Atropisomerism observed in indometacin derivatives

Article abstract of DOI:10.1021/ol103008d

To elucidate the active conformation of indometacin that differentiates between cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), the stereochemistry around the N-benzoylated indole moiety of indometacin was studied. Resolution of stable atropisomers

Full text of DOI:10.1021/ol103008d

ORGANIC
LETTERS
2011
Vol. 13, No. 4
760–763
Atropisomerism Observed in Indometacin
Derivatives
Hideyo Takahashi,* Shintaro Wakamatsu, Hidetsugu Tabata, Tetsuta Oshitari,
Ayako Harada, Keizo Inoue, and Hideaki Natsugari*
School of Pharmaceutical Sciences, Teikyo University, 1091-1, Midori-ku, Sagamihara,
Kanagawa 252-5195, Japan
hide-tak@pharm.teikyo-u.ac.jp; natsu@pharm.teikyo-u.ac.jp
Received December 13, 2010
ABSTRACT
To elucidate the active conformation of indometacin that differentiates between cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), the
stereochemistry around the N-benzoylated indole moiety of indometacin was studied. Resolution of stable atropisomers as representative
conformations was found to be possible by restricting rotation about the N-C70 and/or C70-C10 bond. Only the aR-isomer showed specific
inhibition of COX-1, and COX-2 was not inhibited by either atropisomer.
Indometacin, a “classical” nonsteroidal antiinflamma-
tory drug (NSAID), acts at the cyclooxygenase (COX)
active site and inhibits two isoforms of COX, i.e., COX-1
and COX-2, with little specificity, leading to serious
side effects.1-7 Therefore COX-1/2-selective indometacin
analogues have been extensively explored in attempts to
develop a new generation of NSAIDs.8-11
In the design of new COX-1/2-selective indometacin
analogues, the conformation of the N-benzoyl moiety
has been considered. The benzoyl (phenyl) group can
adopt either a cis or trans conformation with respect to the
indole (Figure 1a). In 1996, Loll et al. proposed that
indometacin interacts with COX-1 in two possible binding
modes corresponding to the cis or trans form.12 Indepen-
dently, the group of Miyashiro and Penning reported that
indometacin adopts a cis conformation in its complex
with COX-2.13 Since then, the cis/trans conformations of
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r
10.1021/ol103008d
Published on Web 01/20/2011
2011 American Chemical Society

published. Here we show presence of atropisomers based
on the C70-C10 axis in the N-benzoyl moiety of 20,60-
disubstituted indometacin derivatives (A, Figure 1b),
which possess high stereochemical stability and are recog-
nized by COX-1. This novel chirality is produced by fixing
the C70-C10 axis alone, and hence the other N-C70 axis
independently rotates to form an equilibrium between cis-
and trans-like conformations in each enantiomer.
Figure 1. (a) Conformations traditionally used for indometacin.
(b) 20,60-Disubstituted indometacin derivative A.
Scheme 1. Synthesis of C-20/C-60 Disubstituted and C-20/C-40/C-
60 Trisubstituted Indometacin Derivatives (1-4), Which Exist
As Equilibrium Mixtures of cis-Like and trans-Like Conformers
indometacin have been regarded as a key feature for
improved drug design.14,15 However, “cis/trans” terminol-
ogy isassociatedwitha planarstructure inwhich the indole
and benzoyl (phenyl) groups are coplanar. Such vague
terminology could prove misleading in the present case.
X-ray crystallographic analysis has shown that indometa-
cin, even in a crystal state, adopts several different con-
formations, in which the indole and benzoyl (phenyl)
groups are twisted relative to each other.16,17
Similarly, in solution the 1-benzoyl-2-methylindole moi-
ety of indometacin is not expected to adopt a completely
planar conformation because of the large steric hindrance
between the phenyl and indolyl groups. The N-benzoyl
moiety has two sp2-sp2 axes along the N-C70 and
C70-C10 bonds that can provide numerous conformations
of indometacin in solution. To elucidate the active con-
formation of indometacin, the resolution of stable atropi-
somers as a representative conformation by restricting the
rotation of the N-C70 and/or C70-C10 bond could provide
a new means to study the molecular origins of COX-1/2
selectivity. At present, atropisomerism is most common in
biphenyl or biaryl C-C bond stereochemistry, although
atropisomers arising from C-N bonds have recently
attracted much attention.18-25 However, to the best of
our knowledge, reports on the chirality of N-benzoylin-
doles, with chirality arising from the two sp2-sp2 axes
of the aromatic N-CO-Ar bonds, have not yet been
Through the examination of various indometacin deri-
vatives, we found that the C-20 and C-60 disubstituted
indometacin derivatives (1-4, Scheme 1) exist in cis-trans
1
equilibrium: the H NMR spectrum of 1 in CDCl3 was
observed as two sets of resonances in a 1:1.7 ratio (see
Supporting Information). In order to assign the cis/trans
conformations, the NOESY method was used for the
trisubstituted (at C-20, C-40, and C-60) benzoyl derivative
2. In the NOESY specrum of the minor form, H7 showed a
correlation peak to H30/50, which confirmed the position of
the benzoyl (phenyl) ring to be cis. On the contrary, for the
major isomer of 2, 2-CH3 showed a correlation peak to
H30/50, which confirmed the position of the benzoyl ring to
be trans (see Supporting Information). We therefore rea-
soned that compound 1 exists as a mixture of cis-like and
trans-like26 forms (cis/trans = 1:1.7) in solution. Com-
pound 1 was observed as an inseparable single peak on
nonchiral HPLC at room temperature, indicating that
conformers are at equilibrium as the result of rotation
around the N-C70 axis.27 For 1-4, either or both con-
formations (cis-like and trans-like) could be adopted when
it binds to an enzyme. However, the ratio of cis-like and
trans-like (1:1.6-1.7) may be taken to indicate a tendency
to adopt the trans-like conformer.
(14) Kakuta, H.; Zheng, X.; Oda, H.; Harada, S.; Sugimoto, Y.;
Sasaki, K.; Tai, A. J. Med. Chem. 2008, 51, 2400–2411.
(15) Kasaya, Y.; Hoshi, K.; Terada, Y.; Nishida, A.; Shuto, S.;
Arisawa, M. Eur. J. Org. Chem. 2009, 4606–4613.
(16) Loll, P. J.; Garavito, R. M.; Carrell, C. J.; Carrell, H. L. Acta
Crystallogr., Sect. C 1996, C52, 455–457.
(17) Xiaoming, C.; Morris, K. R.; Griesser, U. J.; Byrn, S. R.; Stowell,
J. G. J. Am. Chem. Soc. 2002, 124, 15012–15019.
(18) Clayden, J. Tetrahedron 2004, 60, 4335. Tetrahedron Symposia-
in-Print on Atropisomerism.
(19) Clayden, J. Angew. Chem., Int. Ed. 1997, 36, 949–951.
(20) Ikeura, Y.; Doi, T.; Fujishima, A.; Natsugari, H. Chem. Com-
mun. 1998, 2141–2142.
(21) Ikeura, Y.; Ishichi, Y.; Tanaka, T.; Fujishima, A.; Murabayashi,
M.; Kawada, M.; Ishimaru, T.; Kamo, I.; Doi, T.; Natsugari, H. J. Med.
Chem. 1998, 41, 4232–4239.
(22) Natsugari, H.; Ikeura, Y.; Kamo, I.; Ishimaru, T.; Ishichi, Y.;
Fujishima, A.; Tanaka, T.; Kasahara, F.; Kawada, M.; Doi, T. J. Med.
Chem. 1999, 42, 3982–3993.
(23) Tabata, H.; Akiba, K.; Shoukou, L.; Takahashi, H.; Natsugari,
H. Org. Lett. 2008, 10, 4871–4874.
(24) Tabata, H.; Suzuki, H.; Akiba, Kumi; Takahashi, H.; Natsugari,
H. J. Org. Chem. 2010, 75, 5984–5993.
(25) Clayden, J.; Moran, W. J.; Edwards, P. J.; LaPlante, S. R.
(26) These “trans-like/cis-like” terms have the implication that the
carbonyl group takes a type of perpendicular position with respect to the
plane of the phenyl group of the benzoyl moiety.
(27) In order to examine the equilibrium between the cis-/trans-con-
formers, VT-NMR has been measured (see: Supporting Information).
Angew. Chem., Int. Ed. 2009, 48, 6398–6401.
Org. Lett., Vol. 13, No. 4, 2011
761

Figure 2. Structure of 3 determined using X-ray crystallographic
analysis.
After confirming that the N-C70 axis of N-benzoylated
indole derivatives allows rotation in solution, the next step
was to examine the C70-C10 axis. The structural analysis
was supported by single-crystal X-ray analysis of 3, in
which the dihedral angle (φ) C20-C10-C70-O was shown
to be 67.8°, confirming that the benzene ring is essentially
orthogonal to the indole ring (Figure 2). This geometry
convinced us that the atropisomeric property arises from
the C70-C10 axis in C-20 and C-60 disubstituted indole
derivatives. As expected, compound 4, in which Cl and I
are present at C-20 and C-60, respectively, gave important
Figure 3. Conformers including atropisomers of the indometa-
cin analogue 4.
rotation. Judging from the above, these two axes behave
independently for 4 in solution. To the best of our knowl-
edge, this type of atropisomer has not been previously
reported. The separated enantiomersof4 (4Aand 4B) were
hydrolyzed to afford the corresponding carboxylic acid
derivatives 5 (5A, 5B) (Scheme 2). We fortunately suc-
ceeded in obtaining the enantiomer 5A as a single crystal.
1
insight into this. On the basis of the H NMR spectrum,
it is clear that 4 also exists as an equilibrium mixture of
cis-like and trans-like conformation (cis-like:trans-like =
1:1.6) in CDCl3. It was also observed as a single peak on
nonchiral HPLC at room temperature. In short, rotation
about the N-C70 axis of 4 is similar to that of 1. To our
surprise, however, 4 could be resolved by chiral HPLC
(CHIRALPAK IB) into enantiomers at room tempera-
ture, indicating that it exists as a racemate of the atrop-
isomers. It is clear that the steric hindrance provided by the
C-20 and C-60 substituents forms a higher rotational barrier
for the C70-C10 axis than for the N-C70 axis. It should be
noted that each cis-like and trans-like conformer of 4 exists
asthe racemate of aR and aS isomers, as shown inFigure 3.
We managed to obtain each enantiomer of 4 using
preparative chiral HPLC at about 96% ee, with high
stereochemical stability at room temperature (see Support-
ing Information). It was confirmed that each enantiomer
exists as an equilibrium mixture of cis-like and trans-like
conformers. The isolated atropisomers have opposite [R]D
values: 4A (with shorter retention time in HPLC using
CHIRALPAK IB) as 96% ee showed [R]24D -25.7 (c 0.1,
CHCl3) and 4B (with longer retention time in HPLC using
CHIRALPAK IB) as 96% ee showed [R]23D þ23.1 (c 0.15,
CHCl3), confirming that they are enantiomers. The isola-
tion of each enantiomer with high stereochemical stability
provides evidence thatrotation about the C70-C10 axiscan
be fully restricted by substituents at C-20 and C-60. It is
worth noting that the N-C70 axis can allow rotation
even though the neighboring C70-C10 axis is blocked to
Scheme 2. Separation of 4 into Atropisomers 4A and 4B and
Hydrolysis to the Corresponding Carboxylic Acids 5A and 5B
On the basis of its X-ray analysis, 5A was assigned to
be (aS), and hence, 5B to be (aR) (see Supporting
Information). We examined the stereochemical stability
of the enantiomers (5A and 5B) and found that it was
estimated to be high: racemization occurred after storage
for 48 h at 37 °C in EtOH. Thus, the activation free-energy
barrier to rotation (ΔGq) was calculated to be 106 kJ/mol28
(see Supporting Information).
We next examined the activities of the racemate 5
and each pure enantiomer (5A and 5B) against COX-1/2
(28) Petit, M.; Lapierre, A. J. B.; Curran, D. P. J. Am. Chem. Soc.
2005, 127, 14994–14995.
762
Org. Lett., Vol. 13, No. 4, 2011

In addition, it was revealed that the enantiomer 5B (aR)
shows potent activity against COX-1 with an IC50 value of
15.6 ( 1.8 μM. The other enantiomer 5A (aS) showed no
inhibitory activity at a concentration of 200 μM. The
results clearly indicate that COX-1 recognizes the stereo-
chemistry of the axial chirality arising from the C70-C10
axis. We assumed that C-20 and C-60 disubstituted indole
derivative 5 provided an atropisomeric property that
should be a clue for the design of COX-1/2-selective
indometacin derivatives.
In conclusion, one of the active conformations of in-
dometacin for COX-1 among the numerous possible con-
formations has been revealed by the introduction of
different substituents at the 20- and 60-positions of indo-
metacin (twistedconformation). Thisisthe first example of
introducing atropisomerism to N-benzoylated indole deri-
vatives. We hope that our newly discovered atropo-selective
bioactivity triggers the development of pure atropisomers as
new drug candidates in medicinal chemistry.
Table 1. In Vitro Cyclooxygenase Assay of 5 (Racemate and
Atropisomers)
IC50 (μM)
[R]22D (CHCl3)
COX-1
COX-2
>500
5
13.4 ( 3.4
5A (aS)
-21.2 (c 0.13,
as 96% ee)
>200
>500
5B (aR)
þ20.4 (c 0.10,
as 94% ee)
15.6 ( 1.8
>500
indometacin
0.106 ( 0.042
3.50 ( 041
(Table 1). It was found that the racemate 5 or enantiomers
5A and 5B did not show COX-2-inhibitory activity at a
concentration of 500 μM. On the other hand, the racemate
5 exhibited potent COX-1-selective inhibitory activity with
an IC50 value of 13.4 ( 3.4 μM. The result is attractive
because COX-1 inhibitors have been revaluated recently.14
It appears reasonable to assume that the COX-1 versus
COX-2 selectivity of 5 is closely linked with its conforma-
tion. As pointed out above, there is a difference in the
conformation of indometacin relevant to the interactions
with COX-1 and COX-2. Although the interaction of
indometacin with COX-2 has been determined to be only
the cis conformation,13 indometacin interacts with COX-1
in the cis or trans conformation.12 On the basis of 1H NMR
spectroscopy, compound 5 exists as an equilibrium mix-
ture of cis-like and trans-like conformation (cis-like:trans-
like = 1:1.6) in CDCl3. Such an equilibrium biased toward
the trans-like conformer should be advantageous to COX-1.
Acknowledgment. We thank Sagami Chemical Research
Center for X-ray analysis. This work was supported in part
by a Grant-in-Aid for Young Scientists (B) (21790025) from
the Japan Society for the Promotion of Sciences.
Supporting Information Available. NMR data of 1-5,
NOESY data of 2, analytical study of enantiomers of 4
(4A and 4B) and 5 (5A and 5B), stereochemical stability of
the enantiomers of 5 (5A and 5B), crystal data (CIF file)
for 3 and 5A, in vitro cyclooxygenase assay. This material
is available free of charge via the Internet at http://pubs.
acs.org.
Org. Lett., Vol. 13, No. 4, 2011
763