Synthesis and antiproliferative mechanism of action of pyrrolo[3′,2′:6,7] cyclohepta[1,2-d]pyrimidin-2-amines as singlet oxygen photosensitizers

Article abstract of DOI:10.1016/j.ejmech.2016.07.051

A new series of pyrrolo[3′,2′:6,7]cyclohepta[1,2-d]pyrimidin-2-amines, was conveniently prepared using a versatile and high yielding multistep sequence. A good number of derivatives was obtained and the cellular photocytotoxicity was evaluated in?vitro against three different human tumor cell lines with EC₅₀(0.08–4.96?μM) values reaching the nanomolar level. Selected compounds were investigated by laser flash photolysis. The most photocytotoxic derivative, exhibiting a fairly long-lived triplet state (τ ～ 7?μs) and absorbance in the UV–Vis, was tested in the photo-oxidations of 9,10-anthracenedipropionic acid (ADPA) by singlet oxygen. The photosentizing properties are responsible for the compounds’ ability to photoinduce massive cell death with involvement of mitochondria.

Full text of DOI:10.1016/j.ejmech.2016.07.051

European Journal of Medicinal Chemistry 123 (2016) 447e461
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and antiproliferative mechanism of action of pyrrolo
[30,20:6,7] cyclohepta[1,2-d]pyrimidin-2-amines as singlet oxygen
photosensitizers
a
,
,
Virginia Spano 1, Ilaria Frasson b 1, Daniele Giallombardo a, Filippo Doria c,
Barbara Parrino a, Anna Carbone a, Alessandra Montalbano a, Matteo Nadai b,
Patrizia Diana a, Girolamo Cirrincione a, Mauro Freccero c, Sara N. Richter b,
ꢀ
,
*
Paola Barraja a
a
ꢀ
Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF), Universita degli Studi di Palermo, Via Archirafi 32, 90123 Palermo,
Italy
b
c
ꢀ
Dipartimento di Medicina Molecolare, Universita degli Studi di Padova, Via Gabelli 63, 35121 Padova, Italy
ꢀ
Dipartimento di Chimica, Universita degli Studi di Pavia, Viale Taramelli 10, 27100 Pavia, Italy
a r t i c l e i n f o
a b s t r a c t
A new series of pyrrolo[30,20:6,7]cyclohepta[1,2-d]pyrimidin-2-amines, was conveniently prepared using
a versatile and high yielding multistep sequence. A good number of derivatives was obtained and the
cellular photocytotoxicity was evaluated in vitro against three different human tumor cell lines with EC50
Article history:
Received 25 May 2016
Received in revised form
20 July 2016
Accepted 21 July 2016
Available online 25 July 2016
(0.08e4.96
m
M) values reaching the nanomolar level. Selected compounds were investigated by laser
~ 7 s)
flash photolysis. The most photocytotoxic derivative, exhibiting a fairly long-lived triplet state (
t
m
and absorbance in the UVeVis, was tested in the photo-oxidations of 9,10-anthracenedipropionic acid
(ADPA) by singlet oxygen. The photosentizing properties are responsible for the compounds' ability to
photoinduce massive cell death with involvement of mitochondria.
Keywords:
Pyrrolo[30,20:6,7]cyclohepta[1,2-d]
pyrimidin-2-amines
© 2016 Elsevier Masson SAS. All rights reserved.
Photodynamic therapy
Antiproliferative activity
Photosensitizing agents
Reactive oxygen species
1. Introduction
administration followed by light activation at a specific wavelength,
induces a series of photobiologic processes caused by reactive in-
In recent years, many efforts have been done to search for new
compounds with antitumor activity [1e6]. Several naturally
occurring and synthetic structures are capable of photoactivation,
with generation of reactive species or photosensitizing activity.
However, only for a limited number of photoprecursors the pho-
toactivation enables specific reactions with photochemically
generated reactive species [7e9]. In general, the activity as photo-
sensitizers (PSs) producing ROS (reactive oxygen species) is a much
more frequently exploited process in photodynamic therapy (PDT).
These photosensitizers have found application in the treatment of a
wide variety of disorders, among which tumors. In fact, their
termediates and end-products responsible for rapid and irrevers-
ible cyto and vasculo toxicity to tumor tissues. All photosensitizers
applied in experimental and clinical photochemotherapy have
broad absorption spectra stretching from ultraviolet up to
600e800 nm [10]. Even if sensitizers with strong absorbances in
the spectral range 600e800 nm are preferred for photo-
chemotherapy for a deeper penetration of light into tissues, many
tumors and skin disorders are localized superficially. In such cases,
light penetration into tissue is not a limiting factor. Dolmans and
coworkers, in their review on PDT [11], referred about photo-
chemical reactions of furocoumarins activation following UV irra-
diation, called photochemotherapy, based on different pathways,
involving photoaddition to DNA. Their mechanism of action relied
mainly on photoinduction of mono-, bis-functional DNA lesions
and photodamage of cell membrane constituents leading to
* Corresponding author.
E-mail address: paola.barraja@unipa.it (P. Barraja).
The authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2016.07.051
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

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V. Spano et al. / European Journal of Medicinal Chemistry 123 (2016) 447e461
O
X
O
O
X
H
N
H
S
N
X
S
N
N
R
N
R
X
R
1
2
3
R1
O
O
Y
X
H
N
X
H
N
N
R
N
N
N R
N
R
4
5
6
Y = C=O or C-R1
X-X = CH2-CH2 or CH = CH
Chart 1. Structures of thiopyrano[2,3-e]indol-2-ones (1), pyrrolo[30,20:4,5]thiopyrano[3,2-b]pyridin-2-ones (2), pyrrolo[2,3-h]quinolin-2-ones (3), pyrrolo[3,4-h]quinolin-2-ones
(4), pyrrolo[3,2-h]quinolin-2-ones (5), pyrazolo[3,4-h]quinolones (6).
antiproliferative activity and immunosuppressive effects. Never-
theless, the main process occurring upon excitation of a PS in PDT is
the formation of the PS triplet excited state and energy transfer to
molecular oxygen yielding 1O2 and other reactive oxygen species
(ROS) [12e14]. Considering that angular furocoumarins, for
geometrical reasons, prevent the formation of interstrand cross-
links (ISC), a big deal of research has been focused to the identifi-
cation of a reaction mechanism based on ROS generation.
other examples are currently under clinical investigation: for
example LZ-11, recently emerged as effective and selective telo-
meric G-quadruplex ligand [39]. Moreover, tricyclic compounds
incorporating the quinazoline moiety, such as pyrazolo[4,3-h]qui-
nazoline-3-carboxamides, have been largely investigated as kinase
inhibitors [40,41]. In particular, derivatives of this class of com-
pounds bearing a 2-cyclohexylamino substitution have been re-
ported as potent selective inhibitors of cyclin-dependent kinases
(CDKs) [40].
We have focused our attention for a long time in the identifi-
cation of new PS agents with improved properties, and in this
contest we have identified new classes of sulphur (1,2, Chart 1) [15]
and nitrogen heterocycles (3e6, Chart 1) [16e18]. Among them,
some derivatives showed very promising photosensitizing prop-
erties with remarkable phototoxicity in some cases higher than the
reference drug. Their mechanism of action involved localization
into mitochondria with production of ROS responsible of the
cellular death without inducing DNA damage, which is essential in
the modulation of long term side effects, typical of furocoumarins.
In our continuing studies aimed at the study of heterocyclic
scaffold as precursors of new potential anticancer drug candidates
[19e33], we have recently focused our attention on heterocyclic
systems containing the pyrimidine nucleus (Chart 2) as a matter of
fact, pyrimidines and their benzofused derivatives, quinazolines,
constitute the core structure of several antitumor drugs [34,35].
Erlotinib (trade name Tarceva) [36], Gefitinib (trade name Iressa)
[37] and Vandetanib (trade name Caprelsa) [38] are approved drugs
with a quinazoline structure acting as kinase inhibitors and many
We have recently studied pyrroloquinazoline systems of type 7
and 8 (Chart 2) which showed very promising antitumor properties
[42,43].
Surprisingly, these two positional isomers revealed deeply
different behaviour between each other in the inhibition of cell
growth. In fact, pyrrolo[3,2-h]quinazolines of type 7 expressed their
activity only upon light activation, at variance of compounds 8
which were active “in the dark”, showing potent phototoxic effect
reaching submicromolar level (GI50 0.2e15.2 mM). The phototox-
icity of derivatives 7 was related to an apoptotic mechanism of cell
death with the involvement of mitochondria and lysosomes [43].
Within this class of PS agents, the best results were achieved from
derivatives bearing an ethoxycarbonyl functionality adjacent to the
pyrrole nitrogen (R1 ¼ COOEt), which seemed crucial to get the
highest phototoxicity in comparison to the reference drug. On the
basis of these results, a structural modification of this class of
compounds was attempted, by varying the size of the central ring of
the tricyclic system extensively represented in potent antitumor
NHR1
X
X
2
N
N
N
N
3N
R
N
N1
R
4
N
9
8
R2
R1
N R
5
8
7
6
7
X = CH, C=O, C-NH2, C-NHPh
9
Chart 2. Structures of pyrrolo[3,2-h]quinazolines (7), pyrrolo[3,4-h]quinazolines (8), pyrrolo[30,20:6,7]cyclohepta[1,2-d]pyrimidines (9).

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V. Spano et al. / European Journal of Medicinal Chemistry 123 (2016) 447e461
449
H
N
H
N
H
N
EtOOC
EtOOC
EtOOC
O
ii
i
OH
( )3
( )4
OH
O
O
12
11
10
iii
R
N
H
O
O
N
Bn
N
O
N
COOEt
v
for 13c
iv
COOEt
COOMe
14
13b-d
13a
b R= Me; c R= Bn; d R= 4-OMeBn
vi
NHR1
N
NHR1
N
R
R
N
N
O
N
R
N
N
N
viii
CONH2
vii
COOEt
COOEt
15b-d
9a-i (Table 1)
9j-r (Table 1)
a-c, j-l R1= H; d-f, m-o R1= Ph; g-i, p-r R1= cyclohexyl
Scheme 1. Reagents and conditions: (i) AlCl3, glutaric anhydride, DCM, rt, 1 h then 10, rt, 1.5 h, 95%; (ii) TES, TFA, rt, 16 h, 94%; (iii) TFAA, DCM, rt, 3 h, 90%; (iv) NaH, DMF, 0 ꢀC to rt,
1.5 h then alkyl or aralkylhalide at 0 ꢀC to rt 2e6 h, 80e96%; (v) DMFDMA (1:20), reflux, 5 h, 96%; (vi) DMFDMA (1:1), DMF, mw (100 ꢀC, 50 W), 40e80 min, 70e75% (Method A), or
TBDMAM (1:1.5), DMF, mw (100 ꢀC, 50 W), 20e40 min, 95e99% (Method B); (vii) a) (NH2)2eC]NHꢁHNO3, MeONa, EtOH, reflux, 1.45 h, 60e72%; b) (NH2)2eC¼NHR1 (R1 ¼ Ph or
cyclohexyl), DMF, 100 ꢀC, 2 h, 60e85%; (viii) a) NaOH, EtOH, reflux, 3 h b) HOBt, DIPEA, EDC, THF, rt, 10 min then (NH4)2CO3, rt, 16 h, 60e94%.
drugs, generating the pyrrolo[30,20:6,7]cyclohepta[1,2-d]pyrimidine
system of type 9 (Chart 2). The synthesis, focused on derivatives
bearing the carboxyester functionality and their further conversion
to carboxyamides, and the investigation of the mechanism of action
of the new class of compounds is herein reported.
pyrrole nitrogen was obtained with classical procedures in N,N-
dimethylformamide (DMF) with sodium hydride (NaH) as the base,
followed by nucleophilic substitution with methyl iodide, benzyl
bromide or 4-methoxybenzyl chloride giving the corresponding N-
substituted derivatives 13b-d (80e96%).
Direct introduction of the
a-enamino functionality was
attempted at first using the N-benzyl derivative 13c as substrate
and N,N-dimethylformamide dimethyl acetal (DMFDMA) as sol-
vent, under conventional heating. However, besides the introduc-
tion of the enamine functionality, an undesired reaction of
transesterification occured, yielding the 2-carboxymethyl deriva-
tive 14 (96%) as the sole product. Thus, we further investigated such
reactions with DMFDMA under microwave irradiation (mw). Strict
control of irradiation conditions (100 ꢀC, 50 w) was crucial to avoid
the undesired transerification of the ethoxycarbonyl group. Addi-
tionally, reactivity towards the Bredereck's reagent, t-butoxy-bis-
(dimethylamino)methane (TBDMAM), was studied. Results from
good excellent to were obtained in terms of reaction times and
yields using DMF as solvent and stoichiometric amount of
DMFDMA (Method A, 70e75%) or 1.5 equivalents of TBDMAM
(Method B, 95e99%). In fact, in these conditions the desired
enaminoketones formed within 40e80 min with DMFDMA and
20e40 min with TBDMAM, associated in these latter cases, with a
significative improvement of yields and of the workup procedures
as the crude enamines 15, once poured onto crushed ice, precipi-
tated as pure compounds. Annelation of the pyrimidine ring to the
2. Chemistry
The synthesis of 8-oxo-1,4,5,6,7,8-hexahydrocyclohepta[b]pyr-
role 2-carboxylates of type 13, suitable precursors for the title ring
system, was achieved according to synthetic Scheme 1. Up to date
only few example of cyclohepta[b]pyrrole-8-ones were reported in
the literature, prepared in very modest yields [44,45] and 2-
ethoxycarbonyl derivatives were never described before. Thus, we
applied for our purpose a multistep approach, with the aim of
annelating the cycloheptanone moiety on the pyrrole ring.
Thus, we used as starting material the commercially available
ethyl 1H-pyrrole-2-carboxylate 10, which was subjected to Friedel-
Crafts acylation using AlCl3 as Lewis acid, and glutaric anhydride as
acylating agent. The obtained acyl derivative 11 (95%) was subse-
quently subjected to reduction of the carbonyl group with trie-
thylsilane (TES) in trifluoroacetic acid (TFA) to give 12 (94%), which
in turn was cyclized by dehydration in an excess of trifluoroacetic
anhydride (TFAA), leading to the seven membered ring closure,
furnishing the ethyl 8-oxo-1,4,5,6,7,8-hexahydrocyclohepta[b]pyr-
role-2-carboxylate 13a in high yield (90%). Functionalization of the

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V. Spano et al. / European Journal of Medicinal Chemistry 123 (2016) 447e461
Table 1
Table 2
Pyrrolo[30,20:6,7]cyclohepta[1,2-d]pyrimidin-2-amines 9a-r.
Spectrophotometric properties of pyrrolo[30,20:6,7]cyclohepta[1,2-d]pyrimidin-2-
amines 9a-r.
Cpd
R
R1
Yield Cpd
R
R1
Yield
Cpd
lMAX (nm)
εMAX
ε365 (nm)
9a
9b
9c
9d
9e
9f
9g
9h
9i
Me
Bn
4-OMeBn
Me
Bn
4-OMeBn Ph
Me
Bn
H
H
H
Ph
Ph
72%
60%
65%
75%
70%
60%
9j
9k
9l
9m
9n
9o
9p
9q
9r
Me
Bn
4-OMeBn
Me
Bn
4-OMeBn Ph
Me
Bn
H
H
H
Ph
Ph
76%
65%
94%
84%
74%
60%
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
9r
341
341
341
335
334
333
324
324
372
341
342
342
321
324
327
318
322
323
13420
11670
13385
18175
21060
14785
13830
12460
10985
18265
20060
17970
17165
9965
4370
2565
3425
15250
17090
11005
13225
11920
10810
3890
4970
4605
8960
7900
9800
8105
8505
4800
Cyclohexyl 80%
Cyclohexyl 85%
Cyclohexyl 82%
Cyclohexyl 85%
4-OMeBn Cyclohexyl 78%
4-OMeBn Cyclohexyl 93%
cycloheptapyrrole scaffold bearing the proper decoration was
achieved by reacting the so obtained enaminoketones 15 with the
proper guanidine to get the 2-amino substituted pyrrolo[30,20:6,7]
cyclohepta[1,2-d]pyrimidines 9a-i. In particular, 2-amino com-
pounds 9a-c were obtained (60e72%) through reaction of enami-
nones with guanidine nitrate in the presence of sodium methoxide
(MeONa) as the base to promote the nucleophilic attack of the
amino group to the electrophilic center, whereas 2-phenylamino
(9d-f, 60e75%) and 2-cyclohexylamino derivatives (9g-i, 78e85%)
were obtained by reaction with phenylguanidine or cyclo-
hexylguanidine in DMF at 100 ꢀC. Conversion of the ester func-
tionality in position nine of compounds 9a-i to carboxamide was
obtained in two steps: hydrolysis to the corresponding acid in basic
media in ethanol at reflux followed by activation of the carboxyacid
functionality with N-ethyl-N0-(3-dimethylaminopropyl)carbodii-
mide hydrochloride (EDC) and subsequent reaction with ammo-
nium carbonate in the presence of N,N-diisopropylethylamine
(DIPEA) and 1-hydroxybenzotriazole (HOBt). The carboxamide
derivatives 9j-r were obtained with good yields (60e94%) (Table 1).
10780
12355
9000
5350
dark”. Then, we hypotized that their behaviour could be similar to
our class of photoactivable analogues of type 7, thus submitting
them to phototoxicity studies.
To assess the potency of the whole series of pyrrolo[30,20:6,7]
cyclohepta[1,2-d]pyrimidin-2-amines, compounds 9a-r were
tested on three human cell lines, derived from cervical cancer
(HeLa), colorectal adenocarcinoma (HT29) and breast adenocarci-
noma (MCF7) (Table 3). Photoactivity was assessed upon light
irradiation (365 nm for five minutes, 1.5 J/cm2) and reported as the
effective concentration able to kill 50% of the cell population (EC50
)
measured 48 h post administration of compounds 9a-r; in contrast,
activity measured in the dark represented the cytotoxic concen-
tration able to kill 50% of the cell population (CC50). The CC50/EC50
ratio was considered the selectivity index (SI), i.e. the possibility to
kill tumor cells only upon light activation. The majority of com-
pounds, with the only exception of 9j, showed potent phototoxic
3. Results and discussion
3.1. Spectrophotometric properties
activity at low micromolarenanomolar level (EC50 0.06e4.96 mM).
The essential requirement for a PS compound is the absorption
of UVeVis light. The absorption spectra of 9a-r were collected in
phosphate buffer (20 mM, pH 7.0) and ε values were calculated in
correspondence of both lmax and l365 nm. All compounds absorbed
UVeVis light, a fundamental feature for a photosensitizer, with
maximum peaks in the UV-A region (318e372 nm), (Fig. S1).
All compounds generally exhibited a high molar extinction co-
efficient at their lmax (ε > 9000 Mꢂ1 cmꢂ1; i.e.: 13830 Mꢂ1 cmꢂ1 at
324 nm for 9g); however, only compounds 9d-i belonging to the 9-
ethoxycarbonyl series and bearing a substituted amine in position
two showed a high molar extinction coefficient also at l365
(Table 2).
Some hypotheses can be formulated regarding the structur-
eeactivity relationships (SAR). Pyrrolo[30,20:6,7]cyclohepta[1,2-d]
pyrimidin-2-amines are variably substituted at positions 2, 9, and
10 and the best results were undoubtely obtained for the 9-
ethoxycarbonyl series (9a-i) when compared with the 9-
carboxamido analogues (9j-r). In fact, the EC50 values of de-
rivatives 9a-i against all the three tested cell lines were at sub-
micromolarenanomolar level, with the sole exception of 9a, which
nevertheless showed low micromolar activity against HT-29 and
MCF7 cell lines. For both series of compounds, the influence of
substituent at position 10 (methyl, benzyl, 4-methoxybenzyl) does
not play a crucial role in affecting the phototoxicity. Whereas, the
effect of the 2-substitution generally decreases along the following
ranking: cyclohexylamino > aniline > amine groups, which is more
evident for the 9-ethoxycarbonyl series. In fact, within this set of
compounds, two of them stood out for their outstanding activity
3.2. Cytotoxicity and phototoxicity of pyrrolo[30,20:6,7]cyclohepta
[1,2-d]pyrimidin-2-amines
In consideration of the contrasting results previously obtained
on the pyrroloquinazolines of type 7 and 8, the new synthetized
derivatives were screened to evaluate the growth inhibitory effect
in cell proliferation either “in the dark” and upon light irradiation.
Initially, the entire series of compounds was submitted to the Na-
tional Cancer Institute (Bethesda, MD) and the 9-carboxamido de-
rivatives 9j-r were selected and prescreened at one dose
concentration (10ꢂ5 M) on the full panel of approximately 60 hu-
man cancer cell lines derived from 9 human cancer cell types.
However, none of the compounds showed cytotoxic activity, and
for this reason they were not considered for further studies “in the
upon light activation: 9g (EC50 0.08e0.09
0.06e0.10 M) displaying EC50 equal or below 0.1
three cancer cell lines, followed by compound 9i (EC50
0.07e0.14 M). The same order of potency is maintained within the
2-cyclohexylamino derivatives of the 9-carboxamido series. In fact,
9p (EC50 0.82e1.85 M) and 9q (EC50 0.18e0.37 M), although
being active at submicromolar level, were less active of one
magnitude order than their 9-ethoxycarbonyl analogues (compare
9p with 9g and 9q with 9h), followed by compound 9r (EC50
m
M) and 9h (EC50
m
mM against all
m
m
m
0.12e0.41 mM). These results indicate that the cyclohexyl group is

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V. Spano et al. / European Journal of Medicinal Chemistry 123 (2016) 447e461
451
Table 3
Photocytotoxicity (EC50 mM) and cytotoxicity (CC50
mM) in human tumor cell lines, measured 48 h post administration of compounds 9a-r, by irradiation at 365 nm for five
minutes (Dose: 1.5 J/cm2).
Cpd
HeLa
aEC50
HT-29
EC50
MCF7
EC50
bCC50
cSI
CC50
SI
CC50
SI
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
9r
0.72 0.07
0.22 0.08
0.32 0.13
0.10 0.09
0.28 0.01
0.18 0.10
0.08 0.02
0.06 0.01
0.07 0.03
8.75 0.79
2.09 0.12
1.14 0.45
1.12 0.14
0.25 0.16
0.16 0.01
0.82 0.07
0.22 0.19
0.12 0.02
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>139
>455
>312
>1042
>355
>559
>1274
>1667
>1361
>11
>48
>88
>90
>397
>610
>121
>463
>813
1.72 0.19
0.28 0.08
0.29 0.04
0.29 0.07
0.70 0.28
0.07 0.01
0.09 0.03
0.08 0.02
0.14 0.02
>100
2.17 0.63
2.00 0.43
1.85 0.39
0.36 0.03
0.28 0.06
1.50 0.25
0.37 0.02
0.41 0.03
>100
79.20 18.70
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>58
284
1.54 0.016
0.79 0.30
0.67 0.04
0.14 0.03
0.24 0.20
0.86 0.02
0.08 0.02
0.10 0.01
0.11 0.09
66.00 1.3
3.69 0.44
3.26 0.30
4.96 1.27
1.04 0.21
0.28 0.16
1.85 0.64
0.18 0.20
0.18 0.19
>100
73.60 6.65
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
66.48 5.76
>100
75.21 4.87
>100
>65
93
>348
>348
>143
>1471
>1111
>1235
>735
n.d.
>46
>50
>54
>281
>360
>67
>270
>243
>150
>709
>412
>117
>1250
>1053
>952
>1.5
>27
>31
>20
64
>358
41
>569
>549
>100
a
Phototoxic concentration able to kill 50% of the cell population.
Cytotoxic concentration able to kill 50% of the cell population (CC50).
Selectivity index CC50/EC50 ratio (SI).
b
c
the preferred decoration at the pyrimidine moiety in conjunction
with the 9-ethoxycarbonyl substitution. Importantly, all these
compounds were not cytotoxic in the absence of UV
irradiated in a diluted (1e2.5 ꢃ 10ꢂ4 M) aqueous acetonitrile (ACN)
solution by nanosecond laser flash photolysis (LFP) using a Nd:YAG
laser, operating at 355 nm. Upon laser excitation at 355 nm, we
recorded the transient absorption spectra in the range of
350e700 nm for both 9g and 9j (Fig. 1a and b, respectively).
Two maxima absorbance were recorded for 9g (lmax 380 nm and
630 nm, Fig. 1a) and 9j (lmax ꢄ 360 nm and 610 nm, Fig. 1b). The
transient absorption profiles arising from the excitation of 9g
monitored at 380 and 630 nm, decay monoexponentially in air-
(CC50 > 100 mM) thus displaying very promising SI. In particular, the
calculated SI for the best compounds 9g and 9h were 1274ꢂ1111
and 1667ꢂ1053 respectively. As a general trend, all the new com-
pounds were more active against the cervical cancer HeLa cell line
and less active against the breast adenocarcinoma MCF7 cell line.
equilibrated solutions with an identical lifetime of
argon-saturated solutions, the lifetime of the transient specie from
9g became bi-exponential (t1 ¼ 1.8 s, 30%; t2 ¼ 7.3 s, 70%). Such
t
¼ 0.5
ms. In
3.3. Spectroscopic and kinetic characterization of the triplet excited
state
m
m
an oxygen dependent lifetimes undoubtedly indicates that at the
LFP pulse end we are very likely observing the triplet-state ab-
sorption of 9g. A similar behaviour was also recorded for 9j.
The intensity of the signal assigned to the T1 of 9g monitored at
630 nm (A ¼ 5.2 ꢃ 10ꢂ2) is proximately 3.6 time more intense than
that assignet to the T1 of 9j recorded at 610 nm (A ¼ 1.46 ꢃ 10ꢂ2).
Taking into consideration that both 9g and 9j solution exhibit an
identical absorbance (A ¼ 1.02 ꢃ 10ꢂ2) at 355 nm, these data
suggests that 9g populates its T1 more effectively than 9j by irra-
diation. Consequetly 9g may act as a slightly a more efficient 1O2
To clarify the origin of the photocytotoxicity and their mecha-
nism of action we decided to investigate the spectroscopic and
kinetic features of the lowest triplet-excited state (T1) of the newly
synthesized pyrrolocycloheptapyrimidine amines. The choice was
made in an attempt to evaluate them as singlet oxygen sensitizers,
as both the population of the triplet-excited states and their life-
times directly correlate to the efficiency for singlet oxygen pro-
duction. Therefore, we set out to examine these features in detail
for the most and the least cytotoxic pyrrolopyrimidinamines 9g and
9j, respectively. For solubility reasons 9g and 9j have been
Fig. 1. Time-resolved difference absorption spectra (
(H2O:ACN ¼ 1:1). Both solutions exhibit an identical absorbance (A ¼ 1.02) at 355 nm.
D
A) flashing a solution of a) 9g (10ꢂ4 M) and b) (2.5 ꢃ 10ꢂ4 M) 9j, both at 355 nm, in argon-purged aqueous ACN

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3.5. Production of radical oxygen species (ROS) upon UV irradiation
Because physical testing showed both triplet excited state
population and production of singlet oxygen species upon light
activation, we tested if the best (i.e. 9g and 9h) and the least active
compounds (i.e. 9j) were able to produce Radical Oxygen Species
(ROS) in cells. We employed a cell-permeant dichlorofluorescein
(DCF) probe, which is converted to a highly fluorescent dye by ROS.
Fluorescence in the presence/absence of UV irradiation was quan-
titatively evaluated in cells treated with pyrrolo[30,20:6,7]cyclo-
hepta[1,2-d]pyrimidin-2-amines 9a-r; cells without compounds
were used as negative controls. Cells treated with phorbol-12-
myristate-13-acetate (PMA) and light activated were used as a
positive control of intracellular ROS generation [48e52].
In the presence of light, and 2 h after photoactivation, increasing
concentrations of 9g and 9h (1e4 mM) showed a steady increase in
fluorescence, which reached 222% and 198% of the untreated con-
trol at the highest 9g and 9h concentration, respectively (Fig. 3).
Conversely, treatment with 9j or the administration of compounds
in the absence of light did not modify control levels of fluorescence
(Fig. S3). The positive control sample (PMA, 400 nM) reached 120%
and 420% of the untreated control 2 h and 6 h after photoactivation.
These data indicate that production of ROS in cells by 9g and 9h is
much faster than that mediated by the ROS inductor PMA.
Production of ROS was confirmed treating cells with N-ace-
tylcysteine (NAC, 200e500 nM), a scavenger of intracellular ROS
[53]. All samples showed a dose-dependent reduction of ROS upon
treatment with NAC (Fig. 3). These data, indicate that 9g and 9h
promptly and massively produce ROS in cells, with 9g being a
slightly better inducer of cytotoxic reactive oxygen radicals than 9h,
at the highest dose.
Fig. 2. Absorbance comparison of 9g and 9j in ACN:water (2:3) solutions.
sensitizer than 9j.
3.4. Generation and trapping of 1O2 by 9,10-anthracenedipropionic
acid in aqueous ACN
To further corroborate the differential efficiency as 1O2 sensi-
tizer of 9g and 9j, we decided to study them in the photo-oxidation
of 9,10-anthracenedipropionic acid (ADPA) in aqueous ACN (ACN:
water ¼ 2: 3) [46]. We did not use lower amount of ACN nor
concentrated buffering conditions (>10ꢂ2 M) to avoid solubility
issues for both 9g and 9j. Nevertheless, we also managed to mea-
sure the efficiency as 1O2 sensitizer in diluted phosphate buffer
(10ꢂ3 M) in ACN:water ¼ 2:3. ADPA is a water-soluble and effective
1O2 trap. In fact, the photo-oxidation of ADPA by 1O2 has been
followed by the absorption at 400 nm, due to consumption of ADPA
anthracene chromophore, forming a colorless endoperoxide via a
[4 þ 2] Diels-Alder cycloaddition.
3.6. Mechanisms of ROS-induced cell damage
Production of large amounts of cellular ROS can trigger cell
death by several different pathways [54e56]. We thus initially set
250
200
150
100
50
The continuous photobleaching of the anthracene absorption, in
the presence of both 9g and 9j, monitored for 1 h by using 315 nm
excitation wavelength was the evidence of 1O2 photogeneration
(Figs S2a and S2b). Byproduct formation was not detected by UV
absorption during the irradiation, nor by HPLC. We did not detect
any decrease in the anthracene absorbance in the solutions without
the sensitizer. We measured the efficiency of the ADPA consump-
tion irradiating at 315 nm (FR-315: quantum yield) for both 9g and 9j
by ferrioxalate actinometry. Surprisingly, the quantum yields for 9g
and 9j were fairly low and comparable between each other FR-
¼ 0.030 0.003. The quantum yield for ADPA consumption (FR-
315
360) using 9g as 1O2 sensitizer measured irradiating at 360 nm was
similar (FR-360 ¼ 0.028 0.003). Buffering conditions in the same
0
l
solvent did not affect substantially the quantum yield
(FR-
M
M
M
M
M
M
μ
4
M
μ
M
n
o
r
t
μ
μ
μ
μ
μ
n
1
2
4
1
2
0
0
4
o
j 4
g
g
g
h
h
h
9
9
C
9
9
9
9
9
A
M
¼ 0.031 0.004). We were not able to reliably measure the FR-
360
360
P
using 9j and irradiating at 360 nm, as its absorbance was
no scavenger
NAC 200nM
NAC 500nM
negligible in comparison to ADPA.
These data suggest that both 9g and 9j are capable of 1O2 pho-
togeneration with comparable efficiency at 315 nm, which was
slightly lower than that measured for the well-studied lipophilic
Fig. 3. ROS production upon photoactivation of 9g,h,j. The increase in intracellular ROS
levels was determined by a DCF-probe and was calculated as fluorescence increase
compared to untreated irradiated controls. Assays were performed on non-treated
(control), 9g,h,j and PMA treated cells, after 2 h from light exposure, in minimal me-
dium (no scavenger) and in medium containing an antioxidant compound at various
concentration (i.e. N-acetylcysteine, NAC 200 nM and 500 nM). Results from three
independent experiments are shown.
singlet oxygen sensitizer bacteriochlorin a (
F
¼ 0.05) in buffered
conditions [47]. The different photocytotoxycity, reported in Table 3
has to be ascribed to their differential optical properties, as 9g ex-
hibits much higher absorbivity at longer wavelength than 360 nm,
in comparison to 9j (Fig. 2).

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453
out to investigate the type of cell death induced by the most potent
light-activated compound, i.e. 9g and the least active, i.e. 9j by flow
cytometry. Double staining (7 AAD and PE Annexin V) flow cyto-
fluorimetric analysis was performed to detect both the early and
late stages of apoptosis. MCF7 cells were treated with 9g and 9j
endosomes, was used. Cells treated with 9g and light showed
massive acidification/activation of lysosomes, visible as bright
green dots dispersed in the cell cytoplasm after 20 min of incuba-
tion with the compound (Fig. 6eeh). Mild fluorescence in the
cytoplasm likely indicated lysosomal membrane impairment [60].
At 2 h-treatment the lysosome signal decreased, indicating an
advanced and irreversible apoptotic process (Fig. 6h) [61]. PMA
treatment (400 nM, 6 h treatment) induced a weak lysosome signal
(Fig. 6g). In contrast, 9j upon light or no compounds or any con-
ditions in the absence of light did not induce lysosome activation
(Fig. 6a-b-c-f-i). These data altogether indicate that the UV-induced
production of ROS by 9g triggers a rapid and massive apoptotic
process.
(EC50 8 nM and 66 mM, respectively) and the whole cell population
was next analyzed. At 6 h post photoactivation, cells treated with
compound 9g were mostly in the early apoptosis stage (47%), with
smaller amounts in the late and necrosis stages (13% and 11%,
respectively) (Fig. 4). In contrast, control cells and cells treated with
9j displayed only low amount of early apoptotic cells (17% and 22%
respectively).
Since different intracellular signalling pathways, including
mitochondrial dysfunction was assessed with aand lysosome acti-
vation, trigger apoptosis, we checked if these mechanisms were
present in cells treated with photoactivated 9g,j. A mitochondrial
membrane fluorescent probe, was used to monitor mitochondria
morphology and distribution within the cells by confocal imaging
(red signal, Fig. 5 and S4). Untreated and non-irradiated cells dis-
played the typical fragmented pattern of mitochondrial organiza-
tion reported for MCF7 cell line (red signal, Fig. 5 a and S4) [57e59].
Cells irradiated in the absence of compounds as well as cells treated
with the compounds in the dark were characterized by a similar
non-modified mitochondrial network (Fig. 5 d-c-f and S4). In
contrast, light-activated 9g induced a sharp remodelling of mito-
chondria both in shape, with evidence of clustering and swelling,
and in location, with mitochondrial signal accumulated at the
perinuclear level (Fig. 5eeh). This effect was evident after 20 min
(Fig. 5e) and exacerbated at 2 h of compound incubation (Fig. 5h).
Compound 9j in the same conditions did not induce any detectable
mitochondrial perturbation (Fig. 5cef). Treatment with PMA pro-
voked moderate mitochondria fusion after 6 h of treatment
(Fig. 5g).
Taken together, our data indicate that apoptosis is the main
mechanism of cellular death induced by light-activated 9g,j.
Apoptosis is associated with (i) rapid and massive production of
large amounts of ROS, (ii) mitochondrial morphology modifications
and alteration of the transmembrane potential, and (iii) activation
of lysososmes.
4. Conclusion
A new series of pyrrolo[30,20:6,7]cyclohepta[1,2-d]pyrimidin-2-
amines has been successfully prepared by annelation of the py-
rimidine ring on the cycloheptapyrrole moiety by a synthetic
pathway never described before, for this class of compounds, which
demonstated versatile and high yielding. A good number of de-
rivatives was obtained and the cellular cyto/photocytotoxicity was
evaluated in vitro against three human tumor cell lines. The ma-
jority of compounds were able to express potent growth inhibitory
effect under light activation at low micromolarꢂnanomolar level
(EC50 0.08e4.96 mM). In particular, derivatives 9g and 9h, decorated
with a carboxyester functionality in position 9 and a cyclohexyl-
amine group in position 2 stood out for their outstanding activity
The light-activated 9g,j induced apoptotic process, initiated by
the mitochondrial depolarization, was further characterized by
investigation of lysosome activation. A fluorescent dye able to stain
acidic subcellular compartments, such as lysosomes and
upon light activation displaying EC50 equal or below 0.1 mM against
all three cancer cell lines, maintaining nanomolar activity against
the most sensitive cell line (HeLa). This data in conjunction with the
very high SI, is the crucial requirement for a PS agent expressing the
possibility to kill tumor cells only upon light activation. The
different photocytotoxycity, has to be ascribed to their differential
optical properties as for the most potent compound 9g which ex-
hibits much higher absorbivity at longer wavelength than 360 nm.
The latter derivative showed, by laser flash photolysis, a fairly long-
120
100
80
60
40
20
0
lived triplet state (t ~ 7 ms), an absorbance in the near UVeVis,
exhibiting a detectable activity as 1O2 sensitizer. Studies directed to
elucidate the mechanism of action of this new class of photosen-
sitizers indicate that the photosentizing properties are responsible
for their ability to photoinduce apoptosis as the main mechanism of
cellular death associated with rapid and massive production of
large amounts of ROS, and a deep involvement of mitochondria and
lysosomes. Thus, overall the rationalized structural modification of
this class of compounds, represents a successful lead modification,
considering the improvement of photocytotoxycity, especially in
conjunction with the 2-cyclohexylamine group of the pyrimidine
moiety. These findings are of great relevance adding new knowl-
edge in our studies, aimed to the development of new photo-
sensitizing agents with improved features, and constitutes the seed
for future development.
Control
9g
9j
living cells
early apoptosis
late apoptosis
5. Experimental
cellular debris/necrotic cells
5.1. Chemistry. Synthesis and characterization
Fig. 4. Cell population analysis. MCF7 cells were treated with 9g and 9j at EC50 con-
centrations and double stained with 7AAD and PE Annexin after photo-
irradiation. Reported values represent the mean of three independent experiments.
Microwave irradiation (mw) was performed using a CEM
Discover Labmate™ apparatus. All melting points were taken on a
V
6 h

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454
Fig. 5. Mitochondrial morphology modifications induced by light-activated 9g,j. Mitochondrial membranes were visualized with a membrane potential-specific red fluorescent
probe. Column A (control cells): a) non-treated and non-irradiated cells, d) non-treated and irradiated cells and g) PMA-treated cells (400 nM). Images a-d were taken 2 h after
treatment/irradiation; g) image 6 h post PMA administration. Column B (9g-treated cells, 2
mM): b) 9g treated and non-irradiated cells, e) 9g treated and irradiated cells (20 min
post light-treatment), h) treated and irradiated cells (2 h post light-treatment). Column C (9j-treated cells, 2
mM): C1) 9j treated and non-irradiated cells, f) 9j-treated and irradiated
cells (20 min post light-treatment), i) 9j-treated and irradiated cells (2 h post light-treatment). A well defined mitochondrial network morphology was stained in non-treated cells,
irradiated and non-irradiated (a and d), as well as in 9g treated and non-irradiated cells (b). 9j-treated cells, with and without photoactivation, displayed no modifications in
organelles structure and organization (c and f). Cells treated with 9g (2 mM), following photoactivation, underwent significant mitochondrial network impairment and organelles
swelling (h). PMA was used as ROS-inducer positive control: a condensed mitocondrial network was visible 6 h post treatment (g).
Fig. 6. Lysosomes activation induced by UV-activated pyrrolo[30,20:6,7]cyclohepta[1,2-d]pyrimidin-2-amines. Lysosomes were visualized with a green fluorescent probe. Column A
(control cells): a) non-treated and non-irradiated cells, d) non-treated and irradiated cells, g) PMA-treated cells (400 nM) (6 h PMA-incubation). Column B (9g-treated cells, 2
b) 9g treated and non-irradiated cells, e) 9g treated and irradiated cells, (20 min post light-treatment), h) treated and irradiated cells (2 h post light-treatment). Column C (9j-treated
cells, 2 M), c) 9j treated and non-irradiated cells, f) 9j-treated and irradiated cells, (20 min post light-treatment), i) 9j-treated and irradiated cells (2 h post light-treatment). Non-
mM),
m
treated MCF7 cells, irradiated and non-irradiated, showed none or minimal lysosome activation (a and e), as well as cells treated and non-irradiated with 9g and 9j. UV-activated 9j
displays the same lysosomal activation as non-treated irradiated controls. 9g Treatment followed by photoactivation induced a severe lysosomal activation after 20 min. After 2 h
considerable lysosomal membrane depolarization was obtained.

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455
Büchi melting point M ꢂ 560 apparatus. IR spectra were deter-
mined in bromoform with Shimadzu FT/IR 8400S spectrophotom-
eter. 1H and 13C NMR spectra were measured at 200 and 50.0 MHz,
respectively, in DMSO-d6 or CDCl3 solution using a Bruker Avance II
series 200 MHz spectrometer. Column chromatography was per-
formed with Merck silica gel (230e400 mesh ASTM) or a Büchi
Sepacor chromatography module (prepacked cartridge system).
Elemental analyses (C, H, N) were within 0.4% of theoretical values
and were performed with a VARIO EL III elemental analyzer.
Representative NMR spectra of compounds 9b, 9e, 9h, 9k, 9n and
9q are reported in the supporting informations (Figs. S5eS10).
50 MHz, ppm): d 14.1 (q), 21.5 (t), 25.4 (t), 26.0 (t), 41.2 (t), 60.2 (t),
115.7 (d), 125.9 (s), 131.9 (s), 132.7 (s), 160.0 (s), 192.1 (s). Anal calcd
for C12H15NO3 (221.25): C, 65.14; H, 6.83; N, 6.33. Found: C, 65.02;
H, 6.96; N, 6.21.
5.1.4. General procedure for the preparation of ethyl 1-substituted-
8-oxo-1,4,5,6,7,8-hexahydrocyclohepta[b]pyrrole-2-carboxylates
(13b-d)
To a solution of 13a (9 mmol) in anhydrous DMF (17 mL), NaH
(0.24 g, 10 mmol) was added at 0 ꢀC and the reaction was stirred at
room temperature for 1.5 h. Then the suitable alkyl or aralkylhalide
(13.5 mmol) was added at 0 ꢀC and the reaction mixture was stirred
at room temperature up to completeness (TLC). The reaction
mixture was poured onto crushed ice. The precipitate was filtered
and dried, in the absence of precipitate the solution was extracted
with DCM (3 ꢃ 50 mL). The organic layer was dried (Na2SO4) and
the solvent was removed under reduced pressure. The crude
product was purified by chromatography (DCM).
5.1.1. Preparation of 5-[5-(ethoxycarbonyl)-1H-pyrrol-3-yl]-5-
oxopentanoic acid (11)
A suspension of AlCl3 (12 g, 90 mmol) and glutaric anhydride
(3.40 g, 30 mmol) in anhydrous DCM (60 mL) was stirred at room
temperature for 1 h. Then a solution of 10 (2.10 g, 15 mmol) in
anhydrous DCM (30 mL) was added and the reaction mixture was
stirred at room temperature for 1.5 h. Ice and water were added
(80 mL) and the two phases were separated. Then the aqueous
phase was extracted with AcOEt (3 ꢃ 60 mL). The organic layer was
dried (Na2SO4) and the solvent was removed under reduced pres-
sure. The crude product was purified by chromatography (DCꢂM1/
5.1.4.1. Ethyl 1-methyl-8-oxo-1,4,5,6,7,8-hexahydrocyclohepta[b]pyr-
role-2-carboxylate (13b). This compound was obtained from reac-
tion of 13a with iodomethane after 2 h. White solid; yield 94%; m.p.
51e52 ꢀC; IR cmꢂ1: 1705 (CO), 1642 (CO); 1H NMR (DMSO-d6,
AcOEt 1:1). Light brown solid; yield 95%; m.p. 81e82 ꢀC; IR c
:
200 MHz, ppm):
d
1.28 (t, 3H, J ¼ 7.1 Hz, CH3), 1.68e1.77 (m, 4H,
3354 (NH), 3337 m (OH, broad), 1702 (CO), 1696 (CO), 1653 (CO); 1H
2 ꢃ CH2), 2.64 (t, 2H, J ¼ 6.6 Hz, CH2), 2.75 (t, 2H, J ¼ 6.6 Hz, CH2),
NMR (DMSO-d6, 200 MHz, ppm):
d
1.29 (t, 3H, J ¼ 6.7 Hz, CH3),
4.06 (s, 3H, CH3), 4.25 (q, 2H, J ¼ 7.1 Hz, CH2), 6.74 (s, 1H, H-3); 13
C
1.70e1.86 (m, 2H, CH2), 2.27 (t, 2H, J ¼ 6.2 Hz, CH2), 2.80 (t, 2H,
J ¼ 6.2 Hz, CH2), 4.26 (q, 2H, J ¼ 6.7 Hz, CH2), 7.13 (s,1H, H-4), 7.72 (s,
1H, H-2), 12.07 (s, 1H, OH), 12.51 (s, 1H, NH); 13C NMR (DMSO-d6,
NMR (DMSO-d6, 50 MHz, ppm): d 14.1 (q), 20.8 (t), 24.4 (t), 24.8 (t),
34.3 (q), 41.4 (t), 60.2 (t),116.6 (d),126.3 (s),132.8 (s),133.5 (s),160.3
(s), 194.1 (s). Anal calcd for C13H17NO3 (235.28): C, 66.36; H, 7.28; N,
5.95. Found: C, 66.48; H, 7.37; N, 5.79.
50 MHz, ppm): d 14.2 (q), 19.6 (t), 32.9 (t), 37.9 (t), 60.1 (t), 114.0 (d),
123.6 (s), 125.8 (s), 127.8 (d), 160.1 (s), 174.2 (s), 194.6 (s). Anal calcd
for C12H15NO5 (253.25): C, 56.91; H, 5.97; N, 5.53. Found: C, 56.79;
H, 6.08; N, 5.62.
5.1.4.2. Ethyl 1-benzyl-8-oxo-1,4,5,6,7,8-hexahydrocyclohepta[b]pyr-
role-2-carboxylate (13c). This compound was obtained from reac-
tion of 13a with benzyl bromide after 2 h. White solid; yield 96%;
m.p. 46e47 ꢀC; IR cmꢂ1: 1710 (CO), 1645 (CO); 1H NMR (DMSO-d6,
5.1.2. Preparation of 5-[5-(ethoxycarbonyl)-1H-pyrrol-3-yl]
pentanoic acid (12)
200 MHz, ppm):
d
1.21 (t, 3H, J ¼ 7.1 Hz, CH3), 1.60e1.80 (m, 4H,
To a solution of 11 (1.50 g, 6 mmol) in trifluoracetic acid (13 mL),
triethylsilane (3.3 mL, 21 mmol) was added and the reaction
mixture was stirred at room temperature for 16 h. The solvent was
removed under reduced pressure and brine (40 mL) was added to
the residue. The solid formed was filtered off, dried and purified by
chromatography (DCM/AcOEt 1:1). Brown solid; yield 94%; m.p.
85e86 ꢀC; IR cmꢂ1: 3449 (NH), 3355 (OH, broad), 1700 (CO), 1653
2 ꢃ CH2), 2.60 (t, 2H, J ¼ 6.6 Hz, CH2), 2.81 (t, 2H, J ¼ 6.6 Hz, CH2),
4.18 (q, 2H, J ¼ 7.1 Hz, CH2), 6.01 (s, 2H, CH2), 6.84 (s, 1H, H-3), 6.88
(s, 2H, H-20 and H-60), 7.11e7.32 (m, 3H, H-30, H-40 and H-50); 13C
NMR (DMSO-d6, 50 MHz, ppm): d 14.0 (q), 20.7 (t), 24.3 (t), 24.7 (t),
41.4 (t), 48.4 (t), 60.3 (t), 117.7 (d), 125.6 (2 ꢃ d), 126.1 (s), 126.6 (d),
128.3 (2 ꢃ d), 133.0 (s), 133.8 (s), 139.2 (s), 160.1 (s), 194.2 (s). Anal
calcd for C19H21NO3 (311.37): C, 73.29; H, 6.80; N, 4.50. Found: C,
73.11; H, 6.97; N, 4.41.
(CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
CH3), 1.41e1.62 (m, 4H, 2 ꢃ CH2), 2.21 (t, 2H, J ¼ 6.4 Hz, CH2), 2.39 (t,
2H, J ¼ 6.4 Hz, CH2), 4.20 (q, 2H, J ¼ 7.1 Hz, CH2), 6.60 (s, 1H, H-4),
6.79 (s, 1H, H-2), 11.55 (s, 1H, OH), 11.99 (s, 1H, NH); 13C NMR
d
1.26 (t, 3H, J ¼ 7.1 Hz,
5.1.4.3. Ethyl 1-(4-methoxybenzyl)-8-oxo-1,4,5,6,7,8-
hexahydrocyclohepta[b]pyrrole-2-carboxylate (13d). This com-
pound was obtained from reaction of 13a with 4-
methoxybenzylchloride after 6 h. White solid; yield 80%; m.p.
73e74 ꢀC; IR cmꢂ1: 1709 (CO), 1645 (CO); 1H NMR (DMSO-d6,
(DMSO-d6, 50 MHz, ppm):
d 14.4 (q), 24.1 (t), 25.7 (t), 29.9 (t), 33.5
(t), 59,3 (t), 114.5 (d), 121.5 (s), 121.7 (d), 124.5 (s), 160.4 (s), 174.5 (s).
Anal calcd for C12H17NO4 (239.27): C, 60.24; H, 7.16; N, 5.85. Found:
C, 60.16; H, 7.03; N, 5.99.
200 MHz, ppm):
d
1.23 (t, 3H, J ¼ 7.1 Hz, CH3), 1.63e1.79 (m, 4H,
2 ꢃ CH2), 2.60 (t, 2H, J ¼ 5.6 Hz, CH2), 2.79 (t, 2H, J ¼ 5.6 Hz, CH2),
3.69 (s, 3H, CH3), 4.20 (q, 2H, J ¼ 7.1 Hz, CH2), 5.93 (s, 2H, CH2),
6.78e6.81 (m, 4H, H-20, H-30, H-50 and H-60), 6.83 (s, 1H, H-3); 13C
5.1.3. Preparation of ethyl 8-oxo-1,4,5,6,7,8-hexahydrocyclohepta
[b]pyrrole-2-carboxylate (13a)
To a solution of 12 (1 g, 4.2 mmol) in anhydrous DCM (10 mL),
trifluoroacetic anhydride (3.5 mL, 25.2 mmol) was added and the
reaction mixture was stirred for 3 h at room temperature. The
solvent was removed under reduced pressure and a saturated so-
lution of NaHCO3 (40 mL) was added to the residue. The solid
formed was filtered off, dried and purified by chromatography
(DCM). White solid; yield 90%; m.p. 76e77 ꢀC; IR cmꢂ1: 3415 (NH),
NMR (DMSO-d6, 50 MHz, ppm): d 14.0 (q), 20.7 (t), 24.2 (t), 24.7 (t),
41.4 (t), 47.6 (t), 54.9 (q), 60.4 (t), 113.6 (2 ꢃ d), 117.7 (d), 126.1 (s),
127.2 (2 ꢃ d), 131.0 (s), 132.9 (s), 133.9 (s), 158.0 (s), 160.2 (s), 194.4
(s). Anal calcd for C20H23NO4 (341.40): C, 70.36; H, 6.79; N, 4.10.
Found: C, 70.55; H, 6.64; N, 3.95.
5.1.5. Isolation of methyl 1-benzyl-7-[(dimethylamino)
methylidene]-8-oxo-1,4,5,6,7,8-hexahydrocyclohepta[b]pyrrole-2-
carboxylate (14)
A solution of ketone 13c (0.45 g, 1.3 mmol) in DMFDMA (4 mL)
was heated at reflux for 5 h. After cooling, the excess of reagent was
1709 (CO), 1633 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
d 1.28 (t,
3H, J ¼ 6.9 Hz, CH3), 1.60e1.97 (m, 4H, 2 ꢃ CH2), 2.63 (t, 2H,
J ¼ 6.6 Hz, CH2), 2.79 (t, 2H, J ¼ 6.6 Hz, CH2), 4.24 (q, 2H, J ¼ 6.9 Hz,
CH2), 6.67 (s, 1H, H-3), 11.91 (s, 1H, NH); 13C NMR (DMSO-d6,

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456
removed under reduced pressure. Yellow oil; yield 96%; IR cmꢂ1
1708 (CO), 1641 (CO); 1H NMR (CDCl3, 200 MHz, ppm):
1.73e1.87
:
Brown oil; yield 75% (Method A), 95% (Method B); IR cmꢂ1: 1706
(CO), 1635 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
1.22 (t, 3H,
d
d
(m, 2H, CH2), 2.35 (t, 2H, J ¼ 6.9 Hz, CH2), 2.62 (t, 2H, J ¼ 6.9 Hz,
CH2), 3.10 (s, 6H, 2 ꢃ CH3), 3.76 (s, 3H, CH3), 6.05 (s, 2H, CH2),
6.78e7.26 (m, 6H, H-3 and Ar), 7.56 (s, 1H, CH); 13C NMR (CDCl3,
J ¼ 7.1 Hz, CH3), 1.70e1.77 (m, 2H, CH2), 2.25 (t, 2H, J ¼ 6.7 Hz, CH2),
2.54 (t, 2H, J ¼ 6.7 Hz, CH2), 3.08 (s, 6H, 2 ꢃ CH3), 3.68 (s, 3H, CH3),
4.17 (q, 2H, J ¼ 7.1 Hz, CH2), 5.87 (s, 2H, CH2), 6.74 (s, 1H, H-3),
6.76e6.90 (m, 4H, Ar), 7.48 (s,1H, CH); 13C NMR (DMSO-d6, 50 MHz,
50 MHz, ppm):
d
23.5 (t), 30.2 (t), 43.4 (2 ꢃ q), 48.5 (t), 51.3 (q), 60.1
(t), 106.7 (s), 116.9 (d), 125.9 (d), 126.3 (2 ꢃ d), 126.6 (s), 127.6 (s),
128.2 (2 ꢃ d), 136.7 (s), 140.0 (s), 150.4 (d), 161.3 (s), 188.5 (s). Anal
calcd for C21H24N2O3 (352.43): C, 71.57; H, 6.86; N, 7.95. Found: C,
71.65; H, 6.99; N, 7.82.
ppm):
d
14.2 (q), 22.8 (t), 23.0 (t), 29.8 (t), 43.1 (2 ꢃ q), 46.6 (t), 55.0
(q), 59.8 (t), 104.9 (s), 113.7 (2 ꢃ d), 116.9 (d), 123.4 (s), 126.4 (s),
127.4 (2 ꢃ d), 131.7 (s), 136.5 (s), 150.3 (d), 158.1 (s), 160.3 (s), 186.6
(s). Anal calcd for C23H28N2O4 (396.48): C, 69.67; H, 7.12; N, 7.07.
Found: C, 69.75; H, 6.98; N, 7.31.
5.1.6. General procedure for the preparation of ethyl 7-
[(dimethylamino)methylidene]-1-substituted-8-oxo-1,4,5,6,7,8-
hexahydrocyclohepta[b]pyrrole-2-carboxylates (15b-d)
Method A: To a solution of ketones 13b-d (1.3 mmol) in anhy-
drous DMF (2.5 mL), DMFDMA (0.19 mL, 1.4 mmol) was added and
the reaction mixture was irradiated under microwave conditions
(Power 50 W; Pressure 100 psi; Temperature 100 ꢀC) up to
completeness (TLC).
Method B: To a solution of ketones 13b-d (1.3 mmol) in anhy-
drous DMF (2.5 mL), TBDMAM (0.41 mL, 2 mmol) was added and
the reaction mixture was irradiated under microwave conditions
(Power 50 W; Pressure 100 psi; Temperature 100 ꢀC) up to
completeness (TLC).
5.1.7. General procedure for the preparation of ethyl 2-amino-10-
substituted-5,6,7,10-tetrahydropyrrolo[30,20:6,7]cyclohepta[1,2-d]
pyrimidine-9-carboxylates (9a-c)
To a suspension of MeONa (1.08 g, 20 mmol) in anhydrous EtOH
(15 mL), guanidine nitrate (1.22 g, 10 mmol) and a solution of the
suitable enaminoketones 15b-d (2 mmol) in anhydrous EtOH
(20 mL) were added. The reaction mixture was heated under reflux
for 1 h and 45 min. Then the reaction mixture was poured onto
crushed ice and the precipitate was filtered off, dried and purified
by chromatography (DCM/AcOEt 8:2).
5.1.7.1. Ethyl 2-amino-10-methyl-5,6,7,10-tetrahydropyrrolo
In both cases, the reaction mixtures was poured onto crushed
ice. The precipitate was filtered off and dried, in the absence of
precipitate the solution was extracted with AcOEt (3 ꢃ 30 mL). The
organic layer was dried (Na2SO4) and the solvent was removed
under reduced pressure.
[30,20:6,7]cyclohepta[1,2-d]pyrimidine-9-carboxylate
(9a). This
compound was obtained from reaction of 15b. White solid; yield
72%; m.p. 212 ꢀC; IR cmꢂ1: 3520e3412 (NH2), 1696 (CO); 1H NMR
(CDCl3, 200 MHz, ppm):
d
1.36 (t, 3H, J ¼ 7.1 Hz, CH3), 2.03e2.16 (m,
2H, CH2), 2.42 (t, 2H, J ¼ 6.9 Hz, CH2), 2.49 (t, 2H, J ¼ 6.9 Hz, CH2),
4.16 (s, 3H, CH3), 4.30 (q, 2H, J ¼ 7.1 Hz, CH2), 5.01 (bs, 2H, NH2), 6.85
5.1.6.1. Ethyl
7-[(dimethylamino)methylidene]-1-methyl-8-oxo-
(15b).
(s, 1H, H-8), 8.17 (s, 1H, H-4); 13C NMR (CDCl3, 50 MHz, ppm):
d 14.4
1,4,5,6,7,8-hexahydrocyclohepta[b]pyrrole-2-carboxylate
(q), 23.5 (t), 27.4 (t), 31.7 (t), 34.6 (q), 60.0 (t), 116.8 (d), 123.9 (s),
124.9 (s), 126.6 (s), 133.4 (s), 158.4 (d), 159.5 (s), 161.2 (s), 161.4 (s).
Anal calcd for C15H18N4O2 (286.33): C, 62.92; H, 6.34; N, 19.57.
Found: C, 61.87; H, 6.06; N, 19.65.
This compound was obtained from reaction of 13b under mw
conditions (Method A, 40 min; Method B, 20 min). Pale yellow
solid; yield 70% (Method A), 99% (Method B); m.p. 110e111 ꢀC; IR
cmꢂ1: 1700 (CO), 1636 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
d
1.27 (t, 3H, J ¼ 6.9 Hz, CH3), 1.70e1.80 (m, 2H, CH2), 2.32 (t, 2H,
5.1.7.2. Ethyl 2-amino-10-benzyl-5,6,7,10-tetrahydropyrrolo
J ¼ 6.2 Hz, CH2), 2.46 (t, 2H, J ¼ 6.2 Hz, CH2), 3.09 (s, 6H, 2 ꢃ CH3),
3.96 (s, 3H, CH3), 4.22 (q, 2H, J ¼ 6.9 Hz, CH2), 6.67 (s, 1H, H-3), 7.46
[30,20:6,7]cyclohepta[1,2-d]pyrimidine-9-carboxylate
(9b). This
compound was obtained from reaction of 15c. White solid; yield
(s, 1H, CH); 13C (DMSO-d6, 50 MHz, ppm):
d 14.2 (q), 22.8 (t), 23.0
60%; m.p. 150e151 ꢀC; IR cmꢂ1: 3523ꢂ3411 (NH2), 1700 (CO); 1H
(t), 29.6 (t), 33.8 (q), 43.0 (2 ꢃ q), 59.6 (t), 105.2 (s), 115.7 (d), 123.9
NMR (CDCl3, 200 MHz, ppm):
d
1.29 (t, 3H, J ¼ 7.1 Hz, CH3),
(s), 125.5 (s), 136.7 (s), 150.0 (d), 160.4 (s), 186.4 (s). Anal calcd for
2.07e2.14 (m, 2H, CH2), 2.36 (t, 2H, J ¼ 6.9 Hz, CH2), 2.51 (t, 2H,
J ¼ 6.9 Hz, CH2), 4.24 (q, 2H, J ¼ 7.1 Hz, CH2), 4.92 (bs, 2H, NH2), 6.13
(s, 2H, CH2), 6.85 (d, 2H, J ¼ 7.8 Hz, H-20 and H-60), 6.93 (s, 1H, H-8),
7.10e7.17 (m, 3H, H-30, H-40 and H-50), 8.12 (s, 1H, H-4); 13C NMR
C
16H22N2O3 (290.36): C, 66.18; H, 7.64; N, 9.65. Found: C, 66.01; H,
7.49; N, 9.78.
5.1.6.2. Ethyl
7-[(dimethylamino)methylidene]-1-benzyl-8-oxo-
(15c).
(CDCl3, 50 MHz, ppm): d 14.3 (q), 23.5 (t), 27.3 (t), 31.8 (t), 48.7 (t),
1,4,5,6,7,8-hexahydrocyclohepta[b]pyrrole-2-carboxylate
60.0 (t), 117.9 (d), 123.6 (s), 124.7 (s), 126.0 (2 ꢃ d), 126.6 (d), 127.3
(s), 128.2 (2 ꢃ d), 133.4 (s), 139.7 (s), 158.5 (d), 159.5 (s), 160.9 (s),
161.4 (s). Anal calcd for C21H22N4O2 (362.42): C, 69.59; H, 6.12; N,
15.46. Found: C, 69.71; H, 6.01; N, 15.34.
This compound was obtained from reaction of 13c under mw
conditions (Method A, 80 min; Method B, 20 min). Pale yellow
solid; yield 73% (Method A), 99% (Method B); m.p. 130e131 ꢀC; IR
cmꢂ1: 1704 (CO), 1635 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
d
1.26 (t, 3H, J ¼ 7.1 Hz, CH3), 1.72e1.86 (m, 2H, CH2), 2.36 (t, 2H,
5.1.7.3. Ethyl 2-amino-10-(4-methoxybenzyl)-5,6,7,10-
tetrahydropyrrolo[30,20:6,7]cyclohepta[1,2-d]pyrimidine-9-
carboxylate (9c). This compound was obtained from reaction of
15d. White solid; yield 65%; m.p. 78e79 ꢀC; IR cmꢂ1: 3512ꢂ3411
J ¼ 6.9 Hz, CH2), 2.62 (t, 2H, J ¼ 6.9 Hz, CH2), 3.07 (s, 6H, 2 ꢃ CH3),
4.21 (q, 2H, J ¼ 7.1 Hz, CH2), 6.08 (s, 2H, CH2), 6.78 (s, 1H, H-3),
7.10e7.22 (m, 5H, Ar), 7.54 (s, 1H, CH); 13C NMR (DMSO-d6, 50 MHz,
ppm):
d
14.1 (q), 22.8 (t), 23.0 (t), 29.8 (t), 43.0 (2 ꢃ q), 47.4 (t), 59.7
(NH2), 1696 (CO); 1H NMR (CDCl3, 200 MHz, ppm):
d 1.31 (t, 3H,
(t), 104.9 (s), 116.7 (d), 123.5 (s), 125.6 (s), 125.7 (2 ꢃ d), 126.6 (d),
128.3 (2 ꢃ d), 133.8 (s), 139.8 (s), 150.3 (d), 160.2 (s), 186.4 (s). Anal
calcd for C22H26N2O3 (366.45): C, 72.11; H, 7.15; N, 7.64. Found: C,
72.30; H, 6.98; N, 7.82.
J ¼ 7.1 Hz, CH3), 2.06e2.16 (m, 2H, CH2), 2.34 (t, 2H, J ¼ 6.7 Hz, CH2),
2.49 (t, 2H, J ¼ 6.7 Hz, CH2), 3.71 (s, 3H, CH3), 4.25 (q, 2H, J ¼ 7.1 Hz,
CH2), 4.94 (bs, 2H, NH2), 6.06 (s, 2H, CH2), 6.69 (d, 2H, J ¼ 8.7 Hz, H-
30 and H-50), 6.83 (d, 2H, J ¼ 8.7 Hz, H-20 and H-60), 6.90 (s, 1H, H-8),
8.13 (s, 1H, H-4); 13C NMR (CDCl3, 50 MHz, ppm):
d 14.3 (q), 23.4 (t),
5.1. 6. 3. E thyl 7-[(dimethylam ino) methylid ene]-1-(4-
methoxybenzyl)-8-oxo-1,4,5,6,7,8-hexahydrocyclohepta[b]pyrrole-2-
carboxylate (15d). This compound was obtained from reaction of
13d under mw conditions (Method A, 40 min; Method B, 40 min).
27.3 (t), 31.9 (t), 48.0 (t), 55.1 (q), 60.0 (t), 113.6 (2 ꢃ d), 117.9 (d),
123.6 (s), 124.6 (s), 127.3 (s), 127.5 (2 ꢃ d), 131.8 (s), 133.3 (s), 158.3
(s), 158.6 (d), 159.7 (s), 160.9 (s), 161.4 (s). Anal calcd for C22H24N4O3
(392.45): C, 67.33; H, 6.16; N, 14.28. Found: C, 67.56; H, 6.29; N,

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457
14.09.
15b. White solid; yield 80%; m.p.: 141 ꢀC; IR cmꢂ1: 3427 (NH), 1697
(CO); 1H NMR (CDCl3, 200 MHz, ppm):
1.13e1.39 (m, 7H, CH3,
d
5.1.8. General procedure for the preparation of ethyl 10-substituted-
2-(phenylamino)-5,6,7,10-tetrahydropyrrolo[30,20:6,7]cyclohepta
[1,2-d]pyrimidine-9-carboxylates (9d-f) and ethyl 2-
2 ꢃ CH2), 1.57e1.85 (m, 4H, 2 ꢃ CH2), 1.95e2.12 (m, 4H, 2 ꢃ CH2),
2.37e2.51 (m, 4H, 2 ꢃ CH2), 3.78e3.90 (m, 1H, CH), 4.18 (s, 3H, CH3),
4.30 (q, 2H, J ¼ 7.1 Hz, CH2), 4.97 (d, 1H, J ¼ 8.3 Hz, NH), 6.84 (s, 1H,
(cyclohexylamino)-10-substituted-5,6,7,10-tetrahydropyrrolo
[30,20:6,7]cyclohepta[1,2-d]pyrimidine-9-carboxylates (9g-i)
A solution of the suitable enaminoketones 15b-d (1.5 mmol) and
phenylguanidine (in the case of derivatives 9d-f) or cyclo-
hexylguanidine (in the case of derivatives 9g-i) (4.5 mmol) in
anhydrous DMF (8 mL) was heated at 100 ꢀC for 2 h. Then the re-
action mixture was poured onto crushed ice and the precipitate
was filtered off, dried and purified by chromatography (DCM).
H-8), 8.13 (s, 1H, H-4); 13C NMR (CDCl3, 50 MHz, ppm):
d 14.4 (q),
23.7 (t), 24.9 (2 ꢃ t), 25.7 (t), 27.3 (t), 31.7 (t), 33.4 (2 ꢃ t), 34.6 (q),
49.9 (d), 59.9 (t), 116.8 (d), 122.3 (s), 124.6 (s), 126.4 (s), 134.0 (s),
146.3 (s), 158.4 (d), 160.4 (s), 161.3 (s). Anal calcd for C21H28N4O2
(368.47): C, 68.45; H, 7.66; N, 15.21. Found: C, 68.29; H, 7.77; N,
15.08.
5.1.8.5. Ethyl 2-(cyclohexylamino)-10-benzyl-5,6,7,10-
tetrahydropyrrolo[30,20:6,7]cyclohepta[1,2-d]pyrimidine-9-
carboxylate (9h). This compound was obtained from reaction of
15c. White solid; yield 85%; m.p. 162 ꢀC; IR cmꢂ1: 3424 (NH), 1700
5.1.8.1. Ethyl 10-methyl-2-(phenylamino)-5,6,7,10-tetrahydropyrrolo
[30,20:6,7]cyclohepta[1,2-d]pyrimidine-9-carboxylate
(9d). This
compound was obtained from reaction of 15b. Pale yellow solid;
(CO); 1H NMR (CDCl3, 200 MHz, ppm):
d 1.15e1.31 (m, 7H, CH3,
yield 75%; m.p. 159 ꢀC; IR cmꢂ1: 3417 (NH), 1704 (CO); 1H NMR
2 ꢃ CH2), 1.56e1.65 (m, 4H, 2 ꢃ CH2), 1.87e1.96 (m, 2H, CH2),
2.06e2.16 (m, 2H, CH2), 2.34 (t, 2H, J ¼ 6.9 Hz, CH2), 2.51 (t, 2H,
J ¼ 6.9 Hz, CH2), 3.65e3.70 (m, 1H, CH), 4.21 (q, 2H, J ¼ 7.1 Hz, CH2),
4.90 (d, 1H, J ¼ 8.4 Hz, NH), 6.15 (s, 2H, CH2), 6.87 (s, 1H, H-8),
6.90e6.94 (m, 2H, H-20 and H-60), 7.13e7.20 (m, 3H, H-30, H-40 and
(CDCl3, 200 MHz, ppm):
d
1.37 (t, 3H, J ¼ 7.1 Hz, CH3), 2.10e2.17 (m,
2H, CH2), 2.43e2.55 (m, 4H, 2 ꢃ CH2), 4.20 (s, 3H, CH3), 4.32 (q, 2H,
J ¼ 7.1 Hz, CH2), 6.87 (s, 1H, H-8), 7.02 (t, 1H, J ¼ 8.4 Hz, H-400), 7.29
(bs,1H, NH), 7.34 (dd, 2H, J ¼ 8.4, 2.5 Hz, H-200 and H-600), 7.62 (d, 2H,
J ¼ 8.4 Hz, H-300 and H-500), 8.29 (s, 1H, H-4); 13C NMR (CDCl3,
H-50), 8.09 (s, 1H, H-4); 13C NMR (CDCl3, 50 MHz, ppm):
d 14.3 (q),
50 MHz, ppm):
d
14.4 (q), 23.6 (t), 27.5 (t), 32.0 (t), 34.8 (q), 60.0 (t),
23.4 (t), 24.7 (2 ꢃ t), 25.7 (t), 27.3 (t), 32.0 (t), 33.4 (2 ꢃ t), 48.7 (t),
49.5 (d), 59.9 (t), 117.9 (d), 122.0 (s), 124.2 (s), 125.9 (2 ꢃ d), 126.5
(d), 127.0 (s), 128.2 (2 ꢃ d), 133.9 (s), 139.8 (s), 158.5 (d), 159.2 (s),
160.4 (s), 160.9 (s). Anal calcd for C27H32N4O2 (444.57): C, 72.94; H,
7.26; N, 12.60. Found: C, 73.06; H, 7.15; N, 12.73.
116.8 (d), 119.0 (2 ꢃ d), 122.2 (d), 124.8 (s), 124.9 (s), 126.7 (s), 128.9
(2 ꢃ d), 133.5 (s), 139.7 (s), 158.3 (d), 158.5 (s), 159.0 (s), 161.3 (s).
Anal calcd for C21H22N4O2 (362.42): C, 69.59; H, 6.12; N, 15.46.
Found: C, 69.44; H, 6.25; N, 15.58.
5.1.8.2. Ethyl 10-benzyl-2-(phenylamino)-5,6,7,10-tetrahydropyrrolo
[30,20:6,7]cyclohepta[1,2-d]pyrimidine-9-carboxylate (9e). This com-
pound was obtained from reaction of 15c. Yellow solid; yield 70%;
m.p. 148 ꢀC; IR cmꢂ1: 3417 (NH), 1700 (CO); 1H NMR (CDCl3,
5.1.8.6. Ethyl 2-(cyclohexylamino)-10-(4-methoxybenzyl)-5,6,7,10-
tetrahydropyrrolo[30,20:6,7] cyclohepta[1,2-d]pyrimidine-9-
carboxylate (9i). This compound was obtained from reaction of
15d. Yellow solid; yield 78%; m.p. 136 ꢀC; IR cmꢂ1: 3428 (NH), 1704
200 MHz, ppm):
d
1.29 (t, 3H, J ¼ 7.1 Hz, CH3), 2.11e2.20 (m, 2H,
(CO); 1H NMR (CDCl3, 200 MHz, ppm):
d 1.12e1.33 (m, 7H, CH3,
CH2), 2.39 (t, 2H, J ¼ 6.9 Hz, CH2), 2.54 (t, 2H, J ¼ 6.9 Hz, CH2), 4.24
(q, 2H, J ¼ 7.1 Hz, CH2), 6.18 (s, 2H, CH2), 6.83 (dd, 2H, J ¼ 8.4, 2.3 Hz,
Ar), 6.95 (s, 1H, H-8), 7.00e7.31 (m, 7H, Ar and NH), 7.56 (d, 2H,
J ¼ 8.4 Hz, Ar), 8.23 (s, 1H, H-4); 13C NMR (CDCl3, 50 MHz, ppm):
2 ꢃ CH2), 1.55e1.68 (m, 4H, 2 ꢃ CH2), 1.87e2.13 (m, 4H, 2 ꢃ CH2),
2.32 (t, 2H, J ¼ 6.9 Hz, CH2), 2.49 (t, 2H, J ¼ 6.9 Hz, CH2), 3.72 (s, 3H,
CH3), 3.75e3.78 (m,1H, CH), 4.23 (q, 2H, J ¼ 7.1 Hz, CH2), 4.92 (d,1H,
J ¼ 8.4 Hz, NH), 6.08 (s, 2H, CH2), 6.71 (d, 2H, J ¼ 8.4 Hz, H-30 and H-
50), 6.85 (d, 2H, J ¼ 8.4 Hz, H-20 and H-60), 6.90 (s, 1H, H-8), 8.11 (s,
d
14.3 (q), 23.5 (t), 27.5 (t), 32.0 (t), 48.7 (t), 60.1 (t), 117.9 (d), 119.0
(2 ꢃ d), 122.2 (d), 124.6 (s), 124.8 (s), 125.9 (2 ꢃ d), 126.6 (d), 127.5
(s), 128.2 (2 ꢃ d), 128.9 (2 ꢃ d), 133.4 (s), 139.6 (s), 139.7 (s), 158.3
(d), 158.5 (s), 159.1 (s), 160.9 (s). Anal calcd for C27H26N4O2 (438.52):
C, 73.95; H, 5.98; N, 12.78. Found: C, 74.08; H, 5.83; N, 12.90.
1H, H-4); 13C NMR (CDCl3, 50 MHz, ppm):
d 14.3 (q), 23.4 (t), 24.8
(2 ꢃ t), 25.7 (t), 27.2 (t), 32.1 (t), 33.4 (2 ꢃ t), 47.9 (t), 49.5 (d), 55.1
(q), 59.9 (t),113.6 (2 ꢃ d),117.8 (d),122.0 (s),124.0 (s),127.0 (s),127.4
(2 ꢃ d), 131.9 (s), 133.8 (s), 158.3 (s), 158.5 (d), 159.3 (s), 160.5 (s),
160.9 (s). Anal calcd for C28H34N4O3 (474.59): C, 70.86; H, 7.22; N,
11.81. Found: C, 70.71; H, 7.38; N, 11.69.
5.1.8.3. Ethyl 10-(4-methoxybenzyl)-2-(phenylamino)-5,6,7,10-
tetrahydropyrrolo[30,20:6,7] cyclohepta[1,2-d]pyrimidine-9-
carboxylate (9f). This compound was obtained from reaction of
15d. Pale yellow solid; yield 60%; m.p. 132e133 ꢀC; IR cmꢂ1: 3416
5.1.9. General procedure for the preparation of 2-amino-10-
substituted-5,6,7,10-tetrahydropyrrolo[30,20:6,7]cyclohepta[1,2-d]
pyrimidine-9-carboxamides (9j-l), 10-substituted-2-
(NH), 1696 (CO); 1H NMR (CDCl3, 200 MHz, ppm):
d 1.31 (t, 3H,
J ¼ 7.1 Hz, CH3), 2.09e2.19 (m, 2H, CH2), 2.37 (t, 2H, J ¼ 6.8 Hz, CH2),
2.51 (t, 2H, J ¼ 6.8 Hz, CH2), 3.70 (s, 3H, CH3), 4.25 (q, 2H, J ¼ 7.1 Hz,
CH2), 6.12 (s, 2H, CH2), 6.67 (d, 2H, J ¼ 8.9 Hz, H-30 and H-50), 6.77 (d,
2H, J ¼ 8.9 Hz, H-20 and H-60), 6.92 (s,1H, H-8), 7.00 (t,1H, J ¼ 8.3 Hz,
H-400), 7.22e7.32 (m, 3H, H-200, H-600 and NH), 7.57 (d, 2H, J ¼ 8.3 Hz,
H-300 and H-500), 8.25 (s, 1H, H-4); 13C NMR (CDCl3, 50 MHz, ppm):
(phenylamino)-5,6,7,10-tetrahydropyrrolo[30,20:6,7]cyclohepta[1,2-
d]pyrimidine-9-carboxamides (9m-o) and 2-(cyclohexylamino)-10-
substituted-5,6,7,10-tetrahydropyrrolo[30,20:6,7]cyclohepta [1,2-d]
pyrimidine-9-carboxamides (9p-r)
To a solution of NaOH (0.02 g, 0.4 mmol) in EtOH (5 mL), a so-
lution of 9a-i (0.1 mmol) in EtOH (8 mL) was added and the reaction
mixture was heated under reflux for 3 h. After cooling, the solvent
was removed under reduced pressure. The residue was added of
water and the solution was acidified with 6 M HCl. Then the solu-
tion was extracted with DCM (3 ꢃ 50 mL). The organic layer was
dried (Na2SO4) and the solvent was removed under reduced pres-
sure to give the crude acid derivative product which was taken up
immediately onto next step.
d
14.3 (q), 23.4 (t), 27.4 (t), 32.1 (t), 47.9 (t), 55.1 (q), 60.1 (t), 113.6
(2 ꢃ d),117.9 (d),119.0 (2 ꢃ d),122.2 (d),124.2 (s),124.5 (s),124.6 (s),
127.4 (2 ꢃ d), 127.5 (s), 128.9 (2 ꢃ d), 131.7 (s), 133.3 (s), 139.7 (s),
158.3 (d), 158.6 (s), 159.3 (s), 161.0 (s). Anal calcd for C28H28N4O3
(468.55): C, 71.78; H, 6.02; N, 11.96. Found: C, 71.91; H, 5.86; N,
12.11.
5.1.8.4. Ethyl 2-(cyclohexylamino)-10-methyl-5,6,7,10-
tetrahydropyrrolo[30,20:6,7]cyclohepta[1,2-d]pyrimidine-9-
carboxylate (9g). This compound was obtained from reaction of
To a suspension of the above product in anhydrous THF (8 mL),
DIPEA (0.10 mL, 0.55 mmol), HOBt (0.07 g, 0.55 mmol), and EDC
(0.11 g, 0.55 mmol) were added. The reaction mixture was stirred at

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458
room temperature for 10 min, then ammonium carbonate (0.14 g,
1.5 mmol) was added in one portion, and the resulting suspension
was stirred at room temperature for 16 h. The mixture was
concentrated under reduced pressure and a saturated solution of
Na2CO3 (15 mL) was added. The resulting suspension was stirred for
2 h, and the formed solid was filtered off, dried and purified by
chromatography (DCM/AcOEt 8:2).
compound was obtained from reaction of 9e. Pale yellow solid;
yield 74%; m.p. 196 ꢀC; IR cmꢂ1: 3318 (NH), 3273ꢂ3164 (NH2), 1661
(CO); 1H NMR (CDCl3, 200 MHz, ppm):
d 2.10e2.18 (m, 2H, CH2),
2.37 (t, 2H, J ¼ 6.8 Hz, CH2), 2.53 (t, 2H, J ¼ 6.8 Hz, CH2), 5.62 (bs, 2H,
NH2), 6.21 (s, 2H, CH2), 6.58 (s, 1H, H-8), 6.84 (d, 2H, J ¼ 7.4 Hz, Ar),
7.01 (t, 1H, J ¼ 7.4 Hz, Ar), 7.12e7.32 (m, 6H, NH and Ar), 7.58 (d, 2H,
J ¼ 7.9 Hz, Ar), 8.22 (s, 1H, H-4); 13C NMR (CDCl3, 50 MHz, ppm):
d
23.6 (t), 27.5 (t), 32.0 (t), 48.7 (t), 113.7 (d), 119.1 (2 ꢃ d), 122.2 (d),
5.1.9.1. 2-Amino-10-methyl-5,6,7,10-tetrahydropyrrolo[30,20:6,7]
cyclohepta[1,2-d]pyrimidine-9-carboxamide (9j). This compound
was obtained from reaction of 9a. Pale yellow solid; yield 76%; m.p.
196e197 ꢀC; IR cmꢂ1: 3502ꢂ3396 (NH2), 3309ꢂ3212 (NH2), 1662
124.4 (s), 126.2 (2 ꢃ d), 126.7 (d), 127.1 (s), 127.4 (s), 128.2 (2 ꢃ d),
128.9 (2 ꢃ d), 132.5 (s), 139.7 (s), 139.8 (s), 158.1 (d), 158.5 (s), 159.2
(s),163.4 (s). Anal calcd for C25H23N5O (409.48): C, 73.33; H, 5.66; N,
17.10. Found: C, 73.57; H, 5.80; N, 16.92.
(CO); 1H NMR (CDCl3, 200 MHz, ppm):
d 2.04e2.14 (m, 2H, CH2),
2.39e2.51 (m, 4H, 2 ꢃ CH2), 4.14 (s, 3H, CH3), 5.26 (s, 2H, NH2), 6.06
5.1.9.6. 10-(4-Methoxybenzyl)-2-(phenylamino)-5,6,7,10-
tetrahydropyrrolo[30,20:6,7]cyclohepta[1,2-d]pyrimidine-9-
carboxamide (9o). This compound was obtained from reaction of
9f. Pale yellow solid; yield 60%; m.p. 195e196 ꢀC; IR cmꢂ1: 3399
(NH), 3319ꢂ3204 (NH2), 1668 (CO); 1H NMR (CDCl3, 200 MHz,
(s, 2H, NH2), 6.51 (s, 1H, H-8), 8.14 (s, 1H, H-4); 13C NMR (CDCl3,
50 MHz, ppm):
d 23.7 (t), 27.4 (t), 31.6 (t), 34.7 (q), 112.8 (d), 123.6
(s), 126.6 (s), 127.3 (s), 132.4 (s), 158.1 (d), 159.6 (s), 161.5 (s), 163.9
(s). Anal calcd for C13H15N5O (257.29): C, 60.69; H, 5.88; N, 27.22.
Found: C, 60.55; H, 6.01; N, 27.37.
ppm):
d
2.08e2.18 (m, 2H, CH2), 2.36 (t, 2H, J ¼ 6.7 Hz, CH2), 2.50 (t,
2H, J ¼ 6.7 Hz, CH2), 3.69 (s, 3H, CH3), 5.72 (bs, 2H, NH2), 6.14 (s, 2H,
CH2), 6.55 (s,1H, H-8), 6.66 (d, 2H, J ¼ 8.7 Hz, H-30 and H-50), 6.80 (d,
2H, J ¼ 8.7 Hz, H-20 and H-60), 7.01 (t,1H, J ¼ 7.8 Hz, H-400), 7.28e7.33
(m, 3H, H-200, H-600 and NH), 7.61 (d, 2H, J ¼ 7.8 Hz, H-300 and H-500),
5.1.9.2. 2-Amino-10-benzyl-5,6,7,10-tetrahydropyrrolo[30,20:6,7]
cyclohepta[1,2-d]pyrimidine-9-carboxamide (9k). This compound
was obtained from reaction of 9b. White solid; yield 65%, m.p.
191e192 ꢀC; IR cmꢂ1: 3481ꢂ3420 (NH2), 3313ꢂ3250 (NH2), 1662
8.23 (s, 1H, H-4); 13C NMR (CDCl3, 50 MHz, ppm):
d 23.5 (t), 27.5 (t),
(CO); 1H NMR (CDCl3, 200 MHz, ppm):
d
2.01e2.09 (m, 2H, CH2),
32.1 (t), 47.9 (t), 55.1(q), 113.6 (2 ꢃ d), 113.7 (d), 119.0 (2 ꢃ d), 122.2
(d), 124.3 (s), 127.0 (s), 127.4 (s), 127.7 (2 ꢃ d), 128.9 (2 ꢃ d), 131.9 (s),
132.4 (s), 139.7 (s), 158.1 (d), 158.3 (s), 158.6 (s), 159.3 (s), 163.4 (s).
Anal calcd for C26H25N5O2 (439.51): C, 71.05; H, 5.73; N, 15.93.
Found: C, 70.88; H, 5.63; N, 16.07.
2.33 (t, 2H, J ¼ 7.1 Hz, CH2), 2.50 (t, 2H, J ¼ 7.1 Hz, CH2), 4.97 (bs, 2H,
NH2), 5.65 (bs, 2H, NH2), 6.13 (s, 2H, CH2), 6.55 (s, 1H, H-8),
6.85e6.90 (m, 2H, H-20 and H-60), 7.06e7.19 (m, 3H, H-30, H-40 and
H-50), 8.09 (s, 1H, H-4); 13C NMR (CDCl3, 50 MHz, ppm):
d 23.6 (t),
27.3 (t), 31.8 (t), 48.6 (t), 113.7 (d), 123.4 (s), 126.3 (2 ꢃ d), 126.7 (d),
127.1 (s), 127.3 (s), 128.2 (2 ꢃ d), 132.4 (s), 139.8 (s), 158.3 (d), 159.6
(s), 161.3 (s), 163.3 (s). Anal calcd for C19H19N5O (333.39): C, 68.45;
H, 5.74; N, 21.01. Found: C, 68.61; H, 5.65; N, 21.29.
5.1.9.7. 2-(Cyclohexylamino)-10-methyl-5,6,7,10-tetrahydropyrrolo
[30,20:6,7]cyclohepta[1,2-d]
pyrimidine-9-carboxamide
(9p).
This compound was obtained from reaction of 9g. Pale pink solid;
yield 82%; m.p. 205 ꢀC; IR cmꢂ1: 3403 (NH), 3276ꢂ3183 (NH2), 1662
5.1.9.3. 2-Amino-10-(4-methoxybenzyl)-5,6,7,10-tetrahydropyrrolo
(CO); 1H NMR (CDCl3, 200 MHz, ppm):
d
1.15e1.50 (m, 4H, 2 ꢃ CH2),
[30,20:6,7]cyclohepta[1,2-d]pyrimidine-9-carboxamide
(9l). This
1.57e1.91 (m, 4H, 2 ꢃ CH2), 1.98e2.12 (m, 4H, 2 ꢃ CH2), 2.37e2.53
(m, 4H, 2 ꢃ CH2), 3.75e3.84 (m, 1H, CH), 4.18 (s, 3H, CH3), 5.04 (d,
1H, J ¼ 8.1 Hz, NH), 5.70 (bs, 2H, NH2), 6.50 (s, 1H, H-8), 8.12 (s, 1H,
compound was obtained from reaction of 9c. White solid; yield
94%; m.p. 194 ꢀC; IR cmꢂ1: 3479ꢂ3420 (NH2), 3310ꢂ3246 (NH2),
1663 (CO); 1H NMR (CDCl3, 200 MHz, ppm):
d
2.05e2.15 (m, 2H,
H-4); 13C NMR (CDCl3, 50 MHz, ppm):
d
23.8 (t), 24.9 (2 ꢃ t), 25.7 (t),
CH2), 2.33 (t, 2H, J ¼ 6.9 Hz, CH2), 2.49 (t, 2H, J ¼ 6.9 Hz, CH2), 3.71 (s,
3H, CH3), 4.93 (bs, 2H, NH2), 5.55 (bs, 2H, NH2), 6.09 (s, 2H, CH2),
6.53 (s, 1H, H-8), 6.70 (d, 2H, J ¼ 8.7 Hz, H-30 and H-50), 6.86 (d, 2H,
J ¼ 8.7 Hz, H-20 and H-60), 8.12 (s, 1H, H-4); 13C NMR (CDCl3,
27.4 (t), 31.7 (t), 33.4 (2 ꢃ t), 34.8 (q), 49.9 (d), 112.7 (d), 122.1 (s),
126.3 (s), 126.8 (s), 133.1 (s), 158.2 (d), 159.1 (s), 160.4 (s), 163.6 (s).
Anal calcd for C19H25N5O (339.43): C, 67.23; H, 7.42; N, 20.63.
Found: C, 67.06; H, 7.55; N, 20.82.
50 MHz, ppm):
d 23.5 (t), 27.3 (t), 31.8 (t), 47.9 (t), 55.1 (q), 113.5
(2 ꢃ d), 113.7 (d), 123.4 (s), 127.0 (s), 127.3 (s), 127.8 (2 ꢃ d), 130.9 (s),
131.9 (s), 132.3 (s), 158.3 (d), 159.8 (s), 161.3 (s), 163.4 (s). Anal calcd
for C20H21N5O2 (363.41): C, 66.10; H, 5.82; N, 19.27. Found: C, 65.99;
H, 5.71; N, 19.39.
5.1.9.8. 2-(Cyclohexylamino)-10-benzyl-5,6,7,10-tetrahydropyrrolo
[30,20:6,7]cyclohepta[1,2-d]pyrimidine-9-carboxamide
(9q). This
compound was obtained from reaction of 9h. White solid; yield
85%; m.p. 226e227 ꢀC; IR cmꢂ1: 3396 (NH), 3209ꢂ3122 (NH2), 1696
(CO); 1H NMR (CDCl3, 200 MHz, ppm):
d
1.10e1.25 (m, 4H, 2 ꢃ CH2),
5.1.9.4. 10-Methyl-2-(phenylamino)-5,6,7,10-tetrahydropyrrolo
1.61e1.75 (m, 4H, 2 ꢃ CH2), 1.89e2.12 (m, 4H, 2 ꢃ CH2), 2.32 (t, 2H,
J ¼ 6.8 Hz, CH2), 2.50 (t, 2H, J ¼ 6.8 Hz, CH2), 3.62e3.79 (m, 1H, CH),
4.91 (d, 1H, J ¼ 7.8 Hz, NH), 5.53 (bs, 2H, NH2), 6.20 (s, 2H, CH2), 6.55
(s, 1H, H-8), 6.89 (d, 2H, J ¼ 7.0 Hz, H-20 and H-60), 7.14e7.26 (m, 3H,
H-30, H-40 and H-50), 8.08 (s, 1H, H-4); 13C NMR (CDCl3, 50 MHz,
[30,20:6,7]cyclohepta[1,2-d]pyrimidine-9-carboxamide
(9m).
This compound was obtained from reaction of 9d. Pale yellow solid;
yield 84%; m.p. 254 ꢀC; IR cmꢂ1: 3401 (NH), 3254ꢂ3170 (NH2), 1635
(CO); 1H NMR (CDCl3, 200 MHz, ppm):
d 2.10e2.17 (m, 2H, CH2),
2.43e2.55 (m, 4H, 2 ꢃ CH2), 4.21 (s, 3H, CH3), 5.72 (bs, 2H, NH2),
6.52 (s, 1H, H-8), 7.02 (t, 1H, J ¼ 8.3 Hz, H-400), 7.26e7.36 (m, 3H, NH,
H-200 and H-600), 7.63 (dd, 2H, J ¼ 8.3, 1.2 Hz, H-300 and H-500); 8.28 (s,
ppm):
d
23.5 (t), 24.8 (2 ꢃ t), 25.7 (t), 27.3 (t), 32.1 (t), 33.4 (2 ꢃ t),
48.5 (t), 49.5 (d), 113.7 (d), 121.9 (s), 126.2 (2 ꢃ d), 126.5 (s), 126.6
(d), 126.9 (s), 128.2 (2 ꢃ d), 133.0 (s), 139.9 (s), 158.4 (d), 159.2 (s),
160.4 (s), 163.2 (s). Anal calcd for C25H29N5O (415.53): C, 72.26; H,
7.03; N, 16.85. Found: C, 72.44; H, 7.26; N, 16.74.
1H, H-4); 13C NMR (CDCl3, 50 MHz, ppm):
d 23.7 (t), 27.6 (t), 32.0 (t),
34.9 (q), 112.7 (d), 119.1 (2 ꢃ d), 122.2 (d), 124.6 (s), 126.6 (s), 127.0
(s), 128.9 (2 ꢃ d), 132.7 (s), 139.7 (s), 158.2 (d), 158.5 (s), 159.0 (s),
163.5 (s). Anal calcd for C19H19N5O (333.39): C, 68.45; H, 5.74; N,
21.01. Found: C, 68.60; H, 5.58; N, 20.89.
5.1.9.9. 2-(Cyclohexylamino)-10-(4-methoxybenzyl)-5,6,7,10-
tetrahydropyrrolo[30,20:6,7] cyclohepta[1,2-d]pyrimidine-9-
carboxamide (9r). This compound was obtained from reaction of
9i. Pale yellow solid; yield 93%; m.p. 163 ꢀC; IR cmꢂ1: 3403 (NH),
3270ꢂ3160 (NH2), 1663 (CO); 1H NMR (CDCl3, 200 MHz, ppm):
5.1.9.5. 10-Benzyl-2-(phenylamino)-5,6,7,10-tetrahydropyrrolo
[30,20:6,7]cyclohepta[1,2-d]pyrimidine-9-carboxamide
(9n). This

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459
d
1.20e1.38 (m, 4H, 2 ꢃ CH2), 1.57e1.73 (m, 4H, 2 ꢃ CH2), 1.94e2.11
All cultures were grown in a humidified incubator maintained at
(m, 4H, 2 ꢃ CH2), 2.31 (t, 2H, J ¼ 7.0 Hz, CH2), 2.48 (t, 2H, J ¼ 7.0 Hz,
CH2), 3.71 (s, 3H, CH3), 3.72e3.77 (m,1H, CH), 4.93 (d,1H, J ¼ 8.4 Hz,
NH), 5.53 (bs, 2H, NH2), 6.12 (s, 2H, CH2), 6.53 (s, 1H, H-8), 6.70 (d,
2H, J ¼ 8.7 Hz, H-30 and H-50), 6.87 (d, 2H, J ¼ 8.7 Hz, H-20 and H-60),
37 ꢀC with 5% CO2.
5.3.4. Cellular antiproliferative tests and photocytotoxicity
Cytotoxic effects were determined by MTT assay. Derivatives 9a-
r were dissolved and diluted into working concentrations with
DMSO. Cells (1.25 ꢃ 104 cells/well) were plated into 96-microwell
8.09 (s, 1H, H-4); 13C NMR (CDCl3, 50 MHz, ppm):
d 23.5 (t), 24.8
(2 ꢃ t), 25.7 (t), 27.3 (t), 32.1 (t), 33.4 (2 ꢃ t), 47.8 (t), 49.6 (d), 55.1
(q),113.6 (2 ꢃ d),113.7 (d),121.9 (s),126.3 (s),126.9 (s), 127.7 (2 ꢃ d),
132.1 (s), 132.8 (s), 158.3 (s), 158.4 (d), 159.4 (s), 160.5 (s), 163.3 (s).
Anal calcd for C26H31N5O2 (445.56): C, 70.09; H, 7.01; N, 15.72.
Found: C, 70.21; H, 7.15; N, 15.51.
plates to a final volume of 100
mL and allowed an overnight
period for attachment. The following day compounds were added
to each well to reach a 0.5% final concentration of DMSO per well;
each concentration was tested in triplicate. Compounds were
incubated for 30 min, then cells were washed in 1ꢃ PBS (100
mL)
5.2. Photophysical and photooxidation of 9,10-
anthracenedipropionic acid
and fresh medium was added. In photocytotoxicity experiments,
immediately after medium removal, cells were irradiated on ice for
5 min at 365 nm, corresponding to 1.5 J/cm2, in a photochemical
multirays reactor and incubated at 37 ꢀC for 48 h. Control cells
(without any compound but with 0.5% DMSO) were treated in the
exact same conditions. Cell survival was evaluated by MTT assay:
5.2.1. Nanosecond laser flash photolysis
LP 920 Edinburgh Instrument photolysis system has been
employed to carry out all the detection experiments of the transient
species. The minimum response time of the detection system was
10 mL of freshly dissolved solution of MTT (5 mg/mL in PBS) were
of about 10 ns. The laser beam (a Nd/YAG operated at
l
¼ 355 nm)
added to each well, and after 4 h of incubation, MTT crystals were
solubilized in solubilisation solution (10% sodium dodecyl sulphate
(SDS) and 0.01 M HCl). After overnight incubation at 37 ꢀC, absor-
bance was read at 540 nm. Data were expressed as mean values of
at least three individual experiments conducted in triplicate. The
percentage of cell survival was calculated as follows: cell survival ¼
(Awell ꢂ Ablank)/(Acontrol ꢂ Ablank) ꢃ 100, where blank denotes
the medium without cells.
was focused on a 3 mm wide circular area of the cell and the first
5 mm in depth were analyzed at a right angle geometry. The inci-
dent pulse energies used were 5e7 mJ per pulse. The spectra were
reconstructed point from time profiles taken each 5 nm. The
absorbance at 355 nm A355 was ~1.0 over 1 cm. Ar-saturated or air-
equilibrated solutions were used. The sample was renewed after
few laser shots. Temperature was 295 K.
5.2.2. Quantum yield measurement
5.3.5. Generation of active oxygen species
Quantum yields for ADPA consumption (FR) were measured by
irradiating 2 mL 10ꢂ4 M solutions of 9g and 2.5 ꢃ 10ꢂ4 M of 9j
(ACN:H2O ¼ 1:1 solutions) in 1 cm optical path cuvettes in the
presence of 1.1 ꢃ 10ꢂ4 M concentration of ADPA, after flushing with
oxygen. The lamp source was a focalized 150 W high-pressure
mercury arc fitted with a transmittance filter (transmission,
315 nm and 360 nm). Potassium ferrioxalate was used as the acti-
nometer [62,63].
The cell-permeant 20,7'-dichlorodihydrofluorescein diacetate
(H2DCFDA) is a chemically reduced form of fluorescein used as
indicator for reactive oxygen species (ROS) in cells. Oxidation re-
sults in the formation of fluorescent DCF, which is maximally
excited at 495 nm and emits at 520 nm. MCF7 cells (3 ꢃ 104) were
plated in 96 wells plates medium. After 24 h, when cells reached
80e90% of confluence, RPMI medium containing 10% FBS was
removed and compounds were incubated at different concentra-
tions for 30 min in phenol red and serum free medium to avoid
5.3. Biology
subsequent probe oxidation. H2DCFDA probe (10 mM) was added to
cell medium and incubated for 15 min prior to irradiation (five
5.3.1. Spectrophotometric determinations
minutes at 365 nm, 1.5 J/cm2). Control plates that did not undergo
All spectrophotometric measures were performed using UVeVis
spectrophotometer (Lambda25, PerkinElmer, Milan, Italy). For all
the compounds, absorption spectra, molar extinction coefficients
(ε) and determination of maxima peaks (lmax) were carried out in
irradiation were also included in each experiment. NAC (N-acetyl-L-
cysteine) was used as antioxidant control; NAC prevention of ROS
formation was performed at different concentrations. Phorbol-12-
myristate-13-acetate (PMA) was used as a validated ROS-inducer
compound. ROS production was monitored in a Victor X2 Multi-
label plate reader, lamp excitation filter at 485 nm and emission
filter at 535 nm (Perkin Elmer Italia, Milan).
phosphate buffer (20 mM, pH 7) at 20 mM.
5.3.2. Irradiation procedure
Irradiation experiments were performed in a photochemical
multirays reactor, equipped with 10 UV lamps (3660 Å), emitting at
365 nm (Helios Italquartz, Italy). The spectral irradiance of the
source was 5 mw/cm2 as measured with a UVX Radiometer (UVP,
Upland, CA, USA).
5.3.6. Flow cytofluorimetry
The rate of apoptotic cells was measured by the amount of cell
surface exposure of phosphatidylserine. MCF7 cells in exponential
growth were treated with different concentrations of the test
compounds for 6 h. Cells were centrifuged, fixed with ice-cold
ethanol (70%), treated with citrate buffer containing RNAase, and
stained with PE annexin V according to the manufacturer's in-
structions (PE Annexin V Apoptosis Detection Kit I, BD Bioscience).
Simultaneously, cells were stained with the vital dye 7-Amino-
Actinomycin (7-AAD). Samples were analyzed on an LRS 2 instru-
ment using FACS DIVA Software (BD Bioscience) and analyzed with
Flow Jo (Tree Star).
5.3.3. Cell lines
MCF7 human breast cancer cells, Hela human cervical adeno-
carcinoma cells and HT29 human colorectal carcinoma cells were
purchased from ATCC (ATCC #HTB-22, #CCL2 and # HTB-38 LGC
Standards, Milan Italy). MCF7 cells were grown in RPMI 1640
(Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented
with 10% heat-inactivated fetal bovine serum (FBS, Gibco, Thermo
Fisher Scientific, Waltham, MA, USA); Hela and HT29 cells were
maintained in DMEM (Gibco, Thermo Fisher Scientific, Waltham,
MA, USA) supplemented with 10% heat-inactivated fetal bovine
serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA, USA).
5.3.7. Analysis of mitochondrial dysfunction by confocal microscopy
Mitochondria were labelled in live-cells using Mitotracker Or-
ange CMTM-ROS (Thermo Fisher Scientific, Milan, Italy). MCF7 cells

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460
were plated on glass slides (4 ꢃ 105). After 24 h the complete
medium was removed and replaced with phenol red and serum
free medium containing the mitochondrial probe (150 nM), the
probe was incubated 45 min prior to addition of compounds and
photoactivation. After irradiation cells were washed in 1ꢃ PBS,
fixed in paraformaldehyde (PFA, 4%) and mounted in Vectashield
mounting medium (DBA ITALIA, Milan, Italy). Nuclei were stained
with Nuclear Green dye LCS1 (Abcam, lex 503 nm, lem 526 nm).
Phorbol-12-myristate-13-acetate (PMA) was used as a validated
ROS-inducer compound. Images were captured at a Leica SP2
confocal microscope (63ꢃ objective, Green HeNe (Helium Neon)
laser lex 543 nm, lem 560e620 nm and the Blue Argon laser lex
488 nm, lem 500e530 nm for the nuclear staining).
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This work was financially supported by Ministero dell’Is-
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truzione, dell’Universita e della Ricerca. Financial support from the
Italian Association for Cancer Research (AIRC, IG2013-14708), the
University of Pavia, the University of Padova and the University of
Palermo are gratefully acknowledged.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
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