WO2010072807A2

WO2010072807A2 - Inhibitors of cystathionine beta synthase to reduce the neurotoxic overproduction of endogenous hydrogen sulfide

Info

Publication number: WO2010072807A2
Application number: PCT/EP2009/067846
Authority: WO
Inventors: Henri Blehaut; François Bellamy; Christophe Matt; Stéphane Giraud; Damien Charre
Original assignee: FONDATION JEROME LEJEUNE
Current assignee: FONDATION JEROME LEJEUNE
Priority date: 2008-12-23
Filing date: 2009-12-23
Publication date: 2010-07-01
Anticipated expiration: 2011-06-23
Also published as: WO2010072807A3

Abstract

The invention is directed to inhibitors of cystathionine beta synthase which, among other biochemical effects, allow reduction of the neurotoxic overproduction of endogenous hydrogen sulphide. These compounds and pharmaceutical compositions containing them are useful for the prevention and treatment of neurotoxic and cognitive disorders such as cognitive disorders in Down syndrome. The invention also relates to methods for preventing or treating neurotoxic and cognitive disorders including cognitive disorders in Down Syndrome.

Description

Inhibitors of cystathionine beta synthase to reduce the neurotoxic overproduction of endogenous hydrogen sulfide

The invention relates to inhibitors of cystathionine beta synthase (CBS) for treatment and/or prevention of neurotoxic and cognitive disorders including cognitive disorders in Down syndrome.

Down Syndrome or trisomy 21 is the most common genetic cause of mental retardation. The gene encoding the cystathionine beta synthase enzyme is located on chromosome 21 and is overexpressed in children with Down syndrome, or trisomy 21. An enrichement in CBS in the brain, an excess of CBS activity and endogenous hydrogen sulfide (H₂S) overproduction have all been found in Down Syndrome [LEJEUNE Jerome. Commentarii vol. III. n⁰ 9, pages 1-12; ICHINOHE et al. Biochemical and Biophysical Research Communications 338 (2005) 1547-1550; CHADEFAUX et al. Biochemical and Biophysical Research Communications, vol. 128 n⁰ 1, 1985, 40-44; POGRIBNA et al. Genet. 69:88-95, 2001; KAMOUN et al. American Journal of Medical Genetics 116A 310-311 (2003)]. However, compounds or composition for preventing and/or treating cognitive disorders in Down syndrome have not been proposed.

The object of the invention is to provide new methods and compounds that inhibit the activity of CBS and consequently regulate overproduction of hydrogen sulfide (H₂S) in trisomic 21 patients due to an overexpression of CBS. CBS in one of the key enzyme of the transulfuration pathway and is the only enzyme of the brain that produce H₂S. It has been proposed that H₂S may function as a neuromodulator or transmitter in the brain (KIMURA, Hideo. Molecular Neurobiology, vol. 6, 13-19 (2002); KAMOUN, Pierre. Medecine/Sciences. Juin-juillet 2004, vol. 20, n⁰ 6-7, P. 697-700). Antimicrobial activity of toxoflavins and their analogs has been described

(NAGAMATSU et al. Chem. Pharm. Bull. 41(2) 362-368; YONEDA et al. Tetrahedron Letters, n⁰ 13 p. 851-854, 1971; YONEDA et al. Tetrahedron Letters, n⁰ 17 p. 1577-1580, 1973; YONEDA et al. Kumamoto University, Japan, 1973). Analogs of toxoflavine have also been described for treatment of cell proliferative disorders such as atherosclerosis and cancer (WO2004/007499, WO2004/007498). Derivatives of toxoflavins have further been described for treatment of diabetes and metabolic diseases (WO2004/065387).

However, the use of toxoflavin derivatives for treatment of the cognitive disorders in Down syndrome has neither been described nor suggested. Description of the invention

In a first embodiment, the invention is related to a method for preventing or treating neurotoxic or cognitive disorders including administering an effective amount, to a human or a patient in need thereof, of a compound having the formula (I) :

wherein

X is N or N⁺-O^"; Rl is C1-C6 alkyl, aryl, aralkyl or C3-C6 cycloalkyl, heteroaryl or a heterocyclic group, each of which is unsubstituted or substituted with one or more substituent groups;

R2 is C1-C6 alkyl, aryl, aralkyl or C3-C6 cycloalkyl, heteroaryl or a heterocyclic group, each of which is unsubstituted or substituted with one or more substituent groups,

R3 is hydrogen, C1-C6 alkyl, aminoalkyl, C2-C6 alkenyl, aryl, aralkyl, aralkenyl, heteroaryl or a heterocyclic group, each of which is unsubstituted or substituted with one or more substituent groups, or R3 is -CH₂-N(CO)-OR" wherein R" is C1-C6 alkyl; and the pharmaceutically acceptable addition salts thereof.

A preferred group of compounds consists of those compounds of formula (I) wherein one or more of the following restrictions apply:

X is N or N⁺-O^";

Rl is preferably C1-C6 alkyl, aryl, aralkyl or C3-C6 cycloalkyl;

R2 is C1-C6 alkyl or C3-C6 cycloalkyl;

R3 is hydrogen, C1-C6 alkyl, aminoalkyl, C2-C6 alkenyl, aryl, aralkyl, aralkenyl, heteroaryl or a heterocyclic group, each of which is unsubstituted or substituted with one or more substituent groups, or R3 is -CH₂-N(CO)-OR" wherein R" is C1-C6 alkyl.

In a second embodiment, the invention is related to a method for preventing or treating neurotoxic or cognitive disorders including administering an effective amount, to a human or a patient in need thereof, of a compound having the formula (II) :

wherein

X is N or N⁺-O^";

R2 is C1-C6 alkyl , aryl, aralkyl or C3-C6 cycloalkyl, heteroaryl or a heterocyclic group, each of which is unsubstituted or substituted with one or more substituent groups,

A preferred group of compounds consists of those compounds of formula (II) wherein one or more of the following restrictions apply:

X is N or N⁺-O^";

R2 is C1-C6 alkyl or C3-C6 cycloalkyl; R3 is hydrogen, C1-C6 alkyl, aminoalkyl, C2-C6 alkenyl, aryl, aralkyl, aralkenyl, heteroaryl or a heterocyclic group, each of which is unsubstituted or substituted with one or more substituent groups, or R3 is -CH₂-N(CO)-OR" wherein R" is C1-C6 alkyl.

In a third embodiment, the invention relates to a method for preventing or treating neurotoxic or cognitive disorders including administering an effective amount, to a human or a patient in need thereof, of a compound having the formula (III) :

wherein

X is N or N⁺-O^"; Rl is C1-C6 alkyl , aryl, aralkyl or C3-C6 cycloalkyl, heteroaryl or a heterocyclic group, each of which is unsubstituted or substituted with one or more substituent groups; R2 is C1-C6 alkyl , aryl, aralkyl or C3-C6 cycloalkyl, heteroaryl or a heterocyclic group, each of which is unsubstituted or substituted with one or more substituent groups, R3 is hydrogen, C1-C6 alkyl, aminoalkyl, C2-C6 alkenyl, aryl, aralkyl, aralkenyl, heteroaryl or a heterocyclic group, each of which is unsubstituted or substituted with one or more substituent groups, or R3 is -CH₂-N(CO)-OR" wherein R" is C1-C6 alkyl,

R4 is hydrogen, C1-C6 alkyl, aryl, aralkyl or C3-C6 cycloalkyl; and the pharmaceutically acceptable addition salts thereof.

A preferred group of compounds consists of those compounds of formula (III) wherein one or more of the following restrictions apply:

X is N or N⁺-O^";

Rl is preferably C1-C6 alkyl, aryl, aralkyl or C3-C6 cycloalkyl; R3 is hydrogen, C1-C6 alkyl, aminoalkyl, C2-C6 alkenyl, aryl, aralkyl, aralkenyl, heteroaryl or a heterocyclic group, each of which is unsubstituted or substituted with one or more substituent groups, or R3 is -CH₂-N(CO)-OR" wherein R" is C1-C6 alkyl.

R4 is hydrogen. Preferably, Rl is methyl, butyl, benzyl, phenyl or cyclohexyl. Preferably, R2 is methyl, ethyl or cyclohexyl.

Advantageously, R3 is hydrogen, methyl, isopropyl, t-butyl, trifluoromethyl, cyclopropyl, cyclohexyl, tetrahydrofuranyl, phenyl, methoxyphenyl, ethylphenyl, benzyl, pyridyl or thienyl. In preferred embodiments, the substituent groups are selected in the group consisting of halogens, amino, C1-C6 alkyl, C1-C6 alkyl substituted with one or more halogens or C1-C6 alkoxy.

Most preferably, the substituent group is methyl, F, Cl, trifluoromethyl, methoxy or amino. In a preferred embodiment, the methods of the present invention concern the prevention or treatment of cognitive disorders in Down syndrome.

In another embodiment, the invention is related to compounds selected in the group consisting of a compound having the formula (I), (II) or (III) wherein

R3 is hydrogen, C1-C6 alkyl, aminoalkyl, C2-C6 alkenyl, aryl, aralkyl, aralkenyl, heteroaryl or a heterocyclic group, each of which is unsubstituted or substituted with one or more substituent groups, or R3 is -CH₂-N(CO)-OR" wherein R" is C1-C6 alkyl;

R4 is hydrogen, C1-C6 alkyl, aryl, aralkyl or C3-C6 cycloalkyl. The present invention further relates to pharmaceutical compositions comprising:

- an effective amount of a compound according to the invention,

- a pharmaceutically acceptable carrier.

The invention is also directed to methods for preventing or treating neurotoxic or cognitive disorders including administering an effective amount of a compound or a pharmaceutical composition according to the invention to a human or a patient in need thereof.

A further embodiment is a method for the prevention or treatment of cognitive disorders in Down syndrome. Preferably, in the formulas and compounds of the present invention, R" is t-butyl.

The term "C1-C6 alkyl " refers to straight or branched hydrocarbon groups having 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, /-propyl, n-butyl, /-butyl or /-butyl.

The term "aminoalkyl" refers to a C1-C6 alkyl substituted with an amino group.

The term "C2-C6 alkenyl" refers to straight or branched chain hydrocarbon groups of 2 to 6 carbon atoms having at least one double bond.

The term "C1-C6 alkoxy" refers to a C1-C6 alkyl linked to an oxygen atom. Preferred alkoxy groups are methoxy and ethoxy groups.

The term "aryl" refers to an aromatic ring having 5 to 10 carbon atoms. A preferred aryl group is phenyl. The term "aralkyl" refers to a C1-C6 alkyl substituted with an aryl group such as phenyl.

The term "aralkenyl" refers to a C1-C6 alkenyl substituted with an aryl group such as phenyl.

The term "C3-C6 cycloalkyl" refers to cyclic hydrocarbon groups of 3 to 6 carbon atoms. Preferred, cycloalkyl groups are cyclopropyl, eye Io butyl, cuclopentyl and cyclohexyl.

The term "heteroaryl" refers to an aromatic cyclic group having 5 to 6 atoms comprising at least one heteroatom selected from nitrogen atoms, oxygen atoms and/or sulphur atoms. Preferred "heteroaryl" groups are thienyl and pyridyl. The term "heterocyclic group" refers to fully saturated or unsaturated, including aromatic or nonaromatic cyclic groups, for example 5 to 6 membered monocyclic groups or 7 to 11 membered bicyclic ring systems which have at least one heteroatom. Each ring of the heterocyclic group may have at least one heteroatom selected from nitrogen atoms, oxygen atoms and/or sulphur atoms. A preferred monocyclic heterocyclic group is tetrahydrofuranyl. A preferred bicyclic heterocyclic group is 2H-1,3-Benzodioxole.

The term 'halogen" refers to F, Cl, Br and I.

The compounds of formula I, II and III form salts which are also within the scope of this invention. Reference to a compound of the present invention is understood to include reference to salts thereof. The term "salts" refers to acidic or basic salts formed with organic and/or inorganic acids and bases.

The present invention relates to compounds inhibiting cystathionine beta synthase (CBS). As used herein the terms "inhibiting cystathionine beta synthase" refers to a compound that inhibits the enzymatic activity of cystathionine beta synthase (EC 4.2.1.22) or that inhibits condensation of cysteine with homocysteine to form cystathionine and hydrogen sulfide (H₂S) or that inhibits condensation of cysteine with homocysteine to form cystathionine and water. Inhibition of the enzymatic activity may be assessed according to the method published by Chen (Chen et al, J. Biol. Chem, 2004, 279).

The present invention further relates to compounds as described herein for use as a medicament.

Preferably the compounds of the present invention are for prevention or treatment of neurotoxic and cognitive disorders including cognitive disorders in Down Syndrome. The present invention also encompasses pharmaceutical compositions. The present invention provides pharmaceutical compositions comprising: a) an effective amount of a compound inhibiting CBS as described herein, b) a pharmaceutically acceptable carrier.

As used herein, "pharmaceutically-acceptable carriers" includes any and all solvents, dispersion media, coatings, and the like that are physiologically compatible.

The compositions of the invention may be in a variety of forms. These include for example liquid, semi-solid, and solid dosage forms, but the preferred form depends on the intended mode of administration and therapeutic application.

These pharmaceutical compositions are preferably for systemic administration such as oral, subcutaneous, percutaneous and parenteral administration. For example, in preparing the compositions for parenteral administration, the carrier will usually comprises sterile water, at least in large part, though other ingredients, for example, to aid solubility, may be included. Injectable solutions, for example, may be prepared in which the carrier comprise saline solution, glucose solution or a mixture of saline and glucose solution. Injectable solutions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. In the composition suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and/or a suitable wettable agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not cause any significant deleterious effects on the skin.

The compound as described herein may be orally administered. As solid compositions for oral administration, tablets, pills, powders (gelatine capsules, sachets) or granules may be used. In these compositions, the active ingredient according to the invention is mixed with one or more inert diluents, such as starch, cellulose, sucrose, lactose or silica, under an argon stream. These compositions may also comprise substances other than diluents, for example one or more lubricants such as magnesium stearate or talc, a coloring, a coating (sugar-coated tablet) or a glaze.

As liquid compositions for oral administration, there may be used pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents such as water, ethanol, glycerol, vegetable oils or paraffin oil. These compositions may comprise substances other than diluents, for example wetting, sweetening, thickening, flavoring or stabilizing products.

The doses depend on the desired effect, the duration of the treatment and the route of administration used.

The invention is also related to the use of a compound as described herein for the manufacture of a medicament for the prevention and/or treatment of neurotoxic and cognitive disorders including cognitive disorders in Down Syndrome.

The present invention also provides methods for preventing or treating neurotoxic and cognitive disorders such as cognitive disorders in Down Syndrome including administering an effective amount of a compound as described herein to a human or to a patient in need thereof.

Exemplary compounds of the present invention can be readily prepared according to the methods of scheme 1 using readily available starting materials, reagents and conventional synthetic procedures. It is also possible to make use of variants of these process steps, which in themselves are known to and well within the preparatory skill of the medicinal chemist.

Scheme 1 In the method of scheme 1 , the starting diethyl malonate can be reacted with alkyl- or aryl- urea using a base such as sodium ethylate in EtOH giving the substituted barbituric acids (I). The N-substituted barbituric acids can be converted to the corresponding chloro- uraciles (II) by treating (II) in refluxing POCI₃ followed by quenching with ice and water and stirring in CH₂Cl₂ several hours. Chloro-Uracile (II) is then reacted with alkyl- or aryl- hydrazines in refluxing EtOH to afford the alkyl- or aryl-hydrazino-uraciles (III) which precipitate from the reaction mixture. Reactions of compounds (III) with aldehydes in EtOH and with acetic acid afford the hydrazones (IV) which can be used without purifications in the next step. C-Nitrosations of the resulting hydrazones with aqueous sodium nitrite in acetic acid give sometimes a mixture of lH,5H,6H,7H-pyrimido[5,4- e][l,2,4]triazine-5,7-dione (VI) and 4-oxido-5,7-dioxo-lH,5H,6H,7H-pyrimido[5,4- e][l,2,4]triazin-4-ium (V). These two kinds of compounds can be isolated by purification on silica gel or either converted into the corresponding lH,5H,6H,7H-pyrimido[5,4- e][l,2,4]triazine-5,7-dione (VI) by the addition of Na₂S₂O₄ in methanol and water. Furthermore, reaction of the obtained compounds (VI) with n-hexylamine in DMF at 110 0C can provide the compounds 5H,6H,7H,8H-pyrimido[5,4-e][l,2,4]triazine-5,7-dione (VII) after purification on silica gel.

Examples

Example 1 : Preparation of 3-Cyclopropyl-l,6-dimethyl-4-oxy-lH-pyrimido[5,4- e] [ 1 ,2,4^"|triazine-5 ,7-dione Step 1

Compound 1 : l-Methyl-pyrimidine-2,4,6-trione

Compound 1 was obtained using the following protocol:

Methyl urea (15g, lβOmmol, leq) and diethyl malonate (24mL, lβOmmol, leq) were added to a solution of sodium (3.68g, lβOmmol, leq) in EtOH (75OmL). The mixture was refluxed for 72h then brought to room temperature and the solvent was partially evaporated. The residue was taken up in HCl IN and stirred with DCM. The resulting precipitate was filtered off and dried to afford the title compound 1 as a beige solid in 66% yield.

MW: 142.12

Formula: CsH₆N₂Os LCMS: 61% MH⁺=143 RT=2.39

IH NMR (250 MHz, DMSO) δ ppm 11.31 (s, IH), 3.57 (s, 2H), 3.06 (s, 3H) Step 2

Compound 2: 6-Chloro-3-methyl-lH-pyrimidine-2,4-dione

Compound 2 was obtained with a mixture of 8g (56mmol, leq) of compound 1 in 1.56mL (560mmol, lOeq) of POCI3 with water 1.5mL (84mmol, 1.5eq) which was refluxed for 3h. The reaction mixture was quenched with water and ice at 0⁰C and stirred for 5h with DCM. The resulting precipitate was filtered off and dried to afford the title compound 2 as a white solid in 74% yield. MW: 160.56 Formula: C₅H₅ClN₂O₂

LCMS: 90% MH⁺=161/163 RT=2.79

IH NMR (250 MHz, DMSO d6) δ ppm 12.35 (bs, IH), 5.89 (s, IH), 3.08 (s, 3H)

Step 3

Compound 3 : 3-Methyl-6-(N-methyl-hydrazino)-lH-pyrimidine-2,4-dione

Compound 3 was obtained with a solution of 300mg (1.87mmol, leq) of compound 2 and 295μL (5.6mmol, 3eq) of methylhydrazine in 7mL of dry EtOH which was heated under reflux for 3h. After cooling, the precipitated crystalline mass was filtered through a sintered funnel to afford the title product 3 as a white solid in 78% yield. MW: 170.17

Formula: CeHi₀N₄O₂

LCMS: 52%+47% (column artefact) MH⁺=171 RT=I.83+2.35

IH NMR (250 MHz, DMSO D6) δ ppm 5.50 (bs), 4,64 (s, IH), 3,02 (s, 3H), 3,01 (s, 3H

Step 4 Compound 4: 6-{N'-[l-Cyclopropyl-meth-(E)-ylidene]-N-methyl-hydrazino}-3-methyl- lH-pyrimidine-2,4-dione

Compound 4 was obtained from the following protocol:

135μL (1.80mmol, 3eq) of cyclopropanecarboxaldehyde was added to a suspension lOOmg (0.59mmol, leq) of compound 3 in 3mL of EtOH and ImL of AcOH at room temperature with stirring. The mixture was stirred for 8h and then partitioned between DCM and H₂O. The aqueous phase was extracted with DCM and the combined organic phases were washed with water, brine, dried over MgSO₄ and concentrated in vacuo. The yellow solid was isolated in 96% yield. The compound 4 was used without further purification and analyses, except when mentioned, in the next step. MW: 222.25

Formula: C10H14N4O2 LCMS: 76% MH⁺=223 RT=4.49 Step S

Compound 5: 3-Cyclopropyl-l,6-dimethyl-4-oxy-lH-pyrimido[5,4-e] [l,2,4]triazine- 5,7-dione

Compound 5 was obtained with the following protocol:

117mg (1.70mmol, 3eq) of sodium nitrite in ImL of water was added portionwise to a stirred solution of 126mg (0.57mmol, leq) of compound 4 in ImL of acetic acid under cooling at 5-7°C. The reaction mixture was stirred at room temperature for 5h. It was then partitioned between DCM and H₂O, the aqueous phase was extracted with DCM and the combined organic phases were washed with water, brine, dried over MgSO₄ and concentrated in vacuo. The residue was purified by a silica SPE cartridge using cyclohexane/acetone 100:0 to 0:100 or DCM/AcOEt 100:0 to 0:100 which afford lOmg (8%) of compound 6 and 30mg (23%) of compound 5 as yellow solids. MW: 249.23 Formula: C10H11N5O3 LCMS: 83% MH⁺=250 RT= 3.80 IH NMR (250 MHz, CDCl₃) δ ppm 3.93 (s, 3H), 3.42 (s, 3H), 2.63 (m, IH), 1.23 (m, 2H), 1.17-0.97 (m, 2H)

Example 2: preparation of S-Cyclopropyl-Lό-dimethyl-lH-pyrimidofSΛ-elfl^Λltriazine- 5,7-dione Step l Compound 6: 3-Cyclopropyl-l,6-dimethyl-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7-dione

Compound 6 was obtained either following protocol described in example 1, step 5 or from compound 5. 84mg (0.48mmol, 3eq) of Na₂S₂O₄ was added portionwise to a stirred solution of 40mg (O.lβmmol, leq) of compound 5 in ImL of MeOH (ImI for O.βmmol) and ImL of water. The reaction mixture was stirred at room temperature for 5h. It was then partitioned between DCM and H₂O, the aqueous phase was extracted with DCM and the combined organic phases were washed with water, brine, dried over MgSO₄. The residue was concentrated in vacuo to afford the title compounds 6 as a yellow solid in 68% yield. MW: 233.23 Formula: C₁₀H₁₁N₅O₂ LCMS: 91% MH⁺=234 RT=3.98 IH NMR (250 MHz, CDCl₃) δ ppm 4.10 (s, 3H), 3.48 (s, 3H), 2.30 (m, IH), 1.38-1.08 (m, 4H)

Example 3: Preparation of l,6-Dimethyl-4-oxy-3-thiophen-2-yl-lH-pyrimido[5,4- e] [ 1 ,2,4^"|triazine-5 ,7-dione Step 1

Compound 7: 3-Methyl-6-{N-methyl-N'-[l-thiophen-2-yl-meth-(E)-ylidene]- hydrazino}-lH-pyrimidine-2,4-dione

Compound 7 was obtained following protocols described for example 1, step 4 with 95mg (0.56mmol) of compound 3 and 105μL (1.12mmol) of the corresponding aldehyde yielding

80mg (54%) of a red solid.

MW: 264.31

Formula: CnHi₂N₄O₂S

LCMS: 76% MH⁺=265 RT=5.37 IH NMR (250 MHz, DMSO) δ ppm:10.15 (s,lH, NH), 8.18 (s, IH), 7.62 (d, 3J = 5.0 Hz,

IH), 7.50 (d, 3J = 3.5 Hz, IH), 7.12 (dd, 3J = 5.0 Hz, 3J = 3.7 Hz, IH), 5.26 (s, IH), 3.32

(s, 3H), 3.08 (s, 3H)

Step 2

Compound 8: l,6-Dimethyl-4-oxy-3-thiophen-2-yl-lH-pyrimido[5,4-e] [l,2,4]triazine- 5,7-dione

Compound 8 was obtained following protocols described for example 1, step 5 with 30mg (0.1 lmmol) of compound 7 and 24mg (0.33mmol) of sodium nitrite yielding 6mg (20%) of a red solid. MW: 291.29 Formula: CnH₉N₅O₃S

LCMS: 88% MH⁺= 292 RT= 4.94

IH NMR (250 MHz, CDCl₃) δ ppm 8.19 (dd, 3J = 4.0 Hz, 4 J = 1.1 Hz, IH), 7.71 (dd, 3J = 5.1 Hz, 4J = 1.1 Hz, IH), 7.23 (t, 3J = 5.4 Hz, 3J = 5.0 Hz, IH), 4.13 (s, 3H), 3.44 (s, 3H)

Example 4: Preparation of l,3,6-Trimethyl-4-oxy-lH-pyrimido[5,4-el[l,2,41triazine-5,7- dione

Step 1

Compound 9 : 6- [N'-Eth-(E)-ylidene-N-methyl-hydrazino] -3-methyl-lH-pyrimidine- 2,4-dione

Compound 9 was obtained following protocols described for example 1, step 4 with 125mg (0.75mmol) of compound 3 and 125 μL (2.25mmol) of the corresponding aldehyde yielding 115mg (78%) of a beige oily solid. MW: 196.21

Formula: CsHi₂N₄O₂

LCMS: 93% MH⁺=197 RT=2.6

Step 2

Compound 10: l,3,6-Trimethyl-4-oxy-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7-dione

Compound 10 was obtained following protocols described for example 1, step 5 with

115mg (0.59mmol) of compound 9 and 122mg (1.76mmol) of sodium nitrite yielding 35mg (29%) of a yellow solid. MW: 223.19 Formula: C₈H₉N₅O₃

LCMS: 98% MH⁺=224 RT=2.49

IH NMR (250 MHz, CDCl₃) δ ppm 3.99 (s, 3H), 3.42 (s, 3H), 2.55 (s, 3H) Example 5 : 3-Isopropyl- 1 ,6-dimethyl-4-oxy- 1 H-pyrimido[5 ,4-e] [ 1 ,2,4^"|triazine-5 ,7-dione

Step l

Compound 11 : 3-Methyl-6-{N-methyl-N'-[2-methyl-prop-(E)-ylidene]-hydrazino}-lH- pyrimidine-2,4-dione

Compound 11 was obtained following protocols described for example 1, step 4 with lOOmg (0.59mmol) of compound 3 and 160μL (1.76mmol) of the corresponding aldehyde yielding 60mg (45%) of an orange oil. MW: 224.26 Formula: Ci₀Hi₆N₄O₂ LCMS: 60% MH⁺=225 Step 2 Compound 12: 3-Isopropyl-l,6-dimethyl-4-oxy-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7- dione

Compound 12 was obtained following protocols described for example 1, step 5 with 60mg

(0.27mmol) of compound 11 and 56mg (0.80mmol) of sodium nitrite yielding lOmg (16%) of a yellow solid. MW: 251.25 Formula: Ci₀Hi₃N₅O₃

LCMS: 99% MH⁺=252 RT=4.46

RMN ¹H (250 M, CDCl₃) δ ppm 4.00 (s, 3H), 3.58 (hept, ³J = 6.7 Hz, IH), 3.42 (s, 3H),

1.30 (d, ³J = 6.8 Hz, 6H)

Example 6: Preparation of l,6-Dimethyl-4-oxy-3-phenethyl-lH-pyrimido[5,4- e] [ 1 ,2,4^"|triazine-5 ,7-dione Step l

Compound 13: 3-Methyl-6-{N-methyl-N'-[3-phenyl-prop-(E)-ylidene]-hydrazino}-lH- pyrimidine-2,4-dione

Compound 13 was obtained following protocols described for example 1, step 4 with lOOmg (0.59mmol) of compound 3 and 155μL (1.18mmol) of the corresponding aldehyde yielding 100% of a yellow solid. MW: 286.34 Formula: C15H18N4O2

LCMS: 50%+21% (syn/anti) MH⁺=287 RT=5.94, 6.39

IH NMR (250 MHz, CDCl₃) δ ppm 7.38-7.28 (m, 5H), 7.03 (t, 3J = 5.2 Hz, IH), 5.01 (s, IH), 3.29 (s, 3H), 3.11 (s, 3H), 2.96 (m, 2H), 2.73 (m, 2H) Step 2 Compound 14: l,6-Dimethyl-4-oxy-3-phenethyl-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7- dione

Compound 14 was obtained following protocols described for example 1, step 5 with

215mg (0.75mmol) of compound 13 and 156mg (2.26mmol) of sodium nitrite yielding 1 Omg (4%) of a yellow solid.

MW: 313.32

Formula: C₁5H₁5N5O3

LCMS: 100% MH⁺=314 RT= 6.07

IH NMR (250 MHz, CDCl₃) δ ppm 7.51-6.99 (m, 5H), 3.96 (s, 3H), 3.42 (s, 3H), 3.22 (t, 3 J = 7.6 Hz, 2H), 3.04 (t, 3 J = 7.7 Hz, 2H)

Example 7: Preparation of l,6-Dimethyl-3-phenethyl-lH-pyrimido[5,4-el[l,2,41triazine- 5,7-dione Step 1 Compound 15: l,6-Dimethyl-3-phenethyl-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7-dione

Compound 15 was obtained following protocols described for example 1, step 5 with 215mg (0.75mmol) of compound 13 and 156mg (2.26mmol) of sodium nitrite yielding 15mg (7%) of a yellow solid MW: 297.32

Formula: Ci₅Hi₅N₅O₂

LCMS: 100% MH⁺=298 RT=6.12 IH NMR (250 MHz, CDCl₃) δ ppm 7.45-7.01 (m, 5H), 4.09 (s, 3H), 3.50 (s, 3H), 3.33 (t, 3J = 8.3 Hz, 2H), 3.15 (t, 3J = 8.3 Hz, 2H)

Example 8: Preparation of (l,6-Dimethyl-5,7-dioxo-4-oxy-l,5,6,7-tetrahydro- pyrimido[5,4-el[l,2,41triazin-3-ylmethyl)-carbamic acid tert-butyl ester Step l Compound 16: {2-[Methyl-(l-methyl-2,6-dioxo-l,2,3,6-tetrahydro-pyrimidin-4-yl)- hydrazono]-ethyl}-carbamic acid tert-butyl ester

Compound 16 was obtained following protocols described for example 1, step 4 with lOOmg (0.59mmol) of compound 3 and 141μL (0.88mmol) of the corresponding aldehyde yielding 160mg (87%) of a yellow oil.

MW: 311.34

Formula: C13H21N5O4 LCMS: 33%+39% (syn/anti) MH⁺=312 RT=4.43+5.11

IH NMR (250 MHz, CDCl₃) δ ppm 5.11 (d, J = 2.5 Hz, IH), 4.10 (m, IH), 3.72 (m, 2H),

3.32 (s, 3H), 3.18 (s, 3H), 1.45 (s, 9H).

Step 2

Compound 17 : (1 ,6-Dimethyl-5,7-dioxo-4-oxy- 1 ,5,6,7- tetrahydro-pyrimido [5,4- e] [l,2,4]triazin-3-ylmethyl)-carbamic acid tert-butyl ester

Compound 17 was obtained following protocols described for example 1, step 5 with 160mg (0.51mmol) of compound 16 and 106mg (1.53mmol) of sodium nitrite yielding 66mg (40%) of a yellow solid. MW: 338.33

Formula: CπHisNβOs

LCMS: 91% MH⁺=339 RT=4.83

IH NMR (250 MHz, CDCl₃) δ ppm 5.23 (bs, IH), 4.51 (d, 3J = 5.8 Hz, 2H), 4.00 (s, 3H),

3.41 (s, 3H), 1.45 (s, 9H)

Example 9: Preparation of π,6-Dimethyl-5 J-dioxo-l,5A7-tetrahydro-pyrimido[5,4- e^^l,2Λ^triazin-3-ylmethv^)-carbamic acid tert-butyl ester Step 1

Compound 18 : (1 ,6-Dimethyl-5,7-dioxo- 1 ,5,6,7- tetrahydro-pyrimido [5,4- e] [l,2,4]triazin-3-ylmethyl)-carbamic acid tert-butyl ester

Compound 18 was obtained following protocols described for example 1, step 5 with 160mg (0.51mmol) of compound 16 and 106mg (1.53mmol) of sodium nitrite. The yellow solid was collected by triturated it in DCM after extraction yielding 68mg (41%). MW: 322.33 Formula: C₁₃H₁₈N₆O₄ LCMS: 77% MH⁺=323 RT=4.84

IH NMR (300 MHz, CDCl₃) δ ppm 5.35 (BS, NH), 4.57 (fd, J = 5.8 Hz, 2H), 4.13 (s, 3H), 3.50 (s, 3H), 1.45 (s, 9H)

Example 10: Preparation of 3-Aminomethyl-l,6-dimethyl-lH-pyrimido[5,4- e] [ 1 ,2,4^"|triazine-5 ,7-dione hydrochloride Step 1 Compound 19: 3-Aminomethyl-l,6-dimethyl-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7- dione hydrochloride

Compound 19 was obtained from compound 17 (35mg, O.lmmol) which was stirred at room temperature with HCl 4N in dioxane. The reaction mixture was concentrated and then taken up in ether and dried to afford 23mg (88%) of a white solid.

MW: 258.67

Formula: C₈Hi₀N₆O₂ .HCl LCMS: 90% 1.7min

IH NMR (250 MHz, DMSO) δ ppm 8.73 (bs, 3H), 4.32 (m, 2H), 3.95 (s, 3H), 3.25 (s, 3H)

Example 11 : Preparation of 3-Cyclohexyl-l,6-dimethyl-4-oxy-lH-pyrimido[5,4- e] [ 1 ,2,4^"|triazine-5 ,7-dione Step 1

Compound 20: 6-{N'-[l-Cyclohexyl-meth-(E)-ylidene]-N-methyl-hydrazino}-3-methyl- lH-pyrimidine-2,4-dione

Compound 20 was obtained following protocols described for example 1, step 4 with lOOmg (0.59mmol) of compound 3 and 150μL (1.20mmol) of the corresponding aldehyde yielding 232mg (100%) of a yellow oil. MW: 264.33

Formula: C13H20N4O2 LCMS: 55% MH⁺=265 Step 2

Compound 21 : 3-Cyclohexyl-l,6-dimethyl-4-oxy-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7- dione

Compound 21 was obtained following protocols described for example 1, step 5 with

230mg (0.87mmol) of compound 20 and 180mg (2.61mmol) of sodium nitrite yielding lOmg (4%) of a yellow solid. MW: 291.31

Formula: C13H17N5O3

LCMS: 88% MH⁺=292 RT=6.16

IH NMR (300 MHz, CDCl₃) δ ppm 3.98 (s, 3H), 3.41 (s, 3H), 3.27 (m, IH), 1.97-1.25 (m,

10H)

Example 12: Preparation of l,6-Dimethyl-3-trifluoromethyl-lH-pyrimido[5,4- e] [ 1 ,2,4^"|triazine-5 ,7-dione

Step l

Compound 22: 3-Methyl-6-{N-methyl-N'-[2,2,2-trifluoro-eth-(E)-ylidene]-hydrazino}- lH-pyrimidine-2,4-dione

Compound 22 was obtained from trifluoroacetaldehyde methyl hemiacetal (300μL,

3.0mmol, 10eq) which was added to a suspension of compound 3 (50mg, 0.3mmol, leq) in toluene (ImL) at 110⁰C for 5h. The mixture was allowed to cool and then partitioned between DCM and H₂O. The aqueous phase was extracted with DCM and the combined organic phases were washed with water, brine, dried over MgSO₄ and concentrated in vacuo to afford 64mg (88%) of a yellow oil. MW: 250.18 Formula: C₈H₉FSN₄O₂ Step 2

Compound 23: l,6-Dimethyl-3-trifluoromethyl-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7- dione

Compound 23 was obtained following protocols described for example 1, step 5 with 64mg (0.26mmol) of compound 22 and 105mg (1.54mmol) of sodium nitrite yielding lOmg

(15%) of a yellow solid.

MW: 261.16

Formula: C₈H₆FSN₅O₂

LCMS: 90% MH⁺= 262 RT=5.07 IH NMR (250 MHz, CDCl₃) δ ppm 4.20 (s, 3H), 3.50 (s, 3H)

19F NMR (235 MHz, CDCl₃) δ ppm -69.55 (s, 3F), internal reference PhOCF3 -58.30

Example 13: Preparation of l,6-Dimethyl-4-oxy-3-(tetrahydro-furan-3-yl)-lH- pyrimido|^"5,4-e^"||^"l,2,41triazine-5,7-dione Step 1

Compound 24: 3-Methyl-6-{N-methyl-N'-[l-(tetrahydro-furan-3-yl)-meth-(E)- ylidene]-hydrazino}-lH-pyrimidine-2,4-dione

Compound 24 was obtained following protocols described for example 1, step 4 with lOOmg (0.59mmol) of compound 3 and 180μL (0.90mmol) of the corresponding aldehyde yielding 205mg (100%) of a yellow oil.

MW: 252.28

Formula: C_{1 1}H₁₆N₄O₃

LCMS: 72% MH⁺=253 Step 2

Compound 25 : l,6-Dimethyl-4-oxy-3-(tetrahydro-furan-3-yl)-lH-pyrimido[5,4- e] [l,2,4]triazine-5,7-dione

Compound 25 was obtained following protocols described for example 1, step 5 with 205mg (0.81mmol) of compound 24 and 168mg (2.44mmol) of sodium nitrite yielding 20mg (9%) of a yellow solid. MW: 279.26

Formula: C_{1 1}H₁₃N₅O₄

LCMS: 92% MH⁺=280 RT=2.84

IH NMR (250 MHz, CDCl₃) δ ppm 4.32-3.78 (massif, 8H), 3.41 (s, 3H), 2.32 (m, 2H)

Example 14: Preparation of l,6-Dimethyl-3-(tetrahydro-furan-3-yl)-lH-pyrimido[5,4- e] [ 1 ,2,4^"|triazine-5 ,7-dione Step l

Compound 26: l,6-Dimethyl-3-(tetrahydro-furan-3-yl)-lH-pyrimido[5,4- e] [l,2,4]triazine-5,7-dione

Compound 26 was obtained following protocols described for example 2, step 1 with 15mg (0.05mmol) of compound 25 and 26mg (0.15mmol) of sodium dithionite yielding 5mg (39%) of a yellow solid. MW: 263.26 Formula: CnHi₃N₅O₃

LCMS: 83% MH⁺=264 RT=2.88

Example 15: Preparation of 3-Benzyl-l,6-dimethyl-4-oxy-lH-pyrimido[5,4- e] [ 1 ,2,4^"|triazine-5 ,7-dione Step 1

Compound 27: 3-Methyl-6-{N-methyl-N'-[2-phenyl-eth-(E)-ylidene]-hydrazino}-lH- pyrimidine-2,4-dione

Compound 27 was obtained following protocols described for example 1, step 4 with 340mg (2.0mmol) of compound 3 and 702μL (3.0mmol) of the corresponding aldehyde yielding 200mg (37%) of a yellow oil. MW: 272.31 Formula: CI₄HI₆N₄O₂ Step 2

Compound 28: 3-Benzyl-l,6-dimethyl-4-oxy-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7- dione

Compound 28 was obtained following protocols described for example 1, step 5 with 380mg (1.30mmol) of compound 27 and 289mg (4.20mmol) of sodium nitrite yielding 33mg (9%) of a yellow solid. MW: 299.29

Formula: C14H13N5O3

LCMS: 82%MH⁺=300 RT=5.57

Example 16: Preparation of 3-(4-Chloro-benzyl)-l,6-dimethyl-lH-pyrimido[5,4- e] [ 1 ,2,41triazine-5 ,7-dione Step l Compound 29: 6-{N'-[2-(4-Chloro-phenyl)-eth-(E)-ylidene]-N-methyl-hydrazino}-3- methyl-lH-pyrimidine-2,4-dio

Compound 29 was obtained following protocols described for example 1, step 4 with lOOmg (0.59mmol) of compound 3 and 235mg (1.50mmol) of the corresponding aldehyde yielding 266mg (87%) of a yellow oil.

MW: 306.75

Formula: Ci₄Hi₅ClN₄O₂ Step 2

Compound 30: 3-(4-Chloro-benzyl)-l,6-dimethyl-lH-pyrimido[5,4-e] [l,2,4]triazine-

5,7-dione

Compound 30 was obtained following protocols described for example 1, step 5 with 266mg (0.86mmol) of compound 29 and 178mg (2.00mmol) of sodium nitrite yielding 15mg (5%) of a yellow solid.

MW: 317.74 Formula: Ci₄Hi₂ClN₅O₂ LCMS: 98% MH⁺=318 RT=6.45

IH NMR (250 MHz, CDCl₃) δ ppm 7.28 (2d, 3J = 8.5 Hz, 4H), 4.25 (s, 2H), 4.10 (s, 3H), 3.47 (s, 3H)

Example 17: Preparation of 3-(4-Methoxy-benzyl)-l,6-dimethyl-lH-pyrimido[5,4- e] [ 1 ,2,4^"|triazine-5 ,7-dione Step l

Compound 31 : 6-{N'-[2-(4-Methoxy-phenyl)-eth-(E)-ylidene]-N-methyl-hydrazino}-3- methyl-lH-pyrimidine-2,4-dione

Compound 31 was obtained following protocols described for example 1, step 4 with lOOmg (0.59mmol) of compound 3 and 298mg (1.80mmol) of the corresponding aldehyde yielding 149mg (82%) of a yellow oil. MW: 302.34

Formula: C15H18N4O3

LCMS: 73% MH⁺=303 RT=5.44

Step 2

Compound 32: 3-(4-Methoxy-benzyl)-l,6-dimethyl-lH-pyrimido[5,4-e] [l,2,4]triazine- 5,7-dione

Compound 32 was obtained following protocols described for example 1, step 5 with

149mg (0.49mmol) of compound 31 and 102mg (1.40mmol) of sodium nitrite yielding

13mg (8%) of a yellow solid. MW: 313.32

Formula: C₁5H₁5N5O3

LCMS: 95% MH⁺=314 RT=5.71

IH NMR (250 MHz, CDCl₃) δ ppm 7.29 (d, 3J =8.8 Hz, 2H), 6.75 (d, 3J =8.8 Hz, 2H),

4.23 (s, 2H), 4.10 (s, 3H), 3.78 (s, 3H), 3.47 (s, 3H)

Example 18: Preparation of 6-Benzyl-13-dimethyl-lH-pyrimido[5,4-e][l,2,41triazine-5,7- dione

Step 1

Compound 33: l-Benzyl-pyrimidine-2,4,6-trione

Compound 33 was obtained following protocols described for example 1, step 1 with 1.5 g (10 mmol) of benzylurea and 1.52 ml (lOmmol) of diethylmalonate yielding 800mg (31%) of a yellow solid. MW: 218.21

Formula: C11H10N2O3

IH NMR (250 MHz, DMSO) δ ppm 11.41 (s, IH), 7.26 (m, 5H), 4.86 (s, 2H), 3.68 (s, 2H)

Step 2

Compound 34: 3-Benzyl-6-chloro-lH-pyrimidine-2,4-dione

Compound 34 was obtained following protocols described for example 1, step 2 with 670 mg (3.07mmol) of compound 33, 2.8 ml (30mmol) of POCI3 and 82μl of water yielding 150mg (21%) of a brown solid. MW: 236.667 Formula: CnH₉ClN₂O₂

LCMS: 81% MH⁺=237 RT=5.92

IH NMR (250 MHz, DMSO) δ ppm 7.74-6.86 (m, 5H), 5.80 (s, 2H), 4.89 (s, 2H)

Step 3

Compound 35 : 3-Benzyl-6-(N-methyl-hydrazino)-lH-pyrimidine-2,4-dione

Compound 35 was obtained following protocols described for example 1, step 3 with 150 mg (0.63 mmol) of compound 34, lOOμl (1.89mmol) of methylhydrazine yielding 68mg (44%) of a beige solid. MW: 246.27 Formula: Ci₂Hi₄N₄O₂

LCMS: 74% MH⁺=247 RT=4.54

Step 4

Compound 36: 3-Benzyl-6-[N'-eth-(E)-ylidene-N-methyl-hydrazino]-lH-pyrimidine-

2,4-dione

Compound 36 was obtained following protocols described for example 1, step 4 with 70mg

(0.28mmol) of compound 35 and 50μL (0.85mmol) yielding 66mg (87%) of a beige solid.

MW: 272.31

Formula: Ci₄Hi₆N₄O₂

LCMS: 59%+14% (syn/anti) MH⁺=273 RT=5.48+5.74

Step S

Compound 37: 6-Benzyl-l,3-dimethyl-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7-dione

Compound 37 was obtained following protocols described for example 1, step 5 with 66mg

(0.24mmol) of compound 36 and 52mg (0.75mmol) of sodium nitrite yielding 9mg (13%) of a yellow solid.

MW: 283.29

Formula: C14H13N5O2 LCMS: 100% MH⁺=284 RT=5.58

IH NMR (250 MHz, CDCl₃) δ ppm 7.55 (dd, 3J = 7.5 Hz, 4J = 1.9 Hz, 2H), 7.36-7.21 (m,

3H), 5.26 (s, 2H), 4.09 (s, 3H), 2.73 (s, 3H)

Example 19: Preparation of 6-Butyl-l,3-dimethyl-4-oxy-lH-pyrimido[5,4- e] [ 1 ,2,4^"|triazine-5 ,7-dione Step 1 Compound 38: l-Butyl-pyrimidine-2,4,6-trione

Compound 38 was obtained following protocols described for example 1, step 1 was used with 2.0 g (17.22mmol) of urea, 2.6 ml (17.22mmol) of diethylmalonate and 396 mg of sodium in 50 ml of ethanol yielding of 2.5g (79%) of an orange oil.

MW: 184.20

Formula: C8H₁₂N₂O3

LCMS: 61% MH⁺=185 RT=5.04 IH NMR (250 MHz, DMSO d6) δ ppm 5.35 (bs, NH), 3.63 (t, 3J = 8.1 Hz, 2H), 3.45 (s,

IH), 3.33 (s, IH), 1.56-1.05 (m, 4H), 0.85 (m, 3H) Step 2

Compound 39: 3-Butyl-6-chloro-lH-pyrimidine-2,4-dione

H

Compound 39 was obtained following protocols described for example 1, step 2 with 2,5 g (13,57mmol) of compound 38, 12.6ml (135mmol) of POCI3 and 366μL of water yielding 975mg (36%) of an orange solid MW: 202.64 Formula: C₈HnClN₂O₂

IH NMR (250 MHz, DMSO d6) δ ppm 12.34 (bs, IH), 5.87 (s, IH), 3.69 (t, 3J = 7.5 Hz, 2H), 1.67-1.46 (m, 2H), 1.23 (m, 2H), 0.88 (t, 3J = 7.2 Hz, 3H) Step 3

Compound 40: 3-Butyl-6-(N-methyl-hydrazino)-lH-pyrimidine-2,4-dione

Compound 40 was obtained following protocols described for example 1, step 3 with 950 mg (34.7mmol) of compound 39, 745μL (14.0mmol) of methylhydrazine in 8 ml of ethanol yielding 300mg (30%) of a white solid.

MW: 212.25

Formula: C9H16N4O2

LCMS: 29 + 42% (column artefact) MH⁺=213 RT=3.85+4.09 IH NMR (250 MHz, DMSO d6) δ ppm 4.62 (s, IH), 3.66 (t, 3J = 7.4 Hz, 2H), 3.00 (s,

3H), 1.41 (quint, 3J = 7.7 Hz, 2H), 1.22 (Sext, 3J = 7.2 Hz, 2H), 0.85 (t, 3J = 7.2 Hz, 3H)

Step 4

Compound 41 : 3-Butyl-6-[N'-eth-(E)-ylidene-N-methyl-hydrazino]-lH-pyrimidine-2,4- dione

Compound 41 was obtained following protocols described for example 1, step 4 with lOOmg (0.47mmol) of compound 40 and 80μL (1.41mmol) of the corresponding aldehyde yielding 109mg (100%) of a yellow oil. MW: 238.29 Formula: CnHi₈N₄O₂

LCMS: 73+19% (syn/anti) MH⁺=239 RT=5.07+5.57 Step S

Compound 42: 6-Butyl-l,3-dimethyl-4-oxy-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7-dione

Compound 42 was obtained following protocols described for example 1, step 5 with 109mg (0.46mmol) of compound 41 and 95mg (1.37mmol) of sodium nitrite yielding 12mg (10%) of a yellow solid. MW: 265.27 Formula: C_{1 1}H₁₅N₅O₃ LCMS: 71% MH⁺=266 RT=5,04 IH NMR (250 MHz, CDCl₃) δ ppm 3.97 (s, 3H), 2.54 (s, 3H), 1.70-1.51 (m, 4H), 1.38 (m, 2H), 0.93 (t, 3J = 7.3 Hz, 3H)

Example 20: Preparation of l,3-Dimethyl-lH-pyrimido[5,4-el[l,2,41triazine-5,7-dione Step l Compound 43: 6-(N-Methyl-hydrazino)-lH-pyrimidine-2,4-dione

Compound 43 was obtained following protocols described for example 1, step 3 with

400mg (2.73mmol) of chlorouracile and 436μL (8.2mmol) of methylhydrazine yielding

355mg (83%) of a yellow solid. MW: 156.15

Formula: CsH₈N₄O₂

LCMS: 84%+9% (column artefact) MH⁺=157 RT= 1.79+2.2

IH NMR (250 MHz, DMSO) δ ppm 10.36 (bs, IH), 8.30 (bs, IH), 5.05 (bs, 2H), 4.46 (s,

IH), 2.99 (s, 3H, Hi) Step 2

Compound 44: 6-[N'-Eth-(E)-ylidene-N-methyl-hydrazino]-lH-pyrimidine-2,4-dione

Compound 44 was obtained following protocols described for example 1, step 4 with 355mg (2.27mmol) of compound 43 and 385μL (6.82mmol) of the corresponding aldehyde yielding 329mg (80%) of a beige solid. MW: 182.18 Formula: C7H10N4O2

LCMS: 76% MH⁺=I 83 RT=2.48

Step 3

Compound 45: l,3-Dimethyl-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7-dione

Compound 45 was obtained following protocols described for example 1, step 5 with

329mg (1.81mmol) of compound 44 and 249mg (3.61mmol) of sodium nitrite yielding 15mg (5%) of a yellow solid. MW: 193.17 Formula: C₇H₇N₅O₂

LCMS: 37%+61% (column artefact) MH⁺=180 RT=l,83+2,34 IH NMR (250 MHz, CDCl₃) δ ppm 4.14 (s, 3H), 2.77 (s, 3H)

Example 21 : Preparation of 6-Cyclohexyl-l,3-dimethyl-4-oxy-lH-pyrimido[5,4- e] [ 1 ,2,41triazine-5 ,7-dione Step l Compound 46: l-Cyclohexyl-pyrimidine-2,4,6-trione

Compound 46 was obtained following protocols described for example 1, step 1 with 2.Og (14mmol) of urea, 2.13 ml (14mmol) of diethylmalonate and 322mg (14mmol) of sodium yielding 212mg (66%) of a beige solid.

MW: 210.23

Formula: C10H14N2O3

IH NMR (250 MHz, DMSO) δ ppm 11.21 (bs), 4.50 (tt, 3J = 12.1 Hz, 3J = 3.8 Hz, IH), 3.57 (s, 2H), 2.31-1.92 (m, 2H), 1.92-1.39 (m, 4H), 1.38-0.85 (m, 4H)

Step 2

Compound 47: 6-Chloro-3-cyclohexyl-lH-pyrimidine-2,4-dione

Compound 47 was obtained following protocols described for example 1, step 2 with 1.93g (9.18mmol) of compound 46, 8.4ml (92mmol) OfPOCl₃ and 250μl Of H₂O yielding 812mg (39%) of a beige solid. MW: 228.68 Formula: CiOHi₃ClN₂O₂

IH NMR (250 MHz, DMSO) δ ppm 12.80 (bs), 5.83 (s, IH), 4.54 (tt, 3J = 12.4 Hz, 3J = 3.9 Hz, IH), 2.25 (m, 2H), 1.89-1.65 (m, 2H), 1.64-1.46 (m, 3H), 1.39-1.00 (m, 3H) Step 3

Compound 48: 3-Cyclohexyl-6-(N-methyl-hydrazino)-lH-pyrimidine-2,4-dione

Compound 48 was obtained following protocols described for example 1, step 3 with 802 mg (3.5mmol) of compound 47 and 560μl (10.5mmol) of methylhydrazine yielding 802mg (23%) of a yellow solid.

MW: 238.29

Formula: CnHi₈N₄O₂

LCMS: 100% MH⁺=239 RT=4,98

IH NMR (250 MHz, DMSO) δ ppm 4.60 (s, IH), 4.55 (m, IH), 2.99 (s, 3H), 2.30 (m, 2H), 1.81-1.36 (m, 8H)

Step 4

Compound 49: 3-Cyclohexyl-6-[N'-eth-(E)-ylidene-N-methyl-hydrazino]-lH- pyrimidine-2,4-dione

Compound 49 was obtained following protocols described for example 1, step 4 with

186mg (0.80mmol) of compound 48 and 140μL (2.40mmol) of the corresponding aldehyde yielding 200mg (94%) of a yellow oil.

MW: 264.33

Formula: CiSH₂ON₄O₂ Step 5

Compound 50: 6-Cyclohexyl-l,3-dimethyl-4-oxy-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7- dione

Compound 50 was obtained following protocols described for example 1, step 5 with 188mg (0.71mmol) of compound 49 and 147mg (2.13mmol) of sodium nitrite yielding 32mg (16%) of a yellow solid. MW: 291.31 Formula: C₁3H₁7N5O3 LCMS: 100% MH⁺=292 RT=5.80

IH NMR (250 MHz, CDCl₃) δ ppm 4.79 (tt, J = 12.2, 3.7, Hz, IH), 3.95 (s, 3H), 2.62-2.28 (m, 5H), 1.95-1.73 (m, 2H), 1.74-1.50 (m, 2H), 1.47-1.20 (m, 4H)

Example 22: Preparation of 6-Cyclohexyl-l,3-dimethyl-lH-pyrimido[5,4-el[l,2,41triazine- 5,7-dione Step 1 Compound 51 : 6-Cyclohexyl-l,3-dimethyl-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7-dione

Compound 51 was obtained following protocols described for example 1, step 5 with

200mg (0.76mmol) of compound 49 and 53mg (0.76mmol) of sodium nitrite yielding 3mg

(1%) of a yellow solid. MW: 275.31

Formula: C13H17N5O2

LCMS: 100% MH⁺=276 RT=5.86

IH NMR (250 MHz, CDCl₃) δ ppm 4.81 (tt, J = 12.4, 3.3 Hz, IH), 4.07 (s, 3H), 2.73 (s,

3H), 2.45 (m, 2H), 2.10-0.68 (m, 8H)

Example 23: Preparation of ό-Cyclohexyl-S-methyl-SH-pyrimidofSΛ-elfl^Λltriazine-SJ- dione

Step 1

Compound 52: 6-Cyclohexyl-3-methyl-8H-pyrimido[5,4-e] [l,2,4]triazine-5,7-dione

Compound 52 was obtained from a solution of 47mg (0.17mmol) of compound 50 with 17mg (0.17mmol) in DMF which was heated at 110⁰C for 5h. It was then partitioned between AcOEt and H₂O, the aqueous phase was extracted with AcOEt and the combined organic phases were washed with water, brine, dried over MgSO₄ and concentrated in vacuo. The residue was purified by a silica gel using DCM/AcOEt 100:0 to 0:100 which afford the reumycin derivative as a yellow solid, with a yield of 86%. MW: 261.29 Formula: C12H15N5O2 LCMS: 95% MH⁺=262 RT=6,04 IH NMR (250 MHz, CDCl₃) δ ppm 9.71 (bs, IH), 4.85 (tt, 3J = 12.2, 3.7 Hz, IH), 3.03 (s, 3H), 2.44 (m, 2H), 1.95-1.61 (massif, 8H)

Example 24: Preparation of 3-Benzyl-6-cyclohexyl-l-methyl-lH-pyrimido[5,4- e] [ 1 ,2,4^"|triazine-5 ,7-dione Step l

Compound 53 : 3-Cyclohexyl-6-{N-methyl-N'-[2-phenyl-eth-(E)-ylidene]-hydrazino}- lH-pyrimidine-2,4-dione

Compound 53 was obtained following protocols described for example 1, step 4 with

200mg (0.84mmol) of compound 48 and 295μL (2.52mmol) of the corresponding aldehyde yielding 280mg (98%) of a yellow oil.

MW: 340.43

Formula: C19H24N4O2 Step 2

Compound 54: 3-Benzyl-6-cyclohexyl-l-methyl-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7- dione

Compound 54 was obtained following protocols described for example 1, step 5 with 280mg (0.82mmol) of compound 53 and 58mg (0.82mmol) of sodium nitrite yielding

14mg (5%) of a yellow solid.

MW: 351.41

Formula: C19H21N5O2

LCMS: 100% MH⁺=352 RT=7.81 IH NMR (250 MHz, CDCl₃) δ ppm 7.54-7.14 (m, 5H), 4.76 (m, IH), 4.26 (s, 2H), 4.07 (s,

3H), 2.58-2.26 (m, 2H), 1.99-1.01 (m, 8H)

Example 25: Preparation of 3,6-Dicyclohexyl-l-methyl-4-oxy-lH-pyrimido[5,4- e] [ 1 ,2,4^"|triazine-5 ,7-dione Step 1

Compound 55 : 3-Cyclohexyl-6-{N'-[l-cyclohexyl-meth-(E)-ylidene]-N-methyl- hydrazino}-lH-pyrimidine-2,4-dione

Compound 55 was obtained following protocols described for example 1, step 4 with 77mg (0.32mmol) of compound 48 and 118μL (0.97mmol) of the corresponding aldehyde yielding 113mg (100%) of an orange oil. MW: 332.45

Formula: CiSH₂SN₄O₂

LCMS: 24%+26% (syn/anti) MH⁺=333 RT=8.76+9.47

IH NMR (250 MHz, CDCl₃) δ ppm 8.94 (bs, IH), 6.88 (d, J = 5.5 Hz, IH), 4.71 (m, IH), 3.91 (s, IH), 3.10 (s, 3H), 2.51-1.23 (massif, 21H) Step 2

Compound 56: 3,6-Dicyclohexyl-l-methyl-4-oxy-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7- dione

Compound 56 was obtained following protocols described for example 1, step 5 with 113mg (0.34mmol) of compound 55 and 47mg (0.68mmol) of sodium nitrite yielding

17mg (15%) of a yellow solid.

MW: 359.43

Formula: C₁₈H₂₅N₅O₃

LCMS 85% MH⁺=360 RT=8.30

Example 26: Preparation of S^-Dicyclohexyl-l-methyl-lH-pyrimidofSΛ-elfl^Λltriazine-

5,7-dione

Step 1

Compound 57: 3,6-Dicyclohexyl-l-methyl-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7-dione

Compound 57 was obtained following protocols described for example 1, step 5 with

113mg (0.34mmol) of compound 55 and 47mg (0.68mmol) of sodium nitrite yielding 1 lmg (9%) of a yellow solid. MW: 343.43 Formula: Ci₈H₂₅N₅O₂

LCMS: 100% MH⁺=344 RT=8.61 IH NMR (250 MHz, CDCl₃) δ ppm 4.80 (tt, 3J = 12.6, 3.4 Hz, IH), 4.35-4.15 (m, 2H), 4.07 (s, 3H), 3.80-3.55 (m, 2H), 2.93 (tt, 3J = 11.5 Hz, 3.54 Hz, IH), 2.55-2.25 (m, 4H), 1.38-2.15 (m, 12H)

Example 27: Preparation of 6-Cyclohexyl-3-isopropyl-l-methyl-4-oxy-lH-pyrimido[5,4- e] [ 1 ,2,4^"|triazine-5 ,7-dione Step 1

Compound 58: 3-Cyclohexyl-6-{N-methyl-N'-[2-methyl-prop-(E)-ylidene]-hydrazino}- lH-pyrimidine-2,4-dione

Compound 58 was obtained following protocols described for example 1, step 4 with

200mg (0.84mmol) of compound 48 and 230μL (2.52mmol) of the corresponding aldehyde yielding 231mg (94%) of an orange solid.

MW: 292.38 Formula: C15H24N4O2

LCMS: 42%+31% (syn/anti) MH⁺=293 RT=7.42+8.00

IH NMR (250 MHz, CDCl₃) δ ppm 8.93 (bs, NH), 6.91 (d, 3J = 5.4 Hz, IH), 4.95 (fd, J =

2.3 Hz, IH), 4.72 (m, IH), 3.11 (s, 3H), 2.63 (m, IH), 2.40 (m, 2H), 1.90-1.51 (massif,

8H), 1.14 (d, 3J = 6.9 Hz, 6H) Step 2

Compound 59: 6-Cyclohexyl-3-isopropyl-l-methyl-4-oxy-lH-pyrimido[5,4- e] [l,2,4]triazine-5,7-dione

Compound 59 was obtained following protocols described for example 1, step 5 with 231mg (0.79mmol) of compound 58 and 109mg (1.58mmol) of sodium nitrite yielding

9mg (4%) of a yellow solid.

MW: 319.37

Formula: C₁5H₂₁N5O3

HPLC: 92% RT=6.95 IH NMR (250 MHz, CDCl₃) δ ppm 4.78 (tt, J = 12.3, 3.8 Hz, IH), 3.96 (s, 3H), 3.56

(sept, J = 6.8 Hz, IH), 2.44 (m, 2H), 1.91-1.54 (m, 8H), 1.28 (d, J = 6.8 Hz, 6H) Example 28: Preparation of: 6-Cyclohexyl-3-isopropyl-l-methyl-lH-pyrimido[5,4- e] [ 1 ,2,4^"|triazine-5 ,7-dione Step 1

Compound 60: 6-Cyclohexyl-3-isopropyl-l-methyl-lH-pyrimido[5,4-e] [l,2,4]triazine- 5,7-dione

Compound 60 was obtained following protocols described for example 1, step 5 with

231mg (0.79mmol) of compound 58 and 109mg (1.58mmol) of sodium nitrite yielding

5mg (2%) of a yellow solid. MW: 303.37

Formula: C15H21N5O2

HPLC: 100% RT=7.14

IH NMR (300 MHz, CDCl₃) δ ppm 4.81 (tt, J = 12.1, 3.8 Hz, IH), 4.08 (s, 3H), 3.27

(sept, J =6.9 Hz, IH), 2.45 (m, 2H), 1.95-1.61 (m, 8H), 1.37 (d, J = 6.9 Hz, 6H)

Example 29: Preparation of l,3-Dimethyl-6-phenyl-4-oxy-lH-pyrimido[5,4- e] [ 1 ,2,4^"|triazine-5 ,7-dione

Step l

Compound 61 : l-Phenyl-pyrimidine-2,4,6-trione

Compound 61 was obtained following protocols described for example 1, step 1 with 2g

(14.70mmol) of urea, 2.23mL (14.70mmol) of diethylmalonate and 340mg (14.70mmol) of sodium yielding 2.4g (80%) of a cream solid.

MW: 204.19 Formula: Ci₀H₈N₂O₃

LCMS: 100% MH⁺=205 RT=4.04

IH NMR (250 MHz, DMSO) δ ppm 11.47 (s, IH), 7.50-7.44 (m, 3H), 7.23 (d, 3 J = 6.4 Hz,

2H), 3.74 (s, 2H)

Step 2 Compound 62: 6-Chloro-3-phenyl-lH-pyrimidine-2,4-dione

Compound 62 was obtained following protocols described for example 1, step 2 with 1.13g (5.53mmol) of compound 61, 5.2mL (55mmol) of POCI3 and 150μL of water yielding 618mg (50%) of a yellow solid. MW: 222.63

Formula: Ci₀H₇ClN₂O₂

IH NMR (250 MHz, DMSO) δ ppm 12.46 (bs, IH), 7.64-7.31 (m, 3H), 7.24 (dd, 3 J =8.2 Hz ,4J = 2.1 Hz, 2H), 6.00 (s, IH) Step 3 Compound 63: 6-(N-Methyl-hydrazino)-3-phenyl-lH-pyrimidine-2,4-dione

Compound 63 was obtained following protocols described for example 1, step 3 with 2.04g

(9.16 mmol) of compound 62, 1.47 ml (27.5mmol) of methylhydrazine yielding 777mg

(37%) of a beige solid. MW: 232.24

Formula: CnHi₂N₄O₂

IH NMR (250 MHz, DMSO) δ ppm 7.49-7.29 (m, 3H), 7.25 (m, 2H), 5.21 (s, IH), 4.14 (s,

2H), 2.58 (s, 3H)

Step 4 Compound 64: 6-[N'-Eth-(E)-ylidene-N-methyl-hydrazino]-3-phenyl-lH-pyrimidine-

2,4-dione

Compound 64 was obtained following protocols described for example 1, step 4 with lOOmg (0.43mmol) of compound 63 and 170μL (1.3mmol) of the corresponding aldehyde yielding 165mg (100%) of a beige oil.

MW: 272.31

Formula: Ci₄Hi₆N₄O₂

LCMS: 24+28% (syn/anti) MH⁺=273 RT=4.05+4.94

Step S Compound 65: l,3-Dimethyl-6-phenyl-4-oxy-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7- dione

Compound 65 was obtained following protocols described for example 1, step 5 with 234mg (0.91mmol) of compound 64 and 187mg (2.72mmol) of sodium nitrite yielding 8mg (3%) of a yellow solid. MW: 285.26

Formula: C₁₃H_{1 1}N₅O₃

LCMS: 76% MH⁺=289 RT=4.08

Example 30: l,6-Dimethyl-3-thiophen-2-yl-lH-pyrimido[5,4-el[l,2,41triazine-5,7-dione (compound 66)

Compound 66 was obtained following protocols described for example 1, step 5 with 30mg

(0.1 lmmol) of compound 7 and 24mg(0.33mmol) of sodium nitrite yielding 5mg (17%) of a red solid. MW: 275,29

Formula:CiiH₉N5θ₂S

LCMS: 100% MH⁺=276 RT=5.21

IH NMR (250 MHz, CDCl₃) δ ppm 8.04 (dd, 3 J = 3.8 Hz, 4J = 1.2 Hz, IH), 7.55 (dd, 3J =

5.0 Hz, 4J = 1.2 Hz, IH), 7.18 (t, 3J = 5.0 Hz, 3J = 3.8 Hz,lH), 4.19 (s, 3H), 3.52 (s, 3H)

Example 31 : l,3,6-Trimethyl-lH-pyrimido[5,4-el[l,2,41triazine-5,7-dione (compound 67)

Compound 67 was obtained following protocols described for example 1, step 5 with

115mg (0.59mmol) of compound 9 and 122mg (1.76mmol) of sodium nitrite yielding 9mg (7%) of a yellow solid.

MW: 207.19

Formula: CsH₉NsO₂

LCMS: 100% MH⁺=208 RT=2.49

IH NMR (250 MHz, CDCl₃) δ ppm 4.13 (s, 3H), 3.51 (s, 3H), 2.76 (s, 3H) Example 32: S-Cyclohexyl-Lβ-dimethyl-lH-pyrimidofSΛ-elfLlΛltriazine-SJ-dione (compound 68)

Compound 68 was obtained following protocols described for example 1, step 5 with 230mg (0.87mmol) of compound 20 and 180mg (2.61mmol) of sodium nitrite yielding 8mg (4%) of a yellow solid. MW: 275.31 Formula: C13H17N5O2 LCMS: 87% MH⁺=276 RT=6.33 IH NMR (250 MHz, CDCl₃) δ ppm 4.13 (s, 3H), 3.50 (s, 3H), 2.97 (m, IH), 2.07-1.25 (m, 10H)

Example 33j 3-Benzyl-l,6-dimethyl-lH-pyrimido[5,4-el[l,2,41triazine-5,7-dione

(compound 69)

Compound 69 was obtained following protocols described for example 1, step 5 with 153mg (0.56mmol) of compound 27 and 116mg (1.70mmol) of sodium nitrite yielding 4mg (3%) of a yellow solid. MW: 283.29 Formula: Ci₄Hi₃N₅O₂

LCMS: 100% MH⁺=284 RT=5.66

IH NMR (250 MHz, CDCl₃) δ ppm 7.42-7.23 (m, 5H), 4.30 (s, 2H), 4.11 (s, 3H), 3.48 (s,

3H)

Example 34: 3-Isopropyl-l,6-dimethyl-lH-pyrimido[5,4-el[l,2,41triazine-5,7-dione (compound 70)

Compound 70 was obtained following protocols described for example 1, step 5 with 60mg (0.27mmol) of compound 11 and 56mg (0.80mmol) of sodium nitrite yielding 8mg (13%) o f a yellow so lid. MW: 235.25 Formula: C10H13N5O2 LCMS: 91% MH⁺=236 RT=4.56

IH NMR (250 MHz, CDCl₃) δ ppm 4.00 (s, 3H), 3.58 (hept, 3J = 6.8 Hz, IH), 3.42 (s, 3H), 1.28 (d, 3J = 6.8 Hz, 6H)

Example 35: Preparation of 3-tert-Butyl-l,6-dimethyl-lH-pyrimido[5,4-el[l,2,41triazine- 5,7-dione Step l

Compound 71 : 6-{N'-[l-Tertbutyl-meth-(E)-ylidene]-N-methyl-hydrazino}-3-methyl- lH-pyrimidine-2,4-dione

Compound 71 was obtained following protocols described for example 1, step 4 with lOOmg (0.59mmol) of compound 3 and 192μL (1.77mmol) of the corresponding aldehyde yielding 57 mg (40 %) of a yellow oil. MW: 238.29

Formula: CnHi₈N₄O₂

LCMS: 72% MH+=239 RT=5.88

Step 2

Compound 72: 3-tert-Butyl-l,6-dimethyl-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7-dione

Compound 72 was obtained following protocols described for example 1, step 5 with 57mg

(0.24 mmol) of compound 71 and 50mg (0.72 mmol) of sodium nitrite yielding l lmg (18%) of a yellow solid. MW: 249.27 Formula: CnHi₅N₅O₂

LCMS 96% MH+=250 RT=5.42

IH NMR (250 MHz, CDC13) δ ppm 4.12 (s, 3H), 3.49 (s, 3H), 1.43 (s, 9H)

Example 36: Preparation of 3-tert-Butyl-l,6-dimethyl-4-oxy-lH-pyrimido[5,4- el r 1.2.41triazine-5 j-dione Step 1

Compound 73 : -tert-Butyl-l,6-dimethyl-4-oxy-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7- dione

Compound 73 was obtained following protocols described for example 1, step 5 with 57mg (0.24mmol) of compound 71 and 50mg (0.72mmol) of sodium nitrite yielding 20mg (33%) of a yellow solid. MW: 265.27 Formula: C₁₁H₁5N5O3 LCMS 98% MH+=266 RT=5.22

IH NMR (250 MHz, CDC13) δ ppm 3.98 (s, 3H), 3.40 (s, 3H) 1.44 (s, 9H).

Example 37: Preparation of 3-Isopropyl-l,6-dicyclohexyl -lH-pyrimido[5,4- e] [ 1 ,2,4^"|triazine-5 ,7-dione Step 1

Compound 74: 6-cyclohexyl-3-(propan-2-yl)-8H-pyrimido[5,4-e] [l,2,4]triazine-5,7- dione

Compound 74 was obtained following protocols described for example 23, step 1 with 66mg (0.22mmol) of compound 60 and 22mg (0.22mmol) of n-hexylamine yielding 35mg

(55%) of a yellow solid.

MW: 289.34

Formula: C14H19N5O2

LCMS 89% MH+=290 RT=7.40 IH NMR (250 MHz, CDC13) δ ppm 10.23 (bs, NH), 4.85 (tt, J = 12.1, 3.6 Hz, IH), 3.60

(sept., J = 6.9 Hz, IH), 2.43 (m, 2H), 1.94-1.60 (massif, 8H), 1.45 (d, J = 6.9 Hz, 6H)

Step 2

Compound 75: 3-Isopropyl-l,6-dicyclohexyl -lH-pyrimido[5,4-e] [l,2,4]triazine-5,7- dione

Compound 75 was obtained from the following protocol:

111 μL (0.90mmol, 9eq) of bromocyclohexane was added to a solution of 30mg (O.lOmmol, 1 eq) of compound 74 and 28mg (0.20, 2eq) of anhydrous potassium carbonate in 2mL of dioxane. The mixture was heated at 120⁰C for 8Oh and then partitioned between DCM and H₂O. The aqueous phase was extracted with DCM and the combined organic phases were washed with water, brine, dried over MgSO₄ and concentrated in vacuo. The residue was purified by a silica SPE cartridge using cyclohexane/acetone 100:0 to 80:20 which afford 4mg (11%) of compound 75 as a yellow solid. MW: 371.49

Formula: C₂OH₂₉N₅O₂

LCMS 91% MH+=372 RT=9.48

IH NMR (250 MHz, CDC13) δ ppm 5.37 (tt, J = 11.1, 4.0 Hz, IH), 4.81 (tt, J = 12.3, 3.8

Hz, IH), 3.30 (sept , J = 6.9, IH), 2.46 (m, 2H), 2.02-1.44(m, 18H), 1.38 (d, J = 6.9 Hz, 6H)

Example 38: Preparation of 6-Cyclohexyl-l,3-diisopropyl-lH-pyrimido|^"5,4- e] [ 1 ,2,4^"|triazine-5 ,7-dione

Compound 76: 6-Cyclohexyl-l,3-diisopropyl-lH-pyrimido[5,4-e] [l,2,4]triazine-5,7- dione

Compound 76 was obtained from the following protocol:

56 μL (0.60mmol, 6eq) of 2-bromopropane was added to a solution of 30mg (O.lOmmol, 1 eq) of compound 74 and 28mg (0.20, 2eq) of anhydrous potassium carbonate in 2mL of dioxane. The mixture was heated at 120⁰C for 3h and then partitioned between DCM and H₂O. The aqueous phase was extracted with DCM and the combined organic phases were washed with water, brine, dried over MgSO₄ and concentrated in vacuo. The residue was purified by a silica SPE cartridge using cyclohexane/acetone 100:0 to 80:20 which afford 7mg (21%) of compound 76 as a yellow solid. MW: 331.42

Formula: C_nH₂₅N₅O₂ LCMS 100% MH+=332 RT=8.36

IH NMR (250 MHz, CDC13) δ ppm 5,71 (sept, J = 6,6 Hz ,1H), 4,82 (tt, J = 12,2, 3,7 Hz, IH), 3,31 (sept, J = 6,9 Hz, IH), 2,45 (m, 2H), 1,91-1,56 (m, 8H), 1,48 (d, J = 6,6 Hz, 6H), 1,38 (d, J = 6,9 Hz, 6H)

Example 39: Preparation of a mixture of 7-Amino-l,3-Dimethyl-lH-pyrimido[5,4- e] [ 1 ,2,4^"|triazine-5-one and_. 7-Amino-l,3-Dimethyl-4-oxy-lH-pyrimido[5,4- e] [ 1 ,2,4^"|triazine-5-one Step 1 Compound 77: 2-Amino-6-(N-methyl-hydrazino)-4-pyrimidinol hydrate

Compound 77 was obtained with a solution of 300mg (2.06mmol, leq) of 2-Amino-6- chloro-4-pyrimidinol hydrate and 323 μL (6.18mmol, 3eq) of methylhydrazine in 6mL of dry EtOH which was heated under reflux for 2h30. After cooling, the precipitated crystalline mass was filtered through a sintered funnel to afford the title product 77 as a white solid in 72% yield.

MW: 155.16

Formula: C₅H₉N₅O

LCMS 85% MH+=156 RT=I.84 IH NMR (250 MHz, DMSO) δ ppm 6.17 (bs, 2H), 4.99 (s, IH), 4.45 (bs, 2H), 3.11 (s, 3H)

Step 2

Compound 78: 2-Amino-6-[N'-Eth-(E)-ylidene-N-methyl-hydrazino]-4-pyrimidinol hydrate

Compound 78 was obtained from the following protocol:

36μL (0.64mmol, leq) of acetaldehyde was added to a suspension of lOOmg (0.64mmol, leq) of compound 77 in 2mL of EtOH and ImL of AcOH at room temperature with stirring. The mixture was stirred for 24h. The resulting precipitate was filtered off and dried to afford the title compound 78 as a beige solid in 61% yield. MW : 181.20

Formula: C₇Hi 1N₅O

LCMS 47+48% MH+=182 RT=1.91+2.37 IH NMR (250 MHz, MeOD) δ ppm 7.20 (q, J = 4.6 Hz, IH), 5.78 (s, IH), 3.38 (s, 3H), 2.02 (d, J = 5.1 Hz, 3H). Step 3

Compounds 79 and 80 : 7-Amino-l,3-Dimethyl-lH-pyrimido[5,4-e] [l,2,4]triazine-5-one and 7-Amino-l,3-Dimethyl-4-oxy-lH-pyrimido[5,4-e] [l,2,4]triazine-5-one

Compounds 79 and 80 were obtained with the following protocol:

54mg (0.78mmol, 2eq) of sodium nitrite was added portionwise to a stirred solution of 71mg (0.39mmol, leq) of compound 78 in 2mL of acetic acid under cooling at 5-7°C. The reaction mixture was stirred at room temperature for 30min. Et₂O was added to the reaction mixture and the resulting precipitate was filtered off and dried. The product was purified by a silica SPE cartridge using DCM/EtOH 100:0 to 0:100 which afford 8mg

(11%) of the mixture of the two compounds as a brown solid. MW: 192.18 + 208.18

Formula: C₇H₈N₆O + C₇H₈N₆O₂

IH NMR (250 MHz, MeOD) δ ppm 4.10 (s, 3H), 3.96 (s, 3H), 2.70 (s, 3H), 2.51 (s, 3H).

Example 52: CBS enzyme activity assays

The activity of CBS was assessed according to the method published by Chen

(Chen et al, J. Biol. Chem, 2004, 279). The reaction mixture contains Tris-HCl buffer pH

8.6, lead acetate, propargylglycine, SAM and 2 mM of L-cysteine and L-homocysteine.

CBS activity is started by addition of L-cyteine, and microplate is then sealed using a special film and placed in reader thermostatted at 37°C. The reaction of H₂S with lead acetate is then monitored by the increase in absorbance at 390 nm during 40 minutes.

These assays were done using either a bacterial protein extract enriched in CBS or the purified CBS from J. Kraus.

The IC50 of a compound was determined by using concentrations of compound ranging from 0.01 μM to 100 μM.

REFERENCES

- CHADEFAUX et al. Cystathionine beta synthase: gene dosage effect in trisomy 21. Biochemical and Biophysical Research Communications, vol. 128 n⁰ 1, 1985, 40-44

- CHEN et al, J. Biol. Chem, 2004, 279

- ICHINOHE et al. Cystathionine β-synthase is enriched in the brains of Down 's patients. Biochemical and Biophysical Research Communications 338 (2005) 1547- 1550 - KAMOUN, Pierre. H2S, un nouveau neuromodulateur. Medecine/ Sciences . Juin-Juillet 2004, vol. 20, n⁰ 6-7, P. 697-700

- KIMURA. Hydrogen Sulfide as a Neuromodulator . Molecular Neurobiology, vol. 6, 13- 19 (2002)

- LEJEUNE Jerome. Reflexion sur Ia dέbilitέ de V intelligence des enfants trisomiques 21. Commentarii vol. III. n⁰ 9, pages 1-12

- NAGAMATSU et al. Synthesis of 3-Substituted l-Methyl-6-phenylpyrimido[5,4-eJ- l,2,4-triazin-5, 7(lH,6H)-diones and Their 4-Oxides, and Evaluation of Antimicrobial Activity ofToxoflavins and Their Analogs. Chem. Pharm. Bull. 41(2) 362-368

- NAGAMATSU et al. Synthesis of 3-Substituted l-Methyl-6-phenylpyrimido[5,4-eJ- l,2,4-tήazin-5, 7(lH,6H)-diones and Their 4-Oxides, and Evaluation of Antimicrobial

Activity ofToxoflavins and Their Analogs. Chem. Pharm. Bull. 41(2) 362-368

- POGRIBNA et al. Homocysteine Metabolism in Children with down Syndrome: In Vitro Modulation. Am. J. Hum. Genet. 69:88-95, 2001; Endogenous Hydrogen Sulfide Overproduction in Down Syndrome. American Journal of Medical Genetics 116A 310- 311 (2003)

- YONEDA et al. A convenient synthesis of Toxoflavins and toxoflavin-n-oxides . Tetrahedron Letters, n⁰ 13 p. 851-854, 1971

- YONEDA et al. Transformation oftoxoflavin into Fervenulin via 1-demethyltoxoflavin. Tetrahedron Letters, n⁰ 17 p. 1577-1580, 1973 - YONEDA et al. Transmethylation from Toxoflavines to Nucleophiles . Kumamoto University, Japan, 1973

Claims

1. Method for preventing or treating neurotoxic or cognitive disorders including administering an effective amount, to a human or a patient in need thereof, of a compound having the formula (I) :

wherein

X is N or N⁺-O^";

Rl is C1-C6 alkyl, aryl, aralkyl or C3-C6 cycloalkyl, heteroaryl or a heterocyclic group, each of which is unsubstituted or substituted with one or more substituent groups;

2. Method for preventing or treating neurotoxic or cognitive disorders including administering an effective amount, to a human or a patient in need thereof, of a compound having the formula (II) :

wherein

X is N or N⁺-O^"; R2 is C1-C6 alkyl , aryl, aralkyl or C3-C6 cycloalkyl, heteroaryl or a heterocyclic group, each of which is unsubstituted or substituted with one or more substituent groups,

3. Method for preventing or treating neurotoxic or cognitive disorders including administering an effective amount, to a human or a patient in need thereof, of a compound having the formula (III) :

wherein

X is N or N⁺-O^";

Rl is C1-C6 alkyl , aryl, aralkyl or C3-C6 cycloalkyl, heteroaryl or a heterocyclic group, each of which is unsubstituted or substituted with one or more substituent groups; R2 is C1-C6 alkyl , aryl, aralkyl or C3-C6 cycloalkyl, heteroaryl or a heterocyclic group, each of which is unsubstituted or substituted with one or more substituent groups,

R3 is hydrogen, C1-C6 alkyl, aminoalkyl, C2-C6 alkenyl, aryl, aralkyl, aralkenyl, heteroaryl or a heterocyclic group, each of which is unsubstituted or substituted with one or more substituent groups, or R3 is -CH₂-N(CO)-OR" wherein R" is C1-C6 alkyl,

4. Method according to any one of claims 1 and 3, wherein Rl is methyl, butyl, benzyl, phenyl or cyclohexyl.

5. Method according to any one of claims 1-4, wherein R2 is methyl, ethyl or cyclohexyl.

6. Method according to any one of claims 1-5, wherein R3 is hydrogen, methyl, isopropyl, t-butyl, trifluoromethyl, cyclopropyl, cyclohexyl, tetrahydrofuranyl, phenyl, methoxyphenyl, ethylphenyl, benzyl, pyridyl or thienyl.

7. Method according to any one of claims 1-6 wherein the substituent groups are selected in the group consisting of halogens, amino, C1-C6 alkyl, C1-C6 alkyl substituted with one or more halogens or C1-C6 alkoxy.

8. Method according to claim 7 wherein the substituent group is methyl, F, Cl, trifluoromethyl, methoxy or amino.

9. Method according to anyone of claims 1-8 wherein the compound is selected in the group consisting of l,6-Dimethyl-3-thiophen-2-yl-lH-pyrimido[5,4- e][l,2,4]triazine-5,7-dione, l,6-dimethyl-3-(pyridin-3-yl)-lH,5H,6H,7H- pyrimido [5 ,4-e] [ 1 ,2,4]triazine-5 ,7-dione, 3 -Benzyl- 1 ,6-dimethyl- 1 H-pyrimido [5 ,4- e][l,2,4]triazine-5,7-dione, l,6-dimethyl-3-(4-methylphenyl)-lH,5H,6H,7H- pyrimido[5,4-e][l,2,4]triazine-5,7-dione, l,6-dimethyl-3-[4-

(trifluoromethyl)phenyl]-lH,5H,6H,7H-pyrimido[5,4-e][l,2,4]triazine-5,7-dione, l,6-dimethyl-3-phenyl-lH,4H,5H,6H,7H,8H-pyrimido[5,4-e][l,2,4]triazine-5,7- dione, 3-(4-fiuorophenyl)-l,6-dimethyl-lH,5H,6H,7H-pyrimido[5,4- e][l,2,4]triazine-5,7-dione, 3-(4-methoxyphenyl)-l,6-dimethyl-lH,5H,6H,7H- pyrimido[5,4-e][l,2,4]triazine-5,7-dione, l,6-dimethyl-3-(pyridin-2-yl)- lH,5H,6H,7H-pyrimido[5,4-e][l,2,4]triazine-5,7-dione, l,6-dimethyl-3-phenyl- lH,5H,6H,7H-pyrimido[5,4-e][l,2,4]triazine-5,7-dione, l-ethyl-6-methyl-3-[(E)-2- phenylethenyl]-lH,5H,6H,7H-pyrimido[5,4-e][l,2,4]triazine-5,7-dione, 3-(2H-l,3- benzodioxol-5-yl)-l,6-dimethyl-lH,5H,6H,7H-pyrimido[5,4-e][l,2,4]triazine-5,7- dione, 3-(4-chlorophenyl)-l,6-dimethyl-lH,5H,6H,7H-pyrimido[5,4- e][l,2,4]triazine-5,7-dione and l,6-dimethyl-3-(pyridin-4-yl)-lH,5H,6H,7H- pyrimido[5,4-e][l,2,4]triazine-5,7-dione.

10. Method according to any one of claims 1-9 for the prevention or treatment of cognitive disorders in Down syndrome.

11. Compound selected in the group consisting of 3-Cyclopropyl-l,6-dimethyl-4-oxy- lH-pyrimido[5,4-e][l,2,4]triazine-5,7-dione, 3-Cyclopropyl-l,6-dimethyl-lH- pyrimido[5,4-e][l,2,4]triazine-5,7-dione, l,6-Dimethyl-4-oxy-3-thiophen-2-yl-lH- pyrimido[5,4-e][l,2,4]triazine-5,7-dione, l,3,6-Trimethyl-4-oxy-lH-pyrimido[5,4- e][l,2,4]triazine-5,7-dione, 3-Isopropyl-l,6-dimethyl-4-oxy-lH-pyrimido[5,4- e][l,2,4]triazine-5,7-dione, l,6-Dimethyl-4-oxy-3-phenethyl-lH-pyrimido[5,4- e][l,2,4]triazine-5,7-dione, l,6-Dimethyl-3-phenethyl-lH-pyrimido[5,4- e][l,2,4]triazine-5,7-dione, (l,6-Dimethyl-5,7-dioxo-4-oxy- 1,5,6, 7-tetrahydro- pyrimido[5,4-e][l,2,4]triazin-3-ylmethyl)-carbamic acid tert-butyl ester, (1,6- Dimethyl-5,7-dioxo-l,5,6,7-tetrahydro-pyrimido[5,4-e][l,2,4]triazin-3-ylmethyl)- carbamic acid tert-butyl ester, 3-Aminomethyl-l,6-dimethyl-lH-pyrimido[5,4- e] [ 1 ,2,4]triazine-5 ,7-dione hydrochloride, 3-Cyclohexyl- 1 ,6-dimethyl-4-oxy- 1 H- pyrimido[5,4-e][l,2,4]triazine-5,7-dione, l,6-Dimethyl-3-trifluoromethyl-lH- pyrimido[5,4-e][l,2,4]triazine-5,7-dione, l,6-Dimethyl-4-oxy-3-(tetrahydro-furan- 3-yl)-lH-pyrimido[5,4-e][l,2,4]triazine-5,7-dione, l,6-Dimethyl-3-(tetrahydro- furan-3-yl)-lH-pyrimido[5,4-e][l,2,4]triazine-5,7-dione, 3-Benzyl-l,6-dimethyl-4- oxy-lH-pyrimido[5,4-e][l,2,4]triazine-5,7-dione, 3-(4-Chloro-benzyl)-l,6- dimethyl- 1 H-pyrimido[5 ,4-e] [ 1 ,2,4]triazine-5 ,7-dione, 3-(4-Methoxy-benzyl)- 1 ,6- dimethyl- 1 H-pyrimido[5 ,4-e] [ 1 ,2,4]triazine-5 ,7-dione, 6-Benzyl- 1 ,3-dimethyl- 1 H- pyrimido[5,4-e][l,2,4]triazine-5,7-dione, 6-Butyl-l,3-dimethyl-4-oxy-lH- pyrimido[5,4-e][l,2,4]triazine-5,7-dione, l,3-Dimethyl-lH-pyrimido[5,4- e][l,2,4]triazine-5,7-dione, 6-Cyclohexyl-l,3-dimethyl-4-oxy-lH-pyrimido[5,4- e][l,2,4]triazine-5,7-dione, 6-Cyclohexyl-l,3-dimethyl-lH-pyrimido[5,4- e][l,2,4]triazine-5,7-dione, 6-Cyclohexyl-3-methyl-8H-pyrimido[5,4- e] [ 1 ,2,4]triazine-5 ,7-dione, 3 -Benzyl-6-cy clohexyl- 1 -methyl- 1 H-pyrimido [5 ,4- e][l,2,4]triazine-5,7-dione, 3,6-Dicyclohexyl-l-methyl-4-oxy-lH-pyrimido[5,4- e][l,2,4]triazine-5,7-dione, 3,6-Dicyclohexyl-l -methyl- 1 H-pyrimido [5,4- e] [ 1 ,2,4]triazine-5 ,7-dione, 6-Cyclohexyl-3-isopropyl- 1 -methyl-4-oxy- 1 H- pyrimido [5 ,4-e] [ 1 ,2,4]triazine-5 ,7-dione, 6-Cyclohexyl-3 -isopropyl- 1 -methyl- 1 H- pyrimido[5,4-e][l,2,4]triazine-5,7-dione, l,3-Dimethyl-6-phenyl-4-oxy-lH- pyrimido[5,4-e][l,2,4]triazine-5,7-dione, l,6-Dimethyl-3-thiophen-2-yl-lH- pyrimido[5,4-e][l,2,4]triazine-5,7-dione, l,3,6-Trimethyl-lH-pyrimido[5,4- e][l,2,4]triazine-5,7-dione, 3-Cyclohexyl-l,6-dimethyl-lH-pyrimido[5,4- e][l,2,4]triazine-5,7-dione, 3-Isopropyl-l,6-dimethyl-lH-pyrimido[5,4- e][l,2,4]triazine-5,7-dione, 3-tert-Butyl-l,6-dimethyl-lH-pyrimido[5,4- e] [ 1 ,2,4]triazine-5 ,7-dione, 3-tert-Butyl- 1 ,6-dimethyl-4-oxy- 1 H-pyrimido[5 ,4- e][l,2,4]triazine-5,7-dione, 3-Isopropyl-l,6-dicyclohexyl - 1 H-pyrimido [5, 4- e][l,2,4]triazine-5,7-dione, 6-Cyclohexyl-l,3-diisopropyl-lH-pyrimido[5,4- e][l,2,4]triazine-5,7-dione, 7-Amino-l,3-Dimethyl-lH-pyrimido[5,4- e][l,2,4]triazine-5-one and 7-Amino-l,3-Dimethyl-4-oxy-lH-pyrimido[5,4- e] [ 1 ,2,4]triazine-5-one.

12. Pharmaceutical composition comprising: an effective amount of a compound according to claim 11 , a pharmaceutically acceptable carrier.

13. Method for preventing or treating neurotoxic or cognitive disorders including administering an effective amount of a compound according to claim 11 or a pharmaceutical composition according to claim 12 to a human or a patient in need thereof.

14. Method according to claim 13 for the prevention or treatment of cognitive disorders in Down syndrome.