Synthesis and cytotoxic activity of substituted Luotonin A derivatives

Article abstract of DOI:10.1016/j.bmcl.2004.09.039

Luotonin A is a cytotoxic alkaloid that has been shown to inhibit topoisomerase I via stabilization of the binary complex topoisomerase-DNA in the same fashion as camptothecin. The synthesis and the cytotoxic activity on the lung carcinoma cell line H460

Full text of DOI:10.1016/j.bmcl.2004.09.039

Bioorganic & Medicinal Chemistry Letters 14(2004) 5757–5761
Synthesis and cytotoxic activity of substituted
Luotonin A derivatives
Sabrina Dallavalle,a,* Lucio Merlini,a Giovanni Luca Beretta,b
Stella Tinellib and Franco Zuninob
a
`
Dipartimento di Scienze Molecolari Agroalimentari, Sezione di Chimica, Universita di Milano, Via Celoria 2, 20133 Milano, Italy
bDipartimento di Farmacologia Antitumorale Preclinica, Istituto Nazionale per lo Studio, e la Cura dei Tumori, Via Venezian 1,
20133 Milano, Italy
Received 16 July 2004; revised 24 August 2004; accepted 17 September 2004
Available online 5 October 2004
Abstract—Luotonin A is a cytotoxic alkaloid that has been shown to inhibit topoisomerase I via stabilization of the binary complex
topoisomerase–DNA in the same fashion as camptothecin. The synthesis and the cytotoxic activity on the lung carcinoma cell line
H460 of a series of derivatives of Luotonin A is reported. The compounds inhibit topoisomerase I but show weak cytotoxic activity,
thus confirming the peculiarity of ring E of camptothecin for antitumor activity.
ꢀ 2004 Published by Elsevier Ltd.
Luotonin A (1a, quino[20,30:3,4]pyrrolo[2,1-b]quinazo-
lin-11(13H)-one) is an alkaloid isolated1 from Peganum
nigellastrum Bunge that has received increasing atten-
tion in the last few years, due to its structural similarity
with the well-known cytotoxic alkaloid camptothecin (2)
(CPT).2 After the report of its isolation in 1997,1 more
than 10 syntheses of Luotonin A have been reported
in the literature,3 included one from our own group.3h
complex. The formation of a stable ternary complex is
deemed to be the reason for the cytotoxic activity of
CPT. These results raise the question of which structural
features of the ring E of these compounds are indeed
necessary, or important, in stabilizing such a complex,
and of the possible correlation between inhibition of
topoisomerase I and cytotoxic activity.
The interest of synthesizing and testing analogues or
derivatives of Luotonin A with substituents of different
position and polarity in ring E to this purpose was
apparent to us as soon as we synthesized the parent
compound. A very recent publication by Hecht and
co-workers5 that reports the synthesis, stabilizing activ-
ity of the binary complex, and cytotoxicity of some ring
E derivatives of Luotonin A prompts us to disclose our
own results.
1
14
13
O
O
11
2
3
N
N
10
N
5
N
4
N
6
O
9
7
8
1a
2
O
HO
Notwithstanding the similarity with CPT, Luotonin A is
far less active than CPT in tests of antitumor activity.1
However, a recent report by Hecht and co-workers4
has shown that Luotonin A is a topoisomerase I poison.
Luotonin A induces the same sequence selectivity of
DNA cleavage by topoisomerase I as CPT, thus indicat-
ing that, although less potently than CPT, it stabilizes in
a similar way the binary DNA–topoisomerase cleavable
We report here the synthesis, the assessment of the cyto-
toxic activity on a tumor cell line and of the stabilizing
activity of the DNA–topoisomerase I complex of a series
of Luotonin A derivatives substituted in ring E but also
in ring A. Except for one compound (1b) they differ
from those reported recently by Hecht and co-workers5
and therefore may complement and enlarge his results.
We explored some of the syntheses so far reported in the
literature, but none of them seemed the optimal proce-
dure for all derivatives, so that the ring E-substituted
Keywords: Topoisomerase I; Cytotoxic alkaloid; Antitumor agents.
*
Corresponding author. Tel.: +39 0250316815; fax: +39
0250316801; e-mail: sabrina.dallavalle@unimi.it
0960-894X/$ - see front matter ꢀ 2004Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2004.09.039

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S. Dallavalle et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5757–5761
compounds 1a–g (Table 1) were prepared from the tricy-
clic intermediate 33h by condensation with the appropri-
ately substituted anthranilic acid6 or by microwave
reaction with the appropriate isatoic anhydride3g
(Scheme 1). The two derivatives substituted at ring A
4a,b were obtained by Friedla¨nder condensation of the
tricyclic compound 53c,3d,7 with the appropriate 2-ami-
nobenzaldehyde.8 Compound 6, with a reduced ring B,
was obtained in the attempt to reduce the nitro group
of 1c to an amino group.
The compounds prepared were evaluated for their cyto-
toxicity against the human non-small lung carcinoma
cell line H460, using SN-38, that is the active principle
of one of the two camptothecin drugs currently in
clinical practice, Irinotecan, as a reference compound
(Table 1).
This cell model was chosen for its sensitivity to topo-
isomerase I inhibitors, likely related to overexpression
of the target enzyme.9
Table 1. In vitro cytotoxic (IC50 (lM)) activity of Luotonin A
derivatives on H460 cell line
Compound
IC50 (lM) 1h
IC50 (lM) 72h
Moreover, stimulation of the topoisomerase I-mediated
cleavage of DNA was studied for some compounds
(1a,d,e,g, 6) (Fig. 1). This experiment shows that indeed
these Luotonin derivatives induce cleavage at the same
sites as well as camptothecins, although in a lesser ex-
tent. Compounds 1d and 1e were the most potent as topo-
isomerase I poisons. From Table 1, it appears that all
compounds are cytotoxic toward the H460 cell line.10
However, their potency was substantially lower than
that of SN-38. Optimal effects of topoisomerase I inhib-
itors require a persistent stabilization of the cleavable
1a
1b
1c
67
85
7.7
11
23
1d
1e
21
7
13
1f
172
40
45
1g
4a
4b
6
3.8
5.47
81
86
SN-38
0.21
O
N
N
COCl
N
NO2
R1
R1
Fe/AcOH
1a R1 = H
1b R1= OCH3
O
R3
R1
R2
O
N
H
O
O
N
NH
R3
N
N
MW
N
O
R2
3
R1
R2
COOH
NH2
1c R1= H
R2 =NO2 R3= H
-
+
BF4 OEt3
1d R1= CH3 R2 =H
R3= H
R3= Cl
1e R1= H
R2 =H
H2/Pd/C
O
O
N
N
N
N
H
N
N
NH2
R2
6
1f R2 = Cl
1g R2 = OH
R4
R5
CHO
NH2
R4
R5
O
O
N
N
+
O
N
N
N
4a R4= OCH3
R5=OCH3
5
4b R4 , R5= -OCH2O-
Scheme 1. Synthesis of Luotonin A derivatives.

S. Dallavalle et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5757–5761
5759
These data, together with the observation that one of the
most active compound is the 2,3-dimethoxy analogue
4a, lead us to suggest that the biological activity of
Luotonin A derivatives is similar to that of other planar
polycyclic heteroaromatic compounds, such as indeno-
isoquinolines,11 benzo[c][1,7] and [1,8]phenanthro-
lines,12 benzo[i]phenanthridines13 and other analogues
of protoberberine alkaloids, that show noticeable stabi-
lization of the binary DNA–topoisomerase I complex,
but cytotoxicity of the order of magnitude of Luotonin
A derivatives, that is far less than that of camptothecins.
The unique arrangement of functionalities in the E ring
of camptothecins remains so far an unparalleled feature
for antitumor activity.
Luotonin A (1a) was prepared following the procedure
reported in our previous paper.3h
7-Methoxyluotonin A (1b): To a suspension of 84mg
(0.46mmol) of 3 in 4mL of dry THF, 20mg (0.5mmol)
of NaH (60% mineral oil) were added with cooling in
one portion. The mixture was heated at 60ꢁC until the
evolution of gas ceased, then ice cooled and added with
108mg (0.5mmol) of 2-nitro-3-methoxybenzoylchloride.
After heating at 60ꢁC for 3h the solvent was evaporated
to obtain a crude product, which was used without fur-
ther purification. 1H NMR (CDCl3): d 4.05 (s, 3H), 5.15
(s, 2H), 7.05 (d, J = 9.09Hz, 1H), 7.45–7.55 (m, 1H),
7.55–7.75 (m, 2H), 7.83 (d, J = 9.09Hz, 1H), 7.96 (d,
J = 9.09Hz, 1H), 8.35 (d, J = 9.09Hz, 1H), 8.40 (s,
1H). The above compound (88mg, 0.24mmol) was sus-
pended in 8mL of AcOH/abs EtOH 1:1, added with
88mg (1.58mmol) of Fe, refluxed for 2h, the iron was
filtered and washed with AcOEt and chloroform. The
organic layers were washed with satd aq NaHCO3 and
brine, then dried. Evaporation and purification by flash
column chromatography (FC) (AcOEt) gave 1b (26mg,
1
17%), mp > 250ꢁC. H NMR (CDCl3): d 4.10 (s, 3H),
Figure 1. Dose dependent stimulation of topoisomerase I-mediated
DNA cleavage in the presence of Luotonin A and derivatives. The
samples were reacted for 30min with 1, 10, and 50lM compound. The
triangle indicates the increase of drug concentration. (C) Control DNA
(751bp); (T) reaction without the compound; (M) purine markers; (S1)
10lM SN-38; (S2) 50lM SN-38. SN-38 is the reference drug 10-
hydroxy-7-ethylcamptothecin.
5.37 (s, 2H), 7.31 (d, J = 8.82Hz, 1H), 7.55 (dd,
J = 8.82 and 8.82Hz, 1H), 7.70 (dd, J = 8.82 and
8.82Hz, 1H), 7.85 (dd, J = 8.82 and 8.82Hz, 1H),
7.90–8.05 (m, 2H), 8.46 (s, 1H).
1. Generalprocedure for the synthesis under MW
irradiation
complex to favor the formation of the lethal double-
strand DNA lesions during DNA synthesis. Indeed topo-
isomerase I poisons are known to be S-phase specific.
Since, as for camptothecins, the drug interaction with
the DNA–enzyme complex is expected to be reversible,
the drug potency is likely related to the drug affinity
in the ternary complex. The cytotoxic effects of the com-
pounds tested in the present study increased following a
prolonged exposure (72h). The low cytotoxic potency
likely reflected a reduced affinity or increased reversibi-
lity of the complex. However, the appreciable stabiliza-
tion of the cleavable complex observed in vitro with
isolated topoisomerase I suggests an unfavorable beha-
vior in cellular pharmacokinetics of compounds of this
series, likely related to their physico-chemical properties
(e.g., low solubility).
A suspension of 3-oxo-1H-pyrrolo[3,4-b]quinoline3h (3,
50mg, 0.27mmol) in CH2Cl2 was added to activated
K-10 clay (50mg) in a beaker and the solvent was evap-
orated. The appropriate isatoic anhydride (0.27mmol)
in CH2Cl2 was added, stirred for 5min, and the solvent
evaporated. The beaker was exposed to MW irradiation
at 450W for 7–15min under solvent-free conditions.
After conversion, the reaction mixture was added with
CH2Cl2, the clay filtered, the organic layer concentrated
in vacuo, and the crude product purified by FC on silica
gel (Merck, 230–400mesh).
9-Nitroluotonin A (1c): The reaction mixture with 5-
nitroisatoic anhydride was subjected to MW irradiation
for 7min to obtain, after purification by FC (eluent:
CH2Cl2/ethyl acetate 95:5), 1c as a white solid (12mg,

5760
S. Dallavalle et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5757–5761
13%), mp > 300ꢁC dec. 1H NMR (DMSO-d6): d 5.35 (s,
2H), 7.78 (dd, J = 7.72 and 7.72Hz, 1H), 7.92 (dd,
J = 7.72 and 7.72Hz, 1H), 8.10–8.28 (m, 3H), 8.64(dd,
J = 9.19 and 2.57Hz, 1H), 8.80 (s, 1H), 8.96 (d,
J = 2.57Hz, 1H).
1H), 7.55 (s, 1H), 7.72 (dd, J = 8.75 and 8.75Hz, 1H),
7.80 (d, J = 8.75Hz, 1H), 7.87 (dd, J = 8.75 and
8.75Hz, 1H), 8.13 (d, J = 8.75Hz, 1H), 8.22 (d,
J = 8.75Hz, 1H), 8.70 (s, 1H).
8
¨
3. Generalprocedure for the Friedal nder cyclization
7-Methylluotonin A (1d): The reaction mixture with 3-
methylisatoic anhydride14 was subjected to MW irradia-
tion for 12min to obtain, after purification by FC (elu-
ent: from hexane/ethyl acetate 1:1 to ethyl acetate), 1d
To a solution of 53d (20mg, 0.1mmol) in toluene (3mL)
were added the appropriate aminobenzaldehyde
(0.11mmol) and p-toluenesulfonic acid (2mg). The mix-
ture was heated using a Dean–Stark trap for 2–4h. The
solvent was removed in vacuo and the residue purified
by FC.
1
(59mg, yield 35%), mp 282–283ꢁC. H NMR (CDCl3):
d 2.75 (s, 3H), 5.32 (s, 2H), 7.45 (dd, J = 8.19 and
8.19Hz, 1H), 7.70 (dd, J = 8.19 and 8.19Hz, 2H), 7.80
(dd, J = 8.19 and 8.19Hz, 1H), 7.90 (dd, J = 8.19 and
1.84Hz, 1H), 8.26 (dd, J = 8.19 and 1.84Hz, 1H), 8.45
(s, 1H), 8.48 (dd, J = 8.19 and 1.84Hz, 1H).
2,3-Dimethoxyluotonin A (4a): The mixture was heated
for 4h. Evaporation of the solvent and purification by
FC (CH2Cl2/MeOH 97:3) gave 4a (11mg, 32%),
mp > 270ꢁC. H NMR (CDCl3): d 4.07 (s, 6H), 5.29
(s, 2H), 7.14(s, 1H), 7.55 (dd, J = 8.19 and 8.19Hz,
1H), 7.77 (s, 1H), 7.83 (dd, J = 8.19 and 8.19Hz, 1H),
8.08 (d, J = 8.19Hz, 1H), 8.26 (s, 1H), 8.42 (d,
J = 8.19Hz, 1H).
10-Chloroluotonin A (1e): The reaction mixture with 6-
chloroisatoic anhydride was subjected to MW irradia-
tion for 12min to obtain, after FC (eluent: from hex-
ane/ethyl acetate 7:3 to ethyl acetate) and
crystallization from Et2O, 1e (60mg, 33%), mp 313–
315ꢁC dec. 1H NMR (CDCl3): d 5.30 (s, 2H), 7.54
(dd, J = 8.19 and 1.49Hz, 1H), 7.68 (dd, J = 8.19 and
8.19Hz, 2H), 7.84(ddd, J = 8.19, 8.19, and 1.49Hz,
1H), 7.94(dd, J = 8.19 and 1.49Hz, 1H), 8.02 (dd,
J = 8.19 and 1.49Hz, 1H), 8.44 (s, 1H), 8.46 (dd,
J = 8.19 and 1.49Hz, 1H).
1
2,3-Methylenedioxyluotonin A (4b): The mixture was
heated for 2h. Evaporation and purification by FC (hex-
1
ane/AcOEt 2:8) gave 4b (12mg, 36%), mp > 270ꢁC. H
NMR (CDCl3): d 5.26 (s, 2H), 6.18 (s, 2H), 7.16 (s,
1H), (dd, J = 8.19 and 1.49Hz, 1H), 7.55 (ddd,
J = 8.56, 8.19, and 1.49Hz, 1H), 7.71 (s, 1H), 7.83
(ddd, J = 8.56, 8.19, and 1.49Hz, 1H), 8.09 (dd,
J = 8.19 and 1.49Hz, 1H), 8.23 (s, 1H), 8.41 (dd,
J = 8.19 and 1.49Hz, 1H).
2. Generalprocedure for the condensation with substituted
anthranilic acid14
To a solution of 3 (300mg, 1.63mmol) in 60mL of anhy-
drous CH2Cl2 under N2 were added 945mg (4.89mmol)
of BFÀ4 Et3Oþ and the resulting mixture was stirred at
room temperature for 48h. After addition of NaOH
1N (30mL) the aqueous phase was extracted with
CH2Cl2 (20 · 3mL). The collected organic layers were
washed with brine, dried, and evaporated to afford, after
FC (eluent: from AcOEt to AcOEt/MeOH 97:3), 110mg
of 3-ethoxy-1H-pyrrolo[3,4-b]quinoline as a white solid,
yield 32%, mp 138ꢁC. 1H NMR (CDCl3): d 1.60 (t,
J = 7Hz, 3H), 4.68 (q, J = 7Hz, 2H), 4.80 (s, 2H), 7.60
(m, 1H), 7.75 (m, 1H), 7.90 (dd, J = 8.09 and 1.48Hz,
1H), 8.25 (s, 1H), 8.35 (dd, J = 8.09 and 1.48Hz, 1H).
The above compound (100mg, 0.47mmol) was dropped
into an ice-cooled solution of the appropriate anthrani-
lic acid (0.43mmol) in acetone or dioxane. The resulting
solution was refluxed for 1–2days, then it was cooled
with an ice bath and the precipitate was filtered and
purified by FC.
9-Amino-4a,5,13a,14-tetrahydroquino[20,30:3,4]pyr-
rolo[2,1-b]quinazolin-11(13H)-one (6): A suspension of
4c (37mg, 0.11mmol) and Pd/C (7mg) in 10mL of
AcOH was hydrogenated for 6h: filtration and evapora-
tion gave, after FC (eluent: AcOEt/hexane 9:1) 6 as a
yellow solid (18mg, 52%), mp > 250ꢁC. 1H NMR
(DMSO-d6): d 2.21 (dd, J = 9.93 and 15.44Hz, 1H),
2.7 (m, 1H), 2.80 (dd, J = 5.14and 15.44Hz, 1H), 3.80
(dd, J = 2.57 and 11.77Hz, 1H), 4.10 (dd, J = 6.25 and
11.77Hz, 1H), 4.75 (d, J = 6.62Hz, 1H), 6.48
(dd, J = 9.19 and 9.19Hz, 1H), 6.69 (d, J = 9.19Hz,
1H), 6.85–6.95 (m, 2H), 7.05 (dd, J = 9.19 and 1.49
Hz, 1H), 7.16 (d, J = 1.49Hz, 1H), 7.35 (d, J =
9.19Hz, 1H).
4. In vitro studies
A human lung large cell carcinoma cell line, H460
(ATCC HTB 177) was used in this study. Cells were cul-
tured in RPMI-1640 containing 10% fetal calf serum.
Cytotoxicity was assessed by growth inhibition assay
after 1 and 72h drug exposure. Cells in the logarithmic
phase of growth were harvested and seeded in duplicates
into six-well plates. Twenty-four hours after seeding,
cells were exposed 1 or 72h to the drug and, in the case
of 1h exposure, harvested 72h before counting with a
Coulter counter. IC50 is defined as the inhibitory drug
concentration causing a 50% decrease of cell growth
9-Chloroluotonin A (1f): The solution was refluxed 24h
in acetone (3mL) to afford after FC (eluent: EtOAc)
1f (82mg, 57%), mp > 300ꢁC. 1H NMR (CDCl3): d
5.39 (s, 2H), 7.70–8.10 (m, 5H), 8.40 (d, J = 1.49, 1H),
8.51 (dd, J = 7.53 and 1.49Hz, 1H), 8.60 (s, 1H).
9-Hydroxyluotonin A (1g): The solution was refluxed
48h in dioxane (8mL) to afford after FC (eluent:
1
EtOAc/MeOH 95:5) 1g (50mg, 38%), mp > 300ꢁC. H
NMR (CDCl3): d 5.26 (s, 2H), 7.36 (d, J = 8.75Hz,

S. Dallavalle et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5757–5761
5761
H.; Takeuchi, Y. Synlett 2003, 6, 847–848; Toyota, M.;
Komori, C.; Ihar, M. Arkivoc 2003, Part 8, 15–23.
4. Cagir, A.; Jones, S. H.; Eisenhauer, B. M.; Gao, R.;
Hecht, S. M. J. Am. Chem. Soc. 2003, 125, 13628–
13630.
5. Cagir, A.; Jones, S. H.; Gao, R.; Eisenhauer, B. M.;
Hecht, S. M. Bioorg. Med. Chem. Lett. 2004, 14, 2051–
2054.
over that of untreated control. All compounds were dis-
solved in DMSO prior to dilution into the biological
assay.
Topoisomerase I-dependent DNA cleavage assay (see
Refs. 15 and 16).
6. Petersen, S.; Tietze, E. Liebigs Ann. 1959, 623, 166–176.
7. Chang, H. W.; Kim, S.; Jung, H.; Jahng, Y. Heterocycles
2003, 60, 1359–1366.
8. Pendrak, I.; Wittrock, R.; Kingsbury, W. D. J. Org.
Chem. 1995, 60, 2912–2915.
Acknowledgements
We are indebted to MIUR (Progetto COFIN 2003-4
2003039581) for financial support.
9. Giaccone, G.; Gazdar, A. F.; Beck, H.; Zunino, F.;
Capranico, G. Cancer Res. 1992, 52, 1666.
10. Hecht and co-workers5 report data of cytotoxicity against
a yeast strain expressing topoisomerase I, but not against a
tumor cell line.
11. Jayaraman, M.; Fox, B. M.; Hollingshead, M.; Kohlha-
gen, G.; Pommier, Y.; Cushman, M. J. Med. Chem. 2002,
45, 242.
References and notes
1. Ma, Z. Z.; Hano, Y.; Nomura, T.; Chen, Y. J. Hetero-
cycles 1997, 46, 541–546.
2. Thomas, C. J.; Rahier, N. J.; Hecht, S. M. Bioorg. Med.
Chem. 2004, 12, 1585.
3. (a) Wang, H.; Ganesan, A. Tetrahedron Lett. 1998, 39,
9097–9098; (b) Ma, Z. Z.; Hano, Y.; Nomura, T.; Chen,
Y.-J. Heterocycles 1999, 51, 1593–1596; (c) Kelly, T. R.;
Chamberland, S.; Silva, R. A. Tetrahedron Lett. 1999, 40,
2723–2724; (d) Molina, P.; Tarraga, A.; Gonzalez-Tejero,
A. Synthesis 2000, 11, 1523–1525; (e) Toyota, M.; Komori,
C.; Ihara, M. Heterocycles 2002, 56, 101–103; (f) Osborne,
D.; Stevenson, P. J. Tetrahedron Lett. 2002, 43, 5469–
5470; (g) Yadav, J. S.; Reddy, B. V. S. Tetrahedron Lett.
2002, 43, 1905–1907; (h) Dallavalle, S.; Merlini, L.
Tetrahedron Lett. 2002, 43, 1835–1837; (i) Lee, E. S.;
Park, J.-G.; Jahng, Y. Tetrahedron Lett. 2003, 44, 1883–
1886; (j) Harayama, T.; Morikami, Y.; Shigeta, Y.; Abe,
12. Prado, S.; Michel, S.; Tillequin, F.; Koch, M.; Pfeiffer, B.;
´
´
Pierre, S.; Leonce, S.; Colson, P.; Baldeyrou, B.; Lansiaux,
A.; Bailly, C. Bioorg. Med. Chem. 2004, 12, 3943–3953,
and refs. quoted therein.
13. Li, D.; Zhao, B.; Sim, S.-P.; Li, T.-K.; Liu, A.; Liu, L. F.;
LaVoie, E. J. Bioorg. Med. Chem. 2003, 14, 521–528.
14. Hennige, H.; Kreher, R. P.; Konrad, M.; Jelitto, F. Chem.
Ber. 1988, 121, 243–252.
15. Beretta, G.; Binaschi, M.; Zagni, E.; Capuani, L.; Capra-
nico, G. Cancer Res. 1999, 59, 3689–3697.
16. Dallavalle, S.; Merlini, L.; Morini, G.; Musso, L.; Penco,
S.; Beretta, G. L.; Tinelli, S.; Zunino, F. Eur. J. Med.
Chem. 2004, 39, 507–513.