Discovery of isoalloxazine derivatives as a new class of potential anti-Alzheimer agents and their synthesis

Article abstract of DOI:10.1016/j.bioorg.2015.05.005

This article describes discovery of a novel and new class of cholinesterase inhibitors as potential therapeutics for Alzheimer's disease. A series of novel isoalloxazine derivatives were synthesized and biologically evaluated for their potential inhibitory outcome for both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). These compounds exhibited high activity against both the enzymes AChE as well as BuChE. Of the synthesized compounds, the most potent isoalloxazine derivatives (7m and 7q) showed IC₅₀ values of 4.72 μM and 5.22 μM respectively against AChE; and, 6.98 μM and 5.29 μM respectively against BuChE. These two compounds were further evaluated for their anti-aggregatory activity for β-amyloid (Aβ) in presence and absence of AChE by performing Thioflavin-T (ThT) assay and Congo red (CR) binding assay. In order to evaluate cytotoxic profile of these two potential compounds, cell viability assay of SH-SY5Y human neuroblastoma cells was performed. Further, to understand the binding behavior of these two compounds with AChE and BuChE enzymes, docking studies have been reported.

Full text of DOI:10.1016/j.bioorg.2015.05.005

Accepted Manuscript
Discovery of isoalloxazine derivatives as a new class of potential anti-Alzheimer
agents and their synthesis
Ashish M. Kanhed, Anshuman Sinha, Jatin Machhi, Ashutosh Tripathi, Zalak
S. Parikh, Prakash P. Pillai, Rajani Giridhar, Mange Ram Yadav
PII:
S0045-2068(15)00043-7
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.05.005
YBIOO 1815
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
26 January 2015
Please cite this article as: A.M. Kanhed, A. Sinha, J. Machhi, A. Tripathi, Z.S. Parikh, P.P. Pillai, R. Giridhar, M.R.
Yadav, Discovery of isoalloxazine derivatives as a new class of potential anti-Alzheimer agents and their synthesis,
Bioorganic Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bioorg.2015.05.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Discovery of isoalloxazine derivatives as a new class of potential anti-
Alzheimer agents and their synthesis
Ashish M. Kanhed,a Anshuman Sinha,a Jatin Machhi,a Ashutosh Tripathi,b Zalak S. Parikh,b
Prakash P. Pillai,b Rajani Giridhar a and Mange Ram Yadav a*
a
Pharmacy Department, Faculty of Technology & Engineering, The M. S. University of Baroda,
Vadodara –390001, Gujarat, India.
b Division of Neurobiology, Department of Zoology, Faculty of Science, The M. S. University of Baroda,
Vadodara–390001, Gujarat, India.
*To whom all Correspondence should be addressed:
Prof. M. R. Yadav
Pharmacy Department,
Faculty of Technology & Engineering,
The M. S. University of Baroda,
Kalabhavan,
Vadodara-390001, Gujarat,
India.
Phone: +91-265-2434187
Fax: +91-265-2418927
Email: mryadav11@yahoo.co.in
∗ Corresponding author Tel.: +91-265-2434187; fax: +91-265-2418927; e-mail: mryadav11@yahoo.co.in

ABSTRACT
This article describes discovery of a novel and new class of cholinesterase inhibitors as
potential therapeutics for Alzheimer’s disease. A series of novel isoalloxazine derivatives were
synthesized and biologically evaluated for their potential inhibitory outcome for both
acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). These compounds exhibited
high activity against both the enzymes AChE as well as BuChE. Of the synthesized compounds,
the most potent isoalloxazine derivatives (7m and 7q) showed IC50 values of 4.72 µM and 5.22
µM respectively against AChE; and, 6.98 µM and 5.29 µM respectively against BuChE. These
two compounds were further evaluated for their anti-aggregatory activity for β-amyloid (Aβ) in
presence and absence of AChE by performing Thioflavin-T (ThT) assay and Congo red (CR)
binding assay. In order to evaluate cytotoxic profile of these two potential compounds, cell
viability assay of SH-SY5Y human neuroblastoma cells was performed. Further, to understand
the binding behavior of these two compounds with AChE and BuChE enzymes, docking studies
have been reported.
Keywords: Alzheimer’s disease; Isoalloxazine; Cholinesterase’s inhibition; β-amyloid; Cyto-
toxicity.

INTRODUCTION
Alzheimer’s disease (AD) is an overwhelming neurodegenerative disorder characterized
by a progressive and irreversible decline in cognitive functions. It typically develops leisurely,
and gradually worsens as brain cells shrink and die. Eventually, Alzheimer's is fatal, and at
present, there is no cure for it.1 AD affects the cholinergic regions of the central nervous system
(CNS) associated with cognitive functions and awareness.2-4 Presently, majority of the
therapeutic treatments for AD are aimed to inhibit acetylcholinesterase (AChE) to enhance
acetylcholine (ACh) levels in brain.5 AChE inhibitors like tacrine, donepezil, rivastigmine and
NMDA receptor antagonists like memantine are currently available for AD treatment.6 Literature
supports the evidence of gradual fall in the levels of AChE in the brain of AD patients, while
there occurs slight increase in the activity of butyrylcholinesterase7,8 (BuChE). Further, post
mortem tissue analysis of AD patients showed a high level of BuChE in the hallmark lesions of
AD. In rats, selective BuChE inhibitor cymserine was found to elevate ACh levels and it
enhanced long-term CNS potentiation and learning.8 Therefore both these enzymes emerge as
appropriate targets for the development of cholinesterase inhibitors in the treatment of AD.9
Although AChE and BuChE are produced by different genes they are highly homologous
with more than 65% similarity. AChE has two major binding sub-sites, a peripheral anionic site
(PAS) and the other a catalyatic anionic site (CAS) which is located in the deep gorge of the
enzyme structure and is assigned to Ser-His-Glu catalytic triad. The gorge is lined by around 14
aromatic amino acids making the active site more hydrophobic, leading to better interaction with
substrates. The gorge goes through half way in the enzyme and is roughly 20 Å long. Common
to AChE, BuChE also has a catalytic triad consisting of Ser–His–Glu. Majority of the important
features of the active site of BuChE like a triad of Ser-His-Glu, a p-cation-binding site, an
oxyanion hole, and an acyl-binding pocket are similar to AChE. The acyl binding pocket of
BuChE is obviously larger than that of AChE. The active sites of both the enzymes acting as
nucleophiles are situated at the base of a cavity to attack the carbonyl group of the substrates.10
Along with tacrine, its different derivatives,4 homo and heterodimers, and hybrids
involving many heterocycles are reported to be beneficial in the AD.11 Further, small molecules
like dihydroindenones (donepezil),12 guanidines,13 coumarins14-16 and their structural derivatives
have also been reported for the treatment of AD. Considering the structural features of tacrine

and donepezil, along with the knowledge of the active site of the enzymes and considering
parameters like lipophilicity and molecular weight as restrainers, isoalloxazine ring was
derivatized and developed as anti-alzheimer’s agents. Isoalloxazine is basically a pteridine based
tricyclic heteronuclear moiety found mainly in flavins. It is the basic nucleus present in the
structure of vitamin B217,18 and some of its derivatives are reported to be effective against
protozoal infections particularly against human and animal trypanosomiasis and malaria.19
Isoalloxazine derivatives are also reported for the treatment of coccidiosis in animals.19
A
piperazine spacered bis-isoalloxazine compound has been reported to suspend the association of
prion proteins and Alzheimer-specific Aβ peptides.20 Isoalloxazine {benzo[g]pteridine-
2,4(3H,10H)-dione} (7) was synthesized as the lead molecule. Compound (7) was observed to
inhibit both AChE and BuChE with IC50 values of 64.45 µM and 55.57 µM respectively. To
improve its activity against ChEs, orderly substitution on compound (7) at position 10 with
different alkyl and substituted phenylalkyl groups were carried out. This was presumed to
improve hydrophobic interactions with the active sites of both the enzymes.
RESULTS AND DISCUSSION
Chemistry
O
O
N
O
O
N
(i)
(ii)
HN
NH
HN
NH
NH
O
O
N
H
O
O
O
1
2
7
O
N
NO2
NO2
NH2
N
(iii)
(iv)
(v)
NH
H
N
H
N
NH2
A
(2)
F
N
O
A
5a - 5q
A
6a - 6q
A
7a - 7q
4a - 4q
3
A =
Z
H2C
H3C
H3C
n=1 or 2
Scheme 1: Synthesis of isoalloxazine (7) and its derivatives (7a-7q).
(i) CrO3, AcOH; (ii) o-phenylenediamine, H3BO3, AcOH, RT, 8 hrs; (iii) K2CO3, DMF, 60°C, 4-6 hrs; (iv) Zn,
AcOH, MeOH, RT, 6-8 hrs; (v) H3BO3, AcOH, RT, 8-10 hrs.

For the syntheses of isoalloxazine (7) and its derivatives (7a-7q), the followed synthetic
route is outlined in scheme 1. Alloxan monohydrate (2) was prepared by oxidation of barbituric
acid (1) according to the reported procedure.21 The lead molecule (7) was prepared by the
reported procedure,22 by cyclizing compound (2) with o-phenylenediamine in presence of boric
acid and acetic acid at room temperature. In order to obtain different N-alkylated products (7a-
7q) at 10 position, 1-fluoro-2-nitrobenzene (3) was reacted with the required alkyl/arylalkyl
amine (4a – 4q) in presence of K2CO3 as the base in DMF to obtain the desired N-alkyl/arylalkyl
2-nitrophenylamines (5a – 5q). Subsequently, the 2-nitro group of compounds (5a – 5q) was
reduced in presence of Zn and AcOH in methanol as solvent at room temperature to obtain N1-
substituted 1,2-diamine intermediates (6a – 6q). The obtained intermediates were used as such
for the next step. To obtain the desired isoalloxazine derivatives (7a – 7q), the N1-substituted
1,2-diamine intermediates (6a – 6q) were reacted with 2 in presence of H3BO3 and AcOH at
room temperature as per the procedure adopted for compound (7) to get the cyclized compounds
(7a – 7q). Structures of all of the compounds were confirmed on the basis of spectral and
elemental analyses. The yields of the synthesized derivatives (7,7a-7q) were in the range of 80-
90% after recrystallization.
Table 1: In vitro AChE and BuChE inhibitory activity of the synthesized compounds (7, 7a – 7q).
O
N
NH
N
N
O
A
(7 - 7q)
IC50 (µM) values
BuChE
A
Compd
7
Z
AChE
H
64.45
55.57
---
---
H3C
9.32
12.2
40.38
7a
7b
---
H3C
43.72

H2C
---
11.22
11.53
27.24
31.72
7c
H
7d
Z
-do-
-do-
-do-
-do-
-do-
-do-
-do-
-do-
-do-
-do-
3-CH3
4-CH3
11.63
29.39
7.28
42.86
7.89
7e
7f
2-F
21.64
8.26
7g
7h
7i
3-F
11.46
47.26
29.95
18.82
9.34
3-Cl
10.49
7.39
4-Cl
7j
2- OCH3
4- OCH3
2,3-(OCH3)2
3,4-(OCH3)2
8.39
7k
7l
47.22
6.98
7m
7n
4.72
32.55
8.55
H
52.75
23.41
7o
Z
-do-
-do-
4-Cl
29.5
5.22
7.13
5.29
7p
7q
3,4-(OCH3)2
0.056
0.023
0.0086
1.87
Tacrine
Donepezil

Biological Activity
Ellman’s method23 was employed to test the ability of the synthesized compounds to
inhibit human AChE and equine serum BuChE using tacrine hydrochloride hydrate and
donepezil hydrochloride as reference standards. The extent of inhibition was expressed as IC50
(µM) and was summarized in table 1. Taking 7 as the lead molecule, various N-alkyl or N-
phenylalkyl substitutions were made in the tricyclic ring, which improved the activity against
both AChE and BuChE enzymes. All the compounds in the series showed a good range of
enzyme inhibition (AChE IC50 = 64.95 µM to 4.72 µM; BuChE IC50 = 55.57 µM to 5.29 µM).
Simple allyl, propyl and butyl substituents were found to improve hydrophobic interactions with
the enzymes and were comparatively more active on AChE than on BuChE. As compared to the
lead, the benzyl and phenylethyl substituents offered improved bio-activity on both the enzymes.
All the derivatives with benzyl and phenylethyl moieties showed moderate activity on both the
enzymes while compounds (7c, 7e and 7l) (with allyl, 3-methylbenzyl and 4-methoxybenzyl
substituents respectively) were observed to be more active on AChE than on BuChE. Whereas
compounds, (7f, 7j, 7n and 7p) (having 4-methylbenzyl, 4-chlorobenzyl, 3,4-dimethoxybenzyl
and 4-chlorophenylethyl groups respectively) were found to be more active against BuChE as
compared to AChE. Compounds (7m and 7q) (with 2,3-dimethoxybenzyl and 3,4-
dimethoxyphenylethyl groups respectively) showed the highest activity on both the enzymes
[AChE (4.72 µM and 5.22 µM) and BuChE (6.98 µM and 5.29 µM) respectively]. These two
compounds were choosen for further evaluation for their ability to prevent β-amyloid (Aβ)
aggregation in presence and absence of hAChE by Thioflavin-T (ThT) and Congo red (CR)
binding assays.
The two compounds (7m and 7q) selected on the basis of screening results of
cholinesterase inhibition assay were further assessed for their ability to prevent hAChE-induced
Aβ1-42 aggregation using thioflavin-T (ThT) fluorescence assay24 in comparison to tacrine and
donepezil as reference drugs. The results revealed that both the compounds (7m and 7q) at a
concentration of 10 µM showed significant inhibition of Aβ1-42 aggregation (35 % and 20 %
inhibition, respectively) as compared to the positive control (Figure 1A). This study
demonstrated that these compounds (7m and 7q) which were found earlier to be potent

cholinesterase inhibitors possessed moderate potential to inhibit hAChE induced Aβ1-42
aggregation also. The standard drugs tacrine and donepezil caused 26.26% and 38.18%
inhibition at the same concentrations under these conditions.
Aβ1-42 aggregation was measured by Congo red binding assay. Congo red is a dye which
has characteristics to bind with β-sheets of Aβ aggregates.25 The selected compounds (7m and
7q) were further evaluated using this assay along with tacrine and donepezil as reference drugs.
The results demonstrated that the compounds (7m and 7q) at 10 µM concentrations caused
significant inhibition of Aβ1-42 aggregation (38% and 24 % inhibition, respectively) as compared
to the positive control (Figure 1B). This strengthened our assumption that these compounds (7m
and 7q) which caused significant inhibition of hAChE-induced Aβ1-42 aggregation have the
ability to inhibit spontaneous Aβ1-42 aggregation in absence of hAChE as well. Inhibition in
aggregation of Aβ1-42 caused by the two standard drugs tacrine and donepezil was 31.82% and
42.45% under similar experimental conditions.
Figure-1: Percentage inhibition of Aβ1-42 aggregation by the test and reference compounds at 10 µM concentrations
with hAChE in (A) ThT assay and without hAChE in (B) CR binding assay. Data is analysed using GraphPad Prism
version 5. Comparison among the groups was made by one way ANOVA followed with Bonferroni test. Data is
expressed as mean SEM. *** indicates P<0.001 vs control (Aβ).
After obtaining encouraging results from the above three studies it was planned to assess
the biosafety of the two promising compounds (7m and 7q), by performing the cytotoxicity
studies of these compounds using MTT assay.26 Figure-2 represents the percentage viability of
the cells treated with the test compounds compared to the control cells. In this assay, tacrine and
donepezil were also used as reference compounds. At 40 µM concentration, none of the

compounds under study showed any significant toxicity. The results indicate that the compounds
are relatively non-toxic and can be further evaluated using different in vivo animal models for
Alzheimer’s disease (AD) to prove their neuroprotective property.
Figure-2: Percentage cell viability of the test and reference compounds assessed by MTT assay at 40 µM
concentration in SH-SY5Y human neuroblastoma cell line. Data is expressed as mean SEM. NS indicates non-
significant vs control.
Docking Study
To understand the intermolecular interactions of the promising molecules with the target
enzymes leading to the inhibitory activities, molecular docking studies were performed using
Glide module27 (Schrodinger). Receptor-ligand interactions were analyzed by using LIGPLOT
program.28 As discussed earlier, both AChE and BuChE have a catalytic triad, Ser–His–Glu and
certain other important features of the active sites in common. The synthesized molecules (7m
and 7q) along with tacrine and donepazil as standard drugs were docked into the active sites of
AChE and BuChE. To understand the molecular interactions of the ligands with AChE, the said
compounds were docked into the active site of the enzyme of Torpedo Californica (TcAChE)
(PDB Code: 1ACJ) that was then humanized to recognize the sequence of interactions. Tacrine
forms a firm complex with the enzyme by making hydrogen bond with the protonated nitrogen
of acridine ring of tacrine and C=O of His 440 (hAChE His447). The aromatic ring of tacrine
was observed to be sandwiched between Trp84 and Phe330 (hAChE Trp86 and Tyr337). In
donepezil, the 1-benzyl moiety of donepezil was observed to be stabilized in the active site by
Trp84 and Phe330 residues (hAChE Trp86 and Tyr337). The dihydroinden-1-one group of
donepezil was stabilized by Trp279 (hAChE Trp286) residue through hydrophobic interactions.

The 6-methoxy group exhibited H-bonding interaction with Trp279 (hAChE Trp286).
Compounds (7m and 7q) also showed good interaction with the CAS of the enzyme. In
compound (7m) (figure 3A) the tricyclic ring was observed to be stabilized within the
hydrophobic pocket of CAS comprising of Trp84, Glu199, Ser200, Phe330 and His440 (hAChE
Trp86, Glu202, Ser203, Tyr337 and His447) whereas the aromatic ring of the benzo[g]pteridine-
2,4(3H,10H)-dione scaffold was stabilized by π-π stacking with Trp84 and Phe330 (hAChE
Trp86 and Tyr337). The 2,3-dimethoxybenzyl group heading towards the PAS was found to be
stabilized by hydrophobic interactions with Tyr70, Tyr121 and Ser122 (hAChE Tyr72, Tyr124
and Ser125). Additionally the hydrogen bond between -NH of the ligand and Glu199 (hAChE
Glu202) residue was observed to stabilize the ligand-receptor complex. In compound (7q)
(figure 3B) the tricyclic ring was found to be stabilized in a similar fashion to that of 7m within
the hydrophobic pocket comprising of Trp84, Glu199, Ser200, Phe330 and His440 (hAChE
Trp86, Glu202, Ser203, Tyr337 and His447). The aromatic ring of benzo[g]pteridine-
2,4(3H,10H)-dione skeleton stabilized the receptor-ligand complex by forming π-π stacking with
Trp84 (hAChE Trp86). Strong hydrophobic interactions of 3,4-dimethoxyphenethyl group,
which heads towards the PAS of the receptor, were observed with Tyr70, Tyr121, Trp279 and
Phe330 (hAChE Tyr72, Tyr124, Trp286 and Tyr337). Additional stability to the ligand-receptor
complex was bestowed by the hydrogen bond between -NH of the ligand and the Glu199
(hAChE Glu202) residue of the receptor’s active site.
In a similar fashion, to understand the molecular interactions of the promising
compounds (7m and 7q) with the active site of hBuChE, docking studies were conducted within
the active site of hBuChE (PDB Code: 4BDS). Tacrine and donepazil, used as standards in
biological testing, were also considered for this study. The aromatic ring of tacrine stabilized the
ligand-receptor complex by π-π interactions with Trp82. H-bonding between amino N-H of
tacrine and C=O of His438 imparted further stability to the complex. While in case of donepazil,
the benzyl group was found to be stabilized by Trp82. The dihydroinden-1-one group by means
of hydrophobic interactions with Ser198, Trp231 and Phe398 stabilized the ligand-receptor
complex. In case of 7m, the tricyclic ring was observed to be stabilized into the hydrophobic
pocket of Trp82, Tyr332 and His438. The -NH of the tricyclic ring of the ligand further imparted
stability to the complex by forming hydrogen bonding with Ala328. The 2,3-dimethoxybenzyl
group was stabilized by the hydrophobic interactions with the Gly117, Ser198, Pro285 and

Phe329 (Figure 4A). Docking study of compound 7q (Figure 4B) showed more prominent
interactions within the hydrophobic pocket comprising of Trp82, Thr120 and Gly121. Additional
stability to the ligand-receptor complex was imparted by the hydrogen bonding between –NH of
the pteridine moiety and Tyr128 residue present in the enzyme. 3,4-Dimethoxyphenethyl group
further added stability to the complex by hydrophobic interactions with Gly116, Pro285, Leu286
and Phe329.
Overall, it could be concluding from the docking studies that the synthesized compounds
(7m and 7q) showed reasonably good interactions with the active sites of AChE and BuChE. 7m
and 7q showed interactions with the receptor active sites comparable to the standard drugs
tacrine and donepezil (Figure S1 and Figure S2) selected in this study for comparison purpose.
Along with the biological evaluation, this docking approach also supports the potential of the test
compounds (7m and 7q).
Figure 3: Docking interaction of the two compounds with the active site of AChE Eenzyme. (A) Compound
(7m); (B) Compound (7q).

Figure 4: Docking interaction of the two compounds with the active site of BuChE enzyme. (A) Compound
(7m); (B) Compound (7q).
CONCLUSION
In conclusion, this study brings to light the discovery of isoalloxazines as novel leads for
the treatment of Alzheimer’s disease. Along with the parent molecule isoalloxazine, different
derivatives of the lead (7) were synthesized using various alkyl and substituted phenylalkyl
groups and evaluated for their ability to inhibit AChE and BuChE enzymes by using Ellman’s
method. All the compounds showed moderate to good inhibitory activity in µM range against
either or both of the enzymes. Amongst all, compounds (7m and 7q) showed the highest
inhibitory activity against AChE (4.72 µM and 5.22 µM respectively) as well as BuChE (6.98
µM and 5.29 µM respectively) enzymes. The promising compounds (7m and 7q) were evaluated
additionally for their ability to prevent β-amyloid (Aβ) aggregation with and without AChE by
Thioflavin-T (ThT) assay and Congo red (CR) binding assay respectively. Further, cytotoxic
effects of the two compounds using MTT assay were analyzed and it was found that both the
compounds were non-toxic at 40 µM dose level. Molecular docking studies also confirmed that
these compounds targeted both the enzymes and supported the binding potential of the
synthesized compounds to the targets. On the basis of these results, further structural

modifications in isoalloxazine scaffold are in progress to design and synthesize more potential
compounds in this laboratory.
Acknowledgements
AMK and AS thank University Grant Commission (UGC), New Delhi for SRF. Authors acknowledge the
analytical facilities provided by Dr. Vikram Sarabhai Research Center, The M. S. University of Baroda, Vadodara,
India.
Supplementary data
Supplementary data associated with this article can be found in the online version
References
1. Brookmeyer, R.; Johnson, E.; Ziegler-Graham, K.; Arrighi, M. H. Alzheimer's and Dementia 2007, 3, 186–191.
2. Suh, Y. H.; Checler, F. Pharmacol. Rev. 2002, 54, 469−525.
3. Selkoe, D. J. Physiol. Rev. 2001, 81, 741-766.
4. Tumiatti, V.; Minarini, A.; Bolognesi, M. L.; Milelli, A.; Rosini, M.; Melchiorre, C. Curr. Med. Chem. 2010,
17, 1825-1838.
5. Munoz-Torrero, D. Curr. Med. Chem. 2008, 15, 2433-2455.
6. Hamulakova, S.; Janovec, L.; Hrabinova, M.; Spilovska, K.; Korabecny, J.; Kristian, P.; Kuca, K.; Imrich, J. J.
Med. Chem. 2014, 57, 7073-7084.
7. Carolan, C. G.; Dillon, G. P.; Gaynor, J. M.; Reidy, S.; Ryder, S. A.; Khan, D.; Marquez, J. F.; Gilmer, J. F. J.
Med. Chem. 2008, 51, 6400-6409.
8. Butini, S.; Campiani, G.; Borriello, M.; Gemma, S.; Panico, A.; Persico, M.; Catalanotti, B.; Ros, S.; Brindisi,
M.; Agnusdei, M.; Fiorini, I.; Nacci, V.; Novellono, E.; Belinskaya, T.; Saxena, A.; Fattorusso, C. J. Med.
Chem. 2008, 51, 3154-3170.
9. Alipour, M.; Khoobi, M.; Foroumadi, A.; Nadri, H.; Moradi, A.; Sakhteman, A.; Grandi, M.; Shafiee, A.
Bioorg. Med. Chem. 2012, 20, 7214-7222.
10. Chiou, S.; Huang, C.; Huang, C.; Hwang, M.; Lin, G. J. Biochem. Mol. Toxicol. 2009, 23, 303-308.
11. (a) Bornstein, J. J.; Eckroat, T. J.; Houghton, J. L.; Jones, C. K.; Green, K. D.; Garneau-Tsodikova, S. Med.
Chem. Commun. 2011, 2, 406-412; (b) Minarini, A.; Milelli, A.; Tumiatti, V.; Rosini, M.; Simoni, E.;
Bolognesi, M. L.; Andrisano, V.; Bartolini, M.; Motori, E.; Angeloni, C.; Hrelia, S. Neuropharmacology 2012,
62, 997-1003; (c) Tang, H.; Zhao, L.; Zhao, H.; Huang, S.; Zhong, S.; Qin, J.; Chen, Z.; Huang, Z.; Liang, H.
Eur. J. Med. Chem. 2011, 46, 4970-4979; (d) Luo, W.; Li, Y.; He, Y.; Huang, S.; Li, D.; Gu, L.; Huang, Z. Eur.
J. Med. Chem. 2011, 46, 2609-2616.
12. Sugimoto, H.; Yamanishi, Y.; Limura, Y.; Kawakami, Y. Curr. Med. Chem. 2000, 7, 303-339.
13. (a) Ghosh, A. K.; Osswald, H. L. Chem. Soc. Rev. 2014, 43, 6765-6813; (b) Zou, Y.; Li, L.; Chen, W.; Chen, T.;
Ma, L.; Wang, X.; Xiong, B.; Xu, Y.; Shen, J. Molecules 2013, 18, 5706-5722.
14. Piazzi, L.; Cavalli, A.; Colizzi, F.; Belluti, F.; Bartolini, M.; Mancini, F.; Recanatini, M.; Andrisano, V.;
Rampa, A. Bioorg. Med. Chem. Lett. 2008, 18, 423-426.
15. Karimi, G.; Iranshahi, M.; Hosseinalizadeh, F.; Riahi, B.; Sahebkar, A. Pharmacologyonline 2010, 1, 566-574.
16. Fallarero, A.; Oinonen, P.; Gupta, S.; Blom, P.; Galkin, A.; Gopi Mohan, C. P.; Vuorela, M. Pharmacol. Res.
2008, 58, 215-221.
17. Heelis, P. F. Chem. Soc. Rev. 1982, 11, 15-39.
18. Yoneda, F.; Sakuma, Y.; Ichiba, M.; Shinomura, K. J. Am. Chem. Soc. 1976, 98, 830-835.
19. Graham, D. W.; Rogers, E. F. U.S. Patent 4,173,631, 1979.

20. Barthel, A.; Trieschmann, L.; Strohl, D.; Kluge, R.; Bohm, G.; Csuk, R. Arch. Pharm. Chem. Life Sci. 2009,
342, 445-452.
21. Rabjohn, N. In Organic Syntheses, John Wiley & Sons, INC, 1963; Collective Vol. 4, pp 23-24.
22. Chen, S.; Hossain, M. S.; Foss, F. W. Organic Letters 2012, 14, 2806-2809.
23. Ellman, G.L.; Courtney, K.D.; Andres, V.; Feather-Stone, R.M. Biochem. Pharmacol. 1961, 7, 88–95.
24. Biancalana, M.; Koide, S. Biochim. Biophys. Acta 2010, 1804, 1405-1412.
25. Howie, A.J.; Brewer, D.B. Micron. 2009, 40, 285-301.
26. (a) Kakko, I.; Toimela, T.; Tahti, H. Environ. Toxicol. Pharmacol. 2004, 15, 95-102. (b) Vellonen, K.S.;
Honkakoski, P.; Urtti, A. Eur. J. Pharm. Sci. 2004, 23, 181-188.
27. Glide version 5.5 (2009) Schrödinger, LLC, New York, NY.
28. Wallace, A. C.; Laskowski, R. A.; Thornton, J. M. Protein Eng. 1995, 8, 127-134.

Highlights
• Isoalloxazine derivatives as novel class of cholinesterase inhibitors are reported.
• Compounds reported showed good activity against both AChE and BuChE.
• Potential therapeutic activity for Alzheimer’s disease is explained.
• For most potent compounds anti-aggregatory activity for β-amyloid was performed.
• Cyto-toxicity on SH-SY5Y neuroblastoma cells, and docking study were performed.