Synthesis and biological evaluation of novel cytotoxic azanaphthoquinone annelated pyrrolo oximes

Article abstract of DOI:10.1016/j.bmcl.2007.09.054

Two series of azanaphthoquinone annelated pyrrolo oximes have been synthesized. The antiproliferative activities of 10 compounds were evaluated on at least four different cell lines. One series of pyrrolo derivatives showed high cytotoxic activity. The effects on cell cycle and caspase activity were investigated. Compounds 9a and 9b showed an accumulation of cells in G2/M phase. Substantial and dose-dependent caspase activity was found after treatment of cells with 9a and 9b. This indicates an apoptosis inducing property of these compounds.

Full text of DOI:10.1016/j.bmcl.2007.09.054

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6091–6095
Synthesis and biological evaluation of novel cytotoxic
azanaphthoquinone annelated pyrrolo oximes
Karem Shanab,a Nipawan Pongprom,a Eva Wulz,a Wolfgang Holzer,a Helmut Spreitzer,a,*
Peter Schmidt,b Babette Aicher,b Gilbert Mullerb and Eckhard Guntherb
¨
¨
aDepartment of Drug and Natural Product Synthesis, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria
bZentaris GmbH, Drug Discovery and Preclinical Development, Weismuellerstrasse 45, 60314 Frankfurt, Germany
Received 14 May 2007; revised 12 September 2007; accepted 13 September 2007
Available online 18 September 2007
Abstract—Two series of azanaphthoquinone annelated pyrrolo oximes have been synthesized. The antiproliferative activities of 10
compounds were evaluated on at least four different cell lines. One series of pyrrolo derivatives showed high cytotoxic activity. The
effects on cell cycle and caspase activity were investigated. Compounds 9a and 9b showed an accumulation of cells in G2/M phase.
Substantial and dose-dependent caspase activity was found after treatment of cells with 9a and 9b. This indicates an apoptosis induc-
ing property of these compounds.
Ó 2007 Published by Elsevier Ltd.
The anthracene-9,10-dione mitoxantrone (1) has proba-
bly become the most widely used synthetic DNA inter-
calating agent.1 It is active against acute leukemia,
lymphoma, breast cancer, and cervix carcinoma.2
Mitoxantrone binds reversibly to DNA by intercalation,
with the side chains lying in the major groove.3 How-
ever, the cytotoxicity of mitoxantrone is caused largely
by inhibition of topoisomerase II enzyme.4 The use in
clinics of topoisomerase II inhibitors is limited by their
cardiotoxicity and drug resistance.5 The search of new
topoisomerase II inhibitors still constitutes an intensive
field of research.
inactive. The studies on topoisomerase II poisoning ef-
fects showed an impaired interference of 8-aza com-
pounds with the DNA processing enzyme, which
explains the lack of cytotoxicity of these compounds.9
A 9-aza-anthrapyrazole, BBR 3438 (3), is currently in
phase II clinical trials in patients with gastric cancer.10
In our continuous effort to develop novel DNA interac-
tive anticancer compounds based on the azanaphthoqui-
none annelated pyrrole structures,11 we are reporting in
this paper the synthesis of two series of azanaphthoqui-
none pyrrolo oximes of type 9 and 13 with the nitrogen
atom in the pyrrole ring located at position 1 and 2,
respectively. The cytotoxicity against at least four tumor
cell lines is reported and compared between these two
isomers. The active compounds have been further stud-
ied with regard to the effects on the cell cycle and cas-
pase activity (Fig. 1).
The development of aza-bioisosteric chemotypes related
to mitoxantrone lacking the 5,8-dihydroxy substituted
pattern led to the discovery of an aza-anthracene-9,10-
dione BBR 2778 (2), which is currently in phase III clin-
ical trials in patients with non-Hodgkin’s lymphoma.6
This compound showed a better therapeutic index and
lower cardiotoxicity in comparison with mitoxantrone.7
The 9-aza-anthrapyrazoles have also been developed as
analogs of anthrapyrazoles. The position of the nitrogen
has been found to exert a profound effect on the antitu-
mor activity.8 While 9-aza derivatives showed outstand-
ing activity, the 8-aza congeners were essentially
Annelated pyrrolo oximes of type 9 were synthesized
from 5-hydroxyisoquinoline (4) as shown in Scheme 1.
Oxidation of 4 yielded isoquinoline-5,8-dione (5) in high
yield.12 Treatment of 5 with sodium azide gave 88% of 7-
aminoisoquinoline-5,8-dione (6), which was reacted with
Mn(OAc)3/acetaldehyde diethylacetal13 to furnish 1H-
pyrrolo[3,2-g]isoquinoline-4,9-dione (7)14 in 62% yield.
N-Alkylation of 7 was carried out with NaH/N,N-dim-
ethylaminoethyl chloride to obtain the alkylated prod-
uct 8 in 48% yield. The aza-condensation of 8 with a
series of hydroxylamine derivatives15 occurred regiose-
Keywords: Anticancer compounds.
*
Corresponding author. Tel.: +43 1427755621; fax: +43 142779556;
e-mail: helmut.spreitzer@univie.ac.at
0960-894X/$ - see front matter Ó 2007 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2007.09.054

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K. Shanab et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6091–6095
H
N
O
MeO OMe
NH2
O
HN
HN
OH
O
HN
HN
OH
2 HCl
OH
a
NH
N
N
N
2 Maleic acid
NH2
O
O
O
OH
O
10
11
N
H
1
2
R
O
b
H
N
N
N
OH
N
O
N
N
Me
N
2 HCl
N
Me
N
c
N
N
Me
Me
O
HN
CH3
O
O
N
H
3
13a: R = CH2CH2NMe2
13b: R = CH2CH2OH
13c: R = Me
12
Figure 1.
d
13d: R = H
13e: R = CH2COOEt
O
O
OH
Scheme 2. Reagents and conditions: (a) 1—TosMIC, KOt-Bu, THF,
rt, 18 h; 2—PPTS/TsOH, acetone/H2O, 35 °C, 24 h; (b)
ClCH2CH2NMe2ÆHCl, NaH, DMF, 67 °C, 4 h; (c) NH2ORÆHCl,
50% KOH, MeOH, rt; (d) BrCH2COOEt, NaH, DMF, rt.
a
b
N
N
N
NH2
O
O
4
6
5
which was condensed regioselectively with hydroxyl
amines at C-9 to the corresponding oximes 13a–13d in
good yields. The regioselectivity of the aza-condensation
was determined by 2D NMR and NOE experiments.
Irradiation at OMe showed a significant nuclear Over-
hauser enhancement of C(1)-H and C(8)-H, therefore
indicating the condensation at C-9 in the quinone sys-
tem. Oxime 13d underwent reaction with ethyl bromo
acetate to give 13e in 26% yield.22
R
O
c
N
O
O
d
e
N
N
N
N
N
N
H
O
O
O
N
CH3
N
8
7
CH3
H3C
H3C
9a: R = CH2CH2NMe2
9b: R = CH2CH2OH
9c: R = Me
Compounds 9a–9e, 13a–13e as well as reference com-
pounds paclitaxel and doxorubicin were screened for
antiproliferative activity against different cancer cell lines
KB/HeLa (cervical carcinoma), NCI-H460 (large-cell
lung cancer), SKOV-3 (ovarian carcinoma), SF-268
(CNS, glioma), and RKOp27 (colon adenocarcinoma).16
The quinones 8 and 12 were included in the screening in
order to determine whether the oxime formation leads to
improved cytotoxic activities. The concentration of the
compound that inhibits 50% (IC50) of cell proliferation
after 48 h was calculated by nonlinear regression
(GraphPad PrismTM) using the data from at least two
independent tetrazolium-based (XTT) cytotoxicity as-
says.17 Results of the cytotoxicity assays are shown in de-
tail in Table 1. Interestingly compounds 8 and 9a–9e of
the pyrrolo[2,3-g]isoquinoline series displayed signifi-
cantly higher inhibition across all cell lines compared
to compounds 12 and 13a–13e of the pyrrolo[3,4-g]iso-
quinoline series (<50%). Cell lines NCI-H460 and
RKOp27 were found to be most sensitive to compounds
of the pyrrolo[2,3-g]isoquinoline series. In particular the
O-(2-hydroxyethyl)oxime derivative 9b exhibited the
highest inhibition with a mean IC50 of 0.13 lM against
NCI-H460 and 0.17 lM against RKOp27 cells. O-[2-
(Dimethylamino) ethyl]oxime 9a was found to be the sec-
ond most active compound with an IC50 value of
0.23 lM, followed by derivative 9c with 0.79 lM against
NCI-H460. Compound 9d showed moderate activity
with IC50 values of >12 lM against all tested cell lines.
It must be emphasized that oximation of 8 led to about
f
9d: R = H
9e: R = CH2COOEt
Scheme 1. Reagents and conditions: (a) PhI(OCOCF3)2, MeCN/H2O,
0 °C, 2 h; (b) NaN3/H2O, AcOH, THF, 40 °C, 2 h; (c) MeCH(OEt)2,
Mn(OAc)3, AcOH, 60 °C, 18 h; (d) ClCH2CH2NMe2ÆHCl, NaH,
DMF, 67 °C, 4 h; (e) NH2ORÆHCl, 50% KOH, MeOH, rt; (f)
BrCH2COOEt, NaH, DMF, rt.
lectively at C-4 to furnish the corresponding oximes 9a–
9d. The structure elucidation of oxime 9c was accom-
plished by 2D NMR and NOE experiments. Irradiation
at OMe showed a significant nuclear Overhauser
enhancement of C(3)-H and C(5)-H thus indicating the
condensation at C-4 in the quinone system. The regiose-
lectivity of the reaction could be explained by the stron-
ger electrophilicity of the carbonyl group located in
para-position to the nitrogen atom in the pyridine ring.
Compound 9d was converted into 9e by O-alkylation
with ethyl bromoacetate using NaH in DMF.
Pyrrolo oximes of type 13 were prepared as outlined in
Scheme 2, starting from 5,5-dimethoxyisoquinolin-
8(5H)-one (10).14 The first step involves the Michael
addition-cyclization of the unsaturated ketone 10 with
tosylmethyl isocyanide (TosMIC) followed by acidic
hydrolysis of the ketal to give pyrrole 11 in 79% overall
yields.14 N-Alkylation of 11 was carried out in the same
way as mentioned above furnishing 59% yield of 12,

K. Shanab et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6091–6095
6093
Table 1. In vitro cytotoxicity of compounds 8, 9a–9e, 12 and 13a–13e across a panel of cancer cell lines
a
Cells (origin)
Compound
IC50 (lmol)
8
9a
9b
9c
9d
9e
12, 13a–13e
KB/HeLa (cervix)
NCI-H460 (lungs)
SF-268 (CNS)
11.04
3.84
4.71
0.67 0.06
0.23 0.01
1.23 0.14
0.94 0.03
0.41 0.07
0.66 0.04
0.13 0.01
1.39 0.01
0.48 0.02
0.17 0.02
1.21 0.05
>12
>12
>12
>12
>12
>12
>12
>12
>12
>12
n.a.b
n.a.
n.a.
n.a.
n.a.
0.79 0.05
2.43 0.12
2.24 0.06
1.16 0.01
SKOV-3 (ovaries)
RKOp27 (colon)
13.87
2.35
a Lower IC50 values are given as means standard deviation from at least two XTT assay experiments.
b n.a., not active, that is <50% inhibition at a compound concentration of 3.16 lg/ml.
Table 2. Cytotoxicity [lM] of selected compounds against drug-resistant cancer cell lines19
LT12
LT12MDR
P388
P388ADR
L1210
L1210VCR
9a
9b
0.03
0.04
0.07
0.16
0.02
0.02
0.038
0.52
0.40
0.50
0.05
0.07
0.11
0.16
Pac
Dox
0.005
0.031
0.368
>3.4
>3.4
>3.4
0.052
0.221
>3.4
>3.4
Pac, paclitaxel; Dox, doxorubicin.
50000
40000
30000
20000
10000
0
10-fold enhancement of cytotoxic activity. Compounds
9a and 9b were subjected to antiproliferative activity
experiments against wild type and MDR cancer cell
lines18 for further characterization. As can be seen from
the results summarized in Table 2, 9a and 9b show nearly
the same activity against wild type/MDR cell line pairs as
paclitaxel and doxorubicin, all compounds being tested
in the same experimental setting.
9a
3.16µM
9b
0.316µM
Effects on the cell cycle were investigated by the treat-
ment of KB/HeLa cells with different concentrations of
selected compounds for 24 h, using untreated cells as
reference. Propidiumiodide-stained cells were analyzed
by flow cytometry analysis20 and the resulting data of
9a and 9b confirmed the arrest of the cell cycle in the
G2/M phase, Figure 2 (data for 9a).
-10000
10µM
1µM
0.1µM
Figure 3. Results of caspase activity experiments for 9a and 9b.
cells in G2/M phase and activation of apoptosis
pathways.
Caspase 3/7 activity experiments were performed in
U937 cells with DEVD rho-110 substrate after treat-
ment with selected compounds for 24 h. Cells treated
with 9a and 9b exhibited significant, dose-dependent
caspase activity (610 lM) which indicates apoptosis
inducing properties, as shown in Figure 3.
Remarkably, it was demonstrated by standard DNA
intercalating UV experiments that 9a did not intercalate
into DNA (data not shown).21
In conclusion, oximes of pyrrolo[3,2-g]isoquinoline-4,9-
dione derivatives and pyrrolo[4,3-g]isoquinoline-4,
9-dione derivatives have been synthesized and character-
ized. These compounds were screened for cytotoxic
activity against different cell lines. All oximes of the
pyrrolo[3,2-g]isoquinoline series exhibited superior
IC50 values compared with the pyrrolo[4,3-g]isoquino-
line series and the quinone precursors. Two lead com-
pounds 9a and 9b were identified, that exhibited better
antiproliferative effects than paclitaxel and doxorubicin
on multidrug resistant cell lines. Caspase 3/7 induction
indicated the arrest of the cell cycle in the G2/M phase
of the cell cycle. These drugs do not intercalate into
DNA, which suggests that the cytotoxic effects underlie
a different mechanism than classical DNA intercalating
agents. These results provide useful information for fur-
ther studies and for the development of an extended
compound library.
Therefore we conclude that compounds 9a and 9b exert
their antiproliferative/cytotoxic effect by arresting the
Figure 2. Representative FACS analysis diagrams for 9a (right)
showing the increase of cells arrested in G2/M phase compared to
control (left).

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K. Shanab et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6091–6095
cell line development in the past or different experimental
References and notes
settings (e.g., use of the XTT vs MTT detection reagent,
use of different cell counts, etc.). However, the difference in
activity between wild type and resistant cell lines for test
and reference compounds reported herein and the conclu-
sions drawn from these results remain unaffected.
20. For FACS analysis, a FACSCaliburTM cytometer (Becton
Dickinson, Heidelberg, GER) and Mod Fit LT VERITY
(cell cycle analysis software) were used.
21. For DNA intercalation experiments following an in-house
protocol, stock solution of reference and test compounds
at 2–5 lM concentration in 5% DMSO (v/v) were
prepared. After 1:10 (v/v) dilution of the compound stock
in reaction buffer (5 mM NaH2PO4, 5 mM Na2HPO4,
70 mM NaCl, pH 7), the absorbance [Amax] ꢀ1 was
determined in 96-well UV plates (Costar, Acton, MA)
between 230 and 570 nm using a Spectramax 190 plus
reader (Molecular Devices, Sunnyvale, CA). The resulting
curve was compared to a curve generated by using 2 mg/
ml (w/v) calf thymus DNA (Sigma–Aldrich Corp., St.
Louis, MO) solution in reaction buffer as compound
diluent.
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14. Spreitzer, H.; Pichler, A.; Holzer, W.; Kratzel, M.; Slanz,
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Heterocycles 2001, 54, 111.
15. (a) Cerri, A.; Almirante, N.; Barassi, P.; Benicchio, A.;
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22. All of the final structures were confirmed by 1H NMR, 13
NMR, IR, and MS as the following.
C
Compound 6: Mp = 287 °C;1H NMR (DMSO-d6): d (ppm)
5.89 (s, 1H), 7.48 (br s, 2H), 7.75 (d, J = 4.9 Hz, 1H), 8.99
(d, J = 4.9 Hz, 1H), 9.07 (s, 1H); MS: m/z 174 (M+, 100),
147 (11), 130 (2), 118 (7), 106 (23).
Compound 8: Mp = 148 °C; 1H NMR (CDCl3): d (ppm)
2.30 (s, 6H), 2.73 (t, J = 6.5 Hz, 2H), 4.57 (t, J = 6.5 Hz,
2H), 6.77 (d, J = 2.8 Hz, 1H), 7.10 (d, J = 2.8 Hz, 1H),
7.94 (d, J = 5.0 Hz, 1H), 8.98 (d, J = 5.0 Hz, 1H), 9.37 (s,
1H); MS: m/z 269 (M+, 1), 211 (1), 155 (1), 58 (100).
Compound 9a: Mp = 70 °C; 1H NMR (CDCl3): d (ppm)
2.28 (s, 6H), 2.33 (s, 6H), 2.71 (t, J = 6.5 Hz, 2H), 2.80 (t,
J = 5.9 Hz, 2H), 4.57 (m, 4H), 7.12 (m, 2H), 8.10 (d,
J = 5.4 Hz, 1H), 8.71 (d, J = 5.4 Hz, 1H), 9.42 (s, 1H); 13
C
NMR (CDCl3): d (ppm) 45.6, 46.0, 47.3, 58.2, 59.8, 74.9,
117.0, 121.9, 126.0, 126.1, 131.6, 140.7, 140.9, 150.0, 151.6,
174.3; IR (KBr): mmax 3427, 3100, 2947, 2762, 1639, 1576,
1460, 1427, 1379, 1270 cmÀ1; MS: m/z 355 (M+, 1), 284 (1),
282 (2), 269 (3), 267 (1), 266 (1), 222 (1), 167 (1), 149 (5),
125 (1).
16. Schmidt, M.; Lu, Y.; Liu, B.; Fang, M.; Mendelsohn, J.;
Fan, Z. Oncogene 2000, 19, 2423.
1
Compound 9b: Mp = 154 °C; H NMR (CDCl3): d (ppm)
2.25 (s, 6H), 2.62 (t, J = 6.7 Hz, 2H), 3.81 (s, 1H), 4.04 (t,
J = 4.4 Hz, 2H), 4.43 (t, J = 6.8 Hz, 2H), 4.54 (t,
J = 4.4 Hz, 2H), 6.97 (m, 2H), 7.86 (d, J = 5.3 Hz, 1H),
8.55 (d, J = 5.4 Hz, 1H), 9.20 (s, 1H); 13C NMR (CDCl3):
d (ppm) 45.5, 47.0, 59.5, 61.3, 59.8, 78.1, 111.7, 116.7,
121.5, 125.7, 125.8, 131.5, 140.3, 140.9, 148.6, 151.3, 173.7;
IR (KBr): mmax 3486, 3191, 3123, 2940, 2821, 2767, 1637,
1595, 1577, 1534, 1520, 1479, 1429 cmÀ1; MS: m/z 328
(M+, 1), 284 (1), 270 (1), 240 (1), 226 (1), 222 (1), 209 (1),
198 (2), 193 (1), 181 (1), 170 (1).
17. Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks,
A.; Tierney, S.; Nofziger, T. H.; Currens, M. J.; Seniff, D.;
Boyd, M. R. Cancer Res. 1988, 48, 4827.
18. From the readily available wild types LT12, P388, and
L1210, cell lines were established, that were resistant
against standard cancer reference compounds.
Wild type Resistant type
1
Compound 9c: Mp = 140 °C; H NMR (CDCl3): d (ppm)
L1210
P388
LT12
L1210VCR (vincristine resist.)
P388ADR (doxorubicin resist.)
2.28 (s, 6H), 2.71 (t, J = 6.6 Hz, 2H), 4.25 (s, 3H), 4.59 (t,
J = 6.6 Hz, 2H), 7.08 (m, 2H), 8.08 (d, J = 5.4 Hz, 1H),
8.70 (d, J = 5.3 Hz, 1H), 9.40 (s, 1H); 13C NMR (CDCl3):
d (ppm) 45.1, 47.3, 59.8, 64.0, 111.5, 117.0, 121.8, 126.0,
126.1, 131.5, 140.6, 140.7, 148.9, 151.6, 174.3; IR (KBr):
LT12MDR (Ectopic expression of human
MDR1 K. Nooter, University of Rotterdam)
m
max 3422, 3118, 2972, 2934, 2818, 2767, 1639, 1576, 1481,
1425, 1381 cmÀ1; MS: m/z 298 (M+, 1), 208 (1), 195 (1),
180 (1), 155 (1), 154 (1), 140 (1), 127 (1).
19. It might attract attention that the activity of doxorubicin
reported herein (see Table 2) differs from results published
elsewhere (e.g., Furusawa, S.; Nakano, S.; Wu, J.;
Sakaguchi, S.; Takayanagi, M.; Sasaki, K. I.; Satoh S.;
J. Pharm. Pharmacol. 2001, 53, 1029). After carefully re-
evaluating and confirming the results published herein,
reasons for these differences might be found in different
Compound 9d: Mp (decomp.) = 173 °C; 1H NMR
(CDCl3): d (ppm) 2.51 (s, 6H), 3.10 (t, J = 5.5 Hz, 2H),
4.72 (t, J = 5.4 Hz, 2H), 7.03 (m, 2H), 7.57 (d, J = 5.4 Hz,
1H), 8.33 (d, J = 5.4 Hz, 1H), 9.20 (s, 1H); 13C NMR
(CDCl3): d (ppm) 45.1, 46.0, 59.7, 112.5, 116.1, 122.7,
125.7, 126.0, 130.9, 140.1, 141.2, 148.3, 150.8, 174.3; IR

K. Shanab et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6091–6095
6095
(KBr): mmax 3224, 2957, 2923, 1624, 1580, 1456, 1431,
1386, 1269 cmÀ1; MS: m/z 284 (M+, 1), 269 (1), 267 (1),
240 (1), 224 (1), 133 (1), 131 (1).
J = 5.6 Hz, 1H), 9.32 (s, 1H); 13C NMR (CDCl3): d
(ppm) 45.5, 48.7, 59.7, 61.7, 77.4, 113.8, 117.3, 120.0,
123.7, 126.1, 126.7, 141.0, 141.4, 149.5, 151.9, 179.4. IR
(KBr): mmax 3422, 2962, 2816, 1642, 1573, 1539, 1265,
1267 cmÀ1; MS: m/z 329 (M++1, 3.3), 265 (1.8), 208 (4.7),
180 (2.8), 58 (100).
Compound 9e: Mp = 90 °C; 1H NMR (CDCl3): d (ppm)
1.29 (t, J = 7.2 Hz, 3H), 2.29 (s, 6H), 2.71 (t, J = 6.6 Hz,
2H), 4.26 (q, J = 7.2 Hz, 2H), 4.59 (t, J = 6.6 Hz, 2H), 4.97
(s, 2H), 7.11 (d, J = 2.6 Hz, 1H), 7.19 (d, J = 2.6 Hz, 1H),
8.02 (d, J = 5.3 Hz, 1H), 8.70 (d, J = 5.3 Hz, 1H), 9.39 (s,
1H); 13C NMR (CDCl3): d (ppm) 14.1, 45.5, 47.2, 59.7,
61.2, 72.4, 112.2, 117.1, 121.5, 126.0, 126.2, 131.7, 140.2,
140.9, 148.9, 151.8, 168.7, 174.2; IR (KBr): mmax 3426,
3145, 3108, 2941, 2917, 2858, 2821, 2767, 1756, 1638, 1598,
1579, 1539, 1482 cmÀ1; MS: m/z 370 (M+, 1), 322 (1), 297
(1), 269 (1), 267 (1), 225 (1), 222 (1), 209 (1), 208 (1), 181
(1), 167 (1).
Compound 13c: Mp = 121–123 °C; 1H NMR (CDCl3): d
(ppm) 2.28 (s, 6H), 2.73 (t, J = 6.5 Hz, 2H), 4.11 (t,
J = 6.5 Hz, 2H), 4.27 (s, 3H), 7.61 (d, J = 2.0 Hz, 1H), 7.70
(d, J = 2.0 Hz, 1H), 8.13 (dd, J = 5.4, 0.6 Hz, 1H), 8.74 (d,
J = 5.4 Hz, 1H), 9.46 (d, J = 0.6 Hz, 1H); 13C NMR
(CDCl3): d (ppm): 45.6, 48.9, 59.8, 63.8, 114.1, 117.5,
120.3, 123.5, 126.3, 126.4, 140.6, 141.7, 149.7, 152.1, 179.8;
IR (KBr): mmax 3415, 2937, 2822, 1648, 1579, 1537,
1270 cmÀ1; MS: m/z 299 (M++1, 9.1), 265 (3.3), 240
(1.4), 208 (3.6), 180 (2.4), 58 (100).
Compound 12: Mp = 180–181 °C; 1H NMR (CDCl3): d
(ppm) 2.28 (s, 6H), 2.71 (t, J = 6.1 Hz, 2H), 4.09 (t,
J = 6.1 Hz, 2H), 7.53 (d, J = 1.8 Hz, 1H), 7.55 (d,
J = 1.8 Hz, 1H), 8.01 (d, J = 5.0 Hz, 1H), 9.00 (d,
J = 5.0 Hz, 1H), 9.47 (s, 1H); MS: m/z 270 (M++1, 0.9),
225 (2.1), 211 (2.3), 183 (1.1), 155 (1.8), 58 (100).
Compound 13a: Mp = 83-85 °C; 1H NMR (CDCl3): d
(ppm) 2.29 (s, 6H), 2.35 (s, 6H), 2.72 (t, J = 6.5 Hz, 2H),
2.81 (t, J = 5.9 Hz, 2H), 4.10 (t, J = 6.5 Hz, 2H), 4.57 (t,
J = 5.9 Hz, 2H), 7.60 (d, J = 2.0 Hz, 1H), 7.78 (d,
J = 2.0 Hz, 1H), 8.13 (d, J = 5.4 Hz, 1H), 8.74 (d,
J = 5.4 Hz, 1H), 9.47 (s, 1H); 13C NMR (CDCl3): d
(ppm) 45.6, 46.0, 48.8, 58.3, 59.7, 74.3, 114.1, 117.5, 120.2,
123.5, 126.2, 126.6, 140.8, 141.7, 149.7, 152.1, 179.7; IR
(KBr): mmax 2944, 2821, 1644, 1579, 1536, 1267 cmÀ1; MS:
m/z 355 (M+, 0.2), 338 (0.6), 296 (0.3), 282 (2.5), 269 (3.2),
58 (100).
Compound 13d: Mp >350 °C; 1H NMR (DMSO-d6): d
(ppm) 2.18 (s, 6H), 2.64 (t, J = 6.1 Hz, 2H), 4.22 (t,
J = 6.1 Hz, 2H), 7.84 (d, J = 1.7 Hz, 1H), 8.01 (d,
J = 1.7 Hz, 1H), 8.11 (d, J = 5.4 Hz, 1H), 8.75 (d,
J = 5.4 Hz, 1H), 9.25 (s, 1H); 13C NMR (DMSO-d6): d
(ppm) 45.1, 47.4, 59.3, 113.1, 117.0, 118.8, 124.2, 125.6,
126.7, 140.2, 142.0, 148.7, 152.1, 178.6; IR (KBr): mmax
3446, 2919, 2842, 1653, 1554, 1533, 1263 cmÀ1; MS: m/z
284 (M+, 0.4), 265 (0.5), 239 (0.5), 223 (0.3), 180 (0.5), 58
(100).
Compound 13e: Mp = 150–151 °C; 1H NMR (CDCl3): d
(ppm) 1.33 (t, J = 7.1 Hz, 3H), 2.29 (s, 6H), 2.73 (t,
J = 6.5 Hz, 2H), 4.11 (t, J = 6.5 Hz, 2H), 4.30 (q,
J = 7.1 Hz, 2H), 5.00 (s, 2H), 7.62 (d, J = 2.0 Hz, 1H),
7.86 (d, J = 2.0 Hz, 1H), 8.08 (dd, J = 5.4, 0.6 Hz, 1H),
8.74 (d, J = 5.4 Hz, 1H), 9.47 (d, J = 0.6 Hz, 1H); 13C
NMR (CDCl3): d (ppm) 14.2, 45.5, 48.8, 59.7, 61.2, 72.3,
113.7, 117.6, 120.3, 123.7, 126.4, 127.4, 141.2, 142.2, 149.7,
152.3, 169.0, 179.6; IR (KBr): mmax 3446, 2959, 1746, 1650,
1583, 1537, 1220 cmÀ1; MS: m/z 370 (M+, 0.6), 297 (0.7),
265 (1.2), 208 (1.9), 180 (1.3), 58 (100).
Compound 13b: Mp = 127–129 °C; 1H NMR (CDCl3): d
(ppm) 2.28 (s, 6H), 2.70 (t, J = 6.5 Hz, 2H), 4.06 (t,
J = 6.5 Hz, 2H), 4.06 (t, J = 4.4 Hz, 2H), 4.56 (t,
J = 4.4 Hz, 2H), 7.49 (d, J = 2.0 Hz, 1H), 7.68 (d,
J = 2.0 Hz, 1H), 7.94 (d, J = 5.6 Hz, 1H), 8.64 (d,