Design, synthesis, biological evaluation and molecular modelling of substituted pyrrolo[2,1-: A] isoquinolinone derivatives: Discovery of potent inhibitors of AChE and BChE

NJC

View Article Online

View Journal | View Issue

PAPER

Design, synthesis, biological evaluation and

molecular modelling of substituted pyrrolo[2,1-a]

isoquinolinone derivatives: discovery of potent

inhibitors of AChE and BChE†

Cite this: New J. Chem., 2021,

45, 8321

a

b

c

Oscar Parravicini,

Francisco Garibotto,

Diego Cortes

Emilio Angelina,

Roque Spinelli,

a

c

d

de

´

Alvaro S. Siano,

and Ricardo D. Enriz

Laura Vila,

Nuria Cabedo,

e

a

*

We report here the design, synthesis and biological evaluation of a new series of substituted pyrrolo[2,1-a]

isoquinolin-3-one derivatives, some of which have strong inhibitory activity against both AChE and BChE

enzymes. The design of these new inhibitors was carried out taking rivastigmine as the starting structure.

Thus, on the basis of an exhausting molecular modeling study using combined techniques (docking,

dynamic molecular simulations and QTAIM calculations), we obtained new ligands possessing stronger

inhibitory eﬀects than rivastigmine, the reference compound. QTAIM analysis gave us detailed information

about the molecular interactions stabilizing the diﬀerent ligand–enzyme complexes. These calculations

showed the importance of the interaction with the CAS esteratic site for the inhibitory effect of these

compounds. Nevertheless, they also indicated that the combination of interactions with CAS and strong

interactions with the PAS site might be beneficial for the inhibitory effect.

Received 21st January 2021,

Accepted 25th March 2021

DOI: 10.1039/d1nj00345c

rsc.li/njc

few patients having received a formal diagnosis of AD. Further-

more, despite all research eﬀorts, therapeutic options for AD are

1. Introduction

Currently, Alzheimer’s disease (AD) is without doubt a very very limited. At this time, the only treatments available with

serious problem for our society, but it is even more complex for clinical evidence to AD patients are the cholinesterase inhibitors

the near future. About fifty million people are currently living (ChEI) donepezil, rivastigmine and galantamine for mild to

with Alzheimer’s disease (AD).¹According to the latest Center moderate AD, and memantine (an NMDA receptor antagonist)

for Disease Control and Prevention (CDC) statistics, while which has been approved for moderate to severe AD. All four

other diseases have decreased like, for example, cardiovascular compounds have been rigorously tested in clinical trials and

disorders, those involving AD complications have increased. have been proved to improve patients’ reported outcomes, that

In addition, not just the patients are involved in this particular is, cognition, memory, communication and the ability to per-

and diﬃcult disease. The burden experienced by caregivers is form daily activities. However, it is important to remark that the

also substantial, aﬀecting their physical and mental wellbeing.^2,3above drugs provide symptomatic treatment for the disease,

Despite its societal impact, AD is underdiagnosed, with relatively without strong evidence for disease-modifying properties.⁴In

some countries, tacrine is also available for AD treatment.

a

However, it is a substance with a questionable safety profile

and, in fact, it is not involved in general AD therapy.

´

Facultad de Quımica, Bioquımica y Farmacia, Universidad Nacional de San Luis,

´

Instituto Multidisciplinario de Investigaciones Biologicas (IMIBIO-SL), Ejercito de

los Andes 950, 5700 San Luis, Argentina. E-mail: denriz@unsl.edu.ar

The principal role of acetylcholinesterase (AChE) is the

termination of impulse transmission at cholinergic synapses

by the rapid hydrolysis of acetylcholine (ACh);⁵and one of the

earliest and major neurobiological findings in AD is a deficit in

cholinergic neurotransmission in the basal forebrain. Thus, the

reduction of cortical and CSF cholinergic markers, such as

AChE, choline acetyl transferase and ACh itself, is correlated

with both the extent of the neuropathology and the severity of

the cognitive impairment in Alzheimer’s disease and other

degenerative diseases.^6,7Inhibitors of AChE are the principal

^bLaboratorio de Estructura Molecular y Propiedades, IQUIBA-NEA, Universidad

Nacional del Nordeste, CONICET, FaCENA, Av. Libertad 5470, Corrientes 3400,

Argentina

c

´

Laboratorio de Peptidos Bioactivos, Departamento de Quımica Organica, Facultad

´

de Bioquımica y Ciencias Biologicas, Universidad Nacional del Litoral, Santa Fe,

Argentina

d

e

´

Instituto de Investigacion Sanitaria INCLIVA, 46010 Valencia, Spain

´

Departamento de Farmacologıa, Facultad de Farmacia, Universidad de Valencia,

46100 Burjassot, Valencia, Spain

† Electronic supplementary information (ESI) available. See DOI: 10.1039/

d1nj00345c

This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021

New J. Chem., 2021, 45, 8321–8334 | 8321

View Article Online

Paper

NJC

On the other hand, we have recently reported new AChE

inhibitors, and in such articles we have conducted molecular

modelling studies that have allowed us to understand in

some detail the molecular interactions that are involved in

the stabilization of different inhibitor–enzyme complexes

for this molecular target.^9–13In particular, for N-benzyl-2-

phenylethanamines and carbamate-type ligands we have

described the importance of interactions with residues W84,

Y121, G118, S122, E199, S200, W279, F330, Y334 and H440 for

the stabilization of cholinesterase complexes. Moreover, we

carried out a comparative study of AChE and BChE, based on

the accessible area within their active sites, which showed that

the active site of BChE is larger and more flexible than AChE.

Taking advantage of having this information, we asked ourselves

if we would be able to design a new structure with inhibitory

effects on AChE, taking as a starting structure those previously

reported in ref. 8. Thus, we report here the synthesis and

biological evaluation of a new series of substituted pyrrolo[2,1-

a]isoquinolin-3-one derivatives, some of them with strong inhi-

bitory activity against both AChE and BChE enzymes.

Fig. 1 Structural features of substituted pyrrolo[2,1-a]isoquinolin-3-one

derivatives and the FDA-approved cholinesterase inhibitor rivastigmine.

therapeutical option for AD; in fact, donepezil, rivastigmine

and galantamine are the three inhibitors that are FDA-approved

for first-line treatment for AD. Although they share the same

mode of action, they diﬀer in terms of their pharmacologic

characteristics and route of administration, which can aﬀect

their safety and tolerability profile. Rivastigmine, available in

both oral and transdermal patch formulations, is a slowly

reversible dual inhibitor of acetyl and butyryl cholinesterase,

selective for the G1 isoform of acetylcholinesterase, without

hepatic metabolism by the CYP-450 system. Considering the

very limited number of drugs currently available for therapeutic

use in the treatment of AD, it is evident that there is a marked

need to find new structures with similar properties to rivastig-

mine that can be used therapeutically. Our study is focused

mainly on such an objective.

2. Results

2.1 Synthesis

The synthesis of compounds 1–11 has been previously reported.⁸

Four new carbamates 12–15 were synthesized by reaction

between the corresponding hydroxy-pyrroloisoquinolone and

the appropriate phenyl or ethyl isocyanate. 8-Phenylcarbamate-

1,2,3,5,6,10b-hexahydropyrrolo[2,1-a]isoquinolin-3-one (12) was

obtained from 8-hydroxy-pyrroloisoquinolinone (3), and 8-

chloro-9-phenylcarbamate-1,2,3,5,6,10b-hexahydropyrrolo[2,1-a]

isoquinolin-3-one (13) was prepared from 8-chloro-9-hydroxy-

pyrroloisoquinolone (7). Whereas, both 8,9-bis(ethylcarbamate)- (14)

Our research group has previously reported the synthesis of

8-substituted and 8,9-disubstituted pyrrolo[2,1-a]isoquinolin-3-

one derivatives (compounds 1–11, Fig. 1) whose general struc-

tures have a slight structural resemblance with rivastigmine.⁸

Scheme 1 Synthesis of 8-substituted and 8,9-disubstituted pyrrolo[2,1-a]isoquinolin-3-one derivatives. Reagents and conditions: (a) ethyl succinyl

chloride, NaOH 5%, CH₂Cl₂, rt, overnight; (b) POCl₃, CH₃CN, N₂, reflux, 4 h; (c) NaBH₄; MeOH, rt, 2 h; (d) conc. HCl–EtOH (1 : 1) for 2, reflux, 3 h, or BBr₃,

for 6 and 10, CH₂Cl₂, rt, 2 h; (e) ethyl or phenyl isocyanate, acetone, reflux, 3 h.

8322

|

New J. Chem., 2021, 45, 8321–8334

This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021

View Article Online

NJC

Paper

and 8,9-bis(phenylcarbamate)-1,2,3,5,6,10b-hexahydropyrrolo and 4 and 8 were not clear enough. In fact, at this stage of our

[2,1-a]isoquinolin-3-ones (15) were prepared from (ꢀ)-trolline study, we were not clear on the mechanism of action of the

(11) (Scheme 1).

active compounds 7 and 8. Thus, in order to better understand

the behaviour of these new inhibitors at a molecular level, in

our next step we conducted a molecular modelling study in

which we paid particular attention to the molecular interac-

tions that would stabilize the molecular complexes.

2.2 Searching for new inhibitors of AChE

Previously, the research group of the University of Valencia

reported the synthesis of compounds 1–11 (Fig. 1).⁸Some of

these compounds seemed interesting to us from a structural

point of view since they resemble rivastigmine in some way

(particularly compound 8). Therefore, our first step in this

study was to evaluate the inhibitory activity against AChE and

BChE of these eleven compounds. These results are shown in

Fig. S1–S3 (ESI†) and Table 1. As can be seen, none of the

mono-substituted derivatives (compounds 1–4) showed any

inhibitory activity. Regarding the rest of the tested compounds,

compounds 5, 6, 9–11 did not show any significant inhibitory

effect. However, compounds 7 and 8 showed a strong inhibitory

activity against both enzymes, AChE and BChE. This first result

was quite encouraging considering that compounds 7 and 8

displayed a strong inhibitory activity. It should be noted that

both compounds displayed a stronger activity against AChE

than the reference compound (rivastigmine).

2.3 Molecular modelling

To understand more deeply our experimental results, we car-

ried out a molecular modelling study. We conducted this study

in four steps using diﬀerent modelling techniques. In the first

step, we conducted a docking study; in the second one, we

performed simulations using molecular dynamics calculations.

With these data we performed a per residue analysis and in the

last step quantum mechanics calculations were made to eval-

uate in detail the molecular interactions that stabilize the

diﬀerent ligand–receptor complexes. The Torpedo californica

AChE (TcAChE) gorge is a narrow, 20 Å long pocket composed

of two binding sites for the cationic substrate acetylcholine,

termed the catalytic anionic site (CAS) and the peripheral

anionic site (PAS), lying at the base and entrance of the gorge,

respectively.

The lack of activity of compounds 2, 6 and 10 was not at all

surprising since in both cases the presence of the benzyloxy or

methoxy group in the replacement of OH has given, in general,

less active compounds when these molecules and other struc-

turally related ones have been tested against other molecular

targets.^14–16However, for this particular series of compounds,

the presence of a hydroxyl group might not be sufficient for the

ligands to develop inhibitory effects. In this regard, a co-

substitution of a Cl atom together with another polar moiety

could be crucial since 7 and 8 displayed the lowest IC₅₀values.

In the case of compounds 1, 5 and 9, a different behaviour is

expected since these compounds are linear and such great

molecular flexibility can justify their different activities. How-

ever, the dissimilar activities observed for compounds 3 and 7

Between the two sites, the gorge narrows due to a constric-

tion formed by two aromatic residues, F330 and Y121 (bottle-

neck region). Both CAS and PAS are lined mostly by aromatic

residues that interact with the enzyme substrate ACh. When

ACh or an analogue substrate like acetylthiocholine (ATCh)

binds to the PAS, it forms interactions with residues Y70, Y121,

W279, F330, F331 and Y334. A characteristic cation–p inter-

action between a quaternary amine and W279 as well as an H-

bond between the acetyl moiety and Y121 are formed (PDB

codes 2C4H, and 2C58). On the other hand, when ACh/ATCh

binds to the CAS, a reaction occurs and the acetyl group

covalently bound to catalytic serine interacts with residues

from the ‘‘esteratic subsite’’ formed by:

(a) the catalytic triad E327, H440 and S200, (b) the backbone

of residues A201, G118 and G119 that together form the

‘‘oxyanion hole’’ and (c) the aromatic residues W233, F288,

F290 that hold the methyl group in place (PDB codes 2ACE, and

2C58). At the same time, the positively charged choline moiety

interacts with CAS residues W84, Y130, E199, F330, F331, and

Y442 that form the ‘‘anionic subsite’’. As in the PAS, the choline

quaternary amine forms a cation–p interaction with tryptophan

W84. Our docking study indicates that compounds 7 and 8 are

bound at the same site to that previously reported for rivastig-

mine (Fig. 2).¹²However, some differences might be appre-

Table 1 Enzyme inhibition of substituted pyrrolo[2,1-a]isoquinolin-3-one

derivatives against acetyl- and butyryl-cholinesterase. All values were

expressed as IC₅₀ꢀ SD, with a confidence of 95%. The IC₅₀values were

determined by regression analyses of three replicate determinations

IC₅₀(mM)

Compound

AChE

BChE

SI^b

1

2

3

4

5

6

450

—

ciated between compounds

7 and 8. While the spatial

arrangement observed for compound 8 is very similar to that

previously reported for rivastigmine,¹²compound 7 is located a

little bit shifted to the bottom of the gorge (Fig. 2). In contrast,

compounds 1, 5 and 9 led to a high number of possible

conformations, which might be related to their lack of activity.

In turn, molecular dynamics calculations allowed us to

perform a per residue analysis for these compounds. As we

expected, the histogram obtained for compound 8 is closely

7

8

9

10

11

Riv^a

19.25 ꢀ 2.88

11.03 ꢀ 0.34

450

4.26 ꢀ 0.78

1.74 ꢀ 0.31

450

4.52

6.34

—

34.18 ꢀ 6.00

0.08 ꢀ 0.00

427.25

a

b

Rivastigmine (Riv) was used as a positive control. SI (selectivity

index) = IC₅₀(AChE)/IC₅₀(BChE).

This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021

New J. Chem., 2021, 45, 8321–8334 | 8323

View Article Online

Paper

NJC

Fig. 2 Spatial view of AChE–4 (cyan), AChE–7 (magenta) and AChE–8

(green) complexes overimposed for comparison.

related to that of rivastigmine (Fig. 3A and B). The main

interactions of both compounds are those with D72, W84,

G118, Y121, S200, W279, F290, F330, Y334 and H440. It should

be noted that the same interactions are found for compound 7.

Nevertheless, interactions obtained for this compound are

weaker in comparison with that observed for compound 8

(compare Fig. 3A and C). Note that compound 8 displays a

stronger inhibitory activity with respect to compound 7

(Table 1); therefore, these results are in line with our experi-

mental results. In contrast to these results, compound 4 shows

a diﬀerent behaviour from that observed for 8. At least two

aspects show clear diﬀerences between these two compounds.

One is the lack of interaction of compound 4 with S200. This

can be clearly seen in the histogram obtained for this com-

pound (Fig. 3D). It is well known that S200 plays a key role in

the inhibition of AChE.^17,18The other important diﬀerence is

that 4 adopts a completely diﬀerent spatial arrangement than 7

and 8. It should be noted that compound 4 orients the pyrrole

ring towards the bottom of the active site and the chain with

the carbamate substituent towards the surface, in exactly the

opposite form of how compound 8 is located. This can be

appreciated clearly in Fig. 2.

At this stage of our study, we focused our eﬀorts on design-

ing and obtaining new compounds structurally related to

compound 8 that possess inhibitory activity against AChE.

2.4 Design of new inhibitors of AChE and BChE

Fig. 3 Histograms of interaction energies partitioned with respect to the

AChE amino acid sequence when complexed with compound 8 (A),

rivastigmine (B), compound 7 (C) and compound 4 (D). The X-axis denotes

the residue number of AChE and the Y-axis denotes the interaction energy

between the compounds and a specific residue. Negative values and

positive values are favourable or unfavourable to binding, respectively.

From Fig. 2 it is possible to observe that there is some room to

incorporate a bulkier substituent at the carbamate group. Thus,

we design four new ligands (compounds 12–15). In compounds

12 and 13 we replace the ethyl group of 4 and 8 by a phenyl

group, respectively, whereas compounds 14 and 15 are the

diethyl and biphenyl carbamates of compound 11. Next, we

performed MD simulations for these new compounds (com-

pounds 12–15) (Fig. 4) and the results were compared with

8324

|

New J. Chem., 2021, 45, 8321–8334

This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021

View Article Online

NJC

Paper

Table 2 Enzyme inhibition of substituted pyrrolo[2,1-a]isoquinolin-3-one

derivatives against acetyl- and butyryl-cholinesterase. All values were

expressed as IC₅₀ꢀ SD, with a confidence of 95%. The IC₅₀values were

determined by regression analyses of three replicate determinations

IC₅₀(mM)

Compound

AChE

BChE

SI^b

12

13

14

450

—

0.76

—

427.25

11.21 ꢀ 1.51

14.68 ꢀ 2.00

1.84 ꢀ 0.09

450

0.08 ꢀ 0.00

450

15

450

34.18 ꢀ 6.00

Riv^a

a

b

Rivastigmine (Riv) was used as a positive control. SI (selectivity

index) = IC₅₀(AChE)/IC₅₀(BChE).

steric factors since compound 14 would be too bulky to enter

the active site. Similar results were obtained for compound 12

(see Fig. 4B and Fig. S4, ESI†). The same results but even more

noticeably were observed for compound 15 which is a logical

result considering that the steric effects in compound 15 are

stronger than those in compound 14.

From these results, we can expect that 13 displays inhibitory

eﬀects while compounds 12, 14 and 15 do not display or at least

display a weaker activity than 13. Thus, we decided to synthe-

size compounds 12–15 (see details of the synthesis in

Section 2.1).

In the next step, we evaluated the inhibitory activity on both

enzymes and these results are shown in Table 2. It might be

seen that our experimental results are in complete agreement

with the simulations, corroborating the results obtained by

molecular modelling. Note that while compound 13 is one of

the most active of this series, 12, 14 and 15 showed no activity

against AChE. A somewhat surprising result was that obtained

for compound 14 against BChE, since although it did not show

any activity on AChE, it showed a strong inhibitory activity

against BChE (Table 2). To explain these results better, we

performed molecular simulations for compounds 13 and 14

interacting at the active site of BChE. These results are shown

in Fig. 5.

It is interesting to note that the histograms obtained for

compounds 13 and 14 interacting with BChE are very similar

(Fig. 5A and B) and they are closely related with that obtained

for rivastigmine as well (Fig. 5C). These results indicate that in

the case of BChE, compound 14 is able to enter into the bottom

of the binding pocket and, therefore, it can properly interact at

the CAS. Such a situation might be better appreciated in Fig. 6,

which shows the spatial orderings adopted by 14 at the binding

sites of both enzymes (AChE and BChE). These results might

explain, at least in part, the selectivity for BChE observed in

compound 14.

Fig. 4 Histograms of interaction energies partitioned with respect to the

AChE amino acid sequence when complexed with compound 13 (A),

compound 12 (B) and compound 14 (C). The X-axis denotes the residue

number of AChE and the Y-axis denotes the interaction energy between

the compounds and a specific residue. Negative values and positive values

are favourable or unfavourable to binding, respectively.

those obtained for compounds 7 and 8. Whereas the histogram

obtained for compound 13 is closely related to that of com-

pound 8 (compare Fig. 3A and 4A), the histograms obtained for

compounds 12 and 14 (Fig. 4B and C) showed significant

differences. The histogram obtained for compound 14 shows

strong interactions, however the lack of interaction with S200,

which is a key interaction for the inhibitory activity, is very

noticeable. At the same time, those interactions with the

residues from the peripheral anionic site (W279 and Y334)

and from the bottleneck (F330) are clearly increased. This

would be a clear indication that compound 14 could not enter

into the bottom of the pocket but would remain at the surface

of the active site. This different behaviour could be explained by

Finally, considering compound 15, our exploratory docking

results showed that this ligand is too large for both active sites

(AChE and BChE). The inability of compound 15 to fully enter

the binding pocket does not allow the carbamate group to reach

the catalytic triad. This is an expected result since changing two

ethyl groups for two phenyl groups implies a considerable

increase in size.

This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021

New J. Chem., 2021, 45, 8321–8334 | 8325

View Article Online

Paper

NJC

Upon going from A to D in Fig. 7 the ligands are more deeply

buried in the gorge. Compound 14 has two bulky N-ethyl

carbamate groups attached to the tricyclic core that prevent it

from accessing the CAS through the bottleneck. When one of

the bulky carbamate groups in 14 is replaced by chlorine, as in

compound 8, entrance to the CAS through the bottleneck is

allowed. However, compound 8 does not completely enter the

CAS but it remains halfway between both gorge sites.

According to a previous study describing TcAChE substrate

traﬃcking, the substrate analog ATCh first binds to the PAS

with the acetyl group oriented towards the active site and the

quaternary amine forming a cation–p interaction with the

W279 ring. The observed orientation allows the substrate to

slide straight through the gorge without the necessity of

reorienting before productive interaction at the active site, as

the CH₃CO group needs to approach the catalytic serine. In this

sense, binding at the PAS may serve as a ‘‘filter’’ that orients the

ACh molecule before it continues its journey towards the CAS.²⁰

Analogously, in complexes of compounds 14 and 8 the carba-

mate moiety is also pointing down towards the active site while

the electropositive amide from the g-lactam ring is stacked over

the W279 ring (Fig. 7A and B).

On the other hand, the binding of compound 13 to the CAS

also closely resembles that of the substrate: the carbonyl oxygen

atom points toward the oxyanion hole (i.e. the backbone of

S200 and A201 in Fig. 7) and the activated carbon atom is

properly positioned for nucleophilic attack by S200 (PDB code

2ACE). Furthermore, the electron-deficient g-lactam ring of

compound 13 is stacked on top of W84 in the same way as

the substrate quaternary amine is also stacked over the same

ring through a cation–p interaction.

One of the most salient characteristics of these carbamate

derivatives is the overall curved shape provided by the tricyclic

core, which allows a good fit of these compounds within the

CAS. Potent carbamate inhibitor physostigmine also has a

similar curved tricyclic core and its binding mode resembles

that of compound 13 where the curved ring of the molecule is

lying on top of the W84 indole ring.²¹Therefore, agreement

between our structural models and previous structural evidence

of substrate analogue binding to both the CAS and the PAS

enable us to be confident in the reliability of our computational

predictions, at least from a qualitative point of view.

Fig. 5 Histograms of interaction energies partitioned with respect to the

BChE amino acid sequence when complexed with compound 13 (A),

compound 14 (B) and rivastigmine (C). The X-axis denotes the residue

number of BChE and the Y-axis denotes the interaction energy between

the compounds and a specific residue. Negative values and positive values

are favourable or unfavourable to binding, respectively.

2.5.1 Charge density analysis of molecular interactions.

Regarding the stability of the structural models, the stacked

bars in Fig. 8 provide a dissected view of the anchoring of

carbamate compounds to the diﬀerent sites within the

TcAChE gorge.

As indicated by the stacked bars in Fig. 8, compound 14

binds almost completely to the PAS, compounds 7 and 13 are

mostly anchored to the CAS, whereas compound 8 is in between

both sites. An unexpected result revealed by the charge density

data in Fig. 8 is that anchoring to the PAS is actually stronger

than to the CAS, as indicated by the contribution of the

PAS residues to the overall anchoring of compounds 14 and 8

(i.e. yellow stacked bars). Nevertheless, the activity of these

carbamates seems to be conditioned to their ability to reach the

2.5 Molecular interactions analysis from QTAIM calculations

In a recent article, we demonstrated that QTAIM calculations

are a very useful tool to evaluate in an accurate and detailed way

the molecular interactions that stabilize ligand–receptor com-

plexes possessing different degrees of structural complexity. In

fact, one of the systems reported in this paper is AChE.¹⁹On the

basis of those results and with the aim of better understanding

the molecular interactions involved in the different complexes

reported here, we carried out a QTAIM study for the most

representative ones. Thus, we selected compounds 7, 8, 13

and 14 complexed with AChE and compounds 13 and 14

complexed with BChE. Fig. 7 shows the binding mode of the

compounds along the AChE enzyme gorge.

8326

|

New J. Chem., 2021, 45, 8321–8334

This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021

View Article Online

NJC

Paper

Fig. 6 Active sites of Torpedo californica AChE (A) and Homo sapiens BChE (B) when complexed with compound 14, which is represented in ball-and-

stick format and coloured in blue. The gorge of each enzyme is depicted by its molecular surface in semi-transparent grey. The main amino acid residues

from both active sites are also shown. Residues from the CAS including the catalytic triad and oxyanionic subsite are depicted in magenta. Amino acids

from the acyl-binding pocket and the anionic subsite are represented in orange and yellow, respectively. Residues from the PAS and the bottleneck

region are depicted cyan and green, respectively.

Known AChE inhibitors like potent donepezil and galantamine

also target both the PAS and CAS simultaneously (PDB entries

4EY7, 1EVE and 1W6R, 4EY6, respectively).

Charge density molecular graphs in Fig. 9 and 10 show in

more detail the molecular interactions that contribute to the

anchoring of compounds 13 and 8, respectively.

Compound 13 mainly interacts with residues from the CAS

at the gorge base and the carbamoyl group is well positioned

within the esteratic subsite for nucleophilic attack by the

reactive serine residue. Fig. 9 shows that the carbamoyl oxygen

atom is hydrogen bonded to the oxyanion hole as evidenced by

the bond paths connecting this atom to backbone H atoms

from A201, G117 and G119, thus enhancing the electrophilicity

of the carbamoyl carbon atom so that it might then be

eventually attacked by the reactive S200 oxygen atom.

Also, several C–Hꢁ ꢁ ꢁp interactions between the N-phenyl

group and ring atoms from aromatic residues W233, F288

and F290 contribute to binding of the carbamoyl moiety at

the esteratic subsite. Moreover, the terminal g-lactam ring from

the tricyclic core is stacked over W84, forming several C–Hꢁ ꢁ ꢁp

interactions between them that together make an important

contribution to the anchoring of compound 8 (see Fig. 7).

On the other hand, as compound 8 has not fully accessed

the CAS the carbamoyl group is too far from the reactive S200

and it is not properly oriented within the oxyanion hole

(Fig. 10). Misplacement of the carbamoyl group is reflected in

the overall weaker binding to the esteratic subsite compared

with compound 13 (see Fig. 7). However, interactions of the

tricyclic core structure with aromatic residues from the PAS

compensate for the anchoring loss from the CAS and so the

Fig. 7 Binding modes of compounds 14 (A), 8 (B), 7 (C) and 13 (D) within

the TcAChE gorge. Residues from the CAS-esteratic, CAS-anionic and PAS

are depicted in blue, red and yellow, respectively. In order to reach the CAS

at the bottom of the gorge, the ligand must slide through the bottleneck

formed by side-chains of residues Y121 and F330.

CAS regardless of its anchoring strength and, so, compound 14

is inactive since it is not able to enter the CAS. Exclusive

binding to the PAS is only observed when high concentrations

of the substrate analogue are present in the crystallization

solution, suggesting that the PAS is occupied only

transiently,²⁰which is also reflected in the very high IC₅₀value

reported for the binding of compound 14 at the TcAChE gorge.

On the other hand, compound 8 seems to benefit from

anchoring at both sites simultaneously. It is likely that com-

pound 8 was captured in a transient intermediate state along

the way to the enzyme active site at the bottom of the gorge.

Unlike compound 14, there is no reason why 8 could not reach

the gorge base since it is even less bulky than compound 13

(Fig. 7). However, because of the stronger interactions with the

PAS residues, anchoring at this intermediate state (i.e. in

between both sites) is stronger than binding at the bottom of

the gorge (compare the stacked bars for 8 and 13 in Fig. 8).

This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021

New J. Chem., 2021, 45, 8321–8334 | 8327

View Article Online

Paper

NJC

Fig. 8 Sum of charge density values (in atomic units) at the intermolecular bond critical points between TcAChE and the selected compounds. The total

height of stacked bars indicates the overall anchoring strength of the compounds within the gorge which can be decomposed into anchoring to the CAS

esteratic subsite (CAS-esteratic), CAS anionic subsite (CAS-anionic) and PAS site.

Fig. 9 Charge density molecular graph of compound 13 (magenta) at the gorge of TcAChE. Residues from the CAS-esteratic, CAS-anionic and PAS are

depicted in blue, red and yellow, respectively. Topological elements of the charge density associated with the intermolecular interactions are depicted

with yellow lines (Bond Paths, BPs) and small red spheres (Bond Critical Points, BCPs).

overall binding of compound 8 ends up being stronger than for Fig. 11 shows in more detail the interactions of compound 14 at

compound 13.

the BChE gorge.

Moreover, although the overall structures of BChE and AChE

As can be seen in Fig. 11, compound 14 is certainly anchored

are similar, their interactions with the same ligands are gen- at the bottom of the gorge between aromatic residues W82 and

erally diﬀerent due to slight diﬀerences in their residue W231 that make up the walls of the CAS at both ends of its

sequences. Bottleneck aromatic residues F330 and Y121 that longer (longitudinal) axis. However, unlike rivastigmine that

separate the PAS from the CAS in AChE are replaced in BChE by bind its carbamate moiety to the esteratic subsite, our model-

the less bulky A328 and Q119, respectively. As a result, there is ling results suggest that compound 14 binds in the opposite

no longer such a bottleneck between the PAS and CAS in BChE, way, namely with the carbamate moiety placed at the CAS-

such that ligands that could not access the CAS in AChE due to anionic subsite. The fact the compound 14 can bind to the CAS

their size might now be able to do so in BChE. This seems to be of BChE, albeit not in the expected orientation, correlates well

the case for bulky compound 14, as indicated by the interaction with the inhibition data, namely that it is able to inhibit BChE

profile in Fig. 5B which resembles that of rivastigmine (Fig. 5C). but with much less potency than rivastigmine.

8328

|

New J. Chem., 2021, 45, 8321–8334

This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021

View Article Online

NJC

Paper

with W279. In this arrangement, Y121 partially covers the CAS

entrance as can be seen in Fig. 11. In BChE, both residues W279

and Y121 are replaced by A277 and Q119 respectively, so that

they are no longer able to interact with each other and conse-

quently Q119 is displaced away from the CAS entrance. Because

of these substitutions (as well as the substitution of F330 by

A328), larger ligands can access the bottom of the gorge in

HsBChE, as compared with TcAChE.

Besides the role of the specialized structure formed by

residues W279 and Y121 as a ligand size-based filter, these

residues at the PAS of TcAChE are also involved in ligand pre-

orientation by accommodating the hydrolysable tail (i.e. the

carbamate moiety) in such a way that it can slide straight

through the CAS-esteratic subsite, as already discussed pre-

viously. Thus, the lack of a pre-orientational filter at the PAS of

BChE might be allowing the inverted binding mode of com-

pound 14 at the CAS of the enzyme.

Fig. 10 Charge density molecular graph of compound 8 (magenta) at the

gorge of TcAChE. Residues from the CAS-esteratic, CAS-anionic and PAS

are depicted in blue, red and yellow, respectively. Topological elements of

the charge density associated with the intermolecular interactions are

depicted with yellow lines (Bond Paths, BPs) and small red spheres (Bond

Critical Points, BCPs).

Fig. 12 shows the interactions of compound 13 as it binds to

the HsBChE gorge. The binding mode of compound 13 to

TcAChE is also shown in grey for comparison. As can be seen

in Fig. 12, compound 13 is anchored at the CAS of both TcAChE

and HsBChE; however, it is able to reach the bottom of the

gorge base only in the first case. This diﬀerence is likely due to

the substitution of residue V400 in TcAChE (in grey in Fig. 12)

To understand the origin of the binding mode diﬀerences of by the bulkier F398 in HsBChE, which prevents the N-phenyl

the compounds to BChE and AChE, Fig. 11 and 12 shows key carbamate moiety of compound 13 from fully accessing the

active site residues from TcAChE that are mutated in HsBChE. CAS-esteratic subsite. As a consequence, the hydrolysable car-

In particular, Fig. 11 shows residues W279 and Y121 from the bamate moiety of compound 13 is not properly placed for

TcAChE structure (in grey) that have been previously super- nucleophilic attack by the reactive serine in HsBChE, as it does

posed with the HsBChE structure. These residues form a in TcAChE. These modelling results are also in line with the

specialized structure at the PAS of TcAChE in which Y121 is inhibition data, which show that compound 13 is a slightly

held firmly on top of the CAS by T-shaped C–Hꢁ ꢁ ꢁp interactions more potent inhibitor of TcAChE than HsBChE.

Fig. 11 Charge density molecular graph of compound 14 (magenta) at the gorge of HsBChE. Residues from the CAS-esteratic, CAS-anionic and PAS are

depicted in blue, red and yellow, respectively. Residues in grey are not from BChE structure but from a superimposed TcAChE structure. Topological

elements of the charge density associated with the intermolecular interactions are depicted with yellow lines (Bond Paths, BPs) and small red spheres

(Bond Critical Points, BCPs).

This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021

New J. Chem., 2021, 45, 8321–8334 | 8329

View Article Online

Paper

NJC

Fig. 12 Charge density molecular graph of compound 13 (magenta) at the gorge of HsBChE. Residues from the CAS-esteratic, CAS-anionic and PAS are

depicted in blue, red and yellow, respectively. The binding mode of compound 13 to TcAChE as well as residue V400 from TcAChE have been

superimposed for comparison (in grey). Topological elements of the charge density associated with the intermolecular interactions are depicted with

yellow lines (Bond Paths, BPs) and small red spheres (Bond Critical Points, BCPs).

Although the results obtained from molecular modelling were purified by silica gel 60 (40–63 mm, Merck) column

simulations represent additional support for the possible eﬀect chromatography. The quoted yields are of the purified material.

on AChE and BChE, further studies, as for example a kinetic

3.2 Synthesis

analysis, could give a better and more complete understanding

about the inhibitory eﬀect displayed by these compounds.

3.2.1 Synthesis of 8,9-disubstituted pyrrolo[2,1-a]isoquino-

linone derivatives. The general synthetic path for compounds

1–11 has been previously reported. 8-Substituted (12) and 8,9-

disubstituted (13–15) pyrrolo[2,1-a]isoquinolinone carbamate

derivatives were prepared according to ref. 8.

3. Methodology

3.1 General experimental procedures and instrumentation

3.2.2 General procedure for the synthesis of carbamate

derivatives (8, 12–15)

All solvents and reagents were purchased from Scharlab S.L.

(Barcelona, Spain) and Sigma-Aldrich (St Louis, MO) commer-

cial sources. Solvents were dried and freshly distilled under a

nitrogen atmosphere if necessary. ¹H and ¹³C NMR spectra

were recorded using CDCl₃as the reference solvent with two

drops of CD₃OD if necessary in a Bruker AC-300 or AC-500

spectrometer (Bruker Instruments, Kennewick, WA). Chemical

shifts (d) are reported in ppm relative to an internal deuterated

solvent reference. Multiplicities of the ¹³C NMR resonances

were assigned by DEPT experiments. The assignments of all

compounds were made by COSY, DEPT, HSQC and HMBC. All

reactions were monitored by analytical thin-layer chromatogra-

phy (TLC), carried out on silica gel 60 F254 plates (Merck

Group, Darmstadt, Germany), and visualized by UV irradiation

at 254 nm. ESIMS analyses were performed using UHPLC

apparatus (Shimadzu, LCMS-8040) coupled to a tandem mass

8-Hydroxy-1,2,3,5,6,10b-hexahydropyrrolo[2,1-a]isoquinolin-3-

one (3). 8-Chloro-9-hydroxy-1,2,3,5,6,10b-hexahydropyrrolo

[2,1-a]isoquinolin-3-one (7) or 8,9-dihydroxy-1,2,3,5,6,10b-

hexahydropyrrolo[2,1-a]isoquinolin-3-one (11) (1 equiv.) was

dissolved in dry acetone (10 mL). Subsequently, the appropriate

ethyl or phenyl isocyanate (1.5 equiv. or 3 equiv. for 12, 13 or 14,

15 respectively) was added. The reaction mixture was heated

under reflux for 3 h and then concentrated to dryness. The

residue was redissolved in 10 mL of CH₂Cl₂and washed with

H₂O (3 ꢂ 10 mL). The organic layer was dried with anhydrous

Na₂SO₄, filtered and evaporated under reduced pressure. The

crude product was purified by column chromatography on

silica gel (CH₂Cl₂/MeOH 97 : 3).

8-Phenylcarbamate-1,2,3,5,6,10b-hexahydropyrrolo[2,1-a]iso-

spectrometry (MS/MS) triple quadrupole instrument equipped quinoline-3-one (12). Colourless oil (13 mg, 27% yield). ¹H NMR

with an electrospray ionization (ESI) ion source (Shimadzu, (300 MHz, CDCl₃): d = 7.44 (2H, d, J = 8.0 Hz, H-2⁰, H-6⁰),

Kyoto, Japan). High-resolution electron ionization mass spec- 7.34 (1H, t, J = 7.4 Hz, H-4⁰), 7.16–7.08 (2H, m, H-3⁰, H-5⁰), 6.97

trometry (HREIMS) was determined by a TripleTOF 5600 LC/ (1H, d, J = 8.4 Hz, H-10), 6.75 (1H, dd, J = 2.6, 8.4 Hz, H-9),

MS/MS system (AB SCIEX, Framingham, MA, U.S.A.). Residues 6.63 (1H, d, J = 2.6 Hz, H-7), 6.20 (1H, brs, NH), 4.78–4.69

8330

|

New J. Chem., 2021, 45, 8321–8334

This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021

View Article Online

NJC

Paper

(1H, m, H-10b), 4.32–4.21 (1H, m, Ha-5), 3.11–2.47 (6H, m, Hb- 0.25 U mL^ꢃ1of EeAChE (E.C. 3.1.1.7, type VI-S from the

5, H-6, Ha-1, H-2), 1.92–1.80 (1H, m, Hb-1); ¹³C NMR (75 MHz, electric eel, Sigma-Aldrich), 0.24 mM of acetylthiocholine

CDCl₃): d = 173.1 (CO-3), 154.8 (NHCO), 149.1 (C-8), 137.2 (C-1⁰), (ATCh), 0.2 mM of DTNB and the corresponding compound

135.1 (C-6a), 129.4 (C-10a), 129.2 (CH-4⁰), 126.0 (CH-10), 125.9 (50–3.125 mM). The reaction started after the addition of

(CH-3⁰, CH-5⁰), 119.0 (CH-2⁰, CH-6⁰), 115.3 (CH-7), 114.3 (CH-9), the substrate to a 15 min pre-incubated solution of AChE with

56.5 (CH-10b), 37.0 (CH2-5), 31.8 (CH2-2), 28.6 (CH2-6), 27.7 the compound. Absorbance at 405 nm was measured after

(CH2-1); HREIMS: m/z 322.1374 [M]⁺(calcd for C₁₉H₁₈N₂O₃, 5 min using a 96-well microplate reader (Thermo Fisher

322.1310).

FC Multiskan). The AChE inhibition percentage (I%) was cal-

culated with eqn (1). Each compound was tested in triplicate.

The AChE residual activity percentage (equal to 100 ꢃ I%)

values were used to calculate the required concentration to

inhibit 50% of enzyme activity (IC₅₀) with a four-parameter

logistic curve.

8-Chloro-9-phenylcarbamate-1,2,3,5,6,10b-hexahydropyrrolo[2,1-a]

isoquinoline-3-one (13). Colourless oil (7.5 mg, 25% yield). ¹H NMR

(500 MHz, CDCl₃): d = 7.46 (2H, d, J = 8.0 Hz, H-2⁰, H-6⁰), 7.35 (1H,

t, J = 7.4 Hz, H-4⁰), 7.26 (1H, s, H-7), 7.17–7.12 (2H, m, H-3⁰, H-5⁰),

7.03 (1H, s, H-10), 4.75 (1H, t, J = 7.8 Hz, H-10b), 4.30 (1H, ddd,

J = 2.6, 6.2, 12.9 Hz, Ha-5), 3.08–3.01 (1H, m, Hb-5), 2.95–2.87

(1H, m, Ha-6), 2.80–2.75 (1H, m, Hb-6), 2.67–2.62 (1H, m, Ha-1),

2.61–2.55 (1H, m, Ha-2), 2.51–2.45 (1H, m, Hb-2), 1.90–1.86 (1H, m,

Hb-1); ¹³C NMR (125 MHz, CDCl₃): d = 173.1 (CO-3), 150.5 (NHCO),

145.3 (C-9), 137.3 (C-10a), 137.0 (C-1⁰), 132.8 (C-6a), 130.6 (CH-7),

129.2 (CH-4⁰), 125.7 (CH-3⁰, CH-5⁰), 124.2 (C-8), 120.4 (CH-10),

118.7 (CH-2⁰, CH-6⁰), 56.3 (CH-10b), 36.7 (CH2-5), 31.6 (CH2-2),

27.9 (CH2-6), 27.2 (CH2-1); HREIMS: m/z 357.1011 [M]⁺(calcd for

C₁₉H₁₇ClN₂O₃, 357.1000).

% I = (AI ꢃ Ab) ꢂ 100/(A0 ꢃ Ab)

(1)

A0: control (no inhibitor); Ab: blank (no enzyme, no inhibitor);

AI: sample.

The same procedure was applied for the BChE inhibitory

assay, with modifications in the enzyme and substrate used,

which were BChE from human serum and S-butyrylthiocholine

iodide (BTCI; 7 mM), respectively.

In both cases, rivastigmine was used as a positive control.

3.4 Molecular modelling

8,9-Bis(ethylcarbamate)-1,2,3,5,6,10b-hexahydropyrrolo[2,1-a]

isoquinoline-3-one (14). Colourless oil (12 mg, 36% yield).

¹H NMR (300 MHz, CDCl₃+ 2 drops CD₃OD): d = 6.97 (1H, s,

H-10), 6.95 (1H, s, H-7), 5.21–5.17 (2H, m, NH), 4.71 (1H, t, J =

6.7 Hz, H-10b), 4.25 (1H, ddd, J = 12.7, 6.0, 2.8 Hz, Ha-5), 3.34–

3.24 (4H, m, CH2NH), 3.03 (1H, td, J = 1.0, 4.2 Hz, Hb-5), 2.95–

2.80 (1H, m, Ha-6), 2.78–2.67 (1H, m, Hb-6), 2.65–2.40 (3H, m,

Ha-1, H-2), 1.95–1.80 (1H, m, Hb-1), 1.19 (3H, t, J = 7.0 Hz,

CH₃CH₂NH); ¹³C NMR (75 MHz, CDCl₃+ 2 drops CD₃OD): d =

173.7 (CO-3), 157.8 (NHCO), 141.7 (C-9), 141.5 (C-8), 135.1

(C-10a), 131.5 (C-6a), 123.6 (CH-10), 119.7 (CH-7), 56.5 (CH-

10b), 36.8 (CH2-5), 35.9 (CH2NH), 31.5 (CH2-2), 27.8 (CH2-6),

27.0 (CH2-1), 14.7 (CH₃CH₂NH); HREIMS: m/z 362.1714 [M]⁺

(calcd for C₁₈H₂₃N₃O5, 362.1710).

Calculations were performed after synthesizing previously

reported compounds 1–11. Conversely, all simulations regard-

ing new derivatives 12–15 were conducted before their

synthesis.

3.4.1 Molecular docking. The following X-ray structures

available in the Protein Data Bank (http://www.rcsb.org) were

used for molecular modelling studies: Torpedo californica acetyl-

cholinesterase (TcAChE; PDB code: 1DX6)²⁴and Homo sapiens

butyrylcholinesterase (HsBChE; PDB code: 6EUL).²⁵Water and

ligand molecules were removed from both PDB structures

before calculations. Compounds 1–15 and receptor structures

were converted from PDB to PDBQT format using AutoDock-

Tools 1.5.4.²⁶Gasteiger charges were added for all the com-

pounds whereas non-polar hydrogen atoms were merged.

The docking simulations were performed using AutoDock 4.2

software.²⁶In all simulations, the receptor structure was set as

rigid while all the torsions of the ligand were allowed to rotate

during docking. 3D affinity maps were defined as a cubic box

centred at the active site of each molecular target. Grid dimen-

sions (XYZ) were 60 ꢂ 60 ꢂ 60 points separated by 0.375 Å. The

maximum number of energy evaluations was set to 2.5 ꢂ 10⁶,

and the maximum number of generations was 2.7 ꢂ 10⁴

whereas 200 poses were collected. Other parameters were set

to default values. The collected conformations were clustered

into different families, taking into account the root-mean

square deviation (RMSD) of the backbone, and were then

ranked according to the binding free energy. The structure

from the most populated cluster with the lower relative free

energy was chosen for further calculations.

8,9-Bis(phenylcarbamate)-1,2,3,5,6,10b-hexahydropyrrolo[2,1-a]

isoquinoline-3-one (15). Colourless oil (15 mg, 41% yield).

¹H NMR (300 MHz, CDCl₃): d = 7.41–7.03 (10H, m, 2xH-2⁰,

2xH-3⁰, 2xH-4⁰, 2xH-5⁰, 2xH-6⁰), 7.12–7.06 (1H, s, H-10),

7.03 (1H, s, H-7), 4.79–4.66 (1H, m, H-10b), 4.27–4.15 (1H, m,

Ha-5), 3.07–2.82 (2H, m, Hb-5, Ha-6), 2.81–2.69 (1H, m, Hb-6),

2.68–2.41 (3H, m, Ha-1, H-2), 1.98–1.76 (1H, m, Hb-1); ¹³C NMR

(75 MHz, CDCl₃): d = 173.3 (CO-3), 150.8 (NHCO), 141.2, 140.9

(C-8, C-9), 136.4 (C-1⁰), 136.0 (C-6a), 132.3 (C-10a), 129.2 (CH-4⁰),

124.5 (CH-10), 124.0 (CH-3⁰, CH-5⁰), 120.1 (CH-7), 118.7

(CH-2⁰, CH-6⁰), 56.4 (CH-10b), 36.8 (CH2-5), 31.6 (CH2-2), 28.1

(CH2-6), 27.1 (CH2-1); HREIMS: m/z 475.1977 [M + H₂O]⁺(calcd

for C₂₆H₂₃N₃O₅, 475.1976).

3.3 AChE and BChE inhibitory activity

End-point inhibitory activity was tested according to a pre-

3.4.2 Molecular dynamics (MD). MD simulations of all

viously reported method.²²For each sample, an Ellman’s complexes and later analysis of their trajectories were per-

reaction²³was performed in a final volume of 200 mL with formed with the Amber16 package.²⁷Antechamber software

New J. Chem., 2021, 45, 8321–8334 | 8331

View Article Online

Paper

NJC

in the AmberTools package²⁸was used to generate the para- of the Multiwfn software.³⁹This type of calculation has been

meters for MD simulations with ﬀ99SB²⁹and GAFF³⁰force used in recent works since it ensures a reasonable compromise

fields. The best ranked conformation of each complex was then between the wave function quality required to obtain reliable

soaked in truncated octahedral periodic boxes of explicit water values of the derivatives of r(r) and the computer power

boxes using the TIP3P model.³¹Na⁺or Cl^ꢃions were placed available.^{12,19,40–45}

using the Leap module to neutralize the negative and positive

charges of the AChE and BChE complexes, respectively. The

energy of each system was minimized with the sander module

using a steepest-descent algorithm for 1000 steps. Subse-

4. Conclusions

quently, the complexes were equilibrated during 500 ps at We report here a new series of inhibitors of AChE and BChE.

constant volume. The SHAKE algorithm³²was applied allowing Some of the substituted pyrrolo[2,1-a]isoquinolinone deriva-

for an integration time step of 2 fs. The systems were heated up tives reported here exhibit a significant inhibitory eﬀect against

to 300 K, a Langevin thermostat³³was applied and the collision both the AChE and BChE enzymes. Among the series reported

frequency was 1.0 ps^ꢃ1. After minimization and heating, three here, compounds 7, 8, 13 and 14 were those that displayed

MD production simulations of 20 ns were carried out at a 298 K more strong inhibitory activities; most of them possessed

target temperature. Thus, in total 60 ns were simulated for each inhibitory eﬀects even stronger than rivastigmine (the com-

complex. Coordinates were saved for analysis every 10 ps. All pound used as the reference). It should be noted that com-

productions were performed under NVT conditions and the pound 14 was the only one showing a selective inhibitory

particle mesh Ewald method (PME)³⁴was applied using a grid activity against BChE. The molecular modelling study showed

spacing of 1.2 Å, a spline interpolation order of 4 and a real that the larger size of the active site of BChE with respect to

space direct sum cutoﬀ of 10 Å. Post MD analysis was per- AChE could explain the selective activity of compound 14.

formed with the program CPPTRAJ.³⁵

QTAIM analysis gave us detailed information about the mole-

3.4.3 MM-PBSA free energy calculation and per-residue cular interactions stabilizing the diﬀerent ligand–enzyme com-

analysis. MMPBSA calculations were conducted to calculate plexes. Such information could be useful for the study and

the relative binding energies of the complexes.³⁶Snapshots design of new inhibitors of both enzymes.

from the corresponding last 1000 ps of the MD trajectories were

considered. The explicit water molecules and counter ions were

removed from the snapshots.

Author contributions

Afterwards, a per-residue decomposition of the relative free

energy was performed using the mm_pbsa program in the O. P. and R. D. E. contributed to the conceptualization of this

AMBER package. This analysis led us to determine which work. O. P., L. V., N. C. and D. C. carried out the synthesis and

amino acids of the AChE and BChE catalytic sites are involved characterization of the compounds. O. P. and F. G. performed

in the interactions with each ligand.

the molecular modelling studies. O. P., E. A. and R. D. E.

3.4.4 Quantum mechanics calculations and topological conducted the QTAIM calculations. R. S. and A. S. S. carried

analysis of the electron density distribution. MD trajectories out the bioassays studies and their analysis. O. P., E. A., N. C.,

were first clustered based on the root mean square deviation D. C. and R. D. E. wrote and corrected the article.

(RMSD) of the complex backbone atoms employing the

CPPTRAJ tool included in the Amber package. Then, the

representative structure from the most populated clusters was

selected for the QTAIM analysis.

Conflicts of interest

Reduced 3D model systems representing the diﬀerent L–R There are not conflicts to declare.

binding pockets of the selected zones were constructed from

the representative structures obtained by MD simulations by

keeping only receptor residues that interact directly with the

ligands. All the amino acids that are within a radius of 5 Å

Acknowledgements

distance from each ligand atom were considered. Those resi- The manuscript was written through contributions of all

dues that showed significant interactions with the ligand in the authors. The continued funding of the UNSL is strongly appre-

free energy decomposition approach were also included.

ciated. This work has also been partially funded by grants from

The charge density of the reduced model systems was then ANPCyT (PICT 2015-1769). This study was also supported by

computed by DFT methodology with the PBE hybrid functional grants from the Spanish Ministry of Economy and Competi-

and the 6-31G(d) as basis set, as implemented in the Gaussian tiveness (SAF2017-89714-R) and the Carlos III Health Institute

16 package.³⁷The topological analysis of charge density was (PI18/01450) and the European Regional Development Fund.

performed on the diﬀerent reduced models to evaluate the NC is an investigator from the ‘‘Miguel Servet’’ programme

interactions between each ligand and the molecular target in (CP15/00150), from Carlos III Institute of Health, co-funded by

the context of the quantum theory of atoms in molecules the European Social Fund. RDE, FG, EA, RS and AS are

(QTAIM).³⁸These calculations were carried out with the help members of CIC-CONICET-ARGENTINA.

8332

|

New J. Chem., 2021, 45, 8321–8334

View Article Online

NJC

Paper

22 I. Sanchis, R. Spinelli, N. Aschemacher, M. V. Humpola and

A. Siano, Amino Acids, 2020, 52, 387–396.

23 G. L. Ellman, K. D. Courtney, V. Andres and R. M.

Featherstone, Biochem. Pharmacol., 1961, 7, 88–95.

24 H. M. Greenblatt, G. Kryger, T. Lewis, I. Silman and

J. L. Sussman, FEBS Lett., 1999, 463, 321–326.

25 S. N. Dighe, E. De la Mora, S. Chan, S. Kantham, G. McColl,

J. A. Miles, S. K. Veliyath, B. Y. Sreenivas, Z. D. Nassar,

I. Silman, J. L. Sussman, M. Weik, R. P. McGeary,

M. O. Parat, X. Brazzolotto and B. P. Ross, Commun. Chem.,

2019, 2, 1–14.

26 G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner,

R. K. Belew, D. S. Goodsell and A. J. Olson, J. Comput. Chem.,

2009, 30, 2785–2791.

27 D. A. Case, R. M. Betz, W. Botello-Smith, D. S. Cerutti,

I. T. E. Cheatham, T. A. Darden, R. E. Duke, T. J. Giese,

H. Gohlke, A. W. Goetz, N. Homeyer, S. Izadi, P. Janowski,

J. Kaus, A. Kovalenko, T. S. Lee, S. LeGrand, P. Li, C. Lin,

T. Luchko, R. Luo, B. Madej, D. M. York, P. A. K. M. York

and P. A. Kollman, Univ. California, San Fr., 2016, 10779586.

28 J. Wang, W. Wang, P. A. Kollman and D. A. Case, J. Mol.

Graphics Modell., 2006, 25, 247–260.

29 W. D. Cornell, P. Cieplak, C. I. Bayly, I. R. Gould, K. M. Merz,

D. M. Ferguson, D. C. Spellmeyer, T. Fox, J. W. Caldwell and

P. A. Kollman, J. Am. Chem. Soc., 1995, 117, 5179–5197.

30 J. Wang, R. M. Wolf, J. W. Caldwell, P. A. Kollman and

D. A. Case, J. Comput. Chem., 2004, 25, 1157–1174.

31 W. L. Jorgensen, J. Chandrasekhar, J. D. Madura,

R. W. Impey and M. L. Klein, J. Chem. Phys., 1983, 79,

926–935.

References

1 M. Prince, A. Comas-Herrera, M. Knapp, M. Guerchet and

M. Karagiannidou, World Alzheimer Report 2016 Improving

healthcare for people living with dementia coverage, QualIty

and costs now and In the future.

2 H. Brodaty and M. Donkin, Dialogues Clin. Neurosci., 2009,

11, 217–228.

3 M. Ma, D. Dorstyn, L. Ward and S. Prentice, Aging Ment.

Heal., 2018, 22, 1395–1405.

4 L. Patel and G. T. Grossberg, Drugs Aging, 2011, 28, 539–546.

5 P. Taylor, in The Pharmacological Basis of Therapeutics, ed.

A. Gilman, A. Nies, T. Rall and P. Taylor, MacMillan, New

York, United States, 5th edn, 1990, pp. 131–150.

6 D. M. Bowen, C. B. Smith, P. White and A. N. Davison, Brain,

1976, 99, 459–496.

7 P. Davies, Brain Res., 1979, 171, 319–327.

8 L. Moreno, J. Parraga, A. Galan, N. Cabedo, J. Primo and

D. Cortes, Bioorg. Med. Chem., 2012, 20, 6589–6597.

9 F. Carmona-Viglianco, D. Zaragoza-Puchol, O. Parravicini,

A. Garro, R. D. Enriz, G. E. Feresin, M. Kurina-Sanz and

A. A. Orden, New J. Chem., 2020, 44, 9466–9476.

´

10 A. Hudcova, A. Kroutil, R. Kubınova, A. D. Garro,

¨

L. J. Gutierrez, D. Enriz, M. Oravec and J. Csollei, Molecules,

2020, 25, 1751.

11 J. E. Ortiz, A. Garro, N. B. Pigni, M. B. Agu¨ero, G. Roitman,

A. Slanis, R. D. Enriz, G. E. Feresin, J. Bastida and A. Tapia,

Phytomedicine, 2018, 39, 66–74.

12 T. Padrtova, P. Marvanova, K. Odehnalova, R. Kubinova,

O. Parravicini, A. Garro, R. Enriz, O. Humpa, M. Oravec and

P. Mokry, Molecules, 2017, 22, 2048.

13 J. E. Ortiz, N. B. Pigni, S. A. Andujar, G. Roitman,

F. D. Suvire, R. D. Enriz, A. Tapia, J. Bastida and

G. E. Feresin, J. Nat. Prod., 2016, 79, 1241–1248.

32 S. Miyamoto and P. A. Kollman, J. Comput. Chem., 1992, 13,

952–962.

33 J. A. Izaguirre, D. P. Catarello, J. M. Wozniak and R. D. Skeel,

J. Chem. Phys., 2001, 114, 2090.

´

14 J. Parraga, S. A. Andujar, S. Rojas, L. J. Gutierrez, N. El

34 U. Essmann, L. Perera, M. L. Berkowitz, T. Darden, H. Lee

and L. G. Pedersen, J. Chem. Phys., 1995, 103(19), 8577–8593.

35 D. R. Roe and T. E. Cheatham, J. Chem. Theory Comput.,

2013, 9, 3084–3095.

Aouad, M. J. Sanz, R. D. Enriz, N. Cabedo and D. Cortes, Eur.

J. Med. Chem., 2016, 122, 27–42.

15 O. Parravicini, M. L. Bogado, S. Rojas, E. L. Angelina, S. A.

´

Andujar, L. J. Gutierrez, N. Cabedo, M. J. Sanz, M. P. Lopez-

36 S. Genheden and U. Ryde, Expert Opin. Drug Discovery, 2015,

10, 449–461.

Gresa, D. Cortes and R. D. Enriz, J. Mol. Model., 2017, 23, 273.

16 J. Parraga, N. Cabedo, S. Andujar, L. Piqueras, L. Moreno,

A. Galan, E. Angelina, R. D. Enriz, M. D. Ivorra, M. J. Sanz

and D. Cortes, Eur. J. Med. Chem., 2013, 68, 150–166.

17 P. Bar-On, C. B. Millard, M. Harel, H. Dvir, A. Enz, J. L. Sussman

and I. Silman, Biochemistry, 2002, 41, 3555–3564.

18 M. B. Colovic, D. Z. Krstic, T. D. Lazarevic-Pasti,

A. M. Bondzic and V. M. Vasic, Curr. Neuropharmacol.,

2013, 11(3), 315–335.

37 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,

M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone,

G. A. Petersson, H. Nakatsuji, X. Li, M. Caricato,

A. V. Marenich, J. Bloino, B. G. Janesko, R. Gomperts,

B. Mennucci, H. P. Hratchian, J. V. Ortiz, A. F. Izmaylov,

J. L. Sonnenberg, D. Williams-Young, F. Ding, F. Lipparini,

F. Egidi, J. Goings, B. Peng, A. Petrone, T. Henderson,

D. Ranasinghe, V. G. Zakrzewski, J. Gao, N. Rega,

G. Zheng, W. Liang, M. Hada, M. Ehara, K. Toyota,

R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima,

Y. Honda, O. Kitao, H. Nakai, T. Vreven, K. Throssell,

J. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. J. Bearpark,

J. J. Heyd, E. N. Brothers, K. N. Kudin, V. N. Staroverov,

T. A. Keith, R. Kobayashi, J. Normand, K. Raghavachari,

A. P. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi,

J. M. Millam, M. Klene, C. Adamo, R. Cammi,

19 S. Rojas, O. Parravicini, M. Vettorazzi, R. Tosso, A. Garro,

´

L. Gutierrez, S. Andu´jar and R. Enriz, Eur. J. Med. Chem.,

2020, 208, 112792.

20 J.-P. Colletier, D. Fournier, H. M. Greenblatt, J. Stojan,

J. L. Sussman, G. Zaccai, I. Silman and M. Weik, EMBO J.,

2006, 25, 2746–2756.

21 D. Barak, A. Ordentlich, D. Stein, Q. S. Yu, N. H. Greig and

A. Shaﬀerman, Biochem. J., 2009, 417, 213–222.

New J. Chem., 2021, 45, 8321–8334 | 8333

View Article Online

Paper

NJC

J. W. Ochterski, R. L. Martin, K. Morokuma, O. Farkas, 42 L. J. Gutierrez, E. E. Barrera Guisasola, N. Peruchena and

J. B. Foresman and D. J. Fox, Gaussian, Gaussian, Inc.,

Wallingford CT, 2016.

38 R. F. W. Bader, Acc. Chem. Res., 1985, 18, 9–15.

39 T. Lu and F. Chen, J. Comput. Chem., 2012, 33, 580–592.

R. D. Enriz, Mol. Simul., 2016, 42, 196–207.

43 E. L. Angelina, S. A. Andujar, R. D. Tosso, R. D. Enriz and

N. M. Peruchena, J. Phys. Org. Chem., 2014, 27, 128–134.

44 J. E. Ortiz, A. Garro, N. B. Pigni, M. B. Agu¨ero, G. Roitman,

A. Slanis, R. D. Enriz, G. E. Feresin, J. Bastida and A. Tapia,

Phytomedicine, 2018, 39, 66–74.

´

40 L. J. Gutierrez, O. Parravicini, E. Sanchez, R. Rodrıguez,

J. Cobo and R. D. Enriz, J. Biomol. Struct. Dyn., 2018, 1–18.

´

41 E. E. Barrera Guisasola, L. J. Gutierrez, R. E. Salcedo, 45 S. A. Andujar, R. D. Tosso, F. D. Suvire, E. Angelina,

F. M. Garibotto, S. A. Andujar, R. D. Enriz and

N. Peruchena, N. Cabedo, D. Cortes and R. D. Enriz,

´

A. M. Rodrıguez, Comput. Theor. Chem., 2016, 1080, 56–65.

J. Chem. Inf. Model., 2012, 52, 99–112.

8334

|

New J. Chem., 2021, 45, 8321–8334

Article Doi

Design, synthesis, biological evaluation and molecular modelling of substituted pyrrolo[2,1-: A] isoquinolinone derivatives: Discovery of potent inhibitors of AChE and BChE

DOI: 10.1039/d1nj00345c

Source and publish data:

Authors:

Article abstract of DOI:10.1039/d1nj00345c

Full text of DOI:10.1039/d1nj00345c

Products guided by the article

R&D Labs maybe for 108163-97-9

Relevant to this article

Hot Product