crystalline compound. Novelty: ethyl 2-(3-oxo-2H-[1,2,4]triazino[5,6-b]indol-5(3H)-yl)acetate (44) is not described in the literature. M.p.: 248.0 -252.0 °C [MeOH / DCM]. = 1.0 Hz, H-C(

  (solid, cm -1 ): 3151 (w), 3088 (w), 2980 (w), 2857 (m), 1733 (s), 1656 (s), 1595 (s), 1497 (s), 1408 (s), 1221 (s), 1171 (m), 1061 (m), 1012 (m), 965 (w), 919 (w), 788 (m), 746 (m), 659 (m), 604 (m), 575 (m), 486 (s). MS (ESI m/z): 271.0 [M-H] -(negative mode).Anal. calcd for C13H12N4O3 (272.26):C, 57.35; H, 4.44; N, 20.58; Found: C, 57.13; H, 4diethyl 2,2,4]triazino[5,6-b]indole-2,5(3H)-diyl)diacetate (45) is not described in the literature.

M

  .p.: 288.1 -290.0 °C [H2O / H + ].

1 H

 1 -NMR (300 MHz, DMSO-d6): δ 13.39 (very br s, 1H, -COOH), 13.20 (br s, 1H, H-N(2)),7.99 (dd, 1H, J(8,9) = 7.3 Hz, J(7,9) = 1.0 Hz, H-C(9)), 7.64 (ddd, 1H, J(7,8) = 8.0 Hz, J(6,7) = 7.6 Hz, J(7,9) = 1.0 Hz, H-C(7)), 7.59 (dd, 1H, J(6,7) = 7.6 Hz, J(6,8) = 1.0 Hz, H-C(6)),7.38 (ddd, 1H, J(7,8) = 8.0 Hz, J(8,9) = 7.3 Hz, J(6,8) = 1.0 Hz, H-C(8)), 4.94 (s, 2H, -CH2COOH).

13 C

 13 -NMR (75 MHz, DMSO-d6): δ 169.2 (-COOH), 154.9 (-NHCO-), 154.5 (C4a), 144.4 (C5a), 132.7 (C1a), 131.2 (C7), 123.5 (C8), 121.3 (C9), 118.0 (C9a), 111.6 (C6), 42.5 (-CH2-). MS (ESI m/z): 242.9 (75 %) [M-H] -, 198.9 (100 %) [M-CO2-H + ] -(negative mode). Anal. calcd for C11H8N4O3 (244.21): C, 54.10; H, 3.30; N, 22.94; Found: C, 54.42; H, 3of 200 mg (0.56 mmol, 1.00 mol eq) diester 45 in 20 mL of MeOH, 78.0 mg (1.95 mmol, 3.50 mol eq) of NaOH in 5 mL water was added and the mixture refluxed for 1.5 h. After consumption of starting material 45 (confirmed by TLC analysis) the reaction was cooled down in an ice bath and acidified with 1 M aq HCl to pH = 4. After 10 min of stirring, formation of precipitate was observed and the mixture was stirred for another 20 min. Then the precipitate was filtered off and dried under reduce pressure to yield 106 mg (0.35 mmol, 63 %) of diacid OTI-1 as a yellow solid product. Novelty: 2,2'-(3-oxo-2H-[1,2,4]triazino[5,6-b]indole-2,5(3H)-diyl)diacetic acid (OTI-1) is not described in the literature. M.p.: 296.0 -299.0 °C [MeOH].

Novelty: 2 -

 2 (2-(methoxymethyl)-3-oxo-2H-[1,2,4]triazino[5,6-b]indol-5(3H)-yl)acetic acid (OTI-2) was not yet described in the literature. M.p.: 225 -270 °C (dec) [MeOH].

Novelty: 2 -

 2 (2-benzyl-3-oxo-2H-[1,2,4]triazino[5,6-b]indol-5(3H)-yl)acetic acid (OTI-3) is described in the literature. M.p.: 269..

  of methyl 2-(3-oxo-2-pivaloyl-2,3-dihydro-5H-[1,2,4]triazino[5,6-b]indol-5yl)acetate (49)

Figure 70 .

 70 Figure 70. A predicted pose of the inhibitor OTI-7 in the active site of ALR2 (PDB: 4QX4) and a diagram of its intermolecular interactions.

  245 mg(1.22 mmol, 1.00 mol eq) of arylmethanol 52 in 20 mL of DCM (abs), 130 µL(1.42 mmol, 1.16 mol eq) of 3,4-dihydro-2H-pyran (DHP) and 30.6 mg (0.12 mmol, 0.10 mol eq) of PPTS (pyridinium p-toulenesulfonate), was stirred under Ar for 30 min at reflux. Subsequent TLC analysis confirmed presence of a new product and traces of two side products. Then, 20 mL of NaHCO3 (sat aq sol.) was added and resulting mixture extracted with DCM (3 x 15 mL). Combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure yielding 310 mg(1.09 mmol, 89 %) of tetrahydropyrane 53 as a colourless oily product. Novelty: 2-{[2-(bromomethyl)benzyl]oxy}tetrahydro-2H-pyran (53) is described in the literature by its 1 H-NMR and crystal property description. 141 141 Novartis AG; Novartis Pharma GMBH; Masuya, Keiichi -WO2006/74924, 2006, A1.

  , J(3,5 or 4,6) = 1.9 Hz, H-C(4 and 5)), 4.94 (d, 1H, Jgem =12.3 Hz, -CH2OTHP), 4.74 (dd, 1H, J(A2,A3) = 8.5 Hz, J(A2,A3) = 3.0 Hz, H-CA(2)), 4.68 (d, 1H, Jgem = 10.2 Hz, -CH2Br), 4.65 (d, 1H, Jgem = 12.3 Hz, -CH2OTHP), 4.63 (d, 1H, Jgem = 10.2 Hz, -CH2Br), 3.94 (ddd, 1H, Jgem = 11.1 Hz, J(A5,A6) = 8.5 Hz, J(A5,A6) = 3.0 Hz, H-CA(6)), 3.59 (m, 1H, H-CA(6)), 1.92 -1.52 (m, 6H, H-CA(3-5)).

FT

  2939 (m), 2867 (w), 1723 (w), 1454 (m), 1385 (m),1260 (w), 1200 (m), 1118 (s), 1076 (m), 1022 (s), 971 (m), 904 (m), 869 (m), 814 (w), 760 (m), 606 (m), 533 (w) Due to moisture sensitivity of the product 53, MS spectrum and elemental analysis were not measured.

  142

M

  .p.: 247.0 -253.0 °C [DCM], lit.: 248.0 -252.0 °C [MeOH / DCM]. 142 142 Hlavac, M.; Kovacikova, L.; Prnova, M.S.; Addova, G.; Majekova, M.; Hanquet, G.; Bohac, A.; Stefek, M.J.Med. Chem., 2020, 63, 369 -381. of 200.0 mg (0.74 mmol, 1.00 mol eq) 44 in 5 mL DMF (abs), 37.0 mg (0.88 mmol, 1.19 mol eq) of CaH2 was added and the mixture stirred at 80 °C under Ar within 30 min. Then, the reaction was cooled to rt and 310 mg (1.09 mmol, 1.47 mol eq) of tetrahydropyrane 53 was added dropwise and the solution was stirred at 80 °C within 1 h under Ar. After consumption of 44 (confirmed by TLC analysis) the reaction was cooled down, 10 mL of water added and then the mixture stirred for 10 min in an ice bath. Resulting mixture was extracted with DCM (3 x 15 mL), washed with brine (3 x 20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. A crude product was purified by FLC (SiO2, Hex / EA = 1 / 5) to give 286 mg (0.60 mmol, 82 %) of 54 as red oil.Novelty: ethyl 2-{3-oxo-2-{2-{[(tetrahydro-2H-pyran-2-yl)oxy]methyl}benzyl}-2,3-dihydro-

  Scheme 71. Synthesis of benzyl analogues OTI-(7-9).

Chapter 12 .

 12 Scheme 74. New proposed synthesis of aminooxazole 69 by using formamide.

Figure 71 .Figure 72 .

 7172 Figure 71. Structures of proposed analogues OTI-(10-12) with their interaction diagrams.

Figure 73 .

 73 Figure 73. Structures and interaction diagrams of proposed polycyclic derivatives OTI-16 and OTI-17.

  L'aldose réductase (ALR2) est l'une des premières enzymes d'une voie polyol et appartient à la superfamille des enzymes aldo-céto réductases. Elle catalyse la réduction dépendante du NADPH d'une grande variété de composés carbonylés, par ex. le glucose. L'enzyme est un domaine polypeptidique unique composé de 315 résidus d'acides aminés repliés en un motif structurel en tonneau β / α contenant huit brins β parallèles. Le site actif est situé dans une grande cavité à l'extrémité C-terminale du tonneau β et le cofacteur NADPH est situé au sommet du tonneau β / α avec l'anneau nicotinamide se détachant au centre du canon.

3.6.2. Spirohydantoïne et dérivés du spirosuccinimide

  La deuxième classe importante d'inhibiteurs d'ALR2 comprend les dérivés de la spirohydantoïne, où le groupe hydantoïne occupe la poche de liaison anionique de la même manière que le groupe anionique des dérivés carboxyliques. Le sorbinil, un puissant inhibiteur de l'ALR2 avec une sous-structure chromane, s'est avéré un excellent inhibiteur de l'ALR2 pour les tests in vivo et in vitro. Malheureusement, les essais cliniques ont montré que le sorbinil augmente le risque de développer une hypersensibilité telle que des éruptions cutanées, de la fièvre et des myalgies (douleurs musculaires). Ces effets indésirables peuvent être attribués à la faible sélectivité du sorbinil envers l'aldéhyde réductase (ALR1). Sur la base de la structure du sorbinil, plusieurs analogues de la spirohydantoïne ont été développés. L'addition d'un

groupe carbamoyle ou méthyle au niveau du squelette du sorbinil a donné le fidarestat et le M79175, respectivement. Le remplacement de l'anneau chromane par l'isoquinoline-1,3 (2H, 4H)-dione et l'introduction de l'anneau spirosuccinimide conduisent à la synthèse du minalrestat. Un autre inhibiteur similaire nommé ranirestat, a été développé pour le traitement de la neuropathie diabétique et est maintenant en phase 3 des essais cliniques au Japon.

. Inhibitors of VEGFR2 TK

	6.2. Inhibitors of VEGF (PDGF-Rs), CLKs and VEGFR receptors which play a role of both tumor angiogenesis and cell
	proliferation. Therefore, sunitinib was the first cancer drug simultaneously approved for two
	Bevacizumab (trade name: Avastin) became the first angiogenesis inhibitor as a humanized different indications. 21 The main side effects are hand-foot syndrome (redness and pain on the
	recombinant monoclonal antibody. It binds to VEGF-A and blocks angiogenesis. Bevacizumab hands and / or feet) and stomatitis (inflammation of the mouth and lips). 22 Sorafenib (marketed
	was first approved by FDA USA in 2004 for a treatment of colorectal cancer and later for other as Nexavar by Bayer) is an inhibitor of several tyrosin kinases such as PDGFR, VEGFR and serious types of cancers such as lung, breast and brain tumor. 14 The main side effects of Raf family kinases (C-Raf, B-Raf). 23 In 2005 it was approved by FDA USA for the treatment
	bevacizumab are hypertension, nose bleeding, headache and rash. Aflibercept (trade names: of kindey cancer (advanced renal cell carcinoma), later in 2007 for hepatocellular carcinomas
	Eylea and Zaltrap) is a medication primarily used to treat metastatic colorectal cancer and wet (HCC) and in 2013 for the treatment of thyroid carcinoma. Very effective and selective
	macular degeneration (a chronic eye disorder). It is a recombinant protein, that acts as a decoy VEGFR2 TK inhibitor is apatinib, which was approved in 2014 for patient with late-stage receptor with characteristic high affinity toward VEGF and placental growth factor (PIGF). 15,16 gastric carcinoma in China. 24 Other active VEGFR2 TK inhibitors are lenvatinib, vandetanib
	In 2014 aflibercept was indicated for the treatment of patient with visual diseases and in 2019 was approved by FDA for the treatment of diabetic retinopathy. 17 Ramucirumab (trade name: and cediranib. (Figure. 30)
	Cyramza) is a human monoclonal antibody, that binds to the extracelullar domain of VEGFR2
	and blocks the binding of natural growth factors (VEGF-A, VEGF-C and VEGF-D). 18 It was
	approved by FDA for treatment of advanced gastric cancer, non-small-cell lung carcinoma
	(NSCLC), colorectal cancer and in 2019 for hepatocellular carcinoma (HCC). 19
	6.3Imatinib (trade name Gleevec) is a competitive TK inhibitor used for treatment of acute
	lymphoblastic leukemia (FDA in 2013), rare gastrointestinal cancer (FDA in 2012) and
	gastrointestinal stromal tumor (GIST) (FDA in 2002). Imatibit occupies a TK active site of
	fused proteins bcr and abl (termed bcr-abl), which leads to locking of ATP-binding site and
	therefore inhibiting the enzyme activity of the bcr-abl kinase. 20 (Figure. 30) Sunitinib Figure. 30. Structures and activities of developed VEGFR2 tyrosin kinases inhibitors and years of the FDA first
	(marketed as Sutent by Pfitzer) is a multiple receptor tyrosine kinase (RTK) inhibitor which approval.
	was approved by FDA in 2006 for the treatment of renal cell carcinoma (RCC) and
	gastrointestinal stromal tumor (GIST). It inhibits also platelet-derived growth factor receptors
	14 Ferrara, N.; Hillan, K.J.; Novotny, W.; Biochem. Biophys. Res. Commun., 2005, 333, 328 -335.
	15 Pandey, A.K.; Singhi, E.K.; Arroyo, J.P.; Ikizler, T.A.; Gould, E.R.; Brown, J.; Beckman, J.A.;
	Harrison, D.G.; Moslehi, J. Hypertension, 2018, 71, e1 -e8.
	16 https://www.drugbank.ca/drugs/DB08885 (accessed April 15 th , 2020).
	17 https://investor.regeneron.com/news-releases/news-release-details/fda-approves-eylear-aflibercept-injection-
	diabetic-retinopathy (accessed April 15 th , 2020).
	18 Singh, A.D.; Parmar, S. P. T., 2015, 40, 430 -434. 21 www.fda.gov (accessed April 25 th , 2020)
	19 https://www.fda.gov/drugs/resources-information-approved-drugs/fda-approves-ramucirumab-hepatocellular-22 Gan, H.L.; Seruga, B.; Knox, J.J. Expert. Opin. Investig. Drugs, 2009, 18, 821 -834.
	11 Plate, K.H.; Breier, G.; Millauer, B.; Ullrich, A.; Risau, W. Cancer Res., 1993, 53, 5822 -5827. carcinoma (accessed April 15 th , 2020). 23 Smalley, K.S.; Xiao, M.; Villanueva, J.; Nguyen, T.K.; Flaherty, K.T.; Letrero, R.; Van Belle, P.; Elder, D.E.;
	12 Folkman, J.; N. Engl. J. Med., 1971, 285, 1182 -1186. 20 Pazdur, R.; Wagman, D.L.; Camphausen, K.A.; Hoskins, W.J Cancer Managment: A Multidisciplinary Wang, Y.; Nathanson, K.L.; Herlyn, M. Oncogene, 2009, 28, 85 -94.
	13 Chau, C.H.; Figg, W.D. Clin. Cancer Res., 2012, 18, 4868 -4871. Approach (11 th ed.), CMP United Business Media, USA, 2009, ISBN 9781891483622 24 www.cancer.gov (accessed April 25 th , 2020)

Autors described two conditions: overnight at room temperature in CH2Cl2 and 30 min heating at 90 °C in dioxane. (Scheme 5)

	Scheme 5. Synthesis of N,5-diaryloxazole-2-amines T47.
	27 Rauf, A.; Farshori, N.N. Microwave-induced Synthesis of Aromatic Heterocycles 2012 Springer, Dordrecht,
	Heidelberg, London, New York.
	28 Harris, A.P.; Cheung, M.; Hunter III, N.R.; Brown, L.M.; Veal, M.J.; Nolte, T.R.; Wang, L.; Liu, W.; Crosby.
	M.R.; Johnson, H.J.; Epperly, H.A.; Kumar, R.; Luttrel, K.D.; Stafford J. A. J. Med. Chem. 2005, 48, 1610 -
	1619.

  -2-hydroxyphenyl]acetamide(2) is not described in the literature.

	Synthesis of N-[5-(ethylsulfonyl)-2-methoxyphenyl]acetamide (3) 3.95 (s, 3H, CH3O-), 3.19 (q, 2H, J(CH2,CH3) = 7.4 Hz, CH3CH2SO2-), 2.13 (s, 3H, CH3CO-),
	1.09 (t, 3H, J(CH2,CH3) = 7.4 Hz, CH3CH2SO2-).
	13 C-NMR (100 MHz, DMSO-d6): δ 169.6 (CH3CO-), 153.3 C(2), 130.2, 128.4, 124.9, 120.9, Novelty: 5-(ethylsulfonyl)-2-methoxyaniline (4) is described in the literature by its M.p., 1 H-
	111.8, 56.8, 50.2, 24.4 (CH3CO-), 7.8 (CH3CH2SO2-). NMR, 13 C-NMR, , IR, MS and elemental analysis. 72
	M.p.: 96.0 -98.9 [EA] (lit.: 102.9 -103.8 [MeOH]). 72
	FT-IR (solid, cm -1 ): 3405 (m), 3082 (w), 2985 (w), 2927 (w), 1890 (w), 1685 (s), 1592 (s),
	1523 (s), 1468 (s), 1412 (s), 1366 (m), 1340 (w), 1298 (s), 1262 (s), 1241 (s), 1122 (s), 1079
	To a solution of 750 mg (3.09 mmol, 1.00 mol eq) acetamide 2 in 10 mL DMF (abs), 511 mg (s), 1017 (s), 886 (m), 824 (m), 793 (m), 775 (s), 729 (s), 636 (m), 604 (s), 565 (s), 524 (s), 489
	(3.70 mmol, 1.20 mol eq) of K2CO3 was added and the mixture stirred for 5 min at rt under Ar. (s), 441 (m).
	Synthesis of 5-(ethylsulfonyl)-2-methoxyaniline (4)
	Anal. calcd for C10H13NO4S (243.28): C, 49.37; H, 5.39; N, 5.76; Found: C, 49.50; H, 5.48;
	N, 5.82. To a solution of 2.29 g (8.91 mmol, 1.00 mol eq) acetamide 3 in 50 mL of dioxane, 1.57 mL
	(17.8 mmol, 2.00 mol eq) of HCl (conc) was added dropwise by stirring for 3 h at 100 °C. Then
	the reaction mixture was cooled down and volatile parts evaporated by RVO. The obtained

M.p.: 237.9 -240.2 °C

[EA]

.

1 H-NMR (400 MHz, DMSO-d6): δ 11.09 (br s, 1H, -OH), 9.41 (br s, 1H, -NH-),

8.43 (d, 1H, J(4,6)

= 2.

3

Hz, H-C(

6

)),

7.43 (dd, 1H, J(3,4)

= 8.4 Hz, J(4,6)

= 2.

3

Hz, H-C(4)), 7.06 (d, 1H,

J(3,4) = 8.4 Hz, H-C(3)), 3.15 (q, 2H, J(CH2,CH3) = 7.4 Hz, CH3CH2SO2-), 2.13 (s, 3H, CH3CO-), 1.09 (t, 3H, J(CH2,CH3) = 7.4 Hz, CH3CH2SO2-).

13 C-NMR (100 MHz, DMSO-d6): δ 169.7 (CH3CO-), 152.5 C

(2), 128.6, 127.4, 125.0, 121.7, 115.8,

, 24.3 (CH3CO-), 7.9 (CH3CH2SO2-).

FT-IR

(solid, cm -1 ): 3410 (w), 2855 (w), 2611 (w), 2112 (w), 1655 (m), 1581 (m), 1533 (m), 1418 (s), 1370 (m), 1276 (s), 1243 (m), 1143 (m), 1114 (s), 1079 (s), 1023 (m), 922 (w), 825 (w), 784 (m), 736 (s), 645 (m), 609 (s), 577 (m), 529 (s), 489 (s), 456 (m). MS (ESI m/z): 242.0 [M-H] -(negative mode). Afterwards, 231 µl (3.70 mmol, 1.20 mol eq) of MeI was added dropwise over 5 min and the mixture was stirred for another 10 min. Subsequently TLC analysis confirmed a presence of only one new spot. Then 50 mL of brine was added and the mixture extracted with EA (5 x 10 mL). Combined organic layer was dried over Na2SO4, filtered and filtrate concentrated under vacuum to yield 682 mg (2.65 mmol, 86 %) of acetamide 3 in form of a dark solid material. Novelty: N-[5-(ethylsulfonyl)-2-methoxyphenyl]acetamide (3) is not described in the literature. M.p.: 140 -150 °C [EA] (dec).

1 H-NMR (400 MHz, DMSO-d6): δ 9.45 (br s, 1H, -NH-), 8.54 (d, 1H, J(4,6) = 2.3 Hz, H-C(6)), 7.58 (dd, 1H, J(3,4) = 8.4 Hz, J(4,6) = 2.3 Hz, H-C(4)), 7.28 (d, 1H, J(3,4) = 8.4 Hz, H-C(3)), MS (ESI m/z): 256.0 [M-H] -(negative mode).

Anal. calcd for C11H15NO4S (257.30):C, 51.

35; H, 5.88; N, 5.44; Found: C, 51.55; H, 6.15; N, 5.80.

crude product was dissolved in 50 mL of EA and extracted with 1 % solution of NaHCO3 (5 x 15 mL). Combined organic layer was dried over Na2SO4, filtered and the filtrate concentrated under reduced pressure to yield 1.843 g

(8.57 mmol, 96 %)

of aniline 4 in form of a grey solid material. 1 H-NMR (400 MHz, CDCl3): δ 7.19 (dd, 1H, J(3,4) = 8.4 Hz, J(4,6) = 2.3 Hz, H-C(4)), 7.11 (d, 1H, J(4,6) = 2.3 Hz, H-C(6)), 6.80 (d, 1H, J(3,4) = 8.4 Hz, H-C(3)), 4.01 (br s, 2H, -NH2), 3.92 (s, 3H, CH3O-), 3.00 (q, 2H, J(CH2,CH3) = 7.4 Hz, CH3CH2SO2-), 1.18 (t, 3H, J(CH2,CH3) = 7.4 Hz, CH3CH2SO2-).

72 Murár, M.; Addová, G.; Boháč, A. Beilstein J.

Org. Chem., 2013, 9, 173-179.

  After that, TLC analysis confirmed presence of a new product, traces of hydroxyketone 8, starting urea 6 and two side products. Desired oxazole 10 was purified by FLC (SiO2, EA / cHex = 1 / 1) and by crystallization (EA / cHex) to yield 570 mg(1.30 mmol, 67 %) of oxazole

	10.
	Novelty: 4-(3-bromophenyl)-N-[5-(ethylsulfonyl)-2-methoxyphenyl]oxazol-2-amine (10) is
	not yet described in the literature.
	1 H-NMR (400 MHz, CDCl3): δ 8.07 (dd, 1H, J(2,4) = J(2,6) = 1.9 Hz, H-C(2)), 7.84 (ddd, 1H,
	J(5,6) = 7.9 Hz, J(2,6) = 1.9 Hz, J(4,6) = 1.1 Hz, H-C(6)), 7.79 (ddd, 1H, J(4,5) = 7.9 Hz, J(2,4) M.p.: 135.0 -139.1 °C [EA / cHex].
	= 1.9 Hz, J(4,6) = 1.1 Hz. H-C(4)), 7.40 (dd, 1H, J(4,5) = J(5,6) = 7.9 Hz, H-C(5)), 4.86 (s, 2H,
	-COCH2OH), 3.41 (br s, 1H, -OH)
	Synthesis of 4-(3-bromophenyl)-N-[5-(ethylsulfonyl)-2-methoxyphenyl]oxazol-2-amine
	(10)
	1 st step: Acetophenone 8 1.50 g (6.98 mmol, 1.00 mol eq) was dissolved in 50 mL of DCM
	(abs) and cooled to -78 °C. At this temperature 1.62 mL (13.96 mmol, 2.00 mol eq) of 2,6-
	75 lutidine was added with subsequent dropwise addition of 2.35 mL (13.96 mmol, 2.00 mol eq)
	of Tf2O. Reaction mixture was stirred for 2 h while its temperature rose up to rt. Afterwards,
	volatile parts were evaporated with RVE and obtained crude intermediate used in the next step
	without further purification.

7 mmol, 5

.50 mol eq) KOH in 40 mL of MeOH (abs), 3.00 g (15.1 mmol, 1.00 mol eq) of acetophenone 7, dissolved in ca 50 mL of MeOH (abs), was added and cooled to 0 °C. Afterwards, 5.34 g (16.6 mmol, 1.10 mol eq) of PhI(OAc)2 was added portionwise within 10 min and stirred for another 10 min at the same temperature. After 2.5 h of stirring at rt, TLC analysis confirmed presence of a new product with traces of two side products. Reaction mixture was evaporated with RVE, resulting solid material was diluted in 70 mL of EA and 20 mL of H2O and subsequently extracted with H2O (3 x 40 mL). Obtained crude oil was dissolved in 20 mL of MeOH and 15 mL of 2 M HCl (aq) was added. After 30 min of stirring at rt, yellow precipitate was obtained and mixture was stirred for another 30 min in an ice bath. Yellow precipitate was filtered off and washed with cHex to yield 2.34 g

(10.8 mmol, 72 %)

of hydroxyketone 8 as a white solid material.

Novelty: 1-(3-bromophenyl)-2-hydroxyethan-1-one (

8

) is described in literature together with its M.p., 1 H-NMR,

13

C-NMR, IR, HRMS, ESI and LCMS spectra. 75 M.p.: 104.2 -107.1 °C [MeOH], (lit. 104 -106 °C). 2 nd step: Urea 6 500 mg (1.95 mmol, 1.00 mol eq) was dissolved in 40 mL of AN (abs). Previously prepared intermediate (triflate, not shown) was added portionwise every 12 h within 2 days.

1 H-NMR (400 MHz, DMSO-d6): δ 9.78 (br s, 1H, -NH-), 8.90 (d, 1H, J(A4,A6) = 2.3 Hz, H-CA(6)), 8.34 (s, 1H, H-CB(

  Novelty: 2-chlorotetrahydro-2H-pyrane (12) is described in the literature by its 1 H-NMR,13 C-NMR, boiling point, 76 IR, refractive index 77 and density.78

	A mixture of 2.62 g NaH (60 % w/w in min oil) (68.3 mmol, 2.00 mol eq) was dispersed in 20
	3 mmol) of 3,4-dihydro-2H-pyrane (DHP, 11) in a sealed tube at 0 °C for mL of THF (abs) at 0 °C. After that, 2.33 g (34.1 mmol. 1.00 mol eq) of imidazole 13 was
	1 h and another 1 h at room temperature. Resulting dark brown solution was distilled under portionwise added within 15 min and stirred 30 min at the same temperature. Aftewards, 6.16
	reduced pressure (50 °C at 23 mbar) yielding 7.40 g (61.7 mmol, 74 %,) of 2-g (51.3 mmol, 1.50 mol eq) of THP 12, dilluted in 10 mL of THF (abs), was added dropwise to

chlorotetrahydropyrane 12 as colorless liquid. Obtained compound is unstable and was used directly in a next step.

1 H-NMR (400 MHz, CDCl3): 6.30 (dd, 1H, J(2,3ax) = 3.5 Hz, J(2,3eq) = 2.7 Hz, H-C2), 4.06 (ddd, 1H, J(6ax,5ax) = 14.8 Hz, J(6gem) = 11.5 Hz, J(6ax,5eq) = 3.2 Hz, H-C6(ax)), 3.87 (ddd, 1H, J(6gem) = 11.5 Hz, J(6eq,5ax) = 6.5 Hz, J(6eq,5eq) = 3.8 Hz H-C6(eq)), 2.11 -1.60 (m, 6H, H-C

(3- 5)

).

Synthesis of 1-(tetrahydro-2H-pyran-2-yl)-1H-imidazole (14)

76 Meltzer, P.C.; Wang, P.; Blundell, P.; Madras, B.K. J.

Med. Chem., 2003, 46, 1538 -1545. 77

Viola, A.; Collins, J.J.; Filipp, N.; Locke, J.S. J.

Org. Chem., 1993, 58, 5067 -5075. 78

Gazizov, M.B.; Razumov, A.I.; Gizatullina, I.Kh.; Ostanina, L.P. Zh.

Obshch. Khim., 1980, 50, 687.

  -78 °C and 2 h at rt. Afterwards, the reaction was quenched with 20 mL of NH4Cl (sat aq solution), extracted with Et2O (3 x 15 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. Crude oily product was purified by FLC (cHex / EA = 30 / 1) yielding 330 mg (0.95 mmol, 52 %) of pinacol boronate 19 as a colorless oil, which solidified in a refrigerator.

	Novelty: 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-(triisopropylsilyl)-1H-pyrrole (19)
	is described in the literature by its 1 H-NMR, 13 C-NMR, M.P, IR and elemental analysis. 81
	M.p.: 56.0 -59.0 °C [cHex / EA], lit: 59 °C. 81

80 Kotoku, N.; Fuijoka, S.; Nakata, C.; Yamada, M.; Sumii, Y.; Kawachi, T.; Arai, M.; Kobayashi, M.

Tetrahedron, 2011, 67, 6673 -6678. at

  amine (29) is not described in the literature.

	and brine (3 x 15 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to
	yield 60 mg (0.14 mmol, 58 %) of oxazole BM7 as a white powder.
	Novelty: 5-[3-(1H-imidazol-5-yl)phenyl]-N-[5-(ethylsulfonyl)-2-methoxyphenyl]oxazol-2-
	amine (BM7) is not described in the literature.
	M.p.: 210.0 -213.0 °C [EA].
	J(A4,A6) = 2.2 Hz, H-CA(6)), 8.08, 7.74, 7.67 and 7.48 -7.38 (3 x br and 1 x m, 6 x 1H, H-
	CC(2 and 4-6), H-CD(2 and 4), 7.50 (dd, 1H, J(A3,A4) = 8.5 Hz, J(A4,A6) = 2.2 Hz, H-CA(4)),
	7.27 (d, 1H, J(A3,A4) = 8.5 Hz, H-CA(
	86 and 7.62 (2 x
	br s, 2 x 1H, -NH-and H-CD(2)), 7.72 (dd, 1H, J(C2,C4) = (C2,C6) = 1.4 Hz, H-CC(2)), 7.60 and
	7.37 (2 x dd, 2 x 1H, J(C4,C5 or C5,C6) = 7.8 Hz, J(C2,C4 or C2,C6) = 1.4 Hz, H-CC(4 and 6)),
	7.59 (dd, 1H, J(A3,A4) = 8.6 Hz, J(A4,A6) = 2.2 Hz, H-CA(4)), 7.49 (dd, 1H, J(C4,C5) = J(C5,C6)
	= 7.8 Hz, H-CC(5)), 7.16 (br s, 1H, H-CD(4)), 7.03 (d, 1H, J(A3,A4) = 8.6 Hz, H-CA(3)), 5.07
	(dd, 1H, J(E2,E3) = 10.5 Hz, J(E2,E3) = 2.3 Hz, H-CE(2)), 4.19 and 3.62 (2 x m, 2 x 1H, 2 x H-
	CE(6), 4.05 (s, 3H, CH3O-), 3.17 (q, 2H, J(CH2,CH3) = 7.4 Hz, CH3CH2SO2-), 2.18 -1.56 (m,
	6H, H-CE(3-5)), 1.31 (t, 3H, J(CH2,CH3) = 7.4 Hz, CH3CH2SO2-).

M.p.: 125.0 -130.0 °C [EA / MeOH].

1 H-NMR (400 MHz, CDCl3): δ 8.90 (d, 1H, J(A4,A6) = 2.2 Hz, H-CA(6)), 7.

1 H-NMR (500 MHz, DMSO-d6): δ 12.23 (br s, 1H, -NDH), 9.77 (br s, 1H, -NH-), 8.80 (d, 1H,

10.1. Synthesis of the general intermediate N,5-diaryloxazol-2-amine

  formation of an oxazole ring was the most problematic step of the synthesis. According to experience in our research group, this type of reaction provides a product from 50 to 85 % yield after FLC. The reason of relatively low 35 % yield of the required oxazole 36 was probably caused by low reactivity of azide 35 toward PPh3. In the first step of the synthesis (Staudinger

	(36)
	Synthesis was initiated by treating of p-aminoacetophenone 30 with NBS to form brominated reaction) PPh3 attacks terminal nitrogen of an azide group which leads to another nucleophilic
	acetophenone 31. Next step was a conversion of an amino group of 31 to a cyano group by attacking to form four-membered ring. (Scheme 37) After releasing of N2 an aza-ylide
	Sandmeyer reaction with t BuONO and Et2O · BF3 to form diazonium salt. Subsequent slow compound 35a is formed. Complete reaction mechanism is described in the chapter 3 of this
	addition of a mixture of CuCN / NaCN below 5 °C followed by stirring at 60 °C afforded thesis in the Scheme 5. In our case, presence of para -CN substitued as a strong electron
	benzonitrile 32 in 64 % yield. Alpha brominated derivative 34 was obtained within 1.5 h by withdrawing group decreases a nuclephilicity of first nitrogen of an azide group leading to
	treating of 32 with trimethylphenylammonium tribromide (33). Traces of bisbrominated side product was removed by trituration with a mixutre of EA / Hex. Nuclephilic substitution of formation of other side product rather then 35a. The scope of this type of reaction was investigated in our research group. Scheme 39. Synthesis of required cyano derivative BM8 and attemp to prepare the designed amide analogue BM9.

The synthesis of inhibitors BM

(8)(9)(10)(11)[START_REF]Preparation of the 1,2,4-triazine-3-(2H)thione catalysed by HOAc or NaOAc / HOAc An alternative way for the synthesis of 1,2,4-triazine-3-thione in acidic conditions was described in 2000 by J. Hlaváč et al. 124 The anion of the acid T102 obtained from isatine T101 was treated with thiosemicarbazide to give aniline T103 in 70 % overall yield. The final reaction step from T103 to T104 was performed at refluxing AcOH. Based on 1 H-NMR spectrum the ratio of obtained isomers T104 to T105 was 10 : 3. (Scheme 56) Scheme 56. Preparation of isomers T104 and T105 from isatine T101. Another preparation of 1,2,4-triazine-3(2H)-thiones T109 and T111 were described on aceanthrylene-1,2-dione T108 in acid-catalysed reaction 127 as well as on substituted thiosemicarbazone T110 catalysed by AcONa in AcOH at reflux[END_REF](13)(14)(15)(16)

started by preparation of their joint intermediate 36. bromine on aliphatic chain in 34 by NaN3 afforded azide 35 in 95 % yield. The last reaction step was a formation of an oxazole ring by reaction of azide 35, isothiocyanate 5 and PPh3 in refluxing dioxane. (Scheme 36) Scheme 36. Synthesis of the joint intermediate 36. A 82 Scheme 37. Proposed mechanism of decreased reactivity of azide 35 in performed Staudinger reaction.

We also prepared the amide and ester derivatives 36b (33 %) and 36c (37 %) in order to verify low yield of an oxazole ring formation due to a presence of an electron withdrawing group in para position of acetophenone 35b,c. As expected, the products 36b and 36c were formed in a similar low yield as 36. (Scheme 38) Similar results were observed by basic hydrolysis of an ester group of the pyrrole containing compound 37c. Basic hydrolysis of a cyano group in 37c did not provide the desired product BM10, but the same reaction condition applied to unsubstituted ester 38c afforded the carboxylic acid 39c in 80 % yield. (Scheme 40) Scheme 40. Unsuccessful basic hydrolysis of an ester group on pyrrole derivative 37c.

  chelatation. (Scheme 43)

	Scheme 43. A proposed mechanism of non-reactivity of 36b by Suzuki coupling reaction.

Since a pyrrole formed anion in BM8 did not allow to prepare the amide product BM9, we decided to protect -NH-groups in BM8 in order to hydrolyse an orto -CN moiety. Reaction of BM8 with Boc2O and DMAP in THF (abs) provided bis Boc-protected oxazole 64 within 1 h.

Even though resulting intermediate 64 should have been stable in basic environment, hydrolysis did not performed and only starting compound BM8 was recovered. (Scheme 44) Scheme 44. Protection of a BM8 and its unsuccessful hydrolysis.

(11-13).

	10.2.	Synthesis of pyrazole inhibitors BM(11-13)
	The pyrazole derivate BM11 was prepared by Suzuki coupling reaction from the joint
	intermediate 36 and pyrazole boronic acid 21 in DMF / H2O at 100 °C within 50 min under
	microwave irradiation. In a basic catalysed synthesis of amidic and carboxylic acid derivatives
	BM(12-13) we expected similar reduced reactivity of -CN group in a presence of a pyrazole
	anion. Much higher stability of pyrazole in acidic conditions compare to pyrrole analogue
	allowed us to successfuly perform acidic hydrolysis of BM11. Partial hydrolysis of a cyano
	group by a mixure of TFA / H2SO4 (conc) (4 / 1) at 70 °C within 3 h gave an amidic inhibitor
	BM12 in 62 % yield. Harder hydrolysis carried out in an aqueous solution of H2SO4 at 100 °C
	(overnight) provided a carboxylic acid derivative BM13 in 60 % yield. (Scheme 45)
	10.3. Synthesis of imidazole inhibitors BM(14-16)
	Synthesis of proposed imidazole inhibitor BM14 was initiated by Suzuki reaction from
	aminooxazole 36 and previously prepared tributylstannylimidazole 15 under microwave
	irradiation. THP-protected imidazole intermediate 37 was hydrolysed by TFA to get required

Scheme 45. Preparation of required pyrazole inhibitors BMcompound BM14 in 50 % yield. Synthesis of amide derivative BM(15) was also performed by partial acidic hydrolysis with a mixture of TFA / H2SO4. (Scheme 46) Derivative BM16 has not been synthetized yet. Scheme 46. Synthesis of the imidazole inhibitors BM(14-16).

  85,86,87

Figure 52. Comparison of NMR shifts of starting compound 66 and proposed product 70.

Nevertheless, MS spectrum of 68 did not directly support 70 from our hypothesis. Therefore a structure of both side products 9a and 68 remains unknown and other supporting analyses are planned to be performed Another and more important problem in the synthesis of required intermediate 69 is low reactivity of crude triflate 67, containing -CN group compare to less substituted triflate 9, whose structure was proved by next reaction product 10. Despite various stronger reaction conditions (extended reaction time, higher temperature or microvawe irradiation) we were not able to prepare the desired aminooxazole 69 from 67 and only starting material 6 and some decomposed products were present.

85

Il Kong, H.; Crichton, J.E.; Manthorpe, J.M. Tetrahedron

Lett., 2011, 52, 3714 -3717. 86

Hanack, M.; Wilhelm, B.; Subramanian, L.R. Synthesis, 1988, 8, 592 -595. 87 Caldwell, Ch.; Chapman, K.; Hale, J.; Kim, D.; Lynch, Ch.; Maccoss, M.; Mills, S.G.; Willoughby, Ch.; Berk, S.; Kim, R.M. Assignee Merck and Co., Inc., USA, WO 2000059498, 2000, A1

10.6. Experimental part 10.6.1. Synthesis of a general intermediate N,5-diaryloxazol-2-amine

  (36)

	Synthesis of 1-(4-amino-3-bromophenyl)ethanone (31)
	A solution of 500 mg (3.70 mmol, 1.00 mol eq) acetophenone 30 in 10 mL of AN was cooled
	down to 0 °C. Subsequently 692 mg (3.89 mmol, 1.05 mol eq) of NBS in 5 mL of AN was
	added dropwise and stirred 30 min at 0 °C and 2 h at rt. After complete consumption of starting
	Scheme 49. Synthesis of a general intermediate 36.

material 30, TLC analysis confirmed only a presense of a one product. Reaction mixture was evaporated, obtained solid residue dissolved in 20 mL of EA and extracted with NaHCO3 (sat aq solution) (3 x 20 mL) and brine (3 x 20 mL). Combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure yielding 658 mg (3.07 mmol, 83 %) of acetophenone 31 as brown oil, which solidified in a refrigerator. Novelty: 1-(4-amino-3-bromophenyl)ethanone 31 is described in the literature by its M.p., 1 H-NMR, 13 C-NMR and MS spectra. 88 M.p.: 59.0 -62.0 °C [EA] (lit. M.p.: 52 °C [CH2Cl2]). 89 88 Nery, M.S.; Azevedo, M.S.; Cardoso, J.N.; Slana, G.B.C.; Lopes, R.S.C.; Lopes, C.C. Synthesis 2007, 10, 1471 -1474. 89 Sartorius, F.; Trebing, M.; Brückner, C.; Brückner, R. Chem.

Eur. J., 2017, 23, 17463 -17468.

  Arrival of the Fittest (1 th ed.), Penguin Group, New York, 2014, p 100, ISNB: 9781591846468.

to NADH, which is utilised in the oxidative phosphorylation (electron transport) pathway to generate energy-rich ATP. The theoretical yield of ATP through oxidation of one molecule of the D-glucose in glycolysis, Krebs cycle and oxidative phosphorylation is 36 molecules of ATP. 96 (Scheme 51)

91 Ponomarev, V.V.; Migarskaya, L.B. Russ. J.

Phys. Chem. 1960, 34, 1182 -1183. 92

Fairclough, S.H.; Houston, K. Biol.

Psychol. 2004, 2, 177 -190. 93

www.thoughtco.com (accessed April 17 th , 2020) 94 Voet, D.; Voet, J.G.; Pratt, C.W. Fundamental of Biochemistry (2 nd ed.), John Wiley & Sons, Inc. USA, 2006, 547-556, ISNB: 0-471-21495-7. 95 Wagner, A. 96 Porter, P.K.; Brand, M.D. Biochem. J. 1995, 2, 379 -382.

  It is exhibiting 85 % sequence homology to human lens ALR2, while the catalytic active site of both enzymes is considered identical. Inhibitors are usually prepared in H2O or DMSO with the final concentration no more than 1 % of DMSO. The

	inhibition of enzyme activity (IC50) is calculated. For selectivity test, an aldehyde reductase
	(ALR1) isolated from a rat kidney is used. 121
	Chapter 9. Synthesis of ALR2 inhibitors possessing
	triazinoindole scaffold
	reaction mixture for assay contains ALR2, sodium phosphate buffer, NADPH, substrate (D,L-
	glyceraldehyde) and ALR2 inhibitor at different concentrations. ALR2 activity is monitored
	spectrophotometrically by measuring the decrease in absorption of NADPH at 340 nm after
	addition of substrate. From obtained numbers a concentration of inhibitor giving 50 %
	120 Lipinski, C.A.; Lombardo, F.; Dominy B.W.; Feeney, P.J. Adv. Drug Deliv. Rev., 2001, 46, 3 -26. 121 Kim, T.H.; Kim, J.K.; Kang, Y-H.; Lee, J-Y.; Kang, I.J, Lim, S.S. Biomed. Res. Int., 2013, 2013, 1 -8.

  El Ashry, E.S.H.; El Sayed, R.;Hamid, A.; Hagar, M.; Synlett., 2004, 4, 723 -725.

126

Kgokong, J.L.; Smith, P.P.; Matsabisa, G.M. Bioorg.

Med. Chem., 2005, 13, 2935-2942.

  Scheme 65. Synthesis of an oxo CMTI isostere OTI and its dicarboxylic derivative OTI-1.

	13.5.2.	Synthesis of an oxo CMTI isostere (OTI) and its dicarboxylic
	derivative (OTI-1)
	13 C-NMR (150 MHZ, DMSO-d6): δ 179.5 (C=S), 169.0 (-COOH), 148.5 (C4a), 144.4 (C5a),
	135.5 (C1a), 132.3 (C7), 124.1 (C8), 122.2 (C9), 117.5 (C9a), 112.3 (C6), 43.4 (NCH2COOH).
	FT-IR (solid, cm -1 ): 3500 (w), 3099 (w), 3020 (w), 2978 (w), 2892 (w), 1736 (m), 1601 (m),
	1575 (s), 1466 (m), 1438 (w), 1370 (m), 1258 (m), 1167 (s), 1147 (s), 1073 (w), 1018 (m), 945
	(w), 811 (w), 784 (m), 754 (m), 639 (w), 526 (m).
	MS (ESI m/z): 259.0 (10 %) [M-H] -, 214.9 (100 %) [M-CO2-H] -(negative mode).
	Anal. calcd for C11H8N4O2S (260.27): C, 50.76; H, 3.10; N, 21.53 Found: C, 50.92; H, 3.32 ;
	N, 21.84.

  Synthesis of hydroxymethyl derivative OTI-6 started from OTI and protected benzyl alcohol 53, prepared in tree steps. (Scheme 68) Synthesis was initiated by reduction of phtalanhydride 50 with LiAlH4 in THF (abs) at 0 °C to give diol 51 in 94 % yield. Afterwards, a reaction of 51 with HBr (48 %) in toluene at 70 °C provided required mono-52 and small amount of bis brominated products. After FLC separation, desired intermediate 52 was obtained in 56 % yield. Finally, O-protected compound 53 was obtained by reaction of 3,4-dihydropyrane (DHP) in a presence of cat amount of PPTS in refluxing DCM. Then, ester 44 was prepared in 74 % yield by acidic catalyzed esterification of OTI with EtOH (abs) Afterwards, alkylation of ester 44 with 53 by CaH2 in DMF at 80 °C performed an intermediate 54 in 82 % yield. Compound 54 was subsequently hydrolysed to required benzyl alcohol derivative OTI-6 by aq HCl in dioxane at 90 °C in 66 % yield. (Scheme 68)

	14.2. Synthesis of OTI-(7-9)
	Synthesis of proposed ALR2 inhibitors OTI-(7-9) was initiated by reaction of ester 44, which
	was alkylated to 56 by 2-(bromomethyl)benzonitrile 55. Obtained benzonitrile derivative 56
	was used as a joint intermediate for a synthesis of the proposed compounds OTI-(7-9).
	Hydrolysis of an ester group of 56 by aq solution of NaOH in MeOH / H2O at reflux provided
	the benzonitrile compound OTI-7. Partial carbonitrile hydrolysis of 56 with TFA / H2SO4
	(conc) (4 / 1) within 6 h provided benzamide derivative OTI-8. Benzoic acid derivative OTI-9
	was prepared by stronger hydrolysis of 56 with a mixture of H2SO4 (conc) / H2O (1 / 1) at 100
	°C within 8 days. (Scheme 69)
	Scheme 68. Synthesis of OTI-6.

Scheme 67. The structures of proposed N-benzyl(oxotriazinoindole) derivatives OTI-(6-9).

Figure 69. Interaction diagrams of proposed N-benzyl(oxotriazinoindole) derivatives OTI-(6-9). 14.1. Synthesis of OTI-6 Scheme 69. Synthetic pathway to OTI-(7-9).

Table 1 .

 1 Determined inhibitory activity and selectivity of substituted N-benzyl(oxotriazinoindole) inhibitors OTI-(6-9) in comparison to unsubsituted reference analoque OTI-3..

	Results are means ± SD from minimum of three independent repetitions. I is the % of enzyme inhibition observed
	at 100 μM inhibitor concentration. Selectivity factor: SF = IC50 (ALR1) / IC50 (ALR2).

Obtained results confirmed proposed additional interactions of inhibitors OTI-(6-9) in the unoccupied interactive pocket of ALR2. The best ALR2 inhibition (IC50 = 76 nM) and selectivity relative to ALR1 (SF > 1333) was obtained for OTI-7 derivative containing a N-(2cyanobenzyl) group. (Table

Table 1

 1

	14.4.	Experimental part
		14.4.1.	Synthesis of benzyl derivative (OTI-6)

) The reason of lower activity of OTI-(6,8,9) could be assigned to a Scheme 70. Synthesis of a benzyl analogue OTI-6.

Penn, J.S. Retinal and Coroidal Angiogenesis (1 st ed.), SpringerNetherlands, 2008, 119, ISBN 978-1-4020- 6779-2.

 7 Holmes, K.; Roberts, O.L.; Thomas, A.M.; Cross, M.J. CellSignal., 2007Signal., , 19, 2003Signal., -2012. .

Duong-Ly, K.C.; Peterson, J.R. Methods Mol. Biol., 2016, 1360, 87 -95.

Determination of enzymatic IC50 activity on VEGFR2 TK was performed by ProQinase GmbG, Freiburg, Germany, https://www.proqinase.com (accessed August 21th, 2020).

Aliprantis, A. O.; Canary, J. W. J. Am.Chem. Soc., 1994, 116, 6985.

ChemLer, S. R.; Trauner, D.; Danishefsky, S. J. Angew.Chem. Int. Ed., 2001, 40, 4544.

Lima, C.F.R.A.C.; Rodrigues, A.S.M.C.; Silva, V.L.M.; Silva, A.M.S.; Santos, L.M.N.B.F. Chem. Cat. Chem., 2014, 6, 1291 -1302.

Primas, N.; Mahatsekake, C.; Bouillon, A.; Lancelot, J-Ch.; de Oliviera Santos, J.S.; Lohier, J-F.; Rault, S.Tetrahedron, 2008, 64, 4596 -4601.

Synthesis was developed by Maroš Smolíček and Peter Šramel. See in: Peter Šramel, Dissertation Thesis 2017, Comenius University in Bratislava, Slovakia and Université de Strasbourg, France.

H-NMR (400 MHz, CDCl3): δ 9.02 (d, 1H, J(A4,A6) =

2.3 Hz, H-CA(6)),8.32 and 7.53 (2 x

H-NMR (400 MHz, CDCl3): δ, 8.98 (d, 1H, J(A4,A6) =

2.1 Hz, H-CA(6)), 8.84 (2 x d, 2 x 1H,

H-NMR (400 MHz, DMSO-d6): δ 13.02 (br s, 1H, H-ND(1)), 9.77 (br s, 1H, -NH-),9.16 (d,

H-NMR (300 MHz, CDCl3): δ 8.07 (d, 1H, J(2,6) =

2.0 Hz, H-C(2)), 7.74 (dd, 1H, J(5,6) =

H-NMR (600 MHz, DMSO-d6): δ 11.29 (br s, 1H, H-ND(1)), 9.89 (br s, 1H, -NH-),8.75 (d,

H-NMR (600 MHz, DMSO-d6): δ 12.27 (br s, 1H, H-ND(1)), 9.87 (br s, 1H, -NH-),8.80 (d,

H-NMR(400 MHz, CDCl3): δ 8.20 (d, 1H, J(3,5) = 1.6 Hz, H-C(3)),7.92 (dd, 1H, J(5,6) =

Hlavac, M.; Kovacikova, L.; Prnova, M.S.; Addova, G.; Majekova, M.; Hanquet, G.; Bohac, A.; Stefek, M. J.Med. Chem., 2020, 63, 369 -381.

H-NMR (300 MHz, DMSO-d6): δ 14.67 (br s, 1H, -NH-),13.25 (very br, 1H,

H-NMR (300 MHz, DMSO-d6): δ 12.27 and 11.93 (2 x br s, 1H, -CONHCO-and =N-NHCO-

H-NMR (300 MHz, DMSO-d6): δ 13.14 (br s, 1H, -NH-),7.95 (dd, 1H, J(8,9) = 8.0 Hz, J(7,9)

H-NMR (300 MHz, DMSO-d6): δ 13.29 (2 x br s,

x 1H, 2x -COOH)7.94 (dd, 1H, J(8,9) =

13.5.3. Synthesis of OTI derivatives OTI-(2-5) Scheme 66. Synthesis of OTI derivatives OTI-(2-5).

H-NMR (300 MHz, DMSO-d6): δ 7.96 (dd, 1H, J(8,9) = 7.6 Hz, J(7,9) = 1.4 Hz, H-C(9)),

H-NMR (600 MHz, CDCl3): δ 7.43 and 7.39(2 x dd,

x 1H, J(3,4 or 5,6) = 7.5 Hz, J(3,5 or

H-NMR (600 MHz, CDCl3): δ7.93, 7.43, 7.35 and 7.13 (4 x dd, 4 x 1H, J(6,7 or 8,9 or A3,A4

H-NMR (600 MHz, DMSO-d6): δ 13.10 (br s, 1H, -COOH), 8.00 and 7.53 (2 x br s,

x 1H, -

Scientific contribution

Scheme 38. Synthesis of amide and carboxylic acid aminooxazoles 36b and 36c.

Synthesis of the first designed inhibitor BM8 started from 36. Bromooxazole 36 underwent Suzuki coupling reaction with previously prepared pyrrole pinacol boronate 19 to give BM8 in 58 % yield. (Scheme 39) We also tried to prepare the amidic pyrrole containing inhibitor BM9 by hydrolysis of a cyano group according to our previously published procedure. 83 Unfortunately, in this case hydrolysis was unsuccessful. When we applied the same reaction conditions to the intermediate 36 we successfully obtained amide 36b within 15 min in 85 % yield. (Scheme 39) We have seen that pyrrole anion, formed in a basic conditions, disrupts reactivity of a cyano group toward nucleofilic attack. Since the pyrrole is an unstable heterocyclic compound in acidic conditions, hydrolysis performed in a mixture of TFA / H2SO4 caused polymerization of the starting compound BM8. (Scheme 39) 83 Dobiaš, J.; Ondruš, M.; Hlaváč, M.; Murár, M.; Kóňa, J.; Addová, G.; Boháč, A. Chem. Papers, 2019, 73, 71 Combined organic layer was dried over Na2SO4, filtered and concentrated with RVE to approx. 30 mL of its volume. Resulting solution was crystallized in a refrigerator to yield 2.34 g (10.4 mmol, 64 %) of 32 in form of brown-red crystals.

Novelty: 4-acetyl-2-bromobenzonitrile (32) is described in the literature by its 1 13 C-NMR (150 MHz,141.0,134.8,132.8,127.1,126.1,119.7,116.6,.

90 Tagawa, H.; Kubo, S. Chem. Pharm. Bull., 1984, 32, 3047 -3052. None of these carboxylic acids as well as spirohydantoin derivatives as ALR2 inhibitors are approved by FDA. Therefore, in the recent search for better ARIs, the focus has been shifted towards novel leading skeletons.

Discovery of the indole-1-acetic acid ALR2 inhibitor (CMTI)

Substituted indole-1-acetic acids represent another leading skeleton of ARIs. 117 Lidorestat was a promising candidate for clinical studies, but it was withdrawn due to its side effects. 118 (Figure 60) Based on lidorestat, scientists from the Slovak Academy of Sciences proposed fifteen compounds possessing indole-1-acetic acid moiety. 119 These structures were evaluated for their potencial to inhibit reduction of D,L-glyceraldehyd by isolated ALR2 enzyme from rat lenses. Among them, 3-merkapto-5H-1,2,4-triazino [5,6-b]-indole-5-acetic acid, named cemtirestat (CMTI), exhibited the highest biological activity both at the level of isolated enzyme with IC50 = 97 nM (ALR2) and also at an organ level. For determination of selectivity, closely related detoxification enzyme aldehyde reductase (ALR1) from a rat kidney was used. The enzyme ALR1 is considered to be an antitarget. CMTI showed moderate inhibition for the reduction of Novelty: 2H-[1,2,4]triazino [5,6-b]indol-3(5H)-one ( 43) is described in the literature with its M.p., 1 9)), 7.53 (ddd, 1H, J(7,8) = 8.0 Hz, J(6,7) = 7.9

Hz, J(7,9) = 1.3 Hz, H-C( 7)), 7.31 (dd, 1H, J(6,7) = 7.9 Hz, J(6,8) = 1.3 Hz, H-C( 6)), 7.26 (ddd, 1H, J(7,8) = 8.0 Hz, J(8,9) = 7.7 Hz, J(6,8) = 1.3 Hz, H-C( 8)).

13 C-NMR (75 MHz, DMSO-d6): δ 155. 5, 155.2; 146.7, 133.2; 131.1, 122.7; 121.4, 118.5, 112.7. To a solution of 100 mg (0.37 mmol, 1.00 mol eq) 44 in 20 mL of acetone (abs), 320 µl (1.84 mmol, 5.00 mol eq) of DIPEA (abs) and 139 µl (1.84 mmol, 5.00 mol eq) of MOMCl were added dropwise and while stirring at 50 °C under Ar. After 5 h TLC analysis confirmed presence of product 46, traces of unknown side product and only small amount of starting material 44. (after 16 h of heating almost no changes were seen by TLC analysis) Then, the reaction mixture was evaporated by RVE, dissolved in 20 mL of EA and extracted with water Biscarboxylic acid OTI-9 exhibits higher activity than hydroxymethyl and amide analogues

MS

OTI-(6,8).

To sum up, we have developed four novel N-benzyl(oxotriazinoindole) inhibitors OTI- or A5,A6) = 7.8 and 7.2 Hz, J(6,8 or 7,9 or A3,A5 or A4,A6) = 1.0 Hz, H-C(6, 9, A3 and A6)), 7.53, 7.31, 2 x 7.27 (4 x ddd, 4 x 1H, J (7,8 or A4,A5) = 8.0 Hz, J(6,7 or 8,9 or A3,A4 or A5,A6) = 7.8 and 7.2 Hz, J(6,8 or 7,9 or A3,A5 or A4,A6) = 1.0 Hz, H-C(7, 8, A4 and A5)), 5.06 (d,1H,Jgem = 12.3 Hz,5.62 and4.89 (suprisingly 2 x s, 2 x 2H, -CH2COOEt and-CH2Ph Due to moisture instability of the 54, its other spectra and analysis were not recorded and compound 54 was used directly in the next step.

Synthesis of 2-{2-[2-(hydroxymethyl)benzyl]-3-oxo-2,3-dihydro-5H-[1,2,4]triazino[5,6b]indol-5-yl}acetic acid (OTI-6)

To a solution of 50.0 mg (0.11 mmol, 1.00 mol eq) ester 54 in 10 mL of dioxane, 1 mL of H2O and 0.1 mL of HCl (37 % w/w) were added and resulting solution stirred at 90 °C for 8 h. After that, TLC analysis confirmed presence of a one product without side products. Reaction mixture was cooled down, 30 mL of water added and extracted with DCM (3 x 15 mL). Combined organic layer was dried over Na2SO4, filtered and concentrated under reduce pressure yielding 25.0 mg (0.07 mmol, 66 %) of acid OTI-6 as a yellow solid compound. [1,2,4]triazino [5,6-b]indol-5-yl]acetic acid (OTI-7) is not described in the literature. M.p.: 280.9 -284.5 °C [MeOH / H3O + ].

1 H-NMR (600 MHz, DMSO-d6): δ 13.35 (br s, 1H, -COOH), 7.94, 7.89, 7.59 and 7.43 (4 x dd, 4 x 1H, J(6,7 or 8,9 or A3,A4 or A5,A6) = 8.0 or 7.7 Hz, J(6,8 or 7,9 or A3,A5 or A4,A6) = 1.0 Hz, H-C(6, 9, A3, A6)), 7.66, 7.63, 7.51 and 7.35 (4 x ddd, 4 x 1H, J(7,8 or A4,A5) = 8.0 Hz, J(6,7 or 8,9 or A3,A4 or A5,A6) = 7.7 or 7.5 Hz, J(6,8 or 7,9 or A3,A5 or A4,A6) = 1.0 Hz, H-C(7, 8, A4, A5)), 5.56 and 4.94 (2 x s, 2 x 2H, -CH2COOH and -CH2Ar).

13 C-NMR (150 MHz, DMSO-d6): 169.1 154.3,153.4,144.6,140.5,134.0,133.6,133.1,131.7,128.9,128.7,123.7,121.7,117.7,117.6,111.8,111.4,55.2,42.5. A solution of 20.0 mg (0.05 mmol, 1.00 mol eq) ester 56 in 1 mL of CF3COOH (TFA) and 0.25 mL of H2SO4 (conc) was stirred at 70 °C for 6 h. After complete consumption of starting compound 56 (confirmed by TLC analysis), the reaction was cooled down and 20 mL of H2O and 20 mL of brine were added and the resulting mixture extracted with EA (5 x 15 mL).

FT-IR (solid, cm

Combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. A crude product OTI-9 was purified by trituration with a mixture of Hex / EA to give 15.0 mg (0.04 mmol, 77 %) of acid OTI-9 as a solid compound.

Novelty:

2-[2-(2-carbamoylbenzyl)-3-oxo-2,3-dihydro-5H- [1,2,4]triazino [5,6-b] This dissertation thesis resulted in a discovery of 14 inhibitors of VEGFR2 TK and 9 inhibitors of ALR2 while 16 of them were biologically evaluated (IC50, VEGFR2 TK, ALR2 and ALR1).

Key words: tyrosine kinase inhibitors, aldose reductase, VEGFR2 TK, ALR2, aminooxazoles, oxotriazinoindoles