Patent Description:
Infectious diseases caused by microorganisms stand as a major threat to public health. Since antibiotics were first introduced as medicines, these drugs have been used to prevent or treat infections in several applications. Nonetheless, antibacterial resistance has increased dramatically, becoming an emergency in healthcare during the last <NUM> years. Among <NUM> emerging infectious agents that have been identified, <NUM>% have developed resistance to multiple drugs including antibiotics such as vancomycin, methicillin, carbapenems, and cephalosporins. Despite enormous efforts, the number of therapeutically useful compounds that aim for circumventing the resistance is continuously decreasing and no truly novel class of compounds has been introduced into therapy, causing the world to face the "post-antibiotic era". In order to stop the clinical consequences of the development and spread of antimicrobial resistance both the preservation of current antimicrobials through their appropriate use, as well as the discovery and development of new agents are mandatory.

Malaria represents a major threat to the public health worldwide, with over <NUM> million clinical cases in <NUM> with <NUM> thousand of deaths. Though the number of cases has shown a decrease since <NUM>, evidences of slower Plasmodium falciparum parasite clearance have appeared in some countries in Southeast Asia especially at Greater Mekong Subregion (GMS) including Lao PDR, Thailand, Cambodia, Myanmar, and Vietnam. These represent a serious threat to global malaria control and eradication. The frontline therapies for the treatment of symptomatic malaria are artemisinin (<NUM>) combination therapies (ACTs) for P. falciparum infections and in the case of infections with P. vivax, chloroquine (CQ, <NUM>) or ACTs are usually employed. This evidence, along with widespread resistance to other historical antimalarials, highlights the need to identify new chemical diversity, ideally with novel antimalarial modes of action.

Several reports emphasized the discovery of new sophisticated antimicrobials from marine sources as a promising strategy to overcome the ever-increasing drug-resistant infectious diseases. In the last years, fungal alkaloids containing an indolomethyl pyrazino[<NUM>,<NUM>-b] quinazoline-<NUM>,<NUM>-dione scaffold were isolated from marine organisms and presented very interesting antimicrobial activities [<NUM>]. For instance, glyantypine (<NUM>) isolated from Cladosporium sp. PJX-<NUM>, exhibited moderate inhibitory activity against bacteria Vibrio harvevi (MIC = <NUM>µg/mL) and neofiscalin A (<NUM>) found in Neosartorya siamensis KUFC <NUM> exhibited a potent antibacterial activity against Staphylococcus aureus and Enterococcus faecalis (MIC = <NUM>µg/mL) [<NUM>].

Strategies used for the development of novel antimalarial drugs include the discovery of new active molecules from natural products, repurposing of commercially available drugs, development of hybrid compounds, and rational drug design with molecular modifications of existing antimalarial and hits. The malarial chemotherapy has always been successfully influenced by natural products and nature is still an important source of antimalarial drugs. Recently, the analysis of Tres Cantos Antimalarial Set (TCAMS) suggested that indole-based antimalarials are the key core for the development of the next generation of antimalarial drugs since the indole scaffold is known as an important moiety present in several lead drug candidates with new mechanisms of action, such as the spiroindolone (<NUM>), febrifugine (<NUM>), and aminoindole derivatives. For example, TCMDC-<NUM> (<NUM>) exhibited very potent antiplasmodial properties against P. falciparum 3D7 strain (EC<NUM> = <NUM>). However, although TCMDC-<NUM> showed no significant cytotoxicity against human HepG2 hepatoma cell line (EC<NUM> > <NUM>), the presence of the <NUM>-aminoquinolyl moiety (an essential pharmacophore of CQ) might be responsible for its cross-resistance with CQ (<NUM>) and poor-drug-like properties [<NUM>].

Lijuan Liao et al. discloses the isolation and characterization of fumiquinazoline F, and its activity against gram-positive B. subtilis and Gram-negative P. hauseri (<NPL>).

Marley Garci Silva et al. discloses the isolation and antimicrobial activity of fumiquinazoline F against M. luetus and S. aureus (<NPL>).

<CIT> discloses indolo[<NUM>,<NUM>-b]quinazole-<NUM>,<NUM>-diones as antimalarial agents.

Solida Long et al. discloses the neuroprotective and antitumor effects activity of fiscalin B, fumiquinazoline G, and derivatives.

The present disclosure relates to four possible approaches to obtain indole-containing pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>-diones comprising a subclass of alkaloids mostly isolated from marine and terrestrial sources. These structurally unique alkaloids contain simultaneously a quinazoline core which can be found in the structure of the natural febrifugine (<NUM>) and an indole moiety commonly found in several drug lead candidates such as spiroindolone (<NUM>) and TCMDC-<NUM> (<NUM>). This hybrid structure comprises a quinazoline core and an indole core such that the observed inhibitory growth of MRSA may be observed and cross-resistance with CQ and ACTs may be overcome.

The first approach is based on the synthesis of enantiomeric pairs of two members of this quinazolinone family (structural modifications at C-<NUM> and C-<NUM> stereochemistry), including the marine-derived alkaloid fiscalin B (7A).

The second approach is based on the synthesis of other derivatives of these natural alkaloids, but with modification of the C-<NUM> side chain and stereochemistry, by using different amino acids.

The third approach is based on the synthesis of indolomethyl pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>-dione analogs: the introduction of halogen atoms in the aromatic ring of the anthranilic acid (Ant).

The fourth approach is based on the synthesis of ring A variations on the pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>-dione scaffold or with an additional indole moiety.

The present disclosure relates to a compound of formula I
<CHM>
wherein.

In an embodiment, R<NUM> may be H or CH<NUM>.

In an embodiment, R<NUM> may be CH<NUM> or CH(CH<NUM>)<NUM> or CH<NUM>CH<NUM>.

In an embodiment, R<NUM> may be H or Cl or I.

In an embodiment, R<NUM> may be Cl or I, preferably R<NUM> may be CI.

In an embodiment, the compound may be
<CHM>
or
<CHM>
preferably the compound may be
<CHM>.

In an embodiment, the compound may be
<CHM>
or,
<CHM>
or,
<CHM>
or,
<CHM>
or,
<CHM>
preferably the compound may be
<CHM>.

The present disclosure also relates to a compound for use in medicine. Preferably, the compound of formula I is
<CHM>
wherein.

In an embodiment, the compounds may be selected from
<CHM>
or,
<CHM>
or,
<CHM>
and it may be for use in the treatment or prevention of malaria.

In an embodiment, any of the compounds herein disclosed may be for use in the treatment of Gram-positive bacterial infections, preferably caused by Staphylococcus spp. and/or Enterococcus spp. , more preferably caused by Staphylococcus aureus and/or Enterococcus faecalis.

In an embodiment, any of the compounds herein disclosed may be for use in the treatment of Gram-positive bacterial infections, preferably caused by Staphylococcus aureus and Enterococcus faecalis, wherein the compound may be
<CHM>
or,
<CHM>
preferably wherein the compound may be
<CHM>.

In an embodiment, any of the compounds herein disclosed may be for use in the treatment of Gram-positive bacterial infections, preferably caused by Staphylococcus aureus, wherein the compound may be
<CHM>
or,
<CHM>
preferably wherein the compound may be
<CHM>.

The present disclosure also relates to a composition comprising any of the compounds herein disclosed and a pharmaceutically acceptable excipient, wherein any of the compounds herein disclosed is in a therapeutically effective amount.

In an embodiment, the above-mentioned composition may further comprise an antibiotic, preferably wherein the antibiotic is a fluoroquinolone, preferably selected from ciprofloxacin, norfloxacin, pefloxacin, enofloxacin, ofloxacin, levofloxacin, moxifloxacin, or mixtures thereof.

The following figures provide preferred embodiments for the present disclosure and should not be seen as limiting the scope of the disclosure.

The present disclosure relates to antibacterial activity and/or to antimalarial activity of the compounds herein disclosed.

The compounds herein disclosed are synthetized using the approaches (<NUM>st, <NUM>nd, <NUM>rd and <NUM>th approaches) summarized in <FIG>.

The chemistry of compounds of the <NUM>st approach (compounds <NUM>-<NUM>) and <NUM>nd approach (compounds <NUM>, <NUM>, <NUM>, <NUM> and <NUM>) is described in references <NUM> and <NUM>. It was, however, surprisingly found that compounds of the <NUM>st and <NUM>nd approaches may have antimalarial activity, as it will be described below.

Chemistry for the <NUM>nd approach. The eleven new indolomethyl pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>-dione derivatives of the third approach were synthesized by a previously described approach using a microwave assisted multicomponent polycondensation of amino acids (Table <NUM>). The coupling of halogenated commercial anthranilic acids (<NUM>) to N-protected L-α-amino acids (<NUM>), and further dehydrative cyclization using triphenyl phosphite [(PhO)<NUM>P], generated the intermediates benzoxazin-<NUM>-ones <NUM> which, followed by the addition of D-tryptophan methyl ester (<NUM>) under microwave irradiation, furnished the desirable final products <NUM>-<NUM> (<NUM>-<NUM> % yield) with partial epimerization (Table <NUM>). Using this methodology only anti isomers were produced (<NUM>S, <NUM>R) and the different side chains at C-<NUM> were obtained by selecting diverse L-α amino acids - valine, leucine, and isoleucine. The purities of the compounds were determined by reversed-phase liquid chromatography, (RP-LC, C18, MeOH: H<NUM>O; <NUM>:<NUM>) and was found to be higher than <NUM> % while for compound <NUM> and <NUM> purities were of <NUM>%.

In the present disclosure, the general conditions for the synthesis of compounds <NUM>-<NUM> is as follows. In a closed vial, <NUM>-chloro anthranilic acid (<NUM> in which R'=H and R"=Cl, <NUM>, <NUM>µmol) for <NUM>, <NUM>, and <NUM>, or <NUM>,<NUM>-dichloro anthranilic acid, (<NUM> in which R' and R"=Cl, <NUM>, <NUM>µmol) for <NUM>, <NUM>, and <NUM>, or <NUM>-iodoanthranilic acid, (<NUM> in which R'=H and R"=I, <NUM>, <NUM>µmol) for <NUM> and <NUM>, or <NUM>-bromo anthranilic acid, (<NUM> in which R'=H and R"=Br, <NUM>, <NUM>µmol) for <NUM> and <NUM>, or <NUM>,<NUM>-diodo anthranilic acid (<NUM> in which R' and R"=I, <NUM>, <NUM>µmol) for <NUM>; was added N-Boc-L-valine (<NUM> in which R=i-Pr, <NUM>, <NUM>µmol) for <NUM>, <NUM>, <NUM> and <NUM>, or N-Boc-L-leucine (<NUM> in which R=i-Bu, <NUM>, <NUM>µmol) for <NUM>, <NUM>, <NUM>, and <NUM>, or N-Boc-L-isoleucine (<NUM> in which R=s-Bu, <NUM>, <NUM>µmol) for <NUM> and <NUM> (as present in Table <NUM>), and triphenylphosphite (<NUM>µL, <NUM>µmol) were added along with <NUM> of dried pyridine. The vial was heated in heating block with stirring at <NUM> for <NUM>-<NUM>. After cooling the mixture to room temperature, D-tryptophan methyl ester hydrochloride (<NUM>, <NUM>, <NUM>µmol) was added, and the mixture was irradiated in the microwave at a constant temperature at <NUM> for <NUM>. Four reaction mixtures were prepared in the same conditions and treated in parallel. After removing the solvent with toluene, the crude product was purified by flash column chromatography using hexane: EtOAc (<NUM>:<NUM>) as a mobile phase. The preparative TLC was performed using CH<NUM>Cl<NUM>:Me<NUM>CO (<NUM>:<NUM>) as mobile phase. The major compound appeared as a black spot with no fluorescence under the UV light. The desired compounds were collected as yellow solids. Before analysis, compounds were recrystallized from methanol.

In an embodiment, the characterization of (<NUM>,4R)-<NUM>-((<NUM>-indol-<NUM>-yl)methyl)-<NUM>-chloro-<NUM>-isopropyl-<NUM>,<NUM>-dihydro-<NUM>-pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>(<NUM>)-dione (<NUM>) is as follows: Yield: <NUM>, <NUM>%; e. r = <NUM>:<NUM>; mp: <NUM>-<NUM>; <MAT>; (c <NUM>); CHCl<NUM>); vmax(KBr) <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>-<NUM>; <NUM>H NMR (<NUM>, CDCl<NUM>): δ <NUM>. <NUM> (d, <NUM>, J = <NUM>, CH), <NUM> (br, <NUM>, NH-indol), <NUM> (dd, <NUM>,J = <NUM> and <NUM>, CH), <NUM> (d, J = <NUM>, CH), <NUM> (d, <NUM>, J = <NUM>, CH-Trp), <NUM> (d, J = <NUM>, CH-Trp), <NUM> (t, <NUM>, J = <NUM>, CH-Trp), <NUM> (t, <NUM>, J = <NUM>, CH-Trp), <NUM> (d, <NUM>, J = <NUM>, CH-Trp), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH*-Trp), <NUM> (s, <NUM>, NH-amide), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (d, J = <NUM>, CH*-val), <NUM> (dtd, <NUM>, J = <NUM>, <NUM>, and <NUM>, CH-val), <NUM> (d, <NUM>, J = <NUM>, CH<NUM>-val); <NUM>C NMR (<NUM>, CDCl<NUM>): δ <NUM> (C=O), <NUM> (C=O), <NUM> (C=N), <NUM> (C), <NUM> (C-Trp <NUM> (CH), <NUM> (C), <NUM> (CH), <NUM> (C-Trp), <NUM> (CH), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C-Trp), <NUM> (CH*-Trp), <NUM> (CH*-val), <NUM> (CH-val), <NUM> (CH<NUM>-Trp), <NUM> (CH<NUM>-val), <NUM> (CH<NUM>-val); (+)-HRMS-ESI m/z: <NUM> (M + H)+, <NUM> (M + Na)+ (calculated for C<NUM>H<NUM>N<NUM>O<NUM>Cl, <NUM>; C<NUM>H<NUM>N<NUM>O<NUM>ClNa, <NUM>).

In an embodiment, the characterization of (<NUM>S,<NUM>R)-<NUM>-((<NUM>H-indol-<NUM>-yl)methyl)-<NUM>-chloro-<NUM>-isobutyl-<NUM>,<NUM>-dihydro-<NUM>H-pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>(<NUM>)-dione (<NUM>) is as follows: Yield: <NUM>, <NUM>%; e. r = <NUM>:<NUM>; mp: <NUM>-<NUM>; <MAT> (c <NUM>; CHCl<NUM>); vmax (KBr) ) <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>-<NUM>; <NUM>H NMR (<NUM>, CDCl<NUM>): δ <NUM>. <NUM> (d, <NUM>, J = <NUM>, CH), <NUM> (br, <NUM>, NH-indol), <NUM> (dd, <NUM>,J = <NUM> and <NUM>, CH), <NUM> (d, J = <NUM>, CH), <NUM> (d, <NUM>, J = <NUM>, CH-Trp), <NUM> (d, J = <NUM>, CH-Trp), <NUM> (t, <NUM>, J = <NUM>, CH-Trp), <NUM> (t, <NUM>, J = <NUM>, CH-Trp), <NUM> (d, <NUM>, J = <NUM>, CH-Trp), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH*-Trp), <NUM> (s, <NUM>, NH-amide), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, J = <NUM> and <NUM>, CH*-Leu), <NUM> (ddd, <NUM>, J = <NUM>, <NUM>, and <NUM>, CH-Leu), <NUM>-<NUM> (m, <NUM>, CH<NUM>-Leu), <NUM> (d, <NUM>, J = <NUM>, CH<NUM>-Leu), <NUM> (d, <NUM>, J = <NUM>, CH<NUM>-Leu); <NUM>C NMR (<NUM>, CDCl<NUM>): δ <NUM> (C=O), <NUM> (C=O), <NUM> (C=N), <NUM> (C), <NUM> (C-Trp <NUM> (CH), <NUM> (C), <NUM> (CH), <NUM> (C-Trp), <NUM> (CH), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C-Trp), <NUM> (CH*-Trp), <NUM> (CH*-Leu), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>-Trp), <NUM> (CH-Leu), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>-Leu); (+)-HRMS-ESI m/z: <NUM> (M + H)+, <NUM> (M + Na)+ (calculated for C<NUM>H<NUM>N<NUM>O<NUM>Cl, <NUM>; C<NUM>H<NUM>N<NUM>O<NUM>ClNa, <NUM>).

In an embodiment, the characterization of (<NUM>S,<NUM>R)-<NUM>-((1iH-indol-<NUM>-yl)methyl)-<NUM>-((S)-sec-butyl)-<NUM>-chloro-<NUM>,<NUM>-dihydro-<NUM>-pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>(<NUM>)-dione (<NUM>) is as follows: Yield: <NUM>, <NUM>%; e. r = <NUM>:<NUM>; mp: <NUM>-<NUM>; <MAT> (c <NUM> CHCl<NUM>); vmax (KBr) <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>-<NUM>; <NUM>H NMR (<NUM>, CDCl<NUM>): δ <NUM>. <NUM> (d, <NUM>, J = <NUM>, CH), <NUM> (br, <NUM>, NH-indol), <NUM> (dd, <NUM>,J = <NUM> and <NUM>, CH), <NUM> (d, J = <NUM>, CH), <NUM> (d, <NUM>, J = <NUM>, CH-Trp), <NUM> (d, J = <NUM>, CH-Trp), <NUM> (t, <NUM>, J = <NUM>, CH-Trp), <NUM> (t, <NUM>, J = <NUM>, CH-Trp), <NUM> (d, <NUM>, J = <NUM>, CH-Trp), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH*-Trp), <NUM> (s, <NUM>, NH-amide), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (d, J = <NUM>, CH*-Ile), <NUM> (ddd, <NUM>, J = <NUM>, <NUM>, and <NUM>, CH*-Ile), <NUM>-<NUM> (m, <NUM>), <NUM> (d, <NUM>, J = <NUM>, CH<NUM>-Ile), <NUM> (d, <NUM>, J = <NUM>, CH<NUM>-Ile); <NUM>C NMR (<NUM>, CDCl<NUM>): δ <NUM> (C=O), <NUM> (C=O), <NUM> (C=N), <NUM> (C), <NUM> (C-Trp <NUM> (CH), <NUM> (C), <NUM> (CH), <NUM> (C-Trp), <NUM> (CH), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C-Trp), <NUM> (CH*-Ile), <NUM> (CH*-Trp), <NUM> (CH-Leu), <NUM> (CH<NUM>-Trp), <NUM> (CH<NUM>-Ile), <NUM> (CH<NUM>-Ile), <NUM> (CH<NUM>-Ile); (+)-HRMS-ESI m/z: <NUM> (M + H)+, <NUM> (M + Na)+ (calculated for C<NUM>H<NUM>N<NUM>O<NUM>Cl, <NUM>; C<NUM>H<NUM>N<NUM>O<NUM>ClNa, <NUM>).

In an embodiment, the characterization of (<NUM>S,<NUM>R)-<NUM>-((<NUM>-indol-<NUM>-yl)methyl)-<NUM>,<NUM>-dichloro-<NUM>-isopropyl-<NUM>,<NUM>-dihydro-<NUM>H-pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>(<NUM>)-dione (<NUM>) is as follows: Yield: <NUM>, <NUM>%; e. r = <NUM>:<NUM>; mp: <NUM>-<NUM>; <MAT> (c <NUM>; CHCl<NUM>); vmax (KBr) <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>-<NUM>; <NUM>H NMR (<NUM>, DMSO-d<NUM>): δ <NUM> (br, <NUM>, NH-indol), <NUM>. <NUM> (d, <NUM>, J = <NUM>, CH), <NUM> (d, <NUM>, J = <NUM>, CH), <NUM> (d, <NUM>, J = <NUM>, CH-Trp), <NUM> (d, J = <NUM>, CH-Trp), <NUM> (s, <NUM>, NH-amide), <NUM> (t, <NUM>, J = <NUM>, CH-Trp), <NUM> (t, <NUM>, J = <NUM>, CH-Trp), <NUM> (d, <NUM>, J = <NUM>, CH-Trp), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH*-Trp), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (d, J = <NUM>, CH*-val), <NUM>- <NUM> (m, <NUM>, CH-val), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-val); <NUM>C NMR (<NUM>, CDCl<NUM>): δ <NUM> (C=O), <NUM> (C=O), <NUM> (C=N), <NUM> (C), <NUM> (C-Trp), <NUM> (CH), <NUM> (C), <NUM> (CH), <NUM> (C-Trp), <NUM> (CH), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C-Trp), <NUM> (CH*-val), <NUM> (CH*-Trp), <NUM> (CH-val), <NUM> (CH<NUM>-Trp), <NUM> (CH<NUM>-val), <NUM> (CH<NUM>-val; (+)-HRMS-ESI m/z: <NUM> (M + H)+ (calculated for C<NUM>H<NUM>N<NUM>O<NUM>Cl<NUM>, <NUM>).

In an embodiment, the characterization of (<NUM>S,<NUM>R)-<NUM>-((<NUM>H-indol-<NUM>-yl)methyl)-<NUM>,<NUM>-dichloro-<NUM>-isobutyl-<NUM>,<NUM>-dihydro-<NUM>H-pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>(<NUM>)-dione (<NUM>) is as follows: Yield: <NUM>, <NUM>%; e. r = <NUM>:<NUM>; mp: <NUM>-<NUM>; <MAT> (c <NUM>; CHCl<NUM>); vmax(KBr) <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>-<NUM>; <NUM>H NMR (<NUM>, DMSO-d<NUM>): <NUM> ( br, <NUM>, NH-indol), δ <NUM> (d, <NUM>, J = <NUM>, CH), <NUM> (d, <NUM>, J = <NUM>, CH), <NUM> (d, <NUM>, J = <NUM>, CH-Trp), <NUM> (d, J = <NUM>, CH-Trp), <NUM> (br, NH-amide), <NUM> (t, <NUM>, J = <NUM>, CH-Trp), <NUM> (t, <NUM>, J = <NUM>, CH-Trp), <NUM> (d, <NUM>, J = <NUM>, CH-Trp), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH*-Trp) , <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, J = <NUM> and <NUM>, CH*-Leu), <NUM>-<NUM> (m, <NUM> CH<NUM>-Leu), <NUM> (tt, <NUM>, J = <NUM> and <NUM>, CH-Leu), <NUM>-<NUM> (m, <NUM>, CH<NUM>-Leu), <NUM> (d, <NUM>, J = <NUM>, CH<NUM>-Leu), <NUM> (d, <NUM>, J = <NUM>, CH<NUM>-Leu); <NUM>C NMR (<NUM>,DMSO-d<NUM>): δ <NUM> (C=O), <NUM> (C=O), <NUM> (C=N), <NUM> (C), <NUM> (C-Trp), <NUM> (CH), <NUM> (C), <NUM> (C), <NUM> (C-Trp), <NUM> (CH), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C-Trp), <NUM> (CH*-Trp), <NUM> (CH*-Leu), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>-Trp), <NUM> (CH-Leu), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>-Leu); (+)-HRMS-ESI m/z: <NUM> (M + H)+, <NUM> (M + Na)+ (calculated for C<NUM>H<NUM>N<NUM>O<NUM>Cl<NUM>, <NUM>; C<NUM>H<NUM>N<NUM>O<NUM>Cl<NUM>Na, <NUM>).

In an embodiment, the characterization of (<NUM>,4R)-<NUM>-((<NUM>-indol-<NUM>-yl)methyl)-<NUM>-((S)-sec-butyl)-<NUM>,<NUM>-dichloro-<NUM>,<NUM>-dihydro-<NUM>-pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>(<NUM>)-dione (<NUM>) is as follows: Yield: <NUM>, <NUM>%; e. r = <NUM>:<NUM>; mp: <NUM>-<NUM>; <MAT> (c <NUM>; CHCl<NUM>); vmax (KBr) <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>-<NUM>; <NUM>H NMR (<NUM>, CDCl<NUM>): δ <NUM>. <NUM> (d, <NUM>, J = <NUM>, CH), <NUM> (br, <NUM>, NH-indol), <NUM> (d, <NUM>, J = <NUM>, CH), <NUM> (d, <NUM>, J = <NUM>, CH-Trp), <NUM> (d, J = <NUM>, CH-Trp), <NUM> (t, <NUM>, J = <NUM>, CH-Trp), <NUM> (t, <NUM>, J = <NUM>, CH-Trp), <NUM> (d, <NUM>, J = <NUM>, CH-Trp), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH*-Trp), <NUM> (s, <NUM>, NH-amide), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (d, J = <NUM>, CH*-Ile), <NUM> (ddd, <NUM>, J = <NUM>, <NUM>, and <NUM>, CH*-Ile), <NUM>-<NUM> ( m, <NUM>, CH<NUM>-Ile), <NUM> (d, <NUM>, J = <NUM>, CH<NUM>-Ile), <NUM> (d, <NUM>, J = <NUM>, CH<NUM>-Ile); <NUM>C NMR (<NUM>, CDCl<NUM>): δ <NUM> (C=O), <NUM> (C=O), <NUM> (C=N), <NUM> (C), <NUM> (C-Trp) <NUM> (CH), <NUM> (C), <NUM> (C), <NUM> (C-Trp), <NUM> (CH), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C-Trp), <NUM> (CH*-Ile), <NUM> (CH*-Trp), <NUM> (CH-Leu), <NUM> (CH<NUM>-Trp), <NUM> (CH<NUM>-Ile), <NUM> (CH<NUM>-Ile), <NUM> (CH<NUM>-Ile; (+)-HRMS-ESI m/z: <NUM> (M + H)+, <NUM> (M + Na)+ (calculated for C<NUM>H<NUM>N<NUM>O<NUM>Cl<NUM>, <NUM>; C<NUM>H<NUM>N<NUM>O<NUM>Cl<NUM>Na, <NUM>).

In an embodiment, the characterization of (<NUM>S,<NUM>R)-<NUM>-((<NUM>H-indol-<NUM>-yl)methyl)-<NUM>-iodo-<NUM>-isopropyl-<NUM>,<NUM>-dihydro-<NUM>H-pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>(<NUM>H)-dione (<NUM>) is as follows: Yield: <NUM>, <NUM>%; e. r = <NUM>:<NUM>; mp: <NUM>-<NUM>; <MAT> (c <NUM> CHCl<NUM>); vmax (KBr) <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>-<NUM>; <NUM>H NMR (<NUM>, CDCl<NUM>): δ <NUM>. <NUM> (d, <NUM>, J = <NUM>, CH), <NUM> (br, <NUM>, NH-indol), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH), <NUM> (d, <NUM>, J = <NUM>, CH-Trp), <NUM> (d, J = <NUM>, CH) <NUM> (d, J = <NUM>, CH-Trp), <NUM> (ddd, <NUM>, J = <NUM>, <NUM> and <NUM>, CH-Trp), <NUM> (ddd, <NUM>, J = <NUM>, <NUM> and <NUM>, CH-Trp), <NUM> (d, <NUM>, J = <NUM>, CH-indol), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH*-Trp), <NUM> (s, <NUM>, NH-amide), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (d, J = <NUM>, CH*-val), <NUM>-<NUM> (m, <NUM>, CH-val), <NUM> (d, <NUM>, J = <NUM>, CH<NUM>-val); <NUM>C NMR (<NUM>, CDCl<NUM>): δ <NUM> (C=O), <NUM> (C=O), <NUM> (C=N), <NUM> (C), <NUM> (CH), <NUM> (C-Trp <NUM> (CH), <NUM> (CH), <NUM> (C-Trp), <NUM> (CH-indol), <NUM> (CH-Trp), <NUM> (C), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C-indol), <NUM> (C), <NUM> (CH*-val), <NUM> (CH*-Trp) <NUM> (CH-val), <NUM> (CH<NUM>-Trp), <NUM> (CH<NUM>-val), <NUM> (CH<NUM>-val); (+)-HRMS-ESI m/z: <NUM> (M + H)+ (calculated for C<NUM>H<NUM>N<NUM>O<NUM>I, <NUM>).

In an embodiment, the characterization of (<NUM>S,<NUM>R)-<NUM>-((<NUM>H-indol-<NUM>-yl)methyl)-<NUM>-bromo-<NUM>-isopropyl-<NUM>,<NUM>-dihydro-<NUM>-pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>(<NUM>)-dione (<NUM>) is as follows: Yield: <NUM>, <NUM>%; e. r = <NUM>:<NUM>; mp: <NUM>-<NUM>; <MAT> (c <NUM>; CHCl<NUM>); vmax (KBr) <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>-<NUM>; <NUM>H NMR (<NUM>, CDCl<NUM>): δ <NUM>. <NUM> (d, <NUM>, J = <NUM>, CH), <NUM> (br, <NUM>, NH-indol), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH), <NUM> (, J = <NUM>, CH), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH-Trp (<NUM>)), <NUM> (ddd, <NUM>, J = <NUM>, <NUM> and <NUM>, CH-Trp), <NUM> (ddd, <NUM>, J = <NUM>, <NUM> and <NUM>, CH-Trp), <NUM> (d, <NUM>, J = <NUM>, CH-Trp), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH*-Trp), <NUM> (s, <NUM>, NH-amide), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (d, J = <NUM>, CH*-val), <NUM> (m, <NUM>, CH-val), <NUM> (d, <NUM>, J = <NUM>, CH<NUM>-val), <NUM> (d, <NUM>, J = <NUM>, CH<NUM>-val); <NUM>C NMR (<NUM>, CDCl<NUM>): δ <NUM> (C=O), <NUM> (C=O), <NUM> (C=N), <NUM> (C), <NUM> (CH), <NUM>. (C-Trp), <NUM> (C), <NUM> (CH), <NUM> (C-trp), <NUM> (CH-indol), <NUM> (CH-Trp), <NUM> (C), <NUM> (CH-Trp), <NUM> (C), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C-Trp), <NUM> (CH*-trp), <NUM> (CH*-val), <NUM> (CH-val), <NUM> (CH<NUM>-Trp), <NUM> (CH<NUM>-val), <NUM> (CH<NUM>-val); (+)-HRMS-ESI m/z: <NUM> (M + H)+, <NUM> (M + Na)+ (calculated for C<NUM>H<NUM>N<NUM>O<NUM>Br: <NUM>; C<NUM>H<NUM>N<NUM>O<NUM>BrNa: <NUM>).

In an embodiment, the characterization of (<NUM>,4R)-<NUM>-((<NUM>H-indol-<NUM>-yl)methyl)-<NUM>-iodo-<NUM>-isobutyl-<NUM>,<NUM>-dihydro-<NUM>H-pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>(<NUM>)-dione (<NUM>) is as follows: Yield: <NUM>, <NUM>%; e. r = <NUM>:<NUM>; mp: <NUM>-<NUM>; <MAT> (c <NUM>; CHCl<NUM>); vmax (KBr) <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>-<NUM>; <NUM>H NMR (<NUM>, CDCl<NUM>): δ <NUM>. <NUM> (d, <NUM>, J = <NUM>, CH), <NUM> (br, <NUM>, NH-indol), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH), <NUM> (d, J = <NUM>, CH-Trp), <NUM> (d, <NUM>, J = <NUM>, CH), <NUM> (d, J = <NUM>, CH-Trp), <NUM> (t, <NUM>, J = <NUM>, CH-Trp), <NUM> (t, <NUM>, J = <NUM>, CH-Trp), <NUM> (d, <NUM>, J = <NUM>, CH-Trp), <NUM> (s, <NUM>, NH-amide), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH*-Trp) , <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, J = <NUM> and <NUM>, CH*-Leu), <NUM>-<NUM> (m, <NUM>, CH-Leu), <NUM>-<NUM> ( m, <NUM>, CH<NUM>-Leu), <NUM> (d, <NUM>, J = <NUM>, CH<NUM>-Leu), <NUM> (d, <NUM>, J = <NUM>, CH<NUM>-Leu); <NUM>C NMR (<NUM>, CDCl<NUM>): δ <NUM> (C=O), <NUM> (C=O), <NUM> (C=N), <NUM> (C), <NUM> (CH), <NUM> (C-Trp), <NUM> (C), <NUM> (CH), <NUM> (C-Trp), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C-Trp), <NUM> (C), <NUM> (CH*-Trp), <NUM> (CH*-Leu), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>-Trp), <NUM> (CH-Leu), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>-Leu); (+)-HRMS-ESI m/z: <NUM> (M + H)+, <NUM> (M + Na)+ (calculated for C<NUM>H<NUM>N<NUM>O<NUM>I, <NUM>; C<NUM>H<NUM>N<NUM>O<NUM>INa, <NUM>).

In an embodiment, the characterization of (<NUM>S,<NUM>R)-<NUM>-((<NUM>H-indol-<NUM>-yl)methyl)-<NUM>-bromo-<NUM>-isobutyl-<NUM>,<NUM>-dihydro-<NUM>-pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>(<NUM>H)-dione (<NUM>) is as follows: Yield: <NUM>, <NUM>%; e. r = <NUM>:<NUM>; mp: <NUM>-<NUM>; <MAT> (c <NUM>; CHCl<NUM>); vmax (KBr) <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>-<NUM>; <NUM>H NMR (<NUM>, CDCl<NUM>): δ <NUM>. <NUM> (d, <NUM>, J = <NUM>, CH), <NUM> (br, <NUM>, NH-indol), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH-Trp (<NUM>)), <NUM> (d, J = <NUM>, CH-Trp), <NUM> (t, <NUM>, J = <NUM>, CH-Trp), <NUM> (t, <NUM>, J = <NUM>, CH-Trp), <NUM> (d, <NUM>, J = <NUM>, CH-Trp), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH*-Trp), <NUM> (s, <NUM>, NH-amide), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, J = <NUM> and <NUM>, CH*-Leu), <NUM>-<NUM> (m, <NUM>, CH-Leu), <NUM>-<NUM> ( m, <NUM>, CH<NUM>-Leu), <NUM> (d, <NUM>, J = <NUM>, CH<NUM>-Leu), <NUM> (d, <NUM>, J = <NUM>, CH<NUM>-Leu); <NUM>C NMR (<NUM>, CDCl<NUM>): δ <NUM> (C=O), <NUM> (C=O), <NUM> (C=N), <NUM> (C), <NUM> (CH), <NUM> (C-Trp), <NUM> (C), <NUM> (CH), <NUM> (C-Trp), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C), <NUM> (CH), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C-Trp), <NUM> (CH*-Trp), <NUM> (CH*-Leu), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>-Trp), <NUM> (CH-Leu), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>-Leu); (+)-HRMS-ESI m/z: <NUM> (M + H)+, <NUM> (M + Na) + (calculated for C<NUM>H<NUM>N<NUM>O<NUM>Br, <NUM>; C<NUM>H<NUM>N<NUM>O<NUM>BrNa, <NUM>).

In an embodiment, the characterization of (<NUM>S,<NUM>R)-<NUM>-((<NUM>-indol-<NUM>-yl)methyl)-<NUM>,<NUM>-diiodo-<NUM>-isobutyl-<NUM>,<NUM>-dihydro-<NUM>-pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>(<NUM>)-dione (<NUM>) is as follows: Yield: <NUM>, <NUM>%; e. r = <NUM>:<NUM>; mp: <NUM>-<NUM>; <MAT> (c <NUM>; CHCl<NUM>); vmax (KBr) <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>-<NUM>; <NUM>H NMR (<NUM>, DMSO-d<NUM>): <NUM> ( br, <NUM>, NH-indol), δ <NUM>. <NUM> (d, <NUM>, J = <NUM>, CH), <NUM> (d, <NUM>, J = <NUM>, CH), <NUM> (d, <NUM>, J = <NUM>, CH-Trp), <NUM> (d, J = <NUM>, CH-Trp), <NUM> (br, NH-amide), <NUM> (t, <NUM>, J = <NUM>, CH-Trp), <NUM> (t, <NUM>, J = <NUM>, CH-Trp), <NUM> (d, <NUM>, J = <NUM>, CH-indol), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH*-Trp) , <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, J = <NUM> and <NUM>, CH*-Leu), <NUM>-<NUM>\<NUM> (m, <NUM>, CH<NUM>-Leu), <NUM>-<NUM> (m, <NUM> CH<NUM>-Leu), <NUM>-<NUM> (m, <NUM>, J = <NUM> and <NUM>, CH<NUM>-Leu), <NUM> (t, <NUM>, J = <NUM>, CH<NUM>-Leu), <NUM> (d, <NUM>, J = <NUM>, CH<NUM>-Leu); <NUM>C NMR (<NUM>,DMSO-d<NUM>): δ <NUM> (C=O), <NUM> (C=O), <NUM> (C=N), <NUM> (C), <NUM> (C-Trp), <NUM> (C), <NUM> (C-Trp), <NUM> (CH-Trp), <NUM> (C), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C-Trp), <NUM> (CH), <NUM> (CH), <NUM> (CH*-Trp), <NUM> (CH*-Leu), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>-Trp), <NUM> (CH-Leu), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>-Leu); (+)-HRMS-ESI m/z: <NUM> (M + H)+, <NUM> (M + Na)+ (calculated for C<NUM>H<NUM>N<NUM>O<NUM>Cl<NUM>, <NUM>; C<NUM>H<NUM>N<NUM>O<NUM>Cl<NUM>Na, <NUM>).

The quantitative analysis of enantioselective liquid chromatography was carried out as follows. Compounds <NUM>-<NUM> were prepared using HPLC grades n-hexane:EtOH (<NUM>:<NUM>) at a final concentration <NUM>µg/mL. The HPLC system comprised a JASCO model <NUM>-PU intelligent HPLC pump (JASCO corporation, Tokyo, Japan), equipped with a <NUM> injector (Rheodyne LCC, Rohnert Park, CA, USA) fitted with a <NUM>µL LC loop, a JASCO model <NUM>-<NUM> solvent mixer involving a <NUM>-UV intelligent UV/VIS detector, a system equipped with a chiral column (Lux® <NUM> Amylose-<NUM>, <NUM> × <NUM>). The data acquisition was performed using ChromNAC chromatography Data system (version <NUM>. <NUM>) from JASCO Corporation (Tokyo, Japan). The mobile phase consisted of the mixture of n-hexane:EtOH (<NUM>:<NUM>, v/v), at a flow rate of <NUM>/min. The mobile phase was prepared in a volume/volume ratio and degassed in an ultrasonic bath for at least <NUM> before use. The chromatographic analyses were carried out in isocratic mode at <NUM> ± <NUM>, in duplicate. The UV detection was performed at a wavelength of <NUM>. The volume void time was considered to be equal to the peak of solvent front and was taken from each particular run. The enantiomeric ratio (e. r) were determined by the mean percentage of peak area of eluted peaks.

The semipreparative enantioselective resolution was as follows. Compound <NUM>, <NUM> and <NUM> were prepared in the mixture of HLCP grade solvent n-hexane:EtoH (<NUM>:<NUM>) at the concentration <NUM>/mL, and the injection volume was <NUM>-<NUM>µL. The HPLC system is similar to what described in quantitative analysis equipped with an in-house column amylose tris-<NUM>,<NUM>-dimethylphenylcarbamate coated with Nucleosil (<NUM> A, <NUM>, <NUM>%, w/w) packed into a stainless-steel (<NUM> x <NUM> I. size) column, prepared in the UFSCar laboratory. Semipreparative chromatographic separations were first achieved through multiple injection with <NUM>µL at a flow rate of <NUM>/min. The chromatographic analyses were carried out in isocratic mode at <NUM> ± <NUM>. The UV detection was performed at a wavelength of <NUM>. The fraction collected was analyzed using the analytical column to determine their enantiomeric ratio/excess with the condition described above.

Chemistry for the <NUM>th approach. Regarding the fourth approach of indole-containing pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>-diones <NUM>-<NUM>, the compounds were also prepared via the highly effective and environmentally friendly microwave-assisted multicomponent polycondensation of amino acids. This methodology allowed us to prepare the fourth approach of pyrazinoquinazoline alkaloids through treatment of the anthranilic acid (<NUM>) derivatives with N-Boc-L-amino acids (<NUM>) and (PhO)<NUM>P at <NUM> for <NUM>-<NUM>. Thereafter, D-tryptophan methyl ester hydrochloride (<NUM>) was added, and the mixture was stirred under microwave irradiation (<NUM> W) at <NUM> for <NUM> to furnish the final products <NUM>-<NUM> (Table <NUM>).

In the present disclosure, the general conditions for the synthesis of quinazolinone-<NUM>,<NUM>-(<NUM>)-diones compounds <NUM>-<NUM> is as follows. In a closed vial, <NUM>-hydroxy-anthranilic acid (51a, <NUM>, <NUM> mmol) for <NUM>, <NUM>-methyl-anthranilic acid (51b, <NUM>, <NUM> mmol) for <NUM>, <NUM>-methoxy-anthranilic acid (51c, <NUM>, <NUM> mmol) for <NUM>, <NUM>-aminoisonicotinic acid (51d, <NUM>, <NUM> mmol) for <NUM>, <NUM>-aminoisonicotinic acid (51e, <NUM>, <NUM>µmol) for <NUM>, <NUM>-triazole-anthranilic acid (51f, <NUM>, <NUM> mmol) for <NUM>, or <NUM>-aminoordotic acid (<NUM>, <NUM>, <NUM> mmol) for <NUM>, or anthranilic acid (<NUM>, <NUM>, <NUM> mmol) for <NUM> with N-Boc-L-leucine (52a, <NUM>, <NUM> mmol) for <NUM>-<NUM> or N-Boc-L-tryptophan, (52b, <NUM>,<NUM> mmol) for <NUM> and triphenyl phosphite (<NUM>µL, <NUM> mmol) were added along with <NUM> of dried pyridine. The vial was heated in heating block with stirring at <NUM> for <NUM>-<NUM>. After cooling the mixture to room temperature, D-tryptophan methyl ester hydrochloride (<NUM>, <NUM>, <NUM> mmol) was added, and the mixture was divided into <NUM> individual vials, and irradiated in the microwave at the constant temperature at <NUM> for <NUM>. After removing the solvent with toluene, the crude product was purified by flash column chromatography using n-hexane: EtOAc (<NUM>:<NUM>) as a mobile phase. The preparative TLC was performed using CH<NUM>Cl<NUM>:Me<NUM>CO (<NUM>:<NUM>) as mobile phase. The major compound appeared as a black spot with no fluorescence under the UV light. The desirable compounds <NUM>-<NUM> were collected as yellow solids. Before analysis, compounds were recrystallized from methanol.

In an embodiment, the characterization of (<NUM>,4R)-<NUM>-((<NUM>H-indol-<NUM>-yl)methyl)-<NUM>-hydroxy-<NUM>-isobutyl-<NUM>,<NUM>-dihydro-<NUM>-pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>(<NUM>)-dione (<NUM>) is as follows: Yield: <NUM>, <NUM> %; mp: <NUM>-<NUM> (MeOH); <MAT> (c <NUM>; CHCl<NUM>); vmax (KBr) <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>-<NUM>; <NUM>H NMR (<NUM>, CDCl<NUM> ): δ <NUM> (d, <NUM>, J = <NUM>, CH), <NUM> (s, <NUM>, NH-Trp), <NUM> (d, <NUM>, J = <NUM>, OH), <NUM> (d, <NUM>, J <NUM>, CH(<NUM>)), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH-Trp (<NUM>)), <NUM> (t, <NUM>, J <NUM>, CH-Trp), <NUM> (t, <NUM>, J <NUM>, CH-Trp), <NUM> (d, <NUM>, J <NUM>, CH-Trp), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH*-Trp), <NUM> (s, <NUM>, NH-amide), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH*-Leu), <NUM>-<NUM> (m, <NUM>, CH-Leu), <NUM>-<NUM> (m, <NUM>, CH<NUM>-Leu) <NUM> (d, <NUM>, J = <NUM>, CH<NUM>-Leu), <NUM> (d, <NUM>, J = <NUM>, CH<NUM>-Leu) ; <NUM>C NMR (<NUM>, Acetone d<NUM>): δ <NUM> (C=O), <NUM> (C=O), <NUM> (C-OH),<NUM> (C=N), <NUM> (C), <NUM> (C-Trp), <NUM> (CH), <NUM> (C-Trp), <NUM> (CH-Trp), <NUM> (CH), <NUM> (CH-Trp), <NUM> (C), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM>(CH-Trp), <NUM> (CH), <NUM> (C-Trp), <NUM> (CH*-Trp), <NUM> (CH*-Leu), <NUM> (CH-Leu), <NUM> (CH<NUM>-Trp), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>-Leu).

In an embodiment, the characterization of (<NUM>,4R)-<NUM>-((<NUM>H-indol-<NUM>-yl)methyl)-<NUM>-isobutyl-<NUM>-methyl-<NUM>,<NUM>-dihydro-<NUM>-pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>(<NUM>)-dione (<NUM>) is as follows: Yield: <NUM>, <NUM>%; mp:<NUM>-<NUM> (MeOH); <MAT> (c <NUM> CHCl<NUM>); vmax (KBr) <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>-<NUM>; <NUM>H NMR (<NUM>, CDCl<NUM>): δ <NUM>. <NUM> (s, <NUM>, CH), <NUM> (br, <NUM>, NH-Trp), <NUM> (dd, <NUM>, J <NUM>, <NUM>, CH), <NUM> (d, <NUM>, J <NUM>, CH & CH-Trp), <NUM> (d, <NUM>, J <NUM>, CH-Trp), <NUM> (t, <NUM>, J <NUM>, CH-Trp), <NUM> (t, <NUM>, J <NUM>, CH-Trp), <NUM> (d, <NUM>, J <NUM>, CH-Trp), <NUM> (s, <NUM>, NH-amide), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH*-Trp), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH*-Leu), <NUM> (s, <NUM>, CH<NUM>), <NUM> (ddd, <NUM>, J <NUM>, <NUM>, and <NUM>, CH-Leu), <NUM>-<NUM> (m, <NUM>, CH<NUM>-Leu), <NUM> (d, <NUM>, J <NUM>, CH<NUM>-Leu), <NUM> (d, <NUM>, J <NUM>, CH<NUM>-Leu) ; <NUM>C NMR (<NUM>, CDCl<NUM>): δ <NUM> (C=O), <NUM> (C=O), <NUM> (C=N), <NUM> (C), <NUM> (C), <NUM> (CH), <NUM>(C-Trp), <NUM> (CH), <NUM> (CH), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C-Trp), <NUM> (C*-Trp), <NUM> (C*-Leu), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>-Trp), <NUM>. <NUM> (CH-Leu), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>), <NUM> (CH<NUM>-Leu).

In an embodiment, the characterization of (<NUM>S,<NUM>R)-<NUM>-((<NUM>-indol-<NUM>-yl)methyl)-<NUM>-isobutyl-<NUM>-methoxy-<NUM>,<NUM>-dihydro-<NUM>-pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>(<NUM>H)-dione (<NUM>) is as follows: Yield: <NUM>, <NUM> %; mp: <NUM>-<NUM> (MeOH); <MAT> (c <NUM>; CHCl<NUM>); vmax (KBr) <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>-<NUM>; <NUM>H NMR (<NUM>, DMSO-d<NUM>): δ <NUM> (s, <NUM>, NH-Trp), <NUM> (d, <NUM>, J <NUM>, CH), <NUM> (d, <NUM>, J <NUM>,CH-Trp), <NUM> (d, <NUM>, J <NUM>, CH), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH), <NUM> (d, <NUM>, J <NUM>, CH-Trp), <NUM> (s, <NUM>, NH-amide), <NUM> (t, <NUM>, J <NUM>, CH-Trp), <NUM> (t, <NUM>, J <NUM>, CH-Trp), <NUM> (d, <NUM>, J <NUM>, CH-Trp), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH*-Trp), <NUM> (s, <NUM>, O-CH<NUM>), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH*-Leu), <NUM>-<NUM> (m, <NUM>, CH-Leu), <NUM>-<NUM> (m, <NUM>, CH<NUM>-Leu), <NUM> (d, J <NUM>, CH<NUM>-Leu), <NUM> (d, <NUM>, J <NUM>, CH<NUM>-Leu); <NUM>C NMR (<NUM>, DMSO-d<NUM>): δ <NUM> (C=O), <NUM> (C=O), <NUM> (C-O),<NUM> (C=N), <NUM> (C), <NUM> (C), <NUM> (CH), <NUM> (C-Trp), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C), <NUM> (CH-Trp), <NUM> (CH), <NUM> (CH-Trp),<NUM> (CH-Trp), <NUM> (C-Trp), <NUM> (C*-Trp), <NUM> (CH<NUM>), <NUM> (C*-Leu), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>-Trp), <NUM> (CH-Leu), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>-Leu).

In an embodiment, the characterization of (<NUM>,4R)-<NUM>-((<NUM>H-indol-<NUM>-yl)methyl)-<NUM>-isobutyl-<NUM>,<NUM>-dihydro-<NUM>H-pyrazino[<NUM>,<NUM>-a]pyrido[<NUM>,<NUM>-d]pyrimi dine-<NUM>,<NUM>(<NUM>H)-dione (<NUM>) is as follows: Yield: <NUM>, <NUM> %; mp: <NUM>-<NUM> (MeOH); <MAT> (c <NUM>; CHCl<NUM>); vmax (KBr) <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>-<NUM>; <NUM>H NMR (<NUM>, CDCl<NUM>): δ <NUM> (s, <NUM>, CH), <NUM> (d, <NUM>, J <NUM>, CH), <NUM> (s, <NUM>, NH-Trp), <NUM> (d, J <NUM>, CH), <NUM> (d, <NUM>, J <NUM>, CH-Trp), <NUM> (d, <NUM>, J <NUM>, CH-Trp), <NUM>(dt, <NUM>, J <NUM> and0. <NUM>, CH-Trp), <NUM> (dt, <NUM>, J <NUM> and <NUM>, CH-Trp), <NUM> (d, <NUM>, J <NUM>, CH-Trp), <NUM>(s, <NUM>, NH-amide), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH*-Trp), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH*-Leu), <NUM>-<NUM> (m, <NUM>, CH-Leu), <NUM>-<NUM> (m, <NUM>, CH<NUM>-Leu), <NUM> (d, J <NUM>, CH<NUM>-Leu), <NUM> (d, <NUM>, J <NUM>, CH<NUM>-Leu); <NUM>C NMR (<NUM>, CDCl<NUM>): δ <NUM> (C=O), <NUM> (C=O), <NUM> (C=N), <NUM> (CH), <NUM> (CH), <NUM> (C-Trp), <NUM> (C-Trp), <NUM> (CH-Trp),<NUM> (CH-Trp),<NUM> (CH-Trp), <NUM> (CH), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C-Trp), <NUM> (C*-Trp), <NUM> (C*-Leu), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>-Trp), <NUM> (CH-Leu) <NUM> (CH-Leu), <NUM> (CH<NUM>-Leu).

In an embodiment, the characterization of (<NUM>R,<NUM>S)-<NUM>-((<NUM>H-indol-<NUM>-yl)methyl)-<NUM>-isobutyl-<NUM>,<NUM>-dihydro-<NUM>H-pyrazino[<NUM>,<NUM>-a]pyrido[<NUM>,<NUM>-d] pyrimidine-<NUM>,<NUM>(<NUM>H)-dione (<NUM>) is as follows: Yield: <NUM>, <NUM>%; mp: <NUM>-<NUM> (MeOH); <MAT> (c <NUM>; CHCl<NUM>); vmax (KBr) <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>-<NUM>; <NUM>H NMR (<NUM>, CDCl<NUM>): δ <NUM> (d, <NUM>, J <NUM>, CH), <NUM> (d, <NUM>, J <NUM>, CH), <NUM> (s, <NUM>,NH-Trp), <NUM> (d, <NUM>, J <NUM>, CH-Trp), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH), <NUM> (d, <NUM>, J <NUM>, CH-Trp), <NUM> (dt, <NUM>, J <NUM> and <NUM>, CH-Trp), <NUM> (dt, <NUM>, J <NUM> and <NUM>, CH-Trp), <NUM> (d, <NUM>, J <NUM>, CH-Trp), <NUM> (s, <NUM>, NH-amide), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH*-Trp), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH*-Leu), <NUM>-<NUM> (m, <NUM>, CH-Leu), <NUM>-<NUM> (m, <NUM>, CH<NUM>-Leu), <NUM> (d, J <NUM>, CH<NUM>-Leu), <NUM> (d, <NUM>, J <NUM>, CH<NUM>-Leu); <NUM>C NMR (<NUM>, CDCl<NUM>): δ <NUM> (C=O), <NUM> (C=O), <NUM> (C=N), <NUM> (CH), <NUM> (CH), <NUM> (C-Trp), <NUM> (C-Trp), <NUM> (CH-Trp),<NUM> (CH-Trp), <NUM> (CH), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C-Trp), <NUM> (C*-Trp), <NUM> (C*-Leu), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>-Trp), <NUM> (CH-Leu) <NUM> (CH-Leu), <NUM> (CH<NUM>-Leu).

In an embodiment, the characterization of (<NUM>S,<NUM>R)-<NUM>-((<NUM>H-indol-<NUM>-yl)methyl)-<NUM>-isobutyl-<NUM>-(<NUM>-methyl-<NUM>H-tetrazol-<NUM>-yl)-<NUM>,<NUM>-dihydro-<NUM>-pyrazino [<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>(<NUM>)-dione (<NUM>) is as follows: Yield: <NUM>, <NUM> %; mp: <NUM>-<NUM> (MeOH); <MAT> (c <NUM>; CHCl<NUM>); vmax (KBr) <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>-<NUM>; <NUM>H NMR (<NUM>, CDCl<NUM>): δ <NUM> (d, <NUM>, J <NUM>, CH), <NUM> (d, <NUM>, J <NUM>, CH), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH), <NUM> (s, <NUM>, NH-Trp), <NUM> (d, <NUM>, J <NUM>, CH-Trp), <NUM> (d, <NUM>, J <NUM>, CH-Trp), <NUM> (t, <NUM>, J <NUM>, CH-Trp), <NUM> (d, <NUM>, J <NUM>, CH-Trp), <NUM> (d, <NUM>, J <NUM>, CH-Trp), <NUM> (s, <NUM>, NH-amide), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH*-Trp), <NUM> (s, <NUM>, CH<NUM>-N), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH*-Leu),<NUM>-<NUM> (m, <NUM>, CH-Leu), <NUM>-<NUM> (m, <NUM>, CH<NUM>-Leu), <NUM> (d, <NUM>, J <NUM>, CH<NUM>-Leu), <NUM> (d, <NUM>, J <NUM>, CH<NUM>-Leu); <NUM>C NMR (<NUM>, CDCl<NUM>): δ <NUM> (C=O), <NUM> (C=N-Tetrazol), <NUM> (C=O), <NUM> (C=N),<NUM> (C), <NUM> (C-Trp), <NUM> (C-Ctriazole), <NUM> (CH), <NUM> (C-Trp), <NUM> (CH), <NUM> (CH), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C), <NUM> (CH-Trp),<NUM> (CH-Trp), <NUM>(CH-Trp), <NUM> (C-Trp), <NUM> (CH*-Trp), <NUM> (CH*-Leu), <NUM> (CH-Leu), <NUM> (CH<NUM>), <NUM> (CH<NUM>-Trp), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>-Leu).

In an embodiment, the characterization of (<NUM>S,<NUM>R)-<NUM>-((<NUM>-indol-<NUM>-yl)methyl)-<NUM>-isobutyl-<NUM>,<NUM>-dihydro-<NUM>-pyrimido[<NUM>,<NUM>-d]pyrimidine-<NUM>,<NUM>,<NUM>,<NUM> (<NUM>H,<NUM>H)-tetraone (<NUM>) is as follows: Yield: <NUM>, <NUM> %; mp: <NUM>-<NUM> (MeOH); <MAT> (c <NUM>; CHCl<NUM>); vmax (KBr) cm-<NUM>; <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>; <NUM>H NMR (<NUM>, CDCl<NUM>): δ <NUM> (s, <NUM>, NH-Trp), <NUM> (d, <NUM>, J <NUM>, CH-Trp), <NUM> (d, <NUM>, J <NUM>, CH-Trp),<NUM> (dd, J <NUM> and <NUM>, CH-Trp), <NUM>-<NUM> (m, <NUM>, CH-Trp), <NUM> (d, <NUM>, J <NUM>, CH-Trp), <NUM> (s, <NUM>, NH-amide), <NUM> (s, <NUM>, NH-Ant), <NUM> (s, <NUM>, NH-Ant), <NUM> (m, <NUM>,CH*-Trp), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (m, <NUM>, CH*-Leu), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH<NUM>-Trp), <NUM> (m, <NUM>, CH-Leu),<NUM> (m, <NUM>, CH<NUM>-Leu), <NUM> (d, J <NUM>, CH<NUM>-Leu), <NUM> (d, <NUM>, J <NUM>, CH<NUM>-Leu); <NUM>C NMR (<NUM>, CDCl<NUM>): δ <NUM> (C=O), <NUM> (C=O), <NUM> (C=O), <NUM> (C=O), <NUM> (C=N), <NUM><NUM> (C), <NUM> (C-Trp), <NUM> (C-Trp), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C),<NUM> (CH-Trp), <NUM> (CH-Trp),<NUM> (CH-Trp), <NUM> (C-Trp), <NUM> (C*-Trp), <NUM> (C*-Leu), <NUM> (CH<NUM>-Leu), <NUM> (CH<NUM>-Trp), <NUM> (CH-Leu), <NUM> (CH-Leu), <NUM> (CH<NUM>-Leu).

In an embodiment, the characterization of (<NUM>,4R)-<NUM>,<NUM>-bis((<NUM>-indol-<NUM>-yl)methyl)-<NUM>,<NUM>-dihydro-<NUM>-pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>(<NUM>)-dione (<NUM>) is as follows: Yield: <NUM>, <NUM>%; er = <NUM>:<NUM>; mp: <NUM>-<NUM> (MeOH); <MAT> (c <NUM>; CHCl<NUM>); vmax (KBr): <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>-<NUM>; <NUM>H NMR (<NUM>, CDCl<NUM>): δ <NUM> (dd, <NUM>, J8. <NUM> and <NUM>, CH), <NUM> (s, <NUM>, NH-Trp), <NUM> (s, <NUM>, NH-Trp), <NUM> (ddd, <NUM>, J <NUM>, <NUM> and <NUM>, CH), <NUM> (d, <NUM>, J <NUM>, CH), <NUM> (ddd, <NUM>, J <NUM>, <NUM> and <NUM>, CH), <NUM> (d, <NUM>, J <NUM>, CH-Trp), <NUM> (d, <NUM>, J <NUM>, CH-Trp), <NUM> (ddd, <NUM>, J <NUM>, <NUM><NUM>, CH-Trp), <NUM> (t, <NUM>, J <NUM>, CH-Trp), <NUM> (ddd, <NUM>, J <NUM>, <NUM><NUM>, CH-Trp), <NUM> (t, <NUM> J <NUM>, CH-Trp), <NUM> (d, <NUM> J <NUM>, CH-Trp), <NUM> (d, <NUM> J <NUM>, CH-Trp), <NUM> (s, <NUM>, NH-amide), <NUM> (dd, <NUM>, J <NUM>, <NUM>, CH*-Trp), <NUM> (dd, J <NUM>, CH*-Trp), <NUM> (dd, <NUM>, J <NUM> and <NUM>, CH<NUM>-Trp ), <NUM> (dd, <NUM>, J <NUM> and <NUM>- CH2-Trp), <NUM> (dd, <NUM> and <NUM>, CH<NUM>-Trp);<NUM>C NMR (<NUM>, CDCl<NUM>): δ <NUM> (C=O), <NUM> (C=O), <NUM>, (C=N), <NUM> (C), <NUM> (C-Trp), <NUM> (C-Trp), <NUM> (CH), <NUM> (CH), <NUM> (CH), <NUM> (CH), <NUM> (C-Trp), <NUM> (C-Trp), <NUM> (CH), <NUM> (CH), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C-Trp(<NUM>)), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (CH-Trp), <NUM> (C-Trp), <NUM> (CH*-Trp), <NUM> (CH*-Trp), <NUM> (CH<NUM>-Trp), <NUM> (CH<NUM>-Trp).

In the present disclosure, all reagents were from analytical grade. Dried pyridine and triphenylphosphite were purchased from Sigma (Sigma-Aldrich Co. , Gillinghan, UK). Anthranilic acids (<NUM>) and protected amino acids <NUM> and <NUM> were purchased from TCI (Tokyo Chemical Industry Co. , Chuo-ku, Tokyo, Japan). Column chromatography purifications were performed using flash silica Merck <NUM>, <NUM>-<NUM> mesh (EMD Millipore corporation, Billerica, MA, USA) and preparative TLC was carried out on precoated plates Merck Kieselgel <NUM> F<NUM> (EMD Millipore corporation, Billerica, MA, USA), spots were visualized with UV light (Vilber Lourmat, Marne-la-Vallée, France). Melting points were measured in a Köfler microscope and are uncorrected. Infrared spectra were recorded in a KBr microplate in a FTIR spectrometer Nicolet iS10 from Thermo Scientific (Waltham, MA, USA) with Smart OMNI-Transmission accessory (Software <NUM> OMNIC <NUM>). <NUM>H and <NUM>C NMR spectra were recorded in CDCl<NUM> (Deutero GmbH, Kastellaun, Germany) at room temperature unless otherwise mentioned on Bruker AMC instrument (Bruker Biosciences Corporation, Billerica, MA, USA), operating at <NUM> for <NUM>H and <NUM> for <NUM>C). Carbons were assigned according to HSQC and or HMBC experiments. Optical rotation was measured at <NUM> using the ADP <NUM> polarimeter (Bellingham + Stanley Ltd. , Tunbridge Wells, Kent, UK), using the emission wavelength of sodium lamp, concentrations are given in g/<NUM>. High resolution mass spectra (HRMS) were measured on a Bruker FTMS APEX III mass spectrometer (Bruker Corporation, Billerica, MA, USA) recorded as ESI (Electrospray) made in Centro de Apoio Cientifico e Tecnolóxico á Investigation (CACTI, University of Vigo, Pontevendra, Spain). The purity of synthesized compounds was determined by reversed-phase LC with diode array detector (DAD) using C18 column (Kimetex®, <NUM> EV0 C18 <NUM>Å, <NUM> × <NUM>), the mobile phase was methanol: water (<NUM>:<NUM>), and the flow rate was <NUM>/min. Enantiomeric ratio was determined by enantioselective LC (LCMS-2010EV, Shimadzu, Lisbon, Portugal), employing a system equipped with a chiral column (Lux® <NUM> Amylose-<NUM>, <NUM> × <NUM>) and UV-detection at <NUM>, mobile phase was hexane:EtOH (<NUM>:<NUM>) and the flow rate was <NUM>/min. for semipreparative chromatography, a HLPC system consisted of a Shimadzu LC-6AD pump with a <NUM>µL loop was used with an amylose tris-<NUM>,<NUM>-dimethylphenylcarbamate coated with Nucleosil (<NUM> A, <NUM>, <NUM>%, w/w) packed into a stainless-steel (<NUM> x <NUM> I. size) column, prepared in the UFSCar laboratory39A.

The present disclosure also relates to antibacterial activity of the compounds herein disclosed.

In the present disclosure, two Gram-positive - Staphylococcus aureus ATCC <NUM> and Enterococcus faecalis ATCC <NUM>- and two Gram-negative - Escherichia coli ATCC <NUM> and Pseudomonas aeruginosa ATCC <NUM> - reference bacterial strains were used. When it was possible to determine a minimal inhibitory concentration (MIC) value for these strains, clinically relevant strains were also used. These included methicillin-resistant S. aureus (MRSA) <NUM>/<NUM>, isolated from public buses, as well as a isolate sensitive to the most commonly used antibiotic families (S. aureus <NUM>/<NUM>/<NUM>); and two vancomycin-resistant Enterococcus (VRE) strains isolated from river water, E. faecalis B3/<NUM> and E. faecalis A5/<NUM>, which is sensitive to ampicillin. Frozen stocks of all strains were grown on Mueller-Hinton agar (MH - BioKar Diagnostics, Allone, France) at <NUM> for <NUM>. All bacterial strains were sub-cultured on MH agar and incubated overnight at <NUM> before each assay, in order to obtain fresh cultures.

An initial screening of the antibacterial activity of the compounds was performed by the Kirby-Bauer disk diffusion method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Briefly, sterile <NUM> blank paper disks (Oxoid, Basingstoke, England) impregnated with <NUM>µg of each compound were placed on inoculated MH agar plates. A blank disk with DMSO was used as a negative control. MH inoculated plates were incubated for <NUM>-<NUM> hours at <NUM>. At the end of the incubation, the inhibition halos where measured. The minimal inhibitory concentration (MIC) was used to determine the antibacterial activity of each compound, in accordance with the recommendations of the CLSI. Two-fold serial dilutions of the compounds were prepared in Mueller-Hinton Broth <NUM> (MHB2 - Sigma-Aldrich, St. Louis, MO, USA) within the concentration range of <NUM>-<NUM>µg/mL. Cefotaxime (CTX) ranging between <NUM>-<NUM>µg/mL was used as a control. Sterility and growth controls were included in each assay. Purity check and colony counts of the inoculum suspensions were also performed in order to ensure that the final inoculum density closely approximates the intended number (<NUM> × <NUM><NUM> CFU/mL). The MIC was determined as the lowest concentration at which no visible growth was observed. The minimal bactericidal concentration (MBC) was assessed by spreading <NUM>µL of culture collected from wells showing no visible growth on MH agar plates. The MBC was determined as the lowest concentration at which no colonies grew after <NUM>-<NUM> hours incubation at <NUM>. These assays were performed in duplicate.

In order to evaluate the combined effect of the compounds and clinically relevant antimicrobial drugs, a screening was conducted using the disk diffusion method, as previously described. A set of antibiotic disks (Oxoid, Basingstoke, England) to which the isolates were resistant was selected: cefotaxime (CTX, <NUM>µg) for extended spectrum beta-lactamase-producer E. coli SA/<NUM>, oxacillin (OX, <NUM>µg) for S. aureus <NUM>/<NUM>, and vancomycin (VA, <NUM>µg) for E. faecalis B3/<NUM>. Antibiotic disks alone (controls) and antibiotic disks impregnated with <NUM>µg of each compound were placed on MH agar plates seeded with the respective bacteria. Sterile <NUM> blank papers impregnated with <NUM>µg of each compound alone were also tested. A blank disk with DMSO was used as a negative control. MH inoculated plates were incubated for <NUM>-<NUM> hours at <NUM>. Potential synergism was recorded when the halo of an antibiotic disk impregnated with a compound was greater than the halo of the antibiotic or compound-impregnated blank disk alone.

Therefore, an initial screening of the antibacterial activity of the compounds <NUM>-<NUM> against the above-mentioned different reference strains of Gram-positive bacteria, Gram-negative bacteria, as well as clinically relevant multidrug-resistant (MDR) strains was performed by the disk diffusion method. This primary assessment was followed by the determination of minimal inhibitory concentrations (MIC) of reference strains. For active compounds, this determination was also made for MDR strains. In the range of concentrations tested, none of the compounds was active against Gram-negative bacteria, and none of <NUM>-<NUM>, <NUM>, <NUM>, <NUM> and <NUM> was active against any of the tested strains (results not shown). The results of antibacterial activity on Gram-positive strains regarding all other compounds are presented in Table <NUM>.

None of the derivatives exhibit antibacterial activity against Gram-negative bacteria, similarly to the described for the natural isolated neofiscalin A (2A). Regarding antimicrobial activity against Gram-positive bacteria, <NUM>, <NUM>, and <NUM> had an inhibitory effect on both Enterococcus faecalis ATCC <NUM> and Staphylococcus aureus ATCC <NUM> reference strains, while <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> only showed an inhibitory effect on S. aureus ATCC <NUM>. The most effective compounds against S. aureus reference strain were <NUM> and <NUM>, with MIC values of <NUM>µg/mL. All of those compounds presented a bacteriostatic activity, with minimal bactericidal concentrations (MBC) greater than <NUM>µg/mL.

Analog <NUM> was the most effective, with MIC values of <NUM>µg/mL and <NUM>µg/mL against E. faecalis ATCC <NUM> and S. aureus ATCC <NUM>, respectively. When tested against vancomycin-resistant Enterococcus (VRE) that was sensitive to ampicillin, the MICs obtained for <NUM>, <NUM> and <NUM> were higher than those obtained for the reference strain (<NUM>µg/mL as opposed to <NUM>µg/mL). In the range of concentrations tested, all these compounds were ineffective against E. faecalis B3/<NUM>, a VRE strain that was also resistant to ampicillin. Regarding S. aureus, <NUM>, <NUM> and <NUM> inhibited the growth of the strain <NUM>/<NUM>/<NUM> (MIC <NUM>µg/mL), which is sensitive to the most commonly used antibiotic families, but not of methicillin-resistant S. aureus (MRSA) <NUM>/<NUM>. More importantly, compounds <NUM> and <NUM> showed a greater inhibitory capacity on both sensitive (<NUM>/<NUM>/<NUM>) and methicillin-resistant S. aureus (<NUM>/<NUM>) strains, with MIC values of <NUM>µg/mL.

In an embodiment, the synergistic effects with vancomycin and oxacillin were evaluated for MDR strains, but no effect was found. These antibiotics are relevant in the treatment of infections caused by Enterococcus spp. and Staphylococcus aureus, respectively.

The compounds showed activity only for Gram-positive strains and, overall, this activity was greater for reference strains than for clinically relevant strains, whether MDR or not. Regarding Gram-positive strains, the range was not equal for all compounds, with a greater number of compounds being active against S. aureus than E. Whereas for E. faecalis there appeared to exist an inverse relationship between compound activity and resistance against clinically important antibiotics, there was not a clear tendency for S. It would be interesting to further study the promising inhibitory effect of ccompounds <NUM> and <NUM> on MRSA. Noteworthy, the first series of compounds (<NUM>st aapproach) showed no relevant effect in the growth of non-malignant cells.

In an embodiment, in order to evaluate the in vitro activities, such as antibacterial, the most promising derivatives <NUM>, <NUM>, and <NUM>, were obtained in milligram scale by semipreparative enantioselective liquid chromatography, employing a tris-<NUM>,<NUM>-dimethylphenylcarbamate amylose column with multiple injection in a <NUM>µL loop.

The analytical method presented good separation (α > <NUM>) and resolution values (Rs > <NUM>) for all compounds to allow the scale-up to the preparative mode. The semipreparative separation was optimized by adjusting the sample volume from the analytical method. The optimized mobile phase of analytical system (hexane: EtOH, <NUM>:<NUM>) was transferred without any modification to semipreparative mode and <NUM> was chosen as minimum wavelength absorption. The column diameter was enlarged to a scale-up factor of <NUM>. The flow rate was increase from <NUM> to <NUM>/min, and the retention times were between <NUM> to <NUM>. The loading effect in semipreparative mode was examined by keeping the concentration of the feed solution at the maximum (<NUM>/mL) and by varying the volume (<NUM> to <NUM>µL). The mobile phase composition, chromatograms, and chromatographic parameter are summarized in <FIG> at analytical and semipreparative scales.

The elution order, specific rotation, and enantiomeric ratio (e. r) of resolved enantiomers were measured and the data is presented in Table <NUM>. r was greater than <NUM>% for each enantiomer.

The pure enantiomers of <NUM>, <NUM>, and <NUM> were evaluated for antibacterial and antifungal activity. Enantiomer 31a showed a MIC of <NUM>µg/mL for reference strain S. aureus ATCC <NUM>, sensitive clinical isolate S. aureus <NUM>/<NUM>/<NUM>, and methicillin-resistant strain S. aureus <NUM>/<NUM>, while enantiomer 31b showed no effect (Table <NUM>). Noteworthy, these derivatives showed higher potency than the natural product neofiscalin A (<NUM>), (tested by the group with the same conditions)<NUM>-<NUM>. None of the pure enantiomers was active against the fungi tested.

Regarding antibacterial activity, the structure-activity relationship (SAR) study suggested that the presence of a halogen atom at positions C-<NUM> or C-<NUM> plays a crucial role for this activity, since all the non-halogenated compounds were inactive against all the tested strains (<FIG>). In fact, compounds containing chlorine atoms at one or both positions exhibited better antibacterial activity compared to those having bromine and iodine. Higher antibacterial activities were obtained when the halogen atom is present at both C-<NUM> and C-<NUM> positions (e. , compound <NUM>, <NUM>, <NUM> and <NUM>) and/or the presence of longer side chains at C-<NUM>. The enantiopure compound 31a showed significant antibacterial effect against a resistant strain of S. aureus while its antipode (31b) did not. This emphasizes that configuration (<NUM>,4R) is crucial for antibacterial activity of quinazolinone scaffold.

The present disclosure also relates to antimalarial activity of the compounds herein disclosed.

The principle of the in vitro susceptibility test to malaria is to assess the degree of development of parasites P. falciparum in the presence of different concentrations of the compounds. In this assay, P. falciparum 3D7, a CQ-susceptible strain, was used to evaluate the antimalarial activity of the <NUM> quinazolinones. Activity was described in terms of IC50 (concentration that inhibits the growth of <NUM> % of P. falciparum parasite present in the culture) for <NUM> compounds (Table <NUM>). The remaining <NUM>, exhibited non-appreciable antiparasitic activity, they fail to produce dose-response curves and/or displayed > <NUM> % survival at <NUM> (data not shown).

To evaluate the antimalarial potential of the pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>-dione scaffold, the following compounds were screened: compounds <NUM>-<NUM> (<NUM>st approach), having <NUM> types of stereoisomers; compounds <NUM>, <NUM>, <NUM>, <NUM> and <NUM> (<NUM>nd approach), compounds <NUM>, <NUM>, <NUM>, <NUM>, <NUM>-<NUM> (<NUM>rd approach) and compounds <NUM>-<NUM> (<NUM>th approach).

It was observed that anti-isomers <NUM>, 4R, like compounds <NUM> (fiscalin B) and <NUM>, exhibited the highest antimalarial activity while syn-isomers <NUM>, <NUM>S were inactive (compounds <NUM> and <NUM>) and syn-isomers 1R,4R had decreased activity (compounds <NUM> and <NUM>). Furthermore, compounds in the <NUM>st approach (<NUM>-<NUM>) demonstrated that increasing the size of the C-<NUM> substituent increased the antimalarial activity, for example, compound <NUM> with C-<NUM> having an isobutyl the same position. Compounds <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>, preferably <NUM>, <NUM>, <NUM> and <NUM>, showed the highest antimalarial activity against P. falciparum strain 3D7.

To further evaluate the effect of C-<NUM> substituent on the activity, the compounds of the <NUM>nd approach (<NUM>, <NUM>, <NUM>, <NUM> and <NUM>) was evaluated, and SAR indicates that a sulfur substituent at C-<NUM> do not favor activity (compounds <NUM>).

In the investigation of the <NUM>rd approach of compounds (<NUM>, <NUM>, <NUM>, <NUM>, <NUM>-<NUM>) with different substituents on A ring, only compound <NUM> having chlorine atom at position <NUM> and <NUM> showed favourable antimalarial activity with an IC<NUM> value of <NUM> (weaker than compounds <NUM> and <NUM>), while other derivatives (substituted with Br or I) showed to be inactive.

For the <NUM>th approach of pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>-diones (<NUM>-<NUM>), isosteric substitutions with the nitrogen atom at different positions of ring A (positions <NUM>, <NUM>, <NUM>), led to a decrease/inactivation of the antimalarial activity (compounds <NUM> and <NUM>). Compounds <NUM> and <NUM> each bearing a hydroxy or methoxy group at position <NUM> of ring A also showed a decrease in activity. Contrary to other reports of febrifugine derivatives, compound <NUM> with a tetrazole group at position <NUM> also showed a weak activity against P. falciparum.

An important criterion in evaluating active antimalarial compounds is their cytotoxicity in mammalian host cells. Compounds that showed the lowest IC<NUM> values against P. falciparum (<NUM>, <NUM>, and <NUM>) were selected to evaluate their cytotoxicity. The cell lines used for in vitro cytotoxicity assay were the V79 from non-tumor cell line of Chinese hamster lung fibroblasts and CQ (<NUM>) was used as control. The results showed relatively low LD<NUM> values (LD<NUM> concentration that inhibits the growth of <NUM> % of cells present in the culture) when compared to CQ (<NUM>) (Table <NUM>). Nonetheless, the selectivity index (SI; calculated by LD<NUM>/IC<NUM>) for compounds <NUM>, <NUM>, and <NUM> were between <NUM>-<NUM> (Table <NUM>) and within the acceptable safety range (SI values greater than <NUM> indicates that a compound has an acceptable therapeutic window for the development of antimalarial drugs).

In general, the higher the SI, the more promising as an anti-malarial are the compounds, due to its selective action against the parasite.

In an embodiment, the evaluation of hemotoxicity in vitro was performed as follows. The in vitro hemolysis assay evaluates the release of hemoglobin in the medium (as an indicator of lysis of erythrocytes) after exposure to the test compounds. Drug-induced hemolysis can occur by two mechanisms: allergic hemolysis (toxicity caused by an immunological reaction in patients previously sensitized to a drug) and toxic hemolysis (direct toxicity of the drug, its metabolite or an excipient in the formulation) [26B]. This test was intended to determine the potential toxic hemolytic effect of the hit compounds <NUM>, <NUM>, and <NUM> on healthy/non-parasitized erythrocytes (<FIG>). The % of hemolysis induced by the compounds was also determined under standard culture conditions of P. falciparum.

The % of hemolysis of healthy erythrocytes induced by <NUM>, <NUM>, and <NUM> was lower than <NUM>% (<FIG>) and within the range of that of CQ (<NUM>). Compounds <NUM>, <NUM>, and <NUM> and CQ (<NUM>) had no hemolytic activity at ≤ <NUM>. CQ (<NUM>) is considered a non-hemolytic antimalarial drug in healthy human erythrocytes. Compounds <NUM>, <NUM>, and <NUM> did not present hemolytic activity, since the % hemolysis was <<NUM> % (% hemolysis > <NUM> % is considered as indicative of risk of hemolysis).

The assay of inhibition of the polymerization of hemozoin (β-hematin) in vitro was based on the protocol of Basilico et al. with some modifications and was carried out for compounds <NUM>, <NUM>, <NUM> and CQ (<NUM>) by using a hemin solution (ferriprotoporphyrin IX chloride). In this assay, CQ (<NUM>) was used as a positive control to evaluate the quality of the test since compound <NUM> binds to portions of hemozoin produced from the proteolytic process of hemoglobin in infected erythrocytes, thus interfering with hemozoin detoxification. Compounds, <NUM>, <NUM>, and <NUM> did not show to inhibit the polymerization of β-hematin in vitro (<FIG>). Febrifuge (compound <NUM>) significantly inhibits the formation of hemozoin required for the maturation of the parasite Plasmodium spp. in the trophozoite stage via axial ligand or π-π interaction to heme. Even though pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>-diones <NUM>, <NUM>, and <NUM> possess structure similarities with febrifugine (compound <NUM>), results suggested that the mechanism of action of these derivatives might be different from febrifugine (compound <NUM>).

Recently, the cytoplasmic prolyl-tRNA synthetase of P. falciparum (PfcPRS), a member of the aminoacyl-tRNA synthetase (aaRS) family that drive protein translation, has been identified as the functional target of febrifugine (compound <NUM>) and analogues, such as halofuginone (HF), a semisynthetic analogue in clinical trials. Therefore, a putative target for this approach of new antimalarials could be the PfcPRS and this hypothesis was explored with in silico studies. The computational docking study on inhibitory effect of prolyl-tRNA synthetase was carried out as follows. The binding affinity of twenty-nine pyrazinoquinazolinones (<NUM>-<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>-<NUM>) to PRS enzyme target was predicted using computational docking AutodockTools. The positive controls were febrifugine (<NUM>, FF), HF, tetrahydroquinazolinone febrifugine (ThFF), and <NUM>-fluorofebrifugine (6FFF) that were predicted as having high binding affininy to PRS, with docking scores between -<NUM> and <NUM> kcal. mol-<NUM>, wherase the negative control, CQ (<NUM>), revealed a docking score of -<NUM> kcal. The most active antimalarials in vitro, compounds <NUM>, <NUM>, <NUM>, <NUM> and <NUM>, preferably <NUM>, <NUM>, <NUM> and <NUM>, presented docking score from -<NUM> to - <NUM> kcal. mol-<NUM>, predicted as forming complexes with PRS enzyme (Table <NUM>, <FIG>).

Halofuginone (HF) is described as being mimetic of the enzyme substrates L-Pro and adenine-<NUM> of tRNA, binding into the active site pockets simultaneously with ATP. Other quinazolinone-based compounds such as FF, 6FFF, and ThFF have also been described as specific for PfPRS when in the presence of the ATP analogue adenosine <NUM>'-(β,γ-imido)triphosphate (AMPPNP). The structure of the ternary complex of PfPRS-AMPPNP-HF reveals hydrogen interactions with Thr359, Glu361, Arg390, Thr478, and His480, and π-π stacking interactions with Phe335 (<FIG>). Compound <NUM> fits the same binding pocket as HF, binding with some of the same residues as HF. The N atom of the indole ring forms hydrogen bonds with Thr478 and His480, and with AMPPNP phosphate groups; and the pyrazinoquinazolinone ring of <NUM> is mainly stabilized by hydrogen interactions with Glu361 and π-π stacking contacts with Phe335, but does not establish polar interactions with Arg390, suggesting chemical spaces available for additional modifications or derivatizations (<FIG>). Compounds <NUM>, <NUM>, and <NUM> bind in the same positions in the PRS cavity but do not stablish hydrogen interactions with AMPPNP. Hydrogen interactions are formed with residues Glu361, Leu325, and Asn330; π-π stacking interactions are stablished with Phe335 and His331 (<FIG>). The indole ring of <NUM>, <NUM>, and <NUM> dock into a lateral cavity flanked by His-<NUM> that is not occupied by HF (<FIG>). The binding pose of <NUM>, different from the binding poses of <NUM>, <NUM>, and <NUM>, provides a hint on the relevance of chirality in the affinity of the binding to PRS target.

A series of halogenated and non-halogenated indolomethyl pyrazino [<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>-diones was designed and synthesized. Among all the obtained compounds, <NUM> and <NUM> exhibited a potent antibacterial activity against S. aureus strains, with MIC values of <NUM>µg/mL for a reference strain and MIC values of <NUM>µg/mL for a sensitive clinical isolate (S. aureus <NUM>/<NUM>/<NUM>) and a methicillin-resistant strain (S. aureus <NUM>/<NUM>). Isolation of the enantiomers of <NUM> revealed that only enantiomer (<NUM>, 4R), 31a, was active, indicating that stereochemistry is vital for the referred activity. Comparing with the marine natural product neofiscalin A (<NUM>), an unexpected two-fold reduction in the MIC was observed. The presence of five stereocenters in neofiscalin A (<NUM>) makes its synthesis a challenge, while with this one-pot microwave-assisted multicomponent polycondensation of amino acids, highly active compounds were obtained in one single step.

Regarding antimalarial activity, the pyrazino[<NUM>,<NUM>-b]quinazoline-<NUM>,<NUM>-dione scaffold showed productive derivatives which demonstrated good antimalarial activity in vitro against P. falciparum strain 3D7. The compounds were not shown to be cytotoxic in vitro against non-tumor mammalian cells V79. These compounds did not show significant hemolytic activity in healthy human erythrocytes and also did not inhibit β-hematin in vitro. These new antimalarial compounds were hypothetized to interact with the prolyl-tRNA similarly to halofuginone.

In the present disclosure, the antimalarial activity was also evaluated. Each compound was lyophilized and solubilized in DMSO (Sigma-Aldrich) to obtain a final concentration of <NUM>. Some intermediate dilutions were made to achieve the final concentration of <NUM> in the first well of the plate. Chloroquine (CQ; Sigma-Aldrich) was prepared with RPMI-<NUM> (Invitrogen ™) supplemented with AlbuMAXII (Invitrogen ™) to obtain a final concentration of <NUM>.

In an embodiment, the culture of P. falciparum was carried out as follows. Laboratory-adapted P. falciparum 3D7 (chloroquine and mefloquine sensitive) were continuously cultured using the method of Trager and Jensen, with previously described modifications (Nogueira et al, <NUM>). Parasites were cultivated at <NUM> % hematocrit, <NUM> and atmosphere with <NUM> % of CO<NUM>, human serum was replaced with <NUM> % AlbuMAXII (Invitrogen™) in the culture medium. Synchronized cultures were obtained by treatments with a <NUM> % (m/v) solution of D-sorbitol (Sigma-Aldrich).

In the present disclosure, the in vitro susceptibility assay of P. falciparum using SYBR Green I was carried out as follows. All compounds were screened for their in vitro antimalarial activity against chloroquine-susceptible (3D7) P. falciparum strain, using the Whole cell SYBR Green I assay as previously described with modifications. Briefly, early ring stage parasites (><NUM> % of rings, <NUM> % haematocrit and <NUM> % parasitaemia) were tested in duplicate in a <NUM>-well plate and incubated with the compounds for <NUM> (<NUM>, <NUM> % CO<NUM>), parasite growth was assessed with SYBRGreenl (Thermo Fisher Scientific). Each compound was tested in concentrations ranging from <NUM> to <NUM>. Fluorescence intensity was measured with a microplate reader with excitation and emission wavelengths of <NUM> and <NUM>, respectively, and analysed by nonlinear regression using GraphPad Prism <NUM> demo version to determine IC<NUM>.

In the present disclosure, the cytotoxicity in vitro against mammalian cell was carried out as follows. Cytotoxicity was assessed on the mammalian cell line V79 (Chinese hamster lung), using an MTT based assay, as previously described [38B]. Tests were conducted in triplicate for each compound, at a range of concentrations (<NUM> to <NUM>), and with culture media containing <NUM> % DMSO (no drug control); incubation time <NUM>. Absorbance was read at <NUM> on a multi-mode microplate reader to produce a log dose-dependence curve. The LD<NUM> value for each compound was estimated by non-linear interpolation of the dose-dependence curve (GraphPad Software).

In the present disclosure, the evaluation of hemotoxicity in vitro was performed as follows. In a <NUM>-flat bottom plate <NUM> % HTC, <NUM>µL of <NUM> % Triton X-<NUM>, and <NUM>µL of PBS or RPMIc in <NUM> % DMSO was added. Compounds were tested in a <NUM>:<NUM> serial dilution in concentrations ranging from <NUM> to <NUM>. After the incubation of <NUM> minutes, the plate was centrifuged at <NUM> rpm for <NUM> minutes. <NUM> of Supernatant was transferred to a flat bottom plate. The absorbance reading was made at <NUM> in a Mode (Triad, Dynex Technologies). Two independent tests were carried out in triplicate. The results are presented in the form of a percentage of hemolysis-% hemolysis, obtained by the following formula: % Hemolysis = ABS (sample)/abs (C +) × <NUM>. Whereas C + is a Triton X-<NUM> to <NUM> % solution RPMIc.

In the present disclosure, the evaluation of inhibition of polymerization of hemozoin was performed as follows. <NUM>µL of a freshly prepared solution of hemin (ferriprotoporphyrin IX chloride; Sigma-Aldrich) <NUM> dissolved in <NUM> NaOH (Sigma-Aldrich) was mixed with <NUM>µL of acetic acid (Sigma-Aldrich) and <NUM>µL of each tested compound. The mixture was incubated for <NUM> at <NUM> in a U-bottom <NUM>-well plate. Compounds were tested at the following doses: compounds <NUM> and <NUM> at <NUM>, <NUM> and <NUM>, compound <NUM> at <NUM>, <NUM> and <NUM>. After incubation, the resulting solution was spun down for <NUM> at <NUM> rpm, the supernatant discarded and the pellet was washed with <NUM>µL of DMSO (<NUM> washes) after an additional final wash with water (<NUM>µL), the pellet was dissolved in <NUM> NaOH (<NUM>µL). <NUM>µL of the solution was transferred to a flat-bottom <NUM>-well clean plate and mixed with <NUM>µL of water and absorption measured at <NUM> using a multi microplate reader plate reader (Triad, Dynex Technologies).

In the present disclosure, the crystal structure of Prolyl-tRNA Synthetase (PRS) (PDB code: 4YDQ), downloaded from the protein databank (PDB), was used. Structure files of <NUM> test molecule, four positive (halofuginone (HF), febrifugine (FF, <NUM>), <NUM>-fluorofebrifugine (6F-FF), and tetrahydro quinazolinone febrifugine (Th-FF)) and one negative (chloroquine, CQ, <NUM>) controls were created and minimized using the chemical structure drawing tool Hyperchem <NUM> (Hypercube, FL, USA) and prepared for docking using AutodockTools. Structure-based docking was carried out using AutoDock Vina (Molecular Graphics Lab, CA, USA). The active site was defined by a grid box (X: <NUM>Å; Y:<NUM>Å; Z: <NUM>Å) drawn around the PRS crystallographic ligand HF. Default settings for small molecule-protein docking were used throughout the simulations. Top <NUM> poses were collected for each molecule and the lowest docking score value was associated with the more favorable binding conformation. <NUM> (Schrödinger, NY, USA) was used for visual inspection of results and graphical representations. To validate the docking approach for the protein structure used, the respective co-crystallized inhibitor HF was docked to the active site using Autodock Vina (<FIG>).

Compounds synthetized and tested in the present disclosure:.

Claim 1:
Compound of formula I
<CHM>
wherein
R<NUM>, R<NUM>, R<NUM>, R<NUM>, R<NUM>, X and Y are independently selected from each other;
R<NUM> and R<NUM> are selected from H or CH<NUM> or CH(CH<NUM>)<NUM> or CH<NUM>CH<NUM>;
R<NUM> is selected from H or Cl or Br or F or OH or OCH<NUM>;
R<NUM> is selected from H or Cl or Br or I or F or OH or OCH<NUM>;
R<NUM> is H or
<CHM>
and
X and Y are selected from N or C;
or a pharmaceutically acceptable salt or solvate thereof;
provided that
if X and Y are C then R<NUM> is different from H; or
if X and Y are C then R<NUM> is H and R<NUM> is
<CHM>
or
if X is N then R<NUM> is absent;
if Y is N then R<NUM> is absent.