Novel Levetiracetam Derivatives That Are Effective against the

Page 1 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Novel levetiracetam derivatives that are effective against the Alzheimer-like phenotype in mice: Synthesis, in vitro, ex vivo and in vivo efficacy studies Irene Sola,†,‡ Ester Aso,§,#,‡ Daniela Frattini,† Irene López-González, §,# Alba Espargaró,¶ Raimon Sabaté,¶ Ornella Di Pietro,† F. Javier Luque, M. Victòria Clos,⊥ Isidro Ferrer,*,§,# and Diego Muñoz-Torrero*,† †

Laboratori de Química Farmacèutica (Unitat Associada al CSIC), Facultat de Farmàcia, and

Institut de Biomedicina (IBUB), Universitat de Barcelona, Av. Joan XXIII 27-31, E-08028, Barcelona, Spain §

Institut de Neuropatologia, Servei d’Anatomia Patològica, IDIBELL-Hospital Universitari de

Bellvitge, Universitat de Barcelona, L’Hospitalet de Llobregat, Spain #

CIBERNED, Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas,

Instituto Carlos III, Spain ¶

Departament de Fisicoquímica, Facultat de Farmàcia, and Institut de Nanociència i

Nanotecnologia (IN2UB), Universitat de Barcelona, Barcelona, Spain

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



Page 2 of 60

Departament de Fisicoquímica, Facultat de Farmàcia (Campus Torribera), and IBUB,

Universitat de Barcelona, Prat de la Riba 171, E-08921, Santa Coloma de Gramenet, Spain ⊥

Departament de Farmacologia, de Terapèutica i de Toxicologia, Institut de Neurociències,

Universitat Autònoma de Barcelona, E-08193, Bellaterra, Barcelona, Spain

2


Page 3 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ABSTRACT: We have synthesized a series of heptamethylene-linked levetiracetam–huprine and levetiracetam–(6-chloro)tacrine hybrids to hit amyloid, tau and cholinergic pathologies as well as β-amyloid (Aβ)-induced epileptiform activity, some of the mechanisms that eventually lead to cognitive deficits in Alzheimer’s disease patients. These hybrids are potent inhibitors of human acetylcholinesterase and butyrylcholinesterase in vitro and moderately potent Aβ42 and tau anti-aggregating agents in a simple E. coli model of amyloid aggregation. Ex vivo determination of the brain acetylcholinesterase inhibitory activity of these compounds after intraperitoneal injection to C57BL6J mice has demonstrated their ability to enter the brain. The levetiracetam‒huprine hybrid 10 significantly reduced the incidence of epileptic seizures, cortical amyloid burden and neuroinflammation in APP/PS1 mice after a four-week treatment with a 5 mg/kg dose. Moreover, the hybrid 10 rescued transgenic mice from cognitive deficits, thereby emerging as an interesting disease-modifying anti-Alzheimer drug candidate.

3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 60

INTRODUCTION Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder. It affects over 35 million people worldwide and this figure will grow exponentially in the upcoming years along with demographic changes.1 Currently there exists no therapy that can halt or slow AD progression, so that this disease inexorably leads to the death of the patient. Consequently, the increasing prevalence of AD is being accompanied with increasing mortality figures, in sharp contrast to other major causes of death such as cardiovascular disease or cancer, for which the number of associated deaths has been decreasing in the past decade.2 Only five drugs have been approved so far for the treatment of AD, namely the acetylcholinesterase (AChE) inhibitors tacrine, donepezil, rivastigmine and galantamine and the glutamate NMDA receptor antagonist memantine.3 These drugs were developed to alleviate the cognitive and functional decline associated to neurotransmitter deficits that appear long after the initiation of the neurodegenerative process, and, hence, they are regarded as mainly symptomatic treatments. An efficient management of AD relies heavily on the development of novel drugs that can address the earliest disease mechanisms as well as on the parallel discovery of suitable biomarkers and techniques that allow an early diagnosis, thereby enabling the use of putative disease-modifying drugs in the most appropriate time. i.e. when neurodegeneration is not too widespread. Two proteins, β-amyloid peptide (Aβ) and tau, are widely thought to be at the root of the pathogenesis of AD, even though it is not clear whether or not their appearance is a cause or a consequence of the disease. Particularly, huge economic and research investments have focused on the development of disease-modifying drug candidates that address the biology of the 39‒43 amino acid peptide Aβ.4,5 However these endeavors have so far failed to derive any new approved drug.3 Indeed, it has been suggested that the formation and subsequent aggregation of

4


Page 5 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Aβ might not be the sole cause of the disease but just one of the main components of a pathological network, where hyperphosphorylation and aggregation of protein tau, synaptic dysfunction or neuroinflammation, among others, would be also key players.6 This conception of AD has prompted the development of an important number of compounds purposely designed to simultaneously hit several of the key biological targets or events of the AD pathological network.7‒19 In most cases, anti-Alzheimer multitarget compounds are designed to hit Aβ formation and/or aggregation and the enzyme AChE,20,21 which besides its cholinergic function, has been reported to be able to interact with Aβ, thereby promoting its aggregation.22,23 It has been recently suggested that aberrant neuronal network activity might be another key component of the AD network,24 which has been associated to elevated levels of Aβ and causally linked to Aβ-induced synaptic and cognitive deficits.25 Indeed, the incidence of epileptic seizures or epileptiform activity is increased in AD patients relative to the general population,24,26 especially in those individuals with early-onset AD, which is characterized by mutations in some deterministic genes encoding proteins involved in the production of Aβ, namely amyloid precursor protein (APP) and the enzymes involved in the APP cleavage presenilin 1 and presenilin 2. Increased incidence of epileptic seizures also occurs in other conditions which are associated with elevated levels of Aβ such as patients with Down syndrome, which feature three copies of chromosome 21, where the APP gene is located, and the incidence is still higher in individuals with Down syndrome that develop AD.27 Also, transgenic mouse models of AD that overexpress human mutated APP show epileptiform activity along with synaptic and cognitive deficits.28 Very interestingly, chronic treatment with the antiepileptic drug levetiracetam (1, Figure 1) can effectively suppress aberrant network activity and improve memory performance in patients with amnestic mild cognitive impairment (aMCI)29 as well as in hAPPJ20 and

5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 60

APP/PS1 mouse models of AD,30,31 thereby highlighting the critical therapeutic potential of targeting excess neuronal network activity, especially in combination with other copathogenic factors that fuel the progression of AD such as Aβ and tau aggregation and accumulation.32

Figure 1. Structure of levetiracetam.

We have recently developed several structural families of anti-Alzheimer hybrid compounds that contain the structure of 6-chlorotacrine (9-amino-6-chloro-1,2,3,4-tetrahydroacridine) or the high affinity AChE inhibitor huprine Y,33–36 structurally related to tacrine, which exhibit a common multitarget biological profile encompassing inhibition of the aggregation of Aβ42 and tau and inhibition of both cholinesterases, i.e. AChE and butyrylcholinesterase (BChE), apart from modulation of other specific targets or pathological events.37–39 Herein, we describe the synthesis and biological evaluation of a series of hybrid compounds that feature the 2-(2-oxopyrrolidin-1-yl)butyramide moiety of levetiracetam linked to a tacrine, 6-chlorotacrine or huprine Y unit as novel multitarget anti-Alzheimer leads with potential to modify AD progression. The biological profiling of the novel compounds includes: i) the in vitro assessment of their human AChE and BChE inhibitory activity; ii) the evaluation of their inhibitory activity against both Aβ42 and tau aggregation in intact Escherichia coli cells overexpressing these proteins, as a simple in vivo model of amyloid aggregation; iii) the ex vivo evaluation of their brain AChE inhibitory activity after intraperitoneal injection in C57BL6J 6


Page 7 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mice, as an indirect proof of their brain penetration; iv) a preliminary assessment of their acute toxicity in C57BL6J mice; and v) the in vivo efficacy studies after chronic administration to transgenic APP/PS1 mice, a well-established mouse model of AD, encompassing the evaluation of their effect on cognition, spontaneous epileptic seizures, cortical soluble Aβ40 and Aβ42 levels, cortical Aβ burden, and gliosis around Aβ plaques.

RESULTS AND DISCUSSION Chemistry. In this work, we envisaged first the straightforward synthesis of hybrids (±)-5 and (±)-6 (Scheme 1), which bear a tacrine or a 6-chlorotacrine unit linked to the racemic form of 1 (etiracetam), as well as the synthesis of the enantiopure hybrids (S)-5, featuring the (S)-eutomer form of etiracetam as an antiepileptic drug (levetiracetam), and its enantiomer (R)-5 (Scheme 1), to assess potential enantioselective interactions with the targets to be evaluated in vitro, due to the levetiracetam moiety. The eutomer for AChE inhibition of huprine Y and other huprinebased hybrids previously developed in our group is the (7S,11S)-enantiomer, whereas the opposite trend or no enantioselectivity has been found for their interaction with other targets of interest such as BChE or Aβ and tau aggregation.34,37,40 In this light, we also planned the synthesis of enantiopure levetiracetam‒huprine hybrids 10 and 11 (Scheme 2), featuring the (S)form of 1 and either a (7S,11S)- or (7R,11R)-huprine moiety. In all cases, an heptamethylene chain was chosen as the linker between the two pharmacophoric moieties because it should enable the hybrids to span the whole length of the active site gorge of AChE, one of their putative targets, thereby leading to high inhibitory potencies, as it has been reported for a number of tacrine-based dimeric and hybrid compounds.41,42 Indeed, other heptamethylene-linked

7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 60

huprine-based hybrids recently developed in our group have been consistently found to be among the most potent AChE inhibitors of the corresponding structural families.37,40

Scheme 1. Synthesis of the levetiracetam–tacrine hybrids (±)-5, (S)-5, (R)-5 and (±)-6a O N

O

HN

NH2

N

7

(i) (±)-4 CO2H

HN HN

N R 2, R = H 3, R = Cl

O

7

N (±)-5, R = H (±)-6, R = Cl

R

O

O

N HN HN

N (R)-5

7

O O

N

O

N

N

(i) (R)-4 CO2H

2

(i) (S)-4 CO2H

HN HN

O

7

N (S)-5

a

Reagents and conditions: (i) (±)-4, (S)-4 or (R)-4 (1 equiv), ClCO2Et (1 equiv), Et3N (2 equiv), CH2Cl2, 0 ºC, 30 min; then, amine 2 or 3 (1 equiv), CH2Cl2, rt, 3 days, 93% [(±)-5], 60% [(S)-5], 27% [(R)-5], 78% [(±)-6].

The synthesis of hybrids (±)-5 and (±)-6 was carried out by treatment of the readily available racemic 2-(2-oxopyrrolidin-1-yl)butyric acid, (±)-4,43 with 1 equiv of ethyl chloroformate and 2 equiv of Et3N in CH2Cl2, followed by reaction of the resulting mixed anhydride with 1 equiv of

8


Page 9 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the known 7-aminoheptyltacrines 244 and 345 at room temperature for 3 days, followed by silica gel column chromatography purification of the crude products (Scheme 1). Enantiopure carboxylic acids (S)-4 or (R)-4 were prepared through a methodology previously developed in our group, based on the resolution of the racemic acid (±)-4 through formation and silica gel column chromatography separation of its diastereomeric esters with (S)-Nphenylpantolactam followed by separate hydrolysis with LiOH / H2O2 in THF.43 Coupling of aminoheptyltacrine 2 with carboxylic acids (S)-4 and (R)-4 under the same reaction conditions used for the racemic compound, afforded in moderate yields the enantiopure hybrids (S)-5 and (R)-5, respectively (Scheme 1). The synthesis of enantiopure hybrids 10 and 11 was undertaken starting from enantiopure huprines (7S,11S)-7 and (7R,11R)-7, which are readily available even at multigram amounts by chromatographic

resolution.33,46

Alkylation

of

(7S,11S)-7

and

(7R,11R)-7

with

7-

bromoheptanenitrile in the presence of NaOH in DMSO afforded in moderate yields the novel enantiopure nitriles (7S,11S)-8 and (7R,11R)-8, which were subsequently reduced to the novel amines (7S,11S)-9 and (7R,11R)-9 upon treatment with LiAlH4 in Et2O (Scheme 2). Treatment of the mixed anhydride derived from carboxylic acid (S)-4 and ethylchloroformate with amines (7S,11S)-9

and

(7R,11R)-9

afforded

the

enantiopure

levetiracetam‒huprine

hybrids

(2S,7”’S,11”’S)-10 and (2S,7”’R,11”’R)-11 in moderate yields, after silica gel column chromatography purification.

9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 60

Scheme 2. Synthesis of the levetiracetam–huprine hybrids (2S,7”’S,11”’S)-10 and (2S,7”’R,11”’R)-11a

a

Reagents and conditions: (i) NaOH (3.3 equiv) 4 Å molecular sieves, DMSO, rt, 2 h; then 7bromoheptanenitrile (1.2 equiv), DMSO, rt, overnight, 52% [(7S,11S)-8], 55% [(7R,11R)-8]; (ii) LiAlH4 (6 equiv), Et2O, 0 ºC to rt, 20 h at rt, 91% [(7S,11S)-9], 45% [(7R,11R)-9]; (iii) (S)-4 (1 equiv), ClCO2Et (1 equiv), Et3N (2 equiv), CH2Cl2, 0 ºC, 30 min; then, amine (7S,11S)-9 or (7R,11R)-9 (1 equiv), CH2Cl2, rt, 3 days, 30% (10), 56% (11).

The novel levetiracetam-based hybrids were converted into their hydrochloride salts for their chemical characterization and biological profiling.

In Vitro Inhibition of Human Cholinesterases. Together with AChE, BChE is partly responsible for the hydrolysis of the neurotransmitter acetylcholine. Unlike AChE, whose brain levels are significantly reduced when AD progresses, the levels of BChE remain the same or may

10


Page 11 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


be even increased.47 Thus, dual inhibition of AChE and BChE is increasingly pursued in the search for novel AD treatments.48 Accordingly, the inhibitory activity of the novel levetiracetambased hybrids 5, 6, 10 and 11 and of the reference compounds (S)-1, tacrine, 6-chlorotacrine and (7S,11S)- and (7R,11R)-huprine Y against human recombinant AChE (hAChE) and human serum BChE (hBChE) was evaluated by the method of Ellman et al.49 The marketed antiAlzheimer reversible AChE inhibitors donepezil and galantamine were also evaluated as reference compounds. All the levetiracetam-based hybrids turned out to be very potent inhibitors of hAChE, with IC50 values in the low nanomolar range (Table 1). Indeed, in agreement with the expected binding of these hybrids along the active site gorge of AChE, the hybridization strategy led to increased hAChE inhibitory potencies of hybrids 5, 6 and 11 relative to their parent compounds tacrine, 6-chlorotacrine, (7R,11R)-huprine Y, and levetiracetam, which, to our surprise, exhibited an outstanding two-digit nanomolar hAChE inhibitory activity (Table 1). The sole exception was the levetiracetam‒huprine hybrid 10, which, despite its remarkable IC50 value of 4.2 nM, was still 6-fold less potent than its highly potent parent compound (7S,11S)-huprine Y, as we have also found for other (7S,11S)-huprine Y-based hybrids.37,40 All of the hybrids were found to be much more potent than galantamine and two of them, 6 and 10, also 2‒3-fold more potent than donepezil. The order of hAChE inhibitory potencies found for the hybrids (6 ≈ 10 > 5 > 11) is consistent with the known positive effect of the chlorine atom at position 6 of the tacrine unit,50,51 the levetiracetam‒6-chlorotacrine hybrid 6 being 10-fold more potent than the levetiracetam‒tacrine hybrid 5, and with the higher potency of (7S,11S)-huprine derivatives relative to the (7R,11R)-counterparts,34,37,40 hybrid 10 being 13-fold more potent than 11. Regarding the influence of the stereochemistry of the levetiracetam moiety on hAChE inhibitory

11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 60

activity, the levetiracetam‒tacrine hybrid (R)-5 was found to be 3-fold more potent than its enantiomer. Like the reference compounds, including 1, all of the novel levetiracetam-based hybrids were also found to be potent hBChE inhibitors, exhibiting two- to three-digit nanomolar IC50 values (Table 1), and hence, displaying an interesting dual AChE / BChE inhibitory activity. The known SAR trends for BChE inhibition regarding the presence of a chlorine atom at position 6 of the tacrine unit or at the equivalent position 3 of the huprine moiety and the enantiopreference of the huprine moiety are just the opposite than for AChE inhibition. Indeed, it is well-known that introduction of a chlorine atom at position 6 of the tacrine unit is somewhat detrimental for the interaction with BChE due to steric hindrance effects at the enzyme active site52,53 and that (7R,11R)-huprine derivatives are more potent hBChE inhibitors than their (7S,11S)counterparts.34,37,40 Thus, the order of potencies for hBChE inhibition of the levetiracetam-based hybrids (5 > 11 > 6 > 10) is consistent with the expected SAR trends, with the levetiracetam‒tacrine hybrid 5 being 4-fold more potent than the levetiracetam‒6-chlorotacrine hybrid 6, and with hybrid 11 being 4-fold more potent than 10. Additionally, it was found that for BChE inhibition the stereochemistry of the levetiracetam moiety did not seem to play a significant role, with hybrids (S)-5 and (R)-5 being roughly equipotent.

12


Page 13 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. Inhibitory Activities of the Hydrochlorides of Levetiracetam-Based Hybrids and Reference Compounds toward AChE, BChE and Aβ42, and Tau Aggregationa compd

hAChE

hBChE

Aβ42 aggregation

tau aggregation

IC50 (nM)a

IC50 (nM)a

(% inhibition)b

(% inhibition)b

(±)-5

30.7 ± 6.9

21.8 ± 0.5

13.8 ± 2.3

12.8 ± 1.2

(S)-5

89.9 ± 8.3

12.2 ± 1.0

12.6 ± 1.9

14.9 ± 1.6

(R)-5

26.2 ± 1.7

17.5 ± 3.4

Novel Levetiracetam Derivatives That Are Effective against the

Recommend Documents