Synthesis of 4H-1,4-oxazines as transthyretin amyloid fibril inhibitors

Article abstract of DOI:10.1039/c3ob40377g

4H-1,4-oxazines were designed as transthyretin (TTR) amyloid fibril inhibitors based on an analysis of the interactions between known small molecule inhibitors and TTR by molecular docking. A series of 2,4,6-triaryl-4H-1,4- oxazines was synthesized by the

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Synthesis of 4H-1,4-oxazines as transthyretin amyloid fibril
inhibitors
Weipeng Li,a Xiaowei Duan,a Hong Yana,* and Hongxing Xina
R2
R2
R2
O
O
Br2
NH2
POCl3
Br
N
O
N
R1
R1
Na2CO3 reflux
R1
pyridine reflux
0°C
R1
R1
O
O
R1
1
2
1a: R1=H, R2=H; 1b: R1=4-Me, R2=H; 1c: R1=4-Cl, R2=H; 1d: R1=4-OMe, R2=H;
1e: R1=H, R2=3-Me; 1f: R1=4-Me, R2=3-Me; 1g: R1=4-Cl, R2=3-Me; 1h: R1=4-OMe, R2=3-Me;
1i: R1=H, R2=4-Me; 1j: R1=4-Me, R2=4-Me; 1k: R1=4-Cl, R2=4-Me; 1l: R1=4-OMe, R2=4-Me;
1m: R1=H, R2=4-Cl; 1n: R1=4-Me, R2=4-Cl; 1o: R1=4-Cl, R2=4-Cl; 1p: R1=4-OMe, R2=4-Cl;
A series of novel 4H-1,4-oxazines were designed and synthesized as transthyretin amyloid fibril
inhibitors and some of 4H-1,4-oxazines displayed potent biological activity by a fibril formation
assay.

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Cite this: DOI: 10.1039/c0xx00000x
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ARTICLE TYPE
Synthesis of 4H-1,4-oxazines as transthyretin amyloid fibril inhibitors
Weipeng Li,a Xiaowei Duan,a Hong Yana,* and Hongxing Xina
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
5
4H-1,4-oxazines were designed as transthyretin (TTR) amyloid fibril inhibitors based on an analysis of
the interactions between known small molecule inhibitors and TTR by molecular docking. A series of
2,4,6-triaryl-4H-1,4-oxazines was synthesized by the cyclization of N,N-bis(phenacyl)anilines with POCl3
in pyridine. Inhibition of TTR amyloid fibril was evaluated by a fibril formation assay. The results
indicate that 4H-1,4-oxazines significantly inhibit TTR amyloid fibril at a concentration of 7.2 μM.
10 Introduction
Transthyretin (TTR) is a tetrameric protein that is composed of
identical β-sheet sandwich subunits of 127 amino acids.1 TTR
transports holoretinol binding protein and thyroxine (T4) in the
blood and CSF (cerebrospinal fluid).2 Under acidic conditions,
15 TTR dissociates to an alternatively folded monomeric
intermediate, which self-assembles into amyloid fibrils and
deposits. This dissociation of TTR causes several
neurodegenerative and amyloid diseases, including familial
amyloid cardiomyopathy, familial amyloid polyneuropathy and
20 senile systemic amyloidosis.3-5 The pH-mediated dissociation and
conformational changes of TTR can be prevented by T4-mediated
stabilization of the tetramer; hundreds of small molecules in
several distinct classes have been identified as inhibitors of TTR
amyloid fibril formation.6,7 Some nonsteroidal anti-inflammatory
25 drugs (NSAIDs) are TTR amyloid fibril inhibitors; indeed, some
NSAIDs are more efficacious than the natural ligand T4 in fibril
formation assays.8 Several typical NSAIDs, such as well-known
flufenamic acid (FLU) and N-Phenyl Phenoxazine (PHENOX)
(Fig. 1), increase the kinetic barrier to tetramer dissociation
30 through ground-state binding and stabilization.9-14 Based on the
molecular modelling of the interactions between the known
inhibitors and TTR, we have initiated the development of small-
molecule inhibitors of TTR amyloid fibril. The designed 4H-1,4-
oxazines interact with the catalytic site of TTR in the same
35 manner as PHENOX. The functional groups that are present may
form electrostatic interactions with the catalytic site of TTR. To
test this possibility, a series of 2,4,6-triaryl-4H-1,4-oxazines was
synthesized by the cyclization of N,N-bis(phenacyl)anilines with
POCl3 in pyridine (Scheme 1). The synthesized 4H-1,4-oxazines
40 were evaluated regarding the inhibition of TTR amyloid fibril.
Docking simulations were performed against the X-ray
crystallographic structure of TTR to explore possible binding
modes of 4H-1,4-oxazines at the active site.
45
Fig. 1 Natural ligand T4 and structures of typical NSAIDs
R2
R2
O
O
Br2
NH2
Na2CO3 reflux
Br
N
R1
R1
R1
R1
O O
2
R2
R2
N
O
N
POCl3
pyridine reflux
R1
R1
R1
R1
O O
1
2
50 1a: R1=H, R2=H; 1b: R1=4-Me, R2=H; 1c: R1=4-Cl, R2=H; 1d:
R1=4-OMe, R2=H; 1e: R1=H, R2=3-Me; 1f: R1=4-Me, R2=3-Me;
1g: R1=4-Cl, R2=3-Me; 1h: R1=4-OMe, R2=3-Me; 1i: R1=H,
R2=4-Me; 1j: R1=4-Me, R2=4-Me; 1k: R1=4-Cl, R2=4-Me; 1l:
R1=4-OMe, R2=4-Me; 1m: R1=H, R2=4-Cl; 1n: R1=4-Me, R2=4-
55 Cl; 1o: R1=4-Cl, R2=4-Cl; 1p: R1=4-OMe, R2=4-Cl;
Scheme.1 General strategy for the synthesis the compound 1.
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sites. In human blood, the concentration of TTR is approximately
3.6 μM; thus, at least 7.2 μM of compound 1 is needed to achieve
60 saturation of TTR. The stabilization of TTR by small molecules
is believed to result from the stabilization of the ground state and
an increase in the energy barrier to tetramer dissociation.18-23
Results and Discussion
2.1 Synthesis of 2,4,6-triaryl-4H-1,4-oxazines(1)
The 2,4,6-triaryl-4H-1,4-oxazines (1) were synthesized by the
cyclization of N,N-bis(phenacyl)anilines (2) with POCl3 in
pyridine (Scheme 1). These N,N-bis(phenacyl)anilines (2) were
obtained through the reaction of phenacyl bromides with anilines
and Na2CO3 in refluxing n-propyl alcohol (Table 1), according to
a method in the literature.15 Synthesis of 1 was carried out
according to Ravindran’s method for the synthesis of 2,4,6-
5
Table 1 Yields of 1b and 2a
Yield(%)
Compound
R1
R2
2
1
10 triphenyl-4H-1,4-oxazine (1a) by the cyclization of 2a with an
excess of POCl3 in pyridine.16 As shown in Table 1, yields of 2
varied depending on the substituents (R1 and R2): yields were
higher with electron-withdrawing groups at R1 and electron-
donating groups at R2. For example, the yield was higher for 2g
15 (78%) than for 2h (64%) because of the difference in R1 (2g:
R1=4-Cl, 2h: R1=4-OCH3). The yield was higher for 2g (78%)
than for 2o (67%) because of the difference in R2 (2g: R2=3-CH3,
2o: R2=4-Cl). Yields of 1 also varied depending on R1 and R2;
higher yields were obtained when R1 and R2 were electron
20 withdrawing. For example, for R1 = 4-Cl, the yield of 1o (74%)
was higher than that of 1k (65%), 1g (66%) and 1c (60%), while
when R2 = 4-Cl, the yield of 1o (74%) was higher than that of 1n
(62%) and 1m (63%). Strong electron-donating groups (R1=4-
OCH3) made 1 unstable in the reaction system, as these
25 compounds were observable by TLC during the reaction but
disappeared within a few minutes.
H
4-CH3
4-Cl
H
65
62
76
60
72
71
78
64
73
70
80
66
55
54
67
49
50
53
60
-
a
b
c
H
H
4-OCH3
H
H
d
e
3-CH3
3-CH3
3-CH3
3-CH3
4-CH3
4-CH3
4-CH3
4-CH3
4-Cl
55
58
66
-
4-CH3
4-Cl
f
g
h
i
4-OCH3
H
53
57
65
-
4-CH3
4-Cl
j
k
l
2.2 Biological activity of 4H-1,4-oxazines
Potential inhibitors (1a, 1b, 1c, 1e, 1i, 1m and 1n) were evaluated
30 in a 72 h stagnant fibril formation assay, in which TTR was
subjected to acidic partial denaturation at pH 4.4 (acetate buffer,
37°C) in the presence or absence of 1 at concentration of 3.6 μM
and 7.2 μM based on the literatures.10,17 The extent of fibril
inhibition was determined by the optical density (OD), measured
35 on a UV spectrophotometer at 400 nM in the presence of an
inhibitor, relative to the OD reading in the absence of the
inhibitor. Potential inhibitors were tested initially at 3.6 μM and
7.2 μM to determine whether there was sufficient activity to
warrant further assessment. Results represent the inhibition of
40 soluble transthyretin into fibrils over a 72 h period, as a
percentage of inhibiton in the absence of an inhibitor. Hence, 100%
inhibiting ratio reflects complete inhibition, whereas 0%
inhibiting ratio indicates no inhibition. The results are
summarized in Fig. 2.
4-OCH3
H
63
62
74
-
m
n
o
p
4-CH3
4-Cl
4-Cl
4-Cl
4-OCH3
4-Cl
65 a Na2CO3, anilines, n-propyl alcohol at reflux; recrystallized from
a mixture of CH2Cl2/petroleum ether. b POCl3, pyridine, 100°C,
recrystallized from absolute methanol.
2.3 Theoretical analysis of the inhibition of TTR by 4H-1,4-
70 oxazines
45 As shown in Fig. 2, compound 1 caused greater inhibition of TTR
dissociation at 7.2 μM (~80%) than at 3.6 μM (20%). At the
concentration of 7.2 μM, compound 1n caused the highest
inhibition (86%), though 1n displayed the best inhibition, its
activity is comparable to that of FLU (98%) and PHENOX (97%).
50 Meanwhile, compounds 1a, 1i and 1m caused more than 80%
inhibition, while 1b, 1c and 1e were modest inhibitors at less than
80% inhibition. At the concentration of 3.6 μM, compound 1
displayed the lower inhibitor efficacy (~23%) and was
significantly less than that of FLU (70%) and PHENOX (73%).
55 These results are due to the symmetry associated with the T4
binding site: compounds are able to bind in two symmetry-
equivalent modes in each of two symmetry-equivalent binding
To understand the structure-activity relationships of these TTR
amyloid fibril inhibitors and the possible binding modes of the 1,
we performed molecular docking experiments between 1 and the
ligand-binding site of TTR, All test compounds were docked into
75 the binding site of the recently published TTR-T4 crystal
structure (PDB entry code 1ICT) from the Protein Data Bank.24
Before making docking calculations, ligand T4 was extracted
from the crystal structure, the structural water molecules were
removed, and hydrogen atoms were added in standard geometry.
80 Prior to automate docking of the compound 1, T4 was docked
into the TTR crystal structure as a means of testing program
performance. For each compound, 100 docking experiments were
2
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initiated with randomized populations and solutions for
individual runs were clustered if their final docked positions were
within a tolerance of 2Å RMSD. A superposition of the most
highly scored conformation of the ligand onto its crystallographic
reported previously.26 Given that AutoDock successfully
reproduced the binding mode of PHENOX in the active site of
TTR. The calculated inhibition constants (Ki) for PHENOX was
also similar to the experimental value. The output files of
5
geometry yielded a RMSD (root mean square deviation of 1.31Å, 15 Autodock contained the final predicted conformations, RMSD
which was the best docked conformation by <2Å), thus revealing
that Autodock was successful in reproducing the binding mode of
T4 into the active site of TTR.25 PHENOX is a good TTR
amyloid fibril inhibitor and have similar structure to 1. So the
from the bound crystal structure, the lowest energy docked and
the estimated free energy of binding for each cluster and each
individual docking (Table 2).
10 PHENOX was also docked; the result was very similar to that
20
a Blank refers to the control (inhibition = 0%). b The error in % inhibiting ratio is typically +/-5% depending on the age and purity of the
protein stock.
Figure. 2 Inhibiting ratiob of compounds 1 and known inhibitors on TTR
25
Table 2 The docking results of 4H-1,4-oxazines (1)
1k
1l
-5.91
-6.54
-6.39
-6.83
-5.58
-6.68
-6.62
-7.57
46.67
16.14
62.94
62.79
61.72
63,38
63.26
62.19
1.31
Compound
ΔGa
Kib
RMSDd
63.12
62.38
62.16
62.72
61.82
63.11
62.43
62.33
61.76
63.26
1m
20.18
1a
1b
1c
1d
1e
1f
-6.07
-5.84
-6.52
-6.07
-6.23
-6.67
-6.28
-6.66
-6.29
-6.69
35.43
51.98
35.27
16.50
23.17
12.80
24.76
13.20
25.36
12.45
1n
9.78
1o
81.93
1p
12.59
T4
14.03(9.313)c
2.83(4.213)c
PHENOX
62.62
1g
1h
1i
a Binding free energy. b Inhibition constant. c Experiment
value(μM). d Root mean spuare deviation.
30
1j
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Binding affinities are reported as the binding free energies (ΔG)
kcal/mol, Ki = 35.43 μM).
and inhibition constants (Ki). Compound 1 fitted the binding 60 In conclusion, through appropriately substituted phenyl rings, 1
cavity of TTR well, with predicted ΔG ranging from -6.83 to -
5.58 kcal/mol and Ki ranging from 9.78 to 81.93 μM. The
predicted ΔG and Ki of 1a (R1=H, R2=H) were -6.07 kcal/mol and
35.43 μM, respectively, and those of 1n (R1=4-CH3, R2=Cl) were
can induce nearby amino acid residues in TTR to form hydrogen
bonds. Most important, the low energy conformation of 1 may
better occupy the inner and outer hormone binding cavities
through extensive van der Waals interactions with nearby
5
-6.83 kcal/mol and 9.78 μM, respectively. Compound 1n gave 65 residues, which should lead to an increase in binding affinity.
significantly better results than 1a, because of the introduction of
electron-donating and electron-withdrawing
(R1=4-CH3)
Compared with van der Waals forces, hydrogen bonds generate
stronger interactions with amino acid residues, so the predicted
affinity of 1 is lower than that of PHENOX.
10 substituents (R2=Cl) in suitable positions. Values for 1n were
similar to those of PHENOX. In the following, we will analyze
the typical docking results of 1a (R1=H, R2=H) and 1n. The
substituents exert a far-reaching impact on docking, as seen in
comparison with PHENOX (Fig. 4).
15 Compound 1a (R1=H, R2=H) binds to TTR in the ‘forward mode’:
the N-phenyl group is located in the inner binding cavity, and the
other two phenyl rings are positioned at the entry port of the
binding site. Compound 1a exhibited a similar binding mode to
PHENOX, in which the m-CF3-substituted phenyl ring occupies
20 the inner TTR binding cavity, while the phenoxazine ring system
occupies the outer thyroid hormone binding cavity. The phenyl
ring of 1a did not form hydrogen bonds with nearby amino acids,
but the COOH-substituted phenyl ring of PHENOX was
positioned at the entry of the hormone binding channel, allowing
25 the formation of a hydrogen bond between N(З) of residue Lys15
and the carboxylate group. The phenyl rings of 1a did not induce
rotation of amino acid side chains, but Ala108 and Thr119 as well
as Leu110 and Ser117 did undergo conformational changes, as
observed in known PHENOX-TTR complexes.26 The three aryl
30 rings connected in 1a were located between residues Lys15 and
Leu 110 from two adjacent TTR subunits, stretching across the
outer cavity and inner cavity of the hormone binding sites.
Because of the stereostructure of the three phenyl rings, 1a has a
low energy conformation that is capable of filling the hormone
35 binding cavities more completely compared with PHENOX. The
planes of the distal phenyl ring and the oxazine (C-C-N-C) are
angled almost 30° to each other in 1a, while the distal phenyl ring
and the tricyclic ring system are almost orthogonal to each other
in PHENOX. Nonetheless, the oxazine ring of 1a and the tricyclic
40 ring of PHENOX have similar characteristics, and interact with
surrounding amino acids through extensive van der Waals
interactions. In conclusion, no strong hydrogen bonds formed
between 1a and TTR. By contrast, in PHENOX, the COOH-
substituted phenyl ring is positioned at the entry of the hormone
45 binding channel, forming hydrogen bonds between COOH and
Lys15. Thus, the docking results suggest that 1a will have lower
affinity than PHENOX.
70
Fig. 4 Docking modes of 1a(green), 1n(blue) and PHENOX(red)
to TTR.
The binding mode of 1n (R1=4-CH3, R2=4-Cl) to TTR was
similar to that of 1a. The chlorine-substituted phenyl ring of 1n
50 occupies the inner binding cavity, while the CH3-substituted rings
fill the outer binding site. Compound 1n did not form a hydrogen
bond to nearby residues, but the introduction of a methyl group
on the phenyl ring induced the nearby amino acids Val121 and
Thr106 to form hydrogen bonds; moreover, the introduction of a
55 chlorine atom on the other phenyl ring of 1n permitted deeper
immersion into the bottom of the binding site compared to 1a.
For these two reasons, the docking result was better for 1n than
for 1a (1n: ΔG= -6.83 kcal/mol, Ki = 9.78 μM, 1a: ΔG= -6.07
Conclusions
We synthesised a series of 4H-1,4-oxazines by the cyclization of
75 N,N-bis(phenacyl)anilines with POCl3 in pyridine. These
compounds were evaluated in a 72 h stagnant transthyretin
amyloid fibril formation assay, giving over 75% inhibition at 7.2
μM. Docking experiments were performed with the ligand-
binding site of TTR, indicating that 4H-1,4-oxazines will inhibit
80 TTR (predicted ΔG of -6.83 to -5.58 kcal/mol and Ki of 9.78 to
81.93 μM). The representative docking modes of 1a (R1=H,
R2=H) and 1n (R1=4-CH3, R2=Cl) show that they have
4
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65 22. R. L. Wiseman, N. S. Green and J. W. Kelly, Biochemistry, 2005, 44,
9265-9274.
comparable activity to that of PHENOX (97%). This activity is
due to their low energy conformations, as well as extensive van
der Waals interactions between the oxazine ring and surrounding
amino acids and hydrogen bonds between adjacent residues
induced by the substituted phenyl ring.
23. R. L. Wiseman, S. M. Johnson, M. S. Kelker, T. Foss, I. A. Wilson
and J. W. Kelly, J. Am. Chem. Soc., 2005, 127, 5540-5551.
24. A. Wojtczak and P. Neumann, Acta Crystallogr., 2001, 57, 957-967.
70 25. X. H. Zhu, Y. Qin and H. Yan, Chem. Biol. Drug Des., 2010, 76,
330-339.
5
26. T. Klabunde, H. M. Petrassi, V. B. Oza, P. Raman, J. W. Kelly and J.
C. Sacchettini, Nat. Struct. Biol, 2000, 7, 312-321.
27. R. J. Hu, F. Barbault, M. Delamar and R. S. Zhang, Bioorg. Med.
Acknowledgments
75
Chem, 2009, 17, 2400-2409.
This work was supported financially by Key Projects in the
National Science
&
Technology Pillar Program (No.
10 2012ZX10001007-008-002) and Doctoral Fund of Innovation of
Beijing University of Technology.
Notes and references
a College of Life Science and Bio-engineering, Beijing University of
15 Technology, Pingleyuan Street No. 100, Chaoyang District, Beijing
100124, P. R. China. Fax: +86-010-67392001; Tel: +86-010-67396642;
E-mail: hongyan@bjut.edu.cn
† Electronic Supplementary Information (ESI) available: [1H and 13C
NMR, HRMS data of all new compound]. See DOI: 10.1039/b000000x/
20
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