Active conformation of seven-membered-ring benzolactams as new ACAT inhibitors: Latent chirality at N5 in the 1,5-benzodiazepin-2-one nucleus

Article abstract of DOI:10.1002/chem.201103264

Nitrogen chirality: Potent new ACAT inhibitors with seven-membered-ring benzolactams as the core structures were first prepared, and the axial chirality recognized by the enzyme was clarified (e.g., 1; see scheme). The chirality at the axis (aS) of 1 controls the conformation of the entire lactam ring, causing the N5-CH₃ to arrange in a pseudo-equatorial position (i.e., the amine at N5 is a chiral center with the S-configuration) both in the crystal state and in solution. Copyright

Full text of DOI:10.1002/chem.201103264

DOI: 10.1002/chem.201103264
Active Conformation of Seven-Membered-Ring Benzolactams as New ACAT
Inhibitors: Latent Chirality at N5 in the 1,5-Benzodiazepin-2-one Nucleus**
Hidetsugu Tabata,[a] Naoya Wada,[a] Yuko Takada,[a] Jun Nakagomi,[a] Tomohiro Miike,[b]
Hiroaki Shirahase,[b] Tetsuta Oshitari,[a] Hideyo Takahashi,[a] and Hideaki Natsugari*[a]
The seven-membered-ring benzolactams (1: 1,5-benzodi-
ACHTUNGTRENNUNGazepin-2-one (X=NCH3), 1,5-benzothiazepin-4-one (X=S)
and 1-benzazepin-2-one (X=CH2); Scheme 1) have been
used as the core structures of various biologically active
molecules: lofendazam,[1] diltiazem[2] and benazepril[3] are
the typical therapeutic agents developed using these struc-
tures. In our preceding paper,[4] atropisomerism in the het-
erocycles 1 between A and B (Scheme 1) was investigated
Scheme 1. Atropisomers (A and B) of seven-membered-ring benzolac-
tams (1).
ACTHNUTRGNEUNG
to reveal that the axial chirality[5] at the aryl–N(C=O) (sp2–
sp2 axis) plays a key role in the entire conformational
change of the lactam ring. With various types of information
on these heterocycles in hand, we began research to discov-
er acyl-coenzyme A: cholesterol acyltransferase (ACAT) in-
hibitors. Herein, we report that the enzyme clearly recogniz-
es the active conformation (axial chirality)[6] of the newly
found inhibitors (2–4; Scheme 2), and that the axial chirality
of the 1,5-benzodiazepin-2-one nucleus (2) induces a latent
chiral center at amine N5.
Scheme 2. New ACAT inhibitors: benzolactam-anilides (2–4) and the
lead compounds (I).
The ACAT enzyme, which consists of the two isomeric
forms ACAT-1 and -2, plays an important role in intracellu-
lar cholesterol esterification (in the intestine, liver, and mac-
rophages, and so on). Many potent ACAT inhibitors have
been synthesized in the expectation of their clinical utility in
preventing both hypercholesterolemia and atherosclerosis
by blocking cholesterol esterification. Some, for example,
pactimibe[7] and avasimibe,[8] which are nonselective inhibi-
tors of ACAT-1 and -2, entered clinical trials, but their de-
velopment was halted because of insufficient efficacy in pa-
tients. The search continues for potent ACAT inhibitors, and
recent studies[9] have suggested that ACAT-2 inhibition may
be more beneficial than ACAT-1 inhibition in reducing
plasma lipoprotein cholesterol levels. In our preceding
study, potent ACAT inhibitors with isoquinolone and quina-
zolone nuclei (I in Scheme 2) were prepared.[10] The struc-
ture–activity relationship study indicated that the important
moieties for their activity are a biaryl moiety and an N-(phe-
nyl)acetamide group in the proper spatial arrangement.
In the present study, the heterocycles 1 were incorporated
into the structure (Scheme 2). An exploratory study using
1,5-benzodiazepin-2-one (1: X=NCH3, Y=Ph) showed that
a
derivative with N-(4-fluoro-2-(trifluoromethyl)phenyl)-
AHCTUNGTREGaNNNU cetamide moiety (2), which was prepared from the benzo-
lactam 5a by direct N-alkylation or amidation through the
N-acetic acid derivative (6a; Scheme 3), is a good inhibitor
of ACAT with an IC50 value of 3.5 mm (Table 1).[11] Following
this, 1,5-benzothiazepin-4-one (1: X=S, Y =Ph) and 1-benz-
[a] H. Tabata, N. Wada, Y. Takada, J. Nakagomi, Dr. T. Oshitari,
Prof. Dr. H. Takahashi, Prof. Dr. H. Natsugari
School of Pharmaceutical Sciences, Teikyo University
1091-1 Midori-ku, Sagamihara, Kanagawa 252-5195 (Japan)
Fax : (+81)426-85-3728
AHCTUNGTREaGNNNU zepin-2-one (1: X=CH2, Y=Ph) were used to yield highly
potent inhibitors 3 (IC50 =0.43 mm) and 4 (IC50 =0.068 mm),
respectively (Scheme 3, Table 1). Compound 4, which
showed excellent activity, warrants further biological stud-
ies.[11]
E-mail: natsu@pharm.teikyo-u.ac.jp
[b] T. Miike, Dr. H. Shirahase
The benzolactams (2–4) thus obtained were expected to
exist as racemates of the atropisomers due to the axial chir-
Kyoto Pharmaceutical Industries, Ltd.
38 Nishinokyo Tsukinowa-cho, Nakakyo-ku, Kyoto 604-8444 (Japan)
ality at aryl–NACTHNUTRGNEUNG
(C=O).[4] Since the compounds have a pend-
[**] ACAT=acyl-coenzyme A: cholesterol acyltransferase
ant phenyl adjacent to the lactam moiety, the rotation
around the axis is restricted to form relatively stable atro-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201103264.
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Chem. Eur. J. 2012, 18, 1572 – 1576

COMMUNICATION
B) by preparative HPLC on a chiral stationary phase
(Scheme 3, Table 1). The chiral axis of the atropisomers (A
and B) is not fully frozen but rotates slowly with DG° (the
activation free-energy barrier to rotation) values[12] of
105 kJmolÀ1 for 3A (X=S), 103 kJmolÀ1 for 2A (X=
NCH3), and 102 kJmolÀ1 for 4A (X=CH2) (Table 1).
Next, the atropisomers (A and B) of 2–4 were evaluated
for ACAT inhibitory activity to reveal approximately a 6- to
11-fold difference in potency between the enantiomers
(active eutomer A and less active distomer B),[13] with the
median potency in the racemate (Table 1). The results clear-
ly indicate that axial chirality plays an important role in ex-
erting the activity.[6] It is reasonable to assume that the
enzyme preferentially binds to the eutomer (A) with the
same configuration. To confirm this, the chiroptical proper-
ties (optical rotation ([a]D) and circular dichroism (CD))
were measured.[14] The angle of [a]D of 2A was (+), and
that of 3A and 4A (À) (Table 1). Compounds 3A and 4A
exhibited similar CD spectra with negative bands around
230–240 nm (corresponding to absorption maxima of acetan-
ilide (241 nm) and aniline (230 nm)), whereas 2A showed a
markedly different spectrum with a positive band around
230–240 nm (Figure 1 (red lines) and Table 1). These chirop-
tical data suggest that, contrary to our expectation, the abso-
lute configuration of the eutomer 2 A may be opposite to
that of 3A and 4A. This confusing situation was finally re-
solved by X-ray structure analysis of eutomers 2A and 3A,
which revealed that, as was expected from the activity data,
both compounds possess the same stereochemistry with the
(aS)-configuration.[15] The configuration of eutomer 4A,
which showed chiroptical properties similar to those of 3A,
was thus deduced to be (aR).[16]
The X-ray structures of the eutomers 2A–4A are shown
in Figure 2. In a unit cell of compound 2A, two independent
molecules (Mol. I and II) are present which have similar
structures. The structure of 4A in Figure 2 is that with the
(aR)-form[16] extracted from the X-ray CIF data of the race-
mate 4. The conformations of the three (four) structures are
similar in that the lactam ring exists in a twisted-boat form
and the pendant phenyl is in a perpendicular alignment rela-
tive to the benzene ring. On the other hand, the benzanilide
moiety in the side chain adopts a different conformation in
the position and orientation among the three compounds,
which may cause the activity difference.
Scheme 3. Synthesis of racemic benzolactam-anilide derivatives (2–4/2’–
4’) and their separation using chiral HPLC into the atropisomers (A and
B).
Table 1. Properties of 2–4 (racemates and atropisomers): ACAT inhibito-
ry activity, chiroptical properties and thermal stability of the enantio-
mers.
[b]
Compound IC50 [mm] [a]D
CD[c]
DG°[d]
[kJmolÀ1
]
Racemization
(axial
ACAT[a]
(CH3OH)
U
t[e] [h]
chirality)
2: racemate
A (aS)
B (aR)
3: racemate
A (aS)
B (aR)
3.5
2.0
11
0.43
0.19
2.1
+51.0[f]
À58.2[h]
+ +
À À
103[g]
103[i]
5[g]
5[i]
À16.3
+19.7
À À À 105[j]
7[j]
+ + +
4: racemate
A (aR)[k]
B (aS)[k]
0.068
0.038
0.26
À34.3
+36.1
À À À 102[j]
2[l]
+ + +
[a] Hepatic ACAT (rabbit liver microsomes). IC50 of pactimibe[7] (as a
control compound): 1.4 mm. See the Supporting Information for details.
[b] See the Supporting Information for each concentration (c=0.11–
0.27 gmLÀ1). [c] Signs and intensities of Cotton Effect around 230–
240 nm measured in CH3OH. See Figure 1 and Table S2 (the Supporting
Information) for detail. [d] Activation free-energy barrier to rotation
À
X-ray analysis of the 1,5-benzodiazepin-2-one 2A re-
vealed particularly interesting structural features. The nitro-
gen at the 5-position takes a slightly twisted pyramidal
form,[17] and the N5 methyl exists in a pseudo-equatorial
(pseudo-eq) position with the lone pair in a pseudo-axial
(pseudo-ax) position (i.e., the N5-S configuration) in the
crystal state, which should be a stable conformation. At first
glance at the planar structure of 2A, a hydrogen bond[18] be-
tween the amide-NH and lactam-oxygen (NH···O) may be
envisioned. However, the X-ray structure of 2A had a hy-
drogen bond between the amide-NH and N5-nitrogen
(NH···N) (Table 2): bond distance (NH···N), 2.18 ꢁ for Mol.
I, and 2.43 ꢁ for Mol. II. The hydrogen bond NH···S
around the Ar NACHTUNGTRENNUNG
(C=O) bond.[12] [e] Time required for racemization at
508C in toluene or DMF. See the Supporting Information for details.
[f] In CHCl3, +49.8 (c=0.30). [g] Conversion from A to B in DMF. [h] In
CHCl3, À49.9 (c=0.30). [i] Conversion from B to A in DMF. [j] Conver-
sion from A to B in toluene. [k] For the definition of the axial chirality
(aS and aR), see ref. [16]. [l] Racemization occurred for 7 h when heated
at 378C in toluene.
pisomers, whose absolute stereochemistry would be recog-
nized by the ACAT enzyme. The inhibitors (2–4) were suc-
cessfully separated into the respective enantiomers (A and
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1573

H. Natsugari et al.
may further constrain the stable conformation described
above. Since the chemical shift of the amide-NH of 2A is
1
observed at quite a low field (d=9.87 ppm) in the H NMR
spectrum, the same structure is assumed to exist in solution
as well. These results mean that, due to the rigid conforma-
tion, the N5-nitrogen in 2A may exist as a chiral center
(with the S-configuration) not only in the crystal state but
also in solution. The difference in chiroptical properties be-
tween 2A and 3A/4A may be explained by this additional
chirality in 2A in solution. In terms of biological activity,
the hydrogen bond NH···O in 4A may cause the preferred
conformation of the side chain by controlling the spatial po-
sition and orientation of the benzene ring to exhibit excel-
lent activity. At this point, the conformation in 2A with the
hydrogen bond NH···N may not be favored.
To examine the effect of the hydrogen bond on the con-
formation, the amide-NCH3 derivatives of 2–4 (2’–4’) were
prepared (Scheme 3)[19] and separated into the respective
enantiomers (A and B), and the CD spectra were measured
(Figure 1, purple and green lines). Compound 2’A, which
lacks the hydrogen bond, caused a large change in the spec-
tral pattern compared with 2A, whereas the spectra of 3’A/
4’A remained similar to those of 3A/4A. Taking into con-
sideration the markedly different spectral pattern of 2’A
from those of 3’A/4’A, it is reasonable to assume that the
chirality at the N5-nitrogen in 2A remains. This may be fur-
1
ther supported by the H NMR spectra of 2A and 2’A, in
which the coupling patterns of the methylene protons in the
lactam ring remain similar, suggesting that the conformation
of the seven-membered ring itself remains unchanged in 2A
and 2’A regardless of the hydrogen bond.[20] Thus, the (aS)-
atropisomer is assumed to take a predominantly stable iso-
meric form with the N5-CH3 disposed in the pseudo-equato-
rial conformation (i.e., the amine at N5 is a chiral center
with the S-configuration), as shown in Scheme 4 (upper):
that is, the axial chirality (aS) controls the stereochemistry
of the entire lactam ring. In other words, both chiralities
may move together like a gear. It should be noted that the
preferred isomer has stereochemistry lacking steric repul-
sion between N5-CH3 and the substituent (Z) at the lactam
nitrogen, as shown in Scheme 4.
Figure 1. CD spectra of the atropisomers (A and B) of the benzolactam-
anilide derivatives (2–4) and their N-CH3 derivatives (2’–4’).
(2.58 ꢁ) was also observed in 3A. In contrast, 4A had the
hydrogen bond NH···O (2.07 ꢁ). The presence of these hy-
drogen bonds is also supported by the bond angles of N-H-
X/N-H-O (Table 2). The hydrogen bond in 2A (NH···N)
Figure 2. X-ray structures of atropisomers (eutomers: 2A, 3A and 4A) generated from the CIF data. In a unit cell of compound 2 A, two independent
molecules (Molecules I and II) are present. The structure of 4 A is extracted from the CIF data of the X-ray analysis of the racemate of 4.[16] Hydrogen
bonds are indicated with dotted lines.
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Seven-Membered-Ring Benzolactams as New ACAT Inhibitors
COMMUNICATION
ported CD spectra of 7[1] and 2’B showed a similar
pattern,[23] suggesting that the two molecules have a
similar molecular chirality also in solution.
Table 2. Hydrogen bond in 2A, 3A, and 4A: in crystal state (distance and bond
angle) and in solution (chemical shift).
In conclusion, the potent new ACAT inhibitors
2–4 with the seven-membered-ring benzolactams as
the core structures were first prepared, and the
axial chirality recognized by the enzyme was clari-
fied. The unique structural feature of the 1,5-benzo-
diazepin-2-one nucleus (2A) was revealed by CD,
1H NMR spectroscopy and X-ray structural analy-
ses; that is, the chirality at the axis (aS) controls the
conformation of the entire lactam ring, causing N5-
CH3 to arrange in the pseudo-equatorial orientation
(=N5-S configuration). We hope that these stereo-
chemical findings in seven-membered-ring benzo-
lactams will assist in future drug design in which
heterocyclic systems are utilized as the core struc-
ture for biologically active molecules.
NH···X
A
N-H-X
(angle)[b] [8]
NH···O
(distance)[a] [ꢁ]
N-H-O
A
NH[c]
9.87
G
E
(angle)[b] [8] d [ppm]
2A-I[d]
2.18
2A-II[d] 2.43
154
161
156
88
3.57
3.28
3.08
2.07
116
99
110
151
3A
2.58
3.85
9.19
8.22
4A[e]
[a] Distance estimated from the X-ray crystal data. [b] Angle estimated from the X-
ray crystal data. [c] Chemical shift of amide-NH (in CDCl3) in 1H NMR spectrum.
[d] Two independent molecules exist in a unit cell of crystal 2 A (Mol. I and II).
[e] The data obtained from the X-ray analysis of the racemate 4.
Acknowledgements
We are grateful to Sagami Chemical Research Center for X-ray analysis.
This work was supported in part by a Grant-in-Aid for Scientific Re-
search (C) (21590124) and a Grant-in-Aid for Young Scientists (B)
(21790025) from the Japan Society for the Promotion of Sciences.
Keywords: atropisomerism · chirality · circular dichroism ·
lactams · medium-ring compounds
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Scheme 4. Stereochemistry of 1,5-benzodiazepin-2-ones: 2A/2’A and 7.
Any atom that is tetrahedral with four different groups
has the potential to be stereogenic, and pyramidal atoms
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atoms do not invert, these molecules will be chiral. Thus,
small amines have the potential to be stereogenic, although
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entire conformation of the ring is rigidly held so as to adopt
the C4-R-methyl in the most stable conformation. The re-
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[14] See the Supporting Information for CD and UV data and spectra.
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(X=CH2) according to the priority rule. The terms aS and aR
(chiral axis nomenclature) correspond to P and M (helix nomencla-
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[17] See the Supporting Information for the bond angles around the N5-
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[19] Compounds 2’–4’ were shown to exist as a mixture of E- and Z-con-
formers at the N-CH3 amide moiety in the 1H NMR spectra (ratio:
4.4–5.0:1). See the Supporting Information for details.
[11] The assay for the ACAT inhibitory activity shown in Table 1 is that
using rabbit liver microsomes, which is regarded as the assay for
ACAT-2 isoform. By this assay, the intrinsic enzyme inhibitory activ-
ity of the compound is well evaluated. Another assay using esteri-
fied cholesterol (EC) accumulation in THP-1 cell-derived macro-
phages, which is regarded as the assay for ACAT-1 isoform, was also
performed. In this EC accumulation assay, a similar pattern of the
difference in potency between A (eutomer) and B (distomer) was
also observed. See the Supporting Information for details.
[12] The DG° value was determined based on the time-dependent con-
version rate (% ee) estimated from chiral HPLC analysis of a solu-
tion of the enantiomers after allowing them to stand at designated
temperatures (see Supporting Information for measurements); see
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127, 14994–14995.
[20] See the Supporting Information for detailed 1H NMR data on 2A
and 2’A, including a magnified view of the methylene region.
[21] D. Didier, B. Tylleman, N. Lambert, C. M. L. V. Velde, F. Blockhuys,
A. Collas, S. Sergeyev, Tetrahedron 2008, 64, 6252–6262.
[22] M. J. Dearden, C. R. Firkin, J.-P. R. Hermet, P. OꢂBrien, J. Am.
Chem. Soc. 2002, 124, 11870–11871.
[23] The Cotton effects in the CD spectrum [lnm (De)] for 7,[1] 306 (+
1.76), 281 (À1.30), 259 (+1.98) (sh), 241 (+3.68), 231 (À5.21), 212
(+3.80), and those for the de-chloro derivative of 7,[1] 299 (+1.74),
270 (À2.05), 237 (+5.26), 226 (À3.10), 211 (+1.75).
Received: October 17, 2011
Published online: December 28, 2011
1576
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Chem. Eur. J. 2012, 18, 1572 – 1576