Synthesis and characterization of novel 1,2-oxazine-based small molecules that targets acetylcholinesterase

Article abstract of DOI:10.1016/j.bmcl.2014.05.040

Thirteen 2-oxazine-based small molecules were synthesized targeting 5-lipoxygenase (LOX), and acetylcholinesterase (AChE). The test revealed that the newly synthesized compounds had potent inhibition towards both 5-LOX and AChE in lower micro molar concen

Full text of DOI:10.1016/j.bmcl.2014.05.040

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and characterization of novel 1,2-oxazine-based small
molecules that targets acetylcholinesterase
Alexey Yu. Sukhorukov a, Anilkumar C. Nirvanappa b, Jagadish Swamy c, Sema L. Ioffe a,
Shivananju Nanjunda Swamy d, Basappa b, , Kanchugarakoppal S. Rangappa c,
⇑
⇑
a N.D. Zelinsky Institute of Organic Chemistry, Leninsky Prospect, 47, Moscow 119991, Russia
b Laboratory of Chemical Biology, Department of Chemistry, Bangalore University, Palace Road, Bangalore 560001, India
c Department of Chemistry, University of Mysore, Manasagangotri, Mysore 560001, India
d Department of Biotechnology, Sri Jayachamarajendra College of Engineering, JSS, Technical Institutions Campus, Mysore 570006, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Thirteen 2-oxazine-based small molecules were synthesized targeting 5-lipoxygenase (LOX), and
acetylcholinesterase (AChE). The test revealed that the newly synthesized compounds had potent
inhibition towards both 5-LOX and AChE in lower micro molar concentration. Among the tested
compounds, the most active compound, 2-[(2-acetyl-6,6-dimethyl-4-phenyl-5,6-dihydro-2H-1,2-oxa-
zin-3-yl)methyl]-1H-isoindole-1,3(2H)-dione (2a) showed inhibitory activity towards 5-LOX and AChE
Received 1 April 2014
Revised 5 May 2014
Accepted 8 May 2014
Available online xxxx
with an IC50 values of 1.88, and 2.5 lM, respectively. Further, the in silico molecular docking studies
Keywords:
Chemical biology
Oxazine
Acetylcholinesterase
Lipoxygenase
Alzheimer disease
revealed that the compound 2a bound to the catalytic domain of AChE strongly with a highest CDOCKER
score of À1.18kcal/mol when compared to other compounds of the same series. Additionally, 2a showed
a good lipophilicity (logP = 2.66), suggesting a potential ability to penetrate the blood–brain-barrier.
These initial pharmacological data revealed that the compound 2a could serve as a drug-seed in developing
anti-Alzheimer’s agents.
Ó 2014 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) is a progressive age related neurode-
generative disorder and it is clinically characterized by impairment
in memory, visuospatial skills, complex cognition, language,
emotion and personality. Although the exact cause of AD remains
elusive, mounting evidence continues to support the involvement
of inflammation in the development of AD.1 An inflammatory
marker, interleukin-1 known to play a major role in enhancing
the neuronal acetylcholinesterase (AChE) activity.2–4 These physio-
logical mechanism or systemic inflammation process is termed
‘cholinergic anti-inflammatory pathway’ because is mediated by
the neurotransmitter acetylcholine (ACh).5 Based on the compel-
ling evidence that inflammatory processes are involved in the
pathogenesis of AD, research has looked into the use of anti-
inflammatory drugs as a treatment option for patients with AD.
Epidemiological evidence continues to build up indicating that
non-steroidal anti-inflammatory drugs (NSAIDs) may lower the
risk of developing AD.6 A possible mode of action for the effective-
ness of NSAIDs is by the blockage of cyclooxygenase (COX)-2 in the
brain.7–10 Evidently, it has been shown that COX-2 mRNA and
protein are considerably up-regulated in affected areas of AD
brain,11–13 suggesting the involvement of COX-2 in AD. So, we
herein attempted to design and synthesize, 1,2-oxazine-based small
molecules that could show anti-inflammatory activity and also play
a major role in inhibiting the AChE activity that involved in AD. Since
the discovery of 2-amino-1,3-oxazine scaffold was identified as the
selective and better inhibitors of b-site amyloid precursor protein
cleaving enzyme 1, and also projected to be the suitable starting
point for further development of brain penetrating compounds
for potential Alzheimer’s disease treatment.14 In addition, the
neuroprotective effect of 2-ethoxy-4,5-diphenyl-1,3-oxazine-6-one
against H2O2-induced cell death in rat pheochromocytoma cells
was reported.15 Evidently, the design, synthesis and results on 1,
4-oxazines revealed that the oxazine-based small molecules signifi-
cantly inhibited the transthyretin (TTR) amyloid fibril formation.16
In continuation of our effort to synthesize novel anti-inflammatory17
and anti-cholinergic agents,18 we herein report the synthesis,
characterization, anti-inflammatory and anti-cholinergic activity of
novel 1,2-oxazine-based small molecules for the first time.
A library of racemic tetrasubstituted functionalized 1,2-oxazines
(5,6-dihydro-4H-1,2-oxazines 1 and N-acetyl-5,6-dihydro-2H-1,
2-oxazines 2) required for the biological assays was generated
according to the synthetic strategy previously developed by
us19,20 (Scheme 1, Table 1). Stereoselective assembly of 1,2-oxazine
⇑
Corresponding authors. Tel.: +91 8022961346 (B.), tel./fax: +91 8212419363
(KSR)
E-mail addresses: salundibasappa@gmail.com (Basappa), rangappaks@gmail.
com (K.S. Rangappa).
http://dx.doi.org/10.1016/j.bmcl.2014.05.040
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sukhorukov Alexey Yu.;. et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.05.040

2
A. Yu. Sukhorukov et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
R1
O
R1
core was achieved by inverse electron demand Diels–Alder (IED-
R2
R3
CH3
O
R2
R3
CH3
O
DA) reaction of nitroalkenes derived from nitroethane to olefines.
The resulting diastereomerically pure 1,2-oxazine-N-oxides 3 were
subjected to silylation with an excess of trimethylsilyl bromide in
the presence of Et3N to give 3-bromomethyl-substituted 5,6-dihy-
dro-4H-1,2-oxazines 4, which serve as key precursors for the syn-
i
N
N
R4
[4+2]
O
R4
3
ii
thesis of C-3 functionalized 1,2-oxazines
1 and 2. Thus,
R1
nucleophilic substitution of bromide for phthalimide or dimethyl-
malonate anions furnished 1,2-oxazines 1a–f. 1,2-Oxazines 1g–i
with FG = CO2CH3 were obtained by catalytic carbonylation of cor-
responding bromides 3 in methanol being followed our previously
reported protocol.21 Transformation of 5,6-dihydro-4H-1,2-oxa-
zines 1 into and N-acetyl-5,6-dihydro-2H-1,2-oxazines 2 was
achieved by acetylation of the former with AcBr/Ac2O mixture in
high yields (Scheme 1).
iii for 1a
R1
R2
iv for 1b-f
v for 1g-i
R2
R3
Br
4
FG
3
5
6
N
1
O
R3
O
4
2N
R4
R4
1
vi
R1
R2
R3
All compounds were obtained in analytically pure form by col-
umn chromatography on silica gel and crystallization. The structure
and stereochemistry of previously unknown products was
confirmed by 1D and 2D NMR spectroscopy and elemental analysis.
Effect of 2-oxazines on LOX-5 and AChE. Oxazin-2-thione-based
small molecule exhibited LOX and COX-1 inhibitory action.22 In
particular, 5-LOX catalyses the biosynthesis of leukotrienes play a
pivotal role in inflammatory and allergic disorders as well as in car-
diovascular diseases and cancer.23 Additionally, tetrahydro-1,4-
FG
CH3
N
O
R4
O
2
Scheme 1. Synthesis of 3,4,5,6-tetrasubstituted-1,2-oxazine-based small mole-
cules. Reagents and conditions: (i) SnCl4, CH2Cl2, À94 °C to À30 °C; (ii) (CH3)3SiBr,
Et3N, CH2Cl2, À30 °C, 24 h; (iii) potassium phthalamide, DMF, 50–60 °C, 2 h; (iv)
CH2(CO2CH3)2, KOtBu, DMF, 60 °C, 2 h; (v) 15 bar CO/Pd(PPh3)2Cl2, CH3OH, 100 °C,
3 h; (vi) AcBr, Ac2O, CH2Cl2, rt, 2 h.
Table 1
Synthesis of 3,4,5,6-tetrasubstituted-1,2-oxazine-based small molecules
Entry
R1
R2
R3
R4
FG
Yielda (%)
Melting point
O
N
1a
C6H5
H
CH3
CH3
99
149–155 °C
O
H3CO2C
1b
1c
1d
1e
1f
4-CH3O–C6H4–
4-CH3O–C6H4–
C6H5
H
CH3
CH3
H
84
86
90
61
70
76–79 °C
H3CO2C
H3CO2C
–(CH2)4–
–(CH2)3–
100–102 °C
123–131 °C
85–90 °C
H3CO2C
H3CO2C
H
H3CO2C
H3CO2C
C6H5
H
H3CO2C
H3CO2C
CH3
H
CH3
CH3
CH3
71–79 °C
H3CO2C
CO2CH3
CO2CH3
1g
1h
4-CH3O–C6H4–
C6H5
H
CH3
H
89
71
63–65 °C
81–84 °C
–(CH2)4–
1i
C6H5
H
CO2CH3
92
75–78 °C
O
N
2a
C6H5
H
CH3
CH3
84
137–139 °C (dec)
O
H3CO2C
2b
2c
2d
4-CH3O–C6H4–
C6H5
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
97
82
73
90–92 °C
64–69 °C
91–93 °C
H3CO2C
H3CO2C
H3CO2C
H3CO2C
4-Cl–C6H4–
H3CO2C
a
Yield for the last step (average of two experiments).
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A. Yu. Sukhorukov et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Table 2
exhibited 5-LOX inhibitory activity, with the exception of 1f, 1g,
and 2d. Amongst the new compounds 2a, 2b, and 2c exhibited
most potent 5-LOX inhibitory activities, whereas compounds 2a
and 1i were found to inhibit the activity of AChE (Table 3)
effectively. Interestingly, the most active compound 2a showed
inhibitory activity towards both the enzymes 5-LOX and AChE with
5-LOX, and AChE inhibitory potential of 3,4,5,6-tetrasubstituted-1,2-oxazine-based
small molecules
Compounds
IC50 5-LOX (
lM)
IC50 AChE (lM)
1a
1b
1c
1d
1e
1f
1g
1h
1i
2a
2b
2c
30.04
66.87
33.75
81.68
21.09
NS
65.32
NSa
NS
75.35
NS
NS
NS
35.12
6.3
an IC50 values of 1.88, and 2.5 lM, respectively. These results
indicated that the 1,2-oxazine pharmacophore plays an important
role in deciding the preferences for these enzyme sites. The present
work thus confirms that the 1,2-oxazine-based ligands are effec-
tive LOX/AChE inhibitors, which can serve as promising therapeu-
tic agents against Alzheimer disease.
NS
16.09
16.68
1.88
1.23
6.32
NS
2.5
Structure based in silico molecular docking analysis on 1,2-oxa-
zine-based small molecules that target AChE. Many cholinesterase
inhibitors are used for symptomatic treatment of AD.25 The drug,
Aricept (E2020) was known to enhance performance in animal
models of cholinergic hypofunction and has a high affinity for
AChE.26 Prior to the elucidation of three-dimensional structure of
Torpedo californica AChE (TcAChE), E2020 was designed on the
basis of QSAR studies, which inhibited both electric eel and mouse
AChE in the nanomolar range. Later, the co-crystal structure of the
E2020–TcAChE complex at 2.5 Å resolution (PDB ID: 1EVE) was
reported. This structure was taken for our molecular docking
studies.26 The molecular docking studies on 1,2-oxazine-based
small molecules into the crystal structure of tcAChE were
performed using CDOCKER of Accelrys as reported earlier.27 Out
of 10 docked complexes, the high score of CDOCKER energy was
selected and described (Table 3). The analysis of the molecular
docking revealed that the compound 1i, and 2a bound to the active
site of the tcAChE with a greater extent of CDOCKER energies of
11.8, and À1.14 (kcal/mol), when compared to other structurally
compounds (Table 2). E2020 that bound to the active site of AChE
is compared with the 1,2-oxazines. The binding of compound 2a
towards the binding of AChE is depicted in Figure 1. The compound
2a makes primary interactions at the active-site gorge of the
enzyme through its three major functional groups such as phthal-
amide moiety, N-acetyl-2-oxazine ring, and the phenyl rings. These
functional groups responsible for three inter-hydrogen bonding
formation between Tyr334, Asp72, and Asn85 amino acids,
Gly118, and Ser122 amino acids, and also with Gln69, Gly123,
and Tyr121 amino acids. These results indicate that the strong
affinity of compound 2a on AChE could lead to the potent inhibi-
tion of the catalytic activity of the enzyme.
14.35
12.0
46.76
2.4
2d
NEOSTIGMINE
a
NS: not significant.
Table 3
CDOCKER Energies of 3,4,5,6-tetrasubstituted-1,2-oxazine small molecules
Compounds
-CDOCKER energy
(kcal/mol)
-CDOCKER interaction energy
(kcal/mol)
1a
1b
1c
1d
1e
1f
1g
1h
1i
2a
2b
2c
2d
17.6109
46.4932
37.026
43.3683
57.429
52.236
31.4987
39.8809
30.7859
35.6327
28.9761
À11.8795
1.14435
25.3233
20.7983
30.7952
48.0747
47.9319
33.7126
42.8039
41.6439
44.2924
45.1691
53.8689
51.7264
57.8437
oxazines are known to be biologically active against inflammatory
drug metabolizing enzymes.24 The potential of all the synthesized
compounds to inhibit 5-LOX and AChE was determined on pure
enzymes. Since inflammation occurs in the pathological regions
of Alzheimer’s disease (AD) brain, and a few animal models and
clinical studies clearly suggest that AD inflammation significantly
contributes to AD pathogenesis.1 Some of the tested 1,2-oxazines
Figure 1. Stereoview of the interaction map of compound 2a that bound to the active site of AChE (PDB ID: 1EVE). The stick model (carbon-pink and other atoms are in parent
color) of the key amino acids that are interacting with compound 2a (ball and stick model, carbon-green and other atoms are in parent color) was shown.
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A. Yu. Sukhorukov et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
R.; McGeer, P. L.; O’Banion, M. K.; Pachter, J.; Pasinetti, G.; Plata-Salaman, C.;
We herein report the synthesis of new 2-oxazine-based small
Rogers, J.; Rydel, R.; Shen, Y.; Streit, W.; Strohmeyer, R.; Tooyoma, I.; Van
Muiswinkel, F. L.; Veerhuis, R.; Walker, D.; Webster, S.; Wegrzyniak, B.; Wenk,
G.; Wyss-Coray, T. Neurobiol. Aging 2000, 21, 383.
molecules targeting 5-LOX and AChE. Among the tested com-
pounds, the most active compound 2a showed inhibitory activity
2. Mrak, R. E.; Griffin, W. S. Neurobiol. Aging 2001, 22, 903.
3. Mrak, R. E.; Griffin, W. S. Neurobiol. Aging 2001, 22, 915.
4. Wang, Y.; Zhang, J. X.; Du, X. X.; Zhao, L.; Tian, Q.; Zhu, L. Q.; Wang, S. H.; Wang,
J. Z. J. Neurochem. 2008, 106, 2364.
5. Borovikova, L. V.; Ivanova, S.; Zhang, M.; Yang, H.; Botchkina, G. I.; Watkins, L.
R.; Wang, H.; Abumrad, N.; Eaton, J. W.; Tracey, K. J. Nature 2000, 405, 458.
6. Breitner, J. C. Neurobiol. Aging 1996, 17, 789.
7. Hoozemans, J. J.; Veerhuis, R.; Van Haastert, E. S.; Rozemuller, J. M.; Baas, F.;
Eikelenboom, P.; Scheper, W. Acta Neuropathol. 2005, 110, 165.
8. In’t Veld, B. A.; Ruitenberg, A.; Hofman, A.; Stricker, B. H.; Breteler, M. M.;
Mahyar, G. Neurobiol. Aging 2001, 22, 407.
towards 5-LOX and AChE with an IC50 value of 1.88, and 2.5 lM,
respectively. Further, the in silico molecular docking studies
revealed that the compound 2a and 1e bound to the catalytic
domain of AChE strongly with a highest CDOCKER score about
À1.18, and 11.8 (kcal/mol), when compared to other compounds
of the same series. These initial pharmacological data revealed that
the compound 2a could serve as a drug-seed in developing anti-
Alzheimer’s agents.
9. McGeer, P. L.; McGeer, E.; Rogers, J.; Sibley, J. Lancet 1990, 335, 1037.
10. Pasinetti, G. M. J. Alzheimers Dis. 2002, 4, 435.
Acknowledgements
11. Pasinetti, G. M.; Aisen, P. S. Neuroscience 1998, 87, 319.
12. Ho, L.; Pieroni, C.; Winger, D.; Purohit, D. P.; Aisen, P. S.; Pasinetti, G. M. J.
Neurosci. Res. 1999, 57, 295.
13. Yasojima, K.; Schwab, C.; McGeer, E. G.; McGeer, P. L. Brain Res. 1999, 830,
226.
14. Woltering, T. J.; Wostl, W.; Hilpert, H.; Rogers-Evans, M.; Pinard, E.; Mayweg,
A.; Göbel, M.; Banner, D. W.; Benz, J.; Travagli, M.; Pollastrini, M.; Marconi, G.;
Gabellieri, E.; Guba, W.; Mauser, H.; Andreini, M.; Jacobsen, H.; Power, E.;
Narquizian, R. Bioorg. Med. Chem. Lett. 2013, 23, 4239.
15. Ansari, F.; Khodagholi, F.; Amini, M. Eur. J. Pharmacol. 2011, 658, 84.
16. Li, Weipeng; Duan, X.; Yan, H.; Xin, W. Org. Biomol. Chem. 2013, 11, 4546.
17. Basappa; Satish, K. M.; NanjundaSwamy, S.; Mahendra, M.; Shashidhara, P. J.;
Viswanath, B. S.; Rangappa, K. S. Bioorg. Med. Chem. Lett. 2004, 14, 3679.
18. Rangappa, K. S.; Basappa J. Phys. Org. Chem. 2006, 18, 773.
19. Sukhorukov, A. Yu.; Klenov, M. S.; Ivashkin, P. E.; Lesiv, A. V.; Khomutova, Y. A.;
Ioffe, S. L. Synthesis 2007, 97.
This research was supported in part by the Foundation for the
Support of Small-scale Enterprises (project 76GU1/2013) and grant
MK-3918.2013.3 from President’s grant council to A.S., and Univer-
sity Grants Commission (41-257-2012-SR), Vision Group Science
and Technology, Department of Science and Technology (DST)
(NO. SR/FT/LS-142/2012) to Basappa. K.S.R. thanks DST for funding.
Basappa thanks Karnataka University, INDIA for providing Pavate
fellowship.
Supplementary data
20. Ivashkin, P. E.; Sukhorukov, A. Yu.; Eliseev, O. L.; Lesiv, A. V.; Khomutova, Yu A.;
Ioffe, S. L. Synthesis 2007, 3461.
21. Eliseev, O. L.; Ivashkin, P. E.; Ostapenko, A. G.; Lesiv, A. V.; Khomutova, Y. A.;
Ioffe, S. L.; Lapidus, A. L. Synlett 2006, 2239.
22. Bennamane, N.; Nedjar-Kolli, B.; Geronikaki, A. A.; Eleftheriou, P. T.; Kaoua, R.;
Boubekeur, K.; Hoffman, P.; Chaudhary, S. S.; Saxena, A. K. ARKIVOC 2011, 69.
23. Werz, O.; Steinhilber, D. Pharmacol. Ther. 2006, 112, 701.
24. Peters-Golden, M.; Henderson, W. R., Jr. New Engl. J. Med. 1841, 2007, 357.
25. Mehta, M.; Adem, A.; Sabbagh, M. Int. J. Alzheimers Dis. 2012, 728983.
26. Kryger, G.; Silman, I.; Sussman, J. L. Structure 1999, 7, 297.
27. Basappa; Sugahara, K.; Thimmaiah, K. N.; Bid, H. K.; Houghton, P. J.; Rangappa,
K. S. PLoS One 2012, 7, e39444.
Supplementary data (detailed experimental procedures for the
synthesis, pharmacological investigations, and spectral datum)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2014.05.040.
References and notes
1. Akiyama, H.; Barger, S.; Barnum, S.; Bradt, B.; Bauer, J.; Cole, G. M.; Cooper, N.
R.; Eikelenboom, P.; Emmerling, M.; Fiebich, B. L.; Finch, C. E.; Frautschy, S.;
Griffin, W. S.; Hampel, H.; Hull, M.; Landreth, G.; Lue, L.; Mrak, R.; Mackenzie, I.
Please cite this article in press as: Sukhorukov Alexey Yu.;. et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.05.040