Novel autoxidative cleavage reaction of 9-fluoredenes discovered during synthesis of a potential DNA-threading indenoisoquinoline

Article abstract of DOI:10.1021/jo048808f

The indenoisoquinolines are a novel class of cytotoxic non-camptothecin topoisomerase I inhibitors. A potential DNA-threading agent was designed by attaching different amine side chains on the lactam nitrogen as well as on the C11 position of the indenoisoquinoline ring system. It was hypothesized that substituents on the lactam nitrogen could protrude out toward the DNA major groove while those on the C11 project out toward the DNA minor groove in the ternary "cleavage complex." Compound 4 was synthesized in order to test this DNA-threading scenario. It was found unexpectedly that an alkenyl substituent on the C11 position was autoxidatively cleaved under basic conditions to afford a ketone. A possible mechanism for this unusual oxidative cleavage was proposed on the basis of the studies of a 9-fluoredene model compound. The proposed mechanism was further supported by computational studies. Although the designed compound 4 showed potent cytotoxicities in various cancer cell lines, it was less potent than its nonthreading counterparts and was not a topoisomerase I inhibitor.

Full text of DOI:10.1021/jo048808f

Novel Au toxid a tive Clea va ge Rea ction of 9-F lu or ed en es
Discover ed d u r in g Syn th esis of a P oten tia l DNA-Th r ea d in g
In d en oisoqu in olin e
Xiangshu Xiao,† Smitha Antony,‡ Glenda Kohlhagen,‡ Yves Pommier,‡ and Mark Cushman*,†
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmacal
Sciences, Purdue University, West Lafayette, Indiana 47907, and Laboratory of Molecular Pharmacology,
Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892-4255
cushman@pharmacy.purdue.edu
Received J uly 13, 2004
The indenoisoquinolines are a novel class of cytotoxic non-camptothecin topoisomerase I inhibitors.
A potential DNA-threading agent was designed by attaching different amine side chains on the
lactam nitrogen as well as on the C11 position of the indenoisoquinoline ring system. It was
hypothesized that substituents on the lactam nitrogen could protrude out toward the DNA major
groove while those on the C11 project out toward the DNA minor groove in the ternary “cleavage
complex.” Compound 4 was synthesized in order to test this DNA-threading scenario. It was found
unexpectedly that an alkenyl substituent on the C11 position was autoxidatively cleaved under
basic conditions to afford a ketone. A possible mechanism for this unusual oxidative cleavage was
proposed on the basis of the studies of a 9-fluoredene model compound. The proposed mechanism
was further supported by computational studies. Although the designed compound 4 showed potent
cytotoxicities in various cancer cell lines, it was less potent than its nonthreading counterparts
and was not a topoisomerase I inhibitor.
In tr od u ction
proved to be compound 2,4 with an aminopropyl group
at the lactam nitrogen, and 3,6 having an aminopropyl-
idene side chain at the C11 position.
Indenoisoquinoline 1, first described in 1978 as a
byproduct obtained during a total synthesis of nitidine
chloride,1 did not capture much attention until the late
1990s, when it was found to be a non-camptothecin
topoisomerase I inhibitor with moderate cytotoxicity in
cancer cell cultures.2 Fueled by this finding, a number
of indenoisoquinoline analogues have been synthesized
to optimize both the cytotoxicity and topoisomerase I
inhibitory potency.3-9 Two of the most potent analogues
* To whom correspondence should be addressed. Ph: 765-494-1465.
Fax: 765-494-6790.
† Purdue University.
‡ National Cancer Institute, NIH.
(1) Cushman, M.; Cheng, L. J . Org. Chem. 1978, 43, 3781-3783.
(2) Kohlhagen, G.; Paull, K.; Cushman, M.; Nagafuji, P.; Pommier,
Y. Mol. Pharmacol. 1998, 54, 50-58.
(3) Strumberg, D.; Pommier, Y.; Paull, K.; J ayaraman, M.; Nagafuji,
P.; Cushman, M. J . Med. Chem. 1999, 42, 446-457.
(4) Cushman, M.; J ayaraman, M.; Vroman, J . A.; Fukunaga, A. K.;
Fox, B. M.; Kohlhagen, G.; Strumberg, D.; Pommier, Y. J . Med. Chem.
2000, 43, 3688-3698.
(5) J ayaraman, M.; Fox, B. M.; Hollingshead, M.; Kohlhagen, G.;
Pommier, Y.; Cushman, M. J . Med. Chem. 2002, 45, 242-249.
(6) Fox, B. M.; Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier, Y.;
Staker, B. L.; Stewart, L.; Cushman, M. J . Med. Chem. 2003, 46, 3275-
3282.
(7) Nagarajan, M.; Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier,
Y.; Cushman, M. J . Med. Chem. 2003, 46, 5712-5724.
(8) Antony, S.; J ayaraman, M.; Laco, G.; Kohlhagen, G.; Kohn, K.
W.; Cushman, M.; Pommier, Y. Cancer Res. 2003, 63, 7428-7435.
(9) Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman, M.
Bioorg. Med. Chem. 2004, 12, 5147-5160.
According to the hypothetical model of the binding of
indenoisoquinolines in the DNA-topoisomerase I “cleav-
age complex” (Figure 1),7,9 substituents on the lactam
nitrogen project out of the duplex toward the DNA major
groove while those at the C11 position protrude out of
the duplex toward the DNA minor groove. The increased
biological activities of compounds 2 and 3 relative to those
10.1021/jo048808f CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/30/2004
J . Org. Chem. 2004, 69, 7495-7501
7495

Xiao et al.
SCHEME 1
SCHEME 2
of the parent compound 1 were partly explained as being
due to the hydrogen bond formation and electrostatic
interactions between the amino groups on the side chains
and the surrounding DNA base residues and the enzyme
amino acid residues in the ternary cleavage complexes.7,9
A further extension of this concept would be to attach
both aminated side chains in a single molecule 4, which
could conceivably occupy both the DNA major and minor
grooves (Figure 1). Assuming compound 4 could in fact
intercalate between the DNA base pairs in the cleavage
complex, the resulting DNA-threading agent could hy-
pothetically have increased topoisomerase I inhibitory
potency as well as cancer cell cytotoxicity by stabilizing
the ternary cleavage complex.10-12
Resu lts a n d Discu ssion
The free base of compound 4 was expected to be
prepared from global deprotection of Fmoc-protected
compound 12, which in turn could be made from Mc-
Murry coupling between indenoisoquinoline derivative 10
and aldehyde 11 (Scheme 1). Thus, mono-Fmoc-protected
1,3-propanediamine 513 was condensed with piperonal (6)
to form the imine 7. Condensation of imine 7 with 3,4-
dimethoxyhomophthalic anhydride (8)1 gave cis acid 9,
whose relative configuration was determined by 1H
NMR.14 Treatment of the cis acid 9 with thionyl chloride
provided the desired indeno[1,2-c]isoquinoline 10,1 which
was then coupled with aldehyde 1115 under McMurry
conditions6,9 to afford the Fmoc-protected compound 12.
(10) Grummitt, A. R.; Harding, M. M.; Anderberg, P. I.; Rodger, A.
Eur. J . Org. Chem. 2003, 63-71.
(11) Wakelin, L. P. G.; Chetcuti, P.; Denny, W. A. J . Med. Chem.
1990, 33, 2039-2044.
(12) Henichart, J . P.; Waring, M. J .; Riou, J . F.; Denny, W. A.; Bailly,
C. Mol. Pharmacol. 1997, 51, 448-461.
F IGURE 1. Hypothetical model of the orientation of inde-
noisoquinoline 4 relative to DNA in the ternary complex
containing topoisomerase I, DNA, and the inhibitor 4.
(13) Adamczyk, M.; Grote, J . Org. Prep. Proc. Int. 1995, 27, 239-
242.
(14) Cushman, M.; Cheng, L. J . Org. Chem. 1978, 43, 286-288.
7496 J . Org. Chem., Vol. 69, No. 22, 2004

Autoxidative Cleavage of 9-Fluoredenes
SCHEME 3
product 13 (85%) after Boc protection. Only a trace
amount of the desired compound 14 was obtained.
The fact that the C11 exocyclic double bond in related
indenoisoquinolines was stable under acidic conditions6,9
suggested that Boc might be an appropriate protecting
group for these amino groups. Therefore, the Fmoc in 10
was exchanged for Boc to yield 13 in 93% yield, which
was then again McMurry coupled with Boc-protected
aldehyde 156 generating 14 in 39% yield after Boc
reprotection (Scheme 2).9 The hydrochloride 4 was ob-
tained in quantitative yield by acidic Boc deprotection.
To shed some light on the unusual oxidative cleavage
of 12 under alkaline conditions, a model compound 18
was prepared to study the mechanism. McMurry coupling
between 9-fluorenone (16) and aldehyde 17, derived from
Swern oxidation of the corresponding alcohol, provided
the alkene 18 in 94% yield (Scheme 3). The near
quantitative yield of this cross-coupling reaction sug-
gested a titanocarbenoid intermediate.16 When the model
compound 18 was treated with piperidine in CHCl3 in
the presence of air at room temperature, in striking
contrast to compound 12, no decomposition was observed
even after stirring for 7 days. However, when the
stronger base LiOH was employed, appreciable oxidative
cleavage was observed after 4 days. In addition to the
recovered starting material 18 (73% conversion), four
products 16 (62%), 19 (32%), 20 (14%), and 21 (22%) were
Deprotection of Fmoc groups in 12 under basic conditions,
however, gave predominantly the oxidatively cleaved
SCHEME 4
J . Org. Chem, Vol. 69, No. 22, 2004 7497

Xiao et al.
SCHEME 5
F IGURE 2. B3LYP/6-31G(d,p) optimized geometries of com-
pounds 37 and 38.
experimental BDE of 416.8 kcal/mol for methane was
employed for calculations.22 All calculations were per-
formed in Gaussian0323 at the B3LYP/6-31G(d,p) level
of theory. The calculated BDE (298 K) is 318.7 kcal/mol
for 37, 327.5 kcal/mol for 38, and 330.0 kcal/mol for 27
after inclusion of thermal and zero-point corrections. On
the basis of these calculations, the proton indicated in
structure 37 is even more acidic than that in 27, which
is adjacent to a carbonyl and phenyl ring. The weaker
acidity of the proton indicated in 38 in comparison to that
in 37 may explain why compound 18 was left intact when
treated with a weak base such as piperidine while
compound 12 was reactive under the same conditions.
isolated from the reaction mixture (the yields are based
on recovered starting material) after careful silica gel
chromatography. When oxygen was stringently excluded
from the reaction mixture, no cleavage reaction was
observed even after stirring for 7 days.
A plausible mechanism for this unusual oxidative
cleavage is outlined in Scheme 4. Deprotonation of 18
generates an allylic anion 22. Autoxidation17,18 of anion
22 gives rise to peroxide 23, which undergoes a [2,3]
sigmatropic rearrangement (oxygen analogue of the [2,3]
Wittig rearrangement) and protonation yielding hydro-
peroxide 24. The carbon-oxygen bond formation that
occurs in the conversion of 22 to 23 is thought to be
initiated by a one-electron transfer from the carbanion
to triplet oxygen to form a carbon radical and the radical
anion of molecular oxygen, followed by short radical chain
reactions and/or radical pair cage-type reactions.19 Frag-
mentation of 24 produces 9-fluorenone (16) and an
R-hydroxy carbene species 25, rearrangement of which
provides ketone 27.20 A similar autoxidation and frag-
mentation of ketone 27 gives p-anisaldehyde (20).17,18 The
hydroperoxide 31 derived from protonation of peroxide
23 undergoes an oxygen-oxygen bond cleavage in the
presence of hydroxide, affording alcohol 19 and hydrogen
peroxide, the latter of which Baeyer-Villiger oxidizes the
ketone 27, resulting in the formation of p-methoxybenzyl
alcohol (21). It is possible, but not likely in this case, that
p-methoxybenzyl alcohol (21) is derived from Cannizzaro
reaction of p-anisaldehyde (20), because p-methoxyben-
zoic acid was not observed in the reaction mixture. The
intermediacy of ketone 27 instead of aldehyde 17 during
the course of oxidative cleavage is consistent with the
observation that only ketone 27 could produce a trace
amount (2%, isolated yield) of p-anisaldehyde (20), among
other products, when treated with LiOH in the presence
of air. Upon similar treatment, no p-anisaldehyde (20)
was generated from aldehyde 17. This also rules out such
mechanisms as shown in Scheme 521 involving the
formation of aldehyde 17 as the intermediate. Also in
agreement with this mechanism, compound 36, without
any allylic protons, did not show any decomposition under
the same conditions.
The final optimized structures of compounds 37 and
38 are shown in Figure 2. The exo-cyclic double bond in
37 is not coplanar with the aromatic ring system (the
dihedral angle of C1-C2-C3-H4 is 1.8° vs 0.0° in 38),
presumably as a result of the steric clash between the
olefinic proton H4 and the proton labeled “a” in Figure
2. The unusual geometry may contribute to the driving
force for the oxidative cleavage reaction to go to comple-
(15) Xu, G. Z.; Micklatcher, M.; Silvestri, M. A.; Hartman, T. L.;
Burrier, J .; Osterling, M. C.; Wargo, H.; Turpin, J . A.; Buckheit, R.
W.; Cushman, M. J . Med. Chem. 2001, 44, 4092-4113.
(16) Fu¨rstner, A.; Hupperts, A.; Ptock, A.; J anssen, E. J . Org. Chem.
1994, 59, 5215-5229.
(17) Doering, W. v. E.; Haines, R. M. J . Am. Chem. Soc. 1954, 76,
482-486.
(18) Gersmann, H. R.; Bickel, A. F. J . Chem. Soc. B 1971, 2230-
2237.
(19) Russell, G. A.; Bemis, A. G. J . Am. Chem. Soc. 1966, 88, 5491-
5497.
To gain further insight into the mechanism, the
relative acidities of the protons indicated in structures
27, 37, and 38 were compared by calculating their
respective heterolytic bond dissociation enthalpies (BDE)
derived from the isodesmic reaction shown in eq 1. An
(20) Tomioka, H.; Nunome, Y. J . Chem. Soc., Chem. Commun. 1990,
1243-1244.
(21) Nishinaga, A.; Itahara, T.; Shimizu, T.; Matsuura, T. J . Am.
Chem. Soc. 1978, 100, 1820-1825.
(22) Lias, S. G.; Bartmess, J . E.; Liebman, J . F.; Holmes, J . L.; Levin,
R. D.; Mallard, W. G. J . Phys. Chem. Ref. Data 1988, 17, Suppl. 1.
7498 J . Org. Chem., Vol. 69, No. 22, 2004

Autoxidative Cleavage of 9-Fluoredenes
tion to give the indenoisoquinoline compound in which
the whole system is highly conjugated and planar.
formation. A possible mechanism for this transformation
has been proposed on the basis of the experimental
results and computational details of a model study on
the 9-fluoredene compound 18.
Compound 4 was examined for antiproliferative activ-
ity against the human cancer cell lines in the National
Cancer Institute screen, in which the activity of the
compound was evaluated with approximately 55 different
cancer cell lines of diverse tumor origins. J udged by the
mean graph midpoint (MGM) value (1.38 µM), this
compound was cytotoxic, although it was less potent than
either 2 or 3 (see Supporting Information). Interestingly,
compound 4 has no capability to inhibit the topoi-
somerase I as seen in the topoisomerase I-mediated DNA
cleavage assay (data not shown).2 The cytotoxicity exerted
by compound 4 therefore has nothing to do with topoi-
somerase I inhibition. As a consequence of these results,
it can be concluded that the hypothetical ternary complex
involving compound 4 does not form, although the
evidence indicates quite clearly that similar complexes
result with both of the topoisomerase I inhibitors 2 and
3. Since a reasonable model of a ternary complex involv-
ing compound 4 can be derived by molecular modeling,
it seems likely that the additional side chain present in
4 inhibits the process of complex formation rather than
destabilizing the complex itself.
Exp er im en ta l Section
11-(3′-Am in op r op ylid en e)-6-(3′-a m in op r op yl)-5,6-d ih y-
dr o-2,3-dim eth oxy-8,9-m eth ylen edioxy-5-oxo-11H-in den o-
[1,2-c]isoqu in olin e Dih yd r och lor id e (4). HCl (2.0 M in
ether, 1 mL, 2 mmol) was added to a stirred solution of 14 (17
mg, 0.026 mmol) in CHCl3 (2 mL) at room temperature. The
resulting mixture was stirred at room temperature for an
additional 18 h, then filtered, and washed with CHCl3, yielding
a light yellow powder (13.5 mg, 100%): mp > 244 °C (dec); 1H
NMR (300 MHz, DMSO-d6) δ 8.11 (brs, 2 H), 7.89 (brs, 2 H),
7.70 (s, 1 H), 7.58 (s, 1 H), 7.52 (s, 1 H), 7.50 (s, 1 H), 6.98
(brt, 1 H), 6.16 (s, 2 H), 4.60 (t, J ) 5.7 Hz, 2 H), 4.02 (s, 3 H),
3.88 (s, 3 H), 3.35-3.44 (m, 2 H), 3.12-3.22 (m, 2 H), 2.85-
2.96 (m, 2 H), 2.05-2.15 (m, 2 H); ESIMS m/z (rel intensity)
450 (MH+, 100). Anal. Calcd for C25H29N3O5Cl2‚HCl‚0.95CH-
Cl3: C, 46.36; H, 4.64; N, 6.25. Found: C, 46.07; H, 5.23; N,
6.30.
cis-4-Car boxy-2-(9H-flu or en -9-yl-m eth oxycar bon ylam i-
n op r op yl)-3,4-d ih yd r o-6,7-d im et h oxy-3-(3,4-m et h ylen e-
d ioxyp h en yl)-1(2H)-isoqu in olon e (9). Et3N (1.35 mL, 9.72
mmol) and MgSO4 (5.0 g) were added to a stirred solution of
5 (2.3 g, 7.13 mmol) in CHCl3 (250 mL) at room temperature.
Piperonal (6) (972 mg, 6.48 mmol) was added, and the
resulting mixture was stirred for an additional 48 h. The
suspension was filtered and washed with CHCl3. The organic
solution was washed with water (2 × 30 mL) and brine (2 ×
30 mL), dried over Na2SO4, filtered, and concentrated, yielding
a colorless oil (2.97 g) containing 20% of piperonal based on
1H NMR. The mixture was used for next operation without
further purification: 1H NMR (300 MHz, CDCl3) δ 8.15 (s, 1
H), 7.69 (d, J ) 7.5 Hz, 2 H), 7.56 (d, J ) 7.5 Hz, 2 H), 7.39 (t,
J ) 7.5 Hz, 2 H), 7.34 (s, 1 H), 7.28 (t, J ) 7.5 Hz, 2 H), 7.10
(d, J ) 8.4 Hz, 1 H), 6.80 (d, J ) 8.4 Hz, 1 H), 5.94 (s, 2 H),
5.47 (brs, 1 H), 4.34 (d, J ) 6.9 Hz, 2 H), 4.19 (t, J ) 6.9 Hz,
1 H), 3.64 (t, J ) 6.3 Hz, 2 H), 3.36 (q, J ) 6.0 Hz, 2 H), 1.88
(quin, J ) 6.3 Hz, 2 H). Homophthalic anhydride derivative 8
(1.42 g, 6.38 mmol) was added to a stirred solution of imine 7
(2.97 g, containing 20% piperonal, 6.38 mmol) in CHCl3 (20
mL) at room temperature. The resulting mixture was further
stirred at room temperature for 12 h. Et2O (15 mL) was added,
and the precipitate was collected by filtration. The residue was
washed with CHCl3, yielding a light yellow solid (2.07 g,
50%): mp 212-214 °C; 1H NMR (300 MHz, DMSO-d6) δ 7.87
(d, J ) 7.5 Hz, 2 H), 7.66 (d, J ) 7.5 Hz, 2 H), 7.55 (s, 1 H),
7.40 (t, J ) 7.5 Hz, 2 H), 7.31 (t, J ) 7.5 Hz, 2 H), 7.14 (s, 1
H), 6.74 (d, J ) 8.4 Hz, 1 H), 6.52 (d, J ) 8.4 Hz, 1 H), 6.45 (s,
1 H), 5.92 (s, 2 H), 4.99 (d, J ) 6.0 Hz, 1 H), 4.56 (d, J ) 6.0
Hz, 1 H), 4.32 (d, J ) 6.6 Hz, 2 H), 4.19 (t, J ) 6.6 Hz, 1 H),
3.81 (s, 3 H), 3.74 (s, 3 H), 2.99 (brs, 2 H), 2.75 (brs, 2 H), 1.64
(brs, 2 H); ESIMS (rel intensity) m/z 651 (MH+, 100). Anal.
Calcd for C37H34N2O9‚0.45CHCl3: C, 63.86; H, 4.93; N, 3.98.
Found: C, 63.99; H, 5.17; N, 3.72.
In conclusion, a potential DNA-threading agent was
designed and synthesized on the basis of the hypothetical
binding model of indenoisoquinolines in the DNA-topo-
isomerase I “cleavage complex”. Although the designed
compound 4 showed cytotoxicity across different cancer
cell lines, it had no topoisomerase I inhibitory activity.
The overall outcome of the oxidative alkene cleavage
reaction of 18 to afford 16 and intermediate 27 is similar
to that observed in the photochemically induced 2 + 2
addition of alkenes with singlet oxygen to form dioxet-
anes that cleave to two carbonyl compounds.24-31 How-
ever, the reactions clearly proceed by different mecha-
nisms, since the reactions of 12 and 18 involve triplet
oxygen instead of singlet oxygen. The presently reported
alkaline autoxidative cleavage reaction of the C11 alkenyl
side chain in the indenoisoqinoline 12 is a novel trans-
(23) Frisch, M. J .; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J . R.; Montgomery, J . A., J r.; Vreven, T.;
Kudin, K. N.; Burant, J . C.; Millam, J . M.; Iyengar, S. S.; Tomasi, J .;
Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson,
G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.;
Hasegawa, J .; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai,
H.; Klene, M.; Li, X.; Knox, J . E.; Hratchian, H. P.; Cross, J . B.; Adamo,
C.; J aramillo, J .; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin,
A. J .; Cammi, R.; Pomelli, C.; Ochterski, J . W.; Ayala, P. Y.; Morokuma,
K.; Voth, G. A.; Salvador, P.; Dannenberg, J . J .; Zakrzewski, V. G.;
Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.;
Rabuck, A. D.; Raghavachari, K.; Foresman, J . B.; Ortiz, J . V.; Cui,
Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J .; Stefanov, B. B.; Liu, G.;
Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J .;
Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challa-
combe, M.; Gill, P. M. W.; J ohnson, B.; Chen, W.; Wong, M. W.;
Gonzalez, C.; Pople, J . A. Gaussian 03, Revision B.05 ed.; Gaussian,
Inc: Pittsburgh, PA, 2003.
6-(9H-Flu or en -9-yl-m eth oxyca r bon yla m in op r op yl)-2,3-
d im et h oxy-5,11-d ioxo-8,9-m et h ylen ed ioxy-11H -in d en o-
[1,2-c]isoqu in olin e (10). Thionyl chloride (5 mL) was slowly
added to the acid 9 (300 mg, 0.46 mmol) with stirring. The
resulting mixture was then stirred for an additional 6 h. Excess
thionyl chloride was removed under reduced pressure. Benzene
(2 × 5 mL) was added, and the mixture was concentrated in
vacuo. The residue was then subjected to silica gel (40 g) flash
column chromatography, eluting with CHCl3 to obtain a dark
red solid (160 mg, 55%): mp 175-177 °C; 1H NMR (300 MHz,
DMSO-d6) δ 7.86 (d, J ) 7.5 Hz, 2 H), 7.84 (s, 1 H), 7.67 (d, J
) 7.5 Hz, 2 H), 7.46 (s, 1 H), 7.38 (t, J ) 7.5 Hz, 2 H), 7.30 (t,
J ) 7.5 Hz, 2 H), 7.23 (s, 1 H), 7.05 (s, 1 H), 6.14 (s, 2 H), 4.38
(brs, 2 H), 4.30 (d, J ) 6.6 Hz, 2 H), 4.20 (t, J ) 6.6 Hz, 1 H),
3.88 (s, 3 H), 3.83 (s, 3 H), 3.14 (brs, 2 H), 1.98 (brs, 2 H);
(24) Foote, C. S. Pure Appl. Chem. 1971, 27, 635-645.
(25) Kearns, D. R. Chem. Rev. 1971, 71, 395-427.
(26) Bartlett, P. D. Chem. Soc. Rev. 1976, 5, 149-163.
(27) Adam, W. Adv. Heterocycl. Chem. 1977, 21, 437-481.
(28) Frimer, A. A. Chem. Rev. 1979, 79, 359-387.
(29) Adam, W.; Encarnacio´n, L. A. A. Chem. Ber. 1982, 115, 2592-
2605.
(30) Adam, W.; Cilento, G. Angew. Chem., Int. Ed. Engl. 1983, 22,
529-542.
(31) Adam, W.; Baader, W. J . Angew. Chem., Int. Ed. Engl. 1984,
23, 166-167.
J . Org. Chem, Vol. 69, No. 22, 2004 7499

Xiao et al.
ESIMS (rel intensity) m/z 653 (MNa+,100). Anal. Calcd for
(2 × 15 mL), H2O (2 × 15 mL), and brine (2 × 15 mL). The
organic solution was dried over anhydrous Na2SO4, filtered,
and concentrated, and the residue was subjected to silica gel
(40 g) flash column chromatography, eluting with CHCl3,
C
37H30N2O8‚1.4H2O: C, 67.76; H, 5.04; N, 4.27. Found: C,
67.78; H, 5.15; N, 3.93.
11-[3′-(9H -F lu o r e n y lm e t h o x y c a r b o n y l)a m in o p r o -
p ylid en e]-6-[3′-(9H -flu or en ylm et h oxyca r b on yl)a m in o-
p r op yl]-5,6-d ih yd r o-2,3-d im eth oxy-8,9-m eth ylen ed ioxy-
5-oxo-11H-in d en o[1,2-c]isoqu in olin e (12). TiCl4-THF (1:
2) complex (160 mg, 0.48 mmol) and zinc dust (63 mg, 0.96
mmol) were put in a two-necked round-bottomed flask. THF
(7 mL) was added. The resulting suspension was heated under
reflux for 4 h. At this point, a mixture of aldehyde 1115 (56
mg, 0.19 mmol) and indenoisoquinoline 10 (100 mg, 0.16 mmol)
in THF (7 mL) was added via syringe. The reaction mixture
was stirred under reflux for an additional 4 h, and then 4 N
HCl (5 mL) was added to the reaction mixture at 0 °C. The
resulting mixture was stirred at 0 °C for 1 h. CHCl3 (3 × 60
mL) was used to extract the product, and the combined organic
layers were washed with H2O (2 × 10 mL) and brine (2 × 10
mL), dried over Na2SO4, filtered, and concentrated. The
residue was subjected to silica gel (80 g) flash column chro-
matography, eluting with CHCl3, giving a yellow powder (89
mg, 63%): mp 184-186 °C; 1H NMR (300 MHz, CDCl3) δ 7.19-
7.68 (m, 20 H), 6.89 (t, J ) 6.0 Hz, 1 H), 6.14 (t, J ) 6.9 Hz,
1 H, exchangeable with D2O), 5.98 (s, 2 H), 5.12 (brs, 1 H),
4.57 (brs, 2 H), 4.37 (d, J ) 6.9 Hz, 4 H), 4.22 (t, J ) 6.9 Hz,
2 H), 4.00 (s, 3 H), 3.95 (s, 3 H), 3.58 (brs, 2 H), 3.25 (brs, 2
H), 3.04 (brs, 2 H), 2.03 (brs, 2 H); ESIMS m/z (rel intensity)
894 (MH+, 100). Anal. Calcd for C55H47N3O9‚0.85H2O: C, 72.65;
H, 5.40; N, 4.62. Found: C, 73.02; H, 6.17; N, 4.60.
1
yielding a yellow powder (50 mg, 39%): mp 193-195 °C; H
NMR (300 MHz, CDCl3) δ 7.84 (s, 1 H), 7.50 (s, 1 H), 7.32 (s,
1 H), 7.22 (s, 1 H), 6.93 (t, J ) 6.9 Hz, 1 H), 6.07 (s, 2 H), 5.72
(brs, 1 H, exchangeable with D2O), 4.70 (brs, 1 H, exchangeable
with D2O), 4.62 (t, J ) 7.2 Hz, 2 H), 4.07 (s, 3 H), 4.01 (s, 3 H),
3.51 (q, J ) 5.7 Hz, 2 H), 3.18 (q, J ) 5.7 Hz, 2 H), 3.02 (q, J
) 6.6 Hz, 2 H), 2.04 (quin, J ) 5.7 Hz, 2 H), 1.44 (s, 9 H), 1.38
(s, 9 H); ESIMS m/z (rel intensity) 650 (MH+, 100). Anal. Calcd
for C35H43N3O9: C, 64.70; H, 6.67; N, 6.47. Found: C, 64.53;
H, 6.45; N, 6.22.
3-(4-Meth oxyp h en yl)-p r op a n a l (17). A solution of DMSO
(1.4 mL, 20 mmol) in CH2Cl2 (5 mL) was added to a stirred
solution of oxalyl chloride (872 µL, 10 mmol) in CH2Cl2 (20
mL) at -78 °C over 30 min. Upon completion of the addition,
the mixture was stirred at -78 °C for 5 min, followed by
addition of a solution of 3-(4-methoxyphenyl)-1-propanol (1.48
g, 5 mmol) in CH2Cl2 (10 mL) over 30 min at -78 °C. The
resulting mixture was stirred at -78 °C for 40 min. Then, Et3N
(4.2 mL, 30 mmol) was added dropwise over 10 min. The
resulting mixture was allowed to warm to 0 °C and stirred at
0 °C for 1 h. Water (10 mL) was added to quench the reaction.
CHCl3 (200 mL) was added to dilute the reaction mixture. The
organic layer was then separated from the water layer, which
was further washed with water (2 × 15 mL) and brine (2 ×
15 mL). The organic layer was dried over anhydrous Na2SO4,
filtered, and concentrated in vacuo. The residue was subjected
to silica gel (80 g) flash chromatography, eluting with n-hex-
anes-EtOAc (10:1), yielding a colorless oil (800 mg, 98%): 1H
NMR (300 MHz, CDCl3) δ 9.77 (brs, 1 H), 7.09 (d, J ) 8.4 Hz,
2 H), 6.82 (d, J ) 8.4 Hz, 2 H), 3.75 (s, 3 H), 2.88 (t, J ) 7.2
Hz, 2 H), 2.71 (t, J ) 6.9 Hz, 2 H); 13C NMR (75 MHz, CDCl3)
δ 201.7, 158.0, 132.2, 129.1 (2 C), 113.9 (2 C), 55.1, 45.4, 27.1;
EIMS (rel intensity) m/z 164 (M+, 49), 121 (100). Anal. Calcd
for C10H12O2: C, 73.15; H, 7.37. Found: C, 73.26; H, 7.35.
9-[3′-(4-Meth oxyp h en yl)-p r op ylid en e]-flu or en on e (18).
TiCl4-THF (1:2) complex (1.0 g, 3.0 mmol) and zinc dust (390
mg, 6.0 mmol) were put in a two-necked round-bottomed flask.
THF (15 mL) was added. The resulting suspension was heated
under reflux for 4 h. A mixture of aldehyde 17 (197 mg, 1.2
mmol) and 9-fluorenone (16) (180 mg, 1.0 mmol) in THF (10
mL) was added via syringe. The reaction mixture was stirred
under reflux for an additional 3 h. Then, 4 N HCl (15 mL)
was added to the reaction mixture at 0 °C, and the reaction
mixture was stirred at 0 °C for 1 h. EtOAc (150 mL) was added
to dilute the reaction mixture, which was then washed with
water (2 × 15 mL) and brine (2 × 15 mL). The organic solution
was dried over anhydrous Na2SO4, filtered, and concentrated,
and the residue was subjected to silica gel (40 g) flash column
chromatography, eluting with n-hexanes-EtOAc (20:1), yield-
ing a solid as colorless needles (292 mg, 94%): mp 69-70 °C;
1H NMR (300 MHz, CDCl3) δ 7.78 (d, J ) 7.2 Hz, 1 H), 7.68
(d, J ) 7.2 Hz, 1 H), 7.63 (d, J ) 7.5 Hz, 1 H), 7.57 (d, J ) 7.5
Hz, 1 H), 7.18-7.32 (m, 4 H), 7.15 (d, J ) 8.1 Hz, 2 H), 6.82
(d, J ) 8.1 Hz, 2 H), 6.69 (t, J ) 7.2 Hz, 1 H), 3.72 (s, 3 H),
3.07 (q, J ) 7.5 Hz, 2 H), 2.87 (t, J ) 7.5 Hz, 2 H); ESIMS (rel
intensity) m/z 313 (MH+, 100). Anal. Calcd for C23H20O‚
0.15H2O: C, 87.67; H, 6.49. Found: C, 87.58; H, 6.54.
6-(3′-ter t-Bu t oxyca r b on yla m in op r op yl)-5,6-d ih yd r o-
2,3-dim eth oxy-8,9-m eth ylen edioxy-5,11-dioxo-11H-in den o-
[1,2-c]isoqu in olin e (13). Fmoc-protected indenoisoquinoline
derivative 10 (400 mg, 0.64 mmol) was dissolved in CHCl3-
piperidine (1:1, 60 mL) and stirred at room temperature for
24 h. The solvent was removed under reduced pressure, and
the residue was subjected to a short silica gel column, eluting
with CHCl3, to remove the side product derived from the Fmoc
moiety. The product was eluted by CHCl3-MeOH-NH3‚H2O
(100:30:5). The crude product (260 mg) was dissolved in CHCl3
(20 mL), and then Et3N (0.18 mL, 1.28 mmol) and Boc2O (209
mg, 0.96 mmol) were added sequentially to the stirred solution
at 0 °C. The resulting mixture was allowed to warm to room
temperature and stirred at room temperature for 20 h. CHCl3
(150 mL) was added to dilute the reaction mixture, which was
then washed with 1 N HCl (2 × 10 mL), water (2 × 10 mL),
and brine (2 × 10 mL). The organic solution was dried over
anhydrous Na2SO4, filtered, and concentrated, and the residue
was subjected to silica gel (40 g) flash column chromatography,
eluting with CHCl3, yielding a purple powder (301 mg, 93%):
1
mp 215-217 °C; H NMR (300 MHz, CDCl3) δ 8.03 (s, 1 H),
7.63 (s, 1 H), 7.07 (s, 1 H), 7.00 (s, 1 H), 6.08 (s, 2 H), 4.52 (t,
J ) 7.2 Hz, 2 H), 4.03 (s, 3 H), 3.98 (s, 3 H), 3.20-3.30 (m, 2
H), 1.97-2.08 (m, 2 H), 1.44 (s, 9 H); CIMS m/z (rel intensity)
508 (M+,100). Anal. Calcd for C27H28N2O8‚0.55H2O: C, 62.55;
H, 5.66; N, 5.40. Found: C, 62.44; H, 5.61; N, 5.45.
11-[3′-ter t-Bu toxycar bon ylam in opr opyliden e]-6-(3′-ter t-
bu toxycar bon ylam in opr opyl)-5,6-dih ydr o-2,3-dim eth oxy-
8,9-m et h ylen ed ioxy-5-oxo-11H -in d en o[1,2-c]isoq u in o-
lin e (14). TiCl4-THF (1:2) complex (200 mg, 0.60 mmol) and
zinc dust (78 mg, 1.2 mmol) were put in a three-necked round-
bottomed flask. THF (7 mL) was added. The resulting suspen-
sion was heated under reflux for 4 h. At this point, a mixture
of Boc-protected â-alaninal (15)6 (42 mg, 0.24 mmol) and
indenoisoquinoline 13 (100 mg, 0.20 mmol) in THF (7 mL) was
added via syringe. The reaction mixture was stirred under
reflux for an additional 4 h. HCl (4 N, 5 mL) was added to the
reaction mixture at 0 °C, and the reaction mixture was further
stirred at 0 °C for 1 h. Anhydrous K2CO3 was added to
neutralize HCl at 0 °C until pH > 7. Boc2O (536 mg, 2 mmol)
was added to the reaction mixture, and the reaction mixture
was stirred overnight. CHCl3 (200 mL) was added to dilute
the reaction mixture, which was then washed with 1 N HCl
Oxid a tive Clea va ge of 9-[3′-(4-Meth oxyp h en yl)-p r op yl-
id en e]-flu or en on e (18). LiOH‚H2O (107 mg, 2.6 mmol) was
added to a stirred solution of 18 (80 mg, 0.26 mmol) in THF-
MeOH-H2O (2:2:1, 12.5 mL) at room temperature. The
reaction mixture was stirred at room temperature for 4 days.
During the course of the reaction, air was allowed to enter
the reaction system. EtOAc (100 mL) was added to dilute the
reaction mixture, which was washed with HCl (1 N, 2 × 10
mL), H2O (2 × 10 mL), and brine (2 × 10 mL). The organic
solution was dried over anhydous Na2SO4, filtered, and
concentrated, and the residue was subjected to silica gel (40
7500 J . Org. Chem., Vol. 69, No. 22, 2004

Autoxidative Cleavage of 9-Fluoredenes
g) flash column chromatography, gradiently eluting with
n-hexane to n-hexanes-EtOAc (3:1), yielding starting material
18 (21.5 mg, 27%), 16 (21 mg, 62% brsm), 19 (20 mg, 32%
brsm), 20 (3.7 mg, 14% brsm), and 21 (5.7 mg, 22% brsm).
Compound 19 was obtained as a light yellow oil: 1H NMR (300
MHz, CDCl3) δ 7.75 (d, J ) 7.5 Hz, 1 H), 7.69 (d, J ) 7.5 Hz,
1 H), 7.63 (t, J ) 7.5 Hz, 2 H), 7.27-7.36 (m, 4 H), 7.21 (d, J
) 8.1 Hz, 2 H), 6.85 (d, J ) 8.1 Hz, 2 H), 6.66 (d, J ) 8.1 Hz,
1 H), 5.41 (ddd, J ) 8.4, 8.1, 4.2 Hz, 1 H), 3.75 (s, 3 H), 3.03-
3.09 (m, 1 H), 2.89-2.97 (m, 1 H); 13C NMR (75 MHz, CDCl3)
δ 158.6, 141.3, 139.2, 138.9, 136.3, 136.1, 130.8, 130.6, 129.1,
128.5, 128.3, 127.2, 125.2, 120.3, 120.0, 119.6, 114.2, 69.5, 55.3,
42.5; HRCIMS m/z calcd for C23H20O2 + H 329.1542, found
329.1549.
energies. Compounds 27, 37, 38, and their corresponding
anions were optimized at the B3LYP/6-31G(d,p) level.
Ack n ow led gm en t. This work was made possible by
the National Institutes of Health (NIH) through support
of this work with Research Grant UO1 CA089566. The
in vitro testing was conducted through the Develop-
mental Therapeutics Program, DCTD, NCI under Con-
tract NO1-CO-56000.
Su p p or tin g In for m a tion Ava ila ble: Cytotoxicity data of
compound 4 in all of the tested cell lines, experimental
procedure for compound 36, and all B3LYP/6-31G(d,p) opti-
mized structures. This material is available free of charge via
the Internet at http://pubs.acs.org.
DTF Ca lcu la tion s. All the DFT studies were carried out
in Gaussian03.23 Frequency calculations were included to
confirm the true minimum and correct thermal and zero-point
J O048808F
J . Org. Chem, Vol. 69, No. 22, 2004 7501