Synthesis and biological evaluation of (-)- and (+)-debromoflustramine B and its analogues as selective butyrylcholinesterase inhibitors

Article abstract of DOI:10.1021/jm800277g

A series of pyrrolidinoindolines have been synthesized as debromoflustramine B (4a) analogues for their evaluation as cholinesterase inhibitors. Structure-activity studies of this series revealed the optimum pharmacophore elements required for activity and resulted in the discovery of selective butyrylcholinesterase inhibitors with micromolar potency. Biological testing demonstrated that (-)-4a was 7500 times more potent than its enantiomer (+)-4b. The most active inhibitor against BChE in the series was demethyldebromoflustramine B (5a), with an IC₅₀ value of 0.26 μM. X-ray crystallography of 15 and docking studies of selected compounds into human BChE (PDB 1POI) are presented. Molecular modeling studies showed that π-hydrogen bond, classical hydrogen bond, and cation-π interactions are critical for optimum potency.

Full text of DOI:10.1021/jm800277g

J. Med. Chem. 2008, 51, 5271–5284
5271
Synthesis and Biological Evaluation of (-)- and (+)-Debromoflustramine B and Its Analogues
as Selective Butyrylcholinesterase Inhibitors
†
‡
,†
Ernesto Rivera-Becerril,†,‡ Pedro Joseph-Nathan, V´ıctor M. Pe´rez-Alvarez, and Martha S. Morales-R´ıos*
´
Departamento de Qu´ımica y Seccio´n Externa de Farmacolog´ıa, Centro de InVestigacio´n y de Estudios AVanzados del Instituto Polite´cnico
Nacional, Apartado 14-740, Me´xico, D. F., 07000 Me´xico
ReceiVed March 13, 2008
A series of pyrrolidinoindolines have been synthesized as debromoflustramine B (4a) analogues for their
evaluation as cholinesterase inhibitors. Structure-activity studies of this series revealed the optimum
pharmacophoric elements required for activity and resulted in the discovery of selective butyrylcholinesterase
inhibitors with micromolar potency. Biological testing demonstrated that (-)-4a was 7500 times more potent
than its enantiomer (+)-4b. The most active inhibitor against BChE in the series was demethyldebromof-
lustramine B (5a), with an IC50 value of 0.26 µM. X-ray crystallography of 15 and docking studies of
selected compounds into human BChE (PDB 1POI) are presented. Molecular modeling studies showed that
π-hydrogen bond, classical hydrogen bond, and cation-π interactions are critical for optimum potency.
Cholinesterase inhibitors have been used as therapeutic targets
in Alzheimer’s disease (AD)a, one of the most common type
of adult-onset dementia.1 The cholinergic hypothesis postulates
that memory impairments in patients with AD result from a
deficit of cholinergic function in the brain.2 Indeed, the
cholinergic system is the earliest and most profoundly affected
neurotransmitter system in AD, in which decreasing levels of
neurotransmitter acetylcholine (ACh) and its hydrolyzing en-
zyme acetylcholinesterase (AChE) correlate positively with
losses of cholinergic neurons and synapses occurring in the
forebrain, cortex, and hippocampus.3 Studies in AD patients with
severe pathology have shown that these changes occur con-
comitantly with a significant increase of butyrylcholinesterase
(BChE), while AChE is reduced in specific brain regions.4 The
survival of AChE knockout mice with normal levels and
localization of BChE shows that this enzyme is likely to play
a constitutive role in the hydrolysis of ACh in the normal brain.5
Therefore, both enzymes are likely to have involvement in
regulating ACh levels and represent therapeutic targets to
ameliorate the cholinergic deficit. Recent evidence suggests that
BChE may also have a role in the etiology and progression of
AD beyond regulation of synaptic ACh levels.6 Studies examin-
ing the relationship between BChE polymorphisms and the
progression of cognitive impairment in dementia with Lewy
bodies and AD suggests that BChE is present in key brain areas
and may influence the aggregation of neuritic ꢀ-amyloid (Aꢀ)
plaques to form the neurofibrillary plaques causing dementia.7,8
These accumulating data suggest that BChE may be particularly
important in individuals with more severe dementia as BChE
activity increases with disease development. Therefore, the
development of selective BChE inhibitors may be of great
interest to clarify the physiological role of this enzyme in the
normal, aging, and diseased brain and to provide therapeutics
for various diseases, including AD. Current treatment for AD
with the use of reversible dual cholinesterase inhibitors, such
as rivastigmine, support a role for the central inhibition of BChE
in addition to AChE based on the high correlation of the former
with cognitive improvement.9 The use of the more selective
BChE inhibitors tacrine and tetrahydroaminoacridine are limited
by its hepatotoxicity as well as side effects stemming from their
low target specificity.10 Recently, selective, reversible cym-
serine-based BChE inhibitors were synthesized, improving
cognition and modulating neuropathological markers of AD.11
Although highly similar (50-60%), the enzymes AChE and
BChE are encoded by different genes and clearly differ in
substrate specificity and sensitivity to inhibitors, likely because
of a larger void at the BChE active site gorge and/or structural
differences in the catalytic site. The existence of a peripheral
site at the lip of the gorge of equine and human BChEs was
hypothesized using molecular docking techniques, and potent
tacrine-based BChE inhibitors were developed in order to
validate such hypothesis.12 To define the role of BChE in brain,
potent analogues of phenserine were designed based on the
X-ray crystallographic structure of the binding sites for ACh
that differentiate BChE from AChE.13
* To whom correspondence should be addressed. Phone: +(52 55)5747
7112. Fax: +(52 55)5747 7137. E-mail: smorales@cinvestav.mx.
†
Departamento de Qu´ımica, Centro de Investigacio´n y de Estudios
Avanzados del Instituto Polite´cnico Nacional.
‡
Besides the discovery of naturally occurring physostigmine
(1) as a potent cholinesterase inhibitor,14 there is a continuing
search for new classes of natural products capable of alleviating
some of the symptoms of AD (Figure 1). Between them,
huperzine A (2), a Lycopodium alkaloid isolated from the
Chinese herb, Huperzia serrata, is a reversible, potent, and
selective AChE inhibitor.15 Another selective AChE inhibitor,
galanthamine (3), is an alkaloid isolated from the common
snowdrop Galanthus niValis.16 In addition, several different
types of steroids, terpenoids, and tropan alkaloids have been
shown to inhibit BChE.17 At this point, we previously reported
the synthesis of racemic and enantiomerically pure debromof-
Seccio´n Externa de Farmacolog´ıa, Centro de Investigacio´n y de Estudios
Avanzados del Instituto Polite´cnico Nacional.
a Abbreviations: Aꢀ, ꢀ-amyloid plaques; ACh, acetylcholine; AChE,
acetylcholinesterase; AD, Alzheimer’s disease; APCIMS, atmospheric
pressure chemical ionization mass spectrometry; BChE, butyrylcholinest-
erase; ∆Gbind, estimated binding free energy for clusters; DMSO, dimeth-
ylsulfoxide; ESI, electrospray ionization; GC/MS, gas chromatography
coupled to mass spectrometry; HRMS, high-resolution mass spectrometry;
IC50, half-maximal inhibitory concentration; LAH, lithium aluminum
hydride; PDB, protein data bank; pKa, minus the decimal logarithm of acid
dissociationconstants;rmsd,root-mean-squaredeviation;SAR,structure-activity
relationship; TBAHS, tetrabutylammonium bisulfate; THF, tetrahydrofuran;
TLC, thin layer chromatography; TMS, tetramethylsilane; 3D, three-
dimension; CH2-4Py, pyridin-4-ylmethyl. Single-letter and three-letter
abbreviations are used for amino acids.
10.1021/jm800277g CCC: $40.75
2008 American Chemical Society
Published on Web 08/08/2008

5272 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
RiVera-Becerril et al.
Table 1. Structures of C(3a) Prenylated Pyrrolidinoindolines 4a-c and
5a-13 and Their in Vitro Inhibition of BChE
Figure 1. Structures of cholinesterase inhibitor alkaloids 1-4a.
lustramine B (4),18,19 a naturally occurring prenylated pyrroli-
dinoindoline isolated from the cheilostome bryozoan Flustra
foliacea,20 lacking the carbamate pharmacophore of physostig-
mine (1). In this article, we describe in vitro anticholinesterases
evaluation of (-)- and (+)-debromoflustramine B (4a and 4b).
On the basis of a rational approach, a series of racemic structural
analogousweredesignedandadiscussionoftheirstructure-activity
relationships is reported.
Chemistry
The structures of all evaluated compounds are shown in
Tables 1 and 2. In Table 1, the prenyl group at C(3a) (R3) was
kept in all cases, and the substituents R2 and R4 were varied
between prenyl, benzyl, and methyl groups. Further modification
of the scaffold molecule include the replacement of the
substituent prenyl at C(3a) with either an aromatic core, such
as benzyl or pyridin-4-ylmethyl (CH2-4Py), or with an alkyl
group varied from methyl to 2-methylbutyl, as shown in Table
2. The synthesis of the target molecules 4-22 and 27 was
accomplished using our previously developed methodology.18,19
Scheme 1 represents the synthesis of 4c and derivatives 5-9,
14, and 16-22, in which the alkyl groups at C(3a) and N(8)
are the same. Commercially available 3-acetonitrilindole 23a
or its 5-methoxy derivative 23b21 were converted to oxindoles
24a and 24b, respectively, by treatment with a mixture of
DMSO/HCl and served as late-stage intermediates for rapid
analogue preparation. Facile N- and C-alkylation of 24a,b with
the proper alkyl halide under mild phase-transfer conditions gave
dialkylated oxindoles 25a-h, which by subsequent reductive
cyclization and either N(1)-prenylation, N(1)-benzylation, or
one-pot selective N(1)-monomethylation of the resulting pyr-
rolidinoindolines 5a-h allowed access to 4c and a variety of
analogues 5-9, 14, 16-18, and 20-22 in four steps. Compound
19 (R2 ) R3 ) 2-methylbutyl) was prepared from 4c (R2 ) R3
) prenyl) by hydrogenation on 10% Pd/C.
a Human BChE was used. The IC50 values represent the concentration
of inhibitor required to decrease enzyme activity by 50% and are the average
of three independent measurements, each performed in triplicate. b ni )
No inhibition a < 10000 µM.
by reaction of 5i-l with CH2O, MeOH, and then NaBH4 to
give 10-12 and 15.
To test whether the nitrogen atom at 1-position in pyrrolidines
is crucial for good inhibitory activity, we decided to synthesize
the furoindoline analogue 27 (Scheme 3). Thus, reductive
cyclization of the sodium salt of 2-oxo-3-indolyl acetic acid
2619 with LAH/THF after cooling the reaction mixture in an
ice/water bath gave the desired furoindoline 27 in 87% yield,
together with a small amount (<5%) of alcohol 28. It is
noteworthy that similar transformation under reflux produced
alcohol 28 in 93% yield.
Analysis of NMR spectral data and crystal structure deter-
mination of 15 (Figure 2) led to the assignment of the
compounds. A prominent structural feature of 15 arising from
the X-ray study is that the N(8)-benzyl group remains nearly
perpendicular to the indole plane, whereas the pyridinylmethyl
group is projected toward the exo-surface of the indole portion.
This feature persists in solution, as demonstrated by the observed
diagnostic cross-peak between H(8a) (δ 4.30) and one of the
diastereotopic methylene protons (δ 2.84) of the pyridinylmethyl
group in the NOESY spectrum. In the same way, the observed
cross-peaks between H(8a) and the vinylic proton of the prenyl
group at C(3a) in the NOESY spectra measured for pyrrolidi-
noindolines 4c (δ 4.71, 4.99), 5a (δ 4.65, 5.05), and 6 (δ 4.41,
4.99) suggest that these molecules exist in solution in a
The synthesis of differentially 3a,8-dialkylated pyrrolidinoin-
dolines 10-13 and 15 required introduction of the alkyl group
at the N(1) position of the corresponding indole prior to the
oxidation step (Scheme 2). Thus, alkylation of 23a,b by reaction
with the proper alkyl bromide gave 23c-e, oxidation with
DMSO/HCl then furnished 24c-e. These oxindole intermediates
were C(3)-alkylated to give 25i-l and transformed into desired
analogues 10-13 and 15 in two or three steps involving
reductive ring closure to pyrrolidinoindolines 5i-l and N(1)-
benzylation of 5j to give 13, or one-pot N(1)-monomethylation

Debromoflustramine B as SelectiVe BChE Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5273
Table 2. Structures of Pyrrolidinoindolines 14-22 and Their in Vitro
Inhibition of BChE
by alkyl groups resulted in complete loss of activity (16, 19-22
Table 2). Having determined the importance of retaining the
prenyl substituents, we were focused to clarify if both prenyl
groups are required for the best biological response. It was found
that the R2-Bn analogues 11 and 12 (Table 1) displayed greater
or almost equal inhibitory potency than their dibenzylated
counterparts 14 and 17 (Table 2) but exhibited ca. 4- and 2-time
less loss of inhibitory activity as compared to their diprenylated
counterparts 4c and 8 (Table 1). In addition, compound 10,
which replace the prenyl group with methyl at the R2 position,
or compound 15, which replace the benzyl group with pyridi-
nylmethyl at the R3 position, were associated with complete
loss of inhibition activity.
Evaluation of the R4-substituted analogues revealed that this
was also a sensitive area for activity where the R4-benzylated
analogues 7, 9, 13, and 18 resulted in significant (9) or total
loss of inhibition activity, while replacement of methyl with a
prenyl group at the R4 position of racemic parent 4c was well-
tolerated, as demonstrated in the analogue 6, which was roughly
equipotent with 4a. Unlike the SAR studies on carbamate-based
cholinesterase inhibitors, in which the replacement of the N(1)
and N(8) atoms with oxygen did not change significantly the
in vitro activity,14 in compound 27, this type of replacement
abolished the activity (data not shown). In addition, the effect
of adding a methoxy group at the R1 position was examined.
As the data indicated (Tables 1 and 2), such substitution resulted
in quite different outcomes for the activity. Minor effects were
observed for the R1-methoxylated analogues 8 and 12, moder-
ated effects for 17, while the inactive demethoxylated analogue
7 turned to be active 9 upon methoxylation (IC50 ) 49.98 (
7.11 µM), suggesting that the conformation was a critical factor
for receptor affinity.
a Human BChE was used. The IC50 values represent the concentration
of inhibitor required to decrease enzyme activity by 50% and are the average
of the three independent measurements, each performed in triplicate. b ni
) No inhibition at <10000 µM.
contributing conformation in which the C-prenyl group is
positioned over the exo-surface of the pyrrolidinoindoline core.
Discussion
Biological
All synthesized compounds, including enantiopure (4a, 4b)
and racemic (4c) debromoflustramine B, were evaluated in vitro
for their inhibitory action in both AChE and BChE. Compounds
were first screened at 1 mM, and those compounds that showed
good inhibitory activities (% inhibition >30%) were further
tested at nine concentration levels to generate concentration-
dependent curves from which the IC50 values were derived. Each
tested compound was assayed three times for triplicate. Experi-
mental screening of 20 compounds showed nine to be active as
BChE inhibitors, while all of them had very little if any
inhibitory activity on AChE. The effect on BChE inhibitory
activity of alterations made to R1-R4 of the pyrrolidinoindoline
scaffold is summarized in Tables 1 and 2. The role of chirality
was initially examined. Synthetic racemic debromoflustramine
B (4c) was determined to have an IC50 value of 2.59 ( 0.15
µM, whereas the corresponding natural occurring 3aS,8aR-(-)
enantiomer 4a was about 2 times more potent than 4c, while
the 3aR,8aS-(+) enantiomer 4b was found to be inactive, in
agreement with the established enantioselective behavior of the
classical AChE inhibitor physostigmine (1).14,22
Compounds 5a-22 and 27 were evaluated as racemic
mixtures, and some general observations can be made from SAR
studies on R1-R4. The more active inhibitors against BChE in
the series, 4a, 4c, 5a, 6, and 8, are R2 and R3 prenyl substituted.
Removal of the methyl group on the racemic parent compound
4c was beneficial, enhancing the inhibition activity 10-fold (5a,
Table 1). Replacement of the prenyl groups at R2 and R3
positions of 4c by benzyl groups caused a 8.5-fold drop in
inhibitory activity (14, Table 2), while replacement of a single
aromatic CH with nitrogen in the benzyl portion or replacing it
On the basis of the fact that R1 carbamoylated pyrrolidinoin-
dolines with a sterically bulky phenyl group at the R3 position
are known to lack cholinesterase inhibition activity,23 it is
surprising to find R3-prenyl substituted pyrrolidinoindolines
lacking the carbamate group that are micromolar cholinesterase
inhibitors. Close examination of the data is consistent, in any
case, with π-hydrogen bond interactions between the substrate
with the enzyme, as the most plausible mechanism. On the basis
of the SAR results outlined above, we postulated that the double
bond of the prenyl groups at the R2 and R3 positions is
interacting as a very specific π-hydrogen bond acceptor with
the enzyme. Because the π-cloud of aromatic rings can act also
as proton-acceptor site, it could be suggested that H-bonding
may play a significant role in the BChE inhibitory action of
benzylated compounds (11, 12, 14, and 17), whereas the
replacement of prenyl groups by heterocyclic aromatic rings
could result in a diminished ability of the π-cloud to form
H-bond interactions, resulting in complete loss of activity (15
and 16), in line with the π-electron density decrease in the order
benzene > pyridine > pyrimidine.24 In addition, not only does
the π-electron density matter but also the ring flexibility is
important, as this reveals the beneficial presence of the benzylic
methylene spacer, which made it easer to adopt the appropriate
conformation for interaction/binding of the compounds with
BChE to allow enzyme inhibition. To see how the aromatic
core benzyl or pyridinylmethyl compared, we calculated the low-
energy conformations of 14 and 15 and superimposed the
resulting structures (Figure 3a). The two low-energy conforma-
tions superimpose well (the structures are off-set slightly for
illustrative purposes). Moreover, superposition of 14 with

5274 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
RiVera-Becerril et al.
Scheme 1. Synthesis of Pyrrolidinoindolines Equally Substituted at N(1) and C(3a)a
a Reagents and conditions: (a) DMSO/37% aq HCl (1:5), rt, 1 h, 77-90%; (b) 15% aq NaOH, TBAHS, CH2Cl2, MeI or R2Br (R2Br ) R3Br, 2.1 equiv),
rt, 1.5-5 h, 72-91%; (c) LAH, THF, reflux, 1.5 h, 47-76%; (d) for 5a, 5b, 5d: 15% aq NaOH, TBAHS, CH2Cl2, R4Br (1.1 equiv), rt, 5-22 h, 48-65%;
(e) for 5a-h: CH2O, MeOH, rt, 3 h, then NaBH4, 43-67%; (f) H2/10% Pd/C, MeOH, rt, 28 h, 64%.
Scheme 2. Synthesis of Pyrrolidinoindolines Differentially Substituted at N(1) and C(3a)a
a Reagents and conditions: (a) 15% aq NaOH, TBAHS, CH2Cl2, MeI or R2Br (1.1 equiv), rt, 24-72 h, 60-87%; (b) DMSO/37% aq HCl (1:5), rt, 1 h,
58-76%; (c) 15% aq NaOH, TBAHS, CH2Cl2, R3Br (1.1 equiv), rt, 3-5 h, 70-77%; (d) LAH, THF, reflux, 1.5 h, 65-86%; (e) for 5j: 15% aq NaOH,
TBAHS, CH2Cl2, BnBr (1.1 equiv), rt, 24 h, 20%; (f) for 5i-l: CH2O, MeOH, rt, 3 h, then NaBH4, rt, 1 h, 54-79%.
debromoflustramine B (4a) reveals that the alignment of the
π-electrons of the benzyl groups of 14 and the π-electrons of
the prenyl groups of 4a are nearly coincident (Figure 3b). These
results also suggest that the loss in activity observed upon
replacement of a single aromatic CH with nitrogen in the benzyl
moiety is likely due to the diminished H-bond interaction rather
than due to an unfavorable conformational change. Finally, in
our study, the crucial pharmacophore role of the N1(H, CH3)
group, as demonstrated by the SAR analysis, is consistent with
a salt-bridge inhibitory mechanism because the basic N1-methyl
group of physostigmine (pKa ) 8.46) and analogues is presumed
to be largely protonated in the receptor to form a quaternary

Debromoflustramine B as SelectiVe BChE Inhibitor
Scheme 3. Synthesis of Furoindoline 27a
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5275
Figure 3. Minimum-energy conformations of compounds 4a (green),
14 (gray), and 15 (brown) and their aligned structures. (a) Superposition
of 14 and 15. (b) Superposition of 4a and 14.
Table 3. Results of 100 Independent Docking Runs for (-)-4a, (+)-4b,
and (-)-5a
b
c
compd
clustera
focc
∆Gbind
(-)-4a
1
2
1
2
3
4
5
6
1
2
65
5
4
12
7
5
20
8
22
35
-8.5
-8.4
-8.0
-7.9
-7.8
-7.7
-7.7
-7.6
-8.5
-8.2
a Reagents and conditions: (a) NaH, THF, 5 °C 30 min, then LAH, 5 °C
3 h, 87%; (b) NaH, THF, 5 °C 30 min, then LAH, reflux 3 h, 93%.
(+)-4b
(-)-5a
a The assessment of conformational cluster sizes was determined as
function of the root-mean-square deviation (rmsd) of 0.3 Å between
conformers. b The number of results contained in the clusters is given by
the frequency of occurrence, focc.
c ∆Gbind is the estimated free energy of
binding for the clusters and is given in kcal/mol.
algorithm together with an empirical free energy function.
Compounds (-)-4a, (+)-4b, and (-)-5a were docked into the
binding pocket of human BChE (PDB code 1POI),27 and the
results of prevailing clusters of each ligand, ranked according
to the total docking energy (∆Gbind), are summarized in Table
3. The binding modes derived from docking simulations of the
two enatiomers of 4 were characterized by significant different
score values (Table 3), which are consistent with the inhibition
data.
A hierarchical cluster analysis, carried out on 100 independent
dockings runs performed for (-)-4a, revealed that all the
docking poses could be collected in one prevalent geometric
Figure 2. X-ray crystal structure of 15.
group associated with the highest frequency of occurrence (focc
)
ammonium group. The presence of a prenyl group at the R4
position (6) introduces an additional double bond, which might
participate in additional π-hydrogen bond that probably
compensates for the loss of activity caused by an increase in
sterically bulky substitution.
Molecular Modeling Studies. Our SAR studies identified
that N(1), C(3a), and N(8) substitution in the pyrrolidinoindoline
skeleton by bulky π-electron-rich prenyl groups provided the
resulting compounds with a selectivity that favored BChE versus
AChE, in agreement with the larger void at the BChE active
site gorge.25 To explore the ligand’s protein-bound conformation
responsible for biological activity that explains the above-
described SAR studies, a molecular docking study was carried
out on selected conformers of ligands (-)-4a, (+)-4b, and (-)-
5a. Docking was carried out using the automated docking
program AutoDock,26 which allows torsional flexibility in the
ligand and incorporates an efficient Lamarckian genetic search
and therefore with low cost of the loss in conformational entropy
upon binding, within an rmsd value of 0.3 Å. Figure 4a (top
panel) shows the most favorable binding mode identified for
(-)-4a (∆Gbind ) -8.5 kcal/mol, focc ) 65) in the mid-gorge
of hBChE and therefore the bioactive one. As already noted,
the docking results (Figure 5a) reveals a cation-π interaction
between the ammonium nitrogen atom of (-)-4a and Trp82,
consistent with a distance between them of 3.5 Å. The
ammonium N(1) atom also favorably interacted with the anionic
residue Glu197 (5.2 Å). In addition, the C(3a) prenyl group
come close to the oxyanion hole,25 delimited by Gly116,
Gly117, and Ala199, with the prenyl double bond interacting
as π-hydrogen bond acceptor with the hydroxylic residues of
Thr120 (4.6 Å) and Tyr128 (4.2 Å). In the same way, the prenyl
double bond at N(8) is engaged in a π-hydrogen bond
interaction with Tyr332 (4.9 Å) and a weaker interaction with
Tyr440 (6.9 Å). In particular, it is reported that His438 is

5276 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
RiVera-Becerril et al.
Figure 4. Accessible surface of active site gorge of hBChE showing docked BChEIs (-)-4a (a) and (-)-5a (b,c).
Figure 5. Binding mode of BChEIs (-)-4a (a) and (-)-5a (b,c) as outcomes of docking simulations. (a) Glu197 has been omitted for clarity. (b)
Tyr128 has been omitted for clarity. Cation-π interactions are shown as dotted lines, classical hydrogen bond and π-hydrogen bonds are shown
as dashed lines.
important for BChE enzyme function, and it has been proposed
together with Ser198 as catalytic site for cocaine hydrolysis;28
however, the docking simulation of (-)-4a indicates that the
pharmacophore prenyl groups lie in an opposite region in space
to make any significant interaction with the catalytic triad
Ser198-His438-Glu325 (Figure S1 in Supporting Information).
Comparing the above results with the docking simulation and
cluster analyses of enantiomer (+)-4b reveals that this ligand
scored poorly against hBChE (Table 3) and consequently make
poorer interactions than those of their active enantiomer with
Trp82 (anionic site) and with the hydroxylic residues of Thr120,
Tyr128, Tyr332, and Tyr440.
Docking simulations of (-)-5a, which possesses the highest
anti-BChE activity (Table 1), revealed a very clear preference
for two prevailing clusters (Figure 4b,c) within an rmsd value
of 0.3 Å (Table 3). The top-ranking results (∆Gbind ) -8.5
kcal/mol, focc ) 22) clearly show that the N(1)-H is responsible
for anchoring the molecule at the BChE gorge by forming a

Debromoflustramine B as SelectiVe BChE Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5277
General Procedure 1 for the Preparation of Indoles 23c
and 23d. To a solution of the corresponding indole (23a or 23b)
(0.013 mol) in CH2Cl2 (65 mL) were added 15% aq NaOH (30
mL), TBAHS (0.14 g, 0.41 mmol), and benzyl bromide (0.015 mol).
The resulting mixture was stirred at room temperature until TLC
analysis showed complete loss of starting material. After completion
(24-30 h), the organic layer was collected and the aqueous phase
was extracted with CH2Cl2 (2 × 5 mL). The combined organic
phases were washed with brine (2 × 60 mL), dried, and concen-
trated under reduced pressure. The resulting indoles were purified
by flash chromatography on silica gel.
(1-Benzyl-1H-indol-3-yl)acetonitrile (23c). Prepared by stirring
23a for 30 h according to general procedure 1. Upon workup, the
crude product was purified by flash chromatography (1:4 EtOAc/
hexane) to give 23c as colorless crystals (2.78 g, 87%); mp 94-95
°C (Lit. 94-95 °C). The spectral and analytical data were consistent
with those reported.31
strong H-bond with the carboxylate group of Glu197 (H-O
2.0 Å, Figure 5b) treated as deprotonated at physiological pH.
A closer look at the model of (-)-5a (Figure 5b) showed
additional π-hydrogen bond interactions between the prenyl
double bond at C(3a) and Tyr440 (5.0 Å) as well as between
the prenyl double bond at N(8) and the hydroxyl oxygen atom
of catalytic Ser198 (2.4 Å). It seem clear that the H-bond
formation causes rotation of the tricyclic structure by nearly
180°, pointing the C(3a)-prenyl group toward Trp82. A signifi-
cant alternative to this position is given by the second-ranked
solution (∆Gbind ) -8.2 kcal/mol, focc ) 35), which resemble
that of prevailing cluster of (-)-4a. The main cation-π
interaction was observed between the ammonium nitrogen atom
of (-)-5a and Trp82 (3.2 Å) as well as π-hydrogen bond
interactions between the prenyl double bonds at C(3a) and N(8)
with the hydroxylic residues of Thr120, Tyr128, Tyr332, and
Tyr440 (Figure 5c).
(1-Benzyl-5-methoxy-1H-indol-3-yl)acetonitrile (23d). Pre-
pared by stirring 23b for 24 h according to general procedure 1.
Upon workup, the crude product was purified by flash chromatog-
raphy (1:6 EtOAc/hexane) to give 23d as pale-yellow oil (2.74 g,
77%). The elemental composition was consistent with that re-
ported.32 To our knowledge, the NMR data are unreported and
therefore follow. 1H NMR (CDCl3) δ 7.31-7.25 (m, 3H), 7.17
(dd, J ) 8.9, 0.6 Hz, 1H), 7.11 (br s overlap, 1H), 7.09 (br d,
overlap, 2H), 6.99 (br d, J ) 2.4 Hz, 1H), 6.87 (dd, J ) 8.9, 2.4
Conclusion
We have evaluated several substituted modified analogues
of natural occurring debromoflustramine B (-)-4a as cholinest-
erase inhibitors. An efficient parallel array synthesis of pyrro-
lidinoindolines led to rapid SAR development. A variety of alkyl
substitution produces analogues with micromolar cholinesterase
inhibitory activity. On the basis of docking studies, enhanced
potency for the conformationally rather rigid pyrrolidinoindo-
lines containing prenyl substituents at C(3a) and N(8) appears
to be the result of favorable π-hydrogen bond interactions
between the inhibitor and the hydroxylic amino acid residues
of Thr120, Tyr128, Tyr332, Tyr440, and Ser198. In addition,
cation-π interactions and classical hydrogen bond seems to be
critical for optimum potency. The most significant outcomes
from this study is that a clear in vitro activity of (-)-4a as
selective, reversible BChE inhibitor has been demonstrated and
that the prenyl groups are fundamental for properly anchoring
the molecule at the enzyme gorge. This information can be used
to design a second generation of prenylated pyrrolidinoindolines,
tailored to the BChE gorge, to produce a drug with both high
affinity and high degree of selectivity for this enzyme.
Hz, 1H), 5.24 (s, 2H), 3.86 (s, 3H), 3.79 (d, J ) 0.7 Hz, 2H). 13
C
NMR (CDCl3) δ 154.4, 137.0, 131.9, 128.8 (2C), 127.8, 127.2,
127.0, 126.8 (2C), 118.2, 112.9, 111.0, 103.0, 99.9, 55.8, 50.3, 14.4.
EIMS m/z (relative intensity) 276 (M+, 100), 91 (100), 65 (19).
HRMS (ESI/APCIMS) calcd for C18H16N2O + H, 277.1341; found,
277.1338.
General Procedure 2 for the Preparation of Oxindoles
24b-24d. To a solution of the corresponding 3-indolylacetonitrile
(23b, 23c, or 23d) (8.5 mmol) in DMSO (7 mL) was added
dropwise and under stirring 37% aq HCl (35 mL). The resulting
mixture was stirred at room temperature until TLC analysis showed
complete loss of starting material. After completion (1-24 h), the
resulting mixture was cooled to 5 °C, diluted with water (15 mL),
and solid K2CO3 was added until pH ca. 7-8. The mixture was
allowed to warm to room temperature and extracted with EtOAc
(3 × 50 mL). The combined organic layers were washed with brine
(3 × 50 mL), dried, and concentrated under reduced pressure. The
resulting oxindoles were purified by flash chromatography on silica
gel.
Experimental Section
(5-Methoxy-2-oxo-2,3-dihydro-1H-indol-3-yl)acetonitrile
(24b). Prepared by stirring 23b for 1 h according to general
procedure 2. Upon workup, the crude product was purified by flash
chromatography (1:1 EtOAc/hexane) to give 24b33 as pale-yellow
oil (1.32 g, 77%); Rf 0.25 (1:1 EtOAc/hexane). IR (CHCl3) νmax
All reagents are of commercial quality and obtained from Sigma-
Aldrich (St. Louis, MO). Solvents were dried, where necessary,
using standard procedures. Melting points (mp) were determined
using a Fisher-Johns apparatus and are uncorrected. Thin-layer
chromatography (TLC) was performed on precoated plates (Merck
TLC aluminum sheets, silica gel 60 F254) with detection by UV
light or with ceric ammonium sulfate in H2SO4 followed by heating.
Flash chromatography was performed on silica gel 60 (230-400
mesh). IR spectra were obtained using a Perkin-Elmer 16 FPC FT
spectrophotometer. NMR spectra were recorded on Mercury
spectrometers working at 300 and 75 MHz for 1H and 13C,
respectively; chemical shifts are measured in ppm (δ) relative to
internal tetramethylsilane (TMS) and coupling constants (J) are in
Hz. The spectral assignments were confirmed by standard proce-
dures (gHMBC, gHSQC, NOESY). Signals, when declared, are
expressed as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), or br (broad); signals due to NH protons were located
by deuterium exchange with D2O. LRMS were recorded on a Varian
Saturn 2000 or Hewlett-Packard 5989A spectrometers working at
20 and 70 eV, respectively. HRMS were recorded at the University
of California, Riverside CA.
1
3436, 3206, 3020, 2254, 1716, 1494 cm-1. H NMR (CDCl3) δ
8.16 (very br s, 1H), 7.10 (br s, 1H), 6.84 (m, 2H), 3.81 (s, 3H),
3.69 (ddd, J ) 8.9, 4.8, 1.0 Hz, 1H), 3.10 (dd, J ) 16.9, 4.8 Hz,
1H), 2.75 (dd, J ) 16.9, 8.9 Hz, 1H). 13C NMR (CDCl3) δ 176.0,
156.2, 134.3, 127.4, 117.0, 114.2, 111.4, 110.7, 55.8, 42.2, 18.8.
EIMS m/z (relative intensity) 202 (M+, 100), 162 (80), 131 (9).
HRMS (ESI/APCIMS) calcd for C11H10N2O2 + H, 203.0821; found,
203.0818.
(1-Benzyl-2-oxo-2,3-dihydro-1H-indol-3-yl)acetonitrile (24c).
Prepared by stirring 23c for 24 h according to general procedure
2. Upon workup, the crude product was purified by flash chroma-
tography (1:4 EtOAc/hexane) to give 24c as colorless solid (1.29
g, 58%); mp 152-154 °C (Lit. 150-151 °C). The elemental
composition was consistent with that reported.34 To our knowledge,
the NMR data are unreported and therefore follow. 1H NMR
(CDCl3) δ 7.50 (br d, J ) 7.3 Hz, 1H), 7.35-7.20 (m, 5H), 7.24
(tm, J ) 7.9 Hz, 1H), 7.08 (td, J ) 7.5, 0.9 Hz, 1H), 6.77 (br d, J
) 7.9 Hz, 1H), 4.95 and 4.89 (AB, J ) 15.7 Hz, 2H), 3.76 (dd, J
) 8.7, 4.7 Hz, 1H), 3.15 (dd, J ) 16.8, 4.7 Hz, 1H), 2.77 (dd, J )
16.8, 8.7 Hz, 1H). 13C NMR (CDCl3) δ 174.3, 143.2, 135.1, 129.3,
128.8 (2C), 127.8, 127.2 (2C), 125.5, 124.2, 123.1, 117.1, 109.6,
Starting indole 23a is commercially available. Indoles 23b21 and
23e,18a oxindoles 24a,18a 24e,18a 25a,18a 25h,29 and 25k,18a as well
as pyrrolidinoindolines 4a-c,18,19 5a,18a 5h,30 5k,18a 10,18a and
22,31 are known and were synthesized using procedures described
from this laboratory.

5278 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
RiVera-Becerril et al.
44.0, 41.4, 19.9. EIMS m/z (relative intensity) 262 (M+, 100), 222
(36), 91 (75), 65 (12). HRMS (ESI/APCIMS) calcd for C17H14N2O
+ H, 263.1184; found, 263.1185.
7.20-7.10 (m, 5H), 7.09 (tm, J ) 7.1 Hz, 2H), 6.90 (dm, J ) 6.9
Hz, 2H), 6.66 (dd, J ) 8.6, 2.7 Hz, 1H), 6.65 (dm, J ) 7.1 Hz,
2H), 6.35 (d, J ) 8.5 Hz, 1H), 4.94 and 4.48 (AB, J ) 15.9 Hz,
2H), 3.80 (s, 3H), 3.35 and 3.26 (AB, J ) 12.9 Hz, 2H), 3.01 and
2.79 (AB, J ) 16.5 Hz, 2H). 13C NMR (CDCl3) δ 175.7, 156,1,
135.9, 134.7, 134.3, 130.1 (2C), 129.6, 128.6 (2C), 128.1 (2C),
127.3, 127.1, 126.5 (2C), 116.4, 113.6, 111.0, 110.2, 55.8, 51.0,
43.8, 42.0, 26.1. EIMS m/z (relative intensity) 382 (M+, 93), 291
(68), 266 (33), 91 (100), 65 (19). HRMS (ESI/APCIMS) calcd for
C25H22N2O2 + H, 383.1760; found, 383.1767.
(1-Benzyl-5-methoxy-2-oxo-2,3-dihydro-1H-indol-3-yl)aceto-
nitrile (24d). Prepared by stirring 23d for 2 h according to general
procedure 2. Upon workup, the crude product was purified by flash
chromatography (1:1 EtOAc/hexane) to give 24d as colorless solid
(0.96 g, 62%); mp 144-145 °C (acetone/CH2Cl2); Rf 0.48 (1:1
EtOAc/hexane). IR (CHCl3) νmax 3016, 2254, 1710, 1602, 1494
cm-1. 1H NMR (CDCl3) δ 7.34-7.24 (m, 5H), 7.12 (dd, J ) 2.2,
0.8 Hz, 1H), 6.76 (ddd, J ) 8.5, 2.5, 0.8 Hz, 1H), 6.65 (br d, J )
8.8 Hz, 1H), 4.94 and 4.87 (AB, J ) 15.7 Hz, 2H), 3.78 (s, 3H),
3.74 (dd, overlap, J ) 8.8, 4.7 Hz, 1H), 3.15 (dd, J ) 16.8, 4.7
Hz, 1H), 2.79 (dd, J ) 16.8, 8.8 Hz, 1H). 13C NMR (CDCl3) δ
174.0, 156.3, 136.5, 135.2, 128.8 (2C), 127.8, 127.2 (2C), 126.8,
117.1, 113.7, 111.5, 110.1, 55.8, 44.1, 41.8, 19.2. EIMS m/z (relative
intensity) 292 (M+, 100), 252 (38), 91 (76), 65 (12). HRMS (ESI/
APCIMS) calcd for C18H16N2O2 + H, 293.1290; found, 293.1283.
General Procedure 3 for the Preparation of Oxindoles
25b-25l. To a solution of the corresponding oxindole (24a-e)
(0.01 mol) in CH2Cl2 (65 mL) were added 15% aq NaOH (23 mL,
86.2 mmol), TBAHS (0.14 g, 0.41 mmol), and the corresponding
halide [ethyl bromide, n-propyl bromide, prenyl bromide, benzyl
bromide, or 4-(bromomethyl)pyridine] [0.025 mol (24a, 24b), or
0.015 mol (24c-e)]. The resulting mixture was stirred at room
temperature until TLC analysis showed complete loss of starting
material. After completion (2-7 h), the organic layer was collected
and the aqueous phase was extracted with CH2Cl2 (2 × 20 mL).
The combined organic phases were washed with brine (2 × 60
mL), dried, and concentrated under reduced pressure. The resulting
oxindoles were purified by flash chromatography on silica gel. Data
for the new compounds (25b-g, 25i, 25j, and 25l) follow.
[5-Methoxy-1,3-bis(3-methylbut-2-enyl)-2-oxo-2,3-dihydro-
1H-indol-3-yl]acetonitrile (25b). Prepared by stirring 24b with
prenyl bromide for 2 h according to general procedure 3. Upon
workup, the crude product was purified by flash chromatography
(1:4 EtOAc/hexane) to give 25b as pale-yellow oil (2.81 g, 83%);
Rf 0.39 (3:7 EtOAc/hexane). IR (CHCl3) νmax 3028, 2972, 2254,
1705, 1602, 1436 cm-1. 1H NMR (CDCl3) δ 7.05 (d, J ) 2.2 Hz,
1H), 6.83 (dd, J ) 8.6, 2.6 Hz, 1H), 6.73 (br d, J ) 8.3 Hz, 1H),
5.07 (tm, J ) 6.6 Hz, 1H), 4.74 (tm, J ) 7.5 Hz, 1H), 4.38 (br dd,
J ) 15.5, 6.6 Hz, 1H), 4.19 (br dd, J ) 15.5, 6.8 Hz, 1H), 3.81 (s,
3H), 2.86 and 2.62 (AB, J ) 16.7 Hz, 2H), 2.61 (br d, J ) 7.5 Hz,
2H), 1.80 (br s, 3H), 1.71 (br s, 3H), 1.56 (br s, 3H), 1.54 (br s,
3H). 13C NMR (CDCl3) δ 176.0, 156.0, 136.8 (2C), 136.1, 130.7,
118.2, 116.6, 116.1, 113.3, 110.8, 109.4, 55.8, 49.2, 38.2, 34.9,
25.8, 25.6, 24.8, 18.1, 18.0. EIMS m/z (relative intensity) 338 (M+,
100), 270 (46), 215 (51), 175 (15), 69 (17), 41 (22). HRMS (ESI/
APCIMS) calcd for C21H26N2O2 + H, 339.2073; found, 339.2076.
[1,3-Dibenzyl-2-oxo-2,3-dihydro-1H-indol-3-yl]acetonitrile (25c).
Prepared by stirring 24a with benzyl bromide for 5 h according to
general procedure 3. Upon workup, the crude product was purified
by flash chromatography (1:4 EtOAc/hexane) to give 25c as
colorless solid (3.17 g, 90%); mp 139-140 °C (EtOAc/hexane);
Rf 0.54 (3:7 EtOAc/hexane). IR (CHCl3) νmax 3020, 2920, 2256,
[2-Oxo-1,3-bis(pyridin-4-ylmethyl)-2,3-dihydro-1H-indol-3-
yl]acetonitrile (25e). Prepared by stirring 24a with 4-(bromom-
ethyl)pyridine for 6 h according to general procedure 3. Upon
workup, the crude product was purified by flash chromatography
(EtOAc) to give 25e as pale-yellow oil (2.72 g, 77%); Rf 0.17 (4:1
EtOAc/acetone). IR (CHCl3) νmax 3022, 2258, 1718, 1604 cm-1
.
1H NMR (CDCl3) δ 8.43 (dm, J ) 4.4 Hz, 2H), 8.32 (dm, J ) 4.4
Hz, 2H), 7.65 (m, 1H), 7.23 (m, 2H), 6.78 (dm, J ) 4.4 Hz, 2H),
6.54 (dm, J ) 4.4 Hz, 2H), 6.45 (m, 1H), 4.87 and 4.58 (AB, J )
16.6 Hz, 2H), 3.37 and 3.29 (AB, J ) 12.8 Hz, 2H), 3.08 and 2.86
(AB, J ) 16.5 Hz, 2H). 13C NMR (CDCl3) δ 175.4, 150.0 (2C),
149.4 (2C), 143.6, 143.2, 141.9, 129.9, 127.3, 125.1 (2C), 123.9,
123.7, 121.2 (2C), 115.9, 109.6, 50.3, 42.7, 41.1, 26.2. EIMS m/z
(relative intensity) 354 (M+, 100), 314 (11), 262 (76), 93 (47).
HRMS (EIMS) calcd for C22H18N4O, 354.1481; found, 354.1480.
[2-Oxo-1,3-di(n-propyl)-2,3-dihydro-1H-indol-3-yl]acetoni-
trile (25f). Prepared by stirring 24a with n-propyl bromide for 7 h
according to general procedure 3. Upon workup, the crude product
was purified by flash chromatography (2:3 EtOAc/hexane) to give
25f as pale-yellow oil (1.84 g, 72%); Rf 0.54 (3:7 EtOAc/hexane).
1
IR (CHCl3) νmax 3028, 2968, 2256, 1712, 1614 cm-1. H NMR
(CDCl3) δ 7.41 (ddd, J ) 7.4, 1.3, 0.5 Hz, 1H), 7.33 (td, J ) 7.8,
1.2 Hz, 1H), 7.12 (td, J ) 7.6, 1.1 Hz, 1H), 6.91 (br d, J ) 7.6 Hz,
1H), 3.66 (dt, J ) 13.4, 6.7 Hz, 1H) 3.63 (dt, J ) 13.4, 6.7 Hz,
1H), 2.83 and 2.59 (AB, J ) 16.5 Hz, 2H), 1.94 (m, 2H), 1.71
(sextet, J ) 7.4 Hz, 2H), 0.97 (t, J ) 7.4 Hz, 3H), 0.95 (m, overlap,
2H), 0.80 (br t, overlap, J ) 6.7 Hz, 3H). 13C NMR (CDCl3) δ
176.8, 142.9, 129.4, 128.9, 123.2, 122.8, 116.4, 108.7, 48.9, 41.7,
38.3, 26.0, 20.6, 17.3, 13.8, 11.3. EIMS m/z (relative intensity) 256
(M+, 100), 214 (96), 187 (53), 174 (33), 158 (25). HRMS (EIMS)
calcd for C16H20N2O, 256.1575; found, 256.1574.
[1,3-Diethyl-2-oxo-2,3-dihydro-1H-indol-3-yl]acetonitrile (25g).
Prepared by stirring 24a with ethyl bromide for 7 h according to
general procedure 3. Upon workup, the crude product was purified
by flash chromatography (2:3 EtOAc/hexane) to give 25g as pale-
yellow oil (1.91 g, 84%); Rf 0.47 (3:7 EtOAc/hexane). IR (CHCl3)
ν
max 3028, 2980, 2252, 1710, 1614, 1468 cm-1. 1H NMR (CDCl3)
δ 7.40 (ddd, J ) 7.4, 1.1, 0.6 Hz, 1H), 7.34 (td, J ) 7.8, 1.4 Hz,
1H), 7.12 (td, J ) 7.4, 1.1 Hz, 1H), 6.93 (br d, J ) 7.6 Hz, 1H),
3.81 (dq, J ) 14.0, 7.0 Hz, 1H), 3.78 (dq, J ) 14.0, 7.0 Hz, 1H),
2.83 and 2.62 (AB, J ) 16.7 Hz, 2H), 2.00 (q, J ) 7.4 Hz, 2H),
1.27 (t, J ) 7.0 Hz, 3H), 0.60 (t, J ) 7.4 Hz, 3H). 13C NMR
(CDCl3) δ 176.2, 142.5, 128.9, 128.8, 123.1, 122.7, 116.3, 108.4,
49.2, 34.6, 29.2, 25.4, 12.4, 8.1. EIMS m/z (relative intensity) 228
(M+, 100), 200 (14), 188 (88), 160 (54), 132 (10). HRMS (EIMS)
calcd for C14H16N2O, 228.1263; found, 228.1262.
1
1714, 1614 cm-1. H NMR (CDCl3) δ 7.58 (m, 1H), 7.20-7.04
(m, 8H), 6.87 (dm, J ) 8.0 Hz, 2H), 6.64 (dm, J ) 7.6 Hz, 2H),
6.45 (m, 1H), 4.97 and 4.50 (AB, J ) 16.2 Hz, 2H), 3.38 and 3.27
[1-Benzyl-3-(3-methylbut-2-enyl)-2-oxo-2,3-dihydro-1H-indol-
3-yl]acetonitrile (25i). Prepared by stirring 24c with prenyl bromide
for 5 h according to general procedure 3. Upon workup, the crude
product was purified by flash chromatography (1:4 EtOAc/hexane)
to give 25i as yellow crystals (2.54 g, 77%); mp 90-92 °C (acetone/
Et2O); Rf 0.47 (3:7 EtOAc/hexane). IR (CHCl3) νmax 3028, 2916,
2256, 1714, 1614, 1468 cm-1. 1H NMR (CDCl3) δ 7.45 (ddd, J )
7.5, 1.3, 0.7 Hz, 1H), 7.27-7.25 (m, 5H), 7.21 (td, J ) 7.8, 1.5
Hz, 1H), 7.08 (td, J ) 1.2, 7.6 Hz, 1H), 6.72 (br d, J ) 7.8 Hz,
1H), 5.14 and 4.70 (AB, J ) 15.8 Hz, 2H), 4.76 (tm, J ) 7.9 Hz,
1H), 2.92 and 2.68 (AB, J ) 16.6 Hz, 2H), 2.71 (br d, J ) 7.5 Hz,
2H), 1.56 (s, 6H). 13C NMR (CDCl3) δ 176.9, 142.6, 137.1, 135.3,
129.1, 129.0, 128.7 (2C), 127.6, 127.0 (2C), 123.4, 123.0, 116.5,
116.3, 109.5, 49.1, 43.9, 35.0, 25.8, 25.2, 18.0. EIMS m/z (relative
intensity) 330 (M+, 9), 262 (100), 235 (42), 184 (10), 171 (8), 91
(AB, J ) 12.9 Hz, 2H), 3.03 and 2.78 (AB, J ) 16.8 Hz, 2H). 13
C
NMR (CDCl3) δ 176.0, 142.5, 134.5, 134.2, 130.0 (2C), 129.2,
128.6 (2C), 128.2, 128.1 (2C), 127.3, 127.1, 126.5 (2C), 123.8,
122.9, 116.5, 109.8, 50.6, 43.7, 41.9, 26.0. EIMS m/z (relative
intensity) 352 (M+, 100), 261 (53), 235 (22), 91 (84). HRMS (ESI/
APCIMS) calcd for C24H20N2O + H, 353.1654; found, 353.1657.
[1,3-Dibenzyl-5-methoxy-2-oxo-2,3-dihydro-1H-indol-3-yl]ac-
etonitrile (25d). Prepared by stirring 24b with benzyl bromide for
3 h according to general procedure 3. Upon workup, the crude
product was purified by flash chromatography (1:4 EtOAc/hexane)
to give 25d as colorless solid (3.13 g, 82%); mp 150-151 °C
(acetone/Et2O); Rf 0.34 (3:7 EtOAc/hexane). IR (CHCl3) νmax 3014,
2920, 2256, 1710, 1604, 1496 cm-1 1H NMR (CDCl3) δ
.

Debromoflustramine B as SelectiVe BChE Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5279
(33), 69 (36). HRMS (ESI/APCIMS) calcd for C22H22N2O + H,
331.1810; found, 331.1814.
(td, J ) 7.3, 0.8 Hz, 1H), 6.14 (dd, J ) 8.2, 0.7 Hz, 1H), 4.79 (s,
1H), 4.35 and 4.27 (AB, J ) 16.3 Hz, 2H), 3.23 and 2.90 (AB, J
) 13.4 Hz, 2H), 2.96 (ddd, J ) 11.2, 6.8, 1.5 Hz, 1H), 2.65 (ddd,
J ) 11.2, 11.0, 5.4 Hz, 1H), 2.11 (ddd, J ) 11.9, 5.4, 1.5 Hz, 1H),
2.01 (ddd, J ) 11.9, 11.2, 6.8 Hz, 1H), 1.87 (very br s, 1H). 13C
NMR (CDCl3) δ 151.3, 138.6, 138.3, 133.0, 130.1 (2C), 128.4 (2C),
128.0, 127.9 (2C), 126.9 (2C), 126.6, 126.2, 123.6, 116.5, 104.9,
85.9, 57.8, 48.1, 45.9, 45.1, 42.1. EIMS m/z (relative intensity) 340
(M+, 40), 249 (100), 220 (15), 158 (8), 91 (27). HRMS (ESI/
APCIMS) calcd for C24H24N2 + H, 341.2018; found, 341.2024.
3a,8-Dibenzyl-5-methoxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-
b]indole (5d). Prepared from 25d according to general procedure
4. Upon workup, the crude product was purified by flash chroma-
tography (9:1 EtOAc/acetone) to give 5d as pale-yellow oil (0.50
g, 68%); Rf 0.21 (EtOAc). IR (CHCl3) νmax 3344, 3012, 2938, 1664,
[1-Benzyl-5-methoxy-3-(3-methylbut-2-enyl)-2-oxo-2,3-dihy-
dro-1H-indol-3-yl]acetonitrile (25j). Prepared by stirring 24d with
prenyl bromide for 3 h according to general procedure 3. Upon
workup, the crude product was purified by flash chromatography
(3:7 EtOAc/hexane) to give 25j as pale-yellow oil (2.84 g, 79%);
Rf 0.36 (3:7 EtOAc/hexane). IR (CHCl3) νmax 3032, 2928, 2256,
1712, 1602, 1436 cm-1. 1H NMR (CDCl3) δ 7.32-7.23 (m, 5H),
7.06 (d, J ) 2.2 Hz, 1H), 6.73 (dd, J ) 8.6, 2.6 Hz, 1H), 6.60 (d,
J ) 8.6 Hz, 1H), 5.12 and 4.67 (AB, J ) 15.8, Hz, 2H), 4.77 (tm,
J ) 7.5 Hz, 1H), 3.78 (s, 3H), 2.91 and 2.69 (AB, J ) 16.7 Hz,
2H), 2.70 (m, overlap, 2H), 1.58 (br s, 3H), 1.57 (s, 3H). 13C NMR
(CDCl3) δ 176.5, 156.2, 137.1, 135.9, 135.4, 130.4, 128.7 (2C),
127.6, 127.0 (2C), 116.6, 116.3, 113.3, 110.7, 110.0, 55.7, 49.5,
44.0, 34.9, 25.8, 25.3, 18.1. EIMS m/z (relative intensity) 360 (M+,
54), 292 (64), 265 (100), 91 (68). HRMS (ESI/APCIMS) calcd for
C23H24N2O2 + H, 361.1916; found, 361.1913.
1
1598 cm-1. H NMR (CDCl3) δ 7.25-7.17 (m, 6H), 7.02-6.96
(m, 4H), 6.60 (br d, J ) 2.4 Hz, 1H), 6.56 (dd, J ) 8.4, 2.6 Hz,
1H), 6.07 (br d, J ) 8.4 Hz, 1H), 4.75 (s, 1H), 4.29 and 4.16 (AB,
J ) 16.0 Hz, 2H), 3.72 (s, 3H), 3.19 and 2.89 (AB, J ) 13.4 Hz,
2H), 2.96 (ddd, J ) 10.8, 6.8, 2.0 Hz, 1H), 2.67 (ddd, J ) 11.0,
10.8, 5.5 Hz, 1H), 2.11 (ddd, J ) 12.1, 5.5, 2.0 Hz, 1H), 2.00 (ddd,
J ) 12.1, 11.0, 6.8 Hz, 1H), 1.88 (very br s, 1H). 13C NMR (CDCl3)
δ 152.0, 145.9, 138.9, 138.2, 134.6, 130.2 (2C), 128.3 (2C), 127.9
(2C), 127.1 (2C), 126.6, 126.3, 112.5, 111.2, 105.5, 86.7, 58.0, 56.0,
49.4, 45.9, 44.9, 41.5. EIMS m/z (relative intensity) 370 (M+, 86),
279 (100), 250 (16), 188 (9), 173 (8), 91 (25). HRMS (ESI/
APCIMS) calcd for C25H26N2O + H, 371.2123; found, 371.2124.
3a,8-Bis(pyridin-4-ylmethyl)-1,2,3,3a,8,8a-hexahydropyrro-
lo[2,3-b]indole (5e). Prepared from 25e according to general
procedure 4. Upon workup, the crude product was purified by flash
chromatography (3:7 MeOH/acetone) to give 5e as pale-yellow oil
(0.53 g, 77%); Rf 0.27 (3:7 MeOH/acetone). IR (CHCl3) νmax 3290,
3018, 2962, 1602, 1560 cm-1. 1H NMR (CDCl3) δ 8.43 (dm, J )
4.4 Hz, 2H), 8.39 (dm, J ) 4.5 Hz, 2H), 7.07 (dd, J ) 7.8, 1.1 Hz,
1H), 7.02 (td, J ) 7.8, 1.1 Hz, 1H) 6.83 (br d, J ) 4.4 Hz, 2H),
6.73 (br d, J ) 4.4 Hz, 2H), 6.71 (td, J ) 7.3, 1.1 Hz, 1H), 6.05
(br d, J ) 7.8 Hz, 1H), 4.72 (s, 1H), 4.35 and 4.21 (AB, J ) 17.1
Hz, 2H), 3.32 and 2.88 (AB, J ) 13.2 Hz, 2H), 3.07 (ddd, J )
11.3, 6.9, 1.1 Hz, 1H), 2.67 (ddd, J ) 11.6, 11.3, 5.2 Hz, 1H),
2.25 (ddd, J ) 11.8, 5.2, 1.1 Hz, 1H), 2.03 (very br s, 1H), 2.01
(ddd, overlap, J ) 11.8, 11.6, 6.9 Hz, 1H). 13C NMR (CDCl3) δ
150.9, 149.8 (2C), 149.4 (2C), 147.9, 147.2, 131.5, 128.6, 125.2
(2C), 123.7, 121.7 (2C), 117.3, 105.0, 86.1, 57.8, 47.2, 46.0, 44.6,
43.1. EIMS m/z (relative intensity) 342 (M+, 100), 250 (91), 221
(28), 92 (12), (18). HRMS (ESI/APCIMS) calcd for C22H22N4 +
H, 343.1923; found, 343.1925.
[1-Benzyl-2-oxo-3-(pyridin-4-ylmethyl)-2,3-dihydro-1H-indol-
3-yl]acetonitrile (25l). Prepared by stirring 24c with 4-(bromom-
ethyl)pyridine for 5 h according to general procedure 3. Upon
workup, the crude product was purified by flash chromatography
(1:1 EtOAc/hexane) to give 25l as pale-yellow oil (2.47 g, 70%);
Rf 0.21 (1:1 EtOAc/hexane). IR (CHCl3) νmax 3028, 2996, 2256,
1
1716, 1614, 1468 cm-1. H NMR (CDCl3) δ 8.23 (dm, J ) 4.4
Hz, 2H), 7.60 (dd, J ) 7.0, 1.3 Hz, 1H), 7.15 (m, 4H), 7.13 (td, J
) 7.7, 1.3 Hz, 1H), 6.73 (dm, J ) 4.4 Hz, 2H), 6.68 (br dm, J )
4.4 Hz, 2H), 6.53 (br dd, J ) 6.9, 1.7 Hz, 1H), 4.87 and 4.54 (AB,
J ) 15.8 Hz, 2H), 3.35 and 3.26 (AB, J ) 12.7 Hz, 2H), 3.05 and
2.77 (AB, J ) 16.7 Hz, 2H). 13C NMR (CDCl3) δ 175.1, 149.2
(2C), 142.9, 142.2, 134.2, 129.5, 128.6 (2C), 127.4, 127.3, 126.3
(2C), 124.8 (2C), 123.5, 123.1, 116.0, 109.8, 50.1, 43.9, 41.1, 26.5.
EIMS m/z (relative intensity) 353 (M+, 93), 261 (68), 91 (100),
65 (26). HRMS (ESI/APCIMS) calcd for C23H19N3O + H,
354.1606; found, 354.1609.
General Procedure 4 for the Preparation of Pyrroloindo-
lines 5b-5j and 5l. To a precooled (5 °C) stirred suspension of
LAH (0.57 g, 15.0 mmol) in anhydrous THF (20 mL) and under
argon atmosphere was added the corresponding oxindole (25b-25j
or 25l) (2.0 mmol) in THF (20 mL), and the mixture was heated at
reflux for 1.5 h. After cooling, the reaction mixture in an ice/water
bath, the reaction was quenched by adding, in sequence, EtOAc
(10 mL) and water (5 mL). The solids were removed by filtration
and washed with EtOAc (2 × 15 mL). The combined organic phases
were washed with brine, dried, and concentrated under reduced
pressure. The resulting pyrroloindolines were purified by flash
chromatography on silica gel. Data for the new compounds (5b-5g,
5i, 5j, and 5l) follow.
3a,8-Di(n-propyl)-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]in-
dole (5f). Prepared from 25f according to general procedure 4. Upon
workup, the crude product was purified by flash chromatography
(1:19 MeOH/CH2Cl2) to give 5f as pale-ellow oil (0.34 g, 70%);
Rf 0.21 (1:9 MeOH/CH2Cl2). IR (CHCl3) νmax 3664, 3054, 2962,
1666, 1602, 1492 cm-1. 1H NMR (CDCl3) δ 7.03 (td, J ) 7.6, 1.1
Hz, 1H), 6.96 (dd, J ) 7.2, 1.3 Hz, 1H), 6.58 (td, J ) 7.3, 0.7 Hz,
1H), 6.30 (br d, J ) 7.9 Hz, 1H), 4.69 (s, 1H), 3.18 (m, 2H), 2.97
(ddd, J ) 10.8, 6.8, 1.8 Hz, 1H), 2.65 (ddd, J ) 11.0, 10.8, 5.5
Hz, 1H), 2.27 (very br s, 1H), 2.00 (ddd, J ) 11.9, 5.5, 1.5 Hz,
1H), 1.80 (m, 2H), 1.63 (m, 3H), 1.29 (m, 1H), 1.12 (m, 1H), 0.94
(t, J ) 7.3 Hz, 3H), 0.86 (t, J ) 7.3 Hz, 3H). 13C NMR (CDCl3)
δ 151.3, 133.7, 127.6, 123.1, 116.1, 104.5, 87.4, 56.6, 46.8, 45.4,
42.4, 42.2, 20.9, 18.9, 14.6, 11.7. EIMS m/z (relative intensity) 244
(M+, 100), 215 (63), 201 (25), 186 (41), 172 (87). HRMS (ESI/
APCIMS) calcd for C16H24N2 + H, 245.2018; found, 245.2018.
3a,8-Diethyl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole (5g).
Prepared from 25g according to general procedure 4. Upon workup,
the crude product was purified by flash chromatography (4:1 EtOAc/
acetone) to give 5g as pale-yellow oil (0.20 g, 47%); Rf 0.22 (1:1
EtOAc/acetone). IR (CHCl3) νmax 3566, 3018, 2968, 1662, 1604
5-Methoxy-3a,8-bis(3-methylbut-2-enyl)-1,2,3,3a,8,8a-hexahy-
dropyrrolo[2,3-b]indole (5b). Prepared from 25b according to
general procedure 4. Upon workup, the crude product was purified
by flash chromatography (4:1 EtOAc/acetone) to give 5b as pale-
yellow oil (0.43 g, 66%); Rf 0.28 (7:3 EtOAc/acetone). IR (CHCl3)
ν
max 3376, 3020, 2932, 1670, 1592 cm-1. 1H NMR (CDCl3) δ 6.65
(d, J ) 2.4 Hz, 1H), 6.62 (dd, J ) 8.3, 2.6 Hz, 1H), 6.27 (d, J )
8.3, 1H), 5.22 (tm, J ) 6.6 Hz, 1H), 5.06 (tm, J ) 7.2 Hz, 1H),
4.58 (s, 1H), 3.76 (m, overlap, 2H), 3.74 (s, overlap, 3H), 2.99
(ddd, J ) 10.3, 6.7, 2.4 Hz, 1H), 2.75 (ddd, J ) 10.3, 10.3, 6.2
Hz, 1H), 2.41 (br d, J ) 7.3 Hz, 2H), 1.93 (very br s, 1H), 1.92
(m, 2H), 1.71 (br s, 3H), 1.70 (br s, 3H), 1.68 (s, 3H), 1.59 (s,
3H). 13C NMR δ 152.3, 145.6, 136.3, 134.8, 133.8, 121.0, 120.5,
112.1, 110.8, 106.0, 87.7, 56.6, 56.0, 45.5, 44.5, 40.6, 37.7, 25.9,
25.8, 18.1, 18.0. EIMS m/z (relative intensity) 326 (M+, 57), 257
(89), 240 (100), 189 (67), 160 (26), 97 (26), 69 (51). HRMS (ESI/
APCIMS) calcd for C21H30N2O + H, 327.2436; found, 327.2440.
3a,8-Dibenzyl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole (5c).
Prepared from 25c according to general procedure 4. Upon workup,
the crude product was purified by flash chromatography (4:1 EtOAc/
acetone) to give 5c as pale-yellow oil (0.51 g, 75%); Rf 0.20
1
cm-1. H NMR (CDCl3) δ 7.04 (td, J ) 7.8, 1.5 Hz, 1H), 6.97
(ddd, J ) 7.3, 1.3, 0.4 Hz, 1H), 6.60 (td, J ) 7.4, 1.1 Hz, 1H),
6.32 (br d, J ) 7.5 Hz, 1H), 4.70 (s, 1H), 3.32 (dq, J ) 12.2, 7.0
Hz, 1H), 3.29 (dq, J ) 12.2, 7.0 Hz, 1H), 2.99 (ddd, J ) 10.8, 6.8,
(EtOAc). IR (CHCl3) νmax 3346, 3016, 2930, 1660, 1602 cm-1
1H NMR (CDCl3) δ 7.23-7.14 (m, 6H), 7.00-6.92 (m, 6H), 6.63
.

5280 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
RiVera-Becerril et al.
1.8 Hz, 1H), 2.69 (ddd, J ) 10.8, 11.0, 5.7 Hz, 1H), 2.10 (very br
s, 1H), 1.98 (ddd, J ) 11.9, 5.7, 1.8 Hz, 1H), 1.87 (dq, J ) 14.7,
7.4 Hz, 1H), 1.81 (ddd, J ) 11.9, 11.0, 6.8 Hz, 1H), 1.68 (dq, J )
14.7, 7.4 Hz, 1H), 1.17 (t, J ) 7.3 Hz, 3H), 0.81 (t, J ) 7.5 Hz,
3H). 13C NMR (CDCl3) δ 150.9, 133.7, 127.7, 123.2, 116.3, 104.8,
86.4, 57.0, 45.5, 41.8, 39.2, 32.4, 12.3, 9.9. EIMS m/z (relative
intensity) 216 (M+, 100), 201 (19), 186 (34), 172 (57), 158 (76),
144 (37). HRMS (ESI/APCIMS) calcd for C14H20N2 + H, 217.1705;
found, 217.1710.
collected and the aqueous phase was extracted with CH2Cl2 (2 ×
10 mL). The combined organic phases were washed wit brine (2
× 10 mL), dried, and concentrated under reduced pressure. The
resulting N(1)-alkylated pyrroloindolines were purified by flash
chromatography on silica gel. Data for the new compounds (6, 7,
9, 13, and 18) follow.
1,3a,8-Tris(3-methylbut-2-enyl)-1,2,3,3a,8,8a-hexahydropyr-
rolo[2,3-b]indole (6). Prepared by stirring 5a with prenyl bromide
for 22 h according to general procedure 5. Upon workup, the crude
product was purified by flash chromatography (1:4 EtOAc/hexane)
to give 6 as pale-yellow oil (0.29 g, 60%); Rf 0.45 (3:7 EtOAc/
8-Benzyl-3a-(3-methylbut-2-enyl)-1,2,3,3a,8,8a-hexahydropy-
rrolo[2,3-b]indole (5i). Prepared from 25i according to general
procedure 4. Upon workup, the crude product was purified by flash
chromatography (3:2 EtOAc/hexane) to give 5i as pale-yellow oil
(0.53 g, 84%); Rf 0.39 (7:3 acetone/hexane). IR (CHCl3) νmax 3336,
3020, 2932, 1604 cm-1. 1H NMR (CDCl3) δ 7.31-7.21 (m, 5H),
7.04 (dd, J ) 7.4, 0.8 Hz, 1H), 7.01 (td, J ) 7.7, 1.4 Hz, 1H), 6.62
(td, J ) 7.4, 0.8, Hz, 1H), 6.26 (br d, J ) 8.0 Hz, 1H), 5.06 (tm,
J ) 7.3 Hz, 1H), 4.70 (s, 1H), 4.47 (br s, 2H), 3.01 (ddd, J ) 11.0,
6.9, 1.5 Hz, 1H), 2.76 (ddd, J ) 11.0, 11.0, 5.5 Hz, 1H), 2.47 (m,
2H), 2.04 (very br s, 1H), 2.03 (ddd, J ) 12.1, 5.5, 1.5 Hz, 1H),
1.90 (ddd, J ) 12.1, 11.0, 6.9 Hz, 1H), 1.67 (br s, 3H), 1.55 (br s,
3H). 13C NMR (CDCl3) δ 151.1, 139.0, 133.9, 134.0, 128.5 (2C),
127.8, 127.2 (2C), 126.8, 123.2, 120.4, 116.6, 104.7, 87.1, 56,9,
48.5, 45.8, 41.5, 37.6, 25.9, 18.1. EIMS m/z (relative intensity) 318
(M+, 36), 249 (100), 220 (15), 158 (12), 91 (44). HRMS (ESI/
APCIMS) calcd for C22H26N2 + H, 319.2174; found, 319.2176.
8-Benzyl-5-methoxy-3a-(3-methylbut-2-enyl)-1,2,3,3a,8,8a-
hexahydropyrrolo[2,3-b]indole (5j). Prepared from 25j according
to general procedure 4. Upon workup, the crude product was
purified by flash chromatography (3:2 EtOAc/hexane) to give 5j
as pale-yellow oil (0.45 g, 65%); Rf 0.30 (7:3 acetone/hexane). IR
hexane). IR (CHCl3) νmax 3016, 2930, 1604 cm-1 1H NMR
.
(CDCl3) δ 7.03 (td, J ) 7.8, 1.3 Hz, 1H), 6.96 (dd, J ) 7.0, 0.9
Hz, 1H), 6.63 (td, J ) 7.3, 0.9 Hz, 1H), 6.39 (br d, J ) 7.9 Hz,
1H), 5.34 (tm, J ) 6.6 Hz, 1H), 5.15 (tm, J ) 6.5 Hz, 1H), 4.99
(tm, J ) 7.0 Hz, 1H), 4.41 (s, 1H), 3.91 (dd, J ) 16.3, 5.5 Hz,
1H), 3.80 (dd, J ) 16.3, 7.3 Hz, 1H), 3.30 (br d, J ) 6.4 Hz, 2H),
2.75 (ddd, J ) 9.7, 6.6, 3.0 Hz, 1H), 2.55 (ddd, J ) 9.9, 9.7, 5.9
Hz, 1H), 2.43 (br d, J ) 7.0 Hz, 2H), 2.03 (ddd, J ) 12.1, 9.9, 6.6
Hz, 1H), 1.88 (ddd, J ) 12.1, 5.9, 3.0 Hz, 1H), 1.73 (br s, 3H),
1.69 (br s, 6H), 1.65 (br s, 6H), 1.58 (br s, 3H). 13C NMR (CDCl3)
δ 151.8, 135.5, 133.9, 133.8, 133.4, 127.5, 122.8, 122.6, 121.5,
120.9, 117.1, 107.0, 90.4, 56.8, 50.3, 49.1, 45.9, 38.8, 38.3, 25.9,
25.8, 25.7, 18.1, 18.1, 18.0. EIMS m/z (relative intensity) 364 (M+,
9), 296 (100), 227 (11), 212 (26), 199 (38), 130 (20), 69 (18).
HRMS (ESI/APCIMS) calcd for C25H36N2 + H, 365.2951; found,
365.2953.
1-Benzyl-3a,8-bis(3-methylbut-2-enyl)-1,2,3,3a,8,8a-hexahy-
dropyrrolo[2,3-b]indole (7). Prepared by stirring 5a with benzyl
bromide for 5 h according to general procedure 5. Upon workup,
the crude product was purified by flash chromatography (1:9 EtOAc/
hexane) to give 7 as pale-yellow oil (0.33 g, 65%); Rf 0.66 (1:4
EtOAc/hexane). IR (CHCl3) νmax 3016, 2930, 1602 cm-1. 1H NMR
(CDCl3) δ 7.39 (dm, J ) 7.7 Hz, 2H), 7.30 (tm, J ) 8.0 Hz, 2H),
7.22 (tm, J ) 7.2 Hz, 1H), 7.04 (td, J ) 7.7, 1.1 Hz, 1H), 6.98
(dd, J ) 7.3, 1.0 Hz, 1H), 6.64 (td, J ) 7.3, 1.1 Hz, 1H), 6.41 (br
d, J ) 7.7 Hz, 1H), 5.12 (tm, overlap, J ) 6.1 Hz, 1H), 5.10 (tm,
overlap, J ) 7.7 Hz, 1H), 4.48 (s, 1H), 3.92 and 3.84 (AB, J )
14.0 Hz, 2H), 3.80 (br dd, J ) 15.9, 5.9 Hz, 1H), 3.65 (br dd, J )
15.9, 7.4 Hz, 1H), 2.69 (ddd, J ) 9.6, 6.9, 2.2 Hz, 1H), 2.56 (ddd,
J ) 9.6, 9.6, 5.8 Hz, 1H), 2.41 (br d, J ) 7.4 Hz, 2H), 2.08 (ddd,
J ) 11.6, 9.6, 6.9 Hz, 1H), 1.85 (ddd, J ) 11.6, 5.8, 2.2 Hz, 1H),
1.69 (s, 3H), 1.64 (s, 3H), 1.59 (s, 3H), 1.49 (s, 3H). 13C NMR
(CDCl3) δ 152.1, 139.9, 135.8, 133.9, 133.4, 128.2 (4C), 127.5,
126.7, 122.8, 121.4, 120.9, 117.2, 107.1, 89.9, 56.8, 54.9, 50.4,
46.3, 38.6, 38.2, 26.0, 25.7, 18.1, 17.8. EIMS m/z (relative intensity)
386 (M+, 13), 317 (100), 249 (21), 212 (21), 198 (45), 130 (22),
91 (45). HRMS (ESI/APCIMS) calcd for C27H34N2 + H, 387.2800;
found, 387.2797.
(CHCl3) νmax 3342, 3024, 2966 cm-1 1H NMR (CD2Cl2) δ
.
7.33-7.21 (m, 5H), 6.67 (d, J ) 2.8 Hz, 1H), 6.52 (dd, J ) 8.4,
2.7 Hz, 1H), 6.10 (br d, J ) 8.5 Hz, 1H), 5.07 (tm, J ) 7.3 Hz,
1H), 4.66 (s, 1H), 4.40 and 4.33 (AB, J ) 16.0, 2H), 3.69 (s, 3H),
2.98 (ddd, J ) 10.9, 7.1, 1.9 Hz, 1H), 2.65 (ddd, J ) 10.9, 10.7,
5.6 Hz, 1H), 2.45 (m, 2H), 2.01 (very br s, 1H), 1.98 (ddd, J )
12.1, 5.6, 1.9 Hz, 1H), 1.86 (ddd, J ) 12.1, 10.7, 7.1 Hz, 1H),
1.67 (br s, 3H), 1.56 (br s, 3H). 13C NMR (CD2Cl2) δ 152.6, 146.1,
140.0, 136.1, 134.1, 128.7 (2C), 127.7 (2C), 127.0, 120.9, 112. 2,
111.2, 105,1, 88.4, 57.3, 56.2, 49.9, 46.2, 41.7, 37.8, 26.0, 18.2.
EIMS m/z (relative intensity) 348 (M+, 55), 279 (100), 188 (31),
173 (19), 91 (27). HRMS (ESI/APCIMS) calcd for C23H28N2O +
H, 349.2280; found, 349.2282.
8-Benzyl-3a-(pyridin-4-ylmethyl)-1,2,3,3a,8,8a-hexahydropy-
rrolo[2,3-b]indole (5l). Prepared from 25l according to general
procedure 4. Upon workup, the crude product was purified by flash
chromatography (1:1 acetone/hexane) to give 5l as pale-yellow oil
(0.59 g, 86%); Rf 0.15 (7:3 acetone/hexane). IR (CHCl3) νmax 3344,
1
3030, 2964, 1604 cm-1. H NMR (CDCl3) δ 8.36 (dm, J ) 4.4
1-Benzyl-5-methoxy-3a,8-bis(3-methylbut-2-enyl)-1,2,3,3a,8,8a-
hexahydropyrrolo[2,3-b]indole (9). Prepared by stirring 5b with
benzyl bromide for 6 h according to general procedure 5. Upon
workup, the crude product was purified by flash chromatography
(1:4 EtOAc/hexane) to give 9 as pale-yellow oil (0.26 g, 48%); Rf
Hz, 2H), 7.25-7.19 (m, 3H), 7.02 (td, J ) 7.5, 1.3 Hz, 1H), 7.00
(br d, overlap, J ) 7.0 Hz, 1H), 6.89 (br m, 2H), 6.81 (dm, J )
4.4 Hz, 2H), 6.66 (td, J ) 7.3, 0.9 Hz, 1H), 6.18 (dd, J ) 7.7, 0.9
Hz, 1H), 4.71 (s, 1H), 4.32 and 4.25 (AB, J ) 16.0 Hz, 2H), 3.26
and 2.86 (AB, J ) 13.2 Hz, 2H), 3.02 (ddd, J ) 11.2, 6.8, 1.3 Hz,
1H), 2.68 (ddd, J ) 11.2, 11.2, 5.3 Hz, 1H), 2.20 (br ddd, J )
11.4, 5.3, 1.3 Hz, 1H), 2.17 (very br s, 1H), 2.00 (ddd, J ) 11.4,
11.2, 6.8 Hz, 1H). 13C NMR (CDCl3) δ 151.4, 149.3 (2C), 147.2,
138.3, 131.7, 128.5 (2C), 128.4, 126.9 (2C), 126.8, 125.3 (2C),
123.5, 116.8, 105.2, 85.7, 57.4, 48.1, 45.7, 44.6, 42.6. EIMS m/z
(relative intensity) 341 (M+, 68), 249 (100), 221 (26), 207 (12),
91 (49), 65 (18). HRMS (ESI/APCIMS) calcd for C23H23N3 + H,
342.1970; found, 342.1964.
General Procedure 5 for the Preparation of Pyrroloindo-
lines 6, 7, 9, 13, and 18. To a solution of the corresponding
pyrroloindoline (5a, 5b, 5d, or 5j) (1.31 mmol) in CH2Cl2 (20 mL)
were added 15% aq NaOH (4 mL, 15.0 mmol), TBAHS (14.0 mg,
0.041 mmol), and the corresponding halide (prenyl bromide or
benzyl bromide) (1.5 mmol). The resulting mixture was stirred at
room temperature until TLC analysis showed complete loss of
starting material. After completion (5-22 h), the organic layer was
0.56 (3:7 EtOAc/hexane). IR (CHCl3) νmax 3024, 2966, 1670 cm-1
.
1H NMR (CDCl3) δ 7.39 (dm, J ) 6.9 Hz, 2H), 7.30 (tm, J ) 7.2
Hz, 2H), 7.22 (tm, J ) 6.9 Hz, 1H), 6.63 (dd, overlap, J ) 9.3, 2.5
Hz, 1H), 6.63 (d, overlap, J ) 2.2 Hz, 1H), 6.36 (d, J ) 9.4 Hz,
1H), 5.11 (br m, 2H), 4.44 (s, 1H), 3.93 and 3.84 (AB, J ) 14.0
Hz, 2H), 3.74 (s, overlap, 3H), 3.73 (br dd, J ) 16.0, 4.1 Hz, 1H),
3.59 (dd, J ) 16.0, 7.7 Hz, 1H), 2.69 (ddd, J ) 9.4, 6.9, 2.0 Hz,
1H), 2.55 (ddd, J ) 10.2, 9.4, 5.5 Hz, 1H), 2.44 (br d, J ) 6.9 Hz,
2H), 2.05 (ddd, J ) 11.8, 10.2, 6.9 Hz, 1H), 1.83 (ddd, J ) 11.8,
5.5, 2.0 Hz, 1H), 1.70 (s, 3H), 1.64 (s, 3H), 1.59 (s, 3H), 1.46 (s,
3H). 13C NMR (CDCl3) δ 152.7, 146.7, 139.9, 137.5, 133.8, 133.4,
128.2 (2C), 128.1 (2C), 126.7, 121.5, 120.9, 112.2, 110.0, 108.1,
90.4, 56.9, 55.9, 54.5, 50.3, 47.8, 38.7, 38.1, 26.0, 25.7, 18.1, 17.8.
EIMS m/z (relative intensity) 416 (M+, 51), 347 (100), 280 (20),
241 (56), 91 (41), 130 (20), 69 (18). HRMS (ESI/APCIMS) calcd
for C28H36N2O + H, 417.2906; found, 417.2909.

Debromoflustramine B as SelectiVe BChE Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5281
1,8-Dibenzyl-5-methoxy-3a-(3-methylbut-2-enyl)-1,2,3,3a,8,8a-
hexahydropyrrolo[2,3-b]indole (13). Prepared by stirring 5j with
benzyl bromide for 5 h according to general procedure 5. Upon
workup, the crude product was purified by flash chromatography
(1:4 EtOAc/hexane) to give 13 as pale-yellow oil (0.11 g, 20%);
Rf 0.71 (3:7 EtOAc/hexane). IR (CHCl3) νmax 3018, 2932, 1494
8-Benzyl-1-methyl-3a-(3-methylbut-2-enyl)-1,2,3,3a,8,8a-hexahy-
dropyrrolo[2,3-b]indole (11). Prepared from 5i according to
general procedure 6. Upon workup, the crude product was purified
by flash chromatography (7:3 EtOAc/hexane) to give 11 as pale-
yellow oil (0.28 g, 72%). The spectral and analytical data were
consistent with those reported.35
1
cm-1. H NMR (CDCl3) δ 7.29-7.17 (m, 10H), 6.66 (d, J ) 2.6
8-Benzyl-5-methoxy-1-methyl-3a-(3-methylbut-2-enyl)-
1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole (12). Prepared from
5j according to general procedure 6. Upon workup, the crude
product was purified by flash chromatography (7:3 EtOAc/hexane)
to give 12 as pale-yellow oil (0.23 g, 54%); Rf 0.14 (1:1 EtOAc/
Hz, 1H), 6.56 (dd, J ) 8.4, 2.6 Hz, 1H), 6.21 (d, J ) 8.4 Hz, 1H),
5.08 (tm, J ) 7.3 Hz, 1H), 4.50 (s, 1H), 4.31 and 4.15 (AB, J )
16.5 Hz, 2H), 3.77 (br s, 2H), 3.73 (s, 3H), 2.73 (m, 2H), 2.43 (m,
2H), 2.09 (ddd, J ) 12.1, 9.2, 7.9 Hz, 1H), 1.89 (ddd, J ) 12.1,
4.8, 3.3 Hz, 1H), 1.68 (br s, 3H), 1.54 (br s, 3H). 13C NMR (CDCl3)
δ 152.7, 146.7, 139.7, 139.6, 137.0, 133.7, 128.3 (2C), 128.2 (2C),
128.1 (2C), 127.2 (2C), 126.7, 126.6, 120.7, 112.0, 110.3, 107.0,
91.7, 57.0, 56.0, 55.1, 53.6, 50.6, 38.6, 38.0, 26.0, 18.1. EIMS m/z
(relative intensity) 438 (M+, 92), 369 (94), 278 (44), 250 (73), 91
(100). HRMS (ESI/APCIMS) calcd for C30H34N2O + H, 439.2749;
found, 439.2744.
hexane). IR (CHCl3) νmax 3024, 2934, 1596 cm-1 1H NMR
.
(CDCl3) δ 7.34-7.20 (br m, 5H), 6.66 (d, J ) 2.6 Hz, 1H), 6.56
(dd, J ) 8.3, 2.6 Hz, 1H), 6.21 (d, J ) 8.3 Hz, 1H), 4.99 (tm, J )
7.2 Hz, 1H), 4.46 and 4.34 (AB, J ) 16.5 Hz, 2H), 4.25 (s, 1H),
3.72 (s, 3H), 2.70 (ddd, J ) 10.2, 8.4, 5.9 Hz, 1H), 2.65 (ddd, J )
10.2, 6.8, 4.3 Hz, 1H), 2.41 (br d, J ) 7.3 Hz, 2H), 2.38 (s, 3H),
2.07 (ddd, J ) 12.1, 8.4, 6.8 Hz, 1H), 1.95 (ddd, J ) 12.1, 5.9, 4.3
Hz, 1H), 1.66 (d, J ) 1.1 Hz, 3H), 1.56 (br s, 3H). 13C NMR
(CDCl3) δ 152.9, 146.3, 139.4, 137.0, 133.7, 128.4 (2C), 127.3
(2C), 126.7, 120.6, 112.1, 110.2, 107.7, 93.8, 57.3, 55.9, 54.3, 52.8,
38.8, 38.4, 38.3, 25.9, 18.1. EIMS m/z (relative intensity) 362 (M+,
100), 293 (96), 250 (83), 202 (48), 187 (32), 91 (39). HRMS (ESI/
APCIMS) calcd for C24H30N2O + H, 363.2436; found, 363.2435.
3a,8-Dibenzyl-1-methyl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-
b]indole (14). Prepared from 5c according to general procedure 6.
Upon workup, the crude product was purified by flash chromatog-
raphy (7:3 EtOAc/hexane) to give 14 as pale-yellow oil (0.23 g,
55%). The spectral and analytical data were consistent with those
reported.36
1,3a,8-Tribenzyl-5-methoxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-
b]indole (18). Prepared by stirring 5d with benzyl bromide for 5 h
according to general procedure 5. Upon workup, the crude product
was purified by flash chromatography (1:4 EtOAc/hexane) to give
18 as pale-yellow oil (0.36 g, 59%); Rf 0.76 (3:7 EtOAc/hexane).
IR (CHCl3) νmax 3016, 2932, 1600 cm-1 1H NMR (CDCl3) δ
.
7.27-7.12 (m, 11H), 6.99-6.91 (m, 4H), 6.57 (d, overlap, J )
2.4 Hz, 1H), 6.56 (dd, J ) 9.2, 2.6 Hz, 1H), 6.18 (d, J ) 9.2 Hz,
1H), 4.54 (s, 1H), 4.15 and 3.92 (AB, J ) 16.3 Hz, 2H), 3.72 (s,
3H), 3.62 (br s, 2H), 3.07 and 2.88 (AB, J ) 13.4 Hz, 2H), 2.66
(m, 2H), 2.23 (ddd, J ) 11.8, 8.6, 8.3 Hz, 1H), 1.97 (ddd, J )
11.8, 4.0, 4.0 Hz, 1H). 13C NMR (CDCl3) δ 152.6, 146.7, 139.6,
139.4, 138.2, 136.2, 130.4 (2C), 128.3 (2C), 128.2 (2C), 128.1 (2C),
127.9 (2C), 127.2 (2C), 126.7, 126.6, 126.3, 112.7, 110.6, 107.5,
90.7, 58.2, 56.0, 55.4, 53.1, 50.6, 45.4, 38.4. EIMS m/z (relative
intensity) 460 (M+, 100), 370 (60), 279 (16), 251 (28), 91 (49).
HRMS (ESI/APCIMS) calcd for C32H32N2O + H, 461.2593; found,
461.259.
8-Benzyl-1-methyl-3a-(pyridin-4-ylmethyl)-1,2,3,3a,8,8a-hexahy-
dropyrrolo[2,3-b]indole (15). Prepared from 5l according to
general procedure 6. Upon workup, the crude product was purified
by flash chromatography (2:3 EtOAc/hexane) to give 15 as colorless
crystals (0.33 g, 79%); mp 113-115 °C (acetone/CH2Cl2); Rf 0.17
(EtOAc). IR (CHCl3) νmax 3034, 2966, 1602 cm-1 1H NMR
.
(CDCl3) δ 8.36 (br d, J ) 6.0 Hz, 2H), 7.22 (m, 3H), 6.98 (m,
4H), 6.77 (br d, J ) 6.0 Hz, 2H), 6.71 (td, J ) 7.4, 0.7 Hz, 1H),
6.20 (br d, J ) 7.8 Hz, 1H), 4.30 (s, 1H), 4.26 and 4.12 (AB, J )
16.4 Hz, 2H), 3.20 and 2.84 (AB, J ) 13.2 Hz, 2H), 2.72 (ddd, J
) 9.4, 8.4, 5.8 Hz, 1H), 2.67 (ddd, J ) 9.4, 6.5, 4.3 Hz, 1H), 2.36
(s, 3H), 2.21 (ddd, J ) 11.9, 8.4, 6.5 Hz, 1H), 2.12 (ddd, J ) 11.9,
5.8, 4.3 Hz, 1H). 13C NMR (CDCl3) δ 151.7, 149.3 (2C), 147.2,
138.4, 133.3, 128.4 (2C), 128.3, 126.9 (2C), 126.8, 125.3 (2C),
123.2, 117.6, 107.2, 91.8, 57.7, 52.7, 52.1, 45.1, 39.6, 38.9. EIMS
m/z (relative intensity) 355 (M+, 100), 298 (39), 263 (73), 220
(78), 172 (32), 91 (41). HRMS (EI) calcd for C24H25N3, 355.2048;
found, 355.2044.
General Procedure 6 for the Preparation of Pyrroloindo-
lines 8, 10-12, 14-17, and 20-22. To a solution of the
corresponding pyrroloindoline (5b-5l) (1.19 mmol) in MeOH (9
mL) at room temperature was added 37% aq CH2O (0.8 mL, 9.87
mmol). The resulting mixture was stirred at this temperature for
3 h, then cooled to 0 °C, and NaBH4 (0.19 g, 5.13 mmol) was
added portionwise over 5 min. After stirring the mixture for 1 h at
room temperature, the solvent was removed under reduced pressure,
the residue was treated with H2O (14 mL) and Et2O (20 mL). The
aqueous layer was extracted with Et2O (2 × 30 mL) and the
combined organic layers were washed with brine (1 × 40 mL),
dried, and concentrated under reduced pressure to afford the
corresponding N(1)-methylated pyrroloindolines (8, 10-12, 14-17,
and 20-22). Data for the new compounds (8, 12, 15-17, 20, and
21) follow.
1-Methyl-3a,8-bis(pyridin-4-ylmethyl)-1,2,3,3a,8,8a-hexahy-
dropyrrolo[2,3-b]indole (16). Prepared from 5e according to
general procedure 6. Upon workup, the crude product was purified
by flash chromatography (1:1 EtOH/acetone) to give 16 as pale-
yellow oil (0.27 g, 64%); Rf 0.20 (1:1 EtOH/acetone). IR (CHCl3)
5-Methoxy-1-methyl-3a,8-bis(3-methylbut-2-enyl)-1,2,3,3a,8,8a-
hexahydropyrrolo[2,3-b]indole (8). Prepared from 5b according
to general procedure 6. Upon workup, the crude product was
purified by flash chromatography (4:1 EtOAc/hexane) to give 8 as
pale-yellow oil (0.27 g, 67%); Rf 0.19 (EtOAc). IR (CHCl3) νmax
1
νmax 3074, 2966, 1602 cm-1. H NMR (CDCl3) δ 8.45 (dm, J )
4.6 Hz, 2H), 8.38 (dm, J ) 4.4 Hz, 2H), 7.09 (dd, J ) 7.4, 1.1 Hz,
1H), 7.00 (td, J ) 7.8, 1.3 Hz, 1H), 6.90 (br d, J ) 4.4 Hz, 2H),
6.79 (br d, overlap, J ) 4.4 Hz, 2H), 6.76 (td overlap, J ) 7.5, 1.0
Hz, 1H), 6.06 (br d, J ) 7.8 Hz, 1H), 4.26 (s, 1H), 4.17 and 4.07
(AB, J ) 17.5 Hz, 2H), 3.27 and 2.88 (AB, J ) 13.1 Hz, 2H),
2.72 (m, 2H), 2.35 (s, 3H), 2.24 (ddd, J ) 12.0, 6.6, 3.8 Hz, 1H),
1.96 (ddd, J ) 12.0, 5.9, 4.4, Hz, 1H). 13C NMR (CDCl3) δ 151.2,
149.8 (2C), 149.2 (2C), 148.1, 147.1, 133.2, 128.4, 125.2 (2C),
123.3, 121.8 (2C), 118.3, 106.9, 92.8, 57.9, 52.6, 51.4, 45.2, 39.7,
39.1. EIMS m/z (relative intensity) 356 (M+, 98), 299 (32), 264
(90), 221 (100), 172 (58), 92 (16). HRMS (EI) calcd for C23H24N4,
356.2001; found, 356.2004.
1
3022, 2934, 1594 cm-1. H NMR (CDCl3) δ 6.63 (dd, overlap, J
) 7.9, 2.6, 1H), 6.61 (d, overlap, J ) 2.6 Hz, 1H), 6.38 (dd, J )
7.9, 0.9 Hz, 1H), 5.18 (tm, J ) 6.6 Hz, 1H), 4.97 (tm, J ) 7.2 Hz,
1H), 4.26 (br s, 1H), 3.86 (br dd, J ) 16.1, 5.7 Hz, 1H), 3.74 (br
dd, J ) 16.1, 6.8 Hz, 1H), 3.74 (s, overlap, 3H), 2.71 (br ddd, J )
9.4, 6.6, 3.3 Hz, 1H), 2.54 (ddd, J ) 9.4, 9.3, 5.9 Hz, 1H), 2.48 (s,
overlap, 3H), 2.41 (br d, J ) 7.0 Hz, 2H), 2.06 (ddd, J ) 12.1,
9.3, 6.6 Hz, 1H), 1.91 (ddd, J ) 12.1, 5.9, 3.3 Hz, 1H), 1.70 (s,
3H), 1.69 (s, 3H), 1.66 (br s, 3H), 1.58 (br s, 3H). 13C NMR
(CDCl3) δ 153.0, 146.3, 137.1, 134.2, 133.6, 121.5, 120.6, 112.4,
109.9, 108.4, 92.0, 57.3, 55.9, 52.6, 48.3, 38.9, 38.3, 37.3, 25.9,
25.7, 18.1, 18.0. EIMS m/z (relative intensity) 340 (M+, 76), 271
(100), 240 (95), 229 (48), 203 (23), 160 (21), 69 (15). HRMS (ESI/
APCIMS) calcd for C22H32N2O + H, 341.2587; found, 341.2593.
3a,8-Dibenzyl-5-methoxy-1-methyl-1,2,3,3a,8,8a-hexahydro-
pyrrolo[2,3-b]indole (17). Prepared from 5d according to general
procedure 6. Upon workup, the crude product was purified by flash
chromatography (4:1 EtOAc/hexane) to give 17 as pale-yellow oil
(0.31 g, 68%); Rf 0.17 (EtOAc). IR (CHCl3) νmax 3022, 2936, 1598

5282 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
RiVera-Becerril et al.
cm-1. 1H NMR (CDCl3) δ 7.25-7.16 (m, 6H), 7.09-7.06 (m, 2H),
6.92-6.89 (m, 2H), 6.60 (d, J ) 2.2 Hz, 1H), 6.54 (dd, J ) 8.5,
2.3 Hz, 1H), 6.12 (d, J ) 8.5 Hz, 1H), 4.24 (s, 1H), 4.03 and 3.96
(AB, J ) 16.0 Hz, 2H), 3.71 (s, 3H), 3.17 and 2.82 (AB, J ) 13.5
Hz, 2H), 2.69 (ddd, J ) 9.5, 7.7, 6.1 Hz, 1H), 2.59 (ddd, J ) 9.5,
6.6, 4.7 Hz, 1H), 2.27 (br s, 3H), 2.21 (ddd, J ) 11.9, 7.7, 6.6 Hz,
1H), 2.06 (ddd, J ) 11.9, 6.1, 4.7 Hz, 1H). 13C NMR (CDCl3) δ
152.9, 146.4, 139.3, 138.2, 136.3, 130.2 (2C), 128.3 (2C), 127.9
(2C), 127.3 (2C), 126.6, 126.3, 112.7, 110.5, 108.4, 93.2, 58.4, 55.9,
54.1, 52.8, 45.8, 39.1, 38.6. EIMS m/z (relative intensity) 384 (M+,
100), 293 (9), 250 (62), 202 (20), 91 (41), 65 (11). HRMS (ESI/
APCIMS) calcd for C26H28N2O + H, 385.2280; found, 385.2279.
1-Methyl-3a,8-di(n-propyl)-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-
b]indole (20). Prepared from 5f according to general procedure 6.
Upon workup, the crude product was purified by flash chromatog-
raphy (EtOAc) to give 20 as pale-yellow oil (0.18 g, 60%); Rf 0.11
Preparation of Furoindoline 27 and Indoline 28. 3a,8-Bis(3-
methylbut-2-enyl)-2,3,3a,8a-tetrahydro-8H-furo[2,3-b]indole (27).
To a precooled (0 °C) solution of the oxindole-3-acetic acid 2619
(0.17 g, 0.52 mmol) in dry THF (20 mL) was added NaH (30 mg,
1.25 mmol), and the reaction mixture was stirred for 30 min at
room temperature. After cooling at 0 °C, LAH (80 mg, 2.11 mmol)
was added and stirring continued at this temperature for 3 h. The
reaction was quenched by adding, in sequence, EtOAc (10 mL)
and water (10 mL). The solids were removed by filtration and
washed with EtOAc (2 × 15 mL). The combined organic phases
were washed with brine, dried, and concentrated under reduced
pressure. The crude product was purified by flash chromatography
on silica gel (1:9 EtOAc/hexane) to afford furoindoline 27 (134
mg, 87%), followed by alcohol 28 (<5%) as pale-yellow oils.
Compound 27 has spectroscopic data, which closely match those
reported.30
2-[1,3-Bis(3-methylbut-2-enyl)-2,3-dihydro-1H-indol-3-yl]e-
thanol (28). Compound 28 was prepared as described above for
27, except reduction with LAH was effected under reflux for 3 h.
The crude product was purified by flash chromatography on silica
gel (1:4 EtOAc/hexane) to afford alcohol 28 (144 mg, 93%) as
pale-yellow oil; Rf 0.66 (7:3 EtOAc/hexane). IR (CHCl3) νmax 3566,
3048, 2930, 1602 cm-1. 1H NMR (CDCl3) δ 7.12 (td, J ) 7.7, 1.4
Hz, 1H), 6.99 (dd, J ) 7.1, 1.1 Hz, 1H), 6.78 (td, J ) 7.4, 1.1 Hz,
1H), 6.62 (br d, J ) 8.0 Hz, 1H), 5.27 (tm, J ) 7.1 Hz, 1H), 5.14
(tm, J ) 7.4 Hz, 1H), 3.80 (dd, J ) 14.3, 6.6 Hz, 1H), 3.52 (dd,
J ) 14.3, 6.7 Hz, 1H), 3.49 (m, overlap, 1H), 3.40 (very br s, 1H),
3.32 and 3.00 (AB, J ) 9.1 Hz, 2H), 3.19 (ddd, J ) 11.5, 9.9, 4.1
Hz, 1H), 2.51 (dd, J ) 14.4, 8.2 Hz, 1H), 2.34 (dd, J ) 14.4, 4.5
Hz, 1H), 2.07 (ddd, J ) 14.0, 9.6, 4.9 Hz, 1H), 1.74 (br s, 3H),
1.70 (br s, 6H), 1.61 (br s, 3H), 1.61 (m, overlap, 1H). 13C NMR
(CDCl3) δ 151.9, 136.1, 136.0, 134.1, 127.7, 122.8, 119.9, 119.4,
119.2, 108.9, 63.4, 60.2, 46.9, 46.8, 42.9, 38.2, 26.0, 25.7, 18.0,
17.9. EIMS m/z (relative intensity) 299 (M+, 19), 230 (100), 162
(68), 144 (91), 69 (31). HRMS (ESI/APCIMS) calcd for C20H29NO
+ H, 300.2327; found, 300.2327.
X-ray Crystal Structure of 15. The X-ray data were measured
on a Bruker-Nonius CAD4 diffractometer with Cu KR radiation (λ
) 1.54184 Å). The data were collected in the ω-2θ scan mode.
Unit cell refinements were carried out by using the CAD4 Express
v2.0 software. The structure was solved by direct methods using
the SHELXS-97 program included in the WINGX v1.64.05
crystallographic software package. For the structure refinement, the
non-hydrogen atoms were treated anisotropically, and the hydrogen
atoms included in the structure factor calculation were refined
isotropically. Compound 15 crystallized as colorless blocks in the
orthorhombic space group P212121 with cell dimensions a )
9.777(2) Å, b ) 12.432(2) Å, c ) 16.241(2) Å and was refined to
final R indices (all data) R (%) ) 4.9, Rw (%) ) 13.2. The CCDC
deposition number is 686711.
Determination of the Inhibitory Effect on AChE and BChE
Activities. The method of Ellman et al.37 was followed. Physos-
tigmine was used as reference compound. Rat brain homogenate
and human plasma were used as a source of AChE and BChE,
respectively. Nine different concentrations (10-3-10-7 M) of each
compound were used to obtain inhibition of AChE or BChE activity
comprised between 20% and 80%. The assay solution consisted of
a 0.1 M phosphate buffer, pH 8.0, with the addition of 340 µM
substrate (acetylthiocoline iodide or butyrylthiocholine iodide). Test
compounds were added to the assay solution and preincubated at
37 °C with the enzyme for 20 min followed by the addition of
substrate. Assays were done with a blank containing all components
except AChE or BChE to account for nonenzymatic reaction. The
percentage of inhibition due to the presence of test compounds was
calculated. Each concentration was analyzed three times in triplicate,
and IC50 values were determined graphically transforming the
percent of activity into probit format38 and then plotted as function
of its log concentration.
(EtOAc). IR (CHCl3) νmax 3018, 2962, 1604 cm-1 1H NMR
.
(CDCl3) δ 7.03 (td, J ) 7.6, 1.3 Hz, 1H), 6.94 (ddd, J ) 7.3, 1.3,
0.5 Hz, 1H), 6.62 (td, J ) 7.3, 1.0 Hz, 1H), 6.39 (br d, J ) 8.0 Hz,
1H), 4.28 (s, 1H), 3.31 (m, 1H), 3.14 (m, 1H), 2.63 (ddd, J ) 9.4,
6.7, 3.9 Hz, 1H), 2.54 (ddd, overlap, J ) 9.4, 8.8, 5.8 Hz, 1H),
2.50 (s, 3H), 2.00 (ddd, J ) 11.9, 8.8, 6.7 Hz, 1H), 1.92 (ddd, J )
11.9, 5.8, 3.9 Hz, 1H), 1.71 (m, 2H), 1.62 (m, 2H), 1.27 (m, 1H),
1.07 (m, 1H), 0.91 (t, J ) 7.1 Hz, 3H), 0.85 (t, J ) 7.1 Hz, 3H).
13C NMR (CDCl3) δ 151.4, 135.2, 127.4, 122.8, 116.8, 106.5, 92.1,
57.1, 52.7, 50.3, 42.9, 39.4, 38.6, 20.2, 18.8, 14.6, 11.6. EIMS m/z
(relative intensity) 258 (M+, 100), 229 (22), 214 (18), 200 (99),
186 (48), 172 (56), 158 (16). HRMS (EI) calcd for C17H26N2,
258.2096; found, 258.2102.
3a,8-Diethyl-1-methyl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-
b]indole (21). Prepared from 5g according to general procedure 6.
Upon workup, the crude product was purified by flash chromatog-
raphy (9:1 acetone/hexane) to give 21 as pale-yellow oil (0.12 g,
43%); Rf 0.26 (9:1 acetone/hexane). IR (CHCl3) νmax 3020, 2966,
1
1604 cm-1. H NMR (CDCl3) δ 7.05 (td, J ) 7.8, 1.3 Hz, 1H),
6.94 (dd, J ) 7.4, 1.2 Hz, 1H), 6.64 (td, J ) 7.4, 0.9 Hz, 1H), 6.42
(br d, J ) 8.0 Hz, 1H), 4.31 (s, 1H), 3.45 (dq, J ) 14.6, 7.2 Hz,
1H), 3.26 (dq, J ) 14.6, 7.2 Hz, 1H), 2.66 (ddd, J ) 9.3, 6.6, 3.5
Hz, 1H), 2.55 (ddd, J ) 9.3, 9.2, 6.0 Hz, 1H), 2.50 (s, 3H), 2.01
(ddd, J ) 11.9, 9.2, 6.6 Hz, 1H), 1.92 (ddd, J ) 11.9, 6.0, 3.5 Hz,
1H), 1.82 (dq, J ) 14.9, 7.4 Hz, 1H), 1.69 (dq, J ) 14.9, 7.4 Hz,
1H), 1.13 (t, J ) 7.1 Hz, 3H), 0.77 (t, J ) 7.4 Hz, 3H). 13C NMR
(CDCl3) δ 151.3, 135.1, 127.5, 122.8, 117.1, 106.8, 90.8, 57.4, 52.6,
42.7, 39.3, 37.9, 32.8, 11.9, 9.9. EIMS m/z (relative intensity) 230
(M+, 100), 186 (56), 172 (55), 158 (78), 144 (35), 130 (24). HRMS
(EI) calcd for C15H22N2, 230.1783; found, 230.1775.
Preparation of Pyrroloindoline 19. 1-Methyl-3a,8-bis(2-me-
thylbutyl)-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole (19). A
suspension of 4a (60 mg, 0.19 mmol) and 10% Pd-C (60 mg) in
MeOH (7 mL) was stirred under a hydrogen atmosphere (1 atm)
at room temperature for 28 h. The reaction mixture was filtered
through a plug of celite with rinsing by EtOAc. The filtrate was
concentrated under reduced pressure, and the residue was purified
by flash chromatography (EtOAc) to give 19 as pale-yellow oil
(39.0 mg, 64%); Rf 0.15 (EtOAc). IR (CHCl3) νmax 3050, 2958,
1
1604 cm-1. H NMR (CDCl3) δ 7.05 (td, J ) 7.4, 1.4 Hz, 1H),
6.94 (dd, J ) 7.4, 1.4 Hz, 1H), 6.63 (td, J ) 7.4, 1.1 Hz, 1H), 6.39
(br d, J ) 7.7 Hz, 1H), 4.29 (s, 1H), 3.38 (dt, J ) 13.9, 6.9 Hz,
1H), 3.19 (dt, J ) 13.9, 6.9 Hz, 1H), 2.66 (ddd, J ) 9.4, 6.6, 3.6
Hz, 1H), 2.55 (ddd, overlap, J ) 9.4, 8.9, 5.8 Hz, 1H), 2.51 (s,
overlap, 3H), 2.01 (ddd, J ) 11.8, 8.9, 6.6 Hz, 1H), 1.91 (ddd, J
) 11.8, 5.8, 3.6 Hz, 1H), 1.76 (dt, J ) 13.5, 4.6 Hz, 1H), 1.65 (dt,
overlap, J ) 13.5, 4.6 Hz, 1H), 1.58 (m, 1H), 1.47 (m, 2H), 1.45
(m, 1H), 1.14 (m, 1H), 0.95 (br d, overlap, J ) 1.4 Hz, 3H), 0.94
(m, overlap, 1H), 0.92 (br d, overlap, J ) 1.6 Hz, 3H), 0.84 (br d,
J ) 4.7 Hz, 3H), 0.82 (br d, J ) 4.7 Hz, 3H). 13C NMR (CDCl3)
δ 151.4, 135.4, 127.5, 122.8, 116.9, 106.5, 91.6, 56.9, 52.6, 46.6,
39.4, 38.3, 38.1, 35.5, 34.5, 28.5, 26.3, 22.7, 22.6, 22.5, 22.4. EIMS
m/z (relative intensity) 314 (M+, 100), 270 (17), 257 (65), 228
(67), 214 (69), 200 (47), 172 (17), 158 (15), 144 (14). HRMS (EI)
calcd for C21H34N2, 314.2721; found, 314.2722.
Computational Studies. The 3D models of compounds (-)-
4a, (+)-4b, (-)-5a, (-)-14, and (-)-15 were built using ab initio
calculations39 and geometry optimized at the density functional level

Debromoflustramine B as SelectiVe BChE Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5283
of theory (B3LYP/6-31G*) by means of the software Gaussian 03.40
Docking simulations for (-)-4a, (+)-4b, and (-)-5a, were carried
out by means of the AutoDock software (v. 4.0).26 To search the
low-energy docked conformation of the ligand on the receptor, the
empirical free energy function and the Lamarckian genetic algorithm
were used. In this approach, the ligand performs a random walk
around the static protein. At each time step, the ligand is moved
by small increments in global translation and orientation at each
of the rotational torsion angles. Depending on the input parameter
values for the number of generations and the total number of energy
evaluations, configurations are generated for grid points on the
protein surface. The energy of the configurations is calculated based
on a previously defined grid surface. Interaction energies are
calculated with a free-energy-based expression. The docked energies
are listed in increasing order of energy.
The crystal structure of human BChE was obtained from the
RCSB protein database (PDB code 1POI).27 Protonation states were
assumed to be those most common at pH 7, that is, lysines,
arginines, histidines, aspartates, and glutamates, were considered
in ionized form. Water molecules and ions were removed. For the
protein receptor, polar hydrogens were added and Kollman charges
were assigned, whereas the Gasteiger-Marsili partial charges were
used for the ligands. The ligand binding site was defined using a
grid map of 60 × 60 × 60 points (Å3), centered on the hydroxyl
oxygen atom of Ser198, with 0.375 Å grid-point spacing applying
a standard protocol with an initial population of 100 randomly
placed individuals and a maximum number of 2.5 × 107 energy
evaluations per run. All other parameters were maintained at their
default settings. A total of 100 binding conformations were carried
out for each ligand generated by the program and then clusterized
by means of AutoDock Tools.26 Cutoffs for clustering were set at
0.3 Å rmsd and were represented by the result with the most
favorable free energy binding (∆Gbind). The poses reported in
Figures 4 and 5 for compounds (-)-4a and (-)-5a are the low and
the two low-energy conformations, respectively, representative of
the most populated clusters that, according to the Chauvenet
criterion,41 were statistically populated. The fitness score is taken
as the negative of the sum of the energy terms so that larger fitness
scores indicate a better binding. The docked structures were
analyzed using AutoDock Tools for hydrogen bonding and hydro-
phobic interactions.
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Acknowledgment. Financial support from CONACYT-
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Supporting Information Available: GC/MS plots, 1H and 13
NMR spectra for tested compounds 4-22 as well as Figure S1,
illustrating docking experiments of (-)-4 and (-)-5a. This material
is available free of charge via the Internet at http://pubs.acs.org.
C
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