Fluorescent analogs of the marine natural product psammaplin A: Synthesis and biological activity

Article abstract of DOI:10.1039/c2ob25909e

The symmetrical disulfide psammaplin A from the marine sponge Pseudoceratina sp. was synthesized and structurally altered by replacement of one of the α-(hydroxyimino)acyl units by a fluorescent 4-coumarinacetyl moiety. Thus, the first fluorescent analogs of psammaplin A were obtained. Structural variation also covered the substitution pattern of the phenyl ring. Cytotoxicity of psammaplin A against the mouse fibroblast cell line L-929 (IC₅₀ 0.42 μg mL^-1) was about two-fold higher than that of the fluorescent hybrid compounds, whereas the disulfide containing two 4-coumarinacetyl moieties was inactive. Fluorescence microscopy revealed uptake of the 4-coumarinacetyl-α-(hydroxyimino)acyl hybrids into the cytoplasm leading to fluorescence in close proximity of the nuclear envelope, most likely in the Golgi apparatus. We did not observe strong fluorescence inside the nucleus, where most of the target histone deacetylases are located. We conclude that reduction of the disulfide probably takes place outside the nucleus. The psammaplin-derived thiol exhibited potent activity against histone deacetylase in the low nanomolar range, but diminished cytotoxicity. Antimicrobial activity of the new derivatives was also determined.

Full text of DOI:10.1039/c2ob25909e

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PAPER
Fluorescent analogs of the marine natural product psammaplin A: synthesis
and biological activity†
Fabia Hentschel,a Florenz Sasseb and Thomas Lindel*a
Received 11th May 2012, Accepted 6th July 2012
DOI: 10.1039/c2ob25909e
The symmetrical disulfide psammaplin A from the marine sponge Pseudoceratina sp. was synthesized
and structurally altered by replacement of one of the α-(hydroxyimino)acyl units by a fluorescent
4-coumarinacetyl moiety. Thus, the first fluorescent analogs of psammaplin Awere obtained. Structural
variation also covered the substitution pattern of the phenyl ring. Cytotoxicity of psammaplin A against
the mouse fibroblast cell line L-929 (IC50 0.42 μg mL−1) was about two-fold higher than that of the
fluorescent hybrid compounds, whereas the disulfide containing two 4-coumarinacetyl moieties was
inactive. Fluorescence microscopy revealed uptake of the 4-coumarinacetyl-α-(hydroxyimino)acyl hybrids
into the cytoplasm leading to fluorescence in close proximity of the nuclear envelope, most likely in the
Golgi apparatus. We did not observe strong fluorescence inside the nucleus, where most of the target
histone deacetylases are located. We conclude that reduction of the disulfide probably takes place outside
the nucleus. The psammaplin-derived thiol exhibited potent activity against histone deacetylase in the low
nanomolar range, but diminished cytotoxicity. Antimicrobial activity of the new derivatives was also
determined.
depleted cells were insensitive against psammaplin A and con-
cluded that 1 functions as a prodrug being reduced to the thiol
Introduction
The natural product psammaplin A (1) is a symmetrical disulfide
with a cystamine linker functionalized on both sides by tyrosine-
derived α-(hydroxyimino)acyl moieties. The compound was iso-
lated in 1987 from the marine sponge Psammaplysilla (revised
to Pseudoceratina) sp. by the Crews1 and Scheuer2 groups who
reported cytostatic properties and activity against Gram-positive
bacteria, respectively. Jung and co-workers determined ED50 and
IC50 values between 100 nM and 1 μM against several cancer
cell lines,3 decreasing the percentage of human endometrial
cancer cells in the S phase in favor of those in the G0/G1 and
G2/M phases.4 Psammaplin A (1) and its derivatives have
become interesting for epigenetic therapy, since Bair, Crews and
co-workers have identified 1 as a potent inhibitor of histone dea-
cetylase (HDAC, IC50 4.2 2.4 nM), being more active than the
benchmark natural product and hydroxamic acid trichostatin A.5
Ho Jeong Kwon and co-workers6 found that glutathione-
inside the cell. This is supported by recent structure–activity
studies by the Fuchter7 and de Lera8 groups. HDAC1 inhibition
by the thiol proved to be stronger than by the disulfide and
S-methylation of the thiol abolished the activity completely.
Docking studies of psammaplin A-derived thiol 2 with the
protein part of a trichostatin A–HDAC8 complex8 and with
HDAC17b indicate that thiolate may serve as a ligand of Zn2+
(Fig. 1).
aInstitute of Organic Chemistry, TU Braunschweig, Hagenring 30,
38106 Braunschweig, Germany. E-mail: th.lindel@tu-braunschweig.de;
Fax: (+49) 531-391-7744
bDepartment of Chemical Biology, Helmholtz Centre for Infection
Research, Inhoffenstraße 7, 38124 Braunschweig, Germany
†Electronic supplementary information (ESI) available: 1H and 13C
NMR spectra of compounds 1, 4, 7–14, 16, 17, 19, 22–27, 30, 31, 33
and 34. See DOI: 10.1039/c2ob25909e
Fig. 1 Psammaplin A (1), reduced form 2, and coumarin derivative 3.
7120 | Org. Biomol. Chem., 2012, 10, 7120–7133
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Results and discussion
Class I HDACs are located in the nucleus9 and it has remained
unclear whether reduction of the disulfide prodrug to the thiol
takes place before or after the disulfide penetrates the nucleus. To
address that question, we decided to synthesize fluorescent
analogs of psammaplin A and to analyze their distribution in the
cell by fluorescence microscopy. The architecture of the new flu-
orescent psammaplin analogs should allow reduction to a bio-
logically active (2) and a fluorescent unit (3). If no fluorescence
would be detected in the nucleus, reduction could either take
place already outside the nucleus, or the coumarin unit would
leave the nucleus rapidly after reduction. If, however, a fluor-
escent disulfide would bind inside the nucleus without reduction,
this should be clearly visible under the microscope. The cou-
marin tag itself lacks strong biological activity and supports
water solubility.10 It was, of course, also to be tested whether our
hybrid compounds were cytotoxic at all.
Synthesis
The synthesis of tyrosine-derived α-(hydroxyimino)amides from
marine sponges has been reviewed comprehensively in 201011
and continues to be the subject of research.7,8,12 An efficient
route elongates substituted benzaldehydes by the Horner–Wads-
worth–Emmons reaction employing Nakamura’s α-OTBS-func-
tionalized
dimethylphosphonate
6.13
The
resulting
silylenolethers react smoothly to the corresponding oximes upon
treatment with hydroxylamine derivatives.14
We synthesized psammaplin A (1) via the Horner–Wads-
worth–Emmons (HWE) route originally established by Spil-
ling.14a The HWE route has the advantage of making available
derivatives of psammaplin A by varying the benzaldehyde build-
ing block. A similar approach was reported recently by Fuchter
and co-workers.7
Bromination of p-hydroxybenzaldehyde15 afforded an
8 : 1 mixture of 3-bromo and 3,5-dibromo-4-hydroxybenzalde-
hydes, which was benzylated without separation (Scheme 1).
Column chromatography yielded aldehyde 4 (58%) together
with its 3,5-dibrominated analog (7%). HWE reaction of 4 with
phosphonate 614a gave ester 7 as E/Z-isomers (1 : 1, 51%). After
desilylation of 7 with 3HF·NEt3 oxime 9 was formed in situ by
treatment with hydroxylamine hydrochloride (97%). Debenzyla-
tion (H2/Pd–C) of 9 afforded the E/Z-hydroxyimino isomers in a
ratio of 4 : 1 (51%). This monobrominated product was obtained
recently by de Lera8 after bromination with NBS of the corre-
sponding debromo precursor. Saponification of the methyl ester
with lithium hydroxide gave acid 12 (97%, E/Z 4 : 1), two
equivalents of which were coupled in the final step with cysta-
mine dihydrochloride (15) in the presence of NEt3, DCC, and
N-hydroxysuccinimide (NHS) in DMF16 to afford the natural
product psammaplin A (1, 36%, E,E-isomer, δ(benzylic CH2)
28.4 in acetone-d6). Psammaplin derivative 16 lacks the phenolic
hydroxyl groups and was synthesized analogously to 1 starting
from m-bromobenzaldehyde (5), with different amide coupling
conditions employing N-hydroxyphthalimide (NHPI) instead of
NHS and MeOH–dioxane (2 : 3) instead of DMF as solvent.17
We also synthesized the doubly THP-protected didehydroxy-
psammaplin 17 using THPONH2·HCl in the oxime forming step
Scheme 1 Synthesis of psammaplin A (1), didehydroxypsammaplin
16, and THP-protected derivative 17.
(Scheme 1). In all cases, the E-configuration of the oxime partial
structures was major, as determined on the basis of NOESY
experiments showing correlations between the oxime proton and
the benzylic protons at 5-H and/or 6-H. E- and Z-isomers of
THP-protected methyl ester 11 were separated by column chrom-
atography and exhibit characteristic NMR shifts of the methylene
group (acetone-d6, E: δH 3.89, 4.01, δC 31.4 and Z: δH 3.72,
3.76, δC 37.2).
The coumarin unit of our fluorescent psammaplin moieties
was synthesized following the Pechmann protocol by conden-
sation of 1,3-acetonedicarboxylate and m-dimethylaminophenol,
followed by saponification (LiOH–H2O) of the ester affording
4-coumarinacetic acid 18 (21% over 2 steps).18 Acid 18 was coupled
with cystamine dihydrochloride (15) providing the symmetrical
bis(coumarinyl) analog 19 of psammaplin A (45%, Scheme 2).
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Scheme 2 Synthesis of the bis(coumarinyl) analog 19 of psamma-
plin A.
For the synthesis of non-symmetrical psammaplin derivatives,
desymmetrization of cystamine was given preference over
disulfide metathesis,19 because the latter gives only a statistical
1 : 2 : 1 ratio of starting materials and hybrid products. Cystamine
dihydrochloride (15) was mono-Boc protected to afford amine
20 (50%)20 and then coupled with the 4-coumarinacetic acid 18
to disulfide 24 (56%, Scheme 3).21 After Boc removal 24 was
coupled in situ with α-(hydroxyimino) acids 12 and 13, respect-
ively, to afford the fluorescent hybrid psammaplin A derivatives
25 (55%) and 26 (62%), respectively. Mono-Boc-protected
cystamine 20 was also coupled with α-(hydroxyimino) acids 21
and 12 to obtain amides 22 (74%) and 23 (28%). The p-hydroxy
acid 21 (98%) was synthesized from p-hydroxyphenylpyruvic
acid.16
As a side reaction of the amide couplings, decarboxylation
and dehydration of the α-(hydroxyimino) acid to the benzyl-
nitriles was observed, such as formation of 27, which was isolated
in 18% yield. This confirms an earlier finding by Spilling et al.
converting α-(hydroxyimino) esters to the benzylnitrile in high
yield by treatment of the free acid with TFA.14a In the presence
of DCC–NEt3, the nitrile is also known to be formed.
Interestingly, by changing the base from NEt3 to pyrrolidine
(28) in a coupling reaction with cystamine dihydrochloride we
obtained the pyrrolidine-derived tertiary amide 30 (42%) as the
only product. This reaction was then transferred to afford the cor-
responding derivative 31 by coupling the acid 21 with thiazoli-
dine (29)22 (40%, Scheme 4).
Thiol 33 was synthesized by treatment of disulfide 22
with dithiothreitol (DTT, 32, 40%, RP chromatography)12,14a
and was stable in methanol-d4. In acetone-d6 we observed
formation of the symmetrical disulfide 34 (Scheme 5), indicated
by intensity decrease of the signals of the methylene protons
of the thiol (33, δ = 3.42 for NCH2 and δ = 2.63 for SCH2)
in favour of those of the disulfide (34, δ = 3.56 for NCH2 and
δ = 2.87 for SCH2).
Scheme 3 Synthesis of fluorescent psammaplin hybrids 25 and 26 and
of the psammaplin derivatives 22 and 23.
against Gram-positive bacteria. Against Micrococcus luteus the
inhibition zone diameter was 11 mm (20 μg per disk, diameter
6 mm), corresponding to an IC50 of 6.7 μg mL−1 in a serial
dilution assay. Similar activities were determined against Myco-
bacterium phlei (12 mm) and Staphylococcus aureus (12 mm),
whereas Gram-negative Klebsiella pneumoniae and Pseudomo-
nas aeruginosa were not sensitive. Only the E. coli TolC mutant
Biological activity
Antibacterial activity. In the agar diffusion test, we observed
moderate activity of the natural product psammaplin A (1)
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Table 1 Cytotoxicity and HDAC inhibitory activity of the psammaplin
derivatives
Compound
IC50 a (μg mL−1
)
IC50 a (μM)
IC50 b (μM)
1
0.42
0.31
>40
0.93
1.10
1.80
1.40
>40
>40
>40
>40
>40
0.63
0.49
Not active
1.46
1.76
4.19
2.75
Not active
Not active
Not active
Not active
Not active
12.48
0.028
0.25
>1.25
0.011
0.50
1.25
0.10
>1.25
>1.25
>1.25
>1.25
>1.25
0.011
0.34
16
19
25
26
22
23
24
13
27
30
31
33
34
17
Scheme 4 Synthesis of pyrrolidine and thiazolidine derivatives 30
and 31.
3.00
1.40
>40
2.76
Not active
>1.25
a Against the L-929 mouse fibroblast cell line. b Against HDAC.
psammaplin A (1) against Mycobacterium phlei may be related
to results by Bewley and co-workers reporting an IC50 of
2.8 0.5 μg mL−1 of psammaplin A (1) against the mycothiol-
S-conjugate amidase from Mycobacterium tuberculosis.24
Cytotoxicity. A few more of our compounds exhibit cytotoxic
activity. Psammaplin
A (1) was cytotoxic against the
L-929 mouse fibroblast cell line (IC50 0.42 μg mL−1, Table 1).
Didehydroxypsammaplin A (16) showed a similar cytotoxicity
(IC50 0.31 μg mL−1). The fluorescent 4-coumarinacetyl-
α-(hydroxyimino)acyl hybrids 25 and 26 retained cytotoxicity in
the range 1 μg mL−1 (IC50). Boc derivatives with only one
α-(hydroxyimino)acyl moiety were also cytotoxic, among which
phenol 22 (IC50 1.80 μg mL−1) was less active than psammaplin
derivative 23 (IC50 1.40 μg mL−1). Bis- and mono(coumarinyl)
analogs 19 and 24 lacking α-(hydroxyimino)acyl moieties were
not active. Compounds without the disulfide bridge, such as acid
13, nitrile 27, p-hydroxyphenylacetonitrile, pyrrolidine derivative
30 and also thiazolidine derivative 31, were not cytotoxic.
Equally, derivative 17 containing THP-protected oximes was not
Scheme 5 Synthesis of thiol 33 and oxidation to disulfide 34.
was slightly sensitive (8 mm). None of the other psammaplin A
derivatives did show any inhibition at all.
Antibacterial activity of psammaplin A (1) against S. aureus
compares well with earlier work. It was known that antibacterial
activity of psammaplin A (1) is rather selective against Gram-
positive Staphylococcus aureus and, to a lesser extent, Strepto-
coccus pyogenes, but does not inhibit, for instance, Gram-nega-
tive Pseudomonas aeruginosa, as reported already by Scheuer
and co-workers.2 Activity against methicillin-resistant S. aureus
(MRSA) was quantified by Sung-Il Yang and co-workers with
average minimum inhibitory concentrations (MICs) ranging
from 0.78 to 6.25 μg mL−1. Gram-negative Escherichia coli,
Klebsiella oxytoca, and Enterobacter cloaca were not sensitive
against psammaplin A (1).23 Nicolaou and co-workers found
MICs of 5.47 μg mL−1 and 3.90 μg mL−1 against four strains of
S. aureus and five strains of MRSA, respectively.19 In our study,
a Micrococcus species proved to be sensitive against psammaplin
A (1) for the first time. It is somewhat surprising that none of the
other synthesized psammaplin A analogs sharing the α-(hydro-
xyimino)acyl unit was active against Gram-positive bacteria,
since Nicolaou and co-workers had identified several psamma-
plin A analogs with activity against MRSA, in which one of the
α-(hydroxyimino)acyl units had been replaced by oxime-free
moieties by disulfide metathesis.19 The disulfide bridge is
necessary for antibacterial activity. Differing from the activity
pattern of HDAC inhibition (vide infra), Nicolaou identified a
disulfide–thiol pair among which only the disulfide was antibac-
terial, but not the thiol.19b The newly discovered activity of
active. Thiol 33 showed decreased cytotoxicity (IC50 3 μg mL−1
)
when compared to the corresponding disulfide 34. Thus, cyto-
toxicity against L-929 was exhibited only by those psammaplin
A derivatives, which contained a free oxime moiety and either a
disulfide or a thiol. This agrees with previously reported studies.
We also can confirm that for cytotoxicity only one α-(hydroxyi-
mino)acyl moiety is required, because Boc derivatives 22 and 23
were cytotoxic. The phenolic hydroxy group of psammaplin A
(1) was not necessary for cytotoxicity, since didehydroxypsam-
maplin A (16) was as active as the natural product. Absence of
the bromine substituent (34) led to only a minor loss of
cytotoxicity.
HDAC inhibition. Table 1 also lists the HDAC inhibitory
activities of our compounds, which were obtained using a fluoro-
metric HDAC assay using HeLa cell lysates as an HDAC source.
Psammaplin A (1, IC50 0.028 μM) and derivative 16 (IC50
0.25 μM) showed HDAC inhibitory activity in the range of the
potent natural product trichostatin A (IC50 0.030 μM). The most
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potent HDAC inhibitors among our compounds are the cou-
marin–psammaplin hybrid 25 (IC50 0.011 μM) and thiol 33
(IC50 0.011 μM), which was found to be 30-fold more potent
than its disulfide derivative 34 (IC50 0.34 μM). None of the non-
cytotoxic compounds showed HDAC inhibition.
The observed cytotoxicities against L-929 correlate well with
HDAC inhibition. As an exception, thiol 33 showed decreased
cytotoxicity and increased HDAC inhibitory activity compared
to the corresponding disulfide 34, confirming Fuchter’s finding7b
that the thiols are more potent than the corresponding disulfides.
However, thiol 33 was the less active compound in the cytotoxi-
city study.
While this work was in progress, a comprehensive study was
reported by Altucci, de Lera, and co-workers who conclude that
the apoptotic effect of 1 on the human leukemia cell line U937
is due to inhibition of Zn2+-dependent HDACs, mainly of
HDAC1, but also of HDAC4, not of HDAC6.8 Fuchter and co-
workers observed selectivity by a factor of more than 20 in favor
of HDAC1 over HDAC6 and reported recently an IC50 of 45 nM
against HDAC1 for 1 and an IC50 of 0.9 nM for the thiol.7
Fluorescence microscopy. Fluorescent hybrid compounds 25
and 26 are cytotoxic (IC50 1 μg mL−1), interestingly at approxi-
mately 50% of the cytotoxicity of psammaplin A (1). Since the
coumarin part alone is not cytotoxic, as shown by bis- and mono
(coumarinyl) analogs 19 and 24, the cytotoxicity of 25 and 26
must be caused by the α-(hydroxyimino)acyl section. Fig. 2
shows the live cell images obtained of L-929 cells incubated
with 25 and 26, which were both taken up into the cytoplasm
and led to fluorescence in close proximity of the nuclear envel-
ope, probably in the Golgi apparatus. We observed only minor
fluorescence inside the nucleus, where HDAC1 is located.9 Non-
cytotoxic bis- and mono(coumarinyl) analogs 19 and 24 lacking
α-(hydroxyimino)acyl sections also caused fluorescence near the
nuclear envelope (see the ESI†). Thus, the coumarin units of the
hybrid and non-hybrid compounds are probably metabolized in
a similar manner, indicating early cleavage of the disulfide
bonds after penetrating the cells. The cytotoxic N-α-(hydroxy-
imino)acyl cysteamine unit, possibly glutathione-bound, would
penetrate the nuclear envelope and inhibit HDAC1 in the
nucleus.
Fig. 2 Live cell imaging of fluorescent compounds 25 (above) and 26
(below) in L-929 mouse fibroblast cells.
fluorescence is observed in the nucleus being the location of the
thiol target enzyme HDAC1. We postulate that cleavage of the
disulfide to the thiol takes place before entering the nucleus. For
entering the cell, however, the disulfide appears to be necessary,
because cytotoxicity is diminished for the thiol.
Consequently, we are currently working on the synthesis of
psammaplin A analogs with coumarin-containing α-(hydroxy-
imino)acyl units which we expect to be enriched in the nucleus.
Conclusion
In summary, we report the synthesis of the first fluorescent psam-
maplin A (1) derivatives by replacement of one of the tyrosine-
derived α-(hydroxyimino)acyl units by a 4-coumarinacetyl
moiety. Interestingly, the cytotoxicity of psammaplin A (1)
Experimental section
against the mouse fibroblast cell line L-929 (IC50 0.42 μg mL−1
)
General methods
is about two-fold higher than that of the fluorescent hybrid com-
pounds 25 and 26, whereas the disulfide containing two 4-cou-
marinylacetyl moieties is inactive.
Activity of the natural product against Gram-positive bacteria
in the single-digit micromolar range is confirmed, for the first
time against Mycobacterium phlei. However, we did not identify
more potent antibacterial derivatives of psammaplin A (1).
We report the first live cell imaging study of fluorescent psam-
maplin derivatives. The strongest fluorescence is found in close
proximity of the nuclear envelope, whereas only minor
NMR spectra were taken with a Bruker DPX-200 (200.1 MHz
for 1H, 188.3 MHz for 19F), a Bruker AV II-300 (300.1 MHz for
1
1H, 75.5 MHz for 13C), a Bruker DRX-400 (400.1 MHz for H,
100.6 MHz for 13C, 376.3 MHz for 19F), a Bruker AV III-400
(400.1 MHz for 1H, 100.6 MHz for 13C) and a Bruker AV II-600
(600.1 MHz for 1H; 150.9 MHz for 13C), referenced to the
solvent signal or TMS. All measurements were carried out at
300 K. Mass spectra were obtained with a ThermoFinnigan
MAT (MAT95XL) spectrometer and a ThermoFisher Scientific
7124 | Org. Biomol. Chem., 2012, 10, 7120–7133
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(LTQ-Orbitrap Velos) spectrometer. IR spectra were recorded
with a Bruker Tensor 27 spectrometer. UV/Vis spectra were
measured with a Varian Cary 100 Bio UV/Vis-spectrometer. Flu-
orescence spectra were measured with a Varian Cary Eclipse flu-
orescence spectrophotometer. Melting points were measured
with a Büchi 530 melting point apparatus. Chemicals were pur-
chased from commercial suppliers and used without further
purification. Silica gel 60 (40–63 μm, Merck) and silica gel
LiChroprep RP-18 (40–63 μm, Merck) were used for column
chromatography. The used petroleum ether (PE) had a boiling
range from 40 to 60 °C. HPLC separation was carried out with a
Merck Hitachi intelligent pump, fitted with a Phenomenex Luna
C18(2) 5 μ column.
Cq-1), 134.3 (2C, C-2, C-6), 129.0 (2C, o-Ph-C), 128.9 (3C,
m-Ph-C, p-Ph-C), 120.1 (1C, CqBr), 75.4 (1C, CH2); IR (ATR):
ν˜ = 1690 cm−1 (vs), 1362 (s), 1254 (vs), 1186 (s), 948 (s), 923
(s), 741 (s), 721 (s), 692 (s), 663 (s); UV-Vis (MeOH): λmax (log
ε) = 263 nm (3.81), 207 (4.61); MS (EI, 70 eV): m/z (%) = 373/
370/368 (2/5/2) [M+], 289 (7), 287 (7), 281 (12), 279 (22), 277
(10), 261 (4), 259 (3), 253 (15), 251 (36), 249 (16), 223 (7), 181
(3), 152 (3), 143 (4), 141 (4), 92 (21), 91 (100), 65 (30),
63 (13).
(E/Z)-Methyl-3-(4-(benzyloxy)-3-bromophenyl)-2-(tert-butyldi-
methylsilyloxy)acrylate (7). Diisopropylamine (0.71 mL,
5.03 mmol, 1.50 equiv.) was dissolved in dry THF (3.5 mL)
under an argon atmosphere and cooled to 0 °C. Then n-BuLi
(1.6 N solution in hexane, 3.14 mL, 5.03 mmol, 1.50 equiv.)
was added dropwise and the mixture was stirred for 10 min at
0 °C and then for 30 min at −78 °C. A solution of phosphonate
6 (1.05 g, 3.35 mmol, 1.00 equiv.) in dry THF (2.5 mL) was
added slowly and the reaction mixture was stirred for 30 min at
−78 °C. A solution of aldehyde 4 (1.36 g, 4.67 mmol, 1.39
equiv.) in dry THF (4 mL) was added dropwise, the reaction
mixture was stirred for 4 h at −78 °C and warmed to rt within
16 h. It was quenched with 15 mL of saturated aqueous NH4Cl
solution and diluted with 70 mL EtOAc. After washing with
saturated aqueous NH4Cl solution (3 × 15 mL), H2O (3 ×
15 mL) and saturated aqueous NaCl solution (3 × 15 mL), the
organic phase was dried over MgSO4, filtered and evaporated.
The residue was purified by column chromatography on silica
[PE–EA (30 : 1)] to give the E/Z-isomers 7 (1 : 1) as a colorless
oil (0.82 g, 1.72 mmol, 51%); TLC [silica, PE–EtOAc (10 : 1)]:
Syntheses
4-(Benzyloxy)-3-bromobenzaldehyde (4) and 4-(benzyloxy)-
3,5-dibromobenzaldehyde. p-Hydroxybenzaldehyde (2.00 g,
16.4 mmol, 1.00 equiv.) was dissolved in CHCl3–MeOH (10 : 1,
22 mL). A solution of Br2 (0.84 mL, 2.62 g, 16.4 mmol, 1.00
equiv.) in CHCl3 (70 mL) was added slowly and the reaction
mixture was stirred for 22 h at rt. It was washed with H2O
(20 mL), saturated aqueous Na2S2O3 solution (20 mL), and H2O
(4 × 20 mL) until pH neutrality. The organic phase was dried
over MgSO4, filtered and the solvent was evaporated. The result-
ing mixture of 3-bromo and 3,5-dibromo aldehyde was used
directly for benzyl protection without further purification. A
mixture of 3-bromo aldehyde (1.00 g, 4.98 mmol, 0.75 equiv.)
and 3,5-dibromo aldehyde (0.47 g, 1.68 mmol, 0.25 equiv.) was
dissolved in DMF (7.3 mL) under an argon atmosphere. K2CO3
(0.79 g, 5.69 mmol, 0.85 equiv.) and BnCl (0.80 mL, 0.88 g,
6.95 mmol, 1.04 equiv.) were added and the reaction mixture
was heated at reflux for 75 min. After cooling to rt, 40 mL H2O
was added and the precipitate was filtered off. The crude product
was purified by column chromatography on silica [PE–EtOAc
(30 : 1)] to obtain 4 (1.36 g, 4.67 mmol, 58% over 2 steps) and
the 3,5-dibromobenzaldehyde (0.23 g, 0.62 mmol, 7% over 2
steps) as colorless solids; 4: TLC [silica, PE–EtOAc (30 : 1)]: Rf
1
4
Rf = 0.46; H NMR (400 MHz, CDCl3): (Z)-7: δ = 8.12 (d, J =
2.1 Hz, 1H, 2-H), 7.48–7.43 (m, 5H, Ph-H), 7.41–7.29 (m, 6H,
3
Ph-H of E-7 and 6-H of Z-7), 6.89 (d, J = 8.6 Hz, 1H, 5-H),
6.73 (s, 1H, 7-H), 5.17 (s, 2H, CH2Ph), 3.80 (s, 3H, COOCH3),
0.98 (s, 9H, C(CH3)3), 0.16 (s, 6H, Si(CH3)2); (E)-7: δ = 7.49
6
4
(dd, JH,Si = 0.7 Hz, J = 2.3 Hz, 1H, 2-H), 7.41–7.29 (m, 6H,
6
4
Ph-H of E-7 and 6-H of Z-7), 7.15 (ddd, JH,Si = 0.7 Hz, J =
3
3
2.2 Hz, J = 8.5 Hz, 1H, 6-H), 6.85 (d, J = 8.5 Hz, 1H, 5-H),
6.29 (s, 1H, 7-H), 5.15 (s, 2H, CH2Ph), 3.68 (s, 3H, COOCH3),
0.97 (s, 9H, C(CH3)3), 0.21 (s, 6H, Si(CH3)2); 13C NMR
(100 MHz, CDCl3): (Z)-7: δ = 165.8 (1C, CqO), 154.6 (1C, Cq-
4), 139.8 (1C, Cq-8), 136.3 (1C, CqPh), 134.4 (1C, C-2), 130.2
(1C, C-6), 128.6 (2C, 2× m-Ph-C), 128.5 (1C, Cq-1), 128.0 (1C,
p-Ph-C), 127.0 (2C, 2× o-Ph-C), 117.3 (1C, C-7), 113.1 (1C,
C-5), 112.3 (1C, CqBr), 70.8 (1C, CH2Ph), 52.1 (1C, COOCH3),
25.6 (3C, C(CH3)3), 18.6 (1C, Cq(CH3)3), −3.6 (2C, Si(CH3)2);
(E)-7: δ = 165.2 (1C, CqO), 154.2 (1C, Cq-4), 141.6 (1C, Cq-8),
136.4 (1C, CqPh), 133.7 (1C, C-2), 128.9 (1C, C-6), 128.6 (2C,
2× m-Ph-C), 128.5 (1C, Cq-1), 128.0 (1C, p-Ph-C), 127.0 (2C,
2× o-Ph-C), 119.2 (1C, C-7), 113.1 (1C, C-5), 111.9 (1C, CqBr),
70.8 (2C, CH2Ph), 51.7 (1C, COOCH3), 25.9 (3C, C(CH3)3),
18.3 (1C, Cq(CH3)3), −4.8 (2C, Si(CH3)2); IR (ATR): ν˜ =
1704 cm−1 (vs), 1244 (vs), 1127 (vs), 998 (s), 824 (s), 800 (s),
783 (s), 737 (s), 692 (vs); UV-Vis (MeOH): λmax (log ε) =
296 nm (4.28), 203 (4.44); MS (EI, 70 eV): m/z (%) = 478/476
(<1/<1) [M+], 340 (17), 330 (26), 328 (24), 315 (18), 313 (17),
234 (5), 213 (9), 211 (9), 91 (100), 89 (34), 75 (24), 73 (96),
59 (23), 45 (7); HREIMS: calcd for C22H26BrO4Si [M − 15]+:
461.07782, found 461.07837.
1
= 0.08; m.p.: 92–94 °C; H NMR (400 MHz, CDCl3): δ = 9.83
(s, 1H, CHO), 8.10 (d, 4J = 2.0 Hz, 1H, 2-H), 7.77 (dd, 3J = 8.5
Hz, 4J = 2.0 Hz, 1H, 6-H), 7.48–7.45 (m, 2H, o-Ph-H),
7.43–7.38 (m, 2H, m-Ph-H), 7.37–7.32 (m, 1H, p-Ph-H), 7.04
(d, 3J = 8.5 Hz, 1H, 5-H), 5.25 (s, 2H, CH2); 13C NMR
(100 MHz, CDCl3): δ = 189.5 (1C, CHO), 159.7 (1C, Cq-4),
135.4 (1C, CqPh), 134.7 (1C, C-2), 131.0 (1C, C-6), 130.9 (Cq-
1), 128.7 (2C, m-Ph-C), 128.3 (1C, p-Ph-C), 126.9 (2C, o-Ph-
C), 113.2 (1C, C-5), 113.0 (1C, CqBr), 71.0 (1C, CH2); IR
(ATR): ν˜ = 1676 cm−1 (vs), 1589 (s), 1277 (s), 1255 (s), 1189
(s), 980 (s), 810 (s), 737 (s), 695 (s), 665 (s), 646 (s); UV-Vis
(MeOH): λmax (log ε) = 272 nm (4.21), 225 (4.26), 208 (4.40);
MS (EI, 70 eV): m/z (%) = 292/290 (24/26) [M+], 209 (7), 201
(5), 143 (3), 92 (9), 91 (100), 89 (3), 65 (11), 51 (2).
4-(Benzyloxy)-3,5-dibromobenzaldehyde. TLC [silica, PE–
EtOAc (30 : 1)]: Rf = 0.25; m.p.: 79–81 °C; 1H NMR (400 MHz,
CDCl3): δ = 9.87 (s, 1H, CHO), 8.06 (s, 2H, H-2, H-6),
7.60–7.57 (m, 2H, o-Ph-H), 7.48–7.36 (m, 3H, m-Ph-H, p-Ph-
H), 5.12 (s, 2H, CH2); 13C NMR (100 MHz, CDCl3): δ = 188.7
(1C, CHO), 158.1 (1C, Cq-4), 135.9 (1C, CqPh), 134.7 (1C,
This journal is © The Royal Society of Chemistry 2012
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(4.46); MS (EI, 70 eV): m/z (%) = 289/287 (14/14) [M+], 273
(26), 271 (29), 258 (3), 256 (3), 239 (10), 237 (9), 227 (9), 225
(8), 212 (95), 210 (89), 193 (9), 191 (56), 187 (59), 185 (61),
159 (27), 147 (18), 132 (100), 118 (6), 107 (24), 105 (30), 91
(13), 77 (63), 69 (15), 59 (28), 51 (34), 44 (49); HREIMS: calcd
for C10H10BrNO4 [M+]: 286.97877, found 286.97872.
(E)-Methyl-3-(4-(benzyloxy)-3-bromophenyl)-2-(hydroxyimino)-
propanoate (9). Ester 7 (0.82 g, 1.72 mmol, 1.00 equiv.) was
dissolved in 10 mL MeOH–CHCl3 (4 : 1) under an argon atmo-
sphere and 3HF·NEt3 (4.76 mL, 4.71 g, 2.92 mmol, 1.70 equiv.)
was added dropwise. After 30 min at rt was added H2NOH·HCl
(0.20 g, 2.92 mmol, 1.70 equiv.) in portions and the reaction
mixture was stirred for another 19 h at rt. The solvent was evap-
orated and the residue was dissolved in 10 mL DCM and washed
with H2O (2 × 40 mL) and saturated aqueous NaHCO3 solution
(40 mL). The organic phase was dried over MgSO4, filtered and
evaporated. The E-oxime 9 was obtained as a colorless solid
(0.63 g, 1.66 mmol, 97%); TLC [silica, PE–EtOAc (5 : 1)]: Rf =
(E/Z)-3-(3-Bromo-4-hydroxyphenyl)-2-(hydroxyimino)propa-
noic acid (12). To a solution of debenzylated methyl ester
(0.18 g, 0.62 mmol, 1.00 equiv.) in 1.5 mLTHF was added drop-
wise a solution of LiOH–H2O (0.08 g, 1.85 mmol, 3.00 equiv.)
in 5.5 mL H2O. The reaction mixture was stirred for 48 h at rt.
Then 2.40 mL 1 N aqueous HCl was added and the reaction
mixture was stirred for another 10 min at rt before evaporating
the solvent. The residue was dissolved in 20 mL EtOAc and
washed with H2O (3 × 20 mL). The organic phase was dried
over MgSO4, filtered and evaporated. The E/Z-acid 12 (4 : 1)
was obtained as a colorless solid (0.17 g, 0.60 mmol, 97%);
TLC [silica RP-18, MeOH–H2O (1 : 1)]: Rf = 0.43; m.p.:
141–143 °C; 1H NMR (400 MHz, DMSO-d6): (E)-12: δ = 12.81
(sbr, 1H, COOH), 12.28 (sbr, 1H, NOH), 10.08 (sbr, 1H, OH),
7.28 (d, 4J = 1.6 Hz, 1H, 2-H), 7.01–6.97 (m, 1H, 6-H), 6.85 (d,
3J = 8.3 Hz, 1H, 5-H), 3.69 (s, 2H, CH2); (Z)-12: δ = 12.81 (sbr,
1H, COOH), 12.16 (sbr, 1H, NOH), 9.19 (sbr, 1H, OH), 7.28 (d,
4J = 1.6 Hz, 1H, 2-H), 7.01–6.97 (m, 1H, 6-H), 6.65 (d, 3J = 8.5
Hz, 1H, 5-H), 3.69 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-
d6): (E)-12: δ = 165.1 (1C, CqOOH), 152.4 (1C, CqOH), 150.2
(1C, CvNOH), 132.7 (1C, C-2), 128.9 (1C, Cq-1), 128.7 (1C,
C-6), 116.2 (1C, C-5), 108.9 (1C, CqBr), 28.6 (1C, CH2). (Z)-
12: δ = 165.3 (1C, CqOOH), 155.7 (1C, CqOH), 150.2 (1C,
CvNOH), 132.7 (1C, C-2), 128.9 (1C, Cq-1), 129.6 (1C, C-6),
115.1 (1C, C-5), 108.9 (1C, CqBr), 28.9 (1C, CH2); IR (ATR): ν˜
= 1693 cm−1 (s), 1201 (s), 1016 (vs), 801 (s), 695 (s); UV-Vis
(MeOH): λmax (log ε) = 283 nm (3.45), 203 (4.45); MS (EI, 70
eV): m/z (%) = 275/273 (<1/<1) [M+], 213 (24), 211 (25), 201
(1), 199 (1), 187 (2), 185 (2), 133 (15), 132 (100), 106 (4), 104
(8), 102 (5), 78(6), 77 (15), 76 (7), 63 (4), 51 (9), 44 (23).
1
0.08; m.p.: 128–130 °C; H NMR (400 MHz, CDCl3): δ = 9.66
4
(sbr, 1H, NOH), 7.53 (d, J = 2.2 Hz, 1H, 2-H), 7.46–7.44 (m,
2H, o-Ph-H), 7.39–7.35 (m, 2H, m-Ph-H), 7.33–7.29 (m, 1H, p-
4
3
3
Ph-H), 7.18 (dd, J = 2.2 Hz, J = 8.4 Hz, 1H, 6-H), 6.83 (d, J
= 8.5 Hz, 1H, 5-H), 5.12 (s, 2H, OCH2), 3.89 (s, 2H, CH2), 3.84
(s, 3H, OCH3); 13C NMR (100 MHz, CDCl3): δ = 163.6 (1C,
CqOOCH3), 153.9 (1C, Cq-4), 150.8 (1C, CvNOH), 136.5
(1C, CqPh), 134.0 (1C, C-2), 129.4 (1C, Cq-1), 129.2 (1C, C-6),
128.6 (2C, m-Ph-C), 127.9 (1C, p-Ph-C), 127.0 (2C, o-Ph-C),
113.8 (1C, C-5), 112.4 (1C, CqBr), 70.9 (1C, OCH2), 52.9 (1C,
OCH3), 29.3 (1C, CH2); IR (ATR): ν˜ = 1726 cm−1 (s), 1221 (s),
1018 (vs), 1001 (s), 805 (s), 730 (s), 696 (s), 676 (s); UV-Vis
(MeOH): λmax (log ε) = 281 nm (3.37), 204 (4.61); MS (EI, 70
eV): m/z (%) = 379/377 (7/7) [M+], 363 (13), 361 (13), 272
(49), 270 (49), 212 (27), 210 (26), 132 (15), 105 (11), 103 (18),
91 (100), 65 (9); HREIMS: calcd for C17H16BrNO4 [M]+:
377.02572, found 377.02605.
(E/Z)-Methyl-3-(3-bromo-4-hydroxyphenyl)-2-(hydroxyimino)-
propanoate. Benzyl ester 9 (0.55 g, 1.45 mmol, 1.00 equiv.)
was dissolved in 44 mL dioxane–concentrated AcOH (1 : 1). To
the solution was added Pd/C (0.15 g, 0.07 mmol, 0.04 equiv.)
and the reaction mixture was hydrogenated for 41 h under 1 atm
H2 at rt. The reaction mixture was filtered by celite and the
solvent was evaporated. The residue was dissolved in 70 mL
EtOAc and washed with H2O (3 × 12 mL) and saturated
aqueous NaCl solution (20 mL). Then the organic phase was
dried over MgSO4, filtered and evaporated. Purification by
column chromatography on silica [PE–EtOAc (3 : 1)] afforded
the E/Z-isomers (4 : 1) as a colorless solid (0.25 g, 0.74 mmol,
51%); TLC [silica, PE–EtOAc (10 : 1)]: Rf = 0.08; m.p.:
Psammaplin A (1). Acid 12 (0.14 g, 0.51 mmol, 1.00 equiv.)
was dissolved in 10 mL DMF under an argon atmosphere. Then
NHS (0.09 g, 0.77 mmol, 1.50 equiv.) and after 5 min DCC
(0.16 g, 1.77 mmol, 1.50 equiv.) were added and the reaction
mixture was stirred for 4 h at rt. After complete consumption of
acid 12 (TLC [silica RP-18, H2O–MeOH (1 : 1)]) cystamine
dihydrochloride (15) (0.06 g, 0.26 mmol, 0.50 equiv.) and NEt3
(0.14 mL, 0.10 g, 1.03 mmol, 2.00 equiv.) were added and the
reaction mixture was stirred for another 40 h. After filtration
from DCU the solvent was evaporated. The residue was purified
by column chromatography on silica [CHCl3–MeOH (20 : 1)]
yielding psammaplin A (1, E,E-isomer) as a pale yellow oil
(0.06 g, 0.09 mmol, 36%); TLC [silica, CHCl3–MeOH (9 : 1)]:
1
148–150 °C; H NMR (400 MHz, DMSO-d6): (E)-7: δ = 12.48
4
(sbr, 1H, NOH), 10.10 (sbr, 1H, OH), 7.28 (d, J = 2.1 Hz, 1H,
2-H), 7.00 (dd, 4J = 2.1 Hz, 3J = 8.4 Hz, 1H, 6-H), 6.85 (d, 3J =
8.3 Hz, 1H, 5-H), 3.72 (s, 3H, COOCH3), 3.37 (s, 2H, CH2);
(Z)-7: δ = 12.37 (sbr, 1H, NOH), 9.23 (sbr, 1H, OH), 7.28 (d, 4J
= 2.1 Hz, 1H, 2-H), 6.97 (m, 1H, 6-H), 6.65 (m, 1H, 5-H), 3.72
(s, 3H, COOCH3), 3.70 (s, 2H, CH2); 13C NMR (100 MHz,
DMSO-d6): (E)-7: δ = 164.1 (1C, CqOOCH3), 152.5 (1C,
CqOH), 149.5 (1C, CvNOH), 132.7 (1C, C-2), 128.9 (1C, C-
6), 128.4 (1C, Cq-1), 116.3 (1C, C-5), 108.9 (1C, CqBr), 52.2
(1C, COOCH3), 28.8 (1C, CH2); (Z)-7: δ = 164.2 (1C,
CqOOCH3), 155.8 (1C, CqOH), 149.9 (1C, CvNOH), 132.7
(1C, C-2), 129.6 (1C, C-6), 126.3 (1C, Cq-1), 115.2 (1C, C-5),
108.9 (1C, CqBr), 52.1 (1C, COOCH3), 29.1 (1C, CH2); IR
(ATR): ν˜ = 1695 cm−1 (s), 1413 (s), 1250 (s), 1013 (vs), 798 (s),
726 (s); UV-Vis (MeOH): λmax (log ε) = 283 nm (3.46), 203
1
Rf = 0.37; H NMR (400 MHz, acetone-d6): δ = 10.33 (sbr, 2H,
2× OH or NOH), 7.67 (t, 3J = 6.0 Hz, 2H, 2× NH), 7.47 (d, 4J =
2.1 Hz, 2H, 2× 2-H), 7.16 (dd, 4J = 2.1 Hz, 3J = 8.3 Hz, 2H, 2×
3
6-H), 6.89 (d, J = 8.3 Hz, 2H, 2× 5-H), 3.85 (s, 4H, 2× CH2),
3.59 (dt, 3J = 7.0 Hz, 4H, 2× NHCH2), 2.88 (t, 3J = 6.8 Hz, 4H,
2× SCH2); 13C NMR (100 MHz, acetone-d6): δ = 164.1 (2C, 2×
CqO), 153.2 (2C, 2× Cq-OH), 153.2 (2C, 2× CvNOH), 134.3
(2C, 2× C-2), 130.5 (2C, 2× Cq-1), 130.4 (2C, 2× C-6), 117.0
7126 | Org. Biomol. Chem., 2012, 10, 7120–7133
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(2C, 2× C-5), 109.9 (2C, 2× CqBr), 39.2 (2C, 2× NHCH2), 38.4
(2C, 2× SCH2), 28.4 (2C, 2× CH2); IR (ATR): ν˜ = 1655 cm−1
(s), 1529 (s), 1493 (s), 1418 (s), 1207 (vs), 1012 (s), 981 (s),
543 (s); UV-Vis (MeOH): λmax (log ε) = 282 nm (3.71), 204
(4.75); MS (ESI): m/z (%) = 689/687/685 (<2/<2/<2) [M +
Na]+, 561 (<2), 433 (<2), 370 (2), 334 (3), 316 (3), 299 (5), 281
(4), 249 (6), 218 (5), 216 (5), 213 (46), 211 (47), 201 (11), 199
(11), 187 (19), 185 (20), 143 (4), 132 (100), 115 (5), 105 (12),
103 (13), 77 (24), 61 (18), 51 (18), 44 (20); HRESIMS: calcd
3HF·NEt3 (0.37 mL, 0.37 g, 2.29 mmol, 1.70 equiv.) was added
dropwise. The reaction mixture was stirred for 30 min at rt. After
finishing TBS deprotection (TLC [silica, PE–EtOAc (30 : 1)])
HONH2·HCl (0.16 g, 2.29 mmol, 1.70 equiv.) was added in por-
tions and the reaction mixture was stirred for another 15 h. After
evaporation of the solvent the residue was dissolved in 20 mL
DCM and washed with H2O (2 × 15 mL) and saturated aqueous
NaHCO3 solution (15 mL). The organic phase was dried over
MgSO4, filtered and evaporated. The E-oxime 10 was obtained
as a colorless solid (0.35 g, 0.95 mmol, 96%); TLC [silica, PE–
EtOAc (1 : 1)]: Rf = 0.56; m.p.: 98–103 °C; 1H NMR (400 MHz,
DMSO-d6): δ = 12.60 (s, 1H, NOH), 7.42–7.37 (m, 2H, 2-H, 6-
H), 7.25 (t, 3J = 7.7 Hz, 1H, 5-H), 7.19 (d, 3J = 1.2 Hz, 4J = 7.6
Hz, 1H, 4-H), 3.84 (s, 2H, CH2), 3.73 (s, 3H, COOCH3); 13C
NMR (100 MHz, DMSO-d6): δ = 164.0 (1C, CqOOCH3), 148.8
(1C, CvNOH), 139.2 (1C, Cq-1), 131.2 (1C, C-2), 130.6 (1C,
C-5), 129.2 (1C, C-6), 127.6 (1C, C-4), 121.5 (1C, CqBr), 52.2
(1C, COOCH3), 29.7 (1C, CH2); IR (ATR): ν˜ = 1728 cm−1 (s),
1206 (s), 1126 (s), 1009 (vs), 723 (s); UV-Vis (MeOH): λmax
(log ε) = 202 nm (3.49); MS (EI, 70 eV): m/z (%) = 273/271
(10/10) [M+], 257(17), 255 (17), 198 (15), 197 (27), 196 (35),
195 (27), 194 (23), 180 (31), 178 (100), 171 (20), 169 (22), 163
(5), 152 (7), 143 (23), 142 (16), 117 (16), 116 (58), 115 (19),
102 (7), 97 (6), 95 (8), 91 (16), 90 (20), 89 (32), 81 (8), 71 (11),
69 (11), 63 (13), 59 (14), 57 (14), 55 (12), 51 (7), 50 (8), 44
(41), 43 (15); HREIMS: calcd for C10H10BrNO3 [M+]:
270.98386, found 270.98375.
for C22H24Br2N4NaO6S2 [M
684.93809.
+
Na]+: 684.93962, found
(E/Z)-Methyl-3-(3-bromophenyl)-2-(tert-butyldimethylsilyl-
oxy)acrylate (8). Diisopropylamine (0.78 mL, 0.56 g,
5.53 mmol, 1.50 equiv.) was dissolved in 4 mL dry THF under
an argon atmosphere and cooled to 0 °C. After 10 min n-BuLi
(1.6 N solution in hexane, 3.45 mL, 5.53 mmol, 1.50 equiv.)
was added dropwise to the solution and it was stirred for 10 min
at 0 °C and then for 30 min at −78 °C. Dimethylphosphonate 6
(1.15 g, 3.69 mmol, 1.00 equiv.) in 1 mL dry THF was added
dropwise and the reaction mixture was stirred for 30 min at
−78 °C. To the solution was added 3-bromobenzaldehyde (5)
(0.47 mL, 0.75 g, 4.05 mmol, 1.10 equiv.) in 1 mL dry THF
dropwise and the reaction mixture was stirred for 19 h at −78 °C
and warmed to rt within 30 min. It was quenched with 15 mL
saturated aqueous NH4Cl solution and stirred for 10 min at rt.
The mixture was diluted with 75 mL EtOAc and washed with
saturated aqueous NH4Cl solution (3 × 30 mL), H2O (3 ×
30 mL) and saturated aqueous NaCl solution (30 mL). The
organic phase was dried over MgSO4, filtered and the solvent
evaporated. Purification by column chromatography on silica
[PE–EtOAc (50 : 1 → 40 : 1 → 10 : 1 → 5 : 1)] afforded the E/Z-
isomers 8 (3 : 1) as a colorless oil (1.14 g, 3.06 mmol, 83%);
TLC [silica, PE–EtOAc (30 : 1)]: Rf = 0.35; 1H NMR (400 MHz,
DMSO-d6): (Z)-8: δ = 8.00 (t, 4J = 1.7 Hz, 1H, 2-H), 7.47 (d, 3J
(E)-3-(3-Bromophenyl)-2-(hydroxyimino)propanoic acid (13).
The methyl ester 10 (0.72 g, 2.64 mmol, 1.00 equiv.) was dis-
solved in 5.5 mL THF and cooled to 0 °C. A solution of LiOH–
H2O (0.22 g, 5.28 mmol, 2.00 equiv.) in 14 mL H2O was added
dropwise. The reaction mixture was stirred for 11 h at rt, then
7.2 mL 1 N aqueous HCl was added and the mixture was stirred
for another 10 min at rt. After evaporation of THF the formed
precipitate was filtered off and dried in an exsiccator over con-
centrated H2SO4. The E-isomer 13 was obtained as a colorless
solid (0.60 g, 2.31 mmol, 87%); TLC [silica RP-18, MeOH–
3
= 7.8 Hz, 1H, 6-H), 7.39–7.34 (m, 1H, 5-H), 7.21 (d, J = 7.9
Hz, 1H, 4-H), 6.76 (s, 1H, 7-H), 3.82 (s, 3H, COOCH3), 0.96 (s,
9H, C(CH3)3), 0.16 (s, 6H, Si(CH3)2); (E)-8: δ = 7.40–7.37 (m,
1H, CH), 7.37–7.34 (m, 1H, CH), 7.18–7.15 (m, 1H, CH),
7.17–7.14 (m, 1H, CH), 6.32 (s, 1H, 7-H), 3.67 (s, 3H,
COOCH3), 0.98 (s, 9H, C(CH3)3), 0.22 (s, 6H, Si(CH3)2); 13C
NMR (100 MHz, DMSO-d6): (Z)-8: δ = 165.6 (1C, CqO),
141.32 (1C, Cq-8), 136.2 (1C, Cq-1), 132.3 (1C, C-2), 130.9
(1C, C-5), 129.6 (1C, C-4), 128.4 (1C, C-6), 122.3 (1C, CqBr),
117.2 (1C, C-7), 52.2 (1C, COOCH3), 25.8 (3C, C(CH3)3), 18.6
(1C, Cq(CH3)3), −3.9 (2C, Si(CH3)2); (E)-8: δ = 165.1 (1C,
CqO), 142.83 (1C, C-8), 136.7 (1C, Cq-1), 131.5 (1C, CH),
130.1 (1C, CH), 129.4 (1C, CH), 127.2 (1C, CH), 121.9 (1C,
CqBr), 118.6 (1C, C-7), 51.8 (1C, COOCH3), 25.5 (3C, C
(CH3)3), 18.3 (1C, Cq(CH3)3), −4.8 (2C, Si(CH3)2); IR (ATR): ν˜
= 1248 cm−1 (s), 833 (vs), 779 (vs); UV-Vis (MeOH): λmax (log
ε) = 277 nm (4.08), 202 (4.24); MS (ESI): m/z (%) = 765 (7)
[2M + Na]+, 395/393 (100/96) [M + Na]+, 373/371 (4/3) [M+],
341 (3), 257 (1); HRESIMS: calcd for C16H23BrNaO3Si
[M + Na]+: 393.04920, found 393.04945.
1
H2O (1 : 1)]: Rf = 0.28; m.p.: 158–159 °C; H NMR (400 MHz,
DMSO-d6): δ = 12.90 (sbr, 1H, COOH), 12.40 (sbr, 1H, NOH),
4
7.41–7.39 (m, 1H, 6-H), 7.37 (t, J = 1.6 Hz, 1H, 2-H), 7.25
(t, 3J = 7.7 Hz, 1H, 5-H), 7.20 (td, 4J = 1.3 Hz, 3J = 7.7 Hz, 1H,
4-H), 3.81 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6): δ =
165.0 (1C, CqOOH), 149.6 (1C, CvNOH), 139.5 (1C, Cq-1),
131.2 (1C, C-2), 130.5 (1C, C-5), 129.1 (1C, C-6), 127.7 (1C,
C-4), 121.5 (1C, CqBr), 29.5 (1C, CH2); IR (ATR): ν˜ =
1691 cm−1 (s), 1022 (s), 774 (s), 697 (vs); UV-Vis (MeOH):
λmax (log ε) = 203 nm (4.37); MS (EI, 70 eV): m/z (%) = 259/
257 (2/2) [M+], 197 (37), 195 (37), 171 (2), 169 (2), 117 (11),
116 (100), 115 (5), 90 (7), 89 (26), 88 (5), 75 (5), 63 (8), 58 (5),
50 (7), 44 (53); HRESIMS: calcd for C9H8BrNNaO3 [M + Na]+:
279.95798, found 279.95819.
(2E,2′E)-N,N′-(2,2′-Disulfanediylbis(ethane-2,1-diyl))bis(3-(3-
bromophenyl)-2-(hydroxyimino)propanamide) (16). To a solu-
tion of acid 13 (0.07 g, 0.27 mmol, 1.00 equiv.) in 1 mL dioxane
were added NHPI (0.04 g, 0.27 mmol, 1.00 equiv.) and DCC
(0.56 g, 0.27 mmol, 1.00 equiv.) at rt and the reaction mixture
was stirred for 8 h. After complete consumption of acid 13 (TLC
(E)-Methyl-3-(3-bromophenyl)-2-(hydroxyimino)propanoate
(10). The E/Z-olefin ester 8 (0.50 g, 1.35 mmol, 1.00 equiv.)
was dissolved in 5 mL MeOH under an argon atmosphere and
This journal is © The Royal Society of Chemistry 2012
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[silica RP-18, H2O–MeOH (1 : 1)]) a solution of cystamine dihy-
drochloride (15) (0.03 g, 0.13 mmol, 0.48 equiv.) and NEt3
(0.38 mL, 0.28 g, 0.54 mmol, 2.00 equiv.) in 1 mL MeOH was
added and the reaction mixture was stirred for another 48 h. The
solvent was evaporated and purification by column chromato-
graphy on silica [CHCl3–MeOH (40 : 1)] afforded the E,E-
isomer 16 as a colorless oil (0.03 g, 0.05 mmol, 36%); TLC
C(CH3)3), 27.9 (1C, CH2-7); IR (ATR): ν˜ = 1511 cm−1 (vs),
1162 (s); UV-Vis (MeOH): λmax (log ε) = 203 nm (4.20); MS
(ESI): m/z (%) = 881 (63) [2M + Na]+, 452 (100) [M + Na]+,
396 (15), 330 (16), 299 (7); HRESIMS: calcd for
C18H27N3NaO5S2 [M + Na]+: 452.12843, found 452.12857.
(E)-tert-Butyl-2-((2-(3-(3-bromo-4-hydroxyphenyl)-2-(hydro-
xyimino)propanamido)ethyl)disulfanyl)ethylcarbamate (23).
The acid 12 (0.26 g, 0.96 mmol, 1.00 equiv.) was dissolved in
7 mL dry DMF under an argon atmosphere. NHS (0.17 g,
1.46 mmol, 1.50 equiv.) and after 5 min DCC (0.17 g,
1.45 mmol, 1.50 equiv.) were added in portions and the reaction
mixture was stirred for 4 h at rt. After complete consumption of
acid 12 (TLC [silica RP-18, H2O–MeOH (1 : 1)]) was Boc-pro-
tected cystamine 20 (0.27 g, 1.06 mmol, 1.10 equiv.) in 2 mL
dry DMF added dropwise and the reaction mixture was stirred
for 15 h at rt and for 4 days at 30 °C. The solvent was evapor-
ated, dissolved in THF, filtered and the solvent evaporated again.
Purification by column chromatography on silica [CHCl3–
MeOH (70 : 1 → 40 : 1 → 5 : 1)] and on silica RP-18 [MeOH–
H2O (2 : 1)] afforded the E-isomer 23 as a pale yellow oil
(0.14 g, 0.27 mmol, 28%); TLC [silica RP-18, MeOH–H2O
1
[silica, CHCl3–MeOH (9 : 1)]: Rf = 0.47; H NMR (400 MHz,
4
acetone-d6): δ = 7.52–7.51 (m, 2H, 2× 2-H), 7.35 (dd, J = 1.4
Hz, 3J = 7.9 Hz, 2H, 2× 4-H), 7.32 (d, 3J = 7.8 Hz, 2H, 2× 6-H),
3
7.21 (t, J = 7.8 Hz, 2H, 2× 5-H), 3.94 (s, 4H, 2× 7-CH2), 3.58
3
3
(t, J = 6.8 Hz, 4H, 2× NHCH2), 2.89 (t, J = 6.8 Hz, 4H, 2×
SCH2); 13C NMR (100 MHz, acetone-d6): δ = 163.9 (2C, 2×
CqO), 152.5 (2C, 2× CvNOH), 140.6 (2C, 2× Cq-1), 132.9
(2C, 2× C-2), 131.0 (2C, 2× C-5), 130.0 (2C, 2× C-4), 129.0
(2C, 2× C-6), 122.6 (2C, 2× CqBr), 39.1 (2C, 2× NHCH2), 38.4
(2C, 2× SCH2), 29.5 (2C, 2× CH2-7); 1H NMR (400 MHz,
4
CDCl3): δ = 10.16 (sbr, 2H, 2× NOH), 7.45 (t, J = 1.7 Hz, 2H,
2× 2-H), 7.28 (m, 2H, 2× 4-H), 7.25–7.24 (m, 2H, 2× 6-H), 7.21
3
3
(t, J = 7.8 Hz, 2H, 2× NH), 7.07 (t, J = 7.8 Hz, 2H, 2× 5-H),
3
3.92 (s, 4H, 2× 7-CH2), 3.57 (q, J = 6.1 Hz, 4H, 2× NHCH2),
2.74 (t, 3J = 6.2 Hz, 4H, 2× SCH2); 13C NMR (100 MHz,
CDCl3): δ = 163.7 (2C, 2× CqO), 151.8 (2C, 2× CvNOH),
138.4 (2C, 2× Cq-1), 132.1 (2C, 2× C-2), 129.9 (2C, 2× C-5),
129.6 (2C, 2× C-4), 128.1 (2C, 2× C-6), 122.4 (2C, 2× CqBr),
38.3 (2C, 2× NHCH2), 37.7 (2C, 2× SCH2), 28.9 (2C, 2× CH2-
7); IR (ATR): ν˜ = 1655 cm−1 (vs), 1567 (vs), 768 (s); UV-Vis
(MeOH): λmax (log ε) = 673 nm (2.72), 203 (4.69); MS (ESI):
m/z (%) = 1287 (2) [2M + Na]+, 653/655/657 (46/84/39) [M +
Na]+, 631/633/635 (4/10/4) [M+], 471 (11), 325 (11), 225 (100);
1
(2 : 1)]: Rf = 0.20; H NMR (400 MHz, acetone-d6): δ = 11.1
3
(sbr, 1H, NOH), 8.67 (sbr, 1H, OH), 7.65 (t, J = 5.8 Hz, 1H,
4
4
NHC-9), 7.47 (d, J = 2.1 Hz, 1H, 2-H), 7.16 (dd, J = 2.1 Hz,
3J = 8.3 Hz, 1H, 6-H), 6.89 (d, 3J = 8.3 Hz, 1H, 5-H), 6.20 (sbr,
1H, NHC-14), 3.84 (s, 2H, 7-CH2), 3.60 (dt, J = 6.7 Hz, J =
3
3
3
3
13.1 Hz, 2H, 10-CH2), 3.37 (dt, J = 6.5 Hz, J = 12.8 Hz, 2H,
13-CH2), 2.91–2.88 (m, 2H, 11-CH2), 2.87–2.82 (m, 2H, 12-
CH2), 1.40 (s, 9H, C(CH3)3); 13C NMR (100 MHz, acetone-d6):
δ = 164.1 (1C, Cq-9), 156.7 (1C, Cq-14), 153.3 (1C, Cq-OH),
153.2 (1C, CvNOH), 134.3 (1C, C-2), 130.6 (1C, C-6), 130.5
(1C, Cq-1), 117.0 (1C, C-5), 110.0 (1C, CqBr), 78.9 (1C, Cq-
15), 40.5 (1C, CH2-13), 39.3 (1C, CH2-12), 39.3 (1C, CH2-10),
38.3 (1C, CH2-11), 28.6 (3C, C(CH3)3), 28.4 (1C, CH2-7); IR
(ATR): ν˜ = 1660 cm−1 (vs), 1161 (vs); UV-Vis (MeOH): λmax
(log ε) = 282 nm (3.46), 203 (4.50); MS (ESI): m/z (%) = 1039
(17) [2M + Na]+, 532/530 (100/92) [M + Na]+, 510/508 (19/18)
[M+], 410/408 (56/52), 168 (76); HRESIMS: calcd for
C18H26BrN3NaO5S2 [M + Na]+: 530.03895, found 530.03911.
HRESIMS: calcd for C22H24Br2N4NaO4S2 [M
652.94979, found 652.95007.
+
Na]+:
(E)-tert-Butyl-2-((2-(2-(hydroxyimino)-3-(4-hydroxyphenyl)-
propanamido)ethyl)disulfanyl)ethylcarbamate (22). The acid 21
(0.15 g, 0.75 mmol, 1.00 equiv.) was dissolved in 3 mL dry
DMF under an argon atmosphere. NHS (0.13 g, 1.12 mmol, 1.50
equiv.) and after 5 min DCC (0.23 g, 1.12 mmol, 1.50 equiv.)
were added in portions and the reaction mixture was stirred for
2 h at rt. After complete consumption of acid 21 (TLC [silica
RP-18, H2O–MeOH (1 : 1)]) was Boc protected cystamine 20
(0.21 g, 0.82 mmol, 1.10 equiv.) in 5 mL dry DMF added drop-
wise and the reaction mixture was stirred for 4 days at rt. The
solvent was evaporated, dissolved in THF, filtered and the
solvent evaporated again. Purification by column chromato-
graphy on silica [CHCl3–MeOH (50 : 1)] afforded the E-isomer
21 as a pale yellow oil (0.24 g, 0.55 mmol, 74%); TLC [silica,
CHCl3–MeOH (40 : 1)]: Rf = 0.16; 1H NMR (600 MHz, DMSO-
(E)-2-(Hydroxyimino)-3-(4-hydroxyphenyl)-N-(2-mercap-
toethyl)propanamide (33). The Boc-amide 22 (0.26 g,
0.64 mmol, 1.00 equiv.) was dissolved in 10 mL MeOH and 1
M aqueous KOH solution (28 μL) was added. Dithiothreitol (32)
(0.27 g, 1.92 mmol, 3.00 equiv.) was added and the reaction
mixture was stirred for 15 h at rt. The reaction mixture was
cooled to 0 °C and diluted with 0.5 M aqueous HCl solution
(10 mL). After addition of saturated aqueous NaCl solution
(10 mL) it was extracted with DCM (5 × 15 mL). The combined
organic layer was washed with H2O (100 mL) and saturated
aqueous NaCl solution (100 mL), dried over MgSO4, filtered
and evaporated. Purification by column chromatography on
silica RP-18 [H2O–MeOH (2 : 1)] afforded the E-isomer 33 as a
pale yellow oil (0.063 g, 0.25 mmol, 39%); TLC [silica RP-18,
H2O–MeOH (2 : 1)]: Rf = 0.17; 1H NMR (300 MHz, CD3OD): δ
3
d6): δ = 11.75 (s, 1H, NOH), 9.18 (s, 1H, OH), 8.04 (t, J = 5.9
Hz, 1H, NHC-9), 6.99–6.98 (m, 3J + 5J = 8.6 Hz, 3H, 2-H, 6-H,
3
5
NHC-14), 6.63–6.62 (m, J + J = 8.6 Hz, 2H, 3-H, 5-H), 3.68
3
3
(s, 2H, 7-CH2), 3.41 (dt, J = 6.3 Hz, J = 13.5 Hz, 2H, 10-
CH2), 3.19 (dt, 3J = 6.3 Hz, 3J = 13.1 Hz, 2H, 13-CH2),
3
2.81–2.79 (m, 2H, 11-CH2), 2.74 (t, J = 6.9 Hz, 2H, 12-CH2),
1.37 (s, 9H, C(CH3)3); 13C NMR (150 MHz, DMSO-d6): δ =
163.3 (Cq-9), 155.5 (1C, CqOH), 155.4 (1C, Cq-14), 152.2 (1C,
CvNOH), 129.7 (2C, C-2, C-6), 126.7 (1C, Cq-1), 114.9 (2C,
C-3, C-5), 77.7 (1C, Cq(CH3)3), 39.2 (1C, CH2-13), 38.1 (1C,
CH2-10), 37.5 (1C, CH2-12), 36.8 (1C, CH2-11), 28.1 (3C,
3
5
= 8.05 (m, 1H, NH), 7.08 (m, J + J = 8.7 Hz, 2H, 2-H, 6-H),
3
5
6.65 (m, J + J = 8.7 Hz, 2H, 3-H, 5-H), 3.80 (s, 2H, CH2),
3
3
3
3.37 (dd, J = 6.6 Hz, J = 7.3 Hz, 2H, NCH2), 2.58 (dd, J =
7128 | Org. Biomol. Chem., 2012, 10, 7120–7133
This journal is © The Royal Society of Chemistry 2012

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3
6.5 Hz, J = 7.4 Hz, 2H, SCH2); 13C NMR (75 MHz, CD3OD):
1587 cm−1 (vs), 1512 (s), 1450 (s), 1224 (s); UV-Vis (MeOH):
λmax (log ε) = 278 nm (3.29), 202 (4.24); MS (ESI): m/z (%) =
519 (56) [2M + Na]+, 271 (100) [M + Na]+, 249 (66);
HRESIMS: calcd for C13H16N2NaO3 [M + Na]+: 271.10531,
found 271.10528.
δ = 166.1 (1C, CqO), 156.8 (1C, CqOH), 153.8 (1C, CvNOH),
131.1 (2C, C-2, C-6), 128.8 (1C, Cq-1), 116.1 (2C, C-3, C-5),
43.7 (1C, NCH2), 29.0 (1C, CH2), 24.4 (1C, SCH2); IR (ATR):
ν˜ = 1655 cm−1 (s), 1511 (vs), 1212 (s); UV-Vis (MeOH): λmax
(log ε) = 276 nm (3.32), 203 (4.18); MS (ESI): m/z (%) = 531
(86) [2M + Na]+, 277 (100) [M + Na]+, 255 (63) [M + H]+;
HRESIMS: calcd for C11H14N2NaO3S [M + Na]+: 277.06173,
found 277.06176.
(E)-2-(Hydroxyimino)-3-(4-hydroxyphenyl)-1-(thiazolidin-3-
yl)propan-1-one (31). A solution of acid 21 (0.21 g, 1.09 mmol,
1.00 equiv.) in 9 mL dry DMF in a flask with a CaCl tube was
cooled to 0 °C. NHS (0.19 g, 1.64 mmol, 1.50 equiv.) and after
5 min DCC (0.34 g, 1.64 mmol, 1.50 equiv.) were added and the
reaction mixture was stirred for 24 h at rt. Thiazolidine (29)
(0.13 mL, 0.12 g, 1.63 mmol, 2.00 equiv.) was added and the
mixture was stirred for another 20 h. Under ice cooling was
added 5% aqueous KHSO4 solution (25 mL) and extracted with
DCM (7 × 30 mL). The combined organic layer was washed in
each case with 60 mL of H2O, 5% aqueous NaHCO3 solution,
H2O and saturated aqueous NaCl solution, dried over MgSO4,
filtered and evaporated. The residue was dissolved in THF,
filtered off from DCU and evaporated again. After purification
by column chromatography on silica [CHCl3–MeOH (30 : 1) →
(10 : 1)] was obtained the E-derivative 31 as a yellow oil of a
(1 : 1) mixture of the two rotamers (0.12 g, 0.43 mmol, 40%);
TLC [silica, CHCl3–MeOH (10 : 1)]: Rf = 0.36; 1H NMR
(400 MHz, acetone-d6): δ = 10.84 (sbr, 1H, NOH), 8.25 (sbr,
(2E,2′E)-N,N′-(2,2′-Disulfanediylbis(ethane-2,1-diyl))bis(2-
(hydroxyimino)-3-(4-hydroxyphenyl)propanamide) (34). Com-
pound 33 (0.02 g, 0.08 mmol, 1.00 equiv.) was stored in 0.6 mL
acetone-d6 at rt for 90 days. After complete oxidation to the cor-
responding E,E-isomer 34 (1H NMR control) it was isolated by
evaporation of the solvent as a colorless solid (0.02 g,
0.04 mmol, quant.); TLC [silica, CHCl3–MeOH (10 : 1)]: Rf =
1
0.27; m.p.: 137–138 °C; H NMR (600 MHz, DMSO-d6): δ =
11.74 (s, 2H, 2× NOH), 9.18 (sbr, 2H, 2× OH), 8.03 (t, J = 5.9
3
3
5
Hz, 2H, 2× NH), 7.00 (m, J + J = 8.6 Hz, 4H, 2× 2-H, 2× 6-
H), 6.63 (m, 3J + 5J = 8.6 Hz, 4H, 2× 3-H, 2× 5-H), 3.68 (s, 4H,
2× CH2-7), 3.41 (dt, 3J = 6.3 Hz, 3J = 13.6 Hz, 4H, 2×
NHCH2), 2.81–2 (m, 4H, 2× SCH2); 13C NMR (150 MHz,
DMSO-d6): δ = 163.3 (2C, 2× CqO-9), 155.5 (2C, 2× CqOH),
152.2 (2C, 2× CvNOH), 129.6 (4C, 2× C-2, 2× C-6), 126.7
(2C, 2× Cq-1), 114.9 (2C, 2× C-3, 2× C-5), 38.0 (2C, 2×
NHCH2), 36.8 (2C, 2× SCH2), 27.9 (2C, 2× CH2-7); IR (ATR):
ν˜ = 1653 (s), 1511 (vs), 999 (s); UV-Vis (MeOH): λmax (log ε) =
210 nm (4.44), 201 (4.53); MS (ESI): m/z (%) = 1035 (10) [2M
+ Na]+, 529 (100) [M + Na]+, 507 (5), 262 (5), 179 (7);
HRESIMS: calcd for C22H26N4NaO6S2 [M + Na]+: 529.11860,
found 529.11872.
3
5
1H, OH), 7.10 (m, J + J = 8.0 Hz, 2H, 2-H, 6-H), 6.75/6.74
3
5
(m, J + J = 8.6 Hz, 2H, 3-H, 5-H), 4.50/4.50 (s, 2H, 10-CH2),
3
3.87 (s, 2H, 7-CH2), 3.73/3.70 (t, J = 6.5/6.3 Hz, 2H, 12-CH2),
2.97/2.79 (t, 3J = 6.4/6.2 Hz, 2H, 11-CH2); 13C NMR
(100 MHz, acetone-d6): δ = 164.8/164.4 (1C, Cq-9), 157.0/
156.9 (1C, CqOH), 155.2/155.2 (1C, CvNOH), 131.0/131.0
(2C, C-2, C-6), 127.6/127.4 (1C, Cq-1), 116.2 (2C, C-3, C-5),
51.5/49.5 (1C, CH2-12), 50.6/48.3 (1C, CH2-10), 31.4/29.6 (1C,
CH2-11), 30.8/30.7 (1C, CH2-7); IR (ATR): ν˜ = 1591 cm−1 (vs),
1451 (s), 556 (s); UV-Vis (MeOH): λmax (log ε) = 277 nm
(3.33), 203 (4.27); MS (EI, 70 eV): m/z (%) = 266 (18) [M+],
249 (22), 221 (6), 146 (12), 132 (46), 116 (100), 107 (66),
90 (14), 88 (56), 77 (16), 70 (10), 61 (8), 56 (22), 41 (16);
HREIMS: calcd for C12H14N2O3S [M+]: 266.07196, found
266.07208.
(E)-2-(Hydroxyimino)-3-(4-hydroxyphenyl)-1-(pyrrolidin-1-yl)-
propan-1-one (30). A solution of acid 21 (0.16 g, 0.82 mmol,
1.00 equiv.) in 7 mL dry DMF in a flask with a CaCl tube was
cooled to 0 °C. NHS (0.14 g, 1.22 mmol, 1.50 equiv.) and after
5 min DCC (0.25 g, 1.22 mmol, 1.50 equiv.) were added and the
reaction mixture was stirred for 6 h at rt. Pyrrolidine (28)
(0.13 mL, 0.12 g, 1.63 mmol, 2.00 equiv.) was added and the
reaction mixture was stirred for another 20 h. Under ice cooling
was added 5% aqueous KHSO4 solution (20 mL) and extracted
with DCM (3 × 30 mL). The combined organic layer was
washed in each case with 60 mL of H2O, 5% aqueous NaHCO3
solution, H2O and saturated aqueous NaCl solution, dried over
MgSO4, filtered and evaporated. The residue was dissolved in
THF, filtered off from DCU and the solvent evaporated. Purifi-
cation by column chromatography on silica [CHCl3–MeOH
(30 : 1) → (10 : 1)] obtained the E-isomer 30 as a colorless oil
(0.08 g, 0.34 mmol, 42%); TLC [silica, CHCl3–MeOH (20 : 1)]:
Rf = 0.33; H NMR (400 MHz, acetone-d6): δ = 10.68 (sbr, 1H,
NOH), 8.27 (sbr, 1H, OH), 7.11 (m, J + J = 6.9 Hz, 2H, 3-H,
5-H), 6.74 (m, J + J = 6.6 Hz, 2H, 2-H, 6-H), 3.86 (s, 2H,
7-CH2), 3.37 (dt, J = 6.5 Hz, J = 12.5 Hz, 4H, 2× NCH2),
1.80–1.66 (m, 4H, 2× CH2CH2); 13C NMR (100 MHz, acetone-
d6): δ = 164.7 (1C, CqO), 156.9 (1C, Cq-4), 155.5 (1C,
CvNOH), 131.0 (2C, C-3, C-5), 127.8 (1C, Cq-1), 116.1 (2C,
C-2, C-6), 49.0 (1C, NCH2), 46.6 (1C, NCH2), 30.7 (1C, CH2-
7), 26.6 (1C, CH2CH2), 24.5 (1C, CH2CH2); IR (ATR): ν˜ =
(E/Z)-Methyl-3-(3-bromophenyl)-2-(tetrahydro-2H-pyran-2-
yloxyimino)propanoate (11). To a solution of TBS–ether 8
(0.40 g, 1.01 mmol, 1.00 equiv.) in 6 mL ethanol 3HF·NEt3
(0.30 mL, 1.83 mmol, 1.70 equiv.) was added dropwise under an
argon atmosphere. The reaction mixture was stirred for 30 min at
rt. After finishing TBS deprotection (TLC [silica, PE–EtOAc
(30 : 1)]) THPONH2 (0.32 g, 2.69 mmol, 2.50 equiv.) was added
in portions and the reaction mixture was stirred for another 48 h.
After evaporation of the solvent the residue was dissolved in
60 mL DCM and washed with H2O (2 × 30 mL) and saturated
aqueous NaHCO3 solution (30 mL). The organic phase was
dried over MgSO4, filtered and evaporated. After purification by
column chromatography on silica [PE–EtOAc (15 : 1)] were
obtained the Z-isomer 11 (0.03 g, 0.09 mmol, 9%) and the
E-isomer 11 (0.34 g, 0.94 mmol, 88%) separated as colorless
oils; (Z)-11: TLC [silica, PE–EtOAc (15 : 1)]: Rf = 0.19; 1H
NMR (400 MHz, CDCl3): δ = 7.48–7.32 (m, 2H, 2-H, 5-H),
1
3
5
3
5
3
3
3
7.23–7.12 (m, 2H, 4-H, 6-H), 5.37 (t, J = 3.1 Hz, 1H, 10-H),
This journal is © The Royal Society of Chemistry 2012
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3.92–3.86 (m, 1H, 14-OCHH), 3.76 (d, 2J = 15.1 Hz, 1H,
7-CHH), 3.73 (s, 3H, OCH3), 3.72 (d, 2J = 15.0 Hz, 1H, 7-CHH),
3.68–3.63 (m, 1H, 14-OCHH), 1.83–1.53 (m, 6H, 11-CH2, 12-
CH2, 13-CH2); 13C NMR (100 MHz, CDCl3): δ = 163.0 (1C,
CqOOCH3), 151.0 (1C, CqN-8), 136.9 (1C, Cq-1), 132.0 (1C,
C-2), 130.3 (1C, C-5), 130.1 (1C, C-4), 127.6 (1C, C-6), 122.6
(1C, Cq-Br), 100.6 (1C, C-10), 62.1 (1C, OCH2-14), 52.0 (1C,
OCH3), 37.2 (1C, CH2-7), 28.3 (1C, CH2-11), 25.1 (1C, CH2-
13), 18.9 (1C, CH2-12); (E)-11: TLC [silica, PE–EtOAc
(2E,2′E)-N,N′-(2,2′-Disulfanediylbis(ethane-2,1-diyl))bis(3-(3-
bromophenyl)-2-(tetrahydro-2H-pyran-2-yloxyimino)propana-
mide) (17). To a solution of acid 14 (0.25 g, 0.72 mmol, 1.00
equiv.) in 3.5 mL dioxane were added NHPI (0.12 g, 0.72 mmol,
1.00 equiv.) and DCC (0.15 g, 0.72 mmol, 1.00 equiv.). The
reaction mixture was stirred at rt for 4 h. A solution of cystamine
dihydrochloride (15) (0.08 g, 0.35 mmol, 0.48 equiv.) and NEt3
(0.20 mL, 0.15 g, 1.44 mmol, 2.00 equiv.) in 3.5 mL MeOH was
added within 1.5 h and the reaction mixture was stirred for
another 20 h. After evaporation of the solvent the crude product
was isolated by column chromatography on silica [PE–EtOAc
(9 : 1) → (2 : 1) → (1 : 2)] and purified by HPLC on RP-18
[MeOH–H2O (9 : 1) → MeOH]. The E,E-isomer 17 was
obtained as a colorless solid (0.13 g, 0.16 mmol, 45%); TLC
1
(15 : 1)]: Rf = 0.10; H NMR (400 MHz, CDCl3): δ = 7.48 (dd,
3
4J = 1.7 Hz, 1H, 2-H), 7.34 (d, J = 7.5 Hz, 1H, 4-H), 7.23 (d,
3
3J = 7.7 Hz, 1H, 6-H), 7.14 (dd, J = 7.8 Hz, 1H, 5-H), 5.50 (t,
2
3J = 3.2 Hz, 1H, 10-H), 4.01 (d, J = 13.5 Hz, 1H, 7-CHH),
3.89 (d, 2J = 13.5 Hz, 1H, 7-CHH), 3.85 (s, 3H, OCH3),
3.62–3.60 (m, 2H, 14-OCH2), 1.84 (m, 2H, 11-CH2), 1.82–1.67
(m, 2H, 12-CH2), 1.66–1.54 (m, H, 13-CH2); 13C NMR
(100 MHz, CDCl3): δ = 163.9 (1C, CqOOCH3), 151.0 (1C,
CqN-8), 138.1 (1C, Cq-1), 132.3 (1C, C-2), 130.0 (1C, C-5),
129.8 (1C, C-4), 127.7 (1C, C-6), 122.4 (1C, Cq-Br), 101.9 (1C,
C-10), 62.4 (1C, OCH2-14), 53.0 (1C, OCH3), 31.4 (1C, CH2-
7), 28.4 (1C, CH2-11), 24.9 (1C, CH2-13), 18.9 (1C, CH2-12);
IR (ATR): ν˜ = 1720 cm−1 (s), 1202 (s), 1115 (s), 958 (vs),
773 (s); UV-Vis (MeOH): λmax (log ε) = 204 nm (4.63); MS
(ESI): m/z (%) = 735 (100) [2M + Na]+, 396/394 (7/7)
[M + K]+, 380/378 (32/32) [M + Na]+, 375/373 (19/19),
358/356 (12/12) [M+], 274/272 (21/21), 171/169 (15/15);
HRESIMS: calcd for C15H18BrNNaO4 [M + Na]+: 378.03114,
found 378.03107.
1
[silica, PE–EtOAc (2 : 1)]: Rf = 0.35; m.p.: 95–97 °C; H NMR
3
(600 MHz, DMSO-d6): δ = 8.33 (t, J = 5.9 Hz, 2H, 2× NH),
4
3
7.47–7.47 (m, 2H, 2× 2-H), 7.40 (dd, J = 2.3 Hz, J = 6.8 Hz,
3
2H, 2× 4-H), 7.27–7.23 (m, 4H, 2× 5-H, 2× 6-H), 5.36 (t, J =
2.7 Hz, 2H, 2× 10-H), 3.89 (d, J = 13.3 Hz, 2H, 2× 7-CHH),
3.82 (d, J = 13.3 Hz, 2H, 2× 7-CHH), 3.48–3.43 (m, 4H, 2×
2
2
NHCH2), 3.43–3.36 (m, 4H, 2× 14-OCH2), 2.85 (t, 3J = 7.0 Hz,
4H, 2× SCH2), 1.74–1.70 (m, 4H, 2× 11-CH2), 1.70–1.56 (m,
4H, 2× 12-CH2), 1.59–1.42 (m, 4H, 2× 13-CH2); 13C NMR
(150 MHz, DMSO-d6): δ = 162.3 (2C, 2× Cq-9), 152.6 (1C, 2×
Cq-8), 139.0 (2C, 2× Cq-1), 131.7 (2C, 2× C-2), 130.5 (2C, 2×
C-5), 129.1 (2C, 2× C-4), 127.8 (2C, 2× C-6), 121.4 (2C, 2×
CqBr), 100.5 (2C, 2× C-10), 60.9 (2C, 2× OCH2-14), 38.3 (2C,
2× NHCH2), 36.7 (2C, 2× SCH2), 29.7 (2C, 2× CH2-7), 28.0
(2C, 2× CH2-11), 24.5 (2C, 2× CH2-13), 18.2 (2C, 2× CH2-12);
IR (ATR): ν˜ = 1651 cm−1 (s), 1532 (s), 1204 (s), 965 (vs), 706
(s), 687 (s); UV-Vis (MeOH): λmax (log ε) = 202 nm (4.48); MS
(ESI): m/z (%) = 823 (44) [M + Na]+, 471 (21), 247 (100);
(E)-3-(3-Bromophenyl)-2-(tetrahydro-2H-pyran-2-yloxyimino)-
propanoic acid (14). To a solution of E-isomer 11 (0.27 g,
0.75 mmol, 1.00 equiv.) in 2 mL THF was added LiOH–H2O
(0.06 g, 1.51 mmol, 2.00 equiv.) in 5 mL H2O dropwise at 0 °C.
The reaction mixture was stirred for 10 min at 0 °C and for
another 14 h at rt. Then was added 1 N aqueous HCl solution
(2.5 mL) and stirred for 10 min. The solvent was evaporated, the
residue dissolved in EtOAc (20 mL) and washed with H2O (2 ×
15 mL) and saturated aqueous NaCl solution (15 mL). The
organic phase was dried over MgSO4, filtered and evaporated.
The E-isomer 14 was obtained as a pale yellow oil (0.23 g,
0.68 mmol, 90%); TLC [silica RP-18, MeOH–H2O (1 : 1)]: Rf =
HRESIMS: calcd for C32H40Br2N4NaO6S2 [M
821.06482, found 821.06532.
+
Na]+:
N,N′-(2,2′-Disulfanediylbis(ethane-2,1-diyl))bis(2-(7-(dimethyl-
amino)-2-oxo-2H-chromen-4-yl)acetamide) (19). 4-Coumarin-
acetic acid 18 (0.07 g, 0.30 mmol, 1.00 equiv.) was dissolved in
4 mL DCM and cooled to 0 °C. Then HOBt (0.04 g,
0.31 mmol, 1.05 equiv.) was added and after stirring for 10 min
at 0 °C EDCI (0.06 mL, 0.05 g, 0.31 mmol, 1.05 equiv.) was
added and the reaction mixture was stirred for another 30 min at
0 °C. Then cystamine dihydrochloride (15) (0.04 g, 0.17 mmol,
0.57 equiv.) and NEt3 (0.05 mL, 0.03 g, 0.34 mmol, 1.14 equiv.)
were added and the reaction mixture was stirred for 10 min at
0 °C and for 11 h at rt. The solvent was evaporated and the
residue dissolved in 300 mL n-butanol and washed with 1 N
aqueous HCl (3 × 100 mL), saturated aqueous NaHCO3 solution
(3 × 100 mL) and saturated aqueous NaCl solution (3 ×
100 mL). The organic phase was dried over MgSO4, filtered and
evaporated. Purification by column chromatography on silica
[CHCl3–MeOH (20 : 1)] afforded the bis(coumarinyl) compound
19 as a yellow solid (0.05 g, 0.08 mmol, 45%); TLC [silica,
1
4
0.13; H NMR (400 MHz, CDCl3): δ = 7.50 (dd, J = 1.6 Hz,
3
3
1H, 2-H), 7.35 (d, J = 7.8 Hz, 1H, 4-H), 7.25 (d, J = 7.7 Hz,
1H, 6-H), 7.15 (dd, J = 7.8 Hz, 1H, 5-H), 5.47 (m, 1H, 10-H),
3
2
2
3.98 (d, J = 13.3 Hz, 1H, 7-CHH), 3.88 (d, J = 13.3 Hz, 1H,
7-CHH), 3.63–3.52 (m, 2H, 14-OCH2), 1.89–1.56 (m, 6H, 11-
CH2, 12-CH2, 13-CH2); 13C NMR (100 MHz, CDCl3): δ =
162.7 (1C, CqCOOH), 150.3 (1C, CqN-8), 137.4 (1C, Cq-1),
132.4 (1C, C-2), 130.2 (1C, C-5), 130.0 (1C, C-4), 127.8 (1C,
C-6), 122.5 (1C, CqBr), 102.1 (1C, C-10), 62.2 (1C, OCH2-14),
30.3 (1C, CH2-7), 28.1 (1C, CH2-11), 24.8 (1C, CH2-13), 18.45
(1C, CH2-12); IR (ATR): ν˜ = 2946 cm−1 (m), 1718 (m), 1592
(m), 1567 (m), 1427 (m), 1202 (m), 1114 (m), 1071 (m), 1036
(m), 963 (vs), 928 (m), 898 (m), 870 (m), 851 (m), 809 (m), 775
(s); UV-Vis (MeOH): λmax (log ε) = 203 nm (4.41); MS (ESI):
m/z (%) = 382/380 (9/9) [M + K]+, 366/364 (92/100) [M +
Na]+, 361/359 (53/53), 344/342 (18/20) [M+]; HRESIMS: calcd
for C14H16BrNNaO4 [M + Na]+: 364.01549, found 364.01553.
1
CHCl3–MeOH (15 : 1)]: Rf = 0.37; m.p.: 146–148 °C; H NMR
3
(600 MHz, DMSO-d6): δ = 8.43 (t, J = 5.7 Hz, 2H, 2× NH),
3
4
3
7.51 (d, J = 9.0 Hz, 2H, 2× 5-H), 6.68 (dd, J = 2.5 Hz, J =
4
9.0 Hz, 2H, 2× 6-H), 6.53 (d, J = 2.5 Hz, 2H, 2× 8-H), 6.00 (s,
2H, 2× 3-H), 3.60 (s, 4H, 2× 9-CH2), 3.36 (m, 4H, 2× NHCH2),
3
3.00 (s, 12H, 2× (CH3)2), 2.78 (t, J = 6.7 Hz, 4H, 2× SCH2);
7130 | Org. Biomol. Chem., 2012, 10, 7120–7133
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13C NMR (150 MHz, DMSO-d6): δ = 168.0 (2C, 2× CqONH),
160.6 (2C, 2× Cq-2), 155.3 (2C, 2× Cq-8a), 152.7 (2C, 2× Cq-7),
151.0 (2C, 2× Cq-4a), 125.9 (2C, 2× C-5), 109.3 (2C, 2× C-3),
108.9 (2C, 2× C-6), 108.1 (2C, 2× Cq-4), 97.3 (2C, 2× C-8),
39.6 (4C, 4× CH3), 38.6 (2C, 2× CH2-9), 38.0 (2C, 2× NHCH2),
36.9 (2C, 2× SCH2); IR (ATR): ν˜ = 1595 cm−1 (vs), 1528 (s),
1400 (s), 1024 (s); UV-Vis (MeOH): λmax (log ε) = 371 nm
(4.44), 244 (4.38), 207 (4.71); fluorescence (MeOH): λmax [Int.
(a.u.), conc.] = 466 nm (980, 0.014 mg per 10 mL); MS (ESI):
m/z (%) = 1243 (35) [2M + Na]+, 633 (63) [M + Na]+, 611 (7)
[M + H]+, 191 (100); HRESIMS: calcd for C30H34N4NaO6S2
[M + Na]+: 633.18120, found 633.18127.
[silica, CHCl3–MeOH (9 : 1)]) the solvent was evaporated and
the residue vacuum dried for 2 h in a 40 °C water bath, washed
with Et2O (3 × 1 mL) and again vacuum dried. In a separate
flask acid 12 (0.12 g, 0.44 mmol, 1.40 equiv.) was dissolved in
3 mL dry DMF under an argon atmosphere. Then NHS (0.07 g,
0.62 mmol, 2.00 equiv.) was added and after 5 min DCC
(0.13 g, 0.62 mmol, 2.00 equiv.) was added and the reaction
mixture was stirred for 2 h at rt. The deprotected coumarin
amine was dissolved in 2 mL dry DMF and added dropwise.
Afterwards was added dry NEt3 (0.06 mL, 0.42 mmol, 1.30
equiv.) and the reaction mixture was stirred for 39 h at 30 °C and
4 h at 35 °C. After filtration from DCU cold H2O (15 mL) was
added, acidified under ice cooling with 1 N aqueous HCl sol-
ution to pH 3–4 and extracted with DCM (3 × 15 mL). The
organic phase was washed with H2O (15 mL) and saturated
aqueous NaCl solution (15 mL), dried over MgSO4, filtered and
evaporated. Purification by column chromatography on silica
[CHCl3–MeOH (50 : 1)] yielded the E-heterodimer 25 as a
yellow oil (0.11 g, 0.17 mmol, 55%). Under other conditions
and work-up was also isolated the corresponding nitrile 27 as a
yellow solid (0.02 g, 0.08 mmol, 18%); (E)-25: TLC [silica,
tert-Butyl-2-((2-(2-(7-(dimethylamino)-2-oxo-2H-chromen-4-
yl)acetamido)ethyl)disulfanyl)ethylcarbamate (24). 4-Coumarin-
acetic acid 18 (0.33 g, 1.33 mmol, 1.00 equiv.) was dissolved in
15 mL dry DCM and cooled to 0 °C under an argon atmosphere.
Then Boc protected cystamine 20 (0.37 g, 1.46 mmol, 1.10
equiv.) in 10 mL dry DCM was added dropwise. After 10 min at
0 °C were added EDCI (0.38 mL, 0.33 g, 2.13 mmol, 1.16
equiv.) and DMAP (57 mg, 0.47 mmol, 0.35 equiv.) and the
reaction mixture was stirred for another 10 min at 0 °C and for
5 h at rt. The reaction mixture was diluted with 5% KHSO4–ice
solution (20 mL) and extracted with DCM (3 × 20 mL). The
organic phase was washed in each case with 50 mL of H2O, 5%
aqueous NaHCO3 solution, H2O and saturated aqueous NaCl
solution. It was dried over MgSO4, filtered and evaporated.
Purification by column chromatography on silica [EtOAc–PE
(2 : 1 → 4 : 1 → EtOAc)] afforded the coumarin carbamate 24 as
a yellow solid (0.36 g, 0.75 mmol, 56%); TLC [silica, EtOAc–
1
CHCl3–MeOH (9 : 1)]: Rf = 0.37; H NMR (600 MHz, DMSO-
d6): δ = 11.89 (sbr, 1H, NOH), 10.11 (sbr, 1H, OH), 8.41 (t, 3J =
3
5.7 Hz, 1H, 10-CONH), 8.11 (t, J = 5.9 Hz, 1H, 15-CONH),
3
4
7.54 (d, J = 9.0 Hz, 1H, 5-H), 7.28 (d, J = 2.1 Hz, 1H, 19-H),
4
3
3
7.00 (dd, J = 2.1 Hz, J = 8.3 Hz, 1H, 21-H), 6.82 (d, J = 8.3
4
3
Hz, 1H, 20-H), 6.71 (dd, J = 2.6 Hz, J = 9.0 Hz, 1H, 6-H),
4
6.55 (d, J = 2.5 Hz, 1H, 8-H), 6.01 (s, 1H, 3-H), 3.68 (s, 2H,
3
3
17-CH2), 3.62 (s, 2H, 9-CH2), 3.41 (dt, J = 6.3 Hz, J = 13.6
Hz, 2H, 14-CH2), 3.37–3.35 (m, 2H, 11-CH2), 3.01 (s, 6H, N
(CH3)2), 2.82–2.79 (m, 2H, 13-CH2), 2.79–2.77 (m, 2H, 12-
CH2); 13C NMR (150 MHz, DMSO-d6): δ = 167.9 (1C, Cq-10),
163.2 (1C, Cq-15), 160.6 (1C, Cq-2), 155.3 (1C, Cq-8a), 152.7
(1C, Cq-7), 152.3 (1C, CqOH), 151.7 (1C, CvNOH), 151.1
(1C, Cq-4a), 132.7 (1C, C-19), 129.0 (1C, C-21), 128.6 (1C,
Cq-18), 125.9 (1C, C-5), 116.0 (1C, C-20), 109.4 (1C, C-3),
108.9 (1C, C-6), 108.8 (1C, CqBr), 108.1 (1C, Cq-4), 97.4 (1C,
C-8), 39.6 (2C, N(CH3)2), 38.7 (1C, CH2-9), 38.1 (1C, CH2-14),
37.9 (1C, CH2-11), 37.0 (1C, CH2-12), 36.7 (1C, CH2-13), 27.6
(1C, CH2-17); IR (ATR): ν˜ = 1598 cm−1 (vs), 1526 (s), 1400 (s);
UV-Vis (MeOH): λmax (log ε) = 375 nm (4.21), 281 (3.59), 243
(4.26), 204 (4.72); fluorescence (MeOH): λmax [Int. (a.u.), conc.]
= 464 nm (337, 0.017 mg per 10 mL); MS (ESI): m/z (%) =
1297 (21) [2M + Na]+, 661 (100) [M + Na]+, 339 (5), 185 (7);
1
PE (2 : 1)]: Rf = 0.40; m.p.: 112–114 °C; H NMR (400 MHz,
3
CDCl3): δ = 7.51 (d, J = 9.0 Hz, 1H, 5-H), 6.84 (sbr, 1H, 10-
4
3
CONH), 6.61 (dd, J = 2.6 Hz, J = 9.0 Hz, 1H, 6-H), 6.49 (d,
4J = 2.6 Hz, 1H, 8-H), 6.08 (s, 1H, 3-H), 4.99 (sbr, 1H, 15-
CONH), 3.67 (s, 2H, 9-CH2), 3.56 (dt, J = 6.0 Hz, J = 21.1
Hz, 2H, 11-CH2), 3.42 (dt, J = 6.4 Hz, J = 13.9 Hz, 2H, 14-
CH2), 3.05 (s, 6H, N(CH3)2), 2.80 (t, J = 6.0 Hz, 2H, 12-CH2),
3
3
3
3
3
2.76–2.72 (m, 2H, 13-CH2), 1.43 (s, 9H, C(CH3)3); 13C NMR
(100 MHz, CDCl3): δ = 168.4 (1C, Cq-10), 161.8 (1C, Cq-2),
156.0 (2C, Cq-8a, Cq-15), 153.1 (1C, Cq-7), 149.8 (1C, Cq-4a),
125.7 (1C, C-5), 110.4 (1C, C-3), 109.2 (1C, C-6), 108.5 (1C,
Cq-4), 98.3 (1C, C-8), 79.5 (1C, Cq(CH3)3), 40.4 (1C, CH2-9),
40.1 (2C, N(CH3)2), 39.5 (1C, CH2-14), 38.7 (1C, CH2-11),
38.2 (1C, SCH2-12), 37.6 (1C, SCH2-13), 28.4 (3C, C(CH3)3);
IR (ATR): ν˜ = 1616 cm−1 (s), 1530 (vs), 1273 (s), 1162 (s),
1147 (s); UV-Vis (MeOH): λmax (log ε) = 374 nm (4.29), 245
(4.22), 208 (4.55); fluorescence (MeOH): λmax [Int. (a.u.), conc.]
= 463 nm (302, 0.014 mg per 10 mL); MS (ESI): m/z (%) = 504
(100) [M + Na]+, 482 (4) [M+], 448 (13), 413 (64), 382 (15),
301 (37), 185 (49), 171 (78); HRESIMS: calcd for
C22H31N3NaO5S2 [M + Na]+: 504.15973, found 504.15958.
HRESIMS: calcd for C26H29BrN4NaO6S2 [M
659.06041, found 659.06041.
+
Na]+:
2-(3-Bromo-4-hydroxyphenyl)acetonitrile (27). 1H NMR
(400 MHz, acetone-d6): δ = 7.54 (d, 4J = 2.2 Hz, 1H, 2-H), 7.24
4
3
3
(dd, J = 2.2 Hz, J = 8.4 Hz, 1H, 6-H), 7.03 (d, J = 8.3 Hz,
1H, 5-H), 3.88 (s, 2H, 7-CH2); 13C NMR (100 MHz, acetone-
d6): δ = 154.6 (1C, CqOH), 133.6 (1C, C-2), 129.4 (1C, C-6),
124.8 (1C, Cq-1), 119.2 (1C, C-5), 117.6 (1C, CqN), 110.5 (1C,
CqBr), 22.2 (1C, 7-CH2); MS (EI, 70 eV): m/z (%) = 213/211
(30/30) [M+], 132 (100), 104 (7), 77 (12), 51 (7).
(E)-3-(3-Bromo-4-hydroxyphenyl)-N-(2-((2-(2-(7-(dimethyl-
amino)-2-oxo-2H-chromen-4-yl)acetamido)ethyl)disulfanyl)ethyl)-
2-(hydroxyimino)propanamide (25). Carbamate 24 (0.15 g,
0.31 mmol, 1.00 equiv.) was dissolved in 3 mL DCM under an
argon atmosphere and cooled to 0 °C. Then 1 mL TFA was
added dropwise and the reaction mixture was stirred for 10 min
at 0 °C and for 60 min at rt. After finishing deprotection (TLC
(E)-3-(3-Bromophenyl)-N-(2-((2-(2-(7-(dimethylamino)-2-oxo-
2H-chromen-4-yl)acetamido)ethyl)disulfanyl)ethyl)-2-(hydroxy-
imino)propanamide (26). Carbamate 24 (0.20 g, 0.42 mmol,
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 7120–7133 | 7131

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1.00 equiv.) was dissolved in 3 mL DCM under an argon atmo-
sphere and cooled to 0 °C. Then 1 mL TFA was added and the
reaction mixture was stirred for 10 min at 0 °C and for 45 min at
rt. After finishing deprotection (TLC [silica, CHCl3–MeOH
(9 : 1)]) it was evaporated under vacuum, dried for 2 h, washed
with Et2O (3 × 1 mL) and again vacuum dried. In a separate
flask acid 13 (0.11 g, 0.44 mmol, 1.05 equiv.) was dissolved in
2.4 mL dioxane at rt and NHPI (0.07 g, 0.42 mmol, 1.00 equiv.)
and after 5 min DCC (0.09 g, 0.42 mmol, 1.00 equiv.) were
added. After 2 h the deprotected amine was dissolved in 2 mL
MeOH and added dropwise to the reaction mixture and NEM
(0.05 mL, 0.05 g, 0.42 mmol, 1.00 equiv.) was added. The reac-
tion mixture was stirred for 30 h at 30 °C under an argon atmo-
sphere. After filtration from DCU it was diluted with cold H2O
(15 mL), acidified with 1 N aqueous HCl solution to pH 3–4 and
extracted with DCM (3 × 15 mL). The organic phase was
washed with H2O (15 mL) and saturated NaCl solution (15 mL),
dried over MgSO4, filtered and evaporated. Purification by
column chromatography on silica [CHCl3–MeOH (50 : 1)]
afforded the E-heterodimer 26 as a yellow solid (0.16 g,
0.26 mmol, 62%); TLC [silica, CHCl3–MeOH (9 : 1)]: Rf =
0.49; m.p.: 88–91 °C; 1H NMR (400 MHz, acetone-d6): δ =
11.23 (sbr, 1H, NOH), 7.86–7.81 (m, 1H, NH), 7.75–7.71 (m,
suspension (50 000 mL−1) in each well. Blank and solvent con-
trols were incubated under identical conditions. After 5 days
20 μL of MTT in phosphate buffered saline (PBS) were added to
a final concentration of 0.5 mg mL−1. After 2 h the precipitate of
formazan crystals was centrifuged, and the supernatant dis-
carded. The precipitate was washed with 100 μL PBS and dis-
solved in 100 μL of isopropanol containing 0.4% hydrochloric
acid. The microplates were gently shaken for 20 min to ensure
complete dissolution of the formazan and finally measured at
590 nm using a plate reader. All experiments were carried out in
two parallel experiments, the percentage of viable cells was cal-
culated as the mean with respect to the controls set to 100%. An
IC50 value was determined from the resulting dose–response
curves.
Agar diffusion assays. Agar plates containing 15 mL of
medium were inoculated with bacterial or yeast suspensions in a
liquid broth to give a final OD of 0.01 (bacteria) or 0.1 (yeasts).
In the case of molds, spores were collected from well-grown
Petri dishes which were rinsed with 10 mL sterile aqua dest.
1 mL of the spore suspension was added to 100 mL of molten
agar medium. 20 μL of test samples in methanol (1 mg mL−1
)
were applied onto 6 mm cellulose discs. The methanol was
allowed to evaporate and the discs were placed upon the inocu-
lated agar plate. The diameters in mm of the resulting growth
zones were determined after 24 h of incubation at 30 °C.
3
4
1H, NH), 7.60 (d, J = 9.0 Hz, 1H, 5-H), 7.50 (t, J = 1.8 Hz,
1H, 19-H), 7.36–7.33 (m, 1H, 20-H), 7.32–7.29 (m, 1H, 22-H),
3
3
4
7.19 (t, J = 7.8 Hz, J = 9.0 Hz, 1H, 21-H), 6.69 (dd, J = 2.6
3
4
Hz, J = 9.0 Hz, 1H, 6-H), 6.49 (d, J = 2.6 Hz, 1H, 8-H), 6.10
(s, 1H, 3-H), 3.93 (s, 2H, 17-CH2), 3.71 (s, 2H, 9-CH2),
3.60–3.55 (m, 2H, 14-CH2), 3.54–3.49 (m, 2H, 11-CH2), 3.01
(s, 6H, N(CH3)2), 2.89–2.84 (m, 2H, 13-CH2), 2.87–2.81 (m,
2H, 12-CH2); 13C NMR (100 MHz, acetone-d6): δ = 169.0 (1C,
Cq-10), 164.1 (1C, Cq-15), 161.7 (1C, Cq-2), 156.9 (1C, Cq-
8a), 154.1 (1C, Cq-7), 152.6 (1C, CvNOH), 151.4 (1C, Cq-4a),
140.6 (1C, Cq-18), 132.8 (1C, C-19), 131.1 (1C, C-21), 130.1
(1C, Cq-20), 129.0 (1C, C-22), 126.9 (1C, C-5), 122.7 (1C, Cq-
Br), 111.1 (1C, C-3), 109.8 (1C, C-6), 109.5 (1C, Cq-4), 98.6
(1C, C-8), 40.2 (2C, N(CH3)2), 39.9 (1C, CH2-9, from HSQC),
39.4 (1C, CH2-11), 39.3 (1C, CH2-14), 38.9 (1C, SCH2-12),
38.1 (1C, SCH2-13), 29.5 (1C, CH2-17); IR (ATR): ν˜ =
1595 cm−1 (vs), 1525 (s), 1400 (s); UV-Vis (MeOH): λmax (log
ε) = 374 nm (4.24), 244 (4.26), 204 (4.71); fluorescence
(MeOH): λmax [Int. (a.u.), conc.] = 465 nm (233, 0.015 mg per
10 mL); MS (ESI): m/z (%) = 1265 (5) [2M + Na]+, 645/643
(100) [M + Na]+, 623/621 (35) [M+], 346 (9), 301 (20), 179
(13); HRESIMS: calcd for C26H29BrN4NaO5S2 [M + Na]+:
643.06550, found 643.06551.
Serial dilution assay with bacteria. Antibiotic potential was
estimated by measuring OD at 600 nm as a parameter of bac-
terial growth of bacteria in serial dilutions of a compound after
24 h of incubation. The IC50 was calculated from resulting inhi-
bition curves.
HDAC assay. Histone deacetylase inhibition was measured
using the Fluorometric Histone Deacetylase Assay Kit purchased
from Sigma.
Acknowledgements
We thank Wera Collisi, Tatjana Hirsch and Bettina Hinkelmann
for the excellent technical assistance with biological activity
assays. This work has been supported in part by the “Evange-
lische Stiftung e.V. Villigst”. We also thank Merck, BASF and
Honeywell for their support with chemicals.
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