Thiopyrano[2,3-e]indol-2-ones: Angelicin heteroanalogues with potent photoantiproliferative activity

Article abstract of DOI:10.1016/j.bmc.2008.10.002

A new class of compounds, the thiopyrano[2,3-e]indol-2-ones, bioisosters of the angular furocoumarin angelicin, was synthesized with the aim of obtaining new photochemotherapeutic agents. In particular 7,8-dimethyl-thiopyranoindolone 6c s showed a remarka

Full text of DOI:10.1016/j.bmc.2008.10.002

Bioorganic & Medicinal Chemistry 16 (2008) 9668–9683
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Thiopyrano[2,3-e]indol-2-ones: Angelicin heteroanalogues with potent
photoantiproliferative activity
Paola Barraja a, Patrizia Diana a, Alessandra Montalbano a, Anna Carbone a, Girolamo Cirrincione a,
,
*
Giampietro Viola b, Alessia Salvador b, Daniela Vedaldi b, Francesco Dall’Acqua b
a Dipartimento Farmacochimico, Tossicologico e Biologico Università degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy
b Dipartimento di Scienze Farmaceutiche Università degli Studi di Padova, Via Marzolo 5, 35131 Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of compounds, the thiopyrano[2,3-e]indol-2-ones, bioisosters of the angular furocoumarin
angelicin, was synthesized with the aim of obtaining new photochemotherapeutic agents. In particular
7,8-dimethyl-thiopyranoindolone 6c s showed a remarkable phototoxicity and a great dose UVA depen-
dence reaching IC50 values at submicromolar level. This latter photoinduced a massive apoptosis and a
remarkable photodamage to lipids and proteins. Although it did not intercalate DNA, it was able to cause
photooxidation of DNA bases.
Received 17 April 2008
Revised 26 September 2008
Accepted 2 October 2008
Available online 5 October 2008
Keywords:
Thiopyrano[2,3-e]indol-2-ones
Angelicin
Ó 2008 Elsevier Ltd. All rights reserved.
Antiproliferative activity
Photochemotherapeutic agents
Apoptosis
1. Introduction
Another promising approach, mainly aimed to dissociate unde-
siderable side effects from the therapeutic ones, was the synthesis
Derivatives of psoralen 1 are naturally occurring or synthetic
aromatic compounds deriving from the condensation of a couma-
rin nucleus with a furan ring nowadays used in the treatment of
the human skin diseases such as psoriasis, T-cell lymphoma (Cuta-
neous T-cell lymphoma, CTCL) and vitiligo, in combination with
UVA radiation, commonly referred as PUVA (psoralen plus UVA)
therapy.1 These tricyclic aromatic compounds possess a planar
structure that allows their intercalation between nucleic acid base
pairs. Upon UVA irradiation, the intercalated complex is activated,
thus generating photoadducts with pyrimidine bases in cellular
DNA.2 The psoralen monoadducts formed can further photochem-
ically react with a pyrimidine base on the complementary strand of
the DNA thus leading to interstrand cross links (ICL), which are be-
lieved to be the primary cause of photoinduced cell killing.
To improve the photobinding capability and at the same time to
decrease the phototoxicity of photochemotherapeutic agents were
synthesized derivatives of angelicin 2, a natural compound, angu-
lar isomer of psoralen, whose geometry does not permit the simul-
taneous alignment of the two photoreactive sites with pyrimidine
bases, thus allowing only monofunctional photobinding.3
of heteroanalogues of angelicin. Thus, sulphur or nitrogen isosters
such as thioangelicins, thienocoumarins, pyrrolocoumarins, and
furoquinolinones were studied demonstrating in some cases im-
proved interaction with DNA both in the dark and under UVA light
in comparison to angelicin itself.4 We have recently reported the
synthesis and the photochemotherapeutic activity of the new ring
system pyrrolo[2,3-h]quinolin-2-one of type 3 or 4, an angelicin
heteroanalogue, in which nitrogen atoms replaced both oxygens
on the furan and the pyrone ring.5
These compounds were highly phototoxic on a panel of human
tumor cell lines with GI50 in the micromolar and submicromolar le-
vel, being in some instances more cytotoxic than 8-methoxypsora-
len (8-MOP), 5-methoxypsoralen (5-MOP) and angelicin used as
reference drugs. However, studies of linear dichroism (LD) strongly
suggested that the new derivatives did not efficaciously interact
with DNA, thus indicating a different mechanism from that of
furocoumarins.5
Continuing our studies in the search for new angelicin hetero-
analogues and considering our previous satisfactory results on pyr-
roloquinolinones, we focused our attention on the synthesis on the
thiopyrano[2,3-e]indol-2-one ring system of type 5 or 6, respec-
tively (Chart 1). Now, we wish to report the synthesis of such ring
system along with the results of their photobiological activity and
studies directed to elucidate the mode of action.
* Corresponding author. Tel.: +39 916161606; fax: +39 916169999.
E-mail address: gcirrinc@unipa.it (G. Cirrincione).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.002

P. Barraja et al. / Bioorg. Med. Chem. 16 (2008) 9668–9683
9669
tetrahydroindole 8g.6 Alkaline hydrolysis of the ester functionality
of this latter afforded 8h which underwent decarboxylation to give
the 3-methyl substituted tetrahydroindole 8i. Alkylation of the in-
dole nitrogen of derivatives 8g and 8i with iodomethane allowed
the isolation of 8j and 8k, respectively. The synthetic pathway pro-
ceeded through the acylation of the position five of the tetrahydr-
oindoles 8 which was performed by using the Vilsmeier–Haack
reagent (DMF/POCl3). This step proceeded very easily for tetra-
hydroindoles 8b–d,f,j provided the strict control of the tempera-
ture (0 °C) and the very short reaction time (3 min) to avoid tarry
insoluble materials as the corresponding chloroformylated deriva-
tives 9 are unstable and, once isolated (yields 60–95%), have to be
stored at low temperatures (ꢀ18 °C). N-Unsubstituted tetrahydro-
indole 8a, due to the poor solubility in the solvent mixture used for
the chloroformylation, did not succeed in reacting with the Vilsme-
ier–Haack reagent, producing the corresponding chloro-aldehyde
9a in very low yields (3%), and the starting material was recovered
almost quantitatively from the reaction mixture even after pro-
longed reaction time. Although the BOC protective group of 8e in-
creased the solubility of the compound, the corresponding
chloroformylated product 9e was isolated only in 10% yield since
it was very unstable and difficult to handle. In the case of the N-
methyl tetrahydroindole 8c, along with the desired chloroformy-
lated compound 9c, was isolated the chloro-bisformylated deriva-
tive 10 in 20% yield. Instead, in the case of derivative 8k, the
bisformylated compound 11 was the only reaction product isolated
in 90% yield. The formation of this product could not be avoided by
decreasing the reaction temperature (ꢀ40 °C) and/or the reaction
time (1–2 min). When stoichiometric amount of Vilsmeier–Haack
reagent was used, the reaction did not proceed and when prolong-
ing the heating, tarry material was formed and only the 2-formyl
derivative 8l was isolated from the reaction mixture (40%)
(scheme 1). The next step in the synthesis was the nucleophilic
substitution of the chlorine atom of the chloroaldehydes 9b–d,f,j
O
O
O
O
O
O
N
1
2
O
O
X
H
N
X
S
X
N
X
3,4
5,6
X-X = CH2-CH2 or CH=CH
Chart 1.
2. Results and discussion
2.1. Chemistry
The synthetic route to thiopyrano[2,3-e]indol-2-ones started
from 2-acetonyl-1,3-cyclohexanedione 7a which was reacted, in
acetic acid, with ammonium acetate to get the 1,5,6,7-tetrahydro-
indol-4-one 8a or with suitable primary amines to obtain the N-
substituted tetrahydroindolones 8b–d.5b Derivatives 8e and 8f
were obtained by reaction of the N-unsubstituted derivative 8a
with t-butyl dicarbonate or benzensulfonyl chloride, respectively.
To investigate the influence on the biological activity of the methyl
group in the eight position of the tricyclic ring we thought to
switch it to the nine position. Thus, 1,3-cyclohexanedione 7b was
subjected to Knorr reductive condensation by reaction with diethyl
hydroxyl iminomalonate in the presence of zinc dust to give the
O
Me
O
O
O
Me
Me
Me
v
viii
vi
CO2H
CHO
N
N
H
N
Me
N
Me
H
8h
8i
8k
8l
vii
8g, iv
O
O
Cl
O
R2
Me
R
OHC
7a, i
7b, ii
8a, iii
R1
CHO
Me
N
R
N
Me
N
O
R
7a,b
11
8a-d,g
8e-f
a R=CH2Ac
e R=BOC
b R=H
f R=SO2Ph
8g, vi
vii
8c
O
Cl
Cl
Me
R2
CHO
Me
OHC
OHC
vii
CO2Et
R1
N
Me
N
R
N
Me
8j
9a-f,j
10
a R=R2=H, R1=Me; b R=Ph, R1=Me, R2=H; c R=R1=Me, R2=H; d R=Bn, R1=Me, R2=H; e R=BOC, R1=Me, R2=H;
f R=SO2Ph, R1=Me, R2=H; g R=H, R1=CO2Et, R2=Me; j R=R2=Me, R1=CO2Et.
Scheme 1. Reagents and conditions: (i) AcONH4 or RNH2, AcOH, 60 °C; (ii) (CO2Et)2C@NOH/Zn, AcOH, reflux; (iii) compound 8e: t-Butyl dicarbonate, DMAP, benzene/DMF,
reflux; compound 8f benzensulfonylchloride, NaH, DMF, r.t.;(iv) NaOH, EtOH, reflux; (v) HCl, EtOH, r.t.; (vi) NaH, THF, MeI, rt; (vii) DMF/POCl3, DCM, 0 °C then reflux; (viii)
DMF/POCl3, DCM, ꢀ40 °C.

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P. Barraja et al. / Bioorg. Med. Chem. 16 (2008) 9668–9683
SEt
SEt
R2
R2
OHC
OHC
i
ii
9b-f,j, 11
R1
R1
N
R
N
R
12b-d,f,j,l
13b-d,f
iii
iii
O
O
X
O
HO
EtO
S
SEt
X
R2
SEt
X
R2
R
R2
v
iv
R1
R1
R1
X
N
X
N
N
X
R
R
5b-d,f (X-X=CH2-CH2)
6b,c (X-X=CH=CH)
16b-d,f,j,n (X-X=CH2-CH2)
17b-d,f (X-X=CH=CH)
14b-d,f,j,m (X-X=CH2-CH2)
15b-d,f (X-X=CH=CH)
a R=R2=H, R1=Me; b R=Ph, R1=Me, R2=H; c R=R1=Me, R2=H; d R=Bn, R1=Me, R2=H; e R=BOC, R1=Me, R2=H; f
R=SO2Ph, R1=Me, R2=H; j R=R2=Me, R1=CO2Et; l R=R2=Me, R1=CHO; m R=R2=Me, R1=CH=CHCO2Et; n R=R2=Me,
R1=CHCHCO2H.
Scheme 2. Reagents and conditions: (i) HSEt, K2CO3, DMF, r.t.; (ii) DDQ, benzene, reflux; (iii) (EtO)2P(O)CH2O2Et/t-BuOK, DMF, r.t then 50 °C; (iv) KOH, EtOH 50%, reflux; (v)
PPA,
D
.
and 11 which afforded the thioethers 12b–d,f,j,l in excellent yields
(80–95%), upon reaction with the ethanthiolate anion, generated
in situ by using ethanthiol and potassium carbonate as the base.
Oxidation with DDQ of thioethers 12b–d,f gave good yields (65–
85%) of the corresponding stable aromatic aldehydes 13b–d,f.
Transformation of the thioethers 12b–d,f,j,l and 13b–d,f into the
vinylogous esters was attempted, using the Wittig–Horner reaction
with triethyl phosphonoacetate and barium hydroxide catalyst (C-
200) under solid–liquid phase-transfer conditions.7 However, un-
der these conditions, our substrates reacted very slowly and reac-
tions did not reach completeness. Best results were achieved with
the Wittig–Horner reaction, whose anion was generated in situ in
the presence of t-BuOK as base in anhydrous dimethylformamide.
Cooling the reaction mixture during the addition of triethyl phos-
phonoacetate was crucial to avoid self-condensation products of
the generated anion. The esters 14b–d,f,j,m (75–92%) and 15b–
d,f (70–96%) were obtained in good to excellent yields and easily
hydrolyzed to the corresponding acids 16b–d,f,j,n (72–93%) and
17b–d,f (65–93%), respectively. The first attempt to cyclize the
lengths of the compounds exhibited (Table 1) a remarkable batho-
chromic shift with respect to angelicin (299 nm) as reference
compound. The relative fluorescence quantum yields were mea-
sured at 298 K.9 As it can be observed from Table 1, all derivatives
showed fluorescence quantum yields values similar to that of
angelicin. Partition coefficients of the test compounds were deter-
mined by a computational method.10 The results indicated that the
thiopyrano were highly hydrophobic in comparison to angelicin.
2.2.2. Phototoxicity
The phototoxicity of test compounds was investigated on a pa-
nel of cultured cell lines of human tumor such as HL-60 (promye-
locityc leukaemia), Jurkat (T cells leukaemia), HT-1080
(fibrosarcoma) and LoVo (intestinal adenocarcinoma). We also
used an immortalized cell line of human keratinocytes NCTC-
2544 for comparison. Table 2 shows the extent of cell survival ex-
pressed as IC50, which is the concentration (lM) that induces 50%
of the inhibition of cell growth, after irradiation at different UVA
doses (2.5 and 3.75 J cmꢀ2). Control experiments with UVA light
or drugs alone were carried out without significant cytotoxic ef-
fects (data not shown). It can be noted that the compounds exhib-
ited different values of IC50, according to the substitution pattern,
and generally a remarkable UVA dose -dependence.
Almost all the compounds inhibited the proliferation of tumor
cells at submicromolar/micromolar concentration. The most inter-
esting compound was 6c whose IC50 values were comparable, if not
lower, than 8-methoxypsoralen (8-MOP) and Angelicin (ANG), ta-
ken as reference compounds. The most active compound 6c is a
fully aromatic derivative and this characteristic seems to be impor-
tant for the photoactivity since the corresponding dehydroderiva-
tive 5c is devoid of any effect. It is interesting to note that
among the thiopyrano indoles the most active compounds were
the aromatic derivatives at variance with the pyrroloquinolinones
series in which the aromatic derivative was devoid of activity.
a–b unsaturated acids 16 and 17 was performed using trimethyl-
silyl polyphosphate prepared from P2O5 and hesamethyldisiloxane
in chloroform according to the Yamamoto procedure.8 However,
such reagent was unsuitable for our purpose giving tarry reaction
mixtures from which we were not able to isolate the desired tricy-
clic compounds. When the dihydro acids 16b–d,f,j,n were heated
at temperatures ranging from 70 to 130 °C in PPA, cyclization oc-
curred, with the exception of derivative 16j,n allowing thus isola-
tion of the corresponding tetrahydro thiopyrano indoles 5b–d,f
(30–40%). Instead, in the case of the aromatic acids 17 only thio-
pyrano indoles 6b,c (30–32%) were obtained (Scheme 2). Deriva-
tives 6d,f could not be isolated also using ZnCl2 as Lewis acid to
promote the desired ring closure. Attempts of direct oxidation of
the polycondensed system 5 with DDQ in dichloromethane or ben-
zene to get derivatives 6 were not successful.
2.2. Biology
2.2.3. Effect of scavengers on thiopyrano indole 6c induced
phototoxicity
2.2.1. Physico-chemical properties
The test compounds represent isosters of the typical tricyclic
system of angelicin. Both absorption and emission maxima wave-
In an attempt to evaluate the possible mechanism of action of
these derivatives, we carried out a series of experiments, in which
various scavengers were included in the LoVo cell cultures during

P. Barraja et al. / Bioorg. Med. Chem. 16 (2008) 9668–9683
9671
Table 1
Physico-chemical parameters for thiopyrano derivatives 5b–d,f and 6b,c.
*
a
c
Cpd
kmax.absorba
e
(Mꢀ1 cmꢀ1
)
kmax.absorbb (nm)
kexc(nm)
kmax emissb (nm)
UF
cLogPd
4b
4c
4d
4f
6b
6c
ANG
401
382
411
395
348
353
306
19,263
11,450
15,182
13,744
9693
407
414
414
392
354
362
299
367
407
370
345
390
300
343
479
458
477
417
424, 447, 484
447
413
3.5 ꢁ 10ꢀ2
+4.69
+2.82
+4.33
+3.88
+5.48
+3.26
+2.08
n.d.e
3.2 ꢁ 10ꢀ2
n.d.
<10ꢀ3
7849
14,602
3.1 ꢁ 10ꢀ2
1.8 ꢁ 10ꢀ2
Cpd, compound; absorb, absorbance; emiss, emission.
a
Measured in dimethyl sulfoxide in a concentration range of 5–20
Measured in phosphate buffer 10 mM pH 7.2.
Relative fluorescence quantum yield determined in 0.1 N H2SO4 as described in Ref. 9.
Calculated as described in Ref. 10.
lM.
b
c
d
e
n.d., not determined.
Table 2
Photocytotoxicity of test compounds against different human cell lines.
Compound
(IC50
HL-60b
, l
M)a
Jurkat
HT-1080
2.5 J/cm2
LoVo
NCTC-2544
2.5 J/cm2c
3.75 J/cm2
2.5 J/cm2
3.75 J/cm2
3.75 J/cm2
2.5 J/cm2
3.75 J/cm2
2.5 J/cm2
3.75 J/cm2
5b
5c
6.2 0.6
>10
2.7 0.3
>10
4.2 0.4
>10
3.5 0.4
>10
11.0 0.1
>20
10.5 0.1
>20
>20
>20
12.3 0.2
>20
>20
>20
>20
>20
5d
5f
6b
6c
ANG
8-MOP
4.0 0.4
>10
2.6 0.3
0.6 0.06
1.2 0.1
1.4 0.2
2.7 0.3
>10
1.3 0.2
0.3 0.1
0.9 0.2
1.2 0.4
4.0 0.4
7.3 0.7
2.5 0.3
0.5 0.03
0.9 0.2
1.2 0.3
2.2 0.3
5.0 0.4
3.5 0.4
0.2 0.01
n.d.d
11.2 0.2
>20
5.4 0.5
0.9 0.1
15.7 1.9
7.8 0.7
9.8 0.7
>20
2.0 0.2
0.6 0.03
2.6 0.2
1.9 0.2
>20
>20
5.1 0.5
1.0 0.1
1.6 0.2
1.1 0.4
7.2 0.8
>20
4.2 0.4
0.8 0.2
0.9 0.1
0.7 0.1
>20
>20
17.0 1.7
2.1 0.2
4.2 0.5
5.5 0.6
>20
>20
14.8 1.5
1.6 0.2
1.5 0.3
0.9 0.1
n.d.
a
Concentration of compound required to inhibit the cell growth by 50% after 72 h of exposure as determined by MTT assay.
Human cell lines: HL-60 human promyelocityc leukaemia, Jurkat human T-cell leukaemia, HT-1080 fibrosarcoma cells. LoVo intestinal adenocarcinoma, NCTC-2544
b
human keratynocytes.
c
Dose UVA expressed in J cmꢀ2 as measured at 365 nm with a Cole–Parmer radiometer.
n.d., not determined.
d
irradiation using the most active compound 6c. The results of these
studies are presented in Figure 1. We added sodium azide, a triplet
excited state quencher (thus avoiding the formation of singlet oxy-
gen via triplet excited state); superoxide dismutase (SOD), catalase
(CAT) which scavenge OÅꢀ and H2O2, respectively; N-N0-dim-
ethylthiourea (DMTU) and Mannitol (MAN), which scavenge hy-
droxyl radicals (OH?) and the free radical scavengers 2,6-di-t-
butylhydroxyanisole (BHA), tocopherol (TOC) and reduced gluta-
thion (GSH). Scavengers on their own did not have any effect on
cell viability during UVA exposure (data not shown). A strong pro-
tective effect was observed with GSH and BHA, DMTU suggesting
that the mechanism of action of thiopyrano indole 6c was mainly
mediated by free radicals and in particular OHÅ radicals. No protec-
tive effects were observed with NaN3 indicating that singlet oxy-
gen could not been involved in the photocytotoxic activity of the
test compound. Moreover, a modest protection was observed with
SOD, CAT and TOC.
2.2.4. Externalization of phosphatidylserine
With the purpose to investigate the mode of cell death (apopto-
sis or necrosis), we carried out various experiments irradiating the
cells in the presence of 6c since this compound alone in dark has
showed no activity. Apoptosis is characterized by a variety of mor-
phological characteristics. Changes in the plasma membrane are
one of the earliest of these features. In apoptotic cells, the mem-
brane phospholipid phosphatidylserine (PS) is translocated from
the inner to the outer leaflet of the plasma membrane, thereby
exposing PS to the external cellular environment.11 Annexin-V is
a 35–36 kDa Ca2+-dependent phospholipid binding protein which
possesses high affinity for PS, and binds to cells with exposed PS.
We performed a biparametric cytofluorimetric analysis using pro-
pidium iodide (PI) and AnnexinV-FITC, which stain DNA and phos-
phatidylserine (PS) residues, respectively.12 Figure 2 shows the
percentage of Annexin-V positive Jurkat cells after 24 h from UVA
irradiation (2.5 J cmꢀ2) in the presence of 6c at different concentra-
tions. A dose-dependent induction of apoptotic cells was observed
in good agreement with MTT experiments. A small percentage of
necrotic cells (2–3%) was detected only at the highest employed
concentration (data not shown).
80
60
40
20
0
CAT SOD
TOC
NaN DMTU
MAN
GSH
6c BHA
3
Figure. 1. Effect of different scavengers on the phototoxicity induced by com-
pounds 6c in LoVo cells. Cell viability was assayed by MTT test after 72 h from the
2.2.5. Mitochondrial depolarization
As demonstrated above, irradiation of Jurkat cells in the pres-
ence of 6c induced apoptosis, thus this observation prompted us
irradiation (2.5 J cmꢀ2) in the presence of 1.25
l
M 6c and SOD (2000 U I/ml), CAT
(2000 U I/ml), BHA (10ꢀ5 M), Man (10 mM), GSH (1 mM), DMTU (1 mM), NaN3
(10 mM) and Tocopherol (60 M).
l

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P. Barraja et al. / Bioorg. Med. Chem. 16 (2008) 9668–9683
effects of 6c on the production of ROS during apoptosis, we utilized
100
80
60
40
20
0
the fluorescence indicator hydroethidine (HE), whose fluorescence
appeared if reactive oxygen species were generated.17 HE was oxi-
dized by superoxide anion into ethidium bromide, which emitted
red fluorescence. Superoxide is produced by mitochondria due to
a shift from the normal 4-electron reduction of O2 to a 1-electron
reduction when cytochrome c is released from mitochondria. ROS
generation was also measured by the dye 20,70-dichlorodihydroflu-
orescein diacetate (DCFDA), which was oxidized to the fluorescent
compound 20,70-dichlorofluorescein (DCF) by a variety of peroxides
including hydrogen peroxide.17 The results are shown in Figure 4.
It can be noticed that there was a remarkable production of ROS,
as evidenced by the high percentage of HE- and DCFDA-positive
cells, in excellent agreement with mitochondrial membrane
depolarization.
In parallel, we evaluated the damage produced by ROS in mito-
chondria by assessing the oxidation state of cardiolipin, a phospho-
lipid restricted to the inner mitochondrial membrane. We used 10-
N-nonyl acridine orange (NAO), a probe whose fluorescence is
independent from mitochondrial permeability transition.18 The
dye interacts stoechiometrically with intact non-oxidized cardio-
lipin. In good agreement with the production of ROS, treated cells
showed a dramatic increase in the oxidized cardiolipin.
0.625 1.25
2.5
5.0
Dark
UVA
6c [μM]
Figure 2. Percentage of Annexin-V positive cells for 6c after 24 h from the
irradiation. Jurkat cells were irradiated (2.5 J cmꢀ2) in the presence of 6c at the
indicated concentrations and analysed after 24 h by flow cytometry as described in
Section 4.
to investigate the activation of apoptotic process after the treat-
ment with 6c. Impairment in mitochondrial function is an early
event in the executive phase of programmed cell death in different
cell types and appears as the consequence of a preliminary reduc-
tion of the mitochondrial transmembrane potential.13,14 The early
Dwmt disruption would be the result of an opening of mitochon-
2.2.7. Oxidative stress
drial transition pores, and this permeability transition would trig-
ger the release of apoptogenic factors such as Apoptosis Inducing
Factor (AIF) or cytochrome c.
With the aim of assessing the effect of ROS accumulation on cel-
lular redox potential we measured the rate of redox modulator glu-
tathione (GSH) in Jurkat cells irradiated in the presence of 6c at
different concentrations. For this purpose, flow cytometric analysis
was carried out using monobromobimane (mBBr) as specific probe.
We used the lipophilic cation 5,50,6,60-tetrachloro-1,10,3,30-tet-
raethylbenzimidazol-carbocyanine (JC-1)15 to monitor the changes
in
UVA irradiation. Cells were irradiated with the UVA dose of
2.5 J cmꢀ2 and 6c at the concentration of 5
M. Cells were har-
Dwmt induced by the thiopyrano indole 6c in combination with
80
l
vested and analyzed at different times from the irradiation. The re-
HE
sults are presented in Figure 3.
DCFDA
60
A rapid increase of the percentage of cells with a collapsed
Dwmt, can be observed, and this may be a signal of the opening
of the mitochondrial megachannels also called the permeability
transition pores (PTP). These results suggested that mitochondria
were strongly involved in the induction of apotosis photoinduced
by derivative 6c.
40
20
0
2.2.6. Mitochondrial reactive oxygen species production
The mitochondrial membrane depolarization has been associ-
ated with mitochondrial production of reactive oxygen species
(ROS).16 Therefore, we investigated whether ROS production in-
creased after irradiation with the test compounds. To analyze the
Dark
UVA
0.625 1.25
2.5
5.0
6c [μM]
120
100
80
60
40
20
0
UVA alone
6c
100
80
60
40
20
0
Dark
UVA
0.625 1.25
2.5
5.0
6
15
24
6c [μM]
Time (h)
Figure 4. Production of reactive oxygen species (upper panel) and cardiolipin
oxidation (lower panel) in Jurkat cells after 24 h from the irradiation (2.5 J cmꢀ2) at
the indicated concentrations of 6c. Data are represented as means SEM of three
experiments.
Figure 3. Induction of mitochondrial depolarization by 6c (5 lM) at different times
from the irradiation (2.5 J cmꢀ2). Data are represented as means SEM of three
experiments.

P. Barraja et al. / Bioorg. Med. Chem. 16 (2008) 9668–9683
9673
40
100
80
60
40
20
0
30
20
10
0.625 1.25
2.5
5.0
Dark
UVA
0
6c [μM]
UVA
0.625
1.25
2.5
5.0
Dark
Figure 5. Decreased levels of intracellular GSH after 24 h from the irradiation
(2.5 J cmꢀ2) of Jurkat cells in the presence of 6c at the indicated concentrations.
Data are represented as means SEM of three experiments.
6c [μM]
Figure 6. Percentages of cells stained with AO (acridine orange) were reported and
analysed by flow cytometry, after 24 h from the irradiation in the presence of 6c at
the indicated concentrations.
The cell permeable mBBr probe is non fluorescent but forms a fluo-
rescent adduct with GSH (GS-mBBr) in a non enzymatic reaction.19
Cellular appearance of GS-mBBr fluorescence was monitored after
24 h, and results are depicted in Figure 5. The intracellular content
of GSH of Jurkat cells was observed to fall after irradiation in the
presence of 6c in a concentration dependent manner. The reduc-
tion in GSH content is well correlated with the appearance of HE
and DCFDA positive cells after treatment with 6c.
drial apoptotic pathway plays a major role in the cellular death
photoinduced by 6c.
2.2.10. Lipid peroxidation
Being hydrophobic as demonstrated by their partition coeffi-
cients, the thiopyrano indole derivatives may be expected to local-
ize mainly in plasma and/or subcellular membranes, making these
structures particularly sensitive to photodamage.27 The thiobarbi-
turic acid (TBA) assay was used in order to determine whether lipid
peroxidation occurred upon irradiation of Jurkat cells incubated in
the presence of test compound.28 The TBA test was performed on
the supernatant medium and in the cell homogenates 24 h after
irradiation.28 Figure 8 presents the results for 6c as a function of
light doses. Thiobarbituric reactive substances (TBARS) were sig-
nificantly produced when the cells were exposed to the compound
and UVA both in the supernatant and in the cell extract.
2.2.8. Assessment of lysosomal stability
It has been previously shown that mitochondrial and/or lysoso-
mial alterations are involved in cell death caused by many photo-
sensitisers including fluoroquinolones20 and porphyrins.21
Furthermore, in a previous paper, we found out the preferential
disposition of pyrroloquinolinones in lysosomes.5b To investigate
the integrity of lysosomes after irradiation with 6c, we performed
flow cytometric analysis using the fluorescent dye acridine orange
(AO). AO is a lysosomotropic base and a metachromatic fluoro-
chrome exhibiting red fluorescence when highly concentrated, as
in the case of intact lysosomes where AO is retained in its charged
protonated form, and green fluorescence at low concentration as in
damaged lysosomes. In this case, AO relocates to the cytosol where
it is predominantly in the deprotonated form.22,23 The number of
intact lysosomes can be evaluated by assaying red fluorescence
after the AO staining of the cells exposed to the photosensitizer.
As can be observed in Figure 6, a significant extent of lysosomal
damage was photoinduced by 6c 24 h after the irradiation,
although it was lower than the mitochondrial damage described
above. Altogether the results suggest that lysosomes are not pref-
erential sites of photosensitization for this derivative.
2.2.11. Protein photodamage
The photosensitization capacity of 6c towards other compo-
nents of cellular membranes, such as proteins, was estimated by
measuring the photoinduced cross-linking in erythrocyte ghost
proteins.29 Light-induced cross-linking of spectrin, a protein asso-
ciated with the cytoplasmic side of the RBC membrane, was de-
tected by the partial or total disappearance of the two spectrin
bands (220.000 and 245.000 Da) on SDS–PAGE as cross-linked
aggregates were not able to run inside the gel and remained at
the top.
7
6
5
4
3
2
1
0
2.2.9. Caspase-3 assay
Many studies demonstrated that programmed cell death is
associated with activation of the large family of cysteine proteases
(caspases), as key elements involved in the sequence of events that
lead to cell death.24 Caspase-3, in particular, is essential for the
propagation of the apoptotic signal after exposure to many DNA-
damaging agents, such as anticancer drugs, and in most cases of
photodynamic therapies it is activated by the photosensitisers
used.25,26 Therefore, it was interesting to evaluate the effect of 6c
on the activity of caspase-3. Lysates from Jurkat cells were pre-
pared and incubated for 24 h after the irradiation in the presence
of 6c at different concentrations. Then, they were assayed for activ-
ity of caspase-3, using an assay solution in which the tetrapeptide
substrate Ac-DEVD (N-acetyl-Asp-Glu-Val-Asp) was conjugated
with a chromophore (p-NA, p-nitroanilina). The results (Fig. 7)
indicated that 24 h after irradiation, 6c was able to activate cas-
pase-3 in a dose dependent manner, suggesting that the mitochon-
Dark
UVA
0.625 1.25
6c
2.5
[μM]
5.0
Figure 7. Caspase-3 induced activity by 6c. Jurkat cells were exposed to 2.5 J cmꢀ2
of UVA in the presence of increasing concentrations of 6c. After 24 h, cells were
harvested and the lysates were assayed for caspase-3 activity using the caspase-3
substrate Ac-DEVD-pNA. Data are represented as fold increase of activity of the
enzyme in comparison to the non-irradiated control.

9674
P. Barraja et al. / Bioorg. Med. Chem. 16 (2008) 9668–9683
St
0
15 30 45 60 St
4
3
2
1
0
Dark
UVA
6c 10 μM
Aggregates cross-links
Spectrin
Mr (Kda)
220
170
116
76
53
1.25
2.50
3.75
UVA Dose (J cm-2)
3
2
1
0
Dark
UVA
6c 10 μM
100
80
60
40
20
0
UVA alone
6c 10 μM
6c 20 μM
1.25
2.50
3.75
UVA Dose (J cm-2)
30
Time (min)
10
20
40
50
0
60
Figure 8. Lipid peroxidation induced in NCTC-2544 cells after 24 h from the
irradiation at the indicated UVA doses in the presence of 6c at the concentration of
10 lM. The TBA test was performed on the supernatant medium and on the cell
homogenates as described in Section 4. Upper panel: supernatant medium, lower
panel: cell homogenates.
Figure 9. Upper panel: Electrophoretic pattern of the photoinduced cross-link of
spectrin in RBC ghosts irradiated at the indicates times in the presence of 6c at the
concentration of 10 lM. Spectrin band and cross-link aggregates are indicated by an
arrow. St is a mixture of molecular weight standard, whose values are depicted in
the left of the figure. Lower panel: Quantitation by gel densitometry of the relative
abundance of spectrin after irradiation at various UVA doses in the presence of 6c at
different concentrations.
Figure 9 shows the pictures of the SDS–PAGE gels of ghosts irra-
diated in the presence of 6c. The compound caused an UVA dose-
dependent disappearance of the two spectrin subunit bands.
In order to investigate more deeply photosensitizing properties
of the title compounds towards proteins, solutions containing bo-
vine serum albumin (BSA) or ribonuclease A (RNAseA) as models30
and the drug in phosphate buffer were irradiated several times.
The degree of oxidative modifications was measured by monitor-
ing the carbonyl content, an index of photodamage of proteins.31
The results are reported in Figure 10 (upper panels) and demon-
strate that 6c significantly increased the carbonyl content of BSA
and RNAse after irradiation.
Moreover, the amount of the aromatic aminoacid tryptophan
(Trp) was directly analysed by monitoring the characteristic fluo-
rescence of Trp residues.32 As depicted in Figure 10 (lower panels),
a rapid decrease of the emission fluorescence was observed for 6c.
No effects were observed when BSA was irradiated in the same
conditions without 6c. Another protein model used in this study
was RNAseA, which is devoid of Trp but has Tyr residues in its se-
quence.33 As a consequence, its fluorescence emission band, cen-
tred at about 350 nm, was totally due to Tyr residues. RNAseA
emission band decreases less than BSA after irradiation in the pres-
ence of compounds 6c.
400 nm region, the compound give no LD bands in this region indi-
cating that the compound is not intercalated into DNA. Further-
more CD spectra carried out with the same derivatives, showed
that increasing concentrations of 6c does not lead to significant
changes of the CD spectrum of st DNA confirming that the title
compound are loosely bound to DNA. These facts strongly suggest
that compound 6c do not interact efficaciously, without irradia-
tion, with the macromolecule, as indeed demonstrated with natu-
ral furocoumarins.2
Moreover, further experiments were performed in order to
determine whether the new derivatives were able to photosensi-
tize DNA strand breakage. Supercoiled circular DNA allowed the
detection of structural alterations such as strand breaks or oxida-
tively damaged bases. We determined the degree of oxidative base
damage using two base excision repair enzymes: Formamidopyr-
imidine DNA glycosilase (Fpg) and Endonuclease III (Endo III) that
cleave oxidized DNA at the sites of modified purine and pyrimidine
bases, respectively. The excision of damaged base is followed by a
DNA breakage.36
In addition to endonuclease sensitive modifications, the num-
ber of single strand breaks generated by the excited photosensi-
tizer was quantified. Figure 12 (upper panel) shows the results
obtained for 6c tested at different [DNA]/[drug] ratios. The test
compound was not able to induce DNA damage without UVA irra-
diation. For compound 6c, the formation of single strand breaks,
expressed as the percentage of form II (Fig. 11), is not significantly
different from the irradiated control, even at the highest ratio em-
ployed. On the contrary, it was able to photooxidize nucleotidic
bases, as assessed after enzymatic digestion with Fpg. and Endo
2.2.12. DNA interactions and DNA-photodamage
In order to understand the binding nature between compound
6c and DNA, linear (LD) and circular dichroism (CD) measure-
ments34,35 were performed on solutions with salmon testes DNA
(st-DNA) and the title compound at various molar ratios.
Inspection of the LD spectra (Fig. 11, upper panel) in the presence
of st-DNA reveals that despite a strong absorption in the 300–

P. Barraja et al. / Bioorg. Med. Chem. 16 (2008) 9668–9683
9675
160
120
80
40
0
6c 10 μM
6c 20 μM
160
120
80
40
0
6c 10 μM
6c 20 μM
RNAse
BSA
0
5
10
15
0
5
10
15
UVA Dose (J cm-2)
UVA Dose (J cm-2)
100
80
100
80
I0/If
I0/If
UVA alone
6c 5 μM
6c 10 μM
6c 20 μM
UVA alone
6c 5 μM
6c 10 μM
6c 20 μM
60
40
60
RNAse
BSA
20
40
0
5
10
15
0
5
10
15
20
UVA Dose (J cm-2)
UVA Dose (J cm-2)
Figure 10. Photosensitized protein damage by 6c. BSA and RNAse were irradiated at different UVA dose in the presence of 6c in phosphate buffer at the indicated
concentration. Protein oxidation was evaluated by monitoring the carbonyl content (upper panels) and the loss of emission intensity of the proteins (lower panels).
1.5
In order to investigate the possible mechanism of action of the
photosensitized DNA damage, the same experiments were per-
formed in the presence of Mannitol, sodium azide and GSH. It
can be observed (Fig. 12 lower panel) that a significative reduction
of the photoinduced DNA damage was observed with mannitol and
GSH indicating the formation of free radicals species and/or as
intermediates. No protection was observed in the presence of
NaN3 suggesting that singlet oxygen is not involved in the mecha-
nism of action.
c
a
1.0
0.5
X10
c
b
0.0
0.0000
-0.0015
-0.0030
-0.0045
a
c
3. Conclusions
The data reported herein indicate that thiopyrano[2,3-e]indol-
2-ones are endowed with a potent photoantiproliferative activity,
and they could represent a new interesting class of potentially use-
ful compounds in photochemotherapy. The most active compound,
6c, presented a double bond in five-six position and a methyl group
in seven position. Less active were the dihydro derivatives at vari-
ance with the pyrroloquinolinones series in which the dihydrode-
rivatives showed potent photoantiproliferative activity, and the
aromatic compound was not active. An important feature of these
new compounds seems to be their incapability to bind and form
covalent adduct with DNA upon UVA irradiation although, as dem-
onstrated in this paper, they are able to photooxidize DNA bases in
particular pyrimidine ones. This could be important in view of the
long term toxic effect such as skin cancer or mutagenesis exhibited
by psoralen.
A preliminary indication about their mechanism of action de-
rives from the experiments of photocytotoxicity carried out in
the presence of different scavengers. The results demonstrated
the participation of free radicals and in particular the involvement
of the hydroxyl radical.
We have demonstrated using the most phototoxic compound 6c
that after UVA irradiation the most prevalent mode of cell death is
apoptosis. The apoptotic process, involves principally a mitochon-
drial damage as clearly demonstrated by the loss of mitochondrial
potential and cardiolipin oxidation after irradiation of Jurkat cells
in the presence of 6c. In addition the fundamental role played by
250
300
350
400
Wavelength (nm)
b,c
a
20
10
0
a,b
c
-10
-20
250
300
350
400
Wavelength (nm)
Figure 11. Upper panel: Absorbance (A), and Linear dichroism (LD) spectra of
mixtures of salmon testes DNA and compounds 6c at different [Drug]/[DNA] ratio
(a = 0.00, b = 0.02, c = 0.04). Lower panel: Circular dichroism spectra of of mixtures
of salmon testes DNA and compounds 6c at different [Drug]/[DNA] ratio (a = 0.00,
b = 0.02, c = 0.04).
III. In fact, high levels of single strand breaks were detected after
enzyme digestion and in particular after Endo III treatment sug-
gesting a preferential oxidation of pyrimidine bases.

9676
P. Barraja et al. / Bioorg. Med. Chem. 16 (2008) 9668–9683
4.1.1. Preparation of 1,5,6,7-tetrahydro-4H-indole-4-ones (8a–k)
4.1.1.1. 2-Methyl-1,5,6,7-tetrahydro-4H-indole-4-one (8a). Deriv-
100
80
60
40
20
0
No enzyme
+Fpg
+Endo III
ative 8a was prepared by reaction of 2-acetonyl-1,3-cyclohexanedione 7a
with ammonium acetate in acetic acid at 60 °C for 2 h.5b After cooling the
reaction mixture was poured onto crushed ice and the precipitate formed
was filtered, air dried and purified by recrystallization from ethanol to
give 8a (88%): mp 198–200 °C; IR: 3212–2939 (NH), 1624 (CO) cmꢀ1
;
1H NMR: d 1.94 (quint, J = 5.9 Hz, 2H, CH2), 2.09 (s, 3H, CH3), 2.21 (t,
J = 5.9 Hz, 2H, CH2), 2.65 (t, J = 5.9 Hz, 2H, CH2), 5.86 (s, 1H, H-3), 11.01
(s, 1H, NH). 13C NMR: d 12.4 (CH3), 22.1 (CH2), 23.7 (CH2), 37.6 (CH2),
101.7 (CH), 119.6 (C), 128.2 (C), 142.8 (C), 192.3 (CO). Anal. C9H11NO: C,
72.46; H, 7.43; N, 9.39. Found: C, 72.68; H, 7.10; N, 9.08.
Derivatives 8b–d were prepared according to previous
procedures.5b
Dark
1:1
1:2
1:4
UVA
[DNA]/[6c] Ratio
100
80
60
40
20
0
4.1.1.2. 1-t-Butoxycarbonyl-2-methyl-1,5,6,7-tetrahydro-4H-
no Enzyme
+ Fpg
+ Endo III
indole-4-one (8e).
To a solution of 8a (3.5 g, 23 mmol) in
anhydrous benzene (100 mL) and DMF (8 mL), dimethylaminopyr-
idine (DMAP) (0.32 g, 3 mmol) and t-butyl dicarbonate (10.3 g,
46 mmol) were added. The mixture was heated under reflux for
24 h, then cooled and treated with a saturated solution of sodium
hydrogen carbonate. After 30 min stirring, the organic layer was
separated and dried. The solvent was removed and the crude
material was purified by cromatography column (DCM: EtOAc
95:5) to give 8e (85%): mp 87–88 °C; IR: 1739 (CO), 1674
(CO) cmꢀ1
;
1H NMR: d 1.58 (s, 9H, 3ꢁ CH3), 1.95–2.08 (m, 2H,
CH2), 2.34 (t, J = 6.2 Hz, 2H, CH2), 2.35 (s, 3H, CH3), 2.99 (t,
J = 6.2 Hz, 2H, CH2), 6.18 (s, 1H, H-3). 13C NMR: d 15.4 (CH3),
23.2 (CH2), 24.6 (CH2), 27.4 (3ꢁ CH3), 37.0 (CH2), 84.9 (C), 106.5
(CH), 121.6 (C), 131.9 (C), 144.9 (C), 148.9 (CO), 193.4 (CO). Anal.
C14H19NO3: C, 67.45; H, 7.68; N, 5.62. Found: C, 67.20; H, 7.52;
N, 5.80.
UVA
6c
+ MAN
+ NaN3 + GSH
Figure 12. Upper panel: DNA strand breaks photoinduced by the compounds 6c,
expressed as percentage of form II obtained after densitometric analysis of the gel.
pBR322 supercoiled circular DNA was irradiated at the doses of 3.5 J cmꢀ2 at the
DNA]/[6c] ratios of 1, 2 and 4, and then treated with the base excision repair
enzymes Fpg and Endo III, as described in Section 4. Lower panel: Induction of
strand breaks in supercoiled pBR322 plasmid DNA, by 6c at molar [DNA]/[6c] ratio
of 1 after UVA irradiation (3.5 J cmꢀ2) in the presence of different conditions as
indicated in the figure. MAN (10 mM), GSH (1 mM), NaN3 (10 mM). After irradiation
the photosensitised DNA was treated with the enzymes Fpg and Endo III as
described in experimental section. Data are expressed as percentage of form II
obtained after densitometric analysis of the digital picture of the agarose gel, and
represent means SEM of three independent experiments.
4.1.1.3. 2-Methyl-1-(phenylsulfonyl)-1,5,6,7-tetrahydro-4H-
indole-4-one (8f).
To a solution of 8a (2 g, 13 mmol) in anhy-
drous DMF (15 mL) sodium hydride (0.36 g, 15 mmol) was added.
After 1 h stirring, the solution was ice-cooled and treated with ben-
zensulfonylchloride (2 mL, 15 mmol) under stirring (1 h). The reac-
tion mixture was allowed to reach room temperature then it was
poured onto crushed ice and filtered. The solid which separated
was filtered off, air dried and and recrystallized from ethanol to
give 8f (98%): mp 82–83 °C; IR: 1664 (CO) cmꢀ1 1H NMR: d 2.05
;
(q, J = 5.9 Hz, 2 H, CH2), 2.35 (s, 3H, CH3), 2.38 (t, J = 5.9 Hz, 2H,
CH2), 3.11 (t, J = 5.9 Hz, 2H, CH2), 6.31 (s, 1H, H-3), 7.67–7.87 (m,
3H, H-3’, H-4’, H-5’), 7.93 (d, J = 7.9 Hz, 2H, H-2’, H-6’). 13C NMR:
d 14.3 (CH3), 23.0 (CH2), 23.7 (CH2), 36.8 (CH2), 108.1 (CH), 122.5
(C), 126.5 (2ꢁ CH), 130.2 (CH), 132.5 (C), 134.9 (2 ꢁ CH), 138.0
(C), 145.3 (C), 193.3 (CO). Anal. C15H15NO3S: C, 62.27; H, 5.23; N,
4.84. Found: C, 62.40; H, 5.53; N, 4.74.
mitochondria, was further confirmed by the activation of caspase-
3. Other subcellular organelles such as lysosomes appear less sus-
ceptibile to the phototoxic action of compound 6c.
At molecular level the effects of 6c seems to be mediated by a
photodamage to lipid, proteins and DNA. In this context it will be
necessary to carry out further investigation on the photochemical
and photophysical aspect to better clarify their mechanism of
action.
4.1.1.4. 2-Ethoxycarbonyl-3-methyl-1,5,6,7-tetrahydro-4H-
indole-4-one (8g).
This compound was prepared according
4. Experimental
4.1. Chemistry
to a known procedure.6 A solution of the commercially available
1,3-cyclohexanedione 7b (8.8 g, 80 mmol) in acqueous acetic acid
(90%, 100 mL) containing sodium acetate (20.5 g, 250 mmol) was
heated at 90 °C. Zinc powder (17.6 g, 270 mmol) was added fol-
lowed by dropwise addition of a freshly prepared solution of
diethyl hydroxyl iminomalonate. This latter was prepared by stir-
ring at room temperature for 3 h a solution of ethylacetoacetate
(11 g, 80 mmol) in acetic acid (60 mL) with a solution of sodium
nitrite (6 g, 90 mmol) dissolved in water. When the addition of
diethyl hydroxyl iminomalonate was finished, the reaction mix-
ture was heated at 100 °C for 30 min. After 24 h stirring at room
temperature, the mixture was poured onto crushed ice and the
solid collected by filtration. Recrystallization with ethanol fur-
nished derivative 8g (60%): mp 165–167 °C; IR: 3425 (NH),
All melting points were taken on a Buchi–Tottoli capillary
apparatus and were uncorrected; IR spectra were determined
with a Jasco FT/IR 5300 spectrophotometer; 1H and 13C NMR
spectra were measured in DMSO-d6 solutions, unless otherwise
specified (TMS as internal reference), at 200 and 50.3 MHz,
respectively, using a Bruker AC series 200 MHz spectrometer.
Column chromatography was performed with Merck silica gel
230–400 Mesh ASTM or with a SEPACORE chromatography using
a BÜCHI apparatus. Elemental analyses (C, H, N) were within
0.4% of the theoretical values.

P. Barraja et al. / Bioorg. Med. Chem. 16 (2008) 9668–9683
9677
1685 (CO), 1649 (CO) cmꢀ1
;
1H NMR: d 1.29 (t, J = 7.4 Hz, 3H,
4.1.2. General procedure for the chloroformylation of
derivatives 8a–f,j,k
CH3), 1.98 (quint, J = 5.9 Hz, 2H, CH2), 2.33 (t, J = 5.9 Hz, 2H,
CH2), 2.48 (s, 3H, CH3), 2.75 (t, J = 5.9 Hz, 2H, CH2), 4.21 (q,
J = 7.4 Hz, 2H, CH2), 11.88 (s, 1H, NH). 13C NMR: d 11.2 (CH3),
14.3 (CH3), 22.3 (CH2), 23.0 (CH2), 38.6 (CH2), 59.5 (CH2), 118.8
(C), 119.0 (C), 126.7 (C), 146.3 (C), 160.9 (CO), 194.3 (CO). Anal.
C12H15NO3: C, 65.14; H, 6.83; N, 6.33. Found: C, 64.92; H, 7.00;
N, 6.60.
A solution of anhydrous DMF (4 mL, 50 mmol) in anhydrous
DCM (17 mL) was cooled at 0 °C. Phosphorous oxychloride (4 mL,
43 mmol) was dissolved in anhydrous DCM (4 mL) and added
dropwise as to keep the temperature at 0 °C. The mixture was stir-
red for 10 min and then rapidly heated to reflux. Immediately after
the boiling began, a solution of 8a–f,j,k (13 mmol) in anhydrous
DCM (5 mL) was added all at once. The mixture was stirred under
reflux for 3 min and then cooled to 10 °C. A 4 N sodium acetate
solution (40 mL) and DCM (14 mL) were added to the mixture un-
der stirring. The organic layer was decanted, evaporated under re-
duced pressure. Upon adding ice to the oily residue a solid
separated which was filtered, dried and purified by chromatogra-
phy column (DCM).
4.1.1.5. 3-Methyl-1,5,6,7-tetrahydro-4H-indole-4-one-2-car-
boxylic acid (8h).
To a suspension of 8g (7.6 g, 34 mmol) in
ethanol (38 mL), 30% NaOH (10 mL) was added. The mixture was
heated under reflux for 1 h, then cooled and washed with ethyl
ether. The acqueous extract was acidified with 6 N HCl and the
solid precipitated was filtered off and dried to furnish derivative
8h (97%): mp 273–274 °C; IR: 3442 (OH), 3255 (NH), 1670 (CO),
1601 (CO) cmꢀ1 1H NMR: d 1.98 (q, J = 5.8 Hz, 2H, CH2), 2.32 (t,
;
4.1.2.1. From 8a, 4-chloro-2-methyl-6,7-dihydro-1H-indole-5-
J = 5.8 Hz, 2H, CH2), 2.47 (s, 1H, CH3), 2.74 (t, J = 5.8 Hz, 2H, CH2),
11.80 (1H, s, NH), 12.52 (1H, s, OH). 13C NMR: d 10.9 (CH3), 21.9
(CH2), 22.7 (CH2), 38.2 (CH2), 118.6 (C), 119.2 (C), 125.8 (C),
145.5 (C), 162.1 (CO), 194.0 (CO). Anal. C10H11NO3: C, 62.17; H,
5.74; N, 7.25. Found: C, 62.41; H, 5.50; N, 7.10.
carbaldehyde (9a).
Compound 9a was obtained as white so-
lid (3%): mp 124–125 °C; IR: 3276 (NH), 1633 (CO) cm-1; 1H
NMR: d 2.17 (s, 3H, CH3), 2.52–2.66 (m, 4H, 2ꢁ CH2), 5.96 (s, 1H,
H-3), 9.95 (s, 1H, CHO), 11.36 (s, 1H, NH). 13C NMR: d 12.5 (CH3),
20.2 (CH2), 22.5 (CH2), 102.8 (CH), 117.3 (C), 121.0 (C), 130.0 (C),
135.3 (C), 145.2 (C), 187.3 (CHO). Anal. C10H10ClNO: C, 61.39; H,
5.15; N, 7.16. Found: C, 61.00; H, 5.48; N, 6.96.
4.1.1.6. 3-Methyl-1,5,6,7-tetrahydro-4H-indole-4-one (8i).
To
a warm suspension of the carboxylic acid 8h (4.8 g, 24 mmol)
in ethanol (55 mL), 6 N HCl was added dropwise. Stirring was
kept until bubbling ceased (20 min). The solvent was removed
in vacuo and the crude residue was treated with brine. The solid
precipitated was filtered off and dried to furnish derivative 8i
4.1.2.2. From 8b, 4-chloro-2-methyl-1-phenyl-6,7-dihydro-1H-
indole-5-carbaldehyde (9b).
Compound 9b was obtained as
white solid (70%): mp 113–115 °C; IR: 1643 (CO) cmꢀ1
;
1H NMR:
d 2.06 (s, 3H, CH3), 2.48–2.69 (m, 4H, 2 ꢁ CH2), 6.28 (s, 1H, H-3),
7.39 (d, J = 6.4 Hz, 2H, H-2’, H-6’), 7.48–7.62 (m, 3H, H-3’, H-4’ e
H-5’), 10.01 (s, 1H, CHO). 13C NMR: d 12.3 (CH3), 20.1 (CH2), 22.3
(CH2), 104.3 (CH), 117.2 (C), 122.2 (C), 127.1 (2ꢁ CH), 128.6 (CH),
129.5 (2ꢁ CH), 131.2 (C), 136.1 (C), 136.2 (C), 144.2 (C), 187.6
(CHO). Anal. C16H14ClNO: C, 70.72; H, 5.19; N, 5.15. Found: C,
70.52; H, 5.00; N, 5.45.
(60%): mp 194–196 °C; IR: 3213 (NH), 1639 (CO) cmꢀ1
;
1H
NMR: d 1.97 (q, J = 6.1 Hz, 2H, CH2), 2.13 (s, 1H, CH3), 2.26 (t,
J = 6.1 Hz, 2H, CH2), 2.69 (t, J = 6.1 Hz, 2H, CH2), 6.44 (s, 1H, H-
2), 10.96 (s, 1H, NH). 13C NMR: d 11.5 (CH3), 22.4 (CH2), 23.8
(CH2), 38.3 (CH2), 116.1 (CH), 116.7 (C), 117.7 (C), 143.3 (C),
193.7 (CO). Anal. C9H11NO: C, 72.46; H, 7.43; N, 9.39. Found:
C, 72.20; H, 7.68; N, 9.55.
4.1.2.3. From 8c, 4-chloro-1,2-dimethyl-6,7-dihydro-1H-indole-
4.1.1.7. Methylation of derivatives 8g and 8i.
To a suspen-
5-carbaldehyde (9c).
Compound 9c was obtained as white
solid (60%): mp 108–110 °C; IR: 1639 (CO) cmꢀ1
;
1H NMR: d
sion of 8g or 8i (20 mmol) in anhydrous THF (40 mL) sodium hy-
dride (0.5 g, 20 mmol) was added. After 1 h stirring, iodomethane
(1.12 mL, 20 mmol) was added. The mixture was stirred for 5 h
for 8g or 1 h for 8i at room temperature, then poured onto crushed
ice. The solid which separated was filtered off and dried to furnish
derivative 8j or 8k.
2.19 (s, 3H, CH3), 2.61–2.77 (m, 4H, 2 ꢁ CH2), 3.43 (s, 3H, CH3),
6.06 (s, 1H, H-3), 9.95 (s, 1H, CHO). 13C NMR: d 11.6 (CH3),
19.4 (CH2), 22.1 (CH2), 30.3 (CH3), 103.3 (CH), 116.2 (C), 120.8
(C), 131.1 (C), 136.5 (C), 144.8 (C), 187.3 (CHO). Anal.
C11H12ClNO: C, 63.01; H, 5.77; N, 6.68. Found: C, 62.86; H,
5.57; N, 6.60. From the same reaction mixture it was also iso-
lated 4-chloro-1,2-dimethyl-6,7-dihydro-1H-indole-3,5-dicarbal-
dehyde 10 as white solid (20%): mp 148–149 °C; IR: 1650
4.1.1.8. 1,3-Dimethyl-2-ethoxycarbonyl-1,5,6,7-tetrahydro-4H-
indole-4-one (8j). The solid which separated was derivative 8j
(88%): mp 216–217 °C; IR: 1680 (CO), 1653 (CO) cmꢀ1
;
1H NMR:
(CO), 1648 (CO) cmꢀ1 1H NMR: d 1.90 (s, 3H, CH3), 2.60 (t,
;
d 1.30 (t, J = 7.1 Hz, 3H, CH3), 2.02 (quint, J = 6.2 Hz, 2H, CH2),
2.34 (t, J = 6.2 Hz, 2H, CH2), 2.49 (s, 3H, CH3), 2.78 (t, J = 6.2 Hz,
2H, CH2), 3.71 (s, 3H, CH3), 4.24 (q, J = 7.1 Hz, 2H, CH2). 13C NMR:
d 12.3 (CH3), 14.6 (CH3), 21.9 (CH2), 22.7 (CH2), 33.6 (CH3), 38.9
(CH2), 60.2 (CH2), 118.2 (C), 121.0 (C), 128.4 (C), 148.5 (C), 161.7
(CO), 194.7 (CO). Anal. C13H17NO3: C, 66.36; H, 7.28; N, 5.95.
Found: C, 66.00; H, 7.46; N, 6.14.
J = 8.2 Hz, 2H, CH2), 2.80 (t, J = 8.2 Hz, 2H, CH2), 2.89 (s, 3H,
CH3), 9.65 (s, 1H, CHO), 9.95 (s, 1H, CHO). 13C NMR: d 11.1
(CH3), 18.9 (CH2), 21.6 (CH2), 30.5 (CH3), 115.6 (C), 117.8 (C),
124.4 (C), 138.4 (C), 139.3 (C), 142.7 (C), 185.9 (CHO), 188.0
(CHO). Anal. C12H12ClNO2: C, 60.64; H, 5.09; N, 5.89. Found: C,
60.75; H, 4.86; N, 6.09.
4.1.2.4. From 8d, 1-benzyl-4-chloro-2-methyl-6,7-dihydro-1H-
indole-5-carbaldehyde (9d).
Compound 9d was obtained as
4.1.1.9. 1,3-Dimethyl-1,5,6,7-tetrahydro-4H-indole-4-one
white solid (88%): mp 67–69 °C; IR: 1639 (CO) cmꢀ1
;
1H NMR: d
(8k).
The solid which separated was derivative 8k (92%): mp
127–128 °C; IR: 1639 (CO) cmꢀ1
;
1H NMR: d 2.01 (q, J = 6.0 Hz,
2.12 (s, 3H, CH3), 2.64–2.70 (m, 4H, 2 ꢁ CH2), 5.16 (s, 2H, CH2)
6.16 (s, 1H, H-3), 7.00 (d, J = 6.9 Hz, 2H, H-2’, H-6’), 7.29–7.34 (m,
3H, H-3’, H-4’, H-5’), 9.98 (s, 1H, CHO). 13C NMR: d 11.8 (CH3),
19.6 (CH2), 22.3 (CH2), 46.7 (CH2), 104.1 (CH), 116.7 (C), 121.4
(C), 126.1 (2ꢁ CH), 127.3 (CH), 128.8 (2ꢁ CH), 131.0 (C), 136.5
(C), 137.2 (C), 144.5 (C), 187.4 (CHO). Anal. C17H16ClNO: C, 71.45;
H, 5.64; N, 4.90. Found: C, 71.25; H, 5.40; N, 4.60.
2H, CH2), 2.12 (s, 1H, CH3), 2.25 (t, J = 6.0 Hz, 2H, CH2), 2.68 (t,
J = 6.0 Hz, 2H, CH2), 3.46 (s, 1H, CH3), 6.46 (s, 1H, H-2). 13C
NMR: d 11.3 (CH3), 20.9 (CH2), 23.2 (CH2), 32.7 (CH3), 37.9
(CH2), 116.6 (C), 118.0 (C), 121.1 (CH), 143.5 (C), 193.3 (CO). Anal.
C10H13NO: C, 73.59; H, 8.03; N, 8.58. Found: C, 73.90; H, 7.73; N,
8.92.

9678
P. Barraja et al. / Bioorg. Med. Chem. 16 (2008) 9668–9683
4.1.2.5. From 8e, 1-t-butoxycarbonyl-4-chloro-2-methyl-6,7-
dihydro-1H-indole-5-carbaldehyde (9e). Compound 9e was
obtained as white solid (10%): mp 101–102 °C; IR: 1747 (CO),
1H NMR: d 1.19 (t, J = 7.1 Hz, 3H, SCH2CH3), 2.08 (s, 3H, CH3),
2.40–2.64 (m, 4H, 2ꢁ CH2), 2.94 (q, J = 7.1 Hz, 2H, SCH2CH3), 6.31
(s, 1H, H-3), 7.37 (d, J = 7.1 Hz, 2H, H-2’, H-6’), 7.45–7.61 (m, 3H,
H-3’, H-4’, H-5’), 10.30 (s, 1H, CHO). 13C NMR: d 12.5 (CH3), 14.9
(CH3), 20.1 (CH2), 23.0 (CH2), 28.3 (CH2), 105.5 (CH), 118.1 (C),
127.1 (2 ꢁ CH), 128.3 (CH), 129.4 (2 ꢁ CH), 130.2 (C), 131.0 (C),
134.7 (C), 136.3 (C), 148.5 (C), 188.9 (CHO). Anal. C18H19NOS: C,
72.69; H, 6.44; N, 4.71. Found: C, 75.65; H, 5.80; N, 5.42.
1653 (CO) cmꢀ1
;
1H NMR: d 1.57 (s, 9H, 3 ꢁ CH3), 2.37 (s, 3H,
CH3), 2.64 (t, J = 9.5 Hz, 2H, CH2), 3.03 (t, J = 9.5 Hz, 2H, CH2), 6.23
(s, 1H, H-3), 10.02 (s, 1H, CHO). 13C NMR: d 15.4 (CH3), 21.8
(CH2), 22.2 (CH2), 27.3 (3 ꢁ CH3), 85.1 (C), 107.4 (CH), 119.4 (C),
124.2 (C), 132.5 (C), 136.3 (C), 141.8 (C), 148.6 (CO), 187.9 (CHO).
Anal. C15H18ClNO3: C, 60.91; H, 6.13; N, 4.74. Found: C, 61.11; H,
6.25; N, 4.80.
4.1.3.2. 1,2-Dimethyl-4-(ethylsulfanyl)-6,7-dihydro-1H-indole-
5-carbaldehyde (12c).
This product was obtained as yellow
4.1.2.6. From 8f, 4-chloro-2-methyl-1-(phenylsulfonyl)-6,7-
solid (80%) from 9c: mp 60–62 °C; IR: 1624 (CO) cmꢀ1
;
1H NMR:
dihydro-1H-indole-5-carbaldehyde (9f).
obtained as white solid (95%): mp 107–108 °C; IR: 1655 (CO)
cmꢀ1 1H NMR: d 2.38 (s, 3H, CH3), 2.68 (t, J = 9.1 Hz, 2H, CH2),
3.14 (t, J = 9.1 Hz, 2H, CH2), 6.36 (s, 1H, H-3), 7.67–7.87 (m, 3H,
H-3’, H-4’, H-5’), 7.95 (d, J = 7.2 Hz, 2H, H-2’, H-6’), 10.03 (s, 1H,
CHO). 13C NMR: d 14.3 (CH3), 21.5 (CH2), 22.2 (CH2), 109.2 (CH),
120.8 (C), 125.5 (C), 126.6 (2 ꢁ CH), 130.3 (CH), 133.0 (C), 135.0
(2ꢁ CH), 136.1 (C), 137.8 (C), 140.7 (C), 188.2 (CHO). Anal.
C16H14ClNO3S: C, 57.23; H, 4.20; N, 4.17. Found: C, 57.00; H,
4.40; N, 4.40.
Compound 9f was
d 1.12 (t, J = 7.2 Hz, 3H, SCH2CH3), 2.19 (s, 3H, CH3), 2.56–2.70 (m,
4H, 2ꢁ CH2), 2.86 (q, J = 7.2 Hz, 2H, SCH2CH3), 3.42 (s, 3H, CH3),
6.01 (s, 1H, H-3), 10.25 (s, 1H, CHO). 13C NMR: d 11.7 (CH3), 14.8
(CH3), 19.4 (CH2), 22.7 (CH2), 28.2 (CH2), 30.0 (CH3), 104.3 (CH),
117.0 (C), 129.72 (C), 130.1 (C), 134.9 (C), 148.8 (C), 188.6 (CHO).
Anal. C13H17NOS: C, 66.35; H, 7.28; N, 5.95. Found: C, 66.00; H,
7.54; N, 5.70.
;
4.1.3.3. 1-Benzyl-4-(ethylsulfanyl)-2-methyl-6,7-dihydro-1H-
indole-5-carbaldehyde (12d).
This product was obtained as
yellow solid (94%) from 9d: mp 73–75 °C; IR: 1628 (CO) cmꢀ1
;
4.1.2.7. From 8j, 4-chloro-1,3-dimethyl-2-ethoxycarbonyl-6,7-
1H NMR: d 1.15 (t, J = 7.4 Hz, 3H, SCH2CH3), 2.12 (s, 3H, CH3),
2.57–2.65 (m, 4H, 2 ꢁ CH2), 2.91 (q, J = 7.4 Hz, 2H, SCH2CH3), 5.14
(s, 2H, CH2), 6.19 (s, 1H, H-3), 6.99 (dd, J = 7.4, 1.5 Hz, 2H, H-2’,
H-6’), 7.25–7.38 (m, 3H, H-3’, H-4’, H-5’), 10.27 (s, 1H, CHO). 13C
NMR: d 11.8 (CH3), 14.8 (CH3), 19.6 (CH2), 22.8 (CH2), 28.3 (CH2),
46.4 (CH2), 105.0 (CH), 117.6 (C), 126.0 (2ꢁ CH), 127.2 (CH),
128.7 (2ꢁ CH), 130.0 (C), 130.1 (C), 134.9 (C), 137.5 (C), 148.8
(C), 188.6 (CHO). Anal. C19H21NOS: C, 73.27; H, 6.80; N, 4.50.
Found: C, 72.93; H, 6.55; N, 4.72.
dihydro-1H-indole-5-carbaldehyde (9j).
tained as white solid (80%): mp 112–113 °C; IR: 1685 (CO), 1679
(CO) cmꢀ1 1H NMR: d 1.31 (t, J = 7.0 Hz, 3H, CH2CH3), 2.54 (s, 3H,
Compound 9j was ob-
;
CH3), 2.59 (t, J = 7.3 Hz, 2H, CH2), 2.76 (t, J = 7.3 Hz, 2H, CH2), 3.71
(s, 3H, CH3), 4.22 (q, J = 7.0 Hz, 2H, CH2CH3), 10.08 (s, 1H, CHO).
13C NMR: d 12.3 (CH3), 14.1 (CH3), 19.0 (CH2), 21.4 (CH2), 33.3
(CH3), 59.8 (CH2), 114.9 (C), 121.8 (C), 124.6 (C), 126.5 (C), 142.6
(C), 143.9 (C), 160.8 (CO), 188.2 (CHO). Anal. C14H16ClNO3: C,
59.68; H, 5.72; N, 4.97. Found: C, 59.85; H, 5.62; N, 5.04.
4.1.3.4. 4-(Ethylsulfanyl)-2-methyl-1-(phenylsulfonyl)-6,7-
4.1.2.8. From 8k, 4-chloro-1,3-dimethyl-6,7-dihydro-1H-indole-
dihydro-1H-indole-5-carbaldehyde (12f).
obtained as yellow solid (88%) from 9f: mp150–151 °C; IR:1645
(CO) cmꢀ1 1H NMR: d 1.09 (t, J = 7.4 Hz, 3H, SCH2CH3), 2.38 (s,
This product was
2,5-dicarbaldehyde (11).
white solid (90%): mp 182–183 °C; IR: 1643 (CO), 1640 (CO)
cmꢀ1 1H NMR: d 2.57 (s, 3H, CH3), 2.62 (t, J = 8.2 Hz, 2H, CH2),
Compound 11 was obtained as
;
;
3H, CH3), 2.62 (t, J = 8.9 Hz, 2H, CH2), 2.79 (q, J = 7.4 Hz, 2H,
SCH2CH3), 3.05 (t, J = 8.9 Hz, 2H, CH2), 6.41 (s, 1H, H-3), 7.66–7.84
(m, 3H, H-3’, H-4’, H-5’), 7.91 (d, J = 7.8 Hz, 2H, H-2’, H-6’), 10.31
(s, 1H, CHO). 13C NMR: d 14.5 (CH3), 14.6 (CH3), 21.4 (CH2), 22.8
(CH2), 28.3 (CH2), 110.6 (CH), 122.1 (C), 126.4 (2ꢁ CH), 130.2
(CH), 132.3 (C), 134.7 (C), 134.8 (2ꢁ CH), 135.1 (C), 138.1 (C),
144.7 (C), 189.7 (CHO). Anal. C18H19NO3S2: C, 59.81; H, 5.30; N,
3.87. Found: C, 60.02; H, 5.58; N, 4.05.
2.83 (t, J = 8.2 Hz, 2H, CH2), 3.80 (s, 3H, CH3), 9.78 (s, 1H, CHO),
10.01 (s, 1H, CHO). 13C NMR: d 10.2 (CH2), 18.4 (CH3), 21.2 (CH3),
32.6 (CH2), 115.2 (C), 125.3 (C), 129.1 (C), 131.2 (C), 143.1 (C),
144.9 (C), 178.7 (CHO), 188.2 (CHO). Anal. C12H12ClNO2: C, 60.64;
H, 5.09; N, 5.89. Found: C, 60.20; H, 5.15; N, 5.79. When reaction
was carried out with stoichiometric amount of the Vilsmeier re-
agent the only compound isolated was 1,3-dimethyl-2-formyl-
1,5,6,7-tetrahydro-4H-indole-4-one (8l) as white solid (40%): mp
89–90 °C; IR: 1652 (CO), 1637 (CO) cmꢀ1
;
1H NMR: d 2.02 (q,
4.1.3.5. 1,3-Dimethyl-2-ethoxycarbonyl-4-(ethylsulfanyl)-6,7-
J = 6.1 Hz, 2H, CH2), 2.37 (t, J = 6.1 Hz, 2H, CH2), 2.50 (s, 1H, CH3),
2.79 (t, J = 6.1 Hz, 2H, CH2), 3.78 (s, 1H, CH3), 9.76 (s, 1H, CHO).
13C NMR: d 10.0 (CH3), 20.9 (CH2), 22.1 (CH2), 32.7 (CH3), 38.3
(CH2), 121.1 (C), 128.1 (C), 133.1 (C), 150.2 (C), 179.3 (CHO),
194.2 (CO). Anal. C11H13NO2: C, 69.09; H, 6.85; N, 7.32. Found: C,
70.22; H, 6.60; N, 7.10.
dihydro-1H-indole-5-carbaldehyde (12j).
obtained as yellow solid (86%) from 9j: mp 119–121 °C; IR: 1681
(CO), 1633 (CO) cmꢀ1 1H NMR: d 1.11 (t, J = 7.0 Hz, 3H, SCH2CH3),
1.31 (t, J = 7.0 Hz, 3H, OCH2CH3), 2.54–2.71 (m, 6H, 3ꢁ CH2), 2.65 (s,
3H, CH3), 3.74 (s, 3H, CH3), 4.24 (q, J = 7.0 Hz, 2H, OCH2CH3), 10.41
(s, 1H, CHO). 13C NMR: d 11.8 (CH3), 14.2 (CH3), 14.4 (CH3), 19.2
(CH2), 21.2 (CH2), 28.6 (CH2), 33.3 (CH3), 59.6 (CH2), 115.6 (C),
121.3 (C), 126.7 (C), 134.5 (C), 142.0 (C), 147.6 (C), 161.0 (CO),
189.8 (CHO). Anal. C16H21NO3S: C 62.52; H, 6.89; N 4.56. Found:
C 62.45; H, 6.96; N 4.40.
This product was
;
4.1.3. General procedure for the nucleophilic substitution of
the chloro aldehydes (9b–d,f,j and 11)
To an ice cooled solution of 9b–d,f,j and 11 (3.37 mmol) in
anhydrous DMF (24 mL), potassium carbonate (0.77 g, 5.53 mmol)
and ethanthiol (2.45 mL, 33.18 mmol) were added. The mixture
was stirred at room temperature for 24 h and then poured onto
crushed ice. The solid precipitate was filtered off and dried to af-
ford the desired compounds.
4.1.3.6. 1,3-Dimethyl-4-(ethylsulfanyl)-6,7-dihydro-1H-indole-
2,5-dicarbaldehyde (12l).
white solid (80%) from 11: mp 101–102 °C; IR: 1649 (CO), 1620
(CO) cmꢀ1 1H NMR: d 1.14 (t, J = 7.2 Hz, 3H, SCH2CH3), 2.56–2.79
This product was obtained as
;
(m, 6H, 3 ꢁ CH2), 2.57 (s, 3H, CH3), 3.93 (s, 3H, CH3), 9.75 (s, 1H,
CHO), 10.41 (s, 1H, CHO). 13C NMR: d 14.3 (CH3), 18.6 (CH2), 21.8
(CH2), 28.9 (CH2), 32.4 (CH3), 39.3 (CH3), 116.3 (C), 128.9 (C),
135.2 (2 ꢁ C), 144.6 (C), 146.7 (C), 178.3 (CHO), 189.9 (CHO). Anal.
4.1.3.1. 4-(Ethylsulfanyl)-2-methyl-1-phenyl-6,7-dihydro-1H-
indole-5-carbaldehyde (12b).
This product was obtained as
yellow solid (95%) from 9b: mp 93–95 °C; IR: 1628 (CO) cmꢀ1
;

P. Barraja et al. / Bioorg. Med. Chem. 16 (2008) 9668–9683
9679
C14H17NO2S: C, 63.85; H, 6.51; N, 5.32. Found: C, 64.05; H, 6.22; N,
5.12.
DMF (6 mL). The mixture was stirred for 1 h at room temperature
and then added dropwise to a solution of the proper carboxalde-
hyde 12b–d,f,j,l or 13b–d,f (3.37 mmol) dissolved in anhydrous
DMF (5 mL). After stirring at the proper temperature and for the
suitable time, the mixture was poured onto crushed ice and the so-
lid which separated was filtered off and dried. Recrystallization
from ethanol gave the desired compounds.
4.1.4. General procedure for the oxidation of the 4-(ethylsulfa-
nyl)-1-substituted-6,7-dihydro-1H-indole-5-carbaldehydes
(12b–d,f)
A
solution of 12b–d,f (3.61 mmol) in anhydrous benzene
(18 mL) and DDQ (0.82 g, 3.61 mmol) was heated under reflux
for 15 min. The reaction mixture was washed with 1 N NaOH first
and then with brine. The organic layer was separated, dried over
sodium sulfate and the solvent removed in vacuo. Recrystallization
with ethanol furnished the desired products.
4.1.5.1. Ethyl 3-[4-(ethylsulfanyl)-2-methyl-1-phenyl-6,7-dihy-
dro-1H-indole-5-yl]acrylate (14b).
tained as white solid (75%) after 1 h at 50 °C from 12b: mp 103–
105 °C; IR: 1685 (CO) cmꢀ1 1H NMR: d 1.15 (t, J = 7.5 Hz, 3H,
This product was ob-
;
SCH2CH3), 1.22 (t, J = 6.9 Hz, 3H, OCH2CH3), 2.07 (s, 3H, CH3),
2.46–2.65 (m, 4H, 2ꢁ CH2), 2.83 (q, J = 7.5 Hz, 2H, SCH2CH3), 4.14
(q, J = 6.9 Hz, 2H, OCH2CH3), 5.87 (d, J = 15.5 Hz, 1H, CH), 6.17 (s,
1H, H-3), 7.36 (d, J = 6.4 Hz, 2H, H-2’, H-6’), 7.47–7.58 (m, 3H, H-
3’, H-4’, H-5’), 8.33 (d, J = 15.5 Hz, 1H, CH). 13C NMR: d 12.4
(CH3), 14.2 (CH3), 14.9 (CH3), 20.2 (CH2), 25.8 (CH2), 28.1 (CH2),
59.4 (CH2), 105.3 (CH), 114.1 (CH), 118.9 (C), 127.1 (2 ꢁ CH),
128.0 (CH), 128.9 (C), 129.0 (C), 129.3 (2 ꢁ CH), 131.5 (C), 136.6
(C), 136.7 (C), 142.6 (CH), 167.1 (CO). Anal. C22H25NO2S: C, 71.90;
H, 6.86; N, 3.81. Found: C, 72.10; H, 6.60; N, 3.65.
4.1.4.1. 4-(Ethylsulfanyl)-2-methyl-1-phenyl-1H-indole-5-carb-
aldehyde (13b).
This product was obtained as yellow solid
(85%) from 12b: mp 86–88 °C; IR: 1670 (CO) cmꢀ1
;
1H NMR: d
1.15 (t, J = 7.1 Hz, 3H, SCH2CH3), 2.32 (s, 3H, CH3), 2.99 (q,
J = 7.1 Hz, 2H, SCH2CH3), 6.84 (s, 1H, H-3), 7.11 (d, J = 8.5 Hz, 1H,
H-7), 7.49 (d, J = 6.4 Hz, 2H, H-2’, H-6’), 7.54–7.68 (m, 4H, H-3’,
H-4’, H-5’, H-6), 10.75 (s, 1H, CHO); 13C NMR: d 13.1 (CH3), 15.0
(CH3), 30.4 (CH2), 102.1 (CH), 110.7 (CH), 120.7 (CH), 127.6
(2 ꢁ CH), 128.6 (CH), 129.8 (2 ꢁ CH), 130.6 (C), 130.8 (C), 131.6
(C), 136.0 (C), 139.7 (C), 139.8 (C), 191.6 (CHO). Anal. C18H17NOS:
C, 73.19; H, 5.80; N, 4.74. Found: C, 73.00; H, 6.10; N, 4.50.
4.1.5.2. Ethyl
1H-indole-5-yl]acrylate (14c).
white solid (92%) after 1 h at room temperature from 12c: mp
95–97 °C; IR: 1684 (CO) cmꢀ1 1H NMR: d 1.08 (t, J = 6.8 Hz, 3H,
3-[1,2-dimethyl-4-(ethylsulfanyl)-6,7-dihydro-
This product was obtained as
4.1.4.2. 1,2-Dimethyl-4-(ethylsulfanyl)-1H-indole-5-carbalde-
hyde (13c).
This product was obtained as yellow solid (65%)
;
from 12c: mp 60–62 °C; IR: 1662 (CO) cm-1; 1H NMR: d 1.07 (t,
J = 7.4 Hz, 3H, SCH2CH3), 2.45 (s, 3H, CH3), 2.91 (q, J = 7.4 Hz, 2H,
SCH2CH3), 3.72 (s, 3H, CH3), 6.61 (s, 1, H-3), 7.53–7.66 (m, 2H, H-
6, H-7), 10.71 (s, 1H, CHO); 13C NMR: d 12.4 (CH3), 14.9 (CH3),
29.7 (CH3), 30.3 (CH2), 100.6 (CH), 110.3 (CH), 119.6 (CH), 129.7
(C), 130.3 (C), 131.2 (C), 139.3 (C), 140.1 (C), 191.7 (CHO). Anal.
C13H15NOS: C, 66.92; H, 6.48; N, 6.00. Found: C, 66.70; H, 6.28;
N, 6.36.
SCH2CH3), 1.22 (t, J = 6.8 Hz, 3H, OCH2CH3), 2.17 (s, 3H, CH3),
2.58–2.70 (m, 4H, 2ꢁ CH2), 2.76 (q, J = 6.8 Hz, 2H, SCH2CH3), 3.40
(s, 3H, CH3), 4.14 (q, J = 6.8 Hz, 2H, OCH2CH3), 5.85 (d, J = 15.1 Hz,
1H, CH), 5.95 (s, 1H, H-3), 8.32 (d, J = 15.1 Hz, 1H, CH). 13C NMR:
d 11.68 (CH3), 14.23 (CH3), 14.79 (CH3), 19.40 (CH2), 25.57 (CH2),
25.95 (CH2), 29.78 (CH3), 59.30 (CH2), 103.85 (CH), 113.09 (CH),
117.79 (C), 127.57 (C), 128.67 (C), 131.62 (C), 137.20 (C), 142.78
(CH), 167.15 (CO). Anal. C17H23NO2S: C, 66.85; H, 7.59; N, 4.59.
Found: C, 67.00; H, 7.40; N, 4.90.
4.1.4.3. 4-(Ethylsulfanyl)-2-methyl-1-benzyl-1H-indole-5-carb-
aldehyde (13d).
This product was obtained as yellow solid
4.1.5.3. Ethyl 3-[1-benzyl-4-(ethylsulfanyl)-2-methyl-6,7-dihy-
(83%) from 12d: mp 73–75 °C; IR: 1668 (CO) cmꢀ1
;
1H NMR: d
dro-1H-indole-5-yl]acrylate (14d).
tained as white solid (80%) after 24 h at room temperature from
12d: mp 112–113 °C; IR: 1691 (CO) cmꢀ1 1H NMR: d 1.11 (t,
This product was ob-
1.11 (t, J = 7.4 Hz, 3H, SCH2CH3), 2.42 (s, 3H, CH3), 2.95 (q,
J = 7.4 Hz, 2H, SCH2CH3), 5.14 (s, 2H, CH2), 6.72 (s, 1H, H-3), 7.01
(d, J = 7.4 Hz, 2H, H-2’, H-6’), 7.24–7.34 (m, 3H, H-3’, H-4’, H-5’),
7.55–7.66 (m, 2H, H-6, H-7), 10.74 (s, 1H, CHO); 13C NMR: d 12.5
(CH3), 14.9 (CH3), 30.4 (CH2), 46.1 (CH2), 101.5 (CH), 110.6 (CH),
120.0 (CH), 126.1 (2ꢁ CH), 127.2 (CH), 128.6 (2 ꢁ CH), 130.0 (C),
130.6 (C), 131.4 (C), 137.5 (C), 139.2 (C), 139.8 (C), 191.6 (CHO).
Anal. C19H19NOS: C, 73.75; H, 6.19; N, 4.53. Found: C, 73.50; H,
6.00; N, 4.80.
;
J = 7.4 Hz, 3H, SCH2CH3), 1.21 (t, J = 7.4 Hz, 3H, OCH2CH3), 2.11 (s,
3H, CH3), 2.58–2.68 (m, 4H, 2 ꢁ CH2), 2.80 (q, J = 7.4 Hz, 2H,
SCH2CH3), 4.13 (q, J = 7.4 Hz, 2H, OCH2CH3), 5.10 (s, 2H, CH2),
5.85 (d, J = 16.2 Hz, 1H, CH), 6.05 (s, 1H, H-3), 6.99 (d, J = 5.9 Hz,
2H, H-2’, H-6’), 7.25–7.37 (m, 3H, H-3’, H-4’, H-5’), 8.32 (d,
J = 16.2 Hz, 1H, CH). 13C NMR: d 11.7 (CH3), 14.2 (CH3), 14.8
(CH3), 19.7 (CH2), 25.7 (CH2), 28.0 (CH2), 46.2 (CH2), 59.4 (CH2),
104.6 (CH), 113.5 (CH), 118.4 (C), 125.9 (2 ꢁ CH), 127.1 (CH),
128.0 (C), 128.6 (2 ꢁ CH), 131.7 (C), 137.0 (C), 137.1 (C), 137.8
(C), 142.7 (CH), 167.1 (CO). Anal. C23H27NO2S: C, 72.41; H, 7.13;
N, 3.67. Found: C, 72.20; H, 7.00; N, 3.47.
4.1.4.4. 4-(Ethylsulfanyl)-2-methyl-1-phenylsulfonyl-1H-indole-
5-carbaldehyde (13f).
This product was obtained as yellow
solid (77%) from 12f: mp 100–101 °C; IR: 1680 (CO) cmꢀ1
;
1H
NMR: d 1.05 (t, J = 7.2 Hz, 3H, SCH2CH3), 2.68 (s, 3H, CH3), 2.88
(q, J = 7.2 Hz, 2H, SCH2CH3), 6.96 (s, 1H, H-3), 7.59–7.82 (m, 4H,
H-3’, H-4’, H-5’, H-7), 7.96 (d, J = 8.0 Hz, 2H, H-2’, H-6’), 8.23 (1H,
d, J = 8.8 Hz, H-6), 10.68 (s, 1H, CHO); 13C NMR: d 14.6 (CH3),
15.2 (CH3), 30.8 (CH2), 109.1 (CH), 114.5 (CH), 123.2 (2 ꢁ CH),
126.3 (CH), 130.0 (2 ꢁ CH), 130.6 (C), 133.2 (C), 133.6 (C), 134.9
(CH), 137.4 (C), 138.4 (C), 139.6 (C), 191.5 (CHO). Anal.
C18H17NO3S2: C, 60.15; H, 4.77; N, 3.90. Found: C, 59.92; H, 4.50;
N, 3.80.
4.1.5.4. Ethyl-3-[4-(ethylsulfanyl)-2-methyl-1-phenylsulfonyl-
6,7-dihydro-1H-indole-5-yl] acrylate (14f).
was obtained as white solid (90%) after 1 h at room temperature
from 12f: mp 118–119 °C; IR: 1693 (CO) cmꢀ1 1H NMR: d 1.05
This product
;
(t, J = 7.3 Hz, 3H, SCH2CH3), 1.23 (t, J = 7.1 Hz, 3H, OCH2CH3), 2.36
(s, 3H, CH3), 2.63–2.73 (m, 4H, SCH2CH3, CH2), 3.06 (t, J = 8.7 Hz,
2H, CH2), 4.16 (q, J = 7.1 Hz, 2H, OCH2CH3), 6.04 (d, J = 15.7 Hz,
1H, CH), 6.29 (s, 1H, H-3), 7.65–7.79 (m, 3H, H-3’, H-4’, H-5’),
7.88 (d, J = 8.1 Hz, 2H, H-2’, H-6’), 8.25 (d, J = 15.7 Hz, 1H, CH).
13C NMR: d 14.2 (CH3), 14.5 (CH3), 14.6 (CH3), 21.6 (CH2), 25.7
(CH2), 28.2 (CH2), 59.8 (CH2), 111.0 (CH), 117.2 (CH), 123.1 (C),
126.3 (2 ꢁ CH), 130.2 (2 ꢁ CH), 131.6 (C), 132.1 (C), 133.1 (C),
133.3 (C), 134.7 (CH), 138.3 (C), 141.6 (CH), 166.7 (CO). Anal.
4.1.5. General procedure for the synthesis of
esters (14b–d,f,j,m) and (15b–d,f)
a–b unsaturated
Triethyl phosphonoacetate (1.32 mL, 6.74 mmol) was added to
a cooled suspension of t-BuOK (0.8 g, 6.74 mmol) in anhydrous

9680
P. Barraja et al. / Bioorg. Med. Chem. 16 (2008) 9668–9683
C22H25NO4S2: C, 61.23; H, 5.84; N, 3.25. Found: C, 61.50; H, 5.64; N,
3.00.
SCH2CH3), 1.26 (t, J = 7.4 Hz, 3H, OCH2CH3), 2.39 (s, 3H, CH3), 2.84
(q, J = 7.4 Hz, 2H, SCH2CH3), 4.19 (q, J = 7.4 Hz, 2H, OCH2CH3), 5.45
(s, 2H, CH2), 6.49 (d, J = 16.2 Hz, 1H, CH), 6.57 (s, 1H, H-3), 7.00
(d, J = 5.9 Hz, 2H, H-2’, H-6’), 7.22–7.36 (m, 3H, H-3’, H-4’, H-5’),
7.46 (d, J = 8.8 Hz, 1H, H-6), 7.62 (d, J = 8.8 Hz, 1H, H-7), 8.58 (d,
J = 16.2 Hz, 1H, CH). 13C NMR: d 12.4 (CH3), 14.2 (CH3), 14.9
(CH3), 29.8 (CH2), 45.9 (CH2), 59.7 (CH2), 101.3 (CH), 110.9 (CH),
116.1 (CH), 119.4 (CH), 126.1 (2ꢁ CH), 126.3 (C), 127.1 (CH),
128.6 (2ꢁ CH), 131.7 (C), 137.3 (C), 137.3 (C), 138.7 (C), 143.6
(CH), 165.7 (CO), 166.6 (CO). Anal. C23H25NO2S: C, 72.79; H, 6.64;
N, 3.69. Found: C, 72.54; H, 6.40; N, 3.46.
4.1.5.5. Ethyl 3-[1,3-dimethyl-2-ethoxycarbonyl-4-(ethylsulfa-
nyl)-6,7-dihydro-1H-indole-5-yl] acrylate (14j).
uct was obtained as white solid (85%) after
This prod-
1 h at room
temperature from 12j: mp 72–73 °C; IR: 1685 (CO), 1676 (CO)
cmꢀ1 1H NMR: d 1.07 (t, J = 7.3 Hz, 3H, SCH2CH3), 1.23 (t,
;
J = 7.0 Hz, 3H, OCH2CH3), 1.29 (t, J = 7.0 Hz, 3H, OCH2CH3), 2.51–
2.70 (m, 6H, 2ꢁ CH2, SCH2CH3), 2.62 (3H, s, CH3), 3.73 (3H, s,
CH3), 4.14 (q, J = 7.0 Hz, 2H, OCH2CH3), 4.25 (q, J = 7.0 Hz, 2H,
OCH2CH3), 6.00 (d, J = 15.7 Hz, 1H, CH), 8.42 (d, J = 15.7 Hz, 1H,
CH). 13C NMR: d 11.8 (CH3), 14.2 (2ꢁ CH3), 14.3 (CH3), 19.3 (CH2),
25.3 (CH2), 28.4 (CH2), 33.1 (CH3), 59.4 (CH2), 59.6 (CH2), 115.8
(CH), 116.6 (C), 120.2 (C), 125.6 (C), 132.6 (C), 136.0 (C), 140.3
(C), 142.3 (CH), 161.1 (CO), 166.0 (CO). Anal. C20H27NO4S: C,
63.63; H, 7.21; N, 3.71. Found: C, 63.36; H, 7.00; N, 3.98.
4.1.5.10. Ethyl 3-[4-(ethylsulfanyl)-2-methyl-1-phenylsulfonyl-
1H-indole-5-yl]acrylate (15f).
white solid (80%) after 1 h at room temperature from 13f: mp
81–82 °C; IR: 1703 (CO) cmꢀ1 1H NMR: d 1.00 (t, J = 7.2 Hz, 3H,
This product was obtained as
;
SCH2CH3), 1.27 (t, J = 6.9 Hz, 3H, OCH2CH3), 2.65 (s, 3H, CH3), 2.77
(q, J = 7.2 Hz, 2H, SCH2CH3), 4.22 (q, J = 6.9 Hz, 2H, OCH2CH3), 6.62
(d, J = 15.9 Hz, 1H, CH), 6.84 (s, 1 H, H-3), 7.59–7.94 (m, 6H, Ph,
H-6), 8.08 (d, J = 8.7 Hz, 1H, H-7), 8.43 (d, J = 15.9 Hz, 1H, CH). 13C
NMR: d 14.2 (CH3), 14.7 (CH3), 15.2 (CH3), 30.2 (CH2), 60.1 (CH2),
109.6 (CH), 114.6 (CH), 119.0 (CH), 122.8 (2ꢁ CH), 126.3 (CH),
126.8 (C), 130.0 (2ꢁ CH), 133.0 (C), 133.7 (C), 134.8 (CH), 136.2
(C), 137.6 (C), 138.8 (C), 144.0 (CH), 166.2 (CO). Anal. C22H23NO4S2:
C, 61.52; H, 5.40; N, 3.26. Found: C, 61.22; H, 5.72; N, 3.06.
4.1.5.6. Ethyl
1H-indole-2,5-diyl]acrylate (14m).
tained as white solid (85%) after 1 h at room temperature from
12l: mp 120–121 °C; IR: 1691 (CO), 1680 (CO) cmꢀ1 1H NMR: d
3-[1,3-dimethyl-4-(ethylsulfanyl)-6,7-dihydro-
This product was ob-
;
1.08 (t, J = 6.9 Hz, 3H, SCH2CH3), 1.11–1.24 (m, 6H, 2ꢁ OCH2CH3),
2.52 (s, 3H, CH3), 2.60–2.69 (m, 6H, SCH2CH3, 2 ꢁ CH2), 3.61 (s,
3H, CH3), 4.11–4.20 (m, 4H, 2ꢁ OCH2CH3), 5.96 (d, J = 15.6 Hz, 1H,
CH), 6.01 (d, J = 15.5 Hz, 1 H, CH), 7.57 (d, J = 15.9 Hz, 1H, CH),
8.37 (d, J = 15.9 Hz, 1H, CH). 13C NMR:
d
11.6 (CH3), 14.2
4.1.6. General procedure for the hydrolisis of the
unsaturated esters (14b–d,f,j,m) and (15b–d,f)
a–b
(2ꢁ CH3), 14.3 (CH3), 19.4 (CH2), 25.3 (CH2), 28.3 (CH2), 31.6
(CH3), 59.6 (2ꢁ CH2), 110.7 (CH), 115.3 (CH), 117.4 (C), 122.1 (C),
126.1 (C), 131.4 (CH), 132.2 (C), 135.9 (C), 139.4 (C), 142.3 (CH),
167.0 (CO), 167.2 (CO). Anal. C22H29NO4S: C, 65.48; H, 7.24; N,
3.47. Found: C, 65.15; H, 7.00; N, 3.62.
To a solution of KOH (0.34 g, 6 mmol) in 50% of acqueous etha-
nol (25 mL) the proper ester 14b–d,f,j,m or 15b–d,f (3 mmol) was
added and the solution was heated under reflux. After cooling the
solution was washed with ether and the acqueous layer was acid-
ified with 6 N HCl and the precipitate was collected. Recrystalliza-
tion from ethanol afforded the desired compounds.
4.1.5.7. Ethyl
indole-5-yl]acrylate (15b).
white solid (70%) after 20 min at room temperature from 13b:
mp 99–101 °C; IR: 1701 (CO) cmꢀ1 1H NMR: d 1.10 (t, J = 7.0 Hz,
3-[4-(ethylsulfanyl)-2-methyl-1-phenyl-1H-
This product was obtained as
4.1.6.1. 3-[4-(Ethylsulfanyl)-2-methyl-1-phenyl-6,7-dihydro-
;
1H-indole-5-yl]acrylic acid (16b).
This product was obtained
3H, SCH2CH3), 1.27 (t, J = 7.0 Hz, 3H, OCH2CH3), 2.30 (s, 3H, CH3),
2.88 (q, J = 7.0 Hz, 2H, SCH2CH3), 4.23 (q, J = 7.0 Hz, 2H, OCH2CH3),
6.49 (d, J = 16.1 Hz, 1H, CH), 6.70 (s, 1H, H-3), 7.02 (d, J = 8.5 Hz,
1H, H-6), 7.47 (dd, J = 8.0, 1.5 Hz, 2H, H-2’, H-6’), 7.54–7.66 (m,
4H, H-3’, H-4’, H-5’, H-7), 8.59 (d, J = 16.1 Hz, 1H, CH). 13C NMR:
d 13.0 (CH3), 14.2 (CH3), 15.0 (CH3), 29.9 (CH2), 59.8 (CH2), 102.1
(CH), 110.9 (CH), 116.5 (CH), 120.0 (CH), 126.5 (C), 127.6
(2 ꢁ CH), 128.3 (CH), 129.3 (C), 129.8 (2ꢁ CH), 131.9 (C), 136.3
(C), 137.8 (C), 138.7 (C), 143.4 (CH), 166.5 (CO). Anal. C22H23NO2S:
C, 72.30; H, 6.34; N, 3.83. Found: C, 72.00; H, 6.04; N, 4.03.
as white solid (72%) after 30 min reflux from 14b: mp 178–180 °C;
IR: 3539–3253 (OH), 1668 (CO) cmꢀ1; 1H NMR: d 1.15 (t, J = 7.4 Hz,
3H, SCH2CH3), 2.07 (s, 3H, CH3), 2.46–2.64 (m, 4H, 2ꢁ CH2), 2.82 (q,
J = 7.4 Hz, 2H, SCH2CH3), 5.81 (d, J = 16.2 Hz, 1H, CH), 6.16 (s, 1H, H-
3), 7.35 (d, J = 7.4 Hz, 2H, H-2’, H-6’), 7.43–7.58 (m, 3H, H-3’, H-4’,
H-5’), 8.29 (d, J = 16.2 Hz, 1H, CH), 12.02 (s, 1H, OH). 13C NMR: d:
12.5 (CH3), 14.9 (CH3), 20.2 (CH2), 25.9 (CH2), 28.1 (CH2), 105.3
(CH), 115.2 (CH), 119.0 (C), 127.1 (2 ꢁ CH), 128.0 (CH), 128.8 (C),
129.2 (C), 129.3 (2ꢁ CH), 131.3 (C), 135.9 (C), 136.7 (C), 142.3
(CH), 168.4 (CO). Anal. C20H21NO2S: C, 70.77; H, 6.24; N, 4.13.
Found: C, 70.52; H, 6.04; N, 4.00.
4.1.5.8. Ethyl
3-[1,2-dimethyl-4-(ethylsulfanyl)-1H-indole-5-
yl]acrylate (15c).
This product was obtained as white solid
4.1.6.2. 3-[1,2-Dimethyl-4-(ethylsulfanyl)-6,7-dihydro-1H-
(92%) after 30 min at room temperature from 13c: mp 73–75 °C;
indole-5-yl]acrylic acid (16c).
white solid (93%) after 1 h reflux from 14c: mp 160–162 °C; IR:
3197 (OH), 1668 (CO) cmꢀ1 1H NMR: d 1.08 (t, J = 7.4 Hz, 3H,
This product was obtained as
IR: 1697 (CO) cmꢀ1 1H NMR: d 1.03 (t, J = 7.4 Hz, 3H, SCH2CH3),
;
1.26 (t, J = 7.4 Hz, 3H, OCH2CH3), 2.43 (s, 3H, CH3), 2.81 (q,
J = 7.4 Hz, 2H, SCH2CH3), 3.69 (s, 3H, CH3), 4.20 (q, J = 7.4 Hz, 2H,
OCH2CH3), 6.47–6.54 (m, 2H, H-3, CH), 7.48 (d, J = 8.8 Hz, 1H, H-
6), 7.64 (d, J = 8.8 Hz, 1H, H-7), 8.57 (d, J = 16.2 Hz, 1H, CH). 13C
NMR: d 12.4 (CH3), 14.2 (CH3), 14.9 (CH3), 29.5 (CH3), 29.7 (CH2),
59.7 (CH2), 100.4 (CH), 110.6 (CH), 115.8 (CH), 119.0 (CH), 126.0
(C), 128.2(C), 131.5 (C), 137.4 (C), 139.0 (C), 143.7 (CH), 166.7
(CO). Anal. C17H21NO2S: C, 67.30; H, 6.98; N, 4.62. Found: C,
67.10; H, 7.18; N, 4.32.
;
SCH2CH3), 2.17 (s, 3H, CH3), 2.58–2.70 (m, 4H, 2 ꢁ CH2), 2.81 (q,
J = 7.4 Hz, 2H, SCH2CH3), 3.69 (s, 3H, CH3), 5.78 (d, J = 15.6 Hz, 1H,
CH), 5.94 (s, 1H, H-3), 8.27 (d, J = 15.6 Hz, 1H, CH), 11.94 (s, 1H,
OH). 13C NMR: d 11.8 (CH3), 14.9 (CH3), 19.5 (CH2), 25.7 (CH2),
27.9 (CH2), 29.9 (CH3), 103.9 (CH), 114.3 (CH), 117.8 (C), 127.7
(C), 128.6 (C), 131.4 (C), 136.5 (C), 142.6 (CH), 168.6 (CO). Anal.
C15H19NO2S: C, 64.95; H, 6.90; N, 5.05. Found: C, 64.65; H, 7.10;
N, 5.35.
4.1.5.9. Ethyl
indole-5-yl]acrylate (15d).
white solid (96%) after 1 h at room temperature from 13d: mp
74–75 °C; IR: 1699 (CO) cmꢀ1 1H NMR: d 1.07 (t, J = 7.4 Hz, 3H,
3-[1-benzyl-4-(ethylsulfanyl)-2-methyl-1H-
4.1.6.3. 3-[1-Benzyl-4-(ethylsulfanyl)-2-methyl-6,7-dihydro-
This product was obtained as
1H-indole-5-yl]acrylic acid (16d).
as white solid (75%) after 40 min reflux from 14d: mp 159–161 °C;
IR: 3433 (OH), 1664 (CO) cmꢀ1 1H NMR: d 1.12 (t, J = 7.4 Hz, 3H,
This product was obtained
;
;

P. Barraja et al. / Bioorg. Med. Chem. 16 (2008) 9668–9683
9681
SCH2CH3), 2.11 (s, 3H, CH3), 2.60–2.66 (m, 4H, 2 ꢁ CH2), 2.79 (q,
4.1.6.8. 3-[1,2-Dimethyl-4-(ethylsulfanyl)-1H-indole-5-yl]acri-
J = 7.4 Hz, 2H, SCH2CH3), 5.10 (s, 2H, CH2), 5.79 (d, J = 16.2 Hz, 1H,
CH), 6.04 (s, 1H, H-3), 6.99 (d, J = 7.4 Hz, 2H, H-2’, H-6’), 7.22–
7.38 (m, 3H, H-3’, H-4’, H-5’), 8.27 (d, J = 16.2 Hz, 1H, CH), 11.93
(s, 1H, OH). 13C NMR: d 11.8 (CH3), 14.8 (CH3), 19.7 (CH2), 25.8
(CH2), 28.0 (CH2), 46.2 (CH2), 104.6 (CH), 114.6 (CH), 118.4 (C),
126.0 (2 ꢁ CH), 127.1 (CH), 128.1 (C), 128.5 (C), 128.7 (2 ꢁ CH),
131.4 (C), 136.2 (C), 137.9 (C), 142.4 (CH), 168.5 (CO). Anal.
C21H23NO2S: C, 71.36; H, 6.56; N, 3.96. Found: C, 71.60; H, 6.36;
N, 3.70.
lyc acid (17c).
This product was obtained as white solid (93%)
after 1 h reflux from 15c: mp 236–238 °C; IR: 3417 (OH), 1676 (CO)
cmꢀ1; 1H NMR: d 1.03 (t, J = 7.4 Hz, 3H, SCH2CH3), 2.43 (s, 3H, CH3),
2.80 (q, J = 7.4 Hz, 2H, SCH2CH3), 3.69 (s, 3H, CH3), 6.37–6.46 (m,
2H, H-3, CH), 7.46 (d, J = 7.4 Hz, 1H, H-6), 7.61 (d, J = 7.4 Hz, 1H,
H-7), 8.52 (d, J = 16.2 Hz, 1H, CH), 12.21 (s, 1H, OH). 13C NMR: d
12.4 (CH3), 14.9 (CH3), 29.5 (CH3), 29.7 (CH2), 100.3 (CH), 110.5
(CH), 116.9 (CH), 118.9 (CH), 125.7 (C), 128.4 (C), 131.5 (C), 137.3
(C), 138.9 (C), 143.3 (CH), 168.0 (CO). Anal. C15H17NO2S: C, 65.43;
H, 6.22; N, 5.09. Found: C, 65.20; H, 6.40; N, 4.82.
4.1.6.4. 3-[4-(Ethylsulfanyl)-2-methyl-1-phenylsulfonyl-6,7-
dihydro-1H-indole-5-yl]acrylic acid (16f).
obtained as white solid (90%) after 40 min reflux from 14f: mp
203–204 °C; IR: 3620–3355 (OH), 1668 (CO) cmꢀ1 1H NMR: d
This product was
4.1.6.9. 3-[1-Benzyl-4-(ethylsulfanyl)-2-methyl-6,7-1H-indole-
5-yl]acrilic acid (17d). This product was obtained as white solid
(85%) after 1 h reflux from 15d: mp 215–217 °C; IR: 3477 (OH),
;
1.05 (t, J = 7.1 Hz, 3H, SCH2CH3), 2.36 (s, 3H, CH3), 2.62–2.72 (m,
4H, 2 ꢁ CH2), 3.38 (q, J = 7.1 Hz, 2H, SCH2CH3), 5.97 (d,
J = 15.9 Hz, 1H, CH), 6.29 (s, 1H, H-3), 7.65–7.79 (m, 3H, H-3’, H-
4’, H-5’), 7.87 (d, J = 7.7 Hz, 2H, H-2’, H-6’), 8.19 (d, J = 15.9 Hz,
1H, CH), 12.31 (s, 1H, OH). 13C NMR: d 14.5 (CH3), 14.6 (CH3),
21.6 (CH2), 25.7 (CH2), 28.2 (CH2), 111.0 (CH), 118.4 (CH), 123.1
(C), 126.2 (2 ꢁ CH), 130.2 (2ꢁ CH), 131.6 (C), 131.9 (C), 132.3 (C),
133.4 (C), 134.7 (CH), 138.4 (C), 141.2 (CH), 168.1 (CO). Anal.
C20H21NO4S2: C, 59.53; H, 5.25; N, 3.47. Found: C, 59.26; H, 5.52;
N, 3.27.
1670 (CO) cmꢀ1 1H NMR: d 1.07 (t, J = 7.4 Hz, 3H, SCH2CH3), 2.39
;
(s, 3H, CH3), 2.84 (q, J = 7.4 Hz, 2H, SCH2CH3), 5.45 (s, 2H, CH2), 6.40
(d, J = 16.2 Hz, 1H, CH), 6.57 (s, 1H, H-3), 7.01 (d, J = 6.9 Hz, 2H, H-
2’, H-6’), 7.23–7.34 (m, 3H, H-3’, H-4’, H-5’), 7.46 (d, J = 8.8 Hz, 1H,
H-6), 7.59 (d, J = 8.8 Hz, 1H, H-7), 8.53 (d, J = 16.2 Hz, 1H, CH),
12.24 (s, 1H, OH). 13C NMR: d 12.4 (CH3), 14.9 (CH3), 29.8 (CH2),
45.9 (CH2), 101.3 (CH), 110.9 (CH), 117.2 (CH), 119.4 (CH), 126.0
(C), 126.1 (2ꢁ CH), 127.2 (CH), 128.6 (2 ꢁ CH), 128.8 (C), 131.7 (C),
137.2 (C), 137.9 (C), 138.7 (C), 143.2 (CH), 167.9 (CO). Anal.
C21H21NO2S: C, 71.77; H, 6.02; N, 3.99. Found: C, 72.00; H, 6.25; N, 4.06.
4.1.6.5. 3-[1,3-Dimethyl-2-ethoxycarbonyl-4-(ethylsulfanyl)-
4.1.6.10. 3-[4-(Ethylsulfanyl)-2-methyl-1-phenylsulfonyl-1H-
6,7-dihydro-1H-indole-5-yl]acrylic acid (16j).
was obtained as white solid (92%) after 1 h reflux from 14j: mp
144–146 °C; IR: 3410 (OH), 1691 (CO), 1682 (CO) cmꢀ1 1H NMR:
This product
indole-5-yl]acrilic acid (17f).
white solid (65%) after 1 h reflux from 15f: mp 204–205 °C; IR:
3427 (OH), 1684 (CO) cmꢀ1 1H NMR: d 1.01 (t, J = 7.1 Hz, 3H,
This product was obtained as
;
;
d 1.07 (t, J = 7.2 Hz, 3H, SCH2CH3), 1.29 (t, J = 7.2 Hz, 3H, OCH2CH3),
2.41–2.70 (m, 6H, 2 ꢁ CH2, SCH2CH3) 2.65 (s, 3H, CH3), 3.72 (s, 3H,
CH3), 4.26 (q, J = 7.2 Hz, 2H, OCH2CH3), 5.94 (d, J = 15.7 Hz, 1H, CH),
8.36 (d, J = 15.7 Hz, 1H, CH), 12.21 (s, 1H, OH). 13C NMR: d 11.8
(CH3), 14.3 (2 ꢁ CH3), 19.3 (CH2), 25.3 (CH2), 28.3 (CH2), 33.1
(CH3), 59.4 (CH2), 116.6 (C), 116.9 (CH), 120.2 (C), 125.5 (C),
132.7 (C), 135.2 (C), 139.9 (C), 142.0 (CH), 161.2 (CO), 168.4 (CO).
Anal. C18H23NO4S: C, 61.87; H, 6.63; N, 4.01. Found: C, 62.02; H,
6.43; N, 4.36.
SCH2CH3), 2.65 (s, 3H, CH3), 2.77 (q, J = 7.1 Hz, 2H, SCH2CH3), 6.54
(d, J = 15.9 Hz, 1H, CH), 6.84 (s, 1H, H-3), 7.58–7.85 (m, 4H, H-3’,
H-4’, H-5’, H-6), 7.93 (d, J = 7.5 Hz, 2H, H-2’, H-6’), 8.10 (d,
J = 8.4 Hz, 1H, H-7), 8.39 (d, J = 15.9 Hz, 1H, CH), 12.50 (s, 1H,
OH). 13C NMR: d 14.7 (CH3), 15.2 (CH3), 30.2 (CH2), 109.7 (CH),
114.6 (CH), 120.2 (CH), 122.7 (CH), 126.3 (2 ꢁ CH), 126.5 (C),
130.0 (2ꢁ CH), 133.2 (C), 133.7 (C), 134.8 (CH), 136.2 (C), 137.6
(C), 138.8 (C), 141.5 (CH), 167.6 (CO). Anal. C20H19NO4S2: C,
59.83; H, 4.77; N, 3.49. Found: C, 59.69; H, 4.96; N, 3.70.
4.1.6.6. 3-[1,3-Dimethyl-4-(ethylsulfanyl)-6,7-dihydro-1H-
4.1.7. General procedure for the cyclization of the acids 16b–
d,f,j,n and 17b–d,f
A mixture of the proper acid 16b–d,f,j,n or 17b–d,f (3 mmol)
and PPA (20g) was heated for the proper time. Addition of crushed
ice gave a solid precipitate which was filtered off, dried and puri-
fied by column chromatography (DCM: EtOAc 95:5).
indole-2,5-diyl]acrylic acid (16n).
as white solid (92%) after 1 h reflux from 14m: mp 188–189 °C; IR:
2966–2485 (vb, OH), 1668 (CO), 1601 (CO) cmꢀ1 1H NMR: d 1.08
This product was obtained
;
(t, J = 7.2 Hz, 3H, SCH2CH3), 2.52 (s, 3H, CH3), 2.60–2.79 (m, 6H,
SCH2CH3, 2ꢁ CH2), 3.60 (s, 3H, CH3), 5.93 (d, J = 15.5 Hz, 2H,
2 ꢁ CH), 7.57 (d, J = 15.7 Hz, 1H, CH), 8.37 (d, J = 15.7 Hz, 1H, CH),
12.06 (s, 1H, OH), 12.21 (s, 1H, OH). 13C NMR: d 11.6 (CH3), 14.3
(CH3), 19.5 (CH2), 25.4 (CH2), 28.4 (CH2), 31.5 (CH3), 112.0 (CH),
116.5 (CH), 117.4 (C), 121.5 (C), 126.1 (C), 131.3 (CH), 132.1 (C),
135.3 (C), 138.9 (C), 142.1 (CH), 168.4 (2ꢁ CO). Anal. C18H21NO4S:
C, 62.23; H, 6.09; N, 4.03. Found: C, 62.00; H, 5.85; N, 4.34.
4.1.7.1. 8-Methyl-7-phenyl-5,6-dihydrothiopyrano[2,3-e]
indole-2(5H)-one (5b).
low solid (30%) by heating 16b at 95–100 °C for 4 h: mp 175–
177 °C; IR: 1703 (CO) cmꢀ1 1H NMR (CDCl3): d 2.09 (s, 3H, CH3),
This product was obtained as a yel-
;
2.55–2.77 (m, 4H, H-5, H-6), 5.92 (d, J = 9.5 Hz, 1H, H-3), 6.32 (s,
1H, H-9), 7.19–7.27 (m, 3H, H-2’, H-6’, H-4), 7.45–7.55 (m, 3H, H-
3’, H-4’, H-5’). 13C NMR (CDCl3): d 12.7 (CH3), 21.3 (CH2), 25.6
(CH2), 102.2 (CH), 105.4 (C), 107.3 (CH), 113.4 (C), 127.3
(2 ꢁ CH), 128.4 (CH), 129.4 (2 ꢁ CH), 131.4 (C), 135.1 (C), 137.1
(C), 145.9 (CH), 157.6 (C), 163.1 (CO). Anal. C18H15NOS: C, 73.69;
H, 5.15; N, 4.77. Found: C, 73.50; H, 5.36; N, 4.50.
4.1.6.7. 3-[4-(Ethylsulfanyl)-2-methyl-1-phenyl-1H-indole-5-
yl]acrylic acid (17b).
lid (70%) after 30 min reflux from 15b: mp 240–242 °C; IR: 3539–
3361 (OH), 1676 (CO) cmꢀ1 1H NMR: d 1.11 (t, J = 7.1 Hz, 3H,
This product was obtained as white so-
;
SCH2CH3), 2.30 (s, 3H, CH3), 2.88 (q, J = 7.1 Hz, 2H, SCH2CH3), 6.40
(d, J = 16.5 Hz, 1H, CH), 6.70 (s, 1H, H-3), 7.03 (d, J = 8.7 Hz, 1H,
H-6), 7.47 (d, J = 6.3 Hz, 2H, H-2’, H-6’), 7.54–7.67 (m, 4H, H-3’,
H-4’, H-5’, H-7), 8.54 (d, J = 16.5 Hz, 1H, CH), 12.31 (s, 1H, OH).
13C NMR: d 13.0 (CH3), 15.0 (CH3), 29.9 (CH2), 102.1 (CH), 110.9
(CH), 117.6 (CH), 120.0 (CH), 126.2 (C), 127.5 (2ꢁ CH), 128.3
(CH), 129.6 (C), 129.8 (2ꢁ CH), 131.9 (C), 136.4 (C), 137.7 (C),
138.6 (C), 143.0 (CH), 167.9 (CO). Anal. C20H19NO2S: C, 71.19; H,
5.68; N, 4.15. Found: C, 70.96; H, 5.24; N, 4.33.
4.1.7.2. 7,8-Dimethyl-5,6-dihydrothiopyrano[2,3-e]indole-
2(5H)-one (5c).
(40%) by heating 16c at 90–100 °C for 4 h: mp 172–173 °C; IR:
1620 (CO) cmꢀ1 1H NMR: d 2.22 (s, 3H, CH3), 2.77–2.79 (m, 4H,
This product was obtained as a yellow solid
;
H-5, H-6), 3.43 (s, 3H, CH3), 5.99 (s, 1H, H-9), 6.16 (d, J = 10.2 Hz,
1H, H-3), 7.22 (d, J = 10.2 Hz, 1H, H-4). 13C NMR: d 12.1 (CH3),
20.8 (CH2), 29.4 (CH2), 30.3 (CH3), 101.5 (CH), 108.2 (C), 116.5

9682
P. Barraja et al. / Bioorg. Med. Chem. 16 (2008) 9668–9683
(C), 117.4 (CH), 120.2 (C), 130.8 (C), 131.7 (C), 144.6 (CH), 184.4
(CO). Anal. C13H13NOS: C, 67.50; H, 5.66; N, 6.06. Found: C,
67.80; H, 5.69; N, 6.40.
(Sigma–Aldrich Milano Italy), human intestinal adenocarcinoma
cells (LoVo) were grown in Ham’s F12 medium (Sigma–Aldrich
Milano Italy), all supplemented with 115 units/ml of penicillin G
(Invitrogen, Milano, Italy), 115
lg/ml streptomycin (Invitrogen,
4.1.7.3. 7-Benzyl-8-methyl-5,6-dihydrothiopyrano[2,3-
Milano Italy) and 10% fetal calf serum (Invitrogen, Milano, Italy).
e]indole-2(5H)-one (5d). This product was obtained as a yellow
Individual wells of a 96-well tissue culture microtiter plate (Falcon
solid (30%) by heating 16d at 100–130 °C for 30 min: mp 175–
BD) were inoculated with 100 ll of complete medium containing
177 °C; IR: 1684 (CO) cmꢀ1
;
1H NMR (CDCl3): d 2.20 (s, 3H, CH3),
8 ꢁ 103 HL-60, Jurkat cells or 5 ꢁ 103 HT-1080, LoVo and NCTC-
2544 cells. Plates were incubated at 37 °C in a humidified 5% CO2
incubator for 18 h prior to the experiments. Drugs were dissolved
in DMSO and then were diluted with Hank’s Balanced Salt Solution
(HBSS pH 7.2) for phototoxicity experiments. After medium re-
2.68–2.76 (m, 4H, H-5, H-6), 4.06 (s, 2H, CH2), 5.86 (d, J = 8.8 Hz, 1H,
H-3), 7.11–7.33 (m, 7H, Ph, H-4, H-9). 13C NMR (CDCl3): d 11.1
(CH3), 21.7 (CH2), 25.8 (CH2), 30.7 (CH2), 105.2 (C), 106.8 (CH), 111.7
(C), 117.1 (C), 120.4 (C), 125.2 (CH), 128.2 (2ꢁ CH), 128.3 (CH),
128.6 (2ꢁ CH), 132.7 (C), 142.1 (C), 146.0 (CH), 162.0 (CO). Anal.
C19H17NOS:C,74.24;H,5.57;N,4.56.Found:C,74.14;H,5.66;N,4.22.
moval, 100 ll of the drug solution were added to each well and
incubated at 37 °C for 30 min and then irradiated. After irradiation,
the solution was replaced with the medium and plates were incu-
bated for 72 h. Cell viability was assayed by the MTT [(3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)] test, as
described previously.5b Analogous experiments were performed
in the presence of various additives.
4.1.7.4. 8-Methyl-7-phenylsulfonyl-5,6-dihydrothiopyrano[2,3-
e]indol-2(5H)-one (5f).
low solid (40%) by heating 16f at 70–90 °C for 20 min: mp 175–
177 °C; IR: 1685 (CO) cmꢀ1 1H NMR: d 2.59 (s, 3H, CH3), 2.64–
This product was obtained as a yel-
;
2.68 (m, 4H, H-5, H-6), 5.93 (d, J = 9.3 Hz, 1H, H-3), 7.43 (d,
J = 9.3 Hz, 1H, H-4), 7.47–7.63 (m, 4H, H-3’, H-4’, H-5’, H-9), 8.18
(d, J = 6.6 Hz, 2H, H-2’, H-6’). 13C NMR: d 12.6 (CH3), 20.4 (CH2),
24.6 (CH2), 107.1 (C), 108.2 (CH), 110.0 (C), 114.9 (C), 127.6
(2 ꢁ CH), 128.7 (3 ꢁ CH), 132.6 (CH), 135.3 (C), 143.5 (C), 146.7
(CH), 154.5 (C), 161.0 (CO). Anal. C18H15NO3S2: C, 60.48; H, 4.23;
N, 3.92. Found: C, 60.18; H, 4.00; N, 4.12.
4.2.3. Externalization of phosphatidylserine
Surface exposure of phosphatidylserine (PS) by apoptotic cells
was measured by flow cytometry with a Coulter Cytomics FC500
(Coulter) by adding Annexin V-FITC to cells according to the man-
ufacturer’s instructions (Annexin-V Fluos Roche Diagnostic).
Simultaneously, cells were stained with PI. Excitation was set at
488 nm and the emission filters were at 525 nm for FITC fluores-
cence and in the range 560–680 nm for PI fluorescence.
4.1.7.5. 8-Methyl-7-phenyl-thiopyrano[2,3-e]indol-2(7H)-one
(6b).
heating 17b at 110–130 °C for 30 min. mp 175–177 °C; IR: 196–
198 °C; IR: 1624 (CO) cmꢀ1 1H NMR: d 2.31 (s, 3H, CH3), 6.47 (d,
J = 10.7 Hz, 1H, H-3), 6.65 (s, 1H, H-9), 7.09 (d, J = 8.8 Hz, 1H, H-
5), 7.48–7.53 (m, 3H, H-2’, H-6’, H-6), 7.58–7.70 (m, 3H, H-3’, H-
4’, H-5’), 8.10 (d, J = 10.7 Hz, 1H, H-4). 13C NMR: d 13.0 (CH3),
99.3 (CH), 109.5 (CH), 118.4 (C), 119.9 (CH), 123.3 (C), 125.2
(CH), 127.7 (2ꢁ CH), 128.7 (CH), 128.9 (C), 129.9 (2ꢁ CH), 136.1
(C), 136.8 (C), 139.2 (C), 146.5 (CH), 183.2 (CO). Anal. C18H13NOS:
C, 74.20; H, 4.50; N, 4.81. Found: C, 74.00; H, 4.24; N, 5.00.
This product was obtained as a yellow solid (32%) by
4.2.4. Assessment of mitochondrial changes
;
The mitochondrial membrane potential was measured with the
lipophilic cation 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimida-
zol-carbocyanine (JC-1, Molecular Probes Eugene, OR, USA) as de-
scribed.37 Briefly, after different times from irradiation, cells were
collected by centrifugation and resuspended in HBSS containing JC-
1 at the concentration of 2.5 lM. Cells were then incubated at 37 °C
for 10 min, centrifuged and resuspended again in HBSS. The fluores-
cencewasdirectlyrecordedbytheflowcytometer(CoulterCytomics
FC500).
4.1.7.6. 7,8-Dimethyl-thiopyrano[2,3-e]indol-2(7H)-one
The production of Reactive Oxygen Species (ROS) and the oxida-
tion of cardiolipin were measured by flow cytometry using Hydro-
ethidine (HE), Dihydrochlorofluorescein-diacetate (DCFDA,
Molecular Probes Eugene, OR, USA) and 10-N-nonyl-acridine or-
ange (NAO, Molecular Probes Eugene, OR, USA), respectively, as de-
scribed previously.37 After 24 h from the irradiation, cells were
collected by centrifugation and resuspended in HBSS containing
the fluorescence probes HE, DCFDA or NAO at the concentration
(6c).
This product was obtained as a yellow solid (30%) by heat-
ing17cat154–156 °Cfor4 h:mp175–177 °C;IR:1628(CO)cmꢀ1;1H
NMR:d2.45(s,3H,CH3),3.75(s,3H,CH3),6.41(d,J = 10.4Hz,1H,H-3),
6.42 (s, 1H, H-9), 7.51–7.60 (m, 2H, H-5, H-6), 8.09 (d, J = 10.4 Hz, 1H,
H-4). 13C NMR: d 12.3 (CH3), 29.7 (CH3), 97.6 (CH), 109.2 (CH), 117.5
(C), 119.2 (CH), 122.9 (C), 124.2 (CH), 128.5 (C), 136.3 (C), 139.4 (C),
146.6 (CH), 183.2 (CO). Anal. C13H11NOS: C, 68.10; H, 4.84; N, 6.11.
Found: C, 68.40; H, 4.60; N, 6.00.
of 0.1 lM, 2.5 lM and 5.0 lM, respectively. Cells were then incu-
Attempts to cyclize acids 16j,n and 17d,f under the described
conditions led to very complex reaction mixture from which it
was not possible to isolate the corresponding tricyclic compounds.
bated at 37 °C for 30 min, centrifuged and resuspended again in
HBSS. The fluorescence was directly recorded with the flow cytom-
eter using the 488 nm wavelength as excitation and the emission
at 585 for HE and at 530 nm for NAO and DCFDA.
4.2. Biology
4.2.5. Lysosomal stability assessment
4.2.1. Irradiation procedure
Jurkat cells were assessed for lysosomal stability using the acri-
dine orange (AO) uptake method.22,23 After the irradiation in the
presence of test compounds, cells were stained with AO at the con-
centration of 5 lg/mL at 37 °C for 15 min, washed and then ana-
lyzed by flow cytometry using as excitation wavelength 488 nm
HPW 125 Philips lamps, mainly emitting at 365 nm, were used
for irradiation experiments. The spectral irradiance of the source
was 4.0 mW cmꢀ2 as measured, at the sample level, by a Cole–
Parmer Instrument Company radiometer (Niles, IL), equipped with
a 365-CX sensor.
and detecting the emission at 546 nm.
4.2.2. Cellular phototoxicity
4.2.6. GSH assay
Human promyelocytic leukaemia cells (HL-60) and human lym-
phoblastoid cells (Jurkat) were grown in RPMI-1640 medium (Sig-
ma–Aldrich Milano Italy), human fibrosarcoma cells (HT-1080) and
human keratinocytes (NCTC-2544) were grown in DMEM medium
Intracellular reduced glutathione (GSH) levels were assessed
using
a specific probe Monobromobimane (mBBr, Molecular
Probes Eugene, OR, USA). Cells were harvested after 24 h from
the irradiation and then incubated for 10 min at 37 °C with mBBr

P. Barraja et al. / Bioorg. Med. Chem. 16 (2008) 9668–9683
9683
at the concentration of 50
cytometry (FACS Vintage Becton–Dickinson; kex = 354, kem = 450).
l
M. Analysis were performed by flow
TAE buffer (0.04 M Tris–acetate, 1 mM EDTA) at 70 V for 4 h. The
quantification of DNA bands was carried out as described previ-
ously.41 Analogous experiments were performed in the presence
of different scavengers.
4.2.7. Caspase 3 assay
Jurkat cells were irradiated in the presence of the test com-
pounds as described above. After 24 h, cells were harvested,
washed and resuspended in a lysis buffer. The N-acetyl-Asp-Glu-
Val-Asp-pNA, (DEVD-pNA) cleavage activity was measured using
a caspase-3 assay kit (Sigma–Aldrich Milano, Italy) and the recom-
mended protocol was followed. The formation of p-nitroanilide
(pNA) was measured at 405 nm using a microtiter plate reader
(BIORAD, Milano Italy). Data were expressed as fold increase of
enzymatic activity normalized to the total protein content in com-
parison to the non-irradiated controls.
Acknowledgment
This work was financially supported by Ministero dell’Istruzi-
one dell’Università e della Ricerca.
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