ES2529865A1 - Use of compounds derived from pyridazino[1',6':1,2]pyrido[3,4-b]indole and pyridazino[3,2-b]benzimidazole salts as anti-inflammatory agents - Google Patents

Abstract

Use of compounds derived from pyridazine [1 ', 6': 1,2] pyrido [3,4-b] indolinium salts as anti-inflammatory agents. The present invention relates to compounds of the pyridazinopyridoindolinium salt families, to the pharmaceutical compositions containing them, and to the use of these as anti-inflammatory agents through the inhibition of the production of tumor necrosis factor alpha (TNF-α ) by monocytes and proinflammatory macrophages in mammals and in all diseases and conditions in which this mediator has high levels, such as rheumatoid arthritis, osteoarthritis, Crohn's disease, ulcerative colitis, asthma, bronchitis, obstructive chronic airway disease, psoriasis, allergic rhinitis, ankylosing spondylitis, hydradenitis suppurativa, dermatitis and any other inflammatory state that occurs with high levels of TNF-α.

Description

The present invention relates to the use of compounds that inhibit the production of TNF-alpha and are therefore useful as anti-inflammatory agents. Therefore, the invention could be framed in the field of biomedicine.

STATE OF THE PREVIOUS TECHNIQUE

Inflammation is a complex and dynamic physiological process that occurs in response to cellular or tissue damage in an attempt to defend the body against external aggression. However, chronic inflammation may be associated with various pathological processes and may be the origin of them or contribute to an aggravation of the clinical picture.

Although various cell types participate in inflammatory processes, macrophages are the main organizers of the process, contributing greatly to the production of various pro-inflammatory cytokines.

TNF-a is an inflammatory cytokine produced by macrophages, macrophage lineage cells (Kupffer cells), neutrophils, basophils, eosinophils, lymphocytes, NK cells, LAK cells, primed cells, bone marrow cells, fibroblasts, astrocytes, keratinocytes and other cell types. However, macrophages are the main producers of TNF- (1. This cytokine induces apoptosis in several tumor lines and in combination with other proinflammatory cytokines is responsible for alterations in endothelial cells that cause various pathologies that affect the microvasculature. In addition TNF- (l promotes angiogenesis in vivo. In macrophages it stimulates microbicidal functions and the biosynthesis of other cytokines. Although TNF-a is required for a normal immune response, its over-expression may have pathological consequences. It is the main mediator of cachexia in patients with tumors and is primarily responsible for some serious effects during sepsis by Gram-negative bacteria, and it has been shown to play a key role in the development of various autoimmune and / or inflammation-associated diseases.

chronic (rheumatoid arthritis, multiple sclerosis).

The synthesis of TNF-a. it is induced by different stimuli, including IFN-y; but the most potent stimulus in macrophages is the LPS present in the wall of Gram negative bacteria. Recently it has also been described that molecules that are released by body cells when they suffer damage or aggression are also capable

of inducing the production of TNF-a ..

Currently, steroidal anti-inflammatory agents (hormones) and non-steroidal anti-inflammatory agents (NSAIDs) are used for the treatment of various pathologies. However, these agents have various points of action and do not have a specific inhibitory action for TNF- (1. Thus, they are likely to cause effects

Harmful side effects, particularly, the side effects of steroidal anti-inflammatory drugs (corticosteroids) have become a medical problem.

Anti-TNF-alpha are the most successful biological drugs today and are indicated for the treatment of autoimmune diseases such as chronic rheumatoid arthritis, psoriatic arthritis, idiopathic autoimmune arthritis, psoriasis, ankylosing spondylitis, Crohn's disease and ulcerative colitis, in the that have shown good

clinical results; but they have side effects such as hypersensitivity reactions, predisposition to infections that can be serious such as tuberculosis and sepsis, increased risk of tumor development and neuronal deterioration caused by a demyelinating effect.

In recent years, several strategies aimed at selectively inhibiting various proinflammatory cytokines have provided protein-based therapies for inflammatory diseases, validating the therapeutic hypothesis that intervention in cytokine signaling can provide a therapeutic benefit. However, in addition to their very high cost, these protein products have the disadvantage of being administered intravenously.

For all these reasons, attention has been paid to new strategies aimed at

develop a new generation of TNF- inhibitors (1 that allow for example oral administration and have few side effects. In addition to

interfering with the production or signaling of pro-inflammatory cytokines with active oral administration drugs would combine the comfort of conventional drugs with the proven efficacy of biological therapies.

The preparation of pyridazino (1 ', 6': 1, 2] pyrido (3,4-b] indolinium and pyridazino (3,2b) benzimidazolium salts has been described above through a sequence of two reactions from azines and azoles having a methyl group in the position (1 to the nitrogen atom. The first reaction consists of an N-amination with a

10 aminating agent such as HOSA (O-hydroxylaminosulfonic acid) or MSH (hydroxylamine mesitylenesulfonate). This N-amino azinium or azolium salt is subsequently reacted with a 1,2-diketone in the presence of a base to give a double condensation leading to the final salt.

15 DESCRIPTION OF THE INVENTION

The present invention provides new TNF-a inhibitors, and therefore, useful for the prevention and / or treatment of inflammatory diseases in mammals, preferably in humans.

Therefore, the first aspect of the present invention relates to the use of a compound of general formula (1):

25 (1) where: Rl is selected from the list comprising hydrogen, alkyl (Cr Clo), alkenyl (Cr Cl0), alkynyl (C2-Cl0), - (CH2) nC02Rl ', - (CH2) mRl ", - (CH2) pCONHR ('or the group of formula (11):

(eleven )

where R is selected from the list comprising (C1-C20) alkyl, (C2-C20) alkenyl,

5 alkynyl (C, -C20), - (CH,), CO, R, '- O, C (CH,), - 6 - (CH') pCONHR, "'- NHOC (CH,) p-; n , mop have a value between O and 5 and R1 'is selected from hydrogen and alkyl (C1ClO), R1 "is selected from halogen, alkenyl (Cr C4) or alkynyl (C2-C4) and Rt' is selected from between (C, -C4) alkyl, (C2-C4) alkenyl or (C2-C4) alkynyl; R2 and R3, are the same or different, and are selected from alkyl (C, -ClO), cycloalkyl

10 (C3-CS), aryl, heteroaryl or R2 and R3 may be linked together as part of a ring, saturated or aromatic; R4 is selected from the list comprising hydrogen, (C, -C10) alkyl, halogen, nitro, amino, hydroxyl, O-R4 ', where R4' is selected from an alkyl (C, -Cd, aryl, aralkyl or heteroaryl ; Y

X-is a biologically or pharmaceutically acceptable anion;

or any of its pharmaceutically acceptable salts, for the preparation of a medicament for the inhibition of the production of TNF - (. (.

In a preferred embodiment, R, is selected from hydrogen, - (CH2) nC02 R, 'or 20 - (CH') mR, ".

In a more preferred embodiment, R, 'is selected from hydrogen or (C, C, o) alkyl and n is O OR 1, more preferably, R,' is hydrogen, methyl, ethyl or tert-butyl.

In another more preferred embodiment, R, "is a halogen or an alkynyl (Cr C3) and m has a value of 2 to 4, more preferably R," is iodine or ethynyl and m is 3.

When R, is the group of formula (11), the compound of the invention is of general formula (111):

R,

R,

R

R,

~

R,

(111)

where R, R2, R3, ~ and X 'have been described above.

In a preferred embodiment, R is alkyl (CS-C1S), alkenyl (CS-C1S) or alkynyl (CsC1S), more preferably R is deca-4,6-diin-1, 1-Q-diyl.

In another preferred embodiment of the compounds of formula (1) or (111), R2 Y ~, they are the same or different, and are selected from methyl, ethyl or 2-furyl or are linked together to form 1, 8- Naphthodiyl More preferably, R2 and R3 are the same.

In another preferred embodiment of the compounds of formula (1) or (111), ~ is selected from the list comprising hydrogen, (C1-C3) alkyl, halogen, nitro, amino, O- ~ ', where selected from a (C1-C3) alkyl or aryl, more preferably R4 is selected from ammonium, bromine, nitro, methoxy or benzoxyl.

In another preferred embodiment of the compounds of formula (1) or (111), X 'is halide or mesitylenesulfonate, more preferably the halide is bromide, iodide or chloride.

The compound of general formula (1) or (111) is selected from the list comprising: 9-Bromo-2,3-diethyl-12H-pyridazino [1 ', 6': 1, 2] pyrido [3,4-b] indole-S-inium mesitylenesulfonate (Compound 4), 2,3-diethyl-12H-11-nitropyridazino mesitylenesulfonate [1 ', 6': 1, 2] pyrido [3,4-b] indole-5-inium

25 (Compound 7),

11-ammonium-2,3-diethyl-12H-pyridazino [1 ', 6': 1,2] pyrido [3,4-b] indole-5-inium dichloride

(Compound 8), 1O-benzyloxy-12-tert-butoxycarbonyl-2,3-diethylpyridazino mesylenesulfonate [1 ', 6': 1,2] pyrido [3,4-b] indole-5-inium (Compound 13), 2,3-dimethyl-12H-1 O-methoxypyridazino bromide [1 ', 6': 1,2] pyrido [3,4-b] indole-5-inium (Compound 17),

2,3-diethyl-12H-1 O-methoxypyridazino [1 ', 6': 1,2] pyrido [3,4-b] indole-5-ynium mesylethenesulfonate (Compound 20), 2,3-diethyl-12-ethoxycarbonylmethyl-1 0-methoxypyridazino [1 ', 6': 1,2] pyrido [3,4b] indole-5-inium bromide (Compound 22), 2,3-diethyl-1 0-methoxy-12- (3-iodopropyl) -pyridazino (1 ', 6': 1,2) pyrido (3,4-b) indole-5inium (Compound 23) iodide, 12-carboxymethyl-2,3-diethyl-1 O-methoxypyridazino bromide [1 ', 6': 1,2] pyrido [3,4-b] indole-5inium (Compound 24), 14-methoxy-16- (pent-4-inyl) acenaphth [1 ", 2": 3 ', 4'] pyridazine [1 ', 6': 1,2] mesylenesulfonate pyrido [3,4-b] indole-9-inium (Compound 27) mesitilensul! 2,3-di- (2-! uryl) -10-methoxy-12- (pent-4-inyl) pyridazino (1 ', 6': 1,2) pyrido [3,4-b] indole-5-inium (Compound 28) and 12,12 'dimesitilensul! onato - (deca-4,6-diin-1,10-diyl) -bis- (2,3-diethyl-1 0-methoxypyridazino (1', 6 ': 1, 2) pyrido (3 , 4-b) indole-5-inium) (Compound 31).

In a more preferred embodiment the compound is 2,3-dimethyl-12H-10-methoxypyridazino bromide (1 ', 6': 1, 2] pyrido [3,4-b] indole-5-inium (Compound 17).

Any of the compounds described in the present invention, of general formula

(1) or (111), can be used as anti-inflammatory agents that are present through the inhibition of the production of tumor necrosis factor alpha (TNF-a). Therefore, another aspect of the present invention relates to the use of the compounds of formula (1) or

( ) for the preparation of a medicament for the treatment and / or prevention of inflammatory diseases, these inflammatory diseases being known by a person skilled in the art and more preferably can be selected from the list comprising rheumatoid arthritis, psoriatic arthritis, autoimmune idiopathic arthritis, osteoarthritis, Crohn's disease, ulcerative colitis, uveitis, asthma, bronchitis,

obstructive chronic airway disease, psoriasis, allergic rhinitis, ankylosing spondylitis, suppurative hydradenitis, dermatitis and any other inflammatory condition with high levels of TNF-a production, including any inflammatory state that occurs associated with kidney disease.

Another aspect of the present invention relates to compounds selected from the list comprising: 2,3-diethyl-1 O-methoxy-12- (3-iodopropyl) pyridazine iodide [1 ', 6': 1, 2] pyrido [3,4-b] indole-5inium (Compound 23) and 12-carboxymethyl-2,3-diethyl-1 O-methoxypyridazino [1 ', 6': 1, 2] pyrido [3,4-b ] indole-5inium (Compound 24).

Another aspect of the present invention relates to a pharmaceutical composition comprising at least one compound selected from 2,3-diethyl-10-methoxy-12- (3-iodopropyl) pyridazine iodide [1 ', 6': 1, 2] pyrido [3,4-b] indole-5-inium (Compound 23) and 12-carboxymethyl-2,3-diethyl-1 O-methoxypyridazino [1 ', 6': 1, 2] pyrido [3,4 -b] indole-5inium (Compound 24), in addition to a pharmaceutically acceptable carrier. Optionally this pharmaceutical composition also comprises another active ingredient, preferably it can be selected from an anti-inflammatory agent or a TNF-a production inhibitor.

The "pharmaceutically acceptable vehicles" that can be used in said compositions are the vehicles known to a person skilled in the art.

Another aspect of the present invention relates to the use of the compound selected from 2,3-diethyl-10-methoxy-12- (3-iodopropyl) pyridazino iodide [1 ', 6': 1, 2] pyrido [3, 4b] indole-5-inium (Compound 23) and 12-carboxymethyl-2,3-diethyl-10-methoxypyridazino bromide [1 ', 6': 1, 2] pyrido [3,4-b] indole-5-inium ( Compound 24), for the preparation of a medicine.

The pharmaceutical compositions or medicaments described in the present invention can be administered by any appropriate pharmaceutical preparation known to any person skilled in the art, as examples of pharmaceutical preparations any solid composition is included (tablets, pills, capsules, granules, etc.) or liquid (gels, solutions, suspensions or emulsions) for oral, nasal, topical or parenteral administration. Preferably the administration will be oral or parenteral (injectable).

In another aspect, the present invention relates to a method for inhibition of TNF-o production. or for the treatment or prevention of diseases inflammatory, more particularly those described above, in a mammal, preferably a human, comprising the administration of an amount therapeutically effective of at least one compound of formula (1) or (111).

Preferably, the administration of the composition can be performed orally, or parenterally (injectable).

In the sense used in this description, the term "therapeutically effective amount" refers to the amount of the composition calculated to produce the effect.

15 desired and, in general, will be determined, among other causes, by the characteristics of the composition, the age, condition and history of the patient, the severity of the disease, and the route and frequency of administration.

The compounds described in the present invention, of formula (1) or (111), are formed

20 as biologically and pharmaceutically acceptable salts. Useful salt forms are halides, particularly iodide, bromide and chloride, tasylates, methanesulfonates, brosylates, fumarates, maleates, succinates, acetates, mesylethenesulfonate salts, and the like. Other related salts can be formed using similar non-toxic anions that are biologically and pharmaceutically

25 acceptable.

The term "alkyl" refers in the present invention to aliphatic, linear or branched chains, having 1 to 20 carbon atoms, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, tert-butyl, sec-butyl, n-pentyl Preferably the group

Alkyl has between 1 and 10, and more preferably between 1 and 4, carbon atoms. The alkyl groups may be optionally substituted by one or more substituents such as halogen, hydroxyl, azide, aryl carboxylic acid, hydroxyl, amino, amide, ester, ether, thiol, acylamino or carboxamide, which in turn may optionally be substituted. Preferably the alkyl group is not substituted.

The term "alkenyl" refers in the present invention to an alkyl radical, as defined above, and which has at least one carbon-carbon double bond in its chain.

The term "alkynyl" refers in the present invention to an alkyl radical, such as It has been defined above, and it has at least one triple link in its chain carbon-carbon

The term "cycloalkyl" refers, in the present invention, to a chain radical

10 saturated carbocyclic and consisting of carbon atoms, between 3 and 6, more preferably 5 or 6. It may be optionally substituted with 1 or more substituents of the group consisting of alkyl, hydroxy, nitro, amino, halogen, aryl or aralkyl.

The term "aryl" refers in the present invention to a carbocyclic chain

15 aromatic, which has 5 to 18 carbon atoms, being able to be single or multiple ring, in the latter case with separate and / or condensed rings. A non-limiting example of aryl is a phenyl, naphthyl, indenyl, furyl group etc ... The aryl group may be optionally substituted by at least one group selected from a hydroxyl, ester, carboxyl, alkyl or halogen.

The term "aralkyl" refers, in the present invention, to an aliphatic chain of between 1 and 5 carbon atoms in which at least one of the hydrogens has been replaced by an aryl group, with the above meanings, as per example, but not limited to, a benzyl or phenethyl group. These aralkyl groups can, in turn,

25 be optionally substituted by an alkyl, hydroxy, nitro, amino or halogen group. Preferably it is an optionally substituted benzyl group, and more preferably it is an unsubstituted benzyl group.

The term "heteroaryl" refers, in the present invention, to a chain radical.

30 carbocyclic (mono- or bicyclic) and consisting of carbon atoms, between 3 and 6, more preferably 5 or 6, where at least one carbon atom is substituted by a heteroatom selected from the group consisting of nitrogen, oxygen or sulfur . This carbocyclic chain may be unsaturated or partially saturated. It may be optionally substituted by 1 or more substituents of the group consisting of alkyl,

Hydroxy, nitro, amino, halogen, aryl or aralkyl.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1. Cell viability of the human promonocytic THP-1 line preincubated for 24 hours with increasing concentrations of some compounds of the Formulas (1) and

(111) in the presence or absence of 100 ng / ml of LPS.

Fig. 2. Effect of some compounds of Formulas (1) and (111) on the production of LPS-induced TNF-a (100 ng / ml) in the human promonocytic THP-1 cell line.

Fig. 3. Effect of compound 17 on the production of TNF-a induced by LPS (10 ng / ml) in human peripheral blood mononuclear cells (PBMCs).

Fig. 4. Effect of compound 17 on the production of LPS-induced TNF-a (10 ng / ml) in human monocytes isolated from peripheral blood.

Fig. 5. Inhibition of TNF-a production induced by LPS (100 ng / ml) in THP-1 cells by compound 17 and calculation of ICso (4.49 ... ML) in THP-1 cells.

Fig. 6. Inhibition of TNF-a production induced by LPS (10 ng / ml) in human PBMCs by compound 17 and calculation of ICso (3.91 LM) in PBMCs.

Fig. 7. Inhibition of LPS-induced TNF-a production (10 ng / ml) in human monocytes (CD14 + leukocytes) by compound 17 and calculation of ICso (3.22¡LM) in isolated monocytes.

Fig. 8. Analysis of the effect of "in vivo" administration of compound 17.

EXAMPLES

Compounds of this invention that have been published have been prepared according to the procedures described above for them (Fontana, A .; Benito, E. J .; 5 Martín, M. J .; Sánchez, N .; Alajarín, R .; Cowboy, J. J .; Álvarez-Builla, J .; Lambel Giraudet, S .; Leonce, S .; I lost, A .; Caignard, D. Bioorg. Med. Chem. Lett. 2002, 11, 2611-2614; Pastor, J .; Siro, J. G .; García-Navío, J. L .; Cowboy, J. J .; Rodrigo, M. M .; Ballesteros, M .; Álvarez-Builla, J. Bioorg. Med. Ehem. Leff. 1995, 5, 3043-3048; Siro, J. G .; Pastor, J .; García-Navío, J. L .; Cowboy, J. J .; Álvarez-Builla, J .; Gago, F .; from 10 Pascual-Teresa, B .; Pastor, M .; Rodrigo, M. M. J. Org. Chem. 1997, 62, 5476-5483; Siro, J. G .; Pastor, J .; García-Navío, J. L .; Cowboy, J. J .; Álvarez-Builla, J. Tetrahedron 1998, 54, 19229-1936; Enrique J. Benito Molinero, Doctoral Thesis, University of

Alcalá, 2004). For other compounds, procedures similar to those described in this invention have been applied.

Scheme 1. Synthesis process of compounds 2 to 8.

6,

_MS "~ _3A _-" _ iona

__ 6 '~:; ::, __ ~ d ~. ~

"

H CH,

"

, "

I HNO, conc.

,

MSTS

SnCI, .2H, or

-

'---'- ~

"

, "

•

Example 1. Preparation of 6-bromo-1-methylpyrido [3,4-b] indole (Compound 2,

Scheme 1) To a solution of harma no 1 (0.20 g; 1.1 mmol) in THF (tetrahydrofuran) (10 mL), bromine (0.70 g; 4.4 mmol) is added with vigorous stirring at room temperature for 2 hours Next, an aliquot of sodium thiosulfate (10%;

10 mL), the mixture is basified with concentrated ammonium hydroxide, extracted and washed with dichloromethane (3 x 10 mL). The organic phase is concentrated, the residue being purified

obtained by silica gel column chromatography using as eluent a dichloromethane: methanol mixture (10: 1). The product is isolated as a green solid (0.19 g; 66%).

P. f. = 224-226 oC. 'H-NMR (300 MHz; CDCI,): 08.19 (d, lH, J = 5.5 Hz); 8.15 (d, lH, J = 2.0 Hz); 7.68 (d, lH, J = 5.6 Hz); 7.55 (dd, 1 H, J = 8.7 Hz, J = 1.9 Hz); 7.37 (d, 1 H, J = 8.7 Hz); 2.74 (s, 3H) ppm. "C-NMR (75 MHz; CDCI,): o142.1; 139.2; 137.0; 135.0; 130.7; 127.0; 124.1; 123.0; 113.1; 112, 8; 112.0; 19.2 ppm. IR (vm ,,, KBr): 3141; 3051; 2954; 2865; 1621; 1569; 1492; 1439; 1406; 1272; 1247; 977; 821; 800; 607 cm ".

Example 2. Preparation of 2-amino-6-bromo-1-methyl mesitylenesulfonate pyrido [3,4-b] indole-2-inium (Compound 3, Scheme 1) On a solution of 2 (0.26 g; 1 mmol) in dichloromethane (10 mL), with stirring vigorous at room temperature, a solution of 0 is added dropwise

hydroxylamine mesyllenesulfonal (MSH) (0.30 g; 1.4 mmol) in dichloromethane (5 mL).

After 1 hour, diethyl ether is added to facilitate precipitation of the salt formed. Filter and wash successively with dichloromethane (2 x 5 mL) and acetone (2 x 5 mL), then recrystallized from ethanol. The product is obtained as a yellow solid (0.38 g; 80%).

P. f. = 253-255 oC. 'H-NMR (300 MHz; DMSO-d,): o12.91 (s, 1 H, NH); 8.71 (d, 1 H, J = 1.8 Hz); 8.54 (s, 2H, NH); 7.89-7.81 (m, 3H); 7.70 (dd, lH, J = 8.8 Hz, J = 3.0 Hz); 6.70 (s, 2 H); 2.98 (s, 3 H); 2.46 (s, 6H); 2.13 (s, 3H) ppm. IR (vm ", KBr): 3258; 3151; 3082; 1638; 1484; 1451; 1281; 1166; 1088; 1016; 808; 681 cm".

Example 3. Preparation of 8-bromo-2,3-diethyl-11H mesitylenesulfonate

pyridazino [1 ', 6': 1,2) pyrido [3,4-b) indole-5-inium (Compound 4, Scheme 1)

To a solution of 3 (0.24 g; 0.5 mmol) in ethanol (20 mL), 3,4-hexadione (0.07 g; 0.6 mmol) and TEA (triethylamine) (0.06 g; 0.6 mmol). The reaction mixture is heated at reflux for 3 hours and concentrated to dryness. The residue obtained is purified by column chromatography on silica gel using dichloromethane: methanol (10: 1) as eluent. A yellow solid is obtained (0.17 9; 62%).

P. f. = 216-218 oC. 'H-NMR (300 MHz; DMSO-d,): o13.67 (s, 1 H, NH); 9.27 (d, 1 H, J = 7.3 Hz); 8.99 (s, 1 H); 8.90 (d, 1H, J = 6.9 Hz); 8.83 (s, 1 H); 7.87 (s, p, 2H); 6.70 (s, 2 H); 3.17 (e, 2H, J = 7.4 Hz); 3.01 (e, 2H, J = 7.0 Hz); 2.46 (s, 6H); 2.13 (s, 3 H); 1.44 (1.3H, J = 7.0 Hz); 1.40 (1.3H, J = 7.0 Hz) ppm. "C-NMR (75 MHz; CDCI, + CD, OD): o163.7; 154.0; 143.4; 142.4; 140.5; 136.4; 135.9; 132.5; 130, 7; 130.6; 129.9; 128.9; 125.0; 124.3; 121.6; 117.3; 115.0; 114.0; 25.9; 23.9; 22.7; 20.3; 11.2; 10.4 ppm IR (vm ,,, KBr): 3412; 2968; 2932; 1638; 1566; 1521; 1450; 1324; 1283; 1226; 1160; 1084; 1008;

675 cm-1_ Elemental analysis: Calculated for C27H2SN30 3SBr · 1H20; C: 56.45; H: 5.61;

N: 7.32; Found: C: 56.12; H: 5.47; N: 7.87.

Example 4. Preparation of 1-methyl-8-nitropyrid [3,4-b] indole (Compound 5, Scheme 1) On 60% concentrated nitric acid (12 mL), harmano 1 (0.91 g; 5 mmol) is added maintaining the temperature at O oC, and vigorous stirring for 2 hours, after which, the temperature is increased until the white suspension is dissolved, maintaining the stirring for 30 more minutes. Water / ice is added to the suspension and basified with potassium carbonate, filtering the solid precipitate obtained. After chromatography on a silica gel column using a dichloromethane: methanol mixture (20: 1) as eluent, the compound is isolated as a yellow solid (0.37 g; 33%).

P. f. = 215-217 oC. 'H-NMR (300 MHz; CDCI,): o9.93 (s, 1H, NH); 8.47-8.42 (m, 3H); 7.82 (d, 1H, J = 5.1 Hz); 7.37 (t ", 1H, J = 8.1 Hz, J = 7.7 Hz); 2.89 (s, 3H) ppm." CRMN (75 MHz; CDCI, + CD, OD): ;; 142.9; 139.5; 134.4; 133.4; 132.6; 129.1; 127.4; 125.6; 124.3; 119.3; 112.8; 19.7 ppm IR (vm ,,, KBr): 3378; 3091; 1635; 1574; 1475; 1430; 1340; 1305; 1266; 1205; 1180; 803; 737 cm- '.

Example 5. Preparation of 2-amino-1-methyl-8 mesitylenesulfonate

nitropyrid [3,4-b] indole-2-inium (Compound 6, Scheme 1)

On a solution of 5 (0.22 g; 1 mmol) in dichloromethane (10 mL), with vigorous stirring at room temperature, a solution of MSH (0.30 g; 1.4 mmol) in dichloromethane is added dropwise (5 mL). After 1 hour, diethyl ether is added to facilitate total precipitation of the aminated salt formed. Filter and wash successively with dichloromethane (2 x 5 mL) and acetone (2 x 5 mL), then recrystallized from ethanol. A yellow solid is obtained (0.36 g; 82%).

P. f. = 289-291 oC. 'H-NMR (300 MHz; CD, OD): S 8.80 (d, 1 H, J = 7.8 Hz); 8.70 (d, 1 H, J = 7.9 Hz); 8.63-8.54 (m, 2H); 7.64 (t, 1H, J = 8.0 Hz); 6.80 (s, 2 H); 3.24 (s, 3 H); 2.57 (s, 6H); 2.19 (s, 3H) ppm. IR (vm ,,, KBr): 3229; 3090; 1689; 1624; 1523; 1486; 1390; 1310; 1289; 1181; 1086; 1015; 845; 748; 680 cm- '.

Example 6. Preparation of 2,3-diethyl-12H-11-nitropyridazino mesitylenesulfonate [l ', 6': l, 2] pyrido [3,4-b] indole-5-inium (Compound 7, Scheme 1) A a solution of 6 (0.22 g; 0.5 mmol) in ethanol (20 mL), 3,4-hexadione (0.07 g; 0.6 mmol) and TEA (0.06 g; 0, 6 mmol). The reaction mixture is heated to

After refluxing for 3 hours, it is concentrated to dryness and the residue obtained is purified by silica gel column chromatography using a dichloromethane: methanol mixture (10: 1) as eluent. A yellow solid is obtained (0.10 g; 40%).

P. f. = 198-200 oC. 'H-NMR (300 MHz; CO, OO): ¡; 9.60 (s, 1 H); 9.13 (d, 1H, J = 7.6 Hz); 8.84-8.78 (m, 2H); 8.68 (d, 1H, J = 7.8 Hz); 7.63 (t, 1H, J = 8.1 Hz); 6.86 (s, 2H);

10 3.22 (e, 2H, J = 7.0 Hz); 3.08 (e, 2H, J = 7.0 Hz); 2.58 (s, 6H); 2.20 (s, 3 H); 1.60 (t, 3H, J = 7.3 Hz); 1.52 (t, 3H, J = 7.3 Hz) ppm. "C-NMR (75 MHz; CO, OO): ¡; 164.7; 155.6; 144.7; 136.9; 136.6; 134.5; 132.8; 132.6; 132.0 ; 131.5; 130.6; 127.2; 126.6; 125.3; 122.0; 118.8; 118.2; 108.7; 26.9; 25.1; 23.7; 21 , 1; 13.1; 11.7 ppm IR (vm ,,, KBr): 3126; 2976; 2938; 1687; 1617; 1566; 1468; 1425; 1317; 1252; 1206; 1177; 1104; 801; 738

15 cm-l. Elemental analysis: Calculated for C27H28N40SS · 2H20; C: 58.26; H: 5.80; N:

10.07; Found: C: 57.73; H: 5.72; N: 10.40.

Example 7. Preparation of 11-ammonium-2,3-diethyl-12H dichloride

pyridazino [l ', 6': 1,2] pyrido [3,4-b] indole-5-inium (Compound 8, Scheme 1)

A suspension of 7 (0.10 g; 0.2 mmol), SnCI2 -2H20 (0.2 g; 0.8 mmol) and HCI (35%, 0.5 mL) in water (2 mL), is Heat at reflux for 2 hours, after which HCI (35%, 0.2 mL) is added to the mixture. The precipitate obtained is filtered and washed successively with HCI (10%, 1 mL) and diethyl ether (2 x 10 mL). The product is obtained as a yellow solid (0.05 g; 71%) _

25 P. f. = 266-268 oC. 'H-NMR (300 MHz; OMSO-d,): ¡; 9.24 (s, lH); 9.05 (d, 1H, J = 6.9 Hz); 8.70 (d, lH, J = 6.9 Hz); 7.70 (d, lH, J = 7.8 Hz); 7.21 (t, lH, J = 7.8 Hz); 6.98 (d, 1H, J = 7.3 Hz); 4.25 (s, p, 3H, NH); 3.11 (e, 2H, J = 7.4 Hz); 2.95 (e, 2H, J = 7.0 Hz); 1.46 (t, 3H, J = 6.9 Hz); 1.38 (t, 3H, J = 7.3Hz) ppm. IR (vm ,,, KBr): 3420; 1627; 1554;

1483; 1414; 1338; 1253; 1238; 1210; 1011; 821; 749 cm ". Elemental analysis: 30 Calculated for C, 8H, 9N4CI2 · 3H20; C: 51, 58; H: 6.28; N: 13.43; found: C: 52.16;

H: 6.08; N: 12.78.

Scheme 2.-Synthesis process of compounds 10 to 17.

M, "

-

"" "

Example 8. Preparation of 9-N-Boc-harmol (Compound 10, Scheme 2) On a solution of harmol 9 (1 g; 5 mmol) in dichloromethane (50 ml), add

5 successively di-terbutyl carbonate (2.18 g; 10 mmol), TEA (0.50 g; 5 mmol) and

DMAP (4-dimethylaminopyridine) (0.61 g; 5 mmol). After leaving the mixture with stirring at room temperature for 24 hours, the solvent is removed in vacuo, without reaching dryness (20 mL), washing the mixture with water (3 x 10 mL). The organic phase is dried with anhydrous MgSO4, concentrated to dryness, purifying the residue obtained by silica gel column chromatography, using dichloromethane: methanol as eluent (20: 1). The compound is isolated as a white solid (1.14 g;

77%)

P. f. = 167-169 oC. 'H-NMR (300 MHz; CDCI3): o9.16 (s, 1 H); 8.29 (d, 1 H, J = 5.3 Hz); 7.70 (d, lH, J = 8.6 Hz); 7.51 (d, lH, J = 5.2 Hz); 7.18 (d, lH, J = 1.9 Hz); 6.97 (dd, lH,

J = 8.5 Hz, J = 2.0 Hz); 2.73 (s, 3 H); 1.63 (s, 9H) ppm. "C-NMR (75 MHz; CDC !,): or 153.0; 150.7; 141.7; 140.8; 138.0; 135.4; 127.3; 122.0; 119.4; 113.3; 112.6; 104.5; 84.7; 27.6; 20.4 ppm IR (Vro ,,, KBr): 3135; 3066; 2982; 2884; 1755; 1637; 1571; 1450; 1373; 1323; 1282; 1251; 1233; 1146; 949; 897; 749 cm ". Elemental Analysis for C "H" N, 0 3; C: 68.43; H: 6.08; N: 9.39; Found C: 67.99; H: 6.11; N: 8.92.

Example 9. Preparation of 7-benzyloxy-9-tert-butoxycarbonyl-1-methylpyridide [3,4blindole (Compound 11, Scheme 2)

At a suspension of 10 (0.30 g; 1 mmol), powdered potassium hydroxide (0.07 g; 1.2 mmol), anhydrous potassium carbonate (0.17 g; 1.2 mmol) and bis (N dichloride) , N'-benzylN, N'-diethyl) -N, N '- (oxidi-2, 1-ethanediyl) diamonium (BBDE-CI) (0.02 g; 0.05 mmol) in dry acetonitrile (50 mL) With vigorous stirring at room temperature, benzyl bromide (BrBn) (0.20 g; 1.2 mmol) is added. The resulting suspension is vigorously stirred at room temperature for 24 hours. The suspension is then filtered and the salts are washed with acetonitrile. The filtering and washing waters are concentrated to dryness, removing the solvent under reduced pressure. The residue obtained is purified by silica gel column chromatography using dichloromethane: melanol (10: 1) as eluent. The product is obtained as a yellow solid

pale (0.30 g; 77%).

P. f. = 180-182 oC. 'H-NMR (300 MHz; CDCI,): ¡; 8.33 (d, 1H, J = 5.5 Hz); 8.10 (d, 1H, J = 8.4 Hz); 7.82 (d, 1H, J = 5.1 Hz); 7.29-7.22 (m, 3H); 7.16 (d, 1 H, J = 1.8 Hz); 7.11 (dd, 1 H, J = 8.4 Hz, J = 2.0 Hz); 6.99-6.95 (m, 2H); 5.73 (s, 2 H); 2.84 (s, 3 H); 1.60 (s, 9H) ppm. "C-NMR (75 MHz; CDCI): ¡; 151.7; 151.5; 142.2; 141.5; 138.6; 137.5; 136.0; 128.9; 128.7; 127.4; 125.2; 122.0; 118.9; 114.1; 112.7; 102.2; 83, S; 48.2; 27.6; 23.0 ppm. IR (vm ,, , KBr): 3416; 2981; 1752; 1625; 1442; 1407; 1371; 1320; 1284; 1248;

1154; 1136; 817; 721; 649 cm-. Elemental Analysis for C24H24N20 3; C: 74.21; H: 6.23;

N: 7.21; found C: 73.85; H: 6.17; N: 7.44.

Example 10. Preparation of 2-amino-7-benzyloxy-9-tert mesylenesulfonate

butoxycarbonyl-1-methylpyrido [3,4-b] indole-2-inium (Compound 12, Scheme 2)

On a solution of 11 (0.38 g; 1 mmol) in diethyl ether (10 mL) with stirring

vigorous at room temperature, a solution of MSH is added dropwise (0.30

g; 1.4 mmol) in dichloromethane (5 mL). After one hour, diethyl ether is added to facilitate total precipitation of the aminated salt formed. Filter and wash successively with dichloromethane (2 x 5 mL) and diethyl ether (2 x 5 mL), obtaining the product as a white solid (0.50 g; 84%).

P. f. = 166-168 oC (dese.). 'H-NMR (300 MHz; CD, OD): ¡; 8.53 (d, 1 H, J = 7.0 Hz); 8.44 (d, 1H, J = 7.0 Hz); 8.41 (d, 1 H, J = 8.8 Hz); 7.54 (d, 1 H, J = 1.5 Hz); 7.33-7.28 (m, 4H); 7.02 (d, 2H, J = 6.6 Hz); 6.79 (s, 2H); 5.92 (s, 2H); 3.03 (s, 3 H); 2.57 (s, 6H); 2.18 (s,

3H); 1.52 (s, 9H) ppm.IR (v ~ .. KBr): 3420; 3285; 3157; 3064; 2978; 1758; 1630; 1449; 1370; 1324; 1276; 1251; 1209; 1180; 1156; 1135; 1085; 1015; 849; 678 cm. "

Example 11. Preparation of 10-benzyloxy-12-tert mesitylenesulfonate

butoxycarbonyl-2,3-diethylpyridazino [1 ', 6': 1, 2] pyrido [3,4-b] indole-5-inium (Compound

13, Scheme 2) To a solution of 12 (0.30 g; 0.5 mmol) in ethanol (20 mL) is added 3,4-hexadione

5 (0.07 g; 0.6 mmol) and anhydrous sodium acetate (0.05 g; 0.6 mmol). After 30 minutes at reflux, 3,4-hexadione (0.5 mmol) and anhydrous sodium acetate (0.04 g; 0.5 mmol) are added back to the mixture, finally leaving the mixture at reflux for 2 hours, after which is concentrated to dryness and the residue is purified by silica gel column chromatography using dichloromethane: methanol (10: 1) as eluent. Be

10 obtains a yellow solid (0.23 g; 63%).

P. f. = 183-185 oC. 'H-NMR (300 MHz; CD, OD + CDCI,): ¡; 8.82 (d, 1H, J = 7.0 Hz); 8.69 (s, 1 H); 8.67 (d, 1H, J = 7.1 Hz); 8.35 (d, 1H, J = 8.1 Hz); 7.51 (s, 1 H); 7.26-7.13 (m, 6H); 6.57 (s, 2H); 6.42 (s, 2H); 2.97 (e, 2H, J = 7.3 Hz); 2.84 (e, 2H, J = 7.3 Hz); 2.47 (s, 6H); 2.11 (s, 3 H); 1.59 (s, 9H); 1.40 (t, 2H, J = 7.2 Hz); 1.09 (t, 2H, J = 7.0 Hz)

15 ppm "C-NMR (75 MHz; CD, OD + CDCI,): ¡; 163.1; 153.6; 151.1; 145.2; 142.7; 139.2; 138.2; 136.6; 134.6; 131.6; 130.2; 130.0; 129.4; 127.6; 125.2; 125.1; 123.3; 123.2; 117.6; 117.1; 116, 1; 112.7; 103.3; 84.2; 49.1; 27.4; 26.1; 24.2; 22.4; 20.4; 11.0; 10.2 ppm. IR (vm ,,, KBr): 3408; 2978; 2934; 1758; 1622; 1574; 1451; 1411; 1337; 1139;

1086; 1014; 809; 678 cm-1. Elemental Analysis for C39H43N30SS · 3H20; C: 63.65; H: 20 6.71; N: 5.71; found C: 63.76; H: 6.47; N: 5.84.

Example 12. Preparation of 9-tert-butoxycarbonyl-1-methyl-7-methoxypyrid [3,4b] indole (Compound 14, Scheme 2) To a suspension of 10 (0.30 g; 1 mmol), powdered potassium hydroxide ( 0.07 g; 1.2

25 mmol), anhydrous potassium carbonate (0.17 g; 1.2 mmol) and SS DE-el (0.02 g; 0.05 mmol)

in dry acetonitrile (50 mL) with vigorous stirring at room temperature, iodomethane (0.17 g; 1.2 mmol) is added. The resulting suspension is vigorously stirred at room temperature for 24 hours. The suspension is then filtered and the salts are washed with acetonitrile. The filtrate and wash waters are concentrated to dryness, removing the solvent under reduced pressure. The residue obtained is purified by silica gel column chromatography using dichloromethane: methanol (10: 1) as eluent. The product is obtained as a yellow solid.

pale (0.20 g; 65%).

P. f. = 133-135 oC. 'H-NMR (300 MHz; CDCI,): ¡; 8.23 (d, lH, J = 5.2 Hz); 7.98 (d, lH, J = 8.4 Hz); 7.70 (d, 1 H, J = 5.2 Hz); 7.19 (s, 1 H); 7.00 (d, 1 H, J = 8.5 Hz); 4.06 (s, 3 H); 2.95 (s, 3 H); 1.64 (s, 9H) ppm. IR (vm, .. KBr): 3412; 2979; 2929; 1755; 1624; 1450;

1408; 1370; 1248; 1148; 1136; 819 cm-l. Elemental Analysis for ClsH20N203 C: 69.22;

H: 6.45; N: 8.97; Found C: 68.93; H: 6.37; N: 9.12.

Example 13. Preparation of 2-amino-9-tert-butoxycarbonyl-1-methyl-7-methoxypyrido [3,4-b] indole-2-inium mesylenesulfonate (Compound 15, Scheme 2) On a solution of 14 (0.30 g; 1 mmol) in diethyl ether (10 mL) with vigorous stirring at room temperature, a solution of MSH (0.30 g; 1.4 mmol) in dichloromethane (5 mL) is added dropwise. After one hour, diethyl ether is added to facilitate total precipitation of the aminated salt formed. Filter and wash successively with dichloromethane (2 x 5 mL) and diethyl ether (2 x 5 mL), obtaining the product as a white solid (0.46 g; 88%).

P. f. = 157-159 oC (dese.). 'H-NMR (300 MHz; DMSO-d,): ¡; 8.78 (d, lH, J = 6.7 Hz); 8.49 (d, lH, J = 6.6 Hz); 8.34 (d, 1 H, J = 8.8 Hz); 7.94 (s, 2H, NH); 7.60 (d, 1 H, J = 2.0 Hz); 7.23 (dd, 1 H, J = 8.8 Hz, J = 2.2 Hz); 6.71 (s, 2H); 3.94 (s, 3 H); 2.82 (s, 3 H); 2.47 (s, 6H); 2.14 (s, 3 H); 1.69 (s, 9H) ppm. IR (vm ", KBr): 3418; 2983; 1747; 1619; 1457; 1356; 1261; 1209; 1144; 1097; 835; 685 cm · '.

Example 14. Preparation of 12-tert-butoxycarbonyl-2,3-dimethyl-1 O-methoxypyrididazino [1 ', S': 1,2] pyrido [3,4-b] indole-5-inium (Compound lS, Scheme 2)

To a solution 15 (0.27 g; 0.5 mmol) in ethanol (20 mL), 2,3-butanedione (0.05 g; 0.6 mmol) and anhydrous sodium acetate (0.05 g; 0.6 mmol). After 30 minutes at reflux, 2,3-butanedione (0.5 mmol) and anhydrous sodium acetate (0.04 g; 0.5 mmol) are added back to the mixture, finally leaving the mixture for 2 hours at reflux, after which the mixture is concentrated to dryness, purifying the residue obtained by silica gel column chromatography using dichloromethane: methanol (10: 1) as eluent. The product is obtained as a yellow solid (0.11 g; 40%).

P. f. = 160-162 oC (dese.). 'H-NMR (300 MHz; CDCI, + CD, OD): ¡; 9.10 (s, lH); 8.80 (d, 1 H, J = 7.0 Hz); 8.57 (d, 1 H, J = 7.0 Hz); 8.29 (d, lH, J = 8.8 Hz); 7.48 (s, 1 H); 7.17 (d, 1 H, J = 8.4 Hz); 6.75 (s, 2H); 3.96 (s, 3 H); 2.74 (s, 3 H); 2.69 (s, 3 H); 2.52 (s, 6H); 2.15 (s, 3 H); 1.58 (s, 9H) ppm. "C-NMR (75 MHz; CDCI, + CD, OD): ¡; 161.6; 153.4; 149.8; 144.8; 141.2; 137.4; 135.3; 131.2; 130.2; 129.7; 129.5; 128.2; 127.1; 125.9; 123.8; 117.2; 116.8; 112.7; 103.1; 84.7; 55, 5; 27.2; 23.0; 20.6; 19.9; 19.7; 19.3; 18.4 ppm. IR (Vm ,,, KBr): 3408; 2979; 2927; 1758; 1624; 1556; 1458; 1370; 1311; 1252;

5 1184; 1124; 1012; 849; 670 cm-l. Elemental Analysis for CJ1HJsNJS06 · 1H20 C: 62.49;

H: 6.26; N: 7.05; found C: 62.37; H: 6.11; N: 7.11.

Example 15. Preparation of 2,3-dimethyl-12H-10-methoxy bromide

pyridazino [1 ', 6': 1,2] pyrido [3,4-b] indole-5-inium (Compound 17, Scheme 2)

A suspension formed by 16 (0.11 g; 0.2 mmol) and concentrated hydrobromic acid (1 mL) in acetone (10 mL) is refluxed for 2 hours. After this time, the resulting suspension is filtered hot, washing the solid obtained with acetone (2 x 10 mL). The product is obtained as an orange solid (0.065 g; 91%).

P. f. = 204-206 oC. 'H-NMR (300 MHz; DMSO-d,): ¡; 13.33 (s, 1 H); 9.18 (d, 1H, J = 7.0 15 Hz); 8.92 (s, 1 H); 8.77 (d, 1H, J = 7.0 Hz); 8.39 (d, 1H, J = 8.8 Hz); 7.24 (d, 1H, J = 1.8

Hz); 7.11 (dd, 1 H, J = 8.8 Hz, J = 2.2 Hz); 3.95 (s, 3 H); 2.75 (s, 3 H); 2.62 (s, 3 H) ppm. IR (vm .., KBr): 3419; 2984; 1627; 1482; 1445; 1410; 1332; 1274; 1200; 1165; 1094;

1023; 812; 682 cm-l. Elemental Analysis for C17H16N30Br · 1H20 C: 54.26; H: 4.82; N: 11.17; found C: 54.38; H: 5.03; N: 10.94.

Scheme 3.-synthesis process of compounds 19 to 31.

& j. N: N

CH, or,

CH, O

, ",, ~

0y, & "-0y"

Or € <

Or "" "

t "

"

Ethyl brorroacetate

MSTS

-

CH O ~ N-N

C ~ O ~ N: NH2

,,. "" "\

N "" -Et

H CH,

-

%

""

""

"'' '

~. N: N "'' '

CH, O

(

"" ""

". ,,,

v, OCH,

j, N "N

CH, O ~ 6

=, -Q: Q--, "'' '

3,4-hexaoodione N · N ',,,,

: "I 1;., IJ CH, O ~ 6 .. ~, N-NH, ____....., H N_N" N, .. 'A (CH, ¡, / "

(CH, ¡'

, "'/ CH, MSTS _ CH, (CH,), ~~~~ (CH, J, MSTS" "

""

Example 16, Preparation of 2-amino-1-methyl-7-methoxy-9H-pyrido [3,4-b] indole-2-inium mesitylenesulfonate (Compound 19, Scheme 3) To a commercial harmine solution 18 (0.20 g; 0.94 mmol) in dichloromethane (5 mL), with stirring and at room temperature, another solution of Q-mesitylenesulfonate of hydroxylamine (MSH) (0.41 g; 1.32 mmol) in dichloromethane is added dropwise (2 mL) _ The mixture is left under stirring at room temperature for 1 hour, after which ethyl ether is added to the resulting suspension. The solid obtained is filtered and

wash successively with dichloromethane and acetone. A white solid is obtained (0.37 g;

92%).

P. f. = 234-235 oC (Lit. 235-237 OC).

Example 17. Preparation of 2,3-diethyl-12H-10 mesitylenesulfonate

methoxypyridazino [1 ', 6': 1,2) pyrido [3,4-b) indole-5-inium (Compound 20, Scheme 3)

On a suspension of 19 (0.15 g; 0.35 mmol) in ethanol (5 mL), 3.4 is added

hexanedione (0.04 g; 0.35 mmol) and anhydrous sodium acetate (0.03 g; 0.35 mmol). After 24

hours at reflux, and then crushing the oil with a mixture of acetone-ethyl ether, the solid thus obtained is filtered, then washing it with hot acetone. It is recrystallized from acetic acid-acetone and a pale yellow solid is obtained (0.10 g;

56%)

P. f. = 253-254 oC. 'H-NMR (300 MHz; DMSO-d,): S 12.87 (s, lH); 9.12 (d, lH, J = 6.9 Hz); 8.91 (s, lH); 8.72 (d, lH, J = 6.9 Hz); 8.34 (d, lH, J = 8.8 Hz); 7.22 (d, lH, J = 2.0 Hz); 7.07 (dd, lH, J = 8.8 Hz, J = 1.8 Hz); 3.93 (s, 3 H); 3.10 (e, 2H, J = 7.3 Hz); 2.95 (e, 2H, J = 7, 3 Hz); 1.42 (t, 3H, J = 7.3 Hz); 1.37 (t, 3H, J = 7.3 Hz) ppm. IR (vm, ,, KBr): 2979; 2940; 1630; 1569; 1460; 1412; 1336; 1225; 1167; 1087; 1016; 813; 678 cm Elemental analysis: Calculated for C28HllNl04S1 / 2H20; C: 65.35; H: 6.27; N: 8.16; Found: C: 65.07; H: 6.44; N: 7.90.

Example 18. Preparation of 2,3-diethyl-10-methoxypyridazino [1 ', 6': 1,2] pyrido [3,4b) indole (Compound 21, Scheme 3) A suspension of 20 (0.10 g; 0 , 2 mmol) in water (5 mL) was treated with excess triethylamine at room temperature. Immediately, the suspension changed to a darker and more intense coloration. After 1 hour with stirring at room temperature, the suspended solid is filtered, washed with water, until the waters cease to be basic, and dried in vacuo, obtaining the anhydrobase as a red solid.

orange (0.055 g; 90%).

P. f. = 191-193 oC. 'H-NMR (300 MHz; DMSO-d,): S 9.12 (d, lH, J = 6.8 Hz); 8.91 (s, lH); 8.72 (d, lH, J = 6.8 Hz); 8.34 (d, lH, J = 8.9 Hz); 7.22 (d, lH, J = 2.0 Hz); 7.07 (dd, 1 H, J = 8.8 Hz, J = 2.4 Hz); 3.93 (s, 3 H); 3.10 (e, 2H, J = 7.3 Hz); 2.95 (e, 2H, J = 7.3 Hz); 1.42 (t, 3H, J = 7.3 Hz); 1.37 (t, 3H, J = 7.3 Hz) ppm. IR (vm ,,, KBr): 2971; 2935; 1618; 1565; 1462; 1410; 1319; 1201; 1159; 1124; 1085; 1030; 771 cm Analysis

Elementary: Calculated for C'9H19N30; C: 74.73; H: 6.27; N: 13.26; Found: C:

74.40; H: 6.49; N: 13.01.

Example 19. Preparation of 2,3-diethyl-12-ethoxycarbonylmethyl-10-methoxy bromide

5 pyridazino [1 ', 6': 1,2] pyrido [3,4-b] indole-5-inium (Compound 22, Scheme 3) At a suspension of 21 (0.06 g; 0.2 mmol) in Acetonitrile (5 mL), stirred at room temperature, ethyl bromoacetate (0.17 g; 1 mmol) is added dropwise. After 24 hours, the resulting suspension is filtered, obtaining a solid, which is washed successively with acetone (2 x 1 mL) and the minimum amount of ethanol. Finally

10 is recrystallized from an ethanol-ethyl ether mixture, and a yellow solid is obtained (0.028

g; 28%)

P. f. = 310 oC. 'H-NMR (300 MHz; DMSQ-d,): or 9.37 (d, lH, J = 6.6 Hz); 8.94 (s, lH); 8.76 (d, lH, J = 8.8 Hz); 8.48 (d, lH, J = 6.6 Hz); 7.69 (d, lH, J = 1.5 Hz); 7.17 (dd, lH, J = 8.8 Hz, J = 1.5 Hz); 6.02 (s, 2H); 4.23 (e, 2H, J = 7.3 Hz); 3 95 (s, 3 H); 3.14 (e, 2H,

15 J = 7.3 Hz); 2.98 (e, 2H, J = 7.3 Hz); 1.46-1.25 (m, 9H) ppm.IR (v ~ "KBr): 2974; 2939;

2885; 1742; 1624; 1572; 1519; 1412; 1344; 1273; 1163; 830 cm Elementary Analysis:

Calculated for C "H" NJOJ Br · 1H, O; C: 56.33; H: 5.75; N: 8.57; Found: C: 56.14;

H: 6.02; N: 8.67.

Example 20. Preparation of 2,3-diethyl-10-methoxy-12- (3-iodopropyl) pyridazino [1 ', 6': 1,2] pyrido [3,4-b] indole-5-y-nium ( Compound 23, Scheme

3)

A suspension of 21 (86.8 mg; 0.3 mmol) in 1,3-diiodopropane (5 mL) is irradiated in a microwave synthesizer for two periods of 10 minutes with a potency

25 of 300 watts, a precipitate appearing which is filtered hot and washed successively with acetone (2 x 5 mL) and diethyl ether (2 x 5 mL). The precipitate formed is recrystallized from ethanol, obtaining an orange solid (96 mg; 55%).

P. f. > 300 oC. 'H-NMR (300 MHz; DMSQ-d,): ón 9.18 (d, 1 H, J = 7.0 Hz); 9.04 (d, 1 H, J = 7.0 Hz); 8.87 (s, 1 H); 8.37 (d, 1 H, J = 8.8 Hz); 7.20 (s, 1 H); 7.08 (d, 1 H, J = 9.0 Hz);

5.19 (s "'2H); 3.94 (s, 3H); 3.35 (s"' 2H); 2.97 (e, 2H, J = 7.2 Hz); 2.90 (e, 2H, J = 7.2 Hz); 2.59 (s, p, 2H); 1.40 (t, 6H, J = 7.0 Hz) ppm.

Example 21. Preparation of 12-carboxymethyl-2,3-diethyl-10-methoxy bromide

pyridazino [1 ', 6': 1, 2] pyrido [3,4-b] indole-5-inium (Compound 24, Scheme 3)

A mixture of 22 (38.7 mg; 0.08 mmol) and HBr (48%, 1 mL) is heated at reflux for 3 hours. Then, the reaction mixture is allowed to cool and concentrated to dryness. The residue obtained is triturated in diethyl ether to provide an orange solid (30.9 mg; 85%).

P. f. = 234-235 oC. 'H-NMR (300 MHz; DMSO-d,): o9.33 (d, 1 H, J = 6.8 Hz); 8.90 (d, 1 H, J = 6.8 Hz); 8.58 (s, 1 H); 8.47 (d, 1 H, J = 8.9 Hz); 7.68 (s, lH); 7.17 (d, 1 H, J = 8.7 Hz); 5.96 (s, 2H); 3.97 (s, 3 H); 3.15 (e, 2H, J = 7.2 Hz); 2.99 (e, 2H, J = 7.2 Hz); 1.39 (I, p, 6H, J = 6.8 Hz) ppm.

Example 22. Preparation of 1-methyl-7-methoxy-9- (pent-4-inyl) pyrido [3,4-b] indole (Compound 25, Scheme 3) A mixture of harmine 18 (1.06 g; 5 mmol), 5-chloro-1-pentino (0.72 g; 7 mmol), BBDE-CI as phase transfer catalyst (0.11 g; 0.25 mmol), dry powdered potassium hydroxide (0.50 g ; 9 mmol), and anhydrous potassium carbonate (1.24 g; 9 mmol) in dry acetonitrile (50 mL); Stir vigorously at room temperature for 400 hours. The resulting suspension is filtered, washing the insoluble residue with

acetonitrile (3 x 10 mL). The filtrates were concentrated to dryness, the residue was

Purify by silica gel column chromatography using AcOEt (ethyl acetate) as eluent. The product is recrystallized from hexane, obtaining a white solid (0.84 g, 61%).

P. f. = 107-109 oC. 'H-NMR (300 MHz; CDCI,): o8.29 (d, 1 H, J = 5.1 Hz); 7.97 (d, 1 H, J = 8.8 Hz); 7.73 (d, 1 H, J = 5.1 Hz); 6.99 (d, 1 H, J = 2.2 Hz); 6.89 (dd, 1H, J = 8.4 Hz, J = 2.2 Hz); 4.64 (t, 2H, J = 7.7 Hz); 3.95 (s, 3 H); 3.03 (s, 3 H); 2.32 (td, 2H, J = 6.6 Hz, J = 2.5 Hz); 2.13 (t, 1 H, J = 2.5 Hz); 2.11-2.01 (m, 2H) ppm. IR (vm ,,, KBr): 3172; 2982; 2103; 1621; 1565; 1449; 1407; 1244; 1162; 1047; 809 cm ". Elemental analysis:

Calculated for C18H18NZO; C: 77.67; H: 6.52; N: 10.06; Found: C: 77.30; H: 6.67;

N: 9.92.

Example 23. Preparation of 2-amino-1-methyl-7-methoxy-9 (pent-4-inyl) pyrido [3,4-b] indole-2-inium mesylenesulfonate (Compound 26, Scheme 3) At a solution of 25 (0.26 g; 1 mmol) in dichloromethane (5 mL), with constant stirring and at room temperature, a solution of 0-methylethenesulfonate of hydroxylamine (MSH) (1.4 mmol), in dichloromethane (2) is added dropwise mL) The mixture is stirred at room temperature for 30 minutes, after which diethyl ether is added to the mixture to precipitate the salt formed. The solid is filtered and washed

successively with acetone and dichloromethane. It is recrystallized from ethanol to obtain a yellow solid (0.43 g; 88%).

P. f. = 201-202 oC. 'H-NMR (300 MHz; DMSO-d,): o8.54 (d, lH, J = 6.9 Hz); 8.43 (d, lH, J = 6.6 Hz); 8.30 (d, lH, J = 8.8 Hz); 7.67 (s, 2H); 7.37 (d, lH, J = 2.2 Hz); 7.06 (dd, 1 H, J = 8.8 Hz, J = 2.2 Hz); 4.72 (t, 2H, J = 7.7 Hz); 3.95 (s, 3 H); 3.15 (s, 3 H); 2.97 (t, lH, J = 2.5 Hz); 2.31 (td, 2H, J = 6.8 Hz, J = 2.5 Hz); 2.00-1, 94 (m, 2H) ppm. IR (vm .., KBr): 3264; 3191; 3086; 2971; 1622; 1578; 1452; 1233; 1205; 1009; 827; 676

l

cm-_ Elemental Analysis for C27H31N304S; C: 65.70; H: 6.33; N: 8.51; found C:

65.33; H: 6.50; N: 8.26.

Example 24. Preparation of 14-methoxy-16- mesitylenesulfonate (pent4

inil) acenaphth [l ", 2": 3 ', 4'] pyridazino [l ', 6': 1, 2] pyrido [3,4-b] indole-9-inium (Compound

27, Scheme 3) A mixture of 26 (0.10 g; 0.2 mmol), acenaphthoquinone (0.036 g; 0.2 mmol), anhydrous sodium acetate (0.016 g; 0.2 mmol) and ethanol (10 mL) , heated at reflux for 1

hour. The solid obtained is filtered hot, washing successively with hot ethanol (5 mL) and hot acetone (5 mL). Finally it is recrystallized from acetone-acetic acid. An orange solid is obtained (0.085 g; 67%)

P. f. = 273-274 oC. 'H-NMR (300 MHz; DMSO-d,): o9.55 (s, lH); 9.37 (d, lH, J = 6.9 Hz); 8.75 (d, 1 H, J = 6.9 Hz); 8.71 (d, 1 H, J = 6.9 Hz); 8.58 (d, 1 H, J = 7.3 Hz); 8.38 (d, 1 H, J = 8.4 Hz); 8.34 (d, 2H, J = 8.0 Hz); 8.02 (t, p, 2H, J = 7.4 Hz); 7.52 (d, 1 H, J = 1.8 Hz); 7.18 (dd, 1 H, J = 8.8 Hz, J = 2.2 Hz); 5.24 (t, 2H, J = 7.3 Hz); 4.05 (s, 3 H); 2.75 (s, lH); 2.54-2.51 (m, 2H); 2.38-2.29 (m, 2H) ppm. IR (vm. "KBr): 2932; 1621; 1574; 1544;

1409; 1250; 1223; 1161; 829; 678 cm-l. Elemental Analysis for C39H33N304S'2H: zO; C:

69.31; H: 5.52; N: 6.22; found C: 69.07; H: 5.19; N: 5.91.

Example 25. Preparation of 2,3-di- (2-furyl) -10-methoxy-12 mesitylenesulfonate

(pent-4-inyl) pyridazino [1 ', 6': 1, 2] pyrido [3,4-b] indole-5-i nium (Compound 28, Scheme

3)

A mixture of 26 (0.10 g; 0.2 mmol), furyl (0.04 g; 0.2 mmol) and sodium acetate

Anhydrous (0.016 g; 0.2 mmol) in ethanol (10 mL) was heated at reflux for 5 hours. The

The resulting solution was concentrated to dryness and the residue was triturated with acetone-diethyl ether. The solid thus obtained was filtered and washed successively with diethyl ether, water and acetone. An ocher solid (0.04 g; 43%) was obtained.

P. f. = 279-280 oC. 'H-NMR (300 MHz; CD, OD): S 9.27 (s, 1 H); 9.19 (d, 1 H, J = 6.6 Hz); 8.75 (d, 1H, J = 6.9 Hz); 8.36 (d, 1H, J = 8.8 Hz); 7.95 (d, 1H, J = 1.1 Hz); 7.91 (d, 1H, J = 2.1 Hz), 7.51 (d, 1H, J = 1.8 Hz); 7.20 (dd, 1H, J = 8.8 Hz, J = 2.1 Hz); 7.18 (d, 1H, J = 3.6 Hz); 6.81 (dd, 1H, J = 3.6 Hz, J = 1.8 Hz); 6.73 (dd, 1H, J = 3.6 Hz, J = 1.8 Hz); 6.58 (d, 1H, J = 3.7 Hz); 5.14 (t, 2H, J = 7.5 Hz); 4.05 (s, 3 H); 2.50 (t, 1H, J = 2.5 Hz); 2.47 (td, 2H, J = 6.6 Hz, J = 2.5 Hz); 2.34-2.26 (m, 2H) ppm. IR (vm ", KBr): 2957; 1683; 1639; 1621; 1563; 1411; 1210; 1017; 824; 679; 644 cm". Elemental Analysis

for C37H33N306S · 1H20; C: 66.75; H: 5.30; N: 6.31; found C: 66.44; H: 5.38; N:

6.25.

Example 26. Preparation of 1,10-bis- (1-methyl-7-methoxypyrido [3,4-b] indole-9-yl) - <leca

4,6-diino (Compound 29, Scheme 3) A mixture of 25 (0.26 g; 1 mmol) and cupric acetate (0.90 g; 5 mmol) in acetonitrile (20 mL), is refluxed for 48 hours, the reaction mixture is cooled, adding to

the same water (10 mL) and ammonia (25%) (5 mL). The mixture is extracted with

dichloromethane (30 mL), the organic phase is dried with anhydrous magnesium sulfate and concentrated to dryness. The obtained residue is purified by column chromatography on silica gel, using acetone / ethanol (8: 2 v / v) as eluent. The product was recrystallized from acetone, obtaining a pale yellow solid (0.12 g; 45%).

P. f. = 184-185 oC. 'H-NMR (300 MHz; CDC !,): S 8.29 (d, 2H, J = 5.5 Hz); 7.97 (d, 2H, J = 8.8 Hz); 7.74 (d, 2H, J = 5.1 Hz); 6.89 (d, 2H, J = 2.2 Hz); 6.89 (dd, 2H, J = 8.8 Hz, J = 2.2 Hz); 4.64 (t, 4H, J = 7.3 Hz); 3.94 (s, 6H); 3.04 (s, 6H); 2.38 (t, 4H, J = 6.6 Hz); 2.13-2.04 (m, 4H) ppm.IR (vm ", KBr): 2966; 2115; 1617; 1558; 1414; 1238; 1108; 839; 675 cm -1. Elemental analysis: Calculated for C36H34N402; C: 77.95; H: 6.18; N: 10.10; found C: 77.71; H: 6.33; N: 9.64.

Example 27. Preparation of 9.9'- (deca-4,6-diin-1,10-diyl} bis- (2-amino-1-methyl-7-methoxypyrido [3,4-b] indole) dimesylenesulfonate 2-in.) (Compound 30, Scheme

3)

To a suspension of 29 (0.55 g; 1 mmol) in dichloromethane (5 mL), with constant stirring and at room temperature, a solution of 0-methylethenesulfonate hydroxylamine (MSH) (3 mL) in dichloromethane (2 mL) was added dropwise mL) The mixture was stirred at room temperature for 1 hour, adding diethyl ether to favor salt precipitation. The solid was filtered and washed successively with acetone and dichloromethane, recrystallized from ethanol. A white solid is obtained

(0.55 g; 90%).

P. f. = 250-251 oC. 'H-NMR (300 MHz; DMSO-d,): i5 8.54 (d, 2H, J = 6.6 Hz); 8.43 (d, 2H, J = 6.9 Hz); 8.30 (d, 2H, J = 9.2 Hz); 7.67 (s, 4H); 7.35 (d, 2H, J = 1.8 Hz); 7.05 (dd, 2H, J = 8.8 Hz, J = 1.8 Hz); 4.70 (1.4H, J = 7.3 Hz); 3.94 (s, 6H); 3.13 (s, 6H); 2.47-2.43 (m, 4H); 2.02-1, 97 (m, 4H) ppm. IR (vm .., KBr): 3432; 3252; 3139; 2934; 2328; 1624; 1573; 1456; 1249; 1228; 1167; 1085; 1014; 816; 679 cm. "Analysis

Elementary: Calculated for CS4H60N60 SS2; C: 65.83, H: 6.14, N: 8.53; Found: C:

65.57, H: 6.22, N: 8.40.

Example 28. Preparation of 12,12 'dimesitylenesulfonate - (deca4,6-diin-1,10

di il) -bis- (2,3-dieti 1-1 O-methoxy pyridazino [1 ', 6': 1, 2] pyrido [3,4-b] indo 1-5-inio)

(Compound 31, Scheme 3)

A mixture of 30 (0.10 g; 0.1 mmol), 3,4-hexanedione (0.025 g; 0.2 mmol), acetate

Anhydrous sodium (0.008 g; 0.1 mmol) and ethanol (10 mL) was heated at reflux for 24 hours. The resulting solution was concentrated to dryness, the residue was triturated in an acetone-diethyl ether mixture and washed with the minimum amount of cold water (1 mL), finally dried under vacuum. A solid is obtained (0.06 g; 52%).

P. f. = 292-293 oC. 'H-NMR (300 MHz; CD30D, CF3COOD): i5 9.10 (d, 2H, J = 6.6 Hz); 8.73 (s, 2H); 8.64 (d, 2H, J = 6.9 Hz); 8.28 (d, 2H, J = 8.8 Hz); 7.22 (d, 2H, J = 2.5 Hz); 7.13 (dd, 2H, J = 8.8 Hz, J = 2.2 Hz); 5.73 (t, 4H, J = 7.5 Hz); 3.99 (s, 6H); 3.16 (e, 4H, J = 7.3 Hz); 3.05 (e, 4H, J = 7.3 Hz); 2.48-2.45 (m, 4H); 2.25-2.18 (m, 4H); 1.51-1.45 (m, 12H) ppm. IR (vm .., K8r): 2954; 1619; 1558; 1414; 1202; 1178; 1085; 679 cm. "

Elemental analysis: Calculated for C66H72N60SS2 · 1H20; C: 68.37; H: 6.43; N: 7.25;

Found: C: 68.04; H: 6.69; N: 7.43.

Example 29. Determination of the cell viability of the THP-1 line pre-incubated for 24 h with increasing concentrations of various compounds (Fig. 1) The human pro-monocytic THP-1 line was incubated for 24 h in culture medium

RPMI-1640 (Spear) supplemented with 5% fetal bovine serum (FBS) and increasing concentrations of compounds 24 (Fig. 1A), 17 (Fig. 1 B), 7 (Fig. 1 C), 23 (Fig. 10) and 22 (Fig. 1E). To determine the effect of cell stimulation on viability, duplicates of the cultures were stimulated with 100 ng / mL of bacterial lipopolysaccharide (LPS, SIGMA-Aldrich) during the last 4 hours.

Cellular viability was determined in each case by flow cytometry by the percentage of exclusion of propidium iodide added to a final concentration of 0.5 µg / mL. The analysis by flow cytometry was carried out in a FACSCalibur cytometer with the CellQuest Pro software (BO Bioscience, San José, CA, USA).

Compounds 24 and 7 did not affect cell viability in a concentration range of 10l-lM -100I-lM. Compounds 23, 22 and 17 showed toxicity at concentrations greater than 10I-lM.

Example 30. Production of LPS-induced TNF-a (100 ng / ml) in the human promonocytic THP-1 cell line in the presence of increasing concentrations of the different compounds (Fig. 2) THP-1 cells were cultured for 24 h in RPMI / 5% FCS in the absence or presence of increasing concentrations of the different compounds chosen within the ranges that did not show cytotoxicity. They were stimulated with 100 ng / ml of LPS during the last 4 hours of culture. The concentrations of the pro-inflammatory cytokine TNF-a in the culture supernatant were determined by Human TNF ELlSA Set (BO Bioscience) following the manufacturer's instructions. A straight pattern was made with known concentrations of the human recombinant protein. The absorbance was measured at 450 nm in the Synergy 4 plate reader by analyzing the results with Gen 5 software (Biotek).

Values (pg / ml of TNF-a / 105 cells) are shown as mean ± SO of 5 experiments performed with compound 24 (Fig. 2A), mean ± SO of 9 experiments performed with compound 17 (Fig. 2B) , mean of 2 experiments performed with compound 7 (Fig. 2C), mean of 2 experiments performed with compound 23 (Fig. 20) and mean ± SO of 3 experiments performed with compound 22 (Fig. 2E).

The 5 compounds caused a decrease in the production of TNF- (1 induced by LPS. The best results depending on the dose-response were obtained in the presence of compound 17.

Table 1 shows the inhibition of the production of TNF- (1 of the above compounds and some other compounds:

Table 1.-Inhibition (%) of the production of TNF- (1 by compounds of Formulas (1) and (111).

Compound (example) 4 (3) 7 (6) 8 (7) 13 (11) 17 (15) 20 (17) 22 (19) 23 (20) 24 (21) 27 (24) 28 (25) 31 ( 28)

0.1 ~ M -0.97 % TNF- inhibition (1 in THP-1 cells 1 ~ M 5 ~ M 1O ~ M 25 ~ M 50 ~ M 100 ~ M 89 44 56 29 47 88 94 34.9 63.4 87.8 76 51 67 45 94 11 36 72 79 38 29 35 40 94 87 34 90 le "('lM) 4.49 11.5

Example 31. Effect of compound 17 on the production of TNF-a. stimulated by LPS in peripheral blood mononuclear cells (PBMCs) (Fig. 3) Peripheral blood mononuclear cells (PBMCs) were isolated from blood donations from healthy individuals by gradient centrifugation of

15 ficoll (Amersham Biosciences). They were grown in RPM1 / 10% FCS for 24 h in the presence of increasing concentrations of compound 17 and were stimulated with 10 ng / mL of LPS during the last 4 hours of culture. The secreted TNF-a was evaluated in the culture supernatants by ELlSA as in the previous case. The results (pg / ml of TNF-a / 10s cells) are expressed as mean ± SO of 5 experiments

independent.

At low concentrations (0.1I1M-1I1M) the interindividual response was very variable.

However, at 5 and 1 µM an inhibition of TNF production was observed in all cultures.

Example 32. Effect of compound 17 on the production of TNF-a. lPS stimulated in monocytes isolated from peripheral blood of healthy individuals (Fig. 4) Monocytes (C014 + cells) were isolated from PBMCs of healthy individuals using magnetic microspheres associated with human anti-CD14 antibody (Miltenyi Biotec). Cell separations were performed with the AutoMacs automatic system or manually with magnetic columns (Miltenyi Biotec). It was verified by flow cytometry that in all cases the purity was greater than 90% in the separated cell fractions.

Once isolated, they were cultured in RPMI / 10% FeS for 24 h in the presence of increasing concentrations of compound 17 and stimulated with 10 ng / ml of lPS during the last 4 hours of culture. The secreted TNF-a was evaluated in the

culture supernatants by EUSA as in the previous case. The results (pg / ml of TNF-a / 10s cells) are expressed as mean ± SO of 8 independent experiments.

In isolated monocytes inhibition of TNF-a production was observed in the cultures

pretreated with compound 17 at a final concentration of 1 or 10 11M.

Example 33. Calculation of the 50% inhibitory concentration (lCso) for compound 17 in THP-1 cells (Fig. 5) Taking as reference the concentration of TNF-a secreted to the stimulated medium by treating 4 hours with 100 ng / ml of lPS in the absence of compound, the percentage of inhibition has been calculated for each of the concentrations tested of compound 17 in each experiment and the average of the percentage of inhibition for each concentration in the 9 experiments performed has been calculated. With these values a regression line has been drawn up to interpolate 50% of

Inhibition which in this case would correspond to a concentration of 4.49¡LM of compound 17 for 20 h prior to stimulation.

Example 34. Calculation of 50% inhibitory concentration (lC50) for

5 compound 17 in PBMCs (Fig. 6) Taking as a reference the concentration of TNF-a secreted to the stimulated medium by treating 4 hours with 10 ng / mL of LPS in the absence of compound, the percentage of inhibition for each one of the tested concentrations of compound 17 as in the previous case. The concentration calculated in

10 PBMCs is 1.83 LM of compound 17 for 20 h prior to stimulation.

Table 2. -Inhibition (%) of TNF-a production by compound 17

% TNF-a inhibition in PBMCs

Compound (example)

0.1 ~ M 1 ~ M 5 ~ M 1O ~ M 25 ~ M 50 ~ M 100 ~ M le "(~ M)

17 (15)

22.7 38.8 71, 2 69.2 3.91

Example 35. Calculation of the 50% inhibitory concentration (lC50) for compound 17 in human monocytes isolated from peripheral blood (Fig. 7) Taking as reference the concentration of TNF-a secreted to the stimulated medium by the 4-hour treatment with 10 ng / mL of LPS in the absence of compound, the percentage of inhibition has been calculated for each of the concentrations

20 tests of compound 17 as in the previous case. The concentration calculated in human monocytes (CD14 + cells) in culture is 3.22) lM of compound 17 for 20 h prior to stimulation.

Table 3.-Inhibition (%) of TNF-a production by compound 17 25

% TNF-a inhibition in human monocytes

Compound (example)

0.5 ~ M 1 ~ M 51lM 10 ~ M 25 ~ M 50 ~ M 100 ~ M ICso (~ M)

17 (15)

27 40 95.3 3.22

Example 36. Analysis of the effect of in vivo administration of compound 17 (Fig. 8) For in vivo studies female mice of strain C57BL6 (Charles

5 River, Barcelona, Spain). Upon reception the mice were kept in cages ventilated to minimize infection by pathogens. the mice used were 8-9 weeks of age and an approximate weight of 20 gr.

Two doses of compound 17 (2mg / kg in 200 JlL) were administered intraperitoneally.

10 saline) with an interval of 18h. Four hours after the second dose the animals were sacrificed, the spleens were removed and mechanically disintegrated to obtain cell suspensions. Spleen cells were cultured "in vitro" in RPMII10% FaS in the presence or absence of LPS (10ng / mL) for 4h. TNF-a levels. secreted were measured in the culture supernatant by cytometry of

15 flow (eSA). Control vs LPS p = 0.0156 (· p <0.05) Wilcoxon test. LPS vs T78d + LPS p = 0.0205 ("p <0.05) Mann-Whitney test.

As seen in Fig. 8, the cells of mice that have been treated in vivo with compound 17 show a significantly lower production of TNF-a when

20 are stimulated with LPS. This result indicates that the compound is effective when administered in vivo.

Claims (16)

one.

Use of a compound of general formula (1):

5

(one)

where:

R, is selected from the list comprising hydrogen, (C, -C, or) alkyl, alkenyl

(C, -C ,,), alkynyl (C, -C ,,), - (CH,), CO, R, ', - (CH') mR, ", - (CH,), CONHR, '' 'or the group

10

of formula (11):

'1 R R,

I

N

(eleven)

fifteen

where R is selected from the list comprising (C, -C20) alkyl, (C2) alkenyl

C ,,), alkynyl (C, -C ,,), - (CH,), CO, R, '- O, C (CH,), - or - (CH,), CONHR,' ''

NHOC (CH2) p-; n, m or p have a value between O and 5 and R, 'is selected from

hydrogen and alkyl (CrC, o), R, "is selected from halogen, alkenyl (C2-C4)

or (C2-C4) alkynyl and R1 '"is selected from (C, -C4) alkyl, (C2-C4) alkenyl or

twenty

alkynyl (Cr C4);

R2 and R3, are the same or different, and are selected from alkyl (CrC, o),

Cidoalkyl (C3-C6), aryl, heteroaryl or R2 and R3 can be linked together

forming part of a ring, saturated or aromatic;

R4 is selected from the list comprising hydrogen, alkyl (CrC, o), halogen,

25

nitro, amino, hydroxyl, O- ~ ', where R4' is selected from a (C1-C'2) alkyl,

aryl, aralkyl or heteroaryl; Y

X 'is a biologically or pharmaceutically acceptable anion;

or any of its pharmaceutically acceptable salts, for the preparation of

a drug for the inhibition of the production of TNF-a.

2. Use according to claim 1, wherein R1 is selected from hydrogen, (CH2) nC02 R1 'or - (CH2) mR l ". Use according to the preceding claim, wherein R1 'is selected from hydrogen or alkyl (C, -C ,,) and n is O Ó 1. 4. Use according to claim 2, wherein R1 "is selected from halogen or a alkynyl (e2-e3) and m has a value of 2 to 4. 10

5.

Use according to the preceding claim, wherein R1 "is iodine or ethynyl and m is 3.

6.

Use according to claim 1, wherein R1 is the group of formula (11) and said compound is that of general formula (111):

~ N R3 R, R R, ~ N R3 I ~ N-N ~ ~

-

+ X

15 R4 --- " (11 1) wherein R, R2, R3, R4 YX-have been described in claim 1.

Use according to the preceding claim, wherein R is alkyl (CS-C1S), alkenyl (CS-C1S)

or alkynyl (CS-C1S). 8. Use according to the preceding claim, wherein R is deca-4,6-diin-1.1 Q-diyl.

Use according to any one of claims 1 to 8, wherein R2 and R3 are the same or different, and are selected from methyl, ethyl, 2-furyl 01, 8-naphthodiyl.

10.

Use according to the preceding claim, wherein R2 and RJ are the same.

eleven.

Use according to any one of claims 1 to 10, wherein it is selected from amino, bromine, nitro, methoxy or benzoxyl.

12.

Use according to any of the preceding claims, wherein K is halide or mesitylenesulfonate.

13.

Use according to the preceding claim, wherein the halide is bromide.

14.

Use according to claim 1, wherein the compound is selected from: 9-bromo-2,3-diethyl-12H-pyridazino mesitylenesulfonate [1 ', 6': 1, 2] pyrido [3,4-b] indole- 5

inio 2,3-diethyl-12H-11-nitropyridazino [1 ', 6': 1, 2] pyrido [3,4-b] indole-5-ynium mesitylenesul! 11-ammonium-2,3-diethyl-12H-pyridazino [1 ', 6': 1, 2] pyrido [3,4-b] indole-5-inium dichloride, 10-benzyloxy-12-tert-butoxycarbonyl-2,3-diethylpyrididazino [1 ', 6': 1,2] pyrido [3,4-b] indole-5-inio mesylenesulfonate, 2,3-dimethyl-12H-1 0-methoxypyridazino bromide [1 ', 6': 1, 2] pyrido [3,4-b] indole-5-inium, 2,3-diethyl-12H-1 0-methoxypyrididazino [1 ', 6': 1, 2) pyrido [3,4-b] indole-5-inio mesylenesulfonate, 2,3-diethyl-12-ethoxycarbonylmethyl-1-0-methoxypyridazino bromide [1 ', 6': 1, 2] pyrido [3,4]

b] indole-5-inium,

I last

from 2,3-diethyl-1 O-methoxy-12- (3-iodopropyl) pyridazino [1 ', 6': 1,2] pyrido [3,4

b] indole-5-inium,

bromide

from 12-carboxymethyl-2,3-diethyl-1-0-methoxypyridazino [1 ', 6': 1, 2] pyrido [3,4

b] indole-5-inium,

14-methoxy-16- (pent-4inyl) acenaphth [1 ", 2": 3 ', 4'] pyridazino [1 ', 6': 1,2] pyrido [3,4-b] indole-9 mesylethenesulfonate -inium, 2,3-di- (2-furyl) -1 0-methoxy-12- (pent-4inyl) pyridazino [1 ', 6': 1, 2] pyrido [3,4-b] indole mesol -5-inio and 12, 12'-dimesitylenesulfonate - (deca-4,6-diin-1, 1-0-diyl) -bis- (2,3-diethyl-1 0-methoxypyridazino [1 ', 6': 1, 2 ] pyrido [3,4-b] indole-5-inio). 15. Use according to the preceding claim, wherein the compound is 2.3 bromide dimethyl-12H-1 O-methoxypyridazino [1 ', 6': 1, 2] pyrido [3,4-b] indole-5-inium. 16. Use of a compound of general formula (1) or (111) as described in 5 any of the preceding claims, for the preparation of a medication for the treatment and / or prevention of inflammatory diseases. 17. Use according to the preceding claim, wherein the inflammatory diseases are selected from the list comprising rheumatoid arthritis, psoriatic arthritis, arthritis 10 idiopathic autoimmune, osteoarthritis, Crohn's disease, ulcerative colitis, uveitis, asthma, bronchitis, obstructive chronic airway disease, psoriasis, allergic rhinitis, ankylosing spondylitis, suppurative hydradenitis, dermatitis and inflammatory processes associated with kidney disease. 18. Compound selected from the list comprising: 2,3-diethyl-1 0-methoxy-12- (3-iodopropyl) pyridazino [1 ', 6': 1, 2] pyrido [3,4b] indole iodide -5-inium and 12-carboxymethyl-2,3-diethyl-1 0-methoxypyridazino [1 ', 6': 1, 2] pyrido [3,4b] indole-5-inium bromide. 19. Pharmaceutical composition comprising a compound described in claim 18 and a pharmaceutically acceptable carrier. 20. Use of the compound according to claim 18 for the preparation of a medicament.