Total synthesis and antiproliferative activity screening of (±)-aplicyanins A, B and E and related analogues

Article abstract of DOI:10.1021/jm900544z

The first total synthesis of the indole alkaloids (±)-aplicyanins A, B, and E, plus 17 analogues, all in racemic form, is reported. Modifications to the parent compound included changing the number of bromine substituents on the indole, the nature of the substituents on the indole nitrogen (H, Me, or OMe), and/or the oxidation level of the heterocyclic core tetrahydropyrimidine. Each compound was screened against three human tumor cell lines, and 14 of the newly synthesized compounds showed considerable cytotoxicity. The assay results were used to establish structure-activity relationships. These results suggest that the presence of the bromine at position 5 of the indole is critical to activity, as well as the acetyl group on the imine nitrogen does in some compounds. 2009 American Chemical Society.

Full text of DOI:10.1021/jm900544z

J. Med. Chem. 2009, 52, 6217–6223 6217
DOI: 10.1021/jm900544z
Total Synthesis and Antiproliferative Activity Screening of (()-Aplicyanins A, B and E and Related
Analogues†
‡
Miroslav Sısa,‡,^ Daniel Pla,‡,§ Marta Altuna, Andres Francesch, Carmen Cuevas, Fernando Albericio,*,‡,§,# and
ꢀ
ꢀ
ꢁ
,‡,§,3
ꢁ
Mercedes Alvarez*
‡Institute for Research in Biomedicine, Barcelona Science Park;University of Barcelona, Baldiri Reixac 10, E-08028 Barcelona, Spain, §CIBER-
BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Baldiri Reixac 10, E-08028 Barcelona, Spain, and Pharma Mar
S.A., Avenida de los Reyes 1, E-28770 Colmenar Viejo, Madrid, Spain. ^ Current address: Academy of Sciences of the Czech Republic, Palacky
University & Institute of Experimental Botany, Slechtitelu 11, 783 71 Olomouc, Czech Republic. # Department of Organic Chemistry, University of
ꢀ
Barcelona, E-08028 Barcelona, Spain. 3 Laboratory of Organic Chemistry, Faculty of Pharmacy, University of Barcelona, E-08028 Barcelona,
Spain.
Received April 28, 2009
The first total synthesis of the indole alkaloids (()-aplicyanins A, B, and E, plus 17 analogues, all in
racemic form, is reported. Modifications to the parent compound included changing the number of
bromine substituents on the indole, the nature of the substituents on the indole nitrogen (H, Me, or
OMe), and/or the oxidation level of the heterocyclic core tetrahydropyrimidine. Each compound was
screened against three human tumor cell lines, and 14 of the newly synthesized compounds showed
considerable cytotoxicity. The assay results were used to establish structure-activity relationships.
These results suggest that the presence of the bromine at position 5 of the indole is critical to activity, as
well as the acetyl group on the imine nitrogen does in some compounds.
Introduction
known to contain this moiety.6 However, similar compounds
sharing a common 3-(pyrimid-4-yl)indole structure are more
common and have been isolated from different marine inver-
tebrates and characterized. These include meridianins A-G,
from the tunicate Aplidium meridianum,7 the psammopem-
mins, from an Antarctic marine sponge of the genus Psammo-
pemma,8 and the closely related, but more complex structures,
variolins A-D,9,10 from the Antarctic sponge Kirkpatrickia
varialosa (Figure1). Furthermore, the 1-methoxyindole found
in some aplicyanins is unprecedented among known marine
alkaloids.11
Last, given the high cytotoxicity typical of bromoindole
derivatives, the presenceofa bromoindole insome aplicyanins
warrants their investigation as anticancer drugs.12 Herein is
reported the first total synthesis of(()-aplicyanins A, B, and E
and 17 analogues. The analogues differ in the nature of the
substituents of the indole nucleus (H and/or Br), of the
substituent the indole nitrogen (H, Me, or OMe), and in the
oxidation level of the six-member heterocyclic core (2-amino-
1,4,5,6-tetrahydropyrimidine or 2-amino-5,6-dihydro-4-pyri-
midone). The compounds were screened for cytotoxicity
against three human tumor cell lines: A-549, HT-29, and
MDA-MB-231. Structure-activity relationships (SAR) were
established based on the screening results.
Marine invertebrates such as sponges, tunicates, ascidians,
and corals have provided a rich arsenal of new bioactive
compounds. The unprecedented structures of these molecules
make them excellent synthetic targets, and their potent acti-
vity against a broad number of therapeutic indications make
these natural products excellent drug lead candidates.1 A new
family of six indole alkaloids, the aplicyanins, was recently
isolated from the ascidian Aplidium cyaneum.2 They are
cytotoxic to the human tumor cell lines MDA-MB-231
(breast adenocarcinoma), A549 (lung carcinoma), and HT-
29 (colorectal carcinoma) and also exhibit antimitotic acti-
vity.2 The cellular arrest in mitosis may involve interaction of
the drug to either tubulin or in microtubules formation, which
usually leads to apoptosis.3 All aplicyanins contain a 3-(2-
amino-1,4,5,6-tetrahydropyrimidin-4-yl)-5-bromoindole nu-
cleus but differ in their respective amino substituents (R1 =
H or Ac), their N-indole substituents (R2=H or OMe) and in
whether or not they contain a second bromine atom at the 6-
position of indole (R3=H or Br). Some aplicyanins structural
traits, namely a six-membered cyclic guanidine (2-amino-
1,4,5,6-tetrahydropyrimidin-4-yl) and/or a N-methoxyindole,
are singular features. This cyclic guanidine is only present in
very few natural products, all of which are peptides isolated
from extracts of Streptomyces sp. (e.g., muraymycins
A1-D34 and chymostatinols A-C).5 To the best of our
knowledge, aplicyanins are the first marine natural products
Results and Discussion
Chemistry. Initial attempts to the synthesis of aplicyanins
were based on introduction of a three-carbon chain at
position 3 of the appropriately substituted indole. The chain
had to be adequately functionalized for construction of the
2-amino-1,4,5,6-tetrahydropyrimidine ring. This was tested
using two different strategies (Schemes 1 and 2). The
†Dedicated to Professor Peter Stanetty on the occasion of his 65th
anniversary.
*Corresponding author: For M.A. and F.A.: phone, þ34934037086;
fax, þ34934037126; E-mail, mercedes.alvarez@irbbarcelona.org.
r
2009 American Chemical Society
Published on Web 09/18/2009
pubs.acs.org/jmc

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Sısa et al.
6218 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
substituted indoles 1 were either commercially available or
prepared by conventional procedures.
Michael cyclization reaction, the product of the reductive
guanidylation compound (4a) was only obtained in 20%
yield.
Wittig chemistry (Scheme 1) was employed to introduce the
chain, starting from the aldehydes 1a-d and the Wittig ylide
(2x) or phosphonate (2y). Addition of guanidine to the
β-position of the conjugated double bond and further intra-
molecular reductive cyclization was the original plan. The E
stereoisomer was obtained in both cases; however, the stere-
ochemistry of the double bond was irrelevant, as it would
ultimately be lost in the subsequent conjugate addition.
Compounds 3a (71% yield) and 3b (65% yield) were
obtained from 2x and the aldehydes 1a or 1b,13,14 respec-
tively (Scheme 1). Interestingly, the ethylene acetal was
cleaved during silica gel column purification of each com-
pound, providing the corresponding R,β-unsaturated alde-
hydes in good yield. After different attempts at a tandem
The esters 3c-e were obtained in excellent yields by
Horner-Wadsworth-Emmons reaction of 1c-d15 or 1a
and the ethyl phosphonate 2y (Cs2CO3 as a base, dioxane,
70 ꢀC).16 However, the guanidine chemistry again failed:
when ester 3c was reacted with guanidine in methoxyethanol
under microwave irradiation, the only product observed was
the acylguanidine 4c.17 On the basis of these results, it was
decided not to continue with the indolyl acrylates 3d and 3e.
A survey of the literature reveals that the condensation of
R,β-unsaturated esters with guanidine to give tetrahydro-
pyrimidones only involve R,β-unsaturated esters containing
an aryl group bearing electron withdrawing substituents at
the β-position.18
Scheme 2. Synthetic Strategy by Acylation Route
Figure 1. Examples of natural bioactive indole alkaloids.
Scheme 1. Synthetic Strategy by Wittig Routea
a Reagents and conditions: (i) for 3a-b, 2x, NaOEt, THF-EtOH, rt to reflux; for 3c-e, 2y, Cs2CO3, dioxane-DMSO, 70 ꢀC. (ii) tert-BuONa, tert-
BuOH, reflux, then NaBH4, MeOH, 0 ꢀC to rt.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6219
Scheme 3. Synthesis of the Aplicyanins and Related Analogues
An alternate strategy was then tried for introducing the
bifunctionalized three-carbon chain: acylation of the indole
with the acid chloride of N-protected β-alanine (Scheme 2).
Acylation of 5,6-dibromo-1-methoxyindole (1e)19 with
3-phthalimidopropanoyl chloride20 using Et2AlCl in CH2Cl2
gave 5a in only 23% yield. On the basis of the low yield and
the difficulty of eliminating the phthalimido protecting
group by hydrazinolysis, another route was sought.21 How-
ever, using other protecting groups (Alloc or Boc) for the
amine of β-alanine did not improve the acylation.
Acylation of N-methylindole with 3-bromopropanoyl
chloride in the same conditions as above gave the bromoke-
tone 5c in 30% yield. Reaction of 5c with the monoacetyl
guanidine in DMF produced the loss of hydrobromic acid.
The elimination reaction was avoided by using sodium
diformylamide, a weaker base for the amine introduction.22
The partially protected aminoketone 5d was obtained in
excellent yield by reacting 5c and sodium diformylamide in
DMF at 80 ꢀC, followed by monodeformylation with
MeOH. The Tces-protected guanidine group of 5e was
synthesized by acidic treatment of 5d to liberate the free
amine and its further reaction with Tces-protected methyl
carbamimidothioate 6. Despite having tested several condi-
tions23 for the intramolecular cyclization of 5e, only
tetrahydropyrimidine 7 was obtained in 7% yield, which
underscored the limitations of the latter synthetic app-
roach.24
procedure described by Jones et al.26 Reaction between
adducts 8a,b,d,f-i and guanidine carbonate in refluxing
2-methoxyethanol27 gave the 2-aminodihydropyrimidones
9a,b,d,f-i in yields that varied according to the indole
substituent.28 As such, N-methylindole 9a was obtained in
excellent yield (90%), whereas 9f-i, corresponding to bro-
mine substituents at indole positions 5 or 6, were obtained in
lower yields (47-74%), and the N-methoxyderivatives 9b
and 9d were only obtained in 20 and 5% yield, respectively.
The poor results for the N-methoxy derivatives can be
rationalized by two factors. The electron donating character
of methoxy in position 1 of indole diminishes the reactivity of
compounds 8b and 8d toward nucleophilic addition of
guanidine. Thus, these derivatives are relatively nonreactive
and, consequently, require longer times to consume the
starting material in the guanidine addition-cyclization
reaction. Second, they confer instability and low solubility
to the 2-aminodihydropyrimidin-4-ones 9b and 9d.
Reduction of compounds 9a,b,d,f-i with borane-THF29
afforded the aminotetrahydropyrimidines 10a,b,d,f-j in
good yields. The reduction conditions for compounds 9b
and 9d had to be strictly controlled because longer reaction
times or higher temperatures led to the loss of the N-methoxy
group. Reduction of 9b produced a 1:2 mixture of 10b and
10j. Because the N-methoxy group of the indole is acid
sensitive, the amino nitrogen in the 2-iminotetrahydropyri-
midine ring was acylated, in moderate yields, under basic
conditions (Ac2O, Pyr) to give 11.
Having deduced that an electron-poor R,β-unsaturated
ester would be necessary to drive the conjugate addition of
guanidine to the double bond, as opposed to reaction with
the ester group, as in the formation of compounds 4
(Scheme 1), a new strategy was devised: to decrease the
electronic density of the conjugated double bond using a
malonic ester derivative such as Meldrum’s acid (MA)
(Scheme 3).25 Thus, the Meldrum acid-indole adducts
8a,b,d,f-i were prepared in good yields by following the
Nearly all of the products were readily purified by column
chromatography and obtained in relatively high yields, illus-
trating the efficiency of our synthetic route. (()-Aplicyanin
A (10f) and its acetyl derivative (()-aplicyanin B (11f) were
obtained in good overall yield from commercially available
5-bromo-3-formylindole, as were several other derivatives
with a bromine at position 6 and/or a methyl group
at position 1. However, the most complicated analogue,

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6220 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Table 1. Cytotoxicity of Compounds 9, 10, and 11 to Three Human
Tumor Cell Lines (GI50 Values Reported in μM)
17 analogues. The compounds were screened in cytotoxicity
assays against three human tumor cell lines: MDA-MB-231
(breast adenocarcinoma), A549 (lung carcinoma), and HT-29
(colorectal carcinoma).
(()-Aplicyanin A and its acetyl derivative (()-aplicyanin
B were obtained in good overall yield from commercial
5-bromo-3-formylindole, as were several other derivatives
with a bromine substituent at position 6 and/or N-methyl
substituents. However, the most complicated analogue, (()-
aplicyanin E, which contains two bromine atoms at positions
5 and 6 and a methoxy group at position 1 of the indole, was
only obtained in sufficient amount for screening.
Fourteen of the newly synthesized compounds showed
considerable cytotoxic activity against three human tumor
cell lines. These results suggest that the bromine at position 5
of the indole strongly favors antiproliferative activity, as well
as the acetyl group at the imine nitrogen does in some
compounds.
cell lines
compd
9a,b,d-h
9i
10a
MDA-MB-231
A-549
HT-29
naa
na
na
na
na
2.86
1.71
10.9
na
2.67
na
4.29
na
10b (()-aplicyanin E
10d
na
na
10f (()-aplicyanin A
10g
0.27
19.1
25.7
8.79
na
0.27
na
0.11
21.1
13.7
4.56
na
10h
10i
na
9.11
na
10j
11a
14.4
0.98
5.67
0.94
8.02
10.7
0.51
6.86
0.43
6.30
7.40
0.33
2.12
0.31
4.30
11f (()-aplicyanin B
11g
11h
11i
(()-Aplicyanin A results active in the submicromolar range
despite the inactivity of the corresponding natural compound.
This evidence the activity of the unnatural enantiomer versus
the natural one. (()-Aplicyanin B is as active as its corre-
sponding parent (natural) compound in all three cellular lines,
whereas (()-aplicyanin E maintains the activity only in
MDA-MB-231. The decrease of cytotoxycity of the racemic
aplicyanin E in front of the natural one indicates again that
one enantiomer is more active than the other.
Therefore, these results demonstrate the potential of the
aplicyanin structure as a scaffold for anticancer drug disco-
very and the need of developing enantiomeric synthesis to
deepen in structure activity relationships.
a na: not active.
(()-aplicyanin E (10b), which contains two bromine atoms
at positions 5 and 6 and a methoxy group at position 1, was
obtained in just sufficient amount to perform the cytotoxi-
city assay. The NMR spectral data for (()-aplicyanins A
(10f), B (11f), and E (10b) obtained by total synthesis are in
good agreement with the data reported in literature.2
Despite the results of (()-aplicyanin E, the relatively high
yields and easy purification of the other compounds are
testament to the utility of the herein reported strategy for the
synthesis of aplicyanins and their analogues.
Biological Results
Compounds 9, 10, and 11 were tested against three human
tumor cell lines: HT-29 colon, A549 lung, and MDA-MB-231
breast. The cytotoxicities were evaluated for 20 synthesized
compounds, and the most significant results are summarized
in Table 1. Except for compounds 9a,b,d-h, the remaining
compounds in Table 1 are cytotoxic to MDA-MB-231 breast
adenocarcinoma cells:9i strongly inhibits growth of these cells
at micromolar concentrations, whereas the compounds with
the saturated core are more active. The tetrahydropyrimidines
10 are cytotoxic; of these, 10a, 10f [(()-aplicyanin A], and 10i
are the most active against all three cell lines.
The acetylated derivatives 11 also inhibit growth of all three
cell lines at micromolar concentration; (()-aplicyanin B (11f)
and 11h are the most active. The comparison of the assay
results for N-H vs N-acetylated compounds shows that in
compounds 11g,h,i, acetylation increases cytotoxicity, whereas
in 11a,f, acetylation decreases activity.
Experimental Section
General Data. Melting points (mp) were determined in a
Buchi melting point B540. Automatic flash chromatography
was done in an Isco Combiflash medium pressure liquid chro-
matograph with Redisep silica gel columns (47-60 μm). 1H
NMR and 13C NMR spectra were recorded on a Varian
Mercury 400 MHz spectrometer and a Gemini 200 MHz
spectrometer. HRMS were performed on a Bruker Autoflex
high resolution mass spectometer. Microwave-assisted reac-
tions were carried out in a CEM Discover microwave. Reversed
phase analytical HPLC was performed on a Waters Alliance
separation module 2695 and a Waters 996 PDA detector at
254 nm, using the following columns: a Waters Xterra MS C18
column (150 ꢀ 4.6 mm, 5 μm) for runs of 15 min, a Waters
XBridge C18 column (75 mm ꢀ 4.6 mm, 2.5 μm) for runs of
8 min. Purification by semipreparative reversed phase HPLC
were performed on a Symmetry C18 (5 μm, 30 mm ꢀ 100 mm)
column, UV detection at 220 nm, with a flow of 10 mL/min, and
using H2O-0.1% TFA/CH3CN-0.05% TFA as solvent system
with a gradient specified for each case. HPLC analytical results
to support final compound purity in two solvent systems are
summarized in Table 1 of the Supporting Information. Purity
determined by this means was superior or equal to 95% for all
the compounds.
The bromine at position 5 of the indole favor cytotoxic
activity in the three cellular lines tested.
In contrast, the substituent of the indole nitrogen gave
results of difficult generalization. Among the deacetylated
compounds 10, N-methoxy group produced only cytotoxicity
of 10b [(()-aplicyanin E] among MDA-MB-231; 10f [(()-
aplicyanin A] (Nind-H) is the most active compound, 100-fold
superior to 10h (Nind-Me). The acetylated compounds 11h
(Nind-Me) and 11f [(()-aplicyanin B] (Nind-H) are equally
active against the three cell lines.
General Procedure for the Reaction of Formylindoles with 2,3-
Dimethyl-1,3-dioxane-4,6-dione (Meldrum Acid). Acetic acid
(100 μL, 1.0 mmol) and piperidine (155 μL, 1.1 mmol) were
added to a solution of formylindole 1a,b,d,f-i (6.0 mmol) and
2,3-dimethyl-1,3-dioxane-4,6-dione (865.8 mg, 6.0 mmol) in
toluene (20 mL). The mixture was stirred at rt overnight.
Evaporation afforded a yellow solid, which was recrystallized
from ethanol to give pure condensation products.
Conclusions
Herein is reported the total synthesis of the recently dis-
covered marine natural products aplicyanins A, B, and E and
General Procedure for Preparation of Compounds 9. Meldrum
acid adduct 8 (ca. 1.5 mmol) was dissolved in toluene (20 mL).

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6221
Guanidine carbonate (1.25 equiv) was then added, and the
reaction mixture was stirred at 135 ꢀC until the starting material
had been consumed (tracked by HPLC from 2 to 16 h). The
crude product was concentrated in vacuo and washed with
hexane and CH2Cl2. The product was crystallized from ethanol
and then washed with hexane, CH2Cl2, and cold water to give a
slightly yellowish solid. 9b and 9d were purified by semiprepara-
tive reverse phase HPLC (H2O-MeCN; gradient 90:10 to 40:60
in 30 min) to afford the pure compound as yellow oil.
General Procedure for the Reduction of Compounds 9.
Borane-THF complex (1 M THF solution, 3.0-5.0 mmol)
was added to a solution of substrate 9 (1.0 mol) in anhydrous
THF (10 mL) under Ar, and the reaction mixture was heated at
45 ꢀC until the starting material has been consumed (tracked by
HPLC, 2-15 h). The reaction mixture was then cooled to rt and
quenched by stirring with sat. NH4Cl for 30 min. The organic
solution was set aside, and the aqueous phase was extracted with
CH2Cl2 saturated with NH3. The organic extracts were
combined, dried over MgSO4, and concentrated in vacuo
to give the crude materials, which were purified as described
below.
1383, 1207, 1139, 801, 724, 523. 1H NMR (400 MHz, MeOH-d4)
δ 2.20-2.29 (m, 2H), 3.38-3.49 (m, 2H), 4.09 (s, 3H), 4.96 (dd,
J = 6.4 and 6.4 Hz, 1H), 7.10-7.15 (m, 1H), 7.24-7.29 (m, 1H),
7.44-7.48b (m, 2H), 7.61 (d, J=8.0 Hz, 1H). 13C NMR (400
MHz, MeOH-d4) δ 14.2, 20.6, 38.2, 61.4, 66.3, 109.4, 110.9,
119.7, 121.1, 122.2, 123.9, 151.0, 172.9. MS (ESI-TOF) 245
(M þ 1, 100).
2-Amino-4-(5-bromoindol-3-yl)-1,4,5,6-tetrahydropyrimidine
(10f). Compound 9f (200 mg, 0.65 mmol) was reduced and then
purified by semipreparative reverse phase HPLC (H2O-MeCN;
gradient 90:10 to 40:60 in 30 min) to afford 10f (57%) as a yellow
oil. IR (KBr film) 3265, 1680, 1630, 1461, 1305, 1203, 1137, 559,
839, 838, 801, 723, 600, 423. 1H NMR (400 MHz, MeOH-d4) δ
2.20-2.27 (m, 2H), 3.34-3.48 (m, 2H), 4.90-4.96 (m, 1H), 7.24
(dd, J = 8.7 and 1.8 Hz, 1H), 7.29-7.35 (m, 2H), 7.74 (d, J=1.7
Hz, 1H).13C NMR (400 MHz, MeOH-d4) δ27.7, 37.9, 47.4,
113.0, 113.9, 114.7, 121.3, 124.7, 125.3, 127.5, 136.6, 155.2. MS
(ESI-TOF) 293 (MBr79 þ 1, 91), 294 (MBr79 þ 2, 10), 295
(MBr81 þ 1, 100), 296 (MBr81 þ 2, 9). HRMS m/z calcd for
C12H14BrN4 293.0396, found 293.0399.
2-Amino-4-(6-bromoindol-3-yl)-1,4,5,6-tetrahydropyrimidine
(10g). Compound 9g (100 mg, 0.33 mmol) was reduced and then
purified by washing with hexane, CH2Cl2, and cold water to
obtain 10g (86%) as a yellow oil. IR (KBr film) 3257, 2942, 1661,
1628, 1455,1333, 1021, 803. 1H NMR (400 MHz, MeOH-d4) δ
2.19-2.27 (m, 2H), 3.35-3.47 (m, 2H), 4.95 (dd, J = 6.3 and 6.3
Hz, 1H), 7.17 (dd, J = 8.5 and 1.7 Hz, 1H), 7.26 (s, 1H), 7.50 (d,
J=8.5 Hz, 1H), 7.56 (d, J=8.5 Hz, 1H). 13C NMR (400 MHz,
MeOH-d4) δ 28.1, 38.2, 47.0, 62.4, 115.4, 115.6, 116.2, 120.6,
123.3, 124.5, 125.0, 139.1. MS (ESI-TOF) 293 (MBr79 þ 1, 85),
294 (MBr79 þ 2, 6), 295 (MBr81 þ 1, 100), 296 (MBr81 þ 2, 7).
HRMS m/z calcd for C12H14BrN4 293.0396, found 293.0397.
2-Amino-4-(5-bromo-1-methylindol-3-yl)-1,4,5,6-tetrahydro-
pyrimidine (10h). Compound 9h (200 mg, 0.62 mmol) was
reduced and then purified by washing with hexane, CH2Cl2,
and cold water to obtain 10h (89%) as a white solid; mp (MeCN)
314-316 ꢀC. The samples for bioassays were crystallized from
MeOH. IR (KBr film) 3209, 3053, 2969, 2879, 1665, 1621, 1476,
1422, 1323, 1124, 1090, 792, 808, 619, 595. 1H NMR (400 MHz,
MeOH-d4) δ 2.19-2.26 (m, 2H), 3.36-3.49 (m, 2H), 3.79 (s,
3H), 4.93 (dd, J = 7.5 and 5.2 Hz, 1H), 7.26 (s, 1H), 7.29-7.37
(m, 2H), 7.74 (d, J=1.3 Hz, 1H). 13C NMR (400 MHz, MeOH-
d4) δ 28.6, 33.4,38.8, 48.1, 77.0, 109.6, 113.0, 114.1, 122.4, 123.1,
126.2, 129.9, 154.7. MS (ESI-TOF) 307 (MBr79 þ 1, 100), 308
(MBr79 þ 2, 15), 309 (MBr81 þ 1, 90), 310 (MBr81 þ 2, 12).
HRMS m/z calcd for C13H16BrN4 307.0553, found 307.0552.
2-Amino-4-(6-bromo-1-methylindol-3-yl)-1,4,5,6-tetrahydro-
pyrimidine (10i). Compound 9i (200 mg, 0.62 mmol) was
reduced and then purified by washing with hexane, CH2Cl2,
and cold water to obtain 10i (83%) as a yellow oil. The samples
for bioassays were further purified by HPLC (C18 column). IR
(KBr film) 3175, 3099, 3062, 2924, 1660, 1624, 1548, 1476, 1321,
1134, 797. 1H NMR (400 MHz, MeOH-d4) δ 2.19-2.26 (m, 2H),
3.33-3.47 (m, 2H), 3.77 (s, 3H), 4.95 (dd, J = 6.3 and 6.3 Hz,
1H), 7.19-7.23 (m, 2H), 7.51 (d, J=8.5 Hz, 1H), 7.60 (d, J=1.6
MHz, 1H). 13C NMR (400 MHz, MeOH-d4) δ 28.3, 33.0, 38.4,
47.9, 111.2, 114.0, 115.3, 116.7, 121.1, 123.7, 125.7, 129.1, 155.7.
MS (ESI-TOF) 307 (MBr79 þ 1, 100), 308 (MBr79 þ 2, 12), 309
(MBr81 þ 1, 85), 310 (MBr81 þ 2, 10). HRMS m/z calcd for
C13H16BrN4 307.0553, found 307.0555.
General Procedure for the Acylation of 10. Ac2O (1.5 mL) was
added to a solution of compound 10 (0.1 mmol) in pyridine
(5 mL), and the resulting mixture was stirred at rt for 15 h. To the
mixture were added CH2Cl2 (20 mL) and sat. NaHCO3 (10 mL).
The aqueous layer was extracted with CH2Cl2, and the com-
bined organic extracts were washed with 5% HCl, dried over
Na2SO4, filtered, and concentrated in vacuo. The crude pro-
ducts were purified by semipreparative reverse phase HPLC
(H2O-MeCN; gradient 90:10 to 40:60 in 30 min) to afford
yellow oils.2
2-Amino-4-(1-methylindol-3-yl)-1,4,5,6-tetrahydropyrimidine
(10a). Compound 9a (50 mg, 0.21 mmol) was reduced and then
purified by washing with hexane, CH2Cl2, and cold water to
obtain 10a (64%) as a yellow solid; mp (MeCN) 283-285 ꢀC. IR
(KBr film) 3259, 2925, 2854, 1663, 1627, 1466, 1382, 1309, 1197,
742. 1H NMR (400 MHz, MeOH-d4) δ 2.15-2.24 (m, 2H),
3.32-3.43 (m, 2H), 3.79 (s, 3H), 4.92 (t, J=6.4 Hz, 1H), 7.06 (t,
J=7.6 Hz, 1H), 7.14-7.21 (m, 2H), 7.35 (d, J=7.4 Hz, 1H), 7.55
(d, J=7.5 Hz, 1H). 13C NMR (400 MHz, MeOH-d4) δ 28.5, 32.9,
38.5, 48.2, 110.9, 114.9, 119.6, 120.5, 123.2, 126.8, 128.1, 139.1,
155.8. MS (ESI-TOF) 229 (M þ 1, 100); 231 (M þ 3, 27). HRMS
m/z calcd for C13H17N4 229.1453, found 229.1453.
2-Amino-4-(5,6-dibromo-1-methoxyindol-3-yl)-1,4,5,6-tetra-
hydropyrimidine (10b) and 2-Amino-4-(5,6-dibromoindol-3-yl)-
1,4,5,6-tetrahydropyrimidine (10j). Compound 9b (5 mg, 0.01 mmol)
was reduced and then purified by semipreparative reverse phase
HPLC (H2O-MeCN; gradient 90:10 to 40:60 in 30 min) to afford
10b (31%) and 10j (60%) as yellow oils.
10b. IR (KBr film) 3522, 2923, 1685, 1560, 1541, 1457, 1204,
1
1139, 800, 723. H NMR (400 MHz, MeOH-d4) δ 2.09-2.27
(m, 2H), 3.32-3.46 (m, 2H), 4.08 (s, 3H), 4.91 (dd, J = 8.1 and
4.4 Hz, 1H), 7.57 (s, 1H), 7.82 (s, 1H), 7.96 (s, 1H). 13C NMR
(400 MHz, MeOH-d4) δ 27.9, 37.9, 47.1, 66.7, 112.0, 114.2,
116.0, 118.9, 123.1, 124.2, 124.4, 133.2, 155.3. MS (ESI-TOF)
401 (M(Br79)2 þ 1, 50), 403 (MBr79Br81 þ 1, 100), 405 (M(Br81)2
þ 1, 54).
10j. 1H NMR (400 MHz, MeOD-d4): 2.16-2.27 (m, 2H),
3.32-3.47 (m, 2H), 4.08 (s, 3H), 4.92 (dd, J=7.6 and 5.1 Hz,
1H), 7.31 (s, 1H), 7.73 (s, 1H), 7.91 (s, 1H). 13C NMR (400 MHz,
MeOD-d4) 27.7, 37.9, 47.3, 68.6, 88.4, 101.1, 102.3, 117.1, 123.3,
125.7, 134.9. MS (ESI-TOF) 371 (M(Br79)2 þ 1, 47), 372
(M(Br79)2 þ 2, 15), 373 (MBr79Br81 þ 1, 100), 375 (M(Br81)2
þ 1, 50). HRMS m/z calcd for C12H13Br2N4 370.9501, found
370.9503.
2-(Acetylamino)-4-(1-methylindol-3-yl)-1H-3,4,5,6-tetrahydro-
pyrimidine (11a). Compound 10a (20 mg, 0.09 mmol) was
converted into 11a (42%) as a colorless oil. 1H NMR (400
MHz, MeOH-d4) δ 2.19 (s, 3H), 2.29-2.36 (m, 2H), 3.45-3.63
(m, 2H), 3.79 (s, 3H), 5.13 (t, J=6.0 Hz, 1H), 7.07-7.12 (m, 1H),
7.20-7.25 (m, 2H), 7.40 (d, J=8.3 Hz, 1H), 7.59 (d, J=8.0 Hz,
1H). 13C NMR (400 MHz, MeOH-d4) δ 24.1, 26.9, 38.6, 83.8,
87.6, 110.9, 113.7, 119.4, 120.7, 123.3, 124.2, 128.3, 138.7, 195.0.
MS (ESI-TOF) 271 (M þ 1, 100). HRMS m/z calcd for
C15H19N4O 271.1553, found 271.1557.
2-Amino-6-(1-methoxyindol-3-yl)-1,4,5,6-tetrahydropyrimi-
dine (10d). Compound 9d (50 mg, 0.19 mmol) was reduced and
then purified by semipreparative reverse phase HPLC
(H2O-MeCN; gradient 90:10 to 40:60 in 30 min) to afford
10d (83%) as a yellow oil. IR (KBr film) 3425, 2923, 2852, 1678,

ꢀ
´ꢀ
Sısa et al.
6222 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
2-(Acetylamino)-4-(5-bromoindol-3-yl)-1H-3,4,5,6-tetrahydro-
pyrimidine (11f). Compound 10f (200 mg, 0.37 mmol) was
converted into 11f (41%) as a yellow oil. 1H NMR (400 MHz,
MeOH-d4) δ 2.20 (s, 3H), 2.29-2.35 (m, 2H), 3.46-3.63 (m,
2H), 5.11 (t, J=6.1 Hz, 1H), 7.26 (dd, J=8.7 and 1.8 Hz, 1H),
7.32-7.36 (m, 2H), 7.78 (d, J=1.6 Hz, 1H). 13C NMR (400
MHz, MeOH-d4) δ 24.1, 26.9, 38.6, 48.2, 86.2, 113.7, 114.6,
121.7, 125.5, 126.1, 127.8, 129.7, 137.2, 191.5. MS (ESI-TOF)
335 (MBr79 þ 1, 100), 336 (MBr79 þ 2, 12), 337 (MBr81 þ 1, 93),
338 (MBr81 þ 2, 10). HRMS m/z calcd for C14H16BrN4O
335.0502, found 335.0505.
If Tz < T < C (growth inhibition), then the result is 100 ꢀ
([T - Tz]/[C - Tz]).
If T < Tz (net cell death), then the result is 100 ꢀ ([T - Tz]/Tz).
After dose-curve generation, the following parameter is cal-
culated by interpolation: GI50, concentration that causes 50%
growth inhibition.
Acknowledgment. This study was partially supported by
CICYT (grant BQU 2006-03794), Generalitat de Catalunya,
and the Barcelona Science Park.
2-(Acetylamino)-4-(6-bromoindol-3-yl)-1H-3,4,5,6-tetrahydro-
pyrimidine (11g). Compound 10g (50 mg, 0.09 mmol) was
converted into 11g (68%) as a yellow oil. 1H NMR (400 MHz,
MeOH-d4) δ 2.20 (s, 3H), 2.32 (q, J = 5.9 and 5.8 Hz, 2H),
3.45-3.61 (m, 2H), 5.13 (t, J=6.0 Hz, 1H), 7.19 (dd, J = 8.5 and
1.6 Hz, 1H), 7.31 (s, 1H), 7.52 (d, J=8.5 Hz, 1H), 7.58 (d, J=1.3
Hz, 1H). 13C NMR (400 MHz, MeOH-d4) δ 24.2, 27.0, 38.5,
48.3, 87.4, 115.0, 115.8, 116.7, 120.7, 123.8, 125.0, 139.4, 152.3,
174.0. MS (ESI-TOF) 335 (MBr79 þ 1, 100), 336 (MBr79 þ 2,
10), 337 (MBr81 þ 1, 90), 338 (MBr81 þ 2, 9). HRMS m/z calcd
for C14H16BrN4O 335.0502, found 335.0501.
Supporting Information Available: General data, experimen-
tal procedures, and characterization of compounds 3a-e, 4a,
1
5a-e, 7, 8a,b,d,f-i, 9a,b,d,f-i. H and 13C NMR spectra and
HPLC chromatograms of compounds 9-11. This material is
available free of charge via the Internet at http://pubs.acs.org.
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