Amino derivatives of indole as potent inhibitors of isoprenylcysteine carboxyl methyltransferase

Article abstract of DOI:10.1021/jm1002843

The enzyme isoprenylcysteine carboxyl methyltransferase (Icmt) plays an important role in the post-translational modification of proteins that are involved in the regulation of cell growth. The indole acetamide cysmethynil is by far the most potent and widely investigated Icmt inhibitor, but it has modest antiproliferative activity and may have pharmacokinetic limitations due to its lipophilic character. We report here that cysmethynil can be structurally modified to give analogues that are as potent in inhibiting Icmt but with significantly greater antiproliferative activity. Key modifications were the replacement of the acetamide side chain by tertiary amino groups, the n-octyl side chain by isoprenyl and the 5-m-tolyl ring by fluorine. Moreover, these analogues have lower lipophilicities that could lead to improved pharmacokinetic profiles.

Full text of DOI:10.1021/jm1002843

6838 J. Med. Chem. 2010, 53, 6838–6850
DOI: 10.1021/jm1002843
Amino Derivatives of Indole As Potent Inhibitors of Isoprenylcysteine Carboxyl Methyltransferase
‡
‡
‡,
Mei-Lin Go,*,†, Jo Lene Leow,† Suresh Kumar Gorla,†,§ Andreas Peter Schuller, Mei Wang, and Patrick J. Casey
€
‡
†Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543, and Program in Cancer and
§
Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore 169857. Presently at Department of Biology, Brandeis University,
Waltham, Massachusetts 02454. Joint main authors.
Received December 21, 2009
The enzyme isoprenylcysteine carboxyl methyltransferase (Icmt) plays an important role in the post-
translational modification of proteins that are involved in the regulation of cell growth. The indole
acetamide cysmethynil is by far the most potent and widely investigated Icmt inhibitor, but it has modest
antiproliferative activity and may have pharmacokinetic limitations due to its lipophilic character.
We report here that cysmethynil can be structurally modified to give analogues that are as potent in
inhibiting Icmt but with significantly greater antiproliferative activity. Key modifications were the
replacement of the acetamide side chain by tertiary amino groups, the n-octyl side chain by isoprenyl and
the 5-m-tolyl ring by fluorine. Moreover, these analogues have lower lipophilicities that could lead to
improved pharmacokinetic profiles.
1. Introduction
For this reason, the possibility of blocking Ras-induced onco-
genic transformation by inhibiting the enzymes involved in the
post-translational processing of the CaaX motif has been
explored for its therapeutic potential.
Proteins that terminate with a C-terminal CaaX motif (where
C is cysteine, a represents an aliphatic amino acid and X
denotes any amino acid) regulate a number of pathways that
areimportantfor oncogenesis. Theseproteins undergoa series
of post-translational modifications at their C-termini that
are critical to their subcellular localization, stability, and func-
tional activity.1,2 The sequence of reactions begins with the
transfer of a 15-carbon farnesyl or 20-carbon geranylgeranyl
residue from the corresponding isoprenoid diphosphate to
the thiol of the cysteine residue in the CaaX motif.3 This is
followed by the endoproteolytic removal of the C-terminal
aaX tripeptide by a prenyl-CaaX specific protease termed Ras
converting enzyme 1 (Rce1a)4,5 and carboxyl methylation of
the newly exposed C-terminal S-prenylated cysteine by iso-
prenylcysteine carboxyl methyltransferase (Icmt).6-8 As a
result of these modifications, hydrophilic cytosolic proteins
are rendered hydrophobic and primed for interaction with
cellular membranes and other proteins.2,9
The most widely studied example of CaaX proteins is the
Ras family of regulatory proteins. Ras is an important mole-
cular switch for a variety of signaling pathways that regulate
normal cell growth and malignant transformation.10 Activat-
ing mutations in Ras genes are implicated in the pathogenesis
of a large number of solid tumors and hematological malig-
nancies.11 Many cancers may also contain alterations in signal-
ing pathways that lie upstream of Ras, and the resulting hyper-
activity of Ras is thought to contribute to tumorigenesis.12-14
The membrane targeting and transforming abilities of Ras
proteins are dependent on the processing of their terminal
CaaX motif by prenylation and postprenylation events.15,16
Protein farnesyltransferase (FTase), the enzyme that cata-
lyzes the prenylation of the cysteine residue, was an early
target of several drug discovery programs.17,18 FTase inhibi-
tors had significant activityin mouse models, but clinical trials
in patients with Ras-driven tumors failed to demonstrate
convincing activity.19 This was attributed to the likely occur-
rence of an alternative prenylation (geranylgeranylation)
pathway when FTase activity was limiting.20,21 For this
reason, attention has shifted to the postprenylation enzymes
Rce1 and Icmt as potential therapeutic targets in oncogenesis.
Of the two enzymes, Icmt is generally viewed as the more
druggable target as mammalian genomes encode only one
member of the Icmt class of methyltransferases and Icmt lacks
homology to other protein methyltransferases.8 Targeted
inactivation of the Icmt gene in mammalian cells led to the
mislocation of K-Ras and the inability of K-Ras and B-Raf to
promote transformation of fibroblasts.22 In mice, inactivation
of Icmt inhibited the progression of K-Ras-induced myelo-
proliferative disease and lung cancer23 as compared to the
acceleration of Ras-induced myeloproliferative disease when
Rce-1 was inhibited.24
Several small molecule inhibitors of Icmt have been re-
ported in the literature,25-32 but most of these agents are
either not sufficiently potent as inhibitors25-29 or have limited
specificity.30,31 The most promising Icmt inhibitor identified
to date is the indole acetamide cysmethynil (2-(1-octyl-5-m-
tolyl-1H-indol-3-yl) acetamide, Figure 1), which was discov-
ered through the screening of a diverse library of over 70 sub-
families with different structural scaffolds.32 Cancer cells
exposed to cysmethynil showed mislocalization of Ras and
impaired growth factor signaling.32 Cysmethynil also blocked
anchorage independent growth of human colon cancer cells
thatwas reversed byoverexpression of Icmt.32 Cell cycle arrest
*To whom correspondence should be addressed. Phone: 65-65162654.
Fax: 65-67791554. E-mail: phagoml@nus.edu.sg.
aAbbreviations: Rce1: Ras converting enzyme; Icmt: isoprenylcys-
teine carboxyl methyltransferase; FTase: farnesyltransferase ; Adomet:
S-adenosylmethionine ; CMC: critical micelle concentration.
r
pubs.acs.org/jmc
Published on Web 09/01/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6839
to give the octyl analogue 4,38 followed by conversion of the
carboxylic acid to an acid chloride and reaction with a
secondary or heterocyclic amine (Scheme 2). Suzuki coupling
was then initiated at position 5 to introduce the m-tolyl ring.
Scheme3 outlines the reaction sequences for the synthesis of
the tertiary amines of series 4. Starting from 5-bromoindole,
N-alkylation and Suzuki coupling were carried out to give
3-1, the cysmethynil analogue without the acetamide side
chain. 3-1 was reacted with a secondary amine and aqueous
formaldehyde in a Mannich reaction to give the amines of
series 4.
Figure 1. Structure of cysmethynil. Modifications investigated
were at positions 1 (n-octyl), 3 (acetamide), and 5 (m-tolyl).
Series 5 comprised of various analogues of cysmethynil in
which the acetamide side chain was replaced by its homo-
logues (5-1, 5-2) and isosteric groups (methyl/ethyl esters
5-3, 5-4, retroamide 5-5, and sulphonamide 5-6). The
esters were prepared from 2-(5-bromo-1H-indol-3-yl) acetic
acid. The latter was functionalized at position 1, converted to
the ester in an acid catalyzed reaction, and then subjected to
Suzuki coupling (Scheme 4).
Scheme 5 outlines the synthesis of the retroamide 5-5,
sulphonamide 5-6, and the short chain homologue 5-1.
Starting from 5-bromo-1H-indole-3-carbaldehyde, sequential
N-alkylationandSukuzi couplinggave9. The aldehydicgroup
of 9 was converted to the oxime 10 (mixture of E and Z) with
hydroxylamine39 and reacted with sodium borohydride to give
the primary amine analogue 4-1, which was then acetylated
to give the retroamide 5-5 or sulphonylated to give the
sulphonamide 5-6. The short chain homologue of cysmethy-
nil (5-1) was prepared by oxidation40 of the aldehydic group
of 9 to give the carboxylic acid 11, which was converted in situ
to the acidchlorideand then reacted with gaseousammonia to
give 5-1.
The long chain homologue of cysmethynil (5-2) was
obtained by converting 2-(5-bromo-1H-indol-3-yl) acetic acid
to its methyl ester 12, followed by hydride reduction to the
corresponding alcohol 13,41 displacement of the alcohol OH
with the nitrile anion,41 and hydrolysis of the nitrile 14 to the
carboxamide 15. The 5-m-tolyl substituent was introduced
by Suzuki coupling, followed by N-alkylation to give 5-2
(Scheme 6)
Series 6 was synthesized following the earlier reported pro-
cedures. The amines 6-1, 6-2, 6-4 to 6-7, 6-9, and 6-10
were prepared by the Mannich reaction of functionalized
indoles. The reaction pathways are given in Scheme 7. The
acetamide 6-8 was synthesized in the same way as 1-8,
starting with 5-fluoro-1H-indole-3-carbaldehyde but with
1-chloro-3-methylbut-2-ene as alkylating agent. The synthesis
of the tertiary amides 6-11 and 6-12 were carried out following
the reactionsequences described in Scheme2. 6-3 was synthe-
sized from 5-bromo-1H-indole by alkylation with 1-chloro-
4-methylbut-2-ene, followed by Suzuki coupling.
and induction of autophagy were found to contribute to
the cell death that accompanied the inhibition of Icmt by
cysmethynil.33 While cysmethynil is the most promising inhi-
bitor reported to date, we have noted its poor water solubility
and strong binding to plasma proteins. A quick assessment of
its compliance to drug-like filters like the Lipinski’s “Rule of
Five”34 and other criteria35 shows that it exceeds the lipophilic
threshold for drug-likeness (ClogP of cysmethynil is 7) and
just complies with the cutoff value35 for rotatable bonds. Less
lipophilic cysmethynil analogues will have a better solubility-
lipophilicity balance, which may translate to an improved
pharmacokinetic profile and bioavailability.36 On the other
hand, an excessive reduction in lipophilicity may result in
compounds that are not able to gain access to the membrane
bound Icmt and thus fail to bring about adequate inhibition.
In an effort to reconcile these conflicting requirements, we
have synthesized cysmethynil analogues with a 104 fold
variation in lipophilicities and evaluated them for Icmt inhi-
bitory activities as well as antiproliferative activity on breast
cancer MDA-MB-231 cells. Our results showed that cys-
methynil can be structurally modified to give analogues that
are significantly more potent than cysmethynil and yet possess
lower lipophilicities that could lead to improved bioavailability.
2. Chemistry
The synthesized compounds were classified as series 1-6
based on modifications made to positions 1, 3, and 5 of cys-
methynil (Table 1). Except for series 6, where compounds
were modified at two or more positions, only one position was
systematically altered in the other series. Briefly, series 1
comprised compounds that were modified at position 5, while
in series 2, changes were made to the n-octyl side chain at
position 1. Theacetamidesidechainatposition3 was replaced
by tertiary amides in series 3, tertiary amines in series 4, and
related homologues and isosteric groups in series 5.
Scheme 1 gives the reaction sequence for series 1 (except
1-7, 1-8) and series 2. Starting with 5-bromo-1H-indole-3-
carbaldehyde, the aldehydic group was converted to the nitrile
1 in a stepwise process that involved in situ reduction to an
alcohol by sodium borohydride, followed by displacement of
the alcoholic OH by a nitrile anion.37 Nitrile 1 was hydrolyzed
under basic conditions to give the carboxamide 2, which was
then reacted by Suzuki coupling with a suitably substituted
phenylboronic acid. In the final step, N-alkylation of the ring
nitrogen of the indole acetamide was achieved by reaction
with an alkyl halide in the presence of sodium hydride. In the
case of 1-7 and 1-8, the same reactions were carried out on
indole-3-carbaldehyde or 5-fluoro-1H-indole-3-carbaldehyde
but without the Suzuki coupling reaction.
3. Results
3.1. Icmt Inhibitory Activity. Icmt inhibitory activity was
determined32 and expressed in terms of IC50 (concentration
required to reduce by 50%, the rate at which the tritiated
methyl group of the methyl donor S-adenosylmethionine
(AdoMet) was transferred to the substrate biotin-farnesyl-
L-cysteine, BFC). Briefly, the assay involved incubation of
enzyme and test compound for a period of time, followed by
addition of the substrate cocktail of AdoMet and BFC. The
concentrations of AdoMet, BFC, and test compounds ranged
from 0.1 to 100 μM, hence under all conditions exceeded the
The tertiary amides of series 3 were synthesized by N-alkylation
ofthe indolenitrogen of2-(5-bromo-1H-indol-3-yl)acetic acid

6840 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Go et al.
Table 1. IC50 Values for Icmt Inhibition, Antiproliferative Activities on MDA-MB-231 Cells and SlogP Values of Test Compoundsa

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6841
Table 1. Continued
a1SlogP is the logarithm of the octanol/water partition coefficient of the test compound in its protonated state. If the compound has no basic groups
and does not exist in a protonated state, SlogP will be calculated from the existing state of the compound. SlogP values were determined from geometry
minimized structures using the software MOE (2008.1001) (Chemical Computing Group, Montreal, Canada). 2Mean and SD of 3 or more deter-
minations. 3ND = Not determined because compound was insoluble at higher concentrations. 45-Phenyl is absent in 1-7 and 1-8 and replaced by
H and F respectively.

6842 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Go et al.
Scheme 1a
aReagents and conditions: (a) (i) NaBH4, NH2CHO-MeOH, rt, (ii) KCN, 100 °C, 2.5 h; (b) KOH, t-BuOH, reflux, 3 h; (c) substituted phenylboronic
acid, Pd(PPh3)4, NaHCO3, EtOH/toluene, reflux; (d) alkyl/arylalkyl halide, NaH, DMF, rt f 53-58°C, 3-6 h.
Scheme 2a
aReagents and conditions: (a) C8H17Br, NaH, THF, 0 °C f rt, 4 h; (b) (i) SOCl2, benzene, reflux, 4 h; (ii) 2° amine/heterocyclic amine, THF;
(c) m-tolylboronic acid, Pd(PPh3)4, Na2CO3, EtOH/DME, reflux, 5 h.
Scheme 3a
aReagents and conditions: (a) 1-bromooctane, NaH, DMSO, rt, 3 h; (b) substituted phenylboronic acid, Pd(PPh3)4, EtOH/DME, Na2CO3, reflux,
5 h; (c) aq HCHO, 2° amine (R00H), ZnCl2, EtOH, rt, 10 h.
Scheme 4a
aReagents and conditions: (a) C8H17Br, NaH, THF, 0 °C f rt, 4 h; (b) R-OH, cat. H2SO4, reflux, 1-1.5 h; (c) m-tolylboronic acid, Pd(PPh3)4, EtOH/
DME, Na2CO3, reflux, 5 h.
enzyme concentration which was estimated to be in the pM
range. Under these conditions, a side-by-side comparison of
the IC50 values of test compounds for the purpose of rank-
ordering of potencies is considered valid. IC50 values are given
in Table 1, which also tabulates the lipophilicity descriptor
SlogP42 of the compounds. SlogP is the logarithm of the
octanol/water partition coefficient of the compound in its
protonated state, and it is used in preference to ClogP43 in

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6843
Scheme 5a
aReagents and conditions: (a) C8H17Br, NaH, DMSO, rt, 3 h, DMF; (b) m-tolylboronic acid, Pd(PPh3)4, EtOH/DME, Na2CO3, reflux, 5 h;
(c) NH2OH HCl, Py, EtOH; (d) NaBH4, NiCl2 6H2O, MeOH; (e) CH3COCl, Et3N, THF, rt, 1 h; (f ) CH3SO2Cl, Et3N, THF, rt, 1 h; (g) KMnO4,
3
acetone, rt, 5 h; (h) (i) SOCl2, benzene, reflux, 4 h, (ii) NH3, THF, rt, 30 min.
3
Scheme 6a
a Reagents and conditions: (a) MeOH, H2SO4, reflux, 1-1.5 h; (b) LiAlH4, THF, rt, 30 min; (c) (i) MsCl, TEA, CH2Cl2, 0 °C, 30 min, (ii) KCN,
DMSO, 100 °C; (d) KOH, t-BuOH, reflux, 1 h; (e) m-tolylboronic acid, Pd(PPh3)4, NaHCO3, EtOH/toluene, reflux, 1 h; (f ) bromooctane, NaH, DMF,
rt f 53-58°C, 4 h.
this report as most of the test compounds have basic func-
tionalities which are protonated at physiological conditions.
Among the series investigated, the compounds in series 1
are notable in having the narrowest variation in Icmt inhi-
bitory activity. Changes in the substitution on the 5-phenyl
ring (1-1 to 1-6) had minimal effects on activity while
a larger but still modest 7-fold loss in activity was observed
when the 5-phenyl ring was removed altogether (1-7) or
replaced with 5-fluoro (1-8). The implication is that the
substitution at position 5 makes a relatively small contribu-
tion to overall activity and a range of structural modifica-
tions are permitted at this position.
Unlike position 5, changes at position 1 caused marked
changes to Icmt inhibitory activity, as seen from the sharp
20-fold decline in inhibition when position 1 was not sub-
stituted (2-1). Interestingly, the inhibitory activities of the
series 2 compounds were closely aligned to their lipophilic
character as assessed from SlogP values. Replacing n-octyl of

6844 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Go et al.
Scheme 7a
aReagents and conditions: (a) 1-chloro-3-methylbut-2-ene or 1-bromooctane, NaH, DMSO, rt, 3 h; (b) o-, m-, or p-tolylboronic acid, Pd(PPh3)4,
Na2CO3, EtOH/DME, reflux, 5 h; (c) aq HCHO, 2° amine (R1R2NH or diethylamine), ZnCl2, EtOH, rt, 3 h.
cysmethynil with side chains that are of comparable or higher
lipophilicities (m-trifluoromethylbenzyl 2-2, geranyl 2-4)
had minimal effects on inhibitory activity, but introducing a
less lipophilic group like isoprenyl (2-3) caused a larger drop
in activity. A possible explanation may be that as Icmt is
found in the hydrophobic environment of the endoplasmic
reticulum, only inhibitors with sufficient lipophilicities are
able to gain access to the enzyme. Clearly, the n-octyl side
chain of cysmethynil played a key role in imparting the
desired level of lipophilicity.
The role of the acetamide moiety at position 3 was
investigated in series 3, 4, and 5. Its removal gave compound
3-1, which had no measurable Icmt inhibitory activity. The
loss of activity was far greater than that observed for 1-7
(removal of 5-m-tolyl from position 5) or 2-1 (removal of
n-octyl from position 1), indicating that the contribution of
the acetamide exceeded that of the other functionalities. The
poor activity of 3-1 was clearly not due to a loss in lipo-
philicity as the SlogP of 3-1 was almost an order of magni-
tude greater than that of cysmethynil. When the acetamide
side chain was modified to give the tertiary amides of series 3
and the amines of series 4, we found that all the tertiary
amides (3-2 to 3-6) were comparable to cysmethynil in
terms of Icmt inhibition. On the other hand, most of the
amino analogues (except 4-2, 4-4, 4-10) had submicro-
molar IC50 values and were 2-3 times more potent than
cysmethynil. These potent analogues included the primary
amine 4-1, tertiary amines with alkyl substituents (N,N-
diethyl 4-3, N,N-di(n-propyl) 4-5, N-methyl-N-isopropyl
4-6), and those that had basic nitrogen atoms in heterocyclic
rings (pyrrolidine 4-7, piperidine 4-8, N-methylpiperazine
4-9). Interestingly, the least active amino analogue was
the N-morpholino 4-10, which was coincidentally less basic
(pKa ca. 8) than the other heterocyclic amines. These findings
raised the question as to the possible contribution of the
amide or amino side chain to Icmt inhibition. A hydrogen
(H) bond donor role is unlikely as the tertiary amides of series
3 cannot function in this capacity but were still as potent as
cysmethynil. A H bond acceptor role is more probable as the
electron pairs on the carbonyl oxygen of the amides (series 3)
and the basic amino side chain (series 4) can function in this
capacity. For the amines which are largely protonated at
physiological pH, this would necessitate deprotonation to
the free base, an event that may occur in the hydrophobic
membrane environment of the enzyme.
In series 5, the acetamide side chain is replaced by groups
that are its isosteres or homologues. The short chain homo-
logue 5-1 was significantly less inhibitory than the longer
chain homologue 5-2, which was as active as cysmethynil.
The marked difference in activities may imply the need to
maintain a minimum distance between the acetamide and the
indole ring. Of the isosteric groups, the esters (5-4, 5-5)
fared poorly but the retroamide (5-5) and sulphonamide
(5-6) were as active as cysmethynil.
Unlike the compounds in series 1-5, those in series 6 were
modified at two or more positions (1, 3, or 5). These
modifications included n-octyl or isoprenyl at position 1,
an amine or amide side chain at position 3, and substituted
phenyl or fluoro at position 5. In spite of these multiple
modifications, structure-activity trends are generally simi-
lar to those observed earlier where changes are limited to a
single position. For instance, the inactivity of the isoprenyl
analogue 6-3, like that of its n-octyl analogue 3-1, empha-
sized the importance of retaining a side chain at position 3.
The preference for an amino group at this position is again
seen from the improved activities of the amino analogues

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6845
6-4, 6-5, and 6-6 (they have isoprenyl in place of n-octyl at
position 1) as compared to the isoprenyl analogue of cys-
methynil 2-3. In the case of the amino analogues (6-1, 6-2)
and tertiary amides (6-11, 6-12), which have substituents
other than m-tolyl at position 5, they were as active as their
corresponding analogues which retained the m-tolyl ring.
This is in keeping with the view that position 5 exerts a
limited influence on activity.
There is however a notable difference in SAR among com-
pounds that have concurrent changes at positions 1 and 5.
This is observed among the isoprenyl analogues (6-7, 6-10)
that do not have a m-tolyl group at position 5: they were
largely inactive as inhibitors despite the potency-enhancing
amino side chain at position 3. In fact, for compounds
without the 5-phenyl ring, a lipophilic side chain at position
1 plays a determining role as seen from the 5-fluoro ana-
logues 6-9 (n-octyl, amine) and 6-10 (isoprenyl, amine) as
well as 1-8 (n-octyl, amide) and 6-8 (isoprenyl, amide). In
both instances, more potent inhibition was found for the
n-octyl analogues 6-9 (> 6-10) and 1-8 (> 6-8). There
appears to be a need to maintain a certain lipophilicity
threshold for activity, and in the absence of the 5-phenyl
ring, the n-octyl side chain fulfills this role better than the
shorter and less lipophilic isoprenyl side chain.
3.2. Effect on Cell Growth and Proliferation. The com-
pounds were evaluated for their effects on the viability of
MDA-MB-231 human breast cancer cells by the colorimetric
tetrazolium assay. Inhibitory IC50 values were determined
after incubation for 72 h. As seen from Table 1, cell viability
IC50 values are generally an order of magnitude higher than
those for Icmt inhibition. They are spread over a narrow
30-fold concentration range, unlike the wider 100-fold variation
observed for Icmt IC50 values. The two inhibitory activities
are significantly correlated (Spearman’s F=0.707, p=0.001,
two-tail), which means that strong inhibitors of Icmt are also
those that have potent effects on cell viability. Moreover,
structure-activity trends for the two activities share many
similarities. Accordingly, we found that of the three func-
tionalities (5-m-tolyl, n-octyl, acetamide) in cysmethynil,
removal of the m-tolyl ring (1-7) had the least effect on
antiproliferative activity. Likewise, the antiproliferative ac-
tivity of the series 3 tertiary amides (IC50 16-31 μM) were
comparable to cysmethynil (IC50 22 μM), while the series 4
amines were significantly more potent (IC50 3-13 μM). The
correlation with Icmt inhibitory activity is also observed for
the series 5 and 6 compounds. There are, however, excep-
tions. For instance, 6-11 had weak antiproliferative activity
(IC50 50 μM) in spite of strong inhibition of Icmt (IC50 0.8 μM),
and 2-4, 6-4, and 6-5 had better than expected antiproli-
ferative activities compared to their Icmt inhibitory activ-
ities. The isoprenyl analogues (6-4, 6-5) were notable in
having antiproliferative activities that were comparable to
the most potent amines (4-3, 4-8) in series 4.
having minimal adverse effects on activity may be introduced
at this position. Compound 6-9 is a case in point where
introducing a fluoro atom at position 5 caused a desired fall in
lipophilicity without significant loss of Icmt inhibitory activity.
In the case of position 1, its main contribution is related
to the size and lipophilicity of the attached side chain. This is
clearlydemonstratedinseries2, where the correlationbetween
lipophilicity (SlogP) arising from changes at position 1 and
inhibitory IC50 values is striking. While the geranyl side chain
(present in 2-4) is a viable alternative to n-octyl (present in
cysmethynil 1-1), it is associated with a further increase in
lipophilicity. Interestingly, we found that analogues with the
shorter isoprenyl side chain (6-4, 6-5) retained good activ-
ities (including antiproliferative activity), which would imply
that a less lipophilic side chain is permissible but only if ap-
propriate functionalities like the potency-enhancing amino
group is present at position 3. It is also apparent that ana-
logues with an isoprenyl side chain must have a 5-phenyl sub-
stituent if good activity is to be retained, possibly to compen-
sate for the loss of the lipophilic n-octyl side chain. This is seen
from the activities of 6-4 (> 6-7) and 1-8 (> 6-8).
The critical role of position 3 in influencing Icmt inhibition
and cell viability is an important finding of the present investi-
gation. The absence of a side chain at this position caused
dramatic losses in both activities. On the other hand, introdu-
cing an amino functionality improved activities in most
instances. The potency-enhancing effect of the amino side
chain raised the question as to whether interaction with the
enzymeinvolved the protonated or nonprotonated state of the
amine. The amino analogues would undoubtedly be proto-
nated at physiological pH because of their basic character. Of
note is that Icmt is a membrane bound enzyme that acts on
a lipidated cysteine residue, which is likely, at least, to be a
component of the active site located in a hydrophobic environ-
ment. Thus it is not improbable that the protonated amino
analogue loses a proton in the membrane environment to give
the nonprotonated specie which then interacts with the enzyme,
while the more polar features of the compound are accom-
modated in the hydrophilic AdoMet binding site.
Notwithstanding the hydrophobic environment in which
Icmt is found, several compounds that are less lipophilic than
cysmethynil demonstrated comparable Icmt inhibitory activ-
ities. They are the amino analogues 4-9, 6-4, 6-5, and 6-9,
which are 10-100 times less lipophilic than cysmethylnil
based on SlogP values yet comparable to it in terms of Icmt
inhibition and more potent as antiproliferative agents. The
reduced lipophilicities of these compounds are due to the
dibasic N-methylpiperazine ring in 4-9, the isoprenyl side
chain in 6-4, 6-5, and 5-fluoro in 6-9. There is, however,
a threshold level of lipophilicity beyond which activity is com-
promised, and this is apparent from the need to couple the
isoprenyl side chain with 5-phenyl (6-4, 6-5) and 5-fluoro
with n-octyl (6-9) in the target compounds.
There is a concern that the amphiphilic nature of the test
compounds (lipophilic side chain at position 1 coupled with
a polar amide or amino side chain at position 3) will lead to
nonspecific detergent-like effects that may confound the
inhibition of Icmt which is a polytopic membrane protein.
Hence, the critical micelle concentrations(CMC) ofthe parent
compound (cysmethynil, 1-1) and a potent analogue 4-3
were determined under identical buffer conditions used in the
Icmt assay and found to be 28 and 12 μM, respectively. As
these concentrations are several-fold higher than their Icmt
inhibitory IC50 (1-1, 1.5 μM; 4-3, 0.7 μM), nonspecific
4. Discussion
The present study has provided useful insight into the
structure-activity relationship of cysmethynil as an Icmt
inhibitor. Of the three functionalities attached to the indole
ring of cysmethynil, the substituent at position 5 is seen to
have the least influence on Icmt inhibitory activity as only
incremental changes were observed when modifications were
made here. This may in fact work to advantage as function-
alities that can moderate physicochemical properties while

6846 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Go et al.
inhibition involving micelle formation is unlikely to interfere
with the Icmt inhibitory activities of these compounds.
Wefound theKi of cysmethynil (1-1) and4-3 tobe 3.4and
4.5 μM, respectively (unpublished results), which are some-
what higher than the IC50 values (1-1, 1.5 μM; 4-3, 0.7 μM)
reported in Table 1. The inhibition of Icmt by cysmethynil has
been shown to be strongly time-dependent, with more than
10-fold variation in IC50 depending on whether the value was
determined using initial velocity measurements or those on
final enzyme-inhibitor complex.44 In this instance, Ki deter-
minations were performed under early time conditions. Given
the complexity of the interaction of the indole-based com-
pounds with Icmt, kinetics analyses of the interaction of 4-3
and other promising analogues with Icmt warrants further
investigation.
Many of the compounds with strong Icmt inhibitory activ-
ity have pronounced effects on cell proliferation. The func-
tional consequences of Icmt inhibition remain to be fully
elucidated. We have shown in an earlier report that cysmethy-
nil arrested the cell cycle of prostate cancer cells at the G1
phase at concentrations close to its antiproliferative IC50 and
induced autophagic cell death at higher concentrations.33
These effects were not observed with 3-1, which had negli-
gible Icmt inhibition.33 On the other hand, the more potent
Icmt inhibitors 4-3 and 6-4 demonstrated similar effects as
cysmethynil but at significantly lower concentrations.45
Taken together, the available evidence on the biological
consequences of Icmt inhibition strongly supports the poten-
tial of the enzyme as a novel therapeutic target for oncogen-
esis. Hence there is a compelling rationale for developing
specific Icmt inhibitors as pharmacological tools to elucidate
the mechanism of action of the enzyme and the functional
consequences of its inhibition and, more importantly, as
potential therapeutic agents for cancer. We have identified
several compounds that are at least equivalent to cysmethynil
in terms of Icmt inhibition but have significantly greater
antiproliferative activity on MDA-MB-231 cells. The gains
in potency were achieved in many instances without an
increase in lipophilicity, and this may translateto an improved
solubility-lipophilicity balance, leading to a more desirable
pharmacokinetic profile that may include a reduction in
plasma protein binding and improved bioavailability. Hence,
the findings of the present investigation may be usefully
employed to guide future synthetic efforts aimed at deriving
clinically useful potent inhibitors of Icmt.
reported. High resolution mass was determined using a Finnigan
Mat 95/XL-T spectrometer equipped with an electron spray
ionization probe (ESI). Spectroscopic data of final compounds
(series 1-6) are given in Supporting Information. Purity of final
compounds was verified by reverse phase HPLC on two differ-
ent solvent systems (isocratic mode) and found to be g95%,
except for 1-8, 3-6, 4-10, 6-8, and 6-10 (92-93% purity).
Details are given in Supporting Information. Compounds 4-6,
6-4, 6-5, 6-6, and 6-9 were analyzed by combustion analysis
(C and H) on a Perkin-Elmer PRE-2400 elemental analyzer and
had C and H values that fall within (5% of theoretical values.
5.2. Synthesis of Series 1 and 2. 5.2.1. 2-(5-Bromo-1H-indol-3-
yl) Acetonitrile (1). The method of Yamada et al.37 was followed.
Briefly, 5-bromo-1H-indole-3-carbaldehyde (4.5 mmol, 1 equiv)
in formamide-methanol (NH2CHO-MeOH) was reacted with
sodium borohydride (NaBH4; 13.5 mmol, 3 equiv). Potassium
cyanide (KCN; 45 mmol, 10 equiv) was added and the mixture
refluxed (100 °C, 2.5 h). Yield: 81%. 1H NMR (300 MHz,
CD3OD): δ 3.73 (s, 2 H), 7.19-7.33 (m, 3 H), 7.69 (s, 1 H). MS
(APCI): m/z 236.1 [M þ H]þ. 2-(5-Fluoro-1H-indol-3-yl)aceto-
nitrile, the intermediate for 1-8, was prepared in a similar
manner starting from 5-fluoro-1H-indole-3-carbaldehyde (52%
yield). 1H NMR (300 MHz, CDCl3): δ 3.66 (s, 2H), 6.90 (dt, J1=
9 Hz, J2=2.1 Hz, 1 H), 7.08 (s, 1 H), 7.13-7.22 (m, 2 H), 8.4 (1H,
NH). 13C NMR (75 MHz, CDCl3) δ 14.2, 102.8, 104.2, 111.0,
112.4, 118.3, 124.8, 126.2, 132.8, 156.3.
5.2.2. 2-(5-Bromo-1H-indol-3-yl)acetamide (2). Nitrile 1 (3.5
mmol, 1 equiv) was refluxed in tertiary butanol (t-BuOH;10 mL)
containing finely powdered 85% KOH (28 mmol, 8 equiv) for
3 h. The reaction mixture was cooled to room temperature,
diluted with water, and acidified (1 M HCl) The resulting
suspension was filtered under reduced pressure, and the residue
was washed with water and dried in vacuo. The product was
isolated as an off-white/light-brown solid. Yield: 89%. 1H NMR
(300 MHz, DMSO-d6): δ 3.44 (s, 2 H), 7.15-7.38 (m, 4 H), 7.73
(s, 1 H), 11.07 (s, 1 H). MS (APCI): m/z 254.1 [M þ H]þ. 2-(5-
Fluoro-1H-indol-3-yl) acetamide (intermediate for 1-8 and
6-8) was obtained from the corresponding nitrile under similar
reaction conditions in 61% yield. 1H NMR (300 MHz, CDCl3):
δ 3.69 (s, 2 H), 5.29 (s, 2 H), 6.98 (dt, J1=9 Hz, J2=2.4 Hz, 1 H),
7.21 (s, 1 H), 7.25-7.33 (m, 2 H), 8.34 (s, 1 H).
5.2.3. General Procedure for Preparation of 2-(5-(Substituted
Phenyl)-1H-indole-3-yl) Acetamides (3). To a suspension of
bromoindole 2 (2 mmol, 1 equiv) in anhydrous toluene (40 mL)
in an ice bath was added Pd(PPh3)4 (5.7 mol %). The bright-
yellow suspension was stirred for 0.5 h, after which was added
(in one portion) the substituted phenylboronic acid (3 mmol, 1.5
equiv) in absolute EtOH (10 mL), followed immediately by a
saturated NaHCO3 solution (25 mL). After refluxing for 1-6 h,
the biphasic mixture was cooled to room temperature and
poured into a solution of brine. The organic phase was sepa-
rated, and the aqueous phase was extracted with ethyl acetate
(EtOAc). The organic extracts were combined, dried (anhydrous
Na2SO4), the drying agent was removed by filtration, and the
filtrate concentrated under reduced pressure to give the crude
product 3, which was used without further purification for the
N-alkylation reaction.
5.2.4. General Procedure for Preparation of 2-(1-Substituted-
5-(phenyl/substituted phenyl)-1H-indol-3-yl)acetamides (1-1 to
1-6, 2-2 to 2-4). To a stirred suspension of sodium hydride
(NaH, 60% dispersion in mineral oil; 3 mmol, 1.5 equiv) in
anhydrous dimethylformamide (DMF; 5 mL) in ice bath was
added dropwise a solution of 3 (2 mmol, 1 equiv) in anhydrous
DMF (10 mL) over 10 min. After stirring at room temperature
for 1.5 h, the alkyl or arylalkyl halide (6 mmol, 3 equiv) was
added dropwise over 5 min. The reaction mixture was heated in
an oil bath (53-58 °C, 3-6 h), cooled to room temperature, and
poured into ice water. The suspension was stirred (10 min) and
extracted with diethyl ether (Et2O). The organic extracts were
combined, washed with brine, and dried (anhydrous Na2SO4).
5. Experimental Section
5.1. General Details. Reagents (synthetic grade or better) were
obtained from commercial suppliers (Sigma-Aldrich Chemical
Co. Inc., Singapore; Alfa Aesar, MA) and used without further
purification. 1H (300 MHz) and 13C (75 MHz) spectra were
measured on a Bruker Spectrospin 300 Ultrashield spectrometer
magnetic resonance spectrometer. Chemical shifts were re-
ported in ppm and referenced to TMS or residual deuterated
solvents (CHCl3 δH 7.26 ppm, DMSO-d6 δH 2.50 ppm; CD3OD
δ
H 3.31 ppm) for 1H spectra and residual CHCl3 (δC 77.16 ppm)
and DMSO-d6 (δC 39.52 ppm) for proton decoupled 13C NMR
spectra. Coupling constants (J ) were reported in Hertz (Hz).
Reactions were routinely monitored by thin layer chromatog-
raphy (TLC) on precoated plates (Silica Gel 60 F254, Merck)
with ultraviolet light as visualizing agent. Column chromatog-
raphy was carried out with Silica Gel 60 (0.04-0.063 mm).
Nominal mass spectra were captured on an LCQ Finnigan
MAT equipped with an atmospheric pressure chemical ioniza-
tion (APCI) probe and m/z values for the molecular ion were

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6847
The drying agent was removed by filtration and the filtrate
concentrated in vacuo to give the crude product, which was
purified by flash silica gel column chromatography (EtOAc/
CH2Cl2) to give 1-1 to 1-6, and 2-2 to 2-4. Compounds 1-7
and 1-8 were obtained by the same method starting from 1H-
indol-3-ylacetamide and 2-(5-fluoro-1H-indol-3-yl)acetamide,
respectively.
5.3. Synthesis of Series 3. 5.3.1. 2-(5-Bromo-1-octyl-1H-indol-
3-yl)acetic Acid (4). The method of Roy et al.38 was followed.
Briefly, 2-(5-bromo-1H-indol-3-yl)acetic acid (28 mmol, 1 equiv)
was reacted with 1-bromooctane (83 mmol, 3 equiv) in the pre-
sence of NaH (60% dispersion in mineral oil, 138 mmol, 5 equiv)
in tetrahydrofuran (THF; 100 mL) at 0 °C. 4 was obtained in
64% yield. 1H NMR (300 MHz, CDCl3): δ 7.71 (s, 1 H), 7.26 (d,
J=2.1 Hz, 1 H), 7.16 (d, J=8.7 Hz, 1H), 7.07 (s, 1 H), 4.03 (t,
J=7.2 Hz, 2 H), 3.73 (s, 2 H), 1.79 (t, J=9.9 Hz, 2H), 1.28-1.24
(m, 10 H), 0.86 (t, J=6.3 Hz, 3H). 13 C NMR (75 MHz, CDCl3):
δ 177.3, 134.8, 129.2, 128.0, 124.5, 121.5, 112.5, 110.9, 105.6,
46.5, 31.7, 30.7, 30.1, 29.1, 29.0, 26.9, 22.5, 14.0.
5.3.2. General Procedure for the Synthesis of 2-(5-Bromo-1-
octyl-1H-indol-3-yl)-N-substituted Acetamides (5). A mixture of
4 (1 mmol, 1 equiv) and thionyl chloride (SOCl2; 2 mL, 27 equiv)
were refluxed for 4 h in dry benzene (5 mL), after which excess
SOCl2 and benzene were removed by distillation in vacuo. The
crude acid chloride was dissolved in dry THF (4 mL) and added
dropwise to a stirred solution of the amine in dry THF at 0-5 °C.
The reaction mixture was stirred at 0-5 °C for 1 h, after which
THF was removed in vacuo and the residue extracted with
CH2Cl2 and dried (anhydrous Na2SO4). Removal of the solvent
under reduced pressure gave the desired amide, which was puri-
fied by column chromatography with EtOAc/hexane as eluting
solvents. Yields and spectroscopic data of the amides are given
in Supporting Information.
made alkaline by the addition of 4 M NaOH. The mixture was
extracted with EtOAc, the solvent was removed under reduced
pressure, and the residue purified by column chromatography
with CH2Cl2/MeOH or EtOAc/hexane as eluting solvents.
Yields and spectroscopic data of 4-2 to 4-10 are given in
Supporting Information.
5.5. Synthesis of 5-3 and 5-4. 5.5.1. General Method for the
Preparation of Alkyl Esters of 2-(5-Bromo-1-octyl-1H-indol-3-
yl) Acetic Acid (7a, 7b). To a solution of acid 4 (4 mmol, 1 equiv)
in 20 mL of the required alcohol (MeOH or EtoH) was added
10 drops of concentrated H2SO4. The mixture was heated under
reflux for 1-1.5 h, cooled to room temperature, and evaporated
in vacuo. The residue was treated with a saturated solution of
NaHCO3 (50 mL), extracted with EtOAc, and dried (anhydrous
Na2SO4). Removal of the solvent under reduced pressure gave
the crude product, which was purified by column chromatog-
raphy with CH2Cl2 as eluting solvent. Yields and spectroscopic
data of 7a and 7b are given in Supporting Information.
5.5.2. General Method for the Preparation of Alkyl Esters of
2-(1-Octyl-5-m-tolyl-1H-indol-3-yl) Acetic Acid (5-3, 5-4). The
esters 7a, 7b were reacted with m-tolylboronic acid as described
in Section 5.3.3. Yields and spectroscopic details of 5-3 and
5-4 are given in Supporting Information.
5.6. Synthesis of 4-1, 5-1, 5-5, 5-6. 5.6.1. 5-Bromo-1-
octyl-1H-indole-3-carbaldehyde (8). 5-Bromo-1H-indole-3-car-
baldehye was reacted with 1-bromooctane and sodium hydride
as described in Section 5.4.1. Yield: 85%. 1H NMR (300 MHz,
CDCl3): δ 9.91 (s, 1 H), 8.43 (s, 1 H), 7.66 (s, 1 H), 7.36 (d, J=7.5
Hz, 1 H), 7.20 (d, J=8.7, 1 H), 4.11 (t, J=7.2 Hz, 2 H), 1.85 (t,
J=6.3 Hz, 2 H),1.30-1.24 (m,10 H), 0.86 (t, J=6 Hz, 3 H). 13C
NMR (75 MHz, CDCl3): δ 184.1, 138.7, 135.8, 126.8, 126.8,
124.7, 117.3, 116.4, 111.4, 47.4, 32.7, 31.6, 29.6, 29.0, 26.7, 22.5,
14.0.
5.3.3. General Procedure for the Synthesis of the Tertiary
Amides (3-2 to 3-6). The method of Li et al.46 was followed
with some modifications. Briefly, a solution of the acetamide 5
(1 mmol, 1 equiv) in 4 mL of dimethoxyethane (DME) was
added Pd(PPh3)4 (0.05 mmol, 0.05 equiv), followed by stirring
under argon for 15 min. A solution of m-tolylboronic acid
(1 mmol, 1 equiv) in 1.5 mL of EtOH was added, stirred for
15 min, after which 2 M Na2CO3 (4 mL) was added and the mixture
was refluxed (5 h) under argon. On cooling, the organic solvent
was removed and the resulting suspension was extracted with
CH2Cl2 and dried (anhydrous Na2SO4). The residue obtained
on removal of the solvent was purified by column chromatog-
raphy on silica gel with EtOAc/hexane as eluting solvents.
5.4. Synthesis of Series 4. 5.4.1. 5-Bromo-1-octyl-1H-indole
(6). The method of Na et al.47 was followed. Briefly, 5-bromo-
1H-indole (5 mmol, 1 equiv) was reacted with NaH (60% in
mineral oil dispersion, 6 mmol, 1.2 equiv) in anhydrous dimethyl-
sulfoxide (DMSO) at room temperature. After 1 h of stirring at
the same temperature, 1-bromooctane was added and the
mixture stirred for 3 h. Distilled water was added to stop the
reaction and mixture was worked up in the usual manner. Yield:
92%. 1H NMR (300 MHz, CDCl3): δ 0.82 (t, J=6.9 Hz, 3 H),
1.20-1.24 (m, 10 H), 1.76 (t, J=6.9 Hz, 2 H), 4.03 (t, J=7.2 Hz,
2 H), 6.37 (d, J=3 Hz, 1 H), 7.04 (d, J=3 Hz, 1H), 7.17 (s, 1 H),
7.21 (d, J = 1.2 Hz, 1 H), 7.69 (d, J = 1.5 Hz, 1 H). 13C NMR
(75 MHz, CDCl3) δ 14.0, 22.5, 26.9, 29.1(2C), 30.1, 31.7, 46.5,
100.4, 110.7, 112.4, 123.3, 124.0, 128.8, 130.1, 134.6.
5.6.2. 1-Octyl-5-m-tolyl-1H-indole-3-carbaldehyde (9). Alde-
hyde 8 was reacted with m-tolylboronic acid as described in
Section 5.3.3. Yield: 62%. 1H NMR (300 MHz, CDCl3): δ 9.98
(s, 1 H), 8.53 (s, 1 H), 7.66 (s, 1 H), 7.57-7.46 (m, 2 H), 7.39-
7.29 (m, 2 H), 7.13 (d, J=7.2 H, 1 H), 4.1 (t, J=8.7 Hz, 2 H),
2.42 (s, 3 H), 1.86 (t, 2 H), 1.29-1.24 (m, 10 H), 0.86 (t, J=6.6
Hz, 3 H). 13C NMR (75 MHz, CDCl3): δ 184.4, 141.4, 138.9,
138.2, 136.6, 136.4, 128.5, 128.2, 127.6, 125.9, 124.5, 123.5,
120.4, 118.1, 110.3, 47.3, 31.7, 29.7, 29.0 (2C), 26.8, 22.5, 21.5,
14.0.
5.6.3. (E and Z)-1-Octyl-5-m-tolyl-1H-indole-3-carbaldehyde
Oxime (10). The method of Jansen et al.39 was followed. Briefly,
9 (1.03 mmol, 1 equiv) was refluxed (2 h) with hydroxylamine
hydrochloride (NH2OH HCl; 2.57 mmol, 2.5 equiv) and pyr-
3
idine (2.67 mmol, 2.6 equiv) in EtOH (20 mL). The mixture was
acidified with 10% HCl and extracted with Et2O, and the
ethereal layer was washed successively with 10% HCl and water
and dried (anhydrous Na2SO4). Removal of solvent in vacuo
gave a residue that was triturated with petroleum ether and
filtered to give 10 in 74% yield. It was used without further
purification for the next step.
5.6.4. 1-Octyl-5-m-tolyl-1H-indol-3-yl) Methanamine (4-1).
To a stirred solution of nickel chloride (NiC12 6H2O; 0.76
3
mmol, 1 equiv) in MeOH (12 mL) was added 10 (0.8 mmol,
1 equiv) and NaBH4 (4.2 mmol, 5.5 equiv) in one portion. After
5 min of stirring, the black precipitate was removed by vacuum
filtration and the filtrate concentrated in vacuo to approxi-
1
5.4.2. 1-Octyl-5-m-tolyl-1H-indole (3-1). Indole 6 was re-
acted with m-tolylboronic acid as described in Section 5.3.3. to
give 3-1. Yield: 85%.
5.4.3. General Procedure for the Synthesis of N-Substituted
(1-Octyl-5-m-tolyl-1H-indol-3-yl) methyl) ethanamine (4-2 to
4-10). The amine (1 mmol, 1 equiv), zinc chloride (ZnCl2; 1.5
mmol, 1.5 equiv), formaldehyde (HCHO; 1 mmol, 36% aq,
1 equiv), indole 3-1 (1 mmol, 1equiv), and EtOH (3 mL) were
stirred together in a round-bottom flask for 10 h at room tem-
perature. Distilled water was added to the mixture, which was
mately /3 of its original volume. It was poured into aqueous
ammonia solution (15% v/v), extracted with EtOAc, and dried
(anhydrous Na2SO4). Removal of solvent gave a viscous, dark-
colored oil which was purified by flash silica gel column
chromatography with CH2Cl2/MeOH to give 4-1. Yield: 54%.
5.6.5. N-[(1-Octyl-5-m-tolyl-1H-indol-3-yl)methyl]acetamide
(5-5). Acetyl chloride (0.0.628 mmol, 1 equiv) was added to a
solution of amine 4-1 (0.63 mmol, 1 equiv) and triethylamine
(0.94 mmol, 1.5 equiv) in THF (4 mL) at 0 °C. The reaction
mixture was stirred at room temperature (27 °C) for 1 h, after

6848 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Go et al.
which the precipitate was removed by vacuum filtration, the fil-
trate was concentrated under reduced pressure, and the residue
was purified by column chromatography with CH2Cl2 as eluting
solvent. Yield: 65%.
5.6.6. N-((1-Octyl-5-m-tolyl-1H-indol-3-yl)methyl)methane-
sulfonamide (5-6). Methanesulfonyl chloride (0.434 mmol, 1 equiv)
was added to a solution of amine 4-1 (0.434 mmol, 1 equiv) and
triethylamine (0.651 mmol, 1.5 equiv) in THF (4 mL) at 0 °C. The
reaction mixture was stirred at room temperature for 1 h. The
precipitate was removed by filtration, the filtrate was concentrated
under reduced pressure, and the residue was purified by column
chromatography with CH2Cl2 as eluting solvent. The sulfonamide
5-6 was obtained in 61% yield.
5.6.7. 1-Octyl-5-m-tolyl-1H-indole-3-carboxylic Acid (11).
Compound 9 (0.573 mmol, 1 equiv) was dissolved in acetone
(10 mL) and treated with an aqueous solution of potassium
permanganate (KMnO4, 5 mL, 1.5 mmol).40 The reaction mix-
ture was stirred at room temperature for 5 h, treated with 10%
H2O2 until the pink color of KMnO4 was not observed, and then
filtered. The solvent was removed under reduced pressure, and
the resulting residue was filtered again (if necessary), acidified
with 2 M HCl, and extracted with Et2O. The organic solvent was
removed in vacuo, and the residue was purified by column
chromatography with hexane/EtOAc as eluting solvents. Yield:
62%. 1H NMR (300 MHz, CDCl3): δ 8.84 (s, 1 H), 7.92 (s, 1 H),
7.54-7.48 (m, 2 H), 7.41-7.29 (m, 3 H), 7.14 (d, J = 6.6 Hz,
1 H), 4.12 (t, J=6.6 Hz, 2 H), 2.44 (s, 3 H), 1.87 (t, J=6.6 Hz,
3 H), 1.37-1.25 (m, 10 H), 0.86 (t, J=6.6 Hz, 3 H). 13C NMR
(75 MHz, CDCl3): δ 170.3, 142.3, 141.9, 138.4, 138.2, 136.1,
136.0, 135.8, 128.5, 128.3, 127.4, 124.6, 120.3, 110.3, 106.3, 47.2,
31.7, 31.1, 29.8, 29.1, 22.6, 21.5, 21.5, 14.0.
Na2SO4), and evaporated to dryness. The residue was dissolved
in anhydrous DMSO and reacted with KCN (3.25 mmol,
3 equiv) by heating at 100 °C for 1 h. The mixture was diluted
with a water-ice mixture, extracted with CHCl3, and the combined
organic layer was washed with water, dried (anhydrous Na2SO4),
and evaporated to dryness under reduced pressure. The residue
was purified by column chromatography with CHCl3 as eluting
solvent to give 14 in 75% yield. 1H NMR (300 MHz, DMSO-d6): δ
2.78 (t, J=15 Hz, 2 H), 3.58 (t, J=12 Hz, 2 H), 7.14-7.17 (m, 2
H), 7.20 (d, J = 27 Hz, 1 H), 7.68 (s, 1 H), 11.01 (s, 1 H). MS
(APCI): m/z 261.3 [M þH]þ.
5.7.4. 2-(5-Bromo-1H-indol-3-yl)propanamide (15). Amide 15
was prepared from the nitrile 14 by the method described in
Section 5.2.2. Yield: 82%. 1H NMR (300 MHz, CDCl3): δ 2.44
(t, J=15 Hz, 2 H), 2.96 (t, J=15 Hz, 2 H), 7.09-7.58 (m, 4 H).
MS (APCI): m/z 268.1 [M þH]þ.
5.7.5. 3-(5-m-Tolyl-1H-indol-3-yl)propanamide (16). 16 was
prepared from the 15 by the method described in Section 5.2.3.
Yield: 50%. 1H NMR (300 MHz, CDCl3): δ 2.38 (s, 3 H), 2.49
(t, J=18 Hz, 2 H), 3.02 (t, J=15 Hz, 2 H), 7.12-7.61 (m, 8 H).
MS (APCI), m/z 279.4 [M þH]þ.
5.7.6. 3-(1-Octyl-5-m-tolyl-1H-indol-3-yl)propanamide (5-2).
5-2 was prepared from 16 by the method described in Section
5.2.4. 5-2 was obtained in 77% yield.
5.8. Synthesis of Series 6. 5.8.1. General Method for the
Synthesis of 5-Halo-1-(3-methylbut-2-enyl)-1H-indole (17a, 17b).
5-Bromo-1H-indole or 5-fluoro-1H-indole was reacted with
1-chloro-3-methylbut-ene by the method described in Section 5.4.1.
to give the 5-bromo (17a) or 5-fluoro (17b) analogue. Yields and
spectroscopic details of 17a and 17b are given in Supporting
Information.
5.8.2. 1-(3-Methylbut-2-enyl)-5-m-tolyl-1H-indole (6-3). 17a
was reacted with m-tolylboronic acid as described in Section 5.3.3.
6-3 was obtained in 75% yield.
5.8.3. General Procedure for the Synthesis of N-Substituted
(1-(3-Methylbut-2-enyl)-5-m-tolyl-1H-indol-3-yl)methyl)ethan-
amines (6-4, 6-5, 6-6). Indole 6-3 was reacted with the amine
in the presence of ZnCl2 and aqueous HCHO as described in
Section 5.4.3. Yields and spectroscopic details of 6-4, 6-5, and
6-6 are given in Supporting Information.
5.8.4. N-Ethyl-N-((5-fluoro-1-(3-methylbut-2-enyl)-1H-indol-
3-yl)methyl)ethanamine (6-10). 17b was reacted with diethyla-
mine in the presence of ZnCl2 and aqueous HCHO as described
in Section 5.4.3. 6-10 was obtained in 77% yield.
5.6.8. 1-Octyl-5-m-tolyl-1H-indole-3-carboxamide (5-1). A
mixture of 11 (0.4 mmol, 1 equiv) and SOCl2 (1 mL, 13 equiv)
in dry benzene (5 mL) was refluxed for 4 h. Excess SOCl2 and
benzene were removed by distillation under reduced pressure,
and the residue containing the acid chloride was dried in vacuo.
It was dissolved in dry THF (4 mL), and ammonia gas was
bubbled into the stirred solution for 30 min. THF was removed
under vacuum, the residue was extracted with CH2Cl2, dried
(anhydrous Na2SO4), and the solvent removed under reduced
pressure to give carboxamide 5-1. Yield: 74%.
5.7. Synthesis of 5-2. 5.7.1. Methyl-2-(5-bromo-1H-indol-3-
yl)acetate (12). The method of Bascop et al.41 was followed.
Briefly, 2-(5-bromo-1H-indol-3-yl)acetic acid (4 mmol) in 20 mL
of MeOH acidified with ca. 10 drops concentrated H2SO4 was
refluxed (1-1.5 h), cooled to room temperature, and evaporated
in vacuo. The acidic residue was neutralized with saturated
NaHCO3 (50 mL) and extracted with EtOAc. Removal of
solvent gave a residue that was purified by flash silica gel column
5.8.5. 5-Fluoro-1-octyl-1H-indole (18b). 5-Fluoro-1H-indole
was reacted with 1-bromooctane and NaH by the method
described in Section 5.4.1. Yield: 92%. H NMR (300 MHz,
1
CDCl3): δ 7.27-7.19 (m, 2 H), 7.10 (d, J=3 Hz, 1 H), 6.93 (dt,
J1 =8.4 Hz, J2 =2.1 Hz, 1H), 6.41 (d, J=2.7 Hz, 1 H), 4.06 (t,
J=6.9 Hz, 2 H), 1.79 (t, J=6.6 Hz, 2 H), 1.28-1.24 (m, 10 H),
0.86 (t, J = 6 Hz, 3 H). 1H NMR (300 MHz, CDCl3): δ 156.1,
132.6, 129.3, 109.8, 109.4, 105.6, 105.3, 100.7, 46.6, 31.7, 30.2,
29.1, 26.9, 22.6, 14.0.
5.8.6. N-Ethyl-N-[(5-fluoro-1-octyl-1H-indol-3-yl)methyl]-
ethanamine (6-9). 18b was reacted with diethylamine in the
presence of ZnCl2 and aqueous HCHO by the method described
in Section 5.4.3. to give 6-9 in 73% yield.
5.8.7. General Method for the Synthesis of 1-Octyl-5-tolyl-1H-
indoles (19a, 19b). Compound 6 was reacted with o-tolylboronic
acid or p-tolylboronic acid by the method described in Section
5.3.3. Yields and spectroscopic details of 19a and 19b are given
in Supporting Information.
5.8.8. General Method for the Synthesis of N-Ethyl-N-((1-
octyl-5-tolyl-1H-indol-3-yl) methyl)ethanamines 6-1 and 6-2.
19a or 19b was reacted with diethylamine in the presence of ZnCl2
and aqueous HCHO by the method described in Section 5.4.3. to
give 6-1 or 6-2. Yields and spectroscopic details of 6-1 and
6-2 are given in Supporting Information.
1
chromatography (CH2Cl2) to give 12. Yield: 89%. H NMR
(300 MHz, CDCl3): δ 3.66 (s, 3 H), 3.68 (s, 2 H), 7.18-7.21 (m,
4 H). MS (APCI), m/z 269.2 [Mþ H]þ.
5.7.2. 2-(5-Bromo-1H-indol-3-yl)ethanol (13). The method of
Bascop et al.41 was followed. Ester 12 (4 mmol, 1 equiv) in
anhydrous THF (50 mL) was reacted with lithium aluminum
hydride (LiAlH4; 16 mmol, 4 equiv) at 0 °C. Excess LiAlH4 was
destroyed with a saturated solution of Na2SO4 at 0 °C and the
reaction mixture was worked up in the usual way. The alcohol 13
was obtained in 83% yield. 1H NMR (300 MHz, CDCl3): δ 2.97
(t, J=18 Hz, 2 H), 3.86-3.93 (m, 2 H), 4.09 (t, J=21 Hz,1 H),
7.09 (d, J=3 Hz, 1 H), 7.22 (d, J=9 Hz, 1 H), 7.75 (s, 1 H), 8.10
(s, 1 H). MS (APCI): m/z 241.2 [M þH]þ.
5.7.3. 2-(5-Bromo-1H-indol-3-yl)propanenitrile (14). The
method of Bascop et al.41 was followed. Briefly, methanesul-
phonyl chloride (MsCl, 0.55 mL, 2 equiv) was added to a stirred
solution of 13 (3.2 mmol, 1 equiv), triethylamine (0.9 mL,
2 equiv), and anhydrous CH2Cl2 (20 mL) at 0 °C. The reaction
mixture was stirred for 30 min, 0 °C under N2, after which it was
treated with 2 M NaOH and extracted with CH2Cl2. The com-
bined organic layer was washed with water, dried (anhydrous
5.8.9. N-Ethyl-N-((1-(3-methylbut-2-enyl)-1H-indol-3-yl)
methyl) Ethanamine (6-7). Indole was reacted with diethylamine,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6849
ZnCl2, HCHO, and ethanol as described in Section 5.4.3. to give
N-[(1H-Indol-3-yl)methyl]-N-ethylethanamine in 64% yield. H
compounds (various concentrations, prepared in DMSO stock
solutions) were then added to each well (final concentration of
DMSO maintained at 0.5% v/v per well) and incubated for 72 h.
Control wells contained cells and DMSO at the same concen-
tration as those in test wells. At the end of the incubation period,
20 μL of CellTiter 96 AQueous One Solution was added to each
well and the plates were incubated in the dark for 2 h at 37 °C
before absorbance readings at 490 nm were taken. Background
absorbance readings from blank wells that contained only
media and DMSO were subtracted from each well. Each con-
dition was determined in triplicate. Cell viability was expressed
as % of absorbance at 490 nm (treated cells) over that of control
(untreated) cells. The concentration of test compound required
to reduce cell growth by 50% (IC50) was determined from the
sigmoidal curve obtained by plotting % cell viability versus
concentration with GraphPad Prism (Version 4.0, GraphPad
Software, San Diego, CA). IC50 values were the mean of at least
two separate determinations.
5.12. Measurement of CMC of Cysmethynil (1-1) and Com-
pound 4-3. Measurements were carried out by a reported
method50 with modifications. At least seven different concen-
trations of 1-1 or 4-3 were prepared in the same buffer solution
used for determining Icmt inhibitory activity. Surface tension
measurements were made at room temperature (25 °C) on a
torsion balance (White Electrical Instrument Co. Ltd., UK)
equipped with a du Nouy platinum ring. The instrument was
calibrated before each measurement and at least three determi-
nations were made for each concentration. CMC was deter-
mined from the plot of surface tension versus concentration.
1
NMR (300 MHz, CDCl3): δ 1.07 (t, J=6.0 Hz, 6 H), 2.55 (q, J=
6.0 Hz, 4 H), 3.77 (s, 2 H), 6.95 (s, 1 H), 7.08- 7.16 (m, 2 H), 7.23 (d,
J= 6.9 Hz, 1 H), 7.67 (d, J= 7.8 Hz, 1 H). 13C NMR (75 MHz,
CDCl3) δ 11.5, 46.4, 50.0, 111.2, 112.1, 119.0, 119.2, 121.6, 124.2,
128.1, 136.1. The resulting amine was reacted with 1-chloro-3-
methylbut-ene by the method described in Section 5.4.1. to give
6-7 in 62% yield.
5.8.10. 2-(5-Fluoro-1-(3-methylbut-2-enyl)-1H-indol-3-yl)acet-
amide (6-8). 2-(5-Fluoro-1H-indol-3-yl) acetamide (Section 5.2.1.)
was reacted with 1-chloro-3-methylbut-2-ene by the method de-
scribed in Section 5.4.1. 6-8 was obtained in 67% yield.
5.8.11. N,N-Diethyl-2-(5-(3-methoxyphenyl)-1-octyl-1H-indol-
3-yl)acetamide (6-11). Acetamide 5 was reacted with m-meth-
oxyphenylboronic acid as described in Section 5.3.3. 6-11 was
obtained in 80% yield.
5.8.12. 2-(5-(3-Ethoxyphenyl)-1-octyl-1H-indol-3-yl)-N,N-diethyl-
acetamide (6-12). Acetamide 5 was reacted with m-ethoxyphenyl-
boronic acid as described in Section 5.3.3. 6-12 was obtained in
74% yield.
5.9. Materials for Biological Assay. Sf9 (Spodoptera frugiperda
ovarian) membranes containing recombinant Icmt were prepared
as described previously.48 The human breast cancer cell line,
MDA-MB-231, was obtained from American Type Culture Col-
lection (ATCC, Rockville, MD). Streptavidin sepharose beads
were purchased from Amersham Biosciences (Piscataway, NJ),
S-adenosylmethionine p-toluene sulfonate from Sigma-Aldrich
(Singapore), and [3H] S-adenosylmethionine (AdoMet) from
Perkin-Elmer (Waltham, MA). Biotin S-farnesylcysteine (BFC)
was synthesized by the Duke Small Molecule Synthesis Facility,49
and its purity was assessed by mass spectrometry and NMR.
CellTiter 96 AQueous One Solution was obtained from Promega
(Madison, WI). Suppliers for other reagents were: 10% fetal bovine
serum (Hyclone Laboratories, Logan, UT), penicillin and strepto-
mycin (Gibco, Invitrogen, Carlsbad, CA), and DMEM (Sigma
Aldrich, Singapore).
Acknowledgment. This work was supported by the Bio-
medical Research Council grant 06/1/21/19/487 to M.L.G.
and P.J.C. J.L. Leow gratefully acknowledged financial support
(research scholarship) from Ministry of Education, Republic
of Singapore, and National University of Singapore.
5.10. Inhibition of Icmt Activity. The method described by
Winter-Vann et al.32 was followed with some modifications.
Briefly, the standard reaction mixture contained recombinant
Icmt (1.0 μg of Sf9 membrane protein) in a final total volume of
50 μL buffer A (100 mM Hepes buffer pH 7.4, 5 mM MgCl2, and
100 μM EDTA). The test compound (dissolved in DMSO, final
concentration of DMSO in mixture was 2% v/v) was added and
the mixture was incubated at 37 °C for 20 min. After this time,
the substrate mixture, which comprised 5 μM BFC and 2.5 μM
[3H] AdoMet (15 Ci/mmol) in buffer A, was added to initiate the
reaction, which was carried out at 37 °C for 20 min. The final
concentrations of BFC and [3H] AdoMet in the mixture were
1 and 0.5 μM, respectively. The reaction was terminated by the
addition of 10% Tween 20 (5 μL) and streptavidin beads (10 μL
of packed beads) in 500 μL of buffer B, which consisted of
20 mM NaH2PO4 (pH 7.4) and 150 mM NaCl. The interaction
between biotin and streptavidin beads was allowed to proceed
overnight at 4 °C with gentle agitation. The beads were har-
vested by centrifugation (1000g, 5 min) on a tabletop micro-
centrifuge and washed 3ꢀ with 500 μL buffer B. The beads were
then suspended in 500 μL of the same buffer and transferred to
scintillation vials (each with 4.5 mL scintillation fluid) for the
determination of radioactivity. Radioactivity was measured on
the LS6500 multipurpose scintillation counter (Beckmann
Coulter Inc., Fullerton, CA) and analyzed by the LS6000 data
capture/network software version 2.11 (Beckman Coulter).
5.11. Cell Viability Assay. MDA-MB-231 human breast
cancer cells were grown in DMEM supplemented with 10%
fetal bovine serum, 50 U/mL penicillin, and 50 μg/mL strepto-
mycin at 37 °C, 5% CO2. Cells were subcultured at 80-90%
confluency and used within 10-25 passages for the assays. For
the assay, cells were seeded at 2400 cells per well in DMEM
containing 5% FBS in 96-well plates for 24 h. Aliquots of test
Supporting Information Available: Characterization and purity
data of synthesized compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
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