WO2013139929A1 - Novel means and methods for treating diseases of the central nervous system, metabolic and cardiac diseases and aging - Google Patents

Description

Novel means and methods for treating diseases of the central nervous system, metabolic and cardiac diseases and aging

This invention relates to a compound of formula (la) or (II a)

(la) (Ma) wherein either (A) R¹ is I and R² is selected from substituted or unsubstituted piperidinyl, preferably N-substituted or unsubstituted piperidin-4-yl; substituted or unsubstituted cyclohexyl, cyclopentyl, or cyclohexyl alkyl; substituted or unsubstituted aryl-alkyl; alkyl; aryl; - CHO; and hydrogen; or (B) R¹ is selected from halogen; substituted or unsubstituted alkoxy; substituted or unsubstituted alkyl; cyano; and hydrogen; and R² is substituted or unsubstituted piperidinyl, preferably N-substituted or unsubstituted piperidin-4-yl; and R³, R⁴ and R⁵ independently are either H or defined as R¹; R⁶ is H; alkyl or aryl-alkyl, or R² and R⁶ together with the N attached thereto form a 5- or 6-membered ring, preferably pyrrol; each of the m occurrences of R⁷ is independently selected from d to C₄ alkyl, Ci to C alkoxy and halogen, preferably F; and m being 0, 1 , 2, 3 or 4; X is selected from NH; N-alkyl, preferably NCH₃; N- aryl-alkyl; S; and O; wherein in formula (la) R² and X together may alternatively form or comprise a 6-membered ring, said 6-membered ring preferably being piperazine; n is 0, 1 or 2; the substituents being independently selected from alkyl, aryl-alkyl; alkoxy, aryl, hydroxy; halogen; oxo; and thio; alkyl being branched, unbranched and/or cyclic Ci to Ci₀ alkyl, preferably branched or unbranched Ci to C₄ alkyl, more preferably methyl, ethyl, n-propyl, i- propyl, i-butyl or t-butyl; alkoxy being to C₄ alkoxy, preferably methoxy; aryl being phenyl, naphthyl or tetrahydronaphthyl, preferably phenyl; or a compound of any one of Tables 3 to 46; or a salt or hydrate thereof. In this specification, a number of documents are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

Alzheimer's disease (AD), the most common form of dementia, is a growing threat to our aging society. There is still no effective therapy for AD and the current treatments are very inadequate (Brody, H. Alzheimer's disease. Nature 475, S1-S1 (2011 )). The very few approved AD drugs in the market at the best show symptomatic relief and only delay the progression of the disease temporarily. Despite the major breakthroughs in understanding the underlying mechanisms governing the disease progression within the last few decades, not much of progress has been made in the development of effective therapies. The major hallmarks of AD are the accumulation of tangles and plaques of amyloid beta (Αβ) protein in the brain. Current AD drug development attempts mainly focus on targeting these two major hallmarks of the disease. However, those events occur at rather late, usually fatal stages of AD at which the disease is most likely irreversible. This is probably one of the reasons for the consistent recent failure of disease-modifying AD drug candidates targeting amyloid and tangle pathologies in late clinical phases.

Long before the pathological hallmarks and cognitive deficits in AD manifest, the intracellular calcium homeostasis within neurons is likely to be altered as a consequence of aging or due to certain mutations in the Presenilin genes - the most common cause of early onset familial AD (FAD) (Bezprozvanny, I. & Mattson, MP. Neuronal calcium mishandling and the pathogenesis of Alzheimer's disease. Trends in neurosciences 31 , 454-63 (2008); Hayley, M., Perspicace, S., Schulthess, T. & Seelig, J. Calcium enhances the proteolytic activity of BACE1 : An in vitro biophysical and biochemical characterization of the BACE1 -calcium interaction. Biochimica et biophysica acta 1788, 1933-8 (2009); Thibault, O., Gant, J.C. & Landfield, P.W. Expansion of the calcium hypothesis of brain aging and Alzheimer's disease: minding the store. Aging cell 6, 307-17 (2007); Nelson, O., Supnet, C, Liu, H. & Bezprozvanny, I. Familial Alzheimer's disease mutations in presenilins: effects on endoplasmic reticulum calcium homeostasis and correlation with clinical phenotypes. Journal of Alzheimer's disease 21 , 781-93 (2010); Stutzmann, G.E. The pathogenesis of Alzheimers disease is it a lifelong "calciumopathy"? The Neuroscientist: a review journal bringing neurobiology, neurology and psychiatry 13, 546-59 (2007)). Long-term disruption of calcium homeostasis has been shown to both trigger and accelerate amyloid and tangle pathology (Hayley, M., Perspicace, S., Schulthess, T. & Seelig, J. Calcium enhances the proteolytic activity of BACE1 : An in vitro biophysical and biochemical characterization of the BACE1- calcium interaction. Biochimica et biophysica acta 1788, 1933-8 (2009); Green, K.N. & LaFerla, F.M. Linking calcium to Abeta and Alzheimer's disease. Neuron 59, 190-4 (2008); Pierrot, N. et al. Calcium-mediated transient phosphorylation of tau and amyloid precursor protein followed by intraneuronal amyloid-beta accumulation. The Journal of biological chemistry 281 , 39907-14 (2006)). Moreover, calcium dysregulation as a proximal event in AD progression plays a key role in synaptic failure and neuronal loss (Camandola, S. & Mattson, M.P. Aberrant subcellular neuronal calcium regulation in aging and Alzheimer's disease. Biochimica et biophysica acta 1813, 965-73 (2011 )). Notably, the latter irreversible pathological events correlate best with the stages of dementia (Gomez-lsla, T. et al. Profound loss of layer II entorhinal cortex neurons occurs in very mild Alzheimer's disease. The Journal of neuroscience: the official journal of the Society for Neuroscience 16, 4491-500 (1996)). Since intracellular calcium homeostasis is also critically involved in the pathophysiology of many other diseases and aging, compounds which reverse alterations in the intracellular calcium handling will also be beneficial for aging as well as disease conditions such as (but not restricted to) the chronic degeneration of retinal ganglion cells or cardiac arrhythmias. Therefore targeting and restoring the disrupted calcium homeostasis, particularly in intracellular calcium stores, e.g. endoplasmic reticulum (ER), as an early event in the cascade of events leading to cellular dysfunction opens novel avenues to more efficiently treat AD patients as well as other diseases where intracellular calcium handling is of pathophysiological relevance.

Mutations in presenilins (PS1 and PS2) account for the vast majority of early onset familial Alzheimer's disease (FAD) cases. These mutations result in enhancement of inositol 1 ,4,5- trisphosphate (IP₃) receptor sensitivity (Cheung, K.-H. et al. Mechanism of Ca²⁺ disruption in Alzheimer's disease by presenilin regulation of lnsP₃ receptor channel gating. Neuron 58, 871-83 (2008); Cheung, K.-H. et al. Gain-of-function enhancement of IP₃ receptor modal gating by familial Alzheimer's disease-linked presenilin mutants in human cells and mouse neurons. Science signaling 3, ra22 (2010)). This affects the beta amyloid (Αβ) generation in the brain as well as undesirable consequences in other cell types such as cardiac muscle cells. Shuttling of calcium between ER and mitochondria plays an important role in many fundamental biological processes (Kanwar, Y.S. & Sun, L. Shuttling of calcium between endoplasmic reticulum and mitochondria in the renal vasculature. American journal of physiology. Renal physiology 295, F1301-2 (2008)). Presenilin mutations have been shown to modulate shuttling of calcium between ER and mitochondria (Zampese, E. ef a/. Presenilin 2 modulates endoplasmic reticulum (ER)-mitochondria interactions and Ca²⁺ cross-talk. Proceedings of the National Academy of Sciences of the United States of America 108, 2777- 82 (201 1 )). Moreover, mitochondrial dysfunction is an early event in progression of AD, but also in many other neurodegenerative disorders as well as generally in the course of aging (Celsi, F. et al. Mitochondria, calcium and cell death: a deadly triad in neurodegeneration. Biochimica et biophysica acta 1787, 335-44 (2009); Mattson, M.P., Gleichmann, M. & Cheng, A. Mitochondria in neuroplasticity and neurological disorders. Neuron 60, 748-66 (2008)). Improving mitochondrial function is by itself a focus in AD drug development (Reddy, P.H. Amyloid beta, mitochondrial structural and functional dynamics in Alzheimer's disease. Experimental neurology 218, 286-92 (2009)).

One of the hallmarks of AD is the generation of amyloid plaques which are protein deposits found in the brains of AD patients. Αβ, the product of proteolytic cleavage of APP, is the major constituent of amyloid plaques (Hardy, J. A. & Selkoe, D.J. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science (New York, N. Y.) 297, 353-6 (2002); De Strooper, B. Proteases and proteolysis in Alzheimer disease: a multifactorial view on the disease process. Physiological reviews 90, 465-494 (2010)). Currently, most attempts in AD drug development are aiming to target the late Αβ pathology (Bergmans, B.A. & De Strooper, B. gamma-secretases: from cell biology to therapeutic strategies. Lancet neurology 9, 215-26 (2010)). Most approaches aim to inhibit or modulate the proteolytic cleavage of APP or to enhance Αβ clearance by using immunotherapy. However, so far most of these attempts have failed and there is a need for novel targets or approaches in the context of Αβ pathology. In view of the deficiencies of the prior art, the technical problem underlying the present invention can be seen in the provision of alternative or improved means and methods for treating a variety of disorders in conditions, said disorders being characterized by being related to disturbances of Ca²⁺ homeostasis and/or including diseases of the central nervous system such as Alzheimer's disease.

This technical problem has been solved by the subject-matter of the claims. In a first aspect, the present invention relates to a compound of formula (la) or (lla)

wherein either (A) R¹ is I and R² is selected from substituted or unsubstituted piperidinyl, preferably N-substituted or unsubstituted piperidin-4-yl; substituted or unsubstituted cyclohexyl, cyclopentyl, or cyclohexyl alkyl; substituted or unsubstituted aryl-alkyl; alkyl; aryl; - CHO; and hydrogen; or (B) R is selected from halogen; substituted or unsubstituted alkoxy; substituted or unsubstituted alkyl; cyano; and hydrogen; and R² is substituted or unsubstituted piperidinyl, preferably N-substituted or unsubstituted piperidin-4-yl; and R³, R⁴ and R⁵ independently are either H or defined as R¹; R⁶ is H; alkyl or aryl-alkyl, or R² and R⁶ together with the N attached thereto form a 5- or 6-membered ring, preferably pyrrol; each of the m occurrences of R⁷ is independently selected from d to C₄ alkyl, d to C₄ alkoxy and halogen, preferably F; and m being 0, 1 , 2, 3 or 4; X is selected from NH; N-alkyl, preferably NCH₃; N- aryl-alkyl; S; and O; wherein in formula (la) R² and X together may alternatively form or comprise a 6-membered ring, said 6-membered ring preferably being piperazine; n is 0, 1 or 2; the substituents being independently selected from alkyl, aryl-alkyl; alkoxy, aryl, hydroxy; halogen; oxo; and thio; alkyl being branched, unbranched and/or cyclic Ci to Ci₀ alkyl, preferably branched or unbranched d to C₄ alkyl, more preferably methyl, ethyl, n-propyl, i- propyl, i-butyl or t-butyl; alkoxy being Ci to C₄ alkoxy, preferably methoxy; aryl being phenyl, naphthyl or tetrahydronaphthyl, preferably phenyl; or a compound of any one of Tables 3 to 46; or a salt or hydrate thereof. This embodiment is also referred to as "main embodiment". The first aspect presents two alternative formulae (la) and (lla) which differ from each other only with regard to the position of the substituted amine group NR²R⁶. While preference is given to compounds of formula (la), it is at the same time envisaged that compounds of formula (lla) are modulators of calcium homeostasis.

Moreover, and in terms of groups R¹ and R², this aspect provides for two alternatives (A) and (B), wherein each alternative is characterized by specific combinations of R¹ and R². While for alternative (a) R¹ is limited to I, a variety of options is available for R¹ in case of alternative (B). Within said options for R¹ in case of alternative (B), preference is given to Br. Also preferred is that R¹ is I. Another preferred option is that R¹ is CN. Particularly preferred R² groups are in either case N-phenyl-methyl piperidin-4-yl and N- phenyl-ethyl piperidin-4-yl; see compounds ADM-43 and AD -44 in Table 3 as well as the compounds of Table 14. As can be seen from the enclosed experimental data, these substituents provide for particularly outstanding performance in the disease-relevant assays disclosed in the examples. Also preferred is that R² is N-ethyl-piperidin-4-yl or N-n-propyl- piperidin-4-yl. All these preferred R² groups apply equally to both alternatives (A) and (B).

Preferred R² groups in case of (A) are benzyl, phenyl-ethyl and cyclohexyl.

As regards the options for R³, R⁴ and R⁵, preference is given to R³, R⁴ and R⁵ all being H. In that case, the phenyl ring of formulae (la) and (lb) bears one substituent which is R¹.

As will be further detailed below, in one preferred embodiment R⁶ is H. In the alternative, and to the extent R⁶ is alky!, preference is given to methyl. Compound ADM-42 as shown in Table 3 is a specific example of a compound wherein R⁶ is methyl. To the extent R⁶ is aryl-alkyl, preference is given to benzyl.

As regards the number of occurrences of R⁷, particular preference is given to m being 0. Also preferred is that m is 1 or 2. To the extent m is 1 or greater than 1 , preference is given for R⁷ to be methyl and ethyl. Particularly preferred is methyl. Position of R⁷ is preferred at C-2, said n being 0, 1 or 2. Also preferred position for R⁷ is at C-4 for n being 1 or 2; and at C-5 for n being 2.

Formulae (VII-0), (VI 1-1 ) and (VII-2) below indicate the numbering scheme of the cycle bearing NR²R⁶ in compounds of formulae (la), (lb), (Ilia), (lllb), (Va) and (Vb).

(VI 1-0)

(VII-1)

(VII-2)

It is understood that one or more groups R⁷ may be present at any position(s) of the ring into which the free valence of the (R⁷)_m is pointing in formulae (la) and (lib) as well as in any of the further formulae presenting R⁷. A "free position" in that context is a position bearing hydrogen, hydrogen not being specifically indicated in any of the formulae.

To the extent reference is made to substituted and unsubstituted moieties, preference is given to unsubstituted moieties if not indicated otherwise.

As regards alkyi substituents, d to C₄ alkyi is preferred over C₅ to C-|₀ alkyi. Furthermore, generally unbranched alkyi chains are preferred over branched alkyi chains. To the extent alkyi is substituted, preference is given to substituted alkyi moieties carrying one or more halogen atoms, the halogen atoms preferably being F and/or the halogen atoms being the only substituents. A preferred embodiment of R¹ according to option (B) of the main embodiment is -CF₃.

Among aryl substitutents, phenyl is most preferred. Preferred aryl-alkyl substituents include benzyl and phenyl-ethyl.

The preferred alkoxy substituent is OCH₃. Preferred halogens, to the extent they fall under the term "substitutent" as used herein are F and CI.

As regards X, preference is given to NH. Preferred is that n is 1 or 2, in particular 1.

Particularly preferred compounds are shown in Table 14. Among these compounds, it is preferred that R¹ is CF₃ or CN. Also preferred is that R¹ is F or CI. Also preferred is a compound with the generic formula shown in Table 14 wherein R¹ is Br (herein also designated ADM-44; see Table 3).

In a preferred embodiment, the compounds according to the invention meet one, two or all three of the following functional criteria (1 ), (2) and (3). (1 ) First, a compound according to the invention, when administered to HEK293 cells, preferably administered in situ and incubated for 16 hours, decreases the peak amplitude of at least 1.1 -fold, preferably 1.5, 2-, 3-, 4-, 5- or 10-fold amplified agonist-induced calcium release from endoplasmatic reticulum (said agonist preferably being carbachol) in cells expressing familial Alzheimer's disease presenilin mutation, said presenilin mutation preferably being PS1 -M146L, by a factor of at least 1.1 , preferably 1.2, 1.5, 2, 3, 4, 5, 10 or 100, in comparison to negative control, an example of a negative control being DMSO. (2) Secondly, a compound according to the invention, when administered to HEK293 cells, preferably administered in situ and incubated for at least 1 hour, increases the TMRM dye (Sigma Aldrich) fluorescence intensity by at least 1.8-fold, preferably 2-, 3-, 4-, 5-, 10- or 100- fold after accumulation into mitochondria in comparison to negative control, an example of a negative control being DMSO. TMRM dye fluorescence intensity is proportional to the mitochondrial membrane potential. (3) Thirdly, the generation of beta amyloid peptides decreases by a factor of at least 1.3, preferably 1.5, 2, 3, 4, 5, 10 or 100, upon administration of a compound according to the present invention, preferably in situ, followed by 16 hours incubation, compared to a negative control, an example of a negative control being DMSO. More preferred is a reduction of beta amyloid formation below the threshold of detection. Preferred cells producing beta amyloid are described in the examples.

In other words, the compounds according to the invention typically show activity in assays for at least one of the following properties: (i) Stabilization of impaired endoplasmatic reticulum (ER) calcium homeostasis (ii) Improvement of mitochondrial function, more specifically of the mitochondrial membrane potential; and (iii) Lowering Αβ levels. Corresponding assays are described above, known in the art and furthermore described in Examples 19 to 21.

In a preferred embodiment of the first aspect, said compound is a compound of formula (Ilia) or (IVa)

(Ilia) (IVa) wherein each of the m occurrences of R⁷ is independently selected from d to C₄ alkyl; m being 0, 1 , 2, 3 or 4; and the remainder of groups and substituents being as defined above.

As regards the number of occurrences of R⁷, as stated further above, particular preference is given to m being 0. Also preferred is that m is 1 or 2. To the extent m is 1 or greater than 1 , preference is given for R⁷ to be methyl and ethyl. Particularly preferred is methyl. Position of R⁷ is preferred at C-2, said n being 0, 1 or 2. Also preferred position for R⁷ is at C-4 for n being 1 or 2; and at C-5 for n being 2.

Preference is given to compounds of formula (Ilia). Preferred values of m are 0 and 1 , 0 being particularly preferred. To the extent m is 1 or greater than 1 , a particularly preferred alkyl group is methyl. In a more preferred embodiment, said compound is a compound of formula (Va)

(Va)

wherein m is 0 or 1.

In a yet more preferred embodiment, said compound is a compound of formula (Via)

(Via)

In an even more preferred embodiment, R¹ is selected from halogen; substituted or unsubstituted Ci to C₄ alkoxy, preferably OCH₃; substituted or unsubstituted to C₄ alkyl, preferably methyl or CF₃; cyano; and hydrogen, R² is substituted or unsubstituted piperidinyl, preferably N-substituted or unsubstituted piperidin-4-yl; the substituents being alkyl, phenyl- alkyl or halogen; alkyl being Ci to C₄ alkyl, preferably methyl, ethyl, n-propyl, i-propyl or t- butyl; or a salt of hydrate thereof.

In a further preferred embodiment, (1 ) R² is (1.1 ) N-aryl-alkyl piperidin-4-yl, preferably N- phenyl-alkyl piperidin-4-yl, particularly preferred N-phenyl-methyl piperidin-4-yl or N-phenyl- ethyl piperidin-4-yl; (1.2) N-alkyl piperidin-4-yl, preferably N-ethyl piperidin-4-yl or N-prop-1-yl piperidin-4-yl; or (1.3) N-alkyl piperidin-4-ylalkyl or N-aryl-alkyl piperidin-4-ylalkyl, preferably N- benzyl piperidin-4-yl-ethyl; or (2) in conjunction with option (A) of the main embodiment, R² is (2.1 ) alkyl cyclohexyl, preferably 3-methyl cyclohexyl, 4-methyl cyclohexyl, 4-ethyl cyclohexyl or 4-tert-butyl cyclohexyl; aryl-cyclohexyl, preferably 4-phenyl cyclohexyl; alkyl cyclopentyl, preferably 3-methyl cyclopentyl; or substituted cyclohexylalkyl such as 5,5-dimethyl-1 ,3- dioxocyclohex-2-ylmethylenyl; (2.2) aryl alkyl, preferably unsubstituted or substituted phenyl alkyl, more preferably phenyl methyl, phenyl ethyl, 4-phenyl-but-2-yl, 1-(4-methoxy phenyl)- prop-2-yl, 1-(4-hydroxy 3-methoxy phenyl)-prop-2-yl, 4-(4-methoxy phenyl)-but-2-yl, 1-(3,4- dimethoxy phenyl)-prop-2-yl, 1-(2-methoxy phenyl)-prop-2-yl, 1-(2-fluoro phenyl) prop-2-yl; or a bicyclic aryl-alkyl group such as 2-oxindol-3-ylmethylenyl, 1 ,2,3,4-tetrahydronaphthalen-3-yl or dihydroinden-2-yl; or (2.3) branched or unbranched and substituted or unsubstituted alkyl, preferably isobutyl, tert-butyl, neopentyl, norbornyl or adamantyl, propyl, prop-2-yl, 2-hydroxy ethyl. Among the various alternatives provided by this preferred embodiment, particular preference is given to option (1.1 ). In combination therewith, but also in general, preferred R¹ groups are Br, I and, Br being most preferred.

Particularly preferred compounds in relation to various aspects of the present invention are those which have at least one "+" sign in at least one of the three columns on the right-hand side of Tables 1 to 3 below. Tables 1 and 2 show compounds which are preferred for medical uses, such medical uses being further detailed below. Table 3 as well as Tables 4 to 46 show preferred compounds in accordance with the first aspect. The compounds designated ADM- 05, ADM-06, ADM-09, ADM-17, ADM-26, ADM-27, ADM-32 and ADM-35 are less preferred.

Tables 1 to 3: Semiquantitative data for the performance of a variety of compounds in accordance with various aspects of the present invention. For each compound, its effect on calcium homeostasis, the β-amyloid (Αβ) levels (Αβ38, Αβ40 and Αβ42), and the mitochondrial membrane potential has been determined; see the last three columns. The respective assays used are those described herein above as well as in Examples 19 to 21.

Table 1

Table 2



Table 3

22

ADM-11 , ADM-22, ADM-43, ADM-44, ADM-46, ADM-47, ADM-48, ADM-49, ADM-50, ADM-51 and ADM-52 are particularly preferred compounds of the present invention (see also table 45 and 46). Tables 4 to 46 show further preferred compounds according to the invention including the first aspect thereof.

R¹: I, Br, F, CI, CF₃, CN, H₃C orH

R¹: I, Br, F, CI, CF₃, CN, H₃C orH

R¹: I, Br, F, CI, CF₃, CN, H₃C orH

R¹: I, Br, F, CI, CF₃, CN, H₃C orH

R¹: I, Br, F, CI, CF₃, CN or H₃C

R¹: F, CI, Br, I, CF₃, CN, H₃C orH

R¹: I, Br, F, CI, CF₃, CN, H₃C orH

R¹: I, Br, F, CI, CF₃, CN, H₃C orH

R¹: F, CI, CF₃, CN, H₃C or H

I, F, CI, CF₃ or CN

R¹: I, Br, F, CI, CF₃, CN, H₃C orH 



Table R¹: I, F, CI,

H CF₃, CN, H₃C, 34

N0₂ or H

Table R¹: I, Br, F, CI,

CF₃, CN, H₃C, 35 H

N0₂ or H

Table R¹: I, Br, F, CI,

CF₃, CN, H₃C, 36 H

N0₂ or H

Table R¹: I, Br, F, CI,

CF₃, CN, H₃C, 37

N0₂ or H

Table R¹: I, Br, F, CI,

CF₃, CN, H₃C, 38

N0₂ or H

Table R¹: I, Br, F, CI,

CF₃, CN, H₃C, 39

N0₂ or H in

It is understood that any of the compounds according to the invention as disclosed herein, be it specific compounds or generic formulae, expressly include, where applicable, racemic mixtures as well as pure enantiomeric (both R and S forms) as well as, where applicable, pure diastereomeric forms. Also envisaged are mixtures of enantiomeric or diastereomeric forms with varying percentage of the respective pure compound, i.e. mixtures are not confined to racemates.

The synthesis of preferred compounds according to the invention is described in Examples 1 to 18 as enclosed herewith. Provided with the teaching of these examples, and based on common general knowledge, the skilled person can synthesize any of the further compounds according to the present invention without further ado.

In a second aspect, the present invention provides a pharmaceutical composition comprising a compound or salt or hydrate thereof, said compound being as defined in accordance with the first aspect including preferred embodiments thereof or a compound of Table 2.

The pharmaceutical compositions described herein can be administered to a patient to be treated or a subject in which prevention is to be effected at a suitable dose. Administration of the compositions may be effected by different ways, e.g., oral, intravenous, intraperitoneal, subcutaneous, as well as transdermal administration.

The compounds (or salts or hydrates thereof) may, accordingly, be administered orally, parenterally, such as subcutaneously, intravenously, intramuscularly, intraperitoneally, intrathecally, transdermally, transmucosaily, subdurally, locally or topically via iontopheresis, sublingually, by inhalation spray, aerosol or rectally and the like, for example in dosage unit formulations optionally comprising conventional pharmaceutically acceptable excipients.

The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient or subject depends upon many factors, including the patient's or subject's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition described herein may comprise further agents depending on the intended use of the pharmaceutical composition. It will be appreciated by the person of ordinary skill in the art that the compounds of the invention and, if applicable, any additional therapeutic agent may be formulated in one single dosage form, or may be present in separate dosage forms and may be either administered concomitantly (i.e. at the same time) or sequentially. Pharmaceutically useful excipients that may be used in the formulation of the pharmaceutical compositions according to the invention may comprise carriers, vehicles, diluents, solvents such as monohydric alcohols such as ethanol, isopropanol and polyhydric alcohols such as glycols and edible oils such as soybean oil, coconut oil, olive oil, safflower oil cottonseed oil, oily esters such as ethyl oleate, isopropyl myristate; binders, adjuvants, solubilizers, thickening agents, stabilizers, disintergrants, glidants, lubricating agents, buffering agents, emulsifiers, wetting agents, suspending agents, sweetening agents, colourants, flavours, coating agents, preservatives, antioxidants, processing agents, drug delivery modifiers and enhancers such as calcium phosphate, magnesium state, talc, monosaccharides, disaccharides, starch, gelatine, cellulose, methylcellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-p-cyclodextrin, polyvinylpyrrolidone, low melting waxes, ion exchange resins.

Other suitable pharmaceutically acceptable excipients are described in Remington^'s Pharmaceutical Sciences, 15^th Ed., Mack Publishing Co., New Jersey (1991 ).

Dosage forms for oral administration include tablets, capsules, lozenges, pills, wafers, granules, oral liquids such as syrups, suspensions, solutions, emulsions, powder for reconstitution. Dosage forms for parenteral administration include solutions or emulsions for infusion, solutions, suspensions or emulsions for injection pre-filled syringes, and/or powders for reconstitution. Dosage forms for local/topical administration comprise insufflations, aerosols, metered aerosols, transdermal therapeutic systems, medicated patches, rectal suppositories, and/or ovula.

For the purpose of the present invention, a therapeutically effective dosage of a compound of the invention will generally be from about 1 to 1000 mg/day, preferably from about 5 to about 500 mg/day, and most preferably from about 10 to about 200 mg/day, which may be administered in one or multiple doses.

It will be appreciated, however, that specific dose level of the compounds of the invention for any particular patient or subject will depend on a variety of factors such as age, sex, body weight, general health condition, diet, individual response of the patient or subject to be treated time of administration, severity of the disease to be treated, the activity of particular compound applied, dosage form, mode of application and concomitant medication. The therapeutically effective amount for a given situation will readily be determined by routine experimentation and is within the skills and judgement of the ordinary clinician or physician.

Pharmaceutical compositions according to the present invention may comprise one compound according to the present invention as the only pharmaceutically active agent. Alternatively, two or more compounds according to the present invention may be present as the only active agents. In yet further alternative embodiments, in addition to one or more compounds according to the present invention, further pharmaceutically active agents may be present, for example pharmaceutically active agents known in the art to be suitable in the treatment or prevention of one or more of the medical indications disclosed herein. In such a case, and noting that the compounds of the present invention act via a novel mechanism (establishing calcium homeostasis where this is disturbed), synergistic effects are expected to occur. Preferred examples of such further pharmaceutically active agents are gamma secretase modulators (e.g. GSM-01 ) and inhibitors (e.g. LY 450139 and MK-0752), BACE1 inhibitors (e.g. LY 2811376), ADAM 10 activators (e.g. Etazolate and Bryostatin-1 ), PPAR agonists (e.g. Rosaglitazone), antibodies against Αβ (e.g. Bapineuzumab, Gammagard, LY- 2062430 and Ganteneurumab) and against Tau protein (e.g. Anti-phospho tau antibodies), anti-aggregative compounds (e.g. ELND-005 and Methylthioninium chloride), acetylcholinesterase inhibitors (e.g. Donezepil, Rivastigmin and Galantamin), ion channel blockers, in particular NMDA receptor antagonists (e.g. Memantine), Neurotrophins (e.g. NGF), Neuroprotective and anti-oxidative stress agents (e.g. Dimebon and EVT-302). The addition of such further pharmaceutically active agents may provide synergistic beneficial effects and their combined use may be advantageous.

While the above embodiments relating to pharmaceutical compositions are described in terms of compounds according to the present invention as well as salts or hydrates thereof, the pharmaceutical compositions according to the present invention are not so limited. In particular, it is furthermore envisaged to use prodrug forms of compounds according to the present invention. A "prodrug" is a compound that is generally not biologically and/or pharmacologically active. After administration, the prodrug is activated, typically in vivo by enzymatic or hydrolytic cleavage and converted to a biologically and/or pharmacologically active compound which has the intended medical effect. Prodrugs are typically obtained from compounds according to the present invention by chemical modification. Conventional procedures for the selection and preparation of the prodrugs are described, for example, in Design of prodrugs, ed.h. Buntgaard, Elsevier, 1984.

Without wishing to be bound by a specific theory, the present inventors considered that the above disclosed compounds as well as further compounds disclosed below such as those of Table 1 act on a cellular mechanism which mechanism is involved in the regulation of calcium homeostasis, mitochondrial activity and amyloid-β formation or clearance. One such mechanism is considered to be the regulation of AMPK or β-secretase (BACE1 ) expression and/or activity. A corresponding assay is described in Example 22. Figure 4 displays corresponding data, the data showing activity of compounds of the invention on AMPK.

Given that compounds according to the present invention act on such common cellular mechanism, it follows that said compounds are suitable for the treatment of a variety of medical indications, said medical indications being characterized in that calcium homeostasis, synaptic function, and/or AMPK activity is known to be of relevance in such indications. Such indications are the subject of the above disclosed specific medical uses.

A further parameter according to the invention which is indicative of a compound's suitability in the treatment of specific medical indications is the mitochondrial activity. The mitochondrial activity is known to be of particular relevance in central nervous system disorders, brain disorders, neural degenerative disorders, neuropsychiatric diseases, cardiovascular diseases and muscle disorders.

A reduction of beta amyloid (Αβ) amounts and/or β-secretase expression/activity is particularly indicative of compounds suitable for treating or preventing Alzheimer's disease and/or mild cognitive impairment (MCI).

As is known in the art, mild cognitive impairment represents an intermediate state of cognitive function between the changes seen in aging and those fulfilling the criteria for dementia and often Alzheimer's disease.

Accordingly, in a third aspect, the present invention provides a compound or salt or hydrate thereof for use in a method of treating or preventing a disorder, said disorder being selected from central nervous system disorders, brain disorders, neurodegenerative disorders, neuropsychiatric diseases, obesity, metabolic syndrome, cardiovascular diseases, muscle disorders, cancer, inflammatory diseases, autoimmune diseases and age-associated disorders, or for use in a method of extending life-span, said compound being a compound of formula (lb) or (lib)

(lb) (Mb) wherein R¹ is selected from halogen; substituted or unsubstituted alkoxy; substituted or unsubstituted alkyl; cyano; N0₂; and hydrogen; R² is selected from substituted or unsubstituted piperidinyl, preferably N-substituted or unsubstituted piperidin-4-yl; substituted or unsubstituted cyclohexyl, cyclopentyl, or cyclohexyl alkyl; substituted or unsubstituted aryl- alkyl; alkyl; aryl; -CHO; and hydrogen, R³, R⁴ and R⁵ independently are either H or defined as R¹; R⁶ is H; alkyl or aryl-alkyl, or R² and R⁶ together with the N attached thereto form a 5- or 6- membered ring, preferably pyrrol; each of the m occurrences of R⁷ is independently selected from d to C₄ alkyl, d to C₄ alkoxy and halogen, preferably F; and m being 0, 1 , 2, 3 or 4; X is selected from NH; N-alkyl, preferably NCH₃; N-aryl-alkyl; S; and O; wherein in formula (lb) R² and X together may alternatively form or comprise a 6-membered ring, said 6-membered ring preferably being piperazine; n is 0, 1 or 2; the substituents being independently selected from alkyl, aryl-alkyl; alkoxy, aryl, hydroxy; halogen; and oxo; thio; alkyl being branched, unbranched and/or cyclic Ci to C₁₀ alkyl, preferably branched or unbranched Ci to C₄ alkyl, more preferably methyl, ethyl, n-propyl, i-propyl, i-butyl or t-butyl; alkoxy being d to C₄ alkoxy, preferably methoxy; aryl being phenyl, naphthyl or tetrahydronaphthyl, preferably phenyl; or a salt or hydrate thereof.

Formulae (lb) and (Mb) are distinguished from formulae (la) and (I Is), respectively, in that the options for substituents R and R² are different. Similar considerations apply mutatis mutandis to a comparison between formulae (Ilia) and (Illb), and so forth.

Central nervous system disorders, brain disorders, neurodegenerative disorders, neuropsychiatric diseases are preferred. Cancer, obesity and metabolic syndrome are less preferred.

In a preferred embodiment of the third aspect, said compound is a compound of formula (Illb) or (IVb)

(Illb) (IVb) wherein each of the m occurrences of R⁷ is independently selected from d to C alkyl; m being 0, 1 , 2, 3 or 4; and the remainder of groups and substituents being as defined in the second aspect. In a more preferred embodiment, said compound is a compound of formula (Vb)

(Vb)

wherein m is 0 or 1.

In a yet more preferred embodiment, said compound is a compound of formula (VIb)

(VIb)

In a particular preferred embodiment of said compound or salt or hydrate thereof for use, R¹ is selected from halogen; substituted or unsubstituted to C₄ alkoxy, preferably OCH₃; substituted or unsubstituted to C₄ alkyi, preferably CF₃; cyano; N0₂; and hydrogen; R² is selected from substituted or unsubstituted piperidinyl, preferably N-substituted or unsubstituted piperidin-4-yl; substituted or unsubstituted cyclohexyl; and phenyl-alkyl; the substituents being alkyi or phenyl-alkyl; alkyi being Ci to C₄ alkyi, preferably methyl, ethyl, n- propyl, i-propyl or t-butyl; or a salt of hydrate thereof. In a preferred embodiment, R¹ is Br, I, OCH₃, F, CI, CF₃, CN, CH₃ or H.

In a further preferred embodiment R² is (a) N-aryl-alkyl piperidin-4-yl, preferably N-phenyl- alkyl piperidin-4-yl, particularly preferred N-phenyl-methyl piperidin-4-yl or N-phenyl-ethyl piperidin-4-yl; (b) N-alkyl piperidin-4-yl, preferably N-ethyl piperidin-4-yl or N-prop-1-yl piperidin-4-yl; (c) N-alkyl piperidin-4-ylalkyl or N-aryl-alkyl piperidin-4-ylalkyl, preferably N- benzyl piperidin-4-yl-ethyl; (d) alkyi cyclohexyl, preferably 3-methyl cyclohexyl, 4-methyl cyclohexyl, 4-ethyl cyclohexyl or 4-tert-butyl cyclohexyl; aryl-cyclohexyl, preferably 4-phenyl cyclohexyl; alkyl cyclopentyl, preferably 3-methyl cyclopentyl; or substituted cyclohexylalkyl such as 5,5-dimethyl-1 ,3-dioxocyclohex-2-ylmethylenyl; (e) aryl alkyl, preferably unsubstituted or substituted phenyl alkyl, more preferably phenyl methyl, phenyl ethyl, 4-phenyl-but-2-yl, 1- (4-methoxy phenyl )-prop-2-yl, 1-(4-hydroxy 3-methoxy phenyl)-prop-2-yl, 4-(4-methoxy phenyl)-but-2-yl, 1-(3,4-dimethoxy phenyl)-prop-2-yl, 1-(2-methoxy phenyl)-prop-2-yl, 1-(2- fluoro phenyl) prop-2-yl; or a bicyclic aryl-alkyl group such as 2-oxindol-3-ylmethylenyl, 1 ,2,3,4-tetrahydronaphthalen-2-yl or dihydroinden-2-yl; or (f) branched or unbranched and substituted or unsubstituted alkyl, preferably isobutyl, tert-butyl, neopentyl, norbornyl or adamantyl, propyl, prop-2-yl, 2-hydroxy ethyl.

Particularly preferred are options (a) to (c).

In a further preferred embodiment of the pharmaceutical composition according to the second aspect, said compound is selected from the compounds of Table 1 to 46. Further preferred compounds are those described as being preferred in relation to the first aspect of this invention.

Related to the third aspect and its preferred embodiments, the present invention provides in a fourth aspect a method of treating a patient suffering from a disorder or a subject at risk of developing a disorder, said disorder being selected from central nervous system disorders, brain disorders, neurodegenerative disorders, neuropsychiatric diseases, obesity, metabolic syndrome, cardiovascular diseases, muscle disorders, cancer, inflammatory diseases, autoimmune diseases and age-associated disorders, and/or of extending life-span, said method comprising the step of administering to said patient or subject a pharmaceutically effective amount of a compound or salt or hydrate thereof as defined in relation to the second aspect.

In preferred embodiments of the third and fourth aspect, said central nervous system disorders, brain disorders, neural degenerative disorders and neuropsychiatric diseases are selected from mild cognitive impairment (MCI), dementia, Alzheimer's disease (AD), Parkinson's disease (PD), Amyothrophic lateral sclerosis (ALS), dementias with lewy bodies, Huntington's disease (HD), frontotemporal dementia (FTD), parkinsonism, tauopathies, aberrant/insufficient neurogenesis diseases, stroke, seizures, ataxia, migraine, schizophrenia, major depression, bipolar disorder, Down's syndrome, traumatic brain injury, posttraumatic stress disorder, chronic stress, alcohol and/or drug abuse, multiple system atrophy (MSA), progressive supranuclear palsy, corticobasal degeneration (CBD), retinal ganglion degeneration, and prion-related disorders. Particularly preferred are mild cognitive impairment (MCI) and Alzheimer's disease (AD). In a further preferred embodiment, obesity and/or metabolic syndrome are further characterized in that they are associated with insulin resistance, type 2 diabetes, glucose intolerance, fatty liver disease, hypertension, diabetic myopathy, lipotoxicity, dyslipidemia, hyperlipidemia, hypertriglyceridemia, and hypercholesterolemia. Preferred cardiovascular diseases and muscle disorders include atherosclerosis, cardiac hypertrophy, cardiac arrhythmias, myocardial ischemia-reperfusion injury, arrhythmogenic right ventricular dysplasia type 2, catecholaminergic polymorphic ventricular tachycardia, sarcopenia diffuse atrophy, vascular restenosis, hypercalcemia, and ischemia. Preferred forms of cancer include epithelial cancers, skin, lung, prostate, breast and adipose tumors and carcinomas, endometrial cancer, adenocarcinomas, squamous cell carcinomas, and leukaemia.

Inflammatory diseases and autoimmune diseases include lung, pancreas and intestine inflammation, chronic obstructive pulmonary disease, inflammatory bowel disease, pancreatitis, and rheumatoid arthritis.

Age associated disorders include common cancer, prostate enlargement, cardiovascular diseases, stroke, atherosclerosis, hypertension, osteoporosis, type 2 diabetes, mild cognitive impairment (MCI), Alzheimer's disease, Parkinson's disease, age-related macular degeneration and tauopathies. Mild cognitive impairment (MCI) and Alzheimer's disease are preferred.

Tauopathies are a group of neurodegenerative diseases with pathological deposition of abnormal tau protein isoforms in brain and central nervous system.

Any of the above listed more specific indications is a disease or disorder which is independently amenable to treatment or prevention by the compounds of the present invention. In preferred embodiments of the third and fourth aspect, said neurodegenerative disorder is Alzheimer's disease, in particular familial Alzheimer's disease; mild cognitive impairment (MCI); or dementia. In a fifth aspect, the present invention provides the use of a compound or salt or hydrate thereof as defined in relation to the second aspect in the manufacture of a medicament.

If not specified otherwise, a preferred embodiment as described in conjunction with a specific aspect applies mutatis mutandis also to the other aspects of the present invention.

The figures show: Figure 1 :

The effect of particularly preferred compounds according to the invention on mitochondrial membrane potential 4 The moieties displayed in the upper parts of the respective bars are the R² moieties; see the generic formula in the Figure.

Figure 2:

The effect of particularly preferred compounds according to the invention on calcium homeostasis. The moieties displayed in the upper parts of the respective bars are the R² moieties; see the generic formula in the Figure.

Figure 3:

The effect of particularly preferred compounds according to the invention on beta amyloid formation. The moieties displayed in the upper parts of the respective bars are the R² moieties; see the generic formula in the Figure.

Figure 4:

(A) The effect of particularly preferred compounds according to the invention and (B) further tetrahydrocarbazole analogues on pAMPK levels.

Figure 5:

The effects of tetrahydrocarbazole analogues on the FAD-PS1 -mediated disruptive ER calcium release (A) and (B) The activity of tetrahydrocarbazole analogues has been tested at 10 μΜ in PS1- M146L HEK293 cells. The presented values indicate the normalized peak amplitude of CCh- evoked calcium release for cells treated with each compound for 24 hours relative to the peak amplitude of DMSO-treated control (normalized ER calcium). Compounds marked with # symbol possess a certain level of toxicity which interferes with calcium release measurement in this assay. TP (1 μΜ), CPA (20 μΜ), TMB-8 (50 μΜ) and Bepridil (20 μΜ), all lowering the amount of calcium release from ER, were used as positive controls,

(n.s.: non-significant; ^* P<0.05, ^** P<0.01 and ^*** P<0.001 ; N=4). Figure 6:

The effect of tetrahydrocarbazole analogues on mitochondrial membrane potential

(A) Representative TMRM staining images of HEK293 cells pretreated for 1 hour with the indicated tetrahydrocarbazole analogues (10 μΜ) or Dimebon (10 μΜ) as a positive control, relative to DMSO-treated control (scale bars: 10 pm).

(B) Quantification of the average TMRM staining signals showing relative intensity for tetrahydrocarbazole analogues upon 1 hour pretreatment of HEK293 cells (10 μΜ). The bars highlighted with single- and double-stripes represent the most active compounds ADM-11 and ADM-22, which respectively contain N-(1-benzylpiperidin-4-yl) and N-(1-phenethylpiperidin-4- yl) groups at their R² position.

(C) Quantification of average TMRM intensity for synthesized tetrahydrocarbazole derivatives tested at 10 μΜ. The marked single-striped bars represent the analogous structures similar to ADM-11 , containing N-(1-benzylpiperidin-4-yl) at their R² position, and double-striped bars represent derivative compounds similar to ADM-22, which contain N-(1-phenethylpiperidin-4- yl) group at R² position.

(D) Quantification of average dose-dependent TMRM relative intensities for 3 select analogues of the lead structure tested at 6 different concentrations relative to Dimebon. The

EC₅₀ of all analogues tested lies at low micromolar range.

All values are normalized to DMSO value, which is set to 1.

(n.s.: non-significant; ^* P<0.05, ^** PO.01 and ^*** P<0.001 ; N=8). Figure 7:

The effect of tetrahydrocarbazoles on APP processing

(A) Relative Αβ38, Αβ40 and Αβ42 levels, decreased after 16 hours treatment with synthesized tetrahydrocarbazole derivatives at 10 μΜ in HEK293 cells coexpressing APPsw and PS1-M146L. Sulindac sulfide (50 μΜ) a γ-secretase modulator, and DAPT (10 μΜ) a γ- secretase inhibitor, were used as positive controls. Inside of the box, a schematic illustration of APP processing by α-, β- and γ-secretase is presented. (B) Relative Αβ42/Αβ40 ratios calculated from (A). Treatment with the majority of the lead structure derivatives does not alter Αβ42/Αβ40 ratio, whereas positive control Sulindac sulfide significantly lowers Αβ42/Αβ40 ratio.

(C) Relative Αβ38, Αβ40 and Αβ42 levels are decreased after 16 hours treatment with select tetrahydrocarbazole derivatives at 10 μΜ in HEK293 cells overexpressing wild type APP.

(D) Relative Αβ42/Αβ40 ratios calculated from (C). Treatment with select lead structure derivatives tested does not alter Αβ42/Αβ40 ratio. (E) Relative sAPPa and εΑΡΡβ levels after 16 hours compound treatment in wild type HEK293 cells. Treatment with select tetrahydrocarbazole derivatives does not (or only marginally) affect secreted sAPPa levels, whereas secreted εΑΡΡβ fragment levels are remarkably decreased. All values are normalized to the value of DMSO, which is set to 1.

(n.s.: non-significant; * P<0.05, *^* P<0.01 and *** PO.001 ; N=2).

Figure 8:

Dose-dependent effects of tetrahydrocarbazole derivatives on Αβ production

The effect of three select synthesized derivatives of tetrahydrocarbazole lead structure on the production of (A) Αβ38, (B) Αβ40 and (C) Αβ42 peptides tested at 6 different concentrations in APPsw/PS1-M146L-expressing HEK293 cells. The IC₅₀ of all Αβ species for all derivative structures tested lies at low micomolar range. All values are normalized to the value of DMSO, which is set to 1. (n.s.: non-significant; * PO.05, ** P<0.01 and *** PO.001 ; N=4). Figure 9:

The effect of synthesized tetrahydrocarbazole analogues on γ-secretase cleavage activity in HEK293-C99 cells (A) Relative Αβ38, Αβ40 and Αβ42 levels after 16 hours treatment with select tetrahydrocarbazole derivatives at 10 μΜ in HEK293-C99 cells. Sulindac sulfide (50 μΜ), Bepridil (30 μΜ) and DAPT (10 μΜ), respectively, a γ-secretase modulator, an iGSM, and a v- secretase inhibitor, were used as positive controls. All values are normalized to the value of DMSO, which is set to 1. (n.s.: non-significant; ^* P<0.05, ** P<0.01 and ^*** PO.001 ; N=2).

(B) Relative Αβ42/Αβ40 ratios calculated from (A). Treatment with tested analogues does not alter Αβ42/Αβ40 ratios, whereas positive controls Sulindac sulfide and Bepridil, respectively lead to a significant decrease and increase in Αβ42/Αβ40 ratios. All values are normalized to the value of DMSO, which is set to 1. (n.s.: non-significant; ^* P<0.05, ^** P<0.01 and *** PO.001 ; N=2).

Figure 10:

Structure-activity relationship for particularly preferred compounds. M and I stand for mesomeric and inductive effect, respectively.

The following examples illustrate the invention but should not be construed as being limiting. Example 1 :

-Tetrahydro-1 H-carbazol-1 -one

A mixture of ethyl 2-oxocyclohexanecarboxylate (26 ml, 164 mmol) and potassium hydroxide (11.6 g) in water (100 ml) was stirred fur 4 hours at 30°C and then cooled 0°C. In a separate flask a solution of aniline (14 ml, 150 mmol) in water (300 ml) and hydrochloric acid (33 ml) was cooled to 0°C before sodium nitrite (14.4 g) was added. The mixture was stirred at 0°C for 1 hour. After addition of urea (10 g) the solution was neutralized with a saturated solution of sodium hydrogencarbonate in water and stirred until gas formation ceased. Then both mixtures were combined, immediately acidified with acetic acid (25 ml), and stirred at room temperature over night. The resulting precipitate was collected by filtration, then dissolved in formic acid (300 ml), and stirred at 80°C over night. The formic acid was evaporated and the residue was treated with sodium hydroxide solution (2 N, 100 ml) and extracted with ethyl acetate (3 x 100 ml). The combined organic layers were dried over sodium sulfate and the solvents evaporated. The residue was crystallised from methanol/water to yield 2,3,4,9- tetrahydro-1H-carbazol-1-one as a brown solid (13 g, 70.3 mmol, 47 % yield).

1H-NMR (400 MHz, CD₂CI₂): δ (ppm) = 9.38 (br s, 1 H), 7,68 (d, J = 8.1 Hz, 1 H), 7.46 (d, J = 8.4 Hz, 1 H), 7.36 (t, J = 7.6 Hz, 1 H), 7.15 (t, J = 7.5 Hz, 1 H), 3.01 (t, J = 6.1 Hz, 2 H,), 2.65 (t, J = 6.3 Hz, 2 H,), 2.32 - 2.20 (m, 2 H). MS (El): m/z 185 [M^+,J; MS (CI): m/z 186 [M⁺ + H].

Example 2:

-lodo-2,3,4,9-tetrahydro-1 - -carbazol-1 -one

6-lodo-2,3,4,9-tetrahydro-1 /-carbazol-1-one was prepared from 4-iodoaniline and ethyl 2- oxocyclohexanecarboxylate in a similar manner as described in Example 1 to give a brown solid (26% yield).

¹H-NMR (500 MHz, CD₂CI₂): δ (ppm) = 9.17 (br s, 1 H), 8.09 - 8.01 (s, 1 H), 7.60 (dd, J = 8.7, 1.7, 1 H), 7.26 (d, J = 8.7, 1 H), 2.96 (t, J = 6.1 , 2H), 2.64 (t, J = 6.5, 2H), 2.29 - 2.22 (m, J = 6.3, 2H). MS (El): m/z 311 [M^+*]; MS (CI): m/z 312 [M⁺ + H].

Example 3:

6-Bromo-2,3,4,9-tetrah dro-1 H-carbazol-1 -one

6-Bromo-2,3,4,9-tetrahydro-1 -/-carbazol-1 -one was prepared from 4-bromoaniline and ethyl 2-oxocyclohexanecarboxylate in a similar manner as described in Example 1 to give a brown solid (60% yield).

¹H-NMR (400 MHz, CD₂CI₂): δ (ppm) = 9.32 (br s, 1 H), 7.83 (s, 1 H), 7.44 (dd, J = 8.8, 1.9, 1 H), 7.37 (d, J = 8.8, 1 H), 2.97 (t, J = 6.1 , 2H), 2.65 (t, J = 6.5, 2H), 2.31 - 2.21 (m, J = 6.3, 2H). MS (El): m/z 264 [M^+*]; MS (CI): m/z 265 [M⁺ + H]. Example 4:

-Dichloro-2,3,4,9-tetrahydro-1 H-carbazol-1 -one

7,8-Dichloro-2,3,4,9-tetrahydro-1 H-carbazol-1 -one was prepared from 2,3-dichloroaniline and ethyl-2-oxocyclohexanecarboxylate in a similar manner as described in Example 1 to give a brown solid (30% yield).

¹H-NMR (400 MHz, CD₂CI₂): δ (ppm) = 9.17 (br s, 1 H), 7.53 (d, J = 8.5, 1 H), 7.23 (dd, J = 8.5, 0.7, 1 H), 2.98 (t, J = 6.1 , 2H), 2.64 (t, J = 6.5, 2H), 2.30 - 2.22 (m, 2H). MS (El): m/z 254 [M^+']; MS (CI): m/z 255 [M⁺ + H].

Example 5:

-Bromo-2,3,4,9-tetrahydro-1 H-carbazol-1 -amine hydrochloride

A solution of 6-bromo-2,3,4,9-tetrahydro-1 H-carbazol-1 -one (500 mg, 1.9 mmol), ammonium acetate (1.46 g, 19 mmol) and sodium cyanoborohydride (597 mg, 9.5 mmol) in methanol was stirred at 60°C over night. The mixture was diluted with water (25 ml) and extracted with ethyl acetate (3 x 20 ml). The combined organic layers were dried over sodium sulfate and the solvents evaporated. The residue was dissolved in methanol, acidified with hydrochloric acid and the precipitate collected by filtration to yield 6-bromo-2, 3, 4, 9-tetrahydro-1 H-carbazol-1 - amine hydrochloride as a brown solid (265 mg, 0.88 mmol, 46 % yield).

1H-NMR (500 MHz, DMSO-d₆): δ (ppm) = 11.30 (br s, 1 H), 8.68 (br s, 3H), 7.66 (d, J = 1.9, 1 H), 7.38 (d, J = 8.6, 1 H), 7.24 (dd, J = 8.6, .9, 1 H), 4.55 (s, 1 H), 2.71 - 2.56 (m, 2H), 2.22 - 2.12 (m, 1 H), 2.06 - 1.97 (m, 1H), 1.96 - 1.88 (m, 1 H), 1.84 - 1.75 (m, 1 H). MS (El): m/z 265 [M^+*]; MS (CI): m/z 266 [M⁺ + H]. Example 6:

-Bromo-9-methyl-2,3,4,9-tetrahydro-1 H-carbazol-1 -one

A solution of 6-bromo-2,3,4,9-tetrahydro-1 H-carbazol-1 -one (396 mg, 1.5 mmol) in anhydrous acetone (10 ml) was cooled to 0°C and sodium hydride (7.5 mmol) was added. The mixture was stirred at room temperature for 30 minutes, then methyl iodide (1.065 g, 7.5 mmol) was added. After 4 hours of further stirring, the mixture was diluted with water (20 ml) and extracted with ethyl acetate (3 x 20 ml). The combined organic layers were dried over sodium sulfate and the solvents evaporated. Purification was accomplished by silica gel chromatography with dichloromethane/ethyl acetate (3:1 ), followed by recrystallisation from ethyl acetate to yield 6-bromo-9-methyl-2,3,4,9-tetrahydro-1 H-carbazol-1 -one as a pale brown solid (260 mg, 0.94 mmol, 63% yield).

¹H-NMR (500 MHz, CDCI₃): δ (ppm) = 7.78 (dd, J = 1.9, 0.5, 1 H), 7.45 (dd, J = 8.9, 1.9, 1 H), 7.22 (dd, J = 8.9, 0.5, 1 H), 4.04 (s, 3H), 2.96 (t, J = 6.1 , 2H), 2.65 (t, J = 6.5, 2H), 2.25 - 2.17 (m_f J = 6.3, 2H). MS (El): m/z 278 [M^+*]; MS (CI): m/z 279 [M⁺ + H].

Exampie 7:

-Benzyl-2,3,4,9-tetrahydro-1 H-carbazol-1 -amine hydrochloride

2,3,4,9-Tetrahydro-1 H-carbazol-1 -one (200 mg, 1.08 mmol), magnesium perchlorate (12 mg, 0.05 mmol) and benzylamine (286 μΙ, 2.6 mmol) were stirred in dichloroethane for 8 hours, then sodium borohydride (78 mg, 2.1 mmol) was added. After stirring the mixture for another 5 hours, a saturated solution of sodium hydrogencarbonate (10 ml) was added, followed by extraction with ethyl acetate (3 x 15 ml). The combined organic layers were dried over sodium sulfate and the solvents evaporated. Purification was accomplished by silica gel chromatography with isohexane/ethyl acetate (2:1 ) to yield a yellow oil. Hydrogen chloride gas was bubbled through a solution of this oil in diethyl ether, for one minute. The precipitate was collected by filtration to afford /V-benzyl-2,3,4,9-tetrahydro-1 H-carbazol-1 -amine hydrochloride as a white solid (180 mg, 0.58 mmol, 54 % yield).

¹H-NMR (500 MHz, DMSO-d₆): δ (ppm) = 1 1.46 (br s, 1 H), 9.95 (br s, 1 H), 9.80 (br s, 1 H), 7.74 - 7.63 (m, 2H), 7.49 (d, J = 7.6, 1 H), 7.45 - 7.38 (m, 4H, 8-H), 7.15 (t, J = 7.6, 1 H), 7.02 (t, J = 7.5, 1 H), 4.68 (s, 1 H), 4.27 (t, J = 6.1 , 2H), 2.77 - 2.60 (m, 2H), 2.28 - 2.20 (m, 2H), 2.15 - 2.06 (m, 1 H), 1.85 - 1.74 (m, 1 H). MS (El): m/z 276 [M^+*]; MS (CI): m/z 277 [M⁺ + H].

Example 8:

-Benzyl-7,8-dichloro-2,3,4,9-tetrahydro-1 H-carbazol-1 -amine hydrochloride

A/-Benzyl-7,8-dichloro-2,3,4,9-tetrahydro-1 - -carbazol-1 -amine hydrochloride was prepared from 7,8-dichloro-2,3,4,9-tetrahydro-1 - -carbazol-1 -one in a similar manner as described in Example 7 to give a pale brown solid (42% yield).

¹H-NMR (500 MHz, DMSO-d₆): δ (ppm) = 1 1.89 (br s, 1 H), 10.12 (br s, 1 H), 9.92 (br s, 1 H), 7.68 (d, J = 6.5, 2H), 7.49 (d, J = 8.4, 1 H), 7.42 (m, 3H), 7.22 (d, J = 8.4, 1 H), 4.74 (s, 1 H), 4.26 (m, 2H), 2.68 (t, J = 5.4, 2H), 2.37 - 2.25 (m, 1 H), 2.24 - 2.15 (m, 1 H), 2.15 - 2.03 (m, 1 H), 1.84 - 1.70 (m, 1 H). MS (El): m/z 345 [M^+"]; MS (CI): m/z 346 [M⁺ + H].

Example 9:

-Benzyl-6-bromo-2,3,4,9-tetrahydro-1 H-carbazol-1 -amine hydrochloride

/V-Benzyl-6-bromo-2,3,4,9-tetrahydro-1 H-carbazol-1 -amine hydrochloride was prepared from 6-bromo-2,3,4,9-tetrahydro-1 H-carbazol-1 -one in a similar manner as described in Example 7 to give a white solid (23% yield).

¹H-NMR (500 MHz, DMSO-d₆): δ (ppm) = 1 1.69 (br s, 1 H), 9.94 (br s, 1 H), 9.80 (br s, 1 H), 7.73 - 7.61 (m, 3H, 5-H), 7.47 - 7.36 (m, 4H, 8-H), 7.25 (dd, J = 8.6, 1.8, 1 H), 4.68 (s, 1 H), 4.35 - 4.21 (m, 2H), 2.75 - 2.59 (m, 2H), 2.28 - 2.18 (m, 2H), 2.16 - 2.02 (m, 1 H), 1.85 - 1.71 (m, 1 H). MS (El): m/z 355 [Ν/Γ]; MS (CI): m/z 356 [M⁺ + H],

Example 0:

-Benzyl-6-iodo-2,3,4,9-tetrahydro-1 H-carbazol-1 -amine hydrochloride

A-Benzyl-6-iodo-2,3,4,9-tetrahydro-1 H-carbazol-1 -amine hydrochloride was prepared from 6- iodo-2,3,4,9-tetrahydro-1 - -carbazol-1-one in a similar manner as described in Example 7 to give a pale yellow solid (42% yield).

¹H-NMR (500 MHz, DMSO-d₆): δ (ppm) = 11.62 (br s, 1 H), 9.89 (br s, 1 H), 9.75 (br s, 1 H), 7.86 (s, 1 H), 7.67 (d, J = 7.2, 2H), 7.48 - 7.35 (m, 4H), 7.28 (d, J = 8.5, 1 H), 4.67 (s, 1 H), 4.36 - 4.20 (m, 2H), 2.76 - 2.60 (m, 2H), 2.28 - 2.16 (m, 2H), 2.16 - 2.04 (m, 1 H), 1.86 - 1.72 (m, 1 H). MS (El): AT?/Z 402 [M^+*]; MS (CI): m/z 403 [M⁺ + H].

Example 11 :

-Bromo-/V-cyclohexyl-2,3,4,9-tetrahydro-1 H-carbazol-1 -amine

A solution of 6-bromo-2,3,4,9-tetrahydro-1 H-carbazol-1 -one (417 mg, 1.58 mmol), sodium triacetoxyborohydride (1.03 g, 4.86 mmol), cyclohexylamine (556 μΙ, 4.86 mmol) and 0.15 μΙ acetic acid in dichloroethane was stirred over night. The mixture was quenched by adding a solution of saturated sodium hydrogencarbonate (20 ml) and extracted with ethyl acetate (3 x 25 ml). The combined organic layers were dried over sodium sulfate and the solvents evaporated. Purification was accomplished by silica gel chromatography with dichloromethane/ethyl acetate to yield 6-bromo-/V-cyclohexyl-2,3,4,9-tetrahydro-1H-carbazol- 1 -amine as a brown oil (320 mg, 0.92 mmol, 58% yield). ¹H-NMR (400 MHz, CDCI₃): δ (ppm) = 8.50 (br s, 1 H), 7.56 (s, 1 H), 7.18 (d, J = 8.6, 1 H), 7.15 (d, J = 8.6, 1 H), 4.01 (s, 1 H), 2.82 - 2.71 (m, 1 H,), 2.70 - 2.56 (m, 2H), 2.34 - 2.21 (m, 1 H), 2.09 - 1.95 (m, 2H), 1.86 - 1.58 (m, 6H), 1.57 - 1.45 (m, 1 H), 1.40 - 1.02 (m, 5H). MS (El): m/z 347 [M^+"]; MS (CI): m/z 348 [M⁺ + H].

Example 12:

/V-(1-Benzylpiperidin-4-yl)-6-iodo-2,3,4,9-tetrahydro-1 -/-carbazol-1 -amine

^(l-Benzylpiperidin^-yO-e-iodo^^^^-tetrahydro-IH-carbazol-l-amine was prepared from 6-iodo-2,3,4,9-tetrahydro-1H-carbazol-1-one and 4-amino-1-benzylpiperidine in a similar manner as described in Example 7 to give a brown solid (38% yield).

¹H-NMR (500 MHz, CDCI₃): δ (ppm) = 8.41 (br s, 1 H), 7.77 (d, J = 1.6, 1 H), 7.35 (dd, J = 8.5, 1.6, 1 H), 7.33 - 7.30 (m, 4H), 7.27 - 7.23 (m, 1 H), 7.08 (d, J = 8.5, 1 H), 3.98 (t, J = 6.7, 1 H), 3.52 (s, 2H), 2.91 - 2.82 (m, 2H)₍ 2.81 - 2.75 (m, 1 H), 2.66 - 2.58 (m, 2H), 2.31 - 2.24 (m, 1 H), 2.10 - 1.99 (m, 4H), 1.96 - 1.90 (m, 1 H), 1.79 - 1.72 (m, 1 H), 1.70 - 1.65 (m, 1 H), 1.57 - 1.45 (m, 3H). MS (El): m/z 485 [M^+']; MS (CI): m/z 486 [M⁺ + H].

Example 13:

A/- 1-Benzylpiperidin-4-yl)-6-bromo-2,3,4,9-tetrahydro-1H-carbazol-1 -amine

A/-(1-Benzylpiperidin-4-yl)-6-bromo-2,3,4,9-tetrahydro-1/- -carbazol-1 -amine was prepared from 6-bromo-2,3,4,9-tetrahydro-1 /-/-carbazol-1-one and 4-amino-1-benzylpiperidine in a similar manner as described in Example 7 to give a brown solid (25% yield).

H-NMR (400 MHz, CDCI₃): δ (ppm) = 8.48 (br s, 1 H), 7.56 (s, 1 H), 7.34 - 7.31 (m, 4H), 7.31 - 7.27 (m, 1H), 7.21 - 7.15 (m, 2H), 4.00 (t, J = 6.6, 1 H), 3.52 (s, 2H), 2.92 - 2.74 (m, 3H), 2.68 - 2.57 (m, 2H), 2.32 - 2.23 (m, 1 H), 2.13 - 2.00 (m, 4H), 1.99 - 1.91 (m, 1 H), 1.79 - 1.64 (m, 2H), 1.55 - 1.45 (m, 3H). MS (El): m/z 438 fJvT]; MS (CI): m/z 439 [M⁺ + H]. Example 14:

-Bromo-/V-(1-phenethylpiperidin-4-yl)-2,3,4,9-tetrahydro

6-Brom-2,3,4,9-tetrahydro-1 H-carbazol-1 -amine hydrochloride (220 mg, 0.73 mmol), magnesium perchlorate (84 mg, 0.3 mmol) and 1-(2-phenethyl)-4-piperidone (360 mg, 1.77 mmol) were stirred in dichloroethane (20 ml) and methanol (5 ml) over night, then sodium borohydride (1 12 mg, 3 mmol) was added. After stirring the mixture for another 5 hours, a saturated solution of sodium hydrogencarbonate (10 ml) was added, followed by extraction with ethyl acetate (3 x 20 ml). The combined organic layers were dried over sodium sulfate and the solvents evaporated. Purification was accomplished by silica gel chromatography with cyclohexane/ethyl acetate/triethylamine/dimethylethylamine (60:30:10:10) to yield 6-Bromo-/V- (1-phenethylpiperidin-4-yl)-2,3,4,9-tetrahydro-1 H-carbazol-1 -amine as a dark brown resin (20% yield).

¹H-NMR (400 MHz, CDCI₃): δ (ppm) = 8.56 (br s, 1 H), 7.57 (s, 1 H), 7.32 - 7.26 (m, 3H), 7.23 - 7.19 (m, 4H), 4.02 (t, J = 6.6, 1 H), 3.02 - 2.97 (m, 2H), 2.81 - 2.77 (m, 3H), 2.67 - 2.57 (m, 4H), 2.34 - 2.24 (m, 1 H), 2.17 - 2.05 (m, 4H), 2.04 - 2.00 (m, 1 H), 1.80 - 1.71 (m, 2H), 1.58 - 1.47 (m, 3H). MS (El): m/z 452 [M^+*]; MS (CI): m/z 453 [M⁺ + H].

Example 15:

-lodo-2,3,4,9-tetrahydro-1 /-/-carbazol-1 -amine hydrochloride

6-lodo-2,3,4,9-tetrahydro-1 /-/-carbazol-1 -amine hydrochloride was prepared from 6-iodo- 2,3,4,9-tetrahydro-1 H-carbazol-1 -one and ammonium acetate in a similar manner as described in Example 5 to give a pale grey solid (56% yield).

¹H-NMR (400 MHz, DMSO-d₆): δ (ppm) = 1 1.28 (br s, 1 H), 8.68 (br s, 3H), 7.83 (s, 1 H), 7.38 (d, J = 8.4, 1 H), 7.27 (d, J = 8.4, 1 H), 4.54 (s, 1 H), 2.67 - 2.61 (m, 2H), 2.23 - 2.14 (m, 1 H), 2.05 - 1.98 (m, 1 H), 1.95 - 1.88 (m, 1 H), 1.85 - 1.76 (m, 1 H). MS (El): m/z 312 [M^+']; MS (CI): 77/z313[M⁺ + H].

Example 16:

-lodo-/V-(1-phenethylpiperidin-4-yl)-2,3,4,9-tetrahydro-1H-carbazol-1-amin

6-lodo-/V-(1-phenethylpiperidin-4-yl)-2,3,4,9-tetrahydro-1H-carbazol-1 -amine was prepared from 6-iodo-2,3,4,9-tetrahydro-1H-carbazol-1 -amine hydrochloride and 1-(2-phenethyl)-4- piperidone in a similar manner as described in Example 14 to give a dark red resin (18% yield).

¹H-NMR (500 MHz, CDCI₃): δ (ppm) = 8.39 (br s, 1 H), 7.78 (d, J = 1.7, 1 H), 7.37 (dd, J = 8.4, 1.7, 1H), 7.29 - 7.27 (m, 2H), 7.23 - 7.21 (m, 3H), 7.10 (d, J = 8.4, 1H), 4.01 (t, J = 6.7, 1H), 3.03 - 2.96 (m, 2H), 2.88 - 2.82 (m, 3H), 2.67 - 2.60 (m, 4H), 2.33 - 2.28 (m, 1H), 2.25 - 2.11 (m, 4H), 2.04 - 2.00 (m, 1H), 1.77 - 1.73 (m, 2H), 1.62 - 1.52 (m, 3H). MS (ESI): m/z 500 [M⁺ + H]; MS (CI): m/z 500 [M⁺ + H].

Example 17:

/V-Benz l-6-bromo-9-methyl-2,3,4,9-tetrahydro-1H-carbazol-1 -amine

/V-Benzyl-6-bromo-9-methyl-2,3,4,9-tetrahydro-1/-/-carbazol-1-amine was prepared from 6- bromo-9-methyl-2,3,4,9-tetrahydro-1 --carbazol-1-one and benzylamine in a similar manner as described in Example 11 to give a yellow oil (36% yield).

¹H-NMR(500 MHz, CDCI₃): δ (ppm) = 7.63 (d, J= 1.9, 1H), 7.48-7.17 (m, 6H), 7.15 (d, J = 8.7, 1H), 4.92 (t, J = 3.7, 1H), 4.03 (d, J= 13.0, 1H), 3.74 (s, 3H), 3.30 (d, J= 13.0, 1H), 2.81 - 2.73 (m, 1H), 2.60 - 2.50 (m, 1H), 2.10 - 2.00 (m, 2H), 1.92 - 1.88 (m, 3H). HRMS (El): Calcd for C₂oH₂iBrN₂: m/z = 368.0888. Found: m/z = 368.0880.

Exampte 18:

-Benzyl-6-bromo-A/-methyl-2,3,4,9-tetrahydro-1 H-carbazol-1 -amine

To a solution of A/-benzyl-6-bromo-2,3,4,9-tetrahydro-1 H-carbazol-1 -amine hydrochloride (353 mg, 0.9 mmol) and Λ/,/V-diisopropylethylamine (500 μΙ, 2.9 mmol) in anhydrous THF (15 ml) under nitrogen atmosphere, methyl iodide (56.4 μΙ, 0.9 mmol) was added. The mixture stirred for 15 hours at room temperature, then the solvent was evaporated. After dissolving the residue in ethyl acetate and water (25 ml), the compound was extracted with further ethyl acetate (3 x 25 ml). The combined organic layers were dried over sodium sulfate and the solvents evaporated. Purification was accomplished by silica gel chromatography with dichloromethane/ethyl acetate to yield A/-benzyl-6-bromo-A/-methyl-2,3,4,9-tetrahydro-1 /- - carbazol-1 -amine as a yellow oil (235 mg, 0.64 mmol, 71% yield).

1H-NMR (500 MHz, CDCI₃): δ (ppm) = 8.39 (br s, 1 H), 7.57 (s, 1 H), 7.39 - 7.32 (m, 4H), 7.28 - 7.24 (m, 1 H), 7.21 - 7.18 (m, 2H), 4.14 (s, 1 H), 3.67 (d, J = 13.3, 1 H), 3.59 (d, J = 13.3, 1 H), 2.71 - 2.55 (m, 2H), 2.19 (s, 3H), 2.16 - 2.05 (m, 2H), 1.85 - 1 .66 (m, 2H). MS (El): m/z 369 PVT]; MS (CI): m/z 370 [M⁺ + H]. Example 19:

Materials and Methods

Cell culture and cell lines

Human embryonic kidney 293 (HEK293) cells were cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1 % penicillin/streptomycin while being incubated at 37°C, 5% C0₂ and 90% humidity. The stable PS1 lines (generously provided by Dr. S. Lammich) were carrying PS1 variants that were cloned into pcDNA3.1/Zeo(+) and single cells were selected via Zeocin antibiotic resistance (S. Lammich et al., Presenilin-dependent intramembrane proteolysis of CD44 leads to the liberation of its intracellular domain and the secretion of an Abeta-like peptide. The Journal of Biological Chemistry 277, 44754 (Nov 22, 2002)). The PS1 lines were then stably transfected with YC3.6/pcDNA3 construct (kindly provided by Dr. A. Miyawaki) and single cells were respectively isolated by G418 antibiotic resistance leading to generation of double stable lines. The APP- , C99- and APPsw/PS1 -M146L-overexpressing HEK293 lines were kindly provided by Dr. S. Lichtenthaler and Dr. H. Steiner and cultured as previously described (S. Mitterreiter et al., Bepridil and amiodarone simultaneously target the Alzheimer's disease beta- and gamma-secretase via distinct mechanisms. J Neurosci 30, 8974 (Jun 30, 2010); R. M. Page et al., Generation of Abeta38 and Abeta42 is independently and differentially affected by familial Alzheimer disease-associated presenilin mutations and gamma-secretase modulation. The Journal of Biological Chemistry 283, 677 (Jan 1 1 , 2008)).

Calcium homeostasis

The readout for dysregulated ER calcium signaling are the potentiated IP₃ calcium signals in HEK293 cells carrying a disease-causing mutated form of PS1. Agonist-induced IP₃ production by Carbachol (CCh) results in calcium being set free from the ER. Various FAD- PS1 mutants show exaggerated CCh-induced calcium release compared to wild type PS1 expressing cells. A genetically-encoded FRET-based calcium probe (Yellow Cameleon 3.6, Miyawaki Lab, Japan) (Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M. & Miyawaki, A. Expanded dynamic range of fluorescent indicators for Ca²⁺ by circularly permuted yellow fluorescent proteins. Proceedings of the National Academy of Sciences of the United States of America 101 , 10554-9 (2004)) has been introduced into these cells as a tool to monitor both the basal intracellular calcium concentration and the released calcium signals from the ER in real-time by confocal imaging.

HEK293 cells stably expressing PS1 -M146L and YC3.6 were seeded at 13,000 cells/well in 40 μΙ on collagen-coated 384-well CellCarrier plates (PerkinElmer) in growth medium consisting of DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% FCS, 1X penicillin-streptomycin, 1X GlutaMAX and selection antibiotics. After 6 hours, using pipetting robot (Bravo, Agilent Technologies), compounds according to the invention, positive controls (Thapsigargin, CPA, TMB-8 and Bepridil) and the vehicle were added into each well at the final concentration of 10 μΜ and 1 % DMSO, each in 4 replicates. 24 hours later using the pipetting robot, DRAQ5 nuclear marker dye (Biostatus) was added into each well at the final concentration of 500 nM. After 2 hours plates were measured for carbachol-induced calcium release from ER using PerkinElmer Opera high-throughput confocal imaging platform. Using 442 nm laser the YC3.6 was excited and the CFP and YFP signals were separated using suitable filters. Using 640 nm laser DRAQ5 dye was excited and its emission was collected in order to locate all nuclei for every time point. Time-lapse calcium imaging was performed at 1 second intervals. After monitoring the basal calcium concentration for 5 seconds 10 μΙ of carbachol diluted in HBSS was injected into each well during the calcium imaging (at final concentration of 10 μΜ) using the dispenser which was equipped in Opera platform. The calcium rise and decay was monitored for 30 seconds post dispensing. Using the Acapella software (PerkinElmer) an automated analysis tool was developed to translate the fluorescence signals into numerical values. Here, DRAQ5 and YC3.6 signals were used respectively to detect single cell nuclei and single cell boundaries over the entire course of time-lapse calcium imaging. For every single cell in the imaging frame, calcium transients were measured by calculating the YFP/CFP ratio. The peak amplitude of the calcium rise upon carbachol injection was the output of automated analysis software at a single cell level. Irresponsive cells to carbachol were excluded from the analysis by setting an arbitrary defined threshold. The average peak amplitude of all responsive cells in each well was calculated as the final readout of the assay.

Mitochondrial membrane potential

The effect of several compounds of the present invention on the mitochondrial activity has been tested. The mitochondrial membrane potential (Ψ ) is used as a measure for mitochondrial activity which is typically reduced in AD (Rhein, V. et al. Amyloid-beta and tau synergistically impair the oxidative phosphorylation system in triple transgenic Alzheimer's disease mice. Proceedings of the National Academy of Sciences of the United States of America 106, 20057-62 (2009); Santos, R.X. et al. Alzheimer's disease: diverse aspects of mitochondrial malfunctioning. International Journal 3, 570-581 (2010). can be measured, for example, by utilizing a fluorescent cationic dye such as TMRM (tetramethylrhodamine methyl ester). The fluorescent relative light unit (RLU) of TMRM was used as a measure for Ψ_ΓΤ, in HEK293 cells; see Figure 1 and last column of Table 1.

HEK293 cells were used to study the effect of the compounds according to the invention on the mitochondrial membrane potential as a measure of mitochondrial activity. The measurement method for membrane potential with TMRM dye was adapted from Scaduto et al. (Scaduto Jr, R.C. & Grotyohann, L.W. Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophysical journal 76, 469-477 (1999)). HEK293 cells were seeded at the density of 50,000 cells/well in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% FCS, 1X penicillin-streptomycin, 1X GlutaMAX on collagen/poly-L-lysine (PLL)-coated 96-well plates (Advanced-TC plates, Greiner) and incubated for 24 hours. Next the cells were loaded with 50 nM tetramethylrhodamine methyl ester (TMRM) dye in the presence or absence of compounds according to the invention which were pre-incubated on the cells (10 μΜ) one hour prior to adding TMRM dye. After 30 minutes each well was washed 3 times using PBS. Fresh medium containing the corresponding compounds according to the invention (10 μΜ) was added into the wells. Live cell image acquisition was performed using inverted confocal microscope LSM510 with 25x magnification (Carl Zeiss Microimaging GmbH, Jena, Germany) and the images were analyzed using ImageJ software to quantify the intensity of TMRM fluorescence signal. The effect of each compound was analyzed in triplicate. The mitochondria uncoupler, CCCP (50 μΜ) and Dimebon (10 μΜ) were used as controls.

Beta amyloid peptide generation

The effect of several compounds of the present invention on Αβ generation has been measured. Using sandwich ELISA, the level of three different Αβ species (Αβ42, Αβ40 and Αβ38) was determined. HEK293 cells stably expressing disease causing mutations of APPsw and PS1 -M146L and producing high levels of Αβ were used.

Generally, a reduction was observed in the levels of all three Αβ species for many compounds of the invention. Data for Αβ42 reduction are shown in Table 1. In many instances, the compounds which were most active in enhancing mitochondrial membrane potential are also highly active in lowering Αβ levels.

Pools of HEK293 cells stably transfected with APPsw and PS1-M146L were used to study the effect of the compounds according to the invention on Αβ generation. According to Page et. al. (Page, R.M. et al. Generation of Abeta38 and Abeta42 is independently and differentially affected by familial Alzheimer disease-associated presenilin mutations and gamma-secretase modulation. The Journal of biological chemistry 283, 677-83 (2008)), cells were seeded at the density of 200,000 cells/well in collagen/poly-L-lysine (PLL)-coated 24-well plates and incubated for 24 hours in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% FCS, 1X penicillin-streptomycin, 1X GlutaMAX and selection antibiotics. Next the medium was exchanged with 500 μΙ of fresh medium containing either the compounds according to the invention (10 μΜ) or positive controls DAPT (10 μΜ) and sulindac sulfide (50 μΜ) or vehicle, all in duplicates. After 16 hours the conditioned medium was collected and the level of secreted Αβ38, Αβ40 and Αβ42 peptides were quantified using "Human (6E10) Abeta 3-Plex" sandwich ELISA immunoassay Kit (Meso Scale Disovery) according to the instructions of the manufacturer. In brief, 150 μΙ of MSD blocker A was added into each well of the ELISA plate and incubated for 1 hour at the room temperature, followed by 3x washing using MSD wash buffer. Next 25 μΙ of detection antibody was added into each well. Then each of samples or calibrators were added into separate wells of the MSD ELISA plate and incubated for 2 hours at the room temperature, followed by 3x washing using MSD wash buffer. Finally 150 μΙ of MSD read buffer were added into the wells. The light emission after electrochemical stimulation was measured using the Meso Scale Discovery Sector Imager 2400 reader. The corresponding concentrations of Αβ species were calculated using the Meso Scale Discovery Discovery Workbench software. sAPPa and sAPPfi measurements

Levels of sAPPa and εΑΡΡβ fragments were measured using sandwich ELISA adapted from Colombo et al. (Constitutive alpha- and beta-secretase cleavages of the amyloid precursor protein are partially coupled in neurons, but not in frequently used cell lines. Neurobiol Dis 49C, 137 (Aug 24, 2012)). Wild type HEK293 cells were seeded at the density of 200,000 cells/well in collagen/poly-L-lysine (PLL)-coated 24-well plates and incubated for 24 hours in growth medium. Next, the medium was exchanged with 500 μΙ of fresh medium containing either compounds or vehicle. After 16 hours conditioned medium was collected and the levels of secreted sAPPa and εΑΡΡβ fragments were quantified using sAPPa/sAPP sandwich ELISA immunoassay kit (Meso Scale Discovery, MD, USA) according to the instructions of the manufacturer. In brief, 150 μΙ of blocker reagent was added to each well of the ELISA plate and incubated for 1 hour at room temperature, followed by 3x washing using TRIS wash buffer. Next, 25 μΙ of samples or calibration standards were added to separate wells of ELISA plate and incubated for 1 hour at room temperature, followed by 3x washing using TRIS wash buffer. Then 25 μΙ of detection antibody was added to each well and incubated for 1 hour at room temperature, followed by 3x washing using TRIS wash buffer. Finally 150 μΙ of read buffer was added to the wells. The light emission after electrochemical stimulation was measured using Sector Imager 2400 reader (Meso Scale Discovery, MD, USA). Based on the values generated with calibration standards, corresponding concentrations of sAPPa and εΑΡΡβ were calculated using the Meso Scale Discovery Workbench software. All measurements were performed in four replicates.

AMPK activity

Using ELISA we quantified the levels of phosphorylated AMP-activated protein kinase (pAMPK). pAMPK is the activated form of AMPK. We observed that 16 hours treatment of HEK293 cells with our lead structure analogues (10 μΜ) results in increased levels of pAMPK. "Calcium/calmodulin-dependent protein kinase β (CaMKK )" is known to be the upstream kinase responsible for calcium-dependant activation of AMPK (AMPK phosphorylation) (Salminen, A., Kaarniranta, K., Haapasalo, A., Soininen, H. & Hiltunen, M. AMP-activated protein kinase (AMPK): a potential player in Alzheimer's disease. Journal of neurochemistry 460-474 (201 1 ).doi:10.1 1 1 1/j.1471-4159.201 1.07331.)■ Therefore we postulate that the activation of AMPK by our lead structure analogues to be a downstream effect of stabilizing ER calcium homeostasis, most likely through CaMKK . Interestingly, AMPK activation has been shown to be implicated in Αβ clearance (Vingtdeux, V., Chandakkar, P., Zhao, H., Davies, P. & Marambaud, P. Small-Molecule Activators of AMP-Activated Protein Kinase (AMPK), RSVA314 and RSVA405, Inhibit Adipogenesis. Molecular medicine (Cambridge, Mass.) 17, 1022-30 (201 1 )) and mitochondrial activity (Canto, C. et al. AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity. Nature 458, 1056-60 (2009)).

The effect of the compounds according to the invention on AMPK activity was investigated by means of measuring pThr172AMPK levels using sandwich immunoassay (Invitrogen). HEK293 cells were seeded at the density of 200,000 cells/well in collagen/poly-L-lysine (PLL)- coated 24-well plates and incubated for 24 hours in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% FCS, 1X penicillin-streptomycin, 1X GlutaMAX. Next medium was exchanged with 500 μΙ of fresh medium containing either the compounds according to the invention (10 μΜ), or controls Resveratol (50 μΜ), AICAR (2 mM), Compound C (1 μΜ) or the vehicle in triplicates. After 16 hours the medium was removed and wells were washed 3x with ice cold PBS on ice. Next the cells were lysed in 150 μΙ of ice cold lysis buffer and the lystes were used to quantify the AMPK activity level using the method adapted from Moreno-Navarrete et al. (Moreno-Navarrete, J.M. et al. OCT1 Expression in adipocytes could contribute to increased metformin action in obese subjects. Diabetes 60, 168-76 (201 1 )). Phosphorylation of AMPK at the Thr172 is directly associated with AMPK activity. The specificity of this assay for phosphorylated ΑΜΡΚα (pT172) was confirmed by peptide competition. The assay was performed according to the manufacturer's instructions. In brief, into each well of the ELISA plate 100 μΙ of the lysates or the standards was added and incubated for 2 hours at the room temperature, followed by 4x washing using the assay wash buffer. Next 100 μΙ of detection antibody was added into each well and incubated for 1 hour at the room temperature, followed by 4x washing using assay wash buffer. Then 100 μΙ of HRP anti-rabbit antibody was added into the wells and incubated for 30 minutes at the room temperature, followed by 4x washing using the assay wash buffer. Finally, 100 μΙ of stabilized chromagen was added into the wells and the reaction was stopped after 30 minutes by adding the "stop" solution into the wells. The plate was read at 450 nm using FLUOstar OPTIMA plate reader. Based on the absorptions of the standards and their calibration curve, and the levels of pAMPK were calculated.

Example 20:

Results

Tetrahydrocarbazoles attenuate the FAD-PS 1 mediated exaggerated ER calcium release

In order to explore the contribution of different R¹ and R² groups in the preferred formula (Via) to the activity of the lead structure, we tested further tetrahydrocarbazole analogues and related structures (Figure 5A and Table 45). Based on the structure-activity-relationship (SAR) knowledge gained, we synthesized further derivative structures with the aim of reaching an improved efficacy (Figure 5B and Table 46). Replacement of nitro group at R¹ position with other electron-withdrawing substituents, e.g. halogens, trifluoromethyl, and cyano groups, maintains the activity of the lead structure, while other small substituents, e.g. hydrogen, lead to the loss of activity. Aliphatic residues at R² position (e.g. ADM-02, ADM-03, ADM-06, ADM- 39) diminish that effect, while additional attachment of an aromatic motif (e.g. phenyl group) is beneficial to the activity (e.g. ADM-1 1 , ADM-22). Tetrahydrocarbazoles increase the mitochondrial membrane potential

It has been demonstrated that ER and mitochondria are physically and functionally interdependent (M. Lebiedzinska, G. Szabadkai, A. W. Jones, J. Duszynski, M. R. Wieckowski, Interactions between the endoplasmic reticulum, mitochondria, plasma membrane and other subcellular organelles. Int J Biochem Cell Biol 41 , 1805 (Oct, 2009)). Constitutive calcium release from IP₃R to mitochondria is a crucial mechanism involved in mitochondrial function (C. Cardenas et al., Essential regulation of cell bioenergetics by constitutive lnsP₃ receptor Ca²⁺ transfer to mitochondria. Cell 142, 270 (Jul 23, 2010)). Indications suggest that FAD-PS mutations affect the physical interaction between ER and mitochondria (E. Area-Gomez et al., Upregulated function of mitochondria-associated ER membranes in Alzheimer disease. EMBO J 31 , 4106 (Nov 5, 2012)), leading to altered shuttling of calcium between the two organelles and affecting the mitochondrial calcium uptake (E. Zampese er a/., Presenilin 2 modulates endoplasmic reticulum (ER)-mitochondria interactions and Ca²⁺ cross-talk. Proc Natl Acad Sci U S A 108, 2777 (Feb 15, 201 1 )). Thus, in the next set of experiments we questioned whether the modulation of ER calcium homeostasis by the lead structure also affects mitochondrial function. To that end, we used mitochondrial membrane potential as an important parameter for addressing mitochondrial activity. We used TMRM dye, a fluorescent rhodamine derivative, to monitor mitochondrial membrane potential (R. C. Scaduto, Jr., L. W. Grotyohann, Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J 76, 469 (Jan, 1999)). Indeed, pretreatment of HEK293 cells for 1 hour with several lead structure analogues led to a remarkable increase in the mitochondrial membrane potential, measured by the TMRM fluorescence signal (Figure 6A and 6B). At 10 μΜ, the increases in mitochondrial membrane potential after treatment with many of the analogues were comparable or even superior to that for Dimebon, an enhancer of mitochondrial activity (S. Zhang, L. Hedskog, C. A. Petersen, B. Winblad, M. Ankarcrona, Dimebon (latrepirdine) enhances mitochondrial function and protects neuronal cells from death. J Alzheimers Dis 21 , 389 (2010)) (Figure 6A and 6B). We particularly found that compounds 5781464 and 5781441 , respectively containing /V-(1 - benzylpiperidin-4-yl) and A/-(1 -phenethylpiperidin-4-yl) groups at their R² position, were among the most active compounds both in terms of efficacy and potency (Figure 6B and 6D). Therefore, in several lead structure derivatives that we synthesized, the R² position remained incorporating A/-(1-benzylpiperidin-4-yl) or /V-(1-phenethylpiperidin-4-yl) groups, while we varied the groups at R¹ position to explore their contribution to the activity of the lead structure (Table 46). Indeed the latter analogues were also among the most active synthesized compounds in enhancing mitochondrial function (Figure 6C). Therefore, we concluded that the highest activity in terms of improving mitochondrial membrane potential is achieved if the lead structure contains A/-(1 -benzylpiperidin-4-yl) or \/-(1-phenethylpiperidin-4-yl) groups at R² position, given that R¹ position incorporates electron-withdrawing residues. Exemplarily, the EC₅₀ for one of the most promising synthesized derivatives of the lead structure (gea_133; R¹ = cyano) was determined to be at the therapeutically relevant value of 4.84 μΜ (Figure 6D). Moreover, the efficacy of compound ADM-51 was remarkably higher than the one of Dimebon, especially at concentrations beyond 1 μΜ (Figure 6D).

Data are shown in Tables 45 and 46.

Tetrahydrocarbazoles lower Αβ peptide production

Next we studied the impact of tetrahydrocarbazoles on the production of Αβ peptides. Modulation of intracellular calcium homeostasis directly affects Αβ production (K. N. Green, F. M. LaFerla, Linking calcium to Abeta and Alzheimer's disease. Neuron 59, 190 (Jul 31 , 2008)). Thus, we hypothesized that reversing disruptive ER calcium homeostasis may as well result in lowered Αβ production. Indeed, we detected remarkably decreased levels of secreted Αβ38, Αβ40 and Αβ42 peptides upon 16 hours treatment of HEK293 cells expressing either APPsw/PS1-M146L or wildtype APP with the lead structure analogues at 10 μΜ (Figure 7A, 7C). The IC₅₀ of the select analogues in terms of decreasing all three Αβ species lies at low micromolar range (Figure 8A, B, C). However, compound treatment in both cell lines did not affect the Αβ42/Αβ40 ratio for most analogues, suggesting that the identified lead structure is not a γ-secretase modulator (Figures 7B, 7D). In order to investigate the γ-cleavage of APP independently from its β-cleavage, we used HEK293 cells expressing C99, the β-cleaved C- terminal fragment of APP and the substrate for γ-secretase. We observed that treatment of HEK293-C99 cells with the majority of the lead structure derivatives tested, does not (or only marginally) affect the production of Αβ38, Αβ40 and Αβ42 (Figure 9A). Moreover, Αβ42/Αβ40 ratios remained unaffected upon exposure of HEK293-C99 cells with the lead structure analogues (Figure 9B). Taken together, these results corroborate that the detected decrease in Αβ peptide levels is not a γ-secretase-dependent phenomenon. Therefore, we postulated that reduced β-cleavage of APP may contribute to lowered Αβ generation. Hence, we measured the levels of sAPPa and εΑΡΡβ, the first cleavage products of APP, respectively, generated by a-secretase and β-secretase. Indeed, we detected significantly decreased levels of secreted εΑΡΡβ, while sAPPa levels were unaffected (or only mildly reduced) upon treatment of wildtype HEK293 cells with most lead structure derivatives (Figure 7E). These results imply that the attenuated Αβ production caused by the lead structure is mediated through decreased cleavage of APP by β-secretase. The SAR analysis among the lead structure analogues in terms of lowering Αβ production was comparable to their determined SAR in regard to increasing mitochondrial membrane potential. We found that analogues incorporating electron-withdrawing residues at R¹ position, in combination with Λ/-(1- benzypiperidin-4-yl) or /V-(1-phenethylpiperidin-4-yl) at R² position show strongest reduction in Αβ production (Figure 7A). Data are shown in Tables 45 and 46.

Tables 45 and 46 show preferred or exemplary compounds of the invention and their performance in the above described assays. The performance data are relative values, wherein DMSO has been used as a negative control. Depending on the assay, values above or below 1 indicate more activity in the respective assay compared to DMSO, and values reaching 1 indicate less activity similar to DMSO. In the TMRM and AMPK assays, values above 1 are desirable in accordance with the present invention, and in the calcium assay as well as in the Αβ and εΑΡΡβ assays values below 1 are desirable and indicative of compounds useful in accordance with the present invention. Table 45

1 H-carbazol-1 -amine

ADM-02 0.981 1.384 1.058 0.733 0.584 9-

ADM-03 0.966 1.545 0.593 0.726 0.593

N-(3-methylcyclohexyl)-6-nitro-2,3,4,9-

0.967 1.180 1.093 0.777 0.648

0.952 2.1 18 0.961 0.564 0.609

N-(1 -(4-methoxyphenyl)propan-2-yl)-6-nitro- -06 0.789 1.926 0.702 0.503 0.623

N-(1 -ethyl piperidin-4-yl)-6-nitro- 2,3,4,9-tetrahydro-! H-carbazol-1 -amine Compound Structure ID Calcium T R Αβ 42 Αβ 40 Αβ 38 and name

0.962 1.756 0.683 0.503 0.618

0.979 1.551 0.980 0.652 0.812

1.030 1.499 0.875 0.718 0.794

0.982 1.128 1.126 0.853 0.807

0.895 3.256 0.575 0.539 0.486

N-( 1 -benzyl piperidin-4-yl )-6-nitro-2,3,4,9-

ADM-12 0.959 1.157 0.891 0.747 0.585

N-(4-(tert-butyl)cyclohexyl)-6-nitro-2,3,4₍9-t

etrahydro-1 H-carbazol-1 -amine

N-(4-(4-methoxyphenyl)butan-2-yl)-6-nitro- 2,3,4,9-tetrahydro-1 H-carbazol-1 -amine

M-15 0.437 0.641 1.471 0.769 0.815

1 -car azo - -y met y cyc o exanam ne

ADM-16 0.499 1.259 1.068 0.753 0.916

1 -methyl-N-((6-methyl-2,3_/4,9-tetrahydro- 1 H-carbazol-1 -yl)methyl)piperidin-4-amine

ADM-17 0.961 1.329 1.053 0.692 0.772

N-(1 -(3,4-dimethoxyphenyl)propan-2-yl)-6- nitro-2,3,4,9-tetrahydro-1 H-carbazol-1 -amine

ADM-18 0.942 0.850 0.760 0.545 0.582

N-isopropyl-6-nitro-2,3,4,9- -amine

ADM-19 0.942 0.491 0.983 0.827 0.735

N-(4-methylcyclohexyl)-6-nitro-2,3,4,9

tetrahydro-1 H-carbazol-1 -amine

-amine

ADM-21 0.925 0.433 0.846 0.706 0.692 -amine

AD -22 0.914 2.357 0.566 0.531 0.523 -amine

AD -23 0.917 1.046 0.785 0.769 0.857

ADM-24 0.933 1.259 1.038 0.593 0.594

H-carbazol-1 -yl)methyl)piperidin-4-amine

ADM-25 1.047 1.514 0.335 0.355 0.317

2-((6-bromo-2,3,4,9-tetrahydro-

ADM-26 0.791 1.922 0.292 0.278 0.176

6-bromo-N-phenethyl-2,3,4,9-tetrahydro- 1 H-carbazol-1 -amine hydrochloride Compound Structure ID Calcium TMRM Αβ 42 Αβ 40 Αβ 38 and name

ADM-27 0.703 1.508 0.683 0.503 0.357

tetrahydro-1 H-carbazol-1 -amine

ADM-28 0.953 1.008 0.332 0.397 0.421

-benzyl-8-bromo-2,3,3a,4,5,6-hexahydro- 1 H-pyrazlno[3,2,1 -jk]carbazole

AD -29 0.869 0.831 0.492 0.481 0.449

6-bromo-l -(1 H-pyrrol-1 -yl)-2,3,4,9- tetrahydro-1 H-carbazole

ADM-30 1.002 1.207 1.056 0.712 0.874

1 H-carbazol-1 -yl)formamide

1 0.961 0.663 0.840 0.642 0.817

8-

0.989 1.116 0.269 0.271 0.309

6-bromo-2,3,4,9-tetrahydro- 1 H-carbazol-1 -amine

Table 46

Compound Structure ID Calcium TMRM Αβ 2 Αβ 40 Αβ 38 and name

ADM-35 0.991 1.219 1.050 0.835 0.635

N-benzyl-2,3,4,9-tetrahydro- 1 H-carbazol-1 -aminium chloride 1.01 1 1.049 1.100 1.050 0.913

0.879 1.444 0.910 0.765 0.651

0.913 1.444 0.901 0.767 0.629

0.946 0.998 0.779 0.564 0.479

0.914 0.926 0.848 0.608 0.610

1.000 0.724 0.815 0.754 0.745

tetrahydro-1 H-carbazol-1

0.840 2.100 0.542 0.520 0.461

N

b I d l) do

tetrahydro-1 H-carbazol-1 -amine

N-(1 -benzylpiperidin-4-yl}-6-bromo-2,3,4,9- tetrahydro-1 H-carbazol-1 -amine 5 0.997 1.563 0.506 0.489 0.586

0.856 1.929 0.449 0.513 0.441

6-bromo-N-(1-phenethylpiperidin-4-yl)- 2 -amine DM-47 0.871 1.726 0.523 0.480 0.479

6-iodo-N-(1 -phenethylpiperidin-4-yl)-

0.832 1.862 0.327 0.383 0.311

N-(1 -benzylpiperidin-4-yl)-6-chloro-2,3,4,9- tetrahydro-1 H-carbazol-1 -amine

ADM-50 0.763 2.214 0.426 0.478 0.514

N-(1-benzylpiperidin-4-yl)-6-(trifluoromethyl)- 2,3,4,9-tetrahydro-1 H-carbazol-1 -amine Compound Structure _1D Calcium TMRM Αβ 2 Αβ 40 Αβ 38 and name

ADM-49 0.834 2.004 0.295 0.361 0.342

N-(1 -benzylpiperidin-4~yI)-6-f!uoro- 2,3,4,9-tetrahydro-1 H-carbazol-1 -amine

0.899 2.636 0.273 0.308 0.296

0.963 1.646 0.504 0.576 0.514

N-(1-benzylpiperidin-4-yl)-2-bromo-5,6,7,8,9,10- hexahyd rocyclohepta [b] i ndol-6-am ine

Claims

Claims

A compound of formula (la) or (lla)

wherein either

(A) R¹ is I and R² is selected from substituted or unsubstituted piperidinyl, preferably N-substituted or unsubstituted piperidin-4-yl; substituted or unsubstituted cyclohexyl, cyclopentyl, or cyclohexyl alkyl; substituted or unsubstituted aryl-alkyl; alkyl; aryl; -CHO; and hydrogen; or

(B) R¹ is selected from halogen; substituted or unsubstituted alkoxy; substituted or unsubstituted alkyl; cyano; and hydrogen; and

R² is substituted or unsubstituted piperidinyl, preferably N-substituted or unsubstituted piperidin-4-yl;

and

R³, R⁴ and R⁵ independently are either H or defined as R¹;

R⁶ is H; alkyl or aryl-alkyl, or R² and R⁶ together with the N attached thereto form a 5- or 6-membered ring, preferably pyrrol;

each of the m occurrences of R⁷ is independently selected from Ci to C₄ alkyl, to

C₄ alkoxy and halogen, preferably F; and m being 0, 1 , 2, 3 or 4;

X is selected from NH; N-alkyl, preferably NCH₃; N-aryl-alkyl; S; and O; wherein in formula (la) R² and X together may alternatively form or comprise a 6- membered ring, said 6-membered ring preferably being piperazine;

n is 0, 1 or 2;

the substituents being independently selected from alkyl, aryl-alkyl; alkoxy, aryl, hydroxy; halogen; oxo; and thio;

alkyl being branched, unbranched and/or cyclic C-i to C ₀ alkyl, preferably branched or unbranched d to C₄ alkyl, more preferably methyl, ethyl, n-propyl, i-propyl, i-butyl or t-butyl;

alkoxy being Ο to C₄ alkoxy, preferably methoxy;

aryl being phenyl, naphthyl or tetrahydronaphthyl, preferably phenyl;

or a compound of any one of Tables 3 to 46;

or a salt or hydrate thereof.

2. The compound or salt or hydrate thereof according to claim 1 , wherein said compound is a compound of formula (Ilia) or (IVa)

(Ilia) (IVa) wherein each of the m occurrences of R⁷ is independently selected from to C₄ alkyl; m being 0, 1 , 2, 3 or 4; and the remainder of groups and substituents being as defined in claim 1.

3. The compound or salt or hydrate thereof according to claim 2, wherein said compound is a compound of formula (Va)

(Va)

wherein m is 0 or 1.

4. The compound or salt or hydrate thereof according to claim 3, wherein said compound is a compound of formula (Via)

(Via)

5. The compound or salt or hydrate thereof according to claim 4, wherein

R¹ is selected from halogen; substituted or unsubstituted C-_\ to C₄ alkoxy, preferably

OCH₃; substituted or unsubstituted Ci to C₄ alkyl, preferably methyl or CF₃; cyano; and hydrogen;

R² is substituted or unsubstituted piperidinyl, preferably N-substituted or unsubstituted piperidin-4-yl;

the substituents being alkyl, phenyl-alkyl or halogen;

alkyl being d to C₄ alkyl, preferably methyl, ethyl, n-propyl, i-propyl or t-butyl;

or a salt of hydrate thereof.

6. The compound or salt or hydrate thereof according to

(1 ) any one of claims 1 to 5, wherein R² is

(1.1 ) N-aryl-alkyl piperidin-4-yl, preferably N-phenyl-alkyl piperidin-4-yl, particularly preferred N-phenyl-methyl piperidin-4-yl or N-phenyl-ethyl piperidin-4-yl;

(1.2) N-alkyl piperidin-4-yl, preferably N-ethyl piperidin-4-yl or N-prop-1-yl piperidin- 4-yl; or

(1.3) N-alkyl piperidin-4-ylalkyl or N-aryl-alkyl piperidin-4-ylalkyl, preferably N-benzyl piperidin-4-yl-ethyl;

or

(2) claim 1(A) and any one of claims 2 to 5 to the extent claims 2 to 5 refer back to claim 1(A), wherein R² is

(2.1 ) alkyl cyclohexyl, preferably 3-methyl cyclohexyl, 4-methyl cyclohexyl, 4-ethyl cyclohexyl or 4-tert-butyl cyclohexyl; aryl-cyclohexyl, preferably 4-phenyl cyclohexyl; alkyl cyclopentyl, preferably 3-methyl cyclopentyl; or substituted cyclohexylalkyl such as 5,5-dimethyl-1 ,3-dioxocyclohex-2-ylmethylenyl; (2.2) aryl alkyl, preferably unsubstituted or substituted phenyl alkyl, more preferably phenyl methyl, phenyl ethyl, 4-phenyl-but-2-yl, 1-(4-methoxy phenyl)-prop-2-yl, 1-(4-hydroxy 3-methoxy phenyl)-prop-2-yl, 4-(4-methoxy phenyl)-but-2-yl, 1- (3,4-dimethoxy phenyl)-prop-2-yl, 1-(2-methoxy phenyl)-prop-2-yl, 1-(2-fluoro phenyl) prop-2-yl; or a bicyclic aryl-alkyl group such as 2-oxindol-3- ylmethylenyl, 1 ,2,3,4-tetrahydronaphthalen-2-yl or dihydroinden-2-yl; or

(2.3) branched or unbranched and substituted or unsubstituted alkyl, preferably isobutyl, tert-butyl, neopentyl, norbornyl or adamantyl, propyl, prop-2-yl, 2- hydroxy ethyl.

A pharmaceutical composition comprising a compound or salt or hydrate thereof, said compound being as defined in any one of claims 1 to 6 or a compound of Table 2.

A compound or salt or hydrate thereof for use in a method of treating or preventing a disorder, said disorder being selected from central nervous system disorders, brain disorders, neurodegenerative disorders, neuropsychiatric diseases, obesity, metabolic syndrome, cardiovascular diseases, muscle disorders, cancer, inflammatory diseases, autoimmune diseases and age-associated disorders, or for use in a method of extending life-span, wherein said compound is a compound of formula (lb) or (Mb)

(lb) (lib) wherein

R¹ is selected from halogen; substituted or unsubstituted alkoxy; substituted or unsubstituted alkyl; cyano; N0₂; and hydrogen;

R² is selected from substituted or unsubstituted piperidinyl, preferably N- substituted or unsubstituted piperidin-4-yl; substituted or unsubstituted cyclohexyl, cyclopentyl, or cyclohexyl alkyl; substituted or unsubstituted aryl- alkyl; alkyl; aryl; -CHO; and hydrogen

R³, R⁴ and R⁵ independently are either H or defined as R¹;

R⁶ is H; alkyl or aryl-alkyl, or R² and R⁶ together with the N attached thereto form a 5- or 6-membered ring, preferably pyrrol;

each of the m occurrences of R⁷ is independently selected from d to C₄ alkyl, Ci to

C₄ alkoxy and halogen, preferably F; and m being 0, 1 , 2, 3 or 4;

X is selected from NH; N-alkyl, preferably NCH₃; N-aryl-alkyl; S; and O; wherein in formula (lb) R² and X together may alternatively form or comprise a 6- membered ring, said 6-membered ring preferably being piperazine; n is 0, 1 or 2;

the substituents being independently selected from alkyl, aryl-alkyl; alkoxy, aryl, hydroxy; halogen; oxo; and thio;

alkyl being branched, unbranched and/or cyclic Ci to C₁₀ alkyl, preferably branched or unbranched Ci to C alkyl, more preferably methyl, ethyl, n-propyl, i-propyl, i-butyl or t-butyl;

alkoxy being Ci to C₄ alkoxy, preferably methoxy;

aryl being phenyl, naphthyl or tetrahydronaphthyl, preferably phenyl.

9. The compound or salt or hydrate thereof for use according to claim 8, wherein said compound is a compound of formula (1Mb) or (IVb)

(1Mb) (IVb) wherein each of the m occurrences of R⁷ is independently selected from d to C₄ alkyl; m being 0, 1 , 2, 3 or 4; and the remainder of groups and substituents being as defined in claim 8.

10. The compound or salt or hydrate thereof for use according to claim 9, wherein said compound is a compound of formula (Vb)

wherein m is 0 or 1.

11. The compound or salt or hydrate thereof for use according to claim 10, wherein said compound is a compound of formula (Vlb)

(Vlb)

12. The compound or salt or hydrate thereof for use according to any one of claims 8 to 1 1 , wherein

R¹ is selected from halogen; substituted or unsubstituted d to C₄ alkoxy, preferably OCH₃; substituted or unsubstituted Ci to C₄ alkyl, preferably CF₃; cyano; N0₂; and hydrogen;

R² is selected from substituted or unsubstituted piperidinyl, preferably N- substituted or unsubstituted piperidin-4-yl; substituted or unsubstituted cyclohexyl; and phenyl-alkyl;

the substituents being alkyl or phenyl-alkyl;

alkyl being Ci to C₄ alkyl, preferably methyl, ethyl, n-propyl, i-propyl or t-butyl;

or a salt of hydrate thereof.

13. The compound or salt or hydrate thereof for use according to any one of claims 8 to

12, wherein R¹ is Br, I, OCH₃, N0_2> F, CI, CF₃, CN, CH₃ or H.

14. The compound or salt or hydrate thereof for use according to any one of claims 8 to

13, wherein R² is

(a) N-aryl-alkyl piperidin-4-yl, preferably N-phenyl-alkyl piperidin-4-yl, particularly preferred N-phenyl-methyl piperidin-4-yl or N-phenyl-ethyl piperidin-4-yl;

(b) N-alkyl piperidin-4-yl, preferably N-ethyl piperidin-4-yl or N-prop-1-yl piperidin- 4-yl;

(c) N-alkyl piperidin-4-ylalkyl or N-aryl-alkyl piperidin-4-ylalkyl, preferably N-benzyl piperidin-4-yl-ethyl;

(d) alkyl cyclohexyl, preferably 3-methyl cyclohexyl, 4-methyl cyclohexyl, 4-ethyl cyclohexyl or 4-tert-butyl cyclohexyl; aryl-cyclohexyl, preferably 4-phenyl cyclohexyl; alkyl cyclopentyl, preferably 3-methyl cyclopentyl; or substituted cyclohexylalkyl such as 5,5-dimethyl-1 ,3-dioxocyclohex-2-ylmethylenyl;

(e) aryl alkyl, preferably unsubstituted or substituted phenyl alkyl, more preferably phenyl methyl, phenyl ethyl, 4-phenyl-but-2-yl, 1-(4-methoxy phenyl)-prop-2-yl, 1-(4-hydroxy 3-methoxy phenyl)-prop-2-yl, 4-(4-methoxy phenyl)-but-2-yl, 1- (3,4-dimethoxy phenyl)-prop-2-yl, 1-(2-methoxy phenyl)-prop-2-yl, 1-(2-fluoro phenyl) prop-2-yl; or a bicyclic aryl-alkyl group such as 2-oxindol-3- ylmethylenyl, 1 ,2,3,4-tetrahydronaphthalen-2-yl or dihydroinden-2-yl; or

(f) branched or unbranched and substituted or unsubstituted alkyl, preferably isobutyl, tert-butyl, neopentyl, norbornyl or adamantyl, propyl, prop-2-yl, 2- hydroxy ethyl.

15. The compound or salt or hydrate thereof for use according to any one of claims 8 to

14, wherein said compound is selected from the compounds of Tables 1 to 46.