NSAID-derived γ-secretase modulation requires an acidic moiety on the carbazole scaffold

Article abstract of DOI:10.1016/j.bmc.2011.06.062

Modulation of γ-secretase activity holds potential for the treatment of Alzheimer's disease. Most NSAID-derived γ-secretase modulators feature a carboxylic acid, which may impair blood-brain barrier permeation. The structure activity relationship of 33 ca

Full text of DOI:10.1016/j.bmc.2011.06.062

Bioorganic & Medicinal Chemistry 19 (2011) 4903–4909
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
NSAID-derived
c
-secretase modulation requires an acidic moiety on the
carbazole scaffold
Andrea Zall a, Daniel Kieser a, Nicole Höttecke a, Eva C. Naumann a, Binia Thomaszewski a, Katrin Schneider a,
Dirk T. Steinbacher a, Robert Schubenel b, Stefan Masur b, Karlheinz Baumann b, Boris Schmidt a,
⇑
a Clemens Schöpf-Institute of Chemistry and Biochemistry, Technische Universität Darmstadt, Petersenstr. 22, Darmstadt D-64287, Germany
b F. Hoffmann-La Roche Ltd, Pharmaceuticals Division, Preclinical Research CNS, Bldg. 70/345, Basel CH 4070, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Modulation of
NSAID-derived
c
-secretase activity holds potential for the treatment of Alzheimer’s disease. Most
Received 4 May 2011
Revised 21 June 2011
Accepted 22 June 2011
Available online 29 June 2011
c-secretase modulators feature a carboxylic acid, which may impair blood–brain barrier
permeation. The structure activity relationship of 33 carbazoles featuring diverse carboxylic acid isoster-
es or metabolic precursors thereof was established in a cellular amyloid secretion assay. The modulatory
activity was observed for acidic moieties and metabolically labile esters only, which supports our hypoth-
esis of an acid-lysine interaction to be relevant for this type of
c
-secretase modulators.
Keywords:
Alzheimer’s disease
Ó 2011 Elsevier Ltd. All rights reserved.
c
-Secretase modulation
Carboxylic acid isosteres
Carprofen
1. Introduction
which is characterized by increased Ab38 secretion and decreased
Ab42 secretion.9 Photoaffinity labeling experiments with an
R-flurbiprofen derivative suggest a binding site for NSAID-derived
Alzheimer’s disease (AD) is the most common neurodegenera-
tive disease, it is a devastating illness effecting more than 20 mil-
lion patients globally. The brain atrophy associated with AD is
accompanied by the hallmark amyloid plaques and fibrillary tan-
gles. The plaques consist of aggregated oligomeric amyloid-b-pep-
c
-secretase modulators directly on the substrate APP close to the
GxxxG region,10,11 which is responsible for substrate dimerization
and may control the processing by
-secretase.18 We suggested an
c
interaction of the NSAID’s carboxylic acid with a basic amino acid,
for example, lysine624 on APP, which is located in the vicinity of the
GxxxG motif, at the membrane interface.12 We reported on
lipophilic N-alkylated carprofen-derivatives and N-sulfonylated or
N-alkylated carbazolyloxyacetic acids which are structurally
tides (Ab) of various lengths wherein Ab42 is more prone to
aggregate than Ab40 or Ab38
1
.
These Ab-peptides are generated by
sequential processing of the amyloid precursor protein (APP) by
b- and -secretase. The -secretase complex catalyzes the critical
step in the liberation of these Ab isoforms, and is thus a promising
target in the prevention of AD. The membrane located -secretase-
c
c
related to the substrate targeting c-secretase modulator flurbipro-
c
fen (sGSMs).13,14 The N-alkyl chain strongly enhances the modula-
tor activity; this may be partially due to modulator orientation via
a membrane anchoring effect (Scheme 1). The structural differ-
ences between acidic NSAID derived GSMs and the basic GSMs17
stimulated us to explore the structure activity relationship of the
carboxylic acid. This acid may interact with the substrate and
thereby influence the positioning of the substrate in relation to
PS1, which has been shown to be crucial for the cleavage event.19
complex consists of four proteins: nicastrin (Nct), anterior-pharynx
defective-1 (Aph-1), presenilin enhancer (Pen-2) and presenilin
(PS1 or PS2).2 PS1 is a nine transmembrane domain (TMD) protein
which bears two catalytic aspartates Asp257 and Asp385 in the
transmembrane domains 6 and 7.3–5 These catalytic domains
cleave APP within the membrane, implicating an unusual regulated
intramembrane proteolysis (RIP) in the lipophilic membrane.6–8
Several inhibitors for this process have been reported recently.7
However, most of these inhibit the cleavage of other c-secretase
substrates such as Notch, which is responsible for cell proliferation.
2. Methods and results
Some NSAIDs (non-steroidal anti-inflammatory drugs) show a par-
tial inhibition, the so called modulation of the c-secretase cleavage,
Herein we report the replacement of the carboxylic acid on
carprofen-derived GSMs 2 (IC50 (Ab38) = not determined at max
concn 100
(EC50 (Ab38) = 24
carboxylic acid isosteres and carboxylic acid derivatives. This
l
M, IC50 (Ab40) >40
l
M, IC50 (Ab42) = 6.9
l
M) and
3
lM, IC50 (Ab40) >40
lM, IC50 (Ab42) = 19
lM) by
⇑
Corresponding author.
E-mail address: Schmidt_boris@mac.com (B. Schmidt).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.062

4904
A. Zall et al. / Bioorg. Med. Chem. 19 (2011) 4903–4909
Scheme 1. Carprofen (1)9 is the GSM-lead structure of 2 and 3.14
replacement aimed to reduce the topological polar surface area and
the amphiphilicity of the lipophilic acids. This was expected to ef-
fect the blood–brain barrier (BBB) permeation.13,14 Carboxylic acid
isosteres require related environments as the carboxylic acid for
target binding and thus some of them were expected to display
(19, Table 1) displayed an Ab42level reducing effect
(IC50 (Ab42) = 38 M). The salicylic acid locks the extended carbon
l
chain in a Z-configuration resulting in close positioning of the car-
boxylic acid. Equipotent Ab42level reducing activity compared to
19 was achieved by a tetronic acid (20) and a tetramic acid (21),
which are rarely employed as carboxylic acid isosteres. Only a ste-
rically more demanding tetramate (22) displayed a full inhibitory
mode of action (Scheme 3).
c-secretase modulatory effects. We chose several established car-
boxylic acid isosteres such as tetrazole, sulfonic acid, amides, sul-
fone amides and tetronates. We included alcohols, nitriles and
amines as controls to obtain either straight or inverse
c
-secretase
On the contrary, the established isosteres: tetrazole (24) or the
modulators. In addition, we explored the modulator binding site
by the introduction of sterically demanding groups. The synthesis
was carried out according to Scheme 2 (explicit synthesis: see Sec-
tion 3 and Supplementary data). The amphiphilicity of these alkyl-
ated acids may result in micelle formation at higher concentration,
therefore the dose response evaluation was limited to a max concn
2,2,2-trifluoroethanol (12) did not affect
a concentration of 100 M or even caused increased Ab42 secretion.
The linear alcohol 23 and bromide 13 were inactive. However, the
-branched alcohol 8 increases Ab42 secretion. The amines (25,
c-secretase activity up to
l
a
30–32) did not show modulatory activity. The nitrile (14) caused
increased Ab42 secretion, whereas the amide (36) and the acidic
sulfonimide (9) showed reduction of the Ab42 level. These results
indicate the necessity of an acidic moiety on this scaffold to
achieve modulatory activity. This supports our hypothesis that
modulators interact with a basic amine on the substrate, because
all GSMs of this series exhibit a pKa value in the range of 2–5.
Additional substituents in the C-3 position of 3 revealed a toler-
ance for small functional groups like an aldehyde (33), but an addi-
tional propionic acid side chain (37) diminished activity
(Scheme 4). These findings suggest a defined binding pocket for
the modulators.
of 100 lM. This max concn excluded the IC50/EC50 determination of
less active compounds. However, the shape of the dose response
(see Supplementary data for examples) allowed us to classify the
compounds as reported in 2006.14 We observed four different
modes of actions in our cellular assay:
crease of Ab38, decrease of Ab42), inverse
(decrease of Ab38, increase of Ab42), -secretase inhibition (de-
c-secretase modulation (in-
c
-secretase modulation
c
crease of Ab38, decrease of Ab42) and lack of activity.
We kept the length of the supposed lipid anchor at eight carbon
atoms, making a compromise between lipophilicity and activity,
although this anchor does not provide the most active sGSM. The
side chain of 3 (BSc3030) bears a methylene spacer, which is an
important structural feature as an additional methylene dimin-
ished activity. This effect was observed for the carboxylic acid 17
and to a lesser extent for the sulfonic acid 18. Surprisingly, the
dimethylation (29) diminished activity, although moderately po-
The methyl ester 15 displayed unexpected Ab42level reducing
activity (Table 2, entry 4, IC50 (Ab42) = 27
activity compared to the carboxylic acid 3 with an IC50 for Ab42
of 19 M. Further investigation confirmed the tolerance of short
lM), a marginal loss in
l
linear esters. However, the activity decreased with increasing
length of the ester. The ethyl ester 34 showed Ab42 level reducing
tent
c
-secretase activity modulating fenofibrate-derivatives have
activity (IC50 (Ab42) = 31
(26) did not reduce Ab42 secretion. On the contrary, the tertiary
lM), but iso-propyl (35) or iso-butyl ester
been reported.12,17 However, the replacement by a salicylic acid
Scheme 2. Reagents: General synthesis of the carbazole derivatives. (a) Benzylbromide, K2CO3, acetone, rt; (b) octylbromide, NaH, THF, 0 °C-rt; (c) octylbromide, NaH, THF,
and 0 °C-rt.

A. Zall et al. / Bioorg. Med. Chem. 19 (2011) 4903–4909
4905
Table 1
Activity of N-octylcarprofen and N-octylcarbazole derivatives
R2
R1
R3
N
octyl
Entry
Compd
Mode of action
R1
R2
R3
IC50/EC50 lM
Ab38
Ab40
Ab42
1
2
5
8
iGSM
iGSM
H
H
Cl
Cl
n.d.c
n.d.c
n.d.c
50a
21a
OH
n.d.
H
N
3
4
9
GSM
GSI
S
H
H
Cl
H
n.d.
n.d.c
n.d.c
32a
O2
O
11
–OH
n.d.c
>80
CF3
O
5
12
iGSM
H
H
n.d.c
n.d.c
12a
OH
Br
CN
6
7
13
14
iGSM
GSI
H
H
H
H
>40
74
>40
105
>40
O
O
252b
O
8
9
17
18
GSM
GSM
H
H
H
H
>80a
n.d.c
>80
>80b
>80b
O
O
OH
SO2OH
n.d.c
10
11
19
20
GSM
GSM
H
H
H
H
23a
21a
>80
>80
38b
37b
O
CO2H
O
O
O
OH
NH
O
12
13
21
22
GSM
H
H
H
H
8a
72
32
28b
O
OH
N
O
O
GSI
31
24
OH
O
O
OH
14
15
23
24
iGSM
GSI
H
H
H
H
n.d.c
165b
n.d.c
102b
>40
O
N
O
113b
NH
N
N
NH2
16
17
25
29
iGSM
GSM
H
H
H
H
>80
>80
>40
>80a
>40
O
>40a
O
CO2H
NMe2
NCO
18
19
30
31
iGSM
GSM
H
H
H
H
n.d.c
n.d.c
n.d.c
n.d.c
>10a
>80
O
O
(continued on next page)

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A. Zall et al. / Bioorg. Med. Chem. 19 (2011) 4903–4909
Table 1 (continued)
Entry
Compd
Mode of action
R1
R2
R3
IC50/EC50 lM
Ab38
Ab40
Ab42
O
CF3
CF3
20
32
GSI
H
H
n.d.c
n.d.c
>80
N
O
O
21
22
33
37
GSM
GSM
H
H
n.d.c
n.d.c
n.d.c
n.d.c
32
O
CO2H
O
CO2H
>80
O
O
CO2H
OH
NH2
OH
23
24
38
36
GSI
GSI
H
H
n.d.c
n.d.c
n.d.c
n.d.c
80
O
H
104
O
Modulators (GSM) display increased production of Ab38 (EC50) and reduction of Ab42 (IC50). Inverse modulators (iGSM) display increased production of Ab42 (EC50) and
reduction of Ab38 (IC50). -secretase inhibitors (GSI) reduce Ab38 and Ab42
c
.
a
EC50 values are displayed.
IC50 values are displayed.
Not determinable at max concn: 100 lM.
b
c
Scheme 3. Reagents: Synthesis of the higher substituted tetramate (22). (a) Piperonal, Na(AcO)3BH, DCE, rt, 5 h; (b) bromoacetyl bromide, TEA, DCM, rt, 2,5 h; (c) 11, K2CO3,
acetone, rt; (d) KOtBu, THF, rt.
butyl ester (16) increased Ab42 levels at EC50 = 19
phosphonic diethyl ester (27) caused an increased Ab42 secretion
with an EC50 (Ab42) = 13 M. These findings suggest a metabolic
activation related to fenofibrate hydrolysis, for example, an ester-
ase releasing the active carboxylic acid. This hydrolysis seems to
tolerate short linear esters as substrates. This presumption was
l
M. Equally the
carprofen derivative 45 (Fig. 1, IC50 Ab42 = 2.9
Ab40 >40 M, IC50 Ab38 = 5.8
M).14 It does not interfere with
secretase cleavage activity at the -site as determined by de novo
lM, IC50
l
l
c-
l
e
production of AICD.14 This indicated that 45 may not exert Notch
inhibition at the required physiological concentration. The ratio
of the carprofen concentrations in plasma (AUCF) versus cerebro-
confirmed by investigation of the
carprofen esters (4, 6, and 7), which inhibit or inversely modulate
-secretase activity. Consequently, short
a0-linear esters fulfill
criteria for a fenofibrate analog prodrug system in this cellular
assay. This potential prodrug system is still under investigation. Fi-
nally, an additional aldehyde in the C-3 position (28) of the methyl
a
-branched side chain of
spinal fluid (AUCCSF
) over time was reported for rats (AU-
CR = AUCCSF AUCF = 0.23), this relates to an AUCR = 5.31 for
:
c
a,
flurbiprofen.20 Moreover, Péhourcq et al. observed a parabolic rela-
tionship for brain permeation and the lipophilicity of deprotonated
NSAIDs. This relationship stimulated us to select C-10-derivative
45 for in vivo evaluation while the SAR study of the acid derivates
was ongoing. Indeed, 45 showed a slow, yet significant BBB perme-
ation in APPswe Tg mice at a single 10 mg/kg oral dosage suspended
in NaCl and gelatine. The plasma and brain concentration of the
modulator were monitored over a period of 5 h. The brain concen-
tration was found to continuously increase to a plasma/brain ratio
of approximately 2:1 ( Fig. 1). The AUCR <0.1 indicates reduced
brain permeation versus carprofen. Moreover, the significant brain
levels of the acid 45 do not imply localization of the compound at
ester was not tolerated and resulted in straight c-secretase inhibi-
tion. All attempts to hydrolyze the phosphonic ester (27) resulted
in decomposition, excluding the resulting acid from the
investigation.
The brain permeability of carboxylic acids is regarded with sus-
picion despite the identification of the MCT-1 transporter in
human brain.23 Therefore we decided to challenge the paradigm
of poor brain permeability of such acids and selected the lead

A. Zall et al. / Bioorg. Med. Chem. 19 (2011) 4903–4909
4907
Scheme 4. Reagents: Synthesis of C3-substituted carbazole 37. (a) Octyl iodide, NaH, THF, DMF, ꢁ70 °C-rt, 12 h; (b) bromoacetic acid methylester, K2CO3, acetone, rt, 12 h; (c)
N,N-diisopropylformamide, POCl3, H2O, DCE, 80 °C, 2–5 d; (d) phosphonium salt, NaOEt, EtOH, 80 °C, 20 h; (e) Pd/C, H2, EtOH, HCl, rt, 24 h; (f) KOH, MeOH, rt/64 °C, 2 d.
the site required for modulatory activity. Furthermore, no signifi-
cant Ab42 changes were observed at this timepoint. These un-
changed Ab levels were linked to the double transgenic mice
exhibiting NSAID-resistant PS1 mutations recently.15,16 This lack
of activity in murine cell lines was reported for several first gener-
with an auto sampler. The purity of the final compounds was
determined using UV detection (k = 254 nm). The chromatographic
method employed the following: column Zorbax Eclipse XDB-C18;
4.6 ꢀ 150 mm; mobile phase A H2O (0.1% TFA), mobile phase B ace-
tonitrile, flow rate 1 mL/min, gradient eluation 30–100% B over
15 min. According to these methods the purities for all compounds
were P95% if not indicated otherwise in the experimental details.
The in vitro measurements were performed on a cellular Ab
ation c-secretase modulators and PS1 mutations both by Weggen
et al.21 and Steiner et al.22 Thus the less potent and even more lipo-
philic carbazole derivatives were excluded from evaluation in this
mouse model.
lowering assay, in H4 APP
D9 cells to evaluate the compounds
Our findings indicate the necessity of an acidic moiety in a pKa-
for their potencyto modulate Ab secretion. We used the Ab liquid
phase electrochemiluminescence (LPECL) assay to measure Ab iso-
forms.12,13 Cell viability was measured by a colorimetric cell prolif-
eration assay (CellTiter 96TM AQ assay, Promega) utilizing the
bioreduction of MTS (Owen’s reagent) to formazan.
See Supplementary data for details of the in vivo mouse
experiments.
range of 2–5 on this class of
c-secretase modulators. This small
pKa-range supports our hypothetical interaction with lysine624
.
The location of this lysine, directly at the membrane interface close
to the GxxxG dimerization motif, suggests a modulation mecha-
nism as an APP dimerization inhibitor or impacts substrate orien-
tation in the membrane19, shifting the cleavage site to the less
toxic Ab38. The optimum spacer length between the arylic back-
bone and carboxylic acid was determined to be 2–3 carbon or oxy-
gen atoms. However, introduction of a rigid aryl spacer allowed an
elongation of up to four atoms. We identified a potential prodrug
system based on short linear esters, which complements the BBB
permeation of the free acid 45.
3.3. 2-Hydroxy-N-octylcarbazole 11
To a suspension of NaH (0.82 g, 20.48 mmol) in THF (20 mL) un-
der argon atmosphere was added 2-hydroxycarbazole (1.50 g,
8.19 mmol) at a temperature of 0 °C. After 30 min stirring octylbr-
omide (1.50 g, 7.78 mmol) was added and the reaction mixture
was allowed to warm to rt. After 12 h the reaction mixture was
quenched by drop wise addition of H2O until gas formation ceased.
The obtained mixture was extracted three times with CH2Cl2
(30 mL). The combined organic layers were washed with brine,
dried over MgSO4, concentrated in vacuo and purified by silica
gel column chromatography (gradient starting with cyclohexane
to cyclohexane: CH2Cl2 1:2) to give 1.58 g (74%) of 11 (BSc4029)
as a colorless solid.
3. Experimental
3.1. General experimental informations
The 1H and 13C spectra were recorded on a Bruker AC 300
(300 MHz) and AC 500 (500 MHz) spectrometer. Chemical shifts
are reported in d (ppm) adjusted to the central line of the deuter-
ated solvent (MeOD, CDCl3, d6-DMSO, Acetone-d6). Mass spectrom-
HPLC: 9.2 min (96%), 1H NMR (500 MHz, CDCl3): d = 7.96 (d,
J = 8.3 Hz, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.36 (m, 1H), 7.34 (m, 1H),
7.19 (td, J = 7.0, 1.2 Hz, 1H), 6.82 (d, J = 2.2 Hz, 1H), 6.72 (dd, J = 8.3,
2.2 Hz, 1H), 4.81–5.43 (b.s., 1H), 4.18 (t, J = 7.5 Hz, 2H), 1.82 (q,
etry was performed on
a Bruker-Franzen Esquire LC mass
spectrometer (ESI) and a double focused MAT 95 (EI). HPLC analy-
sis was performed on an Agilent 1100 system.
J = 7.5 Hz, 2H), 1.20–1.41 (m, 10H), 0.86 (t, J = 6.8 Hz, 3H) ppm, 13
C
3.2. Purity determination
NMR (125 MHz, CDCl3): d = 154.7, 141.9, 140.6, 124.4, 123.0, 121.2,
119.4, 118.9, 116.9, 108.4, 108.0, 95.0, 43.1, 31.8, 29.4, 29.2, 28.8,
27.3, 22.6, and 14.1 ppm, MS (m/z, 70 eV, ESI) = 318.4 [M+Na].
Analytical reversed phase high-performance liquid chromatog-
raphy was performed on an Agilent 1100 HPLC system, equipped

4908
A. Zall et al. / Bioorg. Med. Chem. 19 (2011) 4903–4909
Table 2
Activity of N-octylcarbazole and N-octylcarprofen esters
R2
R1
R3
N
octyl
Entry
Compd
Mode of action
R1
R2
R3
Cl
IC50/EC50 lM
Ab38
Ab40
Ab42
O
1
4
GSI
H
n.d.b
n.d.b
>80
O
O
O
2
3
6
7
iGSM
iGSM
H
H
Cl
Cl
n.d.b
n.d.b
n.d.b
n.d.b
15a
21a
O
O
O
O
O
4
5
15
16
GSM
H
H
H
H
n.d.b
n.d.b
n.d.b
n.d.b
27
O
O
O
O
iGSM
19a
O
6
26
GSM
H
H
H
n.d.b
n.d.b
>80
O
PO(OEt)2
O
7
8
27
28
iGSM
GSI
H
H
n.d.b
n.d.b
n.d.b
n.d.b
13a
89
O
O
O
O
O
O
O
O
O
O
9
34
35
GSM
H
H
H
H
n.d.b
n.d.b
n.d.b
n.d.b
31
10
iGSM
49a
Modulators (GSM) display increased b production of A38 (EC50) b and reduction of A42 (IC50). Inverse modulators (iGSM) display increased production of ꢀ(EC50 b) and
reduction of A38 (IC50). b-Secretase inhibitors (GSI) reduce Ac38 b and A42
.
a
EC50 values are displayed.
b
Not determinable at max concn: 100 lM.
Figure 1. 45 (BSc3041) Penetrate the BBB in C57/BLg mice at an oral dosage of 10 mg/kg.
3.4. 2-(2-Bromoethoxy)-9-octyl-9H-carbazole 13
ethane (0.180 g, 0.97 mmol) and K2CO3 (0.141 g, 1.02 mmol),
acetone (30 mL) yield 80 mg (51%) 13 as colorless solid.
According to general procedure for ether formation (see Supple-
mentary data): 11 (BSc4029) (0.100 g, 0.34 mmol), 1,2-dibromo-
HPLC: 10.8 min (98%), 1H NMR (300 MHz, CDCl3): d = 8.01 (d,
J = 7.7 Hz, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.44 7.35 (m, 2H),

A. Zall et al. / Bioorg. Med. Chem. 19 (2011) 4903–4909
2. Wolfe, M. S. Biochemistry 2006, 45, 7931.
4909
7.24–7.19 (m, 1H), 6.90 (d, J = 2.2 Hz, 1H), 6.85 (dd, J = 8.5, 2.2 Hz,
1H), 4.43 (t, J = 6.3 Hz, 2H), 4.23 (t, J = 7.3 Hz, 2H), 3.74–3.69 (m,
2H), 1.90–1.82 (m, 2H), 1.44–1.23 (m, 10H), 0.89 (t, J = 7.0 Hz,
3H) ppm, 13C NMR (75 MHz, CDCl3): d = 157.3, 141.6, 140.7,
124.6, 122.9, 121.1, 119.6, 118.9, 117.5, 109.5, 107.3, 94.8, 68.6,
43.1, 31.8, 29.4, 29.3, 29.2, 28.8, 27.3, 22.6, and 14.0 ppm.
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3.5. 2-(9-Octyl-9H-carbazol-2-yloxy)-ethanesulfonic acid 18
To a solution of 13 (BSc4004) (0.020 g, 0.05 mmol) in a mixture
of H2O (0.050 mL) and dioxane (0.050 mL) was added Na2SO3
(0.032 g, 0.25 mmol) and the resulting mixture was heated at
160 °C under microwave irradiation until the reaction was com-
pleted (TLC control). The reaction mixture was concentrated in va-
cuo, the resulting solid dissolved in EtOHabs and concentrated in
vacuo to give 0.007 g (35%) of 18 (BSc4079) as a colorless solid.
HPLC: 7.0 min (93%), 1H NMR (500 MHz, MeOD): d = 7.97–7.94
(m, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.41 (d, J = 8.2 Hz, 1H), 7.37–7.30
(m, 1H), 7.18–7.10 (m, 1H), 6.84 (dd, J = 8.5, 2.2 Hz, 1H), 7.04 (d,
J = 2.1 Hz, 1H), 4.32 (t, J = 7.1 Hz, 2H), 4.53–4.47 (m, 2H), 3.38–
3.33 (m, 2H), 1.89–1.81 (m, 2H), 0.86 (m, 10H), 1.32–1.24 (t,
J = 7.0 Hz, 3H) ppm, 13C NMR (125 MHz, MeOD): d = 143.1, 142.0,
125.3, 124.3, 121.8, 120.2, 119.8, 118.2, 109.7, 109.1, 100.5, 95.2,
65.3, 51.9, 43.7, 32.9, 30.7, 30.3, 29.9, 28.2, 23.6, and 14.4 ppm,
MS (m/z, 70 eV, ESI) = 402 [Mꢁ].
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Supplementary data
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References and notes
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