Synthesis and SERCA activities of structurally simplified cyclopiazonic acid analogues

Article abstract of DOI:10.1016/j.bmc.2011.06.001

The indole alkaloid cyclopiazonic acid (CPA) is one of the few known nanomolar inhibitors of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) besides the anticancer drug thapsigargin and the antiplasmoidal terpenoid artemisinin. Due to its less complex structure CPA represents an attractive lead structure for the development of novel antimalarial drugs or for applications in the field of plant protection. We report here the first syntheses of structurally simplified CPA fragments and discuss their SERCA activities on the basis of published crystal structures of CPA-SERCA complexes.

Full text of DOI:10.1016/j.bmc.2011.06.001

Bioorganic & Medicinal Chemistry 19 (2011) 4669–4678
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and SERCA activities of structurally simplified cyclopiazonic
acid analogues
Sheng Yao a, Daniel Gallenkamp a, Katharina Wölfel b, Bettina Lüke b, Michael Schindler b,
Jürgen Scherkenbeck a,
⇑
a Bergische Universität Wuppertal, Fachgruppe Chemie, Gaußstraße 20, D-42119 Wuppertal, Germany
b Bayer CropScience AG, Alfred-Nobel-Straße 50, D-40789 Monheim, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The indole alkaloid cyclopiazonic acid (CPA) is one of the few known nanomolar inhibitors of
sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) besides the anticancer drug thapsigargin and the
antiplasmoidal terpenoid artemisinin. Due to its less complex structure CPA represents an attractive lead
structure for the development of novel antimalarial drugs or for applications in the field of plant protec-
tion. We report here the first syntheses of structurally simplified CPA fragments and discuss their SERCA
activities on the basis of published crystal structures of CPA–SERCA complexes.
Received 26 April 2011
Revised 31 May 2011
Accepted 1 June 2011
Available online 12 June 2011
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Indole alkaloid
Cyclopiazonic acid
SERCA
Analogues
Structure–activity data
1. Introduction
product ryanodin or flubendiamide, a synthetic benzenedicarboxa-
mide insecticide for controlling lepidopterous insect pests.7
Acyltetramic acids have been known since almost a century but
it was not until the 1960s that this heterocycle was identified as a
key structural element in natural products, such as the SERCA
inhibitor cyclopiazonic acid (CPA, 1), the potent HIV-1 integrase
inhibitor equisetin (3), the antiprotozoal ikarugamycin (4), or the
antibiotic tirandamycin (5) to name only a few (Fig. 1).1–4 The wide
range of biological activities that have been shown in many cases
to be related to the tetramic acid core renders this class of natural
products prime targets for chemists and biologists.
The toxic indole alkaloid cyclopiazonic acid (CPA 1) was first
isolated from cereals and meal infected with fungi of the Aspergil-
lus- or Penicillium genus. CPA is the cause of several food poison-
ings but shows on the other hand promising antiplasmoidal
activities on a level comparable to chloroquin. CPA (1) specifically
inhibits a Ca2+-ATPase of the sarcoendoplasmic reticulum (SERCA)
which is essential for calcium reuptake in the muscle contraction–
relaxation cycle.5,6 Calcium metabolism also represents an impor-
tant target for modern insecticides as exemplified by the natural
Despite of more than one hundred published natural tetramic
acids, the skeleton of cyclopiazonic acid (CPA) remains unique as
the acetyltetramic acid core is part of a multicyclic framework.8
The first synthesis of CPA was reported in 1984, followed by two
other racemic syntheses.9–11 A 15-step asymmetric total synthesis
of CPA based on the racemic synthesis developed by Knight and co-
workers has been accomplished only very recently.11 However, less
complex CPA analogues accessible by short syntheses are a prere-
quisite for the development of synthetic drugs against malaria or
for potential applications in crop protection.
Published CPA–SERCA structures suggest the CPA binding pocket
to consist of three regions: a polar region coordinates the acyltet-
ramic acid with Gln56, Asp59, Asn101 and Mg2+ as main interacting
residues (Fig. 2).13,14 The chelation of Mg2+ by the acyltetramic acid
plays a pivotal role for SERCA inhibition.12 The center of the pocket
binds ring C of CPA and displays a preference for conjugated p-sys-
tems as documented by BHQ (2,5-di-(tert-butyl) hydroquinone), a
small molecule inhibitor of SERCA. An extended hydrophobic region
of the binding pocket houses the lipophilic indole residue and con-
nects the CPA with the thapsigargin (TG) binding site.15 Remarkably,
the indole is fixed to its binding site only by lipophilic interactions,
since the NH-group seems not to be involved in hydrogen bond for-
mation. Thus, the indole-ring can be presumed to be more flexible to
structural variations as the other CPA partial structures.
Abbreviations: ACN, acetonitrile; DMF, dimethylformamide; EGTA, ethylene
glycol tetraacetic acid; IPy2BF4, bis(pyridine)iodine-(I)-tetrafluoroborate; MOPS,
3-(N-morpholino)-propanesulfonic acid; THF, tetrahydrofurane; TFA, trifluoroacetic
acid; DMAP, 4-dimethylaminopyridine; DCC, N,N0-dicyclohexylcarbodiimide.
⇑
Corresponding author. Tel.: +49 2024392654; fax: +49 2024393464.
E-mail address: Scherkenbeck@uni-wuppertal.de (J. Scherkenbeck).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.001

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S. Yao et al. / Bioorg. Med. Chem. 19 (2011) 4669–4678
OH
O
O
O
10
N
HO
O
HN
N
D
E
7
OH
H
11
6a
H
H
O
H
11a
O
6
5a
H
C
H
O
11c
11b
1
A
B
NH
N
2a
2
H
α-Cyclopiazonic acid (1)
β-Cyclopiazonic acid (2)
Equisetin (3)
O
O
H
N
H
H
H
OH
O
O
O
O
O
O
NH
NH
HH
Ikarugamycin (4)
Tirandamycin A (5)
O
OH
Figure 1. Natural products containing acyltetramic acids.
lated requirement for unsaturation in ring C. In addition, CPA ana-
logue 6 has a linear arrangement of rings while the analogous
compound 7 with a pyrrolizidine skeleton shows the CPA typical
angulated shape. The saturated compound 9, formally available
from substance 7 by hydrogenation, is identical with the C,D,E-ring
system of CPA (1).
An additional structural simplification we were interested in is
found in compounds 10 and 11 that carry a nitrogen in position 4
instead of a carbon atom. To our surprise, no natural product with
the corresponding acyl pyrazolidine-3,5-dione core is known and
even reports on the synthesis of monocyclic and in particular bicy-
clic acylpyrazolidine-diones are rare, probably due to the very lim-
ited numbers of synthetic procedures available for this heterocyclic
system.16–18 In view of this situation we have developed a general
and straightforward synthesis for the tricyclic dihydro-pyrazolo-
[1,2-a]pyrazole-1,3-diones 10 and 11.
Figure 2. Homology model of Heliothis virescens SERCA complexed with cyclopi-
azonic acid.
Taking the available molecular-modelling studies and struc-
ture–activity data into account, we suggest compounds 6–9
(Fig. 3) as structurally simplified models for CPA (1). These com-
pounds should be appropriate to elucidate the role of the indole
core for SERCA inhibition.12 Tetramic acids 6 and 7 in which the
cyclohexane ring C is replaced by a phenyl ring, combine structural
elements of BHQ and CPA (1). Both compounds address the postu-
2. Results and discussion
2.1. Chemistry
The key steps in our synthesis of acyltetramic acid 6 include an
ortho-selective iodination of phenylalanine and a cationic cycliza-
tion (Scheme 1). IPy2BF4 is a mild iodination reagent which has
been used previously to iodinate phenylalanine and tyrosine selec-
tively in the ortho position even as residues in peptides and pro-
teins.19–22 With the fully protected phenylalanine 12 in hand the
ortho-iodine derivative 13 was prepared in more than 90% yield
and with an ortho/para ratio of up to 10:1. A Suzuki–Miyaura cou-
pling with isopropenylboronic acid pinacol ester under standard
conditions afforded the isopropenylated phenylalanine 14 in rea-
sonable yields. For the ring-closure to the sterically hindered di-
methyl piperidine 15 we applied a cationic cyclization procedure
with triflic acid to form a benzylic cation by protonation of the dou-
ble bond, which is attacked subsequently by the amino acid nitro-
gen. It has been shown recently that cationic cyclizations follow
an overall 5-endo-trig course and afford excellent cyclization yields
even in the case of sterically hindered rings.11,23 The nitrogen pro-
tecting group plays a critical factor in this type of reaction since it
must be strongly electron-withdrawing to prevent protonation of
the basic amino acid nitrogen, but on the other hand the group
should be cleavable under mild conditions. Therefore, the fre-
quently used sulfonamide protecting groups appear to be problem-
atic. We found that a trifluoroacetyl group, which can be removed
under much milder conditions, gives comparable cyclization yields
(compound 15). Following a two-step deprotection–reprotection
O
OH
N
H
O
O
N
A
B
C
OH
OH
H
O
O
6
7
H
H
O
N
N
H
OH
OH
H
H
H
O
O
9
8
H
8a
4a
9
O
O
N
N
4
1
N
N
H
O
O
OH
11
10
Figure 3. Structurally simplified model compounds for CPA.

S. Yao et al. / Bioorg. Med. Chem. 19 (2011) 4669–4678
4671
O
O
O
I
O
O
O
O
a
O
b
HN
CF3
HN
13
CF3
HN
14
CF3
O
O
B
12
O
O
O
O
O
OH
d, e
c
NH
N
CF3
O
f
N
H
O
O
15
16
6
Scheme 1. Synthesis of the linear CPA analogue 6. Reagents and conditions: (a) IPy2BF4, HBF4, DCM/TFA 10:1, rt, 12 h, 92%; (b) Pd(PPh3)4 10 mol %, Na2CO3 (aq), THF/toluene
1:1, 80 °C, 36 h, 55%; (c) TfOH, CHCl3, rt, 6 h, 63%; (d) NaOH (aq) EtOH, rt, 12 h, 89%; (e) SOCl2, MeOH, 0 °C, rt, 12 h, 69%; (f) acetyl Meldrum’s acid, toluene, 110 °C, 30 min, then
KOtBu, 110 °C, 30 min, 67%.
sequence the tetramic acid moiety was finally built up by a one-pot
reaction of the bicyclic piperidine 16 with acetyl Meldrum’s acid
(Scheme 1).
Unfortunately, all attempts to prepare the pyrrolizidine scaffold
of compound 7 in the same way were unsatisfactory due to a low
regioselectivity and yield in the iodination step of phenylglycine,
the corresponding starting material for compounds 7–9. Thus, a
new synthesis for the pyrrolizidine moiety was developed, utilizing
a Strecker-reaction for the introduction of the amino acid function-
ality.24 The central intermediate 22 is available either from com-
mercially available 2-iodobenzoic acid (17) by formation of the
(a)
NHCOCF3
OMe
CO2H
I
CHO
I
c - f
a,b
h,i
O
I
17
(b)
18
19
22
g
l,m
CF3
j,k
H
N
N
Br
aminoacid ester 19 followed by
a Suzuki–Miyaura coupling
NH2
O
20
21
(Scheme 2a), similar to the synthesis of isopropenyl phenylalanine
14, or via aminonitrile 21 which can be prepared from 1-bromo-2-
isopropenyl-benzene (20) in two steps according to Scheme 2b.
However, the Strecker reaction of iodobenzaldehyde 18
(Scheme 2a) gave only low yields of 2-iodo phenylglycine. It turned
out that an inverted sequence (Scheme 2b) with formation of the
isopropenyl group first followed by a Strecker reaction afforded
significantly better yields of the key intermediate 22. The cationic
cyclization yields of dihydroisoindole 23 were similar to those of
tetrahydroisochinoline 15. Cycloacylation with acetyl Meldrum’s
acid then afforded 2-acetyl-5,9b-dihydro-pyrrolo[2,1-a]isoindole-
1,3-dione 7. All attempts to obtain the saturated analogues 8 or 9
directly from compound 7 by hydrogenation resulted in decompo-
sition of the starting material. More successful was the hydrogena-
tion of the precursor 23. The all-cis stereoisomer 24 was formed
almost exclusively controlled by the stereogenic center in position
1. This was subsequently equilibrated to the more stable cis–trans
isomer 25 with NEt3 in benzene.10 Stronger basic conditions caused
a complete decomposition of the cis-stereoisomer 24. Acylation of
the precursors 23–25 with acetyl Meldrum’s acid in NaOEt/EtOH
gave the CPA analogues 7–9 in yields of up to 74% as a mixture
of E/Z-isomers around the exocyclic double bond.
As starting material for the construction of the 2,3-dihydroin-
dazole scaffold (compounds 10 and 11, Scheme 3), we chose ami-
noacetophenone 26, which has already been used for the synthesis
of a variety of heterocyclic compounds such as indoles, quinazo-
lines and benzodiazepines.25 Hydrazine 27 was prepared from
aminoacetophenone 26 by a twofold Grignard addition followed
by a diazotization and reduction of the intermediate diazonium
salt with stannous chloride.26 In principle, diketopyrazolidine 32
is accessible via two routes (Scheme 3): Firstly, by acidic cycliza-
tion of the Boc-protected hydrazine 30 to dihydroindazole 29
and acylation with malonyl chloride. Secondly, by acylation of
phenylhydrazine 27 with ethylmalonyl chloride, followed by an
acidic ring-closure to dihydroindazole 31 and immediate cycliza-
tion to the tricyclic pyrazolo[1,2-a]indazole 32. The yields of the
O
O
H
H
H
o
p
3a
7a
NH
H
O
NH
NH
H
1
H
O
O
O
23
24
O
O
25
n
61%
71%
n
n
74%
H
H
O
O
O
N
N
N
H
OH
OH
H
OH
H
H
O
O
O
7
8
9
Scheme 2. Syntheses of tetramic acids 7–9. Reagents and conditions: (a) BH3xSMe2,
THF, rt, 12 h, 99%; (b) PCC, CH2Cl2, rt, 2 h, 93%; (c) NH4Cl, NaCN in water, MeOH, rt,
3 h, 25%; (d) 6 N HCl, reflux, 1 h, 100%; (e) SOCl2, MeOH, rt, 16 h, 67%; (f) TFAA,
CH2Cl2, Py, rt, 6 h, 85%; (g) Pd(PPh3)4, isopropenylboronic acid pinacol ester, 2 M
Na2CO3, THF/toluene 1:1, 80 °C, 24 h, 83%; (h) Mg, THF, DMF, 20 °C, 12 h, 60%; (i)
AcOH, NH3, NaCN, 40 °C, 6 h, 57%; (j) HCl(g), MeOH, 2 h, 60%; (k) CH2Cl2, TFAA,
pyridine, 1 h, 91%; (l) TfOH, CH2Cl2, rt, 1 h, 85%; (m) (i) 5 M NaOH, EtOH, reflux,
30 min, (ii) SOCl2, MeOH, 14 h, 83%; (n) (i) acetyl Meldrum’s acid, toluene, reflux
1 h, (ii)1 M NaOEt in EtOH, reflux 2 h; (o) PtO2, H2, 8 bar, 48 h, 73%; (p) NEt3,
benzene, reflux 48 h, 85%.
first route tend to be lower, because dihydroindazole 29 is instable
and quickly decomposes to an aromatic indazole by air oxidation.
Tetramic acid 10 was prepared from precursor 32 by a simple acyl-
ation with acetic acid using DCC as coupling reagent.27 Not com-
pletely unexpected, the hydrogenation of the acetylpyrazolo[1,2-
a]indazole 10 resulted in N–N bond cleavage. Instead, precursor
32 was hydrogenated and then acylated with acetic acid to afford
the pyrazolidinedione 11. Both pyrazolinediones were obtained
as E/Z-mixtures around the exocyclic enolic double bond.

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S. Yao et al. / Bioorg. Med. Chem. 19 (2011) 4669–4678
OH
O
a, b
NH
NH2
NH2
26
27
e
c
d
OH
NH
HO
NH
HN
f
NH
O
HN
O
N
H
28
29
O
O
O
30
f
g
O
i
h
O
O
O
O
N
N
N
84%
N
N
N
H
OH
O
32
10
31
O
O
j
H
H
H
i
60%
O
N
N
N
N
H
O
O
33
11
OH
Scheme 3. Syntheses of acylated pyrazolidinediones 10 and 11. Reagents and conditions: (a) CH3MgI, Et2O, rt, 2 h, 90%; (b) (1) NaNO2, HCl, ꢀ25 °C, 30 min, (2) SnCl2, ꢀ25 °C,
30 min; (c) ethyl malonyl chloride, DMAP, CH2Cl2, 0 °C, 1 h, 70% (for steps b and c); (d) TFA, rt, 20 min; (e) BOC2O, DMAP, 0 °C, 1 h, 66% (for steps b and e); (f) TFA, rt, 2 h; (g)
malonyl chloride, DMAP, rt, 1 h, 57% (for steps f and g); (h) NaOEt, EtOH, reflux, 6 h, 85% (for steps f and h); (i) AcOH, DCC, DMAP, Et3N; (j) PtO2, H2, 7 bar, 48 h, 42%.
2.2. SERCA activities and molecular-modelling studies
in isoCPA (17) with the consequence that the indole system now
moves upwards out of the lipophilic binding channel and approx-
imates the side-chain carboxylate-group of Asp 254 (Indol-
N. . .Asp-O: 2.8 Å), probably forming a new hydrogen bond.12 This
provides an explanation for the considerable SERCA activity of iso-
CPA 34. Compound 8 that has the same stereochemistry as isoCPA
but lacks the indole core, shows only weak SERCA inhibition (Ta-
ble 1). This is because now neither the central binding pocket
can be occupied nor is it possible to form an additional hydrogen
bond like isoCPA (Fig. 4, left).
All CPA derivatives were tested for Heliothis virescens SERCA
activity using a luciferin/luciferase-coupled assay that measures
the ATP-level within the reaction. ATP-consumption of the ATPase
enzyme results in reduced ATP levels for the subsequent luciferase
assay and consequently a loss of luminescence-signal is observed.
Dose-response assays allowed the determination of IC50 values for
each inhibitor (Table 1). Since no experimental protein structure of
H. virescens SERCA is available a homology model was constructed
from the structure of rabbit SERCA complexed with CPA.12,28 Since
chelation of Mg2+ by the tetramic acid plays a pivotal role in CPA
binding all CPA analogues were superimposed with their tetramic
acid moieties.
The simple linear tetramic acid 6 lacking the indole system and
chirality in the corresponding CPA positions 6a and 11a shows an
unexpected SERCA inhibition (IC50 6.3 lM) in the range of all-trans-
CPA 35 and Speradine A (36). A plausible explanation is that the
six-membered piperidine ring directs the phenyl system straight
into the lipophilic binding-niche fixing tetramic acid 6 in a position
very similar to that of BHQ (Fig. 4, middle). A consensus orientation
of BHQ obtained from docking into the SERCA structure has been
published recently.14,29 A superimposition of CPA and dihydro iso-
quinoline 6 onto their tetramic acid moieties together with BHQ in
its published binding pocket orientation convincingly confirms the
similarity between these structures. Both the tert-butyl group of
BHQ and the phenyl ring of the linear tetramic acid 6 cover exactly
the same position as the aromatic ring A of CPA (Fig. 4, middle).
This model also explains the unexpected inactivity of tetramic acid
7 without any difficulties. The five-membered pyrrolidine ring
causes an angulated shape of dihydro isoindole 7 with the conse-
quence that the phenyl ring is now shifted out of the lipophilic
binding niche towards the pyrrol ring B in CPA (Fig. 4, right).
The very low activity (Table 1) of the pyrazolidinediones 10 and
11, however, is difficult to interpret with our model since steric
and electronic properties have been changed by the exchange of
It has already been shown that epimerization of the 11a posi-
tion (isoCPA 34) exerts a minor effect on SERCA inhibition than epi-
merization at position 6a (all-transCPA 35), which results in a
decline of activity by a factor of 500 compared to CPA (1).12 CPA-
precursor 37 is completely inactive and thus confirms that the tet-
ramic acid is indispensable for SERCA inhibition (Table 1). Remark-
ably, speradine A (36) still shows some SERCA inhibition even
though the indole system has been changed considerably by the
introduction of an additional stereogenic center in position 11c,
oxidation of position 1 to a carbonyl group, and methylation of
the indole nitrogen. This again points to a certain structural vari-
ability of the indole system.
Most notable, a pronounced SERCA activity (IC50 0.63 lM) was
found for the tricyclic tetramic acid 9, which is identical with the
C,D,E-ring fragment of CPA (Fig. 4, left). Obviously, the complete
indole system does not contribute to the overall SERCA activity
more than the stereochemistry in position 11a of CPA. The inver-
sion of the configuration in position 11 induces a bowl-like shape

S. Yao et al. / Bioorg. Med. Chem. 19 (2011) 4669–4678
4673
Table 1
For instance, ring C opened CPA analogues containing an indole
ring, which is linked by an acyclic spacer of two or three atoms
to the pyrrolidine core, should reach the lipophilic binding niche
without any problems. Starting from fragment 8 with the iso-ste-
reochemistry, the introduction of polar substituents on ring A
should allow the formation of additional hydrogen bonds to Asp
254 and nearby amino acids located above the plane of CPA ring C.
SERCA (Heliothis virescens) activities of CPA, model compounds and related structures
O
OH
O
OH
O
OH
N
N
N
H
H
H
O
O
O
H
H
H
H
H
H
4. Experimental
4.1. General
NH
α-Cyclopiazonic acid (1)
NH
isoCPA (34)
OH
NH
all-transCPA (35)
O
Solvents and reagents were purchased from commercial
sources. Triethylamine and diisopropylethylamine were dried over
CaH2 and distilled prior to use. Methanol was distilled from mag-
nesium. Chloroform and dichloromethane were dried by passing
the solvents through a basic aluminium oxide filled column.
NH
N
H
H
CO2Me
H
O
H
H
H
H
O
Precoated plates (Silica Gel 60 F254, 250 lm, Merck, Darmstadt,
N
N
H
Germany) were used for thin layer chromatography (TLC). Silica
gel 0.04–0.063 mm from Macherey–Nagel was used for chromato-
graphic purification. HPLC–MS was performed on a Varian 500 Ion-
Speradine A (36)
CPA-Precursor (37)
Trap LC–ESI-MS system or on a Bruker MicroTOF (column: 5 lm
RP18) with ACN/H2O gradient 10/90 to 90/10. High-resolution
MS were measured on a Bruker MicroTOF. Semipreparative HPLC
Compound
SERCA (IC50, lM)
was either performed on
a
Kromasil 100 (C18
5 lm,
BHQ
0.13 0.013
0.01 0.005
0.50 0.1
5.0 0.5
8.03
>50
6.3 0.5
>50
CPA (1)
250 ꢁ 20 mm) or on a Zorbax ODS (250 ꢁ 21.2 mm) column with
a Gilson Abimed System. Analytical 1H and 13C NMR spectra were
recorded at 25 °C on a BRUKER AVANCE 400 (400.13 MHz for 1H
NMR, 100.62 MHz for 13C NMR) or BRUKER AVANCE 600
(600.13 MHz for 1H NMR, 150.90 MHz for 13C NMR) spectrometer
using tetramethylsilane as an internal standard. Spectra are re-
ported in units of ppm and coupling constants are given in Hz.
Due to tautomeric- and E/Z-equilibria caused by the tetramic acid
(ring E) in particular the proton NMR signals were considerably
broadened. This made a complete assignment impossible for some
CPA analogues. Melting points (mp) were determined with a Bue-
chi 535 melting point apparatus and are uncorrected. IR-spectra
were measured on a Nicolet Protégé 460 Spectrometer E.S.P.
isoCPA (34)12
all-transCPA (35)
Speradine A (36)
CPA precursor (37)
6
7
8
9
10
11
19.9
0.63 0.04
40.0
40.0 10
4
9
a chiral carbon atom by a nitrogen. Most probably, also Mg binding
is affected by the hydrazide group introduced in 10 and 11.
4.2. Inhibition assay of SERCA activity
3. Summary and conclusion
Sf9 cells (Invitrogen) were maintained under serum-free condi-
tions at 27 °C with Sf-900 II SFM according to the manufacturers’
manual. For expression, Sf9 cells were coinfected with baculovirus
of the H. virescens full-length SERCA (aa 2-1000, Accession No.
AF115572) with a MOI of 0.3. After three days cells were harvested
and microsomal membranes prepared according to standard
procedures and stored at ꢀ80 °C.30 A typical inhibition assay
contained varying concentrations of the inhibitor (from 3.2 nM to
Six new CPA core structures have been prepared in order to elu-
cidate the role of the indole moiety and the stereochemistry of the
ring conjunctions. In particular for the hitherto unknown tricyclic
hydrazides 10 and 11 a new synthesis was developed. CPA frag-
ment 9, consisting of the CPA rings C, D, and E still shows a remark-
able SERCA inhibition and proves that the indole system is of minor
importance for SERCA inhibition. The unexpected activity of dihy-
dro isoquinoline 6 can be attributed to an orientation in the bind-
ing site of SERCA similar to BHQ. On the other hand, the phenyl
ring of the inactive dihydro isoindole 7 is directed towards the pyr-
role ring B of CPA causing unfavourable interactions with the bind-
ing site. The structure-activity data together with molecular
modelling studies provide promising suggestions for future work.
50 lM final concentration) and 0.4 lg/mL recombinant enzyme
together with 0.05 mM ATP in the reaction buffer (45 mM MOPS
pH 7.5, 5 mM MgSO4, 1 mM KCl, 0.41 mM CaCl2, 0.4 mM EGTA,
5% (w/v) gylerol, 0.01% (w/v) Tween 20, 0.05 mg/mL BSA and
0.002 mM A23187) in a microtiter plate. After 60 min incubation
at room temperature, the ATP depletion was detected by a
Figure 4. Superimpositions of CPA with compounds 8 and 9 (left), BHQ and 6 (middle), 6 and 7 (right). Color scheme: CPA, grey; BHQ, blue; 6, green; 7, orange; 8, yellow; 9,
brown.

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S. Yao et al. / Bioorg. Med. Chem. 19 (2011) 4669–4678
subsequent luciferase reaction employing 193.5 nM luciferin (Bio-
Synth), 38.29 ng/mL luciferase (Promega) and 111 nM coenzyme A.
Luminescence signal was detected on a Tecan infinity M1000 with
an integration time of 100 ms. Assays were performed in quadru-
plicates and after normalization data were fitted to a sigmoid
equation to determine IC50 values using Graph Pad Prism software
(Graph Pad Prim Inc., San Diego, USA). In order to exclude direct
luciferase inhibition, a luciferase-assay lacking the SERCA enzyme
was performed with all observed inhibitors.
to 0 °C, triflic acid (0.153 mL, 1.72 mmol) was added at 0 °C and
the mixture was stirred for 6 h without further cooling. Then the
reaction was quenched by addition of saturated NaHCO3 solution.
The phases were separated and the aqueous layer was extracted
with dichloromethane (2 ꢁ 20 mL). The combined organic phases
were dried with Na2SO4, filtered and evaporated. The crude prod-
uct was purified by column chromatography (cyclohexane–ethyl
acetate 10:1) to yield 15 (340.0 mg, 63%) as an oil. IR (film):
1694, 1756 cmꢀ1 1H NMR (400 MHz, CDCl3): d 1.72 (s, 3H), 2.08
;
(s, 3H), 3.21 (dd, J = 15.6, 4.4 Hz, 1H), 3.37 (dd, J = 15.6, 2.8 Hz,
1H), 3.51 (s, 3H), 5.00 (m, 1H), 7.11 (d, J = 7.1 Hz, 1H), 7.18 (t,
J = 7.4 Hz, 1H), 7.28 (t, J = 7.6 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H); 13C
NMR (100 MHz, CDCl3): d 23.3, 30.6, 32.4, 52.5, 55.7, 62.6, 116.4,
124.8, 126.8, 128.0, 128.1, 129.8, 143.1, 156.1, 170.0; 19F NMR
(376 MHz, CDCl3): d ꢀ69.1; MS (ESI): m/z (%) 284 (31), 316
([M+H]+, 100); HR MS (ESI): calcd for C15H16F3NNaO3 ([M+Na]+):
338.0974, found 338.0973.
4.3. rac-2-(2,2,2-Trifluoroacetylamino)-3-(2-iodo-phenyl)-pro-
pionic acid methyl ester (13)
Bis(pyridine)iodonium(I) tetrafluoroborate was prepared
according to the method developed by Barluenga et al.19,20 Trifluo-
roacetyl protected phenylalanine 12 (2.0 g, 7.27 mmol) was dis-
solved in a mixture of 1450 mL CH2Cl2 and 145 mL TFA in a 2 L
three-necked round-bottom flask. To this solution HBF4 (3.05 mL,
21.8 mmol) was added, followed by addition of IPy2BF4 (4.06 g,
10.90 mmol) in several small portions. After stirring overnight at
40 °C under exclusion of light the reaction was stopped by addition
of water. The organic phase was washed with a 5% Na2S2O3 solu-
tion and two times with water. The organic phase was dried over
Na2SO4, filtrated and evaporated. The pale yellow crude product
was purified by column chromatography (cyclohexane–ethyl ace-
tate 8.5:1.5) to afford 13 (2.691 g, 92%, ortho:para 10:1) as white
crystals. Mp 108–110 °C (lit.19 110–111 °C); IR (KBr): 1749,
4.6. rac-1,1-Dimethyl-1,2,3,4-tetrahydro-isoquinoline-3-car-
boxylic acid methyl ester (16)
Five molars of aqueous NaOH (5.8 mL) were added to a solution
of tetrahydroisoquinoline 15 (276.0 mg, 0.87 mmol) in ethanol
(20 mL). The reaction mixture was heated to reflux for 12 h, cooled
down and concentrated in vacuo. The residue was dissolved in a
small amount of ethanol and filtered through a short silica gel col-
umn. The filtrate was concentrated in vacuo. The residue (175 mg,
89%) was dissolved in methanol (5 mL) and thionyl chloride
(0.32 mL, 4.36 mmol) was added dropwise at 0 °C. The reaction
mixture was stirred at room temperature for 12 h and then evapo-
rated. The residue was dissolved in CH2Cl2 (30 mL) and washed
successively with 1 M aqueous K2CO3 solution and water. The
aqueous phases were combined and extracted two times with
dichloromethane. The combined organic layers were dried with
Na2SO4 and evaporated. Flash chromatography (cyclohexane–ethyl
acetate 3:2) provided compound 16 (110 mg, 69%) as an oil. IR
3310 cmꢀ1
; d 3.22 (dd, J = 14.1,
1H NMR (400 MHz, CDCl3):
8.4 Hz, 1H), 3.41 (dd, J = 14.1, 6.1 Hz, 1H), 3.78 (s, 3H), 4.95 (dt,
J = 8.1 Hz, 1H), 6.85 (s, br, 1H), 6.96 (dt, J = 7.6, 1.5 Hz, 1H), 7.17
(dd, J = 7.6, 1.5 Hz, 1H), 7.30 (t, J = 7.4 Hz, 1H), 7.85 (d, J = 8.1 Hz,
1H); 13C NMR (100 MHz, CDCl3): d 42.3, 52.9, 53.0, 100.9, 115.5,
128.6, 129.3, 130.3, 138.2, 140.0, 156.6, 170.4; 19F NMR
(376 MHz, CDCl3): d ꢀ76.3; MS (ESI): m/z (%) 342 (31), 402
([M+H]+, 90), 419 (100). Anal. Calcd for C12H11F3INO3: C, 35.93;
H, 2.76. Found: C, 35.49; H, 2.59.
(film): 1743, 3331 cmꢀ1 1H NMR (400 MHz, CDCl3): d 1.44 (s,
;
4.4. rac-2-(2,2,2-Trifluoroacetylamino)-3-(2-isopropenyl-phenyl)-
propionic acid methyl ester (14)
3H), 1.53 (s, 3H), 1.72 (br s, 1H), 2.91 (dd, J = 16.2, 11.5 Hz, 1H),
3.04 (dd, J = 16.2, 4.1 Hz, 1H), 3.79 (s, 3H), 3.89 (dd, J = 11.5,
3.9 Hz, 1H), 7.07 (d, J = 7.1 Hz, 1H), 7.12 (dt, J = 7.1, 1.5 Hz, 1H),
7.18 (t, J = 7.4 Hz, 1H), 7.22 (d, J = 7.6 Hz, 1H); 13C NMR
(100 MHz, CDCl3): d 30.9, 31.8, 33.4, 51.9, 52.2, 53.9, 125.7,
126.0, 126.4, 129.2, 132.4, 143.3, 173.8; MS (ESI): m/z (%) 220
([M+H]+, 100); HR MS (ESI) calcd for C13H18NO2 ([M+H]+):
220.1332, found 220.1334.
Phenyliodine 13 (1.210 g, 3.02 mmol) and Pd(PPh3)4 (348 mg,
0.30 mmol) were dissolved in THF/toluene 1:1 (20 mL) in a Schlenk
flask and carefully degassed with Helium, followed by isopropenyl
boronicacid pinacol ester (1.20 mL, 6.04 mmol) and 2 M Na2CO3
(3.02 mL, 6.04 mmol; degassed). The reaction was stirred for 48 h
at 80 °C. After cooling down to room temperature the two layers
were separated and the aqueous layer was extracted with CH2Cl2.
The combined organic layers were dried with Na2SO4, filtrated and
evaporated. The crude product was purified by column chromatog-
raphy (cyclohexane–ethanol 95:5) to yield 14 (520.0 mg, 55%) as
4.7. rac-2-[1-Hydroxy-ethylidene]-5,5-dimethyl-10,10a-
dihydro-5H-pyrrolo[1,2-b]isoquinoline-1,3-dione (6)
Acetyl Meldrum’s acid was prepared as described by
Crimmins31 and recrystallized from cyclohexane. Acetyl Meldrum’s
acid (269 mg, 1.44 mmol) was added to a solution of isoquinoline
16 (158 mg, 0.72 mmol) in freshly dried toluene (5 mL). The reac-
tion was heated to 110 °C and monitored by TLC every 15 min until
the starting material was consumed completely. Then KOtBu
(122 mg, 1.08 mmol) was added and the mixture refluxed for an-
other 2 h. After evaporation of the solvent, the residue was dis-
solved in methanol (2 mL) and the pH was adjusted to 1 by slow
addition of 1 M HCl. The mixture was extracted with dichloro-
methane twice, the combined organic layers were dried with
Na2SO4 and the solvents were removed under reduced pressure.
Column chromatography of the crude product (ethyl acetate–EtOH
9:1) yielded tetramic acid 6 (131 mg, 67%) as an oil. IR (film): 3419,
colorless crystals. Mp 68–70 °C; IR (KBr): 1709, 1752, 3311 cmꢀ1
;
1H NMR (400 MHz, CDCl3): d 2.06 (m, J = 1.0, 1.5 Hz, 3H), 3.08
(dd, J = 14.0, 8.4 Hz, 1H), 3.30 (dd, J = 14.0, 5.9 Hz, 1H), 3.73 (s,
3H), 4.84 (dt, J = 8.1, 1H), 4.92 (dt, J = 1.0 Hz, 1H), 5.28 (dt, J = 1.5,
1H), 6.70 (br s, 1H), 7.13 (m, 2H), 7.22 (m, 2H); 13C NMR
(100 MHz, CDCl3): d 25.0, 35.1, 52.7, 53.6, 115.5, 116.3, 127.3,
127.5, 128.8, 129.8, 131.4, 144.4, 145.3, 156.7, 170.8; 19F NMR
(376 MHz, CDCl3): d ꢀ76.3; MS (ESI): m/z (%) 143 (18), 284 (39),
316 ([M+H]+, 100), 333 ([M+NH4]+, 49). Anal. Calcd for
C15H16F3NO3: C, 57.14; H, 5.12. Found: C, 57.00; H, 5.03.
4.5. rac-1,1-Dimethyl-2-(2,2,2-trifluoro-acetyl)-1,2,3,4-tetra-
hydro-isoquinoline-3-carboxylic acid methyl ester (15)
1601, 1661, 879 cmꢀ1 1H NMR (400 MHz, CDCl3): d 1.79 (s, 3H),
;
Isopropenyl phenylalanine 14 (541.0 mg, 1.72 mmol) was dis-
solved in dry chloroform (50 mL). The solution was cooled down
1.84/1.87 (s, 3H), 2.43 (br s, 3H), 2.62 (t, J = 13.7 Hz, 1H), 3.17
(dd, J = 14.8, 2.5 Hz, 1H), 3.68 (m, 1H), 7.18 (m, 2H), 7.26 (t,

S. Yao et al. / Bioorg. Med. Chem. 19 (2011) 4669–4678
4675
J = 6.1 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H); 13C NMR (100 MHz, CDCl3):
d 26.7, 27.5, 31.5, 33.2, 33.5, 58.6, 58.8, 60.8, 61.3, 103.84, 126.4,
127.3, 127.4, 128.1, 128.3, 129.9, 133.9, 134.1, 145.5, 146.0,
174.5, 175.2, 195.3, 195.8; MS (ESI): m/z (%) 272.1 ([M+H]+, 100);
HR MS (ESI) calcd for C16H16NO3 ([MꢀH]ꢀ): 270.1136, found
270.1137.
was quenched with water (300 mL) after 24 h at 40 °C and ex-
tracted with dichloromethane (3 ꢁ 100 mL). The organic phase
was dried over Na2SO4, concentrated and purified by column chro-
matography (cyclohexane–ethyl acetate 4:1) to yield amino-(2-
isopropenyl-phenyl)-acetonitrile (21) (21.0 g, 57%) as a yellow
oil. IR (film): 1642, 3340 cmꢀ1 1H NMR (400 MHz, CDCl3): d 2.13
;
(s, 3H), 5.18 (s, 1H), 5.00 and 5.34 (br s, each 1H), 7.22 (dd,
J = 2.4, 7.6 Hz, 1H), 7.37 (m, 2H), 7.72 (dd, J = 1.0, 7.6 Hz, 1H),
7.66 (dd, J = 1.5, 9.0 Hz, 1H); 13C NMR (100 MHz, CDCl3): d 25.4,
44.4, 116.7, 121.6, 126.9, 128.1, 128.6, 128.9, 133.8, 140.3, 143.8;
MS (ESI) m/z (%):173.1 ([M+H]+, 34), 156.1 (100); HR MS (ESI):
calcd for C11H13N2 ([M+H]+): 173.1073, found 173.1072.
4.8. rac-(2-Iodo-phenyl)-2,2,2-trifluoroacetylamino-acetic acid
methyl ester (19)
2-Amino-2-(20-iodophenyl) acetic acid hydrochloride, prepared
from 18 according to the method described by Steiger,32 was dis-
solved in MeOH (50 mL), cooled to 0 °C and thionyl chloride
(10 mL, 137.68 mmol) was added dropwise. After stirring 16 h at
room temperature the solvents were removed in vacuo. The resi-
due was dissolved in dichloromethane (30 mL), washed with 1 M
NaHCO3 (3 ꢁ 30 mL) and brine (50 mL). The organic layer was
dried with Na2SO4 and filtered. Chromatographic purification
(cyclohexane–ethyl acetate 1:1) of the residue obtained after evap-
oration of the solvent gave 2-amino-2-(20-iodophenyl) acetic acid
methyl ester (4.6 g, 67%) as a yellow amorphous powder. IR
4.10. rac-(2-Isopropenyl-phenyl)-(2,2,2-trifluoro-acetylamino)-
acetic acid methyl ester (22)
Aminoacetonitrile 21 (8.0 g, 46.45 mmol) was dissolved in
MeOH (630 mL, c = 0.1 M) and cooled to 0 °C. This solution was sat-
urated with HCl gas and stirred for 6 h at 0 °C. Then HCl and meth-
anol were removed in vacuo. The residue was dissolved in
dichloromethane (500 mL) and the solution was washed with sat-
urated Na2CO3 solution (3 ꢁ 250 mL). The aqueous layer was ex-
tracted with dichloromethane (3 ꢁ 250 mL), the combined
organic phases were dried with Na2SO4 and evaporated to yield
pure amino-(2-isopropenyl-phenyl)-acetic acid methyl ester (6 g,
(KBr): 1685, 3340 cmꢀ1 1H NMR (600 MHz, CDCl3): d 3.70 (s,
;
3H), 4.94 (s, 1H), 6.97 (ddd, J = 1.2, 7.4, 7.8 Hz, 1H), 7.30 (m, 2H),
7.85 (d, J = 7.8 Hz, 1H); 13C NMR (150 MHz, CDCl3): d 52.4, 62.6,
99.8, 127.4, 128.8, 129.6, 139.9, 143.2, 173.8; MS (ESI): m/z (%)
291.1 ([M+H]+, 100).
60%) as a brownish oil. IR (film): 1725, 3340, 3220 cmꢀ1 1H
;
Pyridine (7.8 mL, 96.83 mmol) was added to 2-amino-2-(20-
iodophenyl) acetic acid methyl ester (4.6 g, 15.80 mmol) in dichlo-
romethane (100 mL) followed by trifluoroacetic anhydride
(11.00 mL, 79.00 mmol). The reaction was stirred 12 h at room
temperature and then quenched with ice-water. The aqueous
phase was extracted with dichloromethane (2 ꢁ 100 mL). The
combined organic phases were washed with water, dried with
Na2SO4 and then evaporated. The crude product was purified on
a silica gel column (cyclohexane–dichloromethane 1:1) to yield
compound 19 (5.2 g, 85%) as a white amorphous powder. IR
NMR (600 MHz, CD3OD): d 2.15 (s, 3H), 3.79 (s, 3H), 5.05 and
5.43 (br s, each 1H), 5.45 (s, 1H), 7.35 (d, J = 7.8 Hz, 1H), 7.40 (d,
J = 7.8 Hz, 1H), 7.42 (m, 2H); 13C NMR (150 MHz, CD3OD): d 25.7,
53.9, 54.1, 118.0, 127.6, 129.3, 130.0, 130.1, 131.2, 145.1, 146.3,
170.4; MS (ESI) m/z (%): 206.1 ([M+H]+, 100); HR MS (ESI): calcd
for C12H16NO2 ([M+H]+): 206.1176, found 206.1175.
Trifluoroacetic anhydride (4.30 mL, 30.35 mmol) was added
dropwise at 0 °C to a solution of 2-amino-2-(20-isopropenylphenyl)
acetic acid methyl ester (6.23 g, 30.35 mmol) and pyridine (4.7 mL,
58.34 mmol) in dichloromethane (100 mL). The reaction mixture
was stirred for 1 h at room temperature, quenched with ice-water
(200 mL) and extracted with dichloromethane (2 ꢁ 100 mL). The
combined organic phases were dried with Na2SO4 and evaporated.
Column chromatography (cyclohexane–ethyl acetate 9:1) of the
residue afforded trifluoroacetate 22 (8.0 g, 91%) as an off-white
(KBr): 1721, 1746, 3343 cmꢀ1 1H NMR (400 MHz, CDCl3): d 3.79
;
(s, 3H), 5.85 (d, J = 6.7 Hz, 1H), 7.06 (ddd, J = 1.1, 7.4, 7.6 Hz, 1H),
7.27 (ddd, J = 7.6, 7.8, 1.7 Hz, 1H), 7.38 (dd, J = 7.4, 1.7 Hz, 1H),
7.90 (dd, J = 7.8, 1.1 Hz, 1H); 13C NMR (100 MHz, CDCl3): d 53.5,
60.6, 99.1, 115.5, 128.7, 128.8, 130.7, 137.7, 140.6, 156.3, 169.4;
19F NMR (376 MHz, CDCl3): d ꢀ76.2; MS (ESI): m/z (%) 388.1
([M+H]+, 100); HR MS (ESI): calcd for C11H9F3NNaO3([M+Na]+):
409.9471, found 409.9472.
amorphous powder. IR (KBr): 1754, 3333 cmꢀ1
;
1H NMR
(400 MHz, CDCl3): d 2.14 (s, 3H), 3.74 (s, 3H), 5.03 and 5.36 (br s,
each 1H), 5.94 (d, J = 6.8 Hz, 1H), 7.22–7.35 (m, 4H); 13C NMR
(100 MHz, CDCl3): d 25.2, 53.2, 53.4, 114.2, 117.1, 126.3, 127.8,
128.8, 128.9, 131.6, 143.5, 144.5, 156.1, 170.5; 19F NMR
(376 MHz, CDCl3): d ꢀ76.3; MS (ESI): m/z (%) 242.1 (100), 302.1
([M+H]+, 35); HR MS (ESI): calcd for C14H14F3NNaO3 ([M+Na]+):
324.0818, found 324.0814.
4.9. rac-Amino-(2-isopropenyl-phenyl)-acetonitrile (21)
At 20 °C DMF (57 mL, 0.74 mol) was added to a solution of the
Grignard reagent obtained from 1-bromo-2-isopropenyl-benzene
20 (73.0 g, 0.37 mol) and Mg turnings (11.0 g, 0.44 mol) in THF
(450 mL).33 The reaction mixture was stirred overnight at room
temperature then poured into of 3 N aq HCl (500 mL) and extracted
with dichloromethane (2 ꢁ 250 mL). The combined organic phases
were washed with a saturated NaHCO3 solution, water and brine.
After drying over Na2SO4 and evaporation of the solvent, pure 2-
isopropenyl-benzaldehyde (32.0 g, 60%) was obtained by Kugel-
4.11. rac-3,3-Dimethyl-2,3-dihydro-1H-isoindol-1-carboxylic
acid methyl ester (23)
Triflic acid (2.54 mL, 28.80 mmol) was added at 0 °C to a solu-
tion of ester 22 (8.7 g, 28.80 mmol) in chloroform (50 mL). The
reaction was stirred without further cooling for 1 h. Then a 10% so-
dium carbonate solution (50 mL) was added, the phases were sep-
arated and the aqueous layer was extracted with dichloromethane
(3 ꢁ 25 mL). The combined organic phases were dried with Na2SO4,
filtered and evaporated. Column chromatography (cyclohexane–
ethyl acetate 10:1) afforded 3,3-dimethyl-2-(2,2,2-trifluoroace-
tyl)-2,3-dihydro-1H-isoindol-1-carboxylic acid methyl ester
(7.4 g, 85%) as a white amorphous powder. IR (KBr): 1701, 1758,
rohr distillation as a colorless oil. IR (film): 1720, 2820 cmꢀ1 1H
;
NMR (400 MHz, CDCl3): d 2.20 (s, 3H), 4.93 and 5.45 (s, each 1H),
7.36 (dd, J = 0.8, 7.6 Hz, 1H), 7.41 (ddd, J = 0.8, 7.5, 7.8, Hz, 1H),
7.57 (ddd, J = 1.1, 7.5, 7.6 Hz, 1H), 7.94 (dd, J = 1.1, 7.8 Hz, 1H),
10.2 (s, 1H); 13C NMR (100 MHz, CDCl3): d 25.0, 118.9, 127.5,
127.8, 128.6, 133.3, 140.4, 141.9, 148.0, 192.5; MS (EI) m/z (%):
146 ([M]+, 38), 131 (100).
2-Isopropenyl-benzaldehyde (32.0 g, 0.22 mol) was added to an
ice-cooled solution of aqueous ammonia (80 mL, 25% w/v), acetic
acid (37.6 mL, 0.66 mol) and NaCN (13.1 g, 0.26 mol). The reaction
2958, 2934 cmꢀ1 1H NMR (400 MHz, CDCl3): d 1.82 (s, 3H), 1.87
;
(s, 3H), 3.77 (s, 3H), 5.83 (br s, 1H), 7.16 - 7.41 (m, 4H); 13C NMR
(100 MHz, CDCl3): d 26.6, 26.7, 53.1, 65.2, 72.0, 115.7, 121.3,

4676
S. Yao et al. / Bioorg. Med. Chem. 19 (2011) 4669–4678
122.9, 128.4, 129.68, 131.5, 145.8, 155.7, 170.1; 19F NMR
(376 MHz, CDCl3): d -72.1; MS (ESI): m/z (%) 302.1 ([M+H]+, 100);
HR MS (ESI): calcd for C14H14F3NNaO3 ([M+Na]+): 324.0818, found
324.0817.
1H), 1.22 and 1.16 (s, each 3H); 13C NMR (150 MHz, CDCl3): d
21.1, 24.8, 24.9, 25.3, 29.7, 30.4, 42.2, 48.2, 51.8, 60.6, 62.6,
175.6; MS (ESI): m/z (%): 212.1 ([M+H]+, 100); HR MS (ESI): calcd
for C12H22NO2 ([M+H]+): 212.1645, found 212.1647.
3,3-Dimethyl-2-(2,2,2-trifluoroacetyl)-2,3-dihydro-1H-isoin-
dol-1-carboxylic acid methyl ester (2.3 g, 7.60 mmol) was dis-
solved in ethanol (30 mL), 5 M aqueous NaOH (14 mL) was added
and the reaction mixture was heated to reflux for 30 min. After
evaporation of the solvent the residue was stirred with silica gel
(20 g) in ethanol, filtered. The filtrate was concentrated in vacuo.
Thionyl chloride (2.7 mL, 38.30 mmol) was added at 0 °C to a solu-
tion of the residue (1.62 g, 7.60 mmol) in methanol and the reac-
tion mixture was stirred at room temperature for 14 h. The
excess methanol and thionyl chloride were removed by evapora-
tion. The residue was dissolved in dichloromethane (30 mL) and
washed successively with 1 M aqueous Na2CO3 (25 mL) and then
with water (25 mL). The aqueous phases were combined, extracted
with dichloromethane (3 ꢁ 50 mL). And the combined organic lay-
ers were dried with Na2SO4, evaporated and purified by flash chro-
matography (dichloromethane–acetone 10:1) to provide of the
isoindole 23 (1.3 g, 83%) as a colorless amorphous powder. IR
4.14. rac-2-(1-Hydroxyethylidene)-5,5-dimethyl-5,9b-dihydro-
1H-pyrrolo[2,1-a]isoindole-1,3(2H)-dione (7)
Acetyl Meldrum’s acid (92 mg, 0.48 mmol) was added to dihy-
dro-isoindole 23 (50 mg, 0.24 mmol) in toluene (2 mL). The reac-
tion mixture was heated to 80 °C and monitored every 15 min
until complete consumption of the starting material. Then the sol-
vent was evaporated and the residue was dried in high vacuum for
12 h. A 0.5 M sodium ethoxide solution (1 mL, 0.50 mmol) was
added to the raw material (68 mg, 0.23 mmol) of step one in EtOH
(2 mL) at 0 °C. The reaction was refluxed for 2 h, the solvent evap-
orated after that time and the residue dissolved in methanol
(2 mL). Aq HCl (1 M) was added until pH 1. The solution was ex-
tracted with dichloromethane (3 ꢁ 25 mL), the organic phase was
dried with Na2SO4, filtered and concentrated in vacuo. Chromato-
graphic purification of the crude product (ethyl acetate–EtOH
100:8) afforded tetramic acid 7 (40.0 mg, 71%) as a white amor-
(KBr): 1735, 2959, 3448 cmꢀ1 1H NMR (400 MHz, CDCl3): d 1.41
;
(s, 3H), 1.54 (s, 3H), 2.62 (br s, 1H), 3.82 (s, 3H), 5.02 (s, 1H), 7.16
(d, J = 7.5 Hz, 1H), 7.26 (t, J = 7.3, 7.5 Hz, 1H), 7.33 (t, J = 7.3,
7.5 Hz, 1H), 7.44 (d, J = 7.5 Hz, 1H); 13C NMR (100 MHz, CDCl3): d
30.1, 31.0, 52.5, 64.1, 64.3, 121.5, 123.1, 127.1, 128.3, 137.4,
149.7, 173.3; MS (ESI): m/z (%) 206.1 ([M+H]+, 100); HR MS (ESI)
calcd. for C12H16NO2 ([M+H]+): 206.1176, found 206.1177.
phous powder. IR (KBr): 1615, 2965, 3432 cmꢀ1 1H NMR
;
(400 MHz, CDCl3): d 1.19/1.20 (s, 3H), 2.12/2.13 (s, 3H), 2.57/2.58
(s, 3H), 4.94 (s, 1H), 7.19–7.65 (m, 4H); 13C NMR (100 MHz, CDCl3):
d 23.3/25.1, 26.7/28.9, 31.6/32.2, 66.2, 69.4, 119.3, 120.9, 122.0,
127.4, 128.0, 136.2, 148.3, 159.6, 178.2,195.3; MS (ESI): m/z (%)
258.1 ([M+H]+, 100); HR MS (ESI): calcd for C15H14NO3 ([MꢀH]ꢀ):
256.0979, found 256.0978.
4.12. rac-(1R,3aR,7aS)-3,3-Dimethyl-octahydro-isoindole-1-
carboxylic acid methyl ester (24)
4.15. rac-(5aR,9aS,9bR)-2-(1-Hydroxyethylidene)-5,5-
dimethyloctahydro-1H-pyrrolo[2,1-a]isoindole-1,3(2H)-dione
Ester 23 (300 mg, 1.46 mmol) was dissolved in ethanol (5 mL),
platinum(IV) oxide (95%) (16.5 mg, 0.07 mmol) was added and
the reaction was hydrogenated (8 bar) for 48 h at room tempera-
ture. After that time, the mixture was filtrated, the catalyst was
washed with ethanol (containing 5% acetic acid) and the combined
solutions were evaporated. The residue was dissolved in dichloro-
methane (20 mL) and washed with saturated NaHCO3 solution. The
aqueous layer was extracted with dichloromethane (3 ꢁ 25 mL).
The combined organic phases were dried with Na2SO4 and evapo-
rated to yield a raw material (280 mg) which was purified by col-
umn chromatography (cyclohexane–ethyl acetate 7:3) to yield
reisolated starting material 23 (110 mg, 37%) and octahydro-isoin-
dole 24 (130.0 mg, 45%) as a colorless oil. IR (film): 1678,
(8)
A solution of octahydro-isoindole 24 (67 mg, 0.32 mmol) and
acetyl Meldrum’s acid (118 mg, 0.63 mmol, freshly prepared) in
toluene (5 mL) was refluxed for 2 h, cooled to room temperature
and evaporated. The residue was dried in high vacuum for 12 h,
dissolved in 1 M NaOEt in EtOH (1 mL) and refluxed for 2 h at
80 °C. After cooling to room temperature the reaction was poured
in 2 M HCl solution (20 mL) and extracted with dichloromethane
(3 ꢁ 25 mL). The combined organic phases were dried with Na2SO4,
filtered and evaporated. Pure tetramic acid 8 (52.0 mg, 61%) was
obtained after reversed phase column chromatography (acetoni-
trile-water 6:4) as a white amorphous powder. IR (KBr): 1572,
3320 cmꢀ1
;
1H NMR (600 MHz, CDCl3): d 3.83 (d, J = 9.3 Hz, 1H),
1682, 3320 cmꢀ1 1H NMR (600 MHz, acetonitril-d3): d 4.24 (d,
;
3.76 (s, 3H), 2.87 (br s, 1H), 2.74 (m, 1H), 1.73 (m, 1H), 1.59 (m,
2H), 1.59 and 1.23 (m, 1H), 1.73 and 1.52 (m, each 1H), 1.34 and
1.16 (m, each 1H), 1.24 and 1.16 (s, each 3H); 13C NMR
(150 MHz, CDCl3): d22.3, 23.5, 23.7, 24.0, 25.7, 29.9, 41.7, 47.2,
61.4, 61.5, 51.8, 175.2; MS (ESI): m/z (%): 212.1 ([M+H]+, 100), HR
J = 6 Hz, 1H), 2.36 (s, 3H), 2.35 (m, 1H), 2.30 (m, 1H), 1.73 (m,
2H), 1.66 and 1.24 (m, each 1H), 1.67 (s, 3H), 1.54 and 1.04 (m, each
1H), 1.48–1.45 (m, 2H), 1.39 (s, 3H); 13C NMR (150 MHz, acetoni-
tril-d3): d 19.6, 22.2, 22.7, 24.3, 24.4, 24.6, 30.8, 39.5, 51.0, 63.6,
71.7, 106.8, 174.2, 184.4, 194.7; MS (ESI): m/z (%): 264.2 ([M+H]+,
100); HR MS (ESI): calcd. for C15H22NO3 ([M+H]+): 264.1594, found
264.1596.
MS (ESI): calcd. for
212.1647.
C
12H22NO2 ([M+H]+): 212.1645, found
4.13. rac-(1S,3aR,7aS)-3,3-Dimethyl-octahydro-isoindole-1-
4.16. rac-(5aR,9aS,9bS)-2-(1-Hydroxyethylidene)-5,5-
carboxylic acid methyl ester (25)
dimethyloctahydro-1H-pyrrolo[2,1-a]isoindole-1,3(2H)-dione
(9)
A solution of triethylamine (0.068 mL, 0.48 mmol) and octahy-
dro-isoindole 24 (25 mg, 0.12 mmol) in benzene (5 mL) was re-
fluxed for 42 h at 80 °C. Chromatographic purification
(dichloromethane–MeOH 100:2) of the residue which was ob-
tained after removal of the solvents in vacuo afforded stereoisomer
25 (20.0 mg, 85% based on conversion) as a colorless oil. IR (film):
1678, 3320; 1H NMR (600 MHz, CDCl3): d 3.79 (d, J = 10.4 Hz, 1H),
3.74 (s, 3H), 2.59 (m, 1H), 1.65 (m, 1H), 1.53 and 1.37 (m, each 1H),
1.75 and 1.16 (m, each 1H), 1.26 (m, 2H), 1.65 and 1.26 (m, each
A solution of octahydro-isoindole 25 (29 mg, 0.14 mmol) and
acetyl Meldrum’s acid (51 mg, 0.27 mmol) in toluene (5 mL) was
refluxed for 2 h at 110 °C, cooled to room temperature and evapo-
rated. Then a 0.3 M NaOEt solution (1 mL, 0.30 mmol) was added to
the dried residue (12 h high vacuum) dissolved in EtOH (2 mL). The
mixture was refluxed at 80 °C for 2 h, cooled to room temperature
and evaporated. The resulting residue was dissolved in of 2 M HCl
solution (25 mL) and extracted with dichloromethane (3 ꢁ 25 mL).

S. Yao et al. / Bioorg. Med. Chem. 19 (2011) 4669–4678
4677
The combined organic phases were dried with Na2SO4 and evapo-
rated. Chromatographic purification on a reversed phase column
4.20. Ethyl 3-(3,3-dimethyl-1H-indazol-2(3H)-yl)-3-
oxopropanoate (31)
(acetonitrile–water 6:4) yielded tetramic acid 9 (21.0 mg, 74%) as
a white amorphous powder. IR (KBr): 1572, 1682, 3320 cmꢀ1
;
1H
A solution of oxopropanoate 28 (90 mg, 0.32 mmol) in TFA
(2 mL) was stirred at room temperature for 30 min and partitioned
between dichloromethane and saturated NaHCO3 after that time.
The aqueous layer was extracted with dichloromethane
(3 ꢁ 25 mL). The combined organic phases were dried with Na2SO4,
filtered and evaporated. The residue was dried in high vacuum for
12 h and used for the next step without further purification. MS
(ESI) m/z (%): 263.1 ([M+H]+, 100).
NMR (600 MHz, acetonitril-d3): d 4.24 (d, J = 12 Hz, 1H), 2.34 (s,
3H), 2.33 (m, 1H), 2.14 and 1.67 (m, each 1H), 1.71 and 1.44 (m,
each 1H), 1.81 and 1.21 (m, each 1H), 1.59 and 1.44 (m, each
1H), 1.49 and 1.40 (s, each 3H); 13C NMR (150 MHz, acetonitril-
d3): d 19.8, 21.5, 24.4, 24.9, 25.0, 25.9, 26.1, 38.6, 53.3, 64.5, 69.1,
106.5, 176.7, 185.3, 197.0; MS (ESI): m/z (%): 264.2 ([M+H]+,
100); HR MS (ESI): calcd for C15H22NO3 ([M+H]+): 264.1594, found
264.1597.
4.21. 9,9-Dimethylpyrazolo[1,2-a]indazole-1,3(2H,9H)-dione
4.17. Ethyl 3-(2-(2-(2-hydroxypropan-2-yl)phenyl)hydrazinyl)-
3-oxopropanoate (28)
(32)
Method A: A 0.76 M NaOEt solution in ethanol (0.5 mL) was
added to indazole 31 (83 mg, 0.32 mmol) dissolved in EtOH
(1 mL). The solution was refluxed for 6 h. After that time the reac-
tion mixture was partitioned between dichloromethane and satu-
rated NH4Cl solution. The aqueous layer was extracted with
dichloromethane (3 ꢁ 25 mL). The combined organic phases were
dried with Na2SO4, filtered and evaporated. Silica gel chromatogra-
phy (cyclohexane–ethyl acetate 2:1) yielded indazole-dione 32 as a
white amorphous powder (59 mg, 85%).
Ethyl malonyl chloride (0.18 mL, 1.33 mmol) was added drop-
wise to a solution of the crude hydrazine 27 (300.0 mg, 1.26 mmol)
prepared according to a literature procedure26, and DMAP (154 mg,
1.26 mmol) in dichloromethane (2 mL). The reaction was stirred
for 30 min at room temperature and then partitioned between
dichloromethane and water. The aqueous layer was extracted with
dichloromethane (3 ꢁ 25 mL). The combined organic phases were
evaporated and the residue was purified by silica gel chromatogra-
phy (cyclohexane–ethyl acetate 1:1) to yield oxopropanoate 28 as
a yellow amorphous powder (120.0 mg, 70%). IR (KBr): 1689, 3340,
Method B: A 1.0 M malonyl chloride solution (2 mL, 2.00 mmol)
in dichloromethane was added dropwise to dihydroindazole 29
(701 mg, 2.00 mmol) and DMAP (490 mg, 4.00 mmol) dissolved
in dichloromethane (5 mL). The reaction was stirred at room tem-
perature for 30 min and then quenched with saturated NH4Cl solu-
tion. The two layers were separated and the aqueous phase was
extracted with dichloromethane (3 ꢁ 50 mL). The combined organ-
ic layers were dried with Na2SO4, filtered and evaporated. Pure
indazole-dione 32 (250.0 mg, 57%) was obtained after column
chromatography (cyclohexane–ethyl acetate 1:1) as a white amor-
3228 cmꢀ1 1H NMR (600 MHz, CDCl3): d 1.30 (t, J = 7.2 Hz, 3H),
;
1.64 (s, 6H), 3.36 (s, 2H), 4.22 (q, J = 7.2 Hz, 2H), 6.82 (ddd,
J = 1.2, 7.5, 8.0 Hz, 1H), 6.91 (dd, J = 1.2, 8.0 Hz, 1H), 7.17 (m, 1H),
7.21 (m, 1H); 13C NMR (150 MHz, CDCl3): d 13.5, 28.9, 39.8, 40.5,
61.7, 73.3, 112.2, 119.7, 125.2, 127.7, 131.6, 145.3, 166.0, 168.0;
MS (ESI): m/z (%) 263.1 ([[MꢀH2O]+H]+, 100), 303.1 ([M+H]+, 8);
HR MS (ESI): calcd for C14H20N2NaO4 ([M+Na]+): 303.1310, found
303.1312.
phous powder. IR (KBr): 1689, 3228, 3340 cmꢀ1
;
1H NMR
(400 MHz, CDCl3): d 1.84 (s, 6H), 3.52 (s, 2H), 7.19 (m, 2H), 7.35
(ddd, J = 2.2, 7.9, 8.0 Hz, 1H), 7.60 (d, J = 7.9 Hz, 1H); 13C NMR
(100 MHz, CDCl3): d 26.9, 26.9, 42.5, 64.5, 112.4, 121.9, 125.5,
129.1, 131.5, 137.6, 159.2, 161.4; MS (ESI): m/z (%) 217.1
([M+H]+, 100), 239.1 ([M+Na]+, 14); HR MS (ESI): calcd for
4.18. tert-Butyl 2-(2-(2-hydroxypropan-2yl)phenyl)hydrazine
carboxylate (30)
A 10% aq Na2CO3 solution (2 mL) was added to hydrazine 27
(150 mg, 0.90 mmol) and di-tert-butyl dicarbonate (200 mg,
0.90 mmol) in dichloromethane (2 mL). The two phase reaction
mixture was stirred for 2 h at room temperature. Then the dichlo-
romethane phase was separated and the water phase was ex-
tracted with dichloromethane (3 ꢁ 5 mL). The combined organic
layers were dried with Na2SO4 filtered and evaporated. Boc pro-
tected hydrazine 30 (160.0 mg, 66%) was obtained as a yellow
amorphous powder after chromatographic purification (cyclohex-
ane–ethyl acetate 9:1) of the raw material. IR (KBr): 1520, 1689,
C
12H12N2NaO2 ([M+Na]+): 239.0786, found 239.0784.
4.22. rac-(4aS,8aR)-9,9-Dimethylhexahydropyrazolo[1,2-
a]indazole-1,3(2H,4aH)-dione (33)
The mixture of indazole-dione 32 (100 mg, 0.46 mmol) in EtOH
(10 mL, containing 0.1% AcOH) and platinum(IV) oxide (95%)
(6 mg, 0.02 mmol) was hydrogenated (7 bar) at room temperature
for 72 h. After that time the reaction was filtrated, the catalyst was
washed with ethanol (containing 5% acetic acid) and the combined
ethanol phases were evaporated. The residue was dissolved in of
dichloromethane (20 mL) and washed with saturated NaHCO3
solution (2 ꢁ 20 mL). The aqueous layer was extracted with dichlo-
romethane (3 ꢁ 25 mL). The combined organic phases were dried
with Na2SO4, filtered and evaporated. The raw material was puri-
fied by column chromatography (cyclohexane–ethyl acetate 1:1)
to yield product 33 (43.0 mg, 42%) as a white amorphous powder.
3228, 3340 cmꢀ1 1H NMR (600 MHz, CDCl3): d 1.48 (s, 9H), 1.68
;
(s, 6H), 2.97 (br s, 1H), 6.43 (br s, 1H), 6.82 (ddd, J = 1.1, 7.6,
7.8 Hz, 1H), 7.00 (d, J = 7.6 Hz, 1H), 7.17 (m, 2H), 8.06 (br s, 1H);
13C NMR (150 MHz, CDCl3): d 28.3, 28.3, 29.4, 29.4, 29.4, 73.9,
80.7, 112.6, 119.4, 125.4, 128.1, 131.2, 147.3, 156.3; MS (ESI): m/
z (%) 149.1 (100), 267.2 ([M+H]+, 2), 289.2 ([M+Na]+, 7); HR MS
(ESI): calcd for
289.1424.
C
14H22N2NaO3 ([M+Na]+): 289.1422, found
IR (KBr) 1697, 1730, 2930, 3436 cmꢀ1 1H NMR (600 MHz, CDCl3),
;
4.19. 3,3-Dimethyl-2,3-dihydro-1H-indazole (29)
1.48 (s, 3H), 1.58 (s, 3H), 1.25 and 1.79 (m, each 1H), 1.39 and
1.81 (m, each 1H), 1.43 and 1.57 (m, each 1H), 1.70 and 2.73 (m,
each 1H), 2.09 (m, 1H), 3.24 and 3.49 (d, J = 21 Hz, each 1H), 4.24
(d, J = 4.7, 8.3 Hz, 1H), 13C NMR (150 MHz, CDCl3): d 20.4, 21.3,
23.5, 24.2, 25.5, 25.6, 43.0, 50.7, 53.7, 62.5, 163.3, 164.3; MS
(ESI): m/z (%) 223.1 ([M+H]+, 100); HR MS (ESI): m/z (%) calcd for
A solution of hydrazine 30 (820 mg, 4.93 mmol) in trifluoroace-
tic acid (2 mL) was stirred 4 h at room temperature until the start-
ing material has disappeared completely. Then the solvent was
evaporated and the residue dried in high vacuum for 12 h. The
raw product 29 was used without further purification for the next
step. MS (ESI): m/z (%) 132.1 (100), 149.1 ([M+H]+, 40).
C
12H18N2NaO2 ([M+Na]+): 245.1260, found 245.1262.

4678
S. Yao et al. / Bioorg. Med. Chem. 19 (2011) 4669–4678
4.23. 2-(1-Hydroxyethylidene)-9,9-dimethylpyrazolo[1,2-a]
indazole-1,3(2H,9H)-dione (10)
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.06.001.
Indazole-dione 32 (50 mg, 0.23 mmol) and DMAP (5.6 mg,
0.05 mmol) were dissolved in dichloromethane (2 mL). To this solu-
tion acetic acid (0.0146 mL, 0.25 mmol), DCC (57.2 mg, 0.28 mmol)
and triethyl amine (0.0387 mL, 0.28 mmol) were added at 0 °C. The
reaction mixture was stirred for 10 min at 0 °C and then 12 h at
room temperature. After that time the solution was evaporated
and the residue purified by column chromatography (ethyl acetate,
then CH2Cl2–MeOH 100:5, containing 0.1% AcOH) to afford tetramic
acid 10 (51 mg, 84%) as a white amorphous powder. IR (KBr): 1520,
1689, 3228, 3340 cmꢀ1; 1H NMR (400 MHz, CD3OD): d 1.85 (s, 6H),
2.53 (s, 3H), 7.33 (d, J = 7.6 Hz, 1H), 7.22 (t, J = 7.6, 7.6 Hz, 1H), 7.39
(t, J = 7.6, 7.9 Hz, 1H), 7.56 (d, J = 7.9 Hz, 1H); 13C NMR (100 MHz,
CD3OD): d 19.2, 19.2, 26.8, 66.1, 100.6, 112.7, 123.3, 126.1, 130.6,
132.8, 138.5, 179.8, 187.5, 189.5; MS (ESI) m/z (%) 159.1 ([M+H]+,
100), 281.1 ([M+Na]+, 29); HR MS (ESI): calcd for C14H14N2NaO3
([M+Na]+): 281.0896, found 281.0897.
References and notes
1. Meyer, C. E. J. Antibiot. 1971, 24, 558–560.
2. Burmeister, H. R.; Bennett, G. A.; Vesonder, R. F.; Hesseltine, C. W. Antimicrob.
Agents Chemother. 1974, 5, 634–639.
3. Jomon, K.; Kuroda, Y.; Ajisaka, M.; Sasaki, H. J. Antibiot. 1972, 25, 271–280.
4. Holzapfel, C. W. Tetrahedron 1968, 24, 2101–2119.
5. Martínez-Azorín, F. FEBS Lett. 2004, 576, 73–76.
6. Riley, R. T.; Goeger, D. E.; Yoo, H.; Showker, J. L. Toxicol. Appl. Pharmacol. 1992,
114, 261–267.
7. Hamaguchi, H.; Hirooka, T. In Modern Crop Protection Compounds; Krämer, W.,
Schirmer, U., Eds.; Wiley-VCH: Weinheim, 2007; Vol. 3, pp 1122–1137.
8. Ortín, I.; González, J. E.; de la Cuesta, E.; Avendaño, C. Tetrahedron 2010, 66,
8707–8713.
9. Kozikowski, A. P.; Greco, M. N.; Springer, J. P. J. Am. Chem. Soc. 1984, 106, 6873–
6874.
10. Muratake, H.; Natsume, M. Heterocycles 1985, 23, 1111–1117.
11. Haskins, C. M.; Knight, D. W. Chem. Commun. 2005, 3162–3164.
12. Beyer, W. R. C.; Woithe, K.; Lüke, B.; Schindler, M.; Antonicek, H.;
Scherkenbeck, J. Tetrahedron 2011, 67, 3062–3070.
13. Moncoq, K.; Trieber, C. A.; Young, H. S. J. Biol. Chem. 2007, 282, 9748–9757.
14. Laursen, M.; Bublitz, M.; Moncoq, K.; Olesen, C.; Møller, J. V.; Young, H. S.;
Nissen, P.; Morth, J. P. J. Biol. Chem. 2009, 284, 13513–13518.
15. Søhoel, H.; Liljefors, T.; Ley, S. V.; Oliver, S. F.; Antonello, A.; Smith, M. D.; Olsen,
C. E.; Isaacs, J. T.; Christensen, S. B. J. Med. Chem. 2005, 48, 7005–7011.
16. Gilbert, A. M.; Failli, A.; Shumsky, J. S.; Yang, Y.-J.; Severin, A.; Singh, G.; Hu, W.;
Keeney, D.; Petersen, P. J.; Katz, A. H. J. Med. Chem. 2006, 49, 6027–6036.
17. Schnell, B.; Kappe, T. Monatsh. Chem. 1998, 129, 871–885.
18. Schatz, F.; Wagner-Jauregg, Th. Helv. Chim. Acta 1968, 51, 1919–1931.
19. Barluenga, J.; Álvarez-Gutiérrez, J. M.; Ballesteros, A.; González, J. M. Angew.
Chem., Int. Ed. 2007, 46, 1281–1283.
20. Barluenga, J.; Rodriguez, M. A.; Campos, P. J. J. Org. Chem. 1990, 55, 3104–3106.
21. Espuña, G.; Arsequell, G.; Valencia, G.; Barluenga, J.; Pérez, M.; González, J. M.
Chem. Commun. 2000, 1307–1308.
22. Espuña, G.; Andreu, D.; Barluenga, J.; Pérez, X.; Planas, A.; Arsequell, G.;
Valencia, G. Biochemistry 2006, 45, 5957–5963.
4.24. rac-(4aS,8aR)-2-(1-Hydroxyethylidene)-9,9-dimethy
lhexahydropyrazolo[1,2-a]indazole-1,3(2H,4aH)-dione (11)
Pyrazoloindazole 33 (35 mg, 0.16 mmol), DMAP (3.8 mg,
0.03 mmol), DCC (39.0 mg, 0.19 mmol) were dissolved in dichloro-
methane (5 mL), followed by acetic acid (0.010 mL, 0.17 mmol) and
triethyl amine (0.0263 mL, 0.19 mmol) at 0 °C. The reaction mix-
ture was filtered after 12 h at room temperature and the filtrate
was washed with 1 M HCl (2 ꢁ 10 mL). The aqueous layer was ex-
tracted with dichloromethane (3 ꢁ 25 mL), the combined organic
phases were dried with Na2SO4, filtered and the solvents were re-
moved in vacuo. Column chromatography (ethyl acetate, then
CH2Cl2–MeOH 100:5, containing 0.1% AcOH) of the residue affor-
ded tetramic acid 11 (25 mg, 60%) as a white amorphous powder.
23. Schlummer, B.; Hartwig, J. F. Org. Lett. 2002, 4, 1471–1474.
24. Gärtner, M.; Ballmann, J.; Damm, C.; Heinemann, F. W.; Kisch, H. Photochem.
Photobiol. Sci. 2007, 6, 159–164.
IR (KBr): 1670, 3218, 3326 cmꢀ1 1H NMR (600 MHz, CD3OD): d
;
1.97 (s, 6H), 2.36 (s, 3H), 1.43 and 1.52 (m, each 1H), 1.29 and
1.76 (m, each 1H), 1.44 and 1.82 (m, each 1H), 1.75 and 2.74 (m,
each 1H), 2.19 (m, 1H), 4.21 (m, 1H); 13C NMR (150 MHz, CD3OD):
d 21.4, 22.9, 22.9, 25.0, 25.4, 26.0, 27.8, 51.3, 55.6, 63.8, 99.9, 167.5,
179.4, 195.5; MS (ESI) m/z (%) 265.1 ([M+H]+, 100); HR MS (ESI):
calcd for C14H20N2NaO3 ([M+Na]+): 287.1366, found 287.1366.
25. Walser, A.; Silverman, G. J. Heterocycl. Chem. 1973, 10, 883–884.
26. Zenchoff, G. S.; Walser, A.; Fryer, R. I. J. Heterocycl. Chem. 1976, 13, 33–39.
27. Portal, C.; Launay, D.; Merritt, A.; Bradley, M. J. Comb. Chem. 2005, 7, 554–560.
28. Lockyer, P. J.; Puente, E.; Windass, J.; Earley, F.; East, J. M.; Lee, A. G. Biochim.
Biophys. Acta 1998, 1369, 14–18.
29. Lape, M.; Elam, C.; Versluis, M.; Kempton, R.; Paula, S. Proteins 2008, 70, 639–
649.
30. Autry, J. M.; Jones, L. R. J. Biol. Chem. 1997, 272, 15872–15880.
31. Crimmins, M. T.; Washburn, D. G.; Zawacki, F. J. Organic Syntheses 2000, 77,
114–118.
Acknowledgments
32. Steiger, R. E. Org. Synth. 1942, 22, 23–25.
33. Hatano, B.; Sato, H.; Ito, T.; Ogata, T. Synlett 2007, 2130–2132.
Financial support by Bayer CropScience is gratefully acknowl-
edged. The authors thank Ms. M. Dausend and Mr. A. Siebert for
measuring the high-resolution MS and NMR spectra.