Huprines for Alzheimer's disease drug development

Review

10.1517/17460441.3.1.65 © 2008 Informa UK Ltd ISSN 1746-0441 65

Huprines for Alzheimer’s disease drug development Diego Mu ñ oz-Torrero † & Pelayo Camps † †Laboratori de Qu í mica Farmac è utica (Unitat Associada al CSIC), Facultat de Farm à cia, and Institut de Biomedicina de la Universitat de Barcelona (IBUB), Universitat de Barcelona, Av. Diagonal, 643, E-08028-Barcelona, Spain

Background : So far, acetylcholinesterase (AChE) inhibitors have dominated the therapeutic arsenal for Alzheimer’s disease. Although conceptually developed as symptomatic drugs, mounting evidence suggests that these compounds can positively modify the disease progression, which has spurred the development of novel classes of AChE inhibitors. Objective : This article reviews the development of novel classes of high affinity AChE inhibitors following a design strategy based on molecular hybridization by stepwise incorporation of different fragments of the known AChE inhibitors ( − )-huperzine A and tacrine. Methods : This review covers the existing litera-ture dealing with the design, synthesis and structural and pharmacological characterization of the title compounds. Results/conclusion : Three novel classes of AChE inhibitors of increasing structural complexity and affinity have been developed, namely huprines, 13-amidohuprines and huprine − tacrine heterodimers. Particularly, huprines and huprine − tacrine heterodimers exhibit a unique profile encompassing both cholinergic and non-cholinergic disease-modifying effects and, thus, constitute promising anti-Alzheimer drug candidates.

Keywords: 13-amidohuprine , acetylcholinesterase inhibitor , Alzheimer’s disease , huprine , huprine − tacrine heterodimer , neuroprotective agent , prion protein antiaggregating agent

Expert Opin. Drug Discov. (2008) 3(1):65-81

1. Introduction

Alzheimer’s disease (AD), the leading cause of dementia in elderly people, constitutes one of the major health problems in developed countries. This devastating neuro-degenerative disorder affects 25 million people worldwide [1] and its prevalence is expected to increase dramatically during the next decades, along with the trend for an increased average life expectancy and the demographic shift to an ageing population [2] .

The therapeutic arsenal for the treatment of AD remains confined to five drugs conceptually developed to provide a symptomatic relief without altering the underlying disease process [3] . With the sole exception of the glutamate NMDA receptor antagonist memantine, the other commercialized anti-Alzheimer drugs, namely tacrine, donepezil, rivastigmine and galantamine, share as their common mode of action the inhibition of the enzyme acetylcholinesterase (AChE) in the CNS. Because this enzyme is responsible for the breakdown of the neurotransmitter acetylcholine in the synapse, the use of these inhibitors leads to the compensation of the central cholinergic deficit characteristic of AD, which, according to the cholinergic hypothesis, is responsible for the cognitive and functional decline of AD patients [4-6] .

Although the treatment of the very late symptoms of the dementia has been efficiently tackled with these AChE inhibitors, the early diagnosis and therapeutic interventions with new drugs addressing the early underlying mechanisms of

1. Introduction

2. Conjunctive approaches in the

design of novel AChE inhibitors:

historical perspective

3. Development of fi rst

generation huprines

4. Further optimization of the lead

huprines: 13-amidohuprines and

huprine – tacrine heterodimers

5. Conclusion

6. Expert opinion

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Huprines for Alzheimer’s disease drug development

66 Expert Opin. Drug Discov. (2008) 3(1)

AD and, consequently, able to halt or slow the disease progress, still constitute one of the major unmet needs in modern medicine. The β -amyloid peptide (A β ) and tau protein, the main components of the two characteristic neuropathological hallmarks of AD, that is, the senile plaques and the neurofibrillary tangles, respectively, seem to be at the root of the pathogenesis of the disease [7] and, consequently, represent the main targets to develop disease-modifying therapies. Indeed, in spite of some controversy [8] or related alternate hypotheses [9,10] , the prevailing hypo-thesis on the cause of Alzheimer’s disease is the so-called amyloid cascade hypothesis, which establishes that the increased production, aggregation and accumulation of A β in the brain triggers a cascade of events leading to neuronal degeneration and eventually to the clinical symptomatology of the dementia [11,12] . Consequently, most of the academic and industrial research efforts to develop disease-modifying anti-Alzheimer drugs have been focused on the targets associated with the amyloid cascade hypothesis, namely A β formation, aggregation, clearance or metabolism [7,13-16] . In fact, > 40% of the drugs in clinical trials for AD target A β [15] and it is expected that the first amyloid-directed disease-modifying anti-Alzheimer drugs reach the market as soon as 2009 or 2010 [13] .

Recently, some interconnections found between the early events of the amyloid cascade of AD, namely the A β forma-tion and aggregation, and some elements of the cholinergic neurotransmission, namely the M 1 and M 3 muscarinic receptors, α 7 and α 4 β 2 nicotinic receptors and AChE, have led to the notion that some cholinomimetic agents could additionally exhibit disease-modifying effects [17-20] . Thus, the activation of M 1 and M 3 muscarinic receptors triggers signaling cascades involving the protein kinase C (PKC), which in turn, stimulates the non-amyloidogenic processing of the amyloid precursor protein (APP), leading to both the secretion of the soluble APP (sAPP α ), which results from the cleavage of APP by α -secretase, and the inhibition of A β formation [18,19] . Similarly, the release of sAPP α can also result from the nicotinic receptor stimulation [18,19] . Moreover, it has been found that the enzyme AChE can bind through its peripheral site to A β , thus accelerating the A β aggregation [20] . Thus, those inhibitors of AChE able to block the peripheral site of the enzyme could inhibit the A β proaggregating action of AChE. Indeed, the increasing body of preclinical and clinical evidence pointing to a disease-modifying role for presently marketed anti-Alzheimer AChE inhibitors [19,21-24] could be ascribed, at least in part, to the cholinergic control of both the formation and aggregation of A β .

2. Conjunctive approaches in the design of novel AChE inhibitors: historical perspective

The prominent role played by AChE inhibitors in the last decades has continuously spurred the development of novel

classes of AChE inhibitors by different research groups. The development of huprines began in the early 1990s, when the presently marketed AChE inhibitors were still under clinical trials. By that time, the 3D structure of the Torpedo californica AChE ( Tc AChE) had just been solved [25] , but no crystal structure of the complex of the enzyme with any inhibitor was available yet.

For the design of huprines, two known inhibitors were taken as models, namely tacrine, which soon after became the first marketed anti-Alzheimer drug, and ( − )-huperzine A, a very promising natural product isolated from a traditional Chinese medicinal plant, which is currently in Phase III clinical trials in the USA.

The most widespread practice in pharmaceutical research to get new molecules usable as lead compounds is that based on the chemical modification of a known drug [26] . Many analogs of both tacrine [27] and ( − )-huperzine A [28,29] were being synthesized by the time the development of huprines started. No systematic improvement in the AChE inhibitory potency had been observed in the analogs of tacrine, but a few of them exhibited significant improvements (up to 19-fold) [30] in potency relative to the parent tacrine. The structure of ( − )-huperzine A seemed to tolerate little modification, almost every slight modification of its structure resulting in a significant loss of AChE inhibitory activity.

Conversely, the combination into a single molecule of two identical or different structural units or fragments of known drugs with the aim of either increasing the potency of the parent compounds or combining complementary actions has remained a strategy largely unexplored for a long time. In this context, the overall idea guiding the design of huprines was to try to find a single molecule consisting of units or fragments of the known ( − )-huperzine A and tacrine that could span as much as possible the active site of AChE. The desired extended binding of the novel compounds relative to ( − )-huperzine A and tacrine should result in an increased number of interactions with the enzyme and, consequently, in an increased affinity. Following this hybridization strategy and, when the binding mode of ( − )-huperzine A and tacrine with AChE was not yet known, a family of huperzine A − tacrine hybrids was empirically designed ( Figure 1 ), for which the abbreviated name huprines was later suggested. Over several years, the hit-to-lead optimization was brought about, helped with the activity data arising from the biological assays of the initially synthesized huprines. Among the different 40 huprines that were synthesized in racemic form, the so-called huprine Y ( Figure 1 ) and its 9-ethyl analog huprine X were selected as the most promising compounds of this novel structural class. In racemic form, the lead huprines turned out to be > 330- and 260-fold more potent human AChE inhibitors than the parent compounds from which they were designed, ( − )-huperzine A and tacrine, respectively, and in enantiopure eutomer form, ( − )-huprines X and Y were

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Expert Opin. Drug Discov. (2008) 3(1) 67

> 800- and 640-fold more potent than ( − )-huperzine A and tacrine, respectively.

During this time, the X-ray crystal structures of the complexes of Tc AChE with a repertoire of ligands, including ( − )-huperzine A [31] and tacrine [32] were being solved. Apart from the unexpected features found in the 3D structure of Tc AChE [25] , namely the position of the active site, deeply buried at the bottom of a 20 Å -long and narrow gorge, the inspection of the structure of the complex of Tc AChE with the elongated bisquaternary ligand decamethonium permitted the identification of the so-called peripheral anionic site of AChE at the entrance of the active-site gorge [32] . The knowledge of the structural features of AChE and the binding mode of a number of ligands provided the basis

for a rational structure-based approach to the design of novel AChE inhibitors, including several arising from conjunctive approaches.

Thus, in a contemporaneous work to the development of huprines, a different conjunctive strategy starting from tacrine was developed in the context of a collaboration between Pang, Carlier and Han with the same aim of increasing the affinity [33-35] . Following the observation that decamethonium spans the entire active-site gorge and the belief that bisquaternary AChE inhibitors derived their enhanced potency relative to similar monoquaternary ligands from their ability to simultaneously interact with the two binding sites of the AChE [32] , they rationally designed a novel class of high-affinity dual binding site AChE inhibitors

Figure 1 . Stepwise molecular hybridization from ( – )-huperzine A and tacrine to huprines, 13-amidohuprines and huprine–tacrine heterodimers

TacrineIC50 (hAChE) = 205 nM

(−)-Huperzine AIC50 (hAChE) = 260 nM

Huperzine A−tacrine hybrids(huprines)

1, IC50 (hAChE) = 52 nM

Conjunctiveapproach

hAChE: human AChE

Huprine YIC50 (hAChE) = 0.78 nM

Hit-to-leadoptimization

Conjunctiveapproach

Conjunctiveapproach

13-AmidohuprinesIC50 (hAChE) = 41 − 98 nM

Huprine−tacrine heterodimersIC50 (hAChE) = 0.29 − 0.50 nM

Y

NH

N R

N

NHCl

N

NH2

N

NH2

ClN

NH2

ClN

NH2

HNR

HO

HN O

NH2

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by the combination of two tacrine units through an oligomethylene linker of suitable length to allow the simultaneous interaction of the two constituting units with key residues of both the active and peripheral sites. In the best case, that is, with bis (7)-tacrine, an increase of 150-fold in the rat brain AChE inhibitory activity relative to tacrine was achieved.

Several years later, in a program directed to the development of selective insecticides, Viner used a very similar molecular hybridization strategy to that developed for huprines. Knowing the binding mode of tacrine [32] and the Quinn’s transition state mimic m -( N ,N, N -trimethylammonio)-2,2,2-trifluoroacetophenone [36] , Viner rationally developed some hybrids that combined the 4-aminoquinoline moiety of the former with the trifluoroacetophenone moiety of the latter, to simultaneously span the separate, but close, binding sites expected for the two constituting structural motifs [37] . The desired synergistic effect of both moieties was achieved, namely a 3-fold increase in Tc AChE inhibitory activity relative to tacrine.

In 2002, the X-ray crystal structure of the complex of Tc AChE with ( − )-huprine X was solved by Sussman and Silman [38] , which enabled the rational structure-based design of a second generation of huprines with potentially increased affinity. The incorporation of a fragment formally derived from the model ( − )-huperzine A into the structure of the first-generation lead huprine Y, led to a series of 13-amidohuprines ( Figure 1 ) that were expected to be able to simultaneously span almost all of the binding zones that are separately occupied by the models ( − )-huperzine A and tacrine. However, contrary to the initial expectations these second-generation huprines were clearly less potent than the parent huprine Y.

Conversely, the combination of the hybridization strategy leading to huprines with the dual site binding strategy developed by Pang, Carlier and Han allowed to make a step forward to increase the affinity of the first-generation lead huprines. Thus, the connection of huprine Y with an additional tacrine unit through a suitable linker resulted in a series of huprines–tacrine heterodimers ( Figure 1 ) that turned out to be up to 3-fold more potent than the parent huprine Y.

3. Development of fi rst generation huprines

3.1 Empirical design of huprines As previously mentioned, when the project on huprines was started, the 3D structures of the complexes of AChE with ( − )-huperzine A and tacrine had not been solved yet, which precluded any rational design based on the structures of these models. In this context, it was hypothesized that the heteroaromatic moieties of both models, that is, 4-aminoquinoline system of tacrine and the pyridone ring of ( − )-huperzine A would occupy a similar zone within the active site of AChE. Similarly, the carbocyclic moieties

of both models, that is, the carbobicyclic bridged system of ( − )-huperzine A and the cyclohexene ring of tacrine, were assumed to be placed in a similar zone within the active site of the enzyme. If the models were to orient in this way, a larger occupancy of the first zone of AChE active site should be expected for the 4-aminoquinoline system of tacrine, whereas a larger occupancy of the second binding zone should be expected for the carbobicyclic bridged system of ( − )-huperzine A. By assuming this working hypothesis, huperzine A-tacrine hybrids (huprines) were empirically designed by the combination of the two structural motifs leading to the largest occupancy of the two binding zones, that is, the 4-aminoquinoline system of tacrine and the carbobicyclic moiety of ( − )-huperzine A. For its relative ease of synthesis, the first synthesized huprine, (1, Figure 1 ), lacked the ethylidene appendage characteristic of the carbobicyclic moiety of ( − )-huperzine A [39,40] . Huprine 1 turned out to be a 5- and 4-fold more potent human AChE inhibitor than the models ( − )-huperzine A and tacrine, respectively, these preliminary results being indicative of the success of this hybridization strategy as a way to increase the affinity. Moreover, the hit huprine offered clear opportunities to upgrade the AChE inhibitory potency by simply introducing in its huperzine A-like moiety the ethylidene appendage of the model ( − )-huperzine A, an essential structural feature for this inhibitor [28,41,42] , or by introducing in its tacrine-like moiety some substituents that had been described to increase the inhibitory potency of the model tacrine. The effects of these and other structural modifications on the hit huprine 1 were studied during the hit-to-lead optimization process ( Figure 2 ), which required the development of different synthetic methodologies [43] . The structure–activity relationships (SAR) studies with the different synthesized huprines were carried out on the basis of their bovine AChE inhibitory activities. Only in the cases of outstanding potency, the inhibitory activity toward human AChE was determined.

3.2 Structure–activity relationship studies: modifi cation of the huperzine A-like moiety of huprine 1 The hit-to-lead optimization process began with the modification of the huperzine A-like carbobicyclic bridged moiety of huprine 1 . The ethylidene appendage of ( − )-huperzine A had proven to be an essential structural feature. Some changes carried out in huperzine A analogs involving the E → Z isomerization of the ethylidene group, or its homologation or replacement by alkyl groups resulted in a clear drop of the AChE inhibitory activity [28] . The same result had been obtained by the modification of the methyl-substituted unsaturated three-carbon bridge, its saturation resulting in a loss of AChE inhibitory potency, the isomerization of the endocyclic carbon–carbon double bond to an exocyclic position or the replacement of its methyl group by a hydrogen atom or by bulkier groups [28] .

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In sharp contrast with the SAR data known for huperzine A analogs, the introduction of an ( E )-ethylidene group on the methylene bridge of huprine 1 led to a 5-fold decrease in the bovine AChE inhibitory activity of the resulting huprine 2 ( Figure 2 ). Other changes including the introduc-tion of ( Z )-ethylidene, alkyl and alkoxy or carbonyl groups on the methylene bridge of 1 (huprines 3 – 6 ) resulted in more pronounced drops of activity, as it was the case for the replacement of the C7–C11 methylene bridge of huprine 1 by an o -phenylene group (huprine 7 ) [40] .

The structural modification of the C7–C11 three-carbon bridge of huprine 1 , including the saturation of the C8–C9 double bond (huprines 8 – 10 ) [40] , the isomeriza-tion of the C8 – C9 double bond to the endocyclic neighbouring C9 – C10-positions (huprines 11 and 12 ) [44] or replacement of the methyl group by a hydrogen atom or by bulkier substituents (huprines 13 – 20 ) resulted in moderate to great decreases of activity ( Figure 2 ) [40,45] , with the sole exception of the 9-ethyl substituted huprine 14 , which turned out to be a slightly more potent bovine

Figure 2 . Hit-to-lead optimization in huprines . The IC 50 values toward bovine AChE are indicated after the compound numbers. AChE: Acetylcholinesterase.

R1 = F, R3 = H, 22, 31.4 nMR1 = H, R3 = F, 23, 8.51 nMR1 = R3 = F, 24, 2.43 nMR1 = H, R3 = Cl, 25, 4.23 nMR1 = H, R3 = Me, 26, 12.4 nM

R9 = Me, R1 = R3 = H, 1, 65 nMRX = RN = H, 8, 41800 nMRX = OH; RN = H, 9, 1610 nMRX = H; RN = OH, 10, 4300 nM

36, 257 nM

subst. at C-1 and/or C-3Saturation

of ∆8,9

subst. at C-13

C-7/C-11 Methylene o-phenylene

Benzene cyclopentene

R1 = F, R3 = H, 27, 46.4 nMR1 = H, R3 = F, 28, 7.40 nMR1 = R3 = F, 29, 2.62 nMR1 = Cl, R3 = H, 30, 16.2 nMR1 = H, R3 = Cl, 31, 2.77 nMR1 = R3 = Cl, 32, 39.6 nMR1 = Me, R3 = H, 33, 29.8 nMR1 = H, R3 = Me, 34, 12.0 nMR1 = R3 = Me, 35, 3.59 nM

R9 = Me, 11, 511 nMR9 = Et, 12, 165 nM

R9 = H, 13, 4890 nM

R9 = Et, 14, 38.5 nM

R9 = n-Pr, 15, 431 nMR9 = i-Pr, 16, 103 nMR9 = n-Bu, 17, 280 nMR9 = t-Bu, 18, 267 nMR9 = allyl, 19, 150 nMR9 = phenyl, 20, 126 nM

subst. at C-9

subst. at C-1and/or C-3

subst. at C-2

∆8,9 ∆9,10

Isomerization

RA, RS = (E )-ethylidene, 2, 320 nMRA, RS = (Z )-ethylidene, 3, 1150 nMRA = Me, RS = OMe, 4, 1340 nMRA = OMe, RS = Me, 5, 6770 nMRA, RS = O, 6, 2090 nM

7, 2090 nM 21, 5580 nM

(−)-Huperzine A, 74 nMTacrine, 130 nM

Cl

N

NH2

R9

N

NH2

N

NH2

RN

RX

RA

RS

N

NH2

N

NH2

N

NH2

N

9

106 5 4

3

2112

1113

7

8

R9

R3

R1NH2

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AChE inhibitor than the hit huprine 1 [45] . Thus, the replacement of the methyl group at the position 9 of the hit huprine 1 by an ethyl group was the first structural modification that led to an improvement in the potency of the hit.

With the sole exception of latter modification, the structural changes affecting the methyl-substituted C7 – C11 unsaturated bridge of huprine 1 produced the same effects on the AChE inhibitory activity as those reported in huperzine A analogs. Conversely, the presence of an ( E )-ethylidene group on the methylene bridge of huprines and huperzine A analogs led to completely different effects on the activity. In view of these results, only a partial overlap of the carbobicyclic moieties of huprines and ( − )-huperzine A within the active site of AChE was anticipated.

3.3 Structure–activity relationship studies: modifi cation of the tacrine-like moiety of huprine 1 The main kind of modification of the tacrine structure that was known to lead to an enhanced AChE inhibitory activity was the introduction of halogen atoms or methyl groups at particular positions of its benzene ring, namely at positions 6 or 8 [27,30] , whereas substitutions at position 5 and especially at position 7 were clearly detrimental to the inhibitory activity [30] . Other changes affecting the benzene ring of tacrine, such as its saturation, had been reported not to affect significantly the inhibitory potency of the parent tacrine [27,46] .

The introduction of fluorine, chlorine or methyl substituents at positions 1 or 3 (equivalents to positions 8 and 6 of tacrine, respectively) of the hit huprine 1 (huprines 22 , 23 , 25 and 26 , Figure 2 ) or its 9-ethyl-substituted analog 14 (huprines 27 , 28 , 30 , 31 , 33 and 34 ) led to a 1.4- to 23-fold increase in the bovine AChE inhibitory activity relative to 1 [45,47] , the best pattern of substitution involving the presence of a chlorine atom at the position 3. Conversely, the substitution at position 2 (equivalent to position 7 of tacrine; huprine 36 ) was detrimental for the activity [47] .

1,3-Difluoro- and 1,3-dimethyl-substitution (huprines 24 , 29 and 35 , Figure 2 ) had an additive effect on the inhibitory potency, whereas, surprisingly, 1,3-dichloro-substitution (huprine 32 ) was detrimental relative to the monosubstituted counterparts [47] . This result doesn’t seem to make much sense and should be taken with caution. Indeed, it was reported later that dichlorosubstitutions at positions 6 and 8 of tacrine (equivalents to positions 3 and 1 of huprines, respectively) led to a 40-fold increase in the bovine AChE inhibitory activity relative to the unsubstituted tacrine [48] .

The replacement of the benzene ring of huprine 1 by a cyclopentene ring (huprine 21 , Figure 2 ) led to a dramatic loss of potency [40] . Taking into account that the saturation of the benzene ring of tacrine was reported to lead to a slight decrease in potency [46] , the drop in the potency of 21 could be mainly ascribed to an effect of the ring contraction.

Overall, the structural changes affecting the 4-aminoquinoline moiety of huprines and tacrine analogs were found to lead to the same effects on the AChE inhibitory activity, which anticipated a good overlap of the heteroaromatic moieties of huprines and tacrine within the active site of AChE.

3.4 Structure–activity relationship studies: enantioselectivity of the AChE inhibition by huprines Both enantiomers of huprines 1 , 14 , 23 , 25 , 28 , 31 , and 34 were separately obtained by chromatographic resolution of the corresponding racemic compounds [47,49,50] . The levorotatory enantiomer of these huprines, bearing the (7 S ,11 S )-configuration [49] turned out to be a 7- to 424-fold more potent bovine AChE inhibitor than the dextrorotatory enantiomer and exhibited roughly twice lower IC 50 values than the racemic compound.

For the most potent huprines arising from pharmacomodulation of the hit huprine 1 , particularly huprines substituted at the benzene ring, the inhibitory activity toward human AChE was also determined. The same pattern of substitution found optimal for the bovine AChE inhibition, led to an optimal human AChE inhibi-tion, that is, the presence of a chlorine atom at position 3. Moreover, 3-chloro-substituted huprines 25 and 31 were 5- and 4-fold more potent for the inhibition of the human AChE versus the bovine esterase, whereas the general increase in human versus bovine AChE inhibitory activity in the rest of huprines assayed was just 1.1- to 2-fold. Thus, huprines 25 and 31 , the so-called huprines Y and X, constituted very interesting leads for human AChE inhibition, exhibiting IC 50 values in the subnanomolar range [0.78 nM and 0.75 nM for racemic 25 and 31 , respectively, and 0.32 nM for both ( − )- 25 and ( − )- 31] . Overall, the molecular hybridi-zation from ( − )-huperzine A and tacrine followed by the pharmacomodulation of the hit huprine 1 had resulted in an increase in the AChE inhibitory activity of up to 813- and 641-fold, respectively, relative to the model drugs from which they were designed.

3.5 Three dimensional structure of the complex Tc AChE-( – )-huprine X: rationalization of structure–activity relationship data When the hit-to-lead optimization process of huprines was finished, the 3D structures of the complexes of Tc AChE with both ( − )-huperzine A [31] and tacrine [32] were already known. As assumed in the working hypothesis used for the empirical design of huprines, the cyclohexene ring of tacrine and the carbobicyclic system of ( − )-huperzine A occupied a similar binding zone within the active site of AChE, but this was not the case for the 4-aminoquinoline system of tacrine and the pyridone ring of ( − )-huperzine A, which, pointed to opposite directions instead. In spite of this, the binding mode of huprines with AChE, first predicted by molecular modeling studies and subsequently confirmed by X-ray

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diffraction studies on the complex Tc AChE-( − )-huprine X, shares common features with both models in agreement with the hybrid nature of huprines [38] .

The tacrine-like moiety of huprine, that is, the 4-aminoquinoline system, completely overlaps with the corresponding moiety of tacrine within the active site of AChE ( Figure 3 ) and, consequently, establishes the same set of interactions, namely: i) π – π stacking between the aromatic rings of Trp84 and Phe330; ii) hydrogen bond between the protonated pyridine nitrogen atom and the main chain carbonyl oxygen of His440; and iii) water-mediated hydrogen bond of the exocyclic amino group with Asp72, Tyr121 and Ser122. Additionally, the chlorine atom at position 3 of ( − )-huprine X was found to lie in a hydro-phobic pocket interacting with the aromatic rings of Trp432 and Phe330 and with the methyl groups of Met436 and Ile439 ( Figure 4 ). These results explain the parallelism previously observed between the SAR data affecting the heteroaromatic moiety of both huprines and tacrine analogs.

Regarding the huperzine A-like moiety of huprines, that is, the carbobicyclic bridged system, the C7–C11 unsaturated three-carbon bridge occupies the same binding zone than the corresponding moiety of ( − )-huperzine A ( Figure 3 ). However, the C7–C11 methylene bridges of huprines and ( − )-huperzine A point to opposite directions. These observa-tions also explain the relative parallelism observed between the SAR data affecting the three-carbon bridge of huprines and huperzine A analogs, as well as the absolute lack of parallelism between the SAR data affecting the methylene bridge of both kind of compounds. Moreover, the inspection

Figure 3 . Representation of the relative orientation of ( – )-huprine X (yellow), ( – )-huperzine A (green) and tacrine (blue) within the active site of Tc AChE, obtained by superposition of the corresponding X-ray structures of the complexes of Tc AChE with the three inhibitors . Residues Trp84, Gly117, Gly118 and Phe330 in the active site of the three complexes are also shown. AChE: Acetylcholinesterase.

Gly118

Gly117

Trp84

Phe330

of the structure of ( − )-huprine X within the enzyme active site reveals that not much room is available around the methylene bridge for the introduction of substituents at this position without causing steric clashes with Trp84, Gly117, Tyr130 or Glu199 ( Figure 4 ). The slightly higher potency observed for 9-ethyl-substituted huprines relative to their 9-methyl-substituted analogs could be ascribed to a better fit with the hydrophobic pocket formed by Tyr121, Phe290, Phe330 and Phe331 ( Figure 4 ). In turn, the introduction of bulkier substituents at position 9 could result in a clash with these residues and with Ser200.

The lower potency of syn -huprines relative to their anti -counterparts seems to arise from the disruption of the network of water molecules that mediate the hydrogen bonds between the exocyclic amino group and Asp72, Tyr121 and Ser122, as a consequence of the closer proximity of the endocyclic carbon–carbon double bond to this amino group in syn -huprines [44] .

Overall, these studies confirmed the simultaneous occupancy by huprines of the complete binding zone of the model tacrine and a part of the binding zone of the model ( − )-huperzine A. The high AChE inhibitory potency of huprines seems to be due, at least in part, to this extended binding within the enzyme active site, thus confirming the success of the hybridization strategy.

3.6 Pharmacological characterization of the lead huprines The outstanding human AChE inhibitory activity of huprines ( − )- 25 and ( − )- 31 was confirmed by Rosenberry, who found an inhibition constant K I of 33 pM and 26 pM, respectively [50] . Thus, the affinity of ( − )-huprines X and Y toward human AChE was found to be 180- and 1200-fold higher than that of the models ( − )-huperzine A and tacrine, respectively, and 40-fold higher than that of donepezil, the most potent commercialized AChE inhibitor [50] . Moreover, Badia found in ex vivo studies with OF1 mice that huprines ( − )- 25 and ( − )- 31 were able to cross the blood–brain barrier and inhibit the mouse brain AChE by 97% and 77%, respectively, whereas ( − )-huperzine A exhibited a 30% inhibition and tacrine was inactive at the doses used [51] .

The clinical efficacy of tacrine and, particularly, ( − )-huperzine A could be ascribed to their action on multiple biological targets belonging or not to the cholinergic system [27,52] . In order to complete the pharmacological profile of the lead huprines 25 and 31 (huprines Y and X, respectively), additional pharmacological studies on targets other than AChE have been carried out.

As previously mentioned, the stimulation of muscarinic M 1 and M 3 receptors, directly with agonists or indirectly with AChE inhibitors, has been suggested to be of particular interest for the treatment of AD, not only for the improve-ment in cholinergic transmission, but, more importantly, for the resulting stimulation of the processing of APP through

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the non-amyloidogenic α -secretase pathway [18,19] . Thus, tacrine, which can interact with muscarinic receptors [53] , has been shown to suppress the secretion of A β in human neuroblastoma cells [19,54] , whereas ( − )-huperzine A, which has little direct effects on muscarinic receptors compared with tacrine [55] , can also reduce the amyloidogenic processing of APP in both rats and HEK293wt cells by upregulating the PKC [56] . Both racemic huprines X and Y exhibit an agonistic activity on rat hippocampal muscarinic M 1 receptors, with IC 50 values for the displacement of [ 3 H]-pirenzepine of 478 nM and 400 nM, respectively [57,58] . Work is in progress to assess the potential effects of the lead huprines on the processing of APP.

Glutamate NMDA receptor antagonists are thought to be of interest for the treatment of neurodegenerative disorders, such as AD and Huntington’s disease. Indeed, the last marketed anti-Alzheimer drug, memantine, is a low-affinity glutamate NMDA receptor antagonist. Both ( − )-huperzine A and tacrine have been reported to protect cells against glutamate-induced neurotoxicity and reduce the neuronal death through antagonism of glutamate NMDA receptors [59,60] . Similarly, Pall à s, Camins and Sureda found that the racemic huprine Y partially prevented glutamate-induced cell death

in rat cerebellar granule cells cultures and almost completely inhibited the NMDA-evoked intracellular calcium increase, with an IC 50 value of 12 µM, thus evidencing the antagonistic effect of huprine Y on NMDA receptors [61] . Moreover, it was found that huprine Y protected in vivo C57BL/6 mice from the striatal lesions induced by 3-nitropropionic acid, a neurotoxin known to induce brain neurodegeneration resembling that of Huntington’s disease [61] . Conversely, neither racemic huprine Y nor the models ( − )-huperzine A and tacrine were able to protect against the neuronal death induced by colchicine in rat cerebellar granule cells cultures, as a model of apoptosis mediated by neuronal cytoskeleton alteration [62] .

Among the interconnections recently found between the early events of the amyloid cascade of AD and some elements of the cholinergic system, of particular importance was the finding by Inestrosa that AChE, on binding to A β through its peripheral site, accelerates the A β aggregation and increases the A β neurotoxicity as an early event in the neurotoxic cascade of AD [20,63-67] . From this finding it became apparent that the blockade of the AChE peripheral site should result in an inhibition of the AChE-induced A β aggregation and, therefore, in a very interesting interference

Figure 4 . Representation of the orientation of ( – )-huprine X (yellow) within the active site of Tc AChE . Active site residues surrounding the inhibitor are shown in green. The surface of the residue Ser200 (at a distance of 3.8 Å from the huprine ethyl group) is shown in magenta. AChE: Acetylcholinesterase.

Phe331

Phe330

Met436

IIe439

Trp432 Trp84 Tyr130

Gly117

Tyr121

Phe290

4.7

3.83.9

4.3

3.83.5

3.9

3.4Glu199

4.2

4.2

4.43.8

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upstream of the neurotoxic cascade of AD. There seem to exist some similarities between A β and prion proteins, including their ability to form amyloid fibrils [68] , the coexistence of A β and prion pathologies in some AD patients [69] or a common spatial pattern of protein deposition [70] . Indeed, Clos and Salmona found that AChE also promoted in a time- and concentration-dependent manner the aggregation of the prion peptide PrP 106-126 [71] , a segment thought to be central to the pathogenic properties of prion proteins. The co-incubation of PrP 106-126 and AChE with the specific peripheral site AChE inhibitor propidium at a concentration 100 µM resulted in a 87% inhibition of the prion peptide aggregation [71] . At the same concentration, propidium was known to inhibit in a similar extent (82%) the AChE-induced aggregation of A β [72] , whereas active-site AChE inhibitors, such as tacrine, exhibited a very poor inhibition of both A β (7%) and PrP 106-126 (16%) aggregation. Analogously, ( − )-huperzine A inhibited AChE-induced PrP 106-126 aggregation by only 15% [71] . In view of these results, an involvement of the peripheral site of AChE in the prion protein proaggregating action of the enzyme was hypothesized. Surprisingly, the active site inhibitors huprines X and Y, as well as huprine Z ( 23 , Figure 2 ) in racemic form inhibited the PrP 106-126 aggregation by 84%, 69% and 89%, respectively [71] . In line with these surprising results, Andrisano and Bartolini have found very recently that racemic huprine Y at 100 µM inhibits AChE-induced A β aggregation by 38% (unpublished results). The binding geometry and added molecular volume of huprines when bound to the enzyme active site seem to be responsible for the decrease in affinity of peripheral site ligands, such as propidium [50] , but also A β or PrP 106-126. Indeed, the comparison of the volumes of the binding gorges of native Tc AChE or complexed with several inhibitors including ( − )-huperzine A, donepezil and ( − )-huprine X led to the observation that, in general, the binding gorges were reduced relative to the native enzyme except for huprine X, which was found to inflate the binding gorge [73] . The diminished ability of these peptides to bind to the peripheral site of the complex of the enzyme with an huprine would lead to an inhibition of their aggregation.

3.7 Toxicological studies The main toxicity issue associated to the use of tacrine is the elevation in liver function enzymes in AD patients, which is both dose-dependent and reversible after reduction of the dose or withdrawal in the patients [74] . The formation of toxic quinone-type metabolites by the hepatic oxidative metabolism could be responsible for the hepatotoxicity of tacrine [75,76] . Similar to tacrine, ( − )-huperzine A raised the liver coefficient and increased serum levels of aspartate aminotransferase and alanine aminotransferase in rats. However, acute and subacute toxicity studies in several animals showed that the administration of ( − )-huperzine A did not induce histopathological changes in the liver [29,52,77] .

So far, only preliminary acute toxicological studies have been carried out with huprines. The LD 50 doses of ( − )-huprine X and ( − )-huprine Y in mice were 97 and 139 mg/kg by mouth and 8 and 6 mg/kg intraperitoneal, thus exhibiting a similar acute toxicity than tacrine after oral administration (86 mg/kg), but somewhat higher after intraperitoneal administration (27 mg/kg; unpublished results).

3.8 Patent protection Huprines were patented in Spain in 1995 and shortly after the patent was extended to several countries [78] . The priority rights of the patent are owned by a Spanish company, which is awaiting partnering for further development of huprines. Unfortunately, this partnership has not arrived yet, thus precluding preclinical studies with huprines, including pharmacokinetic studies or subacute toxicological studies to discard any potential hepatotoxic effect.

4. Further optimization of the lead huprines: 13-amidohuprines and huprine–tacrine heterodimers

First generation huprines span all the binding zone of the model tacrine, but only a part of the binding zone of the model ( − )-huperzine A. The pioneering work by Pang, Carlier and Han on tacrine dimers ( bis -tacrines) as dual binding site AChE inhibitors underscored the importance of the peripheral site and the active-site gorge as additional binding sites for ligands. The full occupancy of the binding zone of ( − )-huperzine A or the simultaneous binding to the peripheral site were recently addressed by the incorporation of additional fragments into the structure of the lead huprine Y. The knowledge of the three-dimensional structures of the complexes of Tc AChE with ( − )-huperzine A, tacrine and ( − )-huprine X allowed the structure-based rational design of two novel structural families related to huprine Y, namely 13-amidohuprines and huprine–tacrine heterodimers, aimed at increasing the affinity of the parent compound in virtue of their more extended binding within the enzyme.

4.1 The 13-amidohuprines The sole binding zone of the model ( − )-huperzine A not spanned by huprines is that occupied by the pyridone ring. In the superimposed structures of the complexes of Tc AChE with ( − )-huperzine A and ( − )-huprine X, the pyridone ring is very close to the C7–C11 methylene bridge of huprine X ( Figure 3 ). Thus, it became apparent that the introduction of an amido group at the methylene bridge (position 13) of huprines should result in the additional interactions with the enzyme exerted by the amido group of the pyridone ring of ( − )-huperzine A, namely a hydrogen bond between the amide carbonyl group and Tyr130 and a water-mediated hydrogen bond between the amide N − H group and Glu199. However, as previously mentioned, there didn’t seem to be much room available for the introduction of additional

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substituents at the methylene bridge of huprines without causing clashes with Trp84, Gly117, Tyr130 or Glu199 ( Figure 4 ). Indeed, all the attempts to improve the potency of the hit huprine 1 by modifying this part of the molecule (huprines 2 – 7 , Figure 2 ) had failed. The same situation could have been expected for ( − )-huperzine A: the amide carbonyl oxygen of ( − )-huperzine A repels the carbonyl oxygen of Gly117, thus causing the Gly117 − Gly118 peptide bond to undergo a flip by 180° compared with the structures of the native AChE and other inhibitor complexes ( Figure 3 ). This peptide flip, observed in the structures of the complex of Tc AChE with ( − )-huperzine A, its synthetic (+)-enantiomer or its natural homolog ( − )-huperzine B, has been suggested to be necessary for the binding of these compounds to AChE [31,79] . It was thought that an amido group at the position 13 of huprines could be accommo-dated within the enzyme active site if it were able to trigger the same Gly117–Gly118 peptide flip than the pyridone ring of ( − )-huperzine A.

Thus, a new generation of huprines was rationally designed, addressed to simultaneously span all of the binding zones that were separately occupied by the models ( − )-huperzine A and tacrine. The design strategy consisted of introducing an small amido group (formamido or acetamido) at the position 13 of the lead huprine Y, with the two possible diastereomeric arrangements. Through a rather difficult and long synthetic sequence, 13-amidohuprines 38a , 38b , 39a and 39b , as well as the 13-methanesulfonamides 37a and 37b and 13-aminohuprines 40a and 40b were prepared in racemic, but diastereopure forms, the compounds 40a and 40b being advanced intermediates for the preparation of the other amidohuprines ( Figure 5 ) [80,81] .

The bovine AChE inhibitory activity of the 13-amidohuprines was dependent on the diastereomeric arrangement at position 13, the amides of the a series being 2- to 12-fold more potent than their counterparts of the b series [82] . This stereochemical bias could be explained on the basis of a steric clash between the huprine amido group and the Trp84 side chain in the amides of the b series, that would preclude the correct positioning of the inhibitor within the active site. In support to this hypothesis, huprine 40b bearing the smaller amino group at position 13 is almost equipotent to its counterpart of the a series. As previously observed with the parent huprine Y, the inhibitory activity of the most potent 13-amidohuprines as bovine AChE inhibitors, namely 13-methanesulfonamide 37a and 13-formamide 38a , was increased 2.9- and 1.6-fold, respectively, when human AChE was used. However, the main observation arising from these pharmacological studies was that none of these 13-amidohuprines was able to even equal the inhibitory potency of the parent huprine Y [82] .

Molecular modeling studies by Luque with 13-amidohuprines 37a and 38a and the Phe330 → Tyr mutant AChE, that is, taking into account the sole mutation between Tc AChE and the human enzyme at the

active site, evidenced that the two novel huprines were able both to trigger the flip of the Gly117–Gly118 peptide bond and to establish the additional interactions with Tyr130 and Glu199, while keeping all of the interactions due to the huprine Y moiety [82] . Thus, the molecular hybridization from huprine Y and ( − )-huperzine A seemed to lead to the full occupancy of the binding sites separately recognized by the models ( − )-huperzine A and tacrine. A sizable deforma-tion cost associated with the 13-amidohuprine-induced flip of the Gly117–Gly118 peptide bond has been suggested to be responsible for the eventually observed reduced potency of 37a and 38a relative to the parent huprine Y. The oxyanion hole formed by the backbone N–H groups of Gly118, Gly119 and Ser201 plays a critical role in stabilizing the structure of the transition state during the hydrolysis of acetylcholine [83] . Thus, the deformation cost associated with the Gly117–Gly118 peptide flip could be regarded as a mechanism selected by evolution to ensure the preorganization of the oxyanion hole in the active site of AChE.

4.2 Huprine–tacrine heterodimers The utmost A β proaggregating effect of AChE found by Inestrosa and the disease-modifying potential expected for those drugs targeting the enzyme peripheral site have been driving the design of the novel AChE inhibitors in the last years. The development of the concept of the dual site binding by Pang, Carlier and Han has paved the way for the design of novel classes of inhibitors able to simultaneously block the active and peripheral sites of AChE, thus resulting in both a highly increased affinity and A β antiaggregating effects [84-92] . In this context, as a further attempt to bring about the lead optimization in the family of huprines, a unit of the lead huprine Y was combined with a unit of tacrine or 6-chlorotacrine, known to be able to interact with both the active and the peripheral site of AChE [33-35,85-88,93,94] . Both units were connected through a linker of suitable length and nature to allow the resulting heterodimer to span the entire active site gorge simultaneously interacting with both the active and peripheral sites of AChE, or even with the aromatic residues lining the gorge as an extra recognition site.

Taking advantage of the methodology developed for the synthesis of bis -tacrines and the SAR data of this structural family with regards to the length of the linker [33-35] , a short series of huprine–tacrine heterodimers ( 41 – 48 , Figure 5 ) was synthesized, through a two-step synthetic sequence from racemic huprine Y and tacrine or 6-chlorotacrine [95] .

As expected, the combination of the extended binding of huprine Y in the AChE active site with the simultaneous binding to the peripheral site resulted in an increased affinity, the huprines–tacrine heterodimers 41 – 48 , in racemic form, being up to 394- and 224-fold more potent than tacrine and ( − )-huperzine A, respectively and, more interestingly, up to 13-fold more potent than the racemic lead huprine Y, with respect to bovine AChE inhibition [95] . Although no important differences in potency were observed

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among the heterodimers, the presence of an unsubstituted tacrine unit and a tether length equivalent to six or seven methylene groups seemed to lead to the optimal bovine AChE inhibitory activity.

As in the lead huprines and 13-amidohuprines, the inhibitory activity of huprines–tacrine heterodimers is somewhat higher for human than for bovine AChE (up to 6-fold in this case). All the heterodimers exhibited very close IC 50 values for human AChE inhibition, in the 0.29 – 0.50 nM range, which is up to 707- and 897-fold more potent than tacrine and ( − )-huperzine A and up to 3-fold more potent than huprine Y.

Besides AChE, butyrylcholinesterase (BChE) can also metabolize acetylcholine in the CNS. BChE activity progressively increases as AD progresses, while the AChE activity decreases [96,97] . Thus, the inhibition of BChE is also considered of interest for the treatment of AD. Indeed, it has been suggested that the dual inhibition of AChE and BChE could increase the efficacy of the treatment and broaden the indications [97] . In general, huprines and 13-amidohuprines are poor inhibitors of human BChE, with a few exceptions, particularly the lead huprine X (IC 50 = 16 nM). Conversely, all the huprines–tacrine heterodimers turned out to be potent inhibitors of human

Figure 5 . Structures of the different 13-amidohuprines and huprine–tacrine heterodimers . The IC 50 values toward bovine AChE are indicated after the compound numbers. AChE: Acetylcholinesterase.

R = SO2Me, 37a, 118 nMR = COH, 38a, 161 nMR = COMe, 39a, 1687 nMR = H, 40a, 389 nM

R = SO2Me, 37b, 1370 nMR = COH, 38b, 741 nMR = COMe, 39b, 3585 nMR = H, 40b, 482 nM

R = H, Y = bond, 41, 0.40 nMR = H, Y = (CH2), 42, 0.48 nMR = H, Y = (CH2)2, 43, 1.26 nMR = H, Y = N−Me, 44, 0.33 nMR = Cl, Y = bond, 45, 1.06 nMR = Cl, Y = (CH2), 46, 1.97 nMR = Cl, Y = (CH2)2, 47, 2.09 nMR = Cl, Y = N−Me, 48, 0.54 nM

N

NHCl

Y

NH

N R

N

NH2

Cl

H

H

R

N

NH2

Cl

NH

H

R

N

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BChE with IC 50 values in the 5 – 31 nM range [95] , that is, up to 9-fold more potent than tacrine (IC 50 = 44 nM), > 2110-fold more potent than ( − )-huperzine A (IC 50 > 10000 nM) and up to 50-fold more potent than huprine Y (IC 50 = 236 nM). The presence of an unsubstituted tacrine unit and an hepta- or octamethylene linker were the structural features leading to an optimal potency.

The increase in AChE inhibitory activity of huprine–tacrine heterodimers relative to the parent compounds from which they were designed seems to confirm their dual binding site character. Although an interaction of the huprine unit of these heterodimers at the AChE active site and an interaction of the tacrine unit at the peripheral site are expected when the huprine unit is that with the (7 S ,11 S )-configuration (eutomer of huprines), the contrary mode of interaction can not be ruled out when the enantiomeric unit of the huprine and a 6-chlorotacrine unit are present. Molecular modeling studies as well as the separate synthesis and pharmacological evaluation of enantiopure huprine–tacrine heterodimers are presently pursued to assess their binding mode and to try to increase the inhibitory potency of the racemic compounds. The pharmacological profile of this novel class of dual binding site AChE inhibitors will be completed with the determination of their effects on AChE-induced A β and PrP 106-126 aggregation and on acetylcholine receptors. Preliminary results have shown a very high PrP 106-126 antiaggregating effects and a highly potent agonistic action on muscarinic M 1 receptors for some of the huprine–tacrine heterodimers that, as huprines, seem to be able to cross the blood–brain barrier (unpublished results).

5. Conclusion

Three different classes of AChE inhibitors have been developed by the progressive incorporation of structural motifs formally derived from ( − )-huperzine A and tacrine so as to maximize the interactions with the target, thereby increasing the affinity and inhibitory potency.

Two of these structural families, namely huprines and 13-amidohuprines, were designed to simultaneously span as much as possible the binding sites, which are separately occupied by the models ( − )-huperzine A and tacrine. Huprines, which combine the 4-aminoquinoline moiety of tacrine and the carbobicyclic bridged system of ( − )-huperzine A, are so far the most developed family among these three classes. During the hit-to-lead optimization in this family, up to 40 racemic and 14 enantiopure different huprines were synthesized, in most cases in a straightforward manner. Two huprines, namely huprines 25 and 31 (huprines Y and X) were selected as leads. These compounds span the whole binding site of the model tacrine as well as the pocket where the C7–C11 unsaturated three-carbon bridge of ( − )-huperzine A is placed. This extended binding within the active site accounts, in part, for the increased potency of the lead huprines relative to the parent compounds from which they were designed.

However, a larger increase in AChE inhibitory potency seems to be due to the occupancy of an additional hydro-phobic pocket by the chlorine atom at position 3 of the lead huprines, not occupied by the models. Huprines X and Y exhibit a very interesting pharmacological profile, which encompasses a highly potent human AChE inhibitory activity, an ability to cross the blood–brain barrier, a moderate agonistic activity on muscarinic M 1 receptors, significant cognitive-enhancing properties in a variety of behavioral studies in mice (unpublished results) as well as neuroprotec-tive effects derived from their NMDA receptor antagonism and in vitro inhibition of the AChE-induced A β and PrP 106-126 aggregation, thus endowing these huprines with potential to positively modify AD or prion diseases.

The knowledge of the three-dimensional structure of the complex Tc AChE-( − )-huprine X enabled the rational design of 13-amidohuprines, which incorporate an amido group formally derived from the model ( − )-huperzine A into the structure of the lead huprine Y, to further span the rest of the binding zone of ( − )-huperzine A not occupied by first-generation huprines. Although molecular dynamics simula-tions suggested the full occupancy of the binding sites of the two models by the most potent 13-amidohuprines, these second generation huprines were clearly less potent AChE inhibitors than the parent huprine Y. The introduction of the additional amido group in these compounds seems to have a net destabilizing effect on the affinity of 13-amidohuprines as a consequence of the greater deformation cost associated with the conformational flip of the Gly117–Gly118 peptide bond, necessary to accommodate the amido group, relative to the gain in binding affinity resulting from the additional interactions made by this group.

A third structural family, namely huprine–tacrine heterodimers, was rationally designed to span the active site gorge which connects the active and peripheral sites of AChE, combining the extended binding of the lead huprine Y within the AChE active site with the simultaneous inter-action of a tacrine-related unit with the peripheral site of the enzyme. This last step of molecular hybridization from ( − )-huperzine A and tacrine resulted in a clear increase in potency relative to these models and also to the parent huprine Y. Moreover, these heterodimers take on added value that they are potent inhibitors of BChE and they seem to be also potent inhibitors of AChE-induced PrP 106-126 aggregation and potent muscarinic M 1 agonists. If their expected A β antiaggregating effect is confirmed, huprine–tacrine heterodimers will constitute promising anti-Alzheimer drug candidates.

6. Expert opinion

The gain in binding affinity by increasing the molecular complexity of a given drug through the incorporation of additional structural fragments, particularly related to a second known drug, thereby increasing the number of

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contact points between the novel molecule and its biological target, is self-evident. However, more than a decade ago, when the project on huprines began, the use of such conjunctive approaches in this field was rather rare. The molecular hybridization from ( − )-huperzine A and tacrine, two known potent AChE inhibitors, has successfully resulted in two novel classes of much more potent inhibitors, namely huprines and huprine–tacrine heterodimers, with different binding modes (active-site binding and dual-site binding, respectively). Even in a third novel class of inhibitors, the 13-amidohuprines, the hybridization strategy could be argued to have been succesful, because, according to the molecular modeling studies, they are able to span the ( − )-huperzine A and tacrine binding sites that were targeted at the outset, and to increase the number of interactions with the enzyme due to their additional functionalization. Unfortunately, in this case the full occupancy of these two close and adjacent binding zones required a conformational flip of an enzyme peptide bond, whose energetic cost exceeded the gain in binding energy, thus rendering the net balance negative.

The most interesting and best studied of these structural families are huprines. The lead huprines are clearly superior in terms of potency to the presently commercialized AChE inhibitors and complement their oustanding human AChE inhibitory activity with other cholinergic and non-cholinergic actions, the latter suggesting a potential to positively modify the progression of Alzheimer’s or prion diseases. In spite of the promising pharmacological profile of the lead huprines, the lack of interest of the pharmaceutical industry has precluded their preclinical development. This disappointing fate of huprines could be due to the fact that when they were available, several AChE inhibitors were already marketed and efficiently used, while the industry’s interests had shifted from cholinergic- to amyloid-directed drug candidates. Toxicity is a very important issue in the decision-making process for the advancement of a drug candidate to the development stage. Because of the known hepatotoxicity associated to the use of tacrine, which has largely eliminated it from the market, the presence of a tacrine-like moiety in huprines could also have been considered of risk to form reactive metabolites involved in a toxicity similar to that of tacrine, thereby discouraging potential interests. However, the toxicity of tacrine is both dose-dependent and reversible after reduction of the dose or withdrawal. The much higher in vitro human AChE inhibitory potency of the lead huprines relative to tacrine (640-fold more potent) should result in a much lower dose, provided that the pharmacokinetics are similar or at least not much worse than those of tacrine. In turn, the lower dosage could avoid the toxicity problems of tacrine. The formation of toxic quinone-type metabolites could be responsible for the toxicity of tacrine. However, the lead huprines do not have a tacrine, but a 6-chlorotacrine moiety, and the presence of the chlorine atom at the

benzene ring not only blocks one of the putative metabolically oxidizable positions, but also its electron-withdrawing character makes the unsubstituted aromatic positions less prone to be oxidized.

Huprines, as the presently commercialized AChE inhibitors, exhibit neuroprotective properties that are clearly independent of their cholinergic actions. The oversimplistic notion that active-site AChE inhibitors provide only a symptomatic relief of the disease ignoring their potential to positively modify the disease progression should be modified and, consequently, they should be used as early as possible and not only to treat the very late symptoms of dementia as they are being used presently. Meanwhile, AChE inhibitors will continue to form the backbone of symptomatic relief of dementia in AD for the near term. AChE inhibitors will be still necessary, but not enough. After the hopefully imminent advent of amyloid-directed disease-modifying therapies, AChE inhibitor treatment would assuredly become combined with these therapies.

In this context, single molecules that are able to target AChE, thus providing symptomatic relief, as well as other processes involved upstream in the neurotoxic cascade of AD, such as the A β aggregation or oxidative stress, thus halting or delaying the progress of the disease, are parti-cularly attractive. Thus, dual-binding site AChE inhibitors endowed with very high AChE inhibitory potency and in vitro or even in vivo A β antiaggregating effects as well as multipotent AChE inhibitors combining structural motifs that permit the interaction with AChE and other biological targets of interest in the pathogenesis of the disease could constitute a very interesting alternative to the polypharmacy involved in the suggested combination therapies. If one decade ago the use of molecular hybridization in the field of AChE inhibitors was very little explored, it has now become an area of very active research in both the academia and the pharmaceutical industry.

Acknowledgments

We thank Prof. A Badia, Prof. V Clos, M Pera and M Ratia from the Departament de Farmacologia, de Terap è utica i de Toxicologia of the Universitat Aut ò noma de Barcelona (Spain) and Prof. V Andrisano and Dr M Bartolini from the Dipartimento di Scienze Farmaceutiche of the Universit à di Bologna (Italy) for providing unpublished pharmacological results. We also thank Dr A Bidon-Chanal from the Departament de Fisicoqu í mica of the Universitat de Barcelona (Spain) for providing the figures of the 3D X-ray structures of the complexes Tc AChE-inhibitors that appear in this paper.

Declaration of interest

The authors state no conflict of interest and have received no payment in preparation of this manuscript.

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Affi liation Diego Mu ñ oz-Torrero † & Pelayo Camps † †Author for correspondence Laboratori de Qu í mica Farmac è utica (Unitat Associada al CSIC), Facultat de Farm à cia, and Institut de Biomedicina de la Universitat de Barcelona (IBUB), Universitat de Barcelona, Av. Diagonal, 643, E-08028-Barcelona, Spain †Tel: +34 934024533 ; Fax: +34 934035941 ; E-mail: [email protected] †Tel: +34 934024536 ; Fax: +34 934035941 ; E-mail: [email protected]

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Huprines for Alzheimer's disease drug development

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