Synthesis of a Fluorescent-Labeled Bisbenzamidine Containing the Central (6,7-Dimethoxy-4-coumaryl)Alanine Building Block

Article abstract of DOI:10.1002/hc.21269

The synthesis of an amino acid amide is reported, which contains two benzamidine cores, one placed in the amide part the other one within the sulfonyl N-capping group. The amino acid side chain bears the 6,7-dimethoxycoumarin fluorophore. The fluorescent amino acid was prepared by using the von-Pechmann reaction. The two corresponding nitrile groups of a precursor molecule were simultaneously converted to the amidine moieties by the Pinner reaction. The fluorescent properties of the final compound were determined.

Full text of DOI:10.1002/hc.21269

Heteroatom Chemistry
Volume 26, Number 5, 2015
Synthesis of a Fluorescent-Labeled
Bisbenzamidine Containing the Central
(6,7-Dimethoxy-4-coumaryl)Alanine Building
Block
Daniela Ha¨ußler and Michael Gu¨tschow
Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, D-53121 Bonn,
Germany
Received 10 March 2015; revised 16 April 2015; accepted 16 April 2015
peptidomimetic inhibitors for trypsin-like serine
ABSTRACT: The synthesis of an amino acid amide
proteases with primary substrate specificity for argi-
is reported, which contains two benzamidine cores,
nine [2–10].
one placed in the amide part the other one within
Bisbenzamidines possess an extended spectrum
the sulfonyl N-capping group. The amino acid side
of antiprotozoal and antifungal activities against the
chain bears the 6,7-dimethoxycoumarin fluorophore.
parasites Plasmodium falciparum, Trypanosoma bru-
The fluorescent amino acid was prepared by using
cei, Leishmania donovani, and Pneumocystis carinii.
the von-Pechmann reaction. The two corresponding
The mode of action has been attributed to the
nitrile groups of a precursor molecule were simul-
ability of the bisbenzamidine scaffold to interact
taneously converted to the amidine moieties by the
with adenine/thymine-rich sites of double-strand
Pinner reaction. The fluorescent properties of the fi-
DNA by insertion into the minor groove [11–14].
ꢀC
nal compound were determined.
2015 Wiley Pe-
Noteworthy, it has been recently shown that cer-
tain aza-bisbenzamidines exhibit DNA-dependent
fluorescent properties. In such compounds, two
4-aminobenzamidine units are connected by link-
ers [15]. DNA binding of peptides, which are con-
jugated to such aza-bisbenzamidines, has also been
examined [16].
Several synthetic routes to benzamidines have
been reported, such as the reaction of benzo-
nitriles with aluminum amides [17], the catalytic
reduction of amidoximes [18], or the catalytic hy-
drogenation of O-acetylamidoximes [4–7,19]. Benz-
amidines can also be obtained from aromatic
thioimidates [2,3] or imidates [2,9,15,16,20] by the
Pinner reaction, a protocol that was utilized in the
course of this study.
riodicals, Inc. Heteroatom Chem. 26:367–373, 2015;
View this article online at wileyonlinelibrary.com.
DOI 10.1002/hc.21269
INTRODUCTION
The benzamidine moiety, capable of being accom-
modated within protein binding sites via anionic
and hydrogen bond interactions, represents a promi-
nent pharmacophore. In drug design, benzamidines
have been extensively shown to act as appropriate
arginine mimetics to address targets whose natural
ligand contains arginine [1]. For example, benz-
amidines have been frequently incorporated into
Fluorescence labeling is of vital importance in
the life sciences. Miscellaneous types of fluorophores
have been employed for manifold purposes. Besides
BODIPY (boron-dipyrromethene) and fluorescein
Correspondence to: Michael Gu¨tschow; e-mail: guetschow@uni-
bonn.de.
2015 Wiley Periodicals, Inc.
ꢀC
367

368 Ha¨ußler and Gu¨tschow
activation of the 4-carboxylic group of 1 with 1,1’-
carbonyldiimidazole, the attack of the magnesium
malonate at the imidazolide and decarboxylation.
The resulting modified amino acid contains the two
carbonyl groups required of the coumarin hetero-
cyclization.
The second precursor, phenol 4, was obtained
from veratraldehyde and m-chloroperbenzoic acid
in the course of a Bayer–Villiger-type oxidation
[34]. The following von-Pechmann reaction yielded
the fluorescent-labeled amino acid 5 [30–32]. Its
coumarin core originated from 4 and the 1,3-
dicarbonyl unit of 2. In detail, we first hydrogenolyt-
ically deprotected the 1-carboxyl group of 2, then
performed the methanesulfonic acid promoted con-
densation, leading to N-deprotection, and after-
wards reprotected the amino group with di-tert-butyl
dicarbonate.
The linear synthesis of the fluorescent-labeled
bisbenzamidine is outlined in Scheme 2. It was in-
tended to introduce two benzonitrile groups, re-
quired to a subsequent transformation into benz-
amidines. One should be placed in the amide
part of the amino acid amide, the other one
within the N-capping group. For this purpose,
we first activated the carboxylic group of 5 with
isobutyl chloroformate and N-methylmorpholine
and reacted the resulting mixed anhydride with
4-aminomethylbenzonitrile to obtain the amino acid
amide 6. To remove the N-protecting group, gaseous
hydrogen chloride was passed through the solution
of 6 in dioxane and the chloride salt 7 precipitated.
The second nitrile group was introduced with 4-
cyanobenzene sulfonylchloride in the presence of
DIPEA (N,N-diisopropylethylamine) leading to the
sulfonamide 8. It was intended to simultaneously
convert both nitrile groups to the amidines by means
of the Pinner reaction. Gaseous hydrogen chloride
was introduced to activate the cyano groups, which
gave imidate moieties by the nucleophilic attack of
methanol and, in turn, produced with ammonium
acetate the amidine functionalities of the final prod-
uct 10.
SCHEME 1 Synthesis of the fluorescent-labeled amino acid
N-Boc-L-(6,7-dimethoxy-4-coumaryl)alanine (5).
derivatives, coumarins represent a widely used
class of fluorescent dyes and are valued for their
small molecular size and large Stokes shifts [21–
28]. Structure–fluorescence relationships of the
coumarin chemotype revealed high fluorescence as
a result of the introduction of an electron-donating
substituent at the 7-position. In particular, the 6,7-
dimethoxy substitution pattern produces a high flu-
orescence quantum yield. It has been found that the
sensitivity for detection of such coumarin deriva-
tives is particularly high and comparable to that ob-
tained by radiolabeling [30–32]. Here, we report on
a fluorescent-labeled bisbenzamidine containing a
central amino acid building block, which, on its part,
bears the 6,7-dimethoxycoumarin fluorophore.
We have recorded UV/Vis and fluorescent
spectra of 10 in two different solvents. Coumarins
possess desired properties of fluorescent labels,
i.e., a high fluorescent intensity and a large Stokes
shift. Such features were also obtained for the
coumarin-labeled bisbenzamidine 10 as shown in
Fig. 1. This compound exhibited an absorption
maximum of 348 nm (ε = 8800 M−1 cm−1) and
350 nm (ε = 9700 M−1 cm−1) in buffer (pH 8.0) and
acetonitrile, respectively. The Stokes shift was 84
and 72 nm, respectively. For the purpose of com-
parison, we have also recorded coumarin 343 (12).
RESULTS AND DISCUSSION
The synthesis starts with the preparation of the
coumarin-labeled amino acid (Scheme 1). Two pre-
cursors were required. The first precursor (2) was
prepared from Boc-L-aspartic acid 1-benzyl ester (1)
and monomethyl monopotassium malonate. Addi-
tion of magnesium chloride produced the corre-
sponding magnesium salt, which was reacted with
1 as described [33]. The conversion included the
Heteroatom Chemistry DOI 10.1002/hc

Fluorescent-Labeled Bisbenzamidine Containing the Central (6,7-Dimethoxy-4-coumaryl) Alanine Building Block 369
SCHEME 2 Synthesis of the coumarin-labeled bisbenzamidine (10).
exhibited a strong fluorescence increase (ꢀ60-fold,
λex = 387 nm, λem = 472 nm) after addition of
double-strand DNA oligonucleotides containing the
AATTT target sequence [24]. Thus, the amino amide
10 might be inspected in future studies to be simi-
larly useful as a biochemical tool.
SCHEME 3 Synthesis of coumarin 343 (12).
EXPERIMENTAL
Its synthesis is depicted in Scheme 3. Coumarin 343
is a representative of rigidified 7-aminocoumarins,
characterized by their bathochromic shift of both
absorption and emission [21,25–27]. It has been re-
ported that coumarin 343 gives a high fluorescence
quantum yield (φ = 0.7) in water [28] and also in
organic solvents, i.e., ethanol (φ = 0.63) [29].
Melting points were determined using a Bu¨chi B510
oil bath apparatus and are not corrected. Thin-
layer chromatography was carried out on Merck
aluminum sheets, coated with silica gel 60 F254
.
Preparative column chromatography was performed
˚
using Merck silica gel (0.06 – 0.20 mm, 60 A).
1H and 13C NMR spectra were recorded on a
Bruker Advance DRX-500 spectrometer operating at
500 MHz for 1H and 125 MHz for 13C at 30°C.
Chemical shifts δ are given in ppm referring to
the signal center using the solvent peaks for refer-
ence: DMSO-d6 2.49/39.7 ppm. Elemental analyses
were performed with a Vario EL apparatus. LC-DAD
The application of compound 10 in biomed-
ical studies is envisaged. In particular, one can
expect affinity of 10 to the minor grooves of
adenine/thymine-rich double-strand DNA. Notewor-
thy, a fluorescent-labeled symmetrical bisbenz-
amidine with a 7-diethylamino-coumarin moiety
Heteroatom Chemistry DOI 10.1002/hc

370 Ha¨ußler and Gu¨tschow
FIGURE 1 Absorption (10 µM, 1% DMSO) and emission (1 µM, 1% DMSO) spectra of 10 (black) and coumarin 343 (12) (gray).
The molar extinction coefficients and fluorescent units, respectively, were plotted versus the wavelength. Spectra recorded in
buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl) and acetonitrile are shown as solid lines and dashed lines, respectively. Maxima
were as follows, λex = 348 nm, λem = 432 nm (10 in buffer); λex = 350 nm, λem = 422 (10 in acetonitrile); λex = 430 nm, λem
=
486 nm (12 in buffer); λex = 447 nm, λem = 486 nm (12 in acetonitrile).
chromatograms, and ESI-MS spectra were recorded
on an Agilent 1100 HPLC system with Ap-
plied Biosystems API-2000 mass spectrometer. The
method was as follows: A solution of the com-
pounds (10 mg/1 mL) was prepared with buffer
(2 mM NH4OAc) and methanol (1:1) and 10 µL
were injected. Mobile phase A was the aforemen-
tioned buffer; mobile phase B was methanol; gra-
dient 10% B in 20 min to 100% B, 10 min 100%
B. HRMS was recorded on a microOTOF-Q mass
spectrometer (Bruker) with ESI-source coupled with
a HPLC Dionex Ultimate 3000 (Thermo Scientific)
using a EC50/2 Nucleodur C18 Gracity 3 µm col-
umn (Macherey-Nagel). The method was as follows.
1 µL of a sample solution (1 mg/1 mL) was injected.
Mobile phase A was buffer (2 mM NH4OAc); mo-
bile phase B was acetonitrile; 1 min 10% B, gra-
dient 10% B in 9 min to 100% B, 5 min 100%
B. Absorption spectra were recorded on the Varian
Cary 50 Bio (Varian, Agilent Technologies), emis-
sion spectra on the Monaco Safas spectrofluorom-
eter flx after adjustment of a PMT value of 200 V.
Coumarin 343 (12) was obtained by the Knoevenagel
reaction and subsequent heterocyclization from 8-
hydroxyjulolidine-9-carboxaldehyde (11) and iso-
propylidene malonate, following a reported method
to obtain coumarin-3-carboxylic acids [35].
1-benzyl ester (1) (2.910 g, 9 mmol) in dry THF
(12 mL) at 5°C and stirred for 1 h at room tem-
perature. To the resulting monomethyl magnesium
malonate suspension, the imidazolide solution was
added dropwise at room temperature. The reaction
mixture was stirred at room temperature overnight,
concentrated under reduced pressure, and sus-
pended in ethyl acetate (150 mL). The suspension
was washed with aqueous sodium sulfite solution,
aqueous potassium bisulfate solution, and sodium
hydrogencarbonate solution (1 × 100 mL each). The
organic layer was dried, and the solvent was re-
moved under reduced pressure to yield 2 (3.347 g,
8.82 mmol, 98%) as a white crystalline product; mp
1
68–71°C, mp lit. 69–72°C [36]; H NMR (500 MHz,
DMSO-d6, 30°C): δ 1.35 (s, 9H, C(CH3)3), 2.89 (dd,
2
3
3J = 8.0 Hz, J = 17.8 Hz, 1H, 3-H), 3.04 (dd, J =
2
5.2 Hz, J = 17.8 Hz, 1H, 3-H), 3.59–3.66 (m, 5H,
Me, 5-H), 4.38–4.42 (m, 3H, 2-H, CH2Ph), 7.24 (s,
1H, OCONH), 7.31–7.37 (m, 5H, CHarom); 13C NMR
(125 MHz, DMSO-d6, 30°C): δ 28.24 (C(CH3)3), 43.44
(C-3), 48.90 (C-5), 49.24 (C-2), 51.93 (Me), 66.24
(CH2Ph), 78.58 (C(CH3)3), 127.78 (C-2’), 128.11 (C-
4’), 128.47 (C-3’), 135.97 (C-1’), 155.30 (OCONH),
167.41 (C-6), 171.52 (C-1), 200.34 (C-4). Anal. Calcd.
for C19H25NO7: C, 60.15; H, 6.64; N, 3.69. Found C,
59.86; H, 6.65; N, 3.70.
1-Benzyl 6-Methyl-2-(tert-butoxycarbonylamino)-
4-oxohexanedioate (2). Following a literature proce-
dure [32], a suspension of dry magnesium chloride
(0.857 g, 9 mmol) and monomethyl monopotassium
malonate (2.109 g, 13.5 mmol) in dry THF (9 mL)
was stirred for 4 h at 50°C. In a second flask, 1,1’-
carbonyldiimidazole (1.751 g, 10.8 mmol) was added
portionwise to a solution of Boc-L-aspartic acid
3,4-Dimethoxyphenol (4) [34]. To a solution of
3,4-dimethoxybenzaldehyde (1.994 g, 12 mmol) in
CH2Cl2 (50 mL), m-chloroperbenzoic acid (2.278 g,
13.2 mmol) was added and the mixture was stirred
at room temperature for 15 h. The reaction mix-
ture was washed with saturated aqueous Na2SO3
solution (3 × 20 mL) and brine (1 × 30 mL). The
organic layer was dried over Na2SO4, filtered and
Heteroatom Chemistry DOI 10.1002/hc

Fluorescent-Labeled Bisbenzamidine Containing the Central (6,7-Dimethoxy-4-coumaryl) Alanine Building Block 371
concentrated under reduced pressure. MeOH
(30 mL) and K2CO3 (3.317 g, 24 mmol)
were added to the residue. The mix-
(C(CH3)3), 33.34 (C-3), 52.66 (C-2), 56.15 (OMe),
56.33 (OMe), 78.47 (C(CH3)3), 100.56 (C-8’), 106.02
(C-5’), 111.22 (C-4a’), 112.24 (C-3’), 146.02 (C-4’),
149.19 (C-8a’), 152.69 and 153.04 (C-6, C-7), 155.47
(OCONH), 160.34 (C-2’), 172.84 (C-1). LC-MS (ESI,
DAD 220–400 nm): 97% purity; C19H23NO8 requires
393.14; m/z: 394.50 [M + H]+, m/z: 392.28 [M +
H]−. Anal. Calcd. for C19H23NO8 × H2O: C, 55.47;
H, 6.13; N, 3.40. Found: C, 55.54; H, 6.18; N, 3.42.
tert-Butyl (S)-N-1-(4-Cyanobenzylamino)-3-(6,7-
dimethoxy-2-oxo-2H-chromen-4-yl)-1-oxopropan-
ture was stirred at room temperature for
30 min. The solvent was removed under reduced
pressure, and the residue was dissolved in EtOAc
(30 mL), washed with H2O (2 × 20 mL) and brine
(1 × 20 mL), and dried over Na2SO4. The organic
phase was filtered and concentrated under reduced
pressure to yield 4 (1.441 g, 9.35 mmol, 78%) as
brown crystals; mp 85–86°C, mp lit. 80–82°C [34].1H
NMR (500 MHz, DMSO-d6, 30°C): δ 3.63 (s, 3H,
OMe), 3.68 (s, 3H, OMe), 6.23 (dd, 4J = 2.5 Hz, 3J =
8.6 Hz, 1H, 6-H), 6.39 (d, 4J = 2.5 Hz, 1H, 2-H), 6.72
(d, 3J = 8.6 Hz, 1H, 5-H), 8.90 (s, 1H, OH); 13C NMR
(125 MHz, DMSO-d6, 30°C): δ 55.44 O(Me), 56.57
(OMe), 101.01 (C-2), 105.80 (C-6), 113.94 (C-5),
141.91 (C-4), 149.94 (C-3), 152.04 (C-1).
2-yl)carbamate
(6).
N-Boc-L-(6,7-dimethoxy-4-
coumaryl)alanine (5, 1.234 g, 3.3 mmol) was
dissolved in dry THF (70 mL), and the carboxyl
group was activated using N-methylmorpholine
(367 mg, 0.40 mL, 3.63 mmol), isobutyl chlo-
roformate (497 mg, 0.47 mL, 3.63 mmol) and
triethylamine (668 mg, 0.91 mL, 6.6 mmol) at
-25°C. After 15 min, 4-(aminomethyl)benzonitrile
hydrochloride (0.556 g, 3.3 mmol) was added. The
mixture was stirred overnight at room temperature
and evaporated under reduced pressure. The residue
was recrystallized from ethyl acetate to obtain 6
(0.452 g, 0.89 mmol, 27%) as a white solid; mp
231–233°C.1H NMR (500 MHz, DMSO-d6, 30°C): δ
(S)-2-(tert-Butoxycarbonylamino)-3-(6,7-
dimethoxy-2-oxo-2H-chromen-4-yl)propanoic
Acid
(5). Following a literature procedure [32], the
β-ketoester 2 (2.276 g, 6 mmol) was dissolved in a
mixture of ethyl acetate (20 mL), ethanol (20 mL),
and 1N HCl (6 mL). The mixture was subjected to
hydrogenolysis under atmospheric pressure over
Pd on charcoal (10%, 2.276 g/10 g) for 3 h. The
resulting reaction mixture was filtrated through
celite, washed with EtOH, and concentrated under
reduced pressure. The deprotected β-ketoester and
3,4-dimethoxyphenol 4 (1.387 g, 9 mmol) were
dissolved in 99% methanesulfonic acid (14.42 g,
9.7 mL, 150 mmol) at 0°C and stirred at room tem-
perature for 2 h. The deep-violet reaction mixture
was diluted with water (10 mL), and the excess
phenol was extracted with diethyl ether (2 × 70 mL).
Afterwards, the pH was adjusted to 8 by the addition
of 10 N NaOH. 1,4-Dioxane (25 mL) was added, and
the α-amino group was protected with di-tert-butyl
dicarbonate (1.44 g, 1.52 mL, 6.6 mmol) at room
temperature and stirred overnight. The reaction
mixture was adjusted to pH 3 with 2 N HCl, and
the crude product was extracted with ethyl acetate
(1 × 50 mL). The organic phase was evaporated
under reduced pressure, and the crude product was
purified by means of column chromatography using
ethyl acetate:methanol (9:1) + 1% AcOH to yield
the brown solid 5 (1.382 g, 3.36 mmol, 56%) as
monohydrate; mp 117–120°C.1H NMR (500 MHz,
DMSO-d6, 30°C): δ 1.29 (s, 9H, C(CH3)3), 3.00 (dd,
3
2
1.31 (s, 9H, C(CH3)3), 2.97 (dd, J = 9.2 Hz, J =
3
2
13.9 Hz, 1H, 3-H), 3.23 (dd, J = 4.7 Hz, J = 13.9
Hz, 1H, 3-H), 3.84 (s, 3H, OMe), 3.86 (s, 3H, OMe),
4.31–4.36 (m, 1H, 2-H), 4.35 (d, 3J = 5.9 Hz, 2H,
CH2C4H4CN), 6.19 (s, 1H, 3’-H), 7.05 (s, 1H, 8’-H),
7.23 (d, 3J = 8.5 Hz, 1H, OCONH), 7.26 (s, 1H, 5’-H),
3
3
7.40 (d, J = 8.1 Hz, 2H, 2"-H), 7.73 (d, J = 8.1 Hz,
2H, 3"-H), 8.64 (t, 3J = 5.9 Hz, 1H, CONH); 13C NMR
(125 MHz, DMSO-d6, 30°C): δ 28.13 (C(CH3)3),
34.08 (C-3), 42.13 (CH2C4H4CN), 53.86 (C-2), 56.15
(OMe), 56.30 (OMe), 78.61 (C(CH3)3), 100.44 (C-8’),
106.32 (C-5’), 109.64 (C-4"), 111.32 (C-4a’), 112.35
(C-3’), 118.99 (CN), 127.98 (C-2"), 132.23 (C-3"),
145.27 (C-1"), 145.95 (C-4’), 149.17 (C-8a’), 152.65
and 153.13 (C-6’, C-7’), 155.44 (OCONH), 160.34
(C-2’), 170.98 (C-1). LC-MS (ESI, DAD 220–400
nm): 99% purity; C27H29N3O7 requires 507.20; m/z:
508.31 [M + H]+, m/z: 506.43 [M + H]−. HRMS:
m/z: [M + H]+ calcd. 508.2078. Found 508.2102.
(S)-1-(4-Cyanobenzylamino)-3-(6,7-dimethoxy-
2-oxo-2H-chromen-4-yl)-1-oxopropan-2-aminium
Chloride (7). Compound 6 (0.431 g, 0.85 mmol)
was dissolved in 1,4-dioxane (25 mL). Dry gaseous
hydrogen chloride was introduced over a period
of 30 min. The reaction mixture was stirred at
room temperature overnight. A white precipitate
was formed and collected by suction filtration to
yield 7 (0.313 g, 0.71 mmol, 83%), which was used
in the next step without further purification; mp
266–267°C.1H NMR (500 MHz, DMSO-d6, 30°C):
2
3
3J = 10.4 Hz, J = 14.4 Hz, 1H, 3-H), 3.27 (dd, J =
2
3.8 Hz, J = 14.4 Hz, 1H, 3-H), 3.84 (s, 3H, OMe),
3.86 (s, 3H, OMe), 4.24–4.29 (m, 1H, 2-H), 6.18
(s, 1H, 3’-H), 7.06 (s, 1H, 8’-H), 7.23 (s, 1H, 5’-H),
7.25 (d, 3J = 8.9 Hz, 1H, OCONH), 12.54 (s, 1H,
OH); 13C NMR (125 MHz, DMSO-d6, 30°C): δ 28.15
Heteroatom Chemistry DOI 10.1002/hc

372 Ha¨ußler and Gu¨tschow
3
2
was then passed through the solution for 30
min. The mixture was stirred at room temper-
ature for 48 h. The imidate ester hydrochloride
precipitated as a gray solid. It was washed with
diethyl ether and dried in vacuo to give (S)-methyl
4-(N-(3-(6,7-dimethoxy-2-oxo-2H-chromen-4-yl)-
1-(4-(imino(methoxy)methyl)-benzylamino)-1-
oxopropan-2-yl)sulfamoyl)benzimidate (9), which
was directly used and was dissolved in a mixture
of a 7 N ammonia solution in methanol (15 mL)
and dichloromethane (5 mL). Dry ammonium
acetate (0.162 g, 2.1 mmol) was added, and the
reaction mixture was stirred for further 24 h at
room temperature. It was evaporated and ethyl
acetate (50 mL, precooled to –20°C) was added to
the residue. A white precipitate was obtained and
purified by preparative HPLC (MeOH/H2O, 4:6). A
few drops of F3CCO2H were added, and 10 (15 mg,
0.02 mmol, 5%) was obtained after lyophilization,
mp 158–162°C.1H NMR (500 MHz, DMSO-d6, 30°C):
δ 3.26 (dd, J = 8.4 Hz, J = 13.4 Hz, 1H, 3-H), 3.41
(dd, 3J = 6.6 Hz, 2J = 13.4 Hz, 1H, 3-H), 3.87 (s, 3H,
OMe), 3.89 (s, 3H, OMe), 4.14 (dd, 3J = 6.6 Hz, 3J =
8.4 Hz, 1H, 2-H), 4.27–4.37 (m, 2H, CH2C4H4CN),
3
6.16 (s, 1H, 3’-H), 7.08 (s, 1H, 8’-H), 7.35 (d, J =
3
8.3 Hz, 2H, 2"-H), 7.36 (s, 1H, 5’-H), 7.70 (d, J =
8.3 Hz, 2H, 3"-H), 8.77 (s, 3H, NH3+), 9.34 (t,
3J = 6.0 Hz, 1H, CONH); 13C NMR (125 MHz,
DMSO-d6, 30°C): δ 32.90 (C-3), 42.07 (CH2C4H4CN),
51.86 (C-2), 56.36 (OMe), 56.91 (OMe), 100.50
(C-8’), 106.76 (C-5’), 109.88 (C-4"), 111.06 (C-4a’),
113.80 (C-3’), 118.92 (CN), 128.12 (C-2"), 132.26
(C-3"), 144.20 (C-1"), 146.15 (C-4’), 149.40 (C-8a’)
150.00 and 152.93 (C-6’, C-7’), 160.09 (C-2’), 167.64
(C-1). LC-MS (ESI, DAD 220–400 nm): 92% purity;
C22H21N3O5 requires 407.15; m/z: 408.10 [M + H]+.
(S)-N-(4-Cyanobenzyl)-2-(4-cyanophenyl-
sulfonamido)-3-(6,7-dimethoxy-2-oxo-2H-chromen-
4-yl)propanamide
(8).
4-Cyanobenzenesulfonyl
chloride (135 mg, 0.67 mmol) was dissolved in dry
THF (8 mL). Afterwards, compound 7 (0.300 g, 0.67
mmol) and DIPEA (0.27 mL, 207 mg, 1.6 mmol)
were added. The reaction mixture was stirred for 1
h at room temperature. The solvent was removed
under reduced pressure, and the crude product was
purified by column chromatography using ethyl
acetate as an eluent. Compound 8 (0.219 g, 0.38
mmol, 57%) was obtained as a white solid; mp 244–
246°C.1H NMR (500 MHz, DMSO-d6, 30°C): δ 2.87
(dd, 3J = 9.4 Hz, 2J = 13.9 Hz, 1H, 3-H), 3.13 (dd, 3J
3
2
δ 2.93 (dd, J = 8.7 Hz, J = 14.1 Hz, 1H, 3-H), 3.14
3
2
(dd, J = 5.8 Hz, J = 14.1 Hz, 1H, 3-H), 3.82, (s,
3H, OMe), 3.86 (s, 3H, OMe), 4.16–4.22 (m, 2H,
CH2-amidinophenyl), 4.28 (dd, 3J = 5.8 Hz, 3J =
9.8 Hz, 1H, 2-H), 6.10 (s, 1H, 3’-H), 6.96 (s, 1H,
3
8’-H), 7.12 (s, 1H, 5’-H), 7.34 (d, J = 8.4 Hz, 2H,
2"-H or 3"-H), 7.72 (d, 3J = 8.4 Hz, 2H, 2"-H or 3"-H),
7.81 (d, 3J = 8.9 Hz, 2H, 2ꢁꢁꢁ-H or 3ꢁꢁꢁ-H), 7.88 (d,
3J = 8.9 Hz, 2H, 2ꢁꢁꢁ-H or 3ꢁꢁꢁ-H), 8.81–8.85 (m, 2H,
SO2NH, CONH), 9.20 (s, 2H, NH2), 9.25 (s, 2H,
NH2), 9.37 (s, 2H, NH2), 9.52 (s, 2H, NH2); 13C NMR
(500 MHz, DMSO-d6, 30°C): δ 34.52 (C-3), 41.87
(CH2-amidinophenyl), 55.94 (C-2), 56.16 (OMe),
56.28 (OMe), 100.28 (C-8’), 106.23 (C-5’), 110.82
(C-4a’), 112.94 (C-3’), 126.61 (C-2ꢁꢁꢁ), 127.33 (C-2"),
128.16 (C-3ꢁꢁꢁ), 128.73 (C-3"), 131.36 (C-4" and C-4ꢁꢁꢁ),
145.30 and 145.46 (C-1", C-1ꢁꢁꢁ), 145.92 (C-4’), 149.10
(C-8a’), 151.66 and 152.77 (C-6’, C-7’), 160.11 (C-2’),
164.27 and 164.45 (NCN), 169.69 (C-1). Signals
for trifluoroacetate were not detectable. LC-MS
(ESI, DAD 220–400 nm): 97% purity; C29H30N6O7S
requires 606.19; m/z: 607.28 [M + H]+; 605.35
[M + H]−.
2
= 4.9 Hz, J = 13.9 Hz, 1H, 3-H), 3.82 (s, 3H), 3.89
(s, 3H, OMe), 4.09 (dd, 3J = 4.9 Hz, 3J = 9.4 Hz, 1H,
2-H), 4.23–4.31 (m, 2H, CH2C4H4CN), 6.13 (s, 1H,
3’-H), 6.94 (s, 1H, 8’-H), 7.06 (s, 1H, 5’-H), 7.32 (d,
3J = 8.7 Hz, 2H, 2"-H), 7.69 (d, 3J = 8.7 Hz, 2H,
3"-H), 7.73-7.76 (m, 4H, 2ꢁꢁꢁ-H, 3ꢁꢁꢁ-H), 8.78 (t, J =
3
6.0 Hz, 1H, CONH), 8.82 (br s, 1H, SO2NH); 13C
NMR (125 MHz, DMSO-d6, 30°C): δ 34.52 (C-3),
42.01 (CH2C4H4CN), 55.79 (C-2), 56.24 (2 × OMe),
100.25 (C-8’), 106.32 (C-5’), 109.80 (C-4"), 110.62
(C-4a’), 113.27 (C-3’), 114.96 (C-4ꢁꢁꢁ), 117.79 (CN),
118.92 (CN), 126.83 (C-2ꢁꢁꢁ), 127.91 (C-2"), 132.28
(C-3"), 132.79 (C-3ꢁꢁꢁ), 144.60 and 144.77 (C-1", C-
1ꢁꢁꢁ), 145.88 (C-4’), 150.00 (C-8a’), 151.40 and 152.74
(C-6’, C-7’), 159.91 (C-2’), 169.77 (C-1). LC-MS
(ESI, DAD 220–400 nm): 99% purity; C29H24N4O7S
requires 572.14; m/z: 573.24 [M + H]+; m/z: 571.44
[M + H]−. HRMS: m/z: [M + H]+ calcd. 573.1439.
Found 573.1468.
ACKNOWLEDGMENT
The work was supported by the NRW International
Graduate Research School Biotech-Pharma
(S)-4-(N-(1-(4-Amidinobenzylamino)-3-(6,7-
dimethoxy-2-oxo-2H-chromen-4-yl)-1-oxopropan-
2-yl)sulfamoyl)benzamidinium-bis-trifluoroacetate
(10). Compound 8 (0.200 g, 0.35 mmol) was
dissolved in a mixture of CH2Cl2 (20 mL) and
MeOH (10 mL). Dry gaseous hydrogen chloride
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