Synthesis and antitumoral evaluation of indole alkaloid analogues containing an hexahydropyrrolo[1′,2′,3′:1,9a,9]imidazo[1,2-a]indole skeleton

Article abstract of DOI:10.1016/j.bmc.2008.08.070

The scope of acid-mediated cyclative additions of electrophiles to tryptophan-derived α-amino nitriles for the synthesis of 10b-substituted-1,2,4,5,10b,10c-hexahydropyrrolo[1′,2′,3′:1,9a,9]imidazo[1,2-a]indoles analogues of indole alkaloids has been studied. The results demonstrate the high potential of the methodology for the synthesis of 10b-bromo-derivatives, by bromination with NBS, 10b-allyl-derivatives, by bromo-allyl exchange, and 10b-prenyl-derivatives, by reaction with prenyl bromide in the presence of Mg(NO₃)₂·6H₂0. Some of the new pyrroloimidazoindole derivatives displayed moderate μM cytotoxicities in human cancer cell lines and at 10 μg/mL inhibited more than 50% EGFR or HIF-1α.

Full text of DOI:10.1016/j.bmc.2008.08.070

Bioorganic & Medicinal Chemistry 16 (2008) 9313–9322
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antitumoral evaluation of indole alkaloid analogues
containing an hexahydropyrrolo[10,20,30:1,9a,9]imidazo[1,2-a]indole skeleton
Pilar Ventosa-Andrés, Juan A. González-Vera, Ángel M. Valdivielso, M. Teresa García-López,
*
Rosario Herranz
Instituto de Química Médica (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The scope of acid-mediated cyclative additions of electrophiles to tryptophan-derived
a-amino nitriles
for the synthesis of 10b-substituted-1,2,4,5,10b,10c-hexahydropyrrolo[10,20,30:1,9a,9]imidazo[1,2-
a]indoles analogues of indole alkaloids has been studied. The results demonstrate the high potential of
the methodology for the synthesis of 10b-bromo-derivatives, by bromination with NBS, 10b-allyl-deriv-
atives, by bromo-allyl exchange, and 10b-prenyl-derivatives, by reaction with prenyl bromide in the pres-
Received 22 May 2008
Revised 21 August 2008
Accepted 28 August 2008
Available online 31 August 2008
ence of Mg(NO3)2ꢀ6H20. Some of the new pyrroloimidazoindole derivatives displayed moderate
cytotoxicities in human cancer cell lines and at 10 g/mL inhibited more than 50% EGFR or HIF-1
Ó 2008 Elsevier Ltd. All rights reserved.
lM
Keywords:
Indole alkaloids
l
a.
a-Amino nitriles
Cytotoxicity
Inhibition of EGFR
Inhibition of HIF-1
a
1. Introduction
In the context of a research program focused on the synthesis of
privileged scaffolds, we discovered that 10b-unsubstituted-
1,2,4,5,10b,10c-hexahydropyrrolo[10,20,30:1,9a,9]imidazo[1,2-a]in-
doles are easily and efficiently obtained with high stereoselectivity
by acid-promoted domino tautomerization of tryptophan-derived
The novel ring system 1,2,4,5,10b,10c-hexahydropyrrol-
o[10,20,30:1,9a,9]imidazo[1,2-a]indole (Fig. 1, A) can be considered
as a constrained hybrid of 1,2,3,3a,8,8a-hexahydropyrrolo[2,3-
b]indole (B) and 2,3,9,9a-tetrahydroimidazo[1,2-a]indole (C), both
present in a growing number of indole alkaloids with a wide range
of biological activities.1 Thus, the hexahydropyrrolo[2,3-b]indole is
present in the acetylcholinesterase inhibitors physostigmine (1)
and phenserine2 (2), in the antibacterials flustramines3 and molle-
nines,4 in the numerous group of fused piperazine derivatives
[among others: the multidrug resistance reversal agents ardee-
mins5 (3), the mycotoxin brevianamide E,6 the vasodilator amauro-
mine,7 roquefortines,8 okaramines,9 gypsetin,10 or the immunomo
dulators sporidesmins11], in dimeric and polymeric indole
alkaloids such as the somatostatin antagonists psycholeine and
quadrigemine C,12 and as a modified tryptophan residue in several
peptides such as himastatin,13 chloptosin,14 or the Bacillus subtilis
pheromone ComX15 (4).
a
-amino nitriles.1,22 To approach the synthesis of higher substi-
tuted analogues of the mentioned indole alkaloids, we have
recently explored the access to 10b-substituted-hexahydropyrrol-
o[10,20,30:1,9a,9]imidazo[1,2-a]indoles by acid-promoted electro-
phile cyclative additions in the cyclohexanone-derived
a-amino
nitrile 8a (Scheme 1).23,24 These preliminary studies demonstrated
the possibility of introducing halogens (Cl and Br) and the prenyl
and hydroxyl groups into the 10b position of the pyrroloimidazoin-
dole skeleton by acid-promoted ring-closing halogenation, prenyl-
ation and oxidation. On the other hand, a bromo-allyl exchange
gave access to the corresponding 10b-allyl derivative. The resulting
10b-substituted-hexahydropyrrolo[10,20,30:1,9a,9]imidazo[1,2-a]
indoles were evaluated as antitumorals in human cancer cell lines.
The 9,10b-dibromo and the 10b-allyl derivatives 11a and 15a,
shown in Scheme 1, displayed micromolar cytotoxicity in human
lung carcinoma (A549) and colon carcinoma (HT-29) cell lines.23
Based on these preliminary results, now we have studied and com-
municate herein the versatility of cyclative electrophile additions
On the other hand, the tetrahydroimidazo[1,2-a]indole is
present in the cholecystokinin CCK1 receptor antagonist asperlicin
(5),16 the substance P antagonist fiscalin A17 (6), the antifungic
fumiquinazolines,18 tryptoquivalines,19 and in chaetominine (7)20
or kapakahines,21 which contain an additional peri-fused
piperidone ring.
to other tryptophan-derived a-amino nitriles and the antitumoral
evaluation of the resulting products. Amino nitriles derived from
acetone (b), N-benzyl-4-piperidone (c), phenylacetaldehyde (d),
benzaldehyde (e) and pivalaldehyde (f) were selected as starting
materials for this study.
* Corresponding author. Tel.: +34 91 562 2900; fax: +34 91 564 4853.
E-mail address: rosario@iqm.csic.es (R. Herranz).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.070

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P. Ventosa-Andrés et al. / Bioorg. Med. Chem. 16 (2008) 9313–9322
analysis of the crude reaction mixtures showed a significant
increase in the yield of the 10b-bromo-pyrroloimidazoindole 9d
with the decrease of the TFA concentration from 10 to 1% and with
the reduction of the reaction time from 2 h to 15 min, but 9d was
unstable and could not be isolated for its complete characterization.
A similar treatment of the benzaldehyde-derived amino nitriles 8e
led to complex mixtures of decomposition products. Interestingly,
in the bromination of the pivalaldehyde-derived amino nitriles 8f
[an inseparable (2:3) epimeric (R):(S) mixture] with one equiv. of
NBS in solution of 1% of TFA in CH2Cl2, a low yield (22%) of the
10b-bromo-pyrroloimidazoindole 9f was isolated, along with 21%
of an ꢁ(1:1) mixture of the dimeric compounds 13f and 14f, which
could not be resolved. A (2.5:1) mixture of 2-exo-/2-endo-9,10b-di-
bromo-pyrroloimidazoindoles 11f and 12f was obtained and chro-
matographically resolved in low yield from the reaction of 8f with 2
equiv of NBS in 1% solution of TFA in CH2Cl2. Under these condi-
tions, the formation of the dimeric species 13f/14f was not
observed. Dimerization was neither observed in the abovemen-
tioned reactions of the other amino nitriles 8a–e.
The HPLC-MS analysis of the 13f + 14f mixture showed two
peaks in a (1.2:1) area ratio with the respective dimeric mass of
625.70 and 703.52. The latter with the characteristic isotopic mass
pattern of brominated compounds, which was confirmed by the Br
microanalysis of the sample. The 1H NMR spectrum also showed
the presence of two dimeric compounds. Thus, four different
t
singlets appeared, corresponding to the Bu groups, and another
four for the OMe groups. However, only two doublets appeared
at 5.49 and 5.56 ppm for the 8a-H protons of the pyrroloindole
moiety, each one coupled with a doublet at 4.50 and 4.63 ppm,
respectively, corresponding to the NH at position 8. The 3H-indole
moiety was evidenced, in each case, from the presence of one
singlet at 6.88 and 6.94 ppm, corresponding to the 2-H protons,
and from the absence of additional 8-H and 8a-H pairs that should
have appeared in the case of a second pyrroloindole unit. The
absence of signals for 3a-H in the pyrroloindole unit and for 3-H
in the 3H-indole one suggested the linkage points between them.
This assignment was confirmed by the 13C NMR spectrum and
the one and two bonds 1H,13C correlation spectra HSQC and HMBC.
These spectra showed the presence of four quaternary carbons for
the cyano groups (117.07, 117.27 for the pyrroloindole moiety, and
119.21 and 119.26 for those of the 3H-indole units). The bromina-
Figure 1. The pyrrolo[10,20,30:1,9a,9]imidazo[1,2-a]indole ring system and related
indole alkaloids.
2. Results and discussion
2.1. Chemistry
tion position in 14f was also assigned based on HMBC 1H,13
C
As shown in Scheme 1, we first studied the bromination of a-ami-
no nitriles 8b-f, by applying the methodology previously developed
for 8a,23 consisting of treatment with N-bromosuccinimide (NBS, 1
equiv for 10b-monobromation and 2 equiv for 9,10b-dibromination)
in the presence of 10% TFA in CH2Cl2 solution at 40 °C. Under these
reaction conditions, the acetone-derived amino nitrile 8b gave very
similar results to those described for the cyclohexanone derivative
8a, except that three equiv. of NBS were required for its complete
dibromination, obtaining exclusively the respective 2-exo-diastere-
oisomers 9b and 11b in high yields (Table 1).
correlations.
A (3aS)-configuration at the pyrroloindole ring was assigned to
both 13f and 14f based on the 1H chemical shifts of their methoxy-
carbonyl groups (3.72 and 3.74 ppm), characteristic of a 2-exo-con-
figuration.22,23 On the other hand,
a (3R)-configuration was
tentatively assigned to the 3H-indole moiety based on the NOEs
effects observed in the NOESY spectrum of 13f + 14f, shown in
Scheme 2. Particularly, the NOEs between the pyrroloindole 8a-H
and the indole 7-H and 2-H, as well as, between this proton and
one of the 3-H protons of the pyrroloindole. According to the min-
imized energy models for both (3R)- and (3S)-epimers, generated
As described for the acid-promoted tautomerization,22 mixtures
of 2-exo- and 2-endo-pyrroloimidazoindoles were obtained both in
the mono- and dibromination of the more constrained amino
nitrile derived from 4-piperidone 8c. As shown in Table 1, the
diastereomeric ratio was dependent on the reaction time and tem-
perature. Thus, the 2-exo-isomers 9c and 11c were the kinetic con-
trolled products that slowly isomerized to the more stable 2-endo
isomers 10c and 12c, respectively.
Aldehyde-derived amino nitriles 8d–f were much more reactive
toward NBS and complex reaction mixtures of decomposition prod-
ucts were obtained under the reaction conditions developed for the
ketone derivatives 8a–c. Trying to avoid this decomposition, the
influence of the TFA concentration, temperature and time of mono-
bromination on the reaction products was studied in the (R)-epimer
of the phenylacetaldehyde-derived amino nitrile (R)-8d. The HPLC
with the Chem3DÒ program, only the (3R)-epimer in a
conformation would explain the observed NOEs. As the starting
-amino nitrile 8f was used as an inseparable (R,S)-epimeric mix-
ture at C- , it was not possible to assign the configuration at these
p-stacked
a
a
carbons in (13 + 14)f.
The reaction mechanism shown in Scheme 2 could explain the
formation of the dimeric species. As recently reported by
Movassaghi et al. for the synthesis of dimeric pyrroloindole alka-
loids, such as chimonanthine, from 3a-bromo-pyrroloindoles,25
the reaction with NBS would generate the 3a-bromo-pyrroloin-
doles A, where, due to the steric hindrance and the low acidity
medium (1% TFA, versus the 10% used for the ketone-derived ami-
no nitriles), the formation of the free radicals B would compete

P. Ventosa-Andrés et al. / Bioorg. Med. Chem. 16 (2008) 9313–9322
9315
Scheme 1. Synthesis of 10b-bromo- and -allyl-hexahydropyrrolo[10,20,30:1,9a,9]imidazo[1,2-a]indoles.
Table 1
Results of the NBS-mediated bromination of
a-amino nitriles 8
Amino nitrile
NBS (equiv.)
TFA (%)
T (°C)
Time (h)
Productsb
8aa
8aa
8b
8b
8c
8c
8c
8c
8c
(R)-8d
(R)-8d
(R)-8d
8e
8f
8f
1
2
1
3
1
1
1
1
3
1
1
1
1
1
2
10
10
10
10
10
10
10
10
10
10
1
ꢂ40
ꢂ40
ꢂ40
ꢂ40
ꢂ40
ꢂ40
0
3
3
3
2
0.5
24
24
24
2
2
0.25
2
9a (91%)
11a (94%)
9b (83%)
11b (68%)
9c (95%) + 10c (5%)c
9c (82%) + 10c (8%)c
9c (70%) + 10c (30%)c
9c (55%) + 10c (45%)c
25
ꢂ40
ꢂ40
ꢂ40
ꢂ40
ꢂ40
ꢂ40
ꢂ40
11c (65%) + 12c (35%)c
9d (19%)c
9d (98%)c
1
1
1
1
decomposition
decomposition
9f (22%) + [13f + 14f (21%)]
11f (25%) + 12f (10%)
2
2
2
a
Ref. 1.
b
Isolated yields, except for 8c.
Yields determined by HPLC.
c
Scheme 2. Proposed mechanism for the formation of dimmers 13f + 14f and most significant NOEs observed in their NOESY spectrum.

9316
P. Ventosa-Andrés et al. / Bioorg. Med. Chem. 16 (2008) 9313–9322
with the cyclization to the pyrroloimidazoindoles 9f–12f. The free
radical species B would attack to the C-3 of a second amino nitrile
molecule to give the dimeric compounds 13f + 14f. This mixture
did not cyclize to pyrroloimidazoindole derivatives after 15 days
of treatment with 10% solution of TFA in CH2Cl2, being recovered
unaltered.
In the ketone-derived 10b-bromo-pyrroloimidazoindoles 9b,c
and 11b,c, the replacement of the bromo by an allyl group was
studied by applying the reaction conditions developed for the
cyclohexanone-derived analogues 9a and 11a, consisting of
treatment with allyltributyltin in the presence of a 10% of the free
radical initiator AIBN in refluxing xylene23 (Scheme 1). The new
10b-allyl-pyrroloimidazoindoles 15b,c and 17b,c were obtained
in significant lower yields (26–57%) than those reported for 15a
and 17a (70 and 65%). These lower yields were consequence of a
partial (11–30%) dehalogenation to the corresponding 10b-unsub-
stituted-pyrroloimidazoindoles 16b,c and 18b,c and to a higher
partial decomposition of the starting materials.
Finally, as the prenyl or reverse prenyl groups are among the
most recurrent substituents in indole alkaloids, we studied the
scope of the cyclative prenylation of tryptophan-derived a-amino
nitriles in ketone (8b,c) and aldehyde (8d–f) derivatives for the
synthesis of 10b-prenyl-pyrroloimidazoindoles. Our previously
reported prenylation of the cyclohexanone derivative 8a involved
the reaction with prenyl bromide in the presence of
Mg(NO3)2ꢀ6H2O in pH 2.9 AcOH/AcONa buffer at room tempera-
ture.23 This methodology was first applied to the acetone-derived
amino nitrile 8b. Due to the high instability of this amino nitrile
in the AcOH/AcONa buffer, only low yields of the 2-exo- and 2-
endo-3a-prenyl-pyrroloindoles 19 and 20 (Scheme 3) could be iso-
lated. The formation of these compounds indicated that, previously
to the prenylation, the amino nitrile had reverted to the trypto-
phan methyl ester. With the aim of decreasing the degradation
produced by the aqueous acid media, an heterogeneous CH2Cl2/
buffer reaction medium was tried, but in this case, the starting
amino nitrile was recovered unaltered. Next, we tried to use a mis-
cible organic cosolvent and to change the order of addition of the
reagents (addition of the amino nitrile, dissolved into acetonitrile,
after the prenyl bromide). In this way, the 2-exo-10b-prenyl-pyr-
roloimidazoindole 21b (41%) was obtained along with a low yield
of the pyrroloindoles 19 + 20. The 4-piperidone-derived amino ni-
trile 8c resulted completely degraded even under the new reaction
conditions, while, the aldehyde-derived amino nitriles 8d–f gave
low yields of the 10b-prenyl-pyrroloimidazoindoles 21–22, along
with a variable% of 19 + 20.
Scheme 3. Prenylation of a-amino nitriles 8b,d–f.
Interestingly, the pure pivalaldehyde-derived (4R)-2-endo-iso-
mer 22f in the CDCl3 solution of the 1H NMR sample slowly
epimerized at C-4 to give 23f, achieving the equilibrium at an
(1:1) epimeric proportion after 60 days in solution at room tem-
perature. As we have previously suggested,22 this epimerization
at C-4 must proceed through the enamine tautomer of the endocy-
clic amidine group.
In all new pyrroloimidazoindoles herein described, the absolute
configuration at C-10b, C-10c, and C-4 was assigned based on the
1H NMR chemical shift of the methoxycarbonyl group [in 2-endo-
isomers this group appears at (3.00–3.36 ppm), ꢁ0.5 ppm upfield
with respect to the 2-exo-isomers (3.60–3.75 ppm)] and on the
NOE effects observed in the respective 1D NOESY spectra.21–24
1a (HIF-1a), and histone deacetylases. The cytotoxicity was evalu-
ated on three representative human cancer cell lines: breast
(MDA-MB-231), lung (A549), and colon (HT-29), according to the
National Cancer Institute (NCI) protocols. The three cell growth
parameters: GI50 (concentration that produces 50% growth inhibi-
tion), TGI (concentration that produces total growth inhibition),
and LC50 (concentration that produces 50% of cellular death) were
determined from the data analysis, automatically generated by the
HTS laboratory information management system. The results of
the compounds that displayed GI50 values lower than the highest
100 lM concentration are shown in Table 2. The cyclohexa-
none-derived 9,10b-dibromo-pyrroloimidazoindole 11a, the piperi-
done-derived 10b-allyl derivatives 16c and 17c, and the acetone-
derived 10b-prenyl derivative 21b were the most active compounds,
2.2. Antitumoral evaluation
Thenewpyrroloimidazoindolederivativeshereindescribedwere
evaluated as antitumorals in HTS programmes, which included in
parallel screening of cytotoxicity and of inhibition of several molec-
ular targets involved in tumor growth, such as mitosis, epidermal
growth factor receptor (EGFR), b-catenin, hypoxia-inducible factor
displaying moderate cytotoxicities, in the lM range, in the three
tested cell lines. The 10b-bromo derivative 9c showed selectivity
for A549 and HT-29 cells, while, 9f and 21f showed selectivity for
breast MDA-MB-231 cells. These results did not allow us to establish
structure–activity relationships.

P. Ventosa-Andrés et al. / Bioorg. Med. Chem. 16 (2008) 9313–9322
9317
Table 2
Results of the antitumoral evaluation of pyrroloimidazoindole derivatives
Compound
Cytotoxicity (lM)
MDA-MB-231
TGI
A549
TGI
HT-29
TGI
% Inhibitiona
GI50
LC50
GI50
LC50
GI50
LC50
EGFR
HIF-1a
9c
9f
>100
17.6
>100
6.21
>100
11.7
>100
>100
23.5
ND
>100
25.5
>100
8.49
>100
17.4
>100
>100
24.6
ND
>100
25.5
>100
>100
>100
17.4
>100
>100
25.7
ND
17.6
>100
>100
7.24
>100
10.8
>100
>100
23.3
9.23
10.5
7.21
8.22
5.66
22.1
>100
>100
12.0
20.2
>100
>100
10.8
>100
17.4
>100
>100
24.1
9.23
10.5
7.21
12.1
28.3
24.1
>100
>100
24.1
20.2
>100
>100
16.1
>100
17.4
>100
>100
25.2
9.23
10.5
7.21
17.2
28.3
24.1
>100
>100
24.1
17.6
>100
18.8
7.45
>100
7.66
>100
>100
22.4
9.23
10.5
7.21
7.47
4.24
20.2
>100
>100
>100
20.2
>100
20.2
9.31
>100
14.6
>100
>100
23.5
9.23
10.5
7.21
7.84
28.3
24.1
>100
>100
>100
20.2
>100
20.2
14.1
>100
17.4
>100
>100
25.2
9.23
10.5
7.21
8.40
28.3
24.1
>100
>100
>100
31
0
75
70
73
ND
55
94
73
65
ND
ND
ND
ND
99
73
78
78
83
61
10c
11a
11b
11c
11f
13f + 14f
15a
16a
16b
16c
17c
21b
21d
21e
21f
27
83
45
24
0
40
52
ND
ND
ND
15
0
ND
ND
ND
ND
ND
ND
8.22
11.0
>100
>100
15.2
9.39
18.7
28.3
>100
>100
26.2
24.1
18.7
28.3
>100
>100
26.2
24.1
20
13
0
22d
0
a
Inhibition produced by a 10
l
g/mL concentration.
The activity on molecular targets was determined in 96-well
microplates at five fixed concentrations (10, 2, 0.4, 0.08, and
imidazoindole derivatives. On the other hand, the instability of a-
amino nitriles in aqueous AcOH/AcONa buffer is in part responsi-
0.016 lg/mL), using established fluorescence cell-based bioassays.
ble of the low yield of the cyclative prenylation reaction. The
Data are expressed as inhibition and cell survival percentages of
the controls. Active compounds were considered those that
displayed inhibition values higher than 50% and cell survival higher
than 70%. An ELISA immunoassay in HeLa cells was used for the
moderated cytotoxic activity and inhibition of EGFR and HIF-1a
herein demonstrated by some pyrroloimidazoindole derivatives
could be a good indication for the search of novel antitumoral
agents.
evaluation of mitosis inhibition,26 using paclitaxel (1
l
g/mL) and
g/mL) as control inhibitors. The inhibition of EGFR,
b-catenin, and hypoxia-inducible factor 1 (HIF-1 ) was evaluated
vinblastine (1
l
4. Experimental
a
a
in tumoral cells stably transfected with a luciferase (luc) reporter
gene under the control of a specific promoter of the signalling tar-
get.27 HeLa cells transfected with the AP1-luc reporter gene were
used for screeningon the EGFR signallingpathway, using EGF as acti-
vator and the selective inhibitor AG-147828 as control inhibitor.
SW480 cells of human colorectal adenocarcinoma transfected with
the TCF4-luc promoter gene, which confer constitutive activation
of the b-catenin signalling pathway, were used for the screening
on this target. HeLa cells transfected with the HIF1-luc reporter gene
4.1. General methods
All reagents were of commercial quality. Solvents were dried
and purified by standard methods. Analytical TLC was performed
on aluminum sheets coated with a 0.2mm layer of silica gel 60
F254. Silica gel 60 (230–400 mesh) was used for flash chroma-
tography. Preparative radial chromatography was performed on
20 cm diameter glass plates coated with a 1-mm layer of silica
gel PF254. Analytical RP-HPLC was performed on a Novapak
were used for the screening on HIF-1
a, using the iron chelator defer-
C18 (3.9 ꢃ 150 mm, 4
lm) column, with a flow rate of 1 mL/
oxamine (50 M) as HIF-1
l
a
inductor.29 MDA-MB231 (human breast
min, and using a tunable UV detector set at 214 nm. Mixtures
of CH3CN (solvent A) and 0.05% TFA in H2O (solvent B) were
used as mobile phases. HPLC-EMS was performed on an Atlantis
carcinoma) cell extracts were used for the evaluation of activity on
histone deacetylases, using trichostatin A30 (250 nM) as specific
inhibitor. None of the compounds displayed inhibition of mitosis,
b-catenin, and histone deacetylases. However, as shown in Table 2,
the cyclohexanone-derived 9,10b-dibromo and the 10b-allyl deriva-
tives 11a and 15a inhibitedEGFR more than 50% at thehighest tested
T3 C18 (2.1 ꢃ 100 mm, 3
lm) column at 30 °C, with a flow rate of
0.25 mL/min. Gradients (5–80%) of CH3CN with 0.08% of formic
acid (solvent A) in 0.1% of formic acid in H2O (solvent B) were
used as mobile phases. 1H NMR spectra were recorded at 300,
400, or 500 MHz, using TMS as reference, and 13C NMR spectra
were recorded at 75, 100, or 125 MHz. The NMR spectra assign-
ment was based on COSY, HSQC, and HMBC spectra. ESI-MS
spectra were performed, in positive mode, using MeOH as
solvent.
concentration (10
lM) and, at this concentration, several com-
pounds displayed high inhibition percentages of HIF-1
a. In view of
the overall antitumoral evaluation results, 11a, 17c, and 21b could
represent interesting hints for the search of novel antitumoral
agents.
3. Conclusions
4.2. General procedure for the acid-promoted cyclative
monobromination of ketone-derived
Synthesis of 9b,c and 10c
a-amino nitriles.
In summary, the studies herein described confirm the poten-
tial of tryptophan-derived
a-amino nitriles for the highly stereo-
selective generation of diversely substituted 1,2,4,5,10b,10c-
hexahydropyrrolo[10,20,30:1,9a,9]imidazo[1,2-a]indoles by cycla-
tive addition of electrophiles. This potential is limited in alde-
NBS (282 mg, 1.58 mmol) and TFA (500
l
L) were added to a
ꢂ40 °C cooled solution of the corresponding
a-amino nitrile 8b,c
(1.58 mmol) in CH2Cl2 (5 mL), and the mixture was stirred at that
temperature for 3 h. Then, the reaction mixture was poured into
ice (ꢁ10 g), neutralized with concentrated ammonium hydroxide
hyde-derived
a-amino nitriles by their high reactivity toward
NBS and the instability of the corresponding brominated pyrrolo-

9318
P. Ventosa-Andrés et al. / Bioorg. Med. Chem. 16 (2008) 9313–9322
and extracted with CH2Cl2 (20 mL). The organic extracts were suc-
cessively washed with H2O (5 mL) and brine (5 mL), dried over
Na2SO4, and evaporated under reduced pressure. The residue was
purified by circular chromatography, using 30–65% gradient of
EtOAc in hexane as eluant, to give the corresponding 10b-bromo
derivatives 9b,c and 10c, whose more significant analytical and
spectroscopic data are shown in Table 3.
(1.58 mmol) in CH2Cl2 (5 mL), and the mixture was stirred at that
temperature for 3 h. Afterwards, the reaction mixture was
processed as above. In the case of the phenylacetaldehyde and
benzaldehyde derivatives 8d and 8e, the reaction products were
unstable and could not be isolated. In the case of the pivalaldehyde
derivative 8f, the 10b-bromo-derivative 9f (22%), whose analytical
and spectroscopic data are summarized in Table 3, and the dimeric
mixture of (13 + 14)f (21%) were obtained.
4.3. General procedure for the acid-promoted cyclative
monobromination of aldehyde-derived
Synthesis of 9d–f and (13 + 14)f
a
-amino nitriles.
4.3.1. Dimeric mixture (13 + 14)f
Foam (21%). HPLC-MS (ES) [Atlantis T3 C18 (2.1 ꢃ 100 mm,
3 lm), 5–80% gradient of solvent A in 20 min] tR 13.0 [55%, m/z
NBS (282 mg, 1.58 mmol) and TFA (50
ꢂ40 °C cooled solution of the corresponding
lL) were added to a
625.70, (M+1)] and 13.4 [45%, m/z 703.52, (M+1)]. 1H NMR
t
a-amino nitrile 8d–f
(500 MHz, CDCl3) d 0.91, 0.93, 1.06, 1.10 (4s, 36H, Bu), 1.80, 1.95
Table 3
Analytical and spectroscopic data of 10b-bromo-pyrroloimidazoindole derivatives 9–12a
9b
9c
9f
10c
11b
11c
11f
12c
12f
R1
CH3
CH3
H
[(CH2)2]2NBn
tBu
H
H
[(CH2)2]2NBn
CH3
CH3
Br
[(CH2)2]2NBn
tBu
H
Br
[(CH2)2]2NBn
H
R2
tBu
R3
H
H
Br
Br
Br
Config.
2S,10bR,10cR
2S,10bR,10cR
2S,10bR,10cR,4S 2S,10bS,10cS
2S,10bR,10cR
2S,10bR,10cR
2S,10bR,10cR,4S 2S,10bS,10cS
2S,10bS,10cS,4R
Yield (%) 83
55
22
45
68
65
25
35
10
Formula
ES-MS
C16H18BrN3O2 C25H27BrN4O2
C18H22BrN3O2
495
C25H27BrN4O2 C16H17Br2N3O2 C25H26Br2N4O2
C18H21Br2N3O2
575
C25H26Br2N4O2 C18H21Br2N3O2
[M+1]+
364
392
495
441
471
575
471
20
[a
]
ꢂ63.0
ꢂ71.8
ꢂ38.6
ꢂ24.5
ꢂ30
ꢂ60.8
ꢂ42
NDb
+38.6
(c 1.5, MeOH)
3.2
D
(c 1, MeOH)
5.3
(c 1.1, MeOH)
11.8
(c 1.5, MeOH)
2.2
(c 0.9, MeOH) (c 1, MeOH)
(c 1.2, MeOH)
2.7
(c 1.5, MeOH)
2.9
HPLC tR
10.0
(25:75)
14.1
(25:75)
1.8
(50:50)
(A:B)c
(25:75)
(25:75)
(50:50)
(50:50)
(50:50)
(50:50)
1H NMRd
1-H
2-H
7-H
8-H
2.86, 2.94
3.17
7.43
7.33
7.16
7.43
6.02
3.72
1.56
2.82, 3.03
3.17
7.40
7.24–7.34
7.15
7.41
5.99
3.68
1.71–2.77, 3.54,
7.15–7.41
12
2.81,3.07
3.11
7.42
7.32
7.15
7.48
5.89
3.71
1.14
3.31
12
2.81;1.99
3.99
7.20
7.24
6.98
7.40
5.78
2.99
2.84, 2.92
3.15
7.35
7.43
—
7.54
5.99
3.72
1.54
1.26
12
2.80, 3.01
3.15
7.25–7.33
7.42
—
7,53
5.97
3.69
1.70–2.72, 3.53,
7.29–7.39
12
2.73;2.99
3.03
7.37
7.37
—
7.46
5.81
3.65
1.07
3.29
12
2.85,3.37
4.06
7.30–7.34
7.40
—
7.44
5.82
3.17
1.42–2.75,
3.48, 7.32
14
2.80;3.11
4.24
7.42
7.42
—
7.48
5.81
3.36
1.11
3.46
14
9-H
10-H
10c-H
OCH3
R1
1.43–2.90,
3.46, 7.26
12
R2
J1,1
1.27
12.5
J1,2
6, 12.5
5, 12
4,12
0, 9
5, 12
5, 12
4, 12
0, 9
4, 8
13C NMRe
C1
50.3
50.7
47.9
50.1
50.1
50.4
47.8
50.4
48.2
C2
61.7
61.9
66.4
62.9
61.7
61.8
66.3
63.0
68.0
C4
69.0
70.6
81.4
67.8
69.0
70.6
81.4
68.1
77.2
C5
C6a
C7
C8
C9
174.0
143.5
115.1
130.4
125.0
125.3
135.4
61.1
95.2
52.5
25.9
20.7
174.0
142.9
115.4
130.4
125.1
125.2
135.9
61.4
95.5
52.5
29.0, 33.6, 49.1,
49.4
171.8
169.9
114.0
115.5
130.3
125.0
125.1
135.4
62.5
98.7
52.2
27.3, 35.1
—
170.1
175.2
146.8
124.8
130.3
123.8
114.3
135.3
63.0
94.7
51.5
29.4, 34.0,
49.5, 49.7
172.6
174.0
142.7
116.8
133.4
117.3
128.3
137.5
59.9
95.3
52.6
25.9
20.6
174.0
142.1
117.0
133.3
117.3
128.2
138.0
60.1
95.6
52.6
28.9, 33.4, 49.1,
49.4
171.5
169.9
137.7
117.1
128.1
117.4
133.3
137.5
61.2
98.8
52.3
27.3, 35.1
—
169.9
178.9
146.3
128.6
137.8
128.2
116.2
137.8
63.12
95.3
170.5
117.3
128.0
133.2
128.0
133.2
137.9
64.1
99.7
52.0
26.9, 36.2
—
170.9
C10
C10a
C10b
C10c
OCH3
R1
52.2
29.6, 29.9,
49.7, 49.9,
172.9
R2
CO2
171.6
171.4
a
Foams with satisfactory analysis for C, H, N.
Not determined.
b
c
Novapack C18 (3.9 ꢃ 150 mm, 4
l
m). A = CH3CN, B = 0.05% TFA in H2O.
Spectra registered at 300 or 400 MHz in CDCl3, assigned with the help of COSY spectra.
Spectra registered at 75 or 100 MHz in CDCl3, assigned with the help of HSQC and HMBC spectra.
d
e

P. Ventosa-Andrés et al. / Bioorg. Med. Chem. 16 (2008) 9313–9322
9319
(2bs, 2H,
[2d, 2H, J = 7 Hz, CN-CH (3H-indole unit)], 2.96, 3.05 (2m, 4H, b-H),
3.55 [m, 2H, -H (3H-indole unit)], 3.63, 3.67, 3.72, 3.74 (4s, 12H,
a
-NH), 2.80, 3.40 [2m, 4H, 3-H (pyrroloindole)], 2.95, 2.98
4.7. Synthesis of (2S,3aS,8aR)- and (2S,3aR,8aS)-2-
methoxycarbonyl-3a-prenyl-1,2,3,3a,8,8a-
hexahydropyrrolo[2,3-b]indoles (19 and 20)
a
OCH3), 3.95 [dd, 2H, J = 7 and 14 Hz, 2-H (pyrroloindole)], 4.02,
4.20 [2s, 2H, CN-CH (pyrroloindole unit)], 4.50, 4.63 [2d, 2H,
J = 3.5 Hz, 8-NH (pyrroloindole)], 5.49, 5.56 [2d, 2H, J = 3.5 Hz, 8a-
H (pyrroloindole)], 6.72, 6.84 [2s, 2H, 2-H (3H-indole)], 6.96 [t,
1H, J = 7 Hz, 5-H (pyrroloindole)], 7.16 [t, 2H, J = 7 Hz, 5-H (3H-in-
dole)], 7.19, 7.20 [2t, 2H, J = 7 Hz, 6-H (3H-indole)], 7.28, 7.34 [2d,
2H, J = 7 Hz, 4-H (pyrroloindole)], 7.53, 7.72 [2d, 2H, J = 7 Hz, 7-H
(3H-indole)], 7.56 [d, 2H, J = 7 Hz, 4-H (3H-indole)]. 13C NMR
(125 MHz, CDCl3) d 26.0, 26.8, 27.0 [C(CH3)3], 29.0 [Cb (3H-indole
unit)], 34.8, 37.8, 37.2 [C(CH3)3], 40.9, 40.7 [C3 (pyrroloindole)],
52.3, 52.41, 52.0, 52.0 (OCH3), 61.5, 60.5 [CN-CH (3H-indole unit)],
Prenyl bromide (267
vigorously stirred solution of the
0.38 mmol) and magnesium nitrate hexahydrate (489 mg,
1.9 mmol) in acetic acid/sodium acetate buffer (pH 2.9, prepared
from 8 g of sodium acetate, 100 mL of acetic acid, and 20 mL of
l
L, 2.28 mmol) was dropwise added to a
-amino nitrile 8b (108.4 mg,
a
Table 4
Analytical and spectroscopic data of 10b-allyl-pyrroloimidazoindole derivatives 15
and 17a
61.6, 61.6 [C (3H-indole unit)], 64.0, 64.3 [C2 (pyrroloindole)],
a
73.9, 74.2 [C3a (pyrroloindole)], 85.4, 85.8 [C8a (pyrroloindole)],
109.6, 110.3 [C3 (3H-indole)], 111.8, 112.0 [C7 (3H-indole)], 112.3
[C5-Br (pyrroloindole)], 112.7, 113.6 [C7 (pyrroloindole)], 117.1,
117.3, 119.2, 119.3 (CN), 119.2, 119.3 [C4 (3H-indole)], 119.3,
119.7 [C5 (3H-indole)], 121.1 [C5 (pyrroloindole)], 122.1, 122.3
[C6 (3H-indole)], 124.6, 125.1 [C2 (3H-indole)], 125.4, 127.7 [C4
(pyrroloindole)], 129.9 [C3a (3H-indole)], 129.7, 132.0 [C3b (pyrro-
loindole)], 130.8, 133.5 [C6 (pyrroloindole)], 134.6, 134.8 [C7a
(3H-indole)], 147.3, 148.8 [C7a (pyrroloindole)], 172.9, 173.2,
174.1 (CO2).
15b
15c
17b
17c
R1
CH3
CH3
H
[(CH2)2]2NBn
CH3
CH3
Br
[(CH2)2]2NBn
R2
R3
H
Br
Config.
Yield (%)
Formula
2S,10bS,10cR
36
C19H23N3O2
326
2S,10bS,10cR
57
C28H32N4O2
457
2S,10bS,10cR
26
C19H22BrN3O2
404
2S,10bS,10cR
28
C28H31BrN4O2
532
4.4. General procedure for the acid-promoted cyclative
dibromination of ketone-derived
11b,c and 12c
a-amino nitriles. Synthesis of
ES-MS [M+1]+
20
[a]
ꢂ89
ꢂ45
ꢂ145
ꢂ29
D
(c 1, MeOH)
6.2 (25:75)
(c 0.8, MeOH)
7.2 (25:75)
(c 0.8, MeOH)
17.6 (25:75)
(c 0.9, MeOH)
11.7 (25:75)
NBS (846 mg, 4.74 mmol) and TFA (500
ꢂ40 °C cooled solution of the corresponding
l
a
L) were added to a
-amino nitrile 8b,c
HPLC tR (A:B)b
1H NMRc
1-H
2-H
7-H
(1.58 mmol) in CH2Cl2 (5 mL), and the mixture was stirred at that
temperature for 3 h. Afterwards, the reaction mixture was pro-
cessed as above. Significant analytical and spectroscopic data of
the resulting 9,10b-dibromo derivatives (11–12)b,c are summa-
rized in Table 3.
2.22, 2.24
3.24
7.42
7.22
7.09
7.17
5.50
3.69
2.61, 2.78
5.63
2.22, 2.26
3.22
7.38
7.25
7.07
7.14
5.46
3.65
2.64, 2.78
5.58
2.22, 2.23
3.20
7.37
7.27
—
7.34
5.49
3.69
2.75
2.21, 2.3
3.20
7.30–7.34
7.30–7.34
—
7.30–7.34
5.46
3.67
2.64,2.77
5.61
5.11, 5.17
1.71–2.69,
3.52, 7.32
8-H
9-H
10-H
10c-H
OCH3
10-H
20-H
30-H
R1
4.5. Acid-promoted cyclative dibromination of the aldehyde-
derived a-amino nitrile 8f. Synthesis of 11f and 12f
5.62
5.12 5.17
1.53
5.08, 5.15
1.55
1.26
5.05, 5.13
1.69–2.69
3.51, 7.3
NBS (846 mg, 4.74 mmol) and TFA (50
lL) were added to a
R2
1.24
ꢂ40 °C cooled solution of the -amino nitrile 8f (1.58 mmol)
a
in CH2Cl2 (5 mL), and the mixture was stirred at that tempera-
ture for 3 h. Afterwards, the reaction mixture was processed as
above. Significant analytical and spectroscopic data of the result-
ing 9,10b-dibromo derivatives 11f and 12f are summarized in
Table 3.
13C NMRd
C1
C2
C4
C5
C6a
C7
C8
C9
44.8
61.8
69.1
45.3
61.8
70.6
44.7
61.7
69.0
49.3
61.8
70.6
174.0
144.8
114.5
128.6
124.2
123.4
136.8
52.4
90.1
52.3
42.5
133.5
119.2
26.0
20.9
173.2
174.0
144.4
114.8
127.0
124.2
123.3
137.2
53.1
90.4
52.3
42.4
133.67
119.0
29.3, 30.9,
49.4, 49.7
173.5
175.1
144.1
116.1
131.5
116.5
126.4
139.1
53.3
90.2
52.4
42.2
132.9
119.7
25.9
20.9
172.9
175.0
143.7
116.6
133.1
116.5
127.1
139.5
53.2
90.5
53.2
49.3
131.5
119.5
29.7, 33.4,
42.1, 45.2
173.2
4.6. General procedure for the exchange of 10b-bromo to 10b-
allyl. Synthesis of (15–18)b,c
C10
C10a
C10b
C10c
OCH3
AIBN (115 mg, 0.7 mmol) and allyltributyltin (445 lL, 2 mmol)
were added to a solution of the corresponding 10b-bromo-pyrrol-
oimidazoindole 9b,c and 11b,c (1 mmol) in xylene (5 mL), and the
mixture was stirred under reflux for 2 h. Afterwards, the solvent
was removed under reduced pressure and the residue was dis-
solved in CH3CN (10 mL). This solution was washed with hexane
(2 ꢃ 20 mL). The CH3CN phase was evaporated to dryness and
the residue was purified by circular chromatography, using 15–
45% gradient of EtOAc in hexane as eluant, to give the 10b-allyl
derivatives 15b,c and 17b,c (foams), along with 10b-unsubstituted
analogues 16b,c22 and 18b,c. Most significant analytical and spec-
troscopic data of allyl derivatives 15b,c and 17b,c are shown in Ta-
ble 4.
0
C1
0
C2
0
C3
R1
R2
CO2
a
Foams with satisfactory analysis for C, H, N.
b
c
Novapack C18 (3.9 ꢃ 150 mm, 4
lm). A = CH3CN, B = 0.05% TFA in H2O.
Spectra registered at 300 or 400 MHz in CDCl3, assigned with the help of COSY
spectra.
d
Spectra registered at 75 or 100 MHz in CDCl3, assigned with the help of HSQC
and HMBC spectra.

9320
P. Ventosa-Andrés et al. / Bioorg. Med. Chem. 16 (2008) 9313–9322
H2O, 15 mL) under argon. After 2 h of stirring at room temperature,
the reaction mixture was sequentially neutralized with Na2CO3
and extracted with CH2Cl2 (20 mL). The organic extracts were suc-
cessively washed with H2O (5 mL) and brine (5 mL), dried over
Na2SO4, and evaporated to dryness. The residue was purified by
circular chromatography, using 12–50% gradient of EtOAc in hex-
ane as eluant, to give 3a-prenyl-pyrroloindole derivatives 19
(10.9 mg, 10%) and 20 (6.1 mg, 5%) as foams.
Table 5
Analytical and spectroscopic data of 10b-prenyl-pyrroloimidazoindole derivatives 21-23a
21b
21d
21e
21f
22d
22f
23f
R1
CH3
CH3
CH2Ph
H
Ph
H
tBu
H
CH2Ph
H
tBu
H
tBu
H
R2
Config.
2S,10bS,10cR
2S,10bS,10cR,4S
2S,10bR,10cS,4R
22
C26H29N3O2
416
2S,10bS,10cR,4S
2S,10bR,10cS,4R
11
C25H27N3O2
402
2S,10bS,10cR,4S
2S,10bR,10cS,4S
22
C26H29N3O2
416
Yield (%)
Formula
ES-MS
41
10
24
C21H27N3O2
354
C23H31N3O2
382
C23H31N3O2
382
C23H31N3O2
[M+1]+
20
[a
]
ꢂ131.7
ꢂ70.0
ꢂ88.6
ꢂ88
+65.0
(c 0.6, MeOH)
D
(c 0.6,
(c 1, MeOH)
(c 1.5, MeOH)
(c 1.5, MeOH
MeOH)
HPLC tR
20.8 (25:75)
5.7 (50:50)
10.1 (40:60)
2.3 (50:50)
5.5 (50:50)
27.9 (50:50)
24.7 (50:50)
(A:B)b
1H NMRc
1-H
2-H
7-H
8-H
2.24, 2.28
3.26
7.18
7.33
7.14
7.15
5.48
3.70
2.64
4.95
1.62,1.65
1.61
1.25
11.5
2.19, 2.34
3.17
7.45
7.25
7.07
7.15
5.17
3.56
2.30, 2.38
3.42
7.53
7.30
7.12
7.19
5.28
3.75
2.56
2.16, 2.35
3.17
7.49
7.26
7.07
7.14
5.29
3.65
2.58
5.00
1.61,1.66
1.14
3.26
12
2.23, 2.39
3.90
7.33
7.13
6.92
7.01
5.02
3.03
2.21, 2.43
4.04
7.41
7.16
6.94
7.09
5.13
3.10
2.44
4.98
1.53,1.61
1.04
3.28
13.5
2.41
3.86
7.09
6.98
6.57
6.67
4.71
3.25
2.51
5.15
1.56,1.71
3.64
1.06
13
9-H
10-H
10c-H
OCH3
10-H
20-H
40-H
R1
2.59
4.98
2.42
4.91
4.95
1.62, 1.67
3.06, 3.22, 7.14–7.33
3.90
12
1.55, 1.62
7.65, 7.41, 7.34
4.91
1.50;1.60
2.94, 3.02, 720–7.34
4.07
13.5
R2
J1,1
12
J1,2
6, 11.5
6, 12
6, 12
6, 12
1.5, 8.5
3, 9
3, 7
13C NMRd
C1
44.2
42.6
42.9
42.2
42.7
42.0
40.8
C2
62.2
64.7
64.3
66.7
66.1
68.6
65.1
C4
68.8
73.5
74.9
81.7
69.1
78.1
65.5
C5
C6a
C7
C8
C9
C10
C10a
C10b
C10c
OCH3
174.0
144.0
123.8
129.5
123.8
123.8
138.3
53.1
171.6
144.9
114.2
128.2
124.1
123.2
137.5
52.0
170.2
144.6
114.5
128.5
124.4
123.4
137.7
53.7
171.3
145.4
114.7
128.3
123.1
124.1
137.5
54.4
172.5
146.3
113.4
128.4
123.5
123.3
137.6
54.9
171.7
145.8
115.4
128.9
123.4
123.5
138.6
55.1
171.7
149.7
125.5
128.3
109.2
119.5
135.1
57.6
89.4
52.5
92.6
54.0
92.7
52.2
94.1
51.9
93.2
51.4
96.5
51.7
90.8
51.9
0
C1
36.6
26.5
36.5
37.1
37.1
36.5
37.0
0
C2
118.4
136.0
18.4, 25.9
21.0
119.1
135.1
119.0
136.2
18.2, 25.9
127.6, 128.1, 128.8,
135.1
—
117.3
135.1
25.9, 18.2
24.6, 34.9
119.0
135.2
18.2;256.0
—
118.7
134.9
18.0, 25.9
—
118.6
135.8
18.2, 25.9
27.3, 36.5
0
C3
0
C4
18.3, 25.9
28.2, 126.7, 128.4, 129.5,
137.5
—
R1
R2
25.2
—
38.7, 126.6, 128.4,129.5,
138.1
173.8
26.3, 36.7
173.7
—
CO2
172.5
171.6
171.9
171.9
172.1
a
Foams with satisfactory analysis for C, H, N.
b
d
e
Novapack C18 (3.9 ꢃ 150 mm, 4
lm). A = CH3CN, B = 0.05% TFA in H2O.
Spectra registered at 300 or 400 MHz in CDCl3, assigned with the help of COSY spectra.
Spectra registered at 75 or 100 MHz in CDCl3, assigned with the help of HSQC and HMBC spectra.

P. Ventosa-Andrés et al. / Bioorg. Med. Chem. 16 (2008) 9313–9322
9321
4.7.1. (2S,3aS,8aR)-2-Methoxycarbonyl-3a-prenyl-1,2,3,3a,8,8a-
hexahydropyrrolo[2,3-b]indole (19)
ubilized with Tris buffer. Optical densities were read on an auto-
mated spectrophotometer plate reader at a single wavelength of
490 nm. Data analysis was automatically generated by the high
throughput screening LIMS implemented at the laboratory. The
three response parameters GI50 (50% cell growth inhibition), LC50
(50% lethal concentration), and TGI (total growth inhibition) were
extracted from concentration-response curves by linear interpola-
tion, according to the National Cancer Institute (NCI) protocols.32
HPLC [Novapak C18 (3.9 ꢃ 150 mm, 4
lm), (A:B, 50:50)] tR
2.1 min. 1H NMR (400 MHz, CDCl3) d 1.55 (s, 3H, CH3), 1.68 (d,
3H, CH3), 1.74 (bs, 1H, 1-NH), 2.01 (dd, 1H, J = 11 and 12 Hz, 3-
H), 2.38 (dd, 1H, J = 6 and 12 Hz, 3-H), 2.44 (m, 2H, 10-H), 3.71
(dd, 1H, J = 6 and 11, 2-H), 3.71 (s, 3H, OCH3), 4.91 (s, 1H, 8a-H),
5.13 (m, 1H, 20-H), 6.57 (dd, 1H, J = 8 Hz, 7-H), 6.73 (t, 1H,
J = 7.5 Hz, 5-H), 7.04 (dd, 1H, J = 7.5 and 8 Hz, 6-H), 7.04 (d, 1H,
J = 7.5 Hz, 4-H). 13C NMR (100 MHz, CDCl3) d 18.0 and 26.0 (CH3),
4.10. Evaluation of EGFR signalling pathway inhibition
0
36.9 (C1 ), 44.1 (C2), 52.1 (OCH3), 58.7 (C3a), 59.4 (C3), 82.1 (C8a),
0
109.0 (C7), 118.8 (C5), 119.9 (C2 ), 123.6 (C6), 128.1 (C4), 133.1
HeLa cells stably transfected with the AP1-luc reporter gene
[maintained in Dulbecco’s modified Eagle’s medium (DMEM), sup-
(C3b), 134.6 (C3 ), 149.9 (C7a), 174.3 (CO2). ES-MS m/z 286 [M+1]+.
0
Anal. Calcd. for C17H22N2O2: C, 71.30; H, 7.74; N, 9.78. Found: C,
71.54; H, 7.89; N, 9.57.
plemented with 10% fetal bovine serum (FBS), 1% L-glutamine and
100 U/mL of penicillin and streptomycin at 37 °C and 5% of CO2]
were plated at a density of 20,000 cells/well in white opaque 96-
well microplates and let to set for 24 h. Afterwards, prior to stim-
ulation with EGF (25 ng/mL), cells were treated with vehicle alone
(1% DMSO), test compounds at five different concentrations (10, 2,
4.7.2. (2S,3aR,8aS)-2-Methoxycarbonyl-3a-prenyl-1,2,3,3a,8,8a-
hexahydropyrrolo[2,3-b]indole (20)
HPLC [Novapak C18 (3.9 ꢃ 150 mm, 4
lm), (A:B, 50:50)] tR
1.9 min. 1H NMR (400 MHz, CDCl3) d 1.55 (s, 3H, CH3), 1.68 (d,
3H, CH3), 1.74 (br s, 1H, 1-NH), 2.37 (dd, 1H, J = 8 and 13 Hz,
3-H), 2.48 (dd, 1H, J = 4 and 13 Hz, 3-H), 2.53 (m, 2H, 10-H), 3.34
(s, 3H, OCH3), 3.88 (dd, 1H, J = 4 and 8, 2-H), 4.86 (s, 1H, 8a-H),
5.71 (m, 1H, 20-H), 6.55 (dd, 1H, J = 7Hz, 7-H), 6.71 (t, 1H,
J = 7 Hz, 5-H), 7.02 (m, 2H,4-H and 6-H). 13C NMR (100 MHz, CDCl3)
0.4, 0.08, and 0.016
To evaluate first the potential cytotoxic effects of compounds, cells
were incubated with calcein-AM (0.1 M) for 30 min at 37 °C, and
lg/mL) or the EGFR inhibitor AG-1478 (20 lM).
l
the cell survival was quantified using a microplate fluorometer(ex-
485 nm/em-535 nm, green fluorescence). Cells were washed with
phosphate buffered saline (PBS) to remove culture medium and ex-
cess of calcein-AM. Then, luciferase activity was assessed in a
microplate luminometer, using the Promega Bright-Glo Luciferase
Assay System. Results were expressed as percentage of control val-
ues (survival and reporter activity).
0
d 18.0 and 26.0 (CH3), 41.5 (C1 ), 43.0 (C2), 51.9 (OCH3), 57.1 (C3a),
0
50.1 (C3), 82.4 (C8a), 109.6 (C7), 118.1 (C5), 118.9 (C2 ), 123.8 (C6),
0
128.3 (C4), 132.6 (C3b), 134.0 (C3 ), 149.5 (C7a), 173.9 (CO2).
ES-MS m/z 286 [M+1]+.
4.8. General procedure of cyclative prenylation of tryptophan-
4.11. Evaluation of inhibition of HIF-1a
derived a-amino nitriles. Synthesis of 21(b,d–f), 22d and 22f
HeLa cells stably transfected with the HIF1-luc reporter gene
(maintained in DMEM, supplemented with 10% FBS, 1% -gluta-
Prenyl bromide (267
lL, 2.28 mmol) was dropwise added to a
L
vigorously stirred suspension of the corresponding
a
-amino nitrile
mine and 100 U/mL of penicillin and streptomycin at 37 °C and
5% of CO2) were plated at a density of 20,000 cells/well in white
opaque 96-well microplates and let to set for 24 h. Established cul-
tures were pre-treated with vehicle alone (1% DMSO) or test com-
pounds at the defined final concentrations for 30 min and then
8b–f (0.38 mmol) dissolved in CH3CN (2 mL) in a solution of mag-
nesium nitrate hexahydrate (489 mg, 1.9 mmol) in acetic acid/so-
dium acetate buffer (pH 2.9, prepared from 8 g of sodium
acetate, 100 mL of acetic acid, and 20 mL of H2O, 15 mL) under ar-
gon. After 2 h of stirring at room temperature, the reaction mixture
was sequentially neutralized with Na2CO3 and extracted with
CH2Cl2 (20 mL). The organic extracts were successively washed
with H2O (5 mL) and brine (5 mL), dried over Na2SO4, and evapo-
rated to dryness. The residue was purified by circular chromatog-
raphy, using 12–50% gradient of EtOAc in hexane as eluant, to
give the corresponding 10b-prenyl-pyrroloimidazoindoles 21–22
as foams, whose significant analytical and spectroscopic data are
summarized in Table 5.
treated with 50
bated for 24 h. To evaluate first the potential cytotoxic effects of
the compounds, cells were incubated with 0.1 M calcein-AM for
lM deferoxamine (hypoxia mimetic) and incu-
l
30 min at 37 °C, and cell survival quantified using a microplate
fluorometer (ex-485 nm/em-535 nm, green fluorescence). Cells
were washed in PBS to remove culture medium and excess calce-
in-AM, and then, luciferase activity was assessed in a microplate
luminometer using the Promega Bright-Glo Luciferase Assay Sys-
tem. Results were expressed as percentage of control values (sur-
vival and reporter activity).
4.9. Evaluation of cytotoxicity
Acknowledgments
A colorimetric assay, using the sulforhodamine B (SRB) reaction,
was adapted for a quantitative measurement of cell growth and
viability, following the technique described by Skehan et al.31 Cells
(MDA-MB-231, A549 and HT-29) were seeded in 96-well microti-
This work was supported by CICYT (SAF2006-01205). P. Vento-
sa-Andrés and J. A. González-Vera held a postgraduate I3P fellow-
ship from the CSIC. The antitumoral evaluation was carried out by
Pharma Mar, S. A.
ter plates, at 5 ꢃ 103 cells per well in aliquots of 195
lL of RPMI
medium, and they were allowed to attach to the plate surface by
growing in drug free medium for 18 h. Afterwards, samples were
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