Practical synthesis of potential endothelin receptor antagonists of 1,4-benzodiazepine-2,5-dione derivatives bearing substituents at the C 3-, N1- and N4-positions

Article abstract of DOI:10.1039/b514937a

The expedient synthesis of various 1,4-benzodiazepine-2,5-dione compounds, particularly those having substituents at the C₃-, N₁- and N₄-positions is achieved. The important features in these synthetic strategies include: (i) using the coupling reaction of isatoic anhydride with α-amino ester for direct construction of the core structure of 1,4-benzodiazepine-2,5-dione; (ii) using potassium carbonate as the base of choice for selective alkylation at the N₁-site, while using lithiated 2-ethylacetanilide as the required base to furnish the N₄- alkylation; and (iii) using 2-nitrobenzoyl chloride as a synthetic equivalent of anthranilic acid to facilitate the polyethylene resin-bound liquid-phase combinatorial synthesis. The prepared 1,4-benzodiazepine-2,5-dione compounds are evaluated for endothelin receptor antagonism by a functional assay that measures the inhibitory activity against the change of intramolecular calcium ion concentration induced by endothelin-1. The preliminary results indicate that 1,4-benzodiazepine-2,5-diones bearing two flanked aryl substituents at the N₁- and N₄-sites show better inhibitory activity than the corresponding unalkylated and N-monoalkylated compounds. A promising candidate, 1-benzyl-7-chloro-3-isopropyl-4-(3-methoxybenzyl)-1,4-benzodiazepine-2,5-dione (17b), exhibits an IC₅₀ value in low nM range. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b514937a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Practical synthesis of potential endothelin receptor antagonists of
1,4-benzodiazepine-2,5-dione derivatives bearing substituents at the
C3-, N1- and N4-positions†
Ming-Fu Cheng,a Hui-Ming Yu,b,c Bor-Wen Ko,a Yu Chang,a Ming-Yi Chen,d Tong-Ing Ho,a Yeun-Min Tsai*a
and Jim-Min Fang*a,c
Received 20th October 2005, Accepted 5th December 2005
First published as an Advance Article on the web 19th December 2005
DOI: 10.1039/b514937a
The expedient synthesis of various 1,4-benzodiazepine-2,5-dione compounds, particularly those having
substituents at the C3-, N1- and N4-positions is achieved. The important features in these synthetic
strategies include: (i) using the coupling reaction of isatoic anhydride with a-amino ester for direct
construction of the core structure of 1,4-benzodiazepine-2,5-dione; (ii) using potassium carbonate as
the base of choice for selective alkylation at the N1-site, while using lithiated 2-ethylacetanilide as the
required base to furnish the N4-alkylation; and (iii) using 2-nitrobenzoyl chloride as a synthetic
equivalent of anthranilic acid to facilitate the polyethylene resin-bound liquid-phase combinatorial
synthesis. The prepared 1,4-benzodiazepine-2,5-dione compounds are evaluated for endothelin receptor
antagonism by a functional assay that measures the inhibitory activity against the change of
intramolecular calcium ion concentration induced by endothelin-1. The preliminary results indicate
that 1,4-benzodiazepine-2,5-diones bearing two flanked aryl substituents at the N1- and N4-sites show
better inhibitory activity than the corresponding unalkylated and N-monoalkylated compounds. A
promising candidate, 1-benzyl-7-chloro-3-isopropyl-4-(3-methoxybenzyl)-1,4-benzodiazepine-2,5-dione
(17b), exhibits an IC50 value in low nM range.
Introduction
Since the first discovery in the mid 1950s,1 many benzodiazepine
derivatives have been developed to show potent pharmacological
activities,2 such as in anxiolytics, anticonvulsants, anti-HIV agents,
and the drugs active to the central nervous system. The benzodi-
azepine scaffold is considered to be one of the most important
structures in drug discovery due to its high functional group
diversity.3 For example, 1,4-benzodiazepine-2,5-dione and its
analogues exhibit remarkable potency in various biological targets,
including antithrombotics, antibiotics and antitumor activities.4
Furthermore, 1,4-benzodiazepine peptidomimetics are employed
as enzyme inhibitors5 and as the ligands to bind with various
G-protein coupled receptors.6
Endothelins are a group of isopeptides locally produced in
various cell types under different physiological stimuli. Human
endothelin-1 (ET-1) is a 21-amino-acid peptide that exhibits a
Fig. 1 Examples of non-peptide endothelin receptor antagonists.
an indole-type compound PD159433,10 a pyrazole-type com-
pound HMR865,11 and a carbazolothiophene-type compound
JMF222.12
potent vasoconstrictor activity, conceivably through its selective
interaction with specific receptor ETA.7 In recent studies, a
series of compounds having planar core structures flanked by
two aryl side-arms have been explored as effective endothelin
receptor antagonists.8 The structures of some representative
antagonists (Fig. 1) include an indan-type compound SB209670,9
In comparison with the low-energy conformations of ET-
1 derived by 1H NMR analysis, the calculated 3-dimensional
structure of SB209670 suggests that the two phenyl substituents
may mimic the Tyr-13 and Phe-14 residues in ET-1 (Fig. 2).9a The
two carboxyl groups in SB209670 may also mimic the Asp-18
residue and the C-terminus of ET-1, which ligate a Zn2+ ion on
binding with the endothelin receptor. This molecular modeling
experiment thus provides useful information for the selection of
specific side chains on a flat core structure for the subsequent
exploration of other endothelin receptor antagonists.
aDepartment of Chemistry, National Taiwan University, Taipei, 106, Taiwan.
E-mail: ymtsai@ntu.edu.tw, jmfang@ntu.edu.tw
bInstitute of Biochemistry, Academia Sinica, Taipei, 115, Taiwan
cGenomics Research Center, Academia Sinica, Taipei, 115, Taiwan
dDepartment of General Education, Taipei Nursing College, Taipei, Taiwan
† Electronic supplementary information (ESI) available: Synthetic proce-
dures and compound characterization. See DOI: 10.1039/b514937a
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Fig. 2 Structure of human endothelin-1.
We speculated that 1,4-benzodiazepine-2,5-dione derivatives 1
bearing appropriate substituents (Fig. 3) might also function
as endothelin receptor antagonists, because the benzodiazepine
structure consists of a nearly planar core platform similar to
the indan ring in SB209670. For a better binding affinity toward
endothelin receptors, various aryl substituents might be implanted
on the N1- and N4-positions, and carboxyl groups might be
introduced at the desired sites on the benzodiazepine scaffold.
To construct the main framework of 1,4-benzodiazepine-2,5-
dione, two general methods have been applied.13,14 One method
utilizes the condensation reaction of an a-amino acid with 2-
aminobenzoic acid (anthranilic acid), in which the amino group
can be used in the protected or latent forms, e.g., NHFmoc,
azido and nitro groups.13 The other method utilizes the Ugi
four-component reaction of anthranilic acid with aldehyde, amine
Fig. 3 1,4-Benzodiazepine-2,5-dione derivatives 1 with a nearly planar
core ring structure flanked by two aryl side-arms are designed to evaluate
their activities in endothelin receptor antagonism.
and isocyanate.14 Although various 1,4-benzodiazepine-2,5-dione
derivatives can be prepared by the above-mentioned methods,13,14
difficulties may be encountered in placing specific side chains and
carboxyl groups at the N1-, N4- and C3-positions. We report herein
the synthetic approaches to circumvent this problem.
Results and discussion
Synthesis of 1,4-benzodiazepine-2,5-dione compounds
In our first approach (Scheme 1, A), isatoic anhydride is used
as the activated derivative of anthranilic acid.15,16 The reaction
Scheme 1 Approaches using isatoic anhydrides for the synthesis of 1,4-benzodiazepine-2,5-dione derivatives that bear carboxyl group and aryl
substituents at the C3-, N1-, and N4-positions.
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of isatoic anhydride (2a) with p-methoxyaniline gave amide 3,
which was subsequently alkylated with 3-methoxybenzyl chloride
to afford 4 in reasonable yield. Compound 4 was subjected to
amidation with methyl malonyl chloride, giving 5, which was
treated with pyridinium tribromide to introduce a bromine atom
at the methylene carbon. The product 6, without isolation, was
treated with two equivalents of sodium tert-butoxide to effect an
intramolecular alkylation reaction, forming 1,4-benzodiazepine-
2,5-dione 7 in 82% yield (from 5). Though ester 7 and the
corresponding acid 8 are obtained with the desired C3-, N1- and
N4-substituents on the core structure of 1,4-benzodiazepine-2,5-
dione, this approach lacks flexibility in tuning the length of the
carboxyl substituent.
nitrobenzoyl chloride was used as an equivalent of anthranilic acid
in the solution-phase synthesis of 1,4-benzodiazepine-2,5-diones
(Scheme 2, A).
Thus, the (substituted)benzaldehydes 12a–c were coupled with
a-amino acid derivatives, e.g., the esters of L-valine and L-aspartic
acid, via reductive amination reactions using NaBH(OAc)3 as the
reducing agent. The resulting amines 14a–d were then reacted
with 2-nitrobenzoyl chloride (or its analogs) to afford amides
15a–f. The nitro groups in 15a–f were smoothly reduced by zinc
powder, and the resulting anilines, without isolation, cyclized on
treatment with p-toluenesulfonic acid to give 1,4-benzodiazepine-
2,5-diones 16a–f in 75–90% yields. In this process, formation of
the core structure of 1,4-benzodiazepine-2,5-dione was facilitated
because the tertiary amide moiety existed preferably in the C–N
cis conformation to render the cyclization reaction.20 Alkylation
of 16a–f with benzyl bromides was conducted in a dipolar solvent
(CH3CN) using K2CO3 as the base to give the desired products
17a–i. Elaboration of 17a–i was feasible. For example, diesters
17a (R1 = p-CO2Me, R2 = CH2CO2Me, R3 = R4 = H) and 17i
(R1 = m-OCH2CO2Me, R2 = CH2CO2Me, R3 = R4 = H) were
hydrolyzed by LiOH to give the corresponding diacids 17j and
17k, respectively. When 17c (R1 = m-OMe, R2 = CH2CO2Me,
R3 = H, R4 = 8-Cl) was treated with BBr3, a phenol 17l (47%)
was obtained by demethylation of the anisole moiety, along with
an acid 17m (30%) derived from a simultaneous removal of the
methyl group on the ester moiety. Aniline 17d (R1 = m-OMe, R2 =
CH2CO2Me, R3 = H, R4 = 8-NH2) was subjected to amidation
with trifluoroacetic anhydride and bromoacetyl bromide to give
17n and 17o, respectively.
Upon the successful solution-phase synthesis of 1,4-
benzodiazepine-2,5-diones, we also explored a liquid-phase syn-
thesis using PEG5000 monomethyl ether as the support to construct
a library of 1,4-benzodiazepine-2,5-diones (17i and 20a–o).21
As the target 1,4-benzodiazepine-2,5-dione requires an N4-aryl
substituent, we deliberately used the PEG-bound benzaldehydes
18 to couple with the esters of a-amino acids (Scheme 2, B).
By a similar reaction sequence to that shown in the solution-
phase synthesis, the desired 1,4-benzodiazepine-2,5-diones 19a–p
bound to a PEG support were prepared. Removal of the PEG
support with concurrent formation of the methyl ester was realized
by stirring with Na2CO3 in MeOH at room temperature for a
short time (<5 min). Thus, sixteen 1,4-benzodiazepine-2,5-dione
dimethyl ester derivatives were obtained in 80–99% crude yields
by a liquid-phase combinatorial synthesis. The HPLC analyses
indicated that the purity was around 53–77% (corresponding to
90–96% average yield in each synthetic step). The final products
17i and 20a–o were simply purified by silica gel chromatog-
raphy, and fully characterized by physical and spectroscopic
methods.
In another approach (Scheme 1, B), isatoic anhydride was
reacted with an amino acid to construct the skeleton of 1,4-
benzodiazepine-2,5-dione in a direct manner. The C3-substituent
can be varied by using different a-amino acids. For example, 5-
chloroisatoic anhydride (2b) was heated with the hydrochloride salt
of L-aspartic acid dimethyl ester in pyridine to afford the desired
1,4-benzodiazepine-2,5-dione 9 in 60% yield. Selective alkylation
at the N1-position was realized by using K2CO3 as the required
base. Thus, the reaction of 9 with benzyl bromide (1 equiv.) was
promoted by using K2CO3 (1 equiv.) in DMF solution to give
exclusively the N1-alkylation product 10a in 85% yield. A similar
reaction of 9 with m-methoxybenzyl bromide (K2CO3, DMF)
also occurred selectively at the N1-site, giving the monoalkylation
product 10b in 83% yield. The structure of the N1-alkylation
1
products were characterized by H NMR analyses. For example,
compound 9 in DMSO-d6 solution showed the N1–H as a singlet
at d 10.59 and the N4–H as a doublet (J = 5.6 Hz) at d 8.71.
The monoalkylation product 10a in CDCl3 solution exhibited the
N4–H signal as a doublet (J = 5.6 Hz) at d 8.18, but no signal for
N1–H. The regioselectivity in the alkylation might be attributable
to a selective removal of the more acidic N1–H, in comparison
with N4–H, by the weak base K2CO3.17
A novel base was generated by lithiation of 2-ethylacetanilide
with BuLi in THF (−78 ◦C, 1 h), similar to that reported by
Ellman.13a,b,18 This base was successfully utilized to effect the
alkylations of 10a,b at the N4-positions, giving 11a,b, without
complication of O-alkylations.17,18 Therefore, this method demon-
strates a feasible approach to various 1,4-benzodiazepine-2,5-
dione compounds bearing C3-, N1- and N4-substituents.
The combinatorial or parallel synthetic approaches are espe-
cially useful in constructing a molecular library for the study
of structure–activity relationship.19 A combinatorial approach
to the synthesis of 1,4-benzodiazepine-2,5-diones was designed
to incorporate four units of 2-nitrobenzoic acid, benzaldehyde,
benzyl halide and a-amino acid (Fig. 4). To test this approach, 2-
Preliminary functional bioassay
The interaction between endothelin receptor and its ligand,
e.g., ET-1, is coupled with the activation of a G-protein. This
interaction triggers a series of biological events to induce an
increase of intracellular calcium concentration, [Ca2+]i.22 The
measurement of inhibitory potency against the ET-1 induced
[Ca2+]i change thus provides a functional assay for preliminary
evaluation of endothelin receptor antagonists. According to the
Fig. 4 A combinatorial approach to the synthesis of 1,4-benzodiazepine-
2,5-diones.
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Scheme 2 Approach using 2-nitrobenzoyl chloride as the equivalent of anthranilic acid for the synthesis of 1,4-benzodiazepine-2,5-dione derivatives:
(A) solution-phase synthesis, and (B) liquid-phase synthesis.
reported experimental protocol,23 Chinese hamster ovary (CHO–
K1) cells were transfected with the rat ETA-expression plasmid
DNA using a lipofectin reagent.24 The ETA overexpression CHO–
K1 cells were prior incubated with calcium chelating agent fura-
2 applied as its penta(acetoxymethyl) ester,24 and then treated
with ET-1 at 10−7 M. The [Ca2+]i increase was monitored at
510 nm fluorescence emission by a ratiometric method using dual
excitations at 340 and 380 nm wavelengths.25 This increment of
[Ca2+]i was taken as the standard value (100%) to assess the
inhibitory potency of the test compounds against the ET-1 binding
with receptor (Fig. 5).
to the functional assays. In comparison, these endothelin receptor
antagonists are more potent than a cyclopentapeptide inhibitor
BQ123 [cyclo(D-Trp-D-Asp-Pro-D-Val-Leu)],26 which showed only
∼60% inhibition at 10−6 M in our functional assay. Compound
17b showed an IC50 value of slightly lower than 10 nM; however,
SB209670 is an even more effective antagonist showing ∼85%
inhibition at 10 nM under similar assay conditions. Unalkylated
and N-monoalkylated 1,4-benzodiazepine-2,5-diones, e.g. 9 and
10b, are less active than the corresponding N1,N4-dialkylated
compound 11a. This trend of inhibitory activity appeared to
be in agreement with the previous prediction on the need of
two flanked aryl substituents for the endothelin receptor anta-
gonism.9a
The 1,4-benzodiazepine-2,5-dione compounds 11a, 17b and 17n
(Fig. 6) showed high antagonistic activity against ET-1 according
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Fig. 5 Fluorescence measurements of intracellular calcium concentration ([Ca2+]i) by addition of test samples (as shown by the arrow) to the
ETA-overexpressing CHO cells in the presence of fura-2. The induced [Ca2+]i change is taken as a measure of antagonist potency against ET-1, shown in
thin lines. (a) Treatment with a mixture of SB209670 (10−6 M, 100% inhibition) and ET-1 (10−7 M), (b) treatment with a mixture of 17a (10−6 M, 55%
inhibition) and ET-1 (10−7 M), (c) treatment with a mixture of 17n (10−6 M, 100% inhibition) and ET-1 (10−7 M), (d) treatment with a mixture of 17b
(10−8 M, 58% inhibition) and ET-1 (10−7 M).
benzodiazepine-2,5-dione. N1–H is selectively removed by using a
mild base, K2CO3, without the interference of N4–H, and exclusive
N1-alkylation is thus achieved. On the other hand, N4-alkylation
requires a special base of lithiated 2-ethylacetanilide. Using 2-
nitrobenzoyl chloride as an equivalent of anthranilic acid, either
in the solution- or liquid-phase approach, is advantageous for the
construction of 1,4-benzodiazepine-2,5-diones. The preliminary
functional assay of our prepared 1,4-benzodiazepine-2,5-dione
compounds has revealed some potentially useful endothelin recep-
tor antagonists. However, the accurate measurement of inhibition
constants (Ki values) by competitive assay23 with the radioactive
125I-labeled endothelin is pending on collaboration with other
laboratories.
Experimental
General
Fig.
6 Potent endothelin receptor antagonists of 1,4-benzodi-
azepine-2,5-diones that show complete inhibition at 10−6 M against the
[Ca2+]i change induced by ET-1 at 10−7 M.
Melting points are uncorrected. 1H NMR spectra were recorded at
400 MHz; 13C NMR spectra were recorded at 100 MHz. Chemical
shifts (d) are given in parts per million (ppm) relative to residual
solvent [d 7.24 (s) for CHCl3 and d 2.49 (m) for DMSO-d6].
The splitting patterns are reported as s (singlet), d (doublet), t
(triplet) and multiplet (m). Chemical shifts of 13C NMR spectra are
reported relative to CDCl3 (d 77.0 for the central line of triplet) and
DMSO-d6 [d 39.5 (m)]. Mass spectra were recorded at an ionizing
voltage of 70 or 20 eV. Merck silica gel 60F sheets were used for
analytical thin-layer chromatography (TLC). Merck silica gel 60F
Conclusion
We have demonstrated straightforward approaches, including a
liquid-phase combinatorial method, for the expedient synthe-
sis of various novel 1,4-benzodiazepine-2,5-dione compounds,
particularly those having substituents at the C3-, N1- and N4-
positions. The coupling reaction of isatoic anhydrides with amino
acids leads to the direct formation of the core structure of 1,4-
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glass plates (20 cm × 20 cm with 2 mm thickness) were used
for preparative TLC. Column chromatography was performed
on silica gel (70–230 mesh) using gradients of EtOAc/hexane as
eluents. HPLC (Hewlett Packard 1100) analysis was performed on
a vp250/10 Nucleosil 100–7 column (25 cm × 1 cm I.D.) with UV
detection at k = 254 nm using the eluents EtOAc/hexane (3 : 2 or
4 : 1) at a flow rate of 1 mL min−1.
Reactions requiring dry conditions were carried out under
an inert atmosphere using standard techniques. All the reagents
and solvents were reagent grade and were used without further
purification unless otherwise specified. THF was distilled from
sodium benzophenone under N2. Polyethylene glycol monomethyl
ether was dried by azeotropic removal of water with refluxing
acetonitrile.
3.09 (1 H, dd, J = 16.8, 8.8 Hz), 3.70 (3 H, s), 4.30 (1 H, ddd,
J = 8.8, 5.6, 5.2 Hz), 4.97 (1 H, d, J = 16 Hz), 5.14 (1 H, d, J =
16 Hz), 7.10–7.38 (7 H, m), 7.78 (1 H, d, J = 2.4 Hz), 8.18 (1 H,
d, J = 5.6 Hz); dC (CDCl3, 100 MHz) 170.7, 169.1, 167.8, 138.5,
136.1, 132.6, 131.9, 130.0, 129.8, 128.8 (2×), 127.5, 126.6 (2×),
123.7. 52.2, 52.1, 49.5, 33.1; MS (70 eV) m/z 372 (46%, M+), 180
(100), 91 (76); HRMS C19H17N2O4Cl requires 372.0877, found
m/z 372.0904. Anal. C19H17N2O4Cl requires: C, 61.21; H, 4.60;
N, 7.51. Found: C, 60.90; H, 4.83; N, 7.14%.
Representative procedure II for alkylation at N-4 position
To a solution of N-(2-ethylphenyl)acetamide (0.16 g, 0.98 mmol)
in THF (2 mL) was added butyllithium (95 lL, 1 mmol, 1.6 M
solution in hexane) dropwise under argon at −78 ◦C (dry
ice/acetone bath). The mixture was stirred at −78 ◦C for 1 h
to give a yellow solution. Compound 10a (0.36 g, 0.98 mmol)
in THF (1 mL) and DMF (1 mL) was added dropwise. After
stirring for 1 h, a solution of 3-methoxybenzyl bromide (200 lL,
0.99 mmol) and lithium iodide (0.44 g, 0.99 mmol) in DMF (1 mL)
was added in one portion. The reaction mixture was heated to
60 ◦C over a period of 6 h. The mixture was cooled, and partitioned
between CH2Cl2 (30 mL) and 1 N HCl (30 mL). The organic phase
was separated, washed with water (30 mL) and brine (30 mL),
dried (MgSO4), filtered, concentrated, and chromatographed on a
silica gel column by elution with hexane/EtOAc (7 : 3) to afford
compound 11a (0.33 g, 68%).
Representative procedure for the formation of 1,4-benzodiazepine-
2,5-dione by condensation of isatoic anhydride with a-amino ester
A mixture of 5-chloroisatoic anhydride (0.5 g, 2.5 mmol) and L-
aspartic acid dimethyl ester hydroc◦hloride (0.5 g, 2.5 mmol) in
pyridine (5 mL) was heated to 120 C for 18 h. After cooling to
room temperature, the mixture was acidified to pH = 1 by adding
6 N HCl (30 mL), and the acidified aqueous phase was extracted
with EtOAc (20 mL × 3). The combined organic phase was
washed with brine (50 mL), dried over MgSO4, and concentrated
to 20 mL of mixture. The precipitated white solids were collected,
and washed with water and EtOAc to provide benzodiazepine 9
(0.42 g, 60%).
(+)-(S)-1-Benzyl-7-chloro-4-(3-methoxybenzyl)-3-(methoxycar-
bonyl)methyl-1,4-benzodiazepine-2,5-dione (11a). Oil; Rf = 0.35
(+)-(S)-7-Chloro-3-(methoxycarbonyl)methyl-1,4-benzodiazepine-
2,5-dione (9). Solid, mp = 197–203 ◦C; Rf = 0.35 (hexane/
(hexane/EtOAc, 3 : 2); [a]26 = +25.8 (c = 0.75, CDCl3); mmax
D
EtOAc, 2 : 3); [a]25 = +258.5 (c = 1.0, DMSO); mmax (KBr, cm−1)
(film, cm−1) 2952, 1740, 1690; dH (CDCl3, 400 MHz) 2.68 (1 H,
dd, J = 16.4, 4.0 Hz), 3.59 (1 H, dd, J = 16.4, 3.6 Hz), 3.79 (3 H,
s), 3.83 (3 H, s), 4.47 (1 H, d, J = 16 Hz), 4.80 (1 H, dd, J = 4.0,
3.6 Hz), 4.91 (1 H, d, J = 16.0 Hz), 5.13 (1 H, d, J = 16.0 Hz),
5.20 (1 H, d, J = 16.0 Hz), 6.77–7.40 (11 H, m), 7.91 (1 H, d,
J = 2.8 Hz); dC (CDCl3, 100 MHz) 170.4, 168.3, 167.5, 160.0,
142.5, 138.8, 138.5, 136.1, 132.4, 131.7, 130.6, 129.8, 127.5, 126.5,
122.8, 119.3, 119.0, 113.2, 112.8, 112.7, 112.1, 55.2, 53.0, 52.1,
51.5, 47.2, 31.8.; MS (70 eV) m/z 492 (22%, M+), 357 (50), 180
(25), 121 (62), 91 (100); HRMS C27H25N2O5Cl requires 492.1452,
found m/z 492.1458. Anal. C27H25N2O5Cl: C, 65.79; H, 5.11; N,
5.68. Found: C, 65.40; H, 5.20; N, 5.19%.
D
3086, 1736, 1685; dH (DMSO-d6, 400 MHz) 2.71 (1 H, dd, J =
16.8, 6.4 Hz), 2.86 (1 H, dd, J = 16.8, 8.4 Hz), 3.57 (3 H, s), 4.06
(1 H, ddd, J = 8.4, 6.4, 5.6 Hz), 7.14 (1 H, d, J = 8.8 Hz), 7.59
(1 H, dd, J = 8.8, 2.6 Hz), 7.69 (1 H, d, J = 2.6 Hz), 8.71 (1 H,
d, J = 5.6 Hz), 10.59 (1 H, s); dC (DMSO-d6, 100 MHz) 170.4
(2×), 166.4, 135.5, 132.3, 129.7, 128.3, 127.7, 123.1, 51.6, 48.5,
32.4; MS (70 eV) m/z 284 (18%, M+ + 2), 282 (55, M+), 181 (100);
HRMS C12H11N2O435Cl requires 282.0407, found m/z 282.0416.
Anal. C12H11N2O4Cl requires: C, 50.99; H, 3.92; N, 9.91. Found:
C, 50.83; H, 3.77; N, 9.48%.
Representative procedure I for the selective alkylation at the N-1
position
Representative procedure III for reductive amination
Under an atmosphere of argon, a mixture of 9 (1 g, 3.54 mmol)
and K2CO3 (0.4 g, 3.56 mmol) in DMF (4 mL) was stirred at
room temperature (25 ◦C) for 1 h. Benzyl bromide (0.42 mL,
3.54 mmol) was added, and the mixture was stirred for another 4 h.
The reaction was quenched by pouring the mixture into ice water
(50 mL), and the mixture was extracted with EtOAc (40 mL ×
3). The combined organic phase was washed with brine (50 mL),
dried (MgSO4), filtered, and concentrated to afford a pale-yellow
oil. The crude product was chromatographed on a silica gel column
by elution with hexane/EtOAc (6 : 4) to afford 10a (1.12 g, 85%).
A mixture of L-valine methyl ester hydrochloride (2.03 g, 12 mmol)
and m-anisaldehyde (1.25 mL, 10 mmol) in CH2Cl2 (100 mL) was
treated with sodium triacetoxyborohydride (3.18 g, 15 mmol) and
◦
NaOAc (1.23 g, 15 mmol) at 0 C. The suspension was warmed
to room temperature and stirred for 5 h. After the reaction was
finished, brine (50 mL) was added, and the mixture was extracted
with CH2Cl2 (30 mL × 2). The organic layer was dried over MgSO4
and concentrated under reduced pressure. The residue was purified
by flash chromatography on silica gel eluting with EtOAc/hexane
(4 : 6) to give N-(3-methoxybenzyl) valine methyl ester 14b (2.46 g,
98%).
(+)-(S)-1-Benzyl-7-chloro-3-(methoxycarbonyl)methyl-1,4-benzo-
diazepine-2,5-dione (10a). Oil; Rf = 0.40 (hexane/EtOAc, 1 : 1);
(−)-(S)-N-(3-Methoxybenzyl) valine methyl ester (14b). Oil;
[a]26 = +41.1 (c = 0.75, CDCl3); mmax (film, cm−1) 3231, 2945,
Rf = 0.63 (EtOAc/hexane, 3 : 2); [a]23 = −65.4 (CH2Cl2, c =
D
D
1715, 1670; dH (CDCl3, 400 MHz) 2.80 (1 H, dd, J = 16.8, 5.2 Hz),
0.75); mmax (film, cm−1) 3344, 1735, 1604; dH(CDCl3, 400 MHz)
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7.20 (1 H, d, J = 8.2 Hz), 6.90–6.87 (2 H, m), 6.77 (1 H, d, J =
8.2 Hz), 3.80 (1 H, d, J = 13.2 Hz), 3.78 (3 H, s), 3.71 (3 H, s),
3.54(1 H, d, J = 13.2 Hz), 3.00 (1 H, d, J = 6.0 Hz), 1.93–1.86
(1 H, m), 0.95–0.91 (6 H, m); dC(CDCl3, 100 MHz) 175.5, 159.5,
141.6, 129.1, 120.4, 113.4, 112.5, 66.5, 55.2, 52.5, 51.4, 31.8, 19.5,
18.8; HRMS C14H22NO3 (M +H+) requires 252.1600, found m/z
252.1598.
(−)-(S)-1-Benzyl-7-chloro-3-isopropyl-4-(3-methoxybenzyl)-1,4-
benzodiazepine-2,5-dione (17b). Alkylation of 16b (1.30 g,
3.5 mmol) with benzyl bromide (0.42 mL, 3.5 mmol), according
to the representative procedure II, gave 17b (90%). Solid, mp =
◦
46–48 C; Rf = 0.60 (EtOAc/hexane, 1 : 1); [a]23 = −229.9
D
(CH2Cl2, c = 0.35); mmax (KBr, cm−1) 2970, 1673, 1647, 1600, 1472,
1266, 1154, 1052; dH (CDCl3, 400 MHz) 7.81 (1H, d, J = 2.8 Hz),
7.32 (1H, d, J = 8.8 Hz), 7.24–7.06 (3H, m), 7.04 (2H, d, J =
6.0 Hz), 6.99 (2H, d, J = 7.6 Hz), 6.93–6.85 (2H, m), 6.82 (1H, d,
J = 7.2 Hz), 5.08–4.93 (3H, m), 4.52 (1H, d, J = 14.0 Hz), 3.81
(1H, d, J = 11.6 Hz), 3.76 (3H, s), 1.45–1.37 (1H, m), 0.81 (3H,
d, J = 6.8 Hz), 0.61 (3H, d, J = 6.4 Hz); dC (CDCl3, 100 MHz)
169.2, 164.8, 159.6, 137.4, 137.2, 136.4, 132.0, 131.3, 131.2, 130.3,
129.5, 128.7 (2×), 127.4, 126.9 (2×), 122.7, 121.3, 114.3, 113.8,
72.6, 55.3, 51.9, 28.3, 19.9, 19.7; HRMS C27H28ClN2O3 (M +H+)
requires 463.1788, found 463.1788. Anal. C27H27ClN2O3 requires:
C, 70.05; H, 5.88; N, 6.05; O, 10.37. Found: C, 70.40; H, 5.82; N,
6.19%.
Representative procedure IV for amidation of 2-nitrobenzoyl
chloride
To a mixture of amine 14b (2.26 g, 9 mmol) K2CO3 (3.73 g,
27 mmol) and Bu4NI (1.66 g, 4.5 mmol) in CH2Cl2 (90 mL)
was added 4-chloro-2-nitrobenzoyl chloride (2.38 g, 10.8 mmol)
dropwise. After the mixture was stirred at room temperature for
4 h, water (70 mL) was added, and the mixture was extracted
with CH2Cl2 (30 mL × 2). The organic layer was dried over
MgSO4 and concentrated under reduced pressure. The residue
was purified by flash chromatography on silica gel eluting with
EtOAc/hexane (3 : 7 to 1 : 1) to give N-(5-chloro-2-nitrobenzoyl)-
N-(3-methoxybenzyl) valine methyl ester 15b (3.68 g, 94%).
(+)-(S)-1-Benzyl-4-(3-methoxybenzyl)-3-(methoxycarbonyl)-
methyl-8-(trifluoroacetyl-amino)-1,4-benzodiazepine-2,5-dione (17n).
The reductive amination of m-anisaldehyde (0.69 mL, 5.53 mmol)
with dimethyl L-aspartate hydrochloride (1.35 g, 6.63 mmol),
according to the representative procedure III, gave N-(3-methoxy-
benzyl) aspartic acid dimethyl ester 14c (1.45 g, 93%). Amidation
of 14c (3.60 g, 12.8 mmol) with 2,4-dinitrobenzoyl chloride (3.29 g,
15.4 mmol), according to the representative procedure IV, gave N-
(2,4-dinitrobenzoyl)-N-(3-methoxybenzyl) aspartic acid dimethyl
ester 15d (5.53 g, 91%). Reduction and in situ cyclization of 15d
(1.00 g, 2.10 mmol), according to the representative procedure V,
gave 8-amino-4-(3-methoxybenzyl)-3-(methoxycarbonyl)methyl-
1,4-benzodiazepine-2,5-dione 16d (0.81 g, 78%). Alkylation
of 16d (2.00 g, 5.23 mmol) with benzyl bromide (0.76 mL,
6.26 mmol), according to the representative procedure I, gave the
N-1 alkylation product, 8-amino-1-benzyl-4-(3-methoxybenzyl)-
3-(methoxycarbonyl)methyl-1,4-benzodiazepine-2,5-dione 17d
(1.24 g, 50%), accompanied by the 8-benzylamino derivative 17e
(0.68 g, 23%) and the 8-dibenzylamino derivative 17f (0.34 g,
10%).
(−)-(S)-N-(5-Chloro-2-nitrobenzoyl)-N-(3-methoxybenzyl) va-
line methyl ester (15b). Gel; Rf = 0.63 (EtOAc/hexane, 3 : 2);
[a]23 = −19.5 (CH2Cl2, c = 0.60); mmax (film, cm−1) 2970, 1745,
D
1654; dH (CDCl3, 400 MHz) 8.25–8.04 (1 H, m), 7.56–7.32 (2 H, m),
7.24–6.58 (4 H, m), 5.00–4.87 (1 H, m), 4.44–4.36 (1 H, m), 3.82–
3.43 (7 H, m), 2.57–2.52 (0.5 H, m), 2.34–2.27 (0.5 H, m), 1.10–
1.06 (3 H, m), 0.77–0.68 (3 H, m); dC (CDCl3, 100 MHz) 169.7–
166.7 (2×), 159.0–133.2 (5×), 129.7–112.9 (7×), 77.3–63.7, 55.4–
52.0 (2×), 47.0–46.5, 28.8–28.3 (CH), 21.1–19.6 (2×); HRMS
C21H24ClN2O6 (M + H+) requires 435.1323, found m/z 435.1325.
Representative procedure V for reduction of nitro compounds and
in situ cyclization to benzodiazepines
A solution of the nitro compound 15b (1.74 g, 4 mmol) in MeOH
(40 mL) was treated with zinc powder (2.61 g, 40 mmol) and NH4Cl
(1.07 g, 20 mmol). The suspension was stirred at room temperature
for 10 min, and filtered through a pad of Celite. The filtrate
was concentrated to about 40 mL, and p-TsOH monohydrate
(76 mg, 0.4 mmol) was added. The mixture was heated under
reflux for 6 h, cooled, and concentrated under reduced pressure.
The residue was purified by flash chromatography on silica gel
eluting with EtOAc/hexane (1 : 1) to give 7-chloro-3-isopropyl-
4-(3-methoxybenzyl)-1,4-benzodiazepine-2,5-dione 16b (1.12 g,
75%).
Compound 17d (142 mg, 0.3 mmol) was dissolved in trifluo-
◦
roacetic anhydride (3 mL) and stirred at 0 C for 1 h. Excess of
trifluoroacetic anhydride was removed under reduced pressure,
and the residue was extracted with CH2Cl2 (5 mL × 2). The
organic layer was dried over MgSO4, filtered, and concentrated
under reduced pressure to afford pure 17n (143 mg, 0.25 mmol) in
83% yield.
Compound 17n: Solid, mp = 101–103 ◦C; Rf = 0.48
(−)-(S)-7-Chloro-3-isopropyl-4-(3-methoxybenzyl)-1,4-benzodi-
azepine-2,5-dione (16b). Solid, mp = 75–77 ◦C; Rf = 0.43
(EtOAc/hexane, 3 : 2); [a]23 = +9.5 (CH2Cl2, c = 0.17); mmax
D
(KBr, cm−1) 3443, 3294, 2966, 1735, 1686, 1640, 1438, 1282, 1157,
1050; dH (CDCl3, 400 MHz) 8.64 (1H, br), 7.78 (1H, d, J = 8.4 Hz),
7.71 (1H, s), 7.28–7.19 (5H, m), 7.09 (2H, d, J = 7.2 Hz), 6.86–6.79
(3H, m), 5.12 (1H, d, J = 16.0 Hz), 5.07 (1H, d, J = 16.0 Hz), 5.02
(1H, d, J = 16.0 Hz), 4.78 (1H, dd, J = 4.4, 10.4 Hz), 4.38 (1H,
d, J = 16.0 Hz), 3.79 (3H, s), 3.57 (3H, s), 3.30 (1H, dd, J = 10.4,
16.6 Hz), 2.70 (1H, dd, J = 4.4, 16.6 Hz); dC (CDCl3, 100 MHz)
170.6, 168.4, 168.1, 159.9, 155.1 (q, J = 38.0 Hz), 140.6, 139.1,
138.8, 136.0, 131.9, 129.8, 128.7 (2×), 127.6, 126.9 (2×), 126.7,
119.4, 117.7, 116.8, 113.1, 113.0, 112.7, 55.2, 53.1, 52.2, 51.1, 47.2,
31.9; HRMS C29H27F3N3O6 (M + H+) requires 570.1852, found
(EtOAc/hexane, 1 : 1); [a]23 = −199.6 (CH2Cl2, c = 0.47); mmax
D
(KBr, cm−1) 3238, 2970, 1690, 1636; dH (CDCl3, 400 MHz) 9.37
(1 H, br), 7.97 (1 H, s), 7.34 (1 H, d, J = 8.8 Hz), 7.10 (1 H, t,
J = 8.0 Hz), 6.88–6.82 (3 H, m), 6.65 (1 H, d, J = 8.4 Hz), 5.29
(1 H, d, J = 14.4 Hz), 4.30 (1 H, d, J = 14.4 Hz), 3.65 (3 H, s),
3.53 (1 H, d, J = 14.0 Hz), 1.72–1.65 (1 H, m), 0.82 (3 H, d, J =
6.8 Hz), 0.72 (3 H, d, J = 6.4 Hz); dC (CDCl3, 100 MHz) 171.7,
164.6, 159.6, 137.3, 133.2, 132.4, 131.0, 130.1, 129.5, 127.8, 121.3,
120.7, 114.1, 113.3, 71.2, 55.4, 55.1, 27.9, 19.7, 19.5; HRMS
C20H22ClN2O3 (M + H+) requires 373.1319, found 373.1317.
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570.1852. Anal. C29H26F3N3O6 requires: C, 61.16; H, 4.60; N, 7.38.
Found: C, 61.18; H, 4.88; N, 7.04%.
ions to bind with fura-2. The [Ca2+]i was indirectly deduced by the
intensity of 510 nm fluorescence with excitation at 340 nm.25 At
120 s, EGTA [ethylene glycol bis(2-aminoethyl ether) tetraacetic
acid] was added to remove Ca2+ ions, and the concentration of free
fura-2 was measured by the intensity of the 510 nm fluorescence
with excitation at 380 nm. The intracellular calcium concentration,
[Ca2+]i, was calculated according the following equation: [Ca2+] =
Kd × (sf/sb) × [(R − Rmin)/(Rmax − R)] in which, Kd is the
dissociation constant of fura-2 to Ca2+; R, Rmin and Rmax represent,
respectively, the ratio of fluorescence intensity at 340 nm to 380 nm,
that for the concentration of Ca2+ close to zero, and that for the
concentration of Ca2+ close to saturation; sf and sb represent,
respectively, the ratio of fluorescence intensity at 380 nm for the
concentration of Ca2+ close to zero, and that for the concentration
of Ca2+ close to saturation.
Materials and methods for the [Ca2+]i assay
Endothelin-1 (ET-1) and the cyclic peptide antagonists BQ123
[cyclo(D-Trp-D-Asp-L-Pro-D-Val-L-Leu)]26 were synthesized by
using an ABI 433A peptide synthesizer (ABI, USA). Non-
peptide endothelin receptor antagonist SB209670 was synthesized
according to the reported procedure.9 The 125I-labeled ET-1
((3-[125I]iodotyrosyl)endothelin-1, 81.4 TBq mmol−1) was pur-
chased from NEN Life Science Products (USA). The fluorescent
reagent, fura-2 penta(acetoxymethyl) ester, was purchased from
Calbiochem-Novabiochem Corporation (La Jolla, CA).
Construction of CHO–K1 cell line over-expressing endothelin
receptor A and ligand binding assay22c
Acknowledgements
The lipofectin-mediated transfection method described by Tseng
and co-workers24 was used to construct stable CHO cell lines
over-expressing ETA. Cells were grown to 30–40% confluence in
60 mm dishes and transfected with 1 lg of pcDNA-3 expressing
plasmid harboring ETA using lipofectin reagent for 6–8 h in a
serum-free medium. Cells were then returned to 5% FBS, cultured
for 36 h, then replated at reduced density in 150 mm plates in
the presence of 0.75 mg mL−1 (active) G418. The G418 resistant
colonies were selected and screened for ETA by binding of (3-
We thank Prof. Wan-Wan Lin (Department of Pharmacology,
National Taiwan University) and Sheau-Huei Chueh (Department
of Biochemistry, National Defence University) for instructing us
in the [Ca2+]i assay. We also thank National Science Council for
financial support.
References
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