Tolvaptan-Type Vasopressin Receptor Ligands: Important Role of Axial Chirality in the Active Form

Article abstract of DOI:10.1021/acs.jmedchem.7b00422

The anti and syn isomers of tolvaptan-type compounds, N-benzoyl-5-hydroxy-1-benzazepines (5a-c), were prepared in a stereocontrolled manner by biasing the conformation with a methyl group at C9 and C6, respectively, and the enantiomeric forms were separat

Full text of DOI:10.1021/acs.jmedchem.7b00422

Brief Article
pubs.acs.org/jmc
Tolvaptan-Type Vasopressin Receptor Ligands: Important Role of
Axial Chirality in the Active Form
Hidetsugu Tabata, Tetsuya Yoneda, Tetsuta Oshitari, Hideyo Takahashi, and Hideaki Natsugari*
Faculty of Pharma Sciences, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan
S
* Supporting Information
ABSTRACT: The anti and syn isomers of tolvaptan-type
compounds, N-benzoyl-5-hydroxy-1-benzazepines (5a−c),
were prepared in a stereocontrolled manner by biasing the
conformation with a methyl group at C9 and C6, respectively,
and the enantiomeric forms were separated. Examination of
the affinity at the human vasopressin receptors revealed that
the axial chirality (aS) plays a more important role than the
central chirality at C5 in receptor recognition, and the most
preferable form was shown to be (E,aS,5S).
INTRODUCTION
■
The nonpeptide arginine vasopressin (VP) V2 receptor
antagonists1 have been developed as the “vaptan” class of
ligands for the treatment of hyponatremia, congestive heart
failure, and other diseases associated with water homeostasis
disturbance. Among the VP antagonists, tolvaptan2 and
mozavaptan3 (Figure 1) are representative: both contain a
Figure 2. Stereoisomers [E/Z-amide rotamers and (aS)/(aR)-axial
isomers] in 1−3. The Z-amide isomers shown in the brackets were
1
negligible in the H NMR spectrum.
Figure 1. Nonpeptide VP receptor antagonists.
amide isomers, compounds 1−3 all exist predominantly in the
E-form, and the Z-isomer was negligible in the 1H NMR
spectrum.10 Thus, 1−3 exist only as a mixture of (aS)/(aR)-
axial isomers (Figure 2).
preserved scaffold, i.e., 1-benzazepine linked through N-1 to a
substituted benzoyl group (blue lines in the structures in Figure
1), and the characteristic feature is a substituent (OH and
NMe2) added to the 1-benzazepine nucleus at C5. Interest in
The molecules with a substituent at the ortho-position
(C9)11 on the benzene ring (e.g., CH3) were conformationally
“frozen” and could be separated into the (aS)- and (aR)-axial
isomers using chiral HPLC, although the conformational
VP antagonists is still growing in the search for new ligands,
their new indications, and in the biological role of the V1a and
V1b subtype receptors.1 A topic of recent interest was the
change in the molecules without the substituent at C9 was
too rapid for isolation of the isomers (i.e., achiral form) at room
temperature (rt). Those studies led to the finding of the
importance of axial chirality in exerting the biological activity of
the vaptan class of VP receptor ligands, exhibiting the active
approval of tolvaptan as the first drug for autosomal-dominant
polycystic kidney disease (ADPKD) in Japan.4
In our previous papers,5,6 atropisomerism in N-benzoyl-1,5-
benzodiazepines (1, X = NCH3)5 and N-benzoyl-1,5-
benzothiazepines (2, X = S; 3, X = S*=O)6 as VP receptor
structure (eutomer) with the (E,aS)-form of the types 1 and 2
(Figure 2).5,6
ligands was described (Figure 2), in which the E/Z-amide
rotamers7 around the N−C(=O) bond and (aS)/(aR)-axial
isomers8 based on the Ar−N(CO) (sp2−sp2) axis were
considered9 (Figure 2). In general, however, for the E/Z-
Received: March 17, 2017
Published: May 5, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.7b00422
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
Whereas 1 and 2 possess no central chirality, tolvaptan and
mozavaptan (Figure 1) possess central chirality at C5 bearing a
substituent and are developed as racemates. Thus, in these two
vaptans, four stereoisomers originating in two stereogenic
elements at C5 and the axis are presumed to exist, i.e., (aR,5S)/
(aS,5R) and (aS,5S)/(aR,5R) (all adopt the E-amide con-
formation) (Figure 3). The former and latter pairs of
prepared using conventional methods starting from 5-oxo-1-
benzazepine derivatives (6a−c), as shown in Scheme 1. First,
Scheme 1. Preparation of Tolvaptan-Type N-Benzoyl-5-
hydroxy-1-benzazepines (5a−c) and Separation into the
a
Enantiomers
a
Only the major anti (aR*,5S*) isomer of 5c was isolated in the pure
form from the anti/syn mixture.
Figure 3. Anti/syn diastereomers and the enantiomers in (E)-N-4-[(2-
methylbenzoyl)amino]benzoyl-1,5-benzothiazepine S-oxides (3) and
(E)-5-hydroxy-N-(p-toluoyl)-1-benzazepines (4).
N-benzoylation of 6a−c using p-nitrobenzoyl chloride was
followed by catalytic reduction using Pd/C to give the p-
aminobenzoyl compounds 7a−c. Then, amidation of 7a−c with
o-toluoyl chloride followed by reduction of the carbonyl group
at C5 with NaBH4 furnished the racemic N-benzoyl-5-hydroxy-
1-benzazepine derivatives (5a−c).16 As for the E/Z-amide
isomers,7 5a−c all exist predominantly in the E-form, and the
stereoisomers correspond to the anti and syn diastereomers,
respectively: the description “anti/syn” is used for the relative
arrangement of the C5 substituent and the N-benzoyl group,
i.e., anti and syn denote the arrangement on the opposite and
the same side, respectively. To the best of our knowledge,
however, the active stereochemistry of the vaptans (i.e.,
tolvaptan, mozavaptan) including the central chirality at C5
has not been discussed thoroughly to date.12,13
1
Z-isomer was negligible in the H NMR spectrum. Thus, in
these compounds, the anti (aR*,5S*) and syn (aS*,5S*)
diastereomers with the E-amide form are presumed to exist.
In 5a (R1 = R2 = H), the ring inversion via rotation around
the Ar−N(C=O) axis readily occurs to form anti/syn
diastereomers in solution at rt [1:0.31 as determined by the
1H NMR spectrum in DMSO-d6)],17 which are inseparable by
HPLC. Meanwhile, in 5c (R1 = H, R2 = CH3), the rotation
around the axis is restricted at rt by the ortho substituent (C9-
CH3) to form separable anti/syn diastereomers with a ratio of
1:0.17, from which the major anti (aR*,5S*) isomer could be
isolated in the pure form with HPLC using a nonchiral column.
Attempted isolation of the minor syn (aS*,5S*) isomer in the
pure form failed because the retention times of the anti/syn
isomers are too close for separation (isolation) of the minor syn
isomer. The anti/syn stereochemistry of 5a and 5c was
In our previous paper,6 we prepared (E)-N-4-[(2-
methylbenzoyl)amino]benzoyl-1,5-benzothiazepine S-oxides
(3), examined the in vitro affinities at the VP receptors, and
proposed that the most active stereochemistry of 3 might be in
the (aS,5S)-form (3b) (Figure 3). However, the conclusion was
not definite because the affinities (Ki values) were weak (at
micromolar level). On the other hand, recently we have
revisited the basic stereochemistry of N-benzoyl-1-benzazepines
with a substituent (CH3, OH, NMe2) at C5 (e.g., 4 in Figure
3),14 for which the stereochemistry in the solution state was
previously ambiguously reported,15 to reveal new physicochem-
ical aspects of these heterocycles including revision of the
reported15 stereochemistry. In this paper, using the stereo-
chemical information obtained in 4, we synthesized the anti and
syn diastereomers of the tolvaptan-type compounds (5-
hydroxy-N-benzoyl-1-benzazepine derivatives) (5a−c)
(Scheme 1) in stereocontrolled manners, and examination of
the affinities at the human VP receptors for the enantiomers
revealed that axial chirality present in the latent form in the
molecule plays a more important role than the central chirality
at C5 in receptor recognition. The results are presented here.
1
confirmed by comparisons with the H NMR spectral data
on the corresponding N-(p-toluoyl) derivatives of 5-hydroxy-1-
benzazepines (4) previously reported.14
On the other hand, in 5b (R1 = CH3, R2 = H), because of the
steric hindrance caused by the C6 methyl group (allylic 1,3-
strain), the C5 hydroxy group is confined so as to adopt a
sterically less hindered axial (ax) orientation, forming the syn
(aS*,5S*) isomer exclusively (Figure 4). The syn stereo-
chemistry of 5b was confirmed by comparisons with the
analytical data on the 5-hydroxy-6-methyl-N-(p-toluoyl)-1-
benzazepine (4) previously reported.14
CHEMISTRY
■
Preparation of N-Benzoyl-5-hydroxy-1-benzazepine
Derivatives (5a−c) and Separation to the Enantiomers.
N-Benzoyl-5-hydroxy-1-benzazepine derivatives (5a−c) were
The racemic N-benzoyl-5-hydroxy-1-benzazepine derivatives
(5a−c) thus obtained were successfully separated and isolated
B
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Journal of Medicinal Chemistry
Brief Article
Figure 4. Syn (aS*,5S*) form of N-benzoyl-5-hydroxy-6-methyl-1-
benzazepine (5b): because of the steric hindrance (A1,3-strain), the 5-
OH group adopts a sterically less hindered ax orientation to form the
syn isomer.
into the enantiomers with HPLC using a chiral column15
(Scheme 1). The chiroptical properties [optical rotation ([α]D)
and circular dichroism (CD)] of the separated enantiomers
were examined and the data are shown in Figure 5. The
Figure 6. X-ray crystal structures of the optically active compounds
(+)-(E,aS,5R)-5a (= anti) and (+)-(E,aS,5S)-5b (= syn).
thermodynamically unstable, with the 5-hydroxy group in the
ax position in a chairlike form. As for the absolute
stereochemistry of 5c (major diastereomer), although the X-
ray data could not be obtained, (+)-5c was deduced to be
(E,aS,5R) by comparison with the chiroptical properties (CD
and [α]D) of (+)-5a and (+)-5b.19
RESULTS AND DISCUSSION
■
Affinity at the V1a and V2 Receptors (Table 1). The in
vitro affinities at the human VP V1a and V2 receptors of 5a−c
are shown in Table 1. First, 5a−c in the racemic forms were
evaluated. All compounds showed good potency in the binding
experiment with the affinity (Ki) at the nanomolar to
submicromolar level. Interestingly, 5b and 5c, which bear a
methyl group in the benzene ring, have a tendency to bind
Table 1. In Vitro Affinity for Human VP V1a and V2
Receptors of 5a−c
20
Figure 5. Chiroptical properties, [α]D (in CHCl3) and CD (in
CH3OH) spectra of the separated enantiomers of 5a−c.
a,b
chirality
axis
Ki (nM)
c
c
compd [α]D rotation
C5
hV1a
hV2
enantiomers of 5a−c with the (+)-angle of [α]D exhibited
similar CD spectra (blue lines) with positive bands around
250−320 nm, and those with the (−)-angle of [α]D exhibited
similar CD spectra (red lines) with negative bands around
250−320 nm.
Fortunately, the enantiomers (+)-5a and (+)-5b could be
subjected to X-ray structural analysis,18 which revealed that the
absolute stereochemistry of (+)-5a is (E,aS,5R) and that of
(+)-5b is (E,aS,5S) (Figure 6). The X-ray structural analysis of
(+)-5a indicated that, although (+)-5a exists as an inseparable
equilibrium state of a mixture of anti and syn (1:0.31) in
solution (NMR) as described above, in the solid state it takes
the thermodynamically stable anti form with the 5-hydroxy
group in the equatorial position in a chairlike form.
5a
( )
(−)
(+)
( )
(+)
(−)
( )
(−)
(+)
S*
S
aR*/aS* (1:0.31)
aR/aS (1:0.31)
1.58
1.85
1.03
6.75
2.06
7.56
4.87
8.30
R
aS/aR (1:0.31)
5b
S*
S
aS*
aS
160
46
1790
170
54
R
aR
78
d
5c
S*
S
aR*
aR
6.26
4.68
2.84
R
aS
140
a
b
Inhibition assays were performed for 2 h at 25 °C in 1% DMSO. Ki
values shown are the means of duplicate measurements. For details,
see the Experimental Section. Major anti [(aR,5S)/(aS,5R)]
diastereomer. The minor syn [(aS,5S)/(aR,5R)] isomers could not
be isolated because of the separation difficulty from the major anti
isomer.
c
d
In contrast, (+)-5b was shown to exist in both solid and
solution state solely as the syn form, which is apparently
C
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Journal of Medicinal Chemistry
Brief Article
selectively to V1a (at the nanomolar level) rather than to V2
receptors (at the submicromolar level). The excellent V1a-
selective affinity may make them worthy candidates for further
biological studies. Next, the separated enantiomers of 5a−c
were subjected to the binding assay to examine the difference in
potency between the enantiomers. However, contrary to our
expectation, in 5a (R1 = R2 = H) and 5c (R1 = H, R2 = CH3),
the enantiomers and the racemate exhibited similar levels of
affinity (within a 3-fold difference) (Table 1). In contrast, 5b
(R1 = CH3, R2 = H) showed a ca. 40-fold difference in potency
between the enantiomers [active eutomer: (+)-(aS,5S) (syn)-5b
and less active distomer: (−)-(aR,5R) (syn)-5b)] with median
potency in the racemate (Table 1).
Active Molecular Forms Recognized by the VP
Receptors. The clear difference in potency between the
enantiomers of 5b (a ca. 40-fold difference) indicates that the
stereochemistry at the scaffold region, i.e., (E,aS,5S), is
recognized by VP receptors. That the (aS)-form is important
in exerting the activity is in accordance with the VP ligands of
N-benzoyl-1,5-benzodiazepine (1) and N-benzoyl-1,5-benzo-
thiazepine (2) (Figure 2) we previously reported.5,6 As for the
stereochemistry at C5 of 5b, (5S) was shown to be the
preferable form for binding with receptors, and it may be
presumed that the (E,aS,5S)-form is the preferable and the
(E,aR,5R)-form the least preferable among the four stereo-
isomers present in 5a−c.
Figure 7. Conversion of (aR,5S) (= anti)-5c to (aS,5S) (= syn)-5c. For
the conversion profile, see Figure S1 in the Supporting Information.
at the similar level as that of the enantiomer (aS,5R)-5c.21
Meanwhile, as for the enantiomer (aS,5R)-5c, the conversion to
(aR,5R)-form would give little effect on the observed Ki value
because the affinity of the (aR,5R)-form may be too weak.
All the above results support the assumption that the (E,aS)-
stereochemistry of the tolvaptan-type of N-benzoyl-5-hydroxy-
1-benzazepine derivatives (5a−c) is the preferable form and the
(E,aS,5S)-form is the most preferable among the four
stereoisomers for recognition by VP receptors (Figure 8): the
However, the results obtained in 5a and 5c, in which the
enantiomers and the racemate exhibited similar levels of
affinity, led to confusion in the analysis of the active
stereochemistry. We considered the following to explain the
results.
In 5a, the ring inversion via rotation around the axis readily
occurs at rt in the solution state to form inseparable anti/syn
diastereomers in a ratio of 1:0.31 [i.e., (aR,5S)/(aS,5S) for
(5S)-5a and (aS,5R)/(aR,5R) for (5R)-5a, each in a ratio of
1:0.31], meaning that the (aS)-form, which we presume is the
active form, is present in the binding experiment in both the
(5S)- and (5R)-forms of 5a. The observed similar level of
affinity between (5S)-5a and (5R)-5a may imply that, although
the (aS,5S)-form exists in (5S)-5a as the minor diastereomer in
solution, it should contribute to exhibit the similar level of
affinity as that of (5R)-5a because it possesses the strongest
affinity among these four isomers.
On the other hand, in the major anti (aS*,5R*)-5c, while the
affinity of the enantiomer (aS,5R)-5c was envisioned to be
reasonably estimated, the affinity of the other enantiomer
(aR,5S)-5c was unexplainable because it does not take the (aS)-
form, which we presume is the active form. Thus, we speculated
that the affinity observed in (aR,5S)-5c is ascribed to the minor
syn diastereomer (aS,5S)-5c, which would be formed from the
(aR,5S)-5c during the binding experiment (for 2 h at 25 °C).
To confirm the conversion, the energy barrier between the two
diastereomers (aR,5S) and (aS,5S) of 5c was examined. The
time-dependent conversion rate [from (aR,5S)-5c to (aS,5S)-
5c] was estimated from chiral HPLC analysis of a solution of
the enantiomer in toluene after allowing it to stand at 25 °C.
The conversion reached an equilibrium state of (aR,5S)/
(aS,5S) = 1:0.17 after 4 h, and the ΔG⧧ value20 was determined
to be 102 kJ/mol (Figure 7). The results indicate that the
conversion from the (aR,5S)-form to (aS,5S)-form actually
occurred during the binding experiment; if the amount of
(aS,5S)-5c (the most potent isomer) formed from (aR,5S)-5c is
10−15%, the observed Ki value of (aR,5S)-5c is possible to be
Figure 8. Four stereoisomers of the tolvaptan-type VP ligands
(tolvaptan and 5a) with a C5-hydroxy group. For receptor recognition,
the (E,aS,5R)-stereochemistry is preferable and the (E,aS,5S)-form is
the most preferable. For the exact structure of tolvaptan, see Figure 1.
two X-ray crystal structures shown in Figure 6 are the desirable
forms with the (aS)-stereochemistry. Thus, the axial chirality
present in the latent form in the molecule plays a more
important role than the central chirality at C5 in receptor
recognition.
CONCLUSION
■
The anti and syn isomers of the tolvaptan-type compounds N-
benzoyl-5-hydroxy-1-benzazepines (5a−c) were successfully
prepared in a stereocontrolled manner by biasing the
conformation with a methyl group at C9 and C6, respectively,
and the enantiomeric forms were separated. Examination of the
affinity at the VP receptors revealed that the axial chirality (aS)
plays a more important role than the central chirality at C5 in
receptor recognition, and the (E,aS,5S)-form is the most
preferable. Sometimes, and somewhat peculiarly, biologically
active compounds bearing central chirality in their molecules
exhibit similar levels of activity regardless of the chirality caused
by the chiral center. In such cases, the activity may be due to
the latent chirality caused by dynamic conformational changes
at different sites of molecules, as was observed in 5a and 5c in
this investigation. This paper suggests the importance of the
detailed conformational analysis of the entire molecule, and the
D
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Journal of Medicinal Chemistry
Brief Article
Single-Crystal X-ray Analysis. The crystal structures of the
optically active compounds [(+)-(aS,5R)-5a (= anti) and (+)-(aS,5S)-
5b (= syn)] were obtained by single-crystal X-ray analysis (Figure 6).
Typical crystal data for (+)-(aS,5R)-5a and (+)-(aS,5S)-5b are shown
in the Supporting Information.
results may provide useful information for future drug design of
biologically active compounds.
EXPERIMENTAL SECTION22
■
Stereochemical Stability of (−)-(aR,5S)-5c. The partial isomer-
ization of (−)-(aR,5S)-5c (= anti) at 25 °C in toluene to the
diastereomer (a S,5S)-5c (= syn) was examined as previously
described.5 The conversion profile analyzed by a chiral HPLC is
shown in Figure 7. The ΔG⧧ value20 was 102 kJ/mol. The method for
determination of ΔG⧧ is shown in the Supporting Information.
Procedure for in Vitro Binding Assay to Human V1a and V2
Receptors. All test compounds (shown in Table 1) had a purity of
≥99% as determined by HPLC analyses. The assays were performed
according to the reported procedures.23,24 (1) Binding assay to hV1a:23
Human recombinant vasopressin V1a receptors expressed in HEK-293
cells were used in assay buffer (50 mM Tris-HCl, pH 7.4, 5 mM
MgCl2, 0.1% BSA). A 0.26 μg aliquot was incubated with 0.03 nM
5-Hydroxy-N-[4-[(2-methylbenzoyl)amino]benzoyl]-2,3,4,5-
tetrahydro-1-benzazepine (5a). Compound 5a (racemate) was
prepared according to the procedure previously reported3 starting
from 5-oxo-1-benzazepine (6a) via N-(p-aminobenzoyl)-5-oxo-1-
benzazepine (7a) as shown in Scheme 1. Because the physicochemical
data of 5a including the 1H NMR spectral data were only briefly
reported,3,12b in this paper the data were precisely examined to reveal
the exact stereochemistry. The detailed 1H NMR spectral data in
DMSO-d6 is shown in the Supporting Information, which indicates
that 5a exists as an equilibrium mixture of anti/syn isomers with a
1:0.31 ratio in solution.17
5-Hydroxy-6-methyl-N-[4-[(2-methylbenzoyl)amino]-
benzoyl]-2,3,4,5-tetrahydro-1-benzazepine (5b). 6-Methyl com-
pound (5b) was prepared from 6-methyl-5-oxo-1-benzazepine (6b)14
via 7b as shown in Scheme 1. The procedure is as follows. Step 1: To a
stirred solution of 6b (275 mg, 1.57 mmol) in THF (10 mL) at 0 °C
under argon, sodium hydride (60% in oil) (125 mg, 3.14 mmol) was
added. The mixture was stirred at 25 °C for 30 min, cooled to 0 °C,
and treated with 4-nitrobenzoyl chloride (873 mg, 4.71 mmol). After it
was stirred at 25 °C for 15 h, the mixture was treated with H2O and
extracted with ethyl acetate. The extract was washed with brine, dried
over Na2SO4, and concentrated. The concentrate was purified by
column chromatography (silica gel, ethyl acetate/hexane = 1:4) to
afford 6-methyl-N-(4-nitrobenzoyl)-5-oxo-1-benzazepine (S1b) as
white solids (506 mg, 1.56 mmol, 99%). To a stirred solution of
S1b thus obtained (451 mg, 1.39 mmol) in EtOH (18 mL) was added
palladium on carbon (10% Pd) (29 mg). After the mixture was stirred
at 25 °C under a hydrogen atmosphere for 12 h, Pd was removed by
filtration, and the filtrate was concentrated. The concentrate was
purified by column chromatography (silica gel, ethyl acetate/hexane =
1:3) to afford N-(p-aminobenzoyl)-6-methyl-5-oxo-1-benzazepine
(7b) as pale-yellow solids (373 mg, 1.27 mmol, 91%).
Step 2: To a stirred solution of 7b (326 mg, 1.11 mmol) in CH2Cl2
(11 mL) was added EtN3 (185 mg, 1.83 mmol) and o-toluoyl chloride
(205 mg, 1.33 mmol). After the mixture was stirred at 25 °C for 17 h,
water was added to the mixture. The mixture was extracted with
CH2Cl2. The extract was washed with 1 N HCl, saturated NaHCO3,
and brine, dried over Na2SO4, and concentrated. The concentrate was
purified by column chromatography (silica gel, ethyl acetate/hexane =
1:4) to afford 6-methyl-N-(o-methylbenzoyl)aminobenzoyl-5-oxo-1-
benzazepine (S2b) as white solids (454 mg, 1.10 mmol, 99%). To a
solution of S2b thus obtained (171 mg, 0.414 mmol) in MeOH (6.0
mL) was added NaBH4 (24 mg, 0.621 mmol). After the mixture was
stirred at 25 °C for 2 h, solvent was evaporated. The residue was
diluted with CH2Cl2, washed with brine, dried over MgSO4, and
concentrated. The concentrate was purified by column chromatog-
raphy (silica gel, ethyl acetate/hexane = 1:2) to afford 5b (syn form)
(racemate) as colorless crystals (144 mg, 0.347 mmol, 84%).16
5-Hydroxy-9-methyl-N-[4-[(2-methylbenzoyl)amino]-
benzoyl]-2,3,4,5-tetrahydro-1-benzazepine (5c). 9-Methyl deriv-
ative (5c) (racemate) was prepared from N-(p-aminobenzoyl)-9-
methyl-5-oxo-1-benzazepine (6c)14 via 7c according to a similar
procedure described for the preparation of 5b from 6b via 7b.
Compound 5c was formed as an anti/syn mixture with a ratio of ca.
1:0.17, from which only the major anti isomer could be isolated in the
pure form with HPLC using a nonchiral column (YMC SIL-06).16 The
physicochemical data of 5b, 5c, and the intermediates (S1b, S1c, 7b,
7c, S2b, and S2c) are shown in the Supporting Information.
[
125I] phenylacetyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 for
2 h at 25 °C. Nonspecific binding was estimated in the presence of 1
μM (Arg8)-vasopressin. Receptors were filtered and washed, and the
filters were then counted to determine [125I] phenylacetyl-D-Tyr(Me)-
Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 specifically bound.
(2) Binding assay to hV2:24 CHO-K1 cells stably transfected with a
plasmid encoding human vasopressin V2 receptors were used to
prepare membranes in assay buffer (50 mM Tris-HCl, pH 7.4, 10 mM
MgCl2, 0.1% BSA) using standard techniques. A 9 μg aliquot of
membrane was incubated with 4 nM [3H] (Arg8)-vasopressin for 2 h
at 25 °C. Nonspecific binding was estimated in the presence of 1 mM
(Arg8)-vasopressin. Membranes were filtered and washed three times,
and the filters were counted to determine [3H] (Arg8)-vasopressin
specifically bound.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.7b00422.
General experimental procedure, 1H-, 13C-, and 2D-
NMR spectra and physicochemical properties for new
compounds and stereochemical stability of (−)-(aR,5S)-
5c (PDF)
Molecular formula strings (CSV)
X-ray crystal data for (+)-(aS,5R)-5a (CIF)
X-ray crystal data for (+)-(aS,5S)-5b (CIF)
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +81-42-685-3728. Fax: +81-42-685-3728. E-mail:
natsu@pharm.teikyo-u.ac.jp.
ORCID
Hideaki Natsugari: 0000-0002-6442-1598
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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Separation of the N-Benzoyl-5-hydroxy-1-benzazepines
(5a−c) into Enantiomers. The racemic compounds 5a−c were
separated using chiral HPLC [Chiralpak IA (for 5a and 5b), and
Chiral ART amylose-SA (for 5c)].16 As for 5a, the chiral synthesis of
the enantiomers has been achieved.12a,b The separation conditions and
physicochemical properties of the enantiomers are shown in the
Supporting Information.
This work was supported in part by a Grant-in-Aid for Scientific
Research (C) (25460154) and a Grant-in-Aid for Young
Scientists (B) (25860091) from the Japan Society for the
Promotion of Science. H.T. thanks the MEXT-Supported
Program for the Strategic Research Foundation at Private
Universities (2013−2017) for financial support.
E
DOI: 10.1021/acs.jmedchem.7b00422
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Journal of Medicinal Chemistry
Brief Article
is consistent with the report that N-benzoyl-N-methylanilines exist in
an E-structure: Azumaya, I.; Kagechika, H.; Fujiwara, Y.; Itoh, M.;
Yamaguchi, K.; Shudo, K. Twisted intramolecular charge-transfer
fluorescence of aromatic amides: conformation of the amide bonds in
excited states. J. Am. Chem. Soc. 1991, 113, 2833−2838.
(11) The numbering of the benzothiazepine rings of 2 and 3 used in
the main text is different from that of the IUPAC nomenclature rule,
i.e., the numbering of N and S in 2/3 are assigned as 1,4-
benzodiazepine (1) and 1-benzazepine (5), respectively, for
convenience.
(12) The tolvaptan-like compound (5a) in the racemic form was
reported in ref 3 and in the chiral (5R and 5S)-forms appeared in the
patent literature (ref 12a) and in the literature (ref 12b): (a) Ootsubo,
K.; Yamashita, S.; Uchida, M.; Morita, K. Preparation of optically
active 5-hydroxybenzoazepines as vasopressin antagonists. (Jpn Kokai
Tokkyo Koho). JP 0680641, 1994. (b) Matsubara, J.; Kitano, K.;
Otsubo, K.; Kawano, Y.; Ohtani, T.; Bando, M.; Kido, M.; Uchida, M.;
Tabusa, F. Enantioselective synthesis of the metabolites of vasopressin
V2 receptor antagonist OPC-31260 via lipase-catalyzed transester-
ification. Tetrahedron 2000, 56, 4667−4682.
ABBREVIATIONS USED
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ADPKD, autosomal-dominant polycystic kidney disease; VP,
vasopressin; CD, circular dichroism; [α]D, optical rotation; ax,
axial; eq, equatorial; ΔG‡, activation free-energy barrier to
rotation
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12a
7.7 for V2,12a (+)-(5R)-5a, 140 for V1a and 32 for V2.
In the
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determination of the chirality at the axis from our amassed data for the
related structures obtained thus far, i.e., compounds with (+)- and
(−)-angles have (aS) and (aR) stereochemistry, respectively. In 5c,
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(aR*,5S*), determination of the axial chirality leads inevitably to the
determination of the central chirality at C5.
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(21) If the differences of the intrinsic Ki values between (aS,5S)-5c
and (aR,5S)-5c are 20-fold, and the formation of (aS,5S)-5c from
(aR,5S)-5c is 10%, the observed Ki value of (aR,5S)-5c would be ca.
half of the intrinsic value, which may be possible to be similar to that
of the enantiomer (aS,5R)-5c.
(22) For general experimental methods, see the Supporting
Information.
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1
isomer were observed in the H NMR spectrum, the peaks were not
sufficient to assign the structure. Formation of the E-structure in 5a−c
F
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