Asymmetric total synthesis of the indole alkaloid cyclopiazonic acid and first structure-activity data

Article abstract of DOI:10.1016/j.tet.2011.03.002

The indole alkaloid α-cyclopiazonic acid (CPA) is one of the few known inhibitors of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) besides the terpenoids thapsigargin and artemisinin. We report here the first asymmetric total synthesis of cy

Full text of DOI:10.1016/j.tet.2011.03.002

Tetrahedron 67 (2011) 3062e3070
Contents lists available at ScienceDirect
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Asymmetric total synthesis of the indole alkaloid cyclopiazonic acid
and first structureeactivity data
a
b
b
b
b
€
W.R. Christian Beyer , Katharina Woithe , Bettina Luke , Michael Schindler , Horst Antonicek ,
a
,
*
€
Jurgen Scherkenbeck
a
€
Bergische Universitat 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 a-cyclopiazonic acid (CPA) is one of the few known inhibitors of sarco(endo)plasmic
reticulum Ca2þ-ATPase (SERCA) besides the terpenoids thapsigargin and artemisinin. We report here the
first asymmetric total synthesis of cyclopiazonic acid by a modification of the Knight synthesis, currently
the most efficient route to CPA. First structureeactivity data of CPA derivatives and stereoisomers are
presented and will be discussed in connection with the published crystal structures of CPAeSERCA
complexes.
Received 31 January 2011
Received in revised form 24 February 2011
Accepted 1 March 2011
Available online 5 March 2011
Keywords:
Indole alkaloid
Ó 2011 Elsevier Ltd. All rights reserved.
Cyclopiazonic acid
SERCA
Enantioselective synthesis
Structureeactivity data
1. Introduction
promising targets for the development of new drugs against vari-
ous diseases and insect pests.
The indole alkaloid
a
-cyclopiazonic acid (CPA, 1) has been iso-
Although the structure of CPA is known since 1968 only three
racemic syntheses have been published to date and almost nothing
is known on structureeactivity relationships. The Kozikowski
synthesis (1984) comprises almost twenty steps and is based on
a stepwise formation of ring C by a stereoselective Michael addition
lated from stored grain and cereal products infected with the fun-
gus Penicillium cyclopium Westling.1,2 Toxicity and mode of action of
CPA have been studied intensively as a consequence of several food
poisonings caused by ingestion of contaminated feed.3,4 Cyclo-
piazonic acid blocks the calcium access channels of Ca2þ-ATPases of
the sarcoendoplasmic reticulum (SERCA), which are essential for
calcium reuptake in the muscle.5,6 Other known inhibitors of SERCA
enzymes are the terpenoids thapsigargin (3, anticancer activity),
artemisinin (4, antimalaria drug) and the CPA-related indole
alkaloid speradine A (2), which shows significant antibacterial
and histone deacetylase activity (Fig. 1).7e11 In addition, calcium
metabolism is an important target for modern insecticides, such as
the natural product ryanodin or the benzenedicarboxamide flu-
bendiamide, a novel insecticide for controlling lepidopterous insect
pests.12 Apparently, sarcoendoplasmic reticulum Ca2þ-ATPases are
O
OH
O
OH
7 N
E
10
N
H 6a
11
D
H
6
5a
O
H11a
O
C
H
O
H11b
H
H
N
11c
5
1
A
B
2NH
2a
Speradine A (2)
α-Cyclopiazonic acid (1)
O
O
OAc
H15C7OCO
H
OCOC3H7
OH
OH
O
O
Abbreviations: ACN, acetonitrile; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene;
DCM, dichloromethane; DMF, dimethylformamide; EGTA, ethylene glycol
tetraacetic acid; KHMDS, potassium hexamethyldisilazide; MOPS, 3-(N-morpho-
lino)-propanesulfonic acid; Ns, p-nosyl; TBDPS, tert-butyldiphenylsilyl; TfOH,
trifluoromethanesulfonic acid; THF, tetrahydrofurane; Ts, tosyl.
O
O
O
O
H
O
Thapsigargin (3)
Artemisinin (4)
* Corresponding author. E-mail address: scherkenbeck@uni-wuppertal.de
(J. Scherkenbeck).
Fig. 1. Known inhibitors of SERCA.
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.03.002

W.R.C. Beyer et al. / Tetrahedron 67 (2011) 3062e3070
3063
A
SPh
NAc
O
PhS
O
PhS
CO2Et
NHAc
CO2Et
H
CO2Et
NHAc
H
N
DBU,
1. H2, Raney
t
BuOH
N
N
2. PhSH, Mg(OTf)2
CO2Et
CO2Et
5
6
7
CO2Et
B
O
O
N
CO2Et
CH2OMe
CO2Et
N
H
CH2OMe
CO2Et
H
CHO
NTs
CHO
DBU, benzene
reflux
1N HCl
NTs
NTs
8
9
10
Scheme 1. Key steps of the Kozikowski (A) and Natsume syntheses (B) of CPA.
(Scheme 1A) followed by a three step sequence to establish the
pyrrolidine ring D and to introduce the second methyl group.13
Similar to the Kozikowski synthesis, also the Natsume synthesis
(1985) utilizes a Michael addition (Scheme 1B) for the construction
of ring C, but due to the higher temperature of the cyclization
reaction a mixture of tricyclic stereoisomers 9 was obtained.14 The
Knight synthesis, published in 2005, represents a significant
shorter route to CPA.15 The key step here is a simultaneous for-
mation of the ring C- and D-framework by a cationic cyclization of
the bicyclic intermediate 12 (Scheme 2), which is available in
enantiomeric pure form on a multigram scale by a 1,4-addition (dr
94:6) of isopropenylcuprate to the chiral indolyl acrylic acid 11
(Scheme 2), followed by a 1,2-addition of trisylazide (dr 98:2) as
detailed recently by our group.16 Remarkably, both stereogenic
centres are established in the correct configuration by one and the
same Evans auxiliary with high diastereofacial control.
first structureeactivity data of several CPA derivatives and stereo-
isomers. The inhibitory activities against Heliothis virescens SERCA
are discussed on the basis of published X-ray structures of
CPAeSERCA complexes.
2. Results and discussion
2.1. Chemistry
The diastereomeric pyrrolidines 13a and 13b (62%) are formed
as a 1:1 mixture accompanied by lactone 14 (27%) and, depending
on the reaction conditions, minor amounts of the monocyclization
product 15 on addition of triflic acid to the key intermediate 12
(Scheme 2). The latter compound clearly demonstrates a stepwise
cyclization process, initiated from a benzylic carbenium ion formed
by acidic cleavage of the silanyloxy-group.17 The C/D trans-ring
junction in 13b (J6a,9a 12.5 Hz), 14 (J6a,10a 12.7 Hz) and 15 (J3,4 9.1 Hz)
originates from a s-trans conformation in the transition-state of the
We wish to report now the first asymmetric total synthesis of
CPA following the Knight route with some modifications as well as
O
O
NNs
NHNs
H
O
H
H
R
10
NHNs
H
10a
9
CO2Et
3
TBDPSO
OEt
NHNs
6a
CO2Et
4
6a
H
H
H
H
9a
i
+
+
NTs
NTs
NTs
NTs
12
14
15
H
H
13a: R =
13b: R =
ii)
iii)
O
H
O
H
OH
OH
O
N
N
O
R
R2
11a
11a
6a
TBDPSO
N
O
O
1 O
6a
H
iv
R
11b
11b
Ph
NTs
NH
NTs
2
1(CPA): R1 =
, R =
H
H
H
H
16a: R =
16b: R =
H
H
17(isoCPA): R1 =
2
11
H
, R =
H, R2 =
18 (all-transCPA): R1 =
i) TfOH (1.0 eq), DCM, rt, 1h, 62% 13a and 13b, 27% 14; ii) DBU (3 eq.), HSCH2CH2OH (3 eq.), DMF, rt, 24h, 68%,
iii) acetyl Meldrums acid (2.5 eq.), dioxane, 80°C, 90min then KOH in MeOH (0.1 M, 2.5 eq.), 80°C, 60min, 80%;
iv) Cs2CO3 ( 6 eq.), THF/MeOH 1/1, reflux, 3d, 61%.
Scheme 2. Asymmetric synthesis of cyclopiazonic acid (1).

3064
W.R.C. Beyer et al. / Tetrahedron 67 (2011) 3062e3070
Table 1
CPA analogues and SERCA inhibition
R1
O
R2
N
O
N
R3
Compound
1 (Reference)
1 (Synthetic)
17 (isoCPA)
18 (alltransCPA) OH
2 (Speradine A)
R1
OH
OH
OH
R2
R3
SERCA (IC50, mM)
Me
Me
Me
Me
H
H
H
H
0.01ꢀ0.005
0.01ꢀ0.006
0.50ꢀ0.1
5.0ꢀ0.5
8.07
16 (tosylCPA)
OH
OH
OH
OH
OH
OH
Me
Tosyl 0.40ꢀ0.05
19
20
21
22
23
24
25
26
27
28
Cyclopropyl
Cyclohexyl
Benzyl
Benzyl
Phenyl
Me
H
H
H
Me
H
H
H
H
H
H
0.14ꢀ0.02a
>50a
1.9ꢀ0.4a
1.7ꢀ0.2a
>50
Fig. 2. Formation of pyrrolidines 13a and 13b (NOE contacts indicated with arrows).
cyclization reaction (Fig. 2). Due to lower steric strain of trans-fused
six-membered rings a significant amount of cyclic lactone 14 is
formed via acidic ester cleavage and subsequent attack of a car-
boxylic oxygen to the tert-butyl carbenium ion in position 7.18
Furthermore, the carbenium ion turns away from N-8 in the
s-trans conformation which additionally disfavours the formation
of the smaller pyrrolidine ring (13b). The coupling constants in-
dicate a trans-arrangement of H-9 and H-9a in 13b (J9,9a 9.8 Hz) and
a cis-configuration in lactone 14 (J10,10a 5.6 Hz) as a direct conse-
quence of the suggested cyclization mechanism (Fig. 2).
Consistent with this model, the C/D-ring cis-fusion is estab-
lished by a ring-closure via a s-cis conformation between the ole-
finic and the b-proton (Fig. 3). In this case, the carbenium ion is
oriented towards the nitrogen and thus the formation of the
strainless, cis-connected pyrrolidine ring is facilitated. The cis-ring
connection in 13a is supported by a J6a,9a coupling constant of
NH2
NHeNH2
NHeNHe4ClePh Me
NHeOH
NHeOMe
0.50ꢀ0.08a
0.78ꢀ0.08a
7.0ꢀ1.3a
16.0ꢀ2a
8.8ꢀ1.4a
Me
Me
Me
a
Contains minor amounts of iso- and alltrans-CPA.
throughout the final steps, and isoCPA 17 (J11a,11b 5.1 Hz, J11a,11b
5.1 Hz), which is formed obviously by epimerization of H-11a under
the cleavage conditions. Strong NOE’s between H-11a, H-11b and
H-6a as well as between H-11b and Ha-6 are in perfect agreement
with the structure of isoCPA (17). Additionally, spectroscopic data of
CPA (1) and isoCPA (17) were identical with those already
published.21,22
In order to evaluate the significance of the acyl tetramic acid
moiety for SERCA inhibition two series of CPA derivatives were
prepared. In the first series the acetyl group was replaced for larger
acyl residues, such as cyclopropyl, benzyl and phenyl by acylation
of the cyclization product 13 with the corresponding acyl
Meldrum’s acids, which were prepared according to literature
procedures.23e25 In the second series of derivatives the acetyl group
of CPA (1) was reacted with various nitrogen nucleophiles to yield
the corresponding oximes, hydrazones and imines. These de-
rivatives were expected to show different complexation behaviour
of Mg2þ ions and thus should have a major influence on SERCA
inhibition (Table 1).
5.6 Hz. NOESY spectra reveal a
configuration of H-6a and H-9a. Most indicative for this stereo-
chemistry are strong NOEs between H-9 and 7 -Me, H-6a with
H-9a, as well as CH2-ester and 7 -Me (Fig. 2).
b-position for H-9 and thus a trans-
b
a
The subsequent steps towards CPA (1) comprise a denosylation
reaction with mercaptoethanol (68%), construction of the tetramic
acid moiety in a one-pot reaction with acetyl-Meldrum’s acid (80%)
and finally the cleavage of the tosyl group (Scheme 2).19,20 Three
stereoisomers could be isolated from the detosylation reaction: CPA
1 (J11a,11b 11.0 Hz, J6a,11b 5.8 Hz), alltransCPA 18 (J11a,11b 10.8 Hz, J6a,11b
11.5 Hz) both resulting from the 6a epimer-mixture used
Fig. 3. Superimposition of CPA 1/isoCPA 17 (left side) and CPA 1/alltransCPA 18 (right side).

W.R.C. Beyer et al. / Tetrahedron 67 (2011) 3062e3070
3065
For all derivatization reactions the mixture of stereoisomers as
obtained from the synthesis was used since the pure stereoisomers
were available only in small amounts. Unfortunately, the final
preparative HPLC separation of the CPA derivatives turned out to be
difficult due to slowly interconverting tautomers and E/Z isomers in
the acyl tetramic acid causing a considerable peak broadening.
Therefore the derivatives except 23 used for biological testing still
contained some iso- and alltrans-CPA.
detosylation reaction in methanol, it can be concluded that sub-
stituents at the indole nitrogen are well tolerated.26 This clearly
points to a certain structural variability of the indole system in CPA.
2.3. Molecular-modelling studies
The sarcoplasmic reticulum Ca2þ-ATPase is an integral mem-
brane protein consisting of 995 amino acids, arranged in three
cytoplasmic domains containing the ATP binding site and 10
transmembrane helices with two calcium binding sites. Despite the
fact that X-ray structures of SERCA, capturing different states of the
protein, have been published, no experimental protein structure of
H. virescens SERCA is available.27 Hence, a homology model was
constructed, using the protein modelling suite (FUGUE, ORCHES-
TRAR) provided by Tripos (SYBYLX1.1).28 The structure of rabbit
SERCA complexed with CPA was chosen because of the identifica-
tion of a metal ion in the CPA binding niche.29,30 However, by
choosing this template, which marks the dynamical endpoint of
ligand binding, the model is limited to a static picture of the binding
niche. No attempt was made to model the helical movements upon
binding of new ligands. The spatial neighborhood of the CPA
binding niche is well conserved between rabbit and H. virescens.
2.2. Biological data and structureeactivity relationships
All CPA derivatives were tested in a functional H. virescens
biochemical SERCA assay 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 lumines-
cence signal is observed. Dose-response assays allowed the de-
termination of IC50 values for each inhibitor.
Synthetic CPA was found to be equally active (IC50 0.01 mM) as
a natural reference sample (Table 1). isoCPA 17 showed a reduced
SERCA inhibition by a factor of around 50 albeit still on a high level
(IC50 0.5
found to have a drastically reduced activity (5.0
than the tosylate 16 (0.40 M), which is still in the range of isoCPA
mM). In contrast to isoCPA 17 the alltransCPA isomer 18 was
ꢀ
mM), much lower
Within 5 A of CPA 24 amino acids (L253 D254 F256 G257 L260 I307
m
P308 E309 G310 L311 P312 I315 I761 Q56 F57 D59 L61 V62 L65 I97
17. The low activity of alltransCPA 18 is not unexpected, since the
trans-conjunction of the six-membered ring C and pyrrolidine ring
D induces a significant change in the shape of the CPA structure
resulting in drastically reduced SERCA binding.
L98 I99 N101 A102), 5 water molecules and one Mg2þ ion can be
ꢀ
found. Only on position 58, slightly outside the 5 A region, the acid
Glu (rabbit) is replaced by the slightly smaller acid Asp (Heliothis).
Thus, it is not expected that additional uncertainties will be
introduced by analyzing the orientation of different ligands based
on this homology model.
Neither the pyrrolidines 13 nor lactone 14 or the monocycliza-
tion product 15 showed any SERCA inhibition, proofing the tetramic
acid (ring E) essential for full SERCA activity. Thus, modifications,
which negatively interfere with hydrogen bonding to SERCA or
Mg2þ coordination of the acetyl tetramic acid (ring E) are expected
to have a major impact on SERCA inhibition. In fact, CPA analogues
with bulky acyl residues, such as phenyl (23) or cyclohexyl (20)
were shown to be inactive. On the contrary, CPA analogue 19 with
a small cyclopropyl-acyl group expressed only a slightly diminished
activity, indicating at least a minor variability of the acyl binding
pocket. This hypothesis is underlined by the benzyl-derivative 21,
which had a considerably reduced but still significant activity in the
low micromolar range.
The critical role of the acyl-group is also demonstrated by the
second series of CPA derivatives. Reaction of CPA with ammonia and
hydrazine afforded the enamines 24 and 25, respectively. Both CPA
analogues were found to have a somewhat reduced SERCA activity
(IC50 0.5 mM and 0.8 mM) but still on the level of isoCPA 17. CPA
derivatives 26e28 obtained by reacting CPA with hydroxylamine,
O-methyl hydroxylamine, and 4-chlorophenyl hydrazine showed
Previous published crystallographic structures demonstrated
that CPA fills the calcium access channel in the E2 calcium
free-form of the enzyme and stabilizes the M1- and M2-helices in
positions that are incompatible with calcium binding.29,30 The CPA
binding pocket, a cavity between transmembrane segments M1,
M2, M3 and M4 formed by Leu61 and Val62 on M1, Asn101, Ala102 on
M2, Gly257 on M3 and Leu311 and Pro312 on M4, consists of three
regions, each accommodating distinct substructures of CPA. A polar
region including the residues Gln56, Asp59, Asn101 on M1 and M2 as
well as Mg2þ coordinates the acyl tetramic acid by a network of
hydrogen bonds. The centre of the pocket, which houses ring C of
CPA and BHQ (2,5-di-(tert-butyl) hydroquinone), a small-molecule
inhibitor of SERCA, expresses a preference for conjugated cyclic
p-systems. The lipophilic indole group is located in an extended
hydrophobic groove between M3 and M4, primarily formed by
Leu61 and Val62. The indole nucleus locks the M1 and M4 helices
and connects the CPA with the thapsigargin (TG) binding site.29,30
Since the chelation of Mg2þ plays a pivotal role in CPA binding
all CPA analogues were superimposed with their tetramic acid
moieties.
only weak SERCA inhibition (IC50 7e16 mM), but were still more
active than the cyclohexyl- and phenyl-analogues 20 and 23. Ap-
parently, also an intact enol- or an unsubstituted enamine-function
is essential for high SERCA inhibition. Taking these results together,
the acetyl tetramic acid can be regarded as the pharmacophoric
unit of cyclopiazonic acid. It should be emphasized that the minor
amounts of iso- and alltrans-CPA, still left in several CPA derivatives
influence the SERCA inhibition data, which range over almost three
orders of magnitude only marginally and the structureeactivity
relationships provided not at all.
Due to cis-connection between the six-membered ring C and
pyrrolidine ring D natural CPA (11aS-configuration) shows a dis-
tinct bend, which allows the indole system to orient upwardly to
the M4-helix in the binding pocket. The nonpolar interactions be-
tween Leu311, Pro308 on M4 and Leu61, Val62 on M1 with the indole
system lock the M1-against the M4-helix and can be regarded as
vital for the SERCA inhibitory action of CPA. The 11aR-configuration
imposes a bowl-like form on isoCPA (17) with the pyrrolidine ring at
the bottom and the indole ring and tetramic acid forming the walls.
Due to its drastically changed shape, isoCPA (Fig. 3, green colour) is
expected to orient differently in the binding pocket compared to
CPA (Fig. 3, yellow colour). In fact, the indole system moves com-
pletely out of the lipophilic binding channel connecting the TG and
CPA binding site. On the other side, the indole-nitrogen comes now
much closer to the side-chain carboxylate-group of Asp 254
The role of the indole system still remains somewhat specula-
tive. On the one hand the oxoindole speradine A (2), which differs
from CPA (1) only in the indole system, shows a remarkably re-
duced SERCA inhibition (IC50 8.0 mM) by a factor of around 800. On
the other hand it remains unclear whether this loss of activity is
caused by the 1-oxo function and/or the additional stereogenic
centre in position 11c. From both tosylCPA 16 and N-methylindolyl
derivative 22, which was isolated as a byproduct from the
ꢀ
(Indol-N/Asp-O: 2.8 A) and should be able to form an additional

3066
W.R.C. Beyer et al. / Tetrahedron 67 (2011) 3062e3070
hydrogen bond (Fig. 3, left side). This, together with the unhindered
Mg2þ coordination provides an explanation for the only slightly
reduced SERCA activity of isoCPA (17). To find a plausible explana-
tion for the low SERCA inhibitory action of alltransCPA (18) appears
to be more difficult since at a first glance the overall shape of
alltransCPA resembles that of CPA (1). However, alltransCPA (18)
adopts an almost planar and more extended conformation com-
pared to the corrugated CPA (1) with the consequence that the
indole system shifts away from M4 and now points directly to Leu61
and Gly257 on M1 and M3 (Fig. 3, right side). As a result, the for-
mation of nonpolar interactions with the respective residues on M4
is reduced and at the same time the indole ring probably causes
a sterical clash with Leu61 (M1) and possibly Gly257 (M3). The
critical role of Gly257 for CPA binding has already been demon-
strated by previous mutagenesis studies.31
Upon introducing bulky acyl residues (compounds 19e23),
two effects can be observed in our model, both of which might
be responsible for reduced activity. Firstly, contrary to cyclopropyl,
the phenyl, benzyl or cyclohexyl moieties cause a pronounced
shift of the ligand towards the hydrophobic pocket, leading to
a modified hydrogen bond pattern as compared to CPA (Fig. 4).
Secondly, the hydrophobic and bulky substituents interact with the
phenyl side-chain of F57, which in turn could modify the orienta-
tion of the alpha-helix M1 close to its kink. In our model the
cyclopropyl-analogue 19 fits perfectly to CPA, both sterically and
electronically.
3. Experimental
3.1. General
Solvents and reagents were purchased from commercial sour-
ces. 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. Pre-
coated plates (silica gel 60 F254, 250 mm, Merck, Darmstadt,
Germany) were used for thin layer chromatography (TLC). Silica gel
0.04e0.063 mm from MachereyeNagel was used for chromato-
graphic purification. HPLCeMS was performed on a Varian 500
IonTrap LCeESI-MS system or on a Bruker MicroTOF (column: 5 mm
RP18) with ACN/H2O gradient 10/90 to 90/10. High-resolution MS
were measured on a Bruker MicroTOF. Semipreparative HPLC was
either performed on a Kromasil 100 (C18 5
m
m, 250ꢁ20 mm) or on
a Zorbax ODS (250ꢁ21.2 mm) column with a Gilson Abimed Sys-
tem. Analytical 1H and 13C NMR spectra were recorded at 25 ꢂC 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 reported in units of parts per
million and coupling constants are given in hertz. Due to tauto-
meric- 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 ana-
logues. Melting points (mp) were determined with a Buechi 535
melting point apparatus and are uncorrected. IR-spectra were
In compound 23 the phenyl substituent matches the cyclopropyl
ring rather smoothly (Fig. 4, left). However, while cyclopropyl-CPA
19 exhibits exactly the same H-bond pattern as CPA, due to the
larger phenyl group 23 is shifted towards the lipophilic part of the
binding niche and looses the ability to chelate Mg2þ with its two
carbonyl functionalities (Fig. 4, middle). In addition, Mg2þ itself has
moved and disturbs the original H-bonding pattern. Differences in
Mg2þ complexation are not so pronounced for the bulkier benzyl-
and cyclohexyl-compounds, but here the interactions with F57
seem to become more important.
ꢁ ꢁ
measured on a Nicolet Protege 460 Spectrometer E.S.P.
3.2. Inhibition assay of SERCA activity
Sf9 cells (Invitrogen) were maintained under serum-free con-
ditions at 27 ꢂC 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 2e1000, accession number
AF115572) with a MOI of 0.3. After three days cells were harvested
and microsomal membranes prepared according to standard pro-
cedures and stored at ꢃ80 ꢂC.32 A typical inhibition assay contained
Regarding the indole NeH Laursen and Bublitz found a distance
ꢀ
of 5.6 A between the indole nitrogen and the carboxylate-group of
aspartate 254, which is too long for a hydrogen bond and thus was
attributed to a polar interaction.30 In accordance with the biological
data of tosylCPA 16 and the N-methylindole 22 also molecular
modelling considerations suggest that substitutions of the indole
hydrogen by other polar groups should be possible without im-
mediate loss of activity. Additionally, the lipophilic binding channel
connecting the CPA and TG binding site appears to be an attractive
starting point for the design of structurally different and less
complex CPA analogues.
varying concentrations of the inhibitor (from 3.2 nM to 50
concentration) and 0.4 g/mL recombinant enzyme together with
mM final
m
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) gylerole,
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 subsequent luciferase reaction
Fig. 4. Superimpositions of CPA with cyclopropyl-CPA 19 (left), CPA with phenyl-CPA 23 (middle), 19 and 23 (right).

W.R.C. Beyer et al. / Tetrahedron 67 (2011) 3062e3070
3067
employing 193.5 nM luciferin (BioSynth), 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 quadruplicates 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.
7.25e7.23 (m, 3H), 6.97 (d, J¼7.3 Hz, 1H), 5.08 (br d, J¼8.7 Hz, 1H),
4.78 (dd, J¼8.7, 5.6 Hz, 1H), 3.62 (ddd, J¼12.7, 5.6, 1.8 Hz, 1H), 2.98
(dd, J¼15.8, 4.0 Hz, 1H), 2.75 (dd, J¼15.6, 12.0 Hz, 1H), 2.30 (s, 3H),
2.15 (
J
dt, J¼12.2,12.2, 4.0 Hz,1H),1.51 (s, 3H),1.38 (s, 3H); 13C NMR
(125 MHz; CDCl3):
d
¼166.8, 150.1, 145.7, 144.9, 135.0, 132.9, 130.0
(CH), 128.8, 128.7, 128.6 (CH), 127.3 (CH), 125.6 (CH), 124.1
(CH), 122.1 (CH), 120.2 (CH), 114.4, 111.7 (CH), 87.1, 53.3 (CH), 41.0
(CH), 34.4 (CH), 29.0 (CH2), 29.0 (CH3), 23.0 (CH3), 21.5 (CH3); HR-
ESI-MS: m/z¼632.1132 (calcd 632.1132 for C29H27N3NaO8S2); IR:
n
¼1735 cmꢃ1
.
3.3. General procedure for the synthesis
of acyl Meldrum’s acid23
3.3.2. (6aR,9S,9aR)-7,7-Dimethyl-2-(toluene-4-sulfonyl)-
2,6a,7,8,9,9a-hexahydro-6H-isoindolo[4,5,6-cd]indole-9-carboxylic
acid ethyl ester and (6aS,9S,9aR)-7,7-dimethyl-2-(toluene-4-sulfonyl)-
2,6a,7,8,9,9a-hexahydro-6H-isoindolo[4,5,6-cd]indole-9-carboxylic
Pyridine (2 equiv) was added to a solution of Meldrum’s acid
(1 equiv) in DCM (0.7 M). The acyl chloride (1 equiv) was dissolved
in DCM (1.4 M) and was added dropwise to the Meldrum’s acid
solution at 0 ꢂC. The reaction was stirred for 1.5 h at 0 ꢂC and 1.5 h at
room temperature. An additional portion acyl chloride (0.5 equiv)
was added and stirring was continued for 12 h at room tempera-
ture. Then the reaction was stopped by adding HCl (2 M) and
diluted with DCM. The organic phase was separated, dried over
Na2SO4 and evaporated. The crude product was either purified by
column chromatography or crystallized to yield the pure acyl
Meldrum’s acid.
acid ethyl ester. Pyrrolidine 10 (190 mg, 297.9
in DMF (3 mL), DBU (133 L, 894 mol) was added followed by
mercaptoethanol (62 L, 894 mol). The resulting mixture was
mmol) was dissolved
m
m
m
m
stirred for 24 h at room temperature. Then diethyl ether was added
and the solution was washed with saturated sodium bicarbonate
solution, water and brine. The combined aqueous layers were
extracted twice with diethyl ether and the combined organic layers
were dried over Na2SO4. Filtration and evaporation of the solvent
under reduced pressure gave a crude product (240 mg), which
afforded the denosylated product (92 mg, 68%) as yellowish foam
after chromatographic purification (cyclohexane/ethyl acetate 1/1).
Rf (cyclohexane/ethyl acetate 1/1): 0.19. 6aR,9S,9aR-isomer (cis-C/D
3.3.1. (6aR,9S,9aR)-Pyrrolidine (13a), (6aS,9S,9aR)-pyrrolidine (13b)
and (6aS,10S,10aR)-lactone (14). Compound 12 (600 mg, 671
was dissolved in DCM (500 mL) under inert gas atmosphere, TfOH
(62.2 L, 671 mol) was added and the reaction stirred for 1 h at
mmol)
ring connection): 1H NMR (600 MHz; CDCl3):
d
¼7.77 (d, J¼8.4 Hz,
m
m
2H), 7.74 (d, J¼8.2 Hz, 1H), 7.35 (d, J¼0.9 Hz, 1H), 7.29e7.26 (m, 1H),
room temperature. Sodium bicarbonate solution was added and the
mixture was extracted twice with DCM. The combined organic
layers were dried over Na2SO4, filtered and the solvent removed
under reduced pressure. The crude product (690 mg) was purified
by column chromatography (DCM, then DCM/MeOH 20/1) and
yielded a 1:1 mixture of the diastereomeric pyrrolidines 13
(265 mg, 62%) as yellowish foam together with lactone 14 (109 mg,
27%). Rf (cyclohexane/ethyl acetate 1/1): 0.48. A sample of this
mixture was separated by semipreparative HPLC (ACN/H2O 40/60).
6aR,9S,9aR-pyrrolidine 13a (cis-C/D ring connection): 1H NMR
7.22 (d, J¼8.1 Hz, 2H), 7.03 (d, J¼7.4 Hz, 1H), 4.39e4.30 (m, 2H), 3.79
(d, J¼8.0 Hz, 1H), 3.74 ( t, J¼7.5 Hz, 1H), 3.04 (dd, J¼16.8, 6.3 Hz,
J
1H), 2.84 (dd, J¼16.8, 7.8 Hz, 1H), 2.35 (s, 3H), 2.29 ( q, J¼7.3 Hz,
J
1H), 1.41 (t, J¼7.2 Hz, 3H), 1.34 (s, 3H), 0.95 (s, 3H); 13C NMR
(125 MHz; CDCl3):
d
¼174.6, 144.7, 135.5, 134.6, 133.1, 130.4, 129.7
(CH),128.6,126.7 (CH),125.5 (CH),121.1 (CH),120.6 (CH),120.6,111.1
(CH), 65.0 (CH), 61.4 (CH2), 48.3 (CH), 40.0 (CH), 30.0 (CH3), 26.0
(CH2), 24.3 (CH3), 21.5 (CH3), 14.3 (CH3); HR-ESI-MS: m/z¼453.1838
(calcd 453.1843 for C25H29N2O4S); IR:
n
¼1731 cmꢃ1. 6aS,9S,9aR-
Isomer (trans-C/D ring connection): 1H NMR (600 MHz; CDCl3):
(600 MHz; CDCl3):
d
¼8.33 (d, J¼8.8 Hz, 2H), 8.06 (d, J¼8.8 Hz, 2H),
d
¼7.79e7.73 (m, 3H), 7.41 (d, J¼1.7 Hz, 1H), 7.27e7.21 (m, 3H), 7.05
7.78 (d, J¼8.4 Hz, 2H), 7.71 (d, J¼8.3 Hz, 1H), 7.27e7.26 (m, 3H), 7.23
(d, J¼7.6 Hz, 1H), 4.45e4.29 (m, 2H), 3.90 (d, J¼10.1 Hz, 1H), 3.15
(ddd, J¼12.4, 9.9,1.7 Hz,1H), 2.92 (dd, J¼15.6, 3.9 Hz,1H), 2.75 (ddd,
(s, 1H), 6.99 (d, J¼7.3 Hz, 1H), 4.42 (d, J¼5.7 Hz, 1H), 4.34 (q,
J¼7.1 Hz, 1H), 3.72 (
J
t, J¼5.6 Hz, 1H), 3.01 (dd, J¼16.2, 5.0 Hz, 1H),
J¼15.4, 12.6 Hz,1H), 2.36 (s, 3H), 2.03 ( dt, J¼12.4,12.3, 3.8 Hz,1H),
J
2.64e2.56 (m, 2H), 2.38 (s, 3H), 1.65 (s, 3H), 1.42 (t, J¼7.1 Hz, 3H),
1.44 (t, J¼7.1 Hz, 3H), 1.30 (s, 3H), 1.20 (s, 3H); 13C NMR (125 MHz;
1.00 (s, 3H); 13C NMR (125 MHz; CDCl3):
d¼171.8, 150.0, 147.0, 145.1,
CDCl3):
d¼174.1, 144.6, 135.3, 133.5, 132.1, 130.1, 129.6 (CH), 128.5,
135.3, 133.1, 129.9 (CH), 129.1, 128.7 (CH), 128.2, 126.8 (CH), 126.1
(CH), 124.1 (CH), 121.1 (CH), 120.8 (CH), 116.8, 111.6 (CH), 69.3,
66.5 (CH), 62.0 (CH2), 48.2 (CH), 37.8 (CH), 30.0 (CH3), 25.5 (CH2),
23.3 (CH3), 21.6 (CH3), 14.2 (CH3); HR-ESI-MS: m/z¼660.1445 (calcd
126.5 (CH), 125.4 (CH), 121.1, 121.0 (CH), 119.4 (CH), 111.4 (CH), 61.4
(CH), 61.2 (CH2), 58.7, 54.6 (CH), 43.5 (CH), 29.3 (CH3), s28.0 (CH2),
25.0 (CH3), 21.3 (CH3), 14.1 (CH3); HR-ESI-MS: m/z¼453.1843 (calcd
453.1843 for C25H29N2O4S); IR:
n
¼1731 cmꢃ1
;
660.1445 for C31H31N3NaO8S2); IR:
n
¼1747 cmꢃ1. 6aS,9S,9aR-Pyr-
rolidine 13b (trans-C/D ring connection): 1H NMR (600 MHz;
3.3.3. N-Tosyl-CPA(16). Acetyl-Meldrum’s acid (309 mg,1.66 mmol)
CDCl3):
d
¼8.34 (d, J¼8.9 Hz, 2H), 8.16 (d, J¼8.9 Hz, 2H), 7.78 (d,
was added to a solution of the denosylated precursor from the
preceding reaction (300 mg, 663 mmol) in dioxane (15 mL). The re-
J¼8.3 Hz, 2H), 7.71 (d, J¼8.3 Hz, 1H), 7.37 (d, J¼1.9 Hz, 1H),
7.29e7.26 (m, 3H), 7.01 (d, J¼7.3 Hz, 1H), 4.45 (d, J¼9.8 Hz, 1H),
4.53e4.42 (m, 2H), 3.41 (ddd, J¼12.8, 9.8, 1.9 Hz, 1H), 2.84 (dd,
J¼15.5, 4.0 Hz, 1H), 2.76 (dd, J¼15.4, 12.1 Hz, 1H), 2.40 (s, 3H), 1.98
(ddd, J¼12.4, 12.3, 4.0 Hz, 1H), 1.49 (t, J¼7.1 Hz, 3H), 1.43 (s, 3H), 1.40
action was stirred for 90 min at 80 ꢂC. Then a solution of KOH in
MeOH (0.1 M, 16.6 mL, 1.66 mmol) was added and stirring was
continued for another 60 min at 80 ꢂC. The solvent was removed
under reduced pressure and the residue separated between DCM
and aqueous ammonium chloride. The aqueous phase was extracted
twice with DCM and the combined organic layers were dried over
Na2SO4. Filtration and evaporation of the solvent under reduced
pressure gave a crude product (347 mg), which yielded 16 (260,
80%) as a 1:1 mixture of diastereomers after chromatographic
purification (DCM/MeOH 25/1). The diastereomers of a small
sample were separated by semipreparative HPLC (ACN/H2O 70/30).
Rf (DCM/MeOH 95/5): 0.45. (6aR,11aS,11bR)-N-tosyl-CPA 16a (cis-C/D
(s, 3H); 13C NMR (125 MHz; CDCl3):
d
¼172.1, 149.9, 147.9, 145.0,
135.5, 133.5, 130.4, 129.9 (CH), 129.2, 128.8 (CH), 126.8 (CH), 125.9
(CH), 124.2 (CH), 121.4 (CH), 119.0 (CH), 118.6, 111.9 (CH), 67.4,
65.6 (CH), 62.2 (CH2), 54.7 (CH), 41.2 (CH), 27.6 (CH2), 25.7 (CH3),
23.7 (CH3), 21.6 (CH3), 14.2 (CH3); HR-ESI-MS: m/z¼660.1457 (calcd
660.1445 for C31H31N3NaO8S2); IR:
n
¼1751 cmꢃ1. (6aS,10S,10aR)-
Lactone 14: Rf (cyclohexane/ethylacetate 1/1): 0.39; 1H NMR
(600 MHz; CDCl3):
7.89 (d, J¼8.4 Hz, 2H), 7.73 (d, J¼8.3 Hz, 1H), 7.65 (d, J¼1.8 Hz, 1H),
d¼8.24 (d, J¼8.8 Hz, 2H), 8.01 (d, J¼8.9 Hz, 2H),
ring connection): 1H NMR (600 MHz; CDCl3):
d¼7.85 (d, J¼8.4 Hz,

3068
W.R.C. Beyer et al. / Tetrahedron 67 (2011) 3062e3070
2H), 7.78 (d, J¼8.3 Hz, 1H), 7.55 (s, 1H), 7.30e7.27 (m, 1H), 7.25 (d,
J¼8.2 Hz, 2H), 7.05 (d, J¼7.3 Hz,1H), 4.00 (d, J¼10.9 Hz,1H), 3.58 (dd,
J¼10.9, 6.0 Hz, 1H), 3.02 (dd, J¼16.3, 5.8 Hz, 1H), 2.94 (dd, J¼16.1,
IR
n
¼3402, 1694, 1594, 1557 cmꢃ1. 6aS,11S,11aR-Phenyl-CPA (trans-
C/D ring connection): 1H NMR (600 MHz; CDCl3):
d¼8.24 (d,
J¼7.6 Hz, 2H), 8.03 (s br, 1H), 7.63 (t, J¼7.3 Hz, 1H), 7.54
(t, J¼7.6 Hz, 2H), 7.34 (s, 1H), 7.23 (d, J¼7.8 Hz, 1H), 7.18 ( t,
J¼7.4 Hz, 1H), 6.95 (d, J¼6.9 Hz, 1H), 4.19 (d, J¼10.5 Hz, 1H), 3.16
t, J¼11.2 Hz, 1H), 3.03e3.01 (m, 2H), 2.48 (ddd, J¼11.6, 9.7,
6.8 Hz, 1H), 1.68 (s, 3H), 1.68 (s, 3H); 13C NMR (125 MHz; CDCl3):
11.7 Hz, 1H), 2.59 (
Jdt, J¼11.6, 5.9, 5.9 Hz, 1H), 2.49 (s, 3H), 2.36 (s,
J
3H), 1.67 (s, 3H), 1.58 (s, 3H); 13C NMR (125 MHz; CDCl3):
d¼194.3,
185.0, 175.4, 144.7, 135.6, 133.1, 129.8 (CH), 129.3, 128.4, 126.9 (CH),
125.5 (CH), 122.9 (CH), 120.6 (CH), 116.2, 111.6 (CH), 105.4, 70.7 (CH),
63.6, 52.4 (CH), 35.4 (CH), 26.1 (CH3), 26.1 (CH2), 24.4 (CH3), 21.5
(CH3), 19.8 (CH3); HR-ES-MS: m/z¼513.1449 (calcd 513.1455 for
(J
d
¼192.2, 181.6, 176.6, 134.0, 133.5 (CH), 132.3, 130.2, 129.5 (CH),
128.1 (CH), 127.1, 123.1 (CH), 119.6 (CH), 117.1 (CH), 112.1, 108.9
(CH), 103.5, 71.6 (CH), 62.8, 58.9 (CH), 39.2 (CH), 27.9 (CH3), 27.5
(CH2), 19.9 (CH3); HR-ESI-MS: m/z¼399.1702 (calcd 399.1703 for
C27H26N2NaO5S); IR:
n
¼3415, 1709, 1614 cmꢃ1. (6aS,11aS,11bR)-N-
Tosyl-CPA 16b (trans-C/D ring connection): 1H NMR (600 MHz;
CDCl3):
J¼1.7 Hz, 1H), 7.29 (
J¼7.3 Hz, 1H), 4.09 (d, J¼10.7 Hz, 1H), 2.95 (
2.92 (dd, J¼15.6, 4.6 Hz, 1H), 2.88 (dd, J¼15.5, 11.8 Hz, 1H), 2.52 (s,
3H), 2.37 (s, 3H), 2.32, (
dt, J¼11.8, 11.8, 4.6 Hz,1H), 1.60 (s, 3H), 1.53
(s, 3H); 13C NMR (125 MHz; CDCl3):
¼193.6, 185.6, 174.6, 144.7,
d
¼7.85 (d, J¼8.4 Hz, 2H), 7.82 (d, J¼8.2 Hz, 1H), 7.55 (d,
t, J¼7.8 Hz, 1H), 7.25 (d, J¼8.1 Hz, 2H), 7.06 (d,
dt, J¼11.4, 1.7 Hz, 1H),
C25H23N2O3); IR
n
¼3391, 1695, 1593, 1558 cmꢃ1. 6aS,11S,11aR-N-
J
Tosyl-phenyl-CPA: 1H NMR (600 MHz; CDCl3):
d
¼8.23 (d, J¼7.6 Hz,
J
2H), 7.83 (d, J¼8.3 Hz, 2H), 7.82 (d, J¼8.4 Hz, 1H), 7.68 (s, 1H), 7.66
(t, J¼7.3 Hz, 1H), 7.58 (t, J¼7.5 Hz, 2H), 7.29 (t, J¼7.6 Hz, 1H), 7.23
(d, J¼8.0 Hz, 2H), 7.07 (d, J¼7.2 Hz, 1H), 4.17 (d, J¼10.4 Hz, 1H),
J
d
2.99 (
(dd, J¼15.5, 11.9 Hz, 1H), 2.37 (
3H), 1.64 (s, 3H), 1.58 (s, 3H); 13C NMR (125 MHz; CDCl3):
J
t, J¼11.3, 11.3 Hz, 1H), 2.95 (dd, J¼15.5, 4.5 Hz, 1H), 2.90
135.7, 133.7, 130.9 (CH), 129.9, 129.4, 126.9 (CH), 125.6 (CH), 121.3
(CH), 121.2 (CH), 118.0, 111.9 (CH), 105.3, 71.2 (CH), 62.1, 58.0 (CH),
38.4 (CH), 28.0 (CH3), 27.2 (CH2), 21.5 (CH3), 19.8 (CH3), 19.7 (CH3);
HR-ES-MS: m/z¼489.1497 (calcd 489.1490 for C27H25N2O5S); IR:
J
dt, J¼11.9, 11.9, 4.4 Hz, 1H), 2.36 (s,
d
¼191.5,
184.6, 176.6, 144.7, 135.7, 133.7 (CH), 133.7, 132.1, 130.9, 129.8 (CH),
129.5 (CH), 129.4, 128.2 (CH), 126.9 (CH), 125.5 (CH), 121.4 (CH),
121.3 (CH), 118.0, 111.9 (CH), 103.3, 70.6 (CH), 62.6, 57.9 (CH), 38.6
(CH), 27.9 (CH3), 27.2 (CH2), 21.5 (CH3), 19.7 (CH3); HR-ESI-MS: m/
n
¼3362, 1611 cmꢃ1
.
3.3.4. N-Tosyl-cyclopropyl-CPA. N-Tosyl-cyclopropyl-CPA was pre-
pared as described for N-tosyl-CPA (16). The reaction was moni-
tored by TLC and continued until the open chain intermediate was
z¼553.1791 (calcd 553.1792 for C32H29N2O5S); IR
n
¼1695, 1595,
1563 cmꢃ1
.
consumed completely. 1H NMR (600 MHz; CDCl3):
d¼7.96e7.43
3.3.8. Cyclopiazonic acid (1), iso-cyclopiazonic acid (17) and alltrans-
cyclopiazonic acid (18). Compound 16 (50 mg, 102 mol) was dis-
solved in MeOH/THF (1:1, 6 mL) and Cs2CO3 (118 mg, 362 mol)
(m br, 4H), 7.35e6.82 (m br, 4H), 4.14e3.76 (m br, 1H), 3.79e3.08
(m br, 2H), 3.00e2.00 (m br, 7H), 1.75e0.72 (m br, 10H); HR-ES-
m
m
MS: m/z¼515.1645 (calcd for 515.1646 C29H27N2O5S); IR:
n
¼3399,
was added. The resulting solution was stirred for 94 h under
reflux. Then the solvent was removed and the residue separated
between DCM and aqueous ammonium chloride. The aqueous
phase was extracted five times with DCM. The combined organic
layers were dried over Na2SO4, filtered and the solvent was
removed under reduced pressure. Chromatographic purification
(DCM/MeOH 9/1) of the crude material (38 mg) yielded CPA
(20.4 mg, 60%) as a mixture of three stereoisomers, which was
separated by semipreparative HPLC (ACN/H20 65/35þ0.1%
HCOOH). Cyclopiazonic acid:1 Rf (DCM/MeOH 1/1): 0.22; 1H NMR
1608 cmꢃ1
.
3.3.5. N-Tosyl-cyclohexyl-CPA. N-Tosyl-cyclohexyl-CPA was pre-
pared as described for N-tosyl-CPA (16). The reaction was monitored
by TLC and continued until the open chain intermediate was con-
sumed completely. 1H NMR (600 MHz; CDCl3):
d
¼7.91e7.51 (m br,
4H), 7.31e7.20 (m br, 3H), 7.09e7.04 (m br,1H), 4.06e3.48 (m br, 2H),
3.25e2.74 (m br, 3H), 2.38e2.28 (m br, 5H), 1.93e0.88 (m br, 16H);
HR-ESI-MS: m/z¼559.2261 (calcd 559.2261 for C32H35N2O5S); IR:
n
¼3423, 1685, 1611 cmꢃ1
.
(600 MHz; CDCl3):
d
¼8.09 (s, br, 1H), 7.21 (d, J¼8.1 Hz, 1H), 7.17 (s,
1H), 7.16 (dd, J¼8.1, 7.0 Hz, 1H), 6.91 (d, J¼7.0 Hz, 1H), 4.07 (d,
J¼11.1 Hz, 1H), 3.66 (dd, J¼11.0, 5.8 Hz, 1H), 3.09e3.02 (m, 2H),
2.66e2.62 (m, 1H), 2.45 (s, 3H), 1.68 (s, 3H), 1.64 (s, 3H); 13C NMR
3.3.6. N-Tosyl-benzyl-CPA. Additional amounts of KOH/MeOH and
prolonged reaction times were needed for the complete conversion
of the 3-oxo-3-phenyl-propionamide intermediate. 1H NMR
(125 MHz; CDCl3):
d
¼195.1, 184.8, 175.3, 133.4, 128.7, 125.9, 123.1
(600 MHz; CDCl3):
d
¼7.86e7.61 (m br, 3H), 7.47e6.88 (m br, 10H),
(CH), 120.8 (CH), 116.5 (CH), 110.1, 108.7 (CH), 105.6, 71.8 (CH), 63.4,
53.1 (CH), 36.1 (CH), 26.6 (CH2), 26.4 (CH3), 24.4 (CH3), 19.7 (CH3);
HR-ESI-MS: m/z¼335.1404 (calcd 335.1401 for C20H19N2O3); IR:
5.29e3.31 (m br, 3H), 2.89e2.05 (m br, 5H), 1.72e0.74 (m br, 9H);
HR-ESI-MS: m/z¼567.1949 (calcd 567.1948 for C33H31N2O5S); IR:
n
¼3349, 1713, 1673, 1614 cmꢃ1
.
n
¼3394, 1704, 1608 cmꢃ1
.
iso-Cyclopiazonic acid:17 1H NMR
(600 MHz; CDCl3):
d
¼7.91 (s, br, 1H), 7.13e7.12 (m, 2H), 6.90
3.3.7. N-Tosyl-phenyl-CPA and phenyl-CPA (23). Additional amounts
of KOH/MeOH and prolonged reaction times were needed for the
complete conversion of the 3-oxo-3-phenyl-propionamide in-
termediate. Under this reaction conditions a significant cleavage of
the N-tosyl group was observed, yielding directly two phenyl-CPA
diastereomers. The reaction mixture was separated by semi-
preparative HPLC (ACN/H2O 75/25) after work-up as described
above. 6aR,11S,11aR-Phenyl-CPA (cis-C/D ring connection): 1H NMR
(s, 1H), 6.87e6.86 (m, 1H), 4.51 (d, J¼5.6 Hz, 1H), 3.84 (
Jt,
J¼5.1 Hz, 1H), 3.32 (dd, J¼17.4, 6.2 Hz, 1H), 2.98 (d, J¼17.6 Hz, 1H),
2.96 (
J
t, J¼5.6 Hz, 1H), 2.47 (s, 3H), 1.51 (s, 3H), 0.90 (s, 3H); 13C
NMR (125 MHz; CDCl3):
d¼194.4, 184.4, 173.0, 133.5, 128.8, 127.3,
123.3 (CH), 120.0 (CH), 116.3 (CH), 108.5, 108.2 (CH), 106.2, 71.7,
62.6 (CH), 52.9 (CH), 35.6 (CH), 30.0 (CH3), 26.4 (CH2), 21.5 (CH3),
19.9 (CH3); HR-ESI-MS: m/z¼337.1531 (calcd 337.1547 for
C20H21N2O3); IR:
n
¼3444, 1682, 1599 cmꢃ1. alltrans-Cyclopiazonic
(600 MHz; CDCl3):
d
¼8.21 (d, J¼7.7 Hz, 2H), 8.13 (s br, 1H), 7.61 (t,
acid:18 1H NMR (600 MHz; CDCl3):
d
¼8.02 (s, br, 1H), 7.31 (s, 1H),
J¼7.2 Hz, 1H), 7.51 (t, J¼7.5 Hz, 2H), 7.23 (d, J¼7.9 Hz, 1H), 7.19e7.17
(m, 2H), 6.95 (d, J¼6.7 Hz, 1H), 4.18 (d, J¼11.0 Hz, 1H), 3.73 (dd,
J¼10.9, 5.7 Hz, 1H), 3.12e3.11 (m, 2H), 2.71e2.67 (m, 1H), 1.76
7.25 (d, J¼8.1 Hz, 1H), 7.19e7.17 (m, 1H), 6.95 (d, J¼7.0 Hz, 1H), 4.12
(d, J¼10.8 Hz, 1H), 3.11 ( t, J¼11.3 Hz, 1H), 3.01e2.98 (m, 2H), 2.51
J
(s, 3H), 2.44 (ddd, J¼11.7, 10.1, 6.6 Hz, 1H), 1.64 (s, 3H), 1.58 (s, 3H);
HR-ESI-MS: m/z¼335.1402 (calcd 335.1401 for C20H19N2O3); IR:
(s, 3H), 1.71 (s, 3H); 13C NMR (125 MHz; CDCl3):
d¼193.4, 181.1,
177.3, 133.5 (CH), 133.4, 132.2, 129.6 (CH), 128.6, 128.1 (CH), 125.9,
123.0 (CH), 121.0 (CH), 116.5 (CH), 110.1, 108.7 (CH), 103.6 (CH), 71.1
(CH), 64.0 (C), 53.0 (CH), 36.2 (CH), 26.6 (CH2), 26.4 (CH3), 24.3
(CH3); HR-ESI-MS: m/z¼399.1703 (calcd 399.1703 for C25H23N2O3);
n
¼3407, 1659, 1590 cmꢃ1
.
The acyl tetramic acids 19, 20 and 21 were prepared as described
for CPA (1). The reactions were monitored by TLC and continued
until complete conversion of the starting material. Cyclopropyl-CPA

W.R.C. Beyer et al. / Tetrahedron 67 (2011) 3062e3070
3069
19: Rf (DCM/MeOH 9/1): 0.38; ESI-MS: m/z¼745.3 ([(MꢃH)2þNa]ꢃ,
22), 361.2 ([MꢃH]ꢃ, 100); HR-ESI-MS: m/z¼361.1557 (calcd
361.1558 for C22H21N2O3). Cyclohexyl-CPA 20: ESI-MS: m/z¼829.4
([(MꢃH)2þNa]ꢃ, 6), 403.2 ([MꢃH]ꢃ, 100); HR-ESI-MS: m/z¼
405.2179 (calcd 405.2173 for C25H29N2O3). Benzyl-CPA 21: ESI-MS:
m/z¼845.3 ([(MꢃH)2þNa]ꢃ, 3), 411.2 ([MꢃH]ꢃ, 100); ESI-MS: m/
z¼413.2 ([MþH]þ, 35), 280.2 ([C17H16N2O2]þ, 100). During the
tosylate cleavage reaction small amounts of N-methyl-benzyl-CPA
22 were isolated: ESI-MS: m/z¼873.4 ([(MꢃH)2þNa]ꢃ, 2), 425.2
([MꢃH]ꢃ, 100).
for 90 h at room temperature. After filtration the solution was di-
luted with water (10 mL) and HCl (1 M, 10 mL) and extracted with
DCM. The combined organic layers were dried over Na2SO4, filtered
and evaporated under reduced pressure to afford a crude product
(11.9 mg), which yielded 28 (6.6 mg, 41%) after chromatographic
purification (DCM, then DCM/MeOH gradient). Rf (DCM/MeOH
6/1): 0.72; 1H NMR (600 MHz; CDCl3):
d
¼13.60e11.49 (m br, 1H),
8.16e7.96 (3ꢁs, 1H), 7.25e6.86 (m, 4H), 4.66e4.03 (m,
1H), 3.94e3.77 (6ꢁs, 3H), 3.68e2.93 (m, 3H), 3.17e2.93 (m, 2H),
2.64e2.41 (6ꢁs, 3H), 1.66e0.87 (11ꢁs, 6H); HR-ESI-MS: m/
z¼366.1813 (calcd 366.1812 for C21H24N3O3); IR: ¼3307, 1668,
n
3.3.9. CPA-amine (24). CPA (19.3 mg, 57.4
mmol) was dissolved in
1626, 1574 cmꢃ1
.
dioxane (2 mL) and aqueous ammonia solution (25%, 2.2 mL) was
added. The reaction was stirred for 18 h at room temperature, di-
luted with water (10 mL) and HCl (37%, 0.5 mL). The solution was
extracted four times with DCM (5 mL). The combined organic layers
were dried over Na2SO4, filtered and evaporated under reduced
pressure to yield 24 (11.8 mg, 61%). Rf (DCM/MeOH 9/1): 0.48; 1H
4. Conclusion
The first enantioselective synthesis of cyclopiazonic acid (CPA,
1), an inhibitor of sarcoplasmic reticulum Ca2þ-ATPase, has been
accomplished by a modification of the Knight synthesis. Several
CPA analogues were prepared by varying the acyl residues in the
tetramic acid unit and by direct modification of the acetyl group
with nitrogen nucleophiles. The structureeactivity data of these
analogues proof the tetramic acid moiety the pharmacophoric unit
of CPA. Any negative interference with the Mg2þ binding and
hydrogen bond-forming properties of the tetramic acid results in at
least considerable losses of SERCA activity. Nevertheless, structur-
eeactivity data as well as the binding model of CPA suggest
structural modifications at the indole system or ring C to be pos-
sible without immediate loss of activity. Thus, future work should
aim at designing structurally less complex CPA analogues as novel
antimalarial or insecticidal drugs.
NMR (600 MHz; CDCl3):
d
¼10.07e9.67 (m, 1H), 8.19e7.99 (m, 1H),
7.23e7.11 (m, 3H), 6.95e6.92 (m, 1H), 5.94e5.68 (m, 1H), 4.10e4.02
(m, 1H), 3.68e2.37 (m, 4H), 2.56e2.50 (3ꢁs, 3H), 1.68e0.90 (6ꢁs,
6H); HR-ESI-MS: m/z¼336.1707 (calcd 336.1707 for C20H22N3O2);
IR:
n
¼3307, 1669, 1616 cmꢃ1
.
3.3.10. CPA-hydrazine (25). CPA (19.0 mg, 56.5
mmol) was dissolved
in CHCl3/MeOH (1:1, 3 mL) and hydrazine hydrate (55
mL,
1.13 mmol) was added. The solution was stirred for 18 h at room
temperature, diluted with water (10 mL) and HCl (37%, 0.5 mL) and
then extracted four times with DCM (5 mL). The combined organic
layers were dried over Na2SO4, filtered and evaporated under
reduced pressure to give 25 (16.7 mg, 84%). Rf (DCM/MeOH 9/1):
0.46; 1H NMR (600 MHz; CDCl3):
d
¼8.47e8.06 (m, 1H), 7.22e6.85
Acknowledgements
(m, 4H), 4.45e3.61 (m, 2H), 3.08e2.88 (m, 3H), 2.69e2.65 (m, 3H),
1.64e0.86 (6ꢁs, 6H); ESI-MS: m/z¼699.3 ([2MꢃH]ꢃ, 9), 349.2
([MꢃH]ꢃ, 100); HR-ESI-MS: m/z¼351.1817 (calcd 351.1816 for
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.
C20H23N4O2); IR:
n
¼1677, 1586 cmꢃ1
.
3.3.11. CPA-p-chlorphenylhydrazine (26). CPA (10.0 mg, 29.7
was dissolved in CHCl3/MeOH (1:1, 3 mL), p-chlorphenylhydrazine
(106 mg, 595 mol) was added and the suspension was stirred for
mmol)
References and notes
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with water (10 mL) and HCl (1 M, 10 mL) and extracted with DCM.
The combined organic layers were dried over Na2SO4, filtered and
evaporated under reduced pressure to afford a crude product
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NMR (600 MHz; CDCl3):
d
¼8.26e8.03 (3ꢁs br, 1H), 7.18e6.89 (m,
4H), 4.56e1.91 (m, 8H), 1.57e0.79 (m, 6H); ESI-MS: m/z¼701.3
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ꢂ
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ꢂ
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m
mol)
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m

3070
W.R.C. Beyer et al. / Tetrahedron 67 (2011) 3062e3070
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