Synthesis and cytotoxic evaluation of new derivatives of the marine alkaloid variolin B

Article abstract of DOI:10.1021/jm051090r

The marine alkaloid variolin B 2 and eight synthetic derivatives with different substituents at positions C-5 and C-7 have been tested in a panel of sixteen human tumor cell lines to evaluate their cytotoxic potential.

Full text of DOI:10.1021/jm051090r

J. Med. Chem. 2006, 49, 1217-1221
1217
Synthesis and Cytotoxic Evaluation of New Derivatives of the Marine Alkaloid Variolin B
Pilar M. Fresneda,*,† Santiago Delgado,† Andre´s Francesch,‡ Ignacio Manzanares,‡,§ Carmen Cuevas,‡ and Pedro Molina*,†
Departamento de Qu´ımica Orga´nica, Facultad de Qu´ımica, UniVersidad de Murcia, Campus de Espinardo, 30100-Murcia, Spain, and
PharmaMar, S. A., AVda. de los Reyes, 1, 28770-Colmenar Viejo, Madrid, Spain
ReceiVed October 27, 2005
The marine alkaloid variolin B 2 and eight synthetic derivatives with different substituents at positions C-5
and C-7 have been tested in a panel of sixteen human tumor cell lines to evaluate their cytotoxic potential.
Introduction
Marine organisms have provided a vast array of structurally
diverse and biologically interesting structures.1 Several new
clinical structures with novel modes of action have been
identified recently from the intermediate metabolites of marine
organisms, leading to great interest in this field.2 Variolins 1-4
(Figure 1) comprise a group of marine heterocyclic substances
isolated from the rare, and difficult to access, Antarctic sponge,
Kirkpatrickia Variolosa.3 This new class of compounds is
interesting from both structural and biological points of view.
All the variolins have a common tricyclic ring skeleton, which
has no precedents in either terrestrial or marine natural products,
namely a pyrido[3′,2′:4,5]pyrrolo[1,2-c]pyrimidine, with either
a heterocyclic or methoxycarbonyl group attached at position
C-5 of the core. Variolins can be also considered as guanidine-
based alkaloids in which the guanidine moiety is found in the
guise of a 2-aminopyrimidine ring.
Figure 1. Structures of variolins 1-4.
This type of compounds exhibit a potent cytotoxic activity
were inhibited in a range of concentrations lower than those
required to inhibit the activity of CDK4/cyclin D or CDK7/
cyclin H complexes. Variolin B 2 and deoxyvariolin B are a
new class of CDK inhibitors that activate apoptosis in a p53-
independent fashion, and thus they may be effective against
tumors with p53 mutations or deletions.4
against P388 murine leukemia cells line, and also being effective
against Herpes simplex type I.3 Variolin B 2 is the most active
of this family of natural products; oxidation or reduction of the
3-aminopyrimidine ring at position C-5, as in variolin A 1 or
N-3′-methyl-3′,4′,5′,6′-tetrahydrovariolin B 3, decreases the
activity. This is corroborated by the absence of activity of
variolin D 4 in which C-5 carries only a methoxycarbonyl group.
In different human cancer cell lines variolin B 2 and the non
natural analogue deoxyvariolin B inhibited colony formation,
caused cell cycle perturbations, and induced apoptosis at
concentrations ranging from 0.1 to 2 µM. Although variolins
induced an increase in the levels of p53 with an increase in
p21, their cytotoxicities did not appear to be dependent on p53
status, as their potency was comparable in cells with wild-type
p53, or in sublines with inactivated p53. Variolin B 2 and
deoxyvariolin B prevent the cells from entering S phase,
blocking cells in G1, and cause accumulation already evident
4 h after the beginning of treatment. Although intercalation of
deoxyvariolin B in DNA has been demonstrated, neither variolin
B 2 nor deoxyvariolin B produce detectable breaks in DNA.
These results are consistent with the in vitro biochemical assays
that also demonstrated that deoxyvariolin B is not a topo-
isomerase I or II poison. Instead, each of these variolins appears
to inhibit cyclin-dependent kinases (CDKs) in the µM range.
CDK1-cyclin B, CDK2-cyclin A, and CDK2/cyclin E complexes
Consequently, its significant bioactivity, interesting structure,
and also its low natural occurrence have stimulated different
groups of scientific community to elaborate the central tricyclic
core of variolins. At this moment, three total syntheses of
variolin B 2 have been reported in the literature. The first
synthesis of variolin B 2,5 starting from 4-chloro-2-methylthio-
pyrimidine, involved a tandem deoxygenation and cyclization
of a triarylmethanol using a combination of triethylsilane and
trifluoroacetic acid as the key step. The preparation of variolin
B 2 and the non natural deoxyvariolin B from 4-methoxy-7-
azaindole has been reported.6 The method is based on the
dihydropyrimido annelation onto the preformed 7-azaindole ring
using N-tosyldichloromethanimine as cyclization reagent, fol-
lowed by the installation of the pyrimidine ring with palladium-
catalyzed cross-coupling reaction of 2-acetyl-4-(trimethylstannyl)-
pyrimidine and an iodopyridopyrrolopyrimidine derivative.
Results and Discussions
Chemistry. Taking into account the key role of the 2-ami-
nopyrimidine ring on the activity of the variolin B, we report
here the synthesis and biological evaluation of analogues of
variolin B in which this ring either is placed at the C-7 position
or is additionally placed at C-7 in the variolin B, giving rise to
a complex molecule such as 7 in which two 2-aminopyrimidine
rings are attached to the central core of the variolin family.
* To whom correspondence should be addressed. Phone: +34 9 68 36
74 84. Fax: +34 9 68 36 41 49. E-mail: fresneda@um.es.
† Universidad de Murcia.
‡ PharmaMar, S. A.
§ Present address: Institut Catala´ d′Investigacio´ Qu´ımica, Av. Pa¨ısos
Catalans, 16. 43007-Tarragona, Spain.
10.1021/jm051090r CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/14/2006

1218 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Brief Articles
Scheme 1. Synthesis of 7-(2-Aminopyrimidin-4-yl)variolin B 7a
a Reagents and conditions: (a) (i) LiOH, THF-H2O (95%); (ii) MeLi, THF, -15 °C (62%); (b) N,N-dimethylacetamide, POCl3, 70 °C, (61%); (c)
N,N-dimethylformamide di-tert-butylacetal, DMF, 40 °C, 97%; (d) N,N-dimethylformamide di-tert-butylacetal, DMF, 100 °C, 92%; (e) (i) N,N-
.
dimethylformamide di-tert-butylacetal, DMF, 60 °C, (ii) N,N-dimethylformamide di-tert-butylacetal, DMF, 100 °C, 67%; (f) H2N(CdNH)NH2 HCl, K2CO3,
2-methoxyethanol, 110 °C, 79%; (g) NaSMe, DMF, 80 °C, 12 (20%) and 13 (60%); (g) triflic acid, r.t., 63%.
Our strategy7 to the synthesis of variolin B 2 is based on the
key steps: (a) synthesis of appropriately functionalized 2,4-
disubstituted-7-azaindole, (b) construction of the central core
pyrido[3′,2′:4,5] pyrrolo[1,2-c]pyrimidine ring, that typifies this
compound, involving a carbodiimide-mediated pyrimido anne-
lation process, and (c) formation of the 2-aminopyrimidine ring
at position C-5.
This strategy opens extensive possibility synthetic to prepare
new derivatives of this alkaloid. As shown in the Scheme 1,
the intermediate 5, carrying an ethoxycarbonyl group at position
C-7 of the core pyrido[3′,2′:4,5]pyrrolo[1,2-c]pyrimidine ring,
has been used as starting material for the introduction at this
position of either different functionalities or of heterocyclic
rings. In this context, the synthesis of 7-isovariolin B 67b
involves formation of the 2-aminopyrimidine ring at position
C-7 of the tricyclic system, by conversion of the ethoxycarbonyl
group into the corresponding 7-acetyl derivative by reaction with
methyllithium, followed by the Bredereck protocol8 to build the
aminopyrimidine ring.
Here, we have applied the same strategy to synthesize 7-(2-
aminopyrimidin-4-yl)variolin B 7, bearing two 2-aminopyrimi-
dine rings at positions C-5 and C-7 of the tricyclic core of
variolin, starting from 5,7-diacetyl derivative 8 (Scheme 1).
To this end, the intermediate 5 by initial hydrolysis followed
by sequential treatment with methyllithium and the Vilsmeier
acylation with N,N-dimethylacetamide in the presence of POCl3
provided the 5,7-diacetylated product 8 in 61% yield. The
preferential reactivity of the acetyl group at C-7 position with
respect to the same group located at C-5 position is corroborated
by the selective preparation of enaminone 9. We have observed
different reactivity of the acetyl group at positions C-5 and C-7
of pyrido[3′,2′:4,5]pyrrolo[1,2-c]pyrimidine core in the reaction
with N,N-dimethylformamide di-tert-butylacetal, and it was
possible to prepare selectively the monoenaminone 9, only by
control of reactions conditions. When compound 8 reacts with
N,N-dimethylformamide di-tert-butylacetal at 40 °C for 24 h,
the monoenaminone 9 was obtained in 95% yield. The second
condensation was achieved by heating of 9 at 100 °C for 12 h
to give the dienaminone 10 in 92% yield. The direct formation
of the 5,7-dienaminone 10 from the diacetyl derivative 8 was
carried out with N,N-dimethylformamide di-tert-butylacetal in
DMF at 60 °C for 18 h followed by addition of a slight excesss
of the reagent and then heating at 100 °C for 12 h. Under these
conditions, compound 10 was isolated in 67% yield. The
simultaneous formation of the two 2-aminopyrimidine rings was
realized in one step in 79% yield, by treatment of 10 with
guanidine hydrochloride in 2-methoxyethanol in the presence
of anhydrous potassium carbonate to afford 11. The last steps
were the O-methyl and N-benzyl deprotection. To remove the
O-methyl group, compound 11 was treated with an excess of
sodium methanethiolate in dry DMF; however, the unexpected
compound 12, bearing a N,N-dimethylaminomethyl group at
position C-3, was isolated in 20% yield along with the ex-
pected compound 13 in 60% yield. Probably, the formation of
compound 12 involves formation of the intermediate N,N-
dimethyl(methylene)ammonium followed by amino alkylation
into the ortho position. Finally, the N-benzyl protecting group
was removed from 13 by treatment with triflic acid at room

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1219
Scheme 2. Synthesis of Amide 15a
potential. Some of the derivatives tested have similar activity
than the natural compound variolin B 2.
Experimental Section
General Methods. All reactions were carried under N2, and
solvents were dried by standard procedures. Column chromatog-
raphy purifications were performed using silica gel (60 Å c.c.
70-200 µm, SDS) as the stationary phase. All melting points were
determined on a Kofler hot-plate melting point apparatus and are
uncorrected. IR spectra were determined as Nujol emulsions or films
on a Nicolet Impact 400 spectrophotometer and were expressed in
cm-1. NMR spectra were recorded on a Bruker AC200 (200 MHz),
a Varian Unity 300 (300 MHz) or a Bruker Avance 400 (400 MHz)
using CDCl3 or DMSO-d6. Chemical shifts were reported in ppm
(δ scale) relative to Me4Si as an internal standard, and all J values
were in Hz. Assignments were made by DEPT or two-dimensional
NMR experiments. The EI mass spectra were recorded on a Fisons
AUTOSPEC 500 VG spectrometer.
1-[5,7-Bis(acetyl-9-(N-benzylamino)-4-methoxypyrido[3′,2′:
4,5]pyrrolo[1,2-c]pyrimidine (8). To a solution of POCl3 (0.53
mL, 5.72 mmol) in anhydrous CHCl3 (5 mL) was added N,N-
dimethylacetamide (0.59 mL, 6.36 mmol) at 0 °C under N2. The
mixture was stirred at room temperature for 25 min. Then, a solution
of monoacetyl derivative (0.11 g, 0.32 mmol) in CHCl3 (6 mL)
was added, and the reaction mixture was heated at 70 °C for 30 h.
After cooling, the mixture was poured into a saturated aqueous
solution of NaOAc (100 mL) and then extracted with CH2Cl2 (5 ×
30 mL). The combined organic layers were washed with H2O
(2 × 10 mL) and dried (MgSO4). The solvent was removed
under reduced pressure, and the residue was chromatographed on
a silica gel column using EtOAc:CH2Cl2 (1:9) as eluent to give 8
(0.075 g, 61% yield); Rf: 0.37 (SiO2, EtOAc:CH2Cl2, 1:9); mp
225-227 °C (yellow prisms from CH2Cl2:Et2O). Anal. (C22H20N4O3)
C, H, N.
a Reagents and conditions: (a) N,N-dimethyletylendiamine, EDC‚HCl,
THF, CH2Cl2, 0 °C then r.t., 70%.
temperature to give 9-amino-5,7-bis(2-aminopyrimidin-4-yl)-
pyrido[3′,2′:4,5]pyrrolo[1,2-c]pyrimidin-4-ol 7 in 63% yield
(Scheme 1).
Finally, the amide 15 was prepared in 70% yield from the
acid 14,7b by reaction with N,N-dimethylethylendiamine in the
presence of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (EDC‚HCl) (Scheme 2).
All the compounds prepared here, 7-13 and 15, as well as
those previously reported, 2, 6, 16, and 17, were isolated as
solids and purified by column chromatography to give the target
compounds in an analytically pure state (see Experimental
Section).
Biological Results. A panel of sixteen human tumor cell
lines was used to evaluate the cytotoxic potential of the variolin
B derivatives (Figure 2): prostate carcinoma tumor cells
(DU-145 and LN-CaP), SKOV-3 ovary adenocarcinoma, ovarian
cells sensitive (IGROV) or resistant (IGROV-ET) to ET-743,
SK-BR-3 breast adenocarcinoma, MEL-28 malignant melanoma,
H-MEC-1 endothelium cells, A-549 lung carcinoma NSCL,
K-562 chronic myelogenous leukemia, PANC-1 pancreatic
epitheloid carcinoma, HT-29 colon carcinoma cells, LoVo
lymph node metathesis cells and the corresponding LoVo-Dox
cells resistant to doxorubicin, and cervix epitheloid carcinoma
(HeLa) or resistant (HeLa-Apl) to aplidine.
(E)-1-[5-Acetyl-9-(N-benzylamino)-7-[(N,N-dimethylamino)-
2-propenon-1-yl]-4-methoxypyrido[3′,2′:4,5]pyrrolo[1,2-c]pyri-
midine (9). A mixture of the diacetyl derivative 8 (0.105 g, 0.27
mmol), N,N-dimethylformamide di-tert-butylacetal (0.27 g, 1.35
mmol), and anhydrous DMF (15 mL) was stirred at 40 °C for 24
h under N2. After cooling, the solution was poured into a saturated
aqueous solution of NaCl (20 mL) and then extracted with CH2Cl2
(3 × 15 mL). The combined organic layers were dried (MgSO4)
and concentrated to dryness under reduced pressure. The residue
was chromatographed on a silica gel column using CH2Cl2:MeOH
(9:1) as eluent to give 9 (0.114 g, 95% yield); mp 216-218 °C
(from CH2Cl2:Et2O). Anal. (C25H25N5O3) C, H, N.
A conventional colorimetric assay was set up to estimate GI50
values, i.e., the drug concentration which causes 50% cell growth
inhibition after 72 h continuous exposure to the test molecules.
Variolin B 2 is included in the test for comparison. The results
obtained are shown in Table 1, and several general observations
can be made.
a. As in the case of variolin D 4, the biological importance
of the substituent at C-5 in variolins is corroborated by the loss
of activity of some of the new variolin B derivatives tested with
a 3-aminopyrimidine at position C-7 (16, 17, and 7-isovariolin
B 6) but not at C-5.
b. For the rest of derivatives tested, the combination of
substituents at C-5 and C-7 decreases the activity with respect
to variolin B 2.
c. Only compounds 12 and 15, which contain an amino alkyl
chain ((dimethylamino)methyl at C-3 and 3-aminopyrimidine
at position C-7 and 2-(dimethylamino)ethyl carboxamide at C-7,
respectively) in addition to a 3-aminopyrimidine at position C-5,
have a micromolar activity.
(E)-9-(N-Benzylamino)-5,7-bis[3-(N,N-dimethylamino)-2-pro-
penon-1-yl]-4-methoxypyrido [3′,2′:4,5]pyrrolo[1,2-c]pyrimidine
(10). Method A: A mixture of the diacetyl derivative 8 (0.1 g,
0.25 mmol), N,N-dimethylformamide di-tert-butylacetal (0.26 g,
1.29 mmol), and anhydrous DMF (15 mL) was stirred at 60 °C
for 18 h under N2. Then N,N-dimethylformamide di-tert-butyl-
acetal (0.52 g, 2.50 mmol) was added and the reaction mixture
was stirred at 100 °C for 12 h. After cooling, the solution was
poured into a saturated aqueous solution of NaCl (20 mL) and
extracted with CH2Cl2 (3 × 15 mL). The combined organic layers
were dried (MgSO4) and concentrated to dryness under reduced
pressure. The residue was chromatographed on a silica gel column
using CH2Cl2:MeOH (9:1) as eluent to give 10 (0.086 g, 67% yield);
mp 115-116 °C (from CH2Cl2:Et2O).
Method B: A mixture of the 5-acetyl-7-enaminone 9 (0.11 g,
0.25 mmol), N,N-dimethylformamide di-tert-butylacetal (0.5 g, 2.5
mmol), and anhydrous DMF (10 mL), was stirred at 100 °C for 12
h under N2. After cooling, the solution was poured into a saturated
aqueous solution of NaCl (20 mL) and extracted with CH2Cl2 (3
× 15 mL). The combined organic layers were dried (MgSO4) and
concentrated to dryness under reduced pressure. The residue was
chromatographed on a silica gel column using CH2Cl2:MeOH
(9:1) as eluent to give 10 (0.114 g, 92% yield). Anal. (C28H30N6O3)
C, H, N.
d. Derivatives 12 and 15 are scaffolds of new types for further
preparation of new antitumor compounds.
Conclusions
We reported here the synthesis and biological evaluation of
eight new variolin B derivatives as potential cytotoxic agents.
Variolin B 2 and derivatives have been tested in a panel of
sixteen human tumor cell lines to evaluate their cytotoxic

1220 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Brief Articles
Figure 2. Structures of variolin B derivatives tested in vitro cytotoxicity.
Table 1. Data of in Vitro Cytotoxicity (GI50, µM) of the Compounds 2,
6, 7, 11, 12, 13, 15, 16, and 17a
was chromatographed on silica gel column using CH2Cl2:MeOH
(9:1) as eluent to give 13 (0.064 mg, 60% yield); Rf: 0.27 (SiO2,
CH2Cl2:MeOH, 9:1); mp: >300 °C (red prisms from CH2Cl2).
Anal. (C25H20N10O) C, H, N.
9-(N-Benzylamino)-5,7-bis(2-aminopyrimidin-4-yl)-3-[(N,N-
dimethylamino)methyl]-4-hydroxi-pyrido[3′,2′:4,5] pyrrolo[1,2-
c]pyrimidine (12). (0.024 mg, 20% yield); Rf: 0.48 (SiO2, CH2Cl2:
MeOH, 9:1); mp >300 °C (red needles from dioxane:Et2O). Anal.
(C28H27N11O) C, H, N.
cell line
2
6
7
11
12
13
15
16
17
DU-145
0.89 34.1 26.9 21.0
0.05 5.28 26.9 18.6
4.74 21.7 2.38 25.2 26.1
9.13 21.7 2.37 16.0 12.9
n.d. 21.7 0.72 25.2 19.0
LN-caP
SKOV-3
IGROV
1.21 n.d.
n.d. n.d.
1.14 27.9 26.9 21.0
4.03 21.7 2.80 15.1
4.46
IGROV-ET 1.28 31.9 26.9 21.0
3.56 9.90 2.05 25.2 12.9
SK-BR-3
MEL-28
H-MEC-1
A-549
0.85 31.9 14.8
1.20 3.41 26.9 21.0
0.27 n.d. n.d. n.d.
0.98 34.1 26.9 21.0 18.7 21.7 3.38 25.2 14.1
1.55 9.24 26.9 21.0 4.78 21.7 3.36 25.2 26.1
8.24 2.79 21.7 2.13 25.2 13.1
4.12 21.7 4.42 25.2 24.4
n.d. 21.7 0.46 15.0 6.31
9-Amino-5,7-bis(2-aminopyrimidin-4-yl)-4-hydroxi-pyrido-
[3′,2′:4,5]pyrrolo[1,2-c]pyrimidine (7). A solution of compound
13 (57 mg, 0.12 mmol) in triflic acid (3 mL) was stirred at room
temperature for 16 h. After cooling at 0 °C, MeOH (5 mL) was
added and the resulting mixture was stirred at that temperature for
5 min. Afterward, a solution of 30% ammonium hydroxide was
added until basic pH. The solvent was removed off at room
temperature under reduced pressure. To residue was added a
saturated aqueous solution of NaCl (10 mL) and extracted with
EtOAc (3 × 10 mL). The residue was chromatographed on a silica
amine (SiO2-NH2) using CH2Cl2:MeOH (9:1) as eluent to afford
7 (30 mg, 65% yield); mp > 300 °C (red prisms from CH2Cl2:
MeOH).). Anal. (C18H14N10O) C, H, N.
5-(2-Aminopyrimidin-4-yl)-9-(N-benzylamino)-N-[2-(N,N-di-
methylamino)ethyl]-4-methoxypyrido[3′,2′:4,5]pyrrolo[1,2-c]py-
rimidine-7-carboxamide (15). To a solution of acid 14 (30 mg,
0.068 mmol) and N,N-dimethyletylendiamine (10 µL, 0.092 mmol)
in anhydrous THF (4 mL) at 0 °C under N2 was added a solu-
tion of N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hy-
drochloride (14.3 mg, 0.075 mmol) in anhydrous CH2Cl2 (2 mL).
The mixture was stirred at room temperature for 24 h. The solvent
was concentrated to dryness under reduced pressure. The residue
was chromatographed on a silica gel column using CH2Cl2:MeOH
(9:1) as eluent to give 15 (24 mg, 70% yield); mp: 170-171 °C
(CH2Cl2:Et2O).). Anal. (C27H29N9O2) C, H, N.
K-562
PANC-1
HT-29
1.68 21.8 26.9 19.9 17.7 21.7 3.97 25.2 26.1
2.85 34.1 26.9 21.0 18.7 21.7 3.44 25.2 25.1
LOVO
0.80 34.1 26.9 21.0 13.6 21.7 3.62 14.4
3.05 21.7 3.46 11.0
8.74
6.76
LOVO-DOX 1.02 34.1 26.9 21.0
HELA
HELA-APL n.d.
n.d.
6.14 26.9
8.86 26.9
3.03 4.52 n.d. n.d.
3.68 3.88 n.d. n.d.
n.d. n.d.
n.d. n.d.
a n.d. ) not determined.
9-(N-Benzylamino)-5,7-bis(2-aminopyrimidin-4-yl)-4-meth-
oxypyrido[3′,2′:4,5]pyrrolo[1,2-c]pyrimidine (11). A mixture of
the 5,7-dienaminone 10 (0.141 mg, 0.28 mmol), guanidine hydro-
chloride (0.27 g, 2.83 mmol), anhydrous K2CO3 (0.43 g, 3.08
mmol), and dry 2-methoxyethanol (15 mL) was heated at 100 °C
for 40 h. After cooling, the mixture was poured into a saturated
aqueous solution of NH4Cl (30 mL) and extracted with CH2Cl2 (4
× 25 mL). The combined organic layers were dried (MgSO4) and
concentrated to dryness under reduced pressure. The residue was
chromatographed on a desactive silica gel column using first
CH2Cl2 and then CH2Cl2:MeOH (9:1) as eluent to give 11 (0.11
mg 79%); mp 269-271 °C (orange prisms from CH2Cl2:Et2O).
Anal. (C26H22N10O) C, H, N.
9-(N-Benzylamino)-5,7-bis(2-aminopyrimidin-4-yl)-4-hydroxi-
pyrido[3′,2′:4,5]pyrrolo[1,2-c]pyrimidine (13). To a solution of
compound 5,7-dipyrimidine 11 (0.11 mg, 0.224 mmol) in anhydrous
DMF (12 mL) was added sodium methanethiolate (0.157 mg, 2.24
mmol) under N2. The reaction mixture was stirred at 80 °C for 24
h. After cooling, the mixture was poured into a saturated aqueous
solution of NaCl (50 mL) and extracted with EtOAc:dioxane (2:1,
5 × 40 mL). The combined organic layers were dried (MgSO4)
and concentrated to dryness under reduced pressure. The residue
Cell Growth Inhibition Assay: Screening. A colorimetric assay
using sulforhodamine B (SRB) has been adapted for a quantitative
measurement of cell growth and viability, following a previously
described method.10 Cells were seeded in 96-well microtiter plates,
at 5 × 103 cells per well in aliquots of 195 µL of RPMI medium,
and they are allowed to attach to the plate surface by growing in
drug free medium for 18 h. Afterward, samples are added in aliquots

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1221
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of 5 µL (dissolved in DMSO:H2O, 3:7). After 72 h exposure, the
antitumor effect is measured by the SRB methodology: cells are
fixed by adding 50 µL of cold 50% (wt/vol) trichloroacetic acid
(TCA) and incubating for 60 min at 4 °C. Plates are washed with
deionized H2O and dried; 100 µL of SRB solution (0.4wt %/vol in
1% acetic acid) is added to each microtiter well and incubated for
10 min at room temperature. Unbound SRB is removed by washing
with 1% acetic acid. Plates are air-dried and bound stain is
solubilized with Tris buffer. Optical densities are read on an
automated spectrophotometer plate reader at a single wavelength
of 490 nm. Data analysis are generated automatically by LIMS
implementation. Using control OD values (C), test OD values (T),
and time zero OD values (T0), the drug concentration that causes
50% growth inhibition (GI50 value) was calculated from the
equation: 100 × [(T - T0)/C - T0)] ) 50.
Acknowledgment. We thank the Direccio´n General de
Investigacio´n (MCYT, Spain) project number BQU2001-0014,
Foundation Seneca (CARM) projects number PI-48/00740/
FS/01 and 00582/PI/04, and PharmaMar S. A. (Colmenar Viejo,
Spain) for financial support. S.D. would like to thank the
Fundacio´n Se´neca (CARM).
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von vinylogen carbonsa¨uren zu heterocyclen. Chem. Ber. 1965, 98,
1081-1086.
Supporting Information Available: Analytical and spectro-
scopic data of compounds 7-13 and 15. This material is available
free of charge via the Internet at http://pubs.acs.org.
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